IgG2a Monoclonal Antibody Research Articles

A passive haemagglutination test for human anti-mouse antibody (HAMA) responses in patients undergoing immunoscintigraphy

Sera from seven patients with ovarian or colorectal carcinoma who had been given only single injections of 131I or 111In labelled IgG1, IgG2a or IgG2b monoclonal antibodies for immunoscintigraphy 2 weeks to 9 months earlier were tested for their ability to agglutinate sheep red blood cells coated with a mouse monoclonal antibody. Six showed agglutination, and this was still seen when sera were diluted to 1/125 in four patients and as low as 1/3125 in the other two. Sera from rats and mice which had been injected with monoclonal antibody also caused agglutination, which in the case of the mouse sera is due to the presence of anti-idiotypic antibody. This study shows that it is feasible to detect HAMA by a rapid, simple, passive haemagglutination reaction.

Capacity of passively administered antibody to prevent establishment of Brucella abortus infection in mice

In contrast to immunity against some other facultative intracellular parasites, protective immunity against Brucella abortus is mediated in mice by antibodies as well as by cell-mediated immune responses. It was the purpose of this study to determine whether antibody alone would prevent infection with B. abortus. The majority (82%) of CD-1 outbred mice infected with 100 CFU of virulent B. abortus 2308 preincubated with graded quantities of an O polysaccharide-specific IgG2a monoclonal antibody (MAb) were free of infection 1. 2, 4, and 6 weeks later, based on detection limits of 13 brucellae per spleen and 39 per liver. Infection was present in 95% of control animals. Similar results were obtained with a challenge dose of 500 CFU, but with a challenge dose of 5,000 CFU, infection became established even with the highest concentration of MAb used (50 micrograms of MAb per 5,000 brucellae). Pretreatment with an O polysaccharide-specific IgG1 MAb or with convalescent-phase serum diminished but did not prevent establishment of infection by 100 CFU of B. abortus. A majority of culture-negative mice tested 6 weeks after infection were serologically negative, which could have signified either the absence of previous infection or the early elimination of infection. In an in vitro test system, all of the antibody preparations were efficient in opsonizing B. abortus. Effective killing of the organism by unelicited mouse peritoneal macrophages occurred in conventional but not in endotoxin-free medium, suggesting that activated macrophages were required for killing of opsonized B. abortus. These results emphasize the potential importance of antibodies in the immunoprophylaxis of brucellosis and suggest that the design of a successful vaccine will require the induction of antibodies not only of appropriate specificity but also of the optimal isotype for mediating protective functions.

Inter-relationship between immunoglobulin idiotype and metatype

Allogenic anti-metatype (Met) and anti-idiotype (Id) reagents were elicited to the liganded and nonliganded states, respectively, of a high affinity murine monoclonal anti-fluorescein IgM antibody. Through comparisons of the relative immunogenicity and specificity patterns of the resulting antibody reagents, interpretations regarding the relationship between metatopes and idiotopes were rendered. Anti-Id specificity was measured in terms of the degree of ligand inhibition to two different forms of the fluorescyl hapten (i.e. free ligand and conjugated to a macromolecule). Idiotypic analysis of 1823 H and L chains (immunoglobulin heavy and light chains) demonstrated that recognition of 1823 Id determinants required recombination of H and L chains. Anti-Met reagents were evaluated relative to the liganded and nonliganded states of the IgM antibody. Binding studies indicated anti-Met specificity for liganded or affinity labeled Mab (monoclonal antibody) 1823, but not for nonliganded 1823 or the fluorescein ligand. The affinity labeled metatyplc state provided the optimum immunogen yielding an antibody reagent which was rendered specific for the liganded state upon absorption with appropriate immunoglobulin reagents. Antibodies specific for affinity labeled 1823 did not react with liganded 4420, an IgG2a monoclonal anti-fluorescein antibody of similar high affinity but unrelated idiotyplcally. Results were discussed in terms of intrasite, proximal-site and distal-site epitopes.

