The trial was registered at ClinicalTrials.gov (NCT00617409). All patients provided a written informed consent and treatment protocol was approved by University of South Florida Institutional Review Board. After receiving initial chemotherapy, (4 -6 cycles of a standard platinum/etopo side regimen as first- line chemotherapy), patients were enrolled and randomized. Additional eligibility included; stable disease (SD) or better with Eastern Cooperative Oncology Group (ECOG) performance status of 0 to 1, and adequate organ function were screened for initial registration approximately 4 -6 weeks after the completion of first -line chemotherapy. Prophylactic cranial irradiation (PCI) was permitted. Eligible patients were randomized in a 1:1:1 ratio to one of three treatment arms: Arm A ( control patients or observation), Arm B: ( patients treated with Ad.p53- DC vaccine only), or Arm C (patients treated with Ad.p53- DC vaccine in combination with ATRA) . Each vaccine consisted of 2-5 × 10 6 Ad-p53 DCs. Cells were injected intradermally, at two -week intervals for three times (three vaccine doses). Patients were restaged approximately 2 weeks after the 3 rd vaccine dose. Patients without signs of disease progression (PD) underwent a second leukapheresis and then vaccinated 3 more times at 4 -week int ervals. Patients on Arm C also received 150 mg/m 2 ATRA for 3 days prior to each vaccine administration (followed by vaccine administration on the next day). This scheduling was based on our previous data 57 that demonstrate persistence of the ATRA effect on MDSC for minimum 2 weeks. Protocol: Version 8.0. September 19, 2018 Page | 16 All patients were followed until PD at which time they received salvage or second line chemotherapy with s ingle agent paclitaxel (200 mg/m2 IV every 3 weeks, 4- 6 cycles). The primary objective was to evaluate the efficacy of second line paclitaxel in the two experimental regimens (Arms B and C). Objective response (OR) to paclitaxel in each arm was t he endpoint of the study. An optimal two -stage design was used in planning of this study 63 because it has been successfully applied in randomized phase II trials to rank experimental agents 64. The control arm was used to examine consistency with historical control data and to assure that patients entering the phase II trial are comparable to historical controls. . Using the optimal two -stage design, if 3 responses were observed in the first 9 patients, the second stage would accrue 14 additional patients. To assess immune response, PBMCs were collected from patients at different time points during the treatment ( Fig. 1) and kept frozen in liquid nitrogen. All samples from one patient were analyzed simultaneously to reduce inter -experimental variability. PBMCs were thawed, incubated overnight in complete medium (RPMI -1640 supplemented with antibiotics and 10% FBS) and then used in experiments. Cell viability was greater than 80%. T -cell responses were assessed using IFN -γ ELISPOT after infection with a recombinant canarypox virus (ALVAC) containing wild-type p53 or empty vector (obtained from Aventis Pasteur, Toronto, Canada), and incubation for 48 hours. Empty ALVAC virus served as a control. Protocol: Version 8.0. September 19, 2018 Page | 17 The initial infection step was performed in serum free media (supplemented with cytokines) for 90 minutes after which, complete media was added. In IFN -γ ELI SPOT assay 2 × 105 PBMC s were seeded in triplicates or quadruplicates in 96 -well plates pre- coated with an anti - IFN-γ antibody. To ascertain that T cells are functionally competent for each sample we prepared additional controls (unstimulated or PHA (5µg/ml) stimulated cells), a nd the plate was further incubated for 36 hours. The number of IFN -γ producing cells was evaluated as described previously32 using an automated ELISPOT reader (Cellular Technology, Ltd, OH). 54 patients were enrolled during the first stage, 18, 19 and 17 in arms A, B and C respectively. Median age (range) = 62 (43- 74). Male/female = 31/23, ECOG PS 0/1 = 14/40. Median # of vac cines was 3 (range 0 -6) and total # vaccines administered was 114. AEs associated with vaccine were mild and no grade 3 -4 AE was vaccine related. OR to vaccine was observed in 3/36 patients. OR to paclitaxel (or other second line treatment) was seen in 1/11, 2/12 and 3/ 14 patients for arms A, B and C respectively. Only arm C moved to stage two. An additional 15 patients have been accrued to the Arm C second stage of the trial and their study data and results is currently being collected for the proposed pro tocol analysis 62. Pre-treatment levels of MDSC populations in patients from all three arms was similar (p>0.07). Treatment of patients from arm B with vaccine alone did not affect the proportion of MDSC (Fig. 2) , whereas in patients treated with ATRA the presence of Lin -HLA -DR-CD33+ MDSC decreased more than