9.1.3 Storage and Stability The product does not contain a preservative. Vials of nivolumab injection must be stored at 2-8°C (36°- 46°F) and protected from light, freezing, and shaking. After preparation, store the nivolumab infusion either: At room temperature for no more than 8 hours from the time of preparation. This includes room temperature storage of the infusion in the IV container and time for administration of the infusion or under refrigeration at 2°C to 8°C (36°F-46°F) for no more than 24 hours from the time of infusion preparation. Do not freeze. If a temperature excursion is noted, sites should follow institutional policies for notifying Bristol-Myers Squibb and assessing if the drug is still able to be used. 9.1.4 Compatibility Do not co-administer other drugs through the same intravenous line. Flush the intravenous line at end of infusion. No incompatibilities between nivolumab and polyvinyl chloride (PVC), non-PVC/non DEHP (di(2-ethylhexyl)phthalate) IV components, or glass bottles have been observed. 9.1.5 Handling Qualified personnel, familiar with procedures that minimize undue exposure to themselves and the environment, should undertake the preparation, handling, and safe disposal of the chemotherapeutic agent in a self-contained and protective environment. 9.1.6 Availability Nivolumab for injection will be supplied free of charge by BMS. 9.1.7 Preparation Visually inspect drug product solution for particulate matter and discoloration prior to administration. Nivolumab is a clear to opalescent, colorless to pale-yellow solution. Discard the vial if the solution is cloudy, is discolored, or contains extraneous particulate matter other than a few translucent-to-white, proteinaceous particles. Do not shake the vial. Withdraw the required volume of nivolumab and transfer into an intravenous container. Dilute nivolumab with either 0.9% Sodium Chloride Injection, USP or 5% Dextrose Injection, USP, to prepare an infusion with a final concentration greater than or equal to 0.35mg/ml .. Mix diluted solution by gentle inversion. Do not shake. 9.1.8 Administration Administer the infusion over 60 minutes (+/- 10 minutes) through an intravenous line containing a sterile, non-pyrogenic, low protein binding in-line filter (pore size of 0.2 micrometer to 1.2 micrometer). 9.1.9 Ordering Each institution will order nivolumab directly from the supplier Bristol Myers Squibb Pharmaceuticals (BMS). A study specific order form will be supplied by the lead site. 9.1.10 Accountability DF/HCC Protocol #: 18-280 Protocol Version Date: 10.17.2020 27 The investigator, or a responsible party designated by the investigator, should maintain a careful record of the inventory and disposition of the agent using the NCI Drug Accountability Record Form (DARF) or another comparable drug accountability form. (See the NCI Investigator’s Handbook for Procedures for Drug Accountability and Storage.) 9.1.11 Destruction and Return For this study, study drugs (those supplied by BMS or sourced by the investigator) such as partially used study drug containers, vials and syringes may be destroyed on site. On-site destruction is allowed provided the following minimal standards are met:  On-site disposal practices must not expose humans to risks from the drug.  On-site disposal practices and procedures are in agreement with applicable laws and regulations, including any special requirements for controlled or hazardous substances. BMS will be notified of all Nivolumab drug destructions.  Written procedures for on-site disposal are available and followed. The procedures must be filed with the site’s SOPs and a copy provided to BMS upon request.  Records are maintained that allow for traceability of each container, including the date disposed of, quantity disposed, and identification (ID) of the person disposing the containers. The method of disposal, (e.g., incinerator, licensed sanitary landfill, or licensed waste disposal vendor) must be documented. 