Constantin A. Bona

Criteria to define anti-idiotypic antibodies carrying the internal image of an antigen

Anti-idiotypic antibodies which carry the internal image of a foreign antigen, i.e. so-called Ab2 beta antibodies, have been successfully used as vaccines to pathogens, as tools to isolate cellular receptors or as reagents in cancer therapy. An attempt is made to establish structural, immunochemical and and functional criteria to define anti-idiotypic antibodies of the Ab2 beta type.

Autoantibodies in scleroderma and tightskin mice

There is much evidence to suggest that scleroderma in human patients is caused by a fundamental defect in the immune system. In tightskin mice, the scleroderma syndrome is associated with autoimmunity, particularly autoantibodies interacting with scleroderma target antigens.

Biased usage of certain Vk gene families by autoantibodies and their polymorphism in autoimmune mice

Of 79 hybridomas derived from stimulated or unstimulated autoimmune disease prone mouse strains, secreting autoantibodies of various specificities more than 65% use V genes from five Vk families, namely, Vk1, Vk4, Vk8, Vk10 and Vk19. Restriction fragment length polymorphism (RFLP) analysis of genomic DNAs from autoimmune prone mouse strains, tight skin, NZB and SJL show marked differences in the polymorphism of the Vk1, Vk10 and Vk19 gene families.

Promotion of animal growth with a monoclonal anti-idiotype specific to anti-porcine growth hormone antibody

A monoclonal antibody (mAb), designated PS-7.6, was previously shown to enhance the growth-promoting activity of porcine growth hormone (pGH) in an experimental hypophysectomized (hypox) rat model. The long lasting effect of PS-7.6 was postulated to be a result of the induction of anti-idiotypic antibody (anti-id) in these treated animals. An attempt was made in this report to further explore this issue. It was demonstrated that mice following immunization with PS-7.6 were capable of producing anti-id in serum. The antibody titers of mice immunized with a mixture of PS-7.6 and pGH were much higher than that of those being immunized with PS-7.6 alone. A monoclonal anti-id, designated 2A6, was generated and found to recognize the intact PS-7.6 and its F(ab)2 fragment under non-reducing condition in Western analysis. However, it did not interact with reduced PS-7.6, suggesting the necessity of both H and L chains for the expression of a conformational idiotype. In radioimmunoassay, 2A6 competed with pGH for the binding to PS-7.6, but failed to do so with a control anti-pGH mAb recognizing a distinct pGH epitope from that of PS-7.6. Results from a biospecific interaction analysis which monitored the molecular interactions in a real-time fashion confirmed the facts that 2A6 specifically recognized the variable region of PS-7.6 and that the recognition was inhibited by the presence of pGH. Enzyme-linked immunosorbent assay provided further evidence to indicate that 2A6 bound to GH binding protein, i.e. the soluble GH receptor, and pGH prevented this interaction in a dose-dependent manner. The biological effect of 2A6 was evaluated in hypox rats and shown to promote the growth of these GH-deficient animals. Taken together, the present findings clearly demonstrate that 2A6 raised against a growth-enhancing anti-pGH mAb mimics pGH both conformationally and functionally.

Promotion of animal growth with a monoclonal antibody specific to growth hormone receptor.

A monoclonal antibody (mAb), designated 2C3, was raised against the growth hormone receptor (GHR) of rats. In a radioimmunoassay, 2C3 was found to compete with iodinated porcine GH (pGH) tracer for the binding to GHR, suggesting that GHR binding sites for pGH and 2C3 were identical or closely adjacent. The competition curve generated by 2C3 was identical to that generated by cold pGH, suggesting that the binding affinities of 2C3 and pGH to GHR were very similar. Administration of hypophysectomized rats with 2C3 resulted in the growth of these GH-deficient animals for a long period of time, mimicking the somatogenic effect of GH. However, this effect was abolished when 2C3 was injected into animals in the presence of exogenous GHR. A control mAb recognizing a GHR epitope distal from its binding site for GH failed to produce the growth in rats. Taken together, findings from the present study indicate that 2C3 is fully capable of engaging with GHR and subsequently triggering the growth response in rats. This mAb may also prove useful as a biologically active agonist for better understanding the initiation of the physiological process of GHR.

Functional characterization of monoclonal antibodies specific to growth hormone receptor

Upon engagement with appropriate ligands, receptors can be activated to initiate various metabolic and morphological changes in living cells. An attempt was made in this study to generate monoclonal antibodies (mAb) specific to recombinant rat growth hormone receptor (GHR) and subsequently to investigate their ability to act as biologically active ligands. Three mAbs, designated 1A9, 1H2 and 2C3, were produced and all were highly reactive with GHR in an enzyme-linked immunosorbent assay. In contrast to 1H2, 1A9 and 2C3 competed with radioactive growth hormone (GH) tracer for the binding to GHR in a radioreceptor assay, suggesting that the GH-binding sites of GHR were identical, or very close to its epitopes recognized by 1A9 and 2C3. The molecular interaction evaluated by the BIAcore technology further demonstrated the separate GHR epitopes for 1A9 and 2C3. 2C3 apparently targeted the precise GH-binding sites of GHR, while the antigenic determinants for 1A9 were not at the site, but adjacent to it. Functional analysis showed that 2C3 promoted the growth of hypophysectomized rats, whereas others failed to do so. Therefore, findings from the present study suggest that these mAbs recognize distinct GHR epitopes and are useful for investigating the structure-function relationship of GHR. Furthermore, 2C3 may prove important as a biologically active agonist for better understanding of the process of GHR activation relevant to growth.

