Receptor IIIa Research Articles

Abstract 1555: Enhanced HER2-dependent immune activation by margetuximab, an investigational Fc-engineered anti-HER2 mAb, supports combination with checkpoint blockade

Abstract Background: Margetuximab (M) has demonstrated anti-tumor activity in patients with advanced HER2+ gastric cancer and PFS superiority to trastuzumab (T) in pre-treated metastatic HER2+ breast cancer patients (1). Similar to T, M inhibits HER2 signaling; additionally, M enhances engagement with the activating Fcγ receptor (FcR)IIIA, while diminishing interaction with the inhibitory FcRIIB. Given the role of FcRs in coupling innate and adaptive immune responses, clinical studies of M in combination with PD-1 or PD-1 plus LAG-3 blockade have been initiated (2,3). The combination of M with tebotelimab, an investigational bispecific DART® molecule blocking PD-1 and LAG-3, has demonstrated encouraging early evidence of anti-tumor activity in patients with advanced HER2+ malignancies, including those with PD-L1-negative tumors (3). Here we present data supporting the potential mechanisms underlying the anti-tumor activity elicited by M and tebotelimab combination therapy. Methods: PBMCs were challenged with HER2-expressing tumor cell lines in the presence of M or T. Immune modulation was assessed by analysis of immune cell phenotype, gene expression profiles, and cytokine secretome. Functional activity of immune cells following exposure to the combination of M with tebotelimab was evaluated. Results: M induced greater upregulation relative to T of both co-stimulatory (eg 4-1BB) and/or checkpoint molecules (eg PD-L1 and LAG-3) on NK cells and/or monocytes in PBMCs cocultured with HER2-expressing tumor cells. Compared with T, M also induced greater proinflammatory cytokine release, including IFNγ that in turn upregulates PD-L1 and MHC class I expression on co-cultured tumor cells. These effects were not observed with an Fc-null version of M or were inhibited by the addition of FcR-blocking Abs, demonstrating Fc:FcR dependency of M-induced immune activation. Interestingly, CD8 T cells, which lack FcR, also exhibited LAG-3 upregulation in the above cultures, suggesting the effects of M can also be mediated via a cascade mechanism. An IFNγ neutralizing mAb reduced tumor cell-associated PD-L1 and MHC class I upregulation, but not LAG-3 induction on T-cells. PBMCs, pre-treated with a combination of M and tebotelimab in the presence of HER2-expressing tumor cells, demonstrated enhanced cytolytic activity against both the canonical NK cell target, K562, or M-opsonized HER2-positive tumor cells. Conclusions: M, through an Fc-dependent mechanism, can upregulate checkpoint molecules on NK-cells, CD8 T cells and tumor cells, thereby sensitizing them to immune checkpoint blockade. In turn, PD-1 and LAG-3 blockade by tebotelimab enhances M-mediated NK cell cytolytic activity in vitro.

Margetuximab: First Approval.

The second-generation anti-human epidermal growth factor receptor2 protein (HER2) monoclonal antibody margetuximab (MARGENZA™, margetuximab-cmkb) is being developed for the treatment of HER2-positive breast cancer, gastric cancer and gastro-oesophageal junction cancer. The antibody has been engineered for increased binding to activating Fcγ receptor IIIA (CD16A) and decreased binding to inhibitory Fcγ receptor IIB (CD32B) relative to trastuzumab with the aim of improving response rates. Based on the results of the phase III SOPHIA trial margetuximab has been approved in the USA for use in combination with chemotherapy as treatment of previously-treated metastatic HER2-positive breast cancer. This article summarizes the milestones in the development of margetuximab leading to this first approval.

Post-HDX Deglycosylation of Fc Gamma Receptor IIIa Glycoprotein Enables HDX Characterization of Its Binding Interface with IgG.

