untitled h1
11.1.2 toxicity

The safety of immunizations (injections) with the ETBX-011, ETBX-051, ETBX-061 vaccines has not been established and will be determined in this phase I clinical trial. A Phase I/II clinical trial of ETBX-011 (Ad5 [E1-, E2b-]-CEA(6D)) (IND\#14325) that expresses the tumor-associated antigen carcinoembryonic antigen (CEA) has been performed. A summary of the study results on the clinical trial is presented below.  
Schedule, Dosing, and Safety of ETBX-011: The primary objective of the Phase I/II dosing trial was to assess safety and a secondary objective was to evaluate CEA-specific immune responses to CEA and to obtain preliminary data on response rate. The study was performed under an FDAapproved IND (IND14325) and registered at ClinicalTrials.gov (NCT01147965). Participants were recruited from oncology clinics at Duke University Medical Center and Medical Oncology Associates, Spokane, WA and provided informed consent that was approved by IRB's. Patients with a histological confirmed diagnosis of metastatic malignancy who were previously treated with standard therapy known to have a possible survival benefit were enrolled into the study. For this study, the carcinoma must have had over expression of CEA as defined by immunohistochemical staining (at least 50% of the tumor with at least moderate intensity of staining) or a carcinoma known to be universally CEA positive *i.e.* colorectal adenocarcinoma). Patients were not treated until 4 or more weeks after any prior chemotherapy or radiation therapy. They could not have a history of autoimmune disease, serious intercurrent chronic or acute illness, active hepatitis, serologic evidence for HIV infection, or be receiving steroid or immunosuppressive therapy. All patients were >21 years old and had a Karnofsky Performance Score of 70% or higher and a life expectancy of at least 3 months. Pregnant women and nursing mothers were excluded. Dose Limiting Toxicities (DLTs) were defined as any Grade 3 or 4 immediate hypersensitivity reactions, Grade 3 or 4 fever that may possibly be associated with the immunization, Grade ≥2 autoimmune events except for vitiligo or fever for less than 2 days and less than 101.5 ºF, Grade ≥2 allergic reactions (Grade 2 is defined as generalized urticaria as defined by NCI Common Terminology Criteria for Adverse Events (CTCAE version 5.0), or Grade ≥3 non-hematologic toxicity. The Ad5 [E1-, E2b-]-CEA(6D) injections were given subcutaneously in the same thigh. The doses were administered every 3 weeks for 3 treatments as follows: Cohort 1 (3 patients) 109 VP in 0.5 ml; Cohort 2 (3 patients): 1010 VP; Cohort 3 (6 patients) 1011 VP. Following establishment of safety in Phase I, 12 additional patients were entered into a Phase II using the 1011 VP/dose. To evaluate a higher dose, 6 additional patients (cohort 5) received 5x1011 VP/dose.  
Patient Demographics: One patient with CEA expressing bladder and one patient with lung carcinoma was enrolled. Thirty-two patients, median age 57.5 (range 38-77) with metastatic colorectal cancer, who had failed a median of three prior chemotherapeutic regimens (range: 2–5), had a median performance status of 90% (range 70-100%), and had a median of three sites of metastatic disease (range 1-5), were enrolled. The majority was able to receive all three immunizations. Five patients who stopped immunizations early did so due to significant disease progression. The colorectal adenocarcinoma patient demographics compares favorably with previously published studies of patients with chemotherapy-refractory colorectal cancer [44-46].  
Adverse Effects: A total of 94 immunization treatments were administered to all patients. There was no dose limiting toxicity and no serious adverse events that resulted in treatment discontinuation at any dose level. The most common toxicity was a self-limited, injection site reaction. Other reactions that occurred at a low frequency include fever, flu-like symptoms, anorexia, chills, nausea, and headache. These symptoms were also self-limiting and did not require intervention other than symptomatic measures such as acetaminophen. There were no SAE associated with immunizations. A summary of the adverse events reported on 34 patients treated and evaluated for safety are presented below.  
