Fab fragments of monoclonal antibodies protect the human acetylcholine receptor against antigenic modulation caused by myasthenic sera

The human cell line TE671 produces large amounts of muscle nicotinic acetylcholine receptor (AChR). TE671 cells were used to determine the specificity of antibodies which can increase the internalization rate of AChR (antigenic modulation) and to test procedures for protecting AChR against this mechanism. The half-life of AChR both in the absence and the presence of anti-AChR antibodies was very similar to that of AChR on human muscle cell cultures. The relative contribution of different anti-AChR antibody fractions to the total antigenic modulation capacity of human myasthenic sera was investigated by competition experiments between Fab fragments of anti-AChR monoclonal antibodies (MoAbs) and intact antibodies (MoAb or myasthenic sera). Fab fragments, which do not induce antigenic modulation, were allowed to shield the corresponding regions of the AChR. Intact antibodies were subsequently added. It was found that protection of the main immunogenic region (MIR), but not of a region on the beta-subunit, essentially blocked the modulatory effect of the intact anti-MIR MoAbs, and approximately 80% of that of myasthenic sera. These data suggest that anti-MIR antibodies are mainly responsible for the loss of human AChR via antigenic modulation. Furthermore the observation that Fab fragments of anti-MIR MoAbs can efficiently protect AChR against antigenic modulation may have therapeutic implications.     

Tissue distribution and serum kinetics of T101 monoclonal antibody during passive anti-cancer therapy

We have administered fifty-six 24 hr infusions of the anti-human T-cell monoclonal antibody T101 to 10 patients with cutaneous T-cell lymphoma (CTCL) and 6 patients with chronic lymphocytic leukemia (CLL) in doses of 10, 50, 100, 150, and 500 mg. The larger doses of T101 resulted in higher, more persistent serum T101 concentrations, and CTCL patients generally developed higher serum T101 levels than CLL patients given equivalent doses. The presence of host anti-mIgG antibodies prior to infusion was associated with decreased serum concentrations of T101. Treatments that demonstrated measurable serum T101 levels were also associated with in vivo T101 binding and cytodestruction of circulating target cells. Immunofluorescence analysis of bone marrow and lymph node biopsies in CLL, and skin biopsies in CTCL, suggested that T101 had reached extravascular tumor sites. Infusion of 111In-conjugated T101 showed uptake in the liver, spleen, lymph nodes, and (in CTCL) skin infiltrates. Our data demonstrate the tissue distribution of T101 and suggest that immunoconjugates of T101 with toxins, drugs, or radioisotopes may result in better therapeutic responses.     

Antigenic modulation of lymphocytic surface immunoglobulin yielding resistance to complement-mediated lysis. II. Relationship to redistribution of the antigen.

Experiments were carried out on guinea-pig L2C leukaemic lymphocytes to investigate the mechanism of antigenic modulation of their surface immunoglobulin (Ig) defined as the conferring by anti-Ig of resistance to lysis by anti-Ig plus complement. The phenomenon reflects, and is probably a consequence of, redistribution of the Ig molecules by bivalent antibody. Fab fragments of the antibody were completely ineffective. Parallel studies by indirect immunofluorescence of the movement of th surface antigen-antibody complexes revealed that modulation for syngeneic complement was apparent when the complexes were minimally aggregated: capping and extensive endocytosis were not necessary. Modulation for xenogeneic (rabbit) complement required more extensive movement but was still appreciable while complexes persisted on the surface. Sodium azide at 10 mM, which inhibits antibody-induced redistribution of surface molecules, diminished modulation. In experiments omitting pre-incubation with antibody alone, the presence of azide during incubations with anti-Ig plus syngeneic complement increased lysis from a low and variable to a consistently high level; there was no effect on the already high level of lysis occurring with the non-modulating anti-Ia plus syngeneic complement. This effect of azide provides further evidence that antigenic modulation can be a major factor determining a cell's survival when it is confronted simultaneously by antibody and complement.     

