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Abbreviated Title: IL-15 + Avelumab in ccRCC
NIH Protocol \#: 20C0007 CTEP Protocol \#: 10284 Version Date: 06/02/2021 NCT Number: NCT04150562 Phase II Trial of Avelumab (Bavencio®) with IL-15 in Subjects with Clear-Cell Renal Carcinoma  
| NCI Principal Investigator:   | Kevin Conlon, M.D., MS A-F Lymphoid Malignancies Branch (LYMB) Center for Cancer Research (CCR) National Cancer Institute (NCI) Building 10, Room 3B38 National Institutes of Health (NIH) Bethesda, MD 20892 Phone: 240-541-4559 Email: conlonkc@mail.nih.gov   |
|-------------------------------|------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|
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investigational agents

| Investigational Agents:  Drug Name: Avelumab (MSB0010718C, Bavencio®,  NSC #799232)   | Recombinant human IL-15 (rhIL15-NSC #745101)                                                             |                                                                                                              |
|---------------------------------------------------------------------------------------|-------------------------------------------------------------|--------------------------------------------------------------------------------------------------------------|
| IND Number:                                                                           | 100820                                                      |                                                                                                              |
| Sponsor:                                                                              | Center for Cancer Research, NCI                             |                                                                                                              |
| Manufacturer:                                                                         | EMD Serono, Inc. (Company Study/Tracking #:  MS100070-0207) | Biopharmaceutical Development  Program (BDP)/Leidos  Biomedical Research, Inc. under  contract with DCTD,NCI |
| Supplier:                                                                             | EMD Serono, Inc.                                            | Cancer Therapy Evaluation  Program (CTEP), Division of  Cancer Treatment and Diagnosis  (DCTD), NCI          |  
Commercial Agents: None 1
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precis

Background:  
Clear-cell renal cell carcinoma (ccRCC) is among the 10 most frequent diagnostic cancers in the United States with more than an estimated 62,000 new cases in 2016. The prognosis for patients with metastatic disease is poor with survival rates of 8%.  
The immunologic effects of recombinant human Interleukin-15 (rhIL-15), a stimulatory cytokine that promotes the differentiation and activation of NK cells, monocytes and longterm CD8+ memory T-cells, has been assessed in several Phase 1 trials in cancer patients.  
Avelumab is an anti-programmed death ligand-1 (PD-L1) fully human IgG1 antibody that inhibits PD1/PD-L1 interactions while leaving the PD1/PD-L2 pathway intact and enhances immune activation against tumor cells. It has received U.S. FDA accelerated approval for the treatment of patients with metastatic Merkel cell carcinoma (MCC) and urothelial carcinoma.  
Unlike other approved anti-PD-L1/PD1 antibodies, avelumab induces lysis of tumor cells via antibody-dependent cell-mediated cytotoxicity (ADCC), indicating an additional mechanism of action. However, avelumab has not shown ADCC against normal immune cell subsets in humans.  
More than 50% of ccRCC is PD-L1+ with higher expression in unfavorable prognostic tumors. Since the anti-PD-L1 antibody avelumab has shown ADCC activity *in vitro*, agents that may enhance ADCC by increasing number and activity of Fc-binding effector cells - such as rhIL15 - could improve efficacy of avelumab in this disease.
Objectives:
Determine the efficacy of combined continuous intravenous infusion (CIV) rhIL-15 and avelumab treatment in patients with anti-PD-1/PD-L1 refractory metastatic clear cell renal carcinoma (ccRCC) by assessing the overall response rate Eligibility:  
Age ≥ 18 years of age ECOG performance status of ≤ 1 Histologically proven metastatic clear cell renal carcinoma with ≥ 5% expression of PDL1 in the tumor area confirmed by IHC
Patients must have failed or relapsed and have progressive disease after at least 2 prior therapies that include multityrosine kinase inhibitor like axitinib or sunitinib and an antiPD1 or PD-L1 immune checkpoint inhibitor therapy like nivolumab which could have been administered in combination with an anti-CTLA4 agent like ipilimumab Adequate organ and marrow function
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design

Open-label, single-center, non-randomized Phase II study  
Safety Run-in Cohort with 3-6 patients at dose level 2mcg/kg and 4mcg/kg CIV IL-15
(recommended phase II dose) will ensure safety of recommended phase II dose rhIL-15 with fixed dose avelumab with Dose Expansion Cohort at 4mcg/kg dose level Efficacy of the combination will be assessed in a Simon two-stage phase II design with 9 or 17 patients depending on demonstration of clinical activity in the initial group of 9 patients Maximum 4 cycles (28-day cycle) of combination therapy To explore both Safety Run-in Cohort and further evaluation in a Dose Expansion Cohort, the accrual ceiling will be set at 25 patients
3  
| TABLE OF CONTENTS                                                                                                                                       |                                                                                                                                                             |                                                                                                                                                      |
|---------------------------------------------------------------------------------------------------------------------------------------------------------|-------------------------------------------------------------------------------------------------------------------------------------------------------------|------------------------------------------------------------------------------------------------------------------------------------------------------|
| PRÉCIS                                                                                                                                                  |                                                                                                                                                             |                                                                                                                                                      |
| TABLE OF CONTENTS ....................................................................................................................................4 |                                                                                                                                                             |                                                                                                                                                      |
| STATEMENT OF COMPLIANCE                                                                                                                                 |                                                                                                                                                             |                                                                                                                                                      |
| l                                                                                                                                                       | INTRODUCTION .............................................................................................................................................8 |                                                                                                                                                      |
| 1.1                                                                                                                                                     | Study Objectives                                                                                                                                            |                                                                                                                                                      |
| 1.2                                                                                                                                                     | Background and Rationale .....................................................................................................8                             |                                                                                                                                                      |
| 1.3                                                                                                                                                     | Considerations and Rationale for Phase II Design                                                                                                            |                                                                                                                                                      |
| ELIGIBILITY ASSESSMENT AND ENROLLMENT ........................................................................21                                        |                                                                                                                                                             |                                                                                                                                                      |
| 2                                                                                                                                                       | 2.1                                                                                                                                                         | Eligibility Criteria..............................................................................................................................21 |
| 2.2                                                                                                                                                     | Strategies for Recruitment and Retention .............................................................................24                                    |                                                                                                                                                      |
| Screening Evaluation                                                                                                                                    |                                                                                                                                                             |                                                                                                                                                      |
| 2.3                                                                                                                                                     |                                                                                                                                                             |                                                                                                                                                      |
| 2.4                                                                                                                                                     | Participant Registration and Status Update Procedures .....................................................26                                               |                                                                                                                                                      |
| 2.5                                                                                                                                                     | Baseline Evaluation                                                                                                                                         |                                                                                                                                                      |
| STUDY IMPLEMENTATION .                                                                                                                                  |                                                                                                                                                             |                                                                                                                                                      |
| 3                                                                                                                                                       | Study Design .....................................................................................................................................28        |                                                                                                                                                      |
| 3.1                                                                                                                                                     |                                                                                                                                                             |                                                                                                                                                      |
| 3.2                                                                                                                                                     | Drug Administration                                                                                                                                         |                                                                                                                                                      |
| 3.3                                                                                                                                                     | Dose Modifications .                                                                                                                                        |                                                                                                                                                      |
| Study Intervention Compliance                                                                                                                           |                                                                                                                                                             |                                                                                                                                                      |
| 3.4                                                                                                                                                     | ON Study Evaluations                                                                                                                                        |                                                                                                                                                      |
| 3.5                                                                                                                                                     |                                                                                                                                                             |                                                                                                                                                      |
| 3.6                                                                                                                                                     | Post-Treatment Evaluations ..............................................................................................42                                 |                                                                                                                                                      |
| 3.7                                                                                                                                                     | Study Calendar .......................................................................................................................43                    |                                                                                                                                                      |
| Cost And Compensation.........................................................................................................43                        |                                                                                                                                                             |                                                                                                                                                      |
| 3.8                                                                                                                                                     |                                                                                                                                                             |                                                                                                                                                      |
| 3.9                                                                                                                                                     | Criteria for Removal from Protocol Therapy and Off Study Criteria ...........................44                                                             |                                                                                                                                                      |
| CONCOMITANT MEDICATIONS/MEASURES.....................................................................45                                                 |                                                                                                                                                             |                                                                                                                                                      |
| 4                                                                                                                                                       | 4.1                                                                                                                                                         | Acceptable Medications .....................................................................................................45                       |
| Prohibited Medications                                                                                                                                  |                                                                                                                                                             |                                                                                                                                                      |
| 4.2                                                                                                                                                     |                                                                                                                                                             |                                                                                                                                                      |
| s                                                                                                                                                       | CORRELATIVE STUDIES ..................................................................................................................45                    |                                                                                                                                                      |
| 5.1                                                                                                                                                     | SUMMARY                                                                                                                                                     |                                                                                                                                                      |
| 5.2                                                                                                                                                     | Sample Collection and Processing ......................................................................................46                                   |                                                                                                                                                      |
| Biomarker and Research Methods ....................................................................................49                                   |                                                                                                                                                             |                                                                                                                                                      |
| 5.3                                                                                                                                                     |                                                                                                                                                             |                                                                                                                                                      |
| 5.4                                                                                                                                                     | Sample Storage, Tracking, and Disposition .............................................................................50                                   |                                                                                                                                                      |
| 4                                                                                                                                                       |                                                                                                                                                             |                                                                                                                                                      |  
| Version Date: 06/02/2021                                                                                                                               |                                                                                                                                             |                 |
|--------------------------------------------------------------------------------------------------------------------------------------------------------|---------------------------------------------------------------------------------------------------------------------------------------------|-----------------|
| DATA COLLECTION AND EVALUATION .                                                                                                                       |                                                                                                                                             |                 |
| 6                                                                                                                                                      | 6.1                                                                                                                                         | DATA COLLECTION |
| 6.2                                                                                                                                                    | DATA Sharing Plans .                                                                                                                        |                 |
| 6.3                                                                                                                                                    | Response Criteria                                                                                                                           |                 |
| 6.4                                                                                                                                                    | Immune-related Response Criteria (irRECIST) .........................................................................59                     |                 |
| Toxicity Criteria ......................................................................................................................60             |                                                                                                                                             |                 |
| 6.5                                                                                                                                                    |                                                                                                                                             |                 |
| 7                                                                                                                                                      | NIH REPORTING REQUIREMENTS/DATA AND SAFETY MONITORING PLAN.....60                                                                           |                 |
| Definitions ........................................................................................................................................60 |                                                                                                                                             |                 |
| 7.1                                                                                                                                                    |                                                                                                                                             |                 |
| 7.2                                                                                                                                                    | OHSRP Office of Compliance and Training ..........................................................................60                        |                 |
| 7.3                                                                                                                                                    | NIH Required Data and Safety Monitoring Plan ................................................................60                             |                 |
| 8                                                                                                                                                      | SPONSOR SAFETY REPORTING                                                                                                                    |                 |
| 8.1                                                                                                                                                    | Definitions                                                                                                                                 |                 |
| 8.2                                                                                                                                                    | Assessment of Safety Events                                                                                                                 |                 |
| 8.3                                                                                                                                                    | Reporting of Serious Adverse Events .....................................................................................62                 |                 |
| Waiver of expedited reporting to CCR..................................................................................63                               |                                                                                                                                             |                 |
| 8.4                                                                                                                                                    | Safety Reporting to the Pharmaceutical Collaborators .....................................................63                                |                 |
| 8.5                                                                                                                                                    |                                                                                                                                             |                 |
| 8.6                                                                                                                                                    | Reporting Pregnancy .....................................................................................................................63 |                 |
| 8.7                                                                                                                                                    | Regulatory Reporting for Studies Conducted Under CCR-Sponsored IND ...............64                                                        |                 |
| 9                                                                                                                                                      | CLINICAL MONITORING                                                                                                                         |                 |
| 10                                                                                                                                                     | STATISTICAL CONSIDERATIONS ...........................................................................................64                    |                 |
| 10.1                                                                                                                                                   | Statistical Hypotheses                                                                                                                      |                 |
| 10.2                                                                                                                                                   | Sample size determination                                                                                                                   |                 |
| Population for Analyses ..............................................................................................................66               |                                                                                                                                             |                 |
| 10.3                                                                                                                                                   | Statistical Analyses                                                                                                                        |                 |
| 10.4                                                                                                                                                   |                                                                                                                                             |                 |
| COLLABORATIVE AGREEMENTS .                                                                                                                             |                                                                                                                                             |                 |
| 111111                                                                                                                                                 | Cooperative Research and Development Agreement (CRADA) - EMD Serono .......67                                                               |                 |
| 1.1                                                                                                                                                    |                                                                                                                                             |                 |
| 12  HUMAN SUBJECTS PROTECTIONS ...................................................................................................67                   |                                                                                                                                             |                 |
| 12.1                                                                                                                                                   | Rationale For Subject Selection ................................................................................................67          |                 |
| 12.2                                                                                                                                                   | Participation of Children ........................................................................................................67        |                 |
| Risk/Benefit Assessment...........................................................................................................67                   |                                                                                                                                             |                 |
| 12.3                                                                                                                                                   | Consent Process and Documentation .................................................................................68                       |                 |
| 12.4                                                                                                                                                   |                                                                                                                                             |                 |
| 13                                                                                                                                                     | REGULATORY AND OPERATIONAL CONSIDERATIONS ................................................69                                                |                 |  
| 13.1   | Study Discontinuation and Closure 69                                |
|--------|---------------------------------------------------------------------|
| 13.2   | Quality Assurance and Quality Control 69                            |
| 13.3   | Conflict of Interest Policy 69                                      |
| 13.4   | Confidentiality and Privacy 70                                      |
| 14     | PHARMACEUTICAL INFORMATION 70                                       |
| 14.1   | rhIL-15 (NSC #745101) IND# 100820 70                                |
| 14.2   | Avelumab IND#100820 (NSC #799232) 75                                |
| 15     | REFERENCES 83                                                       |
| 16     | APPENDICES 92                                                       |
| 16.1   | APPENDIX A: Performance Status Criteria 92                          |
| 16.2   | APPENDIX B: Assay for ADCC 93                                       |
| 16.3   | APPENDIX C: IL-15 Dilution Instructions 94                          |
| 16.4   | APPENDIX D: Assay For Antibodies To rhIL-15 96                      |
| 16.5   | APPENDIX E: Modified Immune-Related Response Criteria (irRECIST) 97 |
| 16.6   | APPENDIX F: Study Calendar 98                                       |
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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

1.1.1 Primary Objective Determine the efficacy of combined continuous intravenous infusion (CIV) rhIL-15 and avelumab treatment in patients with anti-PD-1/PD-L1 refractory metastatic clear cell renal carcinoma (ccRCC) by assessing the overall response rate.  
1.1.2 Secondary Objectives
Evaluate duration of response, progression-free survival and overall survival following combined CIV IL-15 and avelumab treatment Assess the safety and tolerability of combined CIV IL-15 and avelumab treatment Analyze the changes in peripheral blood lymphocyte subsets before and after treatment with rhIL-15 and avelumab Define the effects of rhIL-15 on the antibody-dependent cell-mediated cytotoxicity
(ADCC) mediated by avelumab, using ex-vivo peripheral blood mononuclear cells (PBMCs)
Examine treatment related changes in tumor deposits by immunohistochemical and molecular analysis of core biopsies obtained before and during treatment with rhIL-15 and avelumab
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1.2 background and rationale

