Panel b represent s patients that received all 3 vaccine s. Panel c represents patients vaccinat ed 3 times with the 2 highest doses of vaccine. There were 23 events during the study. Abbreviated Title: Ad5 -based combination vaccines Version Date: 05.16.2018 20 DCs were capable of activating both of these T -cell lines but not the CEA -specific T-cell line (Table 1). Human DCs were similarly infected with the ETBX -081 vector. As seen in Table 1 A and Table 1 B, T cells specif ic for CEA, MUC1, and Brachyury were each activated to i nduce similar levels of IFN -γ as seen with the use of the individual Ad -5 vectors. Studies were also undertaken to determine whether simultaneous infection of human DCs with the CEA/MUC1/ Brachyury mixture of ETBX -081 could generate T -cell lines specific for all three TAAs. As seen in Table 2, when the T cells were activated by incubation with autologous B cells pulsed with the corresponding peptide, and not a control peptide, specific T -cell activation was observed. For example, the Brachyury -specific T -cell line, generated by infecting human DCs with ETBX -081, was stimulated to produce IFN -γ when incubated with autologous DCs pulsed with Brachyury peptide, but was not activated with the same autologous DCs pulsed with a CEA pepti de. Similar results were seen with CEA and MUC1 T -cell lines generated with ETBX -081– infected DCs. These results indicate the lack of so -called “antigenic competition” in the in vitro use of ETBX -081. C Figure 3: Expression of Brachyury and MUC1 protein in human dendritic cells (DCs) infected with Ad5 [E1-, E2b -]−Brachyury and Ad5 [E1 -, E2b -]−MUC1. SW620 tumor cells were used as positive control. Actin was used as a loading control. (A) Expression of Brachyury was robust in DCs infected with Ad5 [E1 - , E2b -]-Brachyury. (B) MUC1 e xpression was observed in human DCs infected with Ad5 [E1 -, E2b - ]−MUC1 vector as compared to DCs infected with Ad5 [E1 -, E2b -]–null (no transgene). (C) Expression of CEA in A549 cells infecte d with Ad5 [E1 -, E2b -]-CEA. A549 cells were infected with Ad5 [E1 -, E2b -]- CEA and CEA expression was confirmed by western blot analysis. Recombinant CEA was used as a positive control and uninfected A549 cells served as a negative control. The samples are visualized below in the following order A. Negative Control, B. Magic Mark XP Western Marker, C. Negative, D. CEA Reference Material (30ng), E. Ad5 [E1 -, E2b -]-CEA lysate (20uL), F. Ad5 [E1 -, E2b -]-CEA lysate (20uL), G. Negative A549 cells. Abbreviated Title: Ad5 -based combination vaccines Version Date: 05.16.2018 21 Table 1 Table 1 A: Infection of Human Dendritic Cells with Recombinant Adenovirus Vectors Encoding CEA , MUC1 or Brachyury Can Activate Antigen -specific T-cell Lines Dendritic cells (DCs) infected with Antigen -specific T -cell lines CEA MUC1 (HLA -A2) MUC1 (HLA -A24) Brachyury Ad5 [E1 -, E2b -]–null <15.6 <15.6 <15.6 <15.6 Ad5 [E1 -, E2b -]−Brachyury <15.6 -- -- 351.9 Ad5 [E1 -, E2b -]−MUC1 <15.6 335.2 806.4 -- Ad5 [E1 -, E2b -]−CEA 350.0 <15.6 <15.6 -- Uninfected DCs <15.6 <15.6 <15.6 <15.6 T cells only <15.6 <15.6 <15.6 <15.6 Human DCs (6 -day culture in IL -4 and granulocyte -macrophage colony -stimulating factor (GM -CSF) 2x104 cells/well in 0.5 ml of AIM -V) were infected with indicated adenovirus vectors at 20,000 multiplicity of infection (MOI). After 48 hours, DCs were washed and used for stimulation of human antigen -specific T cells. Results are expressed in pg/ml of IFN -γ per 1x105 T cells/ml. Numbers in bold indicate a significant enhancement of IFN -γ secretion compared to corresponding wells with uninfected DCs. [ -- indicates that the assay was not