Indications of neutralising anti-idiotypic antibodies and selective proteolytic fragmentation in polyclonal anti-D IgG preparations.

Proteolytic fragmentation is the only suggested cause of potency losses during storage of liquid human polyclonal anti-D Ig. Besides the effect of fragmentation, we have investigated the potential contribution of neutralising anti-idiotypic antibodies (anti-Ids). Potency changes during storage and/or upon pH reduction in anti-D IgG batches with or without addition of plasminogen and urokinase were quantitatively analysed by the autoanalyser (AA) method or by a special procedure of flow cytometry (FC). Moreover, simultaneous changes of the molecular size distribution pattern have been determined by size exclusion chromatography. In contrast to the AA procedure, the particular FC methodology was found to be almost insensitive to proteolysis comprising up to 30% of total IgG. Data interpretation was based on the assumption that both assays cannot detect Ids with neutralised paratopes. In the absence of detectable neutralisation (functional absence of anti-Ids), it could be demonstrated that the anti-D IgG subpopulation is more sensitive to fragmentation by endogenous protease as compared to the unrelated bulk. However, both methods detected batch- and assay-dependently variable potency losses during storage. Moreover, the increase of potency induced by pH reduction correlated with the increase of monomeric IgG, essentially on the expense of dimers. This finding was interpreted to indirectly indicate the neutralising action of anti-Ids known to be the major driving force of dimer formation in polyclonal IgG. A more or less pronounced pH-dependent potency increase was also detectable in three arbitrarily selected batches of two other manufacturers. The data allows to assume that anti-Id-mediated neutralisation can significantly contribute to losses of anti-D potency. In addition, it turned out that anti-D plasma itself can be the source of anti-Ids.

The hypothetical N-glycan charge: a number that characterizes protein glycosylation.

The production of recombinant glycoprotein therapeutics requires characterization of glycosylation with respect to the lot-to-lot consistency. Here we introduce the ¿hypothetical N-glycan charge Z' as a parameter that allows to characterize the protein glycosylation in a simple, however, efficient manner. The hypothetical N-glycan charge of a given glycoprotein is deduced from the N-glycan mapping profile obtained via HPAE-PAD. In HPAEC, N-glycans are clearly separated according to their charge, i.e., their number of sialic acid residues, providing distinct regions for neutral structures as well as for the mono- di-, tri, and tetrasialylated N-glycans (Hermentin et al., 1992a). Z is defined as the sum of the products of the respective areas (A) in the asialo, monosialo, disialo, trisialo, tetrasialo, and pentasialo region, each multiplied by the corresponding charge: [formula: see text] Thus, a glycoprotein with mostly C4-4* structures will provide Z approximately equal to 400 (e.g., rhu EPO (CHO), Z = 361), a glycoprotein carrying largely C3-3* structures will amount to Z approximately equal to 300 (e.g., bovine fetuin, Z = 290), a glycoprotein with mostly C2-2* structures will have Z approximately equal to 200 (e.g., human serum transferrin, Z = 207, or human plasma AT III, Z = 180), and a glycoprotein carrying only high-mannose type or trunkated structures will provide Z approximately equal to 0 (e.g., bovine pancreas ribonuclease B, Z = 15, and hen ovomucoid, Z = 15, respectively). The determination of Z was validated in multiple repetitive experiments and proved to be highly accurate and reliable. Z may therefore be regarded as a new and characteristic parameter for protein N-glycosylation.

Modulation of the immunoglobulin dysregulation in GvH- and SLE-like diseases by the murine IL-4 receptor (IL-4-R).

As has been reported previously, models of chronic graft-versus-host (GvH) and systemic lupus erythematosus (SLE)-like diseases are characterized by high IgE and IgG1 immunoglobulin (Ig) levels in the serum. An IL-4 induced pathological expansion of Th2 helper cells has been described for both disease models. Due to the immunopharmacological profile of soluble recombinant interleukin-4 receptor (IL-4-R) to bind specifically the corresponding ligand IL-4 and thereby to modulate biological activity upon exogenous administration in various autoimmune disease models, we investigated the immunoregulatory activity of IL-4-R and anti-IL-4 monoclonal antibody (MAb) 11B11 on the development of SLE-like disease in MRL/lpr autoimmune mice and on chronic GvH reaction in BDF1 hybrid mice. Sensitized GvH-BDF1 hybrid mice and SLE in MRL/lpr autoimmune mice were treated in vivo with the IL-4 antagonists to alter the pattern of serum Ig production and to modulate the disease process. These animals were followed for proteinuria, autoantibody production (anti-dsDNA), serum IgE, IgG1 and IgG2a levels, and the survival was monitored. Treatment of these diseased animals resulted in an improved survival rate, lowered the percentage of animals with lymphadenopathy and hepatosplenomegaly, reduced the levels of autoantibodies and inhibited proteinuria of the developing glomerulonephritis in both mouse strains, even in the established diseases. In both models the increase in total IgE and IgG1 levels in serum was strongly inhibited by the IL-4 antagonists, even under therapeutic conditions. But there was no inhibitory activity observed on the IgG2a serum levels.(ABSTRACT TRUNCATED AT 250 WORDS)

Monomeric and dimeric forms of soluble receptors can differ in their neutralization potential.

