The hinge-engineered IgG1-IgG3 hybrid subclass IgGh47 potently enhances Fc-mediated function of anti-streptococcal and SARS-CoV-2 antibodies.

Streptococcus pyogenes can cause invasive disease with high mortality despite adequate antibiotic treatments. To address this unmet need, we have previously generated an opsonic IgG1 monoclonal antibody, Ab25, targeting the bacterial M protein. Here, we engineer the IgG2-4 subclasses of Ab25. Despite having reduced binding, the IgG3 version promotes stronger phagocytosis of bacteria. Using atomic simulations, we show that IgG3's Fc tail has extensive movement in 3D space due to its extended hinge region, possibly facilitating interactions with immune cells. We replaced the hinge of IgG1 with four different IgG3-hinge segment subclasses, IgGhxx. Hinge-engineering does not diminish binding as with IgG3 but enhances opsonic function, where a 47 amino acid hinge is comparable to IgG3 in function. IgGh47 shows improved protection against S. pyogenes in a systemic infection mouse model, suggesting that IgGh47 has promise as a preclinical therapeutic candidate. Importantly, the enhanced opsonic function of IgGh47 is generalizable to diverse S. pyogenes strains from clinical isolates. We generated IgGh47 versions of anti-SARS-CoV-2 mAbs to broaden the biological applicability, and these also exhibit strongly enhanced opsonic function compared to the IgG1 subclass. The improved function of the IgGh47 subclass in two distant biological systems provides new insights into antibody function.

A Novel Dual-Fc Bispecific Antibody with Enhanced Fc Effector Function.

Bispecific antibodies (BsAbs) are undergoing continued development for applications in oncology and autoimmune diseases. While increasing activity by having more than one targeting arm, most BsAb engineering employs single Fc engagement as monoclonal antibodies. Here, we designed a novel immunoglobulin gamma-1 (IgG1)-derived dual-Fc BsAb containing two Fc regions and two distinct asymmetric antigen binding arms comprising a Fab arm and another VHH domain. In conjunction with the knob-into-hole technology, dual-Fc BsAbs could be produced with a high yield and good stability. We explore how Fc engineering effects on dual-Fc constructs could boost the desired therapeutic efficacy. This new format enabled simultaneous bispecific binding to corresponding antigens. Furthermore, compared to the one-Fc control molecules, dual-Fc BsAbs were shown to increase the avidity-based binding to FcγRs to result in higher ADCC and ADCP activities by potent avidity via binding to two antigens and Fc receptors. Overall, this novel BsAb format with enhanced effector functionalities provides a new option for antibody-based immunotherapy.

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.

Chimerism of avian IgY-scFv and truncated IgG-Fc: A novel strategy in cross-species antibody generation and enhancement.

Chicken single-chain fragment variable (IgY-scFv) is a functional fragment and an emerging development in genetically engineered antibodies with a wide range of biomedical applications. However, scFvs have considerably shorter serum half-life due to the absence of antibody Fc region compared with the full-length antibody, and usually requires continuous intravenous administration for efficacy. A promising approach to overcome this limitation is to fuse scFv with immunoglobulin G (IgG) Fc region, for better recognition and mediation by the neonatal Fc receptor (FcRn) in the host. In this study, engineered mammalian ΔFc domains (CH2, CH3, and intact Fc region) were fused with anti-canine parvovirus-like particles avian IgY-scFv to produce chimeric antibodies and expressed in the HEK293 cell expression system. The obtained scFv-CH2, scFv-CH3, and scFv-Fc can bind with antigen specifically and dose-dependently. Surface plasmon resonance investigation confirmed that scFv-CH2, scFv-CH3, and scFv-Fc had different degrees of binding to FcRn, with scFv-Fc showing the highest affinity. scFv-Fc had a significantly longer half-life in mice compared with the unfused scFv. The identified ΔFcs are promising for the development of engineered Fc-based therapeutic antibodies and proteins with longer half-lives. The avian IgY-scFv-mammalian IgG Fc region opens up new avenues for antibody engineering, and it is a novel strategy to enhance the rapid development and screening of functional antibodies in veterinary and human medicine.

