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10.1.3 justification for exclusions

Due to impaired cellular immunity with the concomitant increased risk of serious side effects from vaccinations with infectious agents, the Centers for Disease Control and Prevention recommends that HIV infected patients be excluded, in addition, patients with chronic hepatitis infection, including B and C, because of potential immune impairment. 10.2 PARTICIPATION OF CHILDREN Because no dosing or adverse event data are currently available on the use of the study vaccines in patients <18 years of age, children are excluded from this study. 10.3 PARTICIPATION OF SUBJECTS UNABLE TO GIVE CONSENT Adults unable to give consent are excluded from enrolling in the protocol. However, re-consent may be necessary and there is a possibility, though unlikely, that subjects could become decisionally impaired. For this reason and because there is a prospect of direct benefit from research participation (section **10.4**), all subjects ≥ age 18 will be offered the opportunity to fill in their wishes for research and care, and assign a substitute decision maker on the "NIH Advance Directive for Health Care and Medical Research Participation" form so that another person can make decisions about their medical care in the event that they become incapacitated or cognitively impaired during the course of the study. Note: The PI or AI will contact the NIH Ability to Consent Assessment Team (ACAT) for evaluation as needed for the following: an independent assessment of whether an individual has the capacity to provide consent; assistance in identifying and assessing an appropriate surrogate when indicated; and/or an assessment of the capacity to appoint a surrogate. For those subjects that become incapacitated and do not have pre-determined substitute decision maker, the procedures described in MAS Policy 87-4 and NIH HRPP SOP 14E for appointing a surrogate decision maker for adult subjects who are (a) decisionally impaired, and (b) who do not have a legal guardian or durable power of attorney, will be followed. 10.4 EVALUATION OF BENEFITS AND RISKS/DISCOMFORTS
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adult subjects including those that

Patients will receive evaluation of their disease at the National Cancer Institute's Clinical Center. This protocol may or may not benefit an individual, but the results may help the investigators learn more about the disease and develop new treatments for patients with this disease. Potential adverse reactions attributable to the administration of the vaccine utilized in this trial are discussed in section 11. All care will be taken to minimize side effects, but they can be unpredictable in nature and severity. Patients will be examined and evaluated prior to enrollment. All evaluations to monitor the treatment of patients will be recorded in the patient chart. If patients suffer any physical injury as a result of the participation in this study, immediate medical treatment is available at the Clinical Center, National Cancer Institute, Bethesda, Maryland. Although no compensation is available, any injury will be evaluated and treated in keeping with the benefits or care to which patients are entitled under applicable regulations. In all publications and presentations resulting from this trial, patients' anonymity will be protected to the maximum extent possible. Authorized personnel from the National Cancer Institute (NCI) and Food and Drug Administration (FDA) or other regulatory authorities may have access to research files in order to verify that patients' rights have been safeguarded. In addition, patient names will be given to the Central Registration to register and verify patients' eligibility.
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10.5 consent process and documentation

The investigational nature and research objectives of this trial, the procedures and treatments involved and their attendant risks and discomforts and benefits, and potential alternative therapies will be carefully explained to the patient, and a signed informed consent document will be obtained by a study investigator prior to entry onto the study. The PI or associate investigator will meet with the patient to discuss the protocol treatment and alternative options in detail. It will be stated clearly that participation in the research study is voluntary and that participants can withdraw from the study without losing benefits they would otherwise be entitled to. The patient will be encouraged to ask questions, and additional meetings to discuss the treatment options will be arranged as necessary.
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10.5.1 telephone re-consent procedure

Re-consent on this study may be obtained via telephone according to the following procedure: the informed consent document will be sent to the subject. An explanation of the study will be provided over the telephone after the subject has had the opportunity to read the consent form. The subject will sign and date the informed consent. A witness to the subject's signature will sign and date the consent. The original informed consent document will be sent back to the consenting investigator who will sign and date the consent form with the date the consent was obtained via telephone. A fully executed copy will be returned via mail for the subject's records. The informed consent process will be documented on a progress note by the consenting investigator.
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10.5.2 consent process for non-english