two- fold (p=0.02). The proportion of CD11b+CD14-CD33+ MDSC decreased less dramatically but also significantly (p=0.03). No significant differences were observed in the proportion of conventional DC and Treg 65. Next, we evaluated the p53 -specific immune response using IFN -γ ELISPOT assay. Prior to start of the treatment no patients had detectable p53 specific response. Consecutive measurements did not show positive p53 responses in any of the arm A patients. After three immunizations, 3 out of 15 tested patients (20%) from arm B met the criteria for p53 -specific immune response, which was not significantly different from arm A (p=0.22). In contrast, in arm C 5 out of 12 patients (41.7%) had detectable p53 response, which was significantly higher than in control group (p=0.012) (Fig. 3) 65. The proportion of p53- specific IFN -γ 0246810121416 Arm A Arm B Arm CNumber of patients Non- responders Figure 1 Protocol: Version 8.0. September 19, 2018 Page | 18 positive cells CD4+ and CD8+ T cells was evaluated using intracellular cytokine staining. Prior to the treatment no statistically significant differences between the arms were seen (p>0.07). The immunization significantly increased the proportion IFN - γ positive CD8+ and CD4+ T cells. The proportion of granzyme B (GrzB) positive CD8+ T cells was increased only in patients from arm C but not from arm B65. To evaluate the possible link between the immune response to vaccination and the cl inical outcome, patients in arms B and C were split based on the presence of p53 specific responses after vaccination. Patients who did not develop immune response had median survival of 11.7 months, as compared with 16.4 months in patients who developed p53 immune responses (hazard ratio 0.52, 95% CI 0.22-1.22) (p=0.1). Patients with brief immune response had the same median survival as non- responders (11.1 months vs. 11.7 months). In contrast, patients who had p53 immune response at least two months after completion of the vaccination had median survival of 41 months (hazard ratio 0.26 (0.09- 0.74), p=0.03) suggesting that improving upon the immune response obtained through this strategy could manifest in clinical outcome improvement for patients (internal data not yet published). 4.5 C heckpoint Inhibitors Immunotherapy 4.5.1 Nivolumab Nivolumab is a fully humanized, IgG4 (kappa) isotype monoclonal antibody (mAb) that binds PD -1 on activated immune cells and disrupts engagement of the receptor with its ligands PD -L1 (B7 -H1/CD274) and PD -L2 (B7 -DC/CD273), thereby abrogating inhibitory signals and augmenting the host antitumor response. PD -1, (CD279), a 55- kDa type I transmembrane protein, is a member of the CD28 family of T -cell co -stimulatory receptors that also includes CD28, CTLA 4, ICOS, and BTLA. PD -1 contains an intracellular membran e proximal immunoreceptor tyrosine inhibitory motif (ITIM) and a membrane distal immunoreceptor tyrosine-based switch motif (ITSM). Two ligands specific for PD -1 have been identified: PD -L1 (B7 -H1/CD274) and PD L2 (B7 - DC/CD273). PD -L1 and PD -L2 have been shown to down- regulate T cell activation on binding to PD -1 in both murine and human systems. PD -1 delivers a negative signal by the recruitment of SHP -2 to the phosphorylated tyrosine residue in the ITSM in its cytoplasmic region. PD -1 is primarily expre ssed on activated T cells, B cells, and myeloid cells. Further evidence for a negative regulatory role of PD -1 comes from studies of PD -1-deficient mice. PD 1 -deficient mice develop various autoimmune phenotypes, including dilated cardiomyopathy, a lupus -like syndrome with arthritis and nephritis, and accelerated diabetes mellitus. The emergence of these autoimmune phenotypes is dependent on the genetic background of the mouse strain, and many of these phenotypes emerge at different times and show variabl e penetrance. In addition to the phenotypes of null mutations, PD -1 inhibition by antibody - mediated blockade in several murine models has been found to play a role in the development of autoimmune diseases such as encephalomyelitis, graft -versus -host disea se, and type I diabetes. Taken together, these results suggest that PD-1 blockade has the potential to activate anti -self T -cell responses, but these responses are variable and dependent on Protocol: Version 8.0. September 19, 2018 Page | 19 various host genetic factors. Thus, PD -1 deficiency or inhibition is not accompanied by a universal loss of tolerance to self- antigens. Preclinical animal models of tumors have shown that blockade by PD -1 by mAbs can enhance the anti -tumor immune response and result in tumor rejection. Antitumor activity by PD-1 blockade functions in PD -L1-positive tumors as well as in tumors that are negative for the expression of PD -L1. This suggests that host mechanisms (i.e., expression of PD -L1 in antigen -presenting cells) limit the antitumor response. Consequently, both PD -L1 posit ive and negative tumors may be targeted using this approach. In humans, constitutive PD -L1 expression is normally limited to macrophage -lineage cells, although expression of PD -L1 can be induced on other hematologic cells as well, including activated T cel ls. However, aberrant expression of PD -L1 by tumor cells has been reported in a number of human malignancies. PD -L1 expressed by tumor cells has been shown to enhance apoptosis of activated tumor -specific T cells in vitro . Moreover, the expression of PD -L1 may protect the tumor cells from the induction of apoptosis by effector T cells. Additional details are available in the Nivolumab Investigator Brochure. 