Øistein Ihle

Structural Difference in the Complement Activation Site of Human IgG1 and IgG3

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.

Identification and Characterisation of C1q-Binding Phage Displayed Peptides

Five phage displayed peptide libraries were screened for binders to C1q, the recognition subunit of the classical complement pathway. Two rounds of panning resulted in the isolation and characterisation of several different phage displayed C1q-binding peptides from all five libraries. Two groups of the characterised peptides show sequence similarity with part of the metal ion dependent adhesion site (MIDAS) of integrin A-domains, and the site 187LRNPCPNKEKECQPPF of CD18 (integrin beta2), respectively. These results support binding of complement receptor 3 (CR3, CD11b/CD18, Mac1) to C1q and further suggest C1q binding sites in CR3. We also discuss sequence matches between the characterised peptides and proteins known to interact with C1q, as well as other proteins listed in the SwissProt databank. These findings are of interest for the study of the complement system and may lead to the development of peptides, fusion products or peptido-mimetics with C1q modulating potential.

A mutant human IgG molecule with only one C1q binding site can activate complement and induce lysis of target cells

There are potentially two binding sites for C1q on IgG, one on each CH2 domain of the gamma heavy chains, close to the lower hinge region. It is not clear whether the presence and involvement of both the C1q binding sites is necessary to induce the activation signal of human IgG. In order to clarify this issue, we made a hybrid mutant IgG1/IgG3 molecule where the IgG1 half of the molecule was made unable to activate complement through the introduction of a P329A mutation. The IgG3 half of the molecule was mutated to harbor a hinge region identical to that of IgG1, and for detection a peptide tag derived from p21ras was introduced into the FG loop of the CH1 domain. The hybrid IgG1P329A/IgG3h1‐ras molecules were isolated by Protein A affinity chromatography and shown to activate complement and induce complement‐mediated lysis at the same levels as wild‐type IgG1 and IgG3h1‐ras molecules. Thus, one C1q binding site per IgG is sufficient to induce activation. Wild‐type human IgG molecules might also normally expose only one C1q binding site as already shown for interaction with FcγR, were IgG expose one binding site per molecule.

Construction and functional activities of chimeric mouse-human immunoglobulin G and immunoglobulin M antibodies against the Neisseria meningitidis PorA P1.7 and P1.16 epitopes.

ABSTRACT We studied the in vitro protective activities of human immunoglobulin G1 (IgG1), IgG3, and IgM antibodies against group B meningococci by constructing sets of chimeric mouse-human antibodies (chIgG1, chIgG3, and chIgM, respectively) with identical binding regions against the P1.7 and P1.16 epitopes on PorA. This was done by cloning the V genes of three mouse hybridoma antibodies and subsequently transfecting vectors containing the homologous heavy- and light-chain genes into NSO cells. Cell clones secreting intact human chIgG1, chIgG3, or chIgM antibodies originating from three parent mouse antibodies were isolated. The functional affinities appeared to be similar for all human isotypes and surprisingly also for the pentameric chIgM antibody. chIgG1 exhibited greater serum bactericidal activity (SBA) than chIgG3, while chIgG3 was more efficient in inducing a respiratory burst (RB) associated with opsonophagocytosis than chIgG1 was. On the other hand, chIgM exhibited SBA similar to that of chIgG1, but it exhibited much higher RB activity than chIgG3 and chIgG1 exhibited. The antibodies against the P1.16 epitope were more efficient in terms of SBA than the antibodies against the P1.7 epitope were; thus, 10- to 40-fold-lower concentrations of antibodies against P1.16 than of antibodies against P1.7 were needed to induce SBA. On the other hand, antibodies against these epitopes were equally effective in inducing RB. Our results revealed differences in the functional activities of human chIgG1, chIgG3, and chIgM antibodies against meningococci, which might influence their protective effects against meningococcal disease.

