Background. Imlifidase is an IgG-cleaving endopeptidase conditionally approved in Europe for desensitization of highly sensitized patients before kidney transplantation. We present 5-y outcomes and donor-specific antibody (DSA) levels for clinical trial participants from a single site who received imlifidase for desensitization before incompatible transplantation (NCT02790437). Methods. Imlifidase was administered up to 24 h before living or deceased donor kidney transplantation. DSAs were monitored before transplantation, at days 7 and 28, and at 5 y posttransplant. Results. At 5 y, 7 of 8 participants were alive. One of these 7 had suboptimal graft function secondary to donor-derived disease but remained dialysis independent. Three participants had antibody-mediated rejection (AMR), which occurred in the first 30 d in all cases and was successfully treated. No new episodes of suspected or biopsy-proven AMR occurred after 30 d posttransplant. Seven participants had DSA rebound. DSAs commonly persisted 5 y posttransplant, although they were generally lower strength compared with pre-imlifidase. Dilution studies of sensitized serum enabled the identification of lower AMR risk phenotypes for persisting DSAs. Severe and/or opportunistic infections were not observed at greater than expected frequency. Conclusions. Five-year outcomes of imlifidase-enabled incompatible transplants are overall favorable. DSA rebound is common, but antibody strength lessens in the long term, and longitudinally persisting DSAs did not lead to premature graft failure.

In four phase 2 clinical trials, patients underwent imlifidase-enabled kidney transplantation, converting a positive crossmatch (+XM) to negative. Here, we present data from 39 patients enrolled in the 5-year follow-up extension trial. Patient survival, graft survival, renal function, delayed graft function (DGF) antibody mediated rejection (AMR) frequency, safety and presence of donor specific antibodies (DSAs) are presented. Data from the long-term follow-up trial (17-HMedIdeS-14); NCT 03611621) with planned visits at 1, 2, 3, and 5 years after imlifidase were combined with up to 6-month phase 2 data from 4 pivotal trials (13-HMedIdeS-02, 13-HMedIdeS-03, 14-HMedIdeS-04, 15-HMedIdeS-06). Five-year patient survival was 90% with 3 deaths occurring between 6 months and 1 year, with no deaths occurring subsequently. Death-censored graft survival was 82% (with 3 early graft losses and 3 graft losses between 2 and 5 years). Mean eGFR at 5 years was 50.1 (MDRD), 55.8 (CKD-EPI, 2021) and 52.5 (KRS, 2023) mL/min/1.73 m2 (N = 24 patients) among functioning grafts. DSA rebounded with levels progressively decreasing over time and no increase in DSA seen between 3 and 5 years. No AMR occurred between 3 and 5 years. Furthermore, no additional safety signals assessed as related to imlifidase were reported in this cohort. At 5 years, highly-HLA sensitized patients who underwent imlifidase desensitization prior to transplantation demonstrated excellent patient and graft survival, despite their high-risk immunological profile. Imlifidase offers a viable and effective treatment option for highly sensitized patients to achieve life-saving transplantation and continued optimism is warranted.Clinical TrialClinicalTrials.gov (NCT02790437), EudraCT Number: 2016-002064-13.

BACKGROUND:Antibody-mediated rejection (ABMR) poses a barrier to long-term graft survival and is one of the most challenging events after kidney transplantation. Removing donor specific antibodies (DSA) through therapeutic plasma exchange (PLEX) is a cornerstone of antibody depletion but has inconsistent effects. Imlifidase is a treatment currently utilized for desensitization with near-complete inactivation of DSA both in the intra- and extravascular space. METHODS:This was a 6-month, randomized, open-label, multicenter, multinational trial conducted at 14 transplant centers. Thirty patients were randomized to either imlifidase or PLEX treatment. The primary endpoint was reduction in DSA level during the 5 days following the start of treatment. RESULTS:Despite considerable heterogeneity in the trial population, DSA reduction as defined by the primary endpoint was 97% for imlifidase compared to 42% for PLEX. Additionally, imlifidase reduced DSA to noncomplement fixing levels, whereas PLEX failed to do so. After antibody rebound in the imlifidase arm (circa days 6-12), both arms had similar reductions in DSA. Five allograft losses occurred during the 6 months following the start of ABMR treatment-four within the imlifidase arm (18 patients treated) and one in the PLEX arm (10 patients treated). In terms of clinical efficacy, the Kaplan-Meier estimated graft survival was 78% for imlifidase and 89% for PLEX, with a slightly higher eGFR in the PLEX arm at the end of the trial. The observed adverse events in the trial were as expected, and there were no apparent differences between the arms. CONCLUSION:Imlifidase was safe and well-tolerated in the ABMR population. Despite meeting the primary endpoint of maximum DSA reduction compared to PLEX, the trial was unsuccessful in demonstrating a clinical benefit of imlifidase in this heterogenous ABMR population. TRIAL REGISTRATION:EudraCT number: 2018-000022-66, 2020-004777-49; ClinicalTrials.gov identifier: NCT03897205, NCT04711850.

