Anti-idiotypic monoclonal antibodies to GM1 identify ganglioside binding proteins

Ganglioside stimulated neurite outgrowth may be due to gangliosidebinding to membrane proteins or to intercalation into the membrane.To test that ganglioside binding proteins could be found onneuronal surfaces, antiidiotypic ganglioside monoclonal antibodies(AIG mAbs) were generated to mimic the biological propertiesof the GM1 ganglioside. The AIG mAbs were identified by theirability to bind to a known GM1 binding protein, the ß-subunit of cholera toxin. For the two AIG mAbs studied, AIG5 andAIG20, binding to ß-CT was blocked most strongly byGM1. This data also suggests that AIG5 and AIG20 mimic differentbut overlapping epitopes of the ganglioside GM1. Western blottingand immunoprecipitation of mammalian tissues reveals four potentialganglioside binding proteins of molecular weight 93, 66, 57,and 45 kDa. Immunocytochemistry demonstrates neuronal surfacelabel with the AIG mAbs, which suggests that gangliosides, enrichedon the neuronal surface membrane, are co-localized with putativeganglioside binding proteins. In bioassays, the AIG mAbs promoteneuronal sprouting. This shows that these antibodies can beused to study the biological effects of ganglioside bindingto neuronal surface proteins, and the role of gangliosides inthe activation of neurite outgrowth. agonist antibody anti-idiotypic antibody gangliosides ganglioside binding proteins     

A monoclonal antiidiotypic antibody with rheumatoid factor activity defines a cross-reactive idiotope on murine anticollagen antibodies.

A syngeneic antiidiotypic mAb, C1C3, was characterized as to its binding to monoclonal anti-collagen II (-CII) auto-antibodies reactive with different epitopes of the native CII molecule. Both by direct binding and by inhibition ELISA studies, the anti-idiotypic antibody was shown to react with a cross-reactive idiotope present on Fab fragments of most, but not all, tested anti-CII mAb, whereas the binding to Fab fragments from normal mouse IgG was low. As previously described, C1C3 bound to isolated Fc fragments from normal mouse IgG. The binding to intact normal mouse IgG was, however, weak, and only isolated Fc-gamma fragments, not intact IgG, competed efficiently with Fab fragments of anti-CII antibodies for binding to the antiidiotypic antibody. The antibody was shown to self-associate, i.e., to behave similarly to certain IgG rheumatoid factors obtained from patients with rheumatoid arthritis. The presented data indicate that the described anti-anti-CII mAb may be representative of antibodies involved in the physiologic regulation of autoimmunity to CII and, consequently, may be used as a tool for further studies on idiotypic regulation in CII-induced arthritis.     

Human monoclonal anti-T cell antibody from a patient with juvenile rheumatoid arthritis

Antibody JRAI is a human monoclonal IgM antibody derived from a patient with juvenile rheumatoid arthritis that is cytotoxic to a subpopulation of normal T lymphocytes. JRAI recognizes approximately 80% of normal peripheral blood T cells, 90% of CD4+ cells, and 75% of CD8+ cells, as determined by complement-mediated cytotoxicity. Within the CD8+ population, JRAI preferentially spares OKM1+ and Leu-11+ cells. These CD8+ cells retain suppressor-effector potential and show enriched natural killer cell activity. Within the CD4+ population, JRAI preferentially kills cells within the Leu-8+ subset, which contains suppressor-inducer cells, and spares the Leu-8- subset, which contains the helpers for immunoglobulin synthesis. JRAI appears to recognize a previously undefined human lymphocyte surface molecule expressed differentially on phenotypically and functionally distinct subsets of human T cells.     

