22 5.1.1 Diagnosis/Condition for Entry into the Trial ...................................................... 22 5.1.2 Subject Inclusion Criteria .................................................................................... 22 5.1.3 Subject Exclusion Criteria .................................................................................. 23 5.1.4 Women and Minorities……………………………………………………….24 5.1.5 Women of Childbearing Potential……………………………………………24 5.2 Trial Treatments ...................................................................................................... 25 5.2.1 Dose Selection/Modification .............................................................................. 25 5.2.1.1 Dose Selection …………………………………………………………...25 Protocol: Version 8.0. September 19, 2018 Page | 3 5.2.1.2 Dose Modification………………………………………………….……25 5.2.2 Timing of Dose Administration .......................................................................... 27 5.2.3 Trial Blinding/Masking ....................................................................................... 28 5.3 Randomization or Treatment Allocation ................................................................ 28 5.4 Stratification ............................................................................................................ 28 5.5 Concomitant Medications/Vaccinations (allowed & prohibited) ........................... 28 5.5.1 Acceptable Concomitant Medications ................................................................ 28 5.5.2 Prohibited Concomitant Medications .................................................................. 28 5.6 Rescue Medications & Supportive Care ................................................................. 29 5.6.1 Supportive Care Guidelines ................................................................................ 29 5.7 Diet/Activity/Other Considerations ........................................................................ 30 5.7.1 Diet ...................................................................................................................... 30 5.7.2 Contraception ...................................................................................................... 30 5.7.3 Use in Pregnancy ................................................................................................ 31 5.7.4 Use in Nursing Women ....................................................................................... 31 5.8 Subject Withdrawal/Discontinuation Criteria ......................................................... 31 5.8.1 Discontinuation of Study Therapy after CR ....................................................... 32 5.9 Subject Replacement Strategy ................................................................................ 32 5.10 Clinical Criteria for Early Trial Termination .......................................................... 32 5.11 Assessment of Outcomes ………………………………………………………….33 5.11.1 Safety Assessments…………………………………………………………..33 5.11.2 Clinical Efficacy……………………………………………………………..33 5.11.3 Immunological Efficacy……………………………………………………..34 5.11.4 Biomarker Testing …………………………..…………………………………34 6.0 TRIAL FLOW CHART .............................................................................................. 35 6.1 Second Phase Flow Chart ………………………………………………..….37 7.0 TRIAL PROCEDURES .............................................................................................. 39 7.1 Trial Procedures ...................................................................................................... 39 7.1.1 Administrative Procedures .................................................................................. 39 7.1.1.1 Informed Consent……………………………………………………...39 7.1.1.1.1 General Informed Consent………………………………………….39 7.1.1.2 Inclusion/Exclusion Criteria ……………………..……………………….39 7.1.1.3 Medical History …………………………………………………………..40 7.1.1.4 Prior and Concomitant Medications Review……………………………..40 7.1.1.4.1 Prior Medications…………………………………………………….40 7.1.1.4.2 Concomitant Medications…………………………………...……….40 7.1.1.5 Disease Details and Treatments …………………………………………….40 7.1.1.5.1 Disease Details ……………………………………………………….40 7.1.1.5.2 Prior Treatment Details ………………………………………………40 7.1.1.5.3 Subsequent Anti- Cancer Therapy Status …………………………..40 7.1.1.6 Assignment of Screening Nu mber………………………………………..40 7.1.1.7 Assignment of Randomization Number…………………………………….41 7.1.1.8 Trial Compliance (Medication/Diet/Activity/Other) ….