Presence of antibodies to different subunits of replication protein A in autoimmune sera.

A human cDNA expression library was used to investigate the nature of molecules recognized by serum from a patient with Sjögren syndrome that exhibits a mixed immunofluorescence pattern and reacts with multiple components on an immunoblot. The data demonstrated that this serum contains IgG antibodies specific for the 70- and 32-kDa subunits of replication protein A (RPA; RPA-70 and RPA-32, respectively), a highly conserved multisubunit DNA binding protein. Affinity purification of serum autoantibodies demonstrated a complete lack of cross-reactivity between RPA-70 and RPA-32, suggesting a direct participation of the native protein complex in the autoimmune response in this patient. Purified anti-RPA-70 and anti-RPA-32 antibodies labeled nuclear and cytoplasmic components in an immunofluorescence assay, suggesting that RPA is present in both cellular compartments. Additional sera from 55 patients with different autoimmune conditions were screened against purified RPA-70 and RPA-32 recombinant proteins. One of these 55 sera was positive and reacted with only RPA-32. Twenty sera from healthy control individuals did not react with RPA. These results show that RPA is a target for autoantibodies in human autoimmune diseases, although its precise frequency, occurrence in other autoimmune diseases, and pathological significance remain to be fully elucidated.

Rapid selection of cell subpopulation-specific human monoclonal antibodies from a synthetic phage antibody library.

Peripheral blood leukocytes incubated with a semisynthetic phage antibody library and fluorochrome-labeled CD3 and CD20 antibodies were used to isolate human single-chain Fv antibodies specific for subsets of blood leukocytes by flow cytometry. Isolated phage antibodies showed exclusive binding to the subpopulation used for selection or displayed additional binding to a restricted population of other cells in the mixture. At least two phage antibodies appeared to display hitherto-unknown staining patterns of B-lineage cells. This approach provides a subtractive procedure to rapidly obtain human antibodies against known and novel surface antigens in their native configuration, expressed on phenotypically defined subpopulations of cells. This approach does not depend on immunization procedures or the necessity to repeatedly construct phage antibody libraries.

Phenotypic knockout of the high-affinity human interleukin 2 receptor by intracellular single-chain antibodies against the alpha subunit of the receptor.

The experimental manipulation of peptide growth hormones and their cellular receptors is central to understanding the pathways governing cellular signaling and growth control. Previous work has shown that intracellular antibodies targeted to the endoplasmic reticulum (ER) can be used to capture specific proteins as they enter the ER, preventing their transport to the cell surface. Here we have used this technology to inhibit the cell surface expression of the alpha subunit of the high-affinity interleukin 2 receptor (IL-2R alpha). A single-chain variable-region fragment of the anti-Tac monoclonal antibody was constructed with a signal peptide and a C-terminal ER retention signal. Intracellular expression of the single-chain antibody was found to completely abrogate cell surface expression of IL-2R alpha in stimulated Jurkat T cells. IL-2R alpha was detectable within the Jurkat cells as an immature 40-kDa form that was sensitive to endoglycosidase H, consistent with its retention in a pre- or early Golgi compartment. A single-chain antibody lacking the ER retention signal was also able to inhibit cell surface expression of IL-2R alpha although the mechanism appeared to involve rapid degradation of the receptor chain within the ER. These intracellular antibodies will provide a valuable tool for examining the role of IL-2R alpha in T-cell activation, IL-2 signal transduction, and the deregulated growth of leukemic cells which overexpress IL-2R alpha.

Suppression and promotion of tumor growth by monoclonal antibodies to ErbB-2 differentially correlate with cellular uptake.

Amplification and overexpression of the erbB-2/neu protooncogene are frequently associated with aggressive clinical course of certain human adenocarcinomas, and therefore the encoded surface glycoprotein is considered a candidate target for immunotherapy. We previously generated a series of anti-ErbB-2 monoclonal antibodies (mAbs) that either accelerate or inhibit the tumorigenic growth of erbB-2-transformed murine fibroblasts. The present study extended this observation to a human tumor cell line grown as xenografts in athymic mice and addressed the biochemical differences between the two classes of mAbs. We show that the inhibitory effect is dominant in an antibody mixture, and it depends on antibody bivalency. By using radiolabeled mAbs we found that all of three tumor-inhibitory mAbs became rapidly inaccessible to acid treatment when incubated with tumor cells. However, a tumor-stimulatory mAb remained accessible to extracellular treatments, indicating that it did not undergo endocytosis. In addition, intracellular fragments of the inhibitory mAbs, but not of the stimulatory mAb, were observed. Electron microscopy of colloidal gold-antibody conjugates confirmed the absence of endocytosis of the stimulatory mAb but detected endocytic vesicles containing an inhibitory mAb. We conclude that acceleration of cell growth by ErbB-2 correlates with cell surface localization, whereas inhibition of tumor growth is associated with an intrinsic ability of anti-ErbB-2 mAbs to induce endocytosis. These conclusions are relevant to the selection of optimal mAbs for immunotherapy and may have implications for the mechanism of cellular transformation by an overexpressed erbB-2 gene.

Characterization of subtype-specific antibodies to the human D5 dopamine receptor: studies in primate brain and transfected mammalian cells.

To achieve a better understanding of how D5 dopamine receptors mediate the actions of dopamine in brain, we have developed antibodies specific for the D5 receptor. D5 antibodies reacted with recombinant baculovirus-infected Sf9 cells expressing the D5 receptor but not with the D1 receptor or a variety of other catecholaminergic and muscarinic receptors. Epitope-tagged D5 receptors expressed in mammalian cells were reactive with both D5 antibodies and an epitope-specific probe. A mixture of N-linked glycosylated polypeptides and higher molecular-mass species was detected on immunoblots of membrane fractions of D5-transfected cells and also of primate brain. D5 receptor antibodies intensely labeled pyramidal neurons in the prefrontal cortex, whereas spiny medium-sized neurons and aspiny large interneurons of the caudate nucleus were relatively lightly labeled. Antibodies to the D5 dopamine receptor should prove important in experimentally determining specific roles for the D5 and D1 receptors in cortical processes and diseases.