Protein glycosylation is a common and highly heterogeneous post-translational modification that challenges biophysical characterization technologies. The heterogeneity of glycoproteins makes their structural analysis difficult; in particular, hydrogen-deuterium exchange mass spectrometry (HDX-MS) often suffers from poor sequence coverage near the glycosylation site. A pertinent example is the Fc gamma receptor RIIIa (FcγRIIIa, CD16a), a glycoprotein expressed on the surface of natural killer cells (NK) that binds the Fc domain of IgG antibodies as a trigger for antibody-dependent cell-mediated cytotoxicity (ADCC). Here, we describe an adaptation of a previously reported method using PNGase A for post-HDX deglycosylation to characterize the binding between the highly glycosylated CD16a and IgG1. Upon optimization of the method to improve sequence coverage while minimizing back-exchange, we achieved coverage of four of the five glycosylation sites of CD16a. Despite some back-exchange, trends in HDX are consistent with previously reported CD16a/IgG-Fc complex structures; furthermore, binding of peptides covering the glycosylated asparagine-164 can be interrogated when using this protocol, previously not seen using standard HDX-MS.

A novel endogenous CD16-Expressing Natural Killer Cell for cancer immunotherapy

Natural killer (NK) cells, as a potential source for off-the-shelf cell therapy, attack tumor cells with low risk of severe cytokine release syndrome (CRS) or graft-versus-host disease (GvHD). Fcγ receptor IIIA, also known as CD16, further confers NK cells with antibody-dependent cell-mediated cytotoxicity (ADCC), one mechanism of action of antibody-based immunotherapy. Here, we establish a novel human NK cell line, oNK-1, endogenously expressing CD16 along with high levels of NK activation markers and low levels of NK inhibitory markers. The long-term expansion and CD16 expression of oNK-1 cells were demonstrated. Furthermore, oNK-1 cells elicit superior cytotoxicity against cancer cells than primary NK cells. In conclusion, this study suggests that endogenous CD16-expressing oNK-1 has the potential to develop an effective NK-based therapy.

Modeling of the Biological Activity of Monoclonal Antibodies Based on the Glycosylation Profile.

The influence of the glycosylation profile of IgG on biological activity is known, but it is not clear which glycoforms have the highest impact on the main mechanism of action. The aim of this study was to design a mathematical model for predicting the antibody-dependent cellular cytotoxicity (ADCC) activity and the Fc gamma IIIa receptors' (FcɣRIIIa) relative binding of rituximab drug products based on their glycosylation profile. An additional goal was to identify the glycoforms that have the greatest impact on these mechanisms of action. For these purposes, the glycosylation profile was examined by hydrophilic interaction ultra-performance liquid chromatography (HILIC-UPLC), ADCC was assessed using a Promega kit, and FcɣRIIIa's binding affinity was assessed by surface plasmon resonance (SPR) analysis of a group of >50 rituximab drug products. Based on the results, mathematical models for the ADCC and FcɣRIIIa binding affinity prediction were designed using JMP 13.2.0. The quality of the model and the influence of sample size and heterogeneity on the reliability were verified. The results allow for the evaluation of rituximab drug products' activity based on their glycosylation profile and show that with a sufficiently large and differentiated dataset, it is possible to generate models for different monoclonal antibodies.

Селекция аптамеров к тромбоцитам с применением цифровой эмульсионной ПЦР

The aim of the research. To obtain aptamers-inhibitors of platelet glycoprotein IIb / IIIa receptors, blocking platelet aggregation. Material and methods. Th e selection of aptamers for IIb / IIIa receptors of platelets was carried out according to the SELEX method (Systematic Evolution of Ligands by Exponential Enrichment), modifi ed to select aptamers for a specifi c epitope. Th e method allows selection and in vitro evolution of aptamers with selectivity to a specifi c target from a large library of oligonucleotides. Th e affi nity of aptamers for platelet IIb / IIIa receptors was determined using fl ow cytometry. Results. Pools of aptamers of aptamers with high affi nity for IIb / IIIa platelet receptors were obtained. Th e study of the antiaggregation properties of the pools with the best binding showed that platelet aggregation was minimal when using the aptamers from the pool of the 5th round of selection. Th us, the aptamers of this pool have the greatest potential to be used as an analogue of a synthetic peptide that blocks thromboaggregation. Aptamers from this pool were taken for sequencing in order to obtain sequences of aptamers with the best antiaggregatory properties. Conclusion. Pools of aptamers with high affi nity for IIb / IIIa receptors of platelets and anticoagulant activity were obtained.