performed.] Table 1 B: Infection of Human Dendritic Cells with ETBX -081 Vectors Encoding Transgenes Can Activate Antigen -specific T -cell L ines to Produce IFN -γ Dendritic cells (DCs) infected with Antigen -specific T -cell lines CEA (HLA -A2) MUC1 (HLA -A2) MUC1 (HLA -A24) Brachyury (HLA -A2) ETBX -081 480 236 763 496 Ad5 [E1, E2b] –null <15.6 <15.6 <15.6 <15.6 Uninfected DCs <15.6 <15.6 <15.6 <15.6 T cells only <15.6 <15.6 <15.6 <15.6 Human DCs (6 -day culture in IL -4 and GM -CSF) from an HLA -A2 and –A24 donor were infected with ETBX - 081 vector at 2 x 104 /well (24 -well plate) in 0.5 ml of AIM -V. ETBX -081 vectors were used at 20,000 MOI for 1 hour and then 1.5 ml of AIM -V were added to each well. Infected DCs were incubated for 48 hours and then washed and used for stimulation of human antigen -specific T cells. Results are expressed in pg of IFN -γ per 1 x 105 T cells/ml. Numbers in bold indicate a significant enhancement of IFN -γ secretion compared to corresponding wells with uninfected DCs . Abbreviated Title: Ad5 -based combination vaccines Version Date: 05.16.2018 22 Table 2: Infection of Human Dendritic Cells with ETBX -081 Can Generate Antigen -specific T Cells to Brachyury , MUC1 and CEA and Produce IFN -γ When S timulated with Autologous B Cells Pulsed with the Corresponding Peptides Peptides (10 μg/ml) Antigen -specific T -cell lines CEA MUC1 (A2) MUC1 (A24) Brachyury T-Brachyury <15.6 -- -- 243 T-MUC1 (A2) <15.6 174 -- -- T-MUC1 (A24) <15.6 -- 206 -- T-CEA 211 <15.6 -- -- Human dendritic cells (DCs) from a prostate cancer patient (6 -day culture in IL -4 and granulocyte -macrophage colony -stimulating factor (GM -CSF) 2x104 cells/well in 0.5 ml of AIM -V) were infected with ETBX -081 at 20,000 MOI. After 48 hours, infected DCs were washed and used to generate specific cytotoxic T lymphocytes (CTLs) using autologous peripheral blood monon uclear cells (PBMCs) as effectors. Following 3 cycles of in vitro stimulations, autologous peptides -pulsed B cells were used as antigen -presenting cells. Results are expressed in pg/ml of IFN -γ. [-- indicates that the assay was not performed.] Table 3: Infection of Human DCs with ETBX -081 Can Generate Brachyury -, MUC1 - and CEA -specific CTLs That E fficiently Lyse Tumor Cells Expressing All Three Antigens Antigen -specific T -cell lines SW620 Brachyury+ MUC1+ CEA+ (HLA -A2+/A24+) ASPC -1 Brachyury+ MUC1+ CEA+ (HLA -A1+/A26+) T-Brachyury 64.4 (3.6) 8.3 (2.7) T-MUC1 (P93L) 28.5 (1.3) 2.0 (1.6) T-MUC1 (C6A) 49.3 (3.3) 5.0 (1.8) T-CEA 42.4 (3.7) 4.3 (1.9) Human dendritic cells (DCs) were infected with ETBX -081 at 20,000 MOI. Infected DCs were used to generate specific cytotoxic T lymphocytes (CTLs) using autologous peripheral blood monoclonal cells (PBMCs). Autologous DCs were used as antigen -presenting cel ls for three in vitro stimulations (IVS). Autologous peptide - pulsed B cells pulsed were used to re -stimulate antigen -specific CTLs for two additional IVS. The effector -to- target ratio used was 30:1; CTLs were used at IVS 5. Results are expressed in % speci fic lysis (SD). Studies also investigated whether Brachyury -, MUC1 –, and CEA -specific human T cells generated using DCs infected with ETBX -081 could lyse human carcinoma cells that endogenously express these TAAs. SW620 human colon carcinoma cells express all three TAAs a nd possess the HLA - Abbreviated Title: Ad5 -based combination vaccines Version Date: 05.16.2018 23 A2 and -A24 Class I alleles. ASPC -1 human pancreatic carcinoma cells were used as a negative control since they express the three TAAs but in the context of HLA -A1 and -A26 molecules. The results (Table 3) demonstrated that ETBX -081–infected human