Nonimmunospecific protein-protein interactions of IgG: studies of the binding of IgG to IgG immunoadsorbents.

Nonimmunospecific interactions of IgG and IgG-agarose columns were systematically studied under varying conditions. Nonimmunospecific binding to the columns was primarily due to protein-protein interactions. These nonimmunospecific protein-protein interactions of IgG were enhanced with heat-induced or chemical aggregation of IgG, low pH, low ionic strength (at pH above 4), or low temperature. Conversely, this binding was decreased with proteolytic fragmentation of IgG, high ionic strength (at pH above 4), or temperatures above 4 degrees C. Chemical modification of IgG by acetylation, formalinization, carbamylation, or reaction with 1,2-cyclohexanedione significantly decreased these interactions. These observations suggest that above pH 4, ionic interactions caused the protein-protein binding. Below pH 4, hydrophobic interactions presumably play a major role. These results permit the development of rational methodology for avoiding nonimmunospecific protein-protein interactions in immunologic procedures for detection, isolation, or quantification of rheumatoid factors and other antibodies to IgG.     

The MHC class I-like IgG receptor controls perinatal IgG transport,IgG homeostasis,and fate of IgG-Fc-coupled drugs

Abs of the IgG isotype are efficiently transported from mother to neonate and have an extended serum t(1/2) compared with Abs of other isotypes. Circumstantial evidence suggests that the MHC class I-related protein, the neonatal FcR (FcRn), is the FcR responsible for both in vivo functions. To understand the phenotypes imposed by FcRn, we produced and analyzed mice with a defective FcRn gene. The results provide direct evidence that perinatal IgG transport and protection of IgG from catabolism are mediated by FcRn, and that the latter function is key to IgG homeostasis, essential for generating a potent IgG response to foreign Ags, and the basis of enhanced efficacy of Fc-IgG-based therapeutics. FcRn is therefore a promising therapeutic target for enhancing protective humoral immunity, treating autoimmune disease, and improving drug efficacy.     

Inhibitors to factor IX contain all IgG subclasses except IgG3.

Five per cent of patients with haemophilia B develop inhibitors to factor IX. It is of interest to know the immunoglobulin subclass of these IgG antibodies. We have developed a sensitive method for the characterization of the subclass nature of inhibitors to factor IX. The technique is a crossed immunoelectrophoresis for the isolation of factor IX-inhibitor complexes followed by an enzyme-linked immunoassay using monoclonal antibodies to IgG subclasses for the subclass identification. We studied seven inhibitors with both low and high titres. One patient was studied at a very early stage of inhibitor development. All inhibitors gave a strong reaction with antibody to IgG4. Depending on the titre of the inhibitor, a reaction was also found with antibodies to IgG1 and IgG2. No inhibitor contained any detectable IgG3. IgG4 does not bind complement and it is therefore of importance that IgG4 is the main subclass both in high-titred and in low-titred inhibitors. The inhibitors are polyclonal antibodies, also at an early stage of inhibitor development.     

Profile of total IgG, IgG1, IgG2, IgG3 and IgG4 levels in sera of patients with paracoccidioidomycosis: treatment follow-up using Mexo and rPb27 as antigens in an ELISA

The levels of total of IgG, IgG1, IgG2, IgG3 and IgG4 were evaluated in 54 patients with chronic paracoccidioidomycosis (PCM) before, during and after treatment using an enzyme-linked immunosorbent assay with Mexo and recombinant Pb27 (rPb27) as the antigens. Mexo was effective in distinguishing PCM patients from individuals in the negative control group (NC) based on total IgG and rPb27 performed worse than Mexo when these two groups were compared. IgG1, IgG2, IgG3 and IgG4 could not be used to clearly distinguish PCM patients from those in the NC group using either antigen. There was no clear relationship between antibody levels and the period of treatment. The majority of patients presented with decreased antibody levels during treatment, with no statistically significant differences among the different periods of treatment. Only IgG4 presented a negative correlation between its levels and clinical improvement during treatment. In total, 65% of untreated PCM patients showed reactivity against IgG4 when the Mexo antigen was used and this reactivity decreased over the course of treatment. There was a tendency towards decreasing antibody levels during treatment, but these antibody levels did not necessarily clear after the treatment was stopped. Mexo was useful for PCM diagnosis using total IgG; however, more studies are necessary before this antigen can be used in measuring the levels of total IgG and its subclasses for monitoring patients during treatment.     

