Approval of both the protocol and the consent form must be obtained before any participant is enrolled. Any amendment to the protocol will require review and approval by the IRB before the changes are implemented to the study. In addition, all changes to the consent form will be IRB-approved; an IRB determination will be made regarding whether a new consent needs to be obtained from participants who provided consent, using a previously approved consent form. 8 Abbreviated Title: Neoadjuvant IO for HNSCC Version Date: August 31, 2022 1. INTRODUCTION 1.1 STUDY O BJECTIVES 1.1.1 Primary Objective Determine the rate of pathologic complete response (pCR) or clinical-to-pathological downstaging in patients with previously untreated intermediate/high risk, non HPV-associated squamous cell carcinoma of the head and neck (T1-T4, N0-N3, M0 stage II, III or IV) who receive any of the three proposed treatments: M7824 alone, M7824 plus TriAd vaccine, or M7824 plus TriAd vaccine plus N803. 1.1.2 Secondary Objectives Estimate the rate of grade 3 or 4 immune related adverse events (irAEs) Determine the rate of treatment-related adverse events (AE) causing a delay of 4 weeks or more beyond planned surgery Estimate the response rate of the primary disease by CT imaging (RECIST) Estimate the one- and two-year recurrence free survival One- and two-year overall survival 1.1.3 Exploratory Objectives Define immune infiltrate and localization using multiplex immunofluorescence Define T cell clonality using Deep TCR-seq Determine tumor infiltrating lymphocyte (TIL) responses to shared antigens and antigens predicted from exome and RNA sequencing Assess antigen specific responses using peripheral T cell isolation Multiparameter characterization of PBMC by flow cytometry Assessment of changes in serum cytokines Assessment of changes in TGF signaling in tumor, normal mucosa and skin 1.2 BACKGROUND AND RATIONALE 1.2.1 Poor outcomes in patients with advanced stage, head and neck cancer not associated with HPV infection Definitive standard-of-care treatment for patients with locally-advanced or locoregionally advanced (stage II, III or IV) non-HPV associated head and neck squamous cell carcinoma (HNSCC) of the oral cavity, oropharynx, hypopharynx and larynx includes multi-modality therapy consisting of concurrent chemoradiation (CRT) or surgery followed by adjuvant radiotherapy with or without chemotherapy. In contrast to patients with human papillomavirus (HPV)-positive oropharyngeal cancer, patients with newly diagnosed advanced stage HPV- negative HNSCC have poor outcomes after definitive standard-of-care treatment [1]. Negative p16 status is a surrogate marker for an HPV-negative tumor (p16-positive tumors are HPV- positive) in tumors originating in the oropharynx. Involvement of HPV and therefore p16 status, have not been demonstrated to be biologically relevant in head and neck cancers originating in 9 Abbreviated Title: Neoadjuvant IO for HNSCC Version Date: August 31, 2022 locations other than the oropharynx (e.g. oral cavity, larynx, hypopharynx or nasopharynx) [2]. Therefore, these tumors are considered to be ‘non-HPV associated.’ Despite maximum treatment, locoregional recurrence (LR) or distant metastasis (DM) will develop in 35% of patients with non-HPV-associated HNSCC in the first one year (Figure 1). Five-year overall survival (OS) is less than 50% [3]. Additionally, standard-of-care treatments for advanced stage HNSCC leave cured patients with devastating functional impairments [4]. More efficacious and less toxic treatments are needed for advanced stage, non-HPV associated, HNSCC. Figure 1: Disease-free survival in patients with advanced p16-negative HNSCC treated with radiotherapy (RT) or RT plus chemotherapy. 1.2.2 Immunotherapy has clinical activity in patients with HNSCC HNSCC has features that make it ideal for immunotherapy. The majority of HNSCC specimens demonstrate evidence of T cell infiltration and PD-L1 expression indicative of an underlying immune response [5]. Additionally, HNSCCs harbor many genomic alterations that may give rise to an array of T cell neoantigens [6, 7]. Based on safety and improved overall survival reported in two clinical trials, both pembrolizumab and nivolumab, two programmed death receptor-1 (PD-1) monoclonal antibodies (mAbs), are FDA-approved for patients with recurrent/metastatic HNSCC that have failed systemic platinum-based chemotherapy [5, 8]. These studies demonstrate a manageable safety profile but objective responses in only 15-20% of patients following PD-1 blockade. Optimal timing of immune checkpoint blockade, or combination immunotherapy targeting resistance mechanisms, may enhance response rates and lead to improved patient survival. 