Human in vivo antigenic modulation induced by the anti-T cell OKT3 monoclonal antibody

The anti-pan T cell monoclonal antibody OKT3 was administered daily for 2 weeks in four human renal allograft recipients. The antibody induced a dramatic and immediate depletion of peripheral T cells followed by an in vivo antigenic modulation of the OKT3-defined membrane antigen: after three injections, OKT3-treated patients showed a limited but significant number of OKT3- cells of T cell nature (as defined by OKT4 and OKT8) which recovered the OKT3 receptor after an overnight in vitro incubation in the absence of the monoclonal antibody.     

A polyspecific monoclonal anti-DNA autoantibody also binds to cell-surface protein(s)

A monoclonal anti-DNA autoantibody (EM85) produced in an autoimmune MRL/lpr/lpr mouse was studied. Its antigenic specificity was demonstrated to be directed against single-stranded DNA, double-stranded DNA, and also a variety of polynucleotides. We have recently reported that a murine monoclonal anti-DNA autoantibody produced in autoimmune B/W mice, with specificity for double-stranded DNA, also binds to cell-surface protein(s). We show here that EM85, which recognizes a variety of polynucleotides, also binds to protein(s) on the surface of Raji cells. These data indicate that the antigenic determinant, recognized by this monoclonal anti-DNA antibody, is common to a variety of polynucleotides and cell-surface protein(s).     

Treatment of a low grade T cell proliferation with monoclonal antibody

A therapeutic trial of a pan-T monoclonal antibody is described in a patient with a low grade mature T cell proliferation and haemopoietic suppression. The study shows that non-complement fixing IgG1 antibodies can be highly effective opsonins, and the dose required can be estimated from in vitro studies. It is demonstrated that therapeutic failure occurred due to the appearance of anti-mouse antibodies and not to antigenic modulation or reticuloendothelial blockade. The literature is reviewed and the potential role of serotherapy in malignant disorders is discussed.     

Triggering CD101 molecule on human cutaneous dendritic cells inhibits T cell proliferation via IL-10 production

Since the CD101 molecule is expressed on a major subpopulation of HLA-DR(+), CD1a(+), CD1c(+) cutaneous dendritic cells (DC), we studied the functional role of CD101 on cutaneous DC. Anti-CD101 monoclonal antibody (mAb) inhibited the proliferation of T cells induced by cutaneous DC. There was a synergistic inhibition between anti-CD101 mAb and anti-CD86/anti-CD80 mAb. Anti-CD101 mAb exerted its inhibitory effect when binding to the CD101 expressed on cutaneous DC. No positive role of CD101 putative ligand expressed by T cells in T cell proliferation was demonstrated, as T cells proliferated in response to soluble anti-CD3 mAb in the presence of CD86-transfected cells but not in the presence of CD101-transfected cells. Of major significance is the fact that IL-10 was produced by cutaneous DC after CD101 triggering with anti-CD101 mAb, while IL-10 secretion was up-regulated in mixed cutaneous DC-T cell cultures after CD101 triggering. Furthermore, IL-10-neutralizing mAb could reverse the inhibition induced by anti-CD101 mAb. Our results demonstrate that the CD101 triggering on cutaneous DC inhibits T cell proliferation via IL-10 production, suggesting an important regulatory role played by the CD101 molecule on DC during T cell activation.     

CD44 is not directly involved in the binding of lymphocytes to cultured high endothelial cells from peripheral lymph nodes.

The Ager assay was adapted to a porcine lymphocyte-rat high endothelial cell (HEC) system. Using this in vitro assay, the role of porcine CD44 in lymphocyte binding to HEC was examined. The results show that the presence of soluble CD44 molecules did not inhibit the binding of porcine lymphocytes to the cultured rat HEC. Treatment of lymphocytes with anti-CD44 monoclonal antibodies (mAb), or with papain, which removes a 45,000 MW peptide from the intact CD44 molecule, did not inhibit the binding. Binding to the rat HEC did not induce modulation of CD44 molecules on the cell surface. Furthermore, modulation of the CD44 molecule by biotinylated anti-CD44 antibody followed by streptavidin-phycoerythrin, which had caused the molecule to cap on the cell surface, did not prevent the cells binding to the HEC. Similarly, cells denuded of CD44 by anti-CD44 antibody retained the capacity to bind to HEC. Moreover, the binding cells were mainly those which had been stripped of CD44 by the antigenic modulation. It is concluded that CD44 is not directly involved in the binding of lymphocytes to the cultured HEC from peripheral lymph nodes (PLN).     

