/PhD . 2 At ASU: Karen Anderson, MD/PhD3 V11.2 (1/9/20 ) Page 3 of 50 1 OBJECTIVES This is a n investigator -initiated prospective clinical study of patients with stage I-IV squamous cell carcinoma of the oropharynx (OPSCC) who are to undergo ablative transoral surgery . We propose to test the hypothesis that the Listeria -based HPV vaccine ADX11 -001 induces circulating and tumor -infiltrating antigen -specific T cells in HPV16+ oropharyngeal cancer patients undergoing transoral resection. The results of this trial will assess the ability of ADX11- 001 vaccination to induce a robust HPV -specific cytotoxic lymphocyte (CTL) response in the blood and tumor . 1.1 Primary Aims 1.1.1 To determine the immunogenicity of ADX11 -001 vaccination in patients with HPV+ squamous cell carcinoma of the oropharynx. 1.1.2 To evaluate the tolerability, safety , and nature and degree of toxicity of ADX11- 001 by the numbers of patients with dose limiting toxicities (DLTs) and adverse events as assessed by the CTCAE v4.0 1.2 Primary Endpoints 1.2.1 The primary endpoint will be the change in HPV E6/E 7-specific CD8+ cytotoxic lymphocyte (CTL) responses in the peripheral blood at time of surgery , with respect to baseline in vaccinated patients . 1.2.2 Toxicity (NCI Common Terminology Criteria for Adverse Events (CTCAE) version 4.0) 1.3 Secondary Aim The secondary objective will be to determine v accine- induced HPV E6/E7 -specific CD8+ CTL responses in the blood at various timepoints after surgery , with respect to baseline levels, in vaccinated patients. 1.4 Secondary Endpoint The secondary endpoint will be the change in HPV E6/E7- specific CD8+ cytotoxic lymphocyte (CTL) responses in the peripheral blood at various times after surgery (3W, 6W, 3M, 6M, 12M), with respect to baseline in vaccinated patients. 1.5 Exploratory Aim To analyze chang es in the profile of tumor -infiltrating effector (NK cells, CD4+ and CD8+ T cells) and suppressor (Treg and MDSC) immunocytes by immunohistochemistry in vaccinated and unvaccinated (observational cohort) patients. V11.2 (1/9/20 ) Page 4 of 50 2 BACKGROUND/RATIONALE 2.1 DISEASE AND I NCIDENCE Squamous cell carcinoma is the most frequently occurring malignant tumor of the head and neck and is a major cause of morbidity and mortality worldwide. More than 90% of head and neck squamous cell carcinoma ( HNSCC) originate from the mucosal linings of the oral cavity, pharynx, or larynx. Reports show that the estimated worldwide incidence of HNSCC in 2000 was more than 551,000 with greater than 217,000 deaths. 1 In the United States (US), in 2006 alone, more than 46,000 new cases of HNSCC and 11,000 deaths due to HNSCC were reported. 2 While HNSCC related to environmental carcinogens (tobacco, alcohol) has declined in the US, the incidence of certain subtypes of H NSCC, particularly HNSCC related to the human papilloma virus (HPV) is rising.3 2.2 HPV-MEDIATED HEAD AND NECK CANCER – A TARGET FOR THERAPEUTIC VACCINATION Currently 60- 80% of oropharyngeal cancer ( OPC ) is caused by the human papillomavirus (HPV), particular ly the so -called “high -risk” oncogenic HPV16 and HPV18 genotypes. HPV 16 is strongly implicated in the etiology of OPC4, and is found in up to 90% of virally -related OPC.5 It has become increasingly clear that HPV -mediated OPC is a different disease from c lassical environmentally -related HNSCC, with distinct epidemiology and natural history . While patients with HPV+ tumors typically have an excellent response to surgical or radiation- based therapy6,7, treatment can be associated with significant short - and long- term toxicity, and it is not yet known whether patients with HPV+ OPSCCA have better long- term control. Thus, approaches which target the unique biology of HPV -infected cancer cells, such as therapeutic vaccination, are attractive strategies for potentially de- escalating XRT and chemo/XRT regimens (and thus decreasing toxicity) and enhancing long- term disease control. Prior to being internalized into epithelial cells in the basal layer of the oropharyngeal mucosa, HPV is susceptible to neutralization with specific antibodies, which is the mechanism of action of the preventative vaccine Gardasil. 