FcRn Research Articles

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.

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.

Molecular characterization of emerging Echovirus 11 (E11) shed light on the recombinant origin of a variant associated with severe hepatitis in neonates.

Echovirus 11 (E11) has gained attention owing to its association with severe neonatal infections. Due to the limited data available, the World Health Organization (WHO) considers public health risk to the general population to be low. The present study investigated the genetic variation and molecular evolution of E11 genomes collected from May to December 2023. Whole genome sequencing (WGS) was performed for 16 E11 strains. Phylogenetic analysis on WG showed how all Italian strains belonged to genogroup D5, similarly to other E11 strains recently reported in France and Germany all together aggregated into separate clusters. A cluster-specific recombination pattern was also identified using phylogenetic analysis of different genome regions. Echovirus 6 was identified as the major recombinant virus in 3Cpro and 3Dpol regions. The molecular clock analysis revealed that the recombination event probably occurred in June 2018 (95% HPD interval: Jan 2016-Jan 2020). Shannon entropy analyses, within P1 region, showed how 11 amino acids exhibited relatively high entropy. Five of them were exposed on the canyon region which is responsible for receptor binding with the neonatal Fc receptor. The present study showed the recombinant origin of a new lineage of E11 associated with severe neonatal infections.

Muscle-Specific Receptor Tyrosine Kinase Antibody Positive Myasthenia Gravis

Reportedly, patients with muscle-specific kinase (MuSK) antibody-positive myasthenia gravis (MG) account for approximately 3.0% of all patients with MG in Japan. Compared with patients who have acetylcholine receptor antibody-positive MG, those with MuSK antibody-positive MG show young-onset disease with female predominance, a low rate of ocular involvement (5.9%), and greater severity of dysphagia. The aforementioned types of MG are indistinguishable based on clinical symptoms and electrophysiological tests, and measurement of MuSK antibodies is essential for diagnosis. Thymectomy and complement inhibitors are not indicated for treatment, and acetylcholinesterase inhibitors, steroids, immunosuppressants, plasma exchange, intravenous immunoglobulin therapy, and neonatal Fc receptor inhibitors are used.

OA04 Efficacy and safety of nipocalimab in patients with moderate to severe active rheumatoid arthritis: the multicenter, randomised, double-blinded, placebo-controlled phase 2a IRIS-RA study

