Stepwise dissociation of subcomponents of C1, the first component of human complement, upon activation on an affinity sorbent

An affinity sorbent comprising macroporous glass coated with the polymer with the polymer with immobilized immunoglobulin IgG was used for the isolation from human serum of the first component of the complement and for its separation into subcomponents C1r, C1s and C1q by the one-step procedure. Serum C1 was quantitatively bound to the sorbent at 0 degrees C. The unbound part of the serum can be used as a R1 reagent for determining the hemolytic activity of C1. After activation of bound C1 by heating (30 degrees C, 40 min) the activated subcomponent C1r is eluted from the sorbent. Stepwise elution with EDTA at pH 7.4 or with EDTA + 1 M NaCl at pH 8.5 results in a selective and quantitative elution of the activated subcomponent C1s and subcomponent C1q. Stepwise elution of C1 subcomponents from the affinity sorbent after activation reflects the process of C1 breakdown following its activation on immune complexes.     

Mapping of the C1q binding site on rituxan, a chimeric antibody with a human IgG1 Fc

Rituxan (Rituximab) is a chimeric mAb with human IgG1 constant domains used in the therapy of non-Hodgkin's B cell lymphomas. This Ab targets B cells by binding to the cell-surface receptor, CD20. In our investigation of the mechanism of B cell depletion mediated by Rituximab, we first constructed mutants of Rituximab defective in complement activation but with all other effector functions intact. Our results demonstrate that the previously described C1q binding motif in murine IgG2b constituting residues E318, K320, and K322 is not applicable to a human IgG1 when challenged with either human, rabbit, or guinea pig complement. Alanine substitution at positions E318 and K320 in Rituximab had little or no effect on C1q binding and complement activation, whereas alanine substitution at positions D270, K322, P329, and P331 significantly reduced the ability of Rituximab to bind C1q and activate complement. We have also observed that concentrations of complement approaching physiological levels are able to rescue >60% of the activity of these mutant Abs with low affinity for C1q. These data localize the C1q binding epicenter on human IgG1 and suggest that there are species-specific differences in the C1q binding site of Igs.     

C1q: isolation from human serum in high yield by affinity chromatography and development of a highly sensitive hemolytic assay.

C1q, a subcomponent of the first component of complement, has been isolated from human serum in fully hemolytically active form by affinity column chromatography and gel filtration with Bio-Gel A-5M. The affinity column was prepared by covalent coupling of purified human IgG to CNBr-activated Sepharose 4B. Final yields of C1q ranged from 25 to 40% with 650- 890-fold purification based on recovery of hemolytic activity. The preparations were free of contaminating serum proteins as judged by SDS-polyacrylamide gel electrophoretic and immunochemical criteria. The final C1q preparations were also devoid of any demonstrable C1q-inhibitor activity. A C1q-depleted reagent (C1qD) was obtained from the nonabsorbed protein containing fractions of the human IgG-Sepharose 4B affinity column and utilized in conjunction with sensitized sheep erythrocytes (EA) for the detection and quantitation of C1q hemolytic activity. Employing optimal quantities of C1qD in the hemolytic assay mixture, the highly purified C1q preparations contained 0.5 to 1 x 10(13) effective molecules/mg and 0.5 to 1 x 10(12) effective C1q molecules/ml of human serum. This assay would therefore reproducibly detect less than 1 ng of C1q hemolytic activity.     

Role of complement component C1q in the IgG-independent opsonophagocytosis of group B streptococcus.

We investigated the role of complement component C1q in the IgG-independent opsonophagocytosis of type III group B Streptococcus (GBS) by peripheral blood leukocytes. We report that C1q binds to type III GBS both in normal human serum deficient in IgG specific for type III capsular polysaccharide and in a low-ionic strength buffer. The dissociation constant Kd ranged from 2.0 to 5.5 nM, and the number of binding sites Bmax ranged from 630 to 1360 molecules of C1q per bacterium (CFU). An acapsular mutant strain of GBS bound C1q even better than the wild type, indicating that the polysaccharide capsule is not the receptor for C1q. In serum, binding of C1q to GBS was associated with activation of the classical complement pathway. However, normal human serum retained significant opsonic activity after complete depletion of C1q, suggesting that the serum contains a molecule that is able to replace C1q in opsonization and/or complement activation. Mannan-binding lectin, known to share some functions with C1q, appeared not to be involved, since its depletion from serum had little effect on opsonic activity. Excess soluble C1q or its collagen-like fragment inhibited phagocytosis mediated by normal human serum, suggesting that C1q may compete with other opsonins for binding to receptor(s) on phagocytes. We conclude that, although C1q binds directly to GBS, C1q binding is neither necessary nor sufficient for IgG-independent opsonophagocytosis. The results raise the possibility that additional unknown serum factor(s) may contribute to opsonization of GBS directly or via a novel mechanism of complement activation.     