DCs can generate T cells capable of lysing, in an MHC -restricted manner, human tumor cells that endogenously express Brachyury, CEA, and MUC 1. Studies were also undertaken to determine whether Ad5 [E1 -, E2b -]–Brachyury , Ad5 [E1 -, E2b - ]–MUC1, and Ad5 [E1 -, E2b -]–CEA could each generate TAA -specific T -cell responses in vivo , and whether the ETBX -081 mixture could generate comparable responses. C57Bl/6 mice (n=5 per group) were injected subcutaneously (s.c.) three times at 2 -week intervals with 1010 viral particles (VP) of Ad5 [E1 -, E2b -]–CEA , Ad5 [E1 -, E2b -]–MUC1, Ad5 [E1 -, E2b -]–Brachyury , or ETBX - 081 (1:1:1 mixture of 1010 VP each). An additional group of mice (n=5) received 3x1010 VP of Ad5 [E1-, E2b -]–null (an empty vector control). Two weeks after the final vaccination, splenocytes from vaccinated mice were stimulated with corresponding Brachyury , CEA, or MUC1 peptide pools and analyzed for IFN -γ and IL -2 secreting cells by the enzyme -linked immunospot (ELISPOT) assay. Mice vaccinated with singular constructs or with ETBX -081 responded to Brachyury , CEA, and MUC1 peptides, respectively, with signi ficant increases in IFN -γ and IL -2 spot forming cells (SFCs) as compared to control mice (Figure 4A and B ). There was no significant difference in the average number of IFN -γ SFCs in mice vaccinated with Ad5 [E1 -, E2b-]–Brachyury or Ad5 [E1 -, E2b -]–CEA individually as compared with the ETBX -081 vaccine. There was a significant decrease in IFN -γ SFCs in mice treated with the ETBX -081 vaccine as compared to Ad5 [E1 -, E2b -]–MUC1 alone, although the MUC1 –specific immune response induced by ETBX -081 remained significantly elevated over control mice ( p < 0.0001) (Figure 4A). IL-2 responses were similar in mice treated with ETBX -081 versus single vaccine constructs; moreover, there was a significant increase (p = 0.004) in CEA -specific IL -2 SFCs when mice were vaccinated with the ETBX -081 vaccine versus the Ad5 [E1 -, E2b -]–CEA vaccine alone (Figure 4B). Taken together, these data indicate that combining Ad5 [E1 -, E2b -]–Brachyury, Ad5 [E1 -, E2b-]–CEA, and Ad5 [E1 -, E2b -]–MUC1 in a ETBX -081 vaccine admixture has the effect of generating antigen -specific IFN -γ– and IL -2–producing cells similar to that achieved when using each vaccine alone. Figure 4: Analysis of IFN -γ− and IL -2−expressing splenocytes following vaccination of mice with Ad5 [E1 -, E2b -]−Brachyury, Ad5 [E1 -, E2b -]−CEA, Ad5 [E1 -, E2b -]−MUC1, ETBX -081, and Ad5 [E1-, E2b -]–null. C57Bl/6 mice (n = 5/group) were vaccinated three times at 2 -week intervals with 1010 VP (viral particle) of Ad5 [E1 -, E2b -]−Brachyury (white bar), Ad5 [E1 -, E2b -]−CEA (grey bar), Ad5 [E1 -, E2b -]−MUC1 (black bar) or ETBX -081 (1:1:1 mixture of 1010 VP each of Ad5 [E1 -, E2b - ]−Brachyury, Ad5 [E1 -, E2b -]−CEA, Ad5 [E1 -, E2b -]−MUC1) (diagonal hatched bar). Controls received 3x1010 VP of Adeno -null (horizontal striped bar). Splenocytes were collected 14 days after the final vaccination and assesse d for IFN -γ−secreting cells (A) or IL -2-secreting cells (B) by ELISPOT assay. For positive controls, splenocytes were exposed to Concanavalin A (Con A) (data not shown). Data reported as the number of spot forming cells (SPFs) per 106 splenocytes. The erro r bars depict the SEM. Significant differences (p < 0.05) between columns are reported in p -values, not significant = ns. Abbreviated Title: Ad5 -based combination vaccines Version Date: 05.16.2018 24 Studies were then undertaken to determine whether the ETBX -081 vaccine regimen was as effective as the use of a single recombinant a denovirus construct in eliciting an anti -tumor effect. It