Abstract Background/Aims Rheumatoid arthritis (RA) is a chronic inflammatory disease associated with autoantibodies. Despite the use of targeted therapies, up to half of patients fail to achieve remission or low disease activity (LDA). Anti-citrullinated protein antibodies (ACPAs), key autoantibodies that predict progression of joint destruction in RA, are largely an immunoglobulin G (IgG) isotype. Nipocalimab is a high affinity, fully human, IgG1 monoclonal antibody that is designed to selectively block the neonatal Fc receptor (FcRn), preventing recirculation and thereby lowering IgG levels, potentially including ACPAs and other pathogenic antibodies. In this phase 2a proof-of-concept study (IRIS-RA; NCT04991753), we report the efficacy and safety of nipocalimab in patients with moderate to severe, active RA. Methods Eligible adult patients with moderate to severe RA (≥6 swollen/tender joints), positive for ACPA or rheumatoid factor, and ≥1 advanced therapies, were randomized 3:2 to receive intravenous 15 mg/kg nipocalimab or placebo every 2 weeks for 10 weeks. The primary endpoint was the change from baseline in the Disease Activity Score 28 using C-reactive protein (DAS28-CRP) at Week 12. Secondary endpoints included the proportion of patients who achieved ACR20, ACR50, ACR70, and ACR90 responses, DAS28-CRP remission, and change from baseline in Health Assessment Questionnaire - Disability Index (HAQ-DI) at Week 12. Results In total, 53 patients were enrolled (nipocalimab, n = 33; placebo, n = 20). Most patients were female (67.9%) and white (90.6%) with a median age of 59 (range, 26-74) years. Demographic and baseline disease characteristics were comparable between groups except higher CRP in placebo. At Week 12, patients treated with nipocalimab showed a numerically greater mean (standard deviation [SD]) change in DAS28-CRP score compared to the placebo group (-1.17 [1.34] vs -0.62 [0.96]; mean difference [95% confidence interval], -0.45 [-1.17-0.28]; p=0.224). A numerically higher proportion of patients achieved ACR20, ACR50, ACR70, and ACR90 responses compared to placebo. In the nipocalimab group, 7 (21.2%) patients achieved DAS28-CRP remission compared to 2 (10.0%) patients in the placebo group (treatment difference, 9.9% [-9.5-29.3%]; p=0.456). Similarly, patients in the nipocalimab group had a numerically greater mean (SD) improvement in HAQ-DI score (-0.27 [0.55] vs -0.11 [0.36], respectively). The proportion of patients with treatment-emergent adverse events (TEAEs) were 81.8% vs 60.0% in the nipocalimab vs placebo groups. In the nipocalimab group, 3 serious TEAEs were reported, including burn infection, infusion-related reaction, and deep-vein thrombosis. There were no TEAEs that led to death. Conclusion Treatment with nipocalimab resulted in consistent and numerically higher improvements across ACR20, ACR50, ACR70, and ACR90 responses, as well as DAS28-CRP, DAS28-CRP remission, and HAQ-DI, with acceptable safety profile. Nipocalimab showed activity in patients with moderate to severe RA and warrants further investigation to understand the predictors of nipocalimab response. Disclosure P.C. Taylor: Consultancies; AbbVie, Biogen, Eli Lilly, Fresenius, Galapagos, Gilead Sciences, GlaxoSmithKline, Janssen, Nordic Pharma, Pfizer Inc, Sanofi, Aqtual, and UCB. Grants/research support; Galapagos. G. Schett: None. F. Ibrahim: Corporate appointments; Employee of Janssen Research & Development. B. Zhou: Corporate appointments; Employee of Janssen Research & Development. J. Leu: Corporate appointments; Employee of Janssen Research & Development. S. Liva: Corporate appointments; Employee of Janssen Research & Development. Q. Wang: Corporate appointments; Employee of Janssen Research & Development. R. Rojo Cella: Corporate appointments; Employee of Janssen Research & Development. C.S. Karyekar: Corporate appointments; Employee of Janssen Research & Development. K. Fei: Corporate appointments; Employee of Janssen Research & Development.

Efgartigimod as a novel FcRn inhibitor for autoimmune disease.

Immunoglobulin G (IgG) autoantibodies can lead to the formation of autoimmune diseases through Fab and/or Fc-mediated interactions with host molecules as well as activated T cells. The neonatal Fc receptor (FcRn) binds at acidic pH IgG and albumin, and the mechanism for prolonging serum IgG half-life is making IgG re-entry into circulation by prompting it not to be degraded by lysosomes and back to the cell surface. Given the FcRn receptor's essential role in IgG homeostasis, one of the strategies to promote the quick degradation of endogenous IgG is to suppress the function of FcRn, which is beneficial to the treatment of IgG-driven autoimmune disorders like myasthenia gravis (MG), chronic inflammatory demyelinating polyradiculoneuropathy (CIDP), stiff person syndrome, and immune thrombocytopenia (ITP). We elaborately read the literature about efgartigimod and systematically reviewed the research progress and clinical application of this novel FcRn inhibitor in autoimmune diseases. Efgartigimod is the firstly FcRn antagonist developed and was approved on 17 December 2021 by the United States for the therapy of acetylcholine receptor-positive MG. In January 2022, efgartigimod received its second regulatory approval in Japan. In addition, the market authorization application in Europe was submitted and validated in August 2021. China's National Medical Products Administration officially accepted the marketing application of efgartigimod on July 13, 2022. To suppress the function of FcRn, which is beneficial to the treatment of IgG-driven autoimmune disorders like MG, CIDP, ITP, and stiff person syndrome. We review the rationale, clinical evidence, and future perspectives of efgartigimod for the treatment of autoimmune disease.

