M.D., Metabolism Branch, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892 USA

Patients with human T -cell Iymphotrophic virus I (HTL V -1)-associated leukemia/lymphoma were treated with different forms of IL-2 receptor (IL-2R)-directed therapy that exploit the difference in IL-2R expression between normal and malignant cells. Using unmodified anti- Tac monoclonal antibody, one-third of the patients with adult T -cell leukemia (A TL) treated have undergone a remission, in two cases complete. There was little toxicity observed; however, unmodified monoclonal antibodies are limited by their immunogenicity and their poor effector functions. To address these issues, "humanized" anti- Tac was produced that contains the complementaritydetermining regions from the mouse with the remainder of the molecule derived from human IgG 1k This antibody is dramatically less immunogenic than the murine version, has improved pharmacokinetics, and, in contrast to the parent antibody, manifests antibodydependent cellular cytotoxicity (ADCC). To enhance its effector function, anti- Tac was armed with toxins and alfa- and ß-emitting radionuclides. In a clinical trial of 90y -anti- Tac in A TL patients, at the doses used (5, 10, and 15 mCi 90y -anti- Tac per patient), 10 of the 15 patients with A TL treated to date underwent sustained partial or complete remission. Thus, the clinical application of IL-2R-directed therapy represents anew perspective for the prevention of allograft rejection and for the treatment of graft-versus-host disease, select autoimmune disorders, and leukemia/lymphoma.

INTRODUCTION

The development of the hybridoma technique by Köhler and Milstein (1) rekindled interest in the use of antibodies to treat cancer patients. However, monoclonal antibodies are just beginning to fulfill the promise for immunotherapy inherent in their great specificity for recognizing and binding to abnormal cells. That is, despite wide-ranging interest, the "magic bullet" of antibody therapy that has been a dream since the time of Paul Ehrlich (2) has proven elusive. One of the factors in this low therapeutic efficacy is that the murine monoclonal antibodies are immunogenic when administered to humans. Another critical factor is that most of the monoclonal antibodies employed are not effective cytocidal agents against human neoplastic cells. Furthermore, in most cases the antibodies were not directed against a vital structure present on the surface of malignant cells, such as a growth factor receptor required for tumor cell proliferation and survival. We readdressed this issue by (a) using genetic engineering to create less immunogenic and more effective monoclonal antibodies, (b) arming antibodies with toxins or radionuclides to enhance their effector functions, and ( c ) selecting the IL-2R on abnormal cells as a target for effective monoclonal antibody action. The scientific basis for the use of this target is that resting cells do not express -IL-2R alfa, whereas this receptor subunit is expressed by a proportion of the abnormal cells in certain forms of lymphoid neoplasia (e.g ., adult T -cellleukemia/lymphoma [A TL], cutaneous T -cell lymphoma, hairy cell leukemia, and Hodgkin's disease), in select autoimmune diseases (e .g ., rheumatoid arthritis, sarcoidosis, scleroderma, and tropical spastic paraparesis [TSP]), in individuals undergoing graft-versus-host disease, and by the T cells involved in allograft rejection (3- 7).

RESULTS AND DISCUSSION

STRUCTURE AND FUNCTION OF THE MULTISUBUNIT IL-2R

Successful T -cell-mediated immune responses require that these cells change from a resting to an activated state. Activation requires two sets of signals from cell surface receptors to the nucleus. The first signal is initiated when an appropriately processed and presented foreign antigen interacts with a heterodimeric T -cell receptor (Tcr) for that specific antigen. Following this encounter with antigen, T cells enter a program of cellular activation leading to de novo synthesis and secretion of IL-2 (3-5). Resting T cells do not express high-affinity IL-2R's, but receptors are rapidly expressed after T -cell activation with antigen or mitogen (3, 6-8). The interaction of IL-2 with the induced cellular receptor for this lymphokine then triggers proliferation of these cells, culminating in the emergence of effector T lymphocytes mediating helper, suppressor, or cytotoxic functions. Thus, the expression of the genes encoding IL- 2 and its receptor and the subsequent interaction of this ligand with its receptor are critical events in T -cell activation and are required for full expression of the human immune response. There are three forms of cellular receptor for IL-2 based on their affinity for ligand, including one with a very high IL2 binding affinity (10 high-11 M), one with an intermediate affinity (10 high-9 M), and one with a lower affinity (10 high-8 M) (810). We have used monoclonal antibodies, affinity crosslinking with radiolabeled IL- 2, coprecipitation analysis, flow cytometric resonance energy transfer measurement, and recovery following fluorescence photobleaching to identify multiple subunits of this receptor (6, 11, 12). Initially, a monoclonal antibody (antiTac) that we prepared was used to identify a 55-kDa IL- 2R protein (now termed IL-2R alfa) (6). The receptor protein identified by anti- Tac was characterized as a densely glycosylated and sulfated structure with an apparent molecular mass of 55 kDa that is composed of 251 amino acids as well as an NH2 terminal 21-amino-acid signal peptide cleaved in vivo (13). Using crosslinking methodology with radiolabeled IL-2, we defined a second 70/75-kDa IL-2 binding protein, now termed IL-2Rß (11). We proposed a multi subunit model for the high-affinity IL2R. In this model, an independently existing IL-2R alfa or IL-2Rß subunit would generate low- and intermediateaffinity receptors, respectively, whereas high-affinity receptors would be formed when both receptors are expressed and noncovalently associated in a receptor complex (11). An additional 64-kDa molecule (termed IL2Ry) has been identified that plays a critical role in IL-2 binding, internalization, and signaling mediated by the IL2R (14).