A new approach to the depletion of albumin and immunoglobulin G from human serum

The use of proteomic analysis to find potential diagnostic biomarkers is limited by the presence of serum albumin (HSA) and immunoglobulin (IgG) at high concentrations in patients’ blood; these substances impede the detection of serum proteins with similar molecular weights. Recombinant HSA- and IgG-binding polypeptides are used as ligands in creating sorbents for complete removal of the proteins by affinity chromatography. The binding specificity of the sorbents for HSA and IgG is higher than that of the conventionally used antibodies. A composite sorbent enabling the depletion of HSA and IgG from serum by single-step affinity chromatography was obtained. The developed sorbents were used to prepare serum for proteomic analysis.     

Structural refinement of protein A mimetic peptide

A novel dendrimeric peptide ligand dubbed D-PAM-Φ was designed to achieve a high capacity for human IgG through the decoration of the D-PAM scaffold. The design criteria based on the introduction of small hydrophobic groups on the D-PAM structure were supported by the recently published solid-state structure of D-PAM complexed to the Fc fragment of a recombinant human IgG1 and by molecular dynamic simulations that provided information on the mode of binding of phenylacetyl-D-PAM (D-PAM-Φ). D-PAM-Φ was immobilised on an activated solid support and compared with the parent D-PAM affinity matrix. The newly obtained affinity sorbent was evaluated for its capacity to selectively capture polyclonal human IgG; the binding capacity was approximately 10 mg/ml, an almost 10-fold enhancement with respect to the D-PAM-functionalised matrices without the specificity of binding being reduced. The new ligand was also effective in the capturing of recombinant humanised IgG1 from a clarified cell culture supernatant. Under a typical laboratory-scale affinity chromatography assembly and preliminarily optimised binding conditions, the affinity purification of humanised IgG1 from culture supernatants rendered the desired product, with purity higher than 90%. The results suggest that the application of the computational approach on the structure of the D-PAM-Fc complex may be very valuable in the development of novel lead molecules for the downstream processing of human or humanised antibodies used in therapy.     

Lack of fucose on human IgG1 N-linked oligosaccharide improves binding to human Fcgamma RIII and antibody-dependent cellular toxicity

Lec13 cells, a variant Chinese hamster ovary cell line, were used to produce human IgG1 that were deficient in fucose attached to the Asn(297)-linked carbohydrate but were otherwise similar to that found in IgG1 produced in normal Chinese hamster ovary cell lines and from human serum. Lack of fucose on the IgG1 had no effect on binding to human FcgammaRI, C1q, or the neonatal Fc receptor. Although no change in affinity was found for the His(131) polymorphic form of human FcgammaRIIA, a slight improvement in binding was evident for FcgammaRIIB and the Arg(131) FcgammaRIIA polymorphic form. In contrast, binding of the fucose-deficient IgG1 to human FcgammaRIIIA was improved up to 50-fold. Antibody-dependent cellular cytotoxicity assays using purified peripheral blood monocytes or natural killer cells from several donors showed enhanced cytotoxicity, especially evident at lower antibody concentrations. When combined with an IgG1 Fc protein variant that exhibited enhanced antibody-dependent cellular cytotoxicity, the lack of fucose was synergistic.     

Anti-DNA activity of IgG F(ab')2 from normal human serum.

The components of normal human serum (NHS) which bound DNA in a standard assay for anti-DNA antibody were characterized. IgG was the major detectable protein isolated from NHS by affinity chromatography on DNA-cellulose. A second adsorption of the whole serum IgG with DNA-cellulose did not remove additional gamma-globulin indicating that only a very small fraction of the IgG was capable of binding DNA. This binding activity was largely restricted to denatured DNA. IgG (Fab')2 bound DNA as well as the intact molecules demonstrating the antibody-like nature of the IgG binding. These results suggest that IgG antibody to denatured DNA is a normal component of human serum.     

