Results will not be required prior to enrollment. 34 Short Title: Enzalutamide Vaccine in mCRPC Version Date: 03/29/2022 Lymphocytes will be separated from heparinized blood using density gradient centrifugation. The lymphocytes will then be placed in human AB serum with 10% DMSO and stored in liquid nitrogen. When samples are available from pre- and post-treatment, the ELISPOT assay will be performed. The ELISPOT assay, measuring IFN-gamma production, is used to determine CTL precursor frequency to peptides from PSA, PSMA, MUC-1, PSCA, CEA, and PAP in both pre- and post-vaccination peripheral blood mononuclear cells, as previously described [37, 38]. Briefly, 96-well mL HA plates (Millipore Corporation, Bedford, MA) are coated with 100 l/well of capture MAb against human IFN-gamma at a concentration of 10  g/mL for 12h at RT. Plates are blocked for 30 min with RPMI 1640 plus 10% human Ab serum 2 x 105 PBMC are added to each well. PSA-3A pulsed C1R-A2 cells are added into each well as antigen- presenting cells (APC) at an effector:APC ratio of 1:1. Unpulsed C1R-A2 cells are used as a negative control. HLA-A2 binding flu matrix peptide 59-66 is used as a positive peptide control [39]. We also perform each sample with 6 replicates to control for variability. In addition, each sample is run with a flu peptide control (pre- and post-vaccine) as well as samples from a “normal” control HLA-A2-positive individual with previously determined levels of flu-specific T-cell precursors. Cells are incubated for 24 h and lysed with phosphate-buffered saline (PBS)- Tween (.05%). Biotinylated anti IFN-gamma antibody diluted to 2  g/mL in PBS-Tween containing 1% bovine serum albumin (BSA) is added and incubated overnight in 5% CO 2 at 37C. Plates are washed 3 times and developed with avidin alkaline phosphatase (GIBCO/BRL, Grand Island, NY) for 45 min. After washing the plates 3 times, each well is examined for positive dots. This assay will be performed in the Genitourinary Malignancies Branch, NCI, NIH. The number of dots in each well will be counted by 2 separate investigators in a blinded manner, and the frequency of responding cells will be determined. 4.1.2.2 CD4 T Cell Proliferation Assay It is planned that all patients will undergo exploratory analysis of the ability to detect CD4- positive responses using a whole-protein PSA assay, as well as a peptide mix with 63 different 15-mer peptides by ELISPOT and/or ELISA. 4.1.2.3 Sera Antibody Analysis Serum will be stored at -80 degrees Celsius and there will be planned analysis for generation of antibodies to PSA, BCG, PAP, PSMA, PSCA, and/or MUC-1. 4.1.2.4 Flow cytometry analysis of thymic emigrant To determine recent thymic emigrants, flow cytometry analysis will be performed on peripheral blood mononuclear cells. Cells will be resuspended in staining buffer (phosphate-buffered saline containing 3% fetal bovine serum) and stained for 30 minutes at 4°C with the combination of following antibodies: APC-H7-conjugated anti-CD4, PE-CY7-conjugated anti-CD3; FITC- conjugated CD45RA, PE-conjugated CD31, PerCP-CY5.5-conjugated Ki-67, AF-700- conjugated-CD197, V450-conjugated CD8, APC-conjugated CD103, V500-conjugated CD27 all purchased from BD Pharmingen, San Diego, CA). After that, FoxP3 intra-cellular staining will be performed on the cells stained with anti-CD4 and anti-CD25. They will be fixed and permeabilyzed using a fix/perm kit (eBioscience, San Diego, CA) according to the manufacturer’s manual, and will be labeled with FITC-conjugated anti-Foxp3 antibody (236A/E7 clone) or its isotype control antibody (eBioscience). Flow cytometry will be performed on a Becton Dickinson LSRII (BD Biosciences) device. 35 Short Title: Enzalutamide Vaccine in mCRPC Version Date: 03/29/2022 4.1.2.5 Natural Killer (NK) CELLS The number and phenotype of NK cells will be determined by phenotypic analysis of PMBCs stained for CD56, CD3, CD8, and CD16 by flow cytometry. 4.1.2.6 Regulatory T Cells Regulatory T cells have been shown to inhibit the activation and function of T cells that participate in antigen-specific immune responses. Higher levels of regulatory T-cells have been reported in the peripheral blood mononuclear cells of patients with several types of tumors. The number and phenotype of regulatory T-cells in peripheral blood mononuclear cells from patients in this study will be determined by 7-color flow cytometry analysis. Cells will be resuspended in staining buffer (phosphate-buffered saline containing 3% fetal bovine serum) and stained for CD4, CD25, CD127, FoxP3, CTLA-4, CD45RA, and CD8. The ratios between regulatory T- cells and CD4 effector cells and the ratios between regulatory T-cells and CD8 Effector cells will also be analyzed. 4.1.3 Additional Assays Blood samples may be used for additional research studies, which may include phenotypic and functional analysis of immune-cell subsets and analyses for cytokines (IFN-γ, IL-10, IL-12, IL-2, IL-4, etc.), chemokines, antibodies, tumor associated antigens, and/or other markers All samples will be labeled with the following identifier system. Patient’s enrollment # Trial number Patient’s initials Example: 01-ABC These labels are used only to send the samples from the NIH Clinical Center to the NCI Frederick Central Repository. The NCI Repository will process all samples, appropriately discard the label on the blood tube, and then store the samples with unique Leidos identifiers, to which only NCI study personnel will have the code to link to patient specific clinical information. Samples will be tracked according to Section 4.3 (Sample Storage, Tracking and Disposition). 4.1.4 Circulating Tumor Cells (CTC) 4.1.4.1 Rationale of investigation Methods are in development for the purification and analysis of circulating tumor cells (CTC). One of these novel CTC technologies, developed by Epic Sciences, is summarized as follows: whole blood is aliquoted onto slides, nucleated peripheral blood cells are attached to slides and examined, cytokeratin-positive/CD45-negative cells with an intact nucleus and a malignancy-consistent morphology are identified as CTCs, and their exact positions on the slides recorded. This technology has the advantage of being able to identify cells that may be CTC but cytokeratin negative.