Microsecond Dynamics of Fc-CD16a Recognition: Impact of Mutations, Core Fucosylation, and Fc Asymmetry.

Antibody-dependent cellular cytotoxicity (ADCC) is mediated through the engagement of the Fc segment of antibodies with Fcγ receptors (FcγRs) on immune cells upon binding of tumor or viral antigen. The co-crystal structure of FcγRIII in complex with Fc revealed that Fc binds to FcγRIII asymmetrically with two Fc chains contacting separate regions of the FcγRIII by utilizing different residues. To fully explore this asymmetrical nature of the Fc-FcγR interaction, we screened more than 9,000 individual clones in Fc heterodimer format in which different mutations were introduced at the same position of two Fc chains using a high throughput competition AlphaLISA® assay. To this end, we have identified a panel of novel Fc variants with significant binding improvement to FcγRIIIA (both Phe-158 and Val-158 allotypes), increased ADCC activity in vitro, and strong tumor growth inhibition in mice xenograft human tumor models. Compared with previously identified Fc variants in conventional IgG format, Fc heterodimers with asymmetrical mutations can achieve similar or superior potency in ADCC-mediated tumor cell killing and demonstrate improved stability in the CH2 domain. Fc heterodimers also allow more selectivity toward activating FcγRIIA than inhibitory FcγRIIB. Afucosylation of Fc variants further increases the affinity of Fc to FcγRIIIA, leading to much higher ADCC activity. The discovery of these Fc variants will potentially open up new opportunities of building the next generation of therapeutic antibodies with enhanced ADCC effector function for the treatment of cancers and infectious diseases.

Rationale: Fc engineering has become the focus of antibody drug development. The current mutagenesis and in silico protein design methods are confined by the limited throughput and high cost, while the high-throughput phage display and yeast display technologies are not suitable for screening glycosylated Fc variants. Here we developed a mammalian cell display-based Fc engineering platform.Methods: By using mammalian cell display and next generation sequencing, we screened millions of Fc variants for optimized affinity and specificity for FcγRIIIa or FcγRIIb. The identified Fc variants with improved binding to FcγRIIIa were substituted into trastuzumab and rituximab and the effector function of antibodies were examined in the PBMC-based assay. On the other hand, the identified Fc variants with selectively enhanced FcγRIIb binding were applied to CD40 agonist antibody and the activities of the antibodies were measured on different cell assays. The immunostimulatory activity of CD40 antibodies was also evaluated by OVA-specific CD8+ T cell response model in FcγR/CD40-humanized mice.Results: Using this approach, we screened millions of Fc variant and successfully identified several novel Fc variants with enhanced FcγRIIIa or FcγRIIb binding. These identified Fc variants displayed a dramatic increase in antibody-dependent cellular cytotoxicity in PBMC-based assay. Novel variants with selectively enhanced FcγRIIb binding were also identified. CD40 agonist antibodies substituted with these Fc variants displayed activity more potent than the parental antibody in the in vitro and in vivo models.Conclusions: This approach increased the throughput of Fc variant screening from thousands to millions magnitude, enabled screening variants containing multiple mutations and could be integrated with glycoengineering technology, represents an ideal platform for Fc engineering. The initial efforts demonstrated the capability of the platform and the novel Fc variants could be substituted into nearly any antibody for the next generation of antibody therapeutics.

