PROSTVAC in Combination With Nivolumab in Men With Prostate Cancer

Changes in T-cell infiltration in the tumor after neoadjuvant treatment with PROSTVAC and nivolumab, is defined as difference in density of cytotoxic T lymphocytes (CD8 T) cells and clusters of differentiation 4 (CD4 T) cells infiltrate from baseline to post-treatment (radical prostatectomy performed at week 9), calculated utilizing computer automated staining analysis. The analysis was done per tissue compartment (normal region, intra-tumoral and invasive margin). We hypothesized an increase in T cell infiltration after treatment. Statistically significant changes in T-cell infiltration are those with p\<0.05; to 0.05. The changes are not statistically significant.

Time frame: From baseline to radical prostatectomy (approximately week 9)

Clinically important immune-related adverse events were defined as grade 3 or above inflammation requiring steroids or anti cytokine therapy or not resolving to grade 1 or less within 28 days. These immune-related adverse events were assessed by the Common Terminology Criteria for Adverse Events (CTCAE v4.0). Grade 3 is severe, grade 4 is life-threatening, and grade 5 is death related to adverse event.

Time frame: From treatment start throughout study completion, an average of 1.4 years.

Population: \*2 out of 7 immune related adverse events (irAEs) occurred during administration of both ipilimumab and nivolumab, before a protocol amendment was performed and eliminated ipilimumab from the study treatment.

The best overall response is the best response recorded from the start of the treatment until disease progression/ recurrence (taking as reference for progressive disease the smallest measurements recorded since the treatment started) measured by the Response Evaluation Criteria in Solid Tumors (RECIST). Complete Response (CR): Disappearance of all target lesions. Any pathological lymph nodes (whether target or non-target) must have reduction in short axis to \<10 mm. Partial Response (PR): At least a 30% decrease in the sum of the diameters of target lesions. Progressive Disease (PD): At least a 20% increase in the sum of the diameters of target lesions, taking as reference the smallest sum on study. The appearance of one or more new lesions is also considered progressions. Stable Disease (SD): Neither sufficient shrinkage to qualify for PR nor sufficient increase to qualify for PD, taking as reference the smallest sum of diameters while on study.

Time frame: Baseline throughout study completion (average 1.4 years)

Population: 7/12 participants had measurable disease at baseline and were analyzed for this outcome.

Apparent diffusion coefficient (ADC) mapping in magnetic resonance imaging (MRI) is an indicator of tumor cell density. This outcome aims to capture any changes in ADC within the prostate tissue before and after the treatment administration. ADC changes secondary to immune treatment are defined as increase or decrease in ADC from baseline (pre-treatment) measurements. Low ADC values in tumors reflect areas of cell proliferation, and high ADC values reflects necrotic and acellular areas.

Time frame: From baseline to closest date to prostatectomy, average on week 9.

Population: 4/12 participants were not analyzed because MRI was not performed due to logistical issues.

Blood samples will be collected at baseline and after treatment, and aliquoted onto slides and examined, cytokeratin-positive/lymphocyte common antigen (CD45)-negative cells with an intact nucleus and a malignancy-consistent morphology will be identified as CTCs, and their exact positions on the slides recorded.

Time frame: At baseline and at week 9

Population: This analysis was not performed because the technology developed by Epic Sciences was still in development, thus samples were not sent to the off-site center to perform analysis.

Peripheral blood samples were collected at baseline and after treatment start via apheresis and cryopreserved peripheral blood mononucleate cells (PBMCs) were analyzed through multicolor flow cytometry to assess cell subsets, i.e. cluster of differentiation 4 (CD4+) T cells, cytotoxic T cells (CD8+) T cells, Tregs, B cells, Natural Killer (NK) cells, NK-T cells, conventional Dendritic Cells (cDCs), plasmocytoid DCs (pDCs), myeloid-derived suppressor cells (MDSCs), and monocytes and refined subsets related to their maturation/function. P values were calculated using the Wilcoxon Signed Rank Test. Statistically significant changes in immune cell subsets are defined as those with p\<0.05 and \>50% of participants having a \>25% change in a given subset.

Time frame: Week 4 and week 10 after therapy compared to baseline.

MRI of the prostate was performed to assess for changes in imaging characteristics of prostate cancer pre and post vaccination. MRI changes secondary to immune treatment is defined as increase or decrease in the size of lesions from baseline (pre-vaccine) measurements. Decrease from baseline to after treatment would represent a positive outcome.

Time frame: From baseline to closest date to prostatectomy, average on week 9.

Population: 4/12 participants were not analyzed because MRI was not performed due to logistical issues.

Changes in soluble immune mediating factors were defined as differences in sCD27 concentration between pre- and post-treatment samples and were measured by enzyme-linked immunosorbent assay (ELISA) and Mesoscale Assay. P values were calculated using the Wilcoxon Signed Rank Test. We hypothesized an increase in sCD27 after treatment. Statistically significant changes in soluble analyzes are those with p\<0.05; to 0.05 means no statistically significant changes.