Assessment of IgG-Fc glycosylation from individual RhD-specific B cell clones reveals regulation at clonal rather than clonotypic level.

The type and strength of effector functions mediated by immunoglobulin G (IgG) antibodies rely on the subclass and the composition of the N297 glycan. Glycosylation analysis of both bulk and antigen-specific human IgG has revealed a marked diversity of the glycosylation signatures, including highly dynamic patterns as well as long-term stability of profiles, yet information on how individual B cell clones would contribute to this diversity has hitherto been lacking. Here, we assessed whether clonally related B cells share N297 glycosylation patterns of their secreted IgG. We differentiated single antigen-specific peripheral IgG+ memory B cells into antibody-secreting cells and analysed Fc glycosylation of secreted IgG. Furthermore, we sequenced the variable region of their heavy chain, which allowed the grouping of the clones into clonotypes. We found highly diverse glycosylation patterns of culture-derived IgG, which, to some degree, mimicked the glycosylation of plasma IgG. Each B cell clone secreted IgG with a mixture of different Fc glycosylation patterns. The majority of clones produced fully fucosylated IgG. B cells producing afucosylated IgG were scattered across different clonotypes. In contrast, the remaining glycosylation traits were, in general, more uniform. These results indicate IgG-Fc fucosylation to be regulated at the single-clone level, whereas the regulation of other glycosylation traits most likely occurs at a clonotypic or systemic level. The discrepancies between plasma IgG and culture-derived IgG, could be caused by the origin of the B cells analysed, clonal dominance or factors from the culture system, which need to be addressed in future studies.

High-Level Production of scFv-Fc Antibody Using an Artificial Promoter System with Transcriptional Positive Feedback Loop of Transactivator in CHO Cells.

With the increasing demand for therapeutic antibodies, CHO cells have become the de facto standard as producer host cells for biopharmaceutical production. High production yields are required for antibody production, and developing a high-titer production system is increasingly crucial. This study was established to develop a high-production system using a synthetic biology approach by designing a gene expression system based on an artificial transcription factor that can strongly induce the high expression of target genes in CHO cells. To demonstrate the functionality of this artificial gene expression system and its ability to induce the high expression of target genes in CHO cells, a model antibody (scFv-Fc) was produced using this system. Excellent results were obtained with the plate scale, and when attempting continuous production in semi-continuous cultures using bioreactor tubes with high-cell-density suspension culture using a serum-free medium, high-titer antibody production at the gram-per-liter level was achieved. Shifting the culture temperature to a low temperature of 33 °C achieved scFv-Fc concentrations of up to 5.5 g/L with a specific production rate of 262 pg/(cell∙day). This artificial gene expression system should be a powerful tool for CHO cell engineering aimed at constructing high-yield production systems.

Antigen-dependent modulation of immune responses to antigen-Fc fusion proteins by Fc-effector functions.

Background: Fc-fusion proteins have been successfully developed for therapeutic purposes, but are also a promising platform for the fast generation and purification of immunogens capable of inducing strong humoral immune responses in preclinical immunization studies. As the Fc-portion of immunoglobulins fused to an antigen confers functional properties of the parental antibody, such as dimerization, binding to Fc-receptors and complement activation, several studies reported that Fc-fusion proteins elicit stronger antigen-specific antibody responses than the unfused antigen. However, dimerization or half-life extension of an antigen have also been described to enhance immunogenicity. Methods: To explore the role of Fc-effector functions for the immunogenicity of fusions proteins of viral glycoproteins and Fc fragments, the HIV-1 gp120 and the RBD of SARS-CoV-2 were fused to the wild type muIgG2a Fc fragment or mutants with impaired (LALA-PG) or improved (GASDIE) Fc-effector functions. Results: Immunization of BALB/c mice with DNA vaccines encoding gp120 - Fc LALA-PG induced significantly higher antigen-specific antibody responses than gp120 - Fc WT and GASDIE. In contrast, immunization with DNA vaccines encoding the RBD fused to the same Fc mutants, resulted in comparable anti-RBD antibody levels and similar neutralization activity against several SARS-CoV-2 variants. Conclusion: Depending on the antigen, Fc-effector functions either do not modulate or suppress the immunogenicity of DNA vaccines encoding Fc-antigen fusion proteins.

