Additionally, excess blood may also be utilized to assess new generations of vaccines. 4.4.4 Specimen Processing Peripheral blood from patients are collected into BD Vacutainer CPT Cell Preparation Tubes (BD, Franklin Lakes, NJ) which contain an anticoagulant (sodium heparin or sodium citrate) with FICOLL HYPAQUE density gradient fluid and a polyester gel barrier. The density gradient fluid and the gel barrier allow for the separation of PBMC from the red blood cells by a single step centrifugation process. The PBMC fraction will be collected by centrifugation and suspended in RPMI-1640 (GIBCO, Invitrogen Corporation, Carlsbad, CA) with 10% FCS (Gemini Bio-Products, West Sacramento, CA) and antibiotics. For the propagation of cultures the PBMC will be suspended at 1-2 x 106 cells/ml with or without 50 IU/ml of IL-2 (R&D Systems, Minneapolis, MN) being added at the beginning or at a later time. The cell cultures are placed in a humidified incubator at 37ºC in 5% CO2 and maintained with the addition of media as needed and/or IL-2 every 2-3 days depending on the growth kinetics. CONFIDENTIAL Page 33 of 102 Protocol: HERCEPTIN® + E75/GM-CSF Version 3.1 ML25749 05 March 2017 4.4.5 HLA-A2/A3/A24/A26 Testing The expression of HLA-A2 by the patients is confirmed by staining peripheral blood mononuclear cells (PBMC) with anti-HLA-A2 monoclonal antibody, (clone BB7.2), directly conjugated to phycoerythrin (PE) or fluorescein-isothiocyanate (FITC) (BD Biosciences) at 4ºC for 60 minutes. The expression of HLA-A3, A24, and A26 by the patients is confirmed by staining PBMC with the appropriate biotinylated-anti HLA-allele monoclonal antibody (One Lambda Inc.) for 45 minutes after which the cells are washed and incubated for an additional 15 minutes with streptavidin-phycoerythrin. After incubation the cells are washed and analyzed on a FACSCanto II flow cytometer (BD Biosciences, San Diego, CA). 4.4.6 Dimer Assay Fresh PBMC can be isolated and stained with HLA-A2:Ig DimerX reagent (Dimer) (BD PharMingen). The HLA-A2:Ig DimerX is a recombinant fusion protein of two extracellular MHC Class I HLA-A2 domains that are fused together with the VH regions of mouse IgG1. The Dimer will be “loaded” with the E75 or control peptides (E37 and Flu-M) and then incubated with fresh PBMC. The PBMC will then be stained with FITC-labeled anti-mouse IgG1 and anti-CD8 PE and analyzed with two-color flow cytometry to determine the percentage of CD8+ T cells which specifically recognize the E75 peptide. 4.4.7 ELISPOT Assay Freshly isolated PBMC are cultured/stimulated overnight in complete medium (RPMI + 5 %FCS + PSG) supplemented with IL-7 (20 ng/mL) with the individual peptides at 25 μg/mL (E37, E75, GP2, Flu-M) or (AEN, AE36, AE37, tetanus toxoid) or PMA + Ionomycin in flat-bottom anti-human IFN-γ ELISPOT plates (BD PharMingen) at 5 x 105 cells/well/200 μL in duplicate wells. The plate is incubated at 37ºC overnight after which the wells will be washed and incubated with the biotinylated-anti-IFN-γ mAb for two hours. The wells will be washed again and incubated with streptavidin-conjugated HRP for one hour. After a final wash the AEC-substrate solution will be added to the wells and allowed to develop for approximately 5-10 minutes at which time the wells will be washed with deionized water to stop the reaction. The number of spots present in each well will be enumerated using the CTL ELISPOT analyzer (CTL Analyzers LLC, Cleveland, OH). 4.4.8 Local Reaction Patients will also be assessed for evidence of in vivo immunologic response by evaluation of the injection site 48-72 hours after each inoculation (Appendix A). The injection site reaction will be measured using the sensitive ball point pen method if the patient returns to the study site. A digital photograph of the local reaction may be taken to document the reaction and for future reporting purposes. It will in no way be visually identifiable as that patient. Photographs will be labeled with the patient’s study number and will not include the face or distinguishing birthmarks or tattoos. They will be electronically stored on a password protected computer in a locked CONFIDENTIAL Page 34 of 102 Protocol: HERCEPTIN® + E75/GM-CSF Version 3.1 ML25749 05 March 2017 office in the vaccine or clinical laboratory facility at each site. Per FDA regulations, each patient’s photograph will be kept for two years after submission of a New Drug Application (NDA) and then destroyed. If the patient does not return to the study site, they will be contacted by phone 48-72 hours after inoculation for questioning regarding the reaction at the injection sites. 