Exploiting the neonatal crystallizable fragment receptor to treat kidney disease.

The neonatal Fc receptor (FcRn) was initially discovered as the receptor that allowed passive immunity in newborns by transporting maternal IgG through the placenta and enterocytes. Since its initial discovery, FcRn has been found to exist throughout all stages of life and in many different cell types. Beyond passive immunity, FcRn is necessary for intrinsic albumin and IgG recycling and is important for antigen processing and presentation. Given its multiple important roles, FcRn has been utilized in many disease treatments including a new class of agents that were developed to inhibit FcRn for treatment of a variety of autoimmune diseases. Certain cell populations within the kidney also express high levels of this receptor. Specifically, podocytes, proximal tubule epithelial cells, and vascular endothelial cells have been found to utilize FcRn. In this review, we summarize what is known about FcRn and its function within the kidney. We also discuss how FcRn has been used for therapeutic benefit, including how newer FcRn inhibiting agents are being used to treat autoimmune diseases. Lastly, we will discuss what renal diseases may respond to FcRn inhibitors and how further work studying FcRn within the kidney may lead to therapies for kidney diseases.

Maternal immunoglobulin G affects brain development of mouse offspring.

Maternal immunoglobulin (Ig)G is present in breast milk and has been shown to contribute to the development of the immune system in infants. In contrast, maternal IgG has no known effect on early childhood brain development. We found maternal IgG immunoreactivity in microglia, which are resident macrophages of the central nervous system of the pup brain, peaking at postnatal one week. Strong IgG immunoreactivity was observed in microglia in the corpus callosum and cerebellar white matter. IgG stimulation of primary cultured microglia activated the type I interferon feedback loop by Syk. Analysis of neonatal Fc receptor knockout (FcRn KO) mice that could not take up IgG from their mothers revealed abnormalities in the proliferation and/or survival of microglia, oligodendrocytes, and some types of interneurons. Moreover, FcRn KO mice also exhibited abnormalities in social behavior and lower locomotor activity in their home cages. Thus, changes in the mother-derived IgG levels affect brain development in offsprings.

Differential effects of FcRn antagonists on the subcellular trafficking of FcRn and albumin.

The homeostasis of IgG is maintained by the neonatal Fc receptor, FcRn. Consequently, antagonism of FcRn to reduce endogenous IgG levels is an emerging strategy for treating antibody-mediated autoimmune disorders using either FcRn-specific antibodies or an engineered Fc fragment. For certain FcRn-specific antibodies, this approach has resulted in reductions in the levels of serum albumin, the other major ligand transported by FcRn. Cellular and molecular analyses of a panel of FcRn antagonists have been carried out to elucidate the mechanisms leading to their differential effects on albumin homeostasis. These analyses have identified 2 processes underlying decreases in albumin levels during FcRn blockade: increased degradation of FcRn and competition between antagonist and albumin for FcRn binding. These findings have potential implications for the design of drugs to modulate FcRn function.

FcRn Inhibitor Therapies in Neurologic Diseases.

In the last decade, the landscape of treating autoimmune diseases has evolved with the emergence and approval of novel targeted therapies. Several new biological agents offer selective and target-specific immunotherapy and therefore fewer side effects, such as neonatal Fc receptor (FcRn)-targeting therapy. Neonatal Fc receptor-targeted therapies are engineered to selectively target FcRn through various methods, such as Fc fragments or monoclonal anti-FcRn antibodies. These approaches enhance the breakdown of autoantibodies by blocking the immunoglobulin G recycling pathway. This mechanism reduces overall plasma immunoglobulin levels, including the levels of pathogenic autoantibodies, without affecting the other immunoglobulin class immunoglobulin A, immunoglobulin E, immunoglobulin M, and immunoglobulin D levels. Drugs that inhibit FcRn include efgartigimod, rozanolixizumab, batoclimab, and nipocalimab. These medications can be administered either intravenously or subcutaneously. Numerous clinical trials are currently underway to investigate their effectiveness, safety, and tolerability in various neurological conditions, including myasthenia gravis and other neurological disorders such as chronic inflammatory demyelinating polyneuropathy, myositis, neuromyelitis optica, and myelin oligodendrocyte glycoprotein antibody disease. Positive results from clinical trials of efgartigimod and rozanolixizumab led to their approval for the treatment of generalized myasthenia gravis. Additional clinical trials are still ongoing. Neonatal Fc receptor inhibitor agents seem to be well tolerated. Reported adverse events include headache (most commonly observed with efgartigimod and rozanolixizumab), upper respiratory tract infection, urinary tract infection, diarrhea, pyrexia, and nausea. Additionally, some of these agents may cause transient hypoalbuminemia and hypercholesterolemia notably reported with batoclimab and nipocalimab. In this review, we discuss the available clinical data for FcRN inhibitor agents in treating different neurological autoimmune diseases.

