Search

Rhesus macaques are a common non-human primate model used in the evaluation of human monoclonal antibodies, molecules whose effector functions depend on a conserved N-linked glycan in the Fc region. This carbohydrate is a target of glycoengineering efforts aimed at altering antibody effector function by modulating the affinity of Fcγ receptors. For example, a reduction in the overall core fucose content is one such strategy that can increase antibody-mediated cellular cytotoxicity by increasing Fc-FcγRIIIa affinity. While the position of the Fc glycan is conserved in macaques, differences in the frequency of glycoforms and the use of an alternate monosaccharide in sialylated glycan species add a degree of uncertainty to the testing of glycoengineered human antibodies in rhesus macaques. Using a panel of 16 human IgG1 glycovariants, we measured the affinities of macaque FcγRs for differing glycoforms via surface plasmon resonance. Our results suggest that macaques are a tractable species in which to test the effects of antibody glycoengineering.

The complement system plays an important role in our innate immune system. Complement activation results in clearance of pathogens, immune complex and apoptotic cells. The host is protected from complement-mediated damage by several complement regulators. Factor H (FH) is the most important fluid-phase regulator of the alternative pathway of the complement system. Heterozygous mutations in FH are associated with complement-related diseases such as atypical hemolytic uremic syndrome (aHUS) and age-related macular degeneration.We recently described an agonistic anti-FH monoclonal antibody that can potentiate the regulatory function of FH. This antibody could serve as a potential new drug for aHUS patients and alternative to C5 blockade by Eculizumab. However, it is unclear whether this antibody can potentiate FH mutant variants in addition to wild type FH. Here, the functionality and potential of the agonistic antibody in the context of pathogenic aHUS-related FH mutant proteins was investigated. The binding affinity of recombinant WT FH, and the FH variants, W1183L, V1197A, R1210C, and G1194D to C3b was increased upon addition of the potentiating antibody and similarly, the decay accelerating activity of all mutants is increased. The potentiating anti-FH antibody is able to restore the surface regulatory function of most of the tested FH mutants to WT FH levels. In conclusion, our potentiating anti-FH is broadly active and able to enhance both WT FH function as well as most aHUS-associated FH variants tested in this study.

The binding strength between IgG and Fc gamma R is influenced by the composition of the N-linked glycan at position N297 in the Fc-domain of IgG. Particularly, afucosylation increases the binding affinity of human IgG1 to human Fc gamma RIIIa up to similar to 20 fold, and additional galactosylation of the afucosylated IgG increases the affinity up to similar to 40 fold. The increase in affinity for afucosylated IgG has previously been shown to depend on direct carbohydrate carbohydrate interactions between the IgG-Fc glycan with an N-linked glycan at position 162 unique to hFc gamma RIIIa and hFc gamma RIIIb. Here we report that the N162 glycosylation site is also found in the orthologous mouse Fc gamma R, mFc gamma RIV. The N162-glycan in mFc gamma RIV was also responsible for enhancing the binding to mouse IgG with reduced fucose similar to hFc gamma RIIIa. However, unlike hFc gamma RIIIa, mFc gamma RIV did not bind more avidly to IgG with increased galactose and reduced fucose. Overall, these results suggest the N162-glycan in the human Fc gamma RIII family and its orthologous mouse Fc gamma RIV to be functionally conserved

Ingileif Jonsdottir, Bjorn Nilsson, Kari Stefansson and colleagues perform a genome-wide association study for immunoglobulin levels in Icelandic and Swedish cohorts. They find 38 new variants associated with IgA, IgG, IgM or composite immunoglobulin traits and identify candidate genes underlying the regulation of immunoglobulin levels. Immunoglobulins are the effector molecules of the adaptive humoral immune system. In a genome-wide association study of 19,219 individuals, we found 38 new variants and replicated 5 known variants associating with IgA, IgG or IgM levels or with composite immunoglobulin traits, accounted for by 32 loci. Variants at these loci also affect the risk of autoimmune diseases and blood malignancies and influence blood cell development. Notable associations include a rare variant at RUNX3 decreasing IgA levels by shifting isoform proportions (rs188468174[C>T]: P = 8.3 × 10−55, β = −0.90 s.d.), a rare in-frame deletion in FCGR2B abolishing IgG binding to the encoded receptor (p.Asn106del: P = 4.2 × 10−8, β = 1.03 s.d.), four IGH locus variants influencing class switching, and ten new associations with the HLA region. Our results provide new insight into the regulation of humoral immunity.