If there is an unexpected enrollment of a research participant for whom there is no translated extant IRB approved consent document, the principal investigator and/or those authorized to obtain informed consent will use the Short Form Oral Consent Process as described in MAS Policy M772, OHSRP SOP 12, 45 CFR 46.117 (b) (2), and 21 CFR 50.27 (b) (2). The summary that will be used is the English version of the extant IRB approved consent document. Signed copies of both the English version of the consent and the translated short form will be given to the subject or their legally authorized representative and the signed original will be filed in the medical record. Unless the PI is fluent in the prospective subject's language, an interpreter will be present to facilitate the conversation. Preferably someone who is independent of the subject (i.e., not a family member) will assist in presenting information and obtaining consent. Whenever possible, interpreters will be provided copies of the relevant consent documents well before the consent conversation with the subject (24 to 48 hours if possible). We request prospective IRB approval of the use of the short form process and will notify the IRB at the time of continuing review of the frequency of the use of the Short Form.
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11 pharmaceutical information

11.1 COMBINATION ETBX-071, ETBX-061, AND ETBX-051 VACCINE REGIMEN (IND\# 17811) 11.1.1 Source
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table 4 investigational products

Table 4a  
| Product Name(s):        | ETBX-071 (Ad5 [E1-, E2b-]-PSA Vaccine)                                                                                                                                                                                                                                |
|-------------------------|-----------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|
| Dosage Form:            | Suspension for injection                                                                                                                                                                                                                                              |
| Unit Dose               | 5 x 1011 VP, 1 x 1011 VP, or 5 x 1010 VP.                                                                                                                                                                                                                             |
| Route of Administration | SC injection                                                                                                                                                                                                                                                          |
| Physical Description    | Each ETBX vaccine is supplied as a sterile, clear  solution in a 2-mL single-dose vial. The vaccine is  provided at a concentration of 5 x 1011 VP per 1 mL and  contains ARM formulation buffer (20 mM TRIS, 25  mM NaCl, 2.5% glycerol, pH 8.0). Each vial contains |  
| approximately 1.1 mL of the vaccine. The product  should be stored at ≤ -20°C.   |                                                                                                                                                                                                                                                                                                                                                       |
|----------------------------------------------------------------------------------|-------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|
| Manufacturer                                                                     | Etubics                                                                                                                                                                                                                                                                                                                                               |
| Table 4b  Product Name(s):                                                       | ETBX-061 (Ad5 [E1-, E2b-]-MUC1 Vaccine)                                                                                                                                                                                                                                                                                                               |
| Dosage Form:                                                                     | Suspension for injection                                                                                                                                                                                                                                                                                                                              |
| Unit Dose                                                                        | 5 x 1011 VP, 1 x 1011 VP, or 5 x 1010 VP.                                                                                                                                                                                                                                                                                                             |
| Route of Administration                                                          | SC injection                                                                                                                                                                                                                                                                                                                                          |
| Physical Description                                                             | Each ETBX vaccine is supplied as a sterile, clear  solution in a 2-mL single-dose vial. The vaccine is  provided at a concentration of 5 x 1011 VP per 1 mL and  contains ARM formulation buffer (20 mM TRIS, 25  mM NaCl, 2.5% glycerol, pH 8.0). Each vial contains  approximately 1.1 mL of the vaccine. The product  should be stored at ≤ -20°C. |
| Manufacturer                                                                     | Etubics                                                                                                                                                                                                                                                                                                                                               |
| Table 4c  Product Name(s):                                                       | ETBX-051 (Ad5 [E1-, E2b-]-brachyury Vaccine)                                                                                                                                                                                                                                                                                                          |
| Dosage Form:                                                                     | Suspension for injection                                                                                                                                                                                                                                                                                                                              |
| Unit Dose                                                                        | 5 x 1011 VP, 1 x 1011 VP, or 5 x 1010 VP.                                                                                                                                                                                                                                                                                                             |
| Route of Administration                                                          | SC injection                                                                                                                                                                                                                                                                                                                                          |
| Physical Description                                                             | Each ETBX vaccine is supplied as a sterile, clear  solution in a 2-mL single-dose vial. The vaccine is  provided at a concentration of 5 x 1011 VP per 1 mL and  contains ARM formulation buffer (20 mM TRIS, 25  mM NaCl, 2.5% glycerol, pH 8.0). Each vial contains  approximately 1.1 mL of the vaccine. The product  should be stored at ≤ -20°C. |
| Manufacturer                                                                     | Etubics                                                                                                                                                                                                                                                                                                                                               |  
The ETBX-071, ETBX-061, and ETBX-051 vaccines will be supplied by the manufacturer, Etubics Corporation, through a Cooperative Research and Development Agreement (CRADA). The manufacturing department of Etubics will supply the vaccine, which will be distributed to the sites by Etubics Corporation.
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11.1.2 toxicity