9.2 DC/MM Fusion Vaccine 9.2.1 Isolation of Tumor Cells Patients will undergo aspiration of 30 mL of bone marrow from which myeloma cell preparations will be generated. Bone marrow will be aspirated into sterile heparinized syringes with approximately 5-8 mL aspirated per syringe to avoid significant hemodilution of bone marrow mononuclear cells. Bone marrow aspirate samples will be transferred in sterile syringes to the cell manipulation facility, and mononuclear cells will be isolated by ficoll density gradient centrifugation. Autologous plasma will be obtained by harvesting supernatant following centrifugation of 50 mL of peripheral blood. Bone marrow mononuclear cells will be cultured in media with 10% autologous plasma. An aliquot of the tumor cells will undergo immunohistochemical staining as detailed below. Tumor lysate will be prepared by freeze/thawing or sonication of an aliquot of tumor cells for immunological analysis. Myeloma cells may be frozen in 10% DMSO/90% autologous plasma stored in liquid nitrogen for potential subsequent vaccine generation. 9.2.2 DC Generation Patients will undergo leukapharesis for DC and vaccine generation. The leukapheresis product will be transferred in a sterile container to the cell manipulation facility, and PBMC will be isolated from the leukapheresis product and cultured in the presence of autologous plasma for 1- 2 hours and then cultured for 5-7 days with GM-CSF and IL-4. Cultures are re-fed with cytokines after 3 days. Twenty-five ng/mL of TNFα will be then be added for 48-72 hours to induce DC maturation. An aliquot of the DC preparation will undergo immunocytochemical staining for immunophenotypic analysis. 9.2.3 DC/MM Fusion Vaccine Preparation Tumor cells will be thawed (when relevant). Tumor cells and DC preparations will be co-cultured at ratio of 1:3 and washed in serum-free medium. After low-speed centrifugation, the cell pellet will be re-suspended in 50% solution of polyethylene glycol (PEG) and will be progressively diluted by the slow addition of serum-free medium. An aliquot of the DC, tumor, and fusion cell preparations will undergo immunocytochemical analysis for vaccine characterization. Dependent on cell yields, 3 doses of 1-5 x 106 fusion cells will be prepared. The fusion cells will then be separated into appropriate aliquots of fusion cells, radiated at 30 Gy, and frozen in 10% DMSO/90% autologous plasma in liquid nitrogen. A minimum of 2 doses will be required to proceed with treatment. An aliquot DF/HCC Protocol #: 18-280 Protocol Version Date: 10.17.2020 28 of the vaccine product will be sent for microbiological assessment consisting of endotoxin, mycoplasma and sterility testing as detailed in the CMC section. Release criteria for vaccine administration are: 1) > 50% of DC prep express CD86 2) Viability of DC prep > 70% 3) Fusion efficiency > 15% 4) Fusion viability > 50% 5) Sterility, mycoplasma, and endotoxin assays are negative 9.2.4 Administration Patients will be vaccinated with 1-5x106 fusion cells in the upper thigh region. The site will be alternated for each vaccine administration (right and left extremity). Vaccination will be administered subcutaneously using a 25-gauge 5/8-inch needle. Tumor vaccine will be administered first, followed by GM-CSF injection. 9.2.5 Ordering Vaccine will be ordered from the Stem Cell Lab at BIDMC on the day of treatment. The lab should be notified of all scheduled treatment days at least 1 week in advance. 9.3 GM-CSF GM-CSF is commercially available and will be purchased in bulk from commercial supply by the research pharmacy and billed to grant. On the day of vaccination, the clinical research nurse/physician assistant will administer 100 ug of GM-CSF subcutaneously at the site of the vaccine. Research pharmacy will reconstitute the drug with bacteriostatic water at the appropriate dose for the patient. The patient will be trained to inject the remaining three GM-CSF injections (100ug dose once a day) for self-administration subcutaneously at home. Tumor vaccine will be administered first, followed by GM-CSF injection. 