IdeS effect on Daudi cell proliferation for 48 hours

INTRODUCTION Alloimmune platelet refractoriness (APR), an insufficient response to platelet transfusion due to anti-HLA alloantibodies, affects ~25% of acute myeloid leukemia (AML) patients. In APR, HLA- or crossmatch-compatible platelet units may not be identifiable, precluding transfusion and increasing hemorrhage risk. Single antigen bead testing (SAB) semi-quantitates antibody levels against cognate HLA antigens in units of mean fluorescence intensity (MFI). MFI >3,000 suggests an incompatible platelet antigen for transfusion. Imlifidase, an IgG-specific cysteine protease, cleaves IgG first into single-cleaved IgG (scIgG) with limited Fc-mediated function, then into inactive F(ab') fragments 1. We previously reported successful desensitization and provision of permissive platelet transfusions in an APR patient following desensitization treatment with imlifidase. Here we report pre- and post-desensitization alloantibody (PDAb) levels in correlation with IgG degradation products and response to single donor platelet transfusion. CASE As described previously 2, a 43-year-old highly sensitized (calculated PRA [cPRA] 99.9%) woman with AML and history of life-threatening thrombocytopenic hemorrhage secondary to APR presented for matched-unrelated donor stem cell transplantation. Nationwide, few compatible platelet units were identified to support her through the anticipated platelet nadir. Following conventional desensitization, cPRA did not change significantly. Following imlifidase-desensitization [0.25 mg/kg IV (D-1 and D1)], cPRA decreased significantly. 7 single-donor, antigen-avoidance platelet units produced corrected count increments (CCI) [median (interquartile range) of 6,000 (500-7500)]. The patient experienced no hemorrhage and was discharged stable, post-platelet engraftment on D+19. METHODS With IRB approval, patient data and samples (D-16; D-1 post-conventional desensitization with rituximab and therapeutic plasma exchange; D0 post-imlifidase dose 1; D2 post-imlifidase dose 2; and D7, D14 and D29 follow-up samples) were collected. Serial dilutions of patient D-1 serum were tested with SAB (LABScreen single antigen, One Lambda/ThermoFisher, West Hills, California). SDS-PAGE (gradient gel, non-reduced conditions), Western Blot (antiF(ab') detection), and SAB were performed and acceptable antigens (to molecular specificity, targeted by MFI ≤3,000) were compared over the clinical course. Summative MFIs targeting transfused single donor platelet units were compared with 1-hour CCI. Wilcoxon matched-pairs signed rank for matched, nonparametric values and Chi-squared or Fisher's exact for categorical variables were used. P <0.05 was significant. RESULTS/DISCUSSION The D-1 sample (post-conventional desensitization, pre-imlifidase) showed exquisite HLA sensitization. The highest Class A and B antibodies demonstrated an effective MFI of >4,000,000, each (Fig. A). PDAb and the number of unacceptable antigens decreased significantly in D0 (post-imlifidase) vs. D-1 (pre-imlifidase) samples; Intact IgG and scIgG were eliminated from the D0 and D2 samples (Fig. B). Of 7 single donor platelet transfusions, the 5 producing adequate response were each targeted by a summative MFI ≤4,000 (median 4,000 MFI, range 300-4,000). The 2 units producing inadequate CCI response were targeted by summative MFI >4,000 (5,050, 4,900-5,200), suggesting a threshold summative MFI precluding adequate response. CONCLUSIONS Here we show that the patient's pre-desensitization alloantibody levels significantly exceeded the analytical range of the SAB; PDAb levels declined significantly from baseline, correlating with lack of intact IgG and scIgG following imlifidase; and permissive transfusions (CCI >5,000) were seen to platelets targeted by ≤4,000 summative MFI. These results suggest imlifidase is capable of effective desensitization despite profound baseline sensitization, with rapid cleavage of intact IgG to F(ab'), resulting in the temporary reduction of unacceptable antigens. Larger scale investigation of imlifidase in desensitization of APR patients requiring significant transfusion over a limited period of time, along with investigation into transfusion strategies in desensitized APR, are warranted. REFERENCES Am J Transpl. 2022;22(3):691-697.Blood. 2022;140 (Suppl. 1):8562-3. .

SDS-PAGE separation of plasma treated with 10 ÂÃ,âµg/mL IdeS

Human IgG concentration in mouse serum after a single i.p. administration of 1 g/kg IVIg in SCID mice.