Activation of human T cells by toxic shock syndrome toxin-1: the toxin-binding structures expressed on human lymphoid cells acting as accessory cells are HLA class II molecules

Toxic shock syndrome toxin-1 (TSST-1)-binding assay using 125I-labeled TSST-1 showed the presence of specific TSST-1 binding in a B cell fraction of human peripheral blood mononuclear cells and L cells transfected with DR2 genes or DR4 genes but not in a T cell fraction and control L cells. Fixation with paraformaldehyde, an inhibitor of antigen processing, did not remove TSST-1-binding activity of the transfectants. Binding of 125I-labeled TSST-1 to the transfectants was reduced by an anti-DR monoclonal antibody. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed the presence of a single band with TSST-1-binding activity and the same migration pattern as DR heterodimers. TSST-1-induced T cell responses, proliferation and interleukin 2 (IL2) production were observed in the presence of the transfectants but not in the presence of control L cells, while concanavalin A-induced IL2 production was observed in the presence of either the transfectants or control L cells. Presence of an anti-DR monoclonal antibody inhibited the TSST-1-induced responses. Paraformaldehyde-fixed Daudi cells were effective in supporting TSST-1-induced IL2 production by T cells. These results indicate that HLA class II molecules directly bind intact TSST-1 and perform an essential role as the TSST-1-binding structures on accessory cells in T cell activation by the toxin.     

Pancreatic antigenic complex p64 69: involvement in regulation of insulin secretion and relation to glutamic acid decarboxylase.

The role of pancreatic beta-cell antigenic structures in modulation of insulin secretion in vitro was recently demonstrated by others. Here we report generation of a monoclonal antibody (mAB) ICA-1 to non-species specific beta-cell antigen(s) 64, 67 and 69 kDa. The mAB inhibits glucose stimulated insulin secretion in islet cell cultures. The ability of mAB ICA-1 to immunoprecipitate active glutamic acid decarboxylase from high speed supernatants of pancreatic and brain crude extracts was demonstrated. The 64, 67 and 69 kDa antigenic material was affinity purified from pancreatic islet cell high speed supernatants, active glutamic acid decarboxylase was found in the material. Immunoaffinity purification with mAB ICA-1 of GAD-like pancreatic beta-cell antigenic material has provided evidence of possible involvement of glutamic acid decarboxylase in modulation of insulin secretion.     

Inhibition by Fab and Fab'2 monoclonal anti-Ia antibody fragments of T-lymphocyte proliferative responses.

We investigated the capacity of anti-Ia monoclonal antibody Fab and Fab'2 fragments to inhibit keyhole limpet haemocyanin (KLH) or concanavalin A (Con A)-induced T-cell proliferations. Both types of fragments of anti-I-Ak and anti-I-E/Ck monoclonal antibodies inhibited these responses. On a protein concentration basis, the inhibitory effects of fragmented antibodies were less pronounced than those of undigested molecules. However, once the differences in antigen-binding capacity were compensated, antibody fragments were as efficient inhibitors as undigested molecules. These results suggest (i) that masking of Ia antigenic determinants is the essential mechanism of anti-Ia antibody-mediated inhibitory effect; (ii) that some KLH-specific proliferating T lymphocytes are I-E/Ck restricted; (iii) that the Ia antigen role is not limited to restriction of cell interactions since the Con A-induced proliferation, a non-H-2 restricted response, is inhibited by anti-Ia Fab fragments.     

Monoclonal antibodies reactive with idiotypic and variable-region specific determinants on human immunoglobulins

Monoclonal antibody JG-B1, specific for the human VKIIIb sub-subgroup of L chains, and JG-B4 specific for an idiotypic determinant on Glo, a monoclonal human IgM-VKIIIb anti-IgG, were produced and characterized. The VKIIIb determinant was detected on L chains alone and intact immunoglobulins with VKIIIb L chains. However, the idiotypic determinant was expressed only on IgM-Glo and required association of H and L chains. Binding of the immunogen Glo, to its antigen-IgG partially inhibited anti-idiotype and anti-VKIIIb binding. Cross-inhibition experiments demonstrated that intact pentameric IgM-Glo expressed one-half the number of idiotypic sites as VKIIIb determinants. However, Glo half-molecules expressed equal numbers of idiotypic and VKIIIb determinants. This is the first described monoclonal antibody produced by hybridoma technology which recognizes an antigenic determinant specific for a single variable region in intact immunoglobulin.     