10. BIOMARKER, CORRELATIVE, AND SPECIAL STUDIES 10.1 Correlative Studies 10.1.1 Myeloma-reactive T cells in the peripheral blood  10 mLs of peripheral blood will be collected in a green top tube at the pre-immunotherapy visit, prior to cycles 2-6, at each active monthly follow up visit, and at time of progression.  Specimens will be delivered to the Avigan lab at BIDMC.  Samples will be processed in the Avigan lab. Ficoll centrifugation of the peripheral blood sample will be performed to isolate peripheral blood mononuclear cells (PBMCs). PMBCs will be co-cultured with autologous tumor lysate prepared from the enrollment bone marrow aspirate. Surface antibody staining for CD4 and CD8 positive lymphocytes in addition to intracellular staining for IFN gamma will be performed on this co-culture to quantify IFN gamma producing T cells. DF/HCC Protocol #: 18-280 Protocol Version Date: 10.17.2020 29 10.1.2 Myeloma-reactive T cells in the bone marrow  5 mLs of bone marrow aspirate will be collected in a green top tube at the pre-immunotherapy visit, prior to cycle 4, at one, three, and six month active follow up visits and at time of progression.  Specimens will be delivered to the Avigan lab at BIDMC.  Samples will be processed in the Avigan lab. Ficoll centrifugation of the aspirate sample will be performed to isolate bone marrow mononuclear cells (BMMCs). BMMCs will be co- cultured with autologous tumor lysate prepared from the enrollment bone marrow aspirate. Surface antibody staining for CD4 and CD8 lymphocytes in addition to intracellular staining for IFN gamma will be performed on this co-culture to quantify IFN gamma producing T cells. 10.1.3 Effect of treatment on circulating T cells binding the MUC1, NY-ESO, Survivin, PRAME, and WT1 tetramers in HLA-A2.1 patients  10 mLs of peripheral blood in a green top tube at the pre-immunotherapy visit, prior to cycles 2-6, at each active monthly follow up visit, and at time of progression.  Specimens will be delivered to the Avigan lab at BIDMC.  Samples will be processed in the Avigan lab using a tetramer assay to isolate tetramer-specific T cells. Functional characteristics of the pentamer positive cells will be determined by quantifying expression of IFNγ, granzyme B, IL-4, and IL-10 expression by intracellular FACS staining. 10.1.4 T cell subsets , T cell checkpoint receptors, and MDSCs  10 mLs of peripheral blood in a green top tube at the pre-immunotherapy visit, prior to cycles 2-6, at each active monthly follow up visit, and at time of progression.  5 mLs of bone marrow aspirate will be collected in a green top tube prior to cycle 4, at one, three, and six month active follow up visits and at time of progression.  Specimens will be delivered to the Avigan lab at BIDMC.  Samples will be processed in the Avigan lab. The PB and BM aspirate samples will undergo ficoll centrifugation to obtain PBMCs/BMMCs. From the PBMCs/BMMCs, T cell subsets and MDSCs will be isolated by antibody staining and quantified by flow cytometric analysis. 10.1.5 Optional vaccine site biopsy  Patients will be referred to dermatology clinic for a punch biopsy of vaccine site reaction.  The biopsy specimen will be delivered to the Avigan lab at BIDMC.  Specimens will be stained using immunohistochemistry for T cell, DC, and myeloma markers to assess for immune cell infiltrate and possible tumor infiltrate. 10.1.6 Optional samples for future research studies  10 mLs of PB in a green top tube at the pre-immunotherapy visit, prior to cycles 2-6, at each active monthly follow up visit, and at time of progression.  5 mLs of bone marrow aspirate will be collected in a green top tube prior to cycle 4, at one, three, and six month active follow up visits and at time of progression.  These specimens will be delivered to the Avigan lab at BIDMC.  The specimens will be cryopreserved in the Avigan lab for possible future research studies. 