FcRn regulates antigen presentation in dendritic cells downstream of DEC205-targeted vaccines

Dendritic cell (DC)-targeted vaccination is a new mode of antigen delivery that relies on the use of monoclonal antibodies (mAb) to target antigen to specific DC subsets. The neonatal Fc receptor (FcRn) is a non-classical Fc receptor that binds to immunoglobulin G (IgG) in acidified endosomes and controls its intracellular transport and recycling. FcRn is known to participate in the antigen presentation of immune complexes, however its contribution to DC-targeted vaccination has not previously been examined. Here we have investigated the role of FcRn in antigen presentation using antigen conjugated to IgG mAb which target specific DC receptors, including DEC205 and Clec9A expressed by the conventional DC 1 (cDC1) subset. We show that FcRn is expressed at high levels by cDC1, both at steady-state and following activation and plays a significant role in MHC I cross-presentation and MHC II presentation of antigens that are targeted to cDC1 via mAb specific for DEC205. This effect of FcRn is intrinsic to cDC1 and FcRn impacts the efficacy of anti-DEC205-mediated vaccination against B cell lymphoma. In contrast, FcRn does not impact presentation of antigens targeted to Clec9A and does not regulate presentation of cell-associated antigen. These data highlight a new and unique role of FcRn in controlling the immunogenicity of anti-DEC205-based vaccination, with consequences for exploiting this pathway to improve DC-targeted vaccine outcomes.

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.

Kinetic modeling of the plasma pharmacokinetic profiles of ADAMTS13 fragment and its Fc-fusion counterpart in mice.

Introduction: Fusion of the fragment crystallizable (Fc) to protein therapeutics is commonly used to extend the circulation time by enhancing neonatal Fc-receptor (FcRn)-mediated endosomal recycling and slowing renal clearance. This study applied kinetic modeling to gain insights into the cellular processing contributing to the observed pharmacokinetic (PK) differences between the novel recombinant ADAMTS13 fragment (MDTCS) and its Fc-fusion protein (MDTCS-Fc). Methods: For MDTCS and MDTCS-Fc, their plasma PK profiles were obtained at two dose levels following intravenous administration of the respective proteins to mice. The plasma PK profiles of MDTCS were fitted to a kinetic model with three unknown protein-dependent parameters representing the fraction recycled (FR) and the rate constants for endocytosis (kup, for the uptake into the endosomes) and for the transfer from the plasma to the interstitial fluid (kpi). For MDTCS-Fc, the model was modified to include an additional parameter for binding to FcRn. Parameter optimization was done using the Cluster Gauss-Newton Method (CGNM), an algorithm that identifies multiple sets of approximate solutions ("accepted" parameter sets) to nonlinear least-squares problems. Results: As expected, the kinetic modeling results yielded the FR of MDTCS-Fc to be 2.8-fold greater than that of MDTCS (0.8497 and 0.3061, respectively). In addition, MDTCS-Fc was predicted to undergo endocytosis (the uptake into the endosomes) at a slower rate than MDTCS. Sensitivity analyses identified the association rate constant (kon) between MDTCS-Fc and FcRn as a potentially important factor influencing the plasma half-life in vivo. Discussion: Our analyses suggested that Fc fusion to MDTCS leads to changes in not only the FR but also the uptake into the endosomes, impacting the systemic plasma PK profiles. These findings may be used to develop recombinant protein therapeutics with extended circulation time.