| Adverse Events: ≥ 5% Frequency Based on Incidence of Total Treatments Incidence %  (based on 94  total  treatments)   |        |                    |          |                     |      |
|-----------------------------------------------------------------------------------------------------------------------|--------|--------------------|----------|---------------------|------|
| Injection Site  Reaction                                                                                              | 21     | 2                  | 21       | 22.3                |      |
| Flu-like symptoms                                                                                                     | 10     | 4                  | 5        | 3                   | 10.6 |
| Fever                                                                                                                 | 9      | 6                  | 1        | 4                   | 9.5  |
| Fatigue                                                                                                               | 8      | 6                  | 2        | 8.5                 |      |
| Shortness of Breath                                                                                                   | 6      | 6                  | 6.3      |                     |      |
| Pain                                                                                                                  | 6      | 4                  | 6.3      |                     |      |
| Anorexia                                                                                                              | 5      | 1                  | 5.3      |                     |      |
| Chills                                                                                                                | 5      | 5                  | 1        | 4                   | 5.3  |
| Constipation                                                                                                          | 5      | 5                  | 5.3      |                     |      |
| Edema                                                                                                                 | 5      | 4                  | 5.3      |                     |      |
| Nausea                                                                                                                | 5      | 4                  | 1        | 5.3                 |      |
| Adverse Events as                                                                                                     | # of   | Unrelated/Unlikely | Possible | Probably/  Definite |      |
| of 07/31/12                                                                                                           | Events |                    |          |                     |      |  
| Grade 3 and Grade 4 Adverse Events in ≥ 2% of Patients* Percent (Based on  34 patients evaluated  for safety)   |                         |                         |
|-----------------------------------------------------------------------------------------------------------------|-------------------------|-------------------------|
| Pain (all types)                                                                                                | 2                       | 5.6                     |
| Fatigue                                                                                                         | 1                       | 2.9                     |
| Anemia                                                                                                          | 1                       | 2.9                     |
| Pleural effusion                                                                                                | 1                       | 2.9                     |
| Alkaline Phosphatase                                                                                            | 2.9                     |                         |
| Increase                                                                                                        | 1                       |                         |
| Adverse Events                                                                                                  | Number of Grade 3  (G3) | Number of Grade 4  (G4) |
| Abdominal Bloating                                                                                              | 1                       | 2.9                     |
| Bowel obstruction                                                                                               | 1                       | 2.9                     |
| GI disorder                                                                                                     | 1                       | 2.9                     |
| Acute renal failure                                                                                             | 1                       | 2.9                     |
| *Represents reported adverse events whether or not related to ETBX-011 vaccine                                  |                         |                         |  
Of 34 total patients entered into the trial, 28 received all three treatments and the blood hematology, chemistry, and ANA values at week 0 (prior to first treatment) were compared with those obtained at week 9 (three weeks after the third treatment). There were no biologically significant changes in chemistry, hematology, or ANA values.
untitled h1
11.1.3 formulation and preparation

Each vaccine is supplied as a clear colorless liquid filled in a 2-mL amber vial at a concentration of 5x1011 total virus particles (VP) per 1.0 mL. Each vial is sealed with a rubber stopper and has a white flip off seal. End user of the product will need to flip the white plastic portion of the cap up/off with their thumb to expose the rubber stopper, and then puncture the stopper with an injection needle to withdraw the liquid. The rubber stopper is secured to the vial with an aluminum crimped seal.
untitled h1
11.1.4 stability and storage

Individual vials (in the desired number) of vaccine will be packaged in a cardboard box and will be shipped over dry ice by overnight courier with a temperature monitoring device included. Upon receipt, one will inspect contents of package for any noticeable damages or defects. Unpack the shipment contents and place the cardboard box containing vaccine vials into a freezer with a monitored temperature control. ETBX-011 vials should be stored at -20 ± 10℃; ETBX-051 and ETBX-061 vials should be stored at ≤ -60℃. Receiver must stop the temperature monitoring device by turning off the power switch (instructions for handling and operation of temperature monitoring device will be provided with the package).
untitled h1
11.1.5 etbx vaccine dose preparation

The dose of ETBX vaccine to be injected is 5 x 1011 VP per 1 mL. A dose level of 1 x 1011 VP per mL (dose level -1)is available for de-escalation. Prior to injection, the appropriate vial should be removed from the freezer and allowed to thaw at controlled room temperature (20–25°C, 68-77°F) for at least 20 minutes and not more than 30 minutes, after which it should be kept at 2-8°C (35– 46°F). The vaccine is stable in the vial for at least 8 hours after removal from the freezer when kept refrigerated at 2-8℃ (35-46℉). Once the vaccine has been thawed, it must not be refrozen. Each vial is sealed with a rubber stopper and has a white flip-off seal. The end user of the product will need to flip the white plastic portion of the cap up/off with their thumb to expose the rubber stopper and then puncture the stopper with an injection needle to withdraw the liquid. The rubber stopper is secured to the vial with an aluminum-crimped seal. The thawed vial should be swirled and then, using aseptic technique, the pharmacist or pharmacist designee should withdraw the appropriate volume from the appropriate vial using an appropriately sized syringe. The vaccine dose should be injected as soon as possible (i.e. within 1 hour) using a 1 to 1/2 inch, 20 to 25-gauge needle. If the vaccine cannot be injected within 1 hour, the syringe should be returned to the pharmacy and properly disposed in accordance with institutional policy and procedure, and disposition must be recorded on the investigational product accountability record.  
11.1.5.1 Instructions for Dose Preparation - 5 x 1011 Virus Particles Withdraw 1 mL of contents from the vial, prepare the injection site with alcohol, and administer to the subject by SC injection without any further manipulation (refer to the vial label for a description of the vialed ETBX vaccine concentration).  