The modulation of the burn wound environment by negative pressure wound therapy: Insights from the proteome

Negative pressure wound therapy has been used to promote wound healing in a variety of settings, including as an adjunct to silver-impregnated dressings in the acute management of paediatric burns. Fluid aspirated by the negative pressure wound therapy system represents a potentially insightful research matrix for understanding the burn wound microenvironment and the intervention's biochemical mechanisms of action. The aim of this study was to characterize the proteome of wound fluid collected using negative pressure wound therapy from children with small-area thermal burns. Samples were obtained as part of a randomized controlled trial investigating the clinical efficacy of adjunctive negative pressure wound therapy. They were compared with blister fluid specimens from paediatric burn patients matched according to demographic and injury characteristics. Protein identification and quantification were performed via liquid chromatography tandem mass spectrometry and sequential window acquisition of all theoretical mass spectra data-independent acquisition. Proteins and biological pathways that were unique to or enriched in negative pressure wound therapy fluid samples were evaluated using principal components, partial least squares-discriminant, and gene ontology enrichment analyses. Eight viable samples of negative pressure wound therapy fluid were collected and analyzed with eight matched blister fluid samples. A total of 502 proteins were quantitatively profiled in the negative pressure wound therapy fluid, of which 444 (88.4%) were shared with blister fluid. Several proteins exhibited significant abundance differences between fluid types, with negative pressure wound therapy fluid showing a higher abundance of matrix metalloproteinase-9, arginase-1, low affinity immunoglobulin gamma Fc region receptor III-A, filamin-A, alpha-2-macroglobulin, and hemoglobin subunit alpha. The results lend support to the hypothesis that negative pressure wound therapy augments wound healing through the modulation of factors involved in the inflammatory response, granulation tissue synthesis, and extracellular matrix maintenance. Data are available via ProteomeXchange with identifier PXD023168.

Enhancing CDC and ADCC of CD19 Antibodies by Combining Fc Protein-Engineering with Fc Glyco-Engineering.

Background: Native cluster of differentiation (CD) 19 targeting antibodies are poorly effective in triggering antibody-dependent cell-mediated cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC), which are crucial effector functions of therapeutic antibodies in cancer immunotherapy. Both functions can be enhanced by engineering the antibody’s Fc region by altering the amino acid sequence (Fc protein-engineering) or the Fc-linked glycan (Fc glyco-engineering). We hypothesized that combining Fc glyco-engineering with Fc protein-engineering will rescue ADCC and CDC in CD19 antibodies. Results: Four versions of a CD19 antibody based on tafasitamab’s V-regions were generated: a native IgG1, an Fc protein-engineered version with amino acid exchanges S267E/H268F/S324T/G236A/I332E (EFTAE modification) to enhance CDC, and afucosylated, Fc glyco-engineered versions of both to promote ADCC. Irrespective of fucosylation, antibodies carrying the EFTAE modification had enhanced C1q binding and were superior in inducing CDC. In contrast, afucosylated versions exerted an enhanced affinity to Fcγ receptor IIIA and had increased ADCC activity. Of note, the double-engineered antibody harboring the EFTAE modification and lacking fucose triggered both CDC and ADCC more efficiently. Conclusions: Fc glyco-engineering and protein-engineering could be combined to enhance ADCC and CDC in CD19 antibodies and may allow the generation of antibodies with higher therapeutic efficacy by promoting two key functions simultaneously.

Structural insight and stability of TNFR-Fc fusion protein (Etanercept) produced by using transgenic silkworms.

