Interference with cell-surface molecules important in immune cell interactions
Targeting cell-surface molecules with biologic agents has several advantages over maintenance oral drug therapy. Cell-surface molecules can be readily blocked with monoclonal antibodies or receptor-fusion proteins, and are easily saturated and modulated. The new humanized biologic agents have the added advantage of a long half-life, requiring infrequent administration. In addition, there is a paradigm shift in biologic drug development from short-term induction therapy to chronic administration as a replacement for maintenance oral immunosuppression. The potential advantage of chronic biologic therapy is regimen simplification (monthly or bi-monthly administrations), no requirement for therapeutic drug monitoring, and assured compliance.Figure 1 shows some of the current biologic agents in early clinical trials or being considered for clinical development. Other biologic agents that are being used in single-center trials but are not yet part of a formal drug development program by their sponsor include campath 1H, a humanized anti-CD52 antibody that results in prolonged lymphocytes depletion; rituximab, an anti-CD20 monoclonal antibody that targets B cells; and several humanized mutagenized nonmitogenic anti-CD3 mAbs(11–16). Kirk et al. and Knechtle et al. are conducting single-center trials with campath 1H in combination with sirolimus in an attempt to induce long-term tolerance or at the very least drug minimization. Rituximab is being used off-label in patients with elevated levels of panel-reactive antibodies as well as in patients undergoing acute humoral rejection (13). The new generation of humanized anti-CD3 mAbs are engineered to loose their toxicities through amino acid(s) substitution in the Fc domain in order to reduce binding to Fc receptors. These antibodies include HuM291, Campath 3 and hOKT3 γ1 (the humanized-mutagenized version of murine OKT3)(14–16). hOKT3 γ1 is licensed to Centecor and is being considered for use in new trials in renal transplantation.
The most dramatic failure in the recent past is Biogen's anti-CD154 (hu5C8) (17). Despite impressive experimental evidence, the phase I trial with the humanized hu5C8 was halted following thromboembolic events, as well as failure of immunosuppression efficacy (5/7 patients had rejection episodes). IDEC Pharmaceutical has started clinical trials with another humanized anti-CD154, IDEC131 (targeting a different epitope than hu5C8) in patients with autoimmune diseases, but may extend these studies to organ transplantation pending results in nonhuman primates. It is clear that there is continuing interest in exploring disruption of the CD40 pathway in clinical trials.
A humanized antibody to CD45 (anti-CD45RB) may soon put the spotlight back on CD45 as an important drug target. CD45 was first described in 1978 as a family of glycoproteins expressed on the surface of nucleated hematopoietic cells (18). CD45 is a transmembrane protein tyrosine phosphatase involved in the coupling of signals from the T-cell receptor to the proximal signaling apparatus. Different CD45 isoforms generated by the alternative slicing of exons A, B and C are expressed by T cells with distinct functions. Monoclonal antibodies to the RB isoforms of CD45 have been shown to induce long-term survival and tolerance in various experimental models of solid organs and islet cell transplant(19–21). The mechanism of action of anti-CD45RB mAbs is unclear. However, it may modulate the expression of RB isoforms with different molecular weights (m.w.) (21). T cells with high m.w. CD45RB (CD45RBbright cells) secrete IL-2, while low m.w. CD45RB (CD45RBdim cells) secrete Il-4. Basadonna et al. showed that lymphocytes obtained from animals treated with anti-CD45RB showed decreased CD45RBbright cells and had up-regulation of CD45RBdim cells (19). In addition,in vitro effects of anti-CD45RB mAbs include down-regulation of thel-selectin, up-regulation of CTLA-4, and suppression of TH1 cytokine production. In a seminal study, Lazarovits et al. showed that an anti-CD45RB mAb in two doses resulted in long-term graft survival in murine renal allografts (20). Non-human primate models are currently underway in preparation for a phase I clinical trial in renal transplant recipients by Abgenix.
Efalizumab is a humanized IgG1 monoclonal antibody targeting the CD11a chain of LFA1. Efalizumab binds to LFA1, preventing LFA1–ICAM interaction. Direct blockade of ICAM-1 with a mAb failed to show any benefit in a randomized renal transplant trial, possibly due to redundancy in the ICAMs (22) Anti-CD11a has been shown to block T-cell adhesion, trafficking and activation (23). Pre-transplant therapy with anti-CD11a prolongs survival of murine skin and heart allografts, and monkey-heart allografts (24). Efalizumab has been successfully used in phase III trials in patients with psoriasis. In a phase I/II open label, dose ranging, multidose, multicenter trial, Efalizumab was administered subcutaneously, weekly for 12 weeks following renal transplantation (25).Table 2 shows patient enrollment as well as the maintenance immunosuppression. At 3 months, 3/38 patients (7.8%) had a reversible rejection episode and at 6 months there was one additional rejection for a cumulative rejection rate of 10.4%. Pharmacokinetic and pharmacodynamic studies showed that the lower doses of Efalizumab (0.5 mg/kg) produced saturation and 80% down-modulation of CD11a by 24–48 h following therapy. In a subset of 10 patients who received the high-dose Efalizumab (2 mg/kg) with full-dose cyclosporine and MMF, 3/10 patients developed post-transplant lymphoproliferative disease. Thus Efalizumab appears to be an effective immunosuppressive agent, but it is best used in a lower-dose regimen with less intense maintenance immunosuppression.