4.5.2 Ipilimumab Ipilimumab is a fully humanized, IgG1 (kappa) isotype mAb that binds to the CTLA -4 antigen expressed on a subset of T cells from human and nonhuman primates. CTLA -4 is a negative regulator of T -cell activity. Ipilimumab binds to CTLA -4 and blocks the interaction of CTLA -4 with its ligands, CD80/CD86. Blockade of CTLA -4 has been shown to augment T-cell activation and proliferation, including the activation and proliferation of TILs. Inhibition of CTLA -4 signaling can also reduce Treg cell function, which may contribute to a general increase in T -cell responsiveness, including the anti-tumor response. 4.6 Study Rationale 4.6.1 Rationale for Immuno- Oncology Therapeutic Approaches in SCLC SCLC has classically been associated with immune -mediated paraneoplastic processes, such as cerebellar degeneration, limbic encephalitis and Lambert -Eaton myasthenic syndrome 66. For example, antibodies generated against human neuronal RNA -binding proteins (e.g. Hu), which are expressed on neurons and SCLC tumors, leads to an encephalomyelitis67. Interestingly, SCLC patients that present with these ‘early’ paraneoplastic syndromes have a more favorable prognosis68,69, suggesting that an underlying immune response is being generated against these onconeural antigens. Proof of an active immune environment in SCLC has been described in a few analyses of patient samples. First, analysis of sixty -four SCLC tumors demonst rated that a wide range of CD45+ cells infiltrated the tumor, an average of 40 immune cells/field, and that high CD45+ counts were associated with a better prognosis 70. Secondly, a separate study found that various tumor -infiltrating lymphocyte (TIL) subsets were present in SCLC brain metastases and that programmed death ligan d 1 (PD -L1) was heterogeneously expressed71. Final ly, evaluation of peripheral blood cells in 35 SCLC patients demonstrated a high CD4+ effector T cell to T regulatory (Treg) cell ratio in patients with LD -SCLC vs. ED -SCLC 72. This implies that in some r espect, SCLC pathology integrates with the immune response. Protocol: Version 8.0. September 19, 2018 Page | 20 Furthermore, SCLC is known to have one of the highest mutational loads73, which is thought to be a reflection of myriad insults inflicted by carcinogens from smoking. Additionally, comprehensive genom ic profiling of SCLC tumors has identified that the vast majority lack functional p53 (90%) and Rb1 (65%) 74. This universal genetic aberration facilitates poor genomic stability 75, thus perpe tuating the generation of tumor -associated antigens (TAAs). Interestingly, recent studies have highlighted that the clinical efficacy of cytotoxic T - lymphocyte- associated antigen 4 (CTLA -4) and programmed death 1 (PD -1) blockade in melanoma and non -small cell lung cancer (NSCLC), respectively, are partially driven by the mutational burden of the tumor and the presence of neoantigens 76,77. Thus, the high mutational burden in SCLC may facilitate enhanced immune recognition. Though SCLC pathology appears to be intertwined with the immune response and is predisposed by a substantial mutational load, prior evidence indicates that major histocom patibility complex (MHC) surface expression is reduced in SCLC 78,79, which may preclude robust immune recognition. 4.6.2 Rationale for Nivolumab/Ipilimumab Combination in SCLC In a phase I/II study of nivolumab and nivolumab/ipilimumab for treatment of recurrent SCLC (CA209032), participants who were platinum sensitive or refractory and had progressive disease were enrolled regardless of tumor PD -L1 status or number of prior chemotherapy regimens 80. This open- label study randomized participants to nivolumab 3 mg/kg IV every 2 weeks or nivolumab + ipilimumab (1 + 1 mg/kg, 1 + 3 mg/kg or 3 + 1 mg/kg) IV every 3 weeks for 4 cycles followed by nivolumab 3 mg/kg every 2 weeks. The primary objective was objective response rate. Other objectives were safety, PFS, OS and biomarker analysis. All participants had prior platinum- based first -line treatme nt and progression after the most recent treatment regimen. Baseline characteristics were typical for a SCLC population with respect to age, smoking history, and gender.