Identification of Four Mouse FcRn Splice Variants and FcRn-Specific Vesicles.

Research into the neonatal Fc receptor (FcRn) has increased dramatically ever since Simister and Mostov first purified a rat version of the receptor. Over the years, FcRn has been shown to function not only as a receptor that transfers immunity from mother to fetus but also performs an array of different functions that include transport and recycling of immunoglobulins and albumin in the adult. Due to its important cellular roles, several clinical trials have been designed to either inhibit/enhance FcRn function or develop of non-invasive therapeutic delivery system such as fusion of drugs to IgG Fc or albumin to enhance delivery inside the cells. Here, we report the accidental identification of several FcRn alternatively spliced variants in both mouse and human cells. The four new mouse splice variants are capable of binding immunoglobulins' Fc and Fab portions. In addition, we have identified FcRn-specific vesicles in which immunoglobulins and albumin can be stored and that are involved in the endosomal-lysosomal system. The complexity of FcRn functions offers significant potential to design and develop novel and targeted therapeutics.

Reducing neonatal Fc receptor binding enhances clearance and brain-to-blood ratio of TfR-delivered bispecific amyloid-ß antibody.

Recent development of amyloid-ß (Aß)-targeted immunotherapies for Alzheimer's disease (AD) have highlighted the need for accurate diagnostic methods. Antibody-based positron emission tomography (PET) ligands are well suited for this purpose as they can be directed toward the same target as the therapeutic antibody. Bispecific, brain-penetrating antibodies can achieve sufficient brain concentrations, but their slow blood clearance remains a challenge, since it prolongs the time required to achieve a target-specific PET signal. Here, two antibodies were designed based on the Aß antibody bapineuzumab (Bapi) - one monospecific IgG (Bapi) and one bispecific antibody with an antigen binding fragment (Fab) of the transferrin receptor (TfR) antibody 8D3 fused to one of the heavy chains (Bapi-Fab8D3) for active, TfR-mediated transport into the brain. A variant of each antibody was designed to harbor a mutation to the neonatal Fc receptor (FcRn) binding domain, to increase clearance. Blood and brain pharmacokinetics of radiolabeled antibodies were studied in wildtype (WT) and AD mice (AppNL-G-F). The FcRn mutation substantially reduced blood half-life of both Bapi and Bapi-Fab8D3. Bapi-Fab8D3 showed high brain uptake and the brain-to-blood ratio of its FcRn mutated form was significantly higher in AppNL-G-F mice than in WT mice 12 h after injection and increased further up to 168 h. Ex vivo autoradiography showed specific antibody retention in areas with abundant Aß pathology. Taken together, these results suggest that reducing FcRn binding of a full-sized bispecific antibody increases the systemic elimination and could thereby drastically reduce the time from injection to in vivo imaging.

Antibodies from chlamydia-infected individuals facilitate phagocytosis via Fc receptors.

Non-neutralizing functions of antibodies, including phagocytosis, may play a role in Chlamydia trachomatis (CT) infection, but these functions have not been studied and assays are lacking. We utilized a flow-cytometry-based assay to determine whether serum samples from a well-characterized cohort of CT-infected and naïve control individuals enhanced phagocytosis via Fc-receptor-expressing THP-1 cells, and whether this activity correlated with antibody titers. Fc-receptor-mediated phagocytosis was detected only in CT+ donors. Phagocytosis generally did not correlate well with antibody titer. In addition, we found that complement from both CT+ and negative individuals enhanced phagocytosis of CT into primary neutrophils. These results suggest that anti-CT antibodies can have functions that are not reflected by titer. This method could be used to quantitively measure Fc-receptor-mediated function of anti-CT antibodies or complement activity and could reveal new immune correlates of protection.

