Experimental Agent Preparation A. HER2 ICD peptide- based vaccine 7866/135 08/23/2021 Page 15 of 39 The s ynthetic HER2 peptides to be used in the vaccine (Table 2 ) are 15 amino acids in length with an amino acid sequence identical to the normal HER2 sequence. This HER2 vaccine has been evaluated in a previous study (see Background) and is covered by BB IND 6524 (Sponsor: Mary L Disis, M.D.). 1. Peptide synthesis and characterization: The peptides are manufactured by Multiple Peptide Systems, San Diego, CA. using standard solid phase synthesis techniques employing Boc chemistry. Amino acids are analyzed by TLC and polarimetry prior to use. The peptides are cleaved from the solid support using anhydrous hydrogen fluoride and conventional techniques. Each peptide is purified by HPLC using a Waters Delta- prep system wi th a gradient of acetonitrile (containing 0.1% trifluoroacetic acid) in water (also containing 0.1% trifluoroacetic acid). The peptides are lyophilized to dryness before being passed over an anion exchange column to exchange the trifluoroacetate for aceta te. GMP procedures are followed for the manufacture of these sequences. After formulation is complete, the peptides are characterized by mass spectrometry to confirm their identity, by HPLC for purity using two different elution buffers, and by amino aci d analysis for identity and peptide content. The water content of each peptide was determined by the Karl Fischer procedure and the data obtained used to verify the mass balance of the peptides. These peptides are vialed as the HER2 ICD by the Fred Hutchinson Cancer Research Center, Biologics Product Facility ( a GMP facility ). 2. Peptide formulation and stability : Prior to formulation, the solubility of each peptide in 10 mM sodium acetate buffer (pH 4.0) is determined. T hree peptides were made up in a 10 mM sodium acetate buffer (pH 4.0). The stability of each individual peptide in the acetate buffer, as well as the peptide mixtures, is monitored by HPLC and mass spectrometry. The dissolved peptides are stored at - 20 ± 5˚C prior to use. Microbial and sterility testing are conducted to ensure safety. The peptide formulation is labeled to accurately reflect the product identity, concentration, lot number and fill date. These labels are attached to vials immediately post -fill. B. GM -CSF Recombinant hum an GM -CSF (rhuGM -CSF; Sargramostatin, LEUKINE) will be used as adjuvant admixed with HER2 vaccine, obtained by commercial suppliers and stored at their specifications . GM -CSF is a growth factor that supports the survival, clonal expansion, and differentiation of hematopoietic progenitor cells including dendritic APCs. In general, the use of GM -CSF is associated with little toxicity ; and rhuGM - CSF, when given IV or subcutaneously is well tolerated at doses ranging from 50 -500 mcg/m 2/day. Severe toxicity is extremely rare in patients treated with rhuGM -CSF. For the current study, rhuGM -CSF will be used at a total injection dose of 100 mcg. Administration of rhuGM -CSF may aggravate fluid retention in patients with pre- existent edema , capillary leak syndrome, or pleural or pericardial effusions. In some patients with pre- existing renal or hepatic dysfunction, elevation of the serum creatinine or bilirubin and hepatic enzymes has occurred during rhuGM -CSF administration. Dose reducti on or interruption of rhuGM -CSF administration has resulted in a decrease to pretreatment values. Occasional transient and reversible supraventricular arrhythmia has been reported in uncontrolled studies, particularly in patients with a previous history o f cardiac arrhythmia. Stimulation of marrow precursors with rhuGM -CSF may result in a rapid rise in white blood cell count. If rhuGM -CSF is being used for the purpose of hematopoietic reconstitution, dosing should be stopped if the ANC exceeds 20,000/cm3. The dose being used in this study should not affect ANC levels. RhuGM -CSF may stimulate the growth of myeloid malignancies; therefore