Hassan in collabora tion with Dr. Jeff Schlom at the National Cancer Institute ( NCI) elucidated the immunogenic ep itopes of mesothelin and showed that mesothelin- specific T cells cause lysis of mesothelioma tumor cells . 25 The mesothelin -targeting therapeutic cancer vaccine furthest along in clinical development is CRS -207, a live -attenuated strain of the bacterium Listeria monocytogenes (Lm) encoding human mesothelin (hM eso).26,27 1.1.3 Listeria monocytogenes (Lm)-Based Vaccine Therapy Lm is an attractive platform for presentation of tumor -associated antigens and activation of immune response directed against cancer cells. Lm provides both a potent stimulation of innate immunity and also stimulates an adaptive immune response through recruitment and activation of CD4+ and CD8+ T -cell immunity specific for encoded heterologous antigens . 28-31 Aduro Biotech , Inc. (Aduro) developed a live -attenuated Lm platform strain ( Lm ∆actA/∆inlB), known as ANZ -100 (previously CRS -100), which has deletions of two genes, actA and inlB, that encode the virulence -determinant proteins ActA and Internalin B, respectively27. These two proteins facilitate cell- to-cell spread and invasion of nonphagocytic cells, and deletion of actA and inlB in ANZ -100 results in 1,000- fold attenuation of these processes in mice as compared with wild- type Lm.32, 29 Uptake of ANZ -100 by macrophages and other phagocytic cells in the liver and spleen is still retained and results in a local inflammatory response as well as activation and recruitment of natural killer cells and T cells to the liver. ANZ -100 underwent clinical evaluation in a Phase 1 dose -escalation study in Aduro Biotech , Inc. Protocol ADU -CL-02-v8 CRS -207 with Pemetrexed and Cisplatin Version 8, 08 March 2018 Page 3 adults with carcinoma and liver metastases and was found to be safe and well tolerated in 9 subjects given intravenous doses up to 3 × 108 colony- forming units (CFU). Additionally, no shedding of ANZ-100 in urine , stools , or sputum was observed at any dose. ANZ -100 has additionally been engineered to express mesothelin and the resulting strain has been termed CRS -207. After uptake of CRS -207 by dendritic cells and macrophages, mesothelin is express ed and released into the cytosolic compartment and subsequently processed through the endogenous MHC Class I presentation pathway, resulting in activation of mesothelin- specific T cell - mediated immunity. Other mechanisms to activate mesothelin -specific, cell - mediated immunity may include uptake and cross -presentation of antigens by dendritic cells and other cells after infection by CRS -207 and apoptosis. Nonclinical studies have shown that CRS -207 elicits mesothelin -specific cellular immunity in mice and nonhuman primates and demonstrates therapeutic efficacy in tumor -bearing mice (refer to the Investigator’s Brochure [IB] for details). Findings in a Good Laboratory Practice repeated- dose study in cynomolgus monkeys showed that treatment with up to 3 × 1010 CFU of CRS -207 resulted in no changes related to body weight, food consumption or body temperature and in no findings related to ocular or functional cardiovascular evaluations. CRS -207 was detected in the blood at 24 hours after administration, but w as undetectable at 72 hours. There were transient and dose -dependent decreases in red blood cell, platelet and white blood cell (WBC) counts. Hepatic and renal function changes were transient and generally less than two -fold from that at baseline status. A Phase 1 study (VAC07001; NCT00585845) has been completed with CRS -207 to determine the maximum tolerated dose and to explore the safety profile in subjects with advanced mesothelioma, non–small- cell lung cancer, ovarian cancer or pancreatic adenocarcin oma who had failed standard therapy . 