1.2.1 Disease Characteristics of Clear-cell Renal Carcinoma Clear-cell renal cell carcinoma (ccRCC) is among the 10 most frequent diagnostic cancers in the United States with more than an estimated 62,000 new cases in 2016 [1, 2]. The prognosis for patients with metastatic disease is poor with survival rates of 8% [3, 4]. ccRCC is associated with mutations of VHL, an essential component of the cellular oxygen pathway. VHL, located on chromosome 3p, is inactivated by a mutation in 52% of ccRCC [5]. Several other genes show mutations at lower frequencies than VHLs. These genes include PBRM1 (40%), SETD2 (15%) and BAP1 (15%) which are all part of the chromatin remodeling histone methylation pathway [6]. Like VHL, these three genes are located within a 50-Mb region on the short arm of chromosome 3p.  
1.2.2 Systemic Treatment of ccRCC
In the past 10 years treatment options for metastatic kidney cancer have expanded and improved substantially. Interferon alpha (IFNα) has been replaced by antiangiogenics, tyrosine kinase inhibitors, molecular therapeutics, new cytokines and recently immune checkpoint inhibitors (ICIs) with higher response rates (RRs), longer progression free survival and overall survival [7]. Other drugs used to treat ccRCC include antiangiogenic drugs, that target vascular endothelial growth factor (VEGF), VEGF receptors or platelet derived growth factor (PDGF) and include bevacizumab, axitinib, pazopanib, sorafenib, sunitinib, Cabozantinib and lenvatinib. The molecular target of rapamycin (mTOR) inhibitors utilized in the treatment of ccRCC are everolimus and temsirolimus [8-20]. High-dose IL-2 (HDIL-2) therapy, with a durable complete response rate (CR) of 9% and recognized partial response rate (PR) of 12%, remains a less commonly employed treatment largely due to the fact that this treatment is only appropriate for a minority of very fit patients who are able to safely tolerate this intense regimen. Retrospective analysis of outcome for this treatment has more precisely defined the patient profile with higher likelihood of responding rates but has not identified strategies to expand the number of treatable patients or demonstrated more stringent patient selection has substantial impact on RR or survival in a prospective clinical trial [21-24]. Preclinical animal experiments identified Programmed cell death protein 1 (PD1) expression on CD8+ effector cells as a sign of exhaustion of the viral specific immune responses. The expression of Program death ligand (PD-L1) on tumor cells, sometimes on adjacent stroma cells or other leukocytes in the tumor milieu was also shown to deliver an immune dampening signal through PD-1 on effector lymphocytes. The combination of these 2 findings established the rationale for anti-PD1/PD-L1 treatment of metastatic cancers [25, 26]. Interestingly the preclinical animal data did not demonstrate the striking benefit that has subsequently been seen in the treatment of cancer patients [27, 28]. The initial reports of single agent clinical trials with nivolumab demonstrated response rates in the 20 to 35% range for metastatic melanoma or renal cell carcinoma and 15 to 20% range for patients with relapsed refractory non-small cell lung cancer (NSCLC) [27, 29, 30]. Subsequent publications have demonstrated clinical activity for pembrolizumab, atezolizumab, durvalumab and avelumab (Bavencio) and other anti-PD1/PD-L1 agents in patients with (transitional cell) bladder cancer, Hodgkin's lymphoma (HL), non-Hodgkin's follicular lymphoma (FL) and squamous cell head and neck cancer (SCCH&N) [31-33]. The combination of nivolumab plus ipilimumab, an anticytotoxic lymphocyte-associated protein 4 (CTLA-4) checkpoint inhibitor, has been shown to be more effective than nivolumab alone in several solid tumors including metastatic renal cell carcinoma (mRCC), albeit at the cost of significantly more toxicity [28].  
1.2.3 Expression of PD-L1 in Renal Cell Carcinoma There is a wide range of reported levels of PD-L1 positivity (PD-L1+) depending on the particular PD-L1 antibody used in the immunohistochemical analysis, the histologic subtype of the RCC and whether assessment is scored only for the malignant cells or includes stromal and infiltrating immune effector cells. Conservative estimates for PD-L1 expression are usually based on data obtained from nephrectomy specimens most often reporting that 20% of mRCC are PD-L1+ [34]. Closer appraisal of PD-L1 expression in ccRCC revealed that PD-L1 positivity was associated with female gender, lymph node metastases, distant metastases, higher AJCC stage and advanced stage. Most importantly, the poorer prognostic tumors were more likely to express PD-L1 [35]. In a review of 177 patients more than half of the pathologically stage 3 or 4 (pT3/4) and more than 75% of the G2 tumors were PD-L1+. In an analysis of 85 ccRCC patients who received high dose IL-2, 58% of tumors were PD-L1+ [36]. Another retrospective analysis of 115 surgical specimens similarly 56.5% of the patients' tumors were PD-L1+ [37]. A higher percentage of tumors from patients with sarcomatoid variant mRCC are reported to be PD-L1+ [38].  
1.2.4 Immune Checkpoint Inhibitor Treatment in Renal Cell Carcinoma When the initial clinical data for MDX-1106, now nivolumab were first reported, it was clear that this agent had significant activity in mRCC [27]. Confirming what had been intimated in the firstin-human phase I trial for multiple tumor types, the subsequent report for 296 nivolumab patients showed a response rate (RRs) of 27% (9/33) in the cohort of renal cell patients [30]. The issue of the appropriate dose for nivolumab in the treatment of mRCC was largely settled in a randomized phase II trial that assessed 3 dose levels (0.3, 2 and 10 mg/kg) which demonstrated almost identical RRs of 20, 22 and 20% respectively for the 3 dose levels [39]. A subsequent comparative trial of nivolumab versus everolimus demonstrated a similar level of nivolumab activity with a RR of 25% [40]. The Nivolumab Ipilimumab combination was tested in the CheckMate trials 016 and 214 and showed appreciable activity (40.4 and 42% RRs) albeit with a significantly higher rate of adverse events (AEs) [28]. As a result of these clinical trials, nivolumab either as monotherapy or in combination with ipilimumab for higher risk patients is now FDA approved for the treatment of mRCC. Based on the generally positive results with anti-PD-L1/PD1 treatments including the nivolumab data, many commercial sponsors have developed agents targeting the PD-L1/PD1 axis that also have shown efficacy in mRCC. A phase I trial of atezolizumab in mRCC that included some patients with non-clear cell histology tumors showed an overall RR of 15% and a median progression free survival (PFS) of 5.6 months [41]. Most of the PD-1 and PD-L1 agents have now established effective fixed dosage schedules that eliminates the need for weight-based dose adjustments for individual patients and simplified treatment delivery [42]. There is additional clinical data for combination treatment with anti-PD-L1/PD1 agents and other biological response modifiers (BRMs)or targeted therapies that shows clinical activity in the treatment of mRCC. A phase I trial of pembrolizumab with the 4-1BB/CD137 agonist antibody PF-05082556) showed 1CR, 1 PR and 2 stable disease (SD) in 4 of the 5 mRCC patients treated in the trial [43]. The positive results from a phase 1b trial of Avelumab plus Axitinib [44] showed impressive activity in post nephrectomy treatment naïve mRCC with an overall response rate of 58% (32 of 55 patients) was validated by the recent results of the large (886 patients) randomized phase III Javelin 101 that compared this combination treatment to sunitinib in advanced previously untreated RCC patients and led to FDA approval of this combination as first line treatment for mRCC. The Avelumab plus Axitinib treatment produced a significantly better objective response rate (ORR) 51.4% versus 25.7% (p< 0.001) and progression free survival (PFS) 13.8 versus 8.4 months (p=0.0001) in all patients [45]. It is too early to analyze differences in overall survival for the 2 treatment arms. Treatment related AEs for patients receiving the Avelumab plus Axitinib combination in both trials were similar and substantial with 71% of the patients having > grade 3 AE and discontinuation of treatment of study treatment in 22.8%, but the rate of these events were comparable to the Sunitinib patients (71.5% rate of > grade 3 AEs and 13.4% discontinued treatment). Deaths due to treatment related toxicities in the phase III were almost identical (0.7% versus 0.2%). Additional clinical trials examining combination regimens with Nivolumab, Pembrolizumab, Atezolizumab, Durvalumab and Avelumab in multiple malignancies are currently ongoing [46, 47].  
1.2.5 Avelumab (Bavencio)
When overexpressed in tumors, PD-L1 is a negative prognostic marker due to T-cell anergy induced by the binding of PD-L1 to PD1 [31, 33]. Avelumab (Bavencio) is a fully human IgG1 antibody directed against programmed death ligand-1 (PD-L1) protein. Avelumab inhibits PD1/PD-L1 interactions while leaving the PD1/PD-L2 pathway intact, enhances immune activation against tumor cells and has been evaluated in the treatment of multiple refractory solid tumor patients [31-33, 48-51]. Avelumab (Bavencio) has received accelerated approval in the United States from the FDA for the treatment of patients with metastatic Merkel cell carcinoma (MCC) [52] and urothelial carcinoma [49]. Unlike other approved anti-PD-L1/PD1 antibodies, avelumab has an additional potential mechanism of action that induces lysis of tumor cells *in vitro* via antibody-dependent cell-mediated cytotoxicity (ADCC) that has yet to be confirmed *in vivo* in patients with normal immune T-cell subsets [53] but has been demonstrated by in vitro testing [54]. The standard avelumab treatment regimen is 10 mcg/kg given every 2 weeks. The drug is well tolerated by patients and has a toxicity spectrum similar to other anti-PD-1 or PD-L1 treatments summarized below in **Table 1** and **Table 2** with most notable AEs being fatigue, infusion reactions, followed by skin changes and GI toxicity [54-56].  
1.2.6 Adverse Events in Avelumab Clinical Trials The safety experience in phase I Javelin Solid Tumor protocol (1650 patients) and phase 2 Javelin Merkel 200 clinical trial (88 patients) was recently updated [57]. The most common treatment related adverse events (TRAEs) of any grade were fatigue in 307 patients (17.7%) and infusion related reactions (IRR) in 295 patients (17.0%). **Table 1** from this report shown below lists the most common clinical and laboratory TRAEs. These therapy related events led to permanent discontinuation of treatment in 107 patients (6.2%). The most common cause of treatment discontinuation was immune related events (IRR) seen in 32 patients (1.8%), gamma-glutamyl transferase increase seen in 7 patients (0.4%), increased alanine aminotransferase in 4 patients 0.2%), increased creatine phosphokinase (CPK) in 4 patients (0.2%), and fatigue in 4 patients
(0.2%). One hundred and eight patients (6.2%) had serious TRAEs again most commonly immune related (15 patients; 0.9%), pneumonitis (11 patients; 0.6%), pyrexia (6 patients; 0.3%), and adrenal insufficiency (5 patients; 0.3%). **Table 2** below lists the Immune Related Adverse Events (irAE assessed by the CTC version 4.0 grading criteria) which were seen in a small number of patients and generally were grade 3 AEs
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table 1 avelumab most common

| Most Common Adverse Treatment Related Events N= 1738 TRAE Any Grade No (%) > Grade 3 No (%) Any TRAE 1164 (67.0) 177 (10.2) Fatigue 307 (17.7) 17 (1.0) IRRb 295 (17.0) 10 (0.6) Nausea 150 (8.6) 2 (0.1) Diarrhea 123 (7.1) 5 (0.3) Chills 116 (6.7) 0 Pyrexia 106 (6.1) 0 Decreased appetite 90 (5.2) 3 (0.2) Hypothyroidism 87 (5.0) 3 (0.2) AST increased 38 (2.2) 8 (0.5) Lipase increased 25 (1.4) 17 (1.0) GGT increased 17 (1.0) 10 (0.6)   |
|-----------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|
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table 2 immune-related adverse events

| Immune Related Adverse Events (irAEs) N = 1738 No. (%)   |            |          |         |         |
|----------------------------------------------------------|------------|----------|---------|---------|
| irAEa                                                    | Any Grade  | Grade 3  | Grade 4 | Grade 5 |
| Any irAE                                                 | 247 (14.2) | 32 (1.8) | 4 (0.2) | 3 (0.2) |
| Rash                                                     | 90 (5.2)   | 1 (0.1)  | 0       | 0       |  
11  
| Immune Related Adverse Events (irAEs)   |           |          |         |         |
|-----------------------------------------|-----------|----------|---------|---------|
| Colitis                                 | 26 (1.5)  | 7 (0.4)  | 0       | 0       |
| Pneumonitis                             | 21 (1.2)  | 5 (0.3)  | 1 (0.1) | 1 (0.1) |
| Hepatitis                               | 16 (0.9)  | 11 (0.6) | 0       | 2 (0.1) |
| Endocrinopathies                        | 106 (6.1) | 6 (0.3)  | 0       | 0       |
| Thyroid disorders                       | 98 (5.6)  | 3 (0.2)  | 0       | 0       |
| Adrenal insufficiency                   | 8 (0.5)   | 1 (0.1)  | 0       | 0       |
| Type 1 diabetes mellitus                | 2 (0.1)   | 2 (0.1)  | 0       | 0       |
| All other irAEs                         | 19 (1.1)  | 5 (0.3)  | 3 (0.2) | 0       |
| Blood CPK increased                     | 5 (0.3)   | 1 (0.1)  | 2 (0.1) | 0       |
| Myositis                                | 5 (0.3)   | 1 (0.1)  | 1 (0.1) | 0       |
| Psoriasis                               | 5 (0.3)   | 1 (0.1)  | 0       | 0       |
| Guillain-Barre syndrome                 | 1 (0.1)   | 1 (0.1)  | 0       | 0       |
| Systemic inflammatory response          | 1 (0.1)   | 1 (0.1)  | 0       | 0       |
| syndrome                                |           |          |         |         |
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1.2.7 immune related risks

In addition, some clinically relevant irAEs were reported after the data cutoff or outside of the Pooled Safety Dataset. These included: 3 events of nonfatal graft versus host disease (2 in the liver and 1 in skin) and 1 event of immune thrombocytopenic purpura in Study B9991007 (avelumab in subjects with cHL), 4 events of myasthenia gravis/myasthenic syndrome in the clinical studies, and overall 15 events of pancreatitis including one case of fatal pancreatitis necrotising in Study B9991003 with the combination of avelumab with axitinib in subjects with RCC (Investigator Brochure, 29May 2020).
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1.2.8 efficacy in avelumab clinical

The activity of avelumab in refractory metastatic urothelial carcinoma was assessed in 66 patients as part of the phase 1 Javelin trial [49]. An independent central assessment confirmed an overall response rate (ORR) of 18.2% including 5 patients (11.4%) with a complete response as seen in Figure 1. Three patients (6.8%) had a partial response and 15 patients with stable disease as best response. Patients' responses were predominantly those with PD-L1 positive tumors with seven of eight responding patients (87.5%) having PD-L1 positive tumors compared to only 1 of 24 (4.7%) patients with PD-L1 negative tumors having a response.
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figure 1 clinical activity of

(A) Time to response, duration of treatment, and duration of response to avelumab (eight confirmed responses and four unconfirmed responses as of data cutoff), with programmed death-ligand 1 (PD-L1) expression status indicated (on the basis of a > 5% staining threshold on tumor cells; nonevaluable specimens [n = 7]
included those that were missing, of poor quality, or otherwise not available to provide results). The vertical dotted line represents 1 year from the initiation of treatment. (B) Plot of tumor regression from baseline as measured by Response Evaluation Criteria in Solid Tumors (RECIST) in all assessable patients (n = 38), with PD-L1 expression status indicated (on the basis of a ≥ 5% staining threshold on tumor cells). Eastern Cooperative Oncology Group (ECOG) performance status, presence of non-lymph node metastasis, and albumin and hemoglobin levels at baseline are shown for each patient. The upper dotted line represents progression at 20% and the lower dotted line represents the RECIST boundary for complete response or partial response at 30%. (C) Percentage change in sum of target lesion diameters from baseline over time for all assessable patients (n = 38), defined as those patients with baseline tumor assessments and at least one postbaseline assessment. The upper dotted line represents progression at 20% and the lower dotted line represents the RECIST boundary for complete response or partial response at 30%
In addition to the tumor regressions, Avelumab had an impact on median progression free survival (mPFS) and median overall survival (mOS) in this patient population as seen below in Figure 2.  
  