Background. Imlifidase is an immunoglobulin G (IgG)-specific protease conditionally approved in the EU for desensitization in highly sensitized crossmatch positive kidney transplant patients. Imlifidase efficiently cleaves both heavy chains of IgG in a 2-step process. However, low levels of the intermediate cleavage product, single-cleaved IgG (scIgG), may persist in the circulation. The study objective was to investigate Fc-mediated effector functions of scIgG and its potential impact on common clinical immunologic assays used to assess transplant eligibility. Methods. Imlifidase-generated scIgG, obtained by in vitro cleavage of HLA-sensitized patient serum or selected antibodies, was investigated in different complement- and FcγR-dependent assays and models, including clinical tests used to evaluate HLA-specific antibodies. Results. ScIgG had significantly reduced Fc-mediated effector function compared with intact IgG, although some degree of activity in complement- and FcγR-dependent models was still detectable. A preparation of concentrated scIgG generated from a highly HLA-sensitized individual gave rise to a positive signal in the anti-HLA IgG LABScreen, which uses anti-Fc detection, but was entirely negative in the C1qScreen. The same high-concentration HLA-binding scIgG preparation also generated positive complement-dependent cytotoxicity responses against 80%–100% of donor T and B cells, although follow-up titrations demonstrated a much lower intrinsic activity than for intact anti-HLA IgG. Conclusions. ScIgG has a significantly reduced capacity to mediate Fc-dependent effector functions. However, remaining HLA-reactive scIgG in plasma after imlifidase treatment can cause positive assay results equivalent to intact IgG in clinical assays. Therefore, complete IgG cleavage after imlifidase treatment is essential to allow correct decision-making in relation to transplant eligibility.

Infectious diseases are characterized by a complex interplay between host and pathogen, but how these interactions impact the host proteome is unclear. Here we applied a combined mass spectrometry-based proteomics strategy to investigate how the human proteome is transiently modified by the pathogen Streptococcus pyogenes, with a particular focus on bacterial cleavage of IgG in vivo In invasive diseases, S. pyogenes evokes a massive host response in blood, whereas superficial diseases are characterized by a local leakage of several blood plasma proteins at the site of infection including IgG. S. pyogenes produces IdeS, a protease cleaving IgG in the lower hinge region and we find highly effective IdeS-cleavage of IgG in samples from local IgG poor microenvironments. The results show that IdeS contributes to the adaptation of S. pyogenes to its normal ecological niches. Additionally, the work identifies novel clinical opportunities for in vivo pathogen detection.

Safety, immunogenicity, pharmacokinetics, and efficacy of the IgG-degrading enzyme of Streptococcus pyogenes (IdeS [imlifidase]) were assessed in a single-center, open-label ascending-dose study in highly sensitized patients with chronic kidney disease. Eight patients with cytotoxic PRAs (median cytotoxic PRAs of 64%) at enrollment received 1 or 2 intravenous infusions of IdeS on consecutive days (0.12 mg/kg body weight ×2 [n = 3]; 0.25 mg/kg ×1 [n = 3], or 0.25 mg/kg ×2 [n = 2]). IgG degradation was observed in all subjects after IdeS treatment, with <1% plasma IgG remaining within 48 hours and remaining low up to 7 days. Mean fluorescence intensity values of HLA class I and II reactivity were substantially reduced in all patients, and C1q binding to anti-HLA was abolished. IdeS also cleaved the IgG-type B cell receptor on CD19+ memory B cells. Anti-IdeS antibodies developed 1 week after treatment, peaking at 2 weeks. A few hours after the second IdeS infusion, 1 patient received a deceased donor kidney offer. At enrollment, the patient had a positive serum crossmatch (HLA-B7), detected by complement-dependent cytotoxicity, flow cytometry, and multiplex bead assays. After IdeS infusion (0.12 mg/kg ×2) and when the HLA-incompatible donor (HLA-B7+ ) kidney was offered, the HLA antibody profile was negative. The kidney was transplanted successfully.

The British Journal of Haematology publishes original research papers in clinical, laboratory and experimental haematology. The Journal also features annotations, reviews, short reports, images in haematology and Letters to the Editor.

Autoantibodies binding to peripheral nerves followed by complement deposition and membrane attack complex formation results in nerve damage in Guillain-Barré syndrome (GBS). Strategies to remove the pathogenic autoantibodies or block the complement deposition benefit most patients with GBS. Immunoglobulin G-degrading enzyme of Streptococcus pyogenes (IdeS) is a cysteine protease which cleaves IgG antibodies into F(ab′)2 and Fc fragments. In this study, using a rabbit model of axonal GBS, acute motor axonal neuropathy (AMAN), we demonstrated that IdeS treatment significantly reduced the disruption of Nav channels as well as activated C3 deposition at the anterior spinal root nodes of Ranvier in AMAN rabbits. IdeS significantly promoted the clinical recovery of AMAN rabbits and there were significant lower frequencies of axonal degeneration in anterior spinal roots of AMAN rabbits with IdeS treatment compared to the saline controls. Our data support that IdeS treatment is a promising therapeutic strategy for GBS.