Therapeutic proteins expressed using transgenic animals have been of great interest for several years. Especially, transgenic silkworm has been studied intensively because of its ease in handling, low-cost, high-yield and unique glycosylation patterns. However, the physicochemical property of the therapeutic protein expressed in transgenic silkworm remains elusive. Here, we constructed an expression system for the TNFR-Fc fusion protein (Etanercept) using transgenic silkworm. The TNFR-Fc fusion protein was employed to N-glycan analysis, which revealed an increased amount of afucosylated protein. Evidence from surface plasmon resonance analysis showed that the TNFR-Fc fusion protein exhibit increased binding affinity for Fcγ receptor IIIa and FcRn compared to the commercial Etanercept, emphasizing the profit of expression system using transgenic silkworm. We have further discussed the comparison of higher order structure, thermal stability and aggregation of the TNFR-Fc fusion protein.

Anti-CD20 rituximab IgG1, IgG3, and IgG4 but not IgG2 subclass trigger Ca2+ mobilization and cytotoxicity in human NK cells.

NK cell-mediated Ab-dependent cellular cytotoxicity (ADCC) is increasingly recognized to play an important role in cancer immunotherapy, transplant rejection, and autoimmunity. However, several aspects of the molecular interactions of IgG subclasses with the Fc-gamma receptor IIIA (FcγRIIIA)/CD16a expressed on NK cells remain unknown. The aim of the current study was to further analyze the role of IgG subclasses and FCGR3A V158F single nucleotide polymorphism (SNP) on Ca2+ signaling and NK cell-mediated ADCC against Daudi target cells in vitro. NK cells were isolated from donors with different FCGR3A SNP. The affinity of rituximab IgG subclasses to CD20 expressed on Daudi cells showed similar dissociation constant as tested by flow cytometry. Induction of Ca2+ signaling, degranulation, intracellular cytokine production, and ADCC was demonstrated for IgG1 and IgG3, to a lesser degree also for IgG4, but not for IgG2. Compared to NK cells carrying the low-affinity (FF) variant for the FCGR3A V158F SNP, binding of IgG1 and IgG3 to NK cells carrying the high-affinity (VV) and VF SNP variants was two- to threefold higher. Variations of FCGR3A SNP among the eight tested donors (1 VV, 3FF, and 4VF) revealed no significant differences of Ca2+ signaling and degranulation; however, ADCC was somewhat weaker in donors with the low-affinity FF variation. In conclusion, this is the first study correlating Ca2+ signaling and NK cell-mediated ADCC triggered by the four IgG subclasses with the FCGR3A V158F SNP. Our findings indicate important differences in the interactions of IgG subclasses with FcγRIIIA/CD16a but no major impact of FCGR3A SNP and may therefore help to better correlate the functional properties of particular engineered therapeutic antibodies in vitro with individual differences of their clinical efficacy.

Impact of IgG1 N-glycosylation on their interaction with Fc gamma receptors

The effector functions of the IgGs are modulated by the N-glycosylation of their Fc region. Particularly, the absence of core fucosylation is known to increase the affinity of IgG1s for the Fcγ receptor IIIa expressed by immune cells, in turn translating in an improvement in the antibody-dependent cellular cytotoxicity. However, the impact of galactosylation and sialylation is still debated in the literature. In this study, we have investigated the influence of high and low levels of core fucosylation, terminal galactosylation and terminal α2,6-sialylation of the Fc N-glycans of trastuzumab on its affinity for the FcγRIIIa. A large panel of antibody glycoforms (i.e., highly α2,6-sialylated or galactosylated IgG1s, with high or low levels of core fucosylation) were generated and characterized, while their interactions with the FcγRs were analysed by a robust surface plasmon resonance-based assay as well as in a cell-based reporter bioassay. Overall, IgG1 glycoforms with reduced fucosylation display a stronger affinity for the FcγRIIIa. In addition, fucosylation, and the presence of terminal galactose and sialic acids are shown to increase the affinity for the FcγRIIIa as compared to the agalactosylated forms. These observations perfectly translate in the response observed in our reporter bioassay.

The FcγRIIIA-158 VV genotype increased the risk of post-transplant lymphoproliferative disorder in T-cell-depleted kidney transplant recipients - a retrospective study.