FcRY is a key molecule controlling maternal blood IgY transfer to yolks during egg development in avian species.

Maternal immunoglobulin transfer plays a key role in conferring passive immunity to neonates. Maternal blood immunoglobulin Y (IgY) in avian species is transported to newly-hatched chicks in two steps: 1) IgY is transported from the maternal circulation to the yolk of maturing oocytes, 2) the IgY deposited in yolk is transported to the circulation of the embryo via the yolk sac membrane. An IgY-Fc receptor, FcRY, is involved in the second step, but the mechanism of the first step is still unclear. We determined whether FcRY was also the basis for maternal blood IgY transfer to the yolk in the first step during egg development. Immunohistochemistry revealed that FcRY was expressed in the capillary endothelial cells in the internal theca layer of the ovarian follicle. Substitution of the amino acid residue in Fc region of IgY substantially changed the transport efficiency of IgY into egg yolks when intravenously-injected into laying quail; the G365A mutant had a high transport efficiency, but the Y363A mutant lacked transport ability. Binding analyses of IgY mutants to FcRY indicated that the mutant with a high transport efficiency (G365A) had a strong binding activity to FcRY; the mutants with a low transport efficiency (G365D, N408A) had a weak binding activity to FcRY. One exception, the Y363A mutant had a remarkably strong binding affinity to FcRY, with a small dissociation rate. The injection of neutralizing FcRY antibodies in laying quail markedly reduced IgY uptake into egg yolks. The neutralization also showed that FcRY was engaged in prolongation of half-life of IgY in the blood; FcRY is therefore a multifunctional receptor that controls avian immunity. The pattern of the transport of the IgY mutants from the maternal blood to the egg yolk was found to be identical to that from the fertilized egg yolk to the newly-hatched chick blood circulation, via the yolk sac membrane. FcRY is therefore a critical IgY receptor that regulates the IgY uptake from the maternal blood circulation into the yolk of avian species, further indicating that the two steps of maternal-newly-hatched IgY transfer are controlled by a single receptor.

Functions of IgM fc receptor (FcµR) related to autoimmunity.

Unlike Fc receptors for switched immunoglobulin (Ig) isotypes, Fc receptor for IgM (FcµR) is selectively expressed by lymphocytes. The ablation of the FcµR gene in mice impairs B cell tolerance as evidenced by concomitant production of autoantibodies of IgM and IgG isotypes. In this essay, we reiterate the autoimmune phenotypes observed in mutant mice, ie IgM homeostasis, dysregulated humoral immune responses including autoantibodies, and Mott cell formation. We also propose the potential phenotypes in individuals with FCMR deficiency and the model for FcµR-mediated regulation of self-reactive B cells.

VALIDATION OF A NOVEL ENZYME-LINKED IMMUNOSORBENT ASSAY FOR DETECTING PREGNANCY-SPECIFIC PROTEIN B IN MASAI GIRAFFE (GIRAFFA CAMELOPARDALIS TIPPELSKIRCHI).

The Masai giraffe (Giraffa camelopardalis tippelskirchi) is endangered in the wild, and successful reproduction in managed care is important to help maintain assurance populations of this highly charismatic subspecies. Detection of pregnancy in giraffes using hormonal monitoring requires multiple samples and cannot distinguish pregnancy from pseudopregnancy. A novel enzyme-linked immunosorbent assay that can detect pregnancy-specific protein B (PSPB) for pregnancy diagnosis with a single serum sample was developed from a reticulated giraffe (Giraffa camelopardalis reticulata) placenta. Seventy-eight serum samples were analyzed from three female Masai giraffes before and during five gestation periods that resulted in live calf births. Using an optical density cutoff of 0.2, the assay showed a sensitivity of 93% and specificity of 100% for all samples tested. At 59 d of gestation, sensitivity increased to 100%. The earliest pregnancy detection was at 40 d of gestation. This study documents the successful development of a blood-based PSPB assay for pregnancy diagnosis in Masai giraffe, which can help advance conservation efforts in this endangered species.

Human IgG Fc-engineering for enhanced plasma half-life, mucosal distribution and killing of cancer cells and bacteria.