26 CRS -207 was found to be well tolerated at doses up to 1 × 109 CFU. Adverse events (AEs) such as fevers, chills, and nausea reported as the most common, immediate, transient, mild, and temporally related to CRS -207 administration were self -correcting and were resolved by the time of the subjects’ discharge. Lymphopenia was observed in all doses (1 × 10 8, 3 × 108, 1 × 109 and 1 × 1010 CFU), and transaminase elevations were observed at doses of 3 × 108, 1 × 109 and 1 × 1010 CFU. Both were dose -dependent, although transient and not considered clinically significant. Two CRS -207- related serious adverse events ( SAEs ) were reported. One SAE of moderate constipation occurred in one subject after the second dose of CRS -207 at 1 × 108 CFU. The second SAE, a significant decrease in blood pressure (BP) Aduro Biotech , Inc. Protocol ADU -CL-02-v8 CRS -207 with Pemetrexed and Cisplatin Version 8, 08 March 2018 Page 4 after infusion, occurred in another subject after one dose of CRS -207 at 1 × 1010 CFU. This subject required aggressive fluid management and recovered to baseline status within 24 hours. This dose level was subsequently discontinued and confirmed the maximum tolerated dose (MTD) as 1 × 109 CFU . No shedding of CRS -207 in urine or stools was observed at any dose. While the VAC07001 study enrolled subjects with multiple disease types and was not powered to assess survival, 6 of 17 subjects survived for ≥ 15 months. Of these 6 “long- term” survivors, 3 had pancreatic cancer, 2 had non- small -cell lung cancer, and 1 had mesothelioma. All 6 subjects had prior immunotherapy or subsequent local radiation. Five of 6 subjects received all 4 doses of CRS -207 and all 5 evaluable subjects demonstrated vaccine -induced Lm-specific respon ses. In addition, 4 of the 5 evaluable subjects among the long- term survivors had stable disease by RECIST at day 91 (end of study). As 3 of the 6 “long -term” survivors were pancreatic cancer patients had previously received GVAX pancreas vaccine in separate clinical studies, CRS - 207 in combination with GVAX pancreas vaccine (with low -dose cyclophosphamide [ Cy]) is currently being evaluated in a Phase 2 study in metastatic pancreatic cancer patients who have received or refused chemotherapy (Protocol ADU -CL-01; NCT01417000). Enrollment has been completed in this study and a recent interim analysis showed a signi ficant improvement in median overall survival in subjects who received GVAX pancreas vaccine and CRS -207 compared to subjects who received GVAX pancreas vaccine alone ( 6.0 months vs. 3.4 months; one-sided p=0.0.0057). The se interim data were reviewed by a n independent data monitoring committee who recommended that the study be stopped for efficacy and subjects on the GVAX pancreas vaccine arm offered rollover treatment with GVAX pancreas vaccine and CRS -207. FDA concurred with this decision. Subjects in the study continue to receive treatment and all subjects are being followed for survival, safety and clinical and immune responses. Safety findings have been consistent with prior clinical experience . The most frequent Grade 3/4 related toxicities were f ever, lymphopenia, hypophosphatemia, elevated liver enzymes and fatigue following CRS -207 in <5% of subjects. To date , there have been no treatment -related SAEs, significant laboratory abnormalities or safety signals. Another Phase 2 study for pancreatic cancer , ADU- CL-04, was initiated in 2014. This study enroll s subjects with previously -treated metastatic pancreatic cancer for evaluation of GVAX pancreas vaccine (with low -dose Cy) and CRS -207 Aduro Biotech , Inc. Protocol ADU -CL-02-v8 CRS -207 with Pemetrexed and Cisplatin Version 8, 08 March 2018 Page 5 compared to CRS -207 alone or single -agent chemotherapy alone. As of February 2015, over 200 subjects have been enrolled and there have been three SAEs reported as unexpected and related to CRS -207: Grade 3 confusion, Grade 3 hypoxia and Grade 3 hypertension. A second Phase 2 study in previously -treated metastatic pancreatic cancer was initiated in January 2015 which is evaluating GVAX pancreas vaccine (with low -dose Cy) and CRS -207 with and without anti - PD-1 antibody (nivolumab) in 88 subjects. Six patients have been treated with no significant toxicities or SAEs reported. 