Isolation and function of a human endothelial cell C1q receptor

It has been shown previously that cultured human venous and arterial endothelial cells (EC) bind C1q in a time- and dose-dependent manner. Cultured human endothelial cells express an average number of 5.2 x 10(5) binding sites/cell. In the present study the putative receptor for C1q (C1qR) was isolated from the membranes of 1-5 x 10(9) human umbilical cord EC by affinity chromatography on C1q-Sepharose. During isolation, C1qR was detected by its capacity to inhibit the lysis of EAC1q in C1q-deficient serum. The eluate from C1q-Sepharose was concentrated, dialysed and subjected to QAE-A50 chromatography and subsequently to gel filtration on HPLC-TSK 3000. C1qR filtered at an apparent molecular weight of 60 kDa. Purified C1qR exhibited an apparent molecular weight of 55-62 kDa in the unreduced state and a molecular weight of 64-68 kDa in reduced form. Two IgM monoclonal antibodies (mAb) D3 and D5 were raised following immunization of mice with purified receptor preparations. Both monoclonal antibodies increased the binding of (125)I-C1q to endothelial cells but F(ab')(2) anti-C1qR mAb inhibited the binding of a(125)I-C1q to EC in a dosedependent manner. The D3 mAb recognized a band of 54-60 kDa in Western blots of membranes of human EC and polymorphonuclear leukocytes. Previously, the authors showed that C1q induces the binding of IgM-containing immune complexes to EC. Therefore, it was hypothesized that during a primary immune response generation of IgM-IC may occur, resulting in binding and activation of C1, dissociation of activated C1 by C1 inhibitor and subsequent interaction of IgM-IC bearing C1q with EC-C1qR.     

Binding and complement activation by C-reactive protein via the collagen-like region of C1q and inhibition of these reactions by monoclonal antibodies to C-reactive protein and C1q

Ligand-complexed C-reactive protein (CRP), like aggregated or complexed IgG, can react with C1q and activate the classical C pathway. Whereas IgG is known to bind to the globular region and not to the collagen-like region (CLR) of C1q, the site of interaction of C1q with CRP has not been defined. CRP-trimers were prepared by cross-linking and found to bind to C1q and to activate the C system. Heat-aggregated IgG (Agg-IgG) did not block the binding of CRP-trimers to C1q, nor did CRP-trimers block binding of Agg-IgG to C1q, suggesting that CRP and IgG bind at different sites. ELISA and Western blot analysis showed that CRP-trimers bound to the CLR, whereas Agg-IgG bound only to the globular region; similarly, anti-CLR mAb inhibited binding of CRP-trimers to C1q whereas anti-globular region mAb did not. Reactivity with CRP-trimers as well as with Agg-IgG was retained after reduction/alkylation and SDS treatment of C1q. A group of 22 anti-CRP mAb directed against at least six distinct native-CRP epitopes and eight distinct neo-CRP epitopes was tested for ability to inhibit the CRP-CLR interaction; one mAb, anti-native CRP mAb 8D8, with strong inhibitory activity was identified. Fab' of 8D8 blocked binding of CRP-trimers to intact C1q as well as CLR, and also inhibited CRP (CRP-trimers and CRP-protamine complexes) induced C activation, but had no effect on C1q binding or C activation by Agg-IgG. These results indicate that a conformation-determined region on CRP binds to a sequence-determined region on the CLR of C1q in an interaction which leads to C activation. Anti-CRP and anti-C1q mAb that specifically inhibit this interaction are described.     

C-reactive protein and C1q regulate platelet adhesion and activation on adsorbed immunoglobulin G and albumin

Blood platelets and C-reactive protein (CRP) are both used clinically as markers of ongoing inflammation, and both participate actively in inflammatory responses, although the biological effects are still incompletely understood. Rapidly adhering platelets express receptors for complement factor 1q (C1q) and the Fc part of immunoglobulin G (IgG), and CRP is known to activate/regulate complement via C1q binding, and to ligate FcgammaRs. In the present study, we used normal human IgG pre-adsorbed to a well-characterized methylated surface as a model solid-phase immune complex when investigating the effects of CRP and C1q on platelet adhesion and activation. Protein adsorption was characterized using ellipsometry and polyclonal antibodies, and human serum albumin (HSA) and non-coated surfaces were used as reference surfaces. Platelet adhesion to IgG and HSA was inhibited by both C1q and CRP. Furthermore, CRP (moderately) and C1q (markedly) decreased the spreading of adhering platelets. The combination of C1q and CRP was slightly more potent in reducing cell adhesion to IgG, and also impaired the adhesion to HSA and non-coated surfaces. Platelet production of thromboxane B2 (TXB(2)) was also reduced by C1q both in the presence and absence of CRP, whereas CRP alone had no effect on TXB(2) production. We conclude that CRP and C1q regulate the behaviour of platelets, and that this may be an important immunoregulatory mechanism during inflammatory conditions.     

Comparison of autoantibodies to the collagen-like region of C1q in hypocomplementemic urticarial vasculitis syndrome and systemic lupus erythematosus.

