To date, there is no information that suggests that differences in drug metabolism or disease response would be expected in one group compared with another. Efforts will be made to extend accrual to a representative population, but in this preliminary study, a balance must be struck between patient safety considerations and limitations on the number of individuals exposed to potentially toxic and/or ineffective treatment s on one hand and the need to explore gender and ethnic aspects of clinical research on the other hand. If differences in outcome that correlate with gender and ethnic identity are noted, accrual may be expanded or a follow -up study may be written to inves tigate those differences more fully. 10.1.2 Strategies/Procedures for Recruitment Patient accrual for this protocol will be facilitated by Web -based recruitment strategies. This protocol will be listed on www.clinica ltrials.gov . 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 vac cines 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 pos sibility, 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 for evaluation. For those subjects that become incapacitated and do not have Abbreviated Title: Ad5 -based combination vaccines Version Date: 05.16.2018 60 pre-determined substitute decision maker, the procedures described in MAS Policy 87 -4 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 Patients will receive evaluation of their disease at the National Cancer Institute’s Clinical Cente r. This pro tocol 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 ut ilized 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 na mes will be given to the Central Registration to register and verify patients’ eligibility. 10.5 CONSENT PROCESS AND DOCUMENTATION The investigational nature and research objectives of this trial, the procedures and treatments involved and their attendant risk s 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. If there is an optional biopsy for research in the protocol, then the patient will con sent at the time of the procedure. If the patient refuses the optional biopsy at that time, the refusal will be documented in the medical record. 10.5.1 Telephone re -consent procedure Re-consent on this study may be obtained via telephone according to the follow ing 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 consen t. 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 Abbreviated Title: Ad5 -based combination vaccines Version Date: 05.16.2018 61 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 . 10.5.2 Consent process for non -English speaking subjects 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 M77 - 2, 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 translate d 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 obtaini ng 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 pro cess and will notify the IRB at the time of continuing review of the frequency of the use of the Short Form. 11 PHARMACEUTICAL INFOR MATION 11.1 COMBINATION ETBX -011, ETBX -051, ETBX -061 VACCINE REGIMEN The combination ETBX -011, ETBX -051, ETBX -061 vaccine regimen refers to the combination of three investigational adenoviral vaccines. All utilize the same second generation E1( -), E2 ( -) vector. Subjects will each receive the three vaccines at the same time, at the same doses, through three separate injections admini stered in different limbs ( proximal limb areas ) or separate locations . ETBX -011 is a CEA -targeting vaccine that comprises the Ad5 [E1-, E2b -] vector and a modified CEA (CEA(6D)) gene insert . The investigational product ETBX -061 is a MUC1 -targeting vaccine that comprises the Ad5 [E1-, E2b -] vector and a modified MUC1 (MUC1c) gene insert. The investigational product ETBX -051 is a Brachyury -targeting vaccine that comprises the Ad5 [E1-, E2b-] vector and a modified Brachyury gene insert. (Table 8). Refer to the current version of the Investigator’s Brochure for detailed investigational product information. Table 8: Investigational P roducts Table 8a Product Name(s): ETBX -011 (Ad5 [E1 -, E2b -]-CEA(6D) Vaccine) Dosage Form: Suspension for injection Abbreviated Title: Ad5 -based combination vaccines Version Date: 05.16.2018 62 Dose 5 x 1011 VP (standard dose), or 1 x 1011 VP (DL -1). Route of Administration SC injection Physical Description 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. 3 mL of the vaccine. The product should be stored at -20+10°C until ready for use . Manufacturer Etubics Table 8b Product Name(s): ETBX -061 (Ad5 [E1 -, E2b -]-MUC1 Vaccine) Dosage Form: Suspension for injection Dose 5 x 1011 VP (standard dose), or 1 x 1011 VP (DL -1). Route of Administration SC injection Physical Description 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 contains1 mL of extractable vaccine. The product should be stored at ≤ -60°C until ready for use . Manufacturer Etubics Table 8c Product Name(s): ETBX -051 (Ad5 [E1 -, E2b -]-Brachyury Vaccine) Dosage Form: Suspension for injection Dose 5 x 1011 VP (standard dose), or 1 x 1011 VP (DL -1). Route of Administration SC injection Physical Description 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 1 mL of extractable vaccine. The product should be stored at ≤ -60°C until ready for use . Manufacturer Etubics Abbreviated Title: Ad5 -based combination vaccines Version Date: 05.16.2018 63 11.1.1 Source The ETBX vaccines will be supplied by the manufacturer, Etubics Corporation , through a Cooperative Research and Development Agreement (CRADA). The manufacturing department of Etubics will supply each vaccine in 2-mL single -dose vials. Each single -dose vial contains a sterile, clear suspension of the ETBX vaccine at 5 x 1011 VP per 1 mL intended for single dose administration and contains ARM formulation buffer (20 mM TRIS, 25 mM NaCl, 2.5% glycerol, pH 8.0). Upon receipt of the investigational product, the Investigator or delegated designee will verify that an appropriate shippin g temperature was maintained, conduct an inventor y, and sign the drug receipt form and send a scanned copy to the Sponsor cont act as designated on the form. If a temperature excursion occurs , the Sponsor should be notified immediately , and the investigatio nal product must be quarantined and maintained under the correct storage conditions until further instructions are provided by the Sponsor. The temperature excursion form will need to be comp leted and returned to the Sponsor. The original drug receipt form and packing slip must be retained in the Investigator’s pharmacy records. 11.1.2 Toxicity The safety of immunizations (injections) with the ETBX -011, ETBX -051, ETBX -061 vaccine s 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 respo nses to CEA and to obtain preliminary data on response rate. The study was performed under an FDA - approved IND (IND14325) and registered at ClinicalTrials.gov (NCT01147965) . Participants were recruited from oncology clinics at Duke University Medical Cente r 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 su rvival 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 u niversally 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 wome n 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 viti ligo or fever for less than 2 days and less than 101.5 ºF, Grade ≥2 allergic Abbreviated Title: Ad5 -based combination vaccines Version Date: 05.16.2018 64 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; Coh ort 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 d id 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 immuni zation 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 Adverse Events as of 07/31/12 # of Events Unrelated/Unlikely Possible Probably/ Definite 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 Abbreviated Title: Ad5 -based combination vaccines Version Date: 05.16.2018 65 Grade 3 and Grade 4 Adverse Events in ≥ 2% of Patients* Adverse Events Number of Grade 3 (G3) Number of Grade 4 (G4) 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 *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). Ther e were no biologically significant changes in chemistry, hematology, or ANA values. 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. 11.1.4 Stability and Storage Individual vi als (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). 11.1.5 ETBX Vaccine Dose Preparation and Administration 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 availabl e 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 – Abbreviated Title: Ad5 -based combination vaccines Version Date: 05.16.2018 66 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 wh ite flip -off seal. The e nd 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 syri nge. 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 accorda nce 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 v accine concentration). 11.1.5.3 Administration Each of the three ETBX vaccine s will be administered on Weeks 0, 3, and 6 followed by bi - monthly 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 thi gh, 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 adminis tration. 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 . 11.1.6 Incompatibilities The vaccine s are administered as separate subcutaneous in jection s, with no known incompati bilities. 11.1.7 Other Considerations Abbreviated Title: Ad5 -based combination vaccines Version Date: 05.16.2018 67 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 recommend ed 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. Abbreviated Title: Ad5 -based combination vaccines Version Date: 05.16.2018 68 12 REFERENCES 1. 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