The safety of immunizations (injections) witha combination of three immunotherapeutic vaccines (ETBX-071, ETBX-061, and ETBX-051), 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), 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  |      |
| Events                                                                  | Unrelated/Unlikely | Possible | Probably/ |      |      |
| Adverse Events as                                                       | #                  | of       |           |      |      |
| of 07/31/12                                                             | Definite           |          |           |      |      |  
| 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             |                         |
| Increase                                                | 1                       | 2.9                     |
| Abdominal Bloating                                      | 1                       | 2.9                     |
| Bowel obstruction                                       | 1                       | 2.9                     |
| GI disorder                                             | 1                       | 2.9                     |
| Acute renal failure                                     | 1                       | 2.9                     |
| Adverse Events                                          | Number of Grade 3  (G3) | Number of Grade 4  (G4) |
| 73                                                      |                         |                         |  
*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.
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11.1.3 formulation and preparation

Each vaccine is a clear colorless liquid filled in a 2-mL amber vial containing 1.0 mL of extractable vaccine. There are total of 5x1011 total virus particles (VP) in 1.0 mL of the product. 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.
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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 (<-80 °C) 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 temperature control of d-20 °C. 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).
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11.1.5 administration procedures

Please see section **3.2.4**. Briefly, each ETBX-071, ETBX-061, and ETBX-051 vaccines will be administered on Weeks 0, 3, and 6 followed by booster vaccines every 8 weeks for up to a year in (only dose expansion cohor)t. All study drug administration treatments should occur within ± 7 days of the planned visit date. Each of the three vaccines should be given by SC injection into a proximal limb, preferably in the thigh, after preparation of the site with alcohol. Preferably all three injections on a given day should be given to the anterolateral section of the same thigh and preferably the thigh chosen for injections should alternate between treatment days. Less preferably it is possible to give three injections to different limbs on a given day or to use the same thigh for consecutive treatment days. When the 3 injections are given on a given day to the same limb (e.g. thigh) the injections must be separated by at least 5 cm and it is preferred that ETBX-071 is administerd most proximal, ETBX-061 in the middle, and ETBX-051 most distal.
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11.1.6 incompatibilities

The vaccines are administered as a separate subcutaneous injection, with no known incompatibilities.
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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.
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12 references