11.1.5.2 Instructions for Dose Preparation - 1 x 1011 Virus Particles Withdraw 0.2 mL of contents from the vial, prepare the injection site with alcohol, and administer to the subject by SC injection in the thigh without any further manipulation (refer to the vial label for a description of the vialed ETBX vaccine concentration). 11.1.5.3 Administration Each of the three ETBX vaccines will be administered on Weeks 0, 3, and 6 followed by bimonthly booster vaccines for up to a year. All study drug administration treatments should occur within ± 7 days of the planned visit date. ETBX-011 will be administered by subcutaneous injection in the upper arm after preparation of the site with alcohol. A 1 to ½ inch, 20- to 25- gauge needle should be used for administration. ETBX-051 will be administered by subcutaneous injection in the anterolateral upper thigh after preparation of the site with alcohol. A 1 to ½ inch, 20- to 25- gauge needle should be used for administration. It is preferred to administer ETBX-051 and ETBX-061 in separate thighs. However, if administered in the same thigh, injection sites should be separated by at least 5 cm. ETBX-061 will be administered by subcutaneous injection in the anterolateral upper thigh after preparation of the site with alcohol. A 1 to ½ inch, 20- to 25- gauge needle should be used for administration. It is preferred to administer ETBX-051 and ETBX-061 in separate thighs. However, if administered in the same thigh, injections sites should be separated by at least 5 cm.
untitled h1
11.1.6 incompatibilities

The vaccines are administered as separate subcutaneous injections, with no known incompatibilities.
untitled h1
11.1.7 other considerations

The Ad5 [E1-, E2b-] vector is non-replicating and its genome does not integrate into the human genome. However, since this is a non-replicating recombinant virus, it is recommended that it be handled under Biosafety Level-2 conditions. Any vialed ETBX vaccine material that has been used in the study should be autoclaved or incinerated after use according to institutional policy and according to local, state and federal regulations. Refer to the Material Safety Data Sheets for additional handling instructions.
untitled h1
12 references

1. Balint JP, Gabitzsch ES, Rice A, Latchman Y, Xu Y, Messerschmidt GL, et al. Extended evaluation of a phase 1/2 trial on dosing, safety, immunogenicity, and overall survival after immunizations with an advanced-generation Ad5 [E1-, E2b-]-CEA(6D) vaccine in late-stage colorectal cancer. Cancer Immunol Immunother. 2015;64(8):977-87. 2. Morse MA, Chaudhry A, Gabitzsch ES, Hobeika AC, Osada T, Clay TM, et al. Novel adenoviral vector induces T-cell responses despite anti-adenoviral neutralizing antibodies in colorectal cancer patients. Cancer Immunol Immunother. 2013;62(8):1293-301. 3. Horan Hand P, Colcher D, Salomon D, Ridge J, Noguchi P, Schlom J. Influence of spatial configuration of carcinoma cell populations on the expression of a tumor-associated glycoprotein. Cancer Res. 1985;45(2):833-40. 4. Szpak CA, Johnston WW, Lottich SC, Kufe D, Thor A, Schlom J. Patterns of reactivity of four novel monoclonal antibodies (B72.3, DF3, B1.1 and B6.2) with cells in human malignant and benign effusions. Acta Cytol. 1984;28(4):356-67. 5. Greiner JW, Hand PH, Noguchi P, Fisher PB, Pestka S, Schlom J. Enhanced expression of surface tumor-associated antigens on human breast and colon tumor cells after recombinant human leukocyte alpha-interferon treatment. Cancer Res. 1984;44(8):3208-14. 6. Hand PH, Nuti M, Colcher D, Schlom J. Definition of antigenic heterogeneity and modulation among human mammary carcinoma cell populations using monoclonal antibodies to tumor-associated antigens. Cancer Res. 1983;43(2):728-35. 7. Lottich SC, Szpak CA, Johnston WW, Thor A, Schlom J. Phenotypic heterogeneity of a tumor-associated antigen in adenocarcinomas of the colon and their metastases as demonstrated by monoclonal antibody B72.3. Cancer Invest. 1986;4(5):387-95. 8. Gabitzsch ES, Tsang KY, Palena C, David JM, Fantini M, Kwilas A, et al. The generation and analyses of a novel combination of recombinant adenovirus vaccines targeting three tumor antigens as an immunotherapeutic. Oncotarget. 2015;6(31):31344-59. 9. Wilkinson DG, Bhatt S, Herrmann BG. Expression pattern of the mouse T gene and its role in mesoderm formation. Nature. 1990;343(6259):657-9. 10. Hamilton DH, Litzinger MT, Fernando RI, Huang B, Palena C. Cancer vaccines targeting the epithelial-mesenchymal transition: tissue distribution of brachyury and other drivers of the mesenchymal-like phenotype of carcinomas. Semin Oncol. 2012;39(3):358-66.  
11. Hamilton DH, Fernando RI, Schlom J, Palena C. Aberrant expression of the embryonic transcription factor brachyury in human tumors detected with a novel rabbit monoclonal antibody. Oncotarget. 2015;6(7):4853-62. 12. Roselli M, Fernando RI, Guadagni F, Spila A, Alessandroni J, Palmirotta R, et al. Brachyury, a driver of the epithelial-mesenchymal transition, is overexpressed in human lung tumors: an opportunity for novel interventions against lung cancer. Clin Cancer Res. 2012;18(14):3868-79. 13. Miettinen M, Wang Z, Lasota J, Heery C, Schlom J, Palena C. Nuclear Brachyury Expression Is Consistent in Chordoma, Common in Germ Cell Tumors and Small Cell Carcinomas, and Rare in Other Carcinomas and Sarcomas: An Immunohistochemical Study of 5229 Cases. Am J Surg Pathol. 2015;39(10):1305-12. 14. Kilic N, Feldhaus S, Kilic E, Tennstedt P, Wicklein D, Wasielewski R, et al. Brachyury expression predicts poor prognosis at early stages of colorectal cancer. European journal of cancer (Oxford, England : 1990). 2011;47(7):1080-5. 15. Du R, Wu S, Lv X, Fang H, Wu S, Kang J. Overexpression of brachyury contributes to tumor metastasis by inducing epithelial-mesenchymal transition in hepatocellular carcinoma. Journal of experimental & clinical cancer research : CR. 2014;33:105.  