10 Abbreviated Title: Neoadjuvant IO for HNSCC Version Date: August 31, 2022 1.2.3 Neoadjuvant immunotherapy may have increased clinical activity in HNSCC Treatment with immunotherapy before definitive extirpative surgery (neoadjuvant treatment) may optimally activate anti-tumor immunity. The use of up-front neoadjuvant or induction immunotherapy before standard-of-care treatment for HNSCC is being actively studied in at least nine clinical trials worldwide. Pre-clinical work from our laboratory demonstrates that PD-1 mAb treatment before surgical removal of tumor in mice leads to greater induction of neoantigen-specific memory T cells and anti-tumor immunity compared to PD-1 mAb treatment after surgical removal of tumor in multiple models of head and neck cancer (Figure 2). This data suggests that immunotherapy administered when tumor antigen is present followed by removal of tumor may lead to enhanced immune responses. Immunotherapy in the up-front treatment setting before surgical resection may increase the magnitude of response or increase the percentage of patients that respond. Additionally, surgical specimens offer an opportunity to assess for alterations in clinical-to-pathologic staging and evidence of major pathologic response as trial outcome measures [9]. Ongoing clinical trials at the Dana Farber Cancer Institute and University of Pittsburgh are exploring neoadjuvant pembrolizumab or nivolumab before surgery for advanced oral cavity cancer have set the precedent for this treatment schema. Preliminary results from these studies have demonstrated 1) no grade 3/4 adverse events, 2) no delays in pre-determined time to surgery with surgical treatment occurring two to three weeks following the final dose of immunotherapy, 3) improved disease-specific survival at the one-year mark, and 4) evidence of clinical activity with major pathologic responses in surgery specimens and clinical-to-pathologic downstaging [10] (NCT02296684), [ 11] ( NCT02488759). Thus, neoadjuvant single agent immune checkpoint inhibitor immunotherapy before definitive surgical resection in patients with advanced HNSCC appears be safe and have clinical activity. 11 Abbreviated Title: Neoadjuvant IO for HNSCC Version Date: August 31, 2022 Figure 2: Neoantigen-induced IFN production by tumor draining lymph nodes harvested 40 days after resection of oral carcinomas (left) in a syngeneic model of carcinogen-induced oral cavity squamous cell carcinoma called MOC1 (mouse oral cancer 1). Peripheral T cells were assessed for neoantigen: MHC class I tetramer positivity 40 days after resection of oral carcinomas (right). 1.2.4 Rationale for combination of I/O agents Preclinical studies are now revealing that an effective immuno-oncology strategy is the use of multiple immune-mediating agents, each targeting different components of the immune system. These include: (a) induction of an immune response via vaccination directed against tumor- associated antigens, (b) potentiation of the systemic immune response by the use of immunocytokines (such as the N-803 [also known as ALT-803], an IL-15 super agonist fusion protein) to enhance both NK and CD8+ T-cell responses, and (c) reduction of immunosuppressive entities in the tumor microenvironment (TME) by the use of anti-PD1/PDL1 mAb checkpoint inhibitors, and/or reduction of immunosuppressive cytokines such as TGF-β with the use of a bifunctional anti-PDL1/TGF-β2 “Trap” designated M7824. The LTIB, in collaboration with the Genitourinary Malignancies Branch (GMB), has developed and/or co- developed these agents via a series of cooperative research and development agreements (CRADAs). Each has been interrogated in a series of preclinical studies and in Phase I/II clinical studies in the CCR as well as by other investigators. The preclinical studies and early clinical studies detailed below are demonstrating the relative lack of additional toxicities with enhanced clinical benefit employing combinations of these agents. Furthermore, preclinical studies are now demonstrating that the combined use of agents from each of the immune-mediating categories described above elicits the most immune-mediated anti-tumor activities. 