Journal of Clinical Immunology

, Volume 27, Issue 1, pp 1–18

Anti-Tac (daclizumab, Zenapax) in the Treatment of Leukemia, Autoimmune Diseases, and in the Prevention of Allograft Rejection: A 25-Year Personal Odyssey

Authors

    • Metabolism Branch, Center for Cancer ResearchNational Cancer Institute, NIH
    • Metabolism Branch, Center for Cancer ResearchNational Cancer Institute
Article

DOI: 10.1007/s10875-006-9060-0

Cite this article as:
Waldmann, T.A. J Clin Immunol (2007) 27: 1. doi:10.1007/s10875-006-9060-0

Abstract

Twenty-five years ago, we reported the production of the monoclonal antibody, anti-Tac that identifies the IL-2 receptor alpha subunit and blocks the interaction of IL-2 with this growth factor receptor. In 1997, daclizumab (Zenapax®), the humanized form of this antibody, was approved by the FDA for use in the prevention of renal allograft rejection. In addition, we demonstrated that daclizumab is of value in the treatment of patients with noninfectious uveitis, multiple sclerosis, and the neurological disease human T-cell lymphotropic virus I associated myelopathy/tropical spastic paraparesis (HAM/TSP). Others demonstrated therapeutic efficacy with daclizumab in patients with pure red cell aplasia, aplastic anemia, and psoriasis. Thus, translation of basic insights concerning the IL-2/IL-2 receptor system obtained using the monoclonal antibody daclizumab provided a useful strategy for the prevention of organ allograft rejection and the treatment of patients with select autoimmune diseases or T-cell leukemia/lymphoma.

Key Words

Anti-Tacadult T-cell leukemiadaclizumabIL-2 receptormonoclonal antibody

INTRODUCTION

Twenty-five years ago in 1981, we reported the production of the monoclonal antibody, anti-Tac directed toward the IL-2 receptor alpha subunit (1, 2). In its humanized form, daclizumab (Zenapax) was approved for use in renal allograft protocols by the FDA as the third monoclonal antibody, the first humanized antibody, and the first antibody directed toward a cytokine receptor that was approved for therapy (3). This antibody played a pivotal role in the revolution that has occurred in our understanding of the roles played by the IL-2/IL-2 receptor system in the normal immune response and the disorders in this system that underlie T-cell leukemia, select autoimmune disorders, and allograft rejection. Furthermore, these studies have culminated in the definition of the IL-2 receptor alpha subunit as an exceptionally valuable target for immunotherapy (47). The scientific basis for this choice of target was the demonstration that very few normal resting cells express IL-2R alpha. However, this receptor subunit is expressed by abnormal T-cells in patients with lymphoid malignancies, those T-cells involved in autoimmune diseases as well as T-cells participating in allograft rejection (46). Different forms of IL-2R alpha directed therapy have been employed. These include unmodified antibodies to IL-2R alpha (anti-Tac, daclizumab, Zenapax) and this antibody armed with toxins or alpha and beta-emitting radionuclides (4, 710). The administration of the antibody, anti-Tac, in its humanized form (daclizumab) contributed to the reduction of acute renal allograft rejection episodes (3, 10, 11). In addition to its use in the prevention of organ allograft rejection, we and our collaborators have shown that daclizumab is of value in the treatment of T-cell mediated autoimmune disorders. In particular, daclizumab provided effective therapy for patients with noninfectious uveitis who were able to be weaned off their systemic immunosuppressive medications (12, 13). Furthermore, there was a 78% reduction in the development of gadolinium-enhanced MRI lesions in patients with multiple sclerosis failing beta interferon therapy when they were treated with daclizumab (14). In addition, murine anti-Tac and daclizumab provided effective therapy for subsets of patients with HTLV-I associated adult T-cell leukemia (ATL) and the neurological disease, human T-cell lymphotropic Virus I associated myelopathy/tropical spastic paraparesis (HAM/TSP) (15). Finally, in clinical trials that employed this antibody directed to the IL-2 receptor alpha armed with 90Y, we observed remissions in over 50% of the patients with ATL and 70% of patients who had Hodgkin's disease (8, 16).

In the present report, we focus on our 25-year odyssey with the anti-Tac monoclonal antibody. These studies were directed toward the definition of the roles played by IL-2 in the life and death of lymphocytes, and on the disorders of the IL-2/IL-2 receptor system that underlie many clinical disorders. Finally, an emphasis of this report is on the translation of these basic insights into novel cytokine receptor directed monoclonal antibody mediated approaches for the treatment of T-cell leukemia/lymphoma, autoimmune diseases, and for the prevention of organ allograft rejection.

THE BEGINNING: THE PRODUCTION OF THE ANTI-TAC MONOCLONAL ANTIBODY AND ITS CHARACTERIZATION AS AN ANTIBODY DIRECTED TO A SUBUNIT OF THE IL-2 RECEPTOR

In 1975, Köhler and Milstein's development of the hybridoma technology to produce monoclonal antibodies captured the imagination of medical scientists and rekindled interest in the use of antibodies as agents to treat patients with cancer (17). However, the initial use of monoclonal antibodies was relatively disappointing; the dream of a “magic bullet” involving antibody therapy presented by Paul Ehrlich in his Croonian lecture in 1900 where he stated that “Immunizations such as these which are of great theoretic interest may come to be available for clinical application attacking epithelial new formations, particularly carcinoma by means of specific anti-epithelial sera,” proved elusive (18). At the time when our studies were initiated, in the late 1970s and early 1980s, no therapeutic monoclonal antibody had been approved for use by the FDA. Our own efforts were focused on the question “How do T-cells proliferate and develop effector functions following activation”? It had been recognized that activation of T-cells requires two sets of signals from cell surface receptors to the nucleus (19). The first signal was initiated by the interaction of appropriately processed foreign antigen presented to the T-cell receptor specific for that antigen. Following this interaction, in the context of products of the major histocompatibility locus, T-cells were induced to express interleukin-2 which interacted with specific high-affinity membrane IL-2 receptors. The presence of receptors specific for IL-2 was suggested by Robb and coworkers who utilized purified biosynthetically labeled IL-2 to demonstrate specific saturable, high-affinity binding sites on IL-2 dependent T-cell lines as well as mitogen and alloantigen-activated T-cells (20). Further progress in the analysis of the structure, function, and expression of the human IL-2 receptor was greatly facilitated by the production of the anti-Tac monoclonal antibody in our laboratory by Takashi Uchiyama (1, 2, 21). Uchiyama's production of the anti-Tac monoclonal antibody was a classical example of serendipity. He used as his target a T-cell line we had developed from a patient who carried the diagnosis of the Sézary T-cell leukemia, but who in retrospect had HTLV-I associated adult T-cell leukemia. The goal of our efforts was to produce an anti-CD4 monoclonal antibody during a period when such antibodies were being embargoed and were not made available to the scientific community. However, the antibody we produced, anti-Tac (T-cell activation antigen) did not bind to the CD4 antigen. Indeed, the antibody did not react with the vast majority of resting cells, but only with activated T-cells. The anti-Tac monoclonal antibody was rejected by the First Leukocyte Differentiation Workshop since that workshop did not include activated cells in the panel being analyzed with the different monoclonal antibodies submitted. The anti-Tac monoclonal antibody that we had selected, on the basis of its ability to bind to activated T-cells but not to the majority of resting T-cells, B cells, or monocytes, reflected a pattern of cellular reactivity identical to the distribution of IL-2 receptors that had been reported by Robb and coworkers (20). Therefore, with Warren Leonard and Warner Greene in the laboratory taking leadership positions, we hypothesized that anti-Tac recognized the human receptor for IL-2 (22). Data in support of this hypothesis were: (a) Anti-Tac blocked the IL-2 induced DNA synthesis of IL-2 dependent continuous T-cell lines but did not inhibit the DNA synthesis of IL-2 independent T-cell lines; (b) Anti-Tac blocked more than 95% of the binding of 3H—labeled IL-2 to HuT-102 B-2 cells—a cell line derived from the patient whose adult leukemic T-cells were used in the generation of the anti-Tac monoclonal antibody; (c) Anti-Tac blocked the IL-2 binding to phytohaemagglutinin (PHA) activated lymphoblasts; and (d) IL-2 at high concentrations blocked the binding of 3H-labeled anti-Tac to PHA-activated lymphoblasts. These were important findings since anti-Tac was one of the first, if not the first, monoclonal antibody to define a receptor for one of the cytokines. As outlined later, the anti-Tac monoclonal antibody was used to: (a) define an IL-2 binding receptor subunit that participates in the human receptor for IL-2; (b) molecularly clone cDNAs for the 55-kDa peptide of the human IL-2 receptor; (c) define the cellular distribution of high-affinity IL-2 receptors; (d) determine the immunoregulatory effects that require the interaction of IL-2 with its receptor; (e) analyze disorders of IL-2 receptor expression in disease; and (f) develop novel therapeutic strategies for patients with IL-2 receptor expressing T-cell leukemia, autoimmune disorders, and for individuals receiving organ allografts (46).

