OX40 is a member of the TNF/NGF-receptor family expressed on activated T cells, whose ligand is found on activated T and B cells. In the present study, we show that cross-linking of OX40L on CD40L-stimulated B cells, alpha IgD dextran-stimulated B cells, or both results in a significantly enhanced proliferative response with no change in the cell survival rate. Furthermore, OX40 stimulation increases immunoglobulin heavy chain mRNA levels and immunoglobulin secretion, which could not be blocked by anti-cytokine antibodies. In additional molecular studies, we show that OX40L cross-linking results in the down-regulation of the transcription factor BSAP. This, in turn, leads to a change in the in vivo binding pattern of the immunoglobulin heavy chain gene 3' alpha enhancer, suggesting its activation. This effect may thus be one mechanism for OX40-induced increase in immunoglobulin secretion. In conclusion, our data suggest that the OX40-OX40L interaction is a novel pathway in T cell-dependent B cell proliferation and differentiation.

A collection of cell lines expressing each human epidermal growth factor receptor (HER) family member alone or in all pairwise combinations in a clone of NIH3T3 cells (3T3-7d) devoid of detectable epidermal growth factor receptor family members has been generated. Transformation, as measured by growth in soft agar, occurred only in the presence of appropriate ligand and only in cells expressing two different HER family members. Transfection of oncogenic neu (Tneu), conferred ligand-independent transformation only in cells which co-expressed HER1, HER3, or HER4, but not when expressed alone or with HER2. Cell lines were also tested for their ability to form tumors in animals. None of the cell lines expressing single HER family members was able to form tumors in animals with the exception of HER1, which was weakly tumorigenic. Although unable to form tumors when expressed alone, HER2 was tumorigenic when expressed with HER1 or HER3, but not HER4. Of all complexes analyzed, cells expressing HER1 ؉ HER2 were the most aggressive. The relationship between HER1 activation, intracellular calcium fluxes, and phospholipase C␥1 activation is well established. We found that activation of HER1 was required for the induction of a calcium flux and the phosphorylation of phospholipase C␥1. These activities were independent of, and unaffected by, the co-expression of any other family member. Further, heregulin stimulation of all cell lines including those containing HER1 did not demonstrate any effect on intracellular calcium levels or phospholipase C␥1 phosphorylation. This demonstrates that heregulin induced cellular transformation by activating HER3-and HER4-containing complexes does not require the activation of either phospholipase C␥1 or the mobilization of intracellular calcium.

Immunotoxins are hybrid molecules composed of a cell-surface bin domain and a protein toxin moiety that together target sifc cell popaons for elimination. These Vascular leak syndrome (VLS) is the dose-limiting toxicity found in many clinical trials utilizing immunotoxins, including those prepared with blocked ricin, ricin A chain (in native and deglycosylated forms), and saporin (1-10). VLS is characterized in humans by hypoalbuminemia, weight gain, and edema, resulting from the extravasation offluids and proteins from the vascular system into the periphery. VLS restricts the use of immunotoxins in humans and in many cases necessitates either a significant reduction in dose or a complete cessation of therapy. While peripheral edema is clinically manageable, pulmonary edema can be life threatening. Recently, VLS was found to have contributed to the death of two B-cell lymphoma patients who were treated with anti-CD22-deglycosylated ricin A chain (9). Other toxic effects in patients treated with immunotoxins may be a result of VLS, including tachycardia, nausea, aphasias as a result ofcerebral edema, and myocardial damage (4).Other proteins also have been found to induce VLS in humans. Systemic administration of the cytokine interleukin 2 (IL-2) results in the development ofVLS when approaching doses that may provide antitumor efficacy in patients with metastatic cancer (11,12). VLS has also been observed in patients following treatment with granulocyte/macrophage colony-stimulating factor or the anti-GD3 antibody R24 (13,14).BR96 sFv-PE40 is a single-chain immunotoxin fusion protein that has been shown to cure established human tumor xenografts in both athymic rats and mice (15,16). In this study, a rat model for VLS was established following administration of BR96 sFv-PE40 at doses beyond those required for cures of tumor xenografts in rodent models. The identification of an animal model for VLS that closely approximates the human VLS response to immunotoxins provides an opportunity to evaluate specific drugs for their ability to block immunotoxin-induced VLS and to determine whether they affect the antitumor activity of BR96 sFv-PE40. MATERIALS AND METHODSReagents. The single-chain immunotoxin fusion protein BR96 sFv-PE40 was expressed in Escherichia coli and purified as described (16). Diphenhydramine hydrochloride was purchased from Elkins-Sinn (Cherry Hill, NJ). Cyclosporine A (CsA) was purchased from Sandoz Pharmaceutical. 15-Deoxyspergualin (DSG) was purchased from Nippon Kayaku (Tokyo). Dexamethasone (Dex) was purchased from Anpro Pharmaceuticals (Arcadia, CA).Toxicity Studies. Six-to 8-week-old female Wistar Furth and Rowett, nu/nu (athymic) rats (Harlan-Sprague-Dawley) were i.v. injected with various amounts of BR96 sFv-PE40 (0.25-4 mg/kg). After 24 hr, they were euthanized by exposure to C02, and the tissues were analyzed using gross and microscopic techniques. Cardiac blood was collected from comatose animals and placed either in serum collection tubes for blood chemistry analysis or in EDT...

We demonstrate that murine myeloma cells can efficiently mediate homologous recombination. The murine myeloma cell line J558L was shown to appropriately recombine two transfected DNA molecules in 30% of cells that received and integrated intact copies ofboth molecules. This activity was then exploited to direct major reconstructions of an endogenous locus within a hybridoma cell line. Production of antigenspecific chimeric heavy chain was achieved by targeting the human IgG1 heavy chain constant region (Cyi) exons to the genomic heavy chain locus of a hybridoma cell line secreting antibody specific for a human tumor-associated antigen. The frequency of productive genomic recombinations was 1 in 200 transfectants, with accumulation of the chimeric protein reaching >20 ,ug/ml in culture supernatants.

The EGF receptor family of tyrosine kinase growth factor receptors is expressed in a variety of cell types and has been implicated in the progression of certain human adenocarcinomas. The most recent addition to this family of receptors, HER4, was expressed in NIH 3T3 cells to determine its biological and biochemical characteristics. Cells expressing HER4 were responsive to heregulin beta2 as demonstrated by an increase in HER4 tyrosine phosphorylation and ability to form foci on a cell monolayer. HER4 exhibited in vitro kinase activity and was able to phosphorylate the regulatory subunit of phosphatidylinositol 3-kinase and SHC. Peptide competition studies identified tyrosine 1056 of HER4 as the phosphatidylinositol 3-kinase binding site and tyrosines 1188 and 1242 as two potential SHC binding sites. Interestingly, transfection of HER4 into NIH 3T3 cells conferred responsiveness to EGF with respect to colony formation in soft agar. It was also found that in response to heregulin beta2, endogenous murine HER1 or transfected human HER1 became phosphorylated when HER4 was present. This demonstrates that HER1 and HER4 can exist in a heterodimer complex and likely activate each other by transphosphorylation.