should be noted that unlike human carcinomas of which the majority o verexpress the human TAAs CEA, MUC1 and Brachyury, no mouse models express all three of these antigens. C57BL/6 mice (n = 7/group) were implanted s.c. with 1x106 MC38 cells expressing MUC1 (MC38 -MUC1) in the left flank. Mice were vaccinated weekly with s.c. injections in the opposite flank using 1010 VP of Ad5 [E1 -, E2b -]-MUC1 or ETBX -081, respectively. A control group of mice receiv ed 3x1010 VP of Ad5 [E1 -, E2b -]–null (no transgene). Mice vaccinated with Ad5 [E1 -, E2b -]–MUC1 or ETBX -081 had significantly smaller tumors than control mice on days 25 (p < 0.01) and 29 (p < 0.05) (Figure 5). There was no significant difference (p > 0.1) in anti -tumor effect for the groups of mice vaccinated with Ad5 [E1 -, E2b -]–MUC1 vs. ETBX -081 at all time points. Figure 5: Comparison of immunotherapy of MUC1 -expressing tumors using Ad5 [E1 -, E2b - ]−MUC1 vs ETBX -081. C57Bl/6 mice (n=7/group) were inoculated with 106 MC-38-MUC1 cells subcutaneously in the left flank. Mice were administered 1010 VP (viral particle) of Ad5 [E1 -, E2b - ]−MUC1 or ETBX -081 (1:1:1 mixture of 1010 VP each of Ad5 [E1 -, E2b -]−CEA, Ad5 [E1 -, E2b - ]−MUC1, and Ad5 [E1 -, E2b -]−Brachyury, 3x1010 VP total). A control group of mice received 3x1010 VP of Ad5 [E1 -, E2b -]–null (no tr ansgene). Tumor growth was monitored and volumes calculated. (*) indicates days when Ad5 [E1 -, E2b -]−MUC1 treated mice had significantly smaller (p < 0.05) tumors than control mice and (^) indicates days when ETBX -081–treated mice had significantly smaller (p < 0.05) tumors than control mice. There was no significant difference (p > 0.1) between Ad5 [E1 -, E2b -]−MUC1 vs. ETBX -081–treated mice at any time point. Error bars represent the SEM. Abbreviated Title: Ad5 -based combination vaccines Version Date: 05.16.2018 25 1.2.8 Clinical Experience The combination of ETBX -011, ETBX -061, and E TBX -051 vaccine has not been tested in humans. This Phase I study is the first clinical study of this vaccine combination in subjects with advanced cancer. 1.2.9 Rationale The preclinical data with the combination ETBX -011, ETBX -061, and ETBX -051 vaccine support this Phase I study to evaluate the safety, preliminary efficacy, and immunogenicity of the vaccine . Subsequent trials will involve the use of this vaccine in combination with checkpoint inhibitor monoclonal antibodies (MAbs) and other immu ne modulators. 2 ELIGIBILITY ASSESSME NT AND ENROLLMENT 2.1 ELIGIBILITY CRITERIA 2.1.1 Inclusion Criteria 2.1.1.1 Age ≥ 18 years (male and female). 2.1.1.2 Ability to understand and provide signed informed consent that fulfills Institutional Review Board (IRB)’s guidelines. 2.1.1.3 Subjects with cytologically or histologically confirmed locally advanced or metastatic solid tumor malignancy. 2.1.1.4 Subjects must have completed or had disease progression on at least one prior line of disease -appropriate therapy or not be candidates for therapy of pro ven efficacy for their disease. 2.1.1.5 Subjects may have measurable or nonmeasurable but evaluable disease as defined in section 6.3.1 . Subjects with surgically resected locally advanced or metastatic disease at high risk of relapse are also eligible. 2.1.1.6 Eastern Cooperative Oncology Group (ECOG) performance status < 1 (Appendices 2.1.1.7 Appendix A) 2.1.1.8 Subjects who have received prior CEA, MUC1, and/or Brachyury -targeted immunotherapy (vaccine) are eligible for this trial if this treatment was discontinued at least 4 weeks prior to enrollment. 2.1.1.9 Resolution of clinically significant side effects of prior ch emotherapy, radiotherapy, immunotherapy or surgical procedures to NCI CTCAE Grade ≤ 1 or grade ≤ 2 for neuropathy. 