Hypocomplementemic urticarial vasculitis syndrome (HUVS) is an apparent autoimmune disorder that resembles SLE. We previously showed that C1q precipitins in HUVS sera are IgG autoantibody to human C1q. We have compared HUVS anti-C1q autoantibody to a similar autoantibody in the serum of some patients with SLE. As with anti-C1q autoantibody in SLE sera, the HUVS autoantibody binds only to the collagen-like region (CLR) of C1q. In both HUVS and SLE, IgG2 is the predominant subclass of IgG autoantibody and IgM autoantibody to C1q is uncommon. In both diseases, anti-C1q autoantibodies bind preferentially to surface-adsorbed C1q or CLR fragments compared to these antigens in solution. Finally, when HUVS or SLE autoantibodies were added to CLR-coated wells already bound, respectively, by SLE or HUVS autoantibodies, no increases in CLR binding were observed, suggesting that HUVS and SLE autoantibodies to C1q bind to the same CLR epitope(s).     

An engineered human IgG1 antibody with longer serum half-life

The serum half-life of IgG Abs is regulated by the neonatal Fc receptor (FcRn). By binding to FcRn in endosomes, IgG Abs are salvaged from lysosomal degradation and recycled to the circulation. Several studies have demonstrated a correlation between the binding affinity of IgG Abs to FcRn and their serum half-lives in mice, including engineered Ab fragments with longer serum half-lives. Our recent study extended this correlation to human IgG2 Ab variants in primates. In the current study, several human IgG1 mutants with increased binding affinity to human FcRn at pH 6.0 were generated that retained pH-dependent release. A pharmacokinetics study in rhesus monkeys of one of the IgG1 variants indicated that its serum half-life was approximately 2.5-fold longer than the wild-type Ab. Ag binding was unaffected by the Fc mutations, while several effector functions appeared to be minimally altered. These properties suggest that engineered Abs with longer serum half-lives may prove to be effective therapeutics in humans.     

Effector Function Activities of a Panel of Mutants of a Broadly Neutralizing Antibody against Human Immunodeficiency Virus Type 1

The human antibody immunoglobulin G1 (IgG1) b12 neutralizes a broad range of human immunodeficiency virus-type 1 (HIV-1) isolates in vitro and is able to protect against viral challenge in animal models. Neutralization of free virus, which is an antiviral activity of antibody that generally does not require the antibody Fc fragment, likely plays an important role in the protection observed. The role of Fc-mediated effector functions, which may reduce infection by inducing phagocytosis and lysis of virions and infected cells, however, is less clear. To investigate this role, we constructed a panel of IgG1 b12 mutants with point mutations in the second domain of the antibody heavy chain constant region (CH2). These mutations, as expected, did not affect gp120 binding or HIV-1 neutralization. IgG1 b12 mediated strong antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC) of HIV-1-infected cells, but these activities were reduced or abrogated for the antibody mutants. Two mutants were of particular interest. K322A showed a twofold reduction in FcgammaR binding affinity and ADCC, while C1q binding and CDC were abolished. A double mutant (L234A, L235A) did not bind either FcgammaR or C1q, and both ADCC and CDC functions were abolished. In this study, we confirmed that K322 forms part of the C1q binding site in human IgG1 and plays an important role in the molecular interactions leading to complement activation. Less expectedly, we demonstrate that the lower hinge region in human IgG1 has a strong modulating effect on C1q binding and CDC. The b12 mutants K322A and L234A, L235A are useful tools for dissecting the in vivo roles of ADCC and CDC in the anti-HIV-1 activity of neutralizing antibodies.     

Comparative studies on biological activities of subcomponents C1q of the first component of human, bovine, mouse and guinea-pig complement

Both the haemolytic activity and the binding ability to immunoglobulin G(IgG) (Fc-binding ability) were comparatively assayed among human, bovine, mouse and guinea-pig C1q. The haemolytic activity was measured by using the sensitized sheep erythrocytes with rabbit immunoglobulin M(IgM)- or IgG-haemolysin. The Fc-binding ability was assayed by using immune complexes made of rabbit IgG-antibody against human serum albumin as well as agglutination of latex particles coated with human, bovine or rabbit IgG (IgG-latex). The specific haemolytic activity was comparable with between bovine and mouse C1q, while those of guinea pig and human C1q were significantly lower than those of the others. Only the human and mouse C1q showed significantly positive agglutinating activity of human or bovine IgG-latex. In the case of the use of rabbit IgG-latex, each of these C1q gave much weaker agglutination. On the other hand, the ability of all these C1q to bind to Fc of immune complexes specifically was almost comparable. The discrepancy in specific activities between the haemolysis and the Fc-binding ability may suggest that these two biological activities are not always correlative and that these are independent biological phenomena.     