4.4.9 DTH Reaction Regardless of randomization, a pre- and post-inoculation series DTH response (Appendix A) will be assessed of all patients with 100 mcg of E75 (without GM-CSF) injected intradermally at a site on the back or anterior thigh (opposite side from the vaccination site). See detailed guidance (Appendix G) for vaccine and DTH preparation. The DTH reaction will be measured at 48-72 hours using the sensitive ball point pen method and compared between pre-inoculation and post-inoculation time points. Additionally, DTH responses will also be assessed 3-4 weeks after the second and fourth booster. The low dose of peptide used in the DTH test is not expected to induce a long-term immune response; however, any response that is induced is expected to be transitory in nature. A digital photograph of the local reaction will be taken to document the reaction and for future reporting purposes. It will be labeled with the patient’s study number, will not visually identify the patient, and will not include the face or distinguishing birthmarks or tattoos. Photographs will be electronically stored on a password protected computer in a locked office in the vaccine or clinical laboratory facility at each site. As per FDA regulations, each patient’s photograph will be kept for two years after submission of a NDA and then destroyed. These results will also be recorded in the immune response database. 4.5 DISCONTINUATION OF PROTOCOL-SPECIFIED THERAPY Protocol-specified therapy may be discontinued for any of the following reasons: • Progressive disease • Unacceptable toxicity as described in the protocol. • Please refer to sections 3.4-3.5 (Safety Plan) for the management of cardiac issues (symptomatic and asymptomatic), infusion-associated events, hematologic toxicity and neutropenic infections, as well as delayed hypersensitivity reactions. Adverse events that do not respond to either dose modification (as described) or clinical management of the reaction will result in discontinuation from the study. • Please refer to section 4.6 (Subject Discontinuation) for a list of severe adverse reactions warranting discontinuation from the study. • Patient election to discontinue therapy (for any reason) • Physician’s judgment CONFIDENTIAL Page 35 of 102 Protocol: HERCEPTIN® + E75/GM-CSF Version 3.1 ML25749 05 March 2017 4.6 SUBJECT DISCONTINUATION Those patients who display significant reactions (i.e., severe anaphylactic reaction immediately after vaccine administration) or toxicities will be discontinued from the study as determined by the PI. They will be followed by the study investigator until resolution of the adverse event. Inoculations will be immediately halted if any serious adverse reactions occur to include: death, life-threatening adverse drug experience (i.e., severe anaphylactic reaction immediately after vaccine administration), inpatient hospitalization or prolongation of existing hospitalization, a persistent or significant disability/incapacity, a congenital anomaly/birth defect, or other important medical events that may not result in death, be life-threatening, or require hospitalization but which, when based upon appropriate judgment of the PI, be determined to jeopardize the patient or require medical or surgical intervention to prevent an outcome listed above. Any death or grade 4 adverse drug experience found to be directly related to the experimental vaccine will result in suspension of patient enrollment to the study. If the SAE is determined to be unrelated to the study drug, inoculations may be re-started if the patient desires to continue and it is safe for them to do so as determined by the PI and Sponsor. Per 3.5 (Dose Reductions of Vaccine Component (paragraph one), for robust local, systemic > Grade 3, and > Grade 2 hypersensitivity reactions, as evaluated under CTCAE v4.03, will result in dose reduction or discontinuation as discussed. In the event of cardiac toxicities, the determination for discontinuation is described in section 3.4.2. Although prior history of vaccine therapy (with or without Herceptin TM) in Phase I and II studies has not shown evidence of cardiac toxicities, potential overlapping of study treatment will be monitored by ECHO or MUGA scans done every three months during vaccine therapy. Any symptoms of CHF reported by patients, or observed by study coordinators, will be documented and reported to the investigators for a determination on relatedness to either HerceptinTM or the vaccine component. While this attribution will be difficult on a per patient basis, the incidence between the Herceptin only arm will be compared to the Herceptin + vaccine arm to assess for additive toxicities. The Principal Investigator will determine if safety permits continuation in study regardless of attribution, based on the parameters under Section 3.4 (Safety Plan). If LVEF drops >20% or is below facility limits of normal or patient is symptomatic of CHF and requires treatment, the patient will be discontinued from treatment but remain on the study for disease follow-up. Patients will be followed until the resolution of the toxicity.