  
Kaplan-Meier estimate of PFS (A) and OS (B) for the overall study population (N = 44). Kaplan-Meier estimate of PFS (C) and OS (D) on the basis of programmed death-ligand 1 (PD-L1) expression on tumor cells (n = 37). Vertical lines show censored events. Positive PD-L1 expression was defined as >5% of tumor cells expressing PD-L1 at any intensity. ne, not estimable
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1.2.8.2 ovarian cancer

A preliminary report has demonstrated clinical activity in patients with epithelial ovarian cancer [55] Additional ovarian cancer patients were subsequently treated and produced data for 124 relapsed ovarian cancer patients treated with avelumab. The response rate for this disease cohort is 9.7% (12 patients), all partial responses with 6 ongoing. Stable disease was observed in 44% of these patients that resulted in a disease control rate of 54% (unpublished data).
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1.2.8.3 non-small cell lung cancer

In a phase Ib trial of 184 patients, objective responses were seen in 22 (12%) patients that were 1 CR, 21 PRs and 70 patients had stable disease (38%)[56] and resulted in a disease control rate (DCR) of 50%. The most common treatment-related AEs of any grade were fatigue (25%), infusion reactions (21%), and nausea (13%). Grade 3 or worse treatment-related AEs occurred in 13% of patients, with the two most common being infusion reactions (2%) and increased lipase level (2%).
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1.2.8.4 breast cancer

Avelumab was studied in 168 patients with locally advanced or metastatic breast cancer refractory to or progressing after standard of care including 58 patients with triple negative breast cancer (TNBC) [58]. Response rate in the entire cohort was 3.0% and 5.2% in TNBC. Stable disease was observed in an additional 42 patients (25.0%) resulting in a DCR of 28.0%. Again, responses were predominantly in PD-L1 expressing tumors (unpublished data).
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1.2.8.5 merkel cell carcinoma

Merkel cell carcinoma (MCC) is a rare aggressive neuroendocrine tumor of the skin. The initial report of avelumab treatment of 88 MCC patients showed an objective response of 31.8% including 8 CRs and 20 PRs [52].
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1.2.8.6 renal cell carcinoma

There is preliminary evidence of clinical activity for avelumab in the treatment mRCC [59] as additional trials of avelumab for mesothelioma, gastric cancer, adrenocortical carcinoma and renal cancer are ongoing. Additionally, preclinical data from combination treatment with the chimeric anti-PD-L1/anti-TGFβ fusion molecule M7824 and IL-15 [53] demonstrated significantly augmented ADCC with human effector cells in vitro that support this combination treatment strategy.
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1.2.8.7 gastric adenocarcinoma

Adenocarcinoma of the stomach and the closely related gastroesophageal junction (GEJ) are aggressive tumors that are rarely curable and require new and effective treatment options. The results of the phase 1 trial JAVELIN Solid Tumor JPN and the dose expansion cohort of metastatic gastric or GEJ adenocarcinoma was recently published [60].  
1.2.9 Recombinant Human Interleukin-15 (rhIL-15)
IL-15 is a 14-15kDA member of the 4-alpha-helix bundle family of cytokines that acts through a heterotrimeric receptor involving IL-12/IL-15R beta (β)subunit shared with IL-2, the common gamma chain (γc) shared with IL-2, IL-4, IL-9, IL-21, and IL-15 specific receptor subunit IL-15R alpha (IL-15Rα) or CD215 [61] . IL-15 acts as a cell-surface molecule as part of an immunological synapse with IL-15 and IL-15R alpha produced in trans on adjacent mononuclear cells like monocytes and DCs which have been stimulated with interferon and/or CD40 ligation [62-65]. IL- 15 has been shown in many model systems to be a potent stimulator of T and NK-cell functions and in contrast to IL-2 does not activate Tregs and produces less capillary leak syndrome [61-63, 65, 66]. Multiple murine preclinical studies have demonstrated the efficacy of IL-15 in the treatment of cancer that led to clinical development of this cytokine. An intravenous bolus (IVB) regimen of IL-15 was evaluated in a rhesus macaque's toxicology experiment that administered 20 mcg/kg/day of IL-15 for 12 days and produced a 4 to 8-fold increase in the number of circulating NK cells [67, 68]. Administration of the same dose by continuous intravenous infusion (CIV) for 10 days produced a 10-fold increase in the number of circulating NK cells, a 15-fold increase in circulating monocytes and a massive 80 to 100-fold increase in circulating effector memory CD8 T-cells [69].When this same dose was given as a subcutaneous (SC) injection for 10 days, a 10fold expansion in circulating effector memory CD8 T-cells and modest increases in NK cells was observed [69]. Based on animal and laboratory trials of IL-15, great interest was generated among leading immunotherapeutic experts participating in the NCI Immunotherapy Agent Workshop who ranked IL-15 as the most promising unavailable immunotherapeutic agent to be brought to therapeutic trials.
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1.2.10 clinical experience with single

The first in-human (FIH) phase I trial of rhIL-15 administered the treatment as rapid (30 minute) IV bolus (IVB) to adults with refractory metastatic malignant melanoma and metastatic renal cell cancer [70]. This study was initially planned with a starting dose of 3 mcg/kg/day given for 12 consecutive days. However, after the first patient treated developed grade 3 hypotension and after patient \#5 at this dose level developed grade 3 thrombocytopenia, the protocol was amended to add the two lower doses of 1.0 and 0.3 mcg/kg/day with the expectation of returning to higher dose levels later. When two of four patients treated at the 1.0 mcg/kg/day dose unexpectantly developed asymptomatic grade 3 alanine aminotransferase (ALT) and aspartate aminotransferase (AST) elevations that were dose limiting, all subsequent patients were treated at the 0.3 mcg/kg dose level. All 9 patients with IL-15 at 0.3 mcg/kg/day received 12 doses without a DLT, and the maximum tolerated dose (MTD) of rhIL-15 was defined as 0.3 mcg/kg/day. There was a consistent temporal pattern of post-treatment adverse events in patients receiving 3 mcg/kg/day doses of IL-15 with fever and rigors beginning 2 ½ to 4 hours following the start of IV infusions, soon after drops in blood pressure were seen with nadirs approximately 20 mm/Hg below pretreatment levels that responded slowly to rapid normal saline (NS) infusions. These physiologic changes were concurrent with an up to 50-fold elevation of serum IL-6 and interferon gamma (IFNγ) levels. Flow cytometry of peripheral blood lymphocytes in patients receiving 3 mcg/kg/day revealed a dramatic efflux of NK and memory CD8 T-cells from the circulation within minutes of IL-15 administration, gradual rise in NK cell numbers in the first 4 to 5 days of treatment, followed by an influx and hyperproliferation yielding a 10-fold expansion in the number of NK cells at the end of or after completion of the treatment cycle. A modest increase in the number of CD8+ T-cells was also seen but virtually all of these CD8+ T-cells were activated and expressed high levels of Ki67, CD38 and HLA-DR. In this first-in-human phase I trial there were no clinical responses with stable disease as the best response. However, 5 patients had decreases of between 10% and 30% of their marker lesions that included 2 patients that cleared their lung lesions. 1.2.10.2 Subsequent Clinical Trials with Subcutaneous (SC) or Continuous Intravenous (CIV)
rhIL-15 The clinical toxicities that limited dose escalation in the IVB trial were associated with intense cytokine production within the first few hours of treatment and marked by exceedingly high IL-15 levels. Serum IL-15 concentrations at these levels would be sufficient to signal through the intermediate affinity (IL-2) IL-15Rβ and gamma chain (γc) receptor pair that resulted in the undesirable toxicities seen in the FIH trial and suggested that dosing strategies that limited the Cmax, increased the duration of lower IL-15 levels that were optimal for high-affinity IL-15 αβγ receptor would improve the IL-15 stimulatory signal [71]. Alternative SC and CIV dosing regimens were evaluated first in non-human primate (NHP) rhesus macaques and subsequently in phase I clinical trials. The preclinical NHP experiments assessed the pharmacokinetics (PK) for multiple dose levels including the 20 mcg/kg dose given as an IVB (Cmax 720 pg/mL) and produced more advantageous IL-15 concentration; Cmax of 50 pg/mL for SC injections and Cmax of 4 pg/mL
for CIV infusion with more balanced PK parameters that stimulated greater expansion of NK and CD8+ effector cells [67-69].  
A phase I dose escalation trial (Clinical Trials Identifier: NCT01727076) performed in adults with refractory metastatic solid tumors that assessed a SC rhIL-15 treatment regimen (Monday-Friday for 2 consecutive weeks of a 4-week cycle) was performed under the direction of Cancer Immunotherapy Trials Network (CITN) at dose levels of 0.25, 0.5, 1, 2 and 3 mcg/kg/day[71]. Nineteen patients were treated with 14 patients receiving ≥ 2 cycles of treatment. Patients experienced common cytokine side effects of fevers, chills, fatigue, mild anorexia, transient biochemical laboratory abnormalities and transient lymphopenia. The MTD was 2 mcg/kg/day; with notable serious adverse events of pancreatitis at the 2.0 mcg/kg dose level and grade 3 cardiac chest pain at the 3mcg/kg dose level. Flow-cytometry data demonstrated > 10-fold increase in NK cells, an approximately 3-fold increase in CD8 cells at the 2 highest dose levels and a dramatic increase in the frequency of CD56bright CD3 negative NK cells that peaked about 3 days after the last injection on day 15 of the treatment cycle. Again, stable disease was the best response, but 1 patient remained on treatment for 2 years without progression but relapsed soon after the rhIL-15 was stopped. This protocol defined a safe immunostimulatory regimen that could be given as an outpatient in future combination treatment protocols [71].  
A parallel phase I dose escalation trial (Clinical Trials Identifier NCT01572493) was performed in adults with refractory metastatic cancers the Clinical Center NIH, rhIL-15 was administered to patients with metastatic malignancy by CIV for 10 days at progressively increasing doses to 3 patients each of 0.25, 0.5, 1.0, 2.0 and 4.0 mcg/kg/day. Two dose-limiting toxicities were observed at 4.0 mcg/kg/day, one hepatotoxicity and the other visceral arterial ischemia making 2 mcg/kg the MTD that was well tolerated in the 9 patients treated at this dose [72]. The PK data from the CIV regimen was notably different from the IVB regimen having an appreciably lower Cmax 5662 pg/mL (2 mcg/kg cohort) compared to 30,420 pg/mL (for 3 mcg/kg cohort). The serum IL-15 level was largely maintained in the CIVrhIL-15 treated patients over the first 48 hours compared to the rapid decline seen in the IVB patients with serum half-life of approximately 2 ½ hours. Patients in the CIV trial showed a much lower serum IL-15 level during the last 2 to 3 days of the infusion that was approximately 10% of their Cmax level. which is believed to be related to the high number of high affinity IL-15 receptor bearing effector cells acting as a sink rapidly binding and clearing the free serum IL-15. More limited PK data from the SC treatment protocol showed fairly comparable Cmax 6480 pg/mL (for 3 mcg/kg cohort) 4 hours after injection that is largely gone by 24 hours post injection. As predicted in the NHP toxicology studies the lower but sustained IL-15 levels produced by the SC and CIV regimens produced the large increase in activated effector cells at the higher dose levels in the respective trials The CIV regimen produced characteristic changes in lymphocyte numbers with early lymphopenia
(days 1 to 3) for both T and NK cells, followed by a gradual increase in cell numbers and dramatic rise in cells (30 to 80-fold increase in NK cells, 2 to 6-fold increase in CD8+ cells and 350-fold increase in CD56bright NK cells) during the 48 hours after completion of the treatment as shown below in Figure 3(77). The rate of proliferation of different subsets of NK cells assessed by Ki67 was consistent with their levels of CD122 (IL-2/IL-15Rβ) expression with CD56bright > CD56dim, CD94high, > CD56dim, CD94low. The functional capacity of the dominant CD56bright subset was augmented following IL-15 administration and was associated with an increase in their expression of perforin and granzyme. Although the specific lytic activity of CD56bright cells was not as great as that of CD56dim cells, their single cell lytic and antibody-dependent cellular cytotoxicity
(ADCC) activity were markedly increased by rhIL-15 treatment. This ADCC activity was confirmed in assays with anti-CD20 antibody coated Raji cells, natural cytotoxicity to K562 cells, mediated by NKp30 and NKp46 as well as by MICA/NKG2D-mediated cytotoxicity [73]. These clinical trial results and preclinical data combining ICI, anti-CTLA4 and anti-PD1, with rhIL-15 [74, 75]and antitumor antibodies promote the development of clinical trials with these combinations [76]. The CIVrhIL-15 trial was subsequently amended to evaluate a 5-day (120 hour) regimen with further dose escalation at 3, 4 and 5 mcg/kg/day. Ten additional patients have been safely treated at these dose levels without DLTs or remarkable toxicities. Common adverse events (AEs) as expected have been the fatigue, fevers, chills or occasional rigors, capillary leak syndrome and reversible laboratory abnormalities. As seen in table 2, this 5-day regimen has produced impressive increases in absolute lymphocyte count (maximum ALC 25,000 to 30,000 cells/mL, NK cells (30 to > 80-fold increase) and CD8 cells (5 to 8-fold increase) that generally has been higher than seen in the patients treated at the 2 mcg/kg dose level for 10 days.  
A B  
  
received 1.0 mcg/kg and patients 17-19 received 2.0 mcg/kg. Following termination of the treatment (red arrow) there was a dramatic 30-fold increase in the number of circulating lymphocytes predominantly NK cell count and an over 350-fold increase in the number of circulating CD56bright NK cells in the 10-day cohort, and an up to 44-fold increase (33-fold mean increase) in the number of circulating NK cells in the 5day cohort.  
| CD8+ T-cell increase  (fold)   |     |        |                     |              |                  |      |
|--------------------------------|-----|--------|---------------------|--------------|------------------|------|
| Melanoma                       | 63  | M      | 5                   | 5            | 32.01            | 4.21 |
| Small bowel                    | 69  | M      | 5                   | 10           | 39.13            | 4.74 |
| Colorectal                     | 60  | F      | 5                   | 4            | -                | -    |
| Small bowel                    | 51  | F      | 5                   | 10           | 32.03            | 5.66 |
| Colorectal                     | 66  | F      | 4                   | 10           | 44.90            | 3.65 |
| Renal cell                     | 56  | M      | 4                   | 15           | 43.65            | 8.94 |
| Esophageal                     | 60  | M      | 4                   | 10           | 39.63            | 2.01 |
| Colorectal                     | 67  | F      | 3                   | 20           | 21.40            | 1.65 |
| Colorectal                     | 56  | F      | 3                   | 15           | 23.66            | 2.03 |
| Endometrial                    | 70  | F      | 3                   | 3            | -                | -    |
| Colorectal                     | 47  | F      | 3                   | 10           | 24.15            | 1.66 |
| NK cell                        |     |        |                     |              |                  |      |
| Diagnosis                      | Age | Gender | Dose level (mcg/kg) | No. of doses | increase  (fold) |      |  
The complete absence of anti-IL-15 antibodies seen in patients treated with IV E. coli rhIL-15 which contrasts with the experience in patients receiving any formulation of rhIL-15 as a SC injection is justification for the increased patient effort required to receive CIV rather than SC rhIL-15 treatment. The clinical experience is that all SC rhIL-15 treatment regimens have been reported to generate neutralizing anti-IL-15 antibodies that are associated with a decrement in immune activation after continuing multicycle treatment [71-78].
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1.3 considerations and rationale for

