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A hybrid cDNA coding for a fusion protein between rat interleukin 2 (IL-2) and a truncated heavy chain from rat immunoglobulin M (IgM) was constructed. The rat IL-2 and rat IgM CH2-3-4 hybrid gene was subcloned into a vector (PKCR6) for expression of the fusion molecule in Chinese hamster ovary (CHO) cells. Cells transfected with the hybrid cDNA secrete multimeric forms of the fusion protein (IL-2-Mu). Size analysis of the construct revealed that the majority (95%) of the secreted proteins have a high mw (> 500 kDa). The IL-2-Mu construct bind specifically to cells bearing the IL-2 receptors (IL-2R) with a binding affinity around 5 nM. The specific binding to IL-2R leads to T cell proliferation or, if rabbit complement is added, to T cell lysis. Multimeric forms (> 500 kDa) of the fusion protein mediate complement-dependent lysis but trigger only weak proliferation when compared with the low-mw forms (< 500 kDa). In contrast, the latter only efficiently mediate T cell proliferation without inducing complement-dependent lysis. After intravenous administration of CHO supernatant containing IL-2-Mu, or purified IL-2-Mu proteins into rats, the fusion proteins disappeared from the circulation with a t1/2 of 1 hr. The circulating IL-2-Mu constructs in the rat serum retained their capacity to induce complement-dependent lysis of IL-2R-bearing T cells in vitro. Furthermore, the IL-2-Mu construct was able to suppress the delayed-type hypersensitivity (DTH) reaction (an IL-2R, T helper cell-dependent event) in mice. A weak immune response (antirat IL-2-Mu antibodies) was observed when rats received multiple daily injections of the construct.

The focus of progress in transplantation immunosuppression is to achieve more specific immunosuppression with monoclonal antibodies. We have already shown that the efficacy of 33B3.1, a rat monoclonal Ig2A directed against the human IL-2 receptor, was similar to that of rabbit antithymocyte globulin in the prevention of acute rejection in first kidney transplants. A similar comparative analysis has been made in 40-sec renal transplants. ATG (1 mg/kg/day) or 33B3.1 (10 mg/day) was administered during the first 10 days postgrafting in association with corticosteroids and azathioprine. Cyclosporine was introduced on day 9 and azathioprine/CsA constituted the patient's maintenance treatment after day 45. Rejection treatment consisted of equine antilymphocyte globulin in both cases and of steroid boluses when patients were under Cyclosporine. One patient in each group died. Graft survival was 90%, 85%, and 79% in the ATG group (n = 20) and 100%, 89%, and 89% in the 33B3.1 group (n = 20) at 3, 12, and 24 months, respectively. Of the ATG group patients, 45% and 40% in the 33B3.1 group had at least one rejection episode, half the episodes in the MoAb cohort occurring under 33B3.1, vs. none in the ATG group. Transplant function was similar in both groups. Viral infections appeared to be more frequent with ATG (60%) than with 33B3.1 (12%), with CMV accounting for half of these in the ATG group, and none in the MoAb group. Tolerance of both agents was good. Of the 33B3.1 recipients, 70% developed anti-33B3.1 antibodies. From these data, we conclude that this anti-IL-2 receptor MoAb seems less effective than rabbit ATG as induction treatment in second kidney transplant patients.

25.3, a mouse IgG1 monoclonal antibody (MoAb), directed at the alpha chain of the LFA1 molecule (CD11a) has been used in prophylaxis of rejection in recipients of cadaveric kidney graft. Promising clinical results have been obtained for both tolerance and efficacy [1]. The aim of this trial was to determine the optimal dosage, base on a pharmacokinetic-pharmacodynamic analysis of the data obtained from the 15 patients included in this dose-searching study. Biological parameters, such as circulating levels and functional inhibition (as detected in an adhesion assay of patient lymphocytes), were measured during and after treatment. A Hill relation was calculated between the effect and the concentration measured and led us to select a 15 mg/day dose for further clinical trials, with a loading dose of 30 mg. An additional group receiving this protocol was submitted to the same calculation, and the results from this last group were in agreement with this previous analysis.

The present study analyzed the ability of human decay-accelerating factor (DAF) and CD59 to protect rat endothelial cell (EC) clones from human and primate complement-mediated lysis. By flow cytometry and Scatchard analysis, we show that human DAF and/or CD59 cDNAs under the transcriptional control of elongation factor 1-alpha promoter were expressed at levels similar to or higher than that of a human EC line. Human DAF and CD59 were released from the surface of transfected rat cells by phosphatidylinositol phospholipase C, demonstrating that the two molecules were linked to the cell membrane by means of a glycolipid anchor. The functional activity of the two human C regulatory proteins expressed on rat EC lines was studied using an in vitro assay of C-dependent cytotoxic in which rat EC were incubated with human or nonhuman primate sera as sources of xenogeneic natural antibodies and C. We demonstrate that human and monkey xenogeneic natural antibodies bind to rat cells and induce lysis by a C-dependent mechanism involving mainly the C direct activation pathway. Our data indicate that human DAF and CD59, expressed either alone or in combination, abrogated all EC cytotoxicity, even in the presence of 50% human serum. This protective phenotype was correlated with decreased membrane attack complex (CD59 and/or DAF transfectants) and C3 deposition (DAF transfectants) on EC surface. Antibodies against the transfected molecules abolished the protection against C-mediated lysis.