Group G streptococcal IgG binding molecules FOG and protein G have different impacts on opsonization by C1q

Recent epidemiological data on diseases caused by beta-hemolytic streptococci belonging to Lancefield group C and G (GCS, GGS) underline that they are an emerging threat to human health. Among various virulence factors expressed by GCS and GGS isolates from human infections, M and M-like proteins are considered important because of their anti-phagocytic activity. In addition, protein G has been implicated in the accumulation of IgG on the bacterial surface through non-immune binding. The function of this interaction, however, is still unknown. Using isogenic mutants lacking protein G or the M-like protein FOG (group G streptococci), respectively, we could show that FOG contributes substantially to IgG binding. A detailed characterization of the interaction between IgG and FOG revealed its ability to bind the Fc region of human IgG and its binding to the subclasses IgG1, IgG2, and IgG4. FOG was also found to bind IgG of several animal species. Surface plasmon resonance measurements indicate a high affinity to human IgG with a dissociation constant of 2.4 pm. The binding site was localized in a central motif of FOG. It has long been speculated about anti-opsonic functions of streptococcal Fc-binding proteins. The presented data for the first time provide evidence and, furthermore, indicate functional differences between protein G and FOG. By obstructing the interaction between IgG and C1q, protein G prevented recognition by the classical pathway of the complement system. In contrast, IgG that was bound to FOG remained capable of binding C1q, an effect that may have important consequences in the pathogenesis of GGS infections.     

IgG subclass specificity to C1q determined by surface plasmon resonance using Protein L capture technique

Recombinant monoclonal antibodies (mAbs) have become an important category of biological therapeutics. mAbs share the same structures and biological functions as endogenous IgG molecules. One function is complement-dependent cytotoxicity (CDC) initiation by binding of C1q. Traditionally, ELISA methods have been utilized to measure C1q binding. A new robust capture method was established in this study to measure the binding affinity of C1q to antibodies by surface plasmon resonance (SPR). The utility of this method was demonstrated by determination of the difference in IgG subclass specificity of C1q binding.     

Conformational changes in C1q upon binding to IgG oligomers

The interaction between C1q and immune complexes is inhibited by 1-anilino-8-naphthalenesulfonate (ANS) in the concentration range of 2-4 mM. ANS binds to Clq with a 20-fold higher affinity than to IgG [(1986) Mol. Immunol. 23, 39-44] and therefore it is possible to label only C1q with ANS in the presence of IgG. Under such conditions no inhibition is observed. Addition of monomer IgG to a solution of C1q-bound ANS did not significantly alter the fluorescence of the ANS. However when oligomeric IgG was added there was a 2-fold increase in fluorescence over the same IgG concentration range. When C1q was pretreated with diethylpyrocarbonate there was little change in the fluorescence when IgG oligomers were added to C1q:ANS solutions. These results suggest that C1q undergoes conformational changes upon binding to IgG oligomers.     

Binding of the pentamer/hexamer forms of mannan-binding protein to zymosan activates the proenzyme C1r2C1s2 complex, of the classical pathway of complement, without involvement of C1q

The serum lectin, mannan binding protein (MBP), was isolated in a yield of 40 micrograms/liter from pooled normal human serum by affinity chromatography on mannan-Sepharose, followed by gel-filtration and ion-exchange chromatography and finally by passage down an anti-IgM Sepharose column. A rabbit antiserum was prepared against the purified MBP and an enzyme-linked immunoassay developed that used both the specificity of the polyclonal antibody and the Ca+(+)-dependent carbohydrate binding property of MBP. Assay of the sera from 103 blood-donors showed a wide range of MBP levels, ranging from 0 to 870 micrograms/liter. MBP, after interaction with zymosan, caused efficient activation of a C1r2 125I-C1s2 complex that was prepared by incubation of 125I-C1s2 with serum, from a patient with a complete genetic deficiency of C1q, followed by gel-filtration on Sepharose 6B. The purified MBP is composed of a mixture of trimers, tetramers, pentamers, and hexamers of an approximate 90-kDa structural unit as judged by chromatography, SDS-PAGE and electron microscopy studies. Only the molecules in the pentamer/hexamer fraction, which have a similar overall structure to that of C1q, appeared to cause efficient, zymosan-dependent, activation of C1s within the C1r2C1s2 complex. The pentamer/hexamer form of MBP may therefore play an important role in antibody-independent activation of the C system during the early stages of certain infections.