IgG antinuclear antibodies with cross-reactive rheumatoid factor activity

To investigate whether IgG antinuclear antibodies have cross-reactive rheumatoid factor activity, monoclonal IgG antibodies to DNA and Sm from autoimmune MRL-lpr/lpr mice were assayed by ELISA for binding to IgG antigens. Of the nine anti-DNA and anti-Sm monoclonals tested, six showed significant binding to affinity-purified rabbit IgG (RIgG) and human IgG (HIgG). To confirm that cross-reactivities were due to a single antibody, immunoabsorption of a representative polyspecific monoclonal termed C11 (anti-DNA, anti-Sm) on either Sepharose-DNA or Sepharose-RIgG resulted in marked loss of activity to the three antigens DNA, Sm and RIgG compared with immunoabsorption on Sepharose-bovine serum albumin. The monomolecular nature of the cross-reacting antibody was also suggested by inhibition analysis of C11; DNA inhibited C11 binding to RIgG 64%, whereas Sm inhibited binding to RIgG 33%. Aggregated RIgG and HIgG, however, did not inhibit binding of C11 to DNA, Sm, or solid-phase RIgG, probably reflecting the low affinity of this antibody for fluid phase Ig. Together, these findings suggest that antinuclear autoantibodies of the IgG, as well as the IgM, class have polyspecific IgG binding activity and suggest that IgG antinuclear antibodies may emerge from rheumatoid factor responses.     

Anti-idiotypic monoclonal antibody to a T-cell chronic lymphatic leukemia

Summary A murine anti-idiotypic monoclonal antibody (mAb), F1, (IgG_2a) was produced against the variable part of the T-cell receptor for antigen (T_i, /) on the tumor cells of a patient with T-cell chronic lymphatic leukemia (CD3⁺, 8⁺, 4^–). The molecular weight of the protein reactive with mAb F1, comodulation and coprecipitation with anti-CD3 antibody, and the restricted tumor-cell reactivity strongly support the anti-idiotypic nature of mAb F1. MAb F1 also stained 4% of peripheral blood lymphocytes of healthy donors. MAb F1 did not stimulate the tumor cells to DNA synthesis, but stimulated a fraction of the normal peripheral blood lymphocytes, mAb F1 did not mediate antibody-dependent cellular cytotoxicity or complement lysis to any significant degree in vitro. Three infusions of 1–10 mg anti-idiotypic mAb were given over a period of 4 weeks. The plasma half-life for mAb F1 was 3 h in the first 2 h after infusion and 44 h from 2 h to 120 h after infusion. After each treatment a rapid decrease of circulating tumor cells was seen. During the observation period an 80% reduction of the total circulating tumor cells was noted. After the second infusion, IgM and IgG antimouse antibodies were detected. Side-effects from therapy were fever, chills, nausea, vomiting, diarrhea, tachycardia, increase in systolic blood pressure and shortness of breath. Thus, in T-cell malignancies a major reduction of circulating tumor cells can be accomplished by low doses of anti-idiotypic mAb. Anti-idiotypic mAb might be a therapeutic agent of significant importance.     

Human monoclonal anti-idiotypic antibodies. I. Establishment of immortalized cell lines from a tumor patient treated with mouse monoclonal antibodies

Patients who undergo immunotherapy with a murine anti-colon carcinoma mAb (mAb17-1A) generate high titers of anti-idiotype and anti-isotype antibodies. Specifically selected anti-idiotypic antibodies that elicit in vivo a humoral and a cellular immune response against the nominal Ag can be used as surrogate Ag for immunization. We established from the B lymphocytes of a treated patient a series of EBV-transformed cell lines. Three weeks after immortalization, the cells were selected for production of antibodies (Ab2) against the Fab fragment of the murine mAb17-1A. The selected cells were cloned and screened by ELISA for specific anti-mAb17-1A idiotypic antibodies. Thirty-six out of 89 clones were anti-idiotypes. Cell culture supernatants and the purified Ig derived from 10 clones completely inhibited the specific binding of radiolabeled mAb17-1A to HT-29 colon carcinoma cells thus resembling Ab2-gamma anti-idiotypes. These cell lines which grow now in culture for 18 mo, continuously secrete IgG,K anti-Ab1-idiotype mAb. Human anti-idiotypic mAb might be candidates for vaccines when the nominal Ag itself is not available or cannot be used as such.     