Monoclonal IgG antibodies constitute the fastest growing class of therapeutics. Thus, there is an intense interest to design more potent antibody formats, where long plasma half-life is a commercially competitive differentiator affecting dosing, frequency of administration and thereby potentially patient compliance. Here, we report on an Fc-engineered variant with three amino acid substitutions Q311R/M428E/N434W (REW), that enhances plasma half-life and mucosal distribution, as well as allows for needle-free delivery across respiratory epithelial barriers in human FcRn transgenic mice. In addition, the Fc-engineered variant improves on-target complement-mediated killing of cancer cells as well as both gram-positive and gram-negative bacteria. Hence, this versatile Fc technology should be broadly applicable in antibody design aiming for long-acting prophylactic or therapeutic interventions.

Intranasal Nose-to-Brain Drug Delivery via the Olfactory Region in Mice: Two In-Depth Protocols for Region-Specific Intranasal Application of Antibodies and for Expression Analysis of Fc Receptors via In Situ Hybridization in the Nasal Mucosa.

A region-specific catheter-based intranasal administration method was successfully developed, established, and validated as reported previously. By using this method, drugs can be applicated specifically to the olfactory region. Thereby, intranasally administered drugs could be delivered via neuronal connections to the central nervous system. Here, we present a detailed protocol with a step-by-step procedure for nose-to-brain delivery via the olfactory mucosa.Fc receptors such as the neonatal Fc receptor (FcRn) and potentially Fcγ receptor IIb (FcγRIIb) are involved in the uptake and transport of antibodies via the olfactory nasal mucosa. To better characterize their expression levels and their role in CNS drug delivery via the nose, an in situ hybridization (ISH) protocol was adapted for nasal mucosa samples and described in abundant details.

Fc engineered anti-virus therapeutic human IgG1 expressed in plants with altered binding to the neonatal Fc receptor.

Production of therapeutic monoclonal antibody (mAb) in transgenic plants has several advantages such as large-scale production and the absence of pathogenic animal contaminants. However, mAb with high mannose (HM) type glycans has shown a faster clearance compared to antibodies produced in animal cells. The neonatal Fc receptor (FcRn) regulates the persistence of immunoglobulin G (IgG) by the FcRn-mediated recycling pathway, which salvages IgG from lysosomal degradation within cells. In this study, Fc-engineering of antirabies virus therapeutic mAb SO57 with the endoplasmic reticulum (ER)-retention peptide signal (Lys-Asp-Glu-Leu; KDEL) (mAbpK SO57) in plant cell was conducted to enhance its binding activity to human neonatal Fc receptor (hFcRn), consequently improve its serum half-life. Enzyme-linked immunosorbent assay (ELISA) and Surface plasmon resonance assay showed altered binding affinity of the Fc region of three different mAbpK SO57 variants [M252Y/S254T/T256E (MST), M428L/N434S (MN), H433K/N434F (HN)] to hFcRn compared to wild type (WT) of mAbpK SO57. Molecular modeling data visualized the structural alterations in these mAbpK SO57. All of the mAbpK SO57 variants had HM type glycan structures similar to the WT mAbpK SO57. In addition, the neutralizing activity of the three variants against the rabies virus CVS-11 was effective as the WT mAbpK SO57. These results indicate that the binding affinity of mAbpK SO57 variants to hFcRn can be modified without alteration of N-glycan structure and neutralization activity. Taken together, this study suggests that Fc-engineering of antirabies virus mAb can be applied to enhance the efficacy of therapeutic mAbs in plant expression systems.

Treatment patterns and outcomes among adults with immune thrombocytopenia receiving pharmaceutical second-line therapies: a retrospective cohort study using administrative claims data.

