The U.S. FDA approved enzalutamide for chemotherapy- 12 Short Title: Enzalutamide Vaccine in mCRPC Version Date: 03/29/2022 refractory mCRPC patients in August of 2012 [6]. PREVAIL enrolled 1717 patient with mCRPC who progressed on ADT and randomized 1:1 to enzalutamide 160mg/day (n=872) or placebo (n=845). The enzalutamide arm had an 81% decreased risk of radiographic progression or death (HR: 0.186; 95% CI: 0.15 to 0.23; P<0.0001). The overall survival at 5 years favored patients randomized to the enzalutamide arm 35.5 to 31.4 months with a rate of 26% for the enzalutamide arm compared to 21% for the placebo arm. Figure 2. Enzalutamide demonstrates improved overall survival in mCRPC patients previously treated with docetaxel [5] The ultimate role of enzalutamide in the treatment of prostate cancer will likely be in earlier- stage disease. The minimal side effects of enzalutamide in patients with advanced disease will create strong interest in providing this therapy for chemotherapy- naïve mCRPC and even patients with non-metastatic disease, thus supplanting the use of older ARAs such as bicalutamide which are commonly used in non-metastatic patients. Also, unlike the modern androgen-biosynthesis inhibitor abiraterone, enzalutamide does not require daily prednisone. Enzalutamide therapy will thus avoid prednisone’s potential long-term side effects in earlier stage disease, an important consideration for future treatment strategies in this population. Combination with immune based therapies is attractive because unlike abiraterone, this treatment does not require prednisone which can be immunosuppressive. In addition, given the data which suggest that hormonal therapies do enhance immune response, it would be valuable to gain a better understanding of the immune impact of enzalutamide given the emerging role of immunotherapy in prostate cancer[9]. 1.2.2 Therapeutic Cancer Vaccines in Prostate Cancer The goal of therapeutic cancer vaccines is to generate a targeted immune response leading to immune-mediated anti-tumor activity. Sipuleucel-T is a therapeutic cancer vaccine generated from peripheral blood mononuclear cells obtained from individual patients via leukapheresis. This vaccine is generated after a patient’s peripheral immune cells are collected via leukapheresis, transported to a regional processing center where they are exposed in vitro to a PAP/GM-CSF fusion protein. At the end of this process, the activated cellular product is re- infused into the patient. A full course of therapy repeats this process 3 times every 2 weeks for 1 month [10, 11]. A phase III trial (n = 512) demonstrated an overall survival benefit for the vaccine (25.8 months vs. 21.7 months; P = 0.032) [12]. Based on these overall survival findings, the FDA approved sipuleucel-T for the treatment of asymptomatic or minimally symptomatic mCRPC, making it the first FDA-approved therapeutic cancer vaccine for the treatment of any malignancy. 13 Short Title: Enzalutamide Vaccine in mCRPC Version Date: 03/29/2022 1.2.3 PSA-TRICOM PSA-TRICOM (Prostvac™; developed by the National Cancer Institute [NCI] and licensed to Bavarian Nordic, Mountain View, CA), an off-the-shelf therapeutic cancer vaccine, offers an alternative strategy [13, 14]. (The LTIB and Bavarian Nordic have an ongoing CRADA for the preclinical and clinical development of PSA-TRICOM.) To target prostate-specific antigen (PSA), PSA-TRICOM vaccine employs genetically altered poxviruses to deliver targeting information to immune cells and generate an immune response. Administered subcutaneously, the poxviruses deliver the transgenes for the tumor associated antigens (PSA) to antigen presenting cells through cellular infection. Once these pox viruses are within the cellular cytoplasm, the transgenes are processed. The end result is an antigen presenting cell expressing a PSA peptide within the major histocompatibility complex, resulting in PSA-specific cytolytic T lymphocytes activation [13, 15]. ( Figure 3) This approach does not require expensive, labor- intensive ex vivo preparation of patients’ peripheral blood. PSA-TRICOM is thus potentially more logistically and financially feasible over the long-term than sipuleucel-T [16]. PSA-TRICOM has been investigated in 2 phase II trials in mCRPC, both of which administered the vaccine at monthly intervals until disease progression. An industry-sponsored, placebo- controlled, multicenter trial in 125 mCRPC patients randomized them 2:1 in favor of PSA- TRICOM; the placebo was an empty poxviral vector containing no transgenes. As was seen in the sipuleucel-T studies, patients receiving vaccine showed no change in TTP, yet had an overall survival benefit (25.1 months with PSA-TRICOM vs. 16.6 months with placebo; P = 0.0061) [17]. ( Figure 4) A second phase II study of PSA-TRICOM of 32 mCRPC patients at the NCI demonstrated that the vaccine was able to generate a T-cell specific immune response and patients with the greatest magnitude of this response had superior outcomes [18]. A phase III trial (NCT01322490) of Prostvac monotherapy, however, was reported to have not met its primary endpoint of overall survival in September, 2017. Nonetheless, the combination of Prostvac and enzalutamide remains scientifically rational and this trial will continue. Furthermore, at this point neither this trial nor the phase III trial have suggested a safety concern for Prostvac. 14 Short Title: Enzalutamide Vaccine in mCRPC Version Date: 03/29/2022 Figure 3. Poxviral vaccine strategy: Modified poxvirus contains transgenes for the tumor- associated antigen PSA and 3 T-cell costimulatory molecules [12] Figure 4. PSA-TRICOM improved survival in mCRPC patients in a randomized multicenter phase II trial. [13] Like enzalutamide, PSA-TRICOM is very well-tolerated, with common side effects of grade 1 injection-site reactions or flu-like symptoms [17]. Also like enzalutamide, favorable side effect profile and the potential ability to induce a sustained antitumor immune response, clinical trials are ongoing evaluating strategies to use PSA-TRICOM in earlier disease patients. 1.2.4 Rationale for Combining Enzalutamide with PSA-TRICOM Accumulating data demonstrate that androgen-deprivation therapy (ADT) affects not only prostate cancer growth, but also the immune system and suggest that ADT in prostate cancer can augment the immune response by increasing T-cell infiltration into the prostate [ 9]. The impact of this T-cell trafficking would be even greater if T cells were primed by a vaccine prior to ADT [19]. Furthermore, ADT has been shown to decrease immune tolerance of self antigens that are over-expressed in many cancers, increase the production of new T-cells from the thymus ( Table 1), and enhance the Cytotoxic T-cell repertoire [ 9, 20, 21]. Of the two modern anti- androgen therapies for prostate cancer that have emerged in the last few years, enzalutamide is 15 Short Title: Enzalutamide Vaccine in mCRPC Version Date: 03/29/2022 preferred over abiraterone, because abiraterone requires prednisone which may influence the immune response in a vaccine-based combination regimen. Baseline After ADT Naïve CD4 cells 3.25% of CD3+ cells3.95% of CD3+ cellsp = 0.0060 T-Cell Receptor Excision Circles93 per 100,000 cells147 per 100,000 cellsp = 0.0025 Table 1.