Recombinant soluble forms of transmembrane receptors can be produced in monomeric and dimeric versions. Binding affinity and neutralization potential of these different forms of soluble receptors depend on the quaternary structure of their ligands. Monomeric ligands will be bound with equal affinity by both forms, whereas trimeric ligands, e.g. members of the tumor necrosis factor family of ligands, interact with much higher affinity with dimeric soluble receptors than with monomeric ones.

Immunomodulatory activity of recombinant IL-1 receptor (IL-1-R) on models of experimental rheumatoid arthritis.

Given the role of IL-1 in inflammation and in autoimmune diseases, studies were designed to examine the ability of IL-1 receptor (IL-1-R) to suppress inflammation in a model of chronic degenerative joint disease of adjuvant arthritis (AA) in Lewis rats and to suppress the development of a systemic lupus erythematosus (SLE)-like disease in MRL/lpr mice. IL-1-R was able to prevent the onset of the AA and, even if therapy started after the establishment of AA, the cytokine receptor was still able to reduce the degree of chronic inflammation and arrested its progress. Treating MRL/lpr mice with IL-1-R resulted in a decrease in the amount of autoantibodies and inhibited joint inflammation. Even in the established disease IL-1-R could reduce rheumatoid factors (RF) and autoantibodies, and the signs of a polyarthritis were inhibited.

Immunoregulation of SLE-like disease by the IL-1 receptor: disease modifying activity on BDF1 hybrid mice and MRL autoimmune mice.

Due to the immunopharmacological profile of the recombinant IL-1 receptor (IL-1-R) and its potential to modulate biological activity in various inflammatory autoimmune disease models, we further elucidated its disease modifying activity on the development of a systemic lupus erythematosus (SLE)-like disease in BDF1 hybrid mice and in MRL/lpr autoimmune mice. Treatment of BDF1 mice with the IL-1-R during the induction phase resulted in a strong inhibition of the development of a glomerulonephritis, prolonged the survival time and improved the survival rate. Even a therapeutic effect was demonstrated when this receptor was given after the appearance of clinical symptoms. Treating MRL/lpr mice, which develop spontaneously a SLE-like disease, with the IL-1-R resulted in an inhibition of the developing glomerulonephritis and splenomegaly, in a reduction of swollen lymph nodes and in a decrease of autoantibody formation. Even in the established autoimmune disease of MRL/1 pr mice the IL-1-R reduced proteinuria, the levels of autoantibodies and also improved the survival rate.

Construction, expression and characterization of humanized antibodies directed against the human alpha/beta T cell receptor.

Completely humanized antibodies with specificity for the human alpha/beta TCR have been produced by genetic engineering. The L and H chain V region exons encoding the murine mAb BMA 031 CD regions and human EU framework regions were synthesized and replaced into previously isolated genomic fragments. These fragments were inserted into mammalian expression vectors containing the human kappa and gamma 1 C region exons. Two variants were constructed each containing selected BMA 031 amino acids within the human frameworks. The humanized genes were transfected into Sp2/0 hybridoma cells by electroporation and transfectomas secreting humanized antibody were isolated. Levels of antibody expression up to 7 pg/cell/24 h were obtained. The humanized antibody, BMA 031-EUCIV2, competed poorly with murine BMA 031 for binding to T cells. BMA 031-EUCIV3, however, bound specifically to T cells and competed effectively with both the murine BMA 031 antibody and a previously constructed chimeric BMA 031 antibody for binding to these cells. The relative affinity of BMA 031-EUCIV3 was about 2.5 times lower than BMA 031. The ability to promote antibody dependent cell-mediated cytolysis was significantly enhanced with the engineered antibodies as compared to murine BMA 031. Humanized BMA 031 is a clinically relevant, genetically engineered antibody with potential uses in transplantation, graft vs host disease, and autoimmunity.

A rabies-specific human monoclonal antibody that protects mice against lethal rabies.