2.1.1.10 Adequate hematologic function at screening, as follows: 2.1.1.10.1 Absolute neutrophil count (ANC) ≥1 x 109/L 2.1.1.10.2 Hemoglobin ≥ 9 g/dL 2.1.1.10.3 Platelets ≥ 75,000/ mcL. Abbreviated Title: Ad5 -based combination vaccines Version Date: 05.16.2018 26 2.1.1.11 Adequate renal and hepatic function at screening, as follows: 2.1.1.11.1 Serum creatinine ≤ 1.5 x upper limit of normal (ULN) OR creatinine clearance (CrCl) ≥ 40 mL/min (if using the Cockcroft -Gault formula below): i. Female CrCl = [(140 - age in years) x weigh t in kg x 0.85] / [72 x serum creatinine in mg/dL] ii. Male CrCl = [(140 - age in years) x weight in kg x 1.00]/ 1.00] / [72 x serum creatinine in mg/dL] 2.1.1.11.2 Bilirubin ≤ 1.5 x ULN OR in subjects with Gilbert’s syndrome, a total bilirubin ≤ 3.0 x ULN 2.1.1.11.3 Alanine aminotransferase (ALT) and aspartate aminotransferase (AST) ≤ 2.5 x ULN, unless liver metastases are present, then values must be ≤ 3 x ULN) 2.1.1.12 The effects of the combination ETBX -011, ETBX -051, ETBX -061 vaccine regimen on the developing human fetus are unknown. For this reason, f emale subjects of childbearing potential defined as any female who has experienced menarche and who has not undergone surgical sterilization (hysterectomy or bilateral oophorectomy or tubal ligation) or who is not postmenopausal (menopause being defined clinically as 12 months of amenorrhea in a woman over 45 in the absence of other biological or physiological causes) and male patients who are not surgically sterile (vasectomy etc.), must agree to use acceptable contraceptive met hods for the duration of the study and for one month after the last vaccination . Acceptable forms of contraception include oral contraceptives, intrauterine device, condom or vaginal diaphragm plus spermicidal (gel/foam/cream/vaginal suppository), or total abstinence. 2.1.1.13 Ability to attend required study visits and return for adequate follow up, as required by this protocol. 2.1.2 Exclusion Criteria 2.1.2.1 Pregnant and nursing women . Because there is an unknown but potential risk for adverse events in nursing infants second ary to treatment of the mother with combination ETBX -011, ETBX -051, ETBX -061, breastfeeding should be discontinued if the mother is treated with combination ETBX -011, ETBX -051, ETBX -061. These potential risks may also apply to other agents used in this st udy. 2.1.2.2 There should be a minimum of 4 weeks from any prior investigational drug, chemotherapy, immunotherapy, with the exception of hormonal therapy for prostate and breast cancers, HER2 -directed therapy for HER2+ breast or stomach cancer (3+ IHC or FISH+), drugs targeting EGFR , ALK or ROS1 in EGFR,ALK, ROS1 -mutated lung cancer, respectively, or standard maintenance therap ies for any solid tumor under the condition that subjects are on these therapies for at least two months before start of trial treatment. 2.1.2.3 There should also be a minimum of 4 weeks from any prior radiotherapy except for palliative bone directed therapy. 2.1.2.4 Known active brain or central nervous system metastasis (less than 1 month out from definitive radiotherapy or surgery), or seizures requirin g anticonvulsant treatment, or clinically significant cerebrovascular accident or transient ischemic attack (< 3 months). Abbreviated Title: Ad5 -based combination vaccines Version Date: 05.16.2018 27 2.1.2.5 Subjects with active autoimmune disease requiring systemic immunosuppressive treatment within the past 4 weeks such as but not restricted to inflammatory bowel disease, systemic lupus erythematosus, ankylosing spondylitis, scleroderma, or multiple sclerosis. A history of autoimmune disease which is not active nor has required recent systemic immunosuppressive thera py (< 4 weeks prior to enrollment) is not reason for exclusion. 