Although repetitive patterns of antigens are crucial for certain immune responses, an understanding of how antibodies bind and dynamically interact with various spatial arrangements of molecules is lacking. Hence, we introduce a new method where molecularly precise nanoscale patterns of antigens are displayed using DNA origami and immobilized in a surface plasmon resonance (SPR) setup. Using antibodies with identical antigen binding domains, we find that all subclasses and isotypes studied, bind bivalently to two antigens separated at distances ranging from 3 to 17 nm. The binding affinities of these antibodies change with the antigen distances, with a distinct preference for antigens separated by approximately 16 nm, and considerable differences in spatial tolerance exist between IgM and IgG and between low and high affinity antibodies.

Engineering of the constant Fc part of monoclonal human IgG1 (hIgG1) Abs is an approach to improve effector functions and clinical efficacy of next-generation IgG1-based therapeutics. A main focus in such development is tailoring of in vivo half-life and transport properties by engineering the pH-dependent interaction between IgG and the neonatal Fc receptor (FcRn), as FcRn is the main homeostatic regulator of hIgG1 half-life. However, whether such engineering affects binding to other Fc-binding molecules, such as the classical FcγRs and complement factor C1q, has not been studied in detail. These effector molecules bind to IgG1 in the lower hinge–CH2 region, structurally distant from the binding site for FcRn at the CH2–CH3 elbow region. However, alterations of the structural composition of the Fc may have long-distance effects. Indeed, in this study we show that Fc engineering of hIgG1 for altered binding to FcRn also influences binding to both the classical FcγRs and complement factor C1q, which ultimately results in alterations of cellular mechanisms such as Ab-dependent cell-mediated cytotoxicity, Ab-dependent cellular phagocytosis, and Ab-dependent complement-mediated cell lysis. Thus, engineering of the FcRn–IgG1 interaction may greatly influence effector functions, which has implications for the therapeutic efficacy and use of Fc-engineered hIgG1 variants.

The C1q binding epicentre on IgG molecules involves residues Asp270, Lys322, Pro329 and Pro331 in the CH2 domain. IgG1 and IgG3 are usually the most efficient of the four human IgG subclasses in activating complement and they both share all these residues. To reveal possible differences in the structural requirement for complement activation, we created a number of NIP (5‐iodo‐4‐hydroxy‐3‐nitro‐phenacetyl) specific IgG1 and IgG3 antibodies with parallel mutations in or near the putative C1q binding site. The mutants were tested simultaneously for antibody induced, antibody‐dependent complement‐mediated lysis (ADCML) at high and low antigen concentration on the target cells using sera of human, rabbit and guinea pig as complement source. In addition, we tested the antibodies against target cells decorated with the NP hapten, which has 10‐fold lower affinity for the antibodies compared to the NIP hapten. We also used ELISA methods to measure complement activation. We observed a clear difference between IgG1 and IgG3 localized to residues Asp270, Leu334, Leu335. For all these residues, and especially for Asp270, IgG1 was heavily reduced in complement activation, while IgG3 was only moderated reduced, by alanine substitution. This difference was independent of the long hinge region of IgG3, demonstrated by hinge region truncation of this isotype such that it resembles that of IgG1. This report indicates the presence of structural differences between human IgG1 and IgG3 in the C1q binding site, and points to a specialization of the two isotypes with respect to complement activation.

We compared the bactericidal activity of recombinant sets of chimeric IgG monoclonal antibodies against two important outer membrane meningococcal vaccine antigens: PorA and factor H binding protein (FHbp). The sets contained human Fc portions from IgG1, IgG3, and two IgG3 mutants (IgG3m15 and IgGm17) with hinge regions of 15 and 17 amino acids encoded by hinge exons h2 and h1, respectively (human IgG3 has a hinge region of 62 amino acids encoded by hinge exons h1, h2, h3, and h4, while human IgG1 has a hinge region of only 15 amino acids encoded by one hinge exon) and mouse V regions. IgG1 showed higher bactericidal activity than IgG3 when directed against PorA (an abundant antigen), while IgG3 was more bactericidal than IgG1 when directed against FHbp (a sparsely and variably distributed antigen). On the other hand, the IgG3 hinge-truncated antibodies IgG3m15 and IgGm17 showed higher bactericidal activity than both IgG1 and IgG3 regardless of the target antigen. Thus, the Fc region of IgG3 antibodies appears to have an enhanced complement-activating function, independent of their long hinge region, compared to IgG1 antibodies. The greater activity of the truncated IgG3 hinge mutants indicates that the long hinge of IgG3 seems to downregulate through an unknown mechanism the inherent increased complement-activating capability of IgG3 Fc when the antibody binds to a sparse antigen. Immune protection against invasive meningococcal disease depends on recognition of bacterial surface antigens by antibodies, followed by activation of complement, leading to degradation of the bacteria by bacteriolysis, also named serum bactericidal activity (SBA). The class 1 outer membrane porin protein PorA is abundantly expressed by almost all meningococcal strains (1-3), and antigenic variation among PorA proteins is the basis of serosubtyping (1). PorA can induce bactericidal antibodies in humans and mice when they are immunized with meningococcal outer membrane vesicle (OMV) vaccines (4-8), and monoclonal antibodies (MAbs) against PorA can be protective in an infant rat model (9). Factor H binding protein (FHbp) is a lipoprotein that is sparsely distributed on the outer membrane of many meningococcal strains (10-12). It is an immune system-evading protein protecting the meningococci from complement-mediated lysis by binding the human complement-inhibiting protein factor H (FH) (13). Antibodies to FHbp elicit SBA and confer passive protection in infant rat meningococcal bacteremia models (14, 15). PorA is estimated to make up 25% of the outer membrane of meningococci, while FHbp is estimated to make up 1% (16).Human IgG consists of four subclasses (isotypes), IgG1, IgG2, IgG3, and IgG4, which differ greatly in effector functions, such as interaction with FcR on immune cells and the capacity to activate complement (17-19). By using monoclonal hapten (4-hydroxy-3-nitrophenacetyl [NP/NIP])-specific antibodies of all four IgG isotypes, we have demonstrated that IgG1 and IgG3 are best in inducing complement-mediated cellular lysis an...

The presence of a carbohydrate moiety on asparagine 297 in the Fc part of an IgG molecule is essential for its effector functions and thus influences its vaccine protective effect. Detailed structural carbohydrate analysis of vaccine induced IgGs is therefore of interest as this knowledge can prove valuable in vaccine research and design and when optimizing vaccine schedules. In order to better understand and exploit the protective potential of IgG antibodies, we carried out a pilot study; collecting serum or plasma from volunteers receiving different vaccines and determining the IgG subclass glycosylation patterns against specific vaccine antigens at different time points using LC-ESI-MS analysis. The four vaccines included a pneumococcal capsule polysaccharide vaccine, a meningococcal outer membrane vesicle vaccine, a seasonal influenza vaccine, and a pandemic influenza vaccine. The number of volunteers was limited, but the results following immunization indicated that the IgG subclass which dominated the response showed increased galactose and the level of sialic acid increased with time for most vaccinees. Fucose levels increased for some vaccinees but in general stayed relatively unaltered. The total background IgG glycosylation analyzed in parallel varied little with time and hence the changes seen were likely to be caused by vaccination. The presence of an adjuvant in the pandemic influenza vaccine seemed to produce simpler and less varied glycoforms compared to the adjuvant-free seasonal influenza vaccine. This pilot study demonstrates that detailed IgG glycosylation pattern analysis might be a necessary step in addition to biological testing for optimizing vaccine development and strategies.