A Monoclonal Antibody against the Ligand Binding Site of the Receptor for Mouse Interferon-γ

A rat monoclonal IgG2a antibody, GR-20, has been produced against the receptor for mouse interferon-gamma (MuIFN-gamma). Comparison of competitive binding studies performed with either 125I-labeled GR-20 or recombinant (r) MuIFN-gamma, as well as a variety of other studies, suggested that the epitope recognized by the monoclonal antibody (mAb) is in the domain of the receptor that binds ligand. The binding of GR-20 to cells of the monomyelocytic line WEHI-3 was of high affinity (1-2 nM). Approximately 20,000 binding sites were found per cell, a value that is in close agreement with the number of MuIFN-gamma receptors quantified on cells of the same type by ligand binding studies. The mAb also bound to a variety of other mouse cells, suggesting that the same epitope is shared by receptors for MuIFN-gamma, regardless of cell type. The epitope was not detected on two human cell types that were tested, while cells of a rat cell line shown to be minimally responsive to rMuIFN-gamma gave equivocal binding results when they were interacted with GR-20. Binding of the mAb to the receptor did not mimic the effects of ligand. In fact, the opposite was true: binding blocked the induction of three biological effects of MuIFN-gamma, including priming of macrophages for tumor cell killing, upregulation of the expression of class II major histocompatibility antigens (Ia) on the same cell type, and induction of antiviral activity in L cells. Following intravenous injection, initial removal of GR-20 was precipitous, followed after 1 h by a phase which was more gradual, resulting in 5-10% of biologically active mAb remaining in the circulation after 24 h. Such retention should make this mAb useful in a variety of studies in vivo.

Immunolocalisation and imaging of small cell cancer xenografts by the IgG2a monoclonal antibody SWA11 : Smith A, Waibel R, Westera G, Martin A, Zimmerman AT, Stahel RA. Division of Oncology, Department of Medicine, University Hospital, CH-8091 Zurich. Br J Cancer 1989;59:174-8

Immunolocalisation and imaging of small cell cancer xenografts by the IgG2a monoclonal antibody SWA11 : Smith A, Waibel R, Westera G, Martin A, Zimmerman AT, Stahel RA. Division of Oncology, Department of Medicine, University Hospital, CH-8091 Zurich. Br J Cancer 1989;59:174-8

Interaction of meningococcal group B monoclonal antibody and its Fab fragment with α2-8-linked sialic acid polymers: Requirement of a long oligosaccharide segment for binding

Mouse monoclonal IgG2a antibody (735D4) and other antibodies to the capsular polysaccharide of group B meningococci have been shown to require an unusually long segment of the α 2-8-linked N-acetylneuraminic acid polymer for binding. This property may be due to a conformational nature of the polysaccharide epitope recognized, or alternatively due to the requirement of bivalent binding of the antibody to the polysaccharide. In order to study the binding requirements. Fab fragments were prepared from the monoclonal antibody and their binding to α2-8-linked sialic acid polymers of different lengths was studied. Both the intact antibody and its Fab fragment bound to sialic acid poly- and oligomers to similar extents, the critical chain length being about 10 sialyl units for both molecules. This excluded bivaleney as the explanation for the requirement of a long oligosaccharide segment for binding. Although the binding was enhanced with increasing chain length, the first 10 monosaccharides were calculated to contribute to more than 90% of the total binding energy. This is in agreement with an oligosaccharide segment with defined conformational epitope binding to the antibody combining site. The antibody preparations also bound polysialic acid containing glycopeptides isolated from developing human and rat brain, suggesting, in quantitative binding assay, an average chain length of 10 or more sialic acid residues. The interaction of the antibody with both the bacterial and the tissue derived polysialic acids suggests that the conformational epitope critical for the interaction is formed by both classes of compounds.