This study determined the effect of n-3 polyunsaturated fatty acids (n-3 PUFAs)-enriched hen eggs consumption on immunoglobulin G (IgG) and total plasma protein N-glycan profiles and inflammatory biomarkers level in healthy individuals (N=33) and cardiovascular (CV) patients (N=21). Subjects were divided to Control-Healthy and Control-CV subgroups [consumed three regular hens' eggs/daily (249 mg n-3 PUFAs/day)], and n-3 PUFAs-Healthy and n-3 PUFAs-CV subgroups [consumed three n-3 PUFAs-enriched hen eggs/daily (1053 mg n-3 PUFAs/day)] for 3weeks. Serum-free fatty acids profile and high-sensitivity C-reactive protein, interleukin 6 and 10 (IL-6, IL-10) and tumor necrosis factor alpha were measured. Total plasma protein and IgG N-glycome have been profiled before and after dietary protocols. Serum n-3 PUFAs concentration significantly increased following n-3 PUFAs hen eggs consumption in both n-3 PUFAs-Healthy and n-3 PUFAs-CV. IL-10 significantly increased in both Healthy subgroups, whereas no change occurred in CV subgroups. Derived IgG N-glycan traits: bisecting N-acetylglucosamine (B) significantly decreased in n-3 PUFAs-Healthy, whereas agalactosylation (G0) and core fucosylation (CF) significantly increased in Control-Healthy. Derived total plasma protein N-glycan traits: high branching glycans, trigalactosylation, tetragalactosylation, trisialylation, tetrasialylation and antennary fucosylation significantly decreased, whereas G0, monogalactosylation (G1), neutral glycans (S0), B, CF and oligomannose structures significantly increased in n-3 PUFAs-CV. Digalactosylation significantly decreased, and G0, G1, S0, disialylation, B and CF significantly increased in Control-CV. n-3 PUFAs consumption alters IgG N-glycan traits and IL-10 in healthy individuals, and total plasma protein N-glycan traits in CV patients, by shifting them toward less inflammatory N-glycosylation profile.

BackgroundProtein N-glycosylation plays a key role in cancer biology and may offer insights into tumor behavior and treatment response. This study investigated changes in N-glycosylation of immunoglobulin G (IgG) and total plasma proteins in patients with breast cancer undergoing neoadjuvant chemotherapy.MethodsA prospective cohort of 34 women with early-stage or locally advanced breast cancer was recruited. Plasma samples were collected before and after neoadjuvant chemotherapy. IgG was isolated by immunoaffinity chromatography, and IgG and total plasma N-glycans were enzymatically released, fluorescently labeled, and analyzed using ultra-high performance liquid chromatography. Glycan traits were expressed as relative abundances and summarized into derived structural features. Longitudinal changes were assessed using linear mixed-effects models adjusted for age and body mass index.ResultsChemotherapy induced significant decrease in IgG core fucosylation. This effect varied by treatment: anthracycline-based regimen led to decreased core fucosylation and digalactosylation and increased monogalactosylation. The greatest reduction in core fucosylation was observed in patients treated with docetaxel and cyclophosphamide. HER2-targeted therapy was associated with decreased bisecting N-acetylglucosamine. Plasma glycosylation remained largely stable, though oligomannose glycans increased in patients following chemotherapy. Tumor size was significantly associated with several plasma glycan traits, particularly digalactosylation and oligomannosylation.ConclusionsIgG glycosylation patterns change in a treatment-specific manner during chemotherapy, potentially reflecting immune modulation. Plasma glycan traits are more stable but may reflect tumor burden. These results support the potential of glycan profiling as a biomarker for monitoring the impact of therapy and disease progression in breast cancer.Supplementary InformationThe online version contains supplementary material available at 10.1186/s12885-025-15381-5.

Some effector functions prompted by immunoglobulin G (IgG) antibodies, such as antibody-dependent cell-mediated cytotoxicity (ADCC), strongly depend on the N-glycans linked to asparagine 297 of the Fc region of the protein. A single α-(1,6)-fucosyltransferase (FUT8) is responsible for catalyzing the addition of an α-1,6-linked fucose residue to the first GlcNAc residue of the N-linked glycans. Antibodies missing this core fucose show a significantly enhanced ADCC and increased antitumor activity, which could help reduce therapeutic dose requirement, potentially translating into reduced safety concerns and manufacturing costs. Several approaches have been developed to modify glycans and improve the biological functions of antibodies. Here, we demonstrate that expression of a membrane-associated anti-FUT8 intrabody engineered to reside in the endoplasmic reticulum and Golgi apparatus can efficiently reduce FUT8 activity and therefore the core-fucosylation of the Fc N-glycan of an antibody. IgG1-producing CHO cells expressing the intrabody secrete antibodies with reduced core fucosylation as demonstrated by lectin blot analysis and UPLC-HILIC glycan analysis. Cells engineered to inhibit directly and specifically alpha-(1,6)-fucosyltransferase activity allows for the production of g/L levels of IgGs with strongly enhanced ADCC effector function, for which the level of fucosylation can be selected. The quick and efficient method described here should have broad practical applicability for the development of next-generation therapeutic antibodies with enhanced effector functions.