Post-transplantation lymphoproliferative disorder (PTLD) is a severe complication in organ transplant recipients. The use of T lymphocyte-depleting antibodies (TLDAb), especially rabbit TLDAb, contributes to PTLD, and the V158F polymorphism of Fc gamma receptor IIIA (FcγRIIIA) also named CD16A could affect the concentration-effect relationship of TLDAb. We therefore investigated the association of this polymorphism with PTLD in kidney transplant recipients. We characterized the V158F polymorphism in two case-control cohorts (discovery, n=196; validation, n=222). Then, we evaluated the binding of rabbit IgG to human FcγRIIIA-158V and FcγRIIIA-158F. The V158F polymorphism was not linked to PTLD in the overall cohorts, but risk of PTLD was increased in VV homozygous recipients receiving TLDAb compared with F carriers in both cohorts, especially in recipients receiving TLDAb without muromonab (discovery: HR=2.22 [1.03-4.76], P=0.043, validation: HR=1.75 [1.01-3.13], P=0.049). In vitro, we found that the binding of rabbit IgG to human NK-cell FcγRIIIA was increased when cells expressed the 158-V versus the 158-F allotype. While the 158-V allotype of human FcγRIIIA binds rabbit immunoglobulin-G with higher affinity, the risk of PTLD was increased in homozygous VV kidney transplant recipients receiving polyclonal TLDAb.

Demonstrating Analytical Similarity of Trastuzumab Biosimilar HLX02 to Herceptin® with a Panel of Sensitive and Orthogonal Methods Including a Novel FcγRIIIa Affinity Chromatography Technology.

BackgroundA biosimilar needs to demonstrate its similarity to the originator reference product (RP) in terms of structural and functional properties as well as nonclinical and clinical outcomes.ObjectivesThe aim was to assess the analytical similarity between the trastuzumab biosimilar HLX02 and Europe-sourced Herceptin® (EU-Herceptin®) and China-sourced Herceptin® (CN-Herceptin®) following a quality-by-design (QbD) quality study and tier-based quality attribute evaluation.MethodsA panel of highly sensitive and orthogonal methods, including a novel Fc gamma receptor IIIa (FcγRIIIa) affinity chromatography technique that enables quantitative comparison of glycan effects on effector function, was developed for the assessment. To ensure the full product variability was captured, ten batches of HLX02 were compared with 39 RP batches with expiry dates from August 2017 to March 2021.ResultsThe extensive three-way similarity assessment demonstrated that HLX02 is highly similar to the RPs. Furthermore, the %afucose, %galactose, and FcγRIIIa affinity of the RPs were observed to first decrease and then return to the original level in relation to their expiry dates, and the RP batches can be subgrouped by their FcγRIIIa affinity chromatograms. HLX02 is demonstrated to be more similar to the RPs of the high FcγRIIIa affinity group.ConclusionBesides having an overall high analytical similarity to both EU-Herceptin® and CN-Herceptin®, HLX02 is more similar to Herceptin® with high FcγRIIIa affinity, a result that demonstrates the power of the novel FcγRIIIa affinity chromatography technology in biosimilarity evaluation.Electronic supplementary materialThe online version of this article (10.1007/s40259-020-00407-0) contains supplementary material, which is available to authorized users.

Fc functional antibody responses to adjuvanted versus unadjuvanted seasonal influenza vaccination in community-dwelling older adults