Role of carbohydrate in binding of IgG to the Fc receptor of neonatal rat enterocytes

Monoclonal IgG1, IgG2a, IgG2b and IgG2c were prepared from rat hybridoma cells treated with tunicamycin in order to inhibit N-linked glycosylation. The IgG produced by these cells was about 70% lower in carbohydrate content compared to IgG from equivalent untreated cells, but was similar to the corresponding normal IgG in terms of antigen binding. However, the ability of carbohydrate deficient (CHO-) IgG to bind in vitro to Fc receptor extracted from jejunum of neonatal rats was impaired in most cases and, in all but one case, the amount of CHO- IgG transported from gut lumen to blood in vivo was markedly reduced. No reduction in binding of normal IgG to extracted receptor was observed in the presence of various sugars. It is postulated that N-linked carbohydrate acts to stabilize the structure within the IgG molecule which is responsible for binding to this Fc receptor, possibly in the CH2 domain.

Inhibition of metastases of a human melanoma xenograft by monoclonal antibody to the GD2/GD3 gangliosides.

A human melanoma variant cell line was obtained from a lung metastasis that arose spontaneously after we inoculated melanoma cells sc into a nude mouse. In this model, IgG2a monoclonal antibody (MAb) ME 36.1 defining the GD2/GD3 gangliosides inhibited melanoma growth at the primary site and metastatic spread of the cells, whereas an IgG1 variant of MAb ME 36.1 inhibited lung metastasis formation only. Possible mechanisms of antitumor effects of MAb ME 36.1 are discussed.

Trypanosoma musculi co-express several receptors binding rodent IgM, IgE, and IgG subclasses.

The present work demonstrates the expression of receptors for the Fc portion of rodent Ig by the murine parasite Trypanosoma musculi. By using a rosette assay adapted to the parasite morphology and by flow cytometry analysis, three distinct receptors were identified. A receptor binding rabbit or rat polyclonal IgG and mouse monoclonal IgG1, IgG2a, and IgG2b was found on parasites purified from the blood and the peritoneal cavity of infected mice and on parasites maintained in culture conditions. This IgG receptor was degraded by pepsin. A separate receptor, binding only mouse monoclonal IgG3 was observed on cultured parasites. A receptor binding rabbit, rat, and mouse IgM was found on cultured and peritoneal parasites, but not on blood parasites. This receptor did not bind IgG or IgA but it bound mouse and rat IgE as well as IgM. It was degraded by trypsin. IgG and IgM/IgE receptors were co-expressed on single parasites. They were not of host origin but synthesized by trypanosomes as shown by reexpression in vitro after proteolytic degradation. Their expression was variable with the development of trypanosomes both in vitro and in vivo.

Chemotactic properties of rat immunoglobulins and immune complexes.

The effect of rat immunoglobulins and immune complexes on the locomotor function of rat polymorphonuclear leukocytes (PMN) was investigated in vitro. Rat immunoglobulin G1 (IgG1), IgG2a, IgG2b, and IgA monoclonal antibodies specific for the dinitrophenyl hapten were used. Both monomeric and polymeric IgA showed chemotactic activity in a dose-dependent manner. IgG1 and IgG2b also induced a dose-dependent locomotor response of PMN, but the nature of the induced migration was chemokinetic (enhancing random migration). IgG2a was chemotactic and induced maximal migration at a relatively low concentration. IgG1- and IgG2b-immune complexes induced stronger migration than antibody alone; however, IgA- and IgG2a-immune complexes did not. IgA was shown to modify the chemotactic movement of PMN induced by N-formylmethionyl-leucyl-phenylalanine (FMLP). In the presence of both IgA and FMLP in the lower chamber, the migration towards suboptimal concentrations of FMLP was enhanced. By contrast, IgA in the upper chamber decreased migration towards the optimal or higher concentrations of FMLP. These findings suggest that IgA may work synergistically with luminal chemoattractants to mobilize PMN to the locus of infection on the mucosal surface. In addition, the intense activity of IgG2a alone and IgG1- or IgG2b-immune complexes in inducing PMN migration may play an important role in inflammatory processes. The data indicate that immunoglobulins have a direct effect on PMN mobility.

Factors influencing antibody-mediated cytotoxicity during the immunotherapy of Rauscher-virus-induced myeloid leukemic cells.