N-Glycoprofiling of immunoglobulin G and lactoperoxidase from sheep milk using LC-MS/MS

Monitoring human circulating N-glycome could provide valuable insight into an individual's metabolic status. Therefore, we examined if aberrant carbohydrate metabolism in gestational diabetes mellitus (GDM) associates with alterations in plasma protein, immunoglobulin G (IgG) and immunoglobulin A (IgA) N-glycosylation. Plasma protein, IgG and IgA N-glycans were enzymatically released, purified and chromatographically profiled in 48 pregnant women with normal glucose tolerance and 41 pregnant women with GDM, all sampled at 24-28 weeks of gestation. Linear mixed models adjusting for age and multiple testing (FDR<0.05) were used to investigate the associations between glycosylation features, metabolic markers and GDM status. Fasting insulin exhibited significant associations to numerous glycan traits, including plasma protein galactosylation, sialylation, branching, core fucosylation and bisection, to IgG core fucosylated, bisected (FA2B) and afucosylated disialylated (A2G2S2) glycan and to IgA trisialylated triantennary (A3G3S3) glycan (padj range: 4.37x10-05-4.94x10-02). Insulin resistance markers HOMA2-IR and HOMA2-%B were mostly associated to the same glycan structures as fasting insulin. Both markers showed positive association with high-branched plasma glycans (padj = 1.12x10-02 and 2.03x10-03) and negative association with low-branched plasma glycans (padj = 1.21x10-02 and 2.05x10-03). Additionally, HOMA2-%B index was significantly correlated with glycosylation features describing IgG sialylation. Multiple plasma protein IgG and IgA glycans showed significant associations with total cholesterol and triglyceride levels. None of the tested glycan traits showed a significant difference between GDM and normoglycemic pregnancies. Markers of glucose homeostasis and lipid metabolism in pregnancy show extensive associations to various N-glycosylation features. However, plasma protein, IgG and IgA N-glycans were not able to differentiate pregnant women with and without GDM, possibly due to numerous physiological changes accompanying pregnancy, which confound the impact of GDM on protein glycosylation.

N-Glycan Analysis of Polyclonal IgG from Patients with Multiple Myeloma Enables Classification of Stage Specific Pathologies

Fc engineering is a promising approach to enhance the antitumor efficacy of monoclonal antibodies (mAbs) through antibody-dependent cell-mediated cytotoxicity (ADCC). Glyco- and protein-Fc engineering have been employed to enhance FcγR binding and ADCC activity of mAbs; the drawbacks of previous approaches lie in their binding affinity to both FcγRIIIa allotypes, the ratio of activating FcγR binding to inhibitory FcγR binding (A/I ratio) or the melting temperature (TM) of the CH2 domain. To date, no engineered Fc variant has been reported that satisfies all these points. Herein, we present a novel Fc engineering approach that introduces different substitutions in each Fc domain asymmetrically, conferring optimal binding affinity to FcγR and specificity to the activating FcγR without impairing the stability. We successfully designed an asymmetric Fc variant with the highest binding affinity for both FcγRIIIa allotypes and the highest A/I ratio compared with previously reported symmetrically engineered Fc variants, and superior or at least comparable in vitro ADCC activity compared with afucosylated Fc variants. In addition, the asymmetric Fc engineering approach offered higher stability by minimizing the use of substitutions that reduce the TM of the CH2 domain compared with the symmetric approach. These results demonstrate that the asymmetric Fc engineering platform provides best-in-class effector function for therapeutic antibodies against tumor antigens.