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42. 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. J Pathol. 2006;209(2):157-65. 43. 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. 44. Nieto MA, Cano A. The epithelial-mesenchymal transition under control: global programs to regulate epithelial plasticity. Semin Cancer Biol. 2012;22(5-6):361-8. 45. 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. 46. 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. 47. 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. 48. Hamilton DH, Griner LM, Keller JM, Hu X, Southall N, Marugan J, et al. Targeting Estrogen Receptor Signaling with Fulvestrant Enhances Immune and Chemotherapy-Mediated Cytotoxicity of Human Lung Cancer. Clin Cancer Res. 2016;22(24):6204-16. 49. 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. Ann Surg Oncol. 2013;20 Suppl 3:S509-16. 50. 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. 51. Roper-Hall MJ. Changes in ophthalmic nursing. Nurs Mirror Midwives J. 1968;127(2):23-5. 52. 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.  
53. 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. 54. 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). 55. 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.  
56. Imler JL. Adenovirus vectors as recombinant viral vaccines. Vaccine. 1995;13(13):114351. 57. Tatsis N, Ertl HC. Adenoviruses as vaccine vectors. Mol Ther. 2004;10(4):616-29. 58. Campos SK, Barry MA. Current advances and future challenges in Adenoviral vector biology and targeting. Curr Gene Ther. 2007;7(3):189-204. 59. 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.  
60. 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. 61. 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. 62. 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. 63. 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. 64. 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. 65. Gabitzsch ES, Jones FR. New recombinant Ad5 vector overcomes Ad5 immunity allowing for multiple safe, homologous immunizations. J Clin Cell Immunol. 2011;S4:001. 66. 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.  
67. 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. 68. 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. 69. 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. 70. 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.  
71. 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. 72. 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.  
73. 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. 74. Gabitzsch ES, Xu Y, Balcaitis S, Balint JP, Jr., Jones FR. An Ad5[E1-, E2b-]-HER2/neu vector induces immune responses and inhibits HER2/neu expressing tumor progression in Ad5 immune mice. Cancer Gene Ther. 2011;18(5):326-35. 75. Lepone LM DR, Grenga I, 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. 2016;5:5 I doi: 10.5772/62322.
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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, able                                                                                                                             |                                                                        |                                                                    |                 |           |             |
| 0                                                                                                                                                               | 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). | 80                                                                     | Normal activity with effort; some  signs or symptoms of disease.   |                 |           |             |
| 1                                                                                                                                                               | Cares for self, unable to carry on                                     |                                                                    |                 |           |             |
| 70                                                                                                                                                              | normal activity or to do active  work. Requires occasional assistance, |                                                                    |                 |           |             |
| 60                                                                                                                                                              | but is able to care for most of  his/her needs.                        |                                                                    |                 |           |             |
| 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.                            |                                                                        |                                                                    |                 |           |             |
| 2                                                                                                                                                               | 50                                                                     | Requires considerable assistance  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                                                                                                                                                               | 30                                                                     | Severely disabled, hospitalization  indicated. Death not imminent. |                 |           |             |
| 20                                                                                                                                                              | Very                                                                   | sick,                                                              | hospitalization |           |             |
| indicated. Death not imminent.                                                                                                                                  |                                                                        |                                                                    |                 |           |             |
| 100%                                                                                                                                                            | bedridden.                                                             | Completely                                                         |                 |           |             |
| disabled.                                                                                                                                                       | Cannot carry on any                                                    |                                                                    |                 |           |             |
| self-care. Totally confined to bed  or chair.                                                                                                                   |                                                                        |                                                                    |                 |           |             |
| 4                                                                                                                                                               | 10                                                                     | Moribund,                                                          | fatal           | processes |             |
| progressing rapidly.                                                                                                                                            |                                                                        |                                                                    |                 |           |             |
| 5                                                                                                                                                               | Dead.                                                                  | 0                                                                  | Dead.           |           |             |  
| Yes / No                                                                                                              | ___________mm                                                                            |                                                          |                                                         |                      |          |                      |
|-----------------------------------------------------------------------------------------------------------------------|------------------------------------------------------------------------------------------|----------------------------------------------------------|---------------------------------------------------------|----------------------|----------|----------------------|
| Yes / No                                                                                                              | ___________mm                                                                            |                                                          |                                                         |                      |          |                      |
| Yes / No                                                                                                              | ___________mm                                                                            |                                                          |                                                         |                      |          |                      |
| Yes / No                                                                                                              | ___________mm                                                                            |                                                          |                                                         |                      |          |                      |
| Yes / No                                                                                                              | ___________mm                                                                            |                                                          |                                                         |                      |          |                      |
| Yes / No                                                                                                              | ___________mm                                                                            |                                                          |                                                         |                      |          |                      |