CHAPTER 25 – B Cells Producing Pathogenic Autoantibodies

This chapter describes the autoimmune diseases mediated by pathogenic autoantibodies. Knowledge of the available repertoire of unrearranged variable region genes, and of the selection of these for recombination by normal B cells, allows an insight in the perturbation of V-gene usage in disease. Sequence analysis also reveals whether the B cells have undergone somatic mutation, which is presumed to occur in the germinal center following antigen encounter. Autoreactive antibodies can clearly arise in both stages, and in many cases, may be of no pathological consequence. However, some immunoglobulin M (IgM) autoantibodies with low affinity but high avidity can be dangerous, and innocuous IgM antibodies can become pathogenic following somatic mutation and apparently aberrant antigen selection. Either inappropriate T-cell help, or a failure to delete or modify B cells in the periphery, allows the progression through maturation and isotype switch to high-affinity autoantibody production. Data has been drawn together from murine models and human diseases to illustrate the molecular features of autoantibodies, and to point the possible routes to disease. However, understanding the nature of the consequent pathogenic autoantibodies may provide opportunities for preventing their effects.

Molecular Profile of Monoclonal Antibody Expressing the A48 Regulatory Idiotope and Having Distinct Antigen Specificities

It has been proposed that regulatory idiotopes (RId) are autoimmunogens, germline gene encoded and borne by both the T-cell receptor and antibodies having different antigenic specificities. The anti-0 2-6 fructosan myeloma protein A48 expresses an idiotype defined as regulatory because it is silent in bacterial levan-immunized BALB/c mice but can be expressed on priming with either A48 or anti-A48 Id antibodies after challenging with levan.2 In previous studies, several A48 Id bearing monoclonal antibodies (mAbs) were isolated by first priming newborn or adult BALB/c mice with the IDAlO monoclonal anti-A48 Id and then challenging with bacterial l e ~ a n . ~ Most of these antibodies belong to the V, X24 and V,10 germline gene families that respectively encode the V, and V, of the A48 protein! These antibodies display different types of fructosan or unknown antigenic specificities but nevertheless share a common i d i ~ t y p e . ~ This last observation was made among antibodies sharing other RIds. Thus, we were interested in exploring the molecular basis for RId expression. Herein we screened 70 mAbs directed against different antigens for the expression of the idiotype recognized by IDAIO. From this panel we found five antibodies: 226, M56, PY102, XYlOl, and Y19-10, which bound to IDAIO. 226 and M56 are specific for Sm antigen. PY102 and XYlOl are specific for hemagglutinin H1 of PR8 influenza virus and hemagglutinin H 3 of X3 1 influenza virus, respectively, and Y 19-10 is a rheumatoid factor. As can be seen in FIGURE 1, 226 and M56 completely inhibited the binding of mAb 3-14-9 to IDAIO, albeit a t higher concentrations than the homologous ligand. 3-14-9 is an antipolyfructosan that expresses the A48 RId recognized by IDA10.2 PY 102, XY 101, and Y 19-10 are weak inhibitors (40, 30, and 15% inhibition, respectively). These results are in good agreement with a direct binding assay in which these last three antibodies bound *I-labeled IDAlO only twoto threefold higher than background, but 2 2 6 and M56 bound sixfold higher than background (data not shown). The binding of IDAlO to each antibody is not inhibited by the corresponding antigen, which suggests that the idiotypes expressed on these antibodies are not antigen inhibitable (data not shown). In a separate experiment we found that the isolated heavy and light chains of these five antibodies do not bind IDA10. This was done by polyacrylamide gel electrophoresis under reducing conditions followed by Western blotting. However, intact antibodies electrophoresed and blotted from the same gel under nonreducing conditions bound IDAlO (data not

Molecular and Functional Characterization of Genes Encoding Anti-Thyroglobulin and Anti-Tsh Receptor Antibodies

Autoimmune thyroiditis covers a wide spectrum of diseases ranging from hyperthyroid forms, like Graves’disease to hypothyroidism forms like Hashimoto’s disease.

Restricted Set of V Genes and Extensive Idiotype Crossreactivity of Autoantibodies

The production of antibodies recognizing self-antigens is one of the hallmarks of autoimmune diseases (1). Despite the obvious role of T cells in the production of autoantibodies and the existence of genetic and environmental factors, the investigation of autoreactive B cell clones may bring important clues in the comprehension of the pathogenesis of auto-immunity. One of the rational approaches is to look for the existence of common features among autoantibodies of various specificities. The existence of similarities could imply that common mechanisms are involved in diseases with different clinical manifestations.