……………………41 7.1.2 Clinical Procedures/Assessments ........................................................................ 41 7.1.2.1 Adverse Event (AE) Monitoring……………………………………………41 7.1.2.2 Full Physical Exam……………………………………………………….41 Protocol: Version 8.0. September 19, 2018 Page | 4 7.1.2.3 Directed Physical Exam …………………………………………………….41 7.1.2.4 Vital Signs …………………………………………………………………..42 7.1.2.5 Eastern Cooperative Oncology Group (ECOG) Performan ce Scale……..42 7.1.2.6 Tumor Imaging and Assessment of Disease……………………………...42 7.1.2.7 Laboratory Procedures/Assessments ……………………………………..42 7.1.2.8 P harmacokinetic/Pharmacodynamic Evaluations ………………………...44 7.1.3 Other Procedures - Leukopheresis ....................................................................... 44 7.1.4 Withdrawal/Discontinuation …………………………………………………...44 7.1.5 Tumor Tissue Collection and Correlative Studies Blood Sampling…………...45 7.1.5.1 Evaluation of p53- specific immune response against TAAs ........................ 45 7.1.5.1.1 Analysis of IFN -γ production by PBMC ............................................ 45 7.1.5.1.2 Analysis of cell proliferation and IL-2 production…………………45 7.1.5.2 Determination of the relationship between MDSC numbers and anti -p53 T cell responses ………………………………………………………………………..45 7.1.5.3 Analysis of other immune cells…………………………………………..46 7.1.5.4 Immunohistochemical Stains ……………………………………………..46 7.1.6 Visit Requirements ............................................................................................. 46 7.1.6.1 Screening ……………………………………………………………………46 7.1.6.1.1 Screening Period …………………………………………………..…46 7.1.6.2 Treatment Period ……………………………………………………………46 7.1.6.3 Post- Treatment Visits ……………………………………………………….47 7.1.6.3.1 Safety Follow -Up Visit ……………………………………………..47 7.1.6.4 Follow- up Visits ………………………………………………………….47 7.1.6.4.1 Survival Follow-up………………………………………………...47 7.1.6.5 Second Course Phase (Retreatment Period) ……………………………….47 7.2 Assessing and Recording Adverse Events .............................................................. 47 7.2.1 Definition of an Overdose for This Protocol and Reporting of Overdose to the Sponsor and to BMS ..................................................................................... 49 7.2.2 Reporting of Pregnancy and Lactation to the Sponsor and to BMS ................... 49 7.2.3 Immediate Rep orting of Adverse Events to the Sponsor and to BMS ............... 51 7.2.3.1 Serious Adverse Events…………………………………………………..51 7.2.3.2 Events of Clinical Interest …………………………………………………..51 7.2.4 Evaluating Adverse Events ................................................................................. 51 7.2.5 S ponsor Responsibility for Reporting Adverse Events ...................................... 51 7.3 Safety Stopping Criteria…………………………………………………………51 8.0 STATISTICAL ANALYSIS PLAN ........................................................................... 55 8.1 Statistical Analysis Plan Summary ......................................................................... 55 8.1.1 Study Design……………………………………………………………...……..55 8.2 Statistical Analysis Plan .......................................................................................... 55 8.2.1 Sample size ……………………………………………………………………...55 8.2.2 Analysis of Primary and Secondary Endpoints…………………………………55 8.2.3 Toxicity …………………………………………………………………………55 8.2.4 Laboratory Correlative Studies …………………………………………………55 9.0 LABELING, PACKAGING, STORAGE AND RETURN OF CLINICAL SUPPLIES ................................................................................................................... 56 9.1 Investigational Product ........................................................................................... 56 Protocol: Version 8.0. September 19, 2018 Page | 5 9.1.1 Clinical Supplies provided by BMS as summarized in Table 7……………….56 9.1.2 Clinical Supplies provided by Moffitt Cell Therapy Facility ……………………56 9.2 Packaging and Labeling Information ...................................................................... 56 9.3 Clinical Supplies Disclosure ................................................................................... 56 9.4 Storage and Handling Requirements ...................................................................... 56 9.5 Returns and Reconciliation ..................................................................................... 