IdeS effect on Daudi cell proliferation for 48 hours

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.

Imlifidase is a cysteine proteinase which specifically cleaves IgG, inhibiting Fc‐mediated effector function within hours of administration. Imlifidase converts a positive crossmatch to a potential donor (T cell, B cell, or both), to negative, enabling transplantation to occur between previously HLA incompatible donor‐recipient pairs. To date, 39 crossmatch positive patients received imlifidase prior to a kidney transplant in four single‐arm, open‐label, phase 2 studies. At 3 years, for patients who were AMR+ compared to AMR−, death‐censored allograft survival was 93% vs 77%, patient survival was 85% vs 94%, and mean eGFR was 49 ml/min/1.73 m2 vs 61 ml/min/1.73 m2, respectively. The incidence of AMR was 38% with most episodes occurring within the first month post‐transplantation. Sub‐analysis of patients deemed highly sensitized with cPRA ≥ 99.9%, and unlikely to be transplanted who received crossmatch‐positive, deceased donor transplants had similar rates of patient survival, graft survival, and eGFR but a higher rate of AMR. These data demonstrate that outcomes and safety up to 3 years in recipients of imlifidase‐enabled allografts is comparable to outcomes in other highly sensitized patients undergoing HLA‐incompatible transplantation. Thus, imlifidase is a potent option to facilitate transplantation among patients who have a significant immunologic barrier to successful kidney transplantation.

Aim: The aim of this study was to investigate if human IgM is a cleavable substrate for imlifidase and to explain an observed effect in anti-HLA IgM single antigen bead (SAB) assays in sensitized patients. Methods: Serum samples collected pre-and 24 h post-imlifidase administration from sensitized patients enrolled in a phase II trial were investigated for anti-HLA IgG and IgM using SAB assays, with and without in vitro IgG depletion using a CaptureSelectTM affinity matrix. In addition, pre-dose samples and purified human IgM samples were treated with imlifidase in vitro and evaluated by SDS-PAGE, Western blot (PE-conjugated anti-human IgM) and SAB (IgG, IgM) assays. Results: By comparing the mean fluorescence intensity (MFI) of HLA-beads, pre-and post-imlifidase administration, three IgM-related patterns were observed; IgM-specific HLA-SABs with an increased MFI post-imlifidase, IgM-specific HLA-SABs with a decreased MFI post-imlifidase, and IgM-specific HLA-SABs with a marginal MFI difference between the pre-and post-imlifidase administration. These IgM signal patterns were observed despite neither purified IgM nor serum IgM could be cleaved by imlifidase. After removing IgG, the effects observed on anti-HLA IgM was largely eliminated with the biggest differences seen in patients with very high anti-HLA IgG in pre-dose samples. Conclusion: We demonstrate that imlifidase does not cleave human IgM, including HLA-specific IgM antibodies from highly sensitized subjects. Observed decreases of SAB-HLA IgM signals after imlifidase treatment may result from the cleavage of IgG-IgM complexes which are bound to SAB-HLA. Serum analysis of patients with high levels of anti-HLA IgG will result in a more accurate SAB-HLA IgM reading after IgG depletion.

Background. Highly HLA sensitized patients have limited access to life-saving kidney transplantation because of a paucity of immunologically suitable donors. Imlifidase is a cysteine protease that cleaves IgG leading to a rapid decrease in antibody level and inhibition of IgG-mediated injury. This study investigates the efficacy and safety of imlifidase in converting a positive crossmatch test to negative, allowing highly sensitized patients to be transplanted with a living or deceased donor kidney. Methods. This open-label, single-arm, phase 2 trial conducted at 5 transplant centers, evaluated the ability of imlifidase to create a negative crossmatch test within 24 h. Secondary endpoints included postimlifidase donor-specific antibody levels compared with predose levels, renal function, and pharmacokinetic/pharmacodynamic profiles. Safety endpoints included adverse events and immunogenicity profile. Results. Of the transplanted patients, 89.5% demonstrated conversion of baseline positive crossmatch to negative within 24 h after imlifidase treatment. Donor-specific antibodies most often rebounded 3–14 d postimlifidase dose, with substantial interpatient variability. Patient survival was 100% with graft survival of 88.9% at 6 mo. With this, 38.9% had early biopsy proven antibody–mediated rejection with onset 2–19 d posttransplantation. Serum IgG levels began to normalize after ~3–7 d posttransplantation. Antidrug antibody levels were consistent with previous studies. Seven adverse events in 6 patients were classified as possibly or probably related to treatment and were mild-moderate in severity. Conclusions. Imlifidase was well tolerated, converted positive crossmatches to negative, and enabled patients with a median calculated panel-reactive antibody of 99.83% to undergo kidney transplantation resulting in good kidney function and graft survival at 6 mo.

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.