Montanide isa 51

Vaccination with a long peptide forces uptake and presentation by antigen presenting cells, whereas vaccination with short peptides (9–10 amino acids) could lead to merely coating HLA molecules. Thus, vaccination with long peptides permits inclusion of patients without HLA-type restriction, and is associated with stimulation of both CD4⁺ and CD8⁺ T cells (29 (link)). In this study, patients were administered subcutaneous vaccinations containing 100 μg IO103, a 19-amino-acid peptide (FMTYWHLLNAFTVTVPKDL) from the signal peptide of PD-L1 (PolyPeptide Laboratories, France). The peptide was dissolved in dimethylsulfoxide (DMSO), sterile filtered, and frozen at –20° Celsius (NUNC™ CryoTubes™ CryoLine System™ Internal Thread, Sigma-Aldrich). At ≤2 h before administration, the peptide was thawed and dissolved in sterile water for injection. Immediately before injection, the dissolved peptide was emulsified 1:1 with the adjuvant Montanide ISA-51 (Seppic Inc. Paris, France) to a total volume of 1 ml (30 (link)). Vaccinations were administered by subcutaneous injection every two weeks, repeated six times, and then once every 4 weeks until reaching a total of 15 vaccines.

Six peptides from gp100, tyrosinase, MAGE-A1, and MAGE-A10 proteins were selected based on high immunogenicity in prior clinical trials using MEPs for CD8⁺ T cells14 15 31 (link) restricted by HLA-A1, A2, or A3: long peptides encompassing those MEPs were constructed. Also, the NY- ESO-1 _94-102 peptide, containing a MEP for CD8⁺ T cells restricted by HLA-B35 and B51, was selected based on immunogenicity in a prior trial and.12 (link) The sequences and known immunogenic MEP for each peptide are listed in table 1.
Montanide ISA-51 was purchased from Seppic Inc (Fairfield, New Jersey, USA) as cGMP material in sterile vials. PolyICLC was provided as a clinical grade reagent (Hiltonol; Oncovir, Washington, DC, USA) by the Ludwig Institute for Cancer Research and its Cancer Vaccine Consortium. Resiquimod was provided by 3M Pharmaceuticals (St Paul, Minnesota, USA).

The p53 SLP vaccine consisted of 9 synthetic 25–30 amino acids long overlapping peptides, spanning amino acids 70–235 of the wt-p53 protein. Peptides were prepared at the GMP facility of the Department of Clinical Pharmacy and Toxicology at the LUMC. At the day of immunization the peptides (0.3 mg/peptide) were dissolved in dimethyl sulfoxide (DMSO, final concentration 20%) admixed with 20 mM phosphate buffer (pH 7.5) and emulsified with an equal volume of Montanide ISA-51 (Seppic). At the day of vaccination, the vaccine was prepared as previously described [24 (link)]. The vaccine (2.7 mL) was administered subcutaneously in the upper arm.

Wilms tumor 1 peptide (MPS-173; RMFPNAPYL; PolyPeptide Laboratories, San Diego, CA) vaccines were prepared in the University of Chicago HIM-cGMP facility. Briefly, one vial of WT1 peptide was thawed and mixed with 0.9 ml of sterile water. The diluted WT1 peptide was then either emulsified with Montanide ISA-51 (Seppic, Inc) or mixed with poly-ICLC (Hiltonol, Oncovir) at a 1:1 ratio for injection. The final vaccine product contained 1 mg of WT1 peptide.