57 10.0 D ATA SAFETY MONITORING PLAN ................................................................... 57 10.1 Risk to Subjects ...................................................................................................... 57 10.1.1 Human subject involvement and characteristics ................................................. 57 10.2 Recruitment and Informed Consent ........................................................................ 57 10.3 Protection Against Risk .......................................................................................... 58 10.4 Importance of the Knowledge to be Gained ........................................................... 58 10.5 Data Safety and Monitoring Plan ............................................................................ 58 10.6 Scientific Review Committee (SRC) ...................................................................... 58 10.7 PI Responsibility ..................................................................................................... 59 10.8 The Protocol Monitoring Committee (PMC) .......................................................... 59 10.9 Suspension/Termination ......................................................................................... 59 10.10 Monitoring of the Study and Regulatory Compliance ............................................ 60 10.11 Internal Monitoring Plan ......................................................................................... 60 10.12 Protocol Modifications ........................................................................................... 60 10.13 The Institutional Review Board (IRB) .................................................................... 60 10.14 Patient Privacy ........................................................................................................ 60 10.15 Reco rds Retention ................................................................................................... 61 10.16 Compliance with Trial Registration and Results Posting Requirements ................ 61 11.0 APPENDICES ............................................................................................................ 62 11.1 ECOG Performance Status ..................................................................................... 62 11.2 Common Terminology Criteria for Adverse Events V4.0 (CTCAE) ..................... 62 11.3 Response Evaluation Criteria in Solid Tumors (RECIST) 1.1 Criteria for Evaluating Response in Solid Tumors .................................................................... 62 11.4 Pharmaceutical Information for Ad.p53- DC Vaccine………………………………………………………………………63 11.5 Management Algorithm ………………………………………………………….64 11.6 BMS Guidelines for Adverse Event Reporting …………………………………..72 12.0 REFERENCES…………………………………………………................................79 . Protocol: Version 8.0. September 19, 2018 Page | 6 1.0 TRIAL SUMMARY Abbreviated Title Combination I mmunotherapy in Relapsed SCLC Trial Phase Phase II Clinical Indication SCLC , progressive after first -line (platinum -based) chemotherapy Trial Type Single arm Type of control Historical Route of administration Ipilimumab – Intravenous Nivolumab – Intravenous Ad.p3- DC – Intradermal/S ubcutaneous Trial Blinding Open label Treatment Groups Ipilimumab -Nivolumab + Ad.p53 -DC vaccine Number of trial subjects 41 (39 + 10% screen failures) Estimated enrollment period 2.5 years Estimated duration of trial 3 years Duration of Participation 3 years Protocol: Version 8.0. September 19, 2018 Page | 7 2.0 TRIAL DESIGN 2.1 Trial Design Here we propose a phase II trial where relapsed ( progressive) , SCLC patients after treat ment with first line (platinum- based) chemotherapy ( after disease progression) will receive Ad.p53- DC in combination with ipilimumab and nivolumab to assess the impact of this combination immunotherapy regimen on clinical outcomes. We hypothesize that this combination immunotherapy strategy will improve ove rall disease response or control and/or survival in this population. 