Levels of immunoglobulins of isotype IgG and IgG2 in subjects lacking formation of IgG antibodies against lipopolysaccharide of Chlamydia

In some patients with antibodies against LPS antigen of Chlamydia, specific immunoglobulins G are not present. The findings of isolated anti-LPS IgA/IgM antibodies are to be considered as possibly non-specifically or "false" positive. The aim of this study was to investigate if there is any difference in the level of total immunoglobulins of isotypes IgG and IgG2 in probands with isolated positivity anti-LPS IgA (i.e. without simultaneous presence of specific IgG; n = 34) as compared with a control group of subjects presenting positive anti-LPS IgA and IgG antibodies (n = 44). Antibodies against LPS antigen of Chlamydia were determined by ELISA method ("Chlamydien--rELISA", medac, Germany). Total immunoglobulin levels were determined by nephelometry using the following kits: "Immunoglobulin G Reagent, ARRAY Systems", Beckman Coulter, USA and "Human IgG2 Subclass Beckman ARRAY Kits", The Binding Site, UK. The measured values were related to the age-dependent laboratory standard values and the differences between the tested groups were statistically evaluated (chi(2) test). Decreased total IgG have been demonstrated in 4 (11.8 %) probands and in 5 (11.4 %) subjects of the control group; increased total IgG were found in 2 (5.9 %) probands and in 1 (2.3 %) subject of the control group. Decreased levels of total IgG2 were not determined in any proband and were found in 1 (2.3 %) subject of the control group. Increased levels of IgG2 were registered in 12 (35.3 %) probands and in 15 (34.1 %) control subjects. No statistically significant differences were found between the two groups. It can be concluded that no relationship was proved between the levels of total IgG and IgG2 and the absence of formation of specific anti-Chlamydia-LPS IgG. Further research will be necessary for the elucidation of this phenomenon (e.g. the presence of specific anti-LPS IgG in immunocomplexes).     

Radioimmunoassay of IgG and IgM rheumatoid factors reacting with human IgG.

Although IgG rheumatoid factor may play a central role in the pathogenesis of rheumatoid arthritis, previously there have been no precise methods for its specific measurement in serum and synovial fluid. This paper describes a solid phase radioimmunoassay for the independent quantification of IgM and IgG rheumatoid factor reacting with the Fc fragment of human IgG. As measured by this assay, serum IgG rheumatoid factor levels differed significantly between patients with seropositive and seronegative rheumatoid arthritis and normal control subjects. In addition, several sera and joint fluids from patients with seropositive rheumatoid arthritis, even without vasculitis, were shown by gel chromatography to have acid-dissociable complexes of IgG rheumatoid factor suggestive of IgG-IgG dimer or trimer formation.     

Monovalent IgG4 molecules

Antibodies have become the fastest growing class of biological therapeutics, in part due to their exquisite specificity and ability to modulate protein-protein interactions with a high biological potency. The relatively large size and bivalency of antibodies, however, limits their use as therapeutics in certain circumstances. Antibody fragments, such as single-chain variable fragments and antigen binding-fragments, have emerged as viable alternatives, but without further modifications these monovalent formats have reduced terminal serum half-lives because of their small size and lack of an Fc domain, which is required for FcRn-mediated recycling. Using rational engineering of the IgG4 Fc domain to disrupt key interactions at the CH3-CH3 interface, we identified a number of point mutations that abolish Fc dimerization and created half-antibodies, a novel monovalent antibody format that retains a monomeric Fc domain. Introduction of these mutations into an IgG1 framework also led to the creation of half-antibodies. These half-antibodies were shown to be soluble, thermodynamically stable and monomeric, characteristics that are favorable for use as therapeutic proteins. Despite significantly reduced FcRn binding in vitro, which suggests that avidity gains in a dimeric Fc are critical to optimal FcRn binding, this format demonstrated an increased terminal serum half-life compared with that expected for most alternative antibody fragments.     

On the fragmentation of monoclonal IgG1, IgG2a, and IgG2b from BALB/c mice

Methods for the production and purification of F(ab')2 fragments from BALB/c monoclonal IgG1, IgG2a, and IgG2b with pepsin and other proteases were examined. The overall susceptibility to degradation is IgG2b greater than IgG2a greater than IgG1. Stable F(ab')2 can be produced in good yield from IgG1 with pepsin at pH 3.5 to 4.0 and can be made directly by pepsin treatment of ascites fluids or cell culture supernatants containing IgG1. IgG2a is cleaved in two steps by pepsin, first to F(ab')2 and then to Fab'. With carefully chosen conditions, F(ab')2 can be obtained in acceptable yield. The primary cleavage for the IgG2a heavy chain appears to be on the COOH terminal side of the interheavy chain disulfides, and secondary cleavage is on the NH2-terminal side. For IgG2b the reverse is true, and F(ab')2 has not been obtained in useful amounts; however, the primary cleavage of IgG2b appears to be assymetric with respect to the two heavy chains, and Fab/c fragments that have one Fab plus Fc can be made. Digestion with elastase resulted in the best yield of Fab/c. This finding may provide a method for retaining cytotoxicity in monoclonal antibodies against cell surface antigens while eliminating their capacity to modulate. The cleavage patterns of the three classes of IgG are rationalized in terms of the structure of their hinge regions.     

