Imlifidase-generated Single-cleaved IgG: Implications for Transplantation.

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.

Safety, immunogenicity, pharmacokinetics, and efficacy of degradation of anti-HLA antibodies by IdeS (imlifidase) in chronic kidney disease patients.

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.

Streptococcus pyogenes Infection and the Human Proteome with a Special Focus on the Immunoglobulin G-cleaving Enzyme IdeS.

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.

IgG Endopeptidase in Highly Sensitized Patients Undergoing Transplantation.

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 .).

Enzymatic Inactivation of Endogenous IgG by IdeS Enhances Therapeutic Antibody Efficacy.

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.

IgG-degrading enzyme of Streptococcus pyogenes (IdeS) prevents disease progression and facilitates improvement in a rabbit model of Guillain-Barré syndrome.

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.

HSP90 inhibition blocks ERBB3 and RET phosphorylation in myxoid/round cell liposarcoma and causes massive cell death in vitro and in vivo.

Myxoid sarcoma (MLS) is one of the most common types of malignant soft tissue tumors. MLS is characterized by the FUS-DDIT3 or EWSR1-DDIT3 fusion oncogenes that encode abnormal transcription factors. The receptor tyrosine kinase (RTK) encoding RET was previously identified as a putative downstream target gene to FUS-DDIT3 and here we show that cultured MLS cells expressed phosphorylated RET together with its ligand Persephin. Treatment with RET specific kinase inhibitor Vandetanib failed to reduce RET phosphorylation and inhibit cell growth, suggesting that other RTKs may phosphorylate RET. A screening pointed out EGFR and ERBB3 as the strongest expressed phosphorylated RTKs in MLS cells. We show that ERBB3 formed nuclear and cytoplasmic complexes with RET and both RTKs were previously reported to form complexes with EGFR. The formation of RTK hetero complexes could explain the observed Vandetanib resistence in MLS. EGFR and ERBB3 are clients of HSP90 that help complex formation and RTK activation. Treatment of cultured MLS cells with HSP90 inhibitor 17-DMAG, caused loss of RET and ERBB3 phosphorylation and lead to rapid cell death. Treatment of MLS xenograft carrying Nude mice resulted in massive necrosis, rupture of capillaries and hemorrhages in tumor tissues. We conclude that complex formation between RET and other RTKs may cause RTK inhibitor resistance. HSP90 inhibitors can overcome this resistance and are thus promising drugs for treatment of MLS/RCLS.

The Bacterial Enzyme IdeS Cleaves the IgG-Type of B Cell Receptor (BCR), Abolishes BCR-Mediated Cell Signaling, and Inhibits Memory B Cell Activation.

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.

Complete Removal of Extracellular IgG Antibodies in a Randomized Dose-Escalation Phase I Study with the Bacterial Enzyme IdeS--A Novel Therapeutic Opportunity.

UNLABELLED: 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.

Alpha10 integrin expression is up-regulated on fibroblast growth factor-2-treated mesenchymal stem cells with improved chondrogenic differentiation potential.

Mesenchymal stem cells (MSCs) are multipotent cells that have the capacity to differentiate into various different cell lineages and can generate bone, cartilage and adipose tissue. MSCs are presently characterized using a broad range of different cell-surface markers that are not exclusive to MSCs and not sensitive to culture conditions or differentiation capacity. We show that the integrin subunits alpha10 and alpha11 of the collagen binding integrins alpha10beta1 and alpha11beta1 are expressed by human MSCs in monolayer cultures. We also demonstrate that the expression of alpha10 increases, while alpha1 and alpha11 decrease, during aggregate culture of MSCs in chondrogenic medium. Alpha10beta1 is expressed by chondrocytes in cartilage, whereas alpha11beta1 integrins are predominantly expressed by subsets of the fibroblastic lineage. In extensive monolayer cultures of MSCs, alpha10 expression is down-regulated. We show that this down-regulation is reversed by fibroblast growth factor-2 (FGF-2) treatment. Addition of FGF-2 to MSCs not only results in increased alpha10 expression, but also in decreased alpha11 expression. FGF-2 treatment of MSCs has been shown to keep the cells more multipotent and also induces cell proliferation and Sox-9 up-regulation. We demonstrate improved chondrogenecity as well as increased collagen-dependant migratory potential of FGF-2-treated MSCs having a high alpha10 expression. We also demonstrate expression of alpha10 and alpha11 integrin subunits in the endosteum and periosteum of mice, but very low or not detectable expression levels in freshly aspired human or mouse BM. We show that MSCs with high chondrogenic differentiation potential are highly alpha10 positive and propose alpha10 as a potential marker to predict the differentiation state of MSCs.

The myxoid/round cell liposarcoma fusion oncogene FUS-DDIT3 and the normal DDIT3 induce a liposarcoma phenotype in transfected human fibrosarcoma cells.

