Induction of an anti-human type II collagen response by monoclonal anti-idiotypic antibody

Several distinct epitopes on human type II collagen were defined by using mAb. The presence of species-specific and species-nonspecific (common) epitopes was thus clarified. Anti-idiotypic mAb (Ab2) was developed against one of the antibodies (Ab1) reactive with species-specific epitopes. Thus Ab2 was demonstrated to recognize an idiotope expressed on the Ag-binding site (paratope) of Ab1, since the binding of Ab1 to human type II collagen was blocked by Ab2, and the binding of Ab2 to Ab1 was inhibited by soluble human type II collagen, but not by murine and bovine type II collagens. DBA/1 mice immunized with Ab2 coupled to keyhole limpet hemocyanin produced an antibody (Ab3) specifically reactive with human type II collagen. It was also demonstrated that Ab3 expressed an idiotype similar to that of Ab1. These findings indicate that anti-idiotypic antibody directed against mAb to human type II collagen mimics a species-specific epitope on human type II collagen. The anti-idiotypic antibody bearing internal image of type II collagen will open the way to isolation of the arthritogenic epitope on type II collagen.     

A monoclonal anti-idiotypic antibody against a human monoclonal IgM with specificity for myelin-associated glycoprotein

A human monoclonal IgM lambda antibody, directed against MAG, obtained from a patient with polyneuropathy associated with a gammopathy, was used as an immunogen to generate mouse monoclonal anti-idiotype antibodies. One hybridoma antibody, designated A8F2, reacts uniquely with the M-IgM of the patient, shows high affinity binding to the patient's M-IgM, and dose-dependently inhibits binding of the patient's M-IgM to its specific antigen MAG. Thus, A8F2 is a monoclonal anti-idiotype antibody that recognizes a region of the MAG binding site of the patient's IgM. Use of this anti-idiotype antibody in a competition RIA revealed the presence of naturally occurring anti-idiotype in the patient's serum. Because anti-idiotype antibodies may be part of a mechanism for down-regulation of antibody production, the use of A8F2 to induce a specific immunosuppression should be considered.     

A plant-derived human monoclonal antibody induces an anti-carbohydrate immune response in rabbits

A common argument against using plants as a production system for therapeutic proteins is their inability to perform authentic N-glycosylation. A major concern is the presence of beta 1,2-xylose and core alpha 1,3-fucose residues on complex N-glycans as these nonmammalian N-glycan residues may provoke unwanted side effects in humans. In this study we have investigated the potential antigenicity of plant-type N-glycans attached to a human monoclonal antibody (2G12). Using glyco-engineered plant lines as expression hosts, four 2G12 glycoforms differing in the presence/absence of beta 1,2-xylose and core alpha 1,3-fucose were generated. Systemic immunization of rabbits with a xylose and fucose carrying 2G12 glycoform resulted in a humoral immune response to both N-glycan epitopes. Furthermore, IgE immunoblotting with sera derived from allergic patients revealed binding to plant-produced 2G12 carrying core alpha 1,3 fucosylated N-glycan structures. Our results provide evidence for the adverse potential of nonmammalian N-glycan modifications present on monoclonal antibodies produced in plants. This emphasizes the need for the use of glyco-engineered plants lacking any potentially antigenic N-glycan structures for the production of plant-derived recombinant proteins intended for parenteral human application.     

Sieve element specific labeling with an anti-idiotypic monoclonal antibody mimic of the phytotoxin dothistromin

A monoclonal antibody, 12C9, an anti-idiotypic mimic of dothistromin, a toxin produced by Dothistroma pini, was found to label the cell wall of sieve elements in a number of different plant tissues and species. The antibody labeled apple leaf tissue, tobacco leaf mid vein, leaf and meristem, and Coprosma robusta leaf mid vein. Labeling was restricted to cell walls of sieve elements and did not label the companion cells or the lumen of the cells. The antibody labeled over a wide range of dilutions. This antibody could be used to differentiate sieve elements from other types of phloem. It could also be used to co-localize sieve elements and microorganisms such as phytoplasmas stained with DAPI.     

