Daclizumab (Zenapax) identifies the alpha subunit of the interleukin-2 (IL-2) receptor and blocks the interaction of this cytokine with its growth factor receptor. The scientific basis for the choice of the IL-2 receptor alpha subunit as a target for monoclonal antibody-mediated therapy of leukemia/lymphoma is that very few normal cells express IL-2R alpha, whereas the abnormal T cells in patients with an array of lymphoid malignancies express this receptor. In 1997, daclizumab was approved by the FDA for use in the prevention of renal allograft rejection. In addition, anti-Tac provided effective therapy for select patients with T-cell malignancies and an array of inflammatory autoimmune disorders. Finally, therapy with this antibody armed with 90Y has led to clinical responses in the majority of patients with adult T-cell leukemia. These insights concerning the IL-2/IL-2 receptor system facilitated the development of effective daclizumab antibody therapy for select patients with leukemia/lymphoma.
In 1981, in our laboratory, Uchiyama reported the development of the anti-Tac monoclonal antibody directed toward the interleukin-2 (IL-2) receptor alpha subunit (Uchiyama et al., 1981a, 1981b). The IL-2 receptor subunit identified by the murine monoclonal antibody, anti-Tac, was shown to be a densely glyclosylated, sulfated integral membrane protein with an apparent Mr of 55 000 (Leonard et al., 1982, 1984, 1985). The IL-2 receptor is composed of 272 amino acids including a 21 amino-acid signal peptide, a single hydrophobic membrane region of 19 amino acids and a very short 13 amino-acid cytoplasmic domain (Leonard et al., 1984). The cytoplasmic domain of the IL-2 receptor alpha subunit appeared to be too small for enzymatic function. Furthermore, it did not contain a cytoplasmic tyrosine. Thus, the issue was how the IL-2 receptor signals were transduced to the nucleus if there was only an IL-2R alpha subunit. Furthermore, there was the issue of the structural explanation for the great difference in affinity between high- (10−11 M) and low- 10−8 M) affinity receptors. We resolved these issues in parallel with investigators in the laboratory of Warren Leonard by co-discovering a novel non-Tac 70 kDa IL-2 binding protein, IL-2R beta, that is shared with IL-15 (Sharon et al., 1986; Tsudo et al., 1986). Subsequently, the high-affinity IL-2 receptor was shown to also include the 64 kDa IL-2R γ chain (or γc) shared by IL-4, IL-7, IL-9, IL-15 and IL-21 (Sugamura et al., 1996)). The cellular distribution of the 55 kDa subunit of the IL-2 receptor defined using the anti-Tac monoclonal antibody revealed that <5% of unstimulated human peripheral blood T cells react with the anti-Tac monoclonal antibody (Waldmann, 1989). However, high IL-2R alpha expression was demonstrated on an array of abnormal cells including the malignant cells in patients with adult T-cell leukemia (ATL), cutaneous T-cell lymphoma, anaplastic large-cell lymphoma, hairy cell B-cell leukemia, and the Reed Sternberg and associated polyclonal T cells in Hodgkin's disease as well as acute and chronic granulocytic leukemia cells (Waldmann 1986, 1989). Furthermore, such abnormalities of IL-2R alpha expression have been demonstrated in the autoimmune diseases such as rheumatoid arthritis, systemic lupus erythematosus, aplastic anemia, insulin-dependent diabetes mellitus, Crohn's disease, sarcoidosis, scleroderma, noninfectious uveitis, chronic active hepatitis, multiple sclerosis and tropical spastic paraparesis (TSP) (Waldmann, 1989, 2003). Furthermore, such elevations of IL-2 R alpha expression have been detected in the serum of patients during organ allograft rejection and from those with graft-versus-host disease (Rubin and Nelson, 1990). This discordance in IL-2R alpha expression between normal cells, which we wish to retain that do not express the receptor, and abnormal cells in disease that express the receptor provided the scientific basis for the use of anti-Tac and its humanized form, daclizumab, for an array of clinical conditions including CD25-expressing leukemias and lymphomas, autoimmune diseases and to prevent allograft rejection. In 1997 daclizumab, the humanized form of this antibody was approved by the FDA for use in the prevention of renal allograft rejection (Vincenti et al., 1998; Wiseman and Faulds, 1999). In addition, we and our collaborators demonstrated that daclizumab is of value in the treatment of patients with non-infectious uveitis, multiple sclerosis and the neurological disease, human T-cell lymphotropic virus I (HTLV-I)-associated myelopathy (HAM)/TSP (Lehky et al., 1998; Nussenblatt et al., 1999; Bielekova et al., 2004). Others demonstrated therapeutic efficacy with daclizumab in patients with pure red cell aplasia, aplastic anemia and psoriasis (Krueger et al., 2000; Maciejewski et al., 2003; Sloand et al., 2006). In addition, unmodified daclizumab as well as daclizumab armed with toxins and radionuclides has proven effective in the treatment of select patients with T-cell leukemias and lymphomas and Hodgkin's disease (Waldmann et al., 1988, 1993, 1995; Kreitman et al., 2000). These latter studies will be the primary focus of this review.