2.1.2.6 Subjects with serious intercurrent chronic or acute illness, such as cardiac or pulmonary disease, hepatic disease, or other illness considered by the Investigator as high risk for investigat ional drug treatment. 2.1.2.7 Subjects with clinically significant heart disease, such as congestive heart failure (class II, III, or IV defined by the New York Heart Association functional classification), history of unstable or poorly controlled angina, or histo ry (< 1 year) of ventricular arrhythmia. 2.1.2.8 Subjects with a medical or psychological impediment that would impair the ability of the subject to receive therapy per protocol or impact ability to comply with the protocol or protocol -required visits and procedures. 2.1.2.9 History of second malignancy within 3 years prior to enrollment except for the following: adequately treated non -melanoma skin cancer, cervical carcinoma in situ, superficial bladder cancer or other localized malignancy after discussion with th e medical monitor. 2.1.2.10 Presence of a known active acute or chronic infection, including human immunodeficiency virus (HIV, as determined by enzyme -linked immunosorbent assay [ELISA] and confirmed by western blot) and hepatitis B and hepatitis C virus (HBV/HCV , as determined by HBsAg and hepatitis C serology). 2.1.2.11 Subjects on systemic intravenous or oral corticosteroid therapy with the exception of physiologic doses of corticosteroids ( ≤ the equivalent of prednisone 10 mg/day) or other immunosuppressives such as a zathioprine or cyclosporin A are excluded on the basis of potential immune suppression. For these subjects these excluded treatments must be discontinued at least 2 weeks prior to enrollment for recent short course use ( ≤ 14 days) or discontinued at least 4 weeks prior to enrollment for long term use (> 14 days). In addition, the use of corticosteroids as premedication for contrast -enhanced studies is allowed prior to enrollment and on study. 2.1.2.12 Subjects with known allergy or hypersensitivity to any component of the investigational product will be excluded. 2.1.2.13 Subjects with acute or chronic skin disorders that will interfere with injection into the skin of the extremities or subsequent assessment of potential skin reactions will be excluded. 2.1.2.14 Subjects vaccinated w ith a live (attenuated) vaccine (e.g., FluMist®) or a killed (inactivated)/subunit vaccine (e.g., PNEUMOVAX®, Fluzone®) within 28 days or 14 days, respectively, of the first planned dose of ETBX vaccine. Abbreviated Title: Ad5 -based combination vaccines Version Date: 05.16.2018 28 2.1.3 Recruitment Strategies This study will be listed on available websites ( www.clinicaltrials.gov , https://ccr.cancer.gov/clinical -trials -search -start) and participants will be recruited from the current patient population at NIH. 2.2 SCREENING EVALUATION All screening tests and procedures must be performed within 28 days prior to the first planned dosing of the study drug, unless otherwise noted. The following procedures and evaluations will be perfor med and documented in the subject’s source records. • History and physical exam including ECOG performance status and vital signs • 12-Lead ECG • Clinical laboratory tests (within 16 days prior to drug administration ) − Chemistry: sodium, potassium, chloride, bicarbonate, calcium, glucose, BUN, creatinine, ALT, AST, alkaline phosphatase, total protein, albumin, and total and direct bilirubin. − Hematology: complete blood count (CBC) with differential and