RESUMO
BACKGROUND: Optimizing therapeutic monoclonal antibody (mAb) depends on the incorporation of the necessary effector functions and the development of hypoantigenic "humanized" antibodies by genetic engineering, which then need to be tested in appropriate preclinical trials. METHODS: Constructs of humanized OKT4A containing the complementarity-determining region (CDR) of murine OKT4A and the framework and constant regions of human light (kappa) and heavy chains (IgG1 and IgG4) were prepared and tested in cynomolgus monkeys who received a renal allograft. A prophylactic course of CDR-OKT4A/human (h) IgG1 or CDR-OKT4A/ hIgG4, either as high-dose single bolus (10 mg/kg) or as low-dose multiple infusion (1 mg/kg for 12 days) was given, and the effects on graft survival, immunohistology, circulating cells, and lymph node cells were assessed. RESULTS: The IgG1 isotype induced coating of T cells, modulation of surface CD4 molecules, and profound depletion of CD4+ lymphocytes in peripheral blood, which persisted as long as the animals were followed (up to 7 weeks). The IgG4 isotype induced only cell coating without cell clearance or modulation. In lymph nodes, coating of lymphocytes (approximately 60%) was seen with both isotypes in the earliest sample (6 hr). After 2 days, significant depletion of lymph node CD4 cells was evident, with a decrease in the CD4 to CD8 ratio in the IgG1-treated group; no depletion occurred in the IgG4 group. The emigration of CD4+ cells into the allograft was significantly delayed in the CDR-OKT4A/hIgG1-treated animals when compared with the CDR-OKT4A/hIgG4 group as judged by immunocytochemistry (23.8+/-13.2 days vs. 7.4+/-1.5 days, P<0.001) or interleukin-2-promoted T-cell outgrowth from allograft biopsies (22.2+/-11.0 days vs. 6.3+/-0.5 days, P<0.01). CONCLUSIONS: This study demonstrates that the in vivo effects of CDR-grafted OKT4A are dependent on its isotype. The depleting mAb CDR-OKT4A/hIgG1 significantly delays the entry of CD4+ cells into the graft, inhibiting the early phase of rejection. However, graft rejection occurs when CD4+ cells eventually infiltrate the graft, even in the presence of depressed levels of circulating CD4+ cells. Both isotypes demonstrated therapeutic efficacy: graft survival was prolonged over controls. In the case of CDR-OKT4A/hIgG4, neither lymphocyte depletion, antigenic modulation, nor prevention of infiltration is necessary for a beneficial effect, which indicates that this mAb blocks CD4 function or renders the CD4+ cell less responsive. The lack of depletion is a feature of potential clinical advantage in minimizing the risk of excessive immunosuppression.