Objectives Recent reports describe interactions between the two most prominent RA-related autoantibodies, RFs and ACPAs. The main aim of the present study was to investigate whether RFs preferentially interact with ACPA-IgG over non-ACPA IgG. Additionally, interactions of RFs with IgG with altered galactose content in the Fc domain were examined, since ACPA-IgGs have been shown to have decreased Fc galactose content in RF+ patients. Methods (Auto)antibody interactions were studied in a surface plasmon resonance imaging assay and with ELISA. Target antibodies were isolated from RA patient plasma (polyclonal ACPA- and non-ACPA-IgG) or recombinantly produced to obtain monoclonal IgG with well-defined Fc galactose content. Interacting autoantibodies were studied using autoantibody positive patient sera and two recombinantly produced IgM-RFs. Results The sera from 41 RF+ RA patients showed similar RF binding to ACPA- and non-ACPA-IgG and no differences in binding to IgG with normal, high or low levels of Fc galactosylation. Two monoclonal IgM-RFs, one interacting with the CH2-CH3 interface and one binding close to the C-terminal end of the CH3 domain showed no influence of the Fc glycan on IgG binding by IgM-RF. Conclusion Although interactions between RF and ACPA may play a role in inflammatory processes in RA, RFs do not preferentially interact with ACPA-IgG over non-ACPA-IgG nor with agalatosylated IgG over IgG with normal or high galactosylation.

The extensive glycosylation of HIV-1 envelope (Env) glycoprotein leaves few glycan-free holes large enough to admit broadly neutralizing antibodies (bnAb). Consequently, most bnAbs must inevitably make some glycan contacts and avoid clashes with others. To investigate how Env glycan maturation regulates HIV sensitivity to bnAbs, we modified HIV-1 pseudovirus (PV) using various glycoengineering (GE) tools. Promoting the maturation of α-2,6 sialic acid (SA) glycan termini increased PV sensitivity to two bnAbs that target the V2 apex and one to the interface between Env surface gp120 and transmembrane gp41 subunits, typically by up to 30-fold. These effects were reversible by incubating PV with neuraminidase. The same bnAbs were unusually potent against PBMC-produced HIV-1, suggesting similar α-2,6 hypersialylated glycan termini may occur naturally. Overexpressing β-galactosyltransferase during PV production replaced complex glycans with hybrid glycans, effectively 'thinning' trimer glycan coverage. This increased PV sensitivity to some bnAbs but ablated sensitivity to one bnAb that depends on complex glycans. Other bnAbs preferred small glycans or galactose termini. For some bnAbs, the effects of GE were strain-specific, suggesting that GE had context-dependent effects on glycan clashes. GE was also able to increase the percent maximum neutralization (i.e. saturation) by some bnAbs. Indeed, some bnAb-resistant strains became highly sensitive with GE—thus uncovering previously unknown bnAb breadth. As might be expected, the activities of bnAbs that recognize glycan-deficient or invariant oligomannose epitopes were largely unaffected by GE. Non-neutralizing antibodies were also unaffected by GE, suggesting that trimers remain compact. Unlike mature bnAbs, germline-reverted bnAbs avoided or were indifferent to glycans, suggesting that glycan contacts are acquired as bnAbs mature. Together, our results suggest that glycovariation can greatly impact neutralization and that knowledge of the optimal Env glycoforms recognized by bnAbs may assist rational vaccine design., Author summary Here we engineered various changes in the sizes and shapes of sugars that decorate HIV surface spike proteins and tested the effects of these changes on virus susceptibility to neutralizing antibodies. In so doing, we were able to define the optimal Env-sugars recognized by prototype bnAbs that recognize various canonical epitope clusters on Env spike proteins. Some bnAbs preferred spike proteins decorated with large, complex glycans. Others preferred smaller glycans that improved their access to underlying protein targets. For similar reasons, germline-reverted versions of bnAbs were also generally more effective when the glycans were small. In some cases, bnAbs acquired an ability to bind to sugars as they matured. A comparison of viruses generated in cell lines and primary cells revealed large differences in bnAb sensitivity, raising questions about clinical relevance of cell line-produced virus for checking vaccine responses and, moreover, the use of these cell lines for manufacturing vaccines. Overall, just as car engines may be modified to be supercharged or hybrid for increased power or efficiency, the sugars of HIV coat proteins may also need to be engineered as 'supercharged' and 'hybrid' or otherwise modified in rational vaccine designs to optimize bnAb recognition.