The interleukin-2 receptor: a target for monoclonal antibody treatment of human T-cell lymphotrophic virus I-induced adult T-cell leukemia

Adult T-cell leukemia (ATL) is a malignancy of mature lymphocytes caused by the retrovirus human T-cell lymphotrophic virus-I (HTLV-I). It is an aggressive leukemia with an overall mortality rate of 50% within 5 months; no conventional chemotherapy regimen appears successful in inducing long-term disease-free survival in ATL patients. However, ATL cells constitutively express high-affinity interleukin-2 receptors (IL-2Rs) identified by the anti-Tac monoclonal antibody, whereas normal resting cells do not. To exploit this difference in receptor expression, we administered anti-Tac intravenously (IV) to 19 patients with ATL. In general the patients did not suffer untoward reactions, and in 18 of 19 cases did not have a reduction in normal formed elements of the blood. Seven patients developed remissions that were mixed (1 patient), partial (4 patients), or complete (2 patients), with partial and complete remissions lasting from 9 weeks to more than 3 years as assessed by routine hematologic tests, immunofluorescence analysis, and molecular genetic analysis of T-cell receptor gene rearrangements and of HTLV-I proviral integration. Furthermore, remission was associated with a return to normal serum calcium levels and an improvement of liver function tests. Remission was also associated in some cases with an amelioration of the profound immunodeficiency state that characterizes ATL. Thus the use of a monoclonal antibody that blocks the interaction of IL-2 with its receptor expressed on ATL cells provides a rational approach for treatment of this aggressive malignancy.     

Radioimmunotherapy of interleukin-2R alpha-expressing adult T-cell leukemia with Yttrium-90-labeled anti-Tac [see comments]

Adult T-cell leukemia (ATL) is a malignancy of mature lymphocytes caused by the retrovirus human T-cell lymphotropic virus-I. It is an aggressive leukemia with a median survival time of 9 months; no chemotherapy regimen appears successful in inducing long-term disease- free survival. The scientific basis of the present study is that ATL cells express high-affinity interleukin-2 receptors identified by the anti-Tac monoclonal antibody, whereas normal resting cells do not. To exploit this difference, we administered anti-Tac armed with Yttrium-90 (90Y) to 18 patients with ATL initially (first 9 patients) in a phase I dose-escalation trial and subsequently (second group of 9 patients) in a phase II trial involving a uniform 10-mCi dose of 90Y-labeled anti- Tac. Patients undergoing a remission were permitted to receive up to eight additional doses. At the 5- to 15-mCi doses used, 9 of 16 evaluable patients responded to 90Y anti-Tac with a partial (7 patients) or complete (2 patients) remission. The responses observed represent improved efficacy in terms of length of remission when compared with previous results with unmodified anti-Tac. Clinically meaningful (> or = grade 3) toxicity was largely limited to the hematopoietic system. In conclusion, radioimmunotherapy with 90Y anti- Tac directed toward the IL-2R expressed on ATL cells may provide a useful approach for treatment of this aggressive malignancy.     

Failure of regulation of Tac antigen/TCGF receptor on adult T-cell leukemia cells by anti-Tac monoclonal antibody

Anti-Tac monoclonal antibody, which blocks the membrane binding and action of human T-cell growth factor (TCGF), is strongly proposed to recognize TCGF receptor. We have demonstrated that anti-Tac antibody reacted with leukemic cells from patients with adult T-cell leukemia (ATL) and reacted with T-cell lines established from ATL cells. Although antigenic modulation, or down-regulation, of Tac antigen on activated normal T cells was induced by anti-Tac antibody, the expression of Tac antigen on ATL cells or T-cell lines was not affected when examined by the fluorescence-activated cell sorter (FACS) and the radioassay using 125I-staphylococcal protein A. These results indicate that regulation of Tac antigen-TCGF receptor is different between normal and malignant T cells, suggesting that failure of down- regulation of Tac antigen on leukemic cells by anti-Tac antibody may play an important role in the malignant proliferation of ATL cells.     

Comparison of anti-Tac and anti-transferrin receptor-conjugated liposomes for specific drug delivery to adult T-cell leukemia

Adult T-cell leukemia (ATL) is a rapidly progressive and usually fatal malignancy of mature T cells characterized by the expression of large numbers of interleukin-2 (IL-2) receptors on the cell surface. Anti- Tac, a monoclonal antibody directed against the IL-2 receptor, was conjugated to liposomes and compared with anti-transferrin receptor (anti-TFR) conjugates for specific binding, internalization, and intracellular drug delivery to ATL cells. Two independent assays were used: a fluorimetric assay with liposome encapsulated 1-hydroxypyrene- 3,6,8-trisulfonic acid, a pH-sensitive fluorescent dye, and a growth inhibition assay using methotrexate-gamma-aspartate, a liposome- dependent cytotoxic drug. MT-1 and HUT-102 cell lines derived from patients with ATL were compared with Molt-4, a leukemia cell line that does not express IL-2 receptors in an uninduced state. Fluorimetric studies showed specific binding and internalization of anti-Tac- conjugated liposomes by HUT-102 and MT-1 but not by the Tac-negative cell line Molt-4, demonstrating the lack of nonspecific or Fc receptor- mediated uptake. Anti-TFR-conjugated liposomes were effectively bound and internalized by all three cell lines and consistently showed the highest degree of cellular liposome uptake. Drug-containing liposomes conjugated to anti-Tac were more than tenfold more effective in causing growth inhibition of ATL cells than the nonspecific control conjugates. Anti-Tac conjugates caused minimal growth inhibition of Molt-4 cells over the concentration range effective against the ATL cells. Anti-TFR- coupled liposomes gave better growth inhibition of HUT-102 and MT-1 cells (40- to 60-fold) than anti-Tac conjugates. Both anti-Tac-directed and anti-TFR-directed liposomes are effective for intracellular drug delivery to ATL cells and may represent a useful method of treatment in this disease.     

A model of in vivo cell proliferation of adult T-cell leukemia

We have made a model of in vivo cell proliferation of leukemic cells from adult T-cell leukemia (ATL) patients using severe combined immunodeficiency (SCID) mice. Peripheral blood mononuclear cells (PBMC) or lymph node cells (LNC) depleted of B cells and monocytes were intraperitoneally injected into SCID mice treated with