Sieve element specific labeling with an anti-idiotypic monoclonal antibody mimic of the phytotoxin dothistromin

A monoclonal antibody, 12C9, an anti-idiotypic mimic of dothistromin, a toxin produced by Dothistroma pini, was found to label the cell wall of sieve elements in a number of different plant tissues and species. The antibody labeled apple leaf tissue, tobacco leaf mid vein, leaf and meristem, and Coprosma robusta leaf mid vein. Labeling was restricted to cell walls of sieve elements and did not label the companion cells or the lumen of the cells. The antibody labeled over a wide range of dilutions. This antibody could be used to differentiate sieve elements from other types of phloem. It could also be used to co-localize sieve elements and microorganisms such as phytoplasmas stained with DAPI.     

The idiotypic characterization of the immune response to a defined epitope of a protein antigen and the specific in vivo suppression of the immune response to this epitope by anti-idiotypic antibodies

C10, a monoclonal antibody of C3H.SW (CSW) origin, binds a decapeptide epitope of the tobacco mosaic virus protein (TMVP) representing residues 103-112 of the protein. In vivo administration of syngeneic anti-idiotypic antibodies to C10 (anti-C10) prior to immunization with TMVP suppressed the expression of antibodies to this decapeptide determinant in CSW mice without a significant reduction of the total anti-TMVP titer. The suppression could not be overcome with repeated challenges by antigen even 6 months after administration of anti-C10. Analysis of anti-C10 showed that it contains antibodies to at least two idiotopes found on C10. One of these idiotopes, C10-Idm, is found on a very small fraction of CSW anti-TMVP antibodies capable of binding the decapeptide epitope. The other idiotope, C10-IdX, is found on most of the anti-TMVP antibodies which bind the decapeptide determinant. With synthetic analogues of the decapeptide determinant, a correlation was established between the presence of the C10-IdX and the fine specificity of the decapeptide-binding antibodies. The studies reported herein demonstrate that anti-idiotypic antibodies are potent modulators of the immune response and that the C10-IdX is important in the determination of the fine specificity of antibodies to this decapeptide epitope of TMVP.     

A monoclonal antibody specific for a monocyte-macrophage membrane component blocks the human monocyte response to migration inhibitory factor

In our study of the human monocyte response to migration inhibitory factor (MIF), we screened murine anti-monocyte monoclonal antibodies (MAb) for their ability to affect the MIF response. When blood monocytes were preincubated for 30 min at 37 degrees C with MAb of various cell specificities, it was found that Mo3e-MAb could inhibit the monocyte response to MIF. This effect was observed at an antibody concentration of 0.37 microgram/10(6) monocytes. This blocking activity appeared to be specific for Mo3e-MAb, since MAb specific for Mo1, Mo2, Mo4, alveolar macrophage (PAM-1), and platelet (Plt-1) antigens were unable to block the MIF response. Specificity was also shown by experiments performed with MAb similar to Mo3e-MAb in their immunofluorescence staining patterns. Two MAb of this type were not able to block the MIF response. All of these MAb are of the IgM isotype, and therefore it is unlikely that the blocking activity of Mo3e-MAb is mediated by the Fc receptor. These studies suggest that Mo3e-MAb may represent an anti-MIF receptor antibody. Mo3e-MAb recognizes a protease-sensitive antigen of 80 kD and 50 kD that is weakly expressed on fresh blood monocytes but is up-modulated by stimulation of monocytes with bacterial lipopolysaccharide, muramyl dipeptide, or phorbol compounds. Mo3e is also expressed by human myeloid cell lines, U-937 and HL-60, that have been stimulated in culture by biologically active phorbol diester compounds.     

A human and a mouse anti-idiotypic antibody specific for human T14(+) anti-DNA antibodies reconstructed by phage display

Little is known about human anti-idiotypic antibodies. Phage display methodology was used to reconstruct these antibodies from lupus patients, which recognize a subset (T14(+)) of anti-DNA antibodies. Antigen-specific B cells were isolated from the blood using a peptide based on a complementarity determining region (V(H)CDR3) of the prototypic T14(+) antibody. cDNA fragments of the V(H) and V(L) genes prepared from the cells were expressed as phage displayed single chain Fv (scFv) fragments using the pCANTAB-5E phagemid vector. From a reactive clone obtained, the Ig genes used were identified to be V(H)3, D5-D3, J(H)4b, V(kappa)I and J(kappa)2. The heavy chain was highly mutated, especially in CDR3, which bears mutations mostly of the replacement type; this region is also unusual in being extremely long due to a D-D fusion. In contrast, a mouse hybridoma antibody, made to the same T14(+) peptide and transformed as a scFv fragment, uses a short V(H)CDR3 comprising five amino acids, three of which are tyrosines. Tyrosines may be important for antigen binding because two of these also exist in the human V(H)CDR3. The light chains of both antibodies may also contribute to the specificity of the protein, because their V(L) segments, including the CDRs, are highly homologous to each other.     