8 However, once HPV is internalized, it can integrate into the epithelial cell genome, and is no longer susceptible to antibody -mediated clearance. HPV - infected cells can then only be cleared by mechanisms of cell -mediated immunity, including natural killer (NK) cells, and CD8+ cytotoxic T cells (CTL)9. In patients for whom these mechanisms are inefficient, progression of HPV -infected epithelial cells to malignancy is then driven by the E6 and E7 viral early genes, which inactivate p53 and Rb tumor suppressor genes respectively 10. E6 and E7 are essential to maintenance of the transformed phenotype, and are also often recognized by T cells from HPV -infected patients11-13; thus these proteins are logical targets for monitoring HPV -specific immune responses, as well as therapeutic (cell - mediated, or T cell- based) vaccination. Paradoxically, while patients with HPV -mediated tumors are unable to control and clear the virus, anti -HPV T cell responses can be detected9,11-13. Furthermore, a substantial amount of the HPV genome is devoted to various mechanisms of immune evasion14, and HPV -induced cancers are more common in immunosuppressed patients15,16. This suggests that the immune system does, in fact, recognize viral antigens in HPV -infected cells and the efficacy of this response depends critically on the overall balance of immune activation and immunosuppressive/immunoregulatory mechanisms in the host. V11.2 (1/9/20 ) Page 5 of 50 In an effort to tip the immunologic balance towards beneficial immune activation, preventive and therapeutic vaccines targeting HPV have been developed. Therapeutic peptide vaccination using long peptides specific for HPV16 has recently demonstrated to have immunologic and clinical efficacy in the treatment of vulvar intraepithelial neoplasia (VIN )49. The etiologic similarities between cervical cancer /VIN and HPV + OPSCC, where in both the most common HPV subtype is HPV16, provide a convincing rationale for therapeutic vaccination in HPV+ OPSCCA patients. 2.3 ADXS11 -001 SUMMARY OF SAFETY As of June 30, 2016, 722 doses of ADXS11 -001 have been administered to 290 enrolled subjects at doses of 5 x 107, 3.3 x 108, 1 x 109, 3.3 x 109, and 1 x 1010 CFU. In the Phase 1 study, 100% of subjects (n=15) have experienced flu- like AEs or symptoms associated with cytokine release syndrome. The incorporation of NSAIDs and antiemetic medications pre and post infusions has effectively reduced the incidence of these symptoms from 100% to 37%. In addition, a course of antibiotics is given 3 days after each dose of ADXS11- 001 as a precautionary measure to ensure clearance of th e Lm. From the clinical experience in 29 0 subjects, a clear pattern of mild to moderate treatment -related AEs consistent with cytokine release symptoms (e.g., constitutional symptoms such as fever, chills, rigors, headache, nausea, vomiting, tachycardia, shortness of breath, hypotension, and rash) are commonly seen and typically appear 2 -4 hours after infusion. Symptoms either self -resolve or respond quickly to symptomatic treatment. A summary of AEs in ≥5% of subjects by MedDRA system organ class and preferred term is presented in Table 1. There have been no Grade 3-4 related AEs observed in over 5% of the subject population (see Table 1 ). Table 1 Treatment -Related (Possibly Related, Probably Related, Related) Adverse Events Reported ≥5% with ADXS11- 001 (Safety Population, N=29 0) System Organ Class (SOC), Preferred Term, [N (%)] [1] Grades 1 -4 (N=290) Grade 3 -4 (N=290) Blood And Lymphatic System Disorders Anaemia 178 ( 61.4) 3(1.0) Cardiac Disorders Sinus Tachycardia 16(5. 5) 2 (0.7) Tachycardia 32 (11.0) Gastrointestinal Disorders Abdominal Pain 49 (16.5) - Constipation 47 (16.2) - Diarrhoea 30 (10.3) 1 (0.3) Dysphagia 15 (5.2) - Nausea 107 ( 36.9) - V11.2 (1/9/20 ) Page 6 of 50 System Organ Class (SOC), Preferred Term, [N (%)] [1] Grades 1 -4 (N=290) Grade 3 -4 (N=290) Vomiting 88 (30.3) 4 (1.4) General Disorders And Administration Site Conditions Chills 184 ( 63.5) 5 (1.7) Disease Progression 23 (7.9) - Fatigue 74 (25.5) 1 (0.3) Oedema Peripheral 24 (8.3) - Pain 34 (11.7) - Pyrexia 167 ( 57.6) 3 (1.0) Immune System Disorder Cytokine Release Syndrome 24 (8.3) 13 (4.5) Investigations Alanine Aminotransferase Increased 18 (6.2) 2 (0.7) Aspartate Aminotransferase Increased 21 (7.2) 1 (0.3) Blood Alkaline Phosphatase Increased 24 (8.3) 3 (1.0) Blood Creatinine Increased 25 (8.6) - Gamma -Glutamyltransferase Increased 25 (8.6) 4 (1.4) Haemoglobin Decreased 16 (5.5) - White Blood Cell Count Decreased 29 (10.0) 2 (0.7) Metabolism And Nutrition Disorders Decreased Appetite 33 (11.4) - Hyperglycaemia 19 (6.5) - Hypertriglyceridemia 20 (6.9) - Hypoalbuminaemia 43 (14.8) - Hypokalaemia 18 (6.2) - Hyponatraemia 31 (10.7) 1 (0.3) Musculoskeletal And Connective Tissue Disorders Back Pain 33 (11.4) 1 (0.3) Nervous System Disorders Dizziness 19 (6.5) - Headache 69 (23.8) - V11.2 (1/9/20 ) Page 7 of 50 System Organ Class (SOC), Preferred Term, [N (%)] [1] Grades 1 -4 (N=290) Grade 3 -4 (N=290) Psychiatric Disorders Anxiety 17 (5.9) - Depression 17 (5.9) - Respiratory, Thoracic And Mediastinal Disorders Cough 16 (5.5) - Dyspnoea 24 (8.3) 2 (0.7) Vascular Disorders Hypertension 17 (5.9) 3 (1.0) Hypotension 58 (20.0) 18 (6.2) Note: [1] Percentage is calculated using column header count as denominator for percentage calculation. 3 ADXS11 -001 3.1 Vaccine Design: ADXS11 -001 (ADXS -HPV) is a live attenuated Listeria monocytogenes -listeriolysin O ( Lm- LLO) immunotherapy developed for the treatment of HPV -associated cancers .