platelets − Coagulation panel: PT, INR, and PTT. − Urinalysis. • Serum pregnancy test (β -HCG) for females of childbearing -potential and women < 12 months since the onset of menopause (within 16 days prior to enrollment) • HBV (HBsAg), HCV (anti -HCV), HIV (anti -HIV) screening (within 3 months prior to enrollme nt) • Appropriate t umor imaging and assessment . All baseline tumor measurements should be performed based on the subject’s qualifying scan obtained within 28 days prior to the start of treatment. • Confirmation of diagnosis. A report from any CAP or CLIA cert ified laboratory is acceptable. 2.3 REGISTRATION PROCEDURES Registration will be a two -part process as patients are screened on this protocol. Authorized staff must register an eligible candidate with NCI Central Registration Office (CRO) within 24 hours of signing consent. To initially register a subject after the participant has signed the consent, complete the top portion of the registration Eligibility Checklist from the website (http://home.ccr.cancer.gov/intra/eligibility/welcome.htm) indicating that the patient is being registered for screening and send via encrypted email to: NCI Central Registration Office ncicentralregistration -l@mail.nih.gov. Once eligibility is confirmed after completion of screening studies, complete the remainder of the form whic h is the eligibility checklist, indicating that the patient is being registered for treatment and email the completed registration checklist to the CRO at NCI Central Registration Office ncicentralregistration -l@mail.nih.gov . After confirmation of eligi bility at Central Registration Office, CRO staff will call pharmacy to advise them of the acceptance of the patient on the protocol prior to the release of any investigational agents. Verification of Registration will be forwarded electronically via e -mail to the research team. A recorder is available during non -working hours. Abbreviated Title: Ad5 -based combination vaccines Version Date: 05.16.2018 29 Subjects that do not meet screening criteria should be removed from the study following the procedure in section 3.7.3 . 2.3.1 Treatment Assignment Procedure Cohorts Number Name Description 1 Dose De -Escalation Subjects enrolled to dose de -escalation cohorts 2 Dose Expansion Subjects enrolled at the MTD after the MTD is established Arms Number Name Description 1 Dose De -Escalation Combination ETBX -011, ETBX -051, ETBX -061 adenoviral vaccine regimen dose de -escalation 2 Dose Expansion Combination ETBX -011, ETBX -051, ETBX -061 adenoviral vaccine regimen dose Arm Assignment Subjects in cohort 1 will be directly assigned to arm 1. Subjects in cohort 2 will be directly assigned to arm 2. 2.4 BASELINE EVALUATION All subjects are required to complete baseline evaluations within 1 day prior to the first planned dosing of the study drug (Any screening evaluation done on D -1 of treatment can also serve for the baseline evaluation) . The following procedures and evaluat ions will be performed and documented in the subject’s source records. • History and physical exam including ECOG performance status and vital signs • Serum pregnancy test (β -HCG) for females of childbearing -potential and women < 12 months since the onset of menopause . • Chemistry: sodium, potassium, chloride, bicarbonate, calcium, glucose, BUN, creatinine, ALT, AST, alkaline phosphatase, total protein, albumin, and total and direct bilirubin. • Hematology: CBC with differential and platelets 3 STUDY IMPLEMENTATION 3.1 STUDY DESIGN This is a Phase I trial in subjects with advanced cancer. The combination vaccine regimen is three different adenoviral vaccines (ETBX -011, ETBX -061 and ETBX -051), adminis tered to patients at the same time. All utilize the same second generation E1(-), E2 ( -) vector. 