12 Abbreviated Title: Neoadjuvant IO for HNSCC Version Date: August 31, 2022 Figure 3: Combined effect of Ad-Twist vaccine and M7824 in EMT6 carcinoma model As previous shown in studies published by investigators within the LTIB [12], M7824 combined with tumor-targeted vaccine enhances antitumor activity in metastatic murine cancer models ( Figure 3). Additionally, M7824 can mediate ADCC and the addition of N-803 to M7824further enhances this ADCC activity. [13] ( Figure 4). Figure 4: ALT-803 enhances the NK-mediated lysis of avelumab and M7824 model Employing NSG-β2m‒/‒ mice bearing human breast MDA-MB-231 tumors and reconstituted with human PBMC, the combination of vaccine, N-803, and M7824 resulted in the greatest level of anti-tumor activity compared to the use of individual agents (Figure 5). 13 Abbreviated Title: Neoadjuvant IO for HNSCC Version Date: August 31, 2022 Figure 5: Multimodal therapy in mice reconstituted with PBMCs from patients 1.2.5 M7824 TGF is an immunosuppressive cytokine highly expressed in HNSCC by tumor, stromal and infiltrating immune cells and is a mechanism of resistance to immune checkpoint inhibitor immunotherapy[ 14, 15]. M7824 is a bifunctional fusion protein combining anti-PD-L1 mAb and TGF receptor type 2, serving as a TGF  trap ( Figure 6). By dually blocking PD-L1 and neutralizing TGF, M7824 treatment demonstrates robust anti-tumor immune activation in multiple pre-clinical models of cancer [16]. In a dose-escalation phase 1 study performed at the NIH Clinical Center, M7824 demonstrated a manageable safety profile and induced durable anti- tumor responses, as well as reduction in circulating TGF (Figure 7) [17]. Four of nineteen patients (21%) experienced a grade 3 or 4 adverse event; these included one case of grade 2 bullous pemphigoid, one case of grade 3 asymptomatic lipase increase, one case of grade 2 colitis, and once case of grade 3 hypokalemia. Only grade 2 colitis occurred within 30 days of initiating M7824 treatment. 14 Abbreviated Title: Neoadjuvant IO for HNSCC Version Date: August 31, 2022 Figure 6: M7824. Approximately one-third of patients with advanced non-HPV associated HNSCC harbor tumors without evidence of T cell infiltration [5], suggesting these tumors may lack T cell antigens or have antigen processing or presentation defects. NK cells recognize tumors cells through antigen-independent mechanisms and may play a role in anti-tumor immunity in these cancers. In addition to activating T cells, M7824 can induce NK cell anti-tumor activity through antibody-dependent cell-mediated cytotoxicity (ADCC) and the in vivo anti-tumor activity of M7824 is dependent upon both T cells and NK cells [12, 16]. Thus, M7824 dually activates T and NK cell activity along with local neutralization of immunosuppressive TGF to maximally activate anti-tumor immunity. 15 Abbreviated Title: Neoadjuvant IO for HNSCC Version Date: August 31, 2022 Figure 7: Spider plot of clinical responses and mean serum TGF concentration in patients with recurrent/metastatic cancer receiving M7824. 1.2.6 N-803 N-803 is an IL-15 superagonist complex that expands the numbers and enhances the activity of effector T and NK cells, leading to significant in vivo anti-tumor activity when combined with immune checkpoint blockade in multiple pre-clinical solid tumor models [18]. As discussed above, M7824 enhances ADCC-mediated NK cell cytotoxicity [12]. Specifically, experiments conducted in the LTIB show that N-803 and M7824 work synergistically to enhance NK cell- mediated ADCC ( Figure 8A ) [19]. The combination of N-803 and immune checkpoint blockade (nivolumab) has been used clinically with a manageable safety profile and evidence of clinical activity [20]. No patient receiving this combination treatment experienced a grade 4 adverse event. Grade 3 adverse events occurred in 10% of patients and included fever, fatigue, dizziness and alterations in clinical laboratory assessments. 1.2.7 Clinical Safety and Experience with N-803 and anti-PD-1/L1 blockade at the NCI The combination of N-803 and immune checkpoint blockade (nivolumab) has been used clinically with a manageable safety profile and evidence of clinical activity [20]. No patient receiving this combination treatment experienced a grade 4 adverse event.