| Yes / No                                                                                                              | ___________mm                                                                            | Yes / No                                                 | ___________mm                                           |                      |          |                      |
| Yes / No                                                                                                              | ___________mm                                                                            |                                                          |                                                         |                      |          |                      |
| Yes / No                                                                                                              | ___________mm                                                                            |                                                          |                                                         |                      |          |                      |
| Yes / No                                                                                                              | ___________mm                                                                            |                                                          |                                                         |                      |          |                      |
| Yes / No                                                                                                              | ___________mm                                                                            |                                                          |                                                         |                      |          |                      |
| Yes / No                                                                                                              | ___________mm                                                                            |                                                          |                                                         |                      |          |                      |
| Yes / No                                                                                                              | ___________mm                                                                            |                                                          |                                                         |                      |          |                      |
| Day +6                                                                                                                | Yes / No                                                                                 | Mild Moderate Severe                                     | Yes / No                                                | Mild Moderate Severe |          |                      |
| Day +5                                                                                                                | Yes / No                                                                                 | Mild Moderate Severe                                     | Yes / No                                                | Mild Moderate Severe |          |                      |
| Day +4                                                                                                                | Yes / No                                                                                 | Mild Moderate Severe                                     | Yes / No                                                | Mild Moderate Severe |          |                      |
| To be complete by site:  Injection #: _________ Date of Injection: _____/_____/_____ Subject Number: ________________ | Month/Day/Yr                                                                             | Day +3                                                   | Yes / No                                                | Mild Moderate Severe | Yes / No | Mild Moderate Severe |
| 13.2 APPENDIX B: INJECTION SITE REACTIONS DIARY CARD                                                                  | Please answer all questions below once per day for the first 7 days after each injection | Day 0  (Day of Injection)  Day +1 Day +2                 | Yes / No                                                | Mild Moderate Severe | Yes / No | Mild Moderate Severe |
| Yes / No                                                                                                              | Mild Moderate Severe                                                                     | Yes / No                                                 | Mild Moderate Severe                                    |                      |          |                      |
| Yes / No                                                                                                              | Mild Moderate Severe                                                                     |                                                          |                                                         |                      |          |                      |
| Circle: Yes  / No                                                                                                     | If yes tell us  if the  soreness is  mild,  moderate,  or severe Yes / No                | Mild Moderate Severe                                     |                                                         |                      |          |                      |
| Circle: Yes  / No                                                                                                     | If yes tell us  if the pain is  mild,  moderate,  or severe                              |                                                          |                                                         |                      |          |                      |
| Circle: Yes  / No                                                                                                     | If yes  measure  longest  diameter in  millimeters  (mm)  Circle: Yes  / No              | If yes  measure  longest  diameter in  millimeters  (mm) |                                                         |                      |          |                      |
| 1. Is there  redness at  the injection  site?                                                                         | 2. Is there  firmness or  swelling at  the  injection  site?                             | 3. Is there  soreness at  the  injection  site?          | 4. Have you  experienced  pain at the  injection  site? |                      |          |                      |  
83  
| Record Temp:                                                                                                         | ________________                                                                  | _                                                                                  |                                                                                                                                                                                                                                           |                  |    |                                                                                                                                                                                                     |
|----------------------------------------------------------------------------------------------------------------------|-----------------------------------------------------------------------------------|------------------------------------------------------------------------------------|-------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|------------------|----|-----------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|
| Yes / No                                                                                                             | Name:                                                                             | ___________                                                                        | Dose:                                                                                                                                                                                                                                     | ____________     |    |                                                                                                                                                                                                     |
| Day +6                                                                                                               | Yes / No                                                                          | Mild Moderate Severe                                                               | Record Temp:                                                                                                                                                                                                                              | ________________ | _  | Grading Chills:  Mild Mild sensation of cold, shivering, chattering of teeth  Moderate Moderate tremor of entire body, narcotics indicated  Severe Severe or prolonged, not responsive to narcotics |
| Yes / No                                                                                                             | Name:                                                                             | ___________                                                                        | Dose:                                                                                                                                                                                                                                     | ____________     |    |                                                                                                                                                                                                     |
| Day +5                                                                                                               | Yes / No                                                                          | Mild Moderate Severe                                                               | Record Temp:                                                                                                                                                                                                                              | ________________ | _  |                                                                                                                                                                                                     |
