Background/Purpose: PRTX-100, a highly-purified GMP staphylococcal protein A (SpA), is currently in clinical trials treating patients with active rheumatoid arthritis (RA). It has been reported that binding of SpA to rabbit Vh3 IgM antibodies could deplete complement hemolytic activity and release C3a. Furthermore, complement activation is a postulated mechanism for dosing reactions seen in several patients after overly-rapid injection of SpA. We therefore looked at ex vivo effects of: 1. Adding SpA to pooled normal serum using a highly-sensitive modified CH50 assay; and 2. Adding SpA to fresh healthy donor blood on production of stable metabolites of C3a, C4a, and C5a, which are anaphylatoxins and known vasoactive and immunomodulatory mediators. Additionally, these analytes were measured in plasma from RA patients, before and after dosing with SpA.   

Methods: CH50 studies: Pooled normal donor serum was incubated with various concentrations of SpA or positive controls and then assayed for residual complement activity (measured as CH50). Whole blood studies: Heparinized blood was incubated at 37 °C with 0, 250, 500, or 2000 ng/mL of SpA or with zymosan as a positive control. At 15 and 60 minute intervals, samples were evaluated using a multiplexed cytometric bead array (CBA) assay to quantify C3a, C4a, C5a stable metabolites. Patient studies: EDTA/Futham plasma samples were obtained before and after the first and fifth infusion with SpA, and frozen for CBA analysis of anaphylatoxins. Sequential groups of 6 patients were infused weekly with 1.5, 3.0, 6.0, or 12 µg/kg of SpA. 

Results: In the CH50 study, 3 replicate experiments were performed adding serial two-fold dilutions of SpA from 4000 to 125 ng/mL. All CH50 values averaged between 85% and 108% of serum incubated without SpA, with no SpA dose response observed. CH50 values at 4000 ng/mL SpA were 114, 106, and 98% of the serum control.  Incubation with a complement-activating nanoparticle reduced hemolytic activity to undetectable levels (< 5% of serum control).  Using whole blood from 3 donors, and the CBA assay, C3a increased a maximum of 2.5-fold with SpA addition, compared to no addition.  The mean increase with zymosan addition was 332-fold.  For C4a the maximum increase was 2.3-fold compared with a mean 99-fold for zymosan addition.  For C5a the maximum increase was 2.3-fold that seen for control, compared with a mean 213-fold increase with zymosan addition.  Data for pre- and post-infusion samples for 47 infusions in the first 28 patients dosed, showed 7/28 had 3-fold or greater increase in C3a (range 3 to 14 fold) but only 2/28 had a 2-fold or greater increase in C4a (range 2.4 to 2.7 fold).  The  mean of the ratio between pre- and post-dose values was 2.1, 1.0, and 1.1 for C3a, C4a, and C5a respectively. The maximum fold increases were 14.0, 2.7, and 3.0 respectively.

Conclusion: Ex vivo experiments do not demonstrate activation of complement in serum or whole blood by SpA at concentrations of up to 4000 ng/mL (serum) or 2000 ng/mL (blood). One quarter of patients experienced a 3-fold or greater increase in C3a after SpA treatment, which was not associated with any dosing symptoms.  C4a and C5a were unaffected by treatment.

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/complement-activation-and-anaphylatoxin-generation-in-response-to-staphylococcal-protein-a-exposure-ex-vivo-and-in-vivo-human-studies/