Daclizumab therapy for patients receiving organ allografts
Organ allograft rejection is associated with an elevated serum-soluble IL-2R alpha level that is linked to the activation of T cells mediated by major histocompatibility complex mismatch recognition. Two major phase III studies were used to evaluate the clinical efficacy of daclizumab linked to immunosuppression compared with a placebo with the same immunosuppressive regimen (Vincenti et al., 1998; Nashan et al., 1999). In the two phase III multicenter studies, which were double-blind and randomized, the end points were the incidence of biopsy-proven rejection that occurred in the first 6 months after transplantation. A total of 535 patients was studied. In both studies, the patients receiving daclizumab and immunosuppression had a significantly reduced biopsy confirmed number of episodes of rejection when compared with the patient groups receiving standard immunosuppression plus placebo. In one of the studies, the patients on daclizumab therapy had better graft function, reduced requirement for antithrombocyte or antilymphocyte globulin, lower administered corticosteroid doses, a lower incidence of cytomegalovirus infection, a lower incidence of infectious deaths and a greater 1-year survival than did the patients on placebo (Nashan et al., 1999). On the basis of the efficacy in these multicenter trials and the lack of associated increase in toxicity, in 1997 the FDA provided marketing clearance for the use of daclizumab in the prevention of acute kidney transplant rejection. In subsequent studies, in clinical trials involving a large group of recipients, daclizumab provided a reduction in the rejection episodes in patients receiving renal, liver, cardiac and pancreatic islet transplants (Beniaminovitz et al., 2000; Eckhoff et al., 2000; Shapiro et al., 2000).
Daclizumab therapy of IL-2R alpha (CD25) expressing leukemia/lymphoma
IL-2R alpha, the target of daclizumab, is expressed by the malignant cells from select patients with ATL/lymphoma, cutaneous T-cell lymphomas, anaplastic large-cell lymphoma, hairy cell leukemia, granulocytic neoplasms and the Reed–Sternberg cells and associated polyclonal T cells in Hodgkin's disease. We developed a murine model of human ATL (MET-I) to evaluate the different therapeutic approaches involving daclizumab (Phillips et al., 2000). To establish this murine model of human ATL, we injected human ATL cells into non-obese, diabetic (NOD), severe combined immunodeficient (SCID) mice (Phillips et al., 2000). The transferred disease progressed to death of the mice after 4–6 weeks. Various forms of antibody directed to the IL-2 receptor alpha subunit including daclizumab, the humanized form of anti-Tac, murine anti-Tac and the 7G7/B6 monoclonal antibody that targets this IL-2 receptor subunit at an epitope other than the IL-2 and anti-Tac binding sites, significantly delayed the progression of the leukemia and prolonged the survival of tumor-bearing mice (Phillips et al., 2000). Thus, it appeared that in this MET-I model, daclizumab and the other IL-2R alpha receptor-directed antibodies acted by a mechanism that had not been anticipated. The prevailing view was that the antibodies to the IL-2R alpha receptor have an effective action that is limited to the blockade of the interaction of IL-2 with its growth factor receptor, thereby inducing cytokine deprivation-mediated apoptotic cell death (Depper et al., 1984). However, the 7G7/B6 monoclonal antibody defines an epitope on the IL-2 receptor subunit that is not involved in IL-2 binding. Furthermore, in the MET-I model, the human ATL cells obtained from the spleen of the leukemia-bearing mice did not produce human IL-2 nor did they respond to murine IL-2. We considered an alternative mechanism of action of the anti-CD25 antibodies, by requiring FcR receptor expression on host cells such as monocytes or polymorphonuclear leukocytes. To test this hypothesis, we evaluated daclizumab in the therapy of the MET-I model in both wild-type SCID/NOD mice as well as in SCID/NOD FcR γ−/− mice that lacked effective FcRγI, FcRγIII and FcRγIV receptors (Zhang et