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The initiating factor in the hyperacute rejection of pig organs by human or non-human primates is believed to be related to the presence of preformed "natural" antibodies in the host. In 1991, we demonstrated that human anti-pig antibodies were IgG, IgM and IgA and bound most strongly to oligosaccharides with an alpha galactose (alpha Gal) terminal residue. These included (i) alpha Gal-R (alpha galactose), (ii) alpha Gall-3 beta Gal-R (B disaccharide), (iii) alpha Gall-3 beta Gall-4 beta GlcNAc-R (linear B type 2 trisaccharide) and (iv) alpha Gall-3 beta Gall-4 beta Glc-R (linear B type 6 trisaccharide) where R is (CH2) 8COOCH3. In vitro studies using both the chromium release assay and a live/dead staining technique demonstrated that the cytotoxicity of human sera towards pig cells can be significantly reduced or abolished by immunoadsorption of the serum with immunoaffinity columns of an alpha Gal structure, particularly those with an alpha 1-3 linkage, and not by a large selection of other carbohydrates. Similarly, human anti-pig antibodies can be largely inhibited or "neutralized" by the addition of an alpha 1-3Gal di- or trisaccharide to the serum. Staining of pig vascular endothelium utilizing a panel of carbohydrate-specific lectins and immunoaffinity antibodies demonstrated the presence of three different carbohydrate epitopes, namely (i) alpha Gall-3 beta Gall-4 beta GlcNAc-R (linear B type 2 trisaccharide (ii) alpha NeuAc2-3 beta Gall-4 beta GlcNAc-R (sialyl-N-acetyllactosamine), and (iii) beta Gall-4 beta GlcNAc-R (N-acetyllactosamine). We have investigated organs from several breeds of pig and have concluded that the alpha Gal epitope is either monomorphic or at least has a high incidence in porcine species, since we have not found any pig negative for this antigen. Human vascular endothelial cells have at their surface the same lactosamine-ended precursor and sialylated chains as pigs, but instead of terminal alpha Gal they express the fucosylated polymorphic ABH histo-blood group epitopes. As we have found no evidence that human or baboon plasma contain antibodies directed against sialic acid or lactosamine, and as human tissues contain both of these carbohydrates, it seems unlikely that either of these epitopes plays a role in the vascular rejection that takes place when pig organs are transplanted into primates. Unfortunately, the alpha Gal disaccharide and trisaccharides were not available to us in the large quantities required for extracorporeal immunoadsorption or continuous intravenous infusion in adult baboons.(ABSTRACT TRUNCATED AT 400 WORDS)

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There is a large and increasing number of therapeutic proteins approved for clinical use and many more undergoing preclinical studies and clinical trials in humans. Most of them are human or 'humanized' recombinant molecules. Virtually all therapeutic proteins elicit some level of antibody response, which in some cases, can lead to potentially serious side effects. Therefore, immunogenicity of therapeutic proteins is a concern for clinicians, manufacturers and regulatory agencies. In order to assess immunogenicity of these molecules, appropriate detection, quantitation and characterization of antibody responses are necessary. Immune responses to therapeutic proteins in conventional animal models has not been, except in rare cases, predictive of the response in humans. In recent years there has been a considerable progress in development of computational methods for prediction of epitopes in protein molecules that have the potential to induce an immune response in a recipient. Initial attempts to apply such tools in early development of therapeutic proteins have already been reported. It is expected that computer driven prediction followed by in vitro and/or in vivo testing of any potentially immunogenic epitopes will help in avoiding, or at least minimizing, immune responses to therapeutic proteins.

Autoantibodies to ribosomal P-proteins are present in 12-16% of patients with systemic lupus erythematosus and are associated with neuropsychiatric disease. As the ribosomal P proteins are located in the cytoplasm, the pathogenic effects of their cognate autoantibodies are unclear. In this study affinitypurified anti-P autoantibodies were used to explore the cell surface ofseveral types ofhuman and animal cells. Immunofluorescence as well as EM immunogold analysis demonstrated, on the surface ofhuman hepatoma cells, the presence ofan epitope that is antigenically related to the immunodominant carboxy terminus of P-proteins. The presence of this epitope was also demonstrated on the surface of human neuroblastoma cells and, to a lesser extent, on human fibroblasts. Furthermore, the Western blot technique revealed in purified human and animal plasma membranes a 38-kD protein that is closely related or identical with ribosomal Po protein. The availability of reactive P peptide on the surface ofcells makes possible the direct effect of autoantibodies on the function and viability of cells that express this antigenic target. This delineates one of the possible impacts of anti-P antibodies in disease expression. (J. Clin.