Induction of an immune response through the idiotypic network with monoclonal anti-idiotype antibodies in the carcinoembryonic antigen system.

Anti-idiotype antibodies can mimic the conformational epitopes of the original antigen and act as antigen substitutes for vaccination and/or serological purposes. To investigate this possibility concerning the tumor marker carcinoembryonic antigen (CEA), BALB/c mice were immunized with the previously described anti-CEA monoclonal antibody (MAb) 5.D11 (AB1). After cell fusion, 15 stable cloned cell lines secreting anti-Ids (AB2) were obtained. Selected MAbs gave various degrees of inhibition (up to 100%) of the binding of 125I-labeled CEA to MAb 5.D11. Absence of reactivity of anti-Id MAbs with normal mouse IgG was first demonstrated by the fact that anti-Id MAbs were not absorbed by passage through a mouse IgG column, and second because they bound specifically to non-reduced MAb 5.D11 on Western blots. Anti-5.D11 MAbs did not inhibit binding to CEA of MAb 10.B9, another anti-CEA antibody obtained in the same fusion as 5.D11, or that of several anti-CEA MAbs reported in an international workshop, with the exception of two other anti-CEA MAbs, both directed against the GOLD IV epitope. When applied to an Id-anti-Id competitive radioimmunoassay, a sensitivity of 2 ng/ml of CEA was obtained, which is sufficient for monitoring circulating CEA in carcinoma patients. To verify that the anti-Id MAbs have the potential to be used as CEA vaccines, syngeneic BALB/c mice were immunized with these MAbs (AB2). Sera from immunized mice were demonstrated to contain AB3 antibodies recognizing the original antigen, CEA, both in enzyme immunoassay and by immunoperoxidase staining of human colon carcinoma. These results open the perspective of vaccination against colorectal carcinoma through the use of anti-idiotype antibodies as antigen substitutes.     

Sequence uniqueness and sequence variability as modulating factors of human anti-HCV humoral immune response

We recently compared the HCV polyprotein to the human proteome in order to test whether amino acid sequences unique to the virus could represent immunodominant epitopic determinants of the human humoral immune response against HCV. We identified a relatively limited number of HCV fragments with no/low similarity to the human host that represented exclusive HCV motifs. In this study, the peptides corresponding to low/zero similarity sequences were synthesized and assayed with HCV-infected sera. With different patterns, the synthetic HCV peptides corresponding to low/zero similarity sequences were found to be immunoreactive. In particular, the HCV E1 (315-323) HRMAWDMMM, HCV E2/NS1 (547-555) NWFGCTWMN, and HCV NS5 (2638-2646) YDTRCFDST sequences were immunodominant in the HCV-infected cohort under study. These three peptides correspond to sequences that are endowed with low-similarity to the human proteome, are highly conserved among various HCV strains, and have, potentially, a scarce susceptibility to proteolytic attacks. These data may be of help in defining the multiple factors which concur in the modulation of the human immune response against HCV, eventually providing information for the design of effective anti-HCV vaccines.     

Restriction of the human in vivo immune response against the mouse monoclonal antibody OKT3