The safety database for the rhIL-15 being used in this trial has data from more than 80 patients, mostly monotherapy IV patients, but recently patients have been treated with rhIL-15 in combination with antitumor monoclonal antibodies and checkpoint inhibitors [76, 78]. The side effects of rhIL-15 have been all closely linked to the patients' period of treatment without any episodes of delayed toxicity which commonly resolve soon after IV treatment is discontinued and therefore are distinct from the expected toxicities of avelumab. With the CIVrhIL-15 regimen, fevers, fatigue, myalgias, chills with occasional rigors, dry skin with infrequent rashes are the most common symptomatic side effects. Capillary leak characterized by decreased serum albumin and fluid input exceeding urinary output, modest decrease in blood pressure and possibly some pulmonary capillary leak seen in the 4 or 5 mcg/kg dosing cohorts evidenced by slight drops in oxygen saturation have been observed in the previously treated CIV rhIL-15 patients. Common laboratory abnormalities include early (treatment days 1-4) leukopenia, lymphopenia, thrombocytopenia, transaminitis, anemia, hypoalbuminemia during the whole treatment cycle, late cycle rises in bilirubin in patients treated with doses ≥ 2 mcg/kg and the dramatic spike in lymphocytes that occurs in the 72 hours after completion of the CIV infusion. The norm is for most of these laboratory abnormalities to begin returning or to resolve to baseline while treatment continues. There has been no evidence of increased risk of infection or requirement for antibiotic treatment during the period of cytopenias or increased bleeding during the period of decreased platelets. Decreases in hemoglobin (Hgb), which most likely is related to increased sequestration of red blood cells in the liver and spleen, generally require more time to return to pretreatment levels. This incidence of anemia has possibly been greater in the patients treated with the 5-day regimen and the 3, 4 or 5 mcg/kg dose levels, but this assessment has been complicated by the fact that 8 of the 10 patients treated in these dosing cohorts have GI malignancies and were found to be iron deficient. Some patients were transfused during their 2nd or later cycle of treatment and those who tolerated oral iron replacement therapy had replenishment of their iron stores to improve their Hgb level. A number of the patients previously treated in the rhIL-15 trials had past history of autoimmune phenomena related to their prior cancer treatment with checkpoint inhibitors and none of these patients developed new or recurrent autoimmune toxicities. As seen in **Figure 4**, the rate and severity of irAEs is also generally lower for anti-PD-L1 agents  
 [77], including avelumab (**Table 4**), with the onset of the clinical events of greatest concern occurring generally 2 to 3 months after initiation of treatment. Avelumab has been approved by the FDA for the treatment of metastatic refractory urothelial cancers, has a safety database that exceeds 2000 patients and has a very well characterized AE profile [57]. Figure 4: Distribution of irAEs for anti-PD-L1agents [77]
Distribution of (A) grade 1 to 2 and (B) grade 3 to 5 immune-related adverse events (irAEs) for all tumor types in the main clinical trials with anti– cytotoxic T-cell lymphocyte-4 (anti–CTLA-4), anti–programmed death 1 (PD-1), or anti–PD ligand 1 (PD-L1) antibodies as single therapies. The values quoted are the median (range)
irAE rates for the set of clinical trials as a whole.  
| irAE        | Overall rate %   | Grade 3 through 5%   | Onset of Symptoms   |                     |
|-------------|------------------|----------------------|---------------------|---------------------|
| Median      | Range            |                      |                     |                     |
| Pneumonitis | 1.2              | 0.5                  | 2.5 months          | 3 days - 11 months  |
| Hepatitis   | 0.9              | 0.5 (grade 3 only)   | 3.2 months          | 7 days -15 months   |
| Colitis     | 1.5              | 0.4 (grade 3 only)   | 2.1 months          | 2 days - 11 months  |
| Adrenal     | 0.5              | 0.1 (grade 3 only)   | 2.5 months          | 1 day - 8 months    |
| Thyroid     | 6                | 0.2 (grade 3 only)   | 2.8 months          | 14 days - 13 months |
| Diabetes    | 0.1              | 0.1 (grade 3 only)   | NA                  | NA                  |
| Nephritis   | 0.1              | NA                   | NA                  | NA                  |  
Infusion reaction initially occurred in one quarter of the patients receiving avelumab including 9 patients with grade 3 (0.5%) and 3 patients with grade 4 (0.2%) events that were successfully controlled with antihistamines and IV corticosteroids. The avelumab package insert now recommends routine premedication with an antihistamine during the first 4 infusions. Anti-PD-1 (nivolumab, pembrolizumab or PDR001) antibodies have been given in combination with all versions of rhIL-15, E. coli (NCI produced) single chain agent, heterodimeric IL-15 (Novartis) and the ALT-803 superagonist (Altor now NantKWest). While the number of patients treated with these combinations is still modest, there has not been a new safety signal or evidence of synergistic toxicities. Given the close temporal relationship of CIV rhIL-15 treatment to the transient grade 3 hematologic and occasional metabolic abnormalities, we anticipate that these events will not complicate the recognition of the rare autoimmune events seen with avelumab which would normally become apparent during the second or third cycle of treatment. We believe it is reasonable to use our standard DLT exclusion algorithm even in the initial dose level safety evaluation of patients and will view sustained or delayed grade 3 elevations of liver function tests (LFTs) as DLTs.
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2 eligibility assessment and enrollment

2.1.1 Inclusion Criteria
2.1.1.1 Patients must have histologically proven metastatic clear cell renal carcinoma with ≥ 5%
expression of PD-L1 in the tumor area confirmed by IHC in the NCI Lab of Pathology or performed by an outside CLIA certified laboratory using the FDA licensed Dako 28-8 or 22C3 antibodies PharmDx [79, 80]. Archival tumor sample may be used but if archival tissue is not available or is not adequate, tissue biopsy will be required.  
2.1.1.2 Patients must have failed or relapsed and have progressive disease after at least 2 prior therapies that include multityrosine kinase inhibitor (mTKI) like axitinib or sunitinib and an anti-PD1 or PD-L1 (ICI) therapy like nivolumab which could have been administered in combination with an anti-CTLA4 agent like ipilimumab. Patients who received an ICI in combination with a mTKI would be eligible for the trial if they received another appropriate treatment. Adjuvant or neoadjuvant with either type of agent would not fulfill this requirement only treatment for metastatic disease will be considered to satisfy this criterion.  
2.1.1.3 Disease must be measurable with at least one measurable lesion by RECIST v1.1 criteria
(see Section 6.3) that is different from the lesion biopsied.
2.1.1.4 Age >18 years NOTE: Because no dosing or adverse event data are currently available on the use of rhIL-15 in combination with avelumab in patients <18 years of age, children are excluded from this study, but may be eligible for future pediatric trials 2.1.1.5 ECOG performance status <1 (Karnofsky >80%, see **APPENDIX A**)  
| absolute neutrophil count   | >1,500/mcL                                                                      |
|-----------------------------|---------------------------------------------------------------------------------|
| absolute lymphocyte count   | >500/mcL                                                                        |
| Hemoglobin                  | >10 g/dL                                                                        |
| Platelets                   | >100,000/mcL                                                                    |
| total bilirubin             | <1.5 X institutional upper limit of normal (ULN)                                |
| AST(SGOT)/ALT(SGPT)         | <2.5 X institutional ULN                                                        |
| Serum creatinine            | < 1.5 X institutional ULN                                                       |
| OR Creatinine clearance     | >50 mL/min/1.73 m2  for patients with creatinine levels >1.5  institutional ULN |  
2.1.1.7 Negative serum or urine pregnancy test at screening for women of childbearing potential
(WOCBP).  
NOTE: WOCBP is defined as any female who has experienced menarche and who has not undergone successful surgical sterilization or who is not postmenopausal. WOCBP must have a negative pregnancy test (HCG blood or urine) during screening.  
2.1.1.8 Women of child-bearing potential and men must agree to use adequate contraception
(hormonal or barrier method of birth control; abstinence) prior to study entry, for the duration of study participation, and 1 month after completion of rhIL-15 and avelumab 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.  
2.1.1.9 Ability of subject to understand and the willingness to sign a written informed consent
document.  
2.1.2 Exclusion Criteria 2.1.2.1 Chemotherapy within 4 weeks (6 weeks for nitrosoureas or mitomycin C). 2.1.2.2 Persisting toxicity related to prior therapy of grade > 1, with the exception of the following: alopecia, sensory neuropathy grade ≤ 2, or other grade ≤ 2 not constituting a safety risk based on investigator's judgement.  
2.1.2.3 Patients who are receiving any other investigational agents 2.1.2.4 Current use of immunosuppressive medication, EXCEPT for the following:
Intranasal, inhaled, topical steroids, or local steroid injection (e.g., intra-articular injection)
Systemic corticosteroids at physiologic doses ≤ 10 mg/day of prednisone or equivalent; or, Steroids as premedication for hypersensitivity reactions (e.g., CT scan premedication).  
2.1.2.5 Patients with known brain metastases should be excluded from this clinical trial because of their poor prognosis and because they often develop progressive neurologic dysfunction that would confound the evaluation of neurologic and other adverse events.  
2.1.2.6 Patients with previous malignant disease other than the target malignancy within the last 5 years with the exception of basal or squamous cell carcinoma of the skin or cervical carcinoma in situ.
2.1.2.7 Patients with history of any organ transplantation, 2.1.2.8 Vaccination within 4 weeks of the first dose of avelumab. Vaccination with a live vaccine while on trial is prohibited. **NOTE:** Seasonal influenza vaccines for injection are generally inactivated flu vaccines and are allowed; however intranasal influenza vaccines (e.g., Flu-Mist®) are live attenuated vaccines, and are not allowed.  
2.1.2.9 Patients with history of allergic reactions attributed to compounds of similar chemical or biologic composition to rhIL-15 or avelumab.  
2.1.2.10 Patients with uncontrolled intercurrent illness including, but not limited to, ongoing or active infection requiring systemic therapy, or psychiatric illness/social situations that would limit compliance with study requirements.  
2.1.2.11 Inability or refusal to practice effective contraception during therapy or the presence of pregnancy or active breastfeeding. Based on its mechanism of action, avelumab can cause fetal harm when administered to a pregnant woman. Animal studies have demonstrated that inhibition of the PD-1/PD-L1 pathway can lead to increased risk of immune-mediated rejection of the developing fetus resulting in fetal death. These potential risks may also apply to other agents used in this study.  
2.1.2.12 Patients with active bacterial infections, documented HIV infection or positive screening serology, PCR evidence for active or chronic hepatitis B or hepatitis C, or positive screening HBV/HCV serology without documentation of successful curative treatment (see Section **12.1** for IL-15 administration in HIV positive patients).  
2.1.2.13 Patients with active or history of any autoimmune disease, including asthma requiring chronic inhaled or oral corticosteroids, or with history of asthma requiring mechanical ventilation; patients with a history of mild asthma that are on or can be switched to noncorticosteroid bronchodilator regimens are eligible
2.1.2.14 Cardiovascular disease: Clinically significant (i.e., active) cardiovascular disease:
cerebral vascular accident/stroke (< 6 months prior to enrollment), myocardial infarction (< 6 months prior to enrollment), unstable angina, congestive heart failure (≥ New York Heart Association Classification Class II), or serious cardiac arrhythmia requiring medication 2.1.2.15 Other severe acute or chronic medical conditions including immune colitis, inflammatory bowel disease, immune pneumonitis, pulmonary fibrosis or psychiatric conditions including recent (within the past year) or active suicidal ideation or behavior; or laboratory abnormalities that may increase the risk associated with study participation or study treatment administration or may interfere with the interpretation of study results and, in the judgment of the investigator, would make the patient inappropriate for entry into this study.  
2.2 STRATEGIES FOR RECRUITMENT AND R**ETENTION** Study participants will be recruited from the population of patients screened in the lymphoid malignancies clinic of the National Institutes of Health. These will include both referrals from outside physicians as well as patient self-referrals. This study will be posted on NIH websites and on NIH social media forums. In addition, information will be provided on the ccr.cancer.gov/ Lymphoid-Malignancies-Branch and ClinicalTrials.gov web pages, and on the official Lymphoid Malignancy Branch social media accounts.
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2.3 screening evaluation

2.3.1 Screening activities performed prior to obtaining informed consent 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.
2.3.2 Screening activities performed after a consent for screening has been signed The following activities will be performed only after the subject has signed the consent for study \#01-C-0129 on which screening activities will be performed. 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 consent. The following tests/procedures should be performed to determine treatment eligibility. Assessments and procedures to confirm study eligibility should be completed within 28 days prior to registration (unless otherwise noted). See also the Study Calendar provided in **APPENDIX F.**
2.3.3 Clinical Evaluations Disease history, including: diagnosis, treatment (e.g., systemic treatments, radiation and surgeries), status, and significant prior/ongoing side effects and symptoms Complete medical history including: all active conditions considered to be clinically significant by the treating investigator Physical examination, including: height (screening only), weight, vital signs (i.e.,
temperature, pulse, respiratory rate, oxygen saturation and blood pressure); review of concomitant medications and symptoms/side effects; and, assessment of performance status 2.3.4 Laboratory Evaluations NOTE: Results from outside NIH are accepted.  
Hematological Profile: CBC with differential and reticulocyte count Biochemical Profile: Creatinine (i.e., Acute Care Panel); serum calcium, phosphate, magnesium and albumin (i.e., Mineral Panel); ALT, AST, total and direct (if required) bilirubin (i.e., Hepatic Panel); 24-hour urine creatinine clearance (if needed to measure CrCl in cases where serum creatinine >1.5mg/dl); and LDH
Serum Lipase and Amylase Coagulation panel, including: PT/INR and a PTT
Thyroid function tests, including: thyroid stimulating hormone (TSH) and free thyroxine (T4)
HIV, and hepatitis: Hepatitis B surface antigen (HBsAg), Hepatitis B core antibody, Hepatitis C antibody (HCV) [qualitative], HIV 1/2 antibody (qualitative) **NOTE:** For individuals with a positive hepatitis B core antibody, HBV DNA PCR will be performed to screen for subclinical infection. For individuals with a positive hepatitis C
antibody, HCV RNA RT-PCR will be performed to determine infectious status.  
Serum or urine pregnancy test (B-HCG) in women of childbearing potential Urinalysis (with microscopic examination if abnormal)
Antinuclear antibody (ANA), rheumatoid factor (RF) and anti-thyroid antibody Creatine phosphokinase (CPK), troponin Flow cytometry and lymphocyte sets, and subsets 2.3.5 Imaging Studies NOTE: Results from outside NIH are accepted. Other body areas may be imaged if clinically indicated.  
CT, chest, abdomen, pelvis and neck (if required). CT should be performed with IV
and PO contrast, unless patient is allergic or has renal insufficiency; other imaging may be substituted at the discretion of the investigator [such as MRI].  
PET/CT torso (extremities to be included if there is confirmed or suspected disease involvement) maybe performed in selected cases to better evaluate lesions of uncertain etiology Patients with neurological symptoms or signs should undergo MRI scan of the brain and lumbar puncture.  
2.3.6 Other Procedures Pathological review/confirmation of diagnosis by Laboratory of Pathology, NCI (no time limit) and PD-L1 staining as outlined in Section **5.2.4.1**. If no prior samples are available, punch or core biopsy of an appropriate easily accessible tumor deposit will be performed.  
Patients with significant pulmonary or smoking history, including significant pulmonary involvement by tumor, pleural effusion, history of chronic obstructive pulmonary disease (COPD) or lobar or greater surgical resection of lung will require qualifying pulmonary function tests (PFTs) that include adjusted diffusing capacity (DLCO/Adj), forced expiratory volume in 1 second (FEV1).  
Cardiac Evaluation: Electrocardiogram (EKG), and for patients with significant history of vascular disease or clinical suspicion of cardiac dysfunction, a transthoracic echocardiogram will be obtained to ensure adequate cardiac performance to receive the protocol treatment without increased risk.
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2.4 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.
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2.4.1 treatment assignment procedures note

| Number                                                                                                                                                                                                       | Name           | Description Patients with                                                                                                                                                                                                                                     | relapsed/refractory   | PD-L1-expressing    |                  |
|--------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|----------------|---------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|-----------------------|---------------------|------------------|
| metastatic ccRCC who have progressive disease after at least  2 prior therapies that include multityrosine kinase inhibitor  and an anti-PD1/PD-L1 inhibitor to determine safety of dose                     |                |                                                                                                                                                                                                                                                               |                       |                     |                  |
| 1                                                                                                                                                                                                            | Safety Run-in  | Patients                                                                                                                                                                                                                                                      | with                  | relapsed/refractory | PD-L1-expressing |
| metastatic ccRCC who have progressive disease after at least  2 prior therapies that include multityrosine kinase inhibitor  and an anti-PD1/PD-L1 inhibitor to determine efficacy of  combination treatment |                |                                                                                                                                                                                                                                                               |                       |                     |                  |
| 2                                                                                                                                                                                                            | Dose Expansion |                                                                                                                                                                                                                                                               |                       |                     |                  |
| Number                                                                                                                                                                                                       | Name           | Description                                                                                                                                                                                                                                                   |                       |                     |                  |
| 1                                                                                                                                                                                                            | Safety Run-in  | IL-15 by CIV infusion at escalating doses of 2, and 4  mcg/kg/day on days 1-5 of each 28-day cycle (max 4 cycles)  with avelumab by IV infusion at a dose of 800mg on Day 8  and 22 of each cycle, to determine safety of dose (3-6 patients  per dose level) |                       |                     |                  |
| 2                                                                                                                                                                                                            | Dose Expansion | IL-15 by CIV infusion at 4 mcg/kg/day on days 1-5 of cycles  1-4 with avelumab at 800mg on Day 8 and 22 of each cycle  (Total 17 patients) to determine efficacy of treatment                                                                                 |                       |                     |                  |  
2.4.1.2 Arms  
2.4.1.3 Treatment Assignment Treatment assignment is open-label, and non-randomized/non-stratified. Subjects in Cohort 1 are directly assigned to Arm 1. Subjects in Cohort 2 are directly assigned to Arm 2.
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2.5 baseline evaluation