al., 2004). The IL-2R alpha directed monoclonal antibody, daclizumab, that was effective in wild-type mice was not active in these FcR gamma −/− mice, supporting the view that an action requiring Fc-receptor expression on monocytes or polymorphonuclear leukocytes was probably involved in the therapeutic efficacy in this model of ATL (Zhang et al., 2004). Thus, in terms of its mode of action, daclizumab acts by a number of mechanisms that include blockade of the interaction of IL-2 with its receptor as well as by an FcR receptor requiring mechanism. Recently, yet another mechanism of action has been reported. In particular, in humans the interaction of daclizumab with the IL-2 receptor was associated with a 4- to 20-fold increase in the number of circulating CD56 bright, CD25 expressing, IL-10 secreting natural killer cells that mediate negative immunomodulatory actions (Li et al., 2005; Bielekova et al., 2006).
Clinical trials of daclizumab in IL-2R alpha (CD25) directed monoclonal antibody-mediated therapy of human leukemia/lymphoma
The first clinical trials involving murine anti-Tac were directed toward the treatment of patients with the retrovirus HTLV-I-associated ATL (Waldmann et al., 1988). The retrovirus HTLV-I encodes a transactivating protein, tax, that stimulates the transcription of numerous host genes, including those of IL-2 and IL-2R alpha (Uchiyama et al., 1977; Jeang, 2001). The malignant ATL cells constitutively expressed approximately 10 000–28 000 IL-2R alpha receptor subunits identified by the anti-Tac monoclonal antibody. These observations stimulated us to perform a therapeutic trial with the unmodified murine version of the anti-Tac monoclonal antibody. Seven of the 19 patients developed mixed (one case), partial (four cases) or complete (two cases) responses (Waldmann et al., 1993). In one case, the remission has persisted for more than 17 years after initiation of a short course of antibody therapy. None of the patients treated suffered any untoward reactions. However, six of 10 patients treated with the murine antibody that developed clinical remissions produced antibodies to this therapeutic agent. Additional limitations in the murine anti-Tac antibody were that it had a short in vivo survival of only 50 h in the circulation of humans, precluding its long-term use as an agent to provide the antibody-mediated saturation of receptors that is required to prevent the interaction of the growth factor IL-2 with its receptor. An additional problem was that the antibody did not function in antibody-dependent cellular cytotoxicity (ADCC) with human mononuclear cells and was relatively ineffective as a cytocidal agent.
We joined with Cary Queen to generate a humanized version of the anti-Tac monoclonal antibody (daclizumab) that is reactive with the human IL-2R alpha subunit (Queen et al., 1989; Junghans et al., 1990). In this effort, the human IgG1 framework sequence from the Eu-myeloma antibody was chosen to be as homologous as possible to the original mouse antibody to reduce any deformation of the mouse complementarity determining regions (CDRs). Second, computer modeling was used to identify several framework amino acids from the mouse antibody that interact with the CDRs or directly with antigen. These amino acids were retained as were the murine or more typical human amino acids and were transferred to the human framework along with the murine CDRs. This latter action proved to be critical in generating a high-affinity humanized anti-Tac (daclizumab). The parent murine anti-Tac molecule had an affinity of 9 × 10−9 M to its target IL-2R alpha, whereas the hyperchimeric humanized version had an affinity of 3 × 10−9 M– still very high (Queen et al., 1989). The original humanized anti-Tac monoclonal antibody, daclizumab, and the parent murine version manifested comparable inhibition of the T-cell proliferation in response to the tetanus-toxoid antigen indicating that humanization was not associated with the loss of functional activity. Furthermore, in contrast to the murine version, daclizumab manifested ADCC with human mononuclear cells (Junghans et al., 1990).