The murine monoclonal antibody OKT3 (IgG2a) was administered prophylactically to 17 renal allograft recipients (5 mg/day, i.v.), either alone or in association with corticosteroids (0.25 mg/kg/day) and azathioprine (3 mg/kg/day). In all patients the kinetics of the IgM and IgG anti-OKT3 response was monitored by means of immunofluorescence and ELISA. All patients treated with OKT3 alone showed a rapid and strong sensitization that completely neutralized the therapeutic effectiveness of the monoclonal antibody. The anti-OKT3 sensitization was manifested by accelerated OKT3 clearance and abrupt reappearance of circulating OKT3+ cells before the end of treatment. This immune response was significantly delayed and reduced in its incidence and intensity in patients receiving low dose corticosteroids and azathioprine in association to OKT3; mainly IgM anti-OKT3 antibodies that did not accelerate OKT3 clearance were then observed. The fine specificity of the antibodies produced was studied, using patients whole sera and various mouse IgG2a-affinity chromatography-purified serum fractions. The results obtained showed that the anti-OKT3 response was remarkably restricted to two main categories of antibodies: a) anti-idiotypic antibodies that inhibited OKT3 binding to T cells and abrogated its therapeutic activity and b) anti-mouse IgG2a (anti-isotypic) antibodies that did not neutralize OKT3 immunosuppressive activity. These results suggest that OKT3-immunized patients might still be sensitive to the immunosuppressive effect of other anti-T cell monoclonals that do not share the OKT3 idiotype and possibly isotype.     

Induction of a neutralizing immune response to human respiratory syncytial virus with anti-idiotypic antibodies.

Anti-idiotypic (anti-Id) antibodies were raised in rabbits against monoclonal antibodies that recognized either F glycoprotein 47F or G glycoprotein 63G, 62G, or 74G of the human respiratory syncytial virus Long strain. Anti-Id sera inhibited the virus binding of the immunizing monoclonal antibodies and in some cases the binding of other antibodies reacting with overlapping epitopes. The anti-Id sera also inhibited virus neutralization mediated by the original monoclonal antibodies. Affinity purified anti-Id antibodies were subsequently used to raise a homologous anti-anti-Id response in rabbits. One of the rabbits, inoculated with anti-Id 63G, generated antibodies that reacted with the G protein of respiratory syncytial virus and neutralized the virus to high titers. The antiviral antibodies induced by anti-Id 63G were broadly cross-reactive with strains of the A and B subtypes. However, the specificities of monoclonal antibody 63G and anti-anti-Id 63G were not exactly the same, as indicated by their reaction with escape mutants to antibody 63G. These results demonstrate for the first time the induction of an anti-respiratory syncytial virus response by anti-Id antibodies.     

Production of a monoclonal antibody specific for Salmonella spp.

A monoclonal antibody (MAb) specific for heat-treated Salmonella spp. which shows no cross-reactivity with other enteric bacteria when tested by an ELISA system, has been developed. The MAb isotype is IgG2a and has been successfully purified using protein A. Preliminary work suggests that the MAb recognizes a protein present in the Boivin lipopolysaccharide extracts of Salmonella spp.     

Stimulation of human B cells specific for Candida albicans for monoclonal antibody production

Abstract The stimulation and immortalisation of human peripheral blood B lymphocytes specific for Candida albicans antigen were investigated. An in vitro immunisation system was employed which involved pretreatment of mononuclear cells with l -leucyl l -leucine methyl ester which removes the suppressive effects of CD8 + T cells, NK cells and monocytes. The remaining cells, CD4 + T cells, B cells and dendritic cells, were cultured with antigen and a mixture of cytokines. A mixture of IL-2, −4 and −6 was found to be optimal for antibody production as determined by an Elispot assay. Transformation of the activated B cells by Epstein Barr virus was found to be optimal after 2 days and lines secreting anti- Candida antibodies were established. These lines could form the basis for specific monoclonal antibody production by generating hybridomas, or by a newly described technique whereby cDNA encoding antibody Fab regions is transferred into phage display libraries. The overall strategy might be generally applicable for the generation of human monoclonal antibodies to infectious agents.     

Characterization of a mouse monoclonal antibody specific for O-linked N-acetylglucosamine

beta-O-linked N-acetylglucosamine (O-GlcNAc) is an abundant posttranslational modification of resident nuclear and cytoplasmic proteins in eukaryotes. Increasing evidence suggests that O-GlcNAc plays a regulatory role in numerous cellular processes. Here we report on the production and characterization of a highly specific mouse monoclonal antibody, MAb CTD110.6, that specifically reacts with O-GlcNAc. The antibody recognizes O-GlcNAc in beta-O-glycosidic linkage to both serine and threonine. We could detect no cross-reactivity with alpha-linked Ser/Thr-O-GlcNAc, alpha-linked Ser-O-linked N-acetylgalactosamine (O-GalNAc), or N-linked oligosaccharides on ovalbumin and immunoglobulin G. The monosaccharide GlcNAc, but not GalNAc, abolishes immunoreactivity, further demonstrating specificity toward O-GlcNAc. Furthermore, galactose capping of O-GlcNAc sites also inhibits CTD110.6 immunoreactivity. Enrichment of GlcNAc-containing glycoproteins using the lectin wheat germ agglutinin dramatically enriches for CTD110.6-reactive proteins. The antibody reacts with a large number of proteins from cytoplasmic and nuclear extracts and readily detects in vivo changes in O-GlcNAc modification. These studies demonstrate that CTD110.6 is highly specific toward O-GlcNAc, with no cross-reactivity toward similar carbohydrate antigens or toward peptide determinants.     