2.2 Trial Diagram 3.0 OBJECTIVE(S) & HYPOTHESIS General Scientific Hypothesis: Inhibition of the PD -1 receptor pathway with an anti PD -1 inhibitor ( nivolumab) and the CTLA -4 receptor pathway with an anti CTLA -4 inhibitor (ipilimumab) will enhance the p53 - specific cytotoxic immune response ( IR) elicited by a p53 -based DC vaccine (Ad.p53- DC) and this improvement will translate into improved outcomes ( disease control rate [ DCR ], progression free survival [ PFS], overall survival [ OS], overall response rate [ ORR ]) in patients with relapsed ( progressive ) SCLC, after standard, platinum -doublet, first line chemotherapy. Protocol: Version 8.0. September 19, 2018 Page | 8 3.1 Co-Primary Objective(s) & Hypothesis(es) • To determine the DCR of patients with relapsed ( progressive) SCLC (after standard, first line platinum-doublet chemotherapy) treated with combination immunotherapy (Ad.p53- DC plus ipilimumab -nivolumab) • To determine the safety of combination immunotherapy w ith Ad.p53-DC plus ipilimumab -nivolumab in patients with relapsed (progressive) SCLC 3.2 Secondary Objective(s) & Hypothesis(es) • To determine the PFS, OS and ORR of combination immunotherapy with Ad.p53- DC plus ipilimumab -nivolumab in patients with relapsed ( progressive) SCL C • To determine the immune response (IR) of combination immunotherapy with Ad.p53- DC plus ipilimumab -nivolumab in patients with relapsed ( progressive) SCLC 4.0 BACKGROUND & RATIONALE 4.1 Background/Introduction 4.1.1 Overview of Small Cell Lung Cancer In the United States, 224,390 new cases of lung cancer were diagnosed and 1 58,080 patients with lung cancer died in 2016 1, with small cell lung cancer (SCLC) accounting for 13 -15% of them. SCLC typically disseminates ea rly, with 70 -80% of patients diagnosed with metastatic disease or extensive stage, (ES)-SCLC), and is initially also chemo -responsive, so that considerable survival improvements are achieved with (first- line) chemotherapy2-4. Etoposide- platinum remains the preferred first- line treatment and the standard against which new therapies are measured. By this stand ard, patients with ES -SCLC achieve overall response rates (ORR) of ≥ 70% and complete response rates of 20- 30%, but rarely survive beyond 2 years (10- 20%), with median survival times (MST) ranging from 7- 10 months 2-4. The ORR to second- line therapy is also very disappointing and dependent on the previous chemotherapy response. Patients with “platinum -sensiti ve” disease (ORR = 20 -25%) progress ≥ 90 days after initial chemotherapy, whereas those with “platinum- resistant” (ORR ≤ 10%) disease progress sooner than 90 days after initial chemotherapy5-7. Clearly, new therapies are urgently needed, and evidence suggests that immunotherapy may have a potential role in SCLC, including if used in conjunction with chemotherapy 8-12. 4.1.2 p53 as a Target for Cancer Immunotherapy The p53 tumor suppressor gene plays a central role in the control of cell growth and differentiation. Normally, p53 is a short -lived protein localized in the nuclei of cells. Approximately 50% of all human cancers and >90% of patients with SCLC have altered p53 function, mostly as a result of single -point mutations or abnormalities in degradation of wild - type (wt) p53. This leads to accumulation of mutant (mu) or wt -p53 protein (whereas normal tissues have low to undetectable levels) and expression of p53- derived epitopes on the surface of tumor cells in the context of MHC class I13. Protocol: Version 8.0. September 19, 2018 Page | 9 Furthermore, since mutant forms of p53 can result in oncogenic gain of function14,15 it is unlikely that they can escape anti -p53 cytotoxic T lymphocytes (CTLs) by restoring wt -p53 status (no antigen- loss variants). Thus, p53 has many characteristics of an “ideal” tumor - associated antigen (TAA), which makes it a very attractive candidate target for immune recognition and anti-p53- based cancer immunotherapy. 4.1.3 Anti -p53 Dendritic Cell -Based Immunotherapy Different approaches to p53- based cancer immunotherapy have been explored. The role of wt-p53 peptide sequences in the induction of antitumor CTL responses has been investigated in both human and animal in vitro studies, with encouraging results13,16-20. However, peptide - based approaches assume knowledge of precise HLA types and the peptides present on particular tumors, leading to several limitations21,22, such as technical difficulties with generating custom mutant- specific peptides. These difficulties can be circumvented with the use of TAA proteins and dendritic cells (DCs). DCs are the most potent antigen presenting cells (APCs) and the most effective in inducing a primary CTL response . There are numerous methods of loading DCs with a variety of different TAAs, and animal