IgG degrading enzyme of Streptococcus pyogenes (IdeS) cleaves human IgG in two steps, first cleaving one of the heavy chains resulting in a single-cleaved IgG (scIgG) and then cleaving the second heavy chain into a F(ab')₂ and an Fc-fragment. The objective of this study was to address how scIgG present in serum at different time-points post IdeS treatment affects standard transplantation assays such as the HLA- and C1q-single antigen bead (SAB) assays and the CDC cross-match test.

Methods

Pre- and post-IdeS serum samples were collected from a patient with chronic kidney disease (CKD) dosed with IdeS within a clinical phase II study (Clinicaltrials.gov identifier NCT02224820). Different amounts of IdeS was also added to serum in vitro to generate samples containing varying amounts of IgG, scIgG, F(ab')₂ and Fc fragments. The samples were analyzed by HLA-SAB (LABScreen), C1q-SAB (C1qScreen) and CDC-XM against a mini-panel of potential donors (n = 4).

Results

Mean fluorescent intensity (MFI) remained at a high level in the HLA-SAB assay until basically all IgG was converted to F(ab')₂ fragments. In contrast, there was a dramatic drop in MFI in the C1q-SAB assay already upon conversion to scIgG.

In serum from the uremic patient MFI decreased in the HLA-SAB after dosing with IdeS and the MFI continuously decreased during several hours post dosing correlating with the conversion of scIgG into F(ab')₂. In the C1q-SAB the drop in MFI occurred instantly and C1q fixation was prevented within one hour post IdeS.

In the CDC-XM assay, intact IgG from the CKD patient reacted strongly against the mini-panel of potential donors, and scIgG generated positive cross-matches in this assay, however with a dramatic drop in titer levels compared to intact IgG. Serum containing mainly F(ab')₂ with only small amounts of remaining scIgG was negative in the CDC-XM.

A single dose of 0.25 mg/kg body weight IdeS results in a C_max of approximately 5 [micro]g/mL i.e. this corresponds roughly to the IdeS concentration utilized in this study to achieve mainly F(ab')₂ fragments and only small amounts of scIgG. At this IdeS concentration, both the C1q-SAB and the CDC-XM assay results in negative read-outs, however, the HLA-SAB can still give significant MFI signals.

Conclusions

ScIgG can give a false positive read-out in the HLA-SAB assay, but not in the C1q-SAB assay. In the CDC-XM test, scIgG has reduced activity, however, it may still have significant activity when present in high concentrations.

CITATION INFORMATION: Järnum S., Runström A., Winstedt L., Lorant T., Kjellman C. Effects of IdeS on HLA-SAB, C1q-SAB and CDC-XM Am J Transplant. 2017;17 (suppl 3).

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