Journal: Molecular cancer therapeutics
Article Title: Potent Immune Modulation by MEDI6383, an Engineered Human OX40 Ligand IgG4P Fc Fusion Protein
doi: 10.1158/1535-7163.MCT-17-0200
Figure Lengend Snippet: Schematic diagram of the proposed structure of MEDI6383. Figure 1 shows that MEDI6383 is a human OX40 ligand IgG4P Fc fusion protein consisting of three subunits containing an OX40L receptor binding domain (RBD), a TRAF2 coiled coil domain, and an IgG4 Fc domain containing an S→P mutation at position 228 according to EU numbering of the full length IgG4 heavy chain sequence to prevent inter-Fc strand exchange. (A) The proposed structure of the protein product is a hexameric “dimer of trimers”. The OX40L RBDs are oriented from N-terminal amino acids (top) to the C-terminus (bottom) as indicated. (B) Analytical Size Exclusion Chromatography (SEC) analysis of intact MEDI6383 showed highly monomeric (> 98%) single peak with very low levels of aggregates, no fragments or free chains. (C) SEC combined with Multi-Angle Light Scattering (SEC-MALS) showed an apparent molecular mass of approximately 330,000 daltons which supports the proposed hexameric form. Light trace spanning entire elution time, MEDI6383 sample; darker trace, molecular mass standards.
Article Snippet: FcγR-blocking reagents and controls used to demonstrate FcγR-dependent activity in 2-cell reporter bioassays included: bovine serum albumin (Sigma-Aldrich), hexameric GITRL fusion proteins containing human IgG1 (control hexamer IgG1) or human IgG4P (control hexamer IgG4P) Fc domains ( 12 , 13 ) that do not bind OX40 or activate OX40 reporter cells but can compete with MEDI6383 for FcγR binding (generated at MedImmune), LEAF ™ purified mouse IgG1 κ isotype control antibody clone MOPC-21 (Biolegend), LEAF ™ purified mouse anti-human CD64 antibody clone 10.1, sterile PBS-dialyzed purified mouse anti-human CD32 antibody clone FUN-2 (Biolegend), purified human IgG gamma globulin (Jackson Immunoresearch, West Grove, PA), and sterile human AB serum (Thermo Fisher Scientific, Waltham, MA).
Techniques: Binding Assay, Mutagenesis, Sequencing, Size-exclusion Chromatography