16. Pinto F, Pertega-Gomes N, Pereira MS, Vizcaino JR, Monteiro P, Henrique RM, et al. Tbox transcription factor brachyury is associated with prostate cancer progression and aggressiveness. Clin Cancer Res. 2014;20(18):4949-61. 17. Palena C, Roselli M, Litzinger MT, Ferroni P, Costarelli L, Spila A, et al. Overexpression of the EMT driver brachyury in breast carcinomas: association with poor prognosis. J Natl Cancer Inst. 2014;106(5). 18. Hamilton DH, Roselli M, Ferroni P, Costarelli L, Cavaliere F, Taffuri M, et al. Brachyury, a vaccine target, is overexpressed in triple-negative breast cancer. Endocr Relat Cancer. 2016;23(10):783-96. 19. Tirabosco R, Mangham DC, Rosenberg AE, Vujovic S, Bousdras K, Pizzolitto S, et al. Brachyury expression in extra-axial skeletal and soft tissue chordomas: a marker that distinguishes chordoma from mixed tumor/myoepithelioma/parachordoma in soft tissue. Am J Surg Pathol. 2008;32(4):572-80. 20. Vujovic S, Henderson S, Presneau N, Odell E, Jacques TS, Tirabosco R, et al. Brachyury, a crucial regulator of notochordal development, is a novel biomarker for chordomas. The Journal of pathology. 2006;209(2):157-65. 21. Fernando RI, Litzinger M, Trono P, Hamilton DH, Schlom J, Palena C. The T-box transcription factor Brachyury promotes epithelial-mesenchymal transition in human tumor cells. J Clin Invest. 2010;120(2):533-44. 22. Savagner P. Epithelial-mesenchymal transitions: from cell plasticity to concept elasticity. Curr Top Dev Biol. 2015;112:273-300. 23. Nieto MA, Cano A. The epithelial-mesenchymal transition under control: global programs to regulate epithelial plasticity. Seminars in cancer biology. 2012;22(5-6):361-8. 24. Huang B, Cohen JR, Fernando RI, Hamilton DH, Litzinger MT, Hodge JW, et al. The embryonic transcription factor Brachyury blocks cell cycle progression and mediates tumor resistance to conventional antitumor therapies. Cell Death Dis. 2013;4:e682. 25. Larocca C, Cohen JR, Fernando RI, Huang B, Hamilton DH, Palena C. An autocrine loop between TGF-beta1 and the transcription factor brachyury controls the transition of human carcinoma cells into a mesenchymal phenotype. Mol Cancer Ther. 2013;12(9):1805-15. 26. Hamilton DH, Huang B, Fernando RI, Tsang KY, Palena C. WEE1 inhibition alleviates resistance to immune attack of tumor cells undergoing epithelial-mesenchymal transition. Cancer Res. 2014;74(9):2510-9. 27. David JM, Hamilton DH, Palena C. MUC1 upregulation promotes immune resistance in tumor cells undergoing brachyury-mediated epithelial-mesenchymal transition. Oncoimmunology. 2016;5(4):e1117738. 28. Haro A, Yano T, Kohno M, Yoshida T, Koga T, Okamoto T, et al. Expression of Brachyury gene is a significant prognostic factor for primary lung carcinoma. Annals of surgical oncology. 2013;20 Suppl 3:S509-16. 29. Pinto F, Campanella NC, Abrahao-Machado LF, Scapulatempo-Neto C, de Oliveira AT, Brito MJ, et al. The embryonic Brachyury transcription factor is a novel biomarker of GIST aggressiveness and poor survival. Gastric Cancer. 2016;19(2):651-9. 30. Palena C, Polev DE, Tsang KY, Fernando RI, Litzinger M, Krukovskaya LL, et al. The human T-box mesodermal transcription factor Brachyury is a candidate target for T-cellmediated cancer immunotherapy. Clin Cancer Res. 2007;13(8):2471-8.  
31. Tucker JA, Jochems C, Boyerinas B, Fallon J, Greiner JW, Palena C, et al. Identification and characterization of a cytotoxic T-lymphocyte agonist epitope of brachyury, a transcription factor involved in epithelial to mesenchymal transition and metastasis. Cancer Immunol Immunother. 2014;63(12):1307-17. 32. Madan RA, Mohebtash M, Arlen PM, Vergati M, Rauckhorst M, Steinberg SM, et al.  
Ipilimumab and a poxviral vaccine targeting prostate-specific antigen in metastatic castrationresistant prostate cancer: a phase 1 dose-escalation trial. Lancet Oncol. 2012;13(5):501-8.  