The following should be performed within 28 days prior to the first dose of rhIL-15 unless otherwise noted; test performed as part of screening do not need to be repeated if they were performed within the specified window prior to initiating treatment.  
2.5.1 Clinical Evaluations Medical history (interim)
Physical examination including weight, vital signs (i.e., temperature, pulse, respiratory rate, oxygen saturation and blood pressure); review of concomitant medications and symptoms/side effects; and assessment of performance status (ECOG performance score see **APPENDIX A**).
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2.5.2 laboratory evaluations

NOTE: Results from outside NIH are accepted Required within 7 days:
o Serum or urine pregnancy test (B-HCG) for women of childbearing potential Required within 14 days:
o Hematological Profile: CBC with differential and reticulocyte count  
o Biochemical Profile: Chemistry panels including: Acute Care (sodium, potassium, chloride, CO2, glucose, BUN, creatinine), Mineral Panel (serum calcium, phosphate, magnesium and albumin), Hepatic Panel (alkaline phosphatase, ALT, AST, total and direct bilirubin), and 24-hour urine creatinine clearance (if needed measure CrCl if serum creatinine >1.5mg/dl)
o Others: LDH, troponin o Serum Lipase and Amylase o Thyroid panel: TSH and free T4 o Coagulation panel, including: PT/INR and aPTT o Urinalysis (with microscopic examination if abnormal)
o IL-2R alpha Required within 28 days:
o Antinuclear antibody (ANA), rheumatoid factor (RF) and anti-thyroid antibody 2.5.3 Imaging Studies Every participant should have an evaluation of known sites of disease as part of baseline evaluation. **NOTE:** Results from outside the NIH are accepted if the films provide high quality images that can be used to measure tumor marker lesions.
One or more of the following studies: CT, MRI, FDG-PET
Patients with neurological symptoms or signs should undergo MRI scan* of the brain
*NOTE: The MRIs to be done in this study may involve the use of the contrast agent gadolinium, if clinically indicated. The risks associated with MRIs and contrast are discussed in the consent form.  
2.5.4 Research Correlates NOTE: See Section 5 for additional information. The following sample types will be collected for correlative research studies:
Required:
o Blood samples for lymphocyte subset testing and circulating tumor DNA o Tumor Tissue (archival or fresh) to assess PD-L1 expression for eligibility o Blood samples for ADCC of avelumab, tissue immune cell subset comparison
(as outlined in Section 5)
Optional:
o Pretreatment tumor biopsy to assess immune response to therapy can use the archival tissue or the fresh sample obtained to assess PD-L1 expression for protocol entry. Tissue sample for the optional on treatment biopsy obtained between cycle 1 day 22 and cycle 2 day 4 will be a core biopsy of easily accessible tumor deposits.
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3 study implementation

3.1 STUDY D**ESIGN** In this Phase II trial of combination rhIL-15 and avelumab in patients with relapsed or refractory metastatic PD-L1+ ccRCC, IL-15 will be administered by CIV. The trial will begin with a small safety run-in cohort with a rhIL-15 starting dose of 2 mcg/kg/day, and a second dose level of 4 mcg/kg/day (the desired Phase II dose). There is clinical experience for the combination of antiPD1 plus IL-15, but there is no prior safety experience for the combination of avelumab and E.  
coli derived rhIL-15. Consequently, the first 3 patients will begin CIV rhIL-15 treatment at a dose of 2 mcg/kg/day on days 1-5 and avelumab (800mg, by IV) on day 8 and 22 of the 28-day cycle (i.e., every 2 weeks). If one of these patients has a dose limiting toxicity (DLTs), three additional patients will be treated to establish the safety of the combination. Assuming there are no DLTs, subsequent patients will be treated at the 4 mcg/kg/day dose level. Again, the safety of this dose of rhIL-15 in combination will be established in a 3 to 6 patient cohort to confirm this treatment regimen does not pose unacceptable risk to the enrolled subjects. If a second patient in these safety run-in cohorts at either the 2 or 4 mcg/kg dose level experiences a DLT all treatment and enrollment will be halted to review the data and determine if further clinical evaluation of the combination should be attempted. Treatment will continue for a maximum of 4 cycles or until toxicity (i.e., dose limiting toxicity as found in Section **3.1.1** or toxicity requiring hold as defined in Section 3.3) or progressive disease. After the safety of the 4 mcg/kg dose level is established all subsequent patients will be treated with CIVrhIL-15 at the dose of 4 mcg/kg on days 1-5 (120 hours) and avelumab (800mg, by IV) on day 8 and 22 of each 28-day cycle for a maximum of 4 cycles ( in the absence of toxicity requiring hold as defined in Section 3.3 or progressive disease).  
  
The standard immune related adverse event (irAE) treatment decision algorithm [66] will be followed to address avelumab related AEs and any patient who requires corticosteroid treatment for irAE will discontinue their rhIL-15 treatment. Since the expected frequency of grade 3 or 4 irAEs with single agent avelumab is extremely low (< 1%), irAE requiring corticosteroid treatment during the first cycle of treatment for the run-in cohort of patients will also be considered a DLT. All patients in the run-in cohort (Cohort 1) must complete their first 28-day cycle of treatment without experiencing a DLT before enrollment into the 4 mcg/kg cohort is initiated. The efficacy of the combination will be assessed in a Simon two-stage phase II clinical trial scheme that treats a total of minimum 9 or maximum 17 patients at the 4 mcg/kg dose level depending on demonstration of clinical activity in the initial group of patients. See Section **10.2.1** for details regarding early stopping criteria for the whole trial.  
3.1.1 Dose-Limiting Toxicity A dose-limiting toxicity (DLT) is defined as: any grade 3, 4, or 5 toxicity if not incontrovertibly due to disease progression or an extraneous cause, and deemed possibly, probably or definitely related to IL-15 or avelumab by the PI or designee during the first 28 days of treatment, only for the Safety Run-in cohort (Cohort 1) with the following exceptions.: 3.1.1.1 Hematologic exceptions Grade 3 or 4 lymphocytopenia without clinical signs of infection grade 2 or above.  
o rhIL-15 and avelumab will be continued in the event of asymptomatic grade 3 or 4 lymphocytopenia, unless there are clinical signs of significant infection (persistent fevers, labile blood pressure, localized complaints or findings on physical examination, hypoxia or organ dysfunction).  
Grade 3 or 4 neutropenia without clinical signs of infection grade 2 or above.  
o rhIL-15 and avelumab will be continued in the event of grade 3 neutropenia unless there are clinical signs of significant infection, as listed above.  
Grade 3 leukocytosis (WBC > 100,000/mm3) rhIL-15 and avelumab will be continued in the absence of signs of leukostasis or other toxicities possibly related to the expansion of activated cells  
3.1.1.2 Non-Hematologic exceptions
Transient (< 24 hours) grade 3 hypoalbuminemia, hypokalemia, hypomagnesemia, hyponatremia or hypophosphatemia which responds to medical intervention. rhIL-15 and avelumab will be continued while the metabolic abnormalities are corrected by intravenous or oral supplementation Non-sustained (< 7 days) grade 3 liver function test (ATL, AST, alkaline phosphatase, total or direct bilirubin) abnormalities deemed unrelated or unlikely related to avelumab, in the absence of clinical signs of hepatic dysfunction (lethargy, confusion, anorexia, pruritus, tremor); for patients with baseline grade 1 elevations, any increase ≥ 10 x baseline will be considered dose-limiting and these patients will be closely monitored for liver function abnormalities.rhIL-15 and avelumab will be continued as long as these abnormalities do not persist for more than 7 days or the patient does not exhibit findings consistent with hepatic toxicity Management and dose modifications associated with the above adverse events are outlined in Section 3.3. Occurrence of any of the adverse events listed above after the first 28 days of treatment in the Dose Expansion cohort (Cohort 2) will lead to permanent discontinuation of protocol therapy, but will not be deemed a DLT for purposes of dose escalation. See Section **3.3.3** for more details.  
3.1.2 Dose Escalation and DLT Assessment in Safety Run-in Cohorts Dose escalation in the initial patients treated at the 2 and 4 mcg/kg/day dose levels will proceed according to the following schedule (**Table 5**). Dose escalation will follow the following guidelines (**Table 6**). Dose-limiting toxicity (DLT) is defined above. Each patient will continue treatment at the dose level they were enrolled - there will be no intra-patient dose escalation.  
| Dose Level                                                                                         | rhIL-15   | Avelumab   |
|----------------------------------------------------------------------------------------------------|-----------|------------|
| (mcg/kg)                                                                                           | (mg)      |            |
| Level 1                                                                                            | 2         | 800        |
| Level 2                                                                                            | 4         | 800        |
| *Doses are stated as exact dose in units (e.g., mg/m2 , mcg/kg, etc.) rather than as a percentage. |           |            |  
| Table 6: Dose Escalation Guidelines  Number of Patients with DLT at a  Given Dose Level   | Escalation Decision Rule                                                                                                                                                                                                                                                    |
|-------------------------------------------------------------------------------------------|-----------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|
| 0 out of 3 for dose level 1                                                               | Enter 3 patients at dose level 2.                                                                                                                                                                                                                                           |
| 0 out of 3 for dose level 2                                                               | All subsequent patients will be treated at dose level 2  (4mcg/kg). This is the phase 2 dose.                                                                                                                                                                               |
| 1 out of 3 at either dose level 1 or 2                                                    | Enter at least 3 more patients at this dose level.  If 0 of these 3 patients experience DLT at dose level 1,  proceed to the dose level 2.  If 0 of these 3 patients experience DLT at dose level 2, all  subsequent patients will be treated at dose level 2 (4  mcg/kg) |
| > 2 of 3 or 6 for either dose level 1 or                                                  | Enrollment and all treatment will be halted. The safety data                                                                                                                                                                                                                |
| dose level 2                                                                              | will be analyzed and the protocol either substantially  amended or terminated depending on the results of this safety  review and discussion with CTEP and EMD Serono                                                                                                       |
| 3.2                                                                                       | DRUG ADMINISTRATION                                                                                                                                                                                                                                                         |
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table 6 dose escalation guidelines

3.2 DRUG A**DMINISTRATION** Each cycle is 28 days (4 weeks). The minimum window between initiation of new cycles is 26 days; a cycle delay due to scheduling or other administrative reasons (i.e., reasons other than toxicity/dose management as defined below) is 7 days. Treatment with rhIL-15 will be given as a continuous IV infusion days 1 through 5 (120 hours) Avelumab had previously been given at a dose of 10 mg/kg, but recently was given permission by the FDA to administer the drug as a flat dose of 800 mg over 60 minutes every 2 weeks. All patients treated in this protocol will receive the flat 800 mg avelumab dose. The diluted avelumab solution will be administered over 60 minutes through an intravenous line containing a sterile, non-pyrogenic, low protein binding inline filter (pore size of 0.2 micron). Do not co-administer other drugs through the same intravenous line. rhIL-15 will be given intravenously as a continuous infusion through a central venous (midline) catheter and administered on an inpatient basis during week 1 of the first cycle, and as outpatient during subsequent weeks and cycles unless decided otherwise by the principal investigator based on clinical judgment. Reported adverse events and potential risks are described in Sections 14. Appropriate dose modifications for later cycles of treatment are described in Section 3.3. See Table 7 below for description of drug regimen. For a full detailed product description and administration guidelines, see Section 14. Infusions will be administered via central venous access device (if present; not required to be placed by the study). When administered on an outpatient basis, rhIL-15 will be infused via an ambulatory infusion pump. Criteria to be eligible for outpatient ambulatory treatment at 2 mcg/kg dose level are:  
no physiologic organ system or fatigue AE > grade 1, absence of blood pressure (BP) changes < 20% of baseline or systolic BP <
100 mm Hg, more than 5% decline in room air oxygen saturation.
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table 7 drug regimen

| Agent                                                                                                                                                                                           | Premedications; Precautions          | Dose              | Route           | Schedule      |
|-------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|--------------------------------------|-------------------|-----------------|---------------|
| Premedicate with                                                                                                                                                                                | ** in *** D5W with                   |                   |                 |               |
| acetaminophen and/or                                                                                                                                                                            | IV over  24 hours                    | Days 1-5          |                 |               |
| ibuprofen                                                                                                                                                                                       | 0.1% HSA                             |                   |                 |               |
| rhIL-15                                                                                                                                                                                         | Premedicate with                     |                   |                 |               |
| Avelumab                                                                                                                                                                                        | acetaminophen and an  antihistamine* | ** in 250 mL 0.9% | IV over 1  hour | Days 8 and 22 |
| or 0.45% NS                                                                                                                                                                                     |                                      |                   |                 |               |
| * Mandatory for the first four infusions; subsequently based on clinical judgement ** Doses as appropriate for assigned dose level *** Infusion volume of rhIL-15 per calculation in APPENDIX C |                                      |                   |                 |               |  
3.2.1 Prophylactic and supportive care for IL-15 Patients will be given acetaminophen 500-650mg IV or orally, 30-60 minutes prior to each IL-15 infusion as first line, and ibuprofen 400 or 600mg orally, depending on reactions with acetaminophen as premedication.  
3.2.2 Prophylactic and supportive care for avelumab In order to mitigate infusion related reactions, a premedication with an antihistamine and with acetaminophen 30 to 60 minutes prior to the first 4 infusions of avelumab is mandatory (e.g., 2550 mg diphenhydramine and 500-650 mg acetaminophen IV or orally). At the discretion of the investigator, famotidine 20mg PO or IV (or other H2 receptor blocker of equivalent efficacy) may also be administered 30 minutes prior to avelumab. Premedication should be administered for subsequent avelumab infusions based upon clinical judgment and presence/severity of prior infusion reactions.  
3.2.3 Other modalities or procedures Patients will be observed in the day hospital or the inpatient unit for at least 30 minutes after administration of avelumab for potential infusion-related reactions
Avelumab should be administered in a setting that allows for 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. Steroids (dexamethasone 10 mg), epinephrine (1:1,000 dilution), allergy medications (IV antihistamines), bronchodilators, or equivalents, and oxygen should be available for immediate access.
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3.2.4 clinical monitoring for the