Monomeric complement-activating IgG paraproteins

Three patients presented a unique syndrome of recurrent panniculitis with an IgGkappa paraprotein and depletion of the early components of the classical pathway of complement. The IgGkappa paraproteins were monomers with a normal structure, and with no evidence for aggregation, as assessed by electron microscopy and ultracentrifugation. Both heavy and light chains were of normal molecular size (SDS-PAGE), and the paraproteins were not heavily glycosylated. However, the paraproteins from all three patients had unusual features that included abnormal behavior on gel filtration chromatography and a heavy chain of high pI. When analyzed by fast protein liquid chromatography (Superdex 200), elution of the paraproteins was retarded, particularly when the ionic strength was increased. This retardation was partially reversed in 20% alcohol, and fully reversed in 6 M guanidine-HCl. Neither anti-C1 inhibitor nor anti-C1q autoantibodies were found in any of the patients' sera. However, the paraproteins bound to the globular heads of C1q at normal ionic strength. They activated C4 in normal human serum, but not in C1q-deficient serum. Activation led to the formation of C1s-C1 inhibitor complexes. Taken together, the data suggest that the unusual paraproteins have the capacity to bind C1q, which then leads to activation of C1. The ability of these paraproteins to activate C1, in spite of their being soluble monomers, is likely to be related to their unique physicochemical features.     

貉血清IgG纯化及抗血清的制备

目的 制备高纯度貉血清IgG和兔抗貉IgG抗血清,作为建立多种动物抗体检测技术的储备。方法 采用Hitrap Protein A亲和层析及盐析再沉淀法纯化貉血清IgG,通过PAGE电泳和Western-Blot免疫印迹法对IgG作纯度及免疫活性检测;常规免疫法制备兔抗貉IgG血清。结果 貉血清IgG与Protein G虽有较强的结合力,但同时也结合血清中其他杂蛋白;用二步纯化法可从5 mL貉血清中纯化IgG约7 mg,电泳和免疫印迹测定显示,IgG纯度大于95%,常规免疫法制备抗血清免疫双扩散效价达1∶32。结论 建立了可行的貉血清IgG的纯化方法和高效价的兔抗貉血清IgG抗血清,为貉血清IgG二级抗体酶联物的制备储备了资源。     

Hyperacute rejection by anti-Gal IgG1, IgG2a, and IgG2b is dependent on complement and Fc-gamma receptors

We have previously reported that anti-Gal-alpha1,3Gal (Gal) IgG3 mAbs mediate a classical complement-dependent hyperacute rejection (HAR), while anti-Gal IgG1 mAbs mediate HAR that is dependent on complement, the Fc-gamma receptors FcgammaRII/III (CD32/CD16), and NK cells. IgG2a and IgG2b subclasses can activate complement and have FcgammaR binding properties in vitro. Whether these IgG subclasses can mediate HAR in vivo and the mechanisms by which they would do so are not known. In this study, we isolated spontaneous IgG switch mutants from an anti-Gal IgG1 hybridoma. In vitro complement-mediated hemolytic assays with mouse complement indicate that both anti-Gal IgG2a and IgG2b mAbs were more potent compared with the parent anti-Gal IgG1. In vivo administration of anti-Gal IgG2a and IgG2b mAbs into Gal-/- mice induced HAR of rat cardiac xenografts. HAR induced by anti-Gal IgG2a and IgG2b was dependent on complement activation and the presence of NK cells. Using FcgammaRIII-deficient (Gal-/-CD16-/-) recipients, we observed that HAR mediated by different anti-Gal IgG subclasses was variably dependent on FcgammaRIII, with IgG1>IgG2b>IgG2a=IgG3. Using FcgammaRI-deficient (Gal-/-CD64-/-) recipients, we observed that HAR mediated by anti-Gal IgG1, IgG2a, and IgG2b, but not by anti-Gal IgG3, was dependent on FcgammaRI. Collectively, these studies demonstrate the necessity and sufficiency of complement in IgG3-mediated HAR and the necessity of both complement and FcgammaR, especially FcgammaRI, in IgG1-, IgG2a-, and IgG2b-mediated HAR.     

