No abstract

Anti-CD3 immunotoxins exhibit considerable promise for the induction of transplantation tolerance in pre-clinical large animal models. Recently an anti-human anti-CD3epsilon single-chain immunotoxin based on truncated diphtheria toxin has been described that can be expressed in CHO cells that have been mutated to diphtheria toxin resistance. After the two toxin glycosylation sites were removed, the bioactivity of the expressed immunotoxin was nearly equal to that of the chemically conjugated immunotoxin. This immunotoxin, A-dmDT390-sFv, contains diphtheria toxin to residue 390 at the N-terminus followed by VL and VH domains of antibody UCHT1 linked by a (G(4)S)(3) spacer (sFv). Surprisingly, we now report that this immunotoxin is severely compromised in its binding affinity toward CD3(+) cells as compared with the intact parental UCHT1 antibody, the UCHT1 Fab fragment or the engineered UCHT1 sFv domain alone. Binding was increased 7-fold by adding an additional identical sFv domain to the immunotoxin generating a divalent construct, A-dmDT390-bisFv (G(4)S). In vitro potency increased 10-fold over the chemically conjugated immunotoxin, UCHT1-CRM9 and the monovalent A-dmDT390-sFv. The in vivo potency of the genetically engineered immunotoxins was assayed in the transgenic heterozygote mouse, tgepsilon 600, in which the T-cells express human CD3epsilon as well as murine CD3epsilon. T-cell depletion in the spleen and lymph node observed with the divalent construct was increased 9- and 34-fold, respectively, compared with the monovalent construct. The additional sFv domain appears partially to compensate for steric hindrance of immunotoxin binding due to the large N-terminal toxin domain.

The HLA-D region of the human major histocompatibility complex (MHC) has been shown to be homologous to the murine I region in terms of both structure and function. Both regions encode class II MHC molecules which restrict T-lymphocyte interactions with antigen-presenting cells. We have recently described the MHC restriction and antigen specificities of human T-lymphocyte clones directed at strain A influenza virus. The majority of T-lymphocyte clones recognized antigen in the context of cell surface interaction products encoded by HLA-D/DR genes. However, a few clones recognized antigen presented by cells histoincompatible for D/DR antigens. We report here that some of these clones recognized viral antigens in association with antigens encoded by genes identical with or closely linked to the recently described secondary B-cell (SB) locus of the MHC. This is the first report that SB-restricted antigen recognition may form an integral part of normal, human immune responses.

Of London, London UniversityResponses to Thy-1 were used as a model system to examine parameters which affect the production of antibody-secreting lines derived from somatic cell hybridization. Experiments with the Thy-1.1 response revealed that the frequency of clones producing Thy-1.1 antibodies is a constant of 4 to 6% for each 10000 plaque-forming cells (PFC) input in the fusion cell mixture, regardless of the maturational stage of the response. Therefore, PFC responses to Thy-1 were optimized by studying variables in the choice and dose of antigen, the response kinetics and in the fusion procedures. Thus, to produce Thy-1.1 antibody-secreting cell lines, we used (a) spleen cells at the peak of the PFC response, (b) xenogeneic (rat) rather than allogeneic donors, (c) secondary rather than primary responses and (d) high ratios of NS-1 to spleen cells.For the reproducible production of Thy-1.2 antibody-secreting hybridomas, PFC responses to Thy-1.2 were similarly optimized in AKR mice. Response kinetics and antigen dose were shown to be very critical parameters. By varying the number of cells used for priming, it was revealed that doses only slightly higher than optimal produced a dramatic hyporesponsiveness in the subsequent secondary response. Using the above information, hybrid lines secreting antibody to Thy-1.2 were obtained reproducibly and one line, F7 D 5 , which secretes a cytotoxic IgM antibody was characterized in detail since a monoclonal antibody may differ from conventional antisera for immunochemical and genetic reasons.Serologically, F 7 D 5 Thy-1.2 antibody was found to behave as a conventional Thy-1.2 alloantiserum, At high dilutions however, the antibody can be used to discriminate long-lived T cells (adult thymectomized mice) from newly produced T cells (antilymphocyte antiserum-treated mice). Functionally, in numerous T cell-dependent assays both in vivo and in vitro, including helper, suppressor and cytotoxic T cell functions as well as responses to mitogens and antigens, the F 7 D 5 antibody behaved as a potent and absolute T cell-depleting agent. This cell line and some anti-Thy-l.1producing lines are available for research purposes. + Supported by a Fellowship of the MRC of Canada.