According to a recommendation from WHO (World Health Organisation) for prevention of a possible rabies infection, active vaccination has to be combined with application of immunoglobulin to get a fast protective effect. At present, preparations of purified human or equine rabies-specific immunoglobulin are used. We have generated a human rabies-specific monoclonal antibody (huMAb) by immortalization of human B-cells with Epstein Barr Virus (EBV), followed by fusion with a mouse myeloma cell. The resulting clone TW-1 secrets an IgG1 lambda huMAb which specifically reacts in ELISA with 5 laboratory rabies virus strains of serotype 1 and DUV3 (Duvenhage, serotype 4). Western Blot analysis revealed fine specificity for the G glycoprotein (gp67) of rabies virus. HuMAb TW-1 neutralizes rabies virus in vitro (RFFIT) as well as in vivo and protects rabies infected mice. Compared to polyclonal human rabies immunoglobulins, huMAb TW-1 is advantageous, because of its defined specificity and the very low amounts of total protein needed for therapeutic effects.

Inhibitory effects of the anti-rat T-cell receptor (TCR) monoclonal antibody (MAb) R73 on various experimental autoimmune diseases.

The anti-rat alpha/beta-TCR, MAb-R73 has been investigated as to its disease modifying activity on adjuvant arthritis (AA), on experimental allergic encephalomyelitis (EAE) and on a local graft versus host (GvH) reaction (popliteal lymph nodes = PLN) in Lewis or Brown-Norway rats. R73 was able to prevent the onset of the AA and even if therapy started after the establishment of AA the MAb was still able to reduce the degree of chronic inflammation and arrest its progress. Intravenous MAb-R73 application also reduced the signs of EAE and prevented mortality. This was even seen when the substance was given after the outbreak of the clinical symptoms or when the F(ab)2 fragment of this MAb was used. Also in the model of local GvH reaction R73 acted therapeutically and lowered the PLN weights.

Construction, expression, and biologic activity of murine/human chimeric antibodies with specificity for the human alpha/beta T cell receptor.

Murine/human chimeric antibodies with specificity for the human TCR-alpha/beta have been produced by genetic engineering. The L and H chain V region exons encoding the murine mAb BMA 031 were isolated and inserted into mammalian expression vectors containing the human kappa and gamma 1 or gamma 4 C region exons. The chimeric genes were transfected into murine Sp2/O hybridoma cells by electroporation and transfectomas secreting chimeric antibody were isolated. Secretion levels ranged from 1 to 7 pg/cell/24 h. The chimeric antibodies bound specifically to T cells and competed effectively with the parental murine mAb for binding to these sites. The ability to promote antibody-dependent cell-mediated cytolysis was significantly enhanced in the chimeric antibodies as compared with murine BMA 031. C-dependent cytolysis, however, was not detectable with any of the antibodies. Chimeric BMA 031 is a clinically relevant, genetically engineered antibody with potential uses in transplantation, graft-vs-host disease, autoimmune diseases and other T cell-related disorders.

Determination of affinities of murine and chimeric anti alpha/beta-T-cell receptor antibodies by flow cytometry.

In this report we describe the use of a calibrated flow cytometer to quantify the expression of the human alpha/beta-T-cell-receptor on peripheral blood lymphocytes of healthy human blood donors and on the T-cell line HPB-ALL. To calculate the maximum of antibodies which are needed to obtain antigen saturation per cell and to determine the affinity of murine and chimeric anti-TCR antibodies, we applied Scatchard-plot and double reciprocal Klotz-plot analyses.

Physiochemical features of monoclonal idiotype-anti-idiotype complexes: importance of inter-chain disulfides for size distribution patterns and molecular geometries.

Cyclic tetramers represent the preferentially formed complexes of a murine monoclonal idiotype-anti-idiotype (Id-anti-Id) system consisting of IgG antibodies or (Fab')2 fragments at micromolar concentrations. The cleavage of inter-chain disulfides of both Id and anti-Id caused the predominant generation of cyclic dimers at the expense of larger aggregates, suggesting with regard to already published data that the hinge located interheavy-chain disulfides are essential for the strain.

On the nature of IgG dimers. I. Dimers in human polyclonal IgG preparations: kinetic studies.

Human polyclonal IgG for therapeutic or prophylactic purposes is usually prepared from pooled plasmas taken from more than 1000 donors. After storage in solution under physiological conditions for a sufficiently long period, a considerable percentage of dimers (approx. 10-30% [w/w] at a protein concentration of approx. 160 mg/ml) represents the main component of aggregates in contrast to the essentially monomeric IgGs of monoclonal or single donor origin. Analysing the kinetics of monomer-dimer equilibration suggests assuming approx. 10(6) different antibody (ab) populations interacting independently and simultaneously with a specific partner of the reaction. Concerning the average apparent (functional) equilibrium constant of association, Kapp., a distinction could be made between two main populations characterized by values in the range if approx. 2.5-3.0 X 10(10) M-1 and 1.0 X 10(12) M-1, respectively. The results were obtained by computer simulation, taking an association rate constant, k+1, of 5 X 10(5) M-1 s-1 as a basis. Since the main part of the individual populations was found to interact Fc-independently via Fab-located binding sites, we suppose that the dimers are for the most part complexes of idiotypic (Ids) and anti-idiotypic abs (anti-Ids). Moreover, dimerization seems to be mainly a bivalent binding reaction, at least at the experimental concentrations. The results are in line with the concept of an idiotypic network regulation in man.