Cross-reactive HIV-1-neutralizing human monoclonal antibodies identified from a patient with 2F5-like antibodies

The genes encoding broadly HIV-1-neutralizing human monoclonal antibodies (MAbs) are highly divergent from their germ line counterparts. We have hypothesized that such high levels of somatic hypermutation could pose a challenge for elicitation of the broadly neutralizing (bn) Abs and that identification of less somatically mutated bn Abs may help in the design of effective vaccine immunogens. In a quest for such bn Abs, phage- and yeast-displayed antibody libraries, constructed using peripheral blood mononuclear cells (PBMCs) from a patient with bn serum containing Abs targeting the epitope of the bn MAb 2F5, were panned against peptides containing the 2F5 epitope and against the HIV-1 gp140(JR-FL). Two MAbs (m66 and m66.6) were identified; the more mutated variant (m66.6) exhibited higher HIV-1-neutralizing activity than m66, although it was weaker than 2F5 in a TZM-bl cell assay. Binding of both MAbs to gp41 alanine substitution mutant peptides required the DKW(664-666) core of the 2F5 epitope and two additional upstream residues (L(660,663)). The MAbs have long (21-residue) heavy-chain third complementarity-determining regions (CDR-H3s), and m66.6 (but not m66) exhibited polyspecific reactivity to self- and non-self-antigens. Both m66 and m66.6 are significantly less divergent from their germ line Ab counterparts than 2F5--they have a total of 11 and 18 amino acid changes, respectively, from the closest VH and Vκ germ line gene products compared to 25 for 2F5. These new MAbs could help explore the complex maturation pathways involved in broad neutralization and its relationship with auto- and polyreactivity and may aid design of vaccine immunogens and development of therapeutics against HIV-1 infection.     

Actinomyces graevenitzii bacteremia in a patient with alcoholic liver cirrhosis

We report the first case of Actinomyces graevenitzii septicemia in a patient with alcoholic liver cirrhosis. It was identified as A.?graevenitzii by morphologic and 16S rRNA sequencing. Even though A.?graevenitzii is rarely associated with human infections, it should be considered as a potential causative agent of bacteremia.     

Anti-idiotypic antibodies to a polyomavirus monoclonal antibody recognize cell surface components of mouse kidney cells and prevent polyomavirus infection.

Anti-idiotypic antibodies have been successfully used to identify and isolate the receptor for several cell ligands. To prepare an immunologic probe for identification of the polyomavirus receptor on mouse kidney cells, polyclonal antisera against antipolyomavirus antibodies were prepared in rabbits. Fab fragments of the previously characterized monoclonal antibody E7, which neutralizes polyomavirus infection, were used for immunization (S. J. Marriott and R. A. Consigli, J. Virol. 56:365-372, 1985). Sera containing the greatest anti-idiotype activity were identified by enzyme-linked immunosorbent assay (ELISA) and purified by a series of affinity columns. The anti-idiotypic antibodies recognized the E7 idiotope in an ELISA, and anti-idiotype binding could be inhibited by preincubation of E7 monoclonal antibody with polyomavirus virions. When mixed with anti-idiotype immunoglobulin G (IgG), E7 was no longer capable of binding or immunoprecipitating polyomavirus virions or neutralizing polyomavirus infection. Direct immunofluorescence showed anti-idiotype IgG reactivity with a cell surface component of mouse kidney cells. Anti-idiotype F(ab')2 effectively competed with polyomavirus for binding to mouse kidney cells and displayed binding kinetics similar to those of polyomavirus. Virus infection of mouse kidney cells was blocked in a dose-dependent manner following treatment of the cells with anti-idiotype IgG. The anti-idiotype identified several proteins (95, 50, and 24 to 31 kilodaltons) in an immunoblot of mouse kidney cell membrane proteins but reacted predominantly with a single 50-kilodalton protein in a radioimmunoassay. The anti-idiotype failed to react with virus proteins in three assays, including ELISA, immunoprecipitation, and immunoblotting. The implications of this work for future identification and characterization of the polyomavirus receptor on mouse kidney cells are discussed.     