models show that the approach of using viral vectors to introduce these TAAs into DCs is practical, safe, and effective 23-25. Because cells with mu - p53 usually overexpress the protein and because there is a large human experience in melanoma with targeting overexpressed but not mutant proteins such as MAGE and MART, a much more practical approach would be to develop a strategy that targets the non-mutant portions of the p53 overexpressed in tumors. Therefore, we have focused on utilizing DCs transfected with the full -length p53 gene. The rationale for this approach is based on 1) the assumption that multiple MHC class I and II matching p53 -derived epitopes are present on the surface of DCs, eliminating the need for selecting matching patients as well as providing conditions for activation of CD4 + T cells, and 2) the fact that previous studies have demonstrated that each of the different minimal epitopes combined in a single fusion protein can be recognized by specific CTLs26. 4.2 V accine (Ad.p53- DC) I mmunotherapy Adenovirus (Ad) provides a high- level transduction efficacy for many cell types, regardless of their mitotic status27, and replic ation -defective Ad (deletions in the E1 region) has been safely injected into patients28. Successful transduction of DCs with model antigens has been reported, and transduced DCs have effectively presented the recombinant protein antigens29- 31. In this case, DCs were infected with an adenoviral construct containing wt- p53 to generate Ad.p53- DC. 4.2.1 Manufacturing of Ad.p53- DC DCs were prepared from peripheral blood mononuclear cells (PBMC) collected by leukapheresis, separated over a Ficoll density gradient, washed, and cryostoraged in liquid nitrogen in the Cell Therapy Core at the H. Lee Moffitt Cancer Center . Protocol: Version 8.0. September 19, 2018 Page | 10 Preparation of the DC and their characterization was described previously32,33. Briefly, after thawing, PBMCs were placed in X -VIVO -15 medium (Biowhittaker, Walkersville, MD) in tissue culture flasks at a concentration of 1.3- 1.7 × 106 cells per cm2. After 2 -hr culture non- adherent cells were removed and the flasks were recharged with X -VIVO -15 medium supplemented with 5ng/ml GM -CSF (Immunex), and 5ng/ml IL -4 (R&D Systems, Minneapolis, MN). The flasks were incubated for 48 hours, at which time additional cytokine supplemented medium was added to the flasks. The flasks were then incubated for an additional 72 hours . The non- adherent and loosely adherent cells were collected and infected with Ad.p53 and incubated for 2 hours after which X -VIVO - 15 medium was added for a 10 6 cells/ml concentration and cells incubated for an additional 46 hours. The optimal viral particle to cell ratio used was 15,000:1 as determined for our previous study 32. The vaccine release criteria included: (a) negative Gram’s staining; (b) negative Mycoplasma test by PCR analysis; (c) maximum endotoxin concentration of 5 EU/mL; and (d) a mature DC-p53 expressing phenotype. DC phenotype was defined as lineage (CD3, CD14, CD19, CD20, CD56) negative, HLA -DR positive , CD86 positive, and p53 positive cells. Intracellular staining for p53 was performed using the kit from Caltag, Burlingame, CA. DC vaccines in 1 ml were injected intradermally into 4 separate sites (0.25 ml at each site) in bilateral proximal upper and lower extremities (in the regions of the axillary and inguinal nodal basins) three times after the baseline blood samples were drawn and at 2 week intervals. 4.2.2 Preclinical Animal Studies with Ad.p53 -DC To determine the optimal dose of Ad.p53 vector that produces the highest level of human p53 expression with the least amount of toxicity to the DCs, mice were given a human p53- transduced DC vaccine 34,35. We found that infecting murine DCs with the Ad.p53 vector at a multiplicity of infection (MOI) of 50- 200 viral particles per cell (vp/cell) did not adversely affect DC viability. MOI in excess of 500 significantly reduced DC viability. However, transduction of DCs at an MOI of 100 produced very good transduction efficiency, with 40 -45% of the DCs becoming positive for p53. Similarly, infecting human DCs with the Ad.p53 MethA sarcoma -bearing BALB/c mice were treated with 6x105 activated Ad.c - or Ad.p53 -DCs 4 times on days 0, 5, 11, and 17. Four mice treated with Ad.p53 -DCs rejected their tumor. Tumor growth in 2 remaining mice is shown. Protocol: Version 8.0. September 