Immunoglobulin G (IgG) antibodies coordinate immune effector responses by interacting with effector cells via fragment crystallizable γ (Fcγ) receptors. The IgG Fc domain directs effector responses through subclass and glycosylation variation. Although each Fc variant has been extensively characterized in isolation, during immune responses, IgG is almost always produced in Fc mixtures. How this influences effector responses has not been examined. Here, we measure Fcγ receptor binding to mixed Fc immune complexes. Binding of these mixtures falls along a continuum between pure cases and quantitatively matches a mechanistic model, except for several low-affinity interactions mostly involving IgG2. We find that the binding model provides refined estimates of their affinities. Finally, we demonstrate that the model predicts effector cell-elicited platelet depletion in humanized mice. Contrary to previous views, IgG2 exhibits appreciable binding through avidity, though it is insufficient to induce effector responses. Overall, this work demonstrates a quantitative framework for modeling mixed IgG Fc-effector cell regulation.

The neonatal Fc receptor (FcRn) plays an important and well-known role in immunoglobulin G (IgG) catabolism; however, its role in the disposition of IgG after subcutaneous (SC) administration, including bioavailability, is relatively unknown. To examine the potential effect of FcRn on IgG SC bioavailability, we engineered three anti-amyloid β monoclonal antibody (mAb) reverse chimeric mouse IgG2a (mIgG2a) Fc variants (I253A.H435A, N434H and N434Y) with different binding affinities to mouse FcRn (mFcRn) and compared their SC bioavailability to that of the wild-type (WT) mAb in mice. Our results indicated that the SC bioavailability of mIgG2a was affected by mFcRn-binding affinity. Variant I253A.H435A, which did not bind to mFcRn at either pH 6.0 or pH 7.4, had the lowest bioavailability (41.8%). Variant N434Y, which had the greatest increase in binding affinity at both pH 6.0 and pH 7.4, had comparable bioavailability to the WT antibody (86.1% vs. 76.3%), whereas Variant N434H, which had modestly increased binding affinity at pH 6.0 to mFcRn and affinity comparable to the WT antibody at pH 7.4, had the highest bioavailability (94.7%). A semi-mechanism-based pharmacokinetic model, which described well the observed data with the WT antibody and variant I253A.H435A, is consistent with the hypothesis that the decreased bioavailability of variant I253A.H435A was due to loss of the FcRn-mediated protection from catabolism at the absorption site. Together, these data demonstrate that FcRn plays an important role in SC bioavailability of therapeutic IgG antibodies.

The pH-selective interaction between the immunoglobulin G (IgG) fragment crystallizable region (Fc region) and the neonatal Fc receptor (FcRn) is critical for prolonging the circulating half-lives of IgG molecules through intracellular trafficking and recycling. By using directed evolution, we successfully identified Fc mutations that improve the pH-dependent binding of human FcRn and prolong the serum persistence of a model IgG antibody and an Fc-fusion protein. Strikingly, trastuzumab-PFc29 and aflibercept-PFc29, a model therapeutic IgG antibody and an Fc-fusion protein, respectively, when combined with our engineered Fc (Q311R/M428L), both exhibited significantly higher serum half-lives in human FcRn transgenic mice than their counterparts with wild-type Fc. Moreover, in a cynomolgus monkey model, trastuzumab-PFc29 displayed a superior pharmacokinetic profile to that of both trastuzumab-YTE and trastuzumab-LS, which contain the well-validated serum half-life extension Fcs YTE (M252Y/S254T/T256E) and LS (M428L/N434S), respectively. Furthermore, the introduction of two identified mutations of PFc29 (Q311R/M428L) into the model antibodies enhanced both complement-dependent cytotoxicity and antibody-dependent cell-mediated cytotoxicity activity, which are triggered by the association between IgG Fc and Fc binding ligands and are critical for clearing cancer cells. In addition, the effector functions could be turned off by combining the two mutations of PFc29 with effector function-silencing mutations, but the antibodies maintained their excellent pH-dependent human FcRn binding profile. We expect our Fc variants to be an excellent tool for enhancing the pharmacokinetic profiles and potencies of various therapeutic antibodies and Fc-fusion proteins.

