A strategy is described for improving the efficacy of ricin A chain-containing immunotoxins. Highly purified preparations of ricin A chain or ricin B chain were separately coupled to anti-human immunoglobulin antibodies and the conjugates (immunotoxins) were affinity purified to eliminate free chains. Mixtures of anti-Ig-A and anti-Ig-B immunotoxins markedly synergized in vitro in their ability to kill the Ig-bearing human lymphoma cell line Daudi. In contrast, A-chain- or B-chain-containing immunotoxins of irrelevant specificity did not synergize with anti-Ig-A or anti-Ig-B immunotoxins. This finding indicates that free A or B chains do not play a major role in the synergy and that the synergy is specific. Thus, synergy depends on the specificity of the two antibodies; the lectin binding ability of a B-chain-containing immunotoxin of irrelevant specificity does not suffice. This approach of delivering ricin A chain and B chain separately to a target cell may have significant advantages in killing cells that are not effectively killed by A-chain-containing immunotoxins alone.
The 52 kDa subunit of the peripheral-plasma-membrane insulin-stimulated high-affinity cyclic AMP phosphodiesterase can be specifically detected by the antibody PM1 by Western-blotting procedures and also can be immunoprecipitated from a hepatocyte extract. PM1-mediated immunoprecipitation from hepatocyte extracts showed that insulin treatment of intact 32P-labelled hepatocytes caused the rapid phosphorylation of the peripheral-plasma-membrane cyclic AMP phosphodiesterase. Phosphoamino acid analysis and the use of a phosphotyrosine-specific antibody indicated that phosphorylation occurred on tyrosyl residue(s) of this phosphodiesterase. Prior treatment of hepatocytes with glucagon (10 nM) completely blocked the insulin-mediated tyrosyl phosphorylation of this 52 kDa protein, as detected with both the PM1 and the anti-phosphotyrosine antibodies. Treatment of hepatocytes with glucagon alone did not increase the phosphorylation state of the peripheral-plasma-membrane cyclic AMP phosphodiesterase. The specific anti-phosphotyrosine antibody also detected the insulin-stimulated phosphorylation of proteins of 180 kDa, 95 kDa and 39 kDa. Prior treatment of hepatocytes with glucagon decreased the ability of insulin to phosphorylate the 180 kDa and 39 kDa species, but not the 95 kDa species.
Conjugates of tumor-reactive antibody and toxins (immunotoxins) have been used to eradicate tumor cells in vitro and in vivo. Such immunotoxins are highly effective in killing murine leukemic cells in infiltrated bone marrow and should be useful in the bone marrow rescue approach for the treatment of cancer. Similar immunotoxins injected parentally can help to induce prolonged remissions in leukemic mice, and antigen-containing immunotoxins can induce immunologic unresponsiveness in vitro in normal murine splenocytes. Thus, long-range goals for the parental use of immunotoxins include the killing of cancer cells in vivo and the modulation of the immune response for therapeutic purposes.
Abstract In this report, we describe the dose‐dependent increase in both CD25 and CD23 levels on resting human B cells in response to CD40 ligation, as mediated by soluble CD40 ligand (sCD40L) or anti‐CD40 antibody. In combination with interleukin (IL)‐4, sCD40L had limited additive effects on CD25 expression, but significantly enhanced CD23 expression on tonsillar B cells. Interferon‐γ (IFN‐γ) exerted no inhibitory effect upon increases in CD25 or CD23 driven by CD40 ligation with sCD40L or anti‐CD40 antibody. These data suggest that the induction of CD25 and CD23 genes by IL‐4 is mediated, at least in part, by an IFN‐γ‐sensitive component, whereas gene activation driven via CD40 ligation involves signaling pathways which are not sensitive to IFN‐γ.
Epstein–Barr virus nuclear antigen-1 (EBNA-1) is the only latent protein expressed in all virus-associated tumours. It plays a critical role in viral propagation and in the replication, episomal maintenance and partitioning of the viral genome. However, its tumorigenic potential is debated. We have previously shown that lymphocytes from a tumour-prone, EBNA-1-expressing, transgenic mouse line show increased responsiveness to interleukin-2 (IL-2). It was important to determine whether this property was unique to the transgenic line or whether it is a general consequence of EBNA-1 expression in B cells. In order to distinguish between these possibilities, explanted lymphocytes from two independent transgenic mouse lines were examined. The lymphocytes from both lines showed enhanced proliferation rates compared with controls. The transgenic lymphocytes survived for extended periods in culture, dependent on the dose of IL-2, while IL-15 (the receptor of which shares the β and γ chain components of the IL-2 receptor) induced little effect. In accordance with this, transgenic B cells showed enhanced induction of expression of the IL-2 receptor α chain (CD25), which modulates affinity for the ligand. As this phenotype is evident in lymphocytes from mice of both lines, it is necessarily independent of any transgene insertion site effects and may be attributed to EBNA-1 expression. Furthermore, 10/12 tumour-bearing transgenic mice had elevated IL-2 levels in serum and 4/6 tumours were CD25 positive. IL-2 is normally produced by activated T cells in vivo ; thus, chronic immune activation or modulation could elicit this unique mode of virus-infected cell survival.
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