Finally, the humanized version daclizumab had a prolonged terminal t1/2 of 20 ± 0.6 days in humans.
Daclizumab is being evaluated in clinical trials of patients with CD25-expressing ATL (Morris and Waldmann, 2000). An escalation analysis was used to define the dose required to maintain saturation of the IL-2 receptor. In patients with high numbers of leukemic/lymphoma cells, a dose of 8 mg/kg of daclizumab every 3 weeks was required to maintain saturation of the IL-2R alpha expressed on the lymph node cells of the patients. Daclizumab therapy was associated with partial responses that were predominantly observed in patients with smoldering or chronic ATL who appear to be in the IL-2/IL-2R alpha autocrine phase of their disease.
Daclizumab armed with radionuclide
In an alternative approach, anti-Tac and its humanized form, daclizumab, were armed with radionuclides for use in systemic radioimmunotherapy of leukemia/lymphoma. Our systemic radioimmunotherapy clinical trials have focused on the use of 90Y linked to anti-Tac. Eighteen patients with ATL were treated with a total of 55 doses of 90Y-labeled anti-Tac involving 5–15 Ci of 90Y-labeled anti-Tac per patient dose (Waldmann et al., 1995). Ten of the evaluable patients responded to 90Y-anti-Tac with partial (eight) or complete (two) remissions. The only meaningful (>grade 3) toxicity was limited to the hematopoietic system. Recently, we have also had encouraging results with the humanized form of 90Y daclizumab in the treatment of patients with Hodgkin's disease where there were seven complete responses (CRs) and five partial responses (PRs) observed in the 17 patients with Hodgkin's disease treated with repeated doses of 15 Ci of 90Y (O'Mahony et al., 2006). Thus, it appears that daclizumab armed with the radionuclide 90Y provides meaningful therapy for select patients with ATL and Hodgkin's disease.
A number of approaches could be exploited to optimize the action of daclizumab in the therapy of IL-2R alpha (CD25) expressing lymphoid leukemias and lymphomas. A paradigm is being established that monoclonal antibodies will not be used in monotherapy of human malignancy, but rather will be used in association with an array of agents including chemotherapeutic agents that utilize a different mode of action. In the murine MET-I model, there was considerable synergy when the daclizumab antibody was used in conjunction with Velcade (bortezoumib) or with flavopiridol (Tan and Waldmann, 2002; Zhang et al., 2005). Furthermore, another insight derived from the studies involving systemic radioimmunotherapy in preclinical models of adult human T-cell leukemia should be translated into clinical trials. In particular, patients who have T-cell leukemia with its isolated cells could be treated with antibodies linked to select alpha-emitting radionuclides such as astatine-211 in lieu of the beta-emitting radionuclide yttium-90 whose cross-fire effect is relatively ineffective when directed toward isolated malignant cells (Zhang et al., 2006).
In summary, basic insights concerning the IL-2/IL-2R system, developed using the daclizumab monoclonal antibodies, when coupled with the experience with this humanized antibody in clinical trials are providing an effective monoclonal antibody-mediated approach for the treatment of select patients with IL-2R alpha (CD25) expressing leukemia and lymphoma.
This work was supported by the Intramural Research Program of the National Cancer Institute, NIH. All animal studies were approved by the Animal Care and Use Committee of the National Cancer Institute (NCI) and all clinical investigations received prior approach by the IRB, NCI.
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Arthritis Research & Therapy (2011)