19, 2018 Page | 11 vector at a MOI of 10,000- 20,000 vp/cell did not adversely affect DC viability and showed better transduction efficiency with the higher dose. At a MOI of 40,000 vp/cell, DC viability was adversely affected despite transduction rates similar to the lower doses. Thus, 20,000 vp/cell was the MOI chosen to manufacture Ad.p53- DC. Additionally, T cells recovered from immunized mice contained significant numbers of CTLs that could specifically kill tumor cells previously modified to express the human p53 gene, and Ad.p53- DC-immunized mice developed a significant CTL response to murine p53, probably due to the significant homology between murine and human p53. Ad.p53- DC- induced anti -p53 CTL response resulted in protection of mice from challenges with tum or cells that overexpressed human or murine p53. Furthermore, tumors that were established in mice prior to immunization exhibited significantly slowed growth as a result of the immunization with Ad.p53- DC 34,35. 4.2.3 Preclinical Experiments Using Human T Cells To determine whether this response was sufficient to recognize and eliminate tumor cells in cancer patients , peripheral blood- derived T cells and autologous DCs were obtained from 3 healthy volunteers and 9 cancer patients, all of whom were HLA -A2 positive36. T cells were in vitro primed with Ad.p53- DC and then tested for their ability to kill (HLA -A2-positive) target cells with normal to low or overexpressed levels of p53 protein. Selective CTL killing of p53 -overexpressing cells but not of cells with normal p53 expression levels was generated in blood from all 3 healthy donors and 8 cancer patients. Furthermore, when cells that expressed normal to low levels of p53 were forced to overexpress p53 by gene transfection, they became sensitive to Ad.p53- DC-primed CTLs. Additionally, when an excess of NK cell -sensitive target cells or an anti -CD4 antibody was added to the cytotoxicity assays, very little effect or a small decrease in cytotoxicity activity was seen. Conversely, when anti -CD8 was added, we observed a very significant decrease in cytotoxicity, demonstrating that neither NK nor CD4 cells but CD8 -positive CTLs are the relevant effector cells primed with Ad.p53- DC. 4.3 P hase I/II C linical Trial of Ad.p53- DC in SCLC p53 gene mutations and p53 protein overexpression are present in ≥ 90% of SCLC cases 37,38. With many characteristics of an “ideal” TAA present, p53 is an attractive candidate for cancer immunotherapy. Therefore, an approach where an adenoviral vector carrying the intact human wt -p53 gene is used to overexpress the p53 protein in autologous DCs and that allows endogenous processing mechanisms to select and present the appropriate p53 peptides for each individual’s HLA type encourages the use of these transduced cells (Ad.p53- DC) as a vaccine. 4.3.1 Study Design and Patient Characteristics The trial was designed to assess the clinical and immune respon se of SCLC to Ad.p53- DC 32,33. Fifty -four patients (24 male, 30 female) with extensive stage disease (initial or recurrent) were enrolled. All had previously been treated with chemotherapy. Patients with stable disease or better underwent leukopheresis 8 weeks after the last dose of chemotherapy to manufacture Ad.p53- DC. Patients received 3 doses of Ad.p53- DC intra-dermal ly every 2 Protocol: Version 8.0. September 19, 2018 Page | 12 weeks. If after reassessment, stable disease or better persisted, 3 more mo nthly doses of Ad.p53- DC were given. 4.3.2 Ad.p53- DC Dose and Safety The number of p53+ DCs ( Ad.p53- DC dose) was evaluated using flow cytometry. The initial goal was to escalate the dose from 5 × 106 to 5 × 107 p53+ DC. However, generation of >5 × 106 p53+ DC per dose was difficult to achieve (on average, 7.7 × 107 DCs and 8.6 × 106 p53+ DCs were generated per dose and ≥107 p53+ DCs were generated in < 10% of cases). Thus, to maintain consistency throughout the trial, the single doses of p53+ DC were not escalated beyond 5 × 106 cells. On average, each patient received 3.8 × 106 p53+ DCs per dose. However, 5 patients received <106 p53+ DCs because of production difficulties. Ad.p53- DC toxicities were infrequent and mostly mild. Only 2 patien ts experienced grade 2 toxicities (1 fatigue, 1 arthralgia), and Ad.p53- DC was never withheld. The most frequent toxicities were grade 1 arthralgia/myalgia (9 patients), fatigue and injection site erythema (5 patients each), and injection site pain (4 pati ents). Occurrence of toxicities was independent of the number of Ad.p53- DC doses received. 