33. NCT01519817. An Open Label Phase I Study to Evaluate the Safety and Tolerability of GI-6301 Vaccine Consisting of Whole, Heat-Killed Recombinant Saccharomyces Cerevisiae (Yeast) Genetically Modified to Express Brachyury Protein in Adults With Solid Tumors. 34. NCT02179515. An Open Label Phase I Study to Evaluate the Safety and Tolerability of a Modified Vaccinia Ankara (MVA) Based Vaccine Modified to Express Brachyury and T-Cell Costimulatory Molecules (MVA Brachyury-TRICOM). 35. Heery CR, Singh BH, Rauckhorst M, Marté JL, Donahue RN, Grenga I, et al. Phase I Trial of a Yeast-Based Therapeutic Cancer Vaccine (GI-6301) Targeting the Transcription Factor Brachyury. Cancer Immunol Res. 2015;3(11):1248-56. 36. Cheever MA, Allison JP, Ferris AS, Finn OJ, Hastings BM, Hecht TT, et al. The prioritization of cancer antigens: a national cancer institute pilot project for the acceleration of translational research. Clin Cancer Res. 2009;15(17):5323-37. 37. Zaremba S, Barzaga E, Zhu M, Soares N, Tsang KY, Schlom J. Identification of an enhancer agonist cytotoxic T lymphocyte peptide from human carcinoembryonic antigen. Cancer Res. 1997;57(20):4570-7. 38. Yin L, Ahmad R, Kosugi M, Kufe T, Vasir B, Avigan D, et al. Survival of human multiple myeloma cells is dependent on MUC1 C-terminal transmembrane subunit oncoprotein function. Mol Pharmacol. 2010;78(2):166-74. 39. Yin L, Kufe D. MUC1-C Oncoprotein Blocks Terminal Differentiation of Chronic Myelogenous Leukemia Cells by a ROS-Mediated Mechanism. Genes Cancer. 2011;2(1):56-64. 40. Hollingsworth MA, Swanson BJ. Mucins in cancer: protection and control of the cell surface. Nat Rev Cancer. 2004;4(1):45-60. 41. Kawano T, Ito M, Raina D, Wu Z, Rosenblatt J, Avigan D, et al. MUC1 oncoprotein regulates Bcr-Abl stability and pathogenesis in chronic myelogenous leukemia cells. Cancer Res. 2007;67(24):11576-84. 42. Gendler SJ, Lancaster CA, Taylor-Papadimitriou J, Duhig T, Peat N, Burchell J, et al. Molecular cloning and expression of human tumor-associated polymorphic epithelial mucin. J Biol Chem. 1990;265(25):15286-93. 43. Kufe D, Inghirami G, Abe M, Hayes D, Justi-Wheeler H, Schlom J. Differential reactivity of a novel monoclonal antibody (DF3) with human malignant versus benign breast tumors. Hybridoma. 1984;3(3):223-32. 44. Gulley JL, Arlen PM, Tsang KY, Yokokawa J, Palena C, Poole DJ, et al. Pilot study of vaccination with recombinant CEA-MUC-1-TRICOM poxviral-based vaccines in patients with metastatic carcinoma. Clin Cancer Res. 2008;14(10):3060-9. 45. Limacher JM, Quoix E. TG4010: A therapeutic vaccine against MUC1 expressing tumors. Oncoimmunology. 2012;1(5):791-2. 46. Mohebtash M, Tsang KY, Madan RA, Huen NY, Poole DJ, Jochems C, et al. A pilot study of MUC-1/CEA/TRICOM poxviral-based vaccine in patients with metastatic breast and ovarian cancer. Clin Cancer Res. 2011;17(22):7164-73.  
47. Kufe DW. Functional targeting of the MUC1 oncogene in human cancers. Cancer Biol Ther. 2009;8(13):1197-203. 48. Tsang KY, Palena C, Gulley J, Arlen P, Schlom J. A human cytotoxic T-lymphocyte epitope and its agonist epitope from the nonvariable number of tandem repeat sequence of MUC1. Clin Cancer Res. 2004;10(6):2139-49.  
49. Jochems C, Tucker JA, Vergati M, Boyerinas B, Gulley JL, Schlom J, et al. Identification and characterization of agonist epitopes of the MUC1-C oncoprotein. Cancer Immunol Immunother. 2014;63(2):161-74. 50. Imler JL. Adenovirus vectors as recombinant viral vaccines. Vaccine. 1995;13(13):114351. 51. Tatsis N, Ertl HC. Adenoviruses as vaccine vectors. Mol Ther. 2004;10(4):616-29. 52. Campos SK, Barry MA. Current advances and future challenges in Adenoviral vector biology and targeting. Curr Gene Ther. 2007;7(3):189-204. 53. Seregin SS, Amalfitano A. Overcoming pre-existing adenovirus immunity by genetic engineering of adenovirus-based vectors. Expert Opin Biol Ther. 2009;9(12):1521-31. 54. Chirmule N, Propert K, Magosin S, Qian Y, Qian R, Wilson J. Immune responses to adenovirus and adeno-associated virus in humans. Gene Ther. 1999;6(9):1574-83. 55. Barouch DH, Kik SV, Weverling GJ, Dilan R, King SL, Maxfield LF, et al. International seroepidemiology of adenovirus serotypes 5, 26, 35, and 48 in pediatric and adult populations. Vaccine. 2011;29(32):5203-9. 56. Amalfitano A, Hauser MA, Hu H, Serra D, Begy CR, Chamberlain JS. Production and characterization of improved adenovirus vectors with the E1, E2b, and E3 genes deleted. J Virol. 1998;72(2):926-33. 57. Amalfitano A, Chamberlain JS. Isolation and characterization of packaging cell lines that coexpress the adenovirus E1, DNA polymerase, and preterminal proteins: implications for gene therapy. Gene Ther. 1997;4(3):258-63. 58. Hartigan-O'Connor D, Amalfitano A, Chamberlain JS. Improved production of gutted adenovirus in cells expressing adenovirus preterminal protein and DNA polymerase. J Virol. 1999;73(9):7835-41. 59. Gabitzsch ES, Jones FR. New recombinant Ad5 vector overcomes Ad5 immunity allowing for multiple safe, homologous immunizations. J Clin Cell Immunol. 2011;S4:001. 60. Osada T, Yang XY, Hartman ZC, Glass O, Hodges BL, Niedzwiecki D, et al.  