No abstract

Numerous studies have suggested that lipopolysaccharide (LPS), a major component of the cell wall of gram-negative bacteria, is responsible for the initiation of gram-negative septic shock. Previously, others have designed therapeutic regimens to target the biologically active lipid A region of LPS by either neutralization of the biological properties of LPS or enhancement of clearance of this molecule. One such compound capable of neutralizing lipid A is the antibiotic polymyxin B. However, the clinical utility of polymyxin B is limited by its toxicity. We therefore covalently conjugated this antibiotic to the high-molecular-weight polysaccharide dextran 70, resulting in reduced toxicity of polymyxin B but retention of its endotoxin-neutralizing ability. The studies described in this report were designed to test the in vivo efficacy of this compound in an experimental animal model of gram-negative septic shock. Mice were administered graded doses of Escherichia coli or Pseudomonas aeruginosa along with D-galactosamine and the antibiotic imipenem. We had previously determined that antibiotic chemotherapy provides significant protection against E. coli-mediated lethality with smaller doses of bacteria; however, the antibiotic does not provide protection against larger doses of bacteria, but it is effective at killing the bacterial inoculum in vivo. Administration of the polymyxin B-dextran 70 conjugate provided significant protection when given with an antibiotic but was not effective by itself. A requirement for a pretreatment period prior to E. coli challenge was shown to depend upon the bacterial challenge dose. In other studies using this D-galactosamine sensitization model, we demonstrated that the lipid A-specific conjugate had no effect on lethality caused by Staphylococcus aureus or tumor necrosis factor alpha. The results of these studies indicate that this compound is effective in preventing lethal gram-negative septic shock in mice and may be useful as a potential therapeutic agent in humans as well.

This study provides preliminary evidence of the safety and efficacy of corticosteroid- and calcineurin inhibitor-free immunosuppression in a relevant preclinical transplant model. These findings provide a strong rationale for evaluating this nondiabetogenic regimen in a clinical trial of islet transplants in type 1 diabetic recipients.

Central transplantation tolerance through hemopoietic chimerism initially requires inhibition of allogeneic stem cell or bone marrow (BM) rejection, as previously achieved in murine models by combinations of T cell costimulation blockade. We have evaluated LFA-1 blockade as part of regimens to support mixed hemopoietic chimerism development upon fully allogeneic BALB/c BM transfer to nonirradiated busulfan-treated B6 recipient mice. Combining anti-LFA-1 with anti-CD40 ligand (CD40L) induced high incidences and levels of stable multilineage hemopoietic chimerism comparable to chimerism achieved with anti-CD40L and everolimus (40-O-(2-hydroxyethyl)-rapamycin) under conditions where neither Ab alone was effective. The combination of anti-LFA-1 with everolimus also resulted in high levels of chimerism, albeit with a lower incidence of stability. Inhibition of acute allograft rejection critically depended on chimerism stability, even if maintained at very low levels around 1%, as was the case for some recipients without busulfan conditioning. Chimerism stability correlated with a significant donor BM-dependent loss of host-derived Vβ11+ T cells 3 mo after BM transplantation (Tx). Combinations of anti-CD40L with anti-LFA-1 or everolimus also prevented acute rejection of skin allografts transplanted before established chimerism, albeit not independently of allospecific BMTx. All skin and heart allografts transplanted to stable chimeras 3 and 5 mo after BMTx, respectively, were protected from acute rejection. Moreover, this included prevention of heart allograft vascular intimal thickening (“chronic rejection”).