Myxoid/round cell liposarcoma (MLS/RCLS) is the most common subtype of liposarcoma. Most MLS/RCLS carry a t(12;16) translocation, resulting in a FUS-DDIT3 fusion gene. We investigated the role of the FUS-DDIT3 fusion in the development of MLS/RCLS in FUS-DDIT3- and DDIT3-transfected human HT1080 sarcoma cells. Cells expressing FUS-DDIT3 and DDIT3 grew as liposarcomas in severe combined immunodeficient mice and exhibited a capillary network morphology that was similar to networks of MLS/RCLS. Microarray-based comparison of HT1080, the transfected cells, and an MLS/RCLS-derived cell line showed that the FUS-DDIT3- and DDIT3-transfected variants shifted toward an MLS/RCLS-like expression pattern. DDIT3-transfected cells responded in vitro to adipogenic factors by accumulation of fat and transformation to a lipoblast-like morphology. In conclusion, because the fusion oncogene FUS-DDIT3 and the normal DDIT3 induce a liposarcoma phenotype when expressed in a primitive sarcoma cell line, MLS/RCLS may develop from cell types other than preadipocytes. This may explain the preferential occurrence of MLS/RCLS in nonadipose tissues. In addition, development of lipoblasts and the typical MLS/RCLS capillary network could be an effect of the DDIT3 transcription factor partner of the fusion oncogene.

Oncostatin M signaling in human glioma cell lines.

We have recently found that oncostatin M (OSM) is overexpressed in most human brain tumors. The effects of OSM are unclear with conflicting reports of growth stimulatory or inhibitory effects in various cell types. The aim of this study was to investigate the effects of OSM in 5 glioma cell lines and 7 short-term cultures of human gliomas and in normal cultured human astrocytes. None of the cell lines and short-term cultured tumor cells expressed OSM in vitro. OSM signals through a gp130 containing receptor complex over the JAK/STAT pathway. Immunofluorescence and RT-PCR analysis showed that the tumor cells express gp130 and the other receptor components, LIFRbeta and OSMRbeta. OSM treatment induced phosphorylation of STAT3 and STAT1 indicating presence of a functional JAK/STAT pathway. No OSM effect on proliferation was observed. OSM gave no protective effects against tumor necrosis factor-related apoptosis inducing ligand (TRAIL)-induced cytotoxicity.

LEPREL1, a novel ER and Golgi resident member of the Leprecan family.

We have identified a novel protein, Leprecan-like 1 (LEPREL1), with profound similarity to the Leprecan family of proteoglycans. The genomic organization of the Leprecan gene family was found to be highly conserved. Expression analysis shows that LEPREL1 is expressed in most tissues as a 3.4 kb transcript encoding an 80 kDa protein. A LEPREL1 specific antibody stains many cell types including adipocytes and neuroendocrine cells of the gastrointestinal epithelium. Muscle tissue contains a specific 6.5 kb transcript and a 200 kDa protein. The 3.4 kb LEPREL1 transcript encodes a 708 amino acid protein containing a signal sequence, four tetratricopeptide repeats (TPRs), a leucine zipper, a P-loop, a prolyl 4-hydroxylase alpha domain (P4Halpha), and a C-terminal KDEL ER-retention motif. LEPREL1 is localized to the ER and Golgi network and over-expressing it affects normal protein disulfide isomerase staining patterns in the ER.

Identification and characterization of a human smad3 splicing variant lacking part of the linker region.

Smad3 is one of the signal transducers that are activated in response to transforming growth factor-beta (TGF-beta). We have identified and characterized a splicing variant of smad3. The splicing variant (smad3-Delta3) lacks exon 3 resulting in a truncated linker region. We could detect mRNA expression of smad3-Delta3 in all investigated human tissues. Real-time PCR analyses demonstrated that the fraction of smad3-Delta3 mRNA compared to normal smad3 varies between tissues. The amount of spliced mRNA was estimated to represent 0.5-5% of the normal smad3 mRNA. When smad3-Delta3 is overexpressed in a fibrosarcoma cell line, the Smad3-Delta3 is translocated to the nucleus upon TGF-beta stimulation and binds the Smad responsive element. Using a CAGA luciferase reporter system, we demonstrate that Smad3-Delta3 has transcriptional activity and we conclude that Smad3-Delta3 possesses functional transactivating properties.

The myxoid liposarcoma specific TLS-CHOP fusion protein localizes to nuclear structures distinct from PML nuclear bodies.

CHOP in 12q13, also called GADD153 or DDIT3, encodes a transcription factor of the C/EBP type. As a result of t(12;16) translocations, CHOP is rearranged and fused to TLS in 16p11 in about 90% of myxoid liposarcomas/round cell liposarcomas (MLS/RCLS). The TLS-CHOP protein consists of the N-terminal half of TLS juxtaposed to the N-terminal of the entire CHOP. It is capable of forming dimers with the natural dimer partners of CHOP. Here we report that recombinant TLS-CHOP-green fluorescence protein localizes to nuclear structures, similar to, but distinct from, PML nuclear bodies. The TLS-CHOP-green fluorescent protein nuclear structures are resistant to high salt concentration and nuclease treatment. Transfection of TLS-CHOP to normal fibroblasts causes a rapid down regulation and relocation of PML nuclear bodies. An abnormal extra nuclear localization of PML bodies was also found in TLS-CHOP carrying cell lines established from myxoid liposarcomas. Transfection of TLS-CHOP induced a rapid disappearance of PCNA. TLS-CHOP may disturb the nuclear machinery by binding and sequestering important factors from their natural sites.