A cytotoxic monoclonal IgM antibody (Tü 101) directed against an antigenic determinant shared between the HLA-A allospecificities A2 and A28

A complement-fixing murine monoclonal IgM antibody (TÜ 101) strictly directed against a supertypic determinant present on the HLA-A locus antigens A2 and A28 was defined from a fusion experiment employing T cell blasts as immunizing cells. The specificity of this antibody was established in the microcytoxicity assay on 91 normal Caucasian blood donors, as well as in the SpA-Ig assay on a panel of lympho- and hematopoietic cell lines. TÜ 101 segregates only with its defined HLA allotypes in families. This reagent may be of particular value as a probe for analyzing a molecular relationship of different antigenic determinants on the HLA-A2 and A28 specificities in comparison with two recently defined anti-HLA-A2/A28 monoclonal antibodies and may help to characterize structural variations of these HLA-molecules on a serological and immunochemical basis.     

Passive transfer of experimental myasthenia gravis via antigenic modulation of acetylcholine receptor.

Antigenic modulation of acetylcholine receptor (AChR) is considered to contribute to the reduction of endplate AChR in myasthenia gravis (MG). Yet, the pathogenic significance of this mechanism is unclear. To investigate the in vivo role of AChR antigenic modulation we examined the ability of bivalent F(ab')2 and monovalent Fab fragments of monoclonal antibody (mAb) 35 to passively transfer experimental autoimmune MG (EAMG) in rats. mAb 35 which binds at the main immunogenic region (MIR) of the AChR causes severe EAMG without being involved in channel function. Compared to the intact mAb, F(ab')2 35 proved to be less potent but still capable of inducing moderate EAMG, whereas Fab 35 were totally ineffective. Furthermore, both intact and F(ab')2 35 induced mild EAMG in complement-depleted rats. These results (a) provide evidence that antigenic modulation of endplate AChR is sufficient to generate passive transfer of EAMG and (b) further support the pathogenic potential of the anti-MIR antibodies in MG.     

Monocyte-regulated Hyporesponsiveness of Human Cord Blood Lymphocytes to OKT3-monoclonal-antibody-induced Mitogenesis

OKT3 monoclonal antibody recognizes surface antigenic structures present on all human mature T lymphocytes and is mitogenic for resting peripheral T cells. Recent reports suggest that these structures are linked to the specific antigen receptor of the T cells and play an important role in T-cell activation. We have tested the mitogenic action of OKT3 on resting lymphocytes from human newborns, their mothers, and unrelated adults. We found that the proliferative response of cord T cells to OKT3 is significantly lower than the response of maternal and adult cells at all doses of the antibody tested (5-1000 ng/ml). This difference was not dependent on culture conditions (source of serum, kinetics induced by the OKT3 antibody, or different proportions of adherent cells in peripheral blood mononuclear leukocytes), and could only to some extent be accounted for by differences in the proportions of OKT3-binding cells between these populations. Removal of adherent monocytes largely diminished the OKT3-induced proliferation of maternal and adult cells, by an average of 70-80%. In contrast, it significantly enhanced the proliferation of cord cells. The proliferative response of cord T lymphocytes to the two polyclonal T-cell activators phytohaemagglutinin and concanavalin A was similar to or greater than that of mothers and other adults.     

Opposing effects of protein tyrosine kinase inhibitors on the monoclonal antibody induced internalization of CD3 and CD4 antigens.