Comparative Molecular Characterization and Pharmacokinetics of IgG1-Fc and Engineered Fc Human Antibody Variants to Insulin-like Growth Factor 2 Receptor (IGF2R).

Novel therapeutic approaches are much needed for the treatment of osteosarcoma. Targeted radionuclide therapy (TRT) and radioimmunotherapy (RIT) are promising approaches that deliver therapeutic radiation precisely to the tumor site. We have previously developed a fully human antibody, named IF3, that binds to insulin-like growth factor 2 receptor (IGF2R). IF3 was used in TRT to effectively inhibit tumor growth in osteosarcoma preclinical models. However, IF3's relatively short half-life in mice raised the need for improvement. We generated an Fc-engineered version of IF3, termed IF3δ, with amino acid substitutions known to enhance antibody half-life in human serum. In this study, we confirmed the specific binding of IF3δ to IGF2R with nanomolar affinity, similar to wild-type IF3. Additionally, IF3δ demonstrated binding to human and mouse neonatal Fc receptors (FcRn), indicating the potential for FcRn-mediated endocytosis and recycling. Biodistribution studies in mice showed a higher accumulation of IF3δ in the spleen and bone than wild-type IF3, likely attributed to abnormal spleen expression of IGF2R in mice. Therefore, the pharmacokinetics data from mouse xenograft models may not precisely reflect their behavior in canine and human patients. However, the findings suggest both IF3 and IF3δ as promising options for the RIT of osteosarcoma.

Generation of IL-2-Fc-antibody conjugates by click chemistry.

BACKGROUND: Immunocytokines (ICKs) are antibody directed cytokines produced by genetic fusion of an antibody to a cytokine. METHODS: We now show that antibodies conjugated by click chemistry to interleukin-2 (IL-2)-Fc form fully active conjugates, and in one example, equivalent activity to a genetically produced ICK. RESULTS: An IL-2-Fc fusion protein was optimized for click chemistry at hinge cysteines using protein stabilizing IL-2 mutations at Lys35 and Cys125 and Fc hinge mutations at Cys142 and Cys148. The IL-2-Fc fusion protein with K35E and C125S mutations with 3 intact hinge cysteines, designated as IL-2-Fc Par, was selected based on its minimal tendency to aggregate. IL-2-Fc-antibody clicked conjugates retained high IL-2 activity and bound target antigens comparable to parent antibodies. An IL-2-Fc-anti-CEA click conjugate showed comparable anti-tumor activity to an anti-CEA-IL-2 ICK in immunocompetent CEA transgenic mice bearing CEA positive orthotopic breast tumors. Significant increases in IFNγ+ /CD8+ and decreases in FoxP3+ /CD4+ T-cells were found for the clicked conjugate and ICK therapies, suggesting a common mechanism of tumor reduction. CONCLUSION: The production of antibody targeted IL-2 therapy via a click chemistry approach is feasible with comparable activity to genetically produced ICKs with the added advantage of multiplexing with other monoclonal antibodies.

Enhancement of immunogenicity and neutralizing responses against SARS-CoV-2 spike protein using the Fc fusion fragment.