BackgroundSeasonal influenza vaccination with a standard trivalent influenza vaccine (TIV) induces a modest, and cross-reactive, Fc functional antibody response in older adults. Recent improvements to influenza vaccines include a quadrivalent influenza vaccine (QIV) and a TIV adjuvanted with the squalene-based oil-in-water emulsion MF59. MethodsPre- and post-vaccination serum samples from older adults vaccinated with QIV (n = 27) and adjuvanted TIV (n = 44) were studied using hemagglutination inhibition (HAI) assays and dimeric Fc-gamma receptor IIIa binding ELISAs, as a surrogate of antibody-dependent cellular cytotoxicity (ADCC). ResultsWe found that the unadjuvanted QIV elicited a stronger HAI response against the H1N1 vaccine virus than the adjuvanted TIV. Post-vaccination levels of HA-specific ADCC antibodies were similar for older adults vaccinated with QIV and adjuvanted TIV. The ADCC response to influenza vaccination was largely determined by pre-vaccination or baseline levels of these antibodies, with older adults with low baseline levels of ADCC activity demonstrating greater post-vaccination rises. ConclusionsIn this cohort of community-dwelling older adults, the QIV was at least as good as the adjuvanted TIV in the induction of ADCC and HAI responses. Further studies on how these antibody responses translate to efficacy in preventing influenza infections are warranted.

Characterization of IgG1 Fc Deamidation at Asparagine 325 and Its Impact on Antibody-dependent Cell-mediated Cytotoxicity and Fc\u03b3RIIIa Binding

Antibody-dependent cell-mediated cytotoxicity (ADCC) is an important mechanism of action for many therapeutic antibodies. A therapeutic immunoglobulin (Ig) G1 monoclonal antibody lost more than half of its ADCC activity after heat stress at 40 °C for 4 months. Size-exclusion and ion-exchange chromatography were used to fractionate various size and charge variants from the stressed IgG1. Physicochemical characterization of these fractions revealed that a rarely seen crystallizable fragment (Fc) modification, N325 deamidation, exhibited a positive correlation with the loss of ADCC activity. A further surface plasmon resonance study showed that this modification disrupted the binding between the IgG1 Fc and Fcγ receptor IIIa, resulting in decreased ADCC activity of the IgG1 antibody. Mutants of N325/D and N325/Q were made to confirm the effect of N325 deamidation on ADCC. We hypothesize that N325 deamidation altered the local three-dimensional structure, which might interfere with the binding and interaction with the effector cell. Because of its impact on biological activity, N325 deamidation is a critical quality attribute for products whose mechanism of action includes ADCC. A thorough understanding of the criticality of N325 deamidation and appropriate monitoring can help ensure the safety and efficacy of IgG1 or Fc-fusion products.

The Chinese herb Fructus Broussonetiae aids learning and memory in chronic cerebral hypoperfusion by reducing proinflammatory microglia activation in rats.

The neuroprotective role of Fructus Broussonetiae in a model of chronic cerebral hypoperfusion with cognitive decline was focused on neural plasticity and microglia/macrophage polarization. Chronic cerebral hypoperfusion was induced by bilateral common carotid artery ligation. Fructus Broussonetiae shortened escape latency and added the number of platform crossings of rats, up-regulated the expression of synaptophysin in the gray matter and increased myelin basic protein expression in the white matter. Further mechanistic experiments were conducted to examine microglia activation and M1/M2 polarization. It was shown that Fructus Broussonetiae reduced the activation of microglia revealed by decreased expression of ionized calcium-binding adapter molecule-1, inhibited M1 polarization of microglia and improved microglial M2 polarization shown by down-regulated the expression of inducible nitric oxide synthase and Fc fragment of IgG receptor IIIa and up-regulated the expression of arginase-1. In conclusion, the Chinese herb Fructus Broussonetiae can improve cognitive function following chronic cerebral hypoperfusion by down-regulating the activation of microglia, inhibiting microglial M1 polarization, and improving neural plasticity.

The biological activity of bispecific trastuzumab/pertuzumab plant biosimilars may be drastically boosted by disulfiram increasing formaldehyde accumulation in cancer cells