We have previously demonstrated that the monoclonal antibody (mAb)-induced modulation of CD3 and CD4 antigens from the surface of human peripheral blood lymphocytes is not dependent from protein kinase C activity (Thuillier et al., Eur. J. Immunol. 1990. 20:1197). In the present report we study the effect of genistein and of herbimycin A, two potent inhibitors of protein tyrosine kinases (PTK), on the mAb-induced modulation of CD3 and CD4 surface antigens. Both genistein and herbimycin inhibited the mAb-induced internalization of CD3 and, in contrast, facilitated that of CD4 antigen. These results indicate that the mAb-induced modulation of CD3 is essentially dependent on the PTK pathway, whereas PTK appear to negatively regulate the mAb-induced modulation of CD4.     

A cytotoxic monoclonal IgM antibody (Tü 101) directed against an antigenic determinant shared between the HLA-A allospecificities A2 and A28

A complement-fixing murine monoclonal IgM antibody (TÜ 101) strictly directed against a supertypic determinant present on the HLA-A locus antigens A2 and A28 was defined from a fusion experiment employing T cell blasts as immunizing cells. The specificity of this antibody was established in the microcytoxicity assay on 91 normal Caucasian blood donors, as well as in the SpA-Ig assay on a panel of lympho- and hematopoietic cell lines. TÜ 101 segregates only with its defined HLA allotypes in families. This reagent may be of particular value as a probe for analyzing a molecular relationship of different antigenic determinants on the HLA-A2 and A28 specificities in comparison with two recently defined anti-HLA-A2/A28 monoclonal antibodies and may help to characterize structural variations of these HLA-molecules on a serological and immunochemical basis.     

Absence of the OKT4 epitope on blood T cells and thymus cells in a patient with thymoma, hypogammaglobulinemia, and red blood cell aplasia

Human helper/inducer T-lymphocytes that express the T4 antigen are important in the regulation of B and T cell functions. Several epitopes of the T4 molecule have now been recognized; however, the precise role of these molecules in the function of helper/inducer T cells is unclear. We studied a patient with thymoma, hypogammaglobulinemia, and red blood cell aplasia whose blood lymphocytes and thymus cells did not express the epitope recognized by OKT4 monoclonal antibody but did display the T4 epitopes recognized by OKT4A and Leu3A monoclonal antibodies. The absence of the OKT4 epitope on the patient's thymus cells suggested that the abnormality occurred during early T cell differentiation. The patient had intact delayed hypersensitivity to 4/4 antigens, and his blood lymphocytes proliferated normally to phytohemagglutinin, concanavalin A, pokeweed mitogen, tetanus toxoid, and allogeneic cells. The patient's T cells demonstrated augmented suppressor activity that was localized to the OKT8+ population rather than to the unusual T4 subset. Irradiation abrogated suppressor activity and rendered his T cells capable of providing help for polyclonal B cell differentiation. The data emphasize the limitations of OKT4 as the sole reagent for characterizing the subset of human helper/inducer cells and demonstrate that the expression of the T4 epitope recognized by OKT4 monoclonal antibody is not required for certain helper/inducer T cell functions in vitro and in vivo.     

Characterization of monoclonal antibodies raised against recombinant respiratory syncytial virus nucleocapsid (N) protein: identification of a region in the carboxy terminus of N involved in the interaction with P protein.

To investigate structure and biological properties of the nucleocapsid (N) protein of respiratory syncytial virus (RSV), we have generated a panel of 16 monoclonal antibodies, raised against recombinant N protein, and epitope mapped seven of these to three antigenic sites (Site I aa 16-30; Site II aa 341-350; Site III aa 351-365). Characterization by immunofluorescence and by immunoprecipitation assay demonstrated that a monoclonal antibody to antigenic site I can detect N protein complexed with phospho (P) protein. Antibodies to antigenic sites II and III, which are adjacent to each other near the carboxyl terminus of the N protein, have distinct properties. A site III monoclonal antibody detected N protein in cytoplasmic inclusion bodies and in the cytosol, but not when N was complexed to P protein, while the site II antibody reacted with N protein in the nucleocapsid fraction but did not detect cytosolic N protein. Further investigation into the reactivities of the antibodies after binding of P to N in vitro demonstrated that antigenic sites II and III were blocked by the interaction, indicating an involvement for the carboxy domain of N in the N-P interaction. This was confirmed by the ability of peptides from the carboxy terminus of N to inhibit the N-P interaction in vitro.