AIMS: Vaccination has played an important role in protecting against death and the severity of COVID-19. The recombinant protein vaccine platform has a long track record of safety and efficacy. Here, we fused the SARS-CoV-2 spike S1 subunit to the Fc region of IgG and investigated immunogenicity, reactivity to human vaccinated sera, and neutralizing activity as a candidate protein vaccine. MATERIALS AND METHOD: We evaluated the immunogenicity of CHO-expressed S1-Fc fusion protein and tag-free S1 protein in rabbits via the production of S1-specific polyclonal antibodies. We subsequently compared the neutralizing activities of sera from immunized rabbits and human-vaccinated individuals using a surrogate Virus Neutralization Test (sVNT). KEY FINDINGS: The results indicate that S1-specific polyclonal antibodies were induced in all groups; however, antibody levels were higher in rabbits immunized with S1-Fc fusion protein than tag-free S1 protein. Moreover, the reactivity of human vaccinated sera against S1-Fc fusion protein was significantly higher than tag-free S1 protein. Lastly, the results of the virus-neutralizing activity revealed that vaccination with S1-Fc fusion protein induced the highest level of neutralizing antibody response against SARS-CoV-2. SIGNIFICANCE: Our results demonstrate that the S1 protein accompanied by the Fc fragment significantly enhances the immunogenicity and neutralizing responses against SARS-CoV-2. It is hoped that this platform can be used for human vaccination.

Residue interaction network and molecular dynamics simulation study on the binding of S239D/I332E Fc variant with enhanced affinity to FcγRIIIa receptor.

Engineering of Fc has been adapted as an efficient method for enhanced or reduced affinity towards Fc receptors in the development of therapeutic antibodies. S239D/I332E mutation of Fc induces approximately two logs greater affinity to the FcγRIIIa receptor and has been extensively employed in various Fc engineering studies. It is known that the mutation gives rise to the formation of salt bridges between the mutated residues of Fc and FcγRIIIa, but the overall effect of the mutation in the binding interface of the Fc-FcγRIIIa complex is still unclear. In this study, the molecular interactions in the binding interface of mutant Fc and FcγRIIIa were analyzed and compared with those of wild-type Fc binding through residue interaction network (RIN) analysis and molecular dynamics (MD) simulation. RIN analysis identified specific molecular interactions and Hub residues in the interfaces, and their numbers were increased by introducing the mutation, with maintaining most of the molecular interactions in the wild-type complex. MD simulation study revealed that the numbers of stable electrostatic interactions and stable Hub residues in the mutant complex were higher than those in the wild-type complex. The introduced mutations were shown to form further charge-charge attractive interactions in addition to the identified salt bridges without generating any repulsive interactions. These results are expected to provide further structural insight into Fc variants' design based on the S239D/I332E mutation.

Dulaglutide as a demethylating agent to improve the outcome of breast cancer.

Background: Dulaglutide emerged as a promising therapeutic option for diabetes mellitus Type 2 (DM2). Aims: Owing to epigenetic similarities between the pathophysiology of DM2 and breast cancer (BC), we investigated the antitumor effect of dulaglutide. Materials & methods: To investigate the effect of dulaglutide, we analyzed the expression of methylated gene promoter regions in BC (ESR1, CDH1 and ADAM33). Results: Dulaglutide increased the expression of ESR1, CDH1 and ADAM33 up to fourfold in the MDA-MB-231 lineage by demethylating the gene promoter regions. This effect was translated to in vivo antitumoral activity and revealed significant tumor inhibition by combining the half-dose of methotrexate with dulaglutide. Conclusion: This therapy may mitigate the severe side effects commonly associated with chemotherapy.

A homodimeric IL-15 superagonist F4RLI with easy preparation, improved half-life, and potent antitumor activities.