On Day 8 and Day 22 of Cycle 1 only, vital signs ([VS] blood pressure, pulse, respiratory rate, temperature, oxygen saturation) will be assessed predose of avelumab (within 15 minutes of injection and/or infusion), and then every 15 minutes from the start of avelumab infusion for at least 2 hours. If vital signs are not stable after 2 hours, then continue monitoring every 15 minutes until stable on 2 consecutive repeated measurements and consideration for hospital observation for prolonged vital sign changes or instability if the patient is being treated as an outpatient during subsequent treatment cycles. If a patient requires more than 2 hours of observation during both cycle 1 avelumab treatments, they will be observed for 2 hours with similar vital signs timepoints for cycle 2 day 8 treatment. Once it is clear that a patient can tolerate their Avelumab infusions without any > grade 1 infusion reactions during subsequent treatments, patients will be observed for 30 minutes post completion of their avelumab infusion and vital signs will be obtained per local standard. Subjects enrolled in this trial must be observed for 2 hours post avelumab infusion in an area with resuscitation equipment and emergency agents. At all times during avelumab and rhIL-15, immediate emergency treatment of an infusion-related reaction or a severe hypersensitivity reaction according to institutional standards must be assured. To treat possible hypersensitivity reactions, for instance, dexamethasone 10 mg and epinephrine in a 1:1000 dilution or equivalents should always be available along with equipment for assisted ventilation. Infusion of avelumab will be stopped in case of Grade > 2 hypersensitivity, inflammatory response, or infusion-related reaction.
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3.2.5 clinical monitoring for the

The rhIL-15 is given by continuous infusion, and the safety of combined administration will be confirmed by administering Cycle 1 treatment as an inpatient and the patient may continue treatment as an outpatient if they satisfy the criteria listed above in Section 3.2. It is likely that the majority of patients will receive their CIV rhIL-15 as inpatients and will have regular VS assessment as per nursing unit norms. Patients who experience a decrease in their BP below 100 mmHg or have symptomatic orthostatic changes in the BP will receive NS infusion to increase their BP and alleviate their symptoms. These patients may also receive salt poor albumin (25%) and or transfused packed red blood cells (RBCs) as clinically indicated by their monitoring laboratories. Patients who experience shortness of breath or have decrease in their oxygen saturations below 92% will receive Lasix IV, intermittent low flow oxygen (grade 2 CTC event) and respiratory treatments as clinically indicated to improve their blood oxygen levels. Patients who experience these events will have more frequent VS and clinical assessments as per institutional norms. After Cycle 1 monitoring for dose limiting toxicities, patients may also have brief interruptions (< 2 hours) of their CIV rhIL-15 to allow symptoms to decrease to acceptable levels. 3.2.5.1 Vital Signs during Inpatient treatment Inpatient: patients will have vital signs (BP, HR, RR, temperature and oxygen saturation) obtained as per inpatient unit norms.  
3.2.5.2 Vital Signs for Outpatient treatment Outpatient: patients will have vital signs (BP, HR, RR, temperature and oxygen saturation) obtained at the time of their visit to have their infusion bag changed.
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3.3 dose modifications

3.3.1 rhIL-15-specific Adverse Events Please refer to the Comprehensive Adverse Event and Potential Risk list (CAEPR) for rhIL-15 presented in Section **14.1.8**.  
For patients treated later in the phase II dose expansion cohort (Cohort 2) at 4 mcg/kg, CIVrhIL15 infusion may be interrupted and the dose reduced to 3 mcg/kg for patients who have reversible grade 3 thrombocytopenia, hypoxia, symptomatic hypotension or 3 LFT abnormalities during the CIV rhIL-15 infusion for improved general tolerability. After the safety of the 4 mcg/kg dose level has been established, patients treated in the phase II dose expansion cohort who poorly tolerate the 4 or 3 mcg/kg dose level (significant fatigue or anorexia) will also have the option of further dose reduction to the 2 mcg/kg dose level. Patients who are unable to tolerate the reduced dose of rhIL15 will discontinue cytokine treatment and may continue treatment with Avelumab.  
Dose of rhIL-15 is based on the dose level and patient's weight at the beginning of each cycle and can only be modified for rounding and/or consistency with prior cycles or as specified above, and not for adverse events or renal/hepatic dysfunction. Infusion may continue during correction of electrolyte and other laboratory abnormalities listed in Section **3.1.1**. Infusion may be interrupted for up to two hours each day, but treatment should end 120 (±1) hours after initiation on Day 1.
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3.3.2 avelumab

| NCI-CTCAE Grade                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                          | Treatment Modification for Avelumab                                                                                                                                                                       |
|--------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|-----------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|
| Grade 1 - mild Mild transient reaction; infusion interruption not  indicated; intervention not indicated.                                                                                                                                                                                                                                                                                                                                                                                                                                                                | Decrease the avelumab infusion rate by 50% and monitor  closely for any worsening.                                                                                                                        |
| Grade 2 - moderate Therapy or infusion interruption indicated but  responds promptly to symptomatic treatment (for  example, antihistamines, NSAIDs, narcotics, IV  fluids); prophylactic medications indicated for  24 h.                                                                                                                                                                                                                                                                                                                                              | Temporarily discontinue avelumab infusion. Resume infusion at 50% of previous rate once infusionrelated reaction has resolved or decreased to at least Grade 1  in severity, and monitor closely for any worsening.                                                                                                                                                                                                           |
| Grade 3 or Grade 4 - severe or life-threatening 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. Grade 4: Life-threatening consequences; urgent  intervention indicated.                                                                                                                                                                                                             | Stop avelumab infusion immediately and disconnect infusion  tubing from the subject. Subjects have to be withdrawn immediately from study  avelumab and must not receive any further avelumab  treatment. |
| If avelumab infusion rate has been decreased by 50% or interrupted due to an infusion reaction, it must remain  decreased for the next scheduled infusion. If no infusion reaction is observed in the next scheduled infusion, the  infusion rate may be returned to baseline at the subsequent infusions based on investigator's medical judgment.  If hypersensitivity reaction occurs, the subject must be treated according to the best available medical practice.  IV = intravenous; NCI-CTCAE = National Cancer Institute-Common Terminology Criteria for Adverse |                                                                                                                                                                                                           |  
IV = intravenous; NCI-CTCAE = National Cancer Institute-Common Terminology Criteria for Adverse Event; NSAIDs = nonsteroidal anti-inflammatory drugs
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3.3.2.1 treatment modifications for symptoms

3.3.2.2 Treatment modifications for immune-related adverse reactions (irAE)
All grading scales in the following tables are according to the revised NCI Common Terminology Criteria for Adverse Events (CTCAE) version 5.0.
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table 9 treatment modification for

| Gastrointestinal irAEs                                                                                                               |                                                                     |                                                                                                                               |
|--------------------------------------------------------------------------------------------------------------------------------------|---------------------------------------------------------------------|-------------------------------------------------------------------------------------------------------------------------------|
| Severity of Diarrhea/Colitis                                                                                                         | Initial Management                                                  | Follow-up Management                                                                                                          |
| Grade 1 Diarrhea: increase < 4 stools/day over  Baseline; mild increase in ostomy  output compared to baseline Colitis: asymptomatic | Continue avelumab therapy Symptomatic treatment (e.g.,  loperamide) | Close monitoring for worsening symptoms Educate subject to report worsening immediately If worsens: Treat as Grade 2, 3 or 4. |  
| Gastrointestinal irAEs                                                                                                                                                                                                                                                                                                                                                                                                                                                                        |                                                                                                                                                                                                                                         |                                                                                                                                                                                                                                                                                                                                                          |
|-----------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|-----------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|----------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|
| Severity of Diarrhea/Colitis                                                                                                                                                                                                                                                                                                                                                                                                                                                                  | Initial Management                                                                                                                                                                                                                      | Follow-up Management                                                                                                                                                                                                                                                                                                                                     |
| Grade 2  Diarrhea: 4 to 6 stools per day over  Baseline; moderate increase in ostomy  output compared to baseline; limiting  instrumental ADL IV fluids indicated <  24 hours; not interfering with ADL Colitis: abdominal pain; mucus or  blood in stool                                                                                                                                                                                                                                     | Withhold avelumab therapy                                                                                                                                                                                                               | If improves to Grade ≤ 1:                                                                                                                                                                                                                                                                                                                                |
| Symptomatic treatment                                                                                                                                                                                                                                                                                                                                                                                                                                                                         | Resume avelumab therapy If persists > 5-7 days or recurs: Treat as Grade 3 or 4.                                                                                                                                                        |                                                                                                                                                                                                                                                                                                                                                          |
| Grade 3 to 4 Diarrhea (Grade 3): increase of ≥ 7  stools per day over Baseline;  hospitalization indicated; severe  increase in ostomy output compared to  baseline; limiting self-care ADL  incontinence; IV fluids ≥ 24 h;  interfering with ADL Grade 4: Life-threatening  consequences; urgent intervention  indicated Colitis (Grade 3): severe abdominal  pain, medical intervention indicated,  peritoneal signs Grade 4: life-threatening, urgent  intervention indicated perforation | Withhold avelumab for  Grade 3. Permanently discontinue  avelumab for Grade 4 or  recurrent Grade 3. 1 to 2 mg/kg/day prednisone IV  or equivalent Add prophylactic antibiotics for  opportunistic infections  Consider lower endoscopy | If improves:  Continue steroids until Grade ≤ 1, then taper over  at least 1 month; resume avelumab therapy  following steroids taper (for initial Grade 3).  If worsens, persists > 3 to 5 days, or recurs after  improvement: Add infliximab 5mg/kg (if no contraindication).  Note: infliximab should not be used in cases of  perforation or sepsis. |
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table 10 treatment modifications for

| Dermatological irAEs              |                                                                      |                                                                                                                                                                                                                                                                                                                                                                     |
|-----------------------------------|----------------------------------------------------------------------|---------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|
| Rash                              | Initial Management                                                   | Follow-up Management                                                                                                                                                                                                                                                                                                                                                |
| Grade 1 to 2                      | Continue avelumab                                                    | If persists > 1 to 2 weeks or recurs: Consider  skin biopsy Withhold avelumab Consider skin biopsy Consider 0.5 to 1 mg/kg/day prednisone or  equivalent. Once improving, taper steroids  over at least 1 month, consider prophylactic  antibiotics for opportunistic infections, and  resume avelumab following steroids taper. If worsens:  Treat as Grade 3 to 4 |
| Covering < 30% body surface  area | Symptomatic therapy (for example,  antihistamines, topical steroids) |                                                                                                                                                                                                                                                                                                                                                                     |  
| Dermatological irAEs                                                                                                                                                                                      |                                                                                                      |                           |
|-----------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|------------------------------------------------------------------------------------------------------|---------------------------|
| Rash                                                                                                                                                                                                      | Initial Management                                                                                   | Follow-up Management      |
| Grade 3 to 4                                                                                                                                                                                              | Withhold avelumab for Grade 3                                                                        | If improves to Grade ≤ 1: |
| Permanently discontinue for Grade 4 or  recurrent Grade 3 Consider skin biopsy Dermatology consult 1 to 2 mg/kg/day prednisone or  equivalent  Add prophylactic antibiotics for  opportunistic infections |                                                                                                      |                           |
| Covering > 30% body surface  area; life threatening  consequences                                                                                                                                         | Taper steroids over at least 1 month and add  prophylactic antibiotics for opportunistic  infections |                           |
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table 11 treatment modifications for

| Pulmonary irAEs                                                                                                                                                                   |                                                                                                                                                                                                                                  |                                                                                                                                                                                                              |
|-----------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|----------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|--------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|
| Pneumonitis                                                                                                                                                                       | Initial Management                                                                                                                                                                                                               | Follow-up Management                                                                                                                                                                                         |
| Grade 1                                                                                                                                                                           | Consider withholding avelumab                                                                                                                                                                                                    | Re-assess at least every 3 weeks                                                                                                                                                                             |
| Radiographic changes only                                                                                                                                                         | Monitor for symptoms every 2 to 3 days                                                                                                                                                                                           | If worsens:                                                                                                                                                                                                  |
| Consider Pulmonary and Infectious  Disease consults                                                                                                                               | Treat as Grade 2 or Grade 3 to 4                                                                                                                                                                                                 |                                                                                                                                                                                                              |
| Grade 2                                                                                                                                                                           | Withhold avelumab                                                                                                                                                                                                                | Re-assess every 1 to 3 days                                                                                                                                                                                  |
| Mild to moderate new  symptoms                                                                                                                                                    | Pulmonary and Infectious Disease  consults Monitor symptoms daily, consider  hospitalization 1 mg/kg/day prednisone or equivalent Add prophylactic antibiotics for  opportunistic infections  Consider bronchoscopy, lung biopsy | If improves: When symptoms return to Grade ≤ 1, taper  steroids over at least 1 month and then  resume avelumab following steroids taper If not improving after 2 weeks or worsening:  Treat as Grade 3 to 4 |
| Grade 3 or 4                                                                                                                                                                      | Permanently discontinue avelumab                                                                                                                                                                                                 | If improves to Grade ≤ 1:                                                                                                                                                                                    |
| Grade 3:Severe new symptoms;  New / worsening hypoxia; Grade4: Life-threatening                                                                                                   | Hospitalize                                                                                                                                                                                                                      | Taper steroids over at least 1 month                                                                                                                                                                         |
| Pulmonary and Infectious Disease  consults 1 to 2 mg/kg/day prednisone o equivalent Add prophylactic antibiotics for  opportunistic infections Consider bronchoscopy, lung biopsy | If not improving after 48 hours or worsening: Add additional immunosuppression (for  example, infliximab, cyclophosphamide, IV  immunoglobulin, or mycophenolate mofetil)                                                        |                                                                                                                                                                                                              |
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table 12 treatment modifications for

| Hepatic irAEs                                                                               |                                                                                                                                                                                                                                                                                           |                                                                                                                                                                                                                                                                                                                              |
|---------------------------------------------------------------------------------------------|-------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|
| Liver test elevation                                                                        | Initial Management                                                                                                                                                                                                                                                                        | Follow-up Management                                                                                                                                                                                                                                                                                                         |
| Grade 1 Grade 1 AST or ALT > ULN to  3.0 x ULN and / or total  bilirubin > ULN to 1.5 x ULN | Continue avelumab                                                                                                                                                                                                                                                                         | Continue liver function monitoring If worsens: Treat as Grade 2 or 3 to 4                                                                                                                                                                                                                                                    |
| Grade 2                                                                                     | Withhold avelumab                                                                                                                                                                                                                                                                         | If returns to Grade ≤1:                                                                                                                                                                                                                                                                                                      |
| Increase frequency of monitoring to  every 3 days                                           |                                                                                                                                                                                                                                                                                           |                                                                                                                                                                                                                                                                                                                              |
| AST or ALT > 3.0 to ≤ 5 x  ULN and/or total bilirubin >  1.5 to ≤ 3 x ULN                   | Resume routine monitoring, resume avelumab If elevations persist > 5 to 7 days or worsen: Treat as Grade 3 or 4                                                                                                                                                                           |                                                                                                                                                                                                                                                                                                                              |
| Grade 3 to 4                                                                                | Permanently discontinue avelumab                                                                                                                                                                                                                                                          | If returns to Grade ≤ 1:                                                                                                                                                                                                                                                                                                     |
| AST or ALT > 5 x ULN and/or  total bilirubin > 3 x ULN                                      | Increase frequency of monitoring to  every 1 to 2 days  1 to 2 mg/kg/day prednisone or  equivalent Add prophylactic antibiotics for  opportunistic infections  Consult gastroenterologist/ hepatologist Consider obtaining MRI/CT scan of liver  and liver biopsy if clinically warranted | Taper steroids over at least 1 month and add  prophylactic antibiotics for opportunistic  infections If does not improve in > 3 to 5 days, worsens  or rebounds: Add mycophenolate mofetil 1 gram (g) twice  daily If no response within an additional 3 to 5  days, consider other immunosuppressants per  local guidelines |
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table 13 treatment modifications for