OBJECTIVES: To describe and compare real-world treatment patterns and clinical outcomes among individuals with immune thrombocytopenia (ITP) receiving second-line therapies (rituximab, romiplostim, or eltrombopag). METHODS: A retrospective cohort study was conducted using a large administrative claims database (January 2013-May 2020) among continuously enrolled patients ≥18 years prescribed second-line ITP therapies. The index date was the date of the first claim of the study medications. Treatment patterns and outcomes were measured during the 12-month follow-up period. Inverse probability of treatment weighting (IPTW) was used to balance covariates across treatment groups. Multivariable logistic regression was used to compare treatment patterns and bleeding risk outcomes. RESULTS: A total of 695 patients were included (rituximab, N = 285; romiplostim, N = 212; eltrombopag, N = 198). After IPTW, all baseline covariates were balanced. Compared to eltrombopag, patients in the rituximab cohort were 57% more likely to receive other ITP therapies (systematic corticosteroids or third-line therapies) during the follow-up period (odds ratio [OR] = 1.571, p = .030). There was no significant difference in the odds of receiving a different second-line therapy or experiencing a bleeding-related episode among three groups (p > .050). Patients in the romiplostim cohort were 69% more likely to receive rescue therapy compared to those in the rituximab cohort (OR = 1.688, p = .025). CONCLUSION: Patients with ITP receiving rituximab were more likely to need other ITP therapies but did not experience higher risk of bleeding compared to those receiving eltrombopag or romiplostim. Benefits, risks, cost-effectiveness, and patient preference should all be considered in optimizing second-line therapy for ITP.

The efficacy and safety of FcRn inhibitors in patients with myasthenia gravis: a systematic review and meta-analysis.

BACKGROUND: Myasthenia gravis (MG) is an autoimmune disease that causes local or generalized muscle weakness. Complement inhibitors and targeting of the neonatal Fc receptor (FcRn) to block IgG cycling are two novel and successful mechanisms. METHODS: PubMed, EMBASE, the Cochrane Library, and ClinicalTrials.gov were systematically searched to identify relevant studies published before May 18, 2023. Review Manager 5.3 software was used to assess the data. RESULTS: We pooled 532 participants from six randomized controlled trials (RCTs). Compared to the placebo, the FcRn inhibitors were more efficacy in Myasthenia Gravis Activities of Daily Living (MG-ADL) (MD = - 1.69 [- 2.35, - 1.03], P < 0.00001), MG-ADL responder (RR = 2.01 [1.62, 2.48], P < 0.00001), Quantitative Myasthenia Gravis (QMG) (MD = - 2.45 [- 4.35, - 0.55], P = 0.01), Myasthenia Gravis Composite (MGC) (MD = - 2.97 [- 4.27, - 1.67], P < 0.00001), 15-item revised version of the Myasthenia Gravis Quality of Life (MGQoL15r) (MD = - 2.52 [- 3.54, - 1.50], P < 0.00001), without increasing the risk of safety. The subgroup analysis showed that efgartigimod was more effective than placebo in MG-ADL responders. Rozanolixizumab was more effective than the placebo except in QMG, and batoclimab was more effective than the placebo except in MG-ADL responder. Nipocalizumab did not show satisfactory efficacy in all outcomes. With the exception of rozanolixizumab, all drugs showed non-inferior safety profiles to placebo. CONCLUSION: FcRn inhibitors have good efficacy and safety in patients with MG. Among them, efgartigimod and nipocalimab were effective without causing an increased safety risk. Rozanolixizumab, despite its superior efficacy, caused an increased incidence of adverse events. Current evidence does not suggest that nipocalimab is effective in patients with MG.

A phase 2b, randomized, double-blind, placebo-controlled, clinical trial of atacicept for treatment of IgA nephropathy.

Atacicept is a first-in-class, dual anti-B-cell Activation Factor-A Proliferation-Inducing Ligand fusion protein in clinical evaluation for treatment of IgA nephropathy. To compare efficacy and safety of atacicept versus placebo in patients with IgAN, this randomized, double-blind, placebo-controlled phase 2b clinical trial ORIGIN enrolled 116 individuals with biopsy-proven IgA nephropathy. Participants were randomized to atacicept 150, 75, or 25 mg versus placebo once weekly for up to 36 weeks. Primary and key secondary endpoints were changes in urine protein creatinine ratio based on 24-hour urine collection at weeks 24 and 36, respectively, in the combined atacicept 150 mg and 75 mg group versus placebo. The primary endpoint was met at week 24 as the mean urine protein creatinine ratio was reduced from baseline by 31% in the combined atacicept group versus 8% with placebo, resulting in a significant 25% reduction with atacicept versus placebo. At week 36, the key secondary endpoint was met as the mean urine protein creatinine ratio reduced from baseline by 34% in the combined atacicept group versus a 2% increase with placebo, resulting in a significant 35% reduction with atacicept versus placebo. The reduction in proteinuria was accompanied by stabilization in endpoint eGFR with atacicept compared to a decline with placebo at week 36, resulting in significant between-group geometric mean difference of 11%, approximating an absolute difference of 5.7 mL/min/1.73m2. Endpoint galactose deficient IgA1 levels significantly decreased from baseline by 60% versus placebo. The safety profile of atacicept was like placebo. Thus, our results provide evidence to support a pivotal, phase 3 study of atacicept in IgA nephropathy.