Abstract 2720: Engineering sweeping antibodies for the clearance of tumor-derived, pro-tumorigenic cytokines

Abstract Soluble, tumor-derived, pro-tumorigenic (STP) cytokines have been shown to contribute to cancer cell proliferation and drug resistance. But therapeutic targeting of STP cytokines by monoclonal antibodies is limited by high concentrations and persistent production of antigen. We hypothesize that the ability of sweeping antibodies (swAbs) to actively clear soluble antigens will overcome these limitations. SwAbs differ from conventional antibodies via (1) pH-dependent binding of antigen, and (2) increased binding to the neonatal Fc receptor (FcRn). SwAbs facilitate clearance of soluble antigens by releasing antigen in the low pH environment of FcRn-expressing cells. SwAbs are then recycled via FcRn for repeated clearance of antigen.We are developing swAbs against four STP cytokines contributing to cancer cell proliferation, survival, migration or intra-tumoral angiogenesis: hepatocyte growth factor (HGF), vascular endothelial growth factor (VEGF), transforming growth factor-β (TGF-β) and interleukin 16 (IL-16). We selected four existing neutralizing antibodies against these cytokines with available crystal structures and generated a second antibody targeting interleukin 16 with high affinity (clone 3B6; KD = 13 nM) de novo using phage display. We are using bio-layer interferometry and custom recycling/clearance assays for antibody characterization and pre-clinical validation. To introduce pH dependency into the four antibodies with crystal structures, we performed targeted histidine mutagenesis of residues at the interface with the respective target antigens. For the de novo-generated interleukin-16 antibody, we performed an unbiased histidine scanning approach by mutating residues across all six complementarity-determining regions (CDRs), which resulted in slight increases in pH sensitivity. We then performed a second round of mutagenesis of the most pH-sensitive 3B6 variants by replacing additional residues considered developability liabilities with histidine residues. We found that this approach significantly increased expression and affinity at pH 7 of the resulting 3B6 variants but only modestly increased pH sensitivity. In contrast, replacing a single residue in the heavy CDR1 in clone 14.1, a pre-clinical anti-IL-16 antibody, resulted in substantially increased pH sensitivity (KD(pH=7) = 2 nM; KD(pH<6) = n.b.). We are currently performing pH-sensitivity screenings for the three additional STP-specific antibodies. Our findings indicate that the ability to introduce pH sensitivity is largely dependent on the parental antibody and that some antibodies may not be amenable to this process. In addition, we show that histidine mutagenesis can be an effective approach to enhance expressibility and binding, while also increasing pH sensitivity. This project will answer the question whether anti-STP cytokine swAbs are an effective approach to treat patients with cancer. Citation Format: Jillian M. Baker, Nevil J. Singh, Tim Luetkens. Engineering sweeping antibodies for the clearance of tumor-derived, pro-tumorigenic cytokines [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2720.

Abstract 5975: Characterization and pharmacokinetics of ligand traps targeting the human epidermal growth factor receptor (HER) family

Abstract Antibodies targeting HER receptors have led to successful therapeutics for multiple human malignancies. Targeting the family of HER ligands provides an alternative strategy to inhibit signaling and subsequent proliferation of cancer cells. We have engineered ligand traps that bind the growth factors activating the HER family and have characterized a series of molecules (EN2000-Fc) using Surface Plasmon Resonance (SPR). Five distinct molecules were further evaluated for improved in vivo pharmacokinetics in mice that express human neonatal Fc receptor (FcRn). Mutations in the IgG Fc known to extend the in-vivo half-life of monoclonal antibodies were incorporated into the parental EN2000-Fc to generate 3 more distinct pan HER ligand traps. In addition, an EN2000-Fc variant with a full-length hinge was engineered, which is 5-amino-acid longer N-terminally than the parental molecule. These five molecules were synthesized (> 300 mg of each), purified (>98%), and characterized for in vitro and in vivo testing. Two variants were compared by evaluating the ligand binding affinity to 11 HER ligands using SPR. Both molecules showed comparable affinity against all ligands tested, confirming reproducibility of the pan HER ligand trap engineering technology. Among the ligands, HB-EGF had the highest affinity. High binding affinities to EGF, NRG1, NRG2, betacellulin and TGFα were also measured. The serum pharmacokinetics of the different ligand traps were compared in 6- to 8-week-old female C57BL/6 mice engineered to express human FcRn. Ligand traps were administered by single tail vein injection, and serum samples collected over a 28-day interval were analyzed by ELISA. Ligand traps with engineered Fc-mutations had 3- to 4-fold greater systemic exposure (area-under-the-curve) compared to ligand traps with the parental sequence, demonstrating improved in vivo characteristics of selected engineered fusion proteins. These results provide useful information for product candidate selection and for further preclinical development of a ligand trap targeting HER family receptors. Citation Format: Daniel C. Maneval, Cindy Yang, Cynthia Trevisiol, H. Michael Shepard, Pei Jin. Characterization and pharmacokinetics of ligand traps targeting the human epidermal growth factor receptor (HER) family [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5975.