Generating bispecific human IgG1 and IgG2 antibodies from any antibody pair

Bispecific antibodies and antibody fragments are a new class of therapeutics increasingly utilized in the clinic for T cell recruitment (catumaxomab anti-EpCAM/CD3 and blinatumomab anti-CD19/CD3), increase in the selectivity of targeting, or simultaneous modulation of multiple cellular pathways. While the clinical potential for certain bispecific antibody formats is clear, progress has been hindered because they are often difficult to manufacture, may suffer from suboptimal pharmacokinetic properties, and may be limited due to potential immunogenicity issues. Current state-of-the-art human IgG-like bispecific technologies require co-expression of two heavy chains with a single light chain, use crossover domains to segregate light chains, or utilize scFv (single-chain fragment variable)-Fc fusion. We have engineered both human IgG1 and IgG2 subtypes, with minimal point mutations, to form full-length bispecific human antibodies with high efficiency and in high purity. In our system, the two antibodies of interest can be expressed and purified separately, mixed together under appropriate redox conditions, resulting in a formation of a stable bispecific antibody with high yields. With this approach, it is not necessary to generate new antibodies that share a common light chain, therefore allowing the immediate use of an existing antibody regardless of whether it has been generated via standard hybridoma or display methods. We demonstrate the generality of the approach and show that these bispecific antibodies have properties similar to those of wild-type IgGs, and we further demonstrate the utility of the technology with an example of a CD3/CD20 bispecific antibody that effectively depletes B cells in vitro and in vivo.     

Subclasses of human IgG. I. Production of antisera to IgG subclasses]

Guinea pigs were used for preparing antisera to human IgG subclasses for anti-IgG1, and rabbits--for anti-IgG2, anti-IgG3, and anti-IgG4. Schemes of laboratory animals immunization with myeloma paraproteins of four IgG subclasses were determined. Methods of antisera absorption for bringing them up to strict monospecificity were worked out. Antisera specificity were determined by the precipitation test after Ouchterlony with standard myeloma proteins in the concentration of 1 mg/ml, and in the passive hemagglutination test with erythrocytic antigenic diagnostic agents. Precipitating antisera to four human IgG subclasses were obtained.     

Immunogenetic study of porcine IgG

Three allotypes of IgG were identified in pig. Based on data obtained on electrophoretic mobility as well as on results of the International Comparative Test ISABR for pig blood group, polymorphic proteins and enzymes (1987-1988), the allotypes are specified as markers of two different IgG subclasses and are referred to as IgG1a, IgG2b and IgG2c. The former of these is established as corresponding to the already known IGH3 C1, and the other two had not been earlier described. In herds of pigs being bred in the Georgian SSR, the IgG1a allotype frequencies in Kakhetinskaya, Large White, Landrase and Lithuanian white were 0.84, 0.93, 0.91 and 0.94, respectively, whereas for the IgG2b allotype it ran 0.89, 0.73, 0.79, and 0.69 in the order mentioned. The IgG2c allotype was not registered in samples under examination.     

Serum IgG and Renal Transplantation

In 57 patients with renal allografts the prolonged administration of prednisolone ≥ 1 mg/kg/day and azathioprine ≥ 3 mg/kg/day caused a significant and persistent fall in serum IgG at all levels of creatinine clearance. The fall in IgG was more striking when creatinine clearance was below 25 ml/min. At lower doses of azathioprine and prednisolone serum IgG fell when the creatinine clearance was less than 35 ml/min, the degree of recovery towards normal being dependent on creatinine clearance and dosage. Post-transplant haemodialysis decreased the depression of IgG, and patients with immediately functioning grafts had minimal IgG depression. An inverse relation between IgG and IgM was observed in some patients. Severe infections and toxicity were associated with the greatest reduction in IgG; leucopenia and thrombocytopenia were not consistently reliable guides to toxicity. The deaths of four patients (7%) were associated with severe infections. Falls in IgG were not related to the rejection process. IgG measurement should be used as a guide to immunosuppression and toxicity in renal allograft patients.     

Charge variants in IgG1

Antibody charge variants have gained considerable attention in the biotechnology industry due to their potential influence on stability and biological activity. Subtle differences in the relative proportions of charge variants are often observed during routine biomanufacture or process changes and pose a challenge to demonstrating product comparability. To gain further insights into the impact on biological activity and pharmacokinetics (PK) of monoclonal antibody (mAb) charge heterogeneity, we isolated the major charge forms of a recombinant humanized IgG1 and compared their in vitro properties and in vivo PK. The mAb starting material had a pI range of 8.7-9.1 and was composed of about 20% acidic variants, 12% basic variants, and 68% main peak. Cation exchange displacement chromatography was used to isolate the acidic, basic, and main peak fractions for animal studies. Detailed analyses were performed on the isolated fractions to identify specific chemical modification contributing to the charge differences, and were also characterized for purity and in vitro potency prior to being administered either subcutaneously (SC) or intravenously (IV) in rats. All isolated materials had similar potency and rat FcRn binding relative to the starting material. Following IV or SC administration (10 mg/kg) in rats, no difference in serum PK was observed, indicating that physiochemical modifications and pI differences among charge variants were not sufficient to result in PK changes. Thus, these results provided meaningful information for the comparative evaluation of charge-related heterogeneity of mAbs, and suggested that charge variants of IgGs do not affect the in vitro potency, FcRn binding affinity, or the PK properties in rats.