| Renal irAEs                                      |                                                                                                                                                                                |                                                                                                                     |
|--------------------------------------------------|--------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|---------------------------------------------------------------------------------------------------------------------|
| Creatinine Increase                              | Initial Management                                                                                                                                                             | Follow-up Management                                                                                                |
| Grade 1 Creatinine increased > ULN to  1.5 x ULN | Continue avelumab                                                                                                                                                              | Continue renal function monitoring If worsens: Treat as Grade 2 or 3 to 4                                           |
| Grade 2 to 3                                     | Withhold avelumab                                                                                                                                                              | If returns to Grade ≤1:                                                                                             |
| Creatinine increased > 1.5 and  ≤ 6 x ULN        | Increase frequency of monitoring to  every 3 days 1 to 2 mg/kg/day prednisone or  equivalent. Add prophylactic antibiotics for  opportunistic infections Consider renal biopsy | Taper steroids over at least 1 month, and  resume avelumab following steroids taper. If worsens:  Treat as Grade 4. |  
| Renal irAEs                                                                                                                                     |                                  |                                      |
|-------------------------------------------------------------------------------------------------------------------------------------------------|----------------------------------|--------------------------------------|
| Creatinine Increase                                                                                                                             | Initial Management               | Follow-up Management                 |
| Grade 4                                                                                                                                         | Permanently discontinue avelumab | If improves to Grade ≤ 1:            |
| Creatinine increased > 6 x ULN                                                                                                                  | Monitor creatinine daily         | Taper steroids over at least 1 month |
| 1 to 2 mg/kg/day prednisone or  equivalent. Add prophylactic antibiotics for  opportunistic infections Consider renal biopsy Nephrology consult |                                  |                                      |
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table 14 treatment modifications for

| Cardiac irAEs                                                                                                                                                                             |                                                                                                                                                                                                                                                                                                                                                                                                                             |                                                                                                                                                                      |
|-------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|-----------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|----------------------------------------------------------------------------------------------------------------------------------------------------------------------|
| Myocarditis                                                                                                                                                                               | Initial Management                                                                                                                                                                                                                                                                                                                                                                                                          | Follow-up Management                                                                                                                                                 |
| New onset of cardiac signs or  symptoms and / or new  laboratory cardiac biomarker  elevations (e.g., troponin, CKMB, BNP) or cardiac imaging  abnormalities suggestive of  myocarditis                                                                                                                                                                                           | Withhold avelumab.  Hospitalize. In the presence of life threatening cardiac  decompensation, consider transfer to a  facility experienced in advanced heart  failure and arrhythmia management. Cardiology consult to establish etiology  and rule out immune- mediated  myocarditis. Guideline based supportive treatment as  per cardiology consult.* Consider myocardial biopsy if  recommended per cardiology consult. | If symptoms improve and immune-mediated  etiology is ruled out, re-start avelumab. If symptoms do not improve/worsen, viral  myocarditis is excluded, and immunemediated etiology is suspected or confirmed  following cardiology consult, manage as  immune-mediated myocarditis.                                                                                                                                                                      |
| Immune-mediated myocarditis                                                                                                                                                               | Permanently discontinue avelumab.                                                                                                                                                                                                                                                                                                                                                                                           | Once improving, taper steroids over at least 1  month. If no improvement or worsening, consider  additional immunosuppressants (e.g.,  azathioprine, cyclosporine A) |
| Guideline based supportive treatment as  appropriate as per cardiology consult.*  1 to 2 mg/kg/day. prednisone or  equivalent Add prophylactic antibiotics for  opportunistic infections. |                                                                                                                                                                                                                                                                                                                                                                                                                             |                                                                                                                                                                      |
| *Local guidelines, or e.g., European Society of Cardiology or American Heart Association guidelines European Society of Cardiology guidelines website: https://www.escardio.org/Guidelines/Clinical-PracticeGuidelines American Heart Association guidelines website:  http://professional.heart.org/professional/GuidelinesStatements/searchresults.jsp?q=&y=&t=1001                                                                                                                                                                                           |                                                                                                                                                                                                                                                                                                                                                                                                                             |                                                                                                                                                                      |  
| Table 15: Treatment Modifications for Endocrine Immune-related AEs Endocrine irAEs Endocrine disorder Initial Management Follow-up Management Grade 1 or Grade 2  endocrinopathies (hypothyroidism,  hyperthyroidism, adrenal  insufficiency, type I diabetes  mellitus) Continue avelumab Continue hormone  replacement/suppression and  monitoring of endocrine function  as appropriate. Endocrinology consult if needed Start thyroid hormone replacement therapy (for  hypothyroidism), anti-thyroid treatment (for  hyperthyroidism), corticosteroids (for adrenal  insufficiency) or insulin (for Type I diabetes  mellitus) as appropriate.  Rule-out secondary endocrinopathies (i.e.,  hypopituitarism / hypophysitis) Grade 3 or Grade 4  endocrinopathies (hypothyroidism,  hyperthyroidism, adrenal  insufficiency, type I diabetes  mellitus) Withhold avelumab Resume avelumab once  symptoms and/or laboratory tests  improve to Grade < 1 (with or  without hormone  replacement/suppression). Continue hormone  replacement/suppression and  monitoring of endocrine function  as appropriate. Consider hospitalization Endocrinology consult Start thyroid hormone replacement therapy (for  hypothyroidism), anti-thyroid treatment (for  hyperthyroidism), corticosteroids (for adrenal  insufficiency) or insulin (for type I diabetes  mellitus) as appropriate.  Rule-out secondary endocrinopathies (i.e.,  hypopituitarism / hypophysitis) Hypopituitarism/Hypophysitis If secondary thyroid and/or adrenal insufficiency  is confirmed (i.e., subnormal serum FT4 with  (secondary endocrinopathies) inappropriately low TSH and/or low serum  cortisol with inappropriately low ACTH) :  Refer to endocrinologist for dynamic  testing as indicated and measurement of  other hormones (FSH, LH, GH/IGF-1,  PRL, testosterone in men, estrogens in  women)   Hormone replacement/suppressive therapy  as appropriate   Perform pituitary MRI and visual field  examination as indicated Resume avelumab once  symptoms and hormone tests  improve to Grade < 1 (with or  without hormone replacement). In addition, for hypophysitis with  abnormal MRI, resume avelumab  only once shrinkage of the  pituitary gland on MRI/CT scan  is documented.  Continue hormone  replacement/suppression therapy  as appropriate.If hypophysitis confirmed:  Continue avelumab if mild symptoms with  normal MRI. Repeat the MRI in 1 month  Withhold avelumab if moderate, severe or  life-threatening symptoms of  hypophysitis and/or abnormal MRI.  Consider hospitalization. Initiate  corticosteroids (1 to 2 mg/kg/day  prednisone or equivalent) followed by  corticosteroids taper during at least 1  month.  Add prophylactic antibiotics for  opportunistic infections.   |
|---------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|
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table 15 treatment modifications for

| Table 16: Treatment Modifications for Other Immune-related AEs Other irAEs (not described above) Grade of other irAEs (NCI CTCAE v5) Initial Management Follow-up Management Grade 2 or Grade 3 clinical  signs or symptoms suggestive  of a potential irAE Withhold avelumab pending clinical  If irAE is ruled out, manage as appropriate  investigation according to the diagnosis and consider restarting avelumab If irAE is confirmed, treat as Grade 2 or 3  irAE. Grade 2 irAE  Withhold avelumab If returns to Grade < 1: Or  first occurrence of Grade  3 irAE 1 to 2 mg/kg/day prednisone or  equivalent. Add prophylactic antibiotics for  opportunistic infections Specialty consult as appropriate Taper steroids over at least 1 month, and  resume avelumab following steroids taper. Recurrence of same Grade  Permanently discontinue avelumab 3 irAEs 1 to 2 mg/kg/day prednisone or  equivalent. Add prophylactic antibiotics for  opportunistic infections Specialty consult as appropriate If returns to Grade < 1: Taper steroids over at least 1 month Grade 4 Permanently discontinue avelumab If improves to Grade ≤ 1: 1 to 2 mg/kg/day prednisone or  equivalent and/or other  immunosuppressant as needed. Add prophylactic antibiotics for  opportunistic infections Specialty consult Taper steroids over at least 1 month Requirement for 10 mg  per day or greater  prednisone or equivalent  for more than 12 weeks for  reasons other than  hormonal replacement for  adrenal insufficiency Permanently discontinue avelumab Specialty consult Persistent Grade 2 or 3  irAE lasting 12 weeks or  longer Abbreviations: ACTH=adrenocorticotropic hormone; ADL=activities of daily living; ALT=alanine   |
|---|
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table 16 treatment modifications for

Abbreviations: ACTH=adrenocorticotropic hormone; ADL=activities of daily living; ALT=alanine aminotransferase; AST=aspartate aminotransferase; BNP=B-type natriuretic peptide; CK-MB=creatine kinase MB; CT= computed tomography; FSH=follicle-stimulating hormone; GH=growth hormone; IGF-1=insulin-like growth factor 1; irAE=immune related adverse event; IV=intravenous; LH=luteinizing hormone; MRI=magnetic resonance imaging; NCI CTCAE=National Cancer Institute Common Terminology Criteria for Adverse Events; PRL=prolactin;T4=thyroxine; TSH=thyroid stimulating hormone; ULN=upper limit of normal.
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3.3.3 unacceptable toxicity

Occurrence of any of the adverse events listed in Section **3.1.1** after the first 28 days of treatment in the Dose Expansion cohort (Cohort 2) will lead to permanent discontinuation of protocol therapy, but will not be deemed a DLT for purposes of dose escalation. Any patient who requires corticosteroid treatment for an avelumab related irAE will have their rhIL-15 discontinued regardless of their perceived tolerance of the cytokine. Patients may continue single agent avelumab after halting rhIL-15 treatment if considered appropriate in the standard avelumab irAE decision algorithm.
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3.4 study intervention compliance

Adherence to the protocol will be verified by reviewing the Medical Administration Record (MAR) section of the medical record, which will serve as source documentation.
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3.5 on study evaluations

Prior to avelumab administration (Day 8 and 22), pre-dose assessments must be performed (up to 3 days prior). After Cycle 1, pre-dose assessments may be performed up to 3 days prior to a cycle except where otherwise noted. In the event that the patient's condition is deteriorating, laboratory evaluations should be repeated within 48 hours prior to initiation of the next cycle of therapy. The results from all procedures/tests must be reviewed prior to initiation of each cycle of treatment for consideration of dose modifications and delay of therapy. Treatment with rhIL-15 and avelumab will continue for 4 cycles or until disease progression, unacceptable treatment-related toxicity or other reasons outlined in Section **3.9.1**. Refer to the Study Calendars (Section **APPENDIX F**) for a complete list of procedures to be performed at each scheduled study visit. See also Section 5 for all samples to be collected for correlative research. During treatment, it is expected that all laboratory and clinical assessments be conducted at the NIH (including post-treatment imaging evaluations); results from outside NIH will only be accepted at the discretion of the investigator.
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3.6 post-treatment evaluations

Post treatment evaluations (i.e., End of Treatment Visit) will be performed approximately 30 days after the last dose of protocol treatment. If a subject initiates a new anti-cancer therapy within 30 days after the last dose of trial treatment, the 30-day safety follow-up visit must occur before the first dose of the new therapy, if possible. If the patient cannot return to the Clinical Center for this visit, a request will be made to have a local physician or laboratory collect a CBC with differential and send the results. If this is not possible, patients may be assessed by telephone or email for symptoms. Unless otherwise noted, follow-up will occur at the following time point: every 60 days (± 7 days) for 6 months; then every 90 days (± 14 days) for 2 years; then every six months (± 28 days) for the duration of the study until the start of a new anti-neoplastic therapy, disease progression, death or end of study. Any other evaluations and tests should be performed as clinically indicated. Upon disease progression or initiation of other anti-cancer therapy, contact will be for survival only until the subject is off study (i.e., every 3 months [±4 weeks]) for the duration of the study. See Study Calendar (Section **APPENDIX F**) for additional information. Any adverse events which are present at the time of discontinuation should be followed in accordance with the safety requirements. Patients who are removed from study before completing protocol treatment will be followed until completion of the Safety Follow-up Visit or until death, whichever occurs first. Patients removed from study for unacceptable adverse event(s) will be followed until resolution or stabilization of the adverse event.  
3.6.1 Safety Follow-Up Visit The safety follow-up visit should occur 30 days (±7 days) after the last dose of trial treatment, or before the initiation of a new anti-cancer treatment, whichever comes first. Required testing is as noted in the Study Calendars (Section **APPENDIX F**). All AEs that occur prior to the Safety Follow-Up Visit should be recorded. Patients with an ongoing, treatment-related AE of Grade > 1 will be followed until the resolution of the AE to Grade 0-1, stabilization of the AE in the opinion of the investigator, or until the beginning of a new anti-neoplastic therapy, whichever occurs first.  
SAEs that occur within 30 days of the end of treatment or before initiation of a new anti-cancer treatment should also be followed and recorded.  
3.6.2 Extended Safety Follow-up Given the potential risk for delayed immune-related toxicities, safety follow-up must be performed up to 90 (+14) days after the last dose of avelumab administration. This may be performed either via a clinic visit or via a telephone call/email with subsequent clinic visit requested in case any concerns are noted during the telephone call/email.  
3.6.3 Follow-Up Visits- Prior to Disease Progression Patients who complete trial treatment or discontinue treatment without evidence of disease progression will move into the Follow-up Phase and may be assessed every 60 days (± 7 days) for 6 months; then every 90 days (± 14 days) for 2 years; then every six months (± 28 days) for the duration of the study after finishing treatment by radiologic imaging or other clinical assessments to monitor disease status. Every effort should be made to collect information regarding disease status until the start of new anti-neoplastic therapy, disease progression, death, or end of the study If the patient cannot return to the Clinical Center for any of these visits, a request will be made to have a local physician or laboratory collect a CBC with differential and send the results. If this is not possible, patients may be assessed by telephone or email for symptoms.  
3.6.4 Follow-Up Visits - Survival/Post-Disease Progression Once a subject experiences confirmed disease progression or starts a new anti-cancer therapy, the subject moves into the survival follow-up phase and should be contacted (e.g., phone, email, etc.; in-person visit not required) at least every 3 months for the duration of the study to collect information on new anti-cancer treatments received and to assess for survival status until death, withdrawal of consent, or the end of the study, whichever occurs first (see Study Calendar).
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3.7 study calendar see appendix

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.
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3.8.3 reimbursement