Characterization of a Human Platelet Antigen-1a–Specific Monoclonal Antibody Derived from a B Cell from a Woman Alloimmunized in Pregnancy

Human platelet Ag (HPA)-1a, located on integrin β3, is the main target for alloantibodies responsible for fetal and neonatal alloimmune thrombocytopenia (FNAIT) in the white population. There are ongoing efforts to develop an Ab prophylaxis and therapy to prevent or treat FNAIT. In this study, an mAb specific for HPA-1a, named 26.4, was derived from an immortalized B cell from an alloimmunized woman who had an infant affected by FNAIT. It is the only HPA-1a–specific human mAb with naturally paired H and L chains. Specific binding of mAb 26.4, both native and recombinant forms, to platelets and to purified integrins αIIbβ3 (from platelets) and αVβ3 (from trophoblasts) from HPA-1a+ donors was demonstrated by flow cytometry and surface plasmon resonance technology, respectively. No binding to HPA-1a− platelets or integrins was detected. Moreover, the Ab binds with higher affinity to integrin αVβ3 compared with a second HPA-1a–specific human mAb, B2G1. Further in vitro experimentation demonstrated that mAb 26.4 can opsonize HPA-1a+ platelets for enhanced phagocytosis by monocytes, inhibit binding of maternal polyclonal anti–HPA-1a Abs, and weakly inhibit aggregation of HPA-1a–heterozygous platelets, the latter with no predicted clinical relevance. Thus, mAb 26.4 is highly specific for HPA-1a and could potentially be explored for use as a prophylactic or therapeutic reagent for FNAIT intervention and as a phenotyping reagent to identify women at risk for immunization.

Cloning, sequencing and expression of immunoglobulin variable regions of murine monoclonal antibodies specific for the P1.7 and P1.16 PorA protein loops of Neisseria meningitidis.

The P1.7 and P1.16 epitopes on the PorA protein on the outer membrane of Neisseria meningitidis can induce protective antibodies upon vaccination. Structural analysis of antibodies to these targets can give information on the immune response induced by these epitopes and can reveal any structural similarities among the antibodies. To do so, we have isolated the immunoglobulin (Ig) variable genes from four mouse hybridomas expressing antibodies against the P1.7 and P1.16 epitopes. These V genes were successfully expressed as functional chimeric (ch) mouse/human IgG1 antibodies by subcloning them into expression vectors containing the constant genes of human heavy and light chains. Sequencing the two sets of V genes against P1.16 revealed a high degree of homology, similar to that previously published for P1.7 V genes. The close homology allowed us to interchange heavy and light chains between antibodies in some instances to construct new antibodies that bind the original antigen. This study demonstrates that the immune response in mice against the meningococcal PorA protein epitopes P1.7 as well as P1.16 is limited to few and very similar germline genes, and therefore the P1.7‐ and P1.16‐specific antibodies share high degree of similarities amongst each other. These V genes were used to construct chimeric antibodies with conserved antigen‐binding activity.

Human Secretory IgM Antibodies Activate Human Complement and Offer Protection at Mucosal Surface.

IgM molecules circulate in serum as large polymers, mainly pentamers, which can be transported by the poly‐Ig receptor (pIgR) across epithelial cells to mucosal surfaces and released as secretory IgM (SIgM). The mucosal SIgM molecules have non‐covalently attached secretory component (SC), which is the extracellular part of pIgR which is cleaved from the epithelial cell membrane. Serum IgM antibodies do not contain SC and have previously been shown to make a conformational change from ‘a star’ to a ‘staple’ conformation upon reaction with antigens on a cell surface, enabling them to activate complement. However, it is not clear whether SIgM similarly can induce complement activation. To clarify this issue, we constructed recombinant chimeric (mouse/human) IgM antibodies against hapten 5‐iodo‐4‐hydroxy‐3‐nitro‐phenacetyl (NIP) and in addition studied polyclonal IgM formed after immunization with a meningococcal group B vaccine. The monoclonal and polyclonal IgM molecules were purified by affinity chromatography on a column containing human SC in order to isolate joining‐chain (J‐chain) containing IgM, followed by addition of excess amounts of soluble SC to create SIgM (IgM J+ SC+). These SIgM preparations were tested for complement activation ability and shown to be nearly as active as the parental IgM J+ molecules. Thus, SIgM may offer protection against pathogens at mucosal surface by complement‐mediated cell lysis or by phagocytosis mediated by complement receptors present on effector cells on mucosa.