10.2 Sample Collection time points Sample Collection for Stem Cell Lab Required1 At enrollment (at the time of tumor collection) Bone marrow aspirate for tumor collection (30 mL) X DF/HCC Protocol #: 18-280 Protocol Version Date: 10.17.2020 30 Plasma for Vaccine Production (50 mL) X Infectious Serologies X 1. Total tubes: 3 of the 10mL Na-Hep green top tubes aspirate, 5 of the 10mL Na-Hep green top tubes, 2 of the 6mL red top tubes and 3 of the 6mL pink top tubes peripheral blood Sample Collection for Avigan Lab Required At the pre- immunotherapy visit1 Prior to each cycle1, 3 Months 1-6 Active Follow-up2 Disease Progression1 Bone Marrow Aspirate for Myeloma Reactive T Cells (5 mL) X X (cycle 4 only) X (months 1, 3 and 6 only) X Bone marrow aspirate for tetramer reactivity (5 mL) X X (cycle 4 only) X Bone marrow aspirate for T cell subsets, T cell checkpoint receptors, MDSCs (5 mL) X (cycle 4 only) X (months 1, 3 and 6 only) X Peripheral Blood for Myeloma Reactive T Cells (10 mL) X X X X Peripheral blood for tetramer analysis (10 mL) X X X X Peripheral blood for T cell subsets, T cell checkpoint receptors, MDSCs (10 mL) X X X X 1. Total tubes: 3 of the 10mL Na-Hep green top tubes peripheral blood and 2 of the 10mL Na-Hep green top Aspirate 2. Total tubes: 3 of the 10mL Na-Hep green top tubes peripheral blood 3. These samples do not need to be collection at cycle 1, as they were collected at the pre-immunotherapy visit. Optional Sample Collection for Avigan Lab Required At enrollment (at the time of tumor collection)1 Prior to each cycle1 Months 1-6 Active Follow-up2 Disease Progression1 Optional Peripheral Blood Samples for Future Research (10 mL) X X X X Optional Bone Marrow Aspirate Samples for Future Research (5 mL) X (cycle 4 only) X (months 1, 3 and 6 only) X DF/HCC Protocol #: 18-280 Protocol Version Date: 10.17.2020 31 1. Total tubes: 1 of the 10mL Na-Hep green top tube peripheral blood; 1 of the 10mL Na-Hep green top tube aspirate 2. Total tubes: 1 of the 10mL Na-Hep green top tube peripheral blood 11. STUDY CALENDAR Baseline evaluations are to be conducted within 2 weeks prior to registration. Assessments must be performed prior to administration of any study agent. Study assessments and agents should be administered within + 3 days of the protocol-specified date, unless otherwise noted. Cycles 1-3 Study Assessments Pre- Enrollment At Enrollment (at the time of tumor collection) Pre- Immunotherapy Day 1 Day 15 Day 21 History, physical exam, weight and height X X X X Vital Signs1 X X X Ambulatory O2 sat2 X X X X CBC, differential, platelet count, and blood chemistries X X X X TSH, Free T4, Free T3 X X X Evaluation of Creatinine Clearance X X X X Pregnancy test X X X Quantitative serum immunoglobulins X X X SPEP and immunofixation X X X 24 Hour Urine for UPEP, protein excretion and immunofixation X X X Serum free light chain ratio X X X B2M, CRP X X X Skeletal Survey or alternative imaging X Bone marrow aspirate and biopsy X X6 EKG X X X X Toxicity Assessment X X X X Con Meds X X X X DC/MM Fusion Vaccine and GM-CSF Administration3 X Nivolumab Infusion X X Study Tubes4 X X DF/HCC Protocol #: 18-280 Protocol Version Date: 10.17.2020 32 1. Vitals (BP, HR, Temp, RR, O2 sat) to be taken within 15 minutes prior to start of nivolumab infusion and within 15 minutes following completion. 2. Pulse oximetry should be obtained prior to each dose of nivolumab and at any time a subject has any new or worsening respiratory symptoms. A reading at rest and on exertion should be obtained at each time point. The extent of the exertion should be based on the judgment of the investigator, but should remain consistent for each individual subject throughout the study. If the patient’s status changes the investigator can alter the extent of exertion based on their medical judgment. If a subject shows changes on pulse oximetry or other pulmonary-related signs (hypoxia, fever) or symptoms (eg, dyspnea, cough, fever) consistent with possible pulmonary adverse events, the patient should be immediately evaluated to rule out pulmonary toxicity. An algorithm for the management of suspected pulmonary toxicity can be found in Appendix 2 of the IB. Not all results are required to be collected by BMS.