Time frame: Week 4, week 10, and week 20 after therapy compared to baseline

Changes in soluble immune mediating factors were defined as differences in TNFα, and IL-10 concentration between pre- and post-treatment samples and were measured by enzyme-linked immunosorbent assay (ELISA) and Mesoscale Assay. P values were calculated using the Wilcoxon Signed Rank Test. We hypothesized an increase in TNFα and IL-10 after treatment. Statistically significant changes in soluble analyzes are those with p\<0.05, p0.05 means the changes are not statistically significant.

Time frame: Week 4, week 10, and week 20 after therapy compared to baseline

Intraprostatic Treg cell infiltration was measured by computer automated staining analysis pre and post treatment. Quantification will be reported as number of stained cells per mm\^2 of tissue.

Time frame: Baseline and at time of prostatectomy (approximately week 9)

Complete pathologic response is defined as the absence of detectable malignant cells in the prostatectomy specimen evaluated by standard histologic techniques.

Time frame: From baseline and time of radical prostatectomy (average of week 9)

Clinically important immune-related adverse events were defined as grade 3 or above inflammation requiring steroids or anti cytokine therapy or not resolving to grade 1 or less within 28 days. These immune-related adverse events were assessed by the Common Terminology Criteria for Adverse Events (CTCAE v4.0). Grade 3 is severe, grade 4 is life-threatening, and grade 5 is death related to adverse events.

Time frame: From baseline throughout study completion, an average of 20 weeks.

The number of participants who developed positive PSA-Specific T cell responses secondary to immune treatment will be reported. Peripheral prostatic specific antigen (PSA)-specific T cells at baseline and after therapy were assessed. PSA-specific T cells were those producing cytokine (Interferon gamma (IFNγ), Tumor Necrosis Factor Alpha (TNFα), Interleukin-2 (IL-2) or positive for the degranulation marker cluster of differentiation 107a (CD107a) following in vitro stimulation with PSA-15-mer peptides compared to a negative control peptide pool, measured by intracellular cytokine staining. A positive response will be defined as a \>2-fold increase in PSA-specific T cells after therapy compared to baseline.

Time frame: Weeks 7-10 and 17-22 compared to baseline

Population: 1/12 were not analyzed in the lead-in cohort because there was an insufficient number of viable peripheral blood mononuclear cells (PBMC) to perform the analysis.

Best serologic response secondary to immune treatment was reported. Change in Prostate-specific Antigen (PSA) was used to identify serological responses among participants in the lead-in cohort. Complete Serological Response was defined as PSA level less than 0.2 ng/mL measured for 2 consecutive measurements at least 4 weeks apart. Partial Serological Response was defined as decline of PSA at least 50% measured for 2 consecutive measurements at least 4 weeks apart. Serological Progression: Serological progression will only be measured once PSA has risen above 4 ng/mL and this value must be 50% above the PSA level before commencing treatment. Increase in PSA more than 50% of nadir (lowest PSA on treatment). Values must be measured for 2 consecutive measurements at least 2 weeks apart. The date of the first increase will be recorded as progression. Stable disease: not meeting progressive disease (PD) criteria for ≥12 weeks from treatment start.

Time frame: From baseline throughout study completion, an average of 1.4 years.

Changes in PD-L1 expression is defined as changes in density of cells positive for PDL1 and was analyzed by immunohistochemistry staining using the clone 22C3. Placenta tissue was used as a positive control. Previous studies have shown that high PDL1 expression is associated with poor clinical outcomes in prostate cancer participants. An increase of PDL1 after immunotherapy could indicate a resistance of the tumor to the treatment. Values can go from 0 to 100. 50% of Tumor cells is considered positive for PDL1. The sample will be considered positive if at least 1% or tumor cells express PDL1.

Time frame: Baseline (biopsy before first PROSTVAC administration) and at time of radical prostatectomy (on week 9)

Biochemical recurrence following radical prostatectomy is defined as at least two prostate-specific antigen (PSA) values that are 0.2 ng/mL or higher.

Time frame: From time of prostatectomy through study completion, an average of 10 weeks

Here is the number of participants with serious and/or non-serious adverse events assessed by the Common Terminology Criteria for Adverse Events (CTCAE v4.0). A non-serious adverse event is any untoward medical occurrence. A serious adverse event is an adverse event or suspected adverse reaction that results in death, a life-threatening adverse drug experience, hospitalization, disruption of the ability to conduct normal life functions, congenital anomaly/birth defect or important medical events that jeopardize the patient or subject and may require medical or surgical intervention to prevent one of the previous outcomes mentioned.

Time frame: First study intervention, Study Day 1, to 30 days after participant received last study drug administration. Approximately 1.4 years for the metastatic castration-resistant prostate cancer cohort, and an average of 20 weeks for the neoadjuvant cohort.