Lupus nephritis (LN) is a crucial complication of systemic lupus erythematosus (SLE) and has important clinical implications in guiding treatment. N-glycosylation of immunoglobulin G (IgG) plays a key role in the development of SLE by affecting the balance of anti-inflammatory and proinflammatory responses. This study aimed to evaluate the performance of IgG N-glycosylation for diagnosing LN in a sample of female SLE patients. This case-control study recruited 188 women with SLE, including 94 patients with LN and 94 age-matched patients without LN. The profiles of plasma IgG N-glycans were detected by hydrophilic interaction chromatography with ultra-performance liquid chromatography (HILIC-UPLC). A multivariate logistic regression model was used to explore the associations between IgG N-glycans and LN. A diagnostic model was developed using the significant glycans as well as demographic factors. The performance of IgG N-glycans in the diagnosis of LN was evaluated by receiver operating characteristic (ROC) curve analysis, and the area under the curve (AUC) and its 95% confidence interval (CI) were calculated. There were significant differences in 9 initial glycans (GP2, GP4, GP6, GP8, GP10, GP14, GP16, GP18 and GP23) between women with SLE with and without LN (P < 0.05). The levels of sialylated, galactosylated and fucosylated glycans were significantly lower in the LN patients than in the control group, while bisected N-acetylglucosamine (GlcNAc) glycans were increased in LN patients (P < 0.05). GP8, GP10, GP18, and anemia were included in our diagnostic model, which performed well in differentiating female SLE patients with LN from those without LN (AUC = 0.792, 95% CI: 0.727 to 0.858). Our findings indicate that decreased sialylation, galactosylation, and core fucosylation and increased bisecting GlcNAc might play a role in the development of LN by upregulating the proinflammatory response of IgG. IgG N-glycans can serve as potential biomarkers to differentiate individuals with LN among SLE patients.

Enhancing the yield and activity of defucosylated antibody produced by CHO-K1 cells using Cas13d-mediated multiplex gene targeting

Neonatal respiratory distress syndrome (NRDS) is the leading cause of neonatal death. Changes in plasma immunoglobulin G (IgG) N-glycosylation have been demonstrated in a variety of diseases. However, its implications and clinical significance in NRDS remain to be clarified. To determine the effect of IgG N-glycosylation on NRDS, we recruited 88 NRDS participants and 120 control participants from December 2021 to September 2022. Plasma was collected, IgG was isolated and purified, and the glycogram was analyzed by ultra performance liquid chromatography (UPLC) with fluorescence detector. The occurrence of premature rupture of membranes (PROM) [OR=9.043(1.036-78.966), P=0.046] and the elevation of γ-glutamyltransferase (GGT) [OR=1.015(1.001-1.029), P=0.032] were independent risk factors for the occurrence of NRDS. Furthermore, the area percentages of GP1, GP3, GP4, GP11, GP13, and GP24 were significantly higher in NRDS patients compared with control group. Conversely, GP14 was observed to be significantly lower. Furthermore, an increase in plasma IgG sialylation and core fucosylation was observed in NRDS, whereas the modification with galactosylation was decreased. The model constructed using GP1, GP13, GP14, PROM, and GGT as composite indices demonstrated robust predictive performance (AUC=0.902, 95% CI: 0.851-0.953). Patients with NRDS frequently exhibit alterations in the glycosylation of plasma IgG. These findings provide new insights into the diagnosis of NRDS and clinical treatment.