Interleukin-15 (IL-15) is a promising candidate for cancer immunotherapy due to its potent immune-activating effects. There are several IL-15 molecules currently in clinical trials but facing shortages of poor half-life, circulation instability, or complicated production and quality control processes. The aim of this study is to design a novel IL-15 superagonist to set out the above difficulties, and we constructed F4RLI consisting of the GS-linker spaced IgG4 Fc fragment, soluble IL-15 Rα (sIL-15Rα), and IL-15(N72D). Using a single plasmid transient transfection in HEK293E cells, the matured F4RLI was secreted in the form of homodimer and got purified by an easy step of protein A affinity chromatography. The F4RLI product can significantly stimulate the proliferation of human CD3+CD8+ T cells and NK cells in vitro. Meanwhile, F4RLI greatly extended the half-life and prolonged the exposure of IL-15 in mice nearly by 28- and 200-fold, respectively, in comparison with that of the IL-15 monomer. In vivo, F4RLI vastly expanded mouse splenic CD8+ T lymphocytes, illustrating its potential in tumor immunotherapy. Further studies showed that the combination of F4RLI with the immune checkpoint blocker atezolizumab played a synergistic effect in treating MC38 mouse tumor by increasing the percentage of CD8+ T cells in tumor tissue. Moreover, the combination therapy of F4RLI with the angiogenesis inhibitor bevacizumab resulted in significant tumor growth suppression in a xenograft human HT-29 mouse model. Overall, our results demonstrate a homodimeric IL-15 superagonist F4RLI with advances in manufacturing processes and biopharmaceutical applications for cancer immunotherapy. KEY POINTS: • The homodimeric structure of F4RLI facilitates its easy production processes and quality control. • The fusion with Fc and sIL-15Rα extends the plasma half-life of IL-15 by about 28-fold. • F4RLI can play synergistic antitumor activity with the PD-1/PD-L1 checkpoint inhibitor or angiogenesis inhibitor.

Manipulation of mRNA translation elongation influences the fragmentation of a biotherapeutic Fc-fusion protein produced in CHO cells.

Mammalian cells, particularly Chinese hamster ovary cells, are the dominant system for the production of protein-based biotherapeutics, however, product degradation, particularly of Fc-fusion proteins, is sometimes observed that impacts the quality of the protein generated. Here, we identify the site of fragmentation of a model immunoglobulin G1 Fc-fusion protein, show that the observed clipping and aggregation are decreased by reduced temperature culturing, that the fragmentation/clipping is intracellular, and that reduced clipping at a lower temperature (<37°C) relates to mesenger RNA (mRNA) translation elongation. We subsequently show that reduced fragmentation can be achieved at 37°C by addition of chemical reagents that slow translation elongation. We then modified mRNA translation elongation speeds by designing different transcript sequences for the Fc-fusion protein based on alternative codon usage and improved the product yield at 37°C, and the ratio of intact to a fragmented product. Our data suggest that rapid elongation results in misfolding that decreases product fidelity, generating a region susceptible to degradation/proteolysis, whilst the slowing of mRNA translation improves the folding, reducing susceptibility to fragmentation. Manipulation of mRNA translation and/or the target Fc-fusion transcript is, therefore, an approach that can be applied to potentially reduce fragmentation of clipping-prone Fc-fusion proteins.

Self-assembling peptide nanofiber HIV vaccine elicits robust vaccine-induced antibody functions and modulates Fc glycosylation.

To develop vaccines for certain key global pathogens such as HIV, it is crucial to elicit both neutralizing and non-neutralizing Fc-mediated effector antibody functions. Clinical evidence indicates that non-neutralizing antibody functions including antibody-dependent cellular cytotoxicity (ADCC) and antibody-dependent cellular phagocytosis (ADCP) contribute to protection against several pathogens. In this study, we demonstrated that conjugation of HIV Envelope (Env) antigen gp120 to a self-assembling nanofiber material named Q11 induced antibodies with higher breadth and functionality when compared to soluble gp120. Immunization with Q11-conjugated gp120 vaccine (gp120-Q11) demonstrated higher tier 1 neutralization, ADCP, and ADCC as compared to soluble gp120. Moreover, Q11 conjugation altered the Fc N-glycosylation profile of antigen-specific antibodies, leading to a phenotype associated with increased ADCC in animals immunized with gp120-Q11. Thus, this nanomaterial vaccine strategy can enhance non-neutralizing antibody functions possibly through modulation of immunoglobulin G Fc N-glycosylation.