1.2 RATIONALE FOR COMBINATION OF CRS -207 AND CHEMOTHERAPY The combination of vaccines with chemotherapy has traditionally not been used due to the possibility of post -chemotherapy leukopenia eliminating antitumor directed lymphocy tes. Emerging data, however, suggest that vaccines and chemotherapies may be synergistic by augmenting anti -tumor effectiveness of subsequent chemotherapies .33,34 Proposed mechanisms of synergy for activating relatively dormant T cells include chemotherapy -induced alterations in tumor microenvironment, up- regulation of MHC class I and numerous tumor -associated antigens on the surface of tumor cells, reduction of suppressor cell populations and tumor lysis followed by cross priming .35-37 Synergistic activity of combinations of immunotherapy and chemotherapy is also supported by clinical studies. High chemotherapy response rates were observed in patients with small- cell lung cancer who developed an immunologic response to an Ad.p53- dendritic cell vaccine. 38 Advanced -stage cancer patients who developed immunity to cytochrome P4501B1 following administration of a plasmid/microparticle vaccine showed marked response to a subsequent chemotherapy regimen . 39 Patients with metastatic androgen independent prostate cancer who received recombinant viral vectors containing the PSA and B7.1 genes followed by docetaxel had a longer time to progression than patients who received docetaxel alone 40. Glioblastoma patients vaccinated with autologous tumor- pulsed dendritic cell vaccine who received subsequent chemotherapy exhibited significantly longer t imes to tumor recurrence after chemotherapy relative to their own previous recurrence times, as well as significantly longer post- chemotherapy recurrence times and survival relative to patients receiving isolated vaccination or chemotherapy . 41,42 In mesothelioma patients, three vaccinations at two week intervals with autologous tumor lysate -pulsed dendritic Aduro Biotech , Inc. Protocol ADU -CL-02-v8 CRS -207 with Pemetrexed and Cisplatin Version 8, 08 March 2018 Page 6 cells following chemotherapy with cisplatin/pemetrexed induced tumor specific CD8+ T lymphocyte activity in four of the six patients.43 CRS -207 has been shown to be safe and well -tolerated at the proposed route of administration and dose for this study .26 Five subjects with mesothelioma were enrolled in the Phase 1 study and tolerated the treatment at doses up to 1 × 109 CFU. One subject with mesothelioma survived for 27 months post -first dose of CRS -207 in the Phase 1 study. The proposed dose level for this study (1 × 109 CFU) is the MTD as determined in the Phase 1 study and is the dose being administered in the current Phase 2 pancreatic cancer study. This dose level has shown an acceptable safety profile in the 12 p atients treated thus far (in both Phase 1 and Phase 2). In addition, 3 of 4 evaluable patients at this dose level in the Phase 1 study demonstrated CRS -207- induced mesothelin- specific CD8+ T cell responses (as compared to 1/3 and 2/3 patients at lower dos e levels) and 2 of 6 of the “long- term” survivors received CRS -207 at this dose level. This study will be the first to investigate the safety and immunogenicity of CRS -207 with or without Cy when combined with standard of care chemotherapy in patients with malignant mesothelioma. Safety data from the first 23 subjects treated on this study have shown no additive or synergistic toxicities when administering CRS -207 prior to or after pemetrexed and cisplatin. The most frequent AEs have been mild to moderate infusion reactions ( e.g. nausea, chills, headache, and vomiting ) as well as fatigue, anemia, decreased appetite, night sweats and lymphocyte count decreases related to CRS - 207 and expected AEs related to chemotherapy dosing. No treatment -related unexpected Grade 3 or greater AEs, SAEs or deaths have occurred. Additionally, in the first 25 subjects evaluated for objective clinical responses post -study treatme nt, 15 had best overall response of partial response (PR) , 7 had stable disease (SD), and 3 had progressive disease (PD). No subjects have discontinued treatment due to AEs related to CRS -207. The initial safety and clinical response data warrant ed further evaluation in an expansion phase of this Phase 1B study and the original sample size was increased from 16 to 40 . The sample size has been further increased to 60 in order to evaluate the use of low -dose Cy in this patient population and treatment regimen. Aduro Biotech , Inc. Protocol ADU -CL-02-v8 CRS -207 with Pemetrexed and Cisplatin Version 8, 08 March 2018 Page 7 1.3 RATIONALE FOR USE OF LOW -DOS E CYCLOPHOSPHAMIDE AND CRS -207 Cyclophosphamide (Cy) is currently approved as a high -dose chemotherapy agent, however, when administered at low -doses, it has exhibited immune - modulatory effects, namely in the suppression of regulatory T cells (Tregs).44 In the context of cancer vaccines, the preferential inhibition of Tregs may enhance vaccine -induced tumor-specific immunity, thereby improving vaccine efficacy. Data s uggest that immune -modulating doses of Cy may enhance vaccine -induced antitumor immune responses by inhibiting CD4+ CD25+ regulatory T cell activity. Several studies have demonstrated this concept and shown prolonged survival of patients receiving low -doses of Cy 24-72 hours prior to vaccination with minimal side effects .1,2,45,46 Increased vaccine-induced T cell responses and improved survival has been demonstrated in nonclinical studies of CRS -207 administered with immune - modulatory doses of Cy. In mice administered 50 mg/kg Cy (equivalent to 140 mg/m2 human dose), immune responses measured seven days after administration of CRS -207 demonstrated that the combination of Cy and CRS -207 increased mesothelin -specific T cell responses compared to saline and CRS -207 alone. In a lung tumor model, tumor -bearing mice received 50 or 100 mg/kg Cy (corresponding to 140 to 280 mg/m2 of the human dose equivalent, respectively) followed one day later with 2× 106 CFU CRS -207 or ANZ -100 ( the empty LADD platform strain , also named Lm11) and were monitored for survival over 90 days. The combination of low -dose Cy and CRS -207 increased the rate of survival to 100% compared to CRS -207 alone (60% survival) or Cy alone (0%, mOS = 33.5 days) (Figure 1 ). The combination of Cy and LADD- based vaccines has been shown to improve anti -tumor efficacy compared to LADD -vaccine alone in several models. Aduro Biotech , Inc. Protocol ADU -CL-02-v8 CRS -207 with Pemetrexed and Cisplatin Version 8, 08 March 2018 Page 8 Figure 1 . Cyclophosphamide Followed by CRS -207 Improves Vaccine Potency. Female Balb /c mice (n=10) were implanted i.v. with 2×105 CT26 tumor cells expressing mesothelin. Two days later, mice were administered 50 or 100 mg/kg Cy. One da y later, mice were vaccinated i.v. with 2×106 CFU CRS -207 or Lm11 (empty LADD platform). Mice that received only saline (HBSS) were included as controls. Animals were monitored for survival. mOS = median overall survival. 100~----~--------- 90 80 70 60 50 40 30 20 10 0-+--- .... -------,.-- --"T"""----r------. 0 20 40 60 80 100 Days post tumor challenge mOS (dalr'.s) ...,_HBSS 1S.S -+- Cy (100) 33.S -+- Lmll 17 -+- Cy (100) + Lmll 30 ...,_ CRS-207 undefined -+- Cy (SO) + CRS-207 undefined -+- Cy (100) + CRS-207 undefined Aduro Biotech , Inc. Protocol ADU -CL-02-v8 CRS -207 with Pemetrexed and Cisplatin Version 8, 08 March 2018 Page 9 2.0 OBJECTIVES 2.1 PRIMARY OBJECTIVE To determine the safety of CRS -207 (with or without Cy) when administered in combination with pemetrexed and cisplatin and to evaluate the induction of immune response to mesothelin as measured by IFN-γ enzyme -linked immunosorbent spot (ELISPOT ) assay prio r to treatment and at time points during and after treatment. 2.2 SECONDARY OBJECTIVE S To evaluate objective tumor response, progression free survival, time to progression, overall survival and the predictive value of serum mesothelin for therapeutic response for each treatment regimen . 2.3 EXPLORATORY OBJECTIVES i) to conduct immune subset analysis (e.g. CD4, CD8, T reg) and gene expression profiling of tumor tissue pre - and post -vaccination ; ii) to assess induction of anti - mesothelin humoral immune response; iii) to measure tumor marker kinetics as biomarkers of tumor response ; iv) evaluate the association between improvement in pulmonary function and tumor response; v) evaluate the relationship of tumor response to overall survival. Aduro Biotech , Inc. Protocol ADU -CL-02-v8 CRS -207 with Pemetrexed and Cisplatin Version 8, 08 March 2018 Page 10 3.0 STUDY DESIGN 3.1 BASIC DESIGN CHARACTERISTICS This is a phase 1B study to evaluate the safety and induction of immune response of CRS -207 with or without low-dose Cy in combination with pemetrexed and cisplatin in front- line therapy in subjects with MPM. Up to 16 subjects were originally planned to be enrolled in this study.