The present study was undertaken to determine the factors that influence antibody-mediated cytotoxicity during immunotherapy of virally transformed tumor cells. As model a Rauscher-virus-induced myeloid leukemic cell line of BALB/c origin (RMB-1) was used, which forms disseminated tumors, when inoculated intravenously in BALB/c mice. As previously reported, prolonged survival was obtained when tumor-bearing mice were treated in vivo with a single high dose of a tumor-specific IgG2a monoclonal antibody. This study shows that antibody-dependent cellular cytotoxicity is an important mechanism involved in tumor cell destruction. Since in vitro studies showed that peritoneal macrophages were capable of killing RMB-1 cells in the presence of tumor-specific monoclonal antibody and since in the tumors of mice treated with monoclonal antibody a high influx of macrophages was observed histologically, it is likely that macrophages play an important effector role in elimination of tumor cells. Successful therapy in C5-complement-deficient tumor-bearing mice suggests that complement-dependent cytotoxicity does not play a major role. In nude (T-cell-deficient) mice the therapeutic effect of tumor-specific IgG2a antibody was significantly less than in immunocompetent mice. Although infiltration analysis of tumors of treated and untreated mice showed equally low numbers of helper-T and suppressor/cytotoxic T-cells, the mortality studies of T-cell-deficient and immunocompetent mice indicate that T-cells play a substantial, auxiliary role during antibody-mediated, tumor destruction in our model.

Immunolocalisation and imaging of small cell cancer xenografts by the IgG2a monoclonal antibody SWA11

We describe here a murine monoclonal antibody of the IgG2a isotype which was generated against the SW2 human small cell carcinoma cell line. The antibody, SWA11, was shown to bind to a partially defined antigen preferentially expressed on cell lines of small cell carcinoma origin. In vitro binding studies revealed 6.1 x 10(5) antigenic sites on the SW2 cell line and the Ka to be 1.2 x 10(9)M-1. Following injection into mice bearing 1-2 cm3 SW2 xenografts, SWA11 showed strong selective accumulation in the small cell heterotransplants with a tumour to blood ratio of 7.5:1 at day 4. Other tumour to organ ratios were similarly high at 19:1, 22:1 and 12:1 for liver, kidney and spleen respectively. The absolute amount of SWA11 which localised was 10.5% of total injected material per gram tumour at day 2 and this level did not markedly decrease by day 4. The high ability of SWA11 to localise to SCC xenografts was confirmed by external gamma scintigraphy. The potential application of SWA11 as a model system for in vivo radioimmunotherapy is discussed.

Sensitive Detection of Two Igg Fc Receptors of Mouse Macrophages by Chemiluminescence Analysis

Luminol-enhanced chemiluminescence assay was used to detect the surface expression and the consequent activation of receptors (FcRI and FcRII) of murine macrophages (M phi s). When murine IgG2a was used for the specific detection of FcRI and IgG2b for FcRII, a newly established procedure enabled us to detect the activation of each receptor with as few as 3 X 10(5) M phi s. Briefly, TNP-SRBC coated with monoclonal IgG2a or IgG2b antibodies directed to TNP (sensitized SRBC) were used as reagent, in the presence of 1 X 10(-5) M luminol, and the emission was measured with a liquid scintillation counter. When results obtained by chemiluminescence counting were compared to the results obtained by the rosette formation by adding the same SRBC reagent to peritoneal M phi s obtained after ip injection of Listeria, fortified chemiluminescence counting allowed us to obtain a more definite answer about the activation of each receptor. Under the conditions established, the specific activation of FcRI was obtained by the addition of rIFN alpha A/D to the resident M phi s in vitro and the specific activation of spleen M phi FcRII by iv injection of IAP (Immunosuppressive acidic protein) into mice. These two results supported the independence of the two receptors detected by the assay.