caution must be exercised in its use in these malignancies or myelodysplastic syndromes. RhuGM -CSF is a sterile, whit e, preservative- free lyophilized powder in 250 mcg vials. Reconstituted rhuGM -CSF will be admixed and administered with the HER2 vaccine. C. Vaccine Dose Compounding and Dispensing Table 2. HER2 ICD peptides HER2 ICD Vaccine Sequence p776 -790 GVGSPYVSRLLGICL p927 -941 PAREIPDLLEKGERL p1166 -1180 TLERPKTLSPGKNGV 7866/135 08/23/2021 Page 16 of 39 The HER2 ICD Vaccine is supplied in single -use vials as a sterile, frozen solution. Each single- use 1.1 ml vial contains equal volumes of ICD peptides, p776- 790, p927- 941, p1166- 1180 (625 mcg of each peptide per ml ) for a final concentration of 1.9 mg/ml total ICD peptide mix. The ICD va ccine will be supplied as frozen vials and each vial will carry a label bearing the drug identification and conditions for storage. The University of Washington Investigational Drug Pharmacy (UW IDS) will prepare and dispense the drug for administration per their standard operating procedure as follows : 1. Reconstitute rhuGMCSF 250mcg with 0.5ml sterile preservative free water and add rhuGMCSF 125mcg/0.25ml to a sterile empty 2ml vial . 2. Thaw HER2 ICD Vaccine 1.9mg/ml and w ithdraw 1 ml and add to vial containing the rhuGMCSF. Total volume in vial is 1.25ml . Rotate vial gently to mix. 3. Total dose is H ER2 ICD 1500mcg and GMCSF 100mcg in 0.99ml. Divide dose into 3 syringes, containing 0.33ml each for intradermal injection. D. PSK Product and Placebo This PSK and Placebo characterization and storage are covered by BB IND 114546 (Sponsor: Leanna Standish, PhD, ND). The PSK® and Placebo are to be stored at room temperature (59ºF - 86ºF). 1. Description and Composition of PSK® Clinical Drug Product The PSK® clinical drug product is a granular powder, light tan to dark brown in color, which is packaged in unit dose aluminum pouches (Table 3) by First Priority in Ferndale, WA . There are no excipients, other botanical substances, synthetic or highly purified drugs, biotechnology -derived drugs, or other naturally derived drugs in the drug product. Once reconstituted with water , a clear to slightly turbid brownish solution is obtained for oral administration. 2. Description and Composition of Placebo : The placebo is a granular powder, light tan to dark brown but mottled with white in color, which is packaged in unit dose aluminum pouches by First Priority in Ferndale, WA . There are no botanical substances, synthetic or highly purified drugs, biotechnology -derived drugs, or other naturally derived drugs in the drug product (Table 4). Once reconstituted with water , a clear to slightly turbid brownish solution is obtained for oral administration. 3. Description and Composition of Container Closure System : The primary container closure system for PSK ® clinical drug product and placebo is a laminated aluminum pouch. The aluminum pouch is composed of components that are commonly used in the pharmaceutical industry for solid, oral dosage form products. The container closure system is selected based on c omparable specifications to that used for the marketed drug product. 4. Description and Composition of Dosage Preparation Kit : A shaker cup/lid is provided to facilitate preparation of the oral dosage solution. Briefly, the PSK ® clinical drug product or placebo is emptied into the shaker cup, which has already been filled with approximately 4-8 oz. of warm water . The lid is then applied to seal the cup and shaken vigorously to dissolve the powder. The liquid preparation is to be consumed within 15 minutes. After the initial Table 3. Quantitative Composition of the PSK® Clinical Drug Product in Each Unit Dose Pouch Component Source Amount Function PSK® Bulk Drug Product Kureha 1.5 g Active Ingredient Table 4. Description and Composition of Placebo Component Source Amount Function Mannogem®™ 2080, Granular Mannitol, USP SPI Pharma (Wilmington, DE) 1.2 g Bulking Agent Caramel Color 643a D. D.