On this study, the NCI will cover the cost for some of the expenses. Some of the costs may be paid directly by the NIH and some may be reimbursed to the subject. Someone will work with subjects to provide more information 3.9 CRITERIA FOR REMOVAL FROM PROTOCOL THERAPY AND OFF STUDY C**RITERIA** Prior to removal from study, effort must be made to have all patients complete a safety visit approximately 30 days following the last dose of study therapy. Additional safety visits and followup will continue as per Section 3.6.  
3.9.1 Criteria for removal from protocol therapy In the absence of treatment delays due to adverse event(s), treatment may continue for 4 cycles or until one of the following criteria applies. Patients who meet the following criteria should be discontinued from protocol therapy:
Completion of protocol therapy (i.e., up to 4 cycles)  
Clinical or Disease progression
Intercurrent illness that prevents further administration of treatment  
Unacceptable toxicities as listed in Section **3.1.1** or those toxicities listed in Section **3.3**
that require treatment to be stopped.  
Subject's request to withdraw from protocol therapy Investigator's decision to withdraw the patient Subject's non-compliance with trial treatment or procedure requirements that requires removal in the opinion of the PI
Pregnancy Study is cancelled for any reason The drug manufacturer can no longer proved the study agent
3.9.2 Off-Study Criteria Subject requests to be withdrawn from study Subject is lost to follow-up* Death Study is cancelled for any reason
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3.9.3 lost to follow-up

* A participant will be considered lost to follow-up if he or she fails to return for 3 consecutive scheduled visits without good cause 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 within 1-3 business days (to take into account the weekend) to reschedule the missed visit, 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

Medications or vaccinations specifically prohibited in the exclusion criteria are not allowed during the ongoing trial. If there is a clinical indication for one of these or other medications or vaccinations specifically prohibited during the trial, discontinuation from trial therapy or vaccination may be required. For premedication and supportive care measures, see Section 3.2.1, **3.2.2** and 3.2.3.
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4.1 acceptable medications

All treatments that the investigator considers necessary for a subject's welfare may be administered at the discretion of the investigator in keeping with the community standards of medical care. All concomitant medication will be recorded on the case report form (CRF) including all prescription, over-the-counter (OTC), herbal supplements, and IV medications and fluids. If changes occur during the trial period, documentation of drug dosage, frequency, route, and date may also be included on the CRF.  
All concomitant medications received within 28 days before the first dose of trial treatment and 30 days after the last dose of trial treatment should be recorded. 4.2 PROHIBITED M**EDICATIONS** Patients are prohibited from receiving the following therapies during treatment on this trial:  
Other therapy for the disease under study not specified in this protocol, unless specifically noted as permitted Investigational agents other than rhIL-15 and avelumab Radiation therapy Live vaccines within 30 days prior to the first dose of trial treatment and while participating in the trial. Examples of live vaccines include, but are not limited to, the following:
measles, mumps, rubella, varicella/zoster, yellow fever, rabies, BCG, and typhoid vaccine and Flu Mist.  
Patients who, in the assessment by the investigator, require the use of any of the aforementioned treatments for clinical management should be removed from study treatment. Patients may receive other medications that the investigator deems to be medically necessary.
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5 correlative studies 5.1 summary

This study will attempt to use rhIL-15 to increase NK cell number and activity, thereby enhancing the ADCC of avelumab in treatment of patients with relapsed and refractory metastatic PD-L1+ ccRCC. ADCC capacity of ex vivo PBMCs will be tested on PD-L1-expressing cell lines before, during and after protocol treatment. Differences in immune cell subsets associated with administration will be followed throughout treatment to both study the effects of combined rhIL15/avelumab therapy on the immune system, and to identify potential biomarkers that would be predictive of response.  
| Time Points                                                         | Supervising  Laboratory/  Investigator   |              |        |          |              |                            |              |
|---------------------------------------------------------------------|------------------------------------------|--------------|--------|----------|--------------|----------------------------|--------------|
| Sample                                                              | Collection Details*                      | Baselin      | C1     | C1       | C2-4         | C2-4                       | Follow       |
| e                                                                   | D8                                       | D22          | D1     | D8       | up#          |                            |              |
| Blood Samples                                                       | Immunology                               |              |        |          |              |                            |              |
| Lymphocyte subset                                                   |  2 x 10mL K2EDTA  (lavender-top) tubes  | X            | X      | (X)      | (X)          | (X) 1                      | section, NIH |
| testing                                                             | CC                                       |              |        |          |              |                            |              |
|  3 x 10mL sodium                                                   | (X)2                                     | (X)2         | (X) 2  | Waldmann |              |                            |              |
| ADCC of avelumab                                                    | heparin green-top                        | (Leidos CSL) |        |          |              |                            |              |
| tubes                                                               |                                          |              |        |          |              |                            |              |
|  1 x 10mL K2EDTA  tube  1 x 10mL  Streck/BCT tube                 |                                          |              |        |          |              |                            |              |
| Circulating tumor DNA,  plasma banking                              | X                                        | (X)          | (X)    | X3       | (Leidos CSL) |                            |              |
| Anti-IL-15 antibodies                                               |  1 x 4mL SST tube                       | X            | X      | X1       | (Leidos CSL) |                            |              |
| Tissue Samples Tissue immune cell  subsets by  Immunohistochemistry |  Two core biopsy  samples in formalin   | X**          | (X)*** | (X)***   | (X)          | (Laboratory  of Pathology) |              |  
Time Points Supervising Laboratory/
Investigator
(X) = Optional *Tubes/media may be adjusted at the time of collection based upon materials available or to ensure the best samples are collected for planned analyses. ** Pretreatment tumor biopsy sample: may use the archival tissue or the freshly obtained tumor biopsy used to assess PD-L1 expression for eligibility *** The on-treatment tumor biopsy may be obtained anywhere between cycle 1 day 22 and cycle 2 day 4 at whichever time is most expedient for the patient and Interventional Radiology
\#Subjects who discontinue treatment for a reason other than disease progression and who do not start new treatment should continue to have study bloods collected at the scheduled time points.  
1 At the end of treatment only. 2 For ADCC of avelumab, Baseline and C1D8 samples should be collected for at least one patient per dose level, and for at least three patients at the MTD; C2-6 samples may be collected at the discretion of the PI but are not required.  
3 At each follow-up visit prior to disease progression, as specified in Section **3.6.3**
Sample **Collection Details***
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5.2 sample collection and processing

5.2.1 Blood Samples All blood samples will be drawn by NIH Clinical Center phlebotomy, outpatient clinic, or day hospital staff 5.2.2 Summary The planned analyses described below may be done on leftover and/or shared sample portions from the respective laboratories, as needed. In addition to the prospectively collected samples below, leftover portions of samples sent for routine laboratory testing (e.g., plasma from CBC/hematologies) may also be retrieved for research tests prior to being discarded. The planned prospective analyses are identified below; laboratories may share resources or collaborate on analyses, if appropriate. Portions of all samples may be banked for future research analyses; prospective consent will be obtained during the informed consent process. The blood drawing limits for research purposes are as follows:  
For adult subjects: The amount of blood that may be drawn from adult patients for research purposes shall not exceed 10.5 mL/kg or 550 mL, whichever is smaller, over any eight-week period.
Unless otherwise noted, Clinical Research personnel will use courier or escort service to arrange delivery of samples to destinations listed in subsections below.
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5.2.3 blood samples

5.2.3.1 Lymphocyte subset testing by flow cytometry (FACS)  
Whole blood will be collected for each timepoint in 10mL EDTA tubes that are gently invert tubes 8-10 times immediately after collection to thoroughly mix the sample to prevent clotting and loss of mononuclear cells intended to be analyzed.  
Labels will be affixed to these tubes with the patient's name, date of birth, date and time of the blood draw by the hospital staff who obtained the samples.  
One each of the 2 EDTA tubes drawn at the timepoints summarized in table 2 will be delivered to 2 separate locations:
o Routine LYMB flow cytometry panels will be transported to Immunology Lab, NIH Clinical Center Bldg. 10/ Room 2C410 for analysis. If the Immunology Section Laboratory is unable to perform the complete LYMB flow cytometry panels for the specified timepoint, the analysis maybe omitted or replaced with the more limited "TBNK panel."
o Clinical Research Staff will arrange for the cells collected for more extensive bulk flow cytometry studies performed at the completion of all subjects' protocol treatments to be picked up by the Frederick courier from either Phlebotomy or the 12 Floor Outpatient Clinic (OP12) and conveyed to the CLS laboratory Frederick for controlled freezing and storage .  
5.2.3.2 Antibody-dependent cell cytotoxicity (ADCC) of avelumab
Whole blood will be collected for each timepoint in 10mL sodium heparin tubes that are gently invert tubes 8-10 times immediately after collection .  
Labels will be affixed to these tubes with the patient's name, date of birth, date and time of the blood draw by the hospital staff who obtained the samples.  
Clinical Research personnel will arrange for these samples to be delivered to Clinical Support Laboratory, Leidos Biomedical Research, Inc.in Frederick, MD, for processing per established laboratory techniques storage, and batch analysis by the Waldmann Laboratory.
5.2.3.3 Cell-free DNA (cfDNA), and circulating tumor DNA (ctDNA) and plasma banking  
Collect 10 mL of blood in one cell-free DNA (e.g., Streck BCT/collection tubes) and 10 mL of blood in one K2EDTA tube; gently invert the tubes 8-10 times immediately after collection.  
Labels listing the patient's name, date of birth, date and time of the blood draw will be affixed to all the tubes by the staff person who obtained the samples. each).  
Clinical Research personnel will arrange for these samples to be delivered to Clinical Support Laboratory, Leidos Biomedical Research, Inc.in Frederick, MD for sample processing per established laboratory techniques and maintained within standard operating procedures in the laboratory.
5.2.3.4 Anti-IL-15 antibody testing  
Collect blood in a 4mL SST tube; gently invert tubes 8-10 times immediately after collection.  
Clinical Research personnel will arrange for these samples to be delivered to Clinical Support Laboratory, Leidos Biomedical Research, Inc.in Frederick, MD, for storage and further analysis.  
Samples will be batch processed and analyzed per the procedure outlined in APPENDIX D after the last patient has been enrolled and completed treatment, or sooner if there is clinical suspicion for anti-IL-15 antibody formation.  
5.2.4 Tissue Samples 5.2.4.1 PDL-1 testing for immunohistochemistry (IHC)
If a prospective patient's tumor has not already assessed for PD-L1 expression as listed in the eligibility Section 2.1 archival block(s) or slides (i.e., at least 15 unstained slides, 5-microns) is/are required at screening. Patients with prior tumor biopsies performed at NIH or outside institutions may make specimen blocks available to the NCI Pathology Department for analysis. If no prior biopsies are available at the time of screening, a punch or core biopsy of an appropriate easily accessible tumor deposit will be performed during screening period to confirm PDL-1 status for eligibility. Patients with lymph node and/or visceral involvement will undergo an 18g core biopsy performed by the Department of Radiology and Imaging Sciences' Interventional Radiologists (IR).  
Prior specimen blocks will be sent to NCI Pathology Department via FedEx. For patients with no prior samples who undergo biopsy as part of screening, fresh samples will be placed in transport saline or 10% formalin PBS and sent to the Department of Laboratory Medicine (DLM)/ NCI Laboratory of Pathology (LP) (Section 5.1 and 5.2) for concurrent routine histologic analysis and reporting and IHC testing for PD-L1 expression by tumor area Lab).  
5.2.4.2 Optional Tissue Samples
Archival block(s)/ slides or fresh tumor tissue that was obtained to determine eligibility expression of PD-L1 will also be used for pre-treatment IHC analysis of lymphocyte subsets and other immune molecules as described below in section **5.3.5**. If no prior sample is available, core biopsies will be performed during the screening period by IR and the specimens will be transported to NCI pathology as described previously in section **5.2.4.1**.  
The optional, on-treatment research biopsy will be obtained during the period from cycle 1 day 22 to cycle 2 day 4 and sent to the Laboratory of Pathology as described above in section **5.2.4.1**. and under the analyses described below in section **5.3.5**.
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5.3 biomarker and research methods

The technology platforms that are able to interrogate genomic structure and function are constantly in flux; therefore, the exact nature of the methodologies that will be employed will be assessed at the time that the samples are collected and ready for analysis. The protocol will be amended at that time, if needed, to describe the intended techniques prior to initiating the analyses The following are technologies that are currently in use for each planned analysis:
5.3.1 Flow Cytometry of Peripheral Blood Lymphocytes Immunohistochemical (IHC) analyses of tumor tissue samples, including but not necessarily limited to CD2, CD3, CD4, CD5, CD7, CD8, CD14, CD16, CD20, CD25, CD38, CD45RA, CD45RO, CD56, CD62L, CD69, CD79a/CD79b, CD122, Foxp3, perforin, gamma/delta, CXCR3, CCR4, and NKG2D.  
5.3.2 Immune Subset Analysis Peripheral blood mononuclear cells (PBMC) will be assessed using multiparameter flow cytometry listed in section **5.3.1** and used to define cytotoxic CD8+ T-cells, Tregs, T-effector cells, Th1, Th2 and Th17+ CD4+ T-cells, NK cells and subsets, monocyte subsets, MDSC subsets. Assessment may include functional markers, i.e. PD-1, Tim-3, CTLA-4, PD-L1, HLA-DR, Ki67 and/or CD40.  
5.3.3 ADCC Analysis Peripheral blood mononuclear cells (PBMCs) should be isolated by Ficoll-Hypaque Density Gradient Centrifugation. The viable cells should be viably frozen and stored in liquid nitrogen. The ADCC assay will be performed on the same occasion for all samples of a given patient. Vials of frozen cells will be thawed using standard procedures 18 hours before the assay in accord with our experience with normal donors. 1.5 million of patient's PBMCs obtained before and on day 15 following IL-15 injection will be tested in aliquots as follows:
Tested alone Tested with untreated PD-L1-expressing Raji cells, and with the Raji cells coated with avelumab for 5 hours.  
In addition, we may utilize an ATL cell line in addition to Raji cells. These cell populations will be stained with CD107, CD3, CD56, and CD94.  
5.3.4 cfDNA/ctDNA
Since the methods of molecular monitoring in the peripheral blood is an emerging field with numerous technologies under development, the storage of peripheral blood mononuclear cells (PBMC), serum, and plasma will all be performed allowing for future comparison of the different compartments for analytes that include cell-free circulating tumor DNA (ctDNA), and RNA sequencing of circulating tumor cells. Studies to be performed on these samples include: cfDNA/ctDNA for liquid genotyping as a non-invasive dynamic monitoring of disease as well as monitoring for individual molecular aberrations that may be present for individual patients. Changes in the level of ctDNA have been shown to correlate with response to tyrosine kinase inhibitor (TKI) treatment for mRCC and will be quantitated to assess response to therapy [81, 82]. Analysis of ctDNA for characteristic mutations present in ccRCC will also be performed [83]. Additional samples will be stored for later bulk analysis of chromosomal copy number instability (CNI) which recently has been identified as a possible surrogate marker of response to immunotherapy [84, 85] and to analyze for loss of function PBrm1 mutations that was recently reported to be associated with increased clinical benefit in ccRCC patients after ICI treatment [86].  
5.3.5 Immunohistochemical Analyses of Optional Tissue Samples Standard H&E pathologic examination and IHC listed in section **5.3.1** for peripheral blood lymphocytes, will also be performed on the tissue samples obtained to characterize the *in situ* immune response. In addition, other important immune molecules such as PD-1, Fas, FasL, KIR2DL1, NKG2A, NKG2D, TIGID will be performed using established procedures for the Laboratory of Pathology. In situ cytokine analysis (e.g., IL-6, IL-10, interferon beta, TNF-alpha) using established laboratory procedures.  
5.3.6 Other Analyses Testing for anti-IL-15 antibodies will be on a regular basis or in bulk to determine if any subject developed anti-IL-15 antibodies that may have affected their response to therapy. Since no subject's treatment with CIV rhIL-15 [70, 87]have developed antibodies to the cytokine, this test is no longer required prior to the administration of any cycle of treatment.  
5.3.7 Future Use Any blood, tissue, or other products or portions leftover from other analyses will be stored for future research.