Dianne M. Fishwild

High-avidity human IgG kappa monoclonal antibodies from a novel strain of minilocus transgenic mice.

Human immunoglobulin transgenic mice provide a method of obtaining human monoclonal antibodies (Mabs) using conventional hybridoma technology. We describe a novel strain of human immunoglobulin transgenic mice and the use of this strain to generate multiple high-avidity human sequence IgGκ Mabs directed against a human antigen. The light chain transgene is derived in part from a yeast artificial chromosome clone that includes nearly half of the germline human Vκ region. In addition, the heavy-chain transgene encodes both human μ and human γ1 constant regions, the latter of which is expressed via intratransgene class switching. We have used these animals to isolate human IgGκ Mabs that are specific for the human T-cell marker CD4, have high binding avidities, and are immunosuppressive in vitro. The human Mab-secreting hybridomas display properties similar to those of wild-type mice including stability, growth, and secretion levels. Mabs with four distinct specificities were derived from a single transgenic mouse, consistent with an extensive diversity in the primary repertoire encoded by the transgenes.

Lipopolysaccharide-induced E-selectin expression requires continuous presence of LPS and is inhibited by bactericidal/permeability-increasing protein

Endothelial cells stimulated by LPS express E-selectin, which plays an important role in mediating neutrophil adhesion during inflammation. E-selectin is induced within 1–2 h, peaks at 4–6 h, and gradually returns to basal level by 24 h. rBPI21, a recombinant N-terminal fragment of human bactericidal/permeabilityincreasing protein (BPI), inhibited LPS-induced E-selectin expression when added at the same time as, and up to 6 h after, LPS. Delayed administration of rBPI21 also affected LPS-mediated activation of the nuclear factor, NF-κB. Two to 4 h following LPS addition to endothelial cells, when NF-κB was already activated, addition of rBPI21 resulted in marked reduction of NF-κB detectable at 4 or 6 h. These results indicate that endothelial activation requires continuous presence of LPS, and rBPI21 acts to reverse LPS-mediated endothelial activation by interrupting the on-going LPS signal.

Cytotoxicity against human peripheral blood mononuclear cells and T cell lines mediated by anti-T cell immunotoxins in the absence of added potentiator

Several in vitro assays have indicated that anti‐T cell immunotoxins (IT), composed of monoclonal antibodies (MoAbs)conjugated to ricin A chain (RTA), are maximally effective against T cells only in the presence of potentiators. It was thought that such IT might not be sufficiently cytotoxic to deplete T cells in vivo upon administration to patients. Therefore, we have re‐evaluated the in vitro assays and report herein that even with a short exposure lime (2 h), the two anti‐T cell IT, H65‐RTA (anti‐CD5 MoAb coupled to RTA) and 4MRTA (anti‐CD7 MoAb coupled to RTA30), were specifically cytotoxic for peripheral blood mononuclear cells(PBMC)in the absence of potentiators. Moreover, as has been reported for IT when tested against T cell lines, prolonging the exposure lime of the IT with PBMC from 2 h to as long as 90 h, without added potentiators, enhanced their cytotoxicity from 2‐ to 40‐fold. In contrast, most T cell lines were more sensitive to IT in the presence of potentiator, and IT cytotoxicity was much less enhanced by prolonging the exposure time. Thus, T cell lines may not serve as accurate models to determine the efficacy of IT against PBMC in vitro or in vivo. We conclude that IT‐induced cytotoxicity of PBMC can be demonstrated in vitro at pharmacologically achievable concentrations in the absence of added potentiators.

Characterization of the increased cytotoxicity of gelonin anti-T cell immunoconjugates compared with ricin A chain immunoconjugates.

Ribosomal inactivating proteins such as gelonin (Gel) and ricin A chain (RTA) conjugated to MoAbs bind to specific target cells, and upon internalization inhibit protein synthesis, ultimately resulting in cell death. We report here that Gel anti‐T cell MoAb conjugates are more cytotoxic than RTA conjugates when tested against human peripheral blood mononuclear cells (PBMC). This increased cytotoxicity is observed whether Gel is conjugated to the anti‐T cell MoAb or to an anti‐mouse immunoglobulin Fab′ fragment which then binds to the murine anti‐human T cell MoAb. Gel conjugates are not only effective at lower concentrations, but also produce a greater extent of inhibition of cellular proliferation. Moreover, a 10 min exposure to a Gel conjugate is as effective as a 90 h exposure to an RTA conjugate. When part of anti‐T cell F(ab′)2 or Fab′ conjugates, Gel affects the early steps in cellular intoxication more than RTA, Gel conjugates bind more avidly and accelerate the modulation of antigen. In contrast, when part of whole IgG conjugates, Gel does not affect the binding to or modulation of surface antigen compared with RTA, while it does increase conjugate cytotoxicity. These observations suggest that Gel may be delivered more efficiently into the cytosol than RTA. A divergent intracellular pathway for Gel is also supported by the inability of chemical potentiators, which strongly enhance RTA potency, to affect Gel potency. These properties of Gel might also be advantageous for targeted immunoconjugates made with other MoAbs or receptor‐binding molecules.

Lipopolysaccharide (LPS)-Induced E-Selectin Expression and NF-кB Activation in Endothelial Cells Require Continuous Presence of LPS

Exposure of cultured human umbilical vein endothelial cells to lipopolysaccharide (LPS) causes increased expression of E-selectin (endothelial-leukocyte adhesion molecule-1, ELAM-1-by endothelial cells and consequently increased adherence of peripheral blood neutrophils. After administration of a bolus of LPS, the induction of E-selectin mRNA and cell surface protein in endothelial cells and their adhesiveness for neutrophils could be detected within 1 to 2 h. These induced adhesive activities peak at 4 to 6 h and gradually return to basal level by 24 h (Pober et al., 1986; Pohlman et al., 1986; Bevilacqua et al., 1987), a kinetic profile typical of endothelial cell activation in vitro. While the precise molecular events that transmit the LPS signal are not defined, it is known that the activation of endothelial cells by LPS requires soluble CD14 (Frey et al., 1992; Pugin et al., 1993). Furthermore, LPS-induced transcriptional activation of the E-selectin gene has been shown to require a nuclear factor кB (NF-кB) regulatory element in the E-selectin promoter region (Montgomery et al., 1991; Pugin et al., 1993). LPS-mediated NF-кB activation in endothelial cells occurs within 1 h and lasts for at least 4 h.