Fc receptors are key discriminatory markers of granulocytes subsets in people living with HIV-1.

Introduction: Granulocytes are innate immune cells that play a key role in pathogen elimination. Recent studies revealed the diversity of granulocytes in terms of phenotype and function. In particular, a subset of granulocytes identified as low-density granulocytes (LDG) has been described in physiological conditions and with increased frequencies in several pathological contexts. However, the properties of LDG are still controversial as they vary according to the pathophysiological environment. Here we investigated the heterogeneity of granulocyte populations and the potential differences in phenotype and immunomodulatory capacity between LDG and normal density granulocytes (NDG) in people living with HIV-1 (PLWH). Methods: To this end, we developed an optimized method to purify LDG and NDG from a single blood sample, and performed in-depth, comparative phenotypic characterization of both granulocyte subtypes. We also assessed the impact of purification steps on the expression of cell surface markers on LDG by immunophenotyping them at different stages of isolation. Results: We identified 9 cell surface markers (CD16, CD32, CD89, CD62L, CD177, CD31, CD10, CXCR4 and CD172α) differentially expressed between LDG and NDG. Noteworthy, markers that distinguish the two subsets include receptors for the Fc part of IgG (CD16, CD32) and IgA (CD89). Importantly, we also highlighted that the purification procedure affects the expression of several cell surface markers (i.e.CD63, CD66b, …) which must be taken into account when characterizing LDG. Our work sheds new light on the properties of LDG in PLWH and provides an extensive characterization of this granulocyte subset in which Fc receptors are key discriminatory markers.

Long-term treatment with rilzabrutinib in patients with immune thrombocytopenia.

ABSTRACT: Immune thrombocytopenia (ITP) is an autoimmune disease associated with autoantibody-mediated platelet destruction and impaired platelet production, resulting in thrombocytopenia and a predisposition to bleeding. The ongoing, global phase 1/2 study showed that rilzabrutinib, a Bruton tyrosine kinase inhibitor specifically developed to treat autoimmune disorders, could be an efficacious and well-tolerated treatment for ITP. Clinical activity, durability of response, and safety were evaluated in 16 responding patients who continued rilzabrutinib 400 mg twice daily in the long-term extension (LTE) study. At LTE entry, the median platelet count was 87 × 109/L in all patients, 68 × 109/L in those who had rilzabrutinib monotherapy (n = 5), and 156 × 109/L in patients who received concomitant ITP medication (thrombopoietin-receptor agonists and/or corticosteroids, n = 11). At a median duration of treatment of 478 days (range, 303-764), 11 of 16 patients (69%) continued to receive rilzabrutinib. A platelet count of ≥50 × 109/L was reported in 93% of patients for more than half of their monthly visits. The median percentage of LTE weeks with platelet counts ≥30 × 109/L and ≥50 × 109/L was 100% and 88%, respectively. Five patients discontinued concomitant ITP therapy and maintained median platelet counts of 106 × 109/L at 3 to 6 months after stopping concomitant ITP therapy. Adverse events related to treatment were grade 1 or 2 and transient, with no bleeding, thrombotic, or serious adverse events. With continued rilzabrutinib treatment in the LTE, platelet responses were durable and stable over time with no new safety signals. This trial is registered at www.clinicaltrials.gov as #NCT03395210 and www.clinicaltrialsregister.eu as EudraCT 2017-004012-19.

The problem of immune thrombocytopenia refractory to both eltrombopag and romiplostim.

Immune thrombocytopenia refractory to multiple thrombopoietin receptor agonists remains a challenging clinical problem. This commentary discusses and contextualizes the recent report on this entity from Moulis and colleagues, and how to move forward with these patients. Commentary on: Moulis et al. Difficult-to-treat primary immune thrombocytopenia in adults: Prevalence and burden. Results from the CARMEN-France Registry. Br J Haematol 2024;204:1476-1482.