Optimization of vaccine responses with an E1, E2b and E3-deleted Ad5 vector circumvents preexisting anti-vector immunity. Cancer Gene Ther. 2009;16(9):673-82.  
61. Gabitzsch ES, Xu Y, Balint Jr JP, Hartman ZC, Lyerly HK, Jones FR. Anti-tumor immunotherapy despite immunity to adenovirus using a novel adenoviral vector Ad5 [E1-, E2b- ]-CEA. Cancer Immunol Immunother. 2010;59(7):1131-5. 62. Gabitzsch ES, Xu Y, Yoshida LH, Balint J, Gayle RB, Amalfitano A, et al. A preliminary and comparative evaluation of a novel Ad5 [E1-, E2b-] recombinant-based vaccine used to induce cell mediated immune responses. Immunol Lett. 2009;122(1):44-51. 63. Gabitzsch ES, Xu Y, Yoshida LH, Balint J, Amalfitano A, Jones FR. Novel Adenovirus type 5 vaccine platform induces cellular immunity against HIV-1 Gag, Pol, Nef despite the presence of Ad5 immunity. Vaccine. 2009;27(46):6394-8. 64. Gabitzsch ES, Balint-Junior JP, Xu Y, Balcaitis S, Sanders-Beer B, Karl J, et al. Control of SIV infection and subsequent induction of pandemic H1N1 immunity in rhesus macaques using an Ad5 [E1-, E2b-] vector platform. Vaccine. 2012;30(50):7265-70.  
65. Jones FR, Gabitzsch ES, Xu Y, Balint JP, Borisevich V, Smith J, et al. Prevention of influenza virus shedding and protection from lethal H1N1 challenge using a consensus 2009 H1N1 HA and NA adenovirus vector vaccine. Vaccine. 2011;29(40):7020-6.  
66. Wieking BG, Vermeer DW, Spanos WC, Lee KM, Vermeer P, Lee WT, et al. A nononcogenic HPV 16 E6/E7 vaccine enhances treatment of HPV expressing tumors. Cancer Gene Ther. 2012;19(10):667-74. 67. Rice AE, Latchman YE, Balint JP, Lee JH, Gabitzsch ES, Jones FR. An HPV-E6/E7 immunotherapy plus PD-1 checkpoint inhibition results in tumor regression and reduction in PDL1 expression. Cancer Gene Ther. 2015;22(9):454-62.  
68. Gabitzsch ES, Xu Y, Balint Jr JP, Balcaitis S, Sanders-Beer B, Jones FR. Induction and comparison of SIV immunity in Ad5 naïve and Ad5 immune non-human primates using an Ad5 [E1-, E2b-] based vaccine. Vaccine. 2011;29(45):8101-7. 69. Abe M, Kufe DW. Identification of a family of high molecular weight tumor-associated glycoproteins. J Immunol. 1987;139(1):257-61. 70. Poland PA, Kinlough CL, Rokaw MD, Magarian-Blander J, Finn OJ, Hughey RP. Differential glycosylation of MUC1 in tumors and transfected epithelial and lymphoblastoid cell lines. Glycoconj J. 1997;14(1):89-96. 71. Friedman EL, Hayes DF, Kufe DW. Reactivity of monoclonal antibody DF3 with a high molecular weight antigen expressed in human ovarian carcinomas. Cancer Res. 1986;46(10):5189-94. 72. Ramasamy S, Duraisamy S, Barbashov S, Kawano T, Kharbanda S, Kufe D. The MUC1 and galectin-3 oncoproteins function in a microRNA-dependent regulatory loop. Mol Cell. 2007;27(6):992-1004. 73. Salazar E, Zaremba S, Arlen PM, Tsang KY, Schlom J. Agonist peptide from a cytotoxic t-lymphocyte epitope of human carcinoembryonic antigen stimulates production of tc1-type cytokines and increases tyrosine phosphorylation more efficiently than cognate peptide. Int J Cancer. 2000;85(6):829-38. 74. Lepone LM, Donahue RN, Grenga I, Metenou S, Richards J, Heery CR, et al. Analysis of 123 peripheral human immune cell subsets: defining differences with age and between healthy donors and cancer patients not detected in analysis of standard immune cell types. J Circ Biomark. In press. 75. Scher HI, Halabi S, Tannock I, Morris M, Sternberg CN, Carducci MA, et al. Design and end points of clinical trials for patients with progressive prostate cancer and castrate levels of testosterone: recommendations of the Prostate Cancer Clinical Trials Working Group. J Clin Oncol. 2008;26(7):1148-59. 76. Rustin GJ, Quinn M, Thigpen T, du Bois A, Pujade-Lauraine E, Jakobsen A, et al. Re: New guidelines to evaluate the response to treatment in solid tumors (ovarian cancer). J Natl Cancer Inst. 2004;96(6):487-8. 77. Bubley GJ, Carducci M, Dahut W, Dawson N, Daliani D, Eisenberger M, et al. Eligibility and response guidelines for phase II clinical trials in androgen-independent prostate cancer: recommendations from the Prostate-Specific Antigen Working Group. J Clin Oncol. 1999;17(11):3461-7. 78. Vergote I, Rustin GJ, Eisenhauer EA, Kristensen GB, Pujade-Lauraine E, Parmar MK, et al. Re: new guidelines to evaluate the response to treatment in solid tumors [ovarian cancer]. Gynecologic Cancer Intergroup. J Natl Cancer Inst. 2000;92(18):1534-5.  