CHEMICAL CHARACTERIZATION AND MOLECULAR CLONING OF THE IL-2 RECEPTOR SUBUNIT IDENTIFIED BY ANTI-TAC

The IL-2 binding receptor subunit identified by the anti-Tac monoclonal antibody was shown to be a densely glyclosylated, sulfated, integral membrane protein with an apparent Mr of 55,000 (22, 23). Using the anti-Tac monoclonal antibody to purify the receptor peptide, Warren Leonard, Warner Greene, and other members of our group succeeded in cloning, sequencing, and expressing cDNAs encoding the 33-kDa polypeptide backbone of the 55-kDa IL-2 receptor protein (24). The deduced amino-acid sequence of the IL-2 receptor indicated that this protein is composed of 272 amino acids, including a 21 amino-acid signal peptide (24). The receptor contains two potential N-linked glyclosylation sites and multiple possible O-linked carbohydrate sites. There is a single hydrophobic membrane region of 19 amino acids and a very short 13 amino acid cytoplasmic domain. The cytoplasmic domain of the IL-2 receptor subunit identified by anti-Tac thus appeared to be too small for enzymatic function. Potential phosphate acceptor sites (serine, threonine, but not tyrosine) were present within its intracytoplasmic domain. Thus, this receptor subunit differed from other known growth factor receptors that have large intracytoplasmic domains with tyrosine-kinase activity. Leonard and coworkers demonstrated that the single gene encoding the IL-2 receptor alpha subunit, identified by anti-Tac, consists of eight exons located on chromosome number 10 at p14 (25).

The observation that the 13-amino acid intracytoplasmic domain was too short to function in signaling raised the issue of how the IL-2 receptor signals were transduced to the nucleus. Furthermore, questions were posed concerning the IL-2 receptor that were difficult to answer when only the 55 kDa IL-2R alpha protein identified by anti-Tac was considered. These questions included: What is the structural explanation for the great difference in affinity between high (10−11 M) and low (10−8 M) affinity receptors? How could certain non-Tac expressing cells including resting natural killer cells respond to IL-2? In association with Mitsuru Tsudo, who had joined our Laboratory, we resolved these issues in parallel with investigators in the laboratory of Warren Leonard, by codiscovering a novel non-Tac 70 kDa IL-2 binding protein, IL-2R beta (26, 27). Subsequently, Shugamura and coworkers demonstrated that the 64 kDa IL-2R gamma chain or γc is required for high-affinity IL-2 binding and signaling (28). The beta and gamma subunits are shared by other cytokines, with the IL-2R beta subunit being utilized by IL-15 as well as IL-2, and the common gamma chain a participant not only of the IL-2 receptor but also of the IL-4, IL-7, IL-9, IL-15, and IL-21 receptors (Fig. 1) (28, 29).
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Fig. 1

The structure of the heterotrimeric IL-2 receptor. The heterotrimeric IL-2 receptor involves the private IL-2R alpha subunit, IL-2R beta shared with IL-15, as well as the common gamma (γc) subunit shared with IL-4, IL-7, IL-9, IL-15, and IL-21. The IL-2 receptor uses a signaling pathway that involves Janus kinase-1 (Jak-1), Jak-3, and STAT-5 (signaling transducer and activator of transcription-5).

DISTRIBUTION OF INTERLEUKIN-2 RECEPTORS

The cellular distribution of the 55 kDa subunit of the IL-2 receptor has been defined using the anti-Tac monoclonal antibody for humans or with alternate antibodies to the IL-2R alpha receptor subunit (CD25) for other species (4, 5). Less than 5% of unstimulated human peripheral blood T-cells react with the anti-Tac monoclonal antibody. Furthermore, a proportion of immature thymocytes display this receptor subunit. In addition, anti-Tac reacts with small subsets of T and NK cells with regulatory function (30, 31). In particular, this CD25 receptor subunit is expressed by CD4+ CD25+ Foxp3 expressing T regs that inhibit immune responses (30). Furthermore, CD25 identified by anti-Tac is expressed by a small subset of NK T-cells with regulatory function, the CD56 bright IL-10 producing, negative regulatory NK cells (31). The majority of T-cells can be induced to express IL-2 receptor alpha following interaction with lectins, antigens, or alloantigens or by the addition of monoclonal antibodies to the T-cell antigen receptor complex, by appropriate pairs of anti-CD2 antibodies or an antibody to CD28 (4, 5, 32). On activated T-cells, there is a 5–20-fold greater number of IL-2R alpha subunits than high-affinity receptors. Isolated IL-2R alpha subunits bind IL-2 at 10−8 M whereas the trimeric (alpha, beta, gamma) IL-2 receptor binds IL-2 at high-affinity 10−11 M (4). The peptide identified by anti-Tac is also expressed by activated B cells and activated NK cells (33). Furthermore, this receptor subunit has been detected on activated cells of the monocyte-macrophage series, including cultured monocytes, Kupffer's cells of the liver, cultured lung macrophages, and Langerhan's cells of the skin (34, 35).
Table I.

Causes of Elevated Serum Soluble IL-2R Alpha Concentrations

Allograft rejection

Neoplasia

Autoimmune diseases

Infectious diseases

Renal

Acute myelocytic leukemia

Aplastic Anemia

HIV/AIDS

Liver

Anaplastic large-cell lymphoma

Behcet's syndrome

Pulmonary tuberculosis

Cardiac

Adult T-cell leukemia/lymphoma

Crohn's disease

Rubeola

Bone marrow

Chronic lymphocytic leukemia

Giant cell arteritis

Infectious mononucleosis

 

Chronic myelocytic leukemia

Juvenile rheumatoid arthritis

Sepsis

 

Cutaneous T-cell lymphoma

Kawasaki disease

 
 

Mycosis fungoides

Multiple sclerosis

Other

 

Hairy cell leukemia

Polymyalgia rheumatica

 
 

Hodgkin's disease

Rheumatoid arthritis

End-stage renal disease

 

Non-Hodgkin's lymphomas (B-cell)

Sarcoidosis

IL-2 administration

 

Peripheral T-cell lymphomas

Scleroderma

 
  

Sjögren's syndrome

 
  

Systemic lupus erythematosus

 
  

Vasculitis

 
  

Wegerner's granulomatosis

 

Activation through the T-cell receptor complex that is associated with an increase in intracellular calcium and involvement of protein kinase-C leads to the expression of IL-2R alpha (CD25). IL-2 receptors may also be induced without interaction with the T-cell antigen receptor by the use of a calcium ionophore or by phorbol myristic acetate (36, 37). After activation with mitogenic lectins, the number of IL-2 receptors reaches a maximum at 48–72 h. Following this period, there is a progressive decline in the number of receptors so that at 7–21 days in culture the number of receptors is fewer than 20% of the number during peak expression. Several reports have indicated that IL-2 upregulates the expression of the IL-2 specific receptor alpha subunit identified by anti-Tac (38, 39).