Neutralization of botulinum neurotoxin by a human monoclonal antibody specific for the catalytic light chain

Background

Botulinum neurotoxins (BoNT) are a family of category A select bioterror agents and the most potent biological toxins known. Cloned antibody therapeutics hold considerable promise as BoNT therapeutics, but the therapeutic utility of antibodies that bind the BoNT light chain domain (LC), a metalloprotease that functions in the cytosol of cholinergic neurons, has not been thoroughly explored.

Methods and Findings

We used an optimized hybridoma method to clone a fully human antibody specific for the LC of serotype A BoNT (BoNT/A). The 4LCA antibody demonstrated potent in vivo neutralization when administered alone and collaborated with an antibody specific for the HC. In Neuro-2a neuroblastoma cells, the 4LCA antibody prevented the cleavage of the BoNT/A proteolytic target, SNAP-25. Unlike an antibody specific for the HC, the 4LCA antibody did not block entry of BoNT/A into cultured cells. Instead, it was taken up into synaptic vesicles along with BoNT/A. The 4LCA antibody also directly inhibited BoNT/A catalytic activity in vitro.

Conclusions

An antibody specific for the BoNT/A LC can potently inhibit BoNT/A in vivo and in vitro, using mechanisms not previously associated with BoNT-neutralizing antibodies. Antibodies specific for BoNT LC may be valuable components of an antibody antidote for BoNT exposure.     

Equilibrium binding parameters of an autoimmune monoclonal antibody specific for double-stranded DNA

Two techniques have been developed to estimate binding parameters for Jel 241 under equilibrium conditions. Jel 241 is an autoimmune monoclonal antibody derived from an NZB/NZW mouse which binds to double-stranded DNA. Thermal denaturation profiles of poly[d(AT)] were measured in the presence of increasing concentrations of IgG Jel 241. From these data it was estimated that the IgG occludes 12 base-pairs on duplex DNA, and the binding to double-stranded DNA was at least four orders of magnitude greater than to single-stranded DNA. In addition, intrinsic association constants (K(O)) were measured by a gel filtration technique for the interaction of both Fab and IgG Jel 241 to native calf thymus DNA. K(O) for the IgG was only 60-fold greater than for the Fab fragment for which K(O) was 4.4 X 10(4) M-1 at an NaCl concentration of 150 mM. Also, K(O) for the Fab increased dramatically with decreasing ionic strength, suggesting that there are four phosphates involved in the interaction. These techniques should be applicable to most autoimmune antibodies which bind to nucleic acid polymers.     

Biodistribution study of a monoclonal antibody specific to link proteins from human cartilage

We report the use of monoclonal antibody 6F3 prepared against link proteins from human articular cartilage to elucidate the distribution of these glycoproteins within connective and other tissues. By immunohistochemical analysis, we showed that only the Fab fragment could reach the antigenic site in human articular sections. Cross-reactivity of the antibody 6F3 with link proteins purified from rat articular cartilage allowed us to carry out a biodistribution analysis in vivo in the rat. The time course of whole blood and plasma showed maximal activity 6h after the 20 μg i.v. injection of [¹²⁵I]Fab-6F3. Urinary excretion seems to be a high route of elimination. Moreover, we noticed no radioactive uptake across the blood-brain barrier. A significant fixation of labeled antibody Fab-6F3 was observed in noncartilaginous connective tissues such as aorta, skin and lung. As expected, specific and increased radioactivity was observed in all cartilage tissues, this increase was significantly higher 6 h after the [¹²⁵I]Fab-6F3 injection than in the other connective tissues.