79. Eisenhauer EA, Therasse P, Bogaerts J, Schwartz LH, Sargent D, Ford R, et al. New response evaluation criteria in solid tumours: revised RECIST guideline (version 1.1). European journal of cancer (Oxford, England : 1990). 2009;45(2):228-47.
untitled h1
13 appendices

| 13.1                                                                                                                                                                                       | APPENDIX A: PERFORMANCE STATUS CRITERIA ECOG Performance Status Scale   | Karnofsky Performance Scale                                |             |
|--------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|-------------------------------------------------------------------------|------------------------------------------------------------|-------------|
| Grade                                                                                                                                                                                      | Descriptions                                                            | Percent                                                    | Description |
| 100                                                                                                                                                                                        | Normal, no complaints, no  evidence of disease.                         |                                                            |             |
| Normal activity. Fully active,                                                                                                                                                             |                                                                         |                                                            |             |
| 0                                                                                                                                                                                          | able to carry on all pre-disease  performance without restriction.      | Able to carry on normal activity;                          |             |
| 90                                                                                                                                                                                         | minor signs or symptoms of  disease.                                    |                                                            |             |
| Symptoms, but ambulatory.  Restricted in physically strenuous  activity, but ambulatory and able  to carry out work of a light or  sedentary nature (e.g., light  housework, office work). | Normal activity with effort;                                            |                                                            |             |
| 80                                                                                                                                                                                         | some signs or symptoms of  disease.                                     |                                                            |             |
| 1                                                                                                                                                                                          | Cares for self, unable to carry on                                      |                                                            |             |
| 70                                                                                                                                                                                         | normal activity or to do active  work.                                  |                                                            |             |
| In bed <50% of the time.  Ambulatory and capable of all  self-care, but unable to carry out  any work activities. Up and  about more than 50% of waking  hours.                            | Requires occasional assistance,                                         |                                                            |             |
| 60                                                                                                                                                                                         | but is able to care for most of  his/her needs.                         |                                                            |             |
| 2                                                                                                                                                                                          | Requires considerable assistance                                        |                                                            |             |
| 50                                                                                                                                                                                         | and frequent medical care.                                              |                                                            |             |
| 40                                                                                                                                                                                         | Disabled, requires special care  and assistance.                        |                                                            |             |
| In bed >50% of the time.  Capable of only limited self-care,  confined to bed or chair more  than 50% of waking hours.                                                                     |                                                                         |                                                            |             |
| 3                                                                                                                                                                                          | Severely disabled,                                                      |                                                            |             |
| 30                                                                                                                                                                                         | hospitalization indicated. Death  not imminent.                         |                                                            |             |
| 100% bedridden. Completely  disabled. Cannot carry on any  self-care. Totally confined to bed  or chair.                                                                                   | 20                                                                      | Very sick, hospitalization  indicated. Death not imminent. |             |
| 4                                                                                                                                                                                          | 10                                                                      | Moribund, fatal processes  progressing rapidly.            |             |
| 5                                                                                                                                                                                          | Dead.                                                                   | 0                                                          | Dead.       |
untitled h1
13.2 appendix b injection site

| Day 0                                                            | Day +1            | Day +2        | Day +3        | Day +4        | Day +5        | Day +6        |               |          |
|------------------------------------------------------------------|-------------------|---------------|---------------|---------------|---------------|---------------|---------------|----------|
| (Day of Injection)                                               |                   |               |               |               |               |               |               |          |
| 1. Is there  redness at  the injection  site?                    | Circle: Yes  / No | Yes / No      | Yes / No      | Yes / No      | Yes / No      | Yes / No      | Yes / No      | Yes / No |
| If yes  measure  longest  diameter in  millimeters  (mm)         | ___________mm     | ___________mm | ___________mm | ___________mm | ___________mm | ___________mm | ___________mm |          |
| 2. Is there  firmness or  swelling at  the  injection  site?     | Circle: Yes  / No | Yes / No      | Yes / No      | Yes / No      | Yes / No      | Yes / No      | Yes / No      | Yes / No |
| If yes  measure  longest  diameter in  millimeters  (mm)         | ___________mm     | ___________mm | ___________mm | ___________mm | ___________mm | ___________mm | ___________mm |          |
| 3. Is there  soreness at  the  injection  site?                  | Circle: Yes  / No | Yes / No      | Yes / No      | Yes / No      | Yes / No      | Yes / No      | Yes / No      | Yes / No |
| Mild                                                             |                   |               |               |               |               |               |               |          |
| Moderate Severe                                                  | Mild              |               |               |               |               |               |               |          |
| Moderate Severe                                                  | Mild              |               |               |               |               |               |               |          |