| Yes / No                                                                                                             | Name:                                                                             | ___________                                                                        | Dose:                                                                                                                                                                                                                                     | ____________     |    |                                                                                                                                                                                                     |
| Day +4                                                                                                               | Yes / No                                                                          | Mild Moderate Severe                                                               | Record Temp:                                                                                                                                                                                                                              | ________________ | _  |                                                                                                                                                                                                     |
| Yes / No                                                                                                             | Name:                                                                             | ___________                                                                        | Dose:                                                                                                                                                                                                                                     | ____________     |    |                                                                                                                                                                                                     |
| Day +3                                                                                                               | Yes / No                                                                          | Mild Moderate Severe                                                               | Record Temp:                                                                                                                                                                                                                              | ________________ | _  |                                                                                                                                                                                                     |
| Yes / No                                                                                                             | Name:                                                                             | ___________                                                                        | Dose:                                                                                                                                                                                                                                     | ____________     |    |                                                                                                                                                                                                     |
| Day +2                                                                                                               | Yes / No                                                                          | Mild Moderate Severe                                                               | Grading injection site pain:  Mild Noticeable, does not interfere with activity Moderate Interferes with activity, limiting instrumental activities of daily living  Severe Limiting self-care activities of daily living, incapacitating |                  |    |                                                                                                                                                                                                     |
| Record Temp:                                                                                                         | ________________                                                                  | _                                                                                  |                                                                                                                                                                                                                                           |                  |    |                                                                                                                                                                                                     |
| Yes / No                                                                                                             | Name:                                                                             | ___________                                                                        | Dose:                                                                                                                                                                                                                                     | ____________     |    |                                                                                                                                                                                                     |
| Day +1 Day 0  (Day of Injection)                                                                                     | Yes / No                                                                          | Mild Moderate Severe                                                               | Record Temp:                                                                                                                                                                                                                              | ________________ | _  |                                                                                                                                                                                                     |
| Yes / No                                                                                                             | Name:                                                                             | ___________                                                                        | Dose:                                                                                                                                                                                                                                     | ____________     |    |                                                                                                                                                                                                     |
| Circle: Yes  / No                                                                                                    | Provide name and  dose of  medication                                             |                                                                                    |                                                                                                                                                                                                                                           |                  |    |                                                                                                                                                                                                     |
| 5. Have you  taken any  medication  for injection  site pain?                                                        | Yes / No                                                                          | Mild Moderate Severe                                                               |                                                                                                                                                                                                                                           |                  |    |                                                                                                                                                                                                     |
| Circle: Yes  / No                                                                                                    | If yes tell us  if the chills  are mild,  moderate,  or severe  Circle: Yes  / No | If your  temperatur e is 104OF  for more  than 24  hours, call  your study  doctor |                                                                                                                                                                                                                                           |                  |    |                                                                                                                                                                                                     |
| 7. Record  your daily  temperatur e (Do not  drink  anything  within 5  minutes  prior to  taking your  temperature) |                                                                                   |                                                                                    |                                                                                                                                                                                                                                           |                  |    |                                                                                                                                                                                                     |
| 6. Have you  experienced  chills?                                                                                    |                                                                                   |                                                                                    |                                                                                                                                                                                                                                           |                  |    |                                                                                                                                                                                                     |