The impact of IdeS (imlifidase) on allo-specific, xeno-reactive, and protective antibodies in a sensitized rhesus macaque model

Abstract

Background: Highly sensitized patients face many barriers to kidney transplantation, including higher rates of antibody-mediated rejection after HLA-incompatible transplant. IdeS, an endopeptidase that cleaves IgG nonspecifically, has been trialed as desensitization prior to kidney transplant, and successfully cleaves donor-specific antibody (DSA), albeit with rebound.

Methods: IdeS was generated and tested (2 mg/kg, IV) in two naïve and four allosensitized nonhuman primates (NHP). Peripheral blood samples were collected at regular intervals following IdeS administration. Total IgG, total IgM, and anti-CMV antibodies were quantified with ELISA, and donor-specific antibody (DSA) and anti-pig antibodies were evaluated using flow cytometric crossmatch. B cell populations were assessed using flow cytometry.

Results: IdeS successfully cleaved rhesus IgG in vitro. In allosensitized NHP, robust reduction of total, DSA, anti-pig, and anti-CMV IgG was observed within one day following IdeS administration. Rapid rebound of all IgG antibody populations was observed, with antibody levels returning to baseline around day 14 post-infusion. Total IgM level was not affected by IdeS. Interestingly, a comparable reduction in antibody populations was observed after the second dose of IdeS. However, we have not observed any significant modulation of B cell subpopulations after IdeS.

Conclusions: This study evaluated efficacy of IdeS in the allosensitized NHP in IgG with various specificities, mirroring antibody kinetics in human patients. The efficacy of IdeS on preexisting anti-pig antibodies may be useful in clinical xenotransplantation. However, given the limitation of IdeS on its durability as a monotherapy, optimization of IdeS with other agents targeting the humoral response is further needed.

Keywords: IdeS; desensitization; immunoglobulin G; rhesus macaque; sensitization.

Figure 1.. Confirmation of IdeS function on rhesus IgG in vitro.

Enzymatic activity was assessed by non-reducing SDS PAGE. Titration of IdeS (160–0.5μg) was performed against purified polyclonal rhesus IgG (40μg, animal rh2150) (lanes 5–9). The IdeS cleavage products were comparable to fragments generated after IgG treatment with the commercially available Fabricator enzyme (Genovis) (lane 3). Bands corresponding to intact, single-cleaved IgG (scIgG), F(ab’)2, and Fc/2 antibody fragments are indicated. The enzyme-only (Fabricator) band is shown in lane 4. IdeS-only band is shown in lane 10. Undigested purified rhesus polyclonal IgG is shown in lane 2.

Figure 2.. IdeS dosing and total circulating IgG and IgM following IdeS administration.

(A) Schema of IdeS dosing and blood sampling schedule. IdeS was administered to four (4) rhesus macaques 28 days following skin transplantation, and again 28 days after the first dose. Blood draws were performed at predetermined intervals (0, 1, 4, 7, 14, 21 and 28 days) following IdeS (2mg/kg, IV) administration. No immunosuppression was given after skin transplantation. (B) Robust reduction in total IgG, followed by rebound to baseline were observed following IdeS administration. (C) Total IgM levels did not change following IdeS administration. *p<0.05; **p<0.01; ***p<0.005, ****p<0.001; ns no statistical significance.

Figure 3.. The effect of a single dose of IdeS (2mg/kg, IV) on circulating IgG DSA.

The level of DSA, reported as % reduction, was measured as individual MFI intensity by TFXM (A) and BFXM (B) against rhesus skin donor PBMCs (n=4). Initial increase in DSA in response to skin transplantation was followed by robust reduction of DSA observed after IdeS administration, and subsequent rebound. This rebound did not return to baseline as it coincides with the contraction phase of DSA production following sensitization. *p<0.05; **p<0.01; ***p<0.005, ****p<0.001; ns no statistical significance.

Figure 4.. Xenoreactive anti-pig IgG following IdeS (2mg/kg, IV) administration.

The level of DSA, reported as % reduction, was measured as individual MFI intensity by TFXM (A) and BFXM (B) against WT pig PBMCs. Robust reduction in anti-WT pig IgG, followed by rebound to baseline, was observed following IdeS administration. *p<0.05; **p<0.01; ***p<0.005, ****p<0.001; ns no statistical significance.

Figure 5.. Anti-CMV IgG following IdeS (2mg/kg, IV) administration.

The level of antibody against rhCMV gB was measured with ELISA. Reduction in ant-CMV gB IgG, followed by rebound to baseline were observed following IdeS administration. *p<0.05; **p<0.01; ***p<0.005, ****p<0.001; ns no statistical significance.

Figure 6.. No significant changes in circulating B cell subpopulations after a single dose of IdeS (2mg/kg).

No significant modulation of immune cell populations was observed as compared to the pre-treatment timepoint. Total lymphocyte count (A) was stable following IdeS administration. Total (CD20⁺; B), proliferating (CD20⁺Ki67⁺; C), and IgG⁺ (CD20⁺IgG⁺; D), naïve (CD20⁺CD27⁻IgD⁺; E), both switched (CD20⁺CD27⁺IgD⁻; E) and unswitched (CD20⁺CD27⁺IgD⁺; E) memory, and exhausted (CD20⁺CD27⁻IgD⁻; E) B cell populations were also stable following IdeS administration.

Figure 7.. The impact of repeated IdeS (2mg/kg) administration on total, donor-specific, xeno-reactive, and protective IgG.

Comparable IdeS efficacy was observed following first and second doses. There was no difference in total (A), DSA (B), anti-WT pig (C), and protective (D) IgG following first and second doses. ns no statistical significance.