| Moderate Severe                                                  | Mild              |               |               |               |               |               |               |          |
| Moderate Severe                                                  | Mild              |               |               |               |               |               |               |          |
| Moderate Severe                                                  | Mild              |               |               |               |               |               |               |          |
| Moderate Severe                                                  | Mild              |               |               |               |               |               |               |          |
| Moderate Severe                                                  |                   |               |               |               |               |               |               |          |
| If yes tell us  if the  soreness is  mild,  moderate,  or severe |                   |               |               |               |               |               |               |          |
| 4. Have you  experienced  pain at the  injection  site?          | Circle: Yes  / No | Yes / No      | Yes / No      | Yes / No      | Yes / No      | Yes / No      | Yes / No      | Yes / No |
| Mild                                                             |                   |               |               |               |               |               |               |          |
| Moderate Severe                                                  | Mild              |               |               |               |               |               |               |          |
| Moderate Severe                                                  | Mild              |               |               |               |               |               |               |          |
| Moderate Severe                                                  | Mild              |               |               |               |               |               |               |          |
| Moderate Severe                                                  | Mild              |               |               |               |               |               |               |          |
| Moderate Severe                                                  | Mild              |               |               |               |               |               |               |          |
| Moderate Severe                                                  | Mild              |               |               |               |               |               |               |          |
| Moderate Severe                                                  |                   |               |               |               |               |               |               |          |
| If yes tell us  if the pain  is mild,  moderate,  or severe      |                   |               |               |               |               |               |               |          |
| 5. Have you  taken any  medication  for                          | Circle: Yes  / No | Yes / No      | Yes / No      | Yes / No      | Yes / No      | Yes / No      | Yes / No      | Yes / No |
| Name:                                                            |                   |               |               |               |               |               |               |          |
| ___________                                                      | Name:             |               |               |               |               |               |               |          |
| ___________                                                      | Name:             |               |               |               |               |               |               |          |
| ___________                                                      | Name:             |               |               |               |               |               |               |          |
| ___________                                                      | Name:             |               |               |               |               |               |               |          |
| ___________                                                      | Name:             |               |               |               |               |               |               |          |
| ___________                                                      | Name:             |               |               |               |               |               |               |          |
| ___________                                                      |                   |               |               |               |               |               |               |          |
| Provide  name and                                                |                   |               |               |               |               |               |               |          |  
| Day 0                                                                                                                | Day +1             | Day +2             | Day +3             | Day +4             | Day +5             | Day +6             |                    |              |
|----------------------------------------------------------------------------------------------------------------------|--------------------|--------------------|--------------------|--------------------|--------------------|--------------------|--------------------|--------------|
| (Day of Injection)                                                                                                   |                    |                    |                    |                    |                    |                    |                    |              |
| injection                                                                                                            | dose of            | Dose:              | Dose:              | Dose:              | Dose:              | Dose:              | Dose:              | Dose:        |
| site pain?                                                                                                           | medication         | ____________       | ____________       | ____________       | ____________       | ____________       | ____________       | ____________ |
| 6. Have you  experienced  chills?                                                                                    | Circle: Yes  / No  | Yes / No           | Yes / No           | Yes / No           | Yes / No           | Yes / No           | Yes / No           | Yes / No     |
| Mild                                                                                                                 |                    |                    |                    |                    |                    |                    |                    |              |
| Moderate Severe                                                                                                      | Mild               |                    |                    |                    |                    |                    |                    |              |
| Moderate Severe                                                                                                      | Mild               |                    |                    |                    |                    |                    |                    |              |
| Moderate Severe                                                                                                      | Mild               |                    |                    |                    |                    |                    |                    |              |
| Moderate Severe                                                                                                      | Mild               |                    |                    |                    |                    |                    |                    |              |
| Moderate Severe                                                                                                      | Mild               |                    |                    |                    |                    |                    |                    |              |
| Moderate Severe                                                                                                      | Mild               |                    |                    |                    |                    |                    |                    |              |
| Moderate Severe                                                                                                      |                    |                    |                    |                    |                    |                    |                    |              |
| If yes tell us  if the chills  are mild,  moderate,  or severe                                                       |                    |                    |                    |                    |                    |                    |                    |              |
| 7. Record  your daily  temperatur e (Do not  drink  anything  within 5  minutes  prior to  taking your  temperature) | Circle: Yes  / No  | Record Temp:       | Record Temp:       | Record Temp:       | Record Temp:       | Record Temp:       | Record Temp:       | Record Temp: |
| If your  temperatur e is 104OF  for more  than 24  hours, call  your study  doctor                                   | ________________ _ | ________________ _ | ________________ _ | ________________ _ | ________________ _ | ________________ _ | ________________ _ |              |