4.3.3 Immune Response to Ad.p53 -DC The p53- specific immune response was evaluated by ELISPOT using autologous PBMCs infected with a canary pox virus (ALVAC) containing wt -p53 or empty vector as control. The number of γ- interferon producing cells was evaluated using an automated ELISPOT reader 39. In 12 HLA -A2-positive patients, immune responses were tested with p53- derived or control HLA-A2 matched peptides pulsed onto autologous PBMCs and tetramers. Response was considered significant if ≥ 2 SD higher than the response to ALVAC or peptide cont rols. Increase over baseline (pre-Ad.p53- DC) was considered significant if p53- specific responses (post -Ad.p53- DC) were ≥ 2 SD higher than p53- specific responses pre- Ad.p53- DC and at least 2 SD higher than responses to ALVAC or peptides. Because the generation of a p53- specific T -cell response not only depends on the quality of antigen stimulation but also on the functional activity of T cells and DCs in the host, they were also tested. Full immune response evaluation was possible in 43 patients. Overall, 18 patients (41.8%) had a statistically significant p53 -specific response using ALVAC and 7 of 12 patients (58.3%) using p53- derived peptide. Three patients with a significant response to Ad.p53- DC using the p53- derived peptide were not tested with ALVAC ( technical reasons). The baseline p53-specific immunity level was similar in Ad.p53- DC responsive and non-responsive patients. The level of the p53- specific immune response decreased 2 months after completing Ad.p53- DC, coinciding with the start of addition al chemotherapy. Presence and functional activity of DCs were both decreased, and the p53- specific immune response to Ad.p53- DC did not correlate with T cell functional activity, presence of Tregs, or pre-existing levels of DC activity. Because myeloid -derived suppressive cells (MDSC) are implicated in the host’s immunosuppressive state 40,41, we examined patients for the presence of these cells. Pre-Ad.p53- DC MDSC (Lin-HLA -DR-CD3+) levels were elevated compared Protocol: Version 8.0. September 19, 2018 Page | 13 to control donors (p=0.01). Post -Ad.p53- DC levels increased even further (p=0.002). All patients with normal levels of MDSCs prior to Ad.p53- DC developed a p53- specific immune response compared to only 33% of patients who had elevated levels of MDSC (p=0.012). 4.3.4 Clinical Efficacy of Ad.p53 -DC and Enhanced Effect on Salvage Chemotherapy Two patients (3.7%) achieved a partial response (PR), and 13 had stable disease with Ad.p53- DC. All but 5 patients developed progressive disease (PD), and response data were available for 33 patients who received additional chemotherapy post -Ad.p53- DC progression. The ORR for all 33 patients treated with “second” -line chemotherapy was 51.5% and 45.5% for the 22 platinum -resistant patients. The MST (from the date of the first Ad.p53- DC dose) for platinum -resistant patients was 10.5 months (95% CI = 5.3- 14.4) and 8.8 months (95% CI = 5.2-11.8) for all 54 patients. We also evaluated the relation between immune response to Ad.p53- DC and clinical response to second -line chemotherapy. Eleven of 14 patients (78.6%) with a positive immune response experienced a clinical response to second -line chemotherapy compared to 5 of 15 patients (33.3%) with a negative immune response (p=0.014). Patients with a positive immune response had a trend toward improved survival; however, the difference did not reach statistical significance (MST = 12.6 vs. 8.2 months, p = 0.131) 33. 4.4 I mproved Ad.p53- DC I mmunotherapy Strategy 4.4.1 Role of MDSCs in the Imm une Response It is now fully a ppreciated that tumor- specific immune responses in cancer are inhibited. Large numbers of different factors have been implicated in this process, including regulatory T cells (Treg), myeloid cells, various soluble factors and cytokines, inhibitory molecules expressed by immune and tumor cells, etc. 42-44. Among these factors MDSC play a prominent role. MDSC are a heterogeneous group of pathologically activated immature myeloid cells and myeloid precursors with potent immune suppressive activity 45-48. Two major groups of MDSC have been identified: polymorphonuclear (PMN) and mononuclear (M) MDSC. These cells share common features (immature myeloid cells with immune