Treatment of a patient with B cell lymphoma by I-131 LYM-1 monoclonal antibodies

A patient with Richter's syndrome, a malignant lymphomatous transformation of chronic lymphocytic leukemia, had become moribund with rapidly enlarging masses, granulocytopenia and thrombocytopenia despite the use of conventional chemotherapy and radiotherapy. Greater than ten percent of a test dose of I-131 Lym-1, a murine monoclonal antibody produced against Burkitt's African B cell lymphoma, was accumulated by her tumor. The patient was subsequently treated with a series of injections of I-131 Lym-1 with dramatic clinical response, reduction of tumor volume by x-ray computerized tomography and progression of circulating cellular elements toward normality. Her course over the next ten months was not like that to be expected for Richter's syndrome, which has an average survival of four months. This mode of treatment appears promising.     

Mapping of four light chain-associated idiotopes of a human monoclonal rheumatoid factor

In an effort to analyze both IgM rheumatoid factor (RF) repertoire and regulation of RF production in humans, we developed a panel of four mouse monoclonal antibodies (mAb) defining distinct K light chain-associated idiotopes (id) of a human monoclonal IgM RF (Alt). These mAb (A75, AM1, AM2, AM3) had equivalent reactivities with the immunizing RF during classic inhibition of antigen-binding assays. These anti-id reagents were reacting to neither other tested monoclonal IgM RF nor normal polyclonal IgM. It was possible to distinguish the id defined by the mAb from the results of four sets of experiments: dissociation of Alt RF heavy (H) and light (L) chains showed that A75, AM1, and AM2 reacted to id located on the L chain, whereas AM3 defined a conformational RF id; recombination experiments of H and L chains showed that A75 and AM2 reacted well with both homologous (Alt H + Alt L) and heterologous (Alt L + unrelated H) recombinants, whereas AM1 reacted better with the homologous recombinant than with the heterologous one; the relative affinities of the mAb were drawn from their ability to shift already bound labeled Alt RF from solid phase IgG; and radiolabeling of two mAb (A75 and AM3) and experiments of inhibition of id binding with cold anti-id and cold anti-CK showed that A75 recognized a proximal id (close to the K constant region), whereas AM3 defined a more distal id, AM2 and AM1 being located between A75- and AM3-defined sites. This topographic mapping of K light chain-associated id of a human RF with anti-id of known relative affinities could help in studying idiotypic regulation in humans.     

Antigen-binding activity of monoclonal antibodies after incubation with organic solvents

Effects of four organic solvents--methanol, trifluoroethanol, dimethylsulfoxide, and dimethylformamide (DMF)--on the ferritin-binding activity of three monoclonal mouse antibodies of IgG2a and IgG1 subclasses were studied. The ferritin-binding constants of monoclonal antibodies G10 and F11 (the IgG2a subclass) were increased 2-6-fold after incubation with DMF and removal of the organic solvent by gel filtration. The maximum effect on the F11 antibodies was found in the presence of 5-13% DMF and on the G10 antibodies at 11-40% DMF. The effect remained after the removal of DMF from the incubation medium, and this suggests that the incubation with DMF resulted in irreversible conformational changes of the antibodies and in production of active conformers of the G10 and F11 antibodies. These conformations occurred within 15-60 min. The long-term stability and the fluorescence of the antibodies exposed to DMF suggest that the conformational changes were not global, but involved small and relatively independent structural elements of the antibodies, either of hypervariable CDR loops in variable domains or of the hinge region of the antibodies. The affinity of the C5 antibodies of the mouse IgG1 subclass was decreased after incubation with DMF. The activation was a solvent-specific effect because incubation of the G10 antibodies with methanol and dimethylsulfoxide decreased the affinity for the antigen, and incubation with trifluoroethanol virtually did not affect it. Relatively small changes in the antigen-binding activity of the antibodies were found even after the incubation with 5% organic solvent.     