Prevention of Hypercholesterolemia with "Liposomes in Microspheres" Composite Carriers: A Promising Approach for Intestinal-Targeted Oral Delivery of Astaxanthin.

Cardiovascular diseases are caused by hypercholesterolemia. Astaxanthin (AST) has been reported to exhibit antioxidant and anti-inflammatory properties. However, its bioavailability is poor because of low solubility and instability. In order to improve the bioavailability of AST, we developed an intestinal-responsive composite carrier termed as "liposomes in micropheres" incorporating N-succinyl-chitosan (NSC)-poly(ethylene glycol) (PEG) liposomes that functionalized by neonatal Fc receptors (FcRn) into hydrogels of sodium alginate (SA) and carboxymethyl chitosan (CMCS). In the AST NSC/HSA-PEG liposomes@SA/CMCS microspheres, the AST's encapsulation efficiency (EE) was 96.26% (w/w) and its loading capacity (LC) was 6.47% (w/w). AST NSC/HSA-PEG liposomes had stability in the gastric conditions and achieved long-term release of AST in intestinal conditions. Then, AST NSC/HSA-PEG liposomes@SA/CMCS bind to intestinal epithelial cell targets by the neonatal Fc receptor. In vitro permeation studies show that there was a 4-fold increase of AST NSC/HSA-PEG liposomes@SA/CMCS in AST permeation across the intestinal epithelium. Subsequent in vivo experiments demonstrated that the composite carrier exhibited a remarkable mucoadhesive capacity, allowing for extended intestinal retention of up to 12 h, and it displayed deep penetration through the mucus layer, efficiently entering the intestinal villi epithelial cells, and enhancing the absorption of AST and its bioavailability in vivo. And oral administration of AST NSC/HSA-PEG liposomes@SA/CMCS could effectively prevent hypercholesterolemia caused by a high-fat, high-cholesterol diet (HFHCD). These advancements highlight the potential of NSC/HSA-PEG liposomes@SA/CMCS composite carriers for targeted and oral uptake of hydrophobic bioactives.

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

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. 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. 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. 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.

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.

Sialic Acid Mediated Endothelial and Hepatic Uptake: A Mechanism based Mathematic Model Elucidating the Complex Pharmacokinetics and Pharmacodynamics of Efmarodocokin Alfa, a Variably Glycosylated Fusion Protein