On the nature of IgG dimers. II. Idiotype--anti-idiotype complexes of polyclonal and monoclonal origin: size distribution patterns and molecular geometries.

Electron micrographs of a fraction containing dimers isolated from pooled human polyclonal immunoglobulin G (IgG) suggest essentially a cyclic geometry compatible with bivalently associated monomers. It is obvious that such a structure can be produced by idiotype (Id)--anti-idiotype (anti-Id) interactions where the latter are able to neutralize certain combining site related Id functions. Accordingly, antibody (ab) activities against tetanus toxoid (tt) and rubella antigen (ag) were found to be almost exclusively confined to the monomeric molecules in preparations composed of monomers and dimers only. Moreover, electron micrographs of complexes prepared from a murine monoclonal Id as well as anti-Id reveal the presence of ring complexes, especially of cyclic tetramers. Gel filtration patterns of mixtures containing equimolar concentrations (concns) of such abs (1.6 x 10(-6) M) show, correspondingly for 9 different Id--anti-Id pairs and therefore probably representing a more common feature, mainly the formation of even-numbered complexes, especially tetramers. That is basically in accordance to an equilibrium model developed by Archer and Krakauer but not from a quantitative point of view because non-ideality terms had not been originally included. Despite taking strain energies determined by Schumaker et al. for cyclic complexes of polyclonal rabbit abs and a bivalent hapten into account for computation of size distribution patterns, the predominant formation of dimers was, nevertheless, again predicted by the modified theory in contrast to the experimental results. Fundamental conformity could only be achieved by further decreasing one of the statistical factors, namely the ring closing factor, which theoretically influences the generation of cyclic dimers. Therefore, referring to the experimental results of Schumaker et al., we postulate a strain energy well above 700 cal/mol for cyclic dimers produced by interacting Ids and anti-Ids. In general, the findings confirm predictions based on the interpretation of recent kinetic studies.

C4-binding protein prevents spontaneous cleavage of C3 in sera of patients with hereditary angioedema.

We have studied the effects of polyclonal monospecific Fab' preparations against C1r, C1s, C1INH, C4, C4bp, and fragment Bb of factor B on complement activation in NHS and HAES. Furthermore, we have investigated complement activation in these sera after addition of purified C1s and purified C4bp. Blocking C1INH induced a spontaneous activation of the classical pathway in NHS and to a lesser extent in HAES. Addition of p-C1s resulted in a strong C3 conversion in NHS, but not in HAES. However, after the blocking of C4bp in HAES, addition of p-C1s produced a total C3 consumption. The ration of the protein concentration of C4bp to hemolytically active C4 was eight times higher in HAES than in NHS. This increased ratio may account for the resistance of HAES to the C1s induced C3 cleavage in our in vitro system and the stability of C3 in HAE despite C4 and C2 consumption in vivo.

Fc fragments of human IgG may influence allergic reactions.

The antiallergic effect of Fc fragments prepared from human polyclonal IgG was investigated in several experimental models. In an in vitro assay, isolated ileum of cynomolgus monkeys was sensitized with serum from atopic patients. In six of fifteen monkeys the subsequent addition of specific allergens reproducibly resulted in an ileum contraction measured in the Schultz-Dale apparatus. In all six positive monkeys pre-treatment of the isolated ileum with Fc (papain) before sensitization inhibited ileum contraction. Fc (plasmin) or Fc (pepsin) was less or not effective, aggregated IgG, monomeric IgG, sulfonated IgG, the Fc-free F(ab')2 moiety, or albumin had no significant or no reproducible inhibitory effect. In an in vivo assay passive cutaneous anaphylaxis was studied in 28 cynomolgus monkeys. Different dilutions in PBS of the particular specific allergens were subsequently administered to sensitized skin sites, simultaneously to i.v. injection of Evans blue. The degree of the local allergic (passive cutaneous anaphylactia) reaction was evaluated by measuring the diameter of the blue area at the dermal injection sites. About 50% of sera induced positive reaction in monkeys. Compared to the control (application of PBS), the degree of the positive reactions could be reduced by about 50% if Fc (papain) (optimal dose 25 mg/kg body weight) was injected i.v. either 2 h before or after local sensitization. No significant inhibitory effect could be found with the Fab moiety of IgG. Follow-up studies showed that the inhibitory effect of Fc lasted for about 10 days. The pharmacological basis of the observed antiallergic action of Fc is not yet understood.