ABNORMALITIES OF IL-2 RECEPTOR EXPRESSION IN HTLV-I ASSOCIATED DISORDERS

Rubin and coworkers demonstrated that activated normal peripheral blood mononuclear cells and certain lines of T and B-cell origin release a soluble 45 kDa form of the IL-2 receptor alpha into the culture media (40). Using an enzyme-linked immunoabsorbant assay that employs two monoclonal antibodies that recognize distinct epitopes of human IL-2R alpha, they showed that normal individuals express measurable amounts of the IL-2 receptor subunit in their serum. Certain lymphoreticular malignancies, autoimmune disorders, and allograft rejections are associated with elevated serum levels of this receptor subunit (Table I) (40). The release of the soluble IL-2 receptor appears to be associated with the activation of various cell types and may be observed in association with the disordered regulation of the immune response in diverse diseases. Thus, determination of the serum levels of the IL-2 alpha receptor subunit provides a valuable, noninvasive approach to the analysis of both normal and disease-associated lymphocyte activation in vivo. The use of this technique and the direct demonstration of CD25 with the anti-Tac monoclonal antibody on T-cells of the affected tissues have led to the definition of an array of malignant and benign disorders associated with abnormally elevated levels of IL-2R alpha expression (Table I). In particular, elevated serum-soluble IL-2 receptor alpha levels and in select cases high IL-2R alpha expression on abnormal cells, have been demonstrated with the malignant cells in patients with adult T-cell leukemia, cutaneous T-cell lymphoma, hairy cell B-cell leukemia, Hodgkin's disease, as well as acute and chronic granulocytic leukemia (4, 5, 36). Furthermore, such abnormalities of IL-2R alpha expression have been demonstrated in the autoimmune diseases, rheumatoid arthritis, systemic lupus erythematosus, aplastic anemia, insulin-dependent diabetes mellitus (IDDM), Crohn's disease, sarcoidosis, scleroderma, noninfectious uveitis, chronic active hepatitis, multiple sclerosis, and tropical spastic paraparesis. Furthermore, such elevations of IL-2R alpha expression have been detected in the serum of patients during organ allograft rejection and from those with graft versus host disease (4, 10).

LYMPHOCYTE FUNCTIONS INHIBITED BY ANTI-TAC (DACLIZUMAB)

In early studies utilizing the anti-Tac monoclonal antibody, lymphocyte functions that involve the interaction of IL-2 with its inducible receptor on activated T-cells were defined. The addition of anti-Tac to cultures of human peripheral blood mononuclear cells inhibited the proliferation associated with the addition of soluble, autologous, and allogenic antigens (32). Furthermore, anti-Tac inhibited T-cell proliferation induced by mitogenic lectins. Anti-Tac inhibition of antigen and mitogen-induced T-cell proliferation was reversed by the addition of purified IL-2. Anti-Tac inhibited the generation of cytotoxic T-lymphocytes in allogenic cell cultures, but did not inhibit their action once generated (2). Furthermore, anti-Tac inhibited the generation of inhibitory T-cells activated by lectins or by the plate-bound CD3 monoclonal antibody. In addition, anti-Tac inhibited immunoglobulin production by B cells activated by pokeweed mitogen, wheat germ agglutinin, streptolysin O, or Nocardia water-soluble mitogen (41).

More recent studies, including those involving the genetic deletion of IL-2 and its private subunit, IL-2R alpha, have revealed the redundancy of the cytokine system demonstrating that many of the functions mediated by IL-2 that were defined in early studies are shared by other cytokines (4248). In particular, as might be anticipated from their sharing of IL-2R beta and γc subunits, IL-15 and IL-2 share a number of biological activities including the stimulation of the proliferation of activated CD4+, CD8+, as well as gamma delta subsets of T-cells (44). The two cytokines facilitate the induction of cytotoxic effector cells including cytotoxic T-lymphocytes (CTL) and lymphokine-activated killer cells (LAK) cells. IL-2 and IL-15 also stimulate the proliferation of NK cells and both cytokines induce the proliferation and immunoglobulin synthesis by human B-cells stimulated with anti-IgM or CD40 ligand.

In addition to these common functions, although IL-2 and IL-15 share two receptor subunits they have contrasting functions in many adaptive immune responses (4448). IL-2 is pivotally involved in the maintenance and fitness of CD4+ CD25+ Foxp3 expressing T regs (49). Furthermore, IL-2 plays a critical role in AICD, a process that leads to the elimination of self-reactive T-cells (50). In contrast, in IL-15 transgenic mice, there is an inhibition of IL-2 activation-induced cell death (AICD) (47). Furthermore, IL-15 stimulates the maintenance of CD44hi CD8+ memory phenotype T-cells (4448). These observations from ex vivo functional studies are supported by an analysis of mice with disrupted cytokine and cytokine receptor genes. IL-2 deficient and IL-2R alpha deficient mice develop a marked enlargement of peripheral lymphoid organs associated with polyclonal T- and B-cell expansion which reflects the impairment of T reg maintenance and function, and loss of AICD (42, 43). These IL-2R alpha deficient mice and IL-2R deficient humans develop autoimmune disorders such as hemolytic anemia and inflammatory bowel disease (42, 43, 51). Thus, a special role of IL-2 is to prevent a T-cell mediated immune response to self. By contrast, mice genetically deficient in IL-15 or IL-15R alpha, the private receptor for IL-15, do not develop lymphoid enlargement, increased immunoglobulin levels, or autoimmune diseases. Rather, they display a marked reduction in the number of thymic and peripheral NK cells, NK-T-cells, and intraintestinal intraepithelial lymphocytes (IELs) (52, 53). Thus, a special role of IL-15 is to maintain a sustained, long-lasting immune response to invading pathogens.

EARLY THERAPEUTIC TRIALS OF IL-2R ALPHA DIRECTED MONOCLONAL ANTIBODY THERAPY: FROM MURINE ANTI-TAC TO DACLIZUMAB (ZENAPAX)

The initial use of monoclonal antibodies as therapeutic agents was relatively disappointing. At the time in 1983, when we initiated our clinical trials with murine anti-Tac, no monoclonal antibody had received FDA approval for use. Our observation that very few resting normal cells display the alpha subunit of the IL-2 receptor whereas it is expressed by the malignant T-cells in many leukemias and lymphomas, especially those of HTLV-I associated adult T-cell leukemia as well as by the T-cells involved in an array of autoimmune diseases and by those involved in allograft rejection, provided the scientific basis for the initiation of therapeutic studies with the anti-Tac monoclonal antibody (4, 7). Such IL-2R alpha directed agents could theoretically eliminate Tac expressing leukemic cells or activated T-cells and their precursors involved in other disease states and in allograft rejection, while retaining the Tac negative, normal T-cells that express the full repertoire of antigen receptors required for T-cell immune responses. In the first phase of these efforts, we translated the insights from the Laboratory and validated them in preclinical animal studies. In particular, in collaboration with Robert Kirkman, of Harvard Medical School, we demonstrated the efficacy of anti-Tac in the prevention of renal allograft rejection in cynomolgus monkeys (54). In parallel, in our own Laboratory, we demonstrated that the murine version of the anti-Tac monoclonal antibody delayed cardiac allograft rejection in cynomolgus monkeys (55).
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Fig. 2

Effective therapy provided by anti-Tac for a patient with HTLV-I associated adult T-cell leukemia. A patient with acute adult T-cell leukemia was treated with anti-Tac as indicated by the solid bars. There was an elimination of all circulating leukemic T-cells defined as cells expressing high levels of IL-2R alpha, CD4, CD3dim but not CD7. This complete response has been maintained for the 17-year period of observation after initiation of the short-course of anti-Tac therapy.