Sialic acid (SA) is crucial for protecting glycoproteins from clearance. Efmarodocokin alfa (IL-22Fc), a fusion protein agonist that links IL-22 to the crystallizable fragment (Fc) of human IgG4, contains 8 N-glycosylation sites and exhibits heterogeneous and variable terminal sialylation biodistribution. This presents a unique challenge for Pharmacokinetic (PK) and Pharmacodynamic (PD) analysis and cross-species translation. In this study, we sought to understand how varying SA levels and heterogeneous distribution contribute to IL-22Fc's complex PKPD properties. We initially used homogenous drug material with varying SA levels to examine PKPD in mice. Population PKPD analysis based on mouse data revealed that SA was a critical covariate simultaneously accounting for the substantial between subject variability (BSV) in clearance (CL), distribution clearance (CLd), and volume of distribution (Vd). In addition to the well-established mechanism by which SA inhibits ASGPR activity, we hypothesized a novel mechanism by which decrease in SA increases the drug uptake by endothelial cells. This decrease in SA, leading to more endothelial uptake, was supported by the neonatal Fc receptor (FcRn) dependent cell-based transcytosis assay. The population analysis also suggested in vivo EC50 (IL-22Fc stimulating Reg3β) was independent on SA, while the in-vitro assay indicated a contradictory finding of SA-in vitro potency relationship. We created a mechanism based mathematical (MBM) PKPD model incorporating the decrease in SA mediated endothelial and hepatic uptake, and successfully characterized the SA influence on IL-22Fc PK, as well as the increased PK exposure being responsible for increased PD. Thereby, the MBM model supported that SA has no direct impact on EC50, aligning with the population PKPD analysis. Subsequently, using the MBM PKPD model, we employed 5 subpopulation simulations to reconstitute the heterogeneity of drug material. The simulation accurately predicted the PKPD of heterogeneously and variably sialylated drug in mouse, monkey and human. The successful prospective validation confirmed the MBM's ability to predict IL-22Fc PK across variable SA levels, homogenous to heterogeneous material, and across species (R2=0.964 for clearance prediction). Our model prediction suggests an average of 1 mol/mol SA increase leads to a 50% increase in drug exposure. This underlines the significance of controlling sialic acid levels during lot-to-lot manufacturing.

Rozanolixizumab (Rystiggo) for myasthenia gravis.

Rozanolixizumab-noli (Rystiggo – UCB), a neonatal Fc receptor (FcRn) blocker, has been approved by the FDA for treatment of generalized myasthenia gravis in adults with anti-acetylcholine receptor (AChR) or anti-muscle-specific tyrosine kinase (MuSK) antibodies. It is the first drug to be approved for treatment of generalized myasthenia gravis in patients with anti-MuSK antibodies.

An engineered immunomodulatory IgG1 Fc suppresses autoimmune inflammation through pathways shared with i.v. immunoglobulin.

Immunoglobulin G (IgG) antibodies in the form of high-dose intravenous immunoglobulin (IVIG) exert immunomodulatory activity and are used in this capacity to treat inflammatory and autoimmune diseases. Reductionist approaches have revealed that terminal sialylation of the single asparagine-linked (N-linked) glycan at position 297 of the IgG1 Fc bestows antiinflammatory activity, which can be recapitulated by introduction of an F241A point mutation in the IgG1 Fc (FcF241A). Here, we examined the antiinflammatory activity of CHO-K1 cell-produced FcF241A in vivo in models of autoimmune inflammation and found it to be independent of sialylation. Intriguingly, sialylation markedly improved the half-life and bioavailability of FcF241A via impaired interaction with the asialoglycoprotein receptor ASGPR. Further, FcF241A suppressed inflammation through the same molecular pathways as IVIG and sialylated IgG1 Fc and required the C-type lectin SIGN-R1 in vivo. This contrasted with FcAbdeg (efgartigimod), an engineered IgG1 Fc with enhanced neonatal Fc receptor (FcRn) binding, which reduced total serum IgG concentrations, independent of SIGN-R1. When coadministered, FcF241A and FcAbdeg exhibited combinatorial antiinflammatory activity. Together, these results demonstrated that the antiinflammatory activity of FcF241A requires SIGN-R1, similarly to that of high-dose IVIG and sialylated IgG1, and can be used in combination with other antiinflammatory therapeutics that rely on divergent pathways, including FcAbdeg.

Biological Evaluation of Antibody-Drug Conjugates Produced by Tag-Free Lipoate Ligase A Modification.