Non-cross-reactive monoclonal antibodies to human chorionic gonadotropin generated after immunization with a synthetic peptide

Noncross-reactive monoclonal antibodies specific for human chorionic gonadotropin (hCG) were obtained after pre-selection for submolecular specificity with a synthetic peptide immunogen. Mice were immunized with a synthetic peptide representing a segment unique to the beta-subunit of hCG (amino acid residues 109-145), conjugated to diphtheria toxoid. We then derived nine different hybridomas that secreted monoclonal antibodies reactive with both native hCG and isolated C-terminal peptide, after somatic cell hybridization of immune spleen cells with a nonsecretory myeloma cell line. None of the nine monoclonal antibodies, termed beta-hCG-CTPa1----a9, reacted with hLH, hFSH, or hTSH, although these pituitary hormones display extensive amino acid sequence homology with hCG. The noncross-reactive anti-beta-hCG monoclonal antibodies show apparent association constants on the order of 10(9) to 10(10) M-1. A sandwich-type enzyme-linked immunosorbent assay was set up with cut-off values of around 5 mIU/ml. These antibodies might have important implications for: a) improving the diagnosis and clinical management of pregnancy; b) monitoring the course of development of carcinomas which secrete the hormone, through in vitro assays or in vivo radioimmunodetection; c) evaluating the antibodies' therapeutic potential against such carcinomas; d) studying the biologic functions of the C-terminal segment of beta-hCG; and e) addressing the anti-fertility effect of antibodies raised against that segment.     

Independent associations of anti-cyclic citrullinated peptide antibodies and rheumatoid factor with radiographic severity of rheumatoid arthritis

Several recent publications have established a strong association between anti-cyclic citrullinated peptide antibody (anti-CCP)-positive rheumatoid arthritis (RA) and carriage of shared epitope (SE) alleles. Although anti-CCP have also been associated with more severe RA, the issue of whether this is independent of rheumatoid factor (RF) has not been addressed. To identify associations between RF, anti-CCP, SE status and radiological damage, we studied a large cross-sectional cohort with longstanding RA. Individuals (n = 872) enrolled in the study all fulfilled the American College of Rheumatology criteria for RA, had a minimum disease duration of 3 years, and at least one definite radiographic erosion was present in hands or feet. Radiographs were scored blind at study entry by a single musculoskeletal radiologist using a modified Larsen's score. Anti-CCP and RF levels were determined using enzyme-linked immunosorbent assay, and DRB1 typing was performed using polymerase chain reaction based methodology. Both anti-CCP and RF levels were strongly associated with radiographic severity (P < 0.0001). In subgroups stratified for both anti-CCP and RF status, evidence of independent associations of both antibodies with radiographic outcome was found (P < 0.0001). An association of SE alleles with radiographic severity was present only in RF-negative individuals. Anti-CCP positivity was associated with SE status with evidence of a gene-dose effect, most markedly in RF-negative individuals (P < 0.01). Anti-CCP and RF status are independent severity factors for RA, with SE alleles playing at most a secondary role. Our data support the view that previously described associations between SE and radiological severity, especially in RF-negative patients, may be indirect and due to an association with anti-CCP.     

Recurrent pneumococcal meningitis in a splenectomised HIV-infected patient

Background

Hepatitis B virus infection although preventable by vaccination remains an important health issue throughout the world due to its morbidity, mortality and economical losses. Early seroprotection is desirable for people at high risk of exposure. The aim of this study was to determine whether three-week hepatitis B vaccination (on days 0, 10 and 21) provide seroprotection or not.