Table 2. : Efalizumab dose and concomitant immunosuppression
|Dose of Efalizumab||Group I||Group II|
|0.5 mg/kg||2.0 mg/kg|
|A: Half-dose CsA + sirolimus + prednisone||n = 9||n = 9|
|B: Full-dose CsA + MMF + prednisone||n = 10||n = 10|
|Totals (n = 38)||n = 19||n = 19|
The costimulatory pathway also referred to as signal two (signal one being the antigen-driven pathway via the T-cell receptor) is critical in triggering T-cell activation, proliferation and effector function(26–29). While many coactivation or costimulatory pathways have been described (CD154-CD40, LFA1-ICAM-1, ICOS-B7RP-1) the CD28–B7 interaction remains the most thoroughly characterized and possibly represents the best target of immunosuppression therapy. Despite the failure of anti-CD154 in a single clinical trial, blocking CD28 interaction with B7 (either with CTLA4Ig or with anti-B7 mAbs) will continue to be an important focus of clinical studies.
h1F1 and h3D1 are humanized anti-B7.1 (CD80) and B7.2 (CD86). The DNA encoding the complementarity determining regions from the murine antibodies were molecularly spliced on to the DNA for the human kappa light, and the DNA for the γ2 heavy chain sequences mutagenized to minimize Fc binding.In vitro h1F1 and h3D1 were shown to block CD28-dependent T-cell proliferation and decrease mixed lymphocyte reactions. In nonhuman primate models, h1F1 and h3D1 were able to delay renal allograft rejection, and their effectiveness was not undermined by the use of calcineurin inhibitors or steroids (30). The monoclonal antibodies need to be used in tandem, since either B7.1 or B7.2 is sufficient to stimulate T cells via CD28. A single phase I study in renal transplant recipients was performed in patients receiving maintenance therapy consisting of cyclosporine, mycophenolate mofetil and steroids. Patients received a single pretransplant dose ranging from 0.15 mg/kg to 5 mg/kg. Though the results of the study are yet to be published, the preliminary results appear to show that these monoclonal antibodies were safe and effective. While Wyeth Pharmaceutical at the present time has decided not to proceed with further development of these antibodies, they may yet emerge in the future through licensing agreements.
LEA29Y is a second-generation CTLA4Ig (extracellular domain of CTLA4 and IgG1 Fc domain) with an increase in binding avidity to CD80 (2-fold) and CD86 (4-fold), and approximately 10-fold more effectivenessin vitro than CTLA4Ig on a per dose basis in inhibiting T-cell effector functions. A phase I/II trial is currently underway in primary renal transplants with an immunosuppression regimen based on preclinical studies performed by Drs Chris Larsen and Tom Pearson at Emory University (unpublished results). In the phase I study, 210 primary renal transplant patients are randomized to three treatment groups, group 1 and group 2 are treated with different regimens of LEA29Y, basiliximab (20 mg day 0 and day 4), mycophenolate mofetil 2 g and conventional steroid therapy. Patients randomized to group 3 serve as controls and are treated with a standard regimen consisting of basiliximab (20 mg at day 0 and day 4), cyclosporine, mycophenolate mofetil, and steroids. Patient enrollment in this trial should be completed by December 2002. This study may provide an important clue to the clinical efficacy achieved by blocking a single tract of the costimulatory pathway. It is possible, though, that effective clinical blockade of the costimulatory signal may require disruption of several targets within the pathway (29).
Inhibitors of T-cell proliferation
Effective T-cell activation requires T-cell proliferation. The new anti-interleukin-2 α chain receptor monoclonal antibodies cannot completely block T-cell proliferation as proliferative signals may occur through the intermediate affinity interleukin 2 receptor βγ or through pathways that involve cytokines other than IL2. The current approaches to blocking T-cell expansion are the disruption ofcytokine signaling or the inhibition of nucleotide incorporation required for cellular proliferation. Signaling through the γ chain requires activation of the Janus protein tyrosine kinase, JAK3, which also mediates signals from receptors for IL4, IL7, IL9 and IL15. Since JAK3 is required for the transduction of proliferative signals, inhibitors of JAK3 can be potentially powerful and useful drugs in transplantation (32). Individuals genetically lacking JAK3 have severe immunodeficiency disease (33). Whether JAK3 inhibitors turn out to be prohibitively immunosuppressive remains to be determined. Several JAK3 inhibitors, including one from Pfizer Pharmaceuticals, are development candidates for transplantation.
Novel antimetabolites include Lilly's Gemcitabine, a pyrimidine synthesis inhibitor currently being tested in a miniature swine model for renal transplantation, and FK778. FK778, a new oral immunosuppressive agent under development by Fujisawa Healthcare Inc., is an analog of the active metabolites of leflunomide (34). FK778 has a unique mechanism of action, binding to dihydro-orotate dehydrogenase and inhibiting de novo pyrimidine biosynthesis, thereby blocking T- and B-cell proliferation and strongly suppressing IgM and IgG antibody production. In addition, FK778 appears to have antiviral effects, including the polyoma virus. FK778 is currently in a phase II trial in Europe. A new, rationally designed inhibitor of inosine monophosphate dehydrogenase, VX-497, with a mechanism of action similar to mycophenolate mofetil, has been developed by Vertex and used in clinical studies in patients with psoriasis and hepatitis C. Despite encouraging preclinical studies in renal transplantation in dogs, however, its clinical development in transplantation remains in doubt.