IL-2R EXPRESSION IN MALIGNANCY

We have used the IL-2R as a target for immune intervention. The scientific basis for the approach utilizing the IL-2R alfa subunit as a target for immunotherapy is that resting normal cells do not express the Tac peptide of the IL-2R. In contrast, we and others have shown that this receptor is expressed by a proportion of the abnormal cells in certain forms of lymphoid neoplasia, select autoimmune diseases, and in individuals rejecting allografts (3, 15, 16). That is, a proportion of the abnormal cells in these diseases express the Tac antigen on their surface. Furthermore, Nelson and coworkers (17) have shown that the serum concentration of the soluble form of IL- 2R alfa is elevated in patients with these disorders. In terms of neoplasia, certain T -cell, B-cell, monocytic, and even granulocytic leukemias express the Tac antigen. Specifically, the malignant T cells of patients with human T -celllymphotrophic virus I (HTLV-I)-induced ATL constitutively express IL-2R alfa (18). Furthermore, the malignant T cells of the skin and lymph nodes of patients with cutaneous T -cell lymphoma (mycosis fungoides and the sezary syndrome) express the Tac antigen (19). In addition, virtually all of the malignant cells of patients with hairy cell B-cellleukemia express the Tac antigen, and a proportion of other B-celllymphomas are also Tac-positive (20). Finally, true histiocytic leukemia cells and the Reed-Sternberg cell of Hodgkin's disease also manifest IL-2R alfa (21). In addition to these Tac-expressing leukemias and lymphomas, we have shown that there are certain leukemias (e.g., acute lymphoblastic leukemia and large granular lymphocytic leukemia) that do not express IL-2R alfa, yet express the IL-2Rß subunit of the IL-2R (22). Disorders of IL-2R Expression in HTLV-I-Associated ATL. We focused our initial IL-2R immunotherapeutic studies on A TL, a distinct form of aggressive T -cell leukemia (23). HTLV-I is the primary etiologic agent in ATL (24). ATL is a malignant proliferation of CD3/CD4-expressing T cells that typically infiltrates the skin, lungs, and liver. All populations of leukemic cells we have examined from patients with HTL V -I -associated A TL constitutivel y express high- and low-affinity IL- 2R, including very large numbers of the IL-2R alfa defined by the anti- Tac monoclonal antibody (18). An analysis of HTLV-I and its protein products suggests a potential mechanism for the association between HTLV-I and constitutive IL-2R alfa expression (25). The retrovirus HTL V -I encodes a 42-kDa protein (now termed "tax") that plays an important role in the early phases of HTL V -I -induced malignancy by deregulating the expression of the cellular genes that encode IL-2 and a 55-kDa Tac IL-2R peptide that controls T -cell proliferation (25, 26).