suppressive activity) but differ in morphology, phenotype, and mechanisms of suppressive activity 46. In cancer patients, these MDSC have partially overlapping phenotype that can be identified using several markers, which largely depends on the type of tumor. In most tumors, immune suppressive MDSC are defined as Lin -HLA -DR-CD33+ or CD33+CD14-CD11b+ cells that can be further sub- divided into CD15+ PMN -MDSC and CD15- M-MDSC. In some tumors, Protocol: Version 8.0. September 19, 2018 Page | 14 most notably melanoma, M -MDSC are defined as CD14+HLA -DR-/lo49,50. MDSC are characterized by potent immune suppressive activities in both antigen -specific and non- specific experimental systems. The associ ation between tumor progression and the presence of MSDC in cancer patients was demonstrated in a number of studies. In patients with solid tum ors, a significant correlation between circulating MDSC and clinical cancer stage was observed. Among stage IV pa tients, those with extensive metastatic tumor burden had the highest percent and absolute number of MDSC51. Patients with lower levels of circulating MDSC at baseline and on the last cycle of chemotherapy had significantly higher probability of a pathologic complete response 52. Increased circulating MDSC correlated with clinical stage and pathological grade in patients with bladder cancer 53,54 and pancreatic cancer55. Recently, first report linked clinical response to vaccination to accumulation of MDSC in patients with renal cell cancer56. Experiments in mice demonstrated that elimination of MDSC with antibodies or differe nt compounds could substantially improve antitumor immune responses, which resulted in antitumor effect46. However, no data are available testing the hypothesis that depletion of MDSC can improve the effect of cancer vaccines or another immune therapeutic modality in cancer patients. 4.4.2 Rationale for the Use of ATRA in Combination with Ad.p53- DC Because all -trans-retinoic acid (ATRA) causes differentiation of acute promyelocytic leukemia (APL) cells and lineage features between MDSCs and APL cells are comparable, the effect of ATRA on DC differentiation and function was tested in patients with metastatic renal cell carcinoma 57. As expected, patients had a substantially increased proportion of MDSCs, decreased presence of DCs, and decreased MDSC/DC ratio. Treatment with ATRA significantly decreased the presence of MDSC and improved MDSC/DC ratio to control levels. Patients had profound defects in the ability to respond to tetanus toxoid and to stimulate allog eneic T cells and treatment with ATRA improved those defects as well, although not significantly. Gr-1 + cells (analogous to human MDSC) inhibit antigen- specific T cell response and are present in excess in tumor -bearing mice. ATRA differentiates these cells in vitro and removes their immunosuppressive effect58,59. Furthermore, in vivo administration of ATRA to tumor -bearing mice dramatically reduces the presence of Gr -1+ cells and improves the effect of tumor vaccines60. Similarly, in vitro treatment of human MDSCs with ATRA results in their differentiation (2/3 become DC-like and 1/3 myeloid)61. These data confirm the effect of ATRA on DCs and MDSCs and st rongly suggest a valuable role in cancer immunotherapy. 4.4.3 Randomized Phase II Clinical Trial in SCLC Previously we demonstrated that: (1) Ad.p53- DC was safe and induced immune responses (IR ~ 40 -50%), (2) lack of IR was related to immunosuppressive myeloid derived suppressive cells (MDSCs), (3) a high rate of objective tumor regressions was seen in patients treated with chemotherapy after vaccination, particularly if a positive IR was Protocol: Version 8.0. September 19, 2018 Page | 15 obtained and (4) ATRA reduces the number of MDSCs and enhances IR. However, confirmation of this evidence required a prospective, comparative (randomiz ed) trial62. Thus, we designed and recently completed a randomized phase II study to address the question of whether combination of Ad.p53- DC with ATRA improved the clinical outcome of patients. Our hypotheses were 1) salvage chemotherapy in combination with Ad.p53- DC results in a substantial improvement in clinical outcomes, and 2) ATRA, by reversing the immunosuppressive influence of MDSCs, substantially improves the p53- specific immune response to Ad.p53- DC and hence clinical outcomes . Patients with ES -SCLC were enrolled to this randomized phase II study.