The first clinical trials involving murine anti-Tac were directed toward the treatment of patients with adult T-cell leukemia—a malignancy of mature CD4+ CD25+ lymphocytes that is caused by the retrovirus HTLV-I (7, 56, 57). No chemotherapeutic regimen appeared successful in altering the survival of these patients who have a median survival duration of only 9 months (57). The retrovirus HTLV-I encodes a transactivating protein, tax, that indirectly stimulates the transcription of numerous host genes including those of IL-2 and IL-2R alpha (58). The malignant ATL cells constitutively express approximately 10,000–28,000 IL-2R alpha receptor subunits identified by the anti-Tac monoclonal antibody whereas most of the patients’ normal resting cells do not express this receptor subunit (59). These observations stimulated us to perform a therapeutic trial with the unmodified murine version of the anti-Tac monoclonal antibody (7). Seven of the 19 patients treated developed a mixed (one case), partial (four cases), or complete (two cases) response. In one case, the remission has persisted for more than 17 years after initiation of a short course of therapy (Fig. 2). None of the patients treated suffered any untoward reactions. However 6 of 10 patients with an anti-Tac induced clinical remission produced antibodies to the murine anti-Tac monoclonal antibody.

HUMANIZATION OF THE ANTI-TAC MONOCLONAL ANTIBODY TO GENERATE DACLIZUMAB (ZENAPAX)

Although murine antibodies such as murine anti-Tac may be of value in the therapy of human diseases, their effectiveness is limited because rodent monoclonal antibodies induce an immune response that neutralizes their therapeutic effect. In particular, in a clinical trial directed toward the prophylaxis of renal allograft rejection, treatment with murine anti-Tac led to the development in 7 of 10 patients of antibodies directed to the infused monoclonal antibody (60). In addition, murine anti-Tac had a short 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 the receptors required to prevent their interaction with the growth factor IL-2. An additional problem is that the responses induced by murine anti-Tac are limited, because the antibody does not function in antibody dependent cellular cytotoxicity (ADCC) with human mononuclear cells and does not fix human complement and therefore, is relatively ineffective as a cytocidal agent. To circumvent these limitations, genetically engineered antibody variants were produced that combined the rodent variable or hypervariable regions with the human constant or constant and framework regions. Initially, chimeric versions were produced where human constant regions were joined to mouse variable regions. Such chimeric antibodies represented an improvement over mouse antibodies in human patients as they were presumably less immunogenic and sometimes mediated ADCC more effectively. For example, chimeric anti-Tac that included the human IgG1 constant region mediated ADCC with activated human effector cells, whereas the murine anti-Tac did not (61, 62). However, the mouse variable region can itself be highly immunogenic. Jones, Winter, and colleagues therefore took the further innovative step of combining the complementarity determining regions (CDRs) from a mouse (or rat) antibody with the framework from a human antibody thereby reducing the xenogeneic elements in the humanized antibody to a minimum (63). Unfortunately, in many cases, including that of anti-Tac, the fully humanized antibody generated had significantly less binding affinity for antigen than the original mouse antibody. We joined with Cary Queen in generating a humanized anti-Tac monoclonal antibody reactive with the human IL-2R alpha subunit at high affinity and introduced two ideas that have wider application (61). First, 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 complementary determining regions (CDRs). Second, computer modeling was used to identify several framework amino acids in the mouse antibody that might interact with the CDRs or directly with antigen and those murine or more typical human amino acids at positions 27, 30, 48, 66, 67, 94 and 103 in the VH and at positions 48 and 60 in the VL were transferred to the human framework along with the CDRs. This latter action proved to be critical in generating a high-affinity humanized anti-Tac. The IgG1 isotype was chosen since antibody effector functions were desired. The primary goal in these studies was to maintain the affinity and functional capacity of the mouse monoclonal antibody. The parent murine anti-Tac molecule had an affinity of 9×10−9 M whereas the hyperchimeric humanized version had an affinity of 3×10−9 M—still very high (61). The original humanized anti-Tac monoclonal antibody and the parent murine version manifested comparable inhibition of T-cell proliferation in response to tetanus antigen indicating that humanization was not associated with the loss of functional activity (62).

A major opportunity provided by genetic engineering of monoclonal antibodies is the ability to alter the pharmacokinetics of the immunoglobulin molecule. One may wish to prolong the survival of a monoclonal antibody to increase its period of effective action. In the case of anti-Tac, the pharmacokinetics of the radiolabeled humanized version differed substantially from that of the murine anti-Tac when administered to normal cynomolgus monkeys, with a prolongation of the mean terminal half-time of humanized anti-Tac (daclizumab) to 103 h as compared to 38 h for murine anti-Tac (55). When used in humans, humanized anti-Tac yielded a terminal T1/2 of 20 ± 0.6 days (10).

One of the primary goals in the generation of humanized antibodies is to reduce their immunogenicity. Humanized anti-Tac was dramatically less immunogenic than murine anti-Tac when administered to cynomolgus monkeys undergoing heterotopic cardiac allografting (55). Specifically, all monkeys treated with murine anti-Tac developed antibodies to the administered monoclonal antibody by day 15 (mean onset 11 days). In contrast, none of the animals receiving humanized anti-Tac produced antibodies to this monoclonal antibody version (55). A similar marked reduction in immunogenicity has been observed in humans when an assay for such antibodies that does not yield false positives was utilized.

Finally, treatment with anti-Tac prolonged graft survival in a cynomolgus heterotopic cardiac allograft model (55). In animals that received murine anti-Tac, the allograft survival was increased significantly compared to that of a control group (mean survival 14 ± 1.98 days compared to 9.2±0.5 days; P<0.025). Graft survival was further prolonged by administration of humanized anti-Tac (with a mean survival of 20±0.6 days compared to 9.2±0.5 days in controls P<0.001 or compared to murine anti-Tac 14.0±2 days (P<0.02). There was no toxicity attributable to the administration of either form of anti-Tac. Thus, in primate studies, humanized anti-Tac (daclizumab) exhibited reduced immunogenicity, improved pharmacokinetics, and increased ability to prolong cardiac allograft survival relative to the murine antibody. These studies suggested that humanized anti-Tac might be of value as an adjunctive standard immunosuppressant therapy for patients receiving organ transplants and in the treatment of those with leukemias and lymphomas or autoimmune disorders.

DACLIZUMAB THERAPY IN INDIVIDUALS RECEIVING ORGAN ALLOGRAFTS

Organ allograft rejection is associated with an elevated s-IL-2R alpha in the serum that is linked to the activation of T-cells mediated by MHC mismatched recognition. As noted earlier, murine as well as humanized anti-Tac prolonged renal and cardiac allograft survival in cynomolgus monkeys. In a collaborative study with Kirkman and coworkers, the efficacy of standard triple immunosuppressive therapy (Cyclosporin, Azathioprine, and Prednisone) was compared to murine anti-Tac delivered with the same treatment for the prevention of rejection in 80 patients who received renal allografts (60). The addition of murine anti-Tac significantly delayed the time to first rejection, however there was no difference in actual or actuarial graft or patient survival between the two groups. Therapy with murine anti-Tac appeared to be limited by the development of antiidiotypic antibodies. To circumvent this problem of immunogenicity, a humanized version of anti-Tac (daclizumab, Zenapax) was produced by Cary Queen and coworkers of Protein Design Labs Inc. (61). Two major Phase III studies were used to evaluate the clinical efficacy of daclizumab compared with a placebo (10, 64). Both studies were double-blind, randomized, and multicenter in design. The endpoint in both trials was the incidence of biopsy-proven rejection in the first 6 months after transplantation. In the multicenter Phase III, performed in the United States, which was a randomized double-blind placebo-controlled study, 260 patients undergoing a first cadaveric renal transplant were randomized to standard treatment with Cyclosporin A, Azathioprine, and corticosteroids or alternatively this regimen plus daclizumab (10). Daclizumab was administered intravenously at 1 mg/kg within 24 h preoperatively and postoperatively at weeks 2, 4, 6, and 8 for a total of five doses. This was sufficient to maintain therapeutic serum concentrations (5–10 μg/ml) throughout the period of antibody administration with receptor saturation maintained for 90–120 days. Of the 126 patients receiving daclizumab, 28 (22%) had a biopsy confirmed episode of rejection compared with 35% of 134 in the control patient group that received standard immunosuppression plus placebo (P=0.03). There was however no statistically significant difference in graft survival between the two treatment groups at 1 year. The incidence of adverse events related to treatment was similar in placebo and daclizumab recipients. In particular, daclizumab therapy was not associated with an increase in infections or malignancies including lymphoma. In the second European multicenter Phase III randomized double-blind placebo controlled trial, daclizumab was compared with a placebo in 275 patients who were also receiving a less intensive immunosuppressive regimen of Cyclosporin and corticosteroids (64). At 6 months, 28% of patients on the daclizumab arm had a biopsy confirmed episode of acute rejection compared with 47% of patients in the placebo arm (P=0.001). The patients on the daclizumab treatment arm had better graft function, reduced requirement for antithymocyte or anti-lymphocyte globulin, lower administered corticosteroid doses, a lower incidence of cytomegalovirus infections, a lower incidence of infectious deaths, and a greater 1-year survival than did patients on the placebo arm (99% vs. 94%, p=0.01) (64). In a similar extensive randomized trial, it was shown that basiliximab, a chimeric antibody also directed toward IL-2R alpha was associated with reduced numbers of acute rejection episodes in renal allograft recipients. On the basis of the efficacy in multicenter trials and the lack of associated increase in toxicity, the FDA provided marketing clearance for the use of daclizumab in the prevention of acute kidney transplant rejection in 1997 (3, 65). Thus, although the magnitude of the effects of daclizumab on long-term patient and graft survival are not fully defined, the available data indicate that daclizumab is an important advance in renal transplant immunosuppression, reducing acute graft rejection without affecting the tolerability of standard Cyclosporin-based immunosuppression (65).

Daclizumab treatment has been shown to improve the survival of other organ allografts. Daclizumab therapy was associated with decreased organ rejection and no increased mortality in cardiac transplant patients receiving Mycophenolate Mofetil, Cyclosporin, and corticosteroids (66). Fifty-five patients undergoing a first cardiac allograft were randomized to Cyclosporin, Mycophenolate Mofetil, and Prednisone with or without daclizumab 1 mg/kg administered intravenously every 2 weeks for five doses (66, 67). Acute rejection episodes occurred in 17 of 27 patients on standard immunosuppression and in 5 of 28 patients on the standard immunosuppression plus daclizumab treatment (P=0.04). In an analysis by the Scientific Registry of Transplant Recipients of all adult cardiac transplants performed in the USA between January 1998 and October 2003, patients were selected based on induction treatment, either daclizumab (n=684) or no induction (n=2525) (67). The patients receiving daclizumab had no increased risk of patient death or infectious disease. Furthermore, patients receiving daclizumab had a lower incidence of acute rejection at 6 months (P=0.005), 12 months (P≤0.001), and over 3 years (P=0.006).

In two clinical trials, daclizumab in a two-dose regimen reduced the incidence of liver transplant rejection, facilitated improved renal function, and was not associated with side effects (68, 69).

In addition, Shapiro and coworkers incorporated daclizumab in their glucocorticoid-free immunosuppressive regimen for pancreatic islet cell transplantation in patients with type-1 diabetes and demonstrated that islet transplantation with this regimen can result in Insulin independence (70). Thus, in clinical trials involving large groups of recipients, daclizumab therapy provided a reduction of rejection episodes in patients receiving renal, liver, cardiac, and islet transplants.

In general, trials that used daclizumab to prevent allograft rejection did not take advantage of our knowledge that in order to maintain efficacy it would probably require the administration of daclizumab on a q. 3 or 4-week basis indefinitely to maintain the saturation of the IL-2 receptor required for its continued effective action. Thus, if one wishes to make a major long-term difference in organ and patient survival, it is likely that one must utilize the approach described later for patients with uveitis and multiple sclerosis treated with daclizumab where a strategy of persistent therapy with the agent involves using a dosing scheme that maintains receptor saturation perpetually (12, 14).

DACLIZUMAB THERAPY OF CD25 (IL-2R ALPHA) EXPRESSING LEUKEMIA/LYMPHOMA

IL-2R alpha (CD25), the target of daclizumab, is expressed by the malignant cells from patients with a variety of lymphoproliferative disorders including the malignant cells in adult T-cell leukemia/lymphoma (ATL), cutaneous T-cell lymphomas, hairy cell leukemia, the Reed–Sternberg cells, and associated polyclonal T-cells in Hodgkin's disease, anaplastic large cell lymphoma, and some other B-cell neoplasms (4, 7, 16). To evaluate different therapeutic approaches involving daclizumab (humanized anti-Tac) in ATL, we developed a murine model of human adult T-cell leukemia (71). In particular, an in vivo murine model of human ATL (MET-1 model) in nonobese, diabetic, (NOD)/severe combined immunodeficient (SCID) mice was established by introducing human adult T-cell leukemic cells into the mice. Leukemic cell survival was monitored with surrogate markers including ELISA procedures to quantitate the serum concentrations of soluble human IL-2R alpha and human beta-2 microglobulin. The transferred disease progressed to death in the mice after about 4–6 weeks. Various forms of the antibodies to the IL-2R alpha including humanized anti-Tac (daclizumab), murine anti-Tac, and the 7G7/B6 monoclonal antibody that targets the IL-2 alpha 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 (71). It appeared in this model that the three anti-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. However, this action does not appear to be the mechanism involved in our MET-1 model of ATL. In particular, although both murine and humanized anti-Tac block IL-2 binding to the high-affinity IL-2 receptor, 7G7/B6 binds to a different epitope on the IL-2 alpha receptor subunit that is not involved in IL-2 binding (71). Furthermore, the MET-1 ATL cells obtained from the spleens of leukemic mice do not produce human IL-2, nor do they express IL-2 mRNA. In addition, they do not proliferate in response to murine IL-2. We considered an alternative mechanism, one requiring FcR receptor expression on host cells such as monocytes or polymorphonuclear leukocytes. To test this hypothesis, we joined with Jeffrey Ravetch in a collaboration that involved SCID/NOD FcRγ−/− mice that lack effective FcRγI, FcRγIII, and FcRγIV receptors (Fig. 3) (72). The three anti-IL-2R alpha-directed monoclonal antibodies that were effective in the wild-type mice were not active in these FcRγ−/− 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.
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Fig. 3

Kaplan–Meyer analysis of the effect of daclizumab on the MET-1 murine model of human adult T-cell leukemia in wild-type and FcRγ−/− mice. The MET-1 model was established by introducing human adult T-cell leukemic cells into SCID/NOD mice (71). In untreated animals, the transferred disease progressed to death in the mice after about 4–6 weeks. In the wild-type mice, daclizumab therapy significantly delayed the progression of the leukemia and prolonged the survival of tumor-bearing mice (left panel). In contrast, there was no efficacy on daclizumab therapy in FcRγ−/− 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 (right panel) (72).

In another series of studies, we evaluated the efficacy of a combination of unmodified daclizumab along with chemotherapeutic agents to follow the emerging paradigm that monoclonal antibodies for cancer therapy will not be used as standalone agents but as part of multidrug therapy. In the MET-1 model of adult T-cell leukemia, PS341 (Velcade) or Flavopiridol had only modest efficacy when utilized alone. However, there was a marked synergy in the therapeutic efficacy in the MET-1 murine model of ATL observed when Flavopiridol or Velcade were used in concert with the anti-CD25 monoclonal antibody, daclizumab (73, 74).

Daclizumab is being evaluated in clinical trials of patients with CD25 expressing adult T-cell leukemia (9). A dose escalation analysis was used to define the dose required to maintain saturation of the IL-2 receptor. In this study, it was determined that in these patients with high numbers of leukemic cells that display large numbers of CD25 receptors, a dose of 8 mg/kg of anti-Tac (daclizumab) every 3 weeks was required to maintain saturation of IL-2R alpha on the lymph node cells of the patients (9). This is a dose eightfold greater than that approved for the treatment of patients receiving organ allografts or those treated for autoimmune disorders. Therapy with daclizumab yielded some partial responses. These responses were predominantly observed in patients with smoldering or chronic ATL that appeared to be in the IL-2/IL-2R alpha autocrine phase of their disease. In contrast, very few responses were observed in patients in the IL-2 independent acute and lymphoma forms of ATL, especially in those patients who had failed aggressive chemotherapeutic regimens. As just noted, only a subset of patients with ATL responded to daclizumab therapy. Furthermore, in contrast to the situation in the MET-1 model in mice, the responding patients appeared to be in the autocrine phase of their disease where an interaction of IL-2 with the IL-2 receptor was required for malignant T-cell proliferation and survival. In an effort to test this hypothesis and in an effort to develop an ex vivo test that would help predict which patients with ATL would respond to daclizumab therapy, we cultured the peripheral blood mononuclear cells of patients with ATL ex vivo for 6 days in media with fetal calf serum and studied the spontaneous proliferation in the presence and absence of antibodies to IL-2 or IL-2R alpha (daclizumab). The PBMC of a subset of ATL patients manifested spontaneous proliferation, and this proliferation was inhibited by the addition of daclizumab, an antibody to IL-2R alpha. The hypothesis was that these patients were manifesting an IL-2/IL-2 receptor-mediated autocrine loop that underlies the proliferation. This hypothesis was supported by our demonstration using RT-PCR analysis that large quantities of mRNA encoding IL-2 were produced by these patients’ ATL cells and that their culture supernatants contained biologically meaningful quantities of IL-2 as assessed by ELISA analysis. The patients of this subset manifested a partial response when they were treated with daclizumab supporting the view that the leukemic cells in this subset manifested an IL-2/IL-2 receptor autocrine pattern and that the antibody interrupted this autocrine loop.

DACLIZUMAB CYTOTOXIC CONJUGATES: IMMUNOTOXIN-LINKED MONOCLONAL ANTIBODY

 

A limitation in the use of unmodified monoclonal antibodies such as ant-Tac for the treatment of T-cell leukemia/lymphoma is the fact that they are relatively ineffective as cytocidal agents. This is true in the late stages of HTLV-I associated ATL, stages where the cells continue to express IL-2R alpha, but no longer produce nor require IL-2 for their proliferation. This limited efficacy of unmodified anti-Tac in leukemia/lymphoma therapy led to the alternative approach of using this antibody as a carrier of cytotoxic agents such as toxins or radionuclides. In one series of studies, in conjunction with Krietman and Pastan, we evaluated the clinical efficacy of IL-2R alpha-directed immunotoxins that involved a truncated version of Pseudomonas exotoxin A (PE38) (75). Exon I of Pseudomonas toxin that is involved in ubiquitous unwanted binding was deleted. A single-chain toxin fusion protein anti-Tac (Fv) PE38 in which the variable region (Fv) of anti-Tac was joined in peptide linkage to the truncated toxin PE38 had characteristics in vitro and in vivo that suggested its use as an IL-2R directed agent for humans. This agent anti-Tac Fv PE38 (LMB-2) was evaluated in a Phase I/II clinical trial that involved 59 cycles of therapy in 35 patients who had leukemia/lymphoma that expressed IL-2R alpha (75). In this trial, there were eight responders including four with hairy cell leukemia and one each with chronic lymphocytic leukemia, Hodgkin's disease, cutaneous T-cell lymphoma (CTCL), and ATL.

DACLIZUMAB ARMED WITH BETA-AND-ALPHA-EMITTING RADIONUCLIDES

The actions of toxins conjugated to monoclonal antibodies depend on their ability to be internalized by the malignant cells and translocated to the cytoplasm. In fact, the toxin conjugates do not pass easily from the endosome to the cytosol. Furthermore, large protein toxins are immunogenic and thus provide only a narrow therapeutic window prior to the development of host antibodies directed toward the toxins. To circumvent these limitations, anti-Tac was armed with radionuclides. A number of factors needed to be considered when designing an optimal radioimmunotherapy strategy: (a) the choice of the monoclonal antibody and thus the antigenic target; in our case, anti-Tac targeting IL-2R alpha expressed on malignant cells; (b) the choice of the delivery system used to target the radionuclide to the tumor cell; and (c) the choice of radionuclide. In most clinical trials, intact monoclonal antibodies have been employed to deliver the radionuclide. There are however a number of limitations in this approach. There are physiological and structural barriers that limit the delivery of high molecular weight molecules such as intact antibodies to tumor cells especially those in large tumor masses. Meaningful tumor uptake of antibody may not occur until 24–48 h after injection. Unfortunately, the long serum half-lives of the monoclonal antibodies prolong radiation exposure to normal organs including the radiosensitive bone marrow, which limits the radiation that can be safely administered and delivered to the tumor cell. Finally, because of the slow equilibration of intact monoclonal antibodies with cells in a tumor mass, such therapy is limited to long-lived beta-emitting rather than short-lived alpha-emitting radionuclides. To circumvent these obstacles encountered using intact radiolabeled antibodies, a series of multistep strategies have been described to decouple the administration of the radionuclide from that of the antibody (7678). The strategy proposed by Axworthy and coworkers involves a pretargeting approach using Streptavidin-conjugated anti-Tac followed 24–48 h later by the administration of a “clearing agent” to remove unbound antibody from the circulation (77, 78). This procedure is followed 1–3 h later with the administration of radiolabeled biotin that binds to the Streptavidin linked to the antibody on the tumor cells. The fundamental advantages of this pretargeting approach over conventional radioimmunotherapy are that the uptake of therapeutic radionuclide by the tumor is high and rapid and that any excess radioactivity is efficiently eliminated from the body in the urine. Using this pretargeting technique to treat the MET-1 model of human ATL, we demonstrated dramatic efficacy of the pretargeting approach in the MET-1 model of ATL that was considerably superior to that observed with radiolabled intact antibodies (77, 78).

The third component of an optimal radioimmunotherapy regimen is the nature of the radionuclide used. A variety of radionuclides are available that differ in terms of their energy/range of emissions and half-life. Historically beta-particles have received the most use. The emission path length of beta-emitters is relatively long with a low-energy transfer. The use of such beta-emitting radionuclides is effective with large tumor masses where the beta emissions produce a crossfire effect. However, as the tumor mass decreases, so does the benefit of the crossfire effect. When used with micrometastases or with individual tumor cells as in leukemia, the relative path length of the beta-emission compared to alpha radiation may result in high-energy beta emissions outside of the tumor target volume. In addition, it may not be possible to achieve the number of beta emission transversals that are required to kill the malignant cells in small tumors. Thus, future development of radiolabeled monoclonal antibodies including daclizumab for the treatment of leukemias or micrometastases, may focus on alpha-emitting radionuclides that have a short distance of action and release very high-energy emissions. Therapy with an astatine-labeled anti-CD25 monoclonal antibody provided effective therapy for a murine model of human adult T-cell leukemia (79).

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 initially in a Phase I dose escalation trial and subsequently in a Phase II trial involving 10 μCi of 90Y-labeled anti-Tac per patient dose (8). Patients undergoing a partial or a complete response were permitted to receive up to eight additional doses of 90Y-labeled anti-Tac with a minimum of 6 weeks between courses. At the 5–15 μCi doses used, 10 of the 17 evaluable patients responded to 90Y-anti-Tac with partial (8) or complete (2) remissions. The only meaningful (>Grade 3) toxicity was limited to the hematopoietic system. More recently, we have also had encouraging results with 90Y humanized anti-Tac (daclizumab) in the treatment of patients with Hodgkin's disease. There were 7 CRs and 5 PRs obtained in the 17 patients with Hodgkin's disease treated with repeated doses of 15 μCi of 90Y (16). Thus, it appears that daclizumab armed with the radionuclide 90Y provides meaningful therapy for select patients with ATL and Hodgkin's disease.

HUMANIZED ANTI-TAC (DACLIZUMAB) TREATMENT OF AUTOIMMUNE DISEASES

As noted previously (Table I), increased serum soluble IL-2R alpha (Tac) concentrations have been demonstrated in association with an array of autoimmune disorders including: Wegener's granulomatosis, vasculitis, polymyalgia rheumatica, giant cell arteritis, Kawasaki disease, Behcet's syndrome, aplastic anemia, pure red cell aplasia, Crohn's disease, juvenile rheumatoid arthritis, multiple sclerosis, rheumatoid arthritis, sarcoidosis, scleroderma, Sjogren's syndrome, and systemic lupus erythematosus (4, 5).

Daclizumab has been used effectively without meaningful toxicity in the treatment of select patients with HTLV-I associated myelopathy/tropical spastic paraparesis, HAM/TSP, noninfectious uveitis, multiple sclerosis, pure red cell aplasia, and aplastic anemia (1215, 8085). HAM/TSP is a neurological disease that results from the interaction of retroviral infection and immune activation (15). Immune activation is initiated through the production of HTLV-I encoded tax, a trans-activator of viral genes and several host genes including those of interleukin-2 and the interleukin-2 receptor alpha subunit (IL-2R alpha) (86, 87). Other evidence for immune activation in HAM/TSP is demonstrated by the presence of ex vivo spontaneous proliferation of PBMC and HTLV-I tax-specific CD8 cytotoxic T-lymphocytes in the peripheral blood from HAM/TSP patients (88). Spontaneous lymphoproliferation, which probably reflects the effect of multiple autocrine cytokine loops including an autocrine loop initiated by the activation of IL-2 and IL-2R alpha expression, can be inhibited ex vivo by the addition of either an antibody to IL-2 or to IL-2R alpha (anti-Tac, daclizumab) (86, 87). In collaboration with Lehky, Jacobson, and coworkers, we performed a study where we demonstrated a reduction in the HTLV-I proviral load and spontaneous lymphoproliferation in patients with HAM/TSP who were treated with daclizumab (15). In this study, five doses (1 mg/kg) of humanized anti-Tac antibody were administered to nine patients with HAM/TSP at weeks 0, 2, 6, 10, and 14. Immunological studies on the patients with HAM/TSP treated with daclizumab indicated that there was a selective downregulation in the number of circulating activated T-cells that displayed the IL-2 receptor alpha and a decrease in the ex vivo spontaneous proliferation of PBMC as well as a decrease in HTLV-I viral loads in the peripheral lymphocytes that suggested the selective removal of HTLV-I infected IL-2R alpha (CD25) expressing activated CD4 expressing lymphocytes (15). Thus, daclizumab has been demonstrated to have a potential role in the treatment of this autoimmune neurological disease that is secondary to HTLV-I infection.

With another autoimmune disorder, treatment with daclizumab in a Phase I/II clinical trial was effective in preventing the progression of noninfectious intermediate and posterior uveitis. Circulating T-cells in models of experimental autoimmune uveitis in mice and nonhuman primates bear large numbers of IL-2 receptors on their surfaces (82). In studies focusing on experimental uveitis, Guex-Crosier and colleagues in collaboration with our group demonstrated that the infusion of daclizumab had a positive effect on S-antigen-induced experimental uveitis in nonhuman primates (82). Large numbers of IL-2 receptors have been demonstrated on the circulating T-cells from patients with uveitis. To evaluate the safety and potential activity of daclizumab therapy in the treatment of patients with severe sight-threatening intermediate and posterior noninfectious uveitis, in a collaborative effort led by Robert Nussenblatt, a nonrandom open-label pilot study was performed (12, 13). Patients with uveitis who had been treated with a minimum of Prednisone, Cyclosporin, antimetabolites, or any combination of these agents were eligible. Patients were weaned off their systemic immunosuppressive agent while ultimately receiving daclizumab infusions at 1 mg/kg every 4 weeks. Daclizumab administration appeared to prevent the continued expression of severe sight-threatening intraocular inflammatory disease in eight of 10 patients treated over a multiyear period with improvement in visual acuity. Additional benefits of the IL-2R target of treatment that were observed after the patients were tapered off systemic corticosteroids were improvements in blood pressure and serum cholesterol levels.

Daclizumab therapy was also effective in patients with multiple sclerosis, an immune-mediated disorder of the brain and spinal cord, where at least part of the inflammatory process can be objectively measured by contrast-enhancing lesions on brain MRI. In a collaborative study led by Bielekova and Martin, we demonstrated that daclizumab inhibited disease activity in multiple sclerosis patients failing to respond to interferon beta (14, 85). An open-label Phase II baseline-to-treatment trial of daclizumab was performed in 10 multiple sclerosis patients manifesting an incomplete response to interferon-beta therapy and high-brain inflammatory and clinical disease activity. In this initial study, daclizumab therapy was very well tolerated and led to a 78% reduction in new contrast-enhancing lesions and to a significant improvement in several clinical outcome measures (Fig. 4). Comparable sustained clinical improvement (10 patients) or stabilization (9 patients) was observed in an independent study by Rose and coworkers in 19 patients with MS treated with daclizumab (84). A subsequent study in conjunction with Bielekova and Martin was performed to investigate whether daclizumab retains its beneficial effect when applied as monotherapy (85). Daclizumab monotherapy was highly effective in 9/13 MS patients, whereas the synergistic effect of daclizumab and IFN-β was necessary to stabilize disease activity in 04/13. In this study overall ,15 months of daclizumab therapy led to a significant inhibition of MRI disease activity (by 72%) and to a significant amelioration of clinical disability. Prolonged multidose therapy with daclizumab was associated with persistent efficacy. A Phase II multicenter study involving over 200 patients has been initiated.
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Fig. 4

Daclizumab therapy inhibits disease activity in multiple sclerosis patients failing to respond to beta interferon. New contrast-enhancing lesions (CEL) observed in 10 patients with multiple sclerosis receiving beta-interferon therapy alone is indicated at the left in months—3 to 0 and when daclizumab was added to beta-interferon from months 0 through 7 (14). There was little efficacy observed for the first 4–6 weeks of daclizumab therapy. However, after this period, there was a 78% reduction in new contrast-enhancing lesions on brain MRI during daclizumab therapy (modified and reproduced with permission from Proc Natl Acad Sci USA 101:8705–8708, 2004 (14)).

Daclizumab has also been studied by others in small open-label trials involving patients with diverse autoimmune disorders. To examine the efficacy of daclizumab in the treatment of pure red cell aplasia—a disease characterized by anemia, reticulocytopenia, and absence of bone marrow erythroid precursors, 15 patients were treated with 1 mg/kg of this drug every 2 weeks for a total of five infusions (80). Six of 15 patients (40%) responded to treatment. All responders became transfusion-independent and achieved normal or near-normal hemoglobin values and normal reticulocyte counts.

Daclizumab was also evaluated in patients with moderate pancytopenia defined as a depression of two of the three blood counts, absolute neutrophil count of 1200/mm3 or less, platelet count 70,000/mm3 or less, or a hemoglobin level of 8.5 g/dL or less (81). Daclizumab at 1 mg/kg was administered every 2 weeks for 3 months. Six of the 16 (38%) evaluable patients responded to treatment. Two patients with previously chronic disease showed a complete return of normal hematological counts that were sustained for more than 2 years following treatment. Two previously transfusion-dependent patients became transfusion independent; and one patient with many neutropenia-related infections had a normal neutrophil count following treatment (81).

Nineteen patients with psoriasis, studied in two centers, received daclizumab at an initial dose of 2 mg/kg, then 1 mg/kg at weeks 2, 4, 8, and 12. In those patients with pretreatment psoriasis area and severity index with a score of less than 36, there was a mean reduction in severity by 30% at 8 weeks (83).

In summary, daclizumab therapy at receptor saturating doses of antibody, as evaluated in small open-label trials, has provided efficacy with minimal toxicity in patients with HAM/TSP, multiple sclerosis, noninfectious uveitis, pure red cell aplasia, moderate aplastic anemia, and psoriasis. Efficacy required persistence of the therapy, with a recurrence following reduction of circulating daclizumab levels below that required to saturate the IL-2 receptor alpha. On the basis of these encouraging results, large multicenter trials have been initiated using daclizumab in the treatment of patients with T-cell mediated uveitis and multiple sclerosis.

DIVERSE MECHANISMS OF ACTION OF DACLIZUMAB IN PATIENTS WITH AUTOIMMUNE DISEASES

A number of mechanisms underlie the immunosuppressive effects of daclizumab therapy in autoimmune diseases (89, 90). When administered at doses sufficient to saturate the IL-2 receptor alpha subunit, it inhibited the interaction of IL-2 with its high-affinity receptor. This, in turn, inhibited the IL-2 induced tyrosine phosphorylation of the IL-2R beta and gamma subunits that are mediated by IL-2. Furthermore, in coimmunoprecipitation experiments, daclizumab inhibited the IL-2 induced association of the IL-2R beta and common gamma chains—a prerequisite for their mutual phosphorylation (90). In addition, utilizing the cytokine-dependent cell line Kit 225 as well as PHA-activated lymphoblasts, daclizumab markedly inhibited phosphorylation of the Jak-1, Jak-3, and STAT-5 A/B components of the IL-2R dependent signaling pathway (89). Furthermore, the administration of daclizumab was associated with downregulation of the internalization of IL-2R alpha but not IL-2R beta or the common gamma chain (10, 89, 90). Thus, one mode of action of daclizumab is to interrupt IL-2 mediated activation of cells that express the high-affinity IL-2 receptor.

As noted earlier, daclizumab manifests ADCC with human mononuclear cells. Furthermore, in the MET-1 murine model of adult T-cell leukemia, daclizumab has an action in addition to its role in the blockade of the interaction of IL-2 with its growth factor, in that the antibody was ineffective in mice that were deficient in the agonist FcRγI, FcRγIII, and FcRγIV receptors, supporting the view that an action requiring Fc-receptor expression on monocytes or polymorphonuclear leukocytes contributed to the action of daclizumab (72).

Recently, an additional mechanism of action has been reported; i.e., daclizumab interaction with the human IL-2 receptor was associated with an expansion in the number of CD56bright natural killer cells that mediate immunomodulatory effects in patients with active uveitis and multiple sclerosis (91, 92). NK cells have been divided by Cooper and coworkers into two subsets based on their cell-surface density of CD56 (CD56bright and CD56dim) each with distinct phenotypic properties (31). A CD56dim NK cell subset reflects cytotoxic cells that express higher levels of Ig-like NK receptors and Fc gamma receptor III (CD16) than does the CD56bright NK cell subset. The CD56bright subset in contrast has the capacity to produce abundant cytokines following activation of monocytes, but has low cytotoxic activity. The CD56bright subset expresses CD25 identified by daclizumab whereas this receptor subunit is not expressed by the CD56dim NK cell subset. Furthermore, the CD56bright NK cells produce a number of immunoregulatory cytokines including IL-10 that act as checkpoints or brakes on the immune system (31, 92). In studies by Li and coworkers, administration of daclizumab to patients with active uveitis led to a four to twentyfold expansion of the CD56bright regulatory NK cells (92). Furthermore, patients with active uveitis had a significantly lower level of CD56bright NK cells compared with normal donors. In addition, the induced CD56bright cells secreted large quantities of IL-10 whereas the CD56dim did not—suggesting that the induction of CD56bright cells might have a beneficial effect on the activity of the uveitis. In a parallel study led by Bielekova, administration of daclizumab was associated with a significant expansion of CD56bright NK cells in vivo and this effect correlated highly with the treatment response (91). There was a positive correlation between the expansion of CD56bright NK and the contraction of CD4+ and CD8+ T-cell numbers in vivo providing supporting evidence for NK-cell mediated negative immunoregulation of activated T-cells during daclizumab therapy. In support of these two studies, a regulatory role of NK cells has been demonstrated in experimental autoimmune encephalomyelitis (EAE) in mice (93). In these studies when mice were deprived of NK cells by antibody treatment before administration of myelin oligodendrocyte glycoprotein (MOG) sub-35-55 peptide, the mice developed a more serious form of EAE associated with relapse (93). These studies support the view that a subset of natural killer cells inhibits the activity in immune neurological diseases such as experimental autoimmune encephalomyelitis (EAE). The data presented in the two reports on uveitis and multiple sclerosis support the existence of an immunoregulatory pathway wherein CD56bright NK cells activated through the action of daclizumab inhibit disordered T-cell action. This immunoregulatory role has potential importance as a mechanism of action of daclizumab that is of relevance in the treatment of autoimmune diseases and transplant rejection.

FUTURE DIRECTIONS

The inclusion of daclizumab has been of value in protocols to prevent acute organ allograft rejection. However, the protocol approved by the FDA for patients receiving kidney allografts which involves only five 1 mg/kg antibody doses at 2-week intervals is not optimal. Daclizumab is not a cytotoxic monoclonal antibody but requires the continued saturation of the IL-2R alpha receptor for its persistent action. Thus, when therapy with this agent is terminated, an immune response to the foreign transplantation antigens on the organ develops, thereby explaining the modest efficacy of therapy on prolongation of organ and patient survival. To determine if this limitation could be circumvented, it would be of value to evaluate a dosing strategy that involves the indefinite q. 3 to 4-week administration of daclizumab using a protocol analogous to those that have been effective in maintaining long-term suppression of disease progression in T-cell mediated uveitis and multiple sclerosis.

An additional limitation in the use of daclizumab is that it does not inhibit the action of IL-15, a cytokine that does not use IL-2R alpha, but utilizes its own private IL-15R alpha in addition to IL-2/IL-15R beta and the common gamma chain that it shares with IL-2. Furthermore, daclizumab does not inhibit the actions of IL-2 on resting NK cells that display IL-2/IL-15R beta and γc but not IL-2R alpha the target of daclizumab. These limitations could be addressed by simultaneously administrating daclizumab and an antibody, Hu-Mik-Beta-1 that is directed to the cytokine receptor (IL-2R/IL-15R beta) shared by IL-2 and IL-15.

A number of approaches could be exploited to optimize the action of daclizumab in the therapy of CD25 (IL-2R alpha) expressing lymphoid leukemias and lymphomas. Combinations of unmodified daclizumab with an array of agents including chemotherapeutic agents that utilize a different mode of action could be evaluated in order to follow the paradigm that monoclonal antibodies for cancer therapy are most effective as part of a multidrug therapy regimen. Insights derived from effective systemic radioimmunotherapy in preclinical models of human T-cell malignancy in mice should be translated into human clinical trials. In particular, in patients who have T-cell leukemia, select alpha-emitting radionuclides such as astatine-211 are being introduced in lieu of the beta-emitting Yttrium-90 whose crossfire effect is relatively ineffective when directed toward isolated malignant cells. Furthermore, multistep systemic radioimmunotherapy strategies such as pretargeting that decouple the administration of the radionuclide from that of the antibody, to augment the proportion of the radionuclide delivered to the tumor, and accelerate the delivery of the radionuclide following its administration could be studied.

Daclizumab has been shown to be of considerable value in a number of T-cell mediated autoimmune disorders that involve an abnormality of the IL-2/IL-2 receptor system. In particular, continuous therapy with daclizumab has shown efficacy in select patients with the neurological disorder HAM/TSP, multiple sclerosis, noninfectious uveitis, pure red cell aplasia, aplastic anemia, and psoriasis. Therefore, evaluation of this agent is indicated for the other autoimmune disorders that involve disorders of the IL-2/IL-2R alpha system with elevated serum IL-2R alpha levels that are enumerated in Table I, such as rheumatoid arthritis. In support of this proposal, daclizumab was effective in a rhesus monkey model of collagen-induced arthritis (94).

In summary, basic insights concerning the IL-2/IL-2 receptor system developed using daclizumab over the past 25 years when coupled with the experiences with this humanized antibody in clinical trials are providing effective daclizumab-mediated approaches for the treatment of patients with leukemia/lymphoma, autoimmune disorders and for the prevention of organ allograft rejection.

ACKNOWLEDGMENTS

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 had prior approval of the IRB, NCI.

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© Springer Science+Business Media, LLC 2007