Methods

The 120 subjects enrolled into the study were divided into two groups and vaccinated by the classic (months 0, 1, and 2) or the accelerated (days 0, 10, and 21) schedules and antibody response determined on days 30, 60, and 90 and, if below 10 mIU/ml^-1, again on day 180. For each individual in the classic group (B) three subjects were enrolled in the accelerated group (A). Recombinant hepatitis B vaccine (Gen-Hevac B, Pasteur) was given as 20 micrograms intramuscular injections via the deltoid muscle. A booster dose on day 365 was administered for each group. Family members of hepatitis B carriers and volunteer health personnel were enrolled into group A. To the B group only volunteers who wanted vaccination against hepatitis B were included.

Results

After three doses of vaccine, Anti-HBs titers reached protective levels in both groups. The number of vaccinees with seroprotective levels of Anti-HBs (≥10 mIU/ml^-1) on day 30 was 53 (58.9%) in group A and 9 (30.0%) in group B (p < 0.05). On day 60, there was no difference between group A and B, with response rates of 84.4% (n = 76) and 80.0% (n = 24) respectively (p > 0.05). On day 90 there was no difference between group B and group A; with 26 (86.7%) and 79 (87.7%) responders respectively. In both groups those with Anti-HBs levels <10 mIU/ml^-1 attained protective levels by day 180.

Conclusion

In this study, the three-week vaccination provided protective antibody titers within a shorter time compared to the classic schedule. Therefore, in order to provide rapid antibody production against hepatitis B virus, the accelerated vaccination schedule seems to be a good preference.     

Human monoclonal IgG rheumatoid factor has structural homology with bacterial Fc receptor proteins

A cloned lymphoblast cell line, hRF-1, that secreted human monoclonal IgG4 rheumatoid factor autoantibody was produced by Epstein-Barr virus transformation of lymphocytes from rheumatoid arthritis synovium. The binding of hRF-1 rheumatoid factor to IgG globulins of different mammalian species was similar to the binding specificity of Staphylococcus aureus protein A (SpA) and to antibodies found in the sera from patients with rheumatoid arthritis. hRF-1 also had the same binding pattern to human IgG subclasses as SpA. Direct competition was observed between SpA and hRF-1 in binding IgG Fc. These results provide evidence for structural homology between a bacterial Fc receptor protein (SpA) and the monoclonal IgG rheumatoid factor.     

Anti-epidermal growth factor receptor monoclonal antibodies affecting signal transduction

Monoclonal antibodies prepared against tyrosine phosphorylated epidermal growth factor receptor (EGFR) were tested for their effects on transmembrane signal transduction in A431 tumor cells. Monoclonal antibodies (mab) defined by SDS-sensitive epitopes, i.e., epitopes with conformational specificity, were most effective. Mab 5--125 reacting with a site of the extracellular EGFR domain blocked EGF-binding and cell proliferation in vitro, as well as tumor growth in vivo. However, this mab appeared not to be internalized upon binding to EGFR and did not trigger EGFR autophosphorylation. In contrast, mab 5-D43, also defined by an SDS-sensitive epitope and reacting with an extracellular EGFR site, did not block EGF binding but was readily internalized after binding to EGFR of untreated A431 cells. This mab induced EGFR tyrosine phosphorylation in cell lysates and tyrosine-specific autophosphorylation of insolubilized EGFR immune complexes. Cell growth in vitro was greatly stimulated in the presence of mab 5-D43. Since interaction of mab 5-D43 with EGFR induced most EGF-specific functions, although it did not bind to the EGF-specific site of EGFR, we have to assume that binding of mab 5-D43 to EGFR induced a conformational shift that activated the cytoplasmic EGFR kinase site. On the other hand, activation and/or accessibility of the EGFR kinase site could be blocked by mab 1-594, which is defined by an SDS-insensitive protein epitope of the cytoplasmic EGFR domain. Blocking of the EGFR kinase site by mab 1-594 also abolished EGF-induced tyrosine phosphorylation of endogenous cellular substrates with molecular masses of 145, 97, 85, 37, and 32 kDa, as well as of exogenous substrates such as GAT copolymer. © 1993 Wiley-Liss, Inc.