Effect of glioma-derived immunoglobulin on biological function of glioma cells.

INTRODUCTION: Glioma is the most common and most invasive primary central nervous system tumour, and it is urgent to develop new specific therapeutic targets. Studies have confirmed that epithelial-derived tumour cells promote tumour cell proliferation and metastasis by secreting a large number of immunoglobulins (Igs), but the role of tumour-derived Igs in glioma has never been reported. METHODS: The Gene Expression Profiling Interactive Analysis and Chinese Glioma Genome Atlas databases were used to analyse the Ig transcription and its correlation with the prognosis of patients with glioma. Immunohistochemistry and immunofluorescence were used to detect the protein expression of IgG and IgM in the glioma tissues of patients and glioma cell lines. When IgG was knocked down by small interfering RNA or knocked out by CRISPR-Cas9, the function of proliferation and migration of glioma cells were analysed by CCK-8, clone formation, wound healing, and transwell assays. Changes in proteins and their phosphorylation in signalling pathways were detected by western blotting. The nude mouse subcutaneous tumour-bearing model was established to analyse the effect of IgG in vivo. RESULTS: The transcriptional level of IgG was pretty high in glioma tissues and was positively correlated with high WHO grade, recurrence, and poor prognosis. The expression of IgG and IgM was found in tumour tissues and human glioma cell lines U87 and U251, and the main expression form was secreted. Decreased IgG inhibited the proliferation and migration of glioma cells. Knockout or knockdown of IgG downregulated the phosphorylation of the key molecules in the MAPK and PI3K/Akt pathway through the HGF/SF-Met or FAK/Src pathway. In vivo tumourigenesis mouse model confirmed that reduced IgG expression inhibited glioma growth. CONCLUSION: Ig was expressed in glioma tissues and cell lines, and a high expression level predicted a poor prognosis of patients. Glioma-derived IgG promoted glioma cell proliferation and migration through the HGF/SF-Met or FAK/Src pathway.

The Chicken A and E Blood Systems Arise from Genetic Variation in and around the Regulators of Complement Activation Region.

The tightly linked A and E blood alloantigen systems are 2 of 13 blood systems identified in chickens. Reported herein are studies showing that the genes encoding A and E alloantigens map within or near to the chicken regulator of complement activation (RCA) gene cluster, a region syntenic with the human RCA. Genome-wide association studies, sequence analysis, and sequence-derived single-nucleotide polymorphism information for known A and/or E system alleles show that the most likely candidate gene for the A blood system is C4BPM gene (complement component 4 binding protein, membrane). Cosegregation of single-nucleotide polymorphism-defined C4BPM haplotypes and blood system A alleles defined by alloantisera provide a link between chicken blood system A and C4BPM. The best match for the E blood system is the avian equivalent of FCAMR (Fc fragment of IgA and IgM receptor). C4BPM is located within the chicken RCA on chicken microchromosome 26 and is separated from FCAMR by 89 kbp. The genetic variation observed at C4BPM and FCAMR could affect the chicken complement system and differentially guide immune responses to infectious diseases.

Impact of glycoengineering and antidrug antibodies on the anticancer activity of a plant-made lectin-Fc fusion protein.