The concept of tag-free protein modification has attracted considerable interest in chemical biology because of its flexible and straightforward reaction process. In 2021, a groundbreaking approach using lipoate ligase A (LplA) for tag-free enzymatic modification of antibodies was unveiled, demonstrating its potential for the generation of precise antibody conjugates. In this study, to further explore LplA-mediated antibody-drug conjugate (ADC) synthesis, we performed initial biological evaluations of ADCs synthesized using LplA. Using the anti-HER2 antibody trastuzumab, we introduced octanoic acid azide using LplA and subsequently obtained an ADC using click chemistry with the drug DBCO-VC-PAB-MMAE. The bioactivity of the synthesized anti-HER2-ADC was evaluated using HER2-positive SKBR-3 and HER2-negative MCF7 cells. Its toxicity and selectivity were found to be comparable to those of the FDA-approved Kadcyla. In addition, a stability study involving rat and human plasma demonstrated the stability of the LplA-mediated ADC. Additionally, the affinity for the neonatal Fc receptor (FcRn) was retained after conjugation. These preliminary in vitro evaluations suggested that LplA-derived ADCs can have considerable pharmaceutical potential. Our results can set the stage for further in vivo evaluations and safety assessments. We suggest that the integration of tag-free LplA methods into the production of ADCs can offer a novel and promising approach for biopharmaceutical manufacturing.

Cathepsin B Processing Is Required for the In Vivo Efficacy of Albumin-Drug Conjugates.

Targeted drug delivery approaches that selectively and preferentially deliver therapeutic agents to specific tissues are of great interest for safer and more effective pharmaceutical treatments. We investigated whether cathepsin B cleavage of a valine-citrulline [VC(S)]-containing linker is required for the release of monomethyl auristatin E (MMAE) from albumin-drug conjugates. In this study, we used an engineered version of human serum albumin, Veltis High Binder II (HBII), which has enhanced binding to the neonatal Fc (fragment crystallizable) receptor (FcRn) to improve drug release upon binding and FcRn-mediated recycling. The linker-payload was conjugated to cysteine 34 of albumin using a carbonylacrylic (caa) reagent which produced homogeneous and plasma stable conjugates that retained FcRn binding. Two caa-linker-MMAE reagents were synthesized─one with a cleavable [VC(S)] linker and one with a noncleavable [VC(R)] linker─to question whether protease-mediated cleavage is needed for MMAE release. Our findings demonstrate that cathepsin B is required to achieve efficient and selective antitumor activity. The conjugates equipped with the cleavable [VC(S)] linker had potent antitumor activity in vivo facilitated by the release of free MMAE upon FcRn binding and internalization. In addition to the pronounced antitumor activity of the albumin conjugates in vivo, we also demonstrated their preferable tumor biodistribution and biocompatibility with no associated toxicity or side effects. These results suggest that the use of engineered albumins with high FcRn binding combined with protease cleavable linkers is an efficient strategy to target delivery of drugs to solid tumors.

Current Understanding of Immune Thrombocytopenia: A Review of Pathogenesis and Treatment Options.

The management of immune thrombocytopenia (ITP) and the prediction of patient response to therapy still represent a significant and constant challenge in hematology. ITP is a heterogeneous disease with an unpredictable evolution. Although the pathogenesis of ITP is currently better known and its etiology has been extensively studied, up to 75% of adult patients with ITP may develop chronicity, which represents a significant burden on patients' quality of life. A major risk of ITP is bleeding, but knowledge on the exact relationship between the degree of thrombocytopenia and bleeding symptoms, especially at a lower platelet count, is lacking. The actual management of ITP is based on immune suppression (corticosteroids and intravenous immunoglobulins), or the use of thrombopoietin receptor agonists (TPO-RAs), rituximab, or spleen tyrosine kinase (Syk) inhibitors. A better understanding of the underlying pathology has facilitated the development of a number of new targeted therapies (Bruton's tyrosine kinase inhibitors, neonatal Fc receptors, strategies targeting B and plasma cells, strategies targeting T cells, complement inhibitors, and newer TPO-RAs for improving megakaryopoiesis), which seem to be highly effective and well tolerated and result in a significant improvement in patients' quality of life. The disadvantage is that there is a lack of knowledge of the predictive factors of response to treatments, which would help in the development of an optimized treatment algorithm for selected patients.