Isolation and characterization of A V H domain fragment from monoclonal rat IgG of IgG1 and IgG2a subclasses

Digestion with trypsin of monoclonal rat IgG of IgG1 and IgG2a subclasses produced two fragments, isolated only in dissociating media. The larger fragment (mol. wt. 120,000 Da) was comprised of the two light chains covalently bound to shortened γ chains. Amino acid sequence of the shortened γ chain indicated that the site of cleavage is located at the beginning of the Cγ 1 domain at a position homologous to residue 139 of mouse γ heavy chain of IgG1 MOPC 21. The smaller fragment (mol. wt. 13,000 Da) was found to consist of the entire variable domain of the heavy chain and probably a short stretch of the Cγ 1 domain. The unique susceptibility of rat monoclonal IgG1 and IgG2a is likely to be the result of the presence of a lysine residue in a loop of Cγ 1 domain, which therefore is accessible to trypsin. Tryptic cleavage of rat monoclonal antibodies of IgG1 and IgG2a subclasses can be considered as a simple method to produce a fragment related to the V H domain.

Site-specific radiolabeling of monoclonal antibodies with biotin/streptavidin

Sulfhydryl groups of a murine IgG2a monoclonal antibody, 5G6.4, were site-specifically biotinylated with N-iodo-acetyl- N′-biotinylhexylenediamine (compound 1) following partial reduction of disulfide bonds. Antibody labeling using fluoresceinated or [ 125I]streptavidin were consistent with sulfhydryl group-specific incorporation of label with good retention of immunoreactivity, mainly in the form of high molecular weight oligomers (molecular weight ≈ > 400,000). In vivo localization of the radioantibody complex to xenografts of ovarian carcinoma was achieved. This approach appears to be a promising method of labeling monoclonal antibodies.

Release of IgD-binding factor by T cells under the influence of interleukin 2, interleukin 4, or cross-linked IgD.

Helper T cells with receptors specific for IgD have immunoaugmenting properties. We have now detected soluble IgD-binding factor in cell supernatants immobilized on nitrocellulose paper by their ability to bind 125I-labeled IgD. IgD-binding factor is released by normal splenic T cells stimulated with recombinant interleukin 2, recombinant interleukin 4, or crosslinked IgD in amounts paralleling the induction of IgD receptors on the cells. IgD receptors are constitutively produced by antigen-specific helper T-cell hybridomas 2H10 and A3.4C6. Incubation of these hybridoma cells with recombinant interleukin 2 increases release of IgD-binding factor while reducing expression of IgD receptors. Specificity of the binding factor for IgD is established by (i) competitive inhibition; (ii) the ability of the binding factor to bind radiolabeled IgD and not monoclonal IgE, IgG2a, or polyclonal IgG; and (iii) the removal of the binding factor on passage through an IgD-Sepharose column and recovery in a subsequent acid eluate.

Identification of platelet membrane thrombospondin binding molecules using an anti-thrombospondin antibody

A rat monoclonal IgG 2a antibody, 5G11, was raised against native human platelet thrombospondin (TSP). Western blot analysis revealed that 5G11 bound (i) to TSP before and after disulfide reduction, and (ii) to a 15-kDa fragment released after prolonged trypsin digestion. Crossed immunoelectrophoresis confirmed that the binding epitope was expressed in the presence of Ca 2+ and after treatment of TSP with EDTA. Since 5G11 had no effect on platelet aggregation, the antibody was used to immunoprecipitate Ca 2+-dependent and Ca 2+-independent TSP-binding molecules on the surface of thrombin-activated surface-labeled 125I-platelets. The experimental basis was that ligand-receptor interactions are of high affinity and that anti-ligand antibodies should precipitate the ligand-receptor complex. With platelets activated in the presence of EDTA, 5G11 predominantly precipitated a 125I-labeled band of M r 88 000, identified as glycoprotein (GP) IV. In contrast, in the presence of 2 mM Ca 2+ and 1 mM Mg 2+, 5G11 precipitated a complex of five radiolabeled proteins, among which GPIIb, GPIIIa and GPIV were the most prominent.