BACKGROUND:Donor-specific antibodies create an immunologic barrier to transplantation. Current therapies to modify donor-specific antibodies are limited and ineffective in the most highly HLA-sensitized patients. The IgG-degrading enzyme derived from Streptococcus pyogenes (IdeS), an endopeptidase, cleaves human IgG into F(ab')2 and Fc fragments inhibiting complement-dependent cytotoxicity and antibody-dependent cellular cytotoxicity, which suggests that IdeS might be useful for desensitization. We report on the combined experience of two independently performed open-label, phase 1-2 trials (conducted in Sweden and the United States) that assessed the efficacy of IdeS with regard to desensitization and transplantation of a kidney from an HLA-incompatible donor.METHODS:We administered IdeS to 25 highly HLA-sensitized patients (11 patients in Uppsala or Stockholm, Sweden, and 14 in Los Angeles) before the transplantation of a kidney from an HLA-incompatible donor. Frequent monitoring for adverse events, outcomes, donor-specific antibodies, and renal function was performed, as were renal biopsies. Immunosuppression after transplantation consisted of tacrolimus, mycophenolate mofetil, and glucocorticoids. Patients in the U.S. study also received intravenous immune globulin and rituximab after transplantation to prevent antibody rebound.RESULTS:Recipients in the U.S. study had a significantly longer cold ischemia time (the time elapsed between procurement of the organ and transplantation), a significantly higher rate of delayed graft function, and significantly higher levels of class I donor-specific antibodies than those in the Swedish study. A total of 38 serious adverse events occurred in 15 patients (5 events were adjudicated as being possibly related to IdeS). At transplantation, total IgG and HLA antibodies were eliminated. A total of 24 of 25 patients had perfusion of allografts after transplantation. Antibody-mediated rejection occurred in 10 patients (7 patients in the U.S. study and 3 in the Swedish study) at 2 weeks to 5 months after transplantation; all these patients had a response to treatment. One graft loss, mediated by non-HLA IgM and IgA antibodies, occurred.CONCLUSIONS:IdeS reduced or eliminated donor-specific antibodies and permitted HLA-incompatible transplantation in 24 of 25 patients. (Funded by Hansa Medical; ClinicalTrials.gov numbers, NCT02224820 , NCT02426684 , and NCT02475551 .).

Abstract Endogenous plasma IgG sets an immunologic threshold that dictates the activity of tumor-directed therapeutic antibodies. Saturation of cellular antibody receptors by endogenous antibody limits antibody-dependent cell-mediated cytotoxicity (ADCC) and antibody-dependent cellular phagocytosis (ADCP). Here, we show how enzymatic cleavage of IgG using the bacterial enzyme IdeS can be utilized to empty both high and low affinity Fcγ-receptors and clear the entire endogenous antibody pool. Using in vitro models, tumor animal models as well as ex vivo analysis of sera collected during a previous clinical trial with IdeS, we show how clearing of competing plasma antibody levels with IdeS unblocks cellular antibody receptors. We show that therapeutic antibodies against breast cancer (trastuzumab), colon cancer (cetuximab), and lymphomas (rituximab and alemtuzumab) can be potentiated when endogenous IgG is removed. Overall, IdeS is shown to be a potent tool to reboot the human antibody repertoire and to generate a window to preferentially load therapeutic antibodies onto effector cells and thereby create an armada of dedicated tumor-seeking immune cells. Mol Cancer Ther; 16(9); 1887–97. ©2017 AACR.

IdeS is a streptococcal protease that cleaves IgG antibodies into F(ab’)2 and Fc fragments with a unique degree of specificity, thereby providing a novel treatment opportunity of IgG-driven autoimmune conditions and antibody mediated transplant rejection. Here we report the results from a first in man, double blinded and randomized study with single ascending doses of IdeS in healthy, male subjects. Twenty healthy subjects were given intravenous single ascending doses of IdeS. With impressive efficacy IdeS cleaved the entire plasma IgG-pool only minutes after dosing. IgG reached nadir 6-24 hours after dosing and then slowly recovered. The half-life of IdeS was 4.9 (±2.8) hours at 0.24 mg/kg with the main fraction eliminated during 24 hours. Already two hours after IdeS-dosing, the phagocytic capacity of IgG/IgG-fragments was reduced to background levels. Importantly, IdeS has the capacity to inactivate Fc-mediated effector function in vivo, was considered safe with no serious adverse events, and without dose limiting toxicity in this study. The complete, rapid, but temporary removal of IgG provides a new potent therapeutic opportunity in IgG-mediated pathogenic conditions. Trial Registration ClinicalTrials.gov NCT01802697

Ag binding to the BCR is a critical step in B cell development and activation, initiating a cascade of signaling events ultimately leading to proliferation, differentiation, or cell death. A bacterial enzyme, IgG-degrading enzyme of Streptococcus pyogenes (IdeS), was shown to specifically cleave IgG molecules below the hinge region of soluble IgG and when IgG is bound to Ag, resulting in one F(ab′)2 molecule and one homodimeric Fc fragment. Whether IdeS could also cleave the IgG molecule when it is present in the BCR attached to the B cell membrane in a complex with CD79a and CD79b is unknown. In this article, we present human in vitro and ex vivo data showing that IdeS cleaves the IgG present in the BCR complex and very efficiently blocks Ag binding to the BCR. As a consequence of IdeS cleaving the BCR, signaling cascades downstream of the BCR are blocked, and memory B cells are temporarily silenced, preventing them from responding to antigenic stimulation and their transition into Ab-producing cells.