Plants are an efficient production platform for manufacturing glycoengineered monoclonal antibodies and antibody-like molecules. Avaren-Fc (AvFc) is a lectin-Fc fusion protein or lectibody produced in Nicotiana benthamiana, which selectively recognizes cancer-associated high-mannose glycans. In this study, we report the generation of a glycovariant of AvFc that is devoid of plant glycans, including the core α1,3-fucose and ß1,2-xylose residues. The successful removal of these glycans was confirmed by glycan analysis using HPLC. This variant, AvFcΔXF , has significantly higher affinity for Fc gamma receptors and induces higher levels of luciferase expression in an antibody-dependent cell-mediated cytotoxicity (ADCC) reporter assay against B16F10 murine melanoma cells without inducing apoptosis or inhibiting proliferation. In the B16F10 flank tumour mouse model, we found that systemic administration of AvFcΔXF , but not an aglycosylated AvFc variant lacking affinity for Fc receptors, significantly delayed the growth of tumours, suggesting that Fc-mediated effector functions were integral. AvFcΔXF treatment also significantly reduced lung metastasis of B16F10 upon intravenous challenge whereas a sugar-binding-deficient mutant failed to show efficacy. Lastly, we determined the impact of antidrug antibodies (ADAs) on drug activity in vivo by pretreating animals with AvFcΔXF before implanting tumours. Despite a significant ADA response induced by the pretreatment, we found that the activity of AvFcΔXF was unaffected by the presence of these antibodies. These results demonstrate that glycoengineering is a powerful strategy to enhance AvFc's antitumor activity.

Presence and possible impact of Fcγ receptors on resident kidney cells in health and disease.

Fcγ receptors (FcγRs) bind the Fc fragment of immunoglobulin G (IgG), mostly after IgG opsonizes a bacterial or viral antigen or danger/damage-associated molecule. Consequently, classic FcγRs initiate phagocytosis of the IgG-antigen immune complex and stimulate an immune reaction against the threat. Signals from activating FcγRs (FcγRI, FcγRIIa/c, FcγRIIIa/b) are balanced by inhibitory FcγRIIb and likely also by two FcR-like proteins (FCRL4 and FCRL5). The neonatal Fc receptor (FcRn) recirculates IgG and increases its half-life. The last FcγR that has been identified in humans, tripartite motif-containing protein 21 (TRIM21), acts toward pathogen destruction via the proteasomal or autophagic pathway. The expression of FcγRs occurs almost exclusively in immune cells. However, podocytes, key cells in the glomerular filtration barrier, also possess several features of an immune cell and express receptors for IgG. The presence of FcγRs in glomeruli was analyzed in the Human Protein Atlas project. FcγR occurrence in whole glomeruli or in particular resident kidney cells was also showed in a few original articles. In human podocytes only FcRn has been studied extensively, and the presence and role of other FcγRs remain obscure. Research on the genetic background of kidney diseases revealed a connection between FcγRs and several nephropathies. Investigations of FcγR expression in podocytes appear to be of great clinical importance. The present review discusses the latest literature on FcγRs in kidney cells (especially podocytes), with an emphasis on their involvement in kidney health and disease.

Effect of the ADCC-Modulating Mutations and the Selection of Human IgG Isotypes on Physicochemical Properties of Fc.

Monoclonal antibodies, particularly IgGs and Ig-based molecules, are a well-established and growing class of biotherapeutic drugs. In order to improve efficacy, potency and pharmacokinetics of these therapeutic drugs, pharmaceutical industries have investigated significantly in engineering fragment crystallizable (Fc) domain of these drugs to optimize the interactions of these drugs and Fc gamma receptors (FcγRs) in recent ten years. The biological function of the therapeutics with the antibody-dependent cellular cytotoxicity (ADCC) enhanced double mutation (S239D/I332E) of isotype IgG1, the ADCC reduced double mutation (L234A/L235A) of isotype IgG1, and ADCC reduced isotype IgG4 has been well understood. However, limited information regarding the effect of these mutations or isotype difference on physicochemical properties (PCP), developability, and manufacturability of therapeutics bearing these different Fc regions is available. In this report, we systematically characterize the effects of the mutations and IgG4 isotype on conformation stability, colloidal stability, solubility, and storage stability at accelerated conditions in two buffer systems using six Fc variants. Our results provide a basis for selecting appropriate Fc region during development of IgG or Ig-based therapeutics and predicting effect of the mutations on CMC development process.