Anti-IL-2-R monoclonal antibody in allograft recipients

A rat IgG2a monoclonal antibody (33B3.1) directed at the human Tac antigen and interacting with the high affinity binding site of IL-2 on its receptor was tested for treatment of kidney allograft recipients. This antibody is efficient in preventing graft rejection in the weeks following transplantation. Its activity compares to polyclonal rabbit anti-thymocyte globulin but it has fewer side effects. The dose of 33B3.1 given is critical for its preventive effect. A majority of the recipients developed IgM and/or IgG against rat Ig. Rejection episodes occurring during 33B3.1 therapy were associated with an early rise in host anti-33B3.1 antibodies and a drop in their monoclonal antibody (MoAb) circulating levels. When 33B3.1 was given to treat ongoing rejection, it had only an inconsistent and/or delayed effect. Various factors are discussed, such as circulating free 33B3.1 levels, host anti-33B3.1 immune response and the presence of inhibiting concentrations of soluble Tac chain, which can interplay with the protective effect of the anti-interleukin-2-receptor monoclonal antibody. The use of bioreagents which interfere only with determinants present on host activated lymphocytes may represent a new step toward selective immunosuppression in allograft recipients.

Inhibition of differentiation and affinity purification with a monoclonal antibody to a myeloid cell differentiation-inducing protein

The normal myeloid hematopoietic regulatory proteins include four growth-inducing proteins called colony-stimulating factors (CSF), including interleukin-3 (IL-3), or macrophage and granulocyte inducers, type 1 (MGI-1), and another type of protein (MGI-2) with no myeloid cell growth-inducing activity that induces differentiation of normal myeloid precursor cells and certain clones of myeloid leukemic cells. An IgG2a monoclonal antibody was prepared and it neutralized two forms of MGI-2 (MGI-2A and MGI-2B) produced by mouse Krebs ascites tumor cells. The monoclonal antibody was used for affinity purification of MGI-2. This antibody also neutralized MGI-2 produced by normal mouse macrophages, normal myeloblasts incubated with IL-3, and MGI-2 produced by the lungs and found in the serum of mice injected with lipopolysaccharide (LPS). The anti-MGI-2 antibody did not inhibit the activity of any one of the four myeloid growth-inducing proteins (CSF or IL-3 = MGI-1), IL-1, tumor necrosis factor, or lymphotoxin. This antibody also inhibited induction of differentiation of myeloid leukemic cells by LPS, which is mediated by the endogenous production of MGI-2, but did not inhibit induction of differentiation in these leukemic cells by dexamethasone or cytosine arabinoside, which is not mediated by MGI-2. Anti-MGI-2 antibody thus inhibited differentiation when MGI-2 was added externally to cells or when it was mediated by endogenously produced MGI-2.

Inhibition of Differentiation and Affinity Purification With a Monoclonal Antibody to a Myeloid Cell Differentiation-Inducing Protein

The normal myeloid hematopoietic regulatory proteins include four growth-inducing proteins called colony-stimulating factors (CSF), including interleukin-3 (IL-3), or macrophage and granulocyte inducers, type 1 (MGI-1), and another type of protein (MGI-2) with no myeloid cell growth-inducing activity that induces differentiation of normal myeloid precursor cells and certain clones of myeloid leukemic cells. An IgG2a monoclonal antibody was prepared and it neutralized two forms of MGI-2 (MGI-2A and MGI-2B) produced by mouse Krebs ascites tumor cells. The monoclonal antibody was used for affinity purification of MGI-2. This antibody also neutralized MGI-2 produced by normal mouse macrophages, normal myeloblasts incubated with IL-3, and MGI-2 produced by the lungs and found in the serum of mice injected with lipopolysaccharide (LPS). The anti-MGI-2 antibody did not inhibit the activity of any one of the four myeloid growth-inducing proteins (CSF or IL-3 = MGI-1), IL-1, tumor necrosis factor, or lymphotoxin. This antibody also inhibited induction of differentiation of myeloid leukemic cells by LPS, which is mediated by the endogenous production of MGI-2, but did not inhibit induction of differentiation in these leukemic cells by dexamethasone or cytosine arabinoside, which is not mediated by MGI-2. Anti-MGI-2 antibody thus inhibited differentiation when MGI-2 was added externally to cells or when it was mediated by endogenously produced MGI-2.