Studies of breast cancer therapy have examined the improvement of bispecific trastuzumab/pertuzumab antibodies interacting simultaneously with two different epitopes of the human epidermal growth factor receptor 2 (HER2). Here, we describe the creation and production of plant-made bispecific antibodies based on trastuzumab and pertuzumab plant biosimilars (bi-TPB-PPB). Using surface plasmon resonance analysis of bi-TPB-PPB antibodies binding with the HER2 extracellular domain, we showed that the obtained Kd values were within the limits accepted for modified trastuzumab and pertuzumab. Despite the ability of bi-TPB-PPB antibodies to bind to Fcγ receptor IIIa and HER2 oncoprotein on the cell surface, a proliferation inhibition assay did not reveal any effect until α1,3-fucose and β1,2-xylose in the Asn297-linked glycan were removed. Another approach to activating bi-TPB-PPB may be associated with the use of disulfiram (DSF) a known aldehyde dehydrogenase 2 (ALDH2) inhibitor. We found that disulfiram is capable of killing breast cancer cells with simultaneous formaldehyde accumulation. Furthermore, we investigated the capacity of DSF to act as an adjuvant for bi-TPB-PPB antibodies. Although the content of ALDH2 mRNA was decreased after BT-474 cell treatment with antibodies, we only observed cell proliferation inhibiting activity of bi-TPB-PPB in the presence of disulfiram. We concluded that disulfiram can serve as a booster and adjuvant for anticancer immunotherapy.

Impact of FcγR variants on the response to alemtuzumab in multiple sclerosis.

Allelic variants of genes encoding for the Fc gamma receptors IIIA and IIA have been associated with the clinical response to cell‐depleting antibodies in lymphoma patients. Here, we tested the hypothesis that FCGR3A and FCGR2A high‐affinity polymorphisms predict clinical outcomes to alemtuzumab therapy in 85 patients with relapsing‐remitting multiple sclerosis. No differences in clinical and MRI‐based efficacy parameters, the development of severe infusion‐associated reactions and secondary autoimmune diseases during a 2 year follow‐up was observed based on FCGR3A or FCGR2A polymorphisms. This study does not support the use of FCGR genetic variants to predict clinical outcomes to alemtuzumab.

Effects of Glycan Structure on the Stability and Receptor Binding of an IgG4-Fc.

A series of well-defined N-glycosylated IgG4-Fc variants were utilized to investigate the effect of glycan structure on their physicochemical properties (conformational stability and photostability) and interactions with an Fc γ receptor IIIA (FcγRIIIA). High mannose (HM, GlcNAc2Man(8+n) [n= 0-4]), Man5 (GlcNAc2Man5), GlcNAc1, and N297Q IgG4-Fc were prepared in good quality. The physical stability of these IgG4-Fc variants was examined with differential scanning calorimetry and intrinsic fluorescence spectroscopy. Photostability was assessed after photoirradiation between 295 and 340 nm (λ max= 305 nm), and HPLC-MS/MS analysis of specific products was performed. The size of glycans at Asn297 affects the yields of light-induced Tyr side-chain fragmentation products, where the yields decreased in the following order: N297Q > GlcNAc1 > Man5 > HM. These yields correlate with the thermal stability of the glycoforms. The HM and Man5 glycoforms display increased affinity for FcγRIIIA by at least 14.7-fold compared with GlcNAc1 IgG4-Fc. The affinities measured for the HM and Man5 IgG4-Fc (0.39-0.52 μM) are similar to those measured for fucosylated IgG1. Dependent on the mechanisms of action of IgG4 therapeutics, such glycoforms may need to be carefully monitored. The nonglycosylated N297Q IgG4-Fc did not present measurable affinity to FcγRIIIA.

Binding affinities of human IgG1 and chimerized pig and rabbit derivatives to human, pig and rabbit Fc gamma receptor IIIA.

While pigs and rabbits are used as models for human immune diseases, FcγR binding is poorly characterized in both test species. To evaluate antibody binding to FcγRIIIA, a receptor involved in antibody-dependent cellular cytotoxicity, chimerized antibodies were generated by grafting the variable regions of a human IgG1 onto scaffolds from both species. The affinities of the parent and chimeric antibodies to the FcγRIIIA proteins from all three species were determined. While the human IgG1 and rabbit IgG had similar affinities for each FcγRIIIA with notable differences across species, pig IgG1 only bound pig FcγRIIIA with appreciable affinity. Also, the functional pig and rabbit proteins described here can be used in future experiments, such as pharmacology and mechanism of action studies.