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 Abbreviated Title: Ad5 -based combination vaccines Version Date: 05.16.2018 30 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 ins ert. (Table 8). 3.1.1 Dose Limiting Toxicity For the purposes of dose esca lation a dose limiting toxicity (DLT) is defined as: • Any Grade 3 or greater toxicity that is possibly related to the vaccine and as defined by Common Terminology Criteria for Adverse Events (CTCAE ) Version 5.0 with the exception of transient (≤ 24 hours) Grade 3 flu -like symptoms or fever, which is controlled with medical management or transient (≤ 24 hours) Grade 3 fatigue, skin reactions or rash , headach e, nausea, emesis that resolves to Grade ≤ 1 or asymptomatic grade 3 amylase/lipase elevation. For purposes of dose escalation the DLT evaluation period will be for 3 week s following the first dose of drug. Subjects experiencing a DLT will be removed from protocol therapy . 3.1.2 Dose De -Escalation Up to six patients will be enrolled at dose level 1 . If ≤1 of 6 patients experience a DLT, initiation of the dose expansion phase will occur. If ≥2 of 3 or 6 experience DLT in the initial dose level , then dose de -escalation will occur. Up to six patients will be enrolled at the lower dose level ( -1) (1x1011 VP). If ≤1 of 6 patients experience a DLT, then the maximum tolerated (MTD) will be declared at this dose, and initiation of the dose expansion phase will occur. If ≥2 of 3 or 6 experience DLT at dose level -1, then an amendment may be written to evaluate a further dose de - escalation. In the second part, dose expansion will occur when the MTD has been determined. An additional 4 subjects will be enrolled in the dose expansion component of the trial, f or a total of 1 0 subjects treated at the MTD . A schematic overview of the study is sho wn in Figure 6. In the initial component of the study, 3 to 6 subjects will be sequentially enrolled starting at the standard dose at dose level 1 . During enrollment at each dose level , there will be a minimum of 3 days between enrolling successive subjects. DLTs will be monitored conti nuously. Dose levels are shown in Table 4. No intra -patient dose escalations are permitted. Table 4: Dose Levels for Each ETBX VP Dose Level (DL) ETB X VP Standard (1) 5 x 1011 -1 1 x 1011 Standard dose (DL 1) (5 x 1011 VP): • If ≤ 1 of the initial 6 subjects experience a DLT, dose expansion will commence. • If >1 of the initial 3 subjects, or if ≥ 2 of the 6 total subjects experience a DLT, enrollment at DL -1 will commence. Abbreviated Title: Ad5 -based combination vaccines Version Date: 05.16.2018 31 Dose de -escalation DL -1 (1 x 1011 VP): • If ≤ 1 of the initial 6 subjects experience a DLT, this dose level will be defined as the MTD and dose expansion will commence. • If >1 of the initial 3 subjects, or if ≥ 2 of the 6 total subjects experience a DLT, then a protocol amendment may be written to evaluate a further dose de -escalation .. Abbreviated Title: Ad5 -based combination vaccines Version Date: 05.16.2018 32 Figure 6: Study Design and Treatment Schema Abbreviated Title: Ad5 -based combination vaccines Version Date: 05.16.2018 33 3.2 ADMINISTRATION AND DURATION OF TREATMENT Each of the three ETBX vaccine s will be administered on Weeks 0, 3, and 6 followed by booster vaccines every 8 weeks for up to a year. Treatment may be shorter for patients who experience progressive disease (unequivocal or confirmed) or unacceptable toxicity, withdraw consent, or if the Investigator feels it is no longer in their best interest to continue treatment.