IL-2R alfa AS A TARGET FOR THERAPY IN PATIENTS WITH HTLV-I-ASSOCIATED ATL

Unmodified Anti-Tac Monoclonal Antibody. No conventional treatment program is successful in inducing long-term disease-free survival in A TL patients, with an overall mortality of approximately 50% within 5 months. However, the HTLV-I-induced ATL cells constitutively express the IL-2R alfa chain identified by the anti- Tac monoclonal antibody, whereas normal resting cells do not. This observation provided the scientific basis for IL-2Rdirected immunotherapy with this monoclonal antibody. IL-2R-directed immunotherapeutic agents could theoreticall y eliminate IL- 2R alfa-expressing leukemic cells or abnormally activated T cells involved in other disease states while retaining the Tac-nonexpressing normal T cells and their precursors that express the antigen receptors for T -cell-mediated immune responses. In our initial studies, we administered unmodified murine anti- Tac to patients with ATL (15, 16). The leukemic cells of each patient with A TL reacted with anti- Tac. Our goal was to inhibit the interaction of IL-2 with its growth factor receptor expressed on the malignant cells. The 20 patients treated in this study did not have untoward reactions related to the immunotherapy. Only patients undergoing a remission produced antibodies to the monoclonal antibody. Seven of the 20 treated patients had transient mixed (1 patient), partial ( 4 patients), or complete (2 patients) remission, lasting from 1 to more than 30 months after anti- Tac therapy. This was assessed by elimination of measurable skin and lymph nodal disease, normalization of serum calcium levels, and routine hematologic and phenotypic tests of circulating cells. Further, elimination of clonal malignant cells was shown by molecular genetic analysis of HTL V -I proviral integration and by definition of monoclonal T -cell antigen receptor gene rearrangements. Although this approach to the treatment of A TL was encouraging, 13 of the 20 patients did not manifest at least a partial remission. Furthermore, six of the seven responding patients ultimately relapsed. This relapse was not associated with the loss of IL- 2R alfa expression by the A TL cells. However, it did reflect the fact that certain A TL cells no longer produce nor require IL- 2 for their proliferation. Since anti- Tac functions, in part, by preventing the interaction of IL-2 with IL-2R alfa, it is no longer effective at this stage of the patient's disease. Nevertheless, IL-2R alfa is still expressed on the leukemIc cells, but not on the normal cells of the patients, thus providing a target for immunotherapy with anti- Tac armed with toxins or radionuclides.

Humanized Antibodies to IL-2R. There are two problems with murine monoclonal antibodies in general: their immunogenicity and their ineffectiveness at recruiting hosteffector functions. We have addressed these issues by producing "humanized" antibodies. These humanized antiTac molecules, produced in conjunction with Cary Queen, retain the complementarity-detem1ining region from the mouse, but virtually all the remainder of the molecule is derived from human IgG 1 K. On the basis of computer modeling of the structure of this antibody, murine elements close to the complementarity-determining regions were identified, and those that were believed to be important to maintain the appropriate conformation of this antibody were retained (27, 28).

One primary goal in these studies is to maintain the affinity and functional capacity of the mouse monoclonal antibody. The parent anti- Tac molecule had an affinity of 9 x 10 high 9/M, whereas the hyperchimeric "humanized" version has an affinity of 3 x 10 high 9/M, still very high (27). The parent monoclonal and the humanized version showed comparable inhibition of T -cell proliferation in response to tetanus antigen (28). The humanized version of anti- Tac had improved pharmacokinetics when compared to the murine version, with an in vivo survival that is 2.5- fold longer (terminal t112' 103 hr vs. 38 hr). In addition, humanized anti- Tac was less immunogenic than murine anti- Tac when administered to cynomolgus monkeys undergoing heterotopic cardiac allografting (29). A final goal of this project is to make an antibody that is abetter effector of cell killing than is murine anti- Tac. Therefore, we were greatly encouraged by the observation that although the parent mouse anti- Tac could not function in antibody-dependent cellular cytotoxicity (ADCC) with human mononuclear cells, the hyperchimeric IgG 1 anti -Tac manifests ADCC with human mononuclear cells (28). Clinical trials will be initiated using humanized anti- Tac in the treatment of patients with leukemia/lymphoma or with graft-versus-host disease. M onoclonal Antibody-Cytotoxic Agent Conjugates: IL-2R Directed Immunotoxins. To continue to take advantage of the difference in IL- 2R expression between normal and leukemic cells and to improve the effectiveness of IL-2Rdirected therapy, different approaches were initiated to modify the antibody for clinical purposes. In one approach, anti- Tac or IL-2 was used to deliver modified toxins to IL-2R-expressing cells without incurring severe systemic toxicity. Hwang and coworkers (30) generated. a genetically modified form of Pseudomonas exotoxm (PE40), from which the first domain responsible for ubiquitous cell binding was deleted. To generate a homogeneous product, a single-chain antibody fusion protein [e.g., anti- Tac (Fv)-PE40] in which the variable regions of anti- Tac are joined in protein linkages wIth PE40 was constructed and expressed in E. coli. Anti- Tac (Fv)-PE40 is very cytotoxic to IL-2R-bearing human cell lines as well as fresh cells derived from patients with IL-2R alfa-expressing A TL, but is not cytotoxic to receptor negative cells (31). IL-2-PE40, a chimeric protein composed of human IL-2 genetically linked to the amino terminus of PE40, was constructed to provide an alternative (lymphokine-mediated) method of delivering the truncated toxin to the surface of IL- 2R -expressing cells (32).

