Pan HLA I Chimeric Human IgG Reveal Subclass Differences in Complement Fixing Assays.

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Purpose: The purpose of this study was to compare hIgG1 and hIgG2 HLA I antibodies in their capacity to activate complement in in vitro assays, and to determine whether any correlation existed between antibody strength (MFI in single antigen assay) and ability to fix C1q. Methods: We generated chimeric pan HLA I antibodies by fusing W6/32 variable regions to human IgG1 and human IgG2 constant regions. Binding to HLA I and activation of complement was assessed by solid phase (SAB Labscreen and C1qScreen (OneLambda); C3d bead assay (Immucor/Lifecodes) and cell-based assays (flow cytometry, cell-based ELISA). Results: HLA I hIgG1 and hIgG2 bound highly to all alleles and loci of HLA I on single antigen (SAB) beads. While the distribution differed among alleles, depending on antigen density (with Cw15 having the highest, and Cw4 the lowest, MFI), there was no significant difference between subclasses (R2=0.967). Both chimers also bound to native antigen on endothelial cells (EC). HLA I hIgG1 caused C1q deposition on all LSA1 beads in a concentration dependent manner. There was an association between SAB MFI and C1q binding that varied depending on HLA locus; on B locus beads (R2>0.9) but not C locus beads. C1q positivity (MFI>1000) correlated with MFI in SAB tests of >12000 MFI. C1q was also deposited on all HLA I beads in response to high dose HLA I hIgG2, although hIgG2 was significantly less potent than hIgG1 at fixing C1q. The degree of C1q deposition by HLA I hIgG2 varied significantly (MFI >25000 to <1000) from bead to bead at the same concentration of antibody, particularly on C locus beads. hIgG2 showed poor correlation between SAB MFI and C1q-fixing capacity. Both HLA I hIgG1 and hIgG2 also resulted in functional complement activation, as measured by C3d deposition on single antigen beads, C3b deposition on EC, and CDC-XM positivity in a concentration-dependent manner. hIgG1 was significantly more effective at complement activation than hIgG2. Conclusions: hIgG2, while less potent than hIgG1, fixed complement in C1q, C3b and CDC assays at high concentrations, challenging the notion that hIgG2 DSA is less pathogenic. Moreover, C1q deposition in solid phase assays was quite variable from bead to bead, at the same concentration of pan HLA I antibody, suggesting that antigen density or denatured state of antigen significantly influences sensitivity in complement assays. In general, HLA-C locus beads were the most variable in assays measuring hIgG and C1q binding. DISCLOSURE:Reed, E.: Grant/Research Support, Pfizer, Novartis, True North Therapeutics, Immucor.