Monoclonal Antibodies Armed with alfa- and ß-Emitting Radionuclides. The action of toxins conjugated to monoclonal antibodies depends on their ability to be internalized by the cell 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 short therapeutic window prior to the development of host antibodies directed toward the toxin. In the future these problems will probably be resolved. However, to circumvent these limitations, radiolabeled monoclonal antibodies were developed as alternative immunoconjugates to deliver a cytotoxic agent to target cells. There are a number of advantages over other approaches in the use of radiolabeled monoclonal antibody conjugates for therapy. One is that with the appropriate choice of radionuclide, radiolabeled monoclonal antibodies kill cells at distances of several cell diameters; therefore, a radiolabeled monoclonal antibody binding to an antigen-expressing cell may kill adjacent antigen-nonexpressing cells, thereby overcoming the tumor cell antigenic heterogeneity that presents a problem for most other monoclonal-antibody-mediated approaches. Furthermore, the radiolabeled antibody need not be internalized to kill the tumor cell. Nuclear chemistry has provided a selection of radioisotopes that could be linked . to Immunoprotems. In studies performed in collaboration with Otto Gansow, we turned to alfa- and ß-emitting radionuclides as alternative cytotoxic agents that could be conjugated to anti- Tac. In initial studies, a series of chelating agents was developed that did not compromise antibody specificity and did not permit premature release of the radionuclide in viva. Our choice of isotopes is based on the desire to have agents with a short distance of action that will act on the cell in question and on a small number of bystander cells without unwanted toxicity. In one case, we bound the ß-emitting 90y to anti- Tac using chelates that did not permit elution of radiolabeled yttrium from the monoclonal antibody (33). Monkeys that received xenografts or allografts of cynomolgus hearts showed a marked prolongation of graft survival following administration of 90y -labeled anti- Tac (29). Following preclinical efficacy and toxicity studies, we initiated a dose escalation trial of 90y anti- Tac for the treatment of patients with HTL V -I-associated Tac-expressing A TL. At the doses utilized (5, 10, and 15 mCi per patient) 10 of the 15 patients underwent partial (8 patients) or complete (2 patients) remission. Thus, antiTac armed with a radionuclide provides meaningful therapy for a form of leukemia that was previously universally fatal.

Future development of isotopic monoclonal antibody chelates may focus on alfa-emitting radionuclides, which may be the most effective agents at killing tumor targets without damaging distant normal tissues. Radionuclides emitting alfa particles release high-energy emissions (6-9 MeV, 10 times as great as ß or yemitters) over a short distance ( 40--80 µm) and are efficient at killing individual target cells, such as those found in leukemia, without significantly penetrating normal tissues. U nder hypoxic conditions that permit little cellular repair , alfa irradiation is efficient at killing cells. Suitable alfa-emitting nuclides available for immunotherapy that are under our investigation include 212Bi, 212pb, and 211 At.

A major future direction for our IL-2R-directed therapeutic program will be to arm the humanized version of the antiTac monoclonal antibody with alfa-emitting radionuclides. Two chelates have been developed (DOT A and CHX) that permit the linkage of 212Bi to humanized anti- Tac. We have shown that 212Bi conjugated to anti- Tac by use of these bifunctional chelates is well suited to effectively kill Tac-expressing A TL cells at doses that have only a modest effect on IL-2R alfa-nonexpressing lines. Activity levels of 0.5 µCi targeted by 212Bi-Iabeled anti- Tac eliminated more than 98% of the proliferative capacity of the IL-2Rexpressing HTL V -I -associated A TL line, HUT -102- B 2, with only a modest effect on an IL-2R-nonexpressing line (34 ). This specific therapeutic effect was blocked by an excess of unlabeled anti- Tac but not human IgG. On the basis of in vitra binding studies and in viva pharmacokinetic analyses in mice, the two chelating agents fulfilled the criteria for a suitable radioimmunotherapeutic agent. Thus, one of the most promising directions for the future development of armed monoclonal antibodies for the treatment of human cancer involves the linkage of alfa-emitting radionuclides such as 212Bi to humanized monoclonal antibodies. Such conjugates may prove to be relatively nonimmunogenic agents that are effective in eliminating malignant cells when used alone or as part of multimodality treatment with conventional chemotherapy.

In summary, our present understanding of the IL-2/IL-2R system has opened the possibility for more specific immune intervention strategies. The IL-2R is proving to be an extraordinarily useful therapeutic target. The clinical applications of anti-IL-2R-directed therapy, especially the use of humanized antibodies armed with radionuclides, represents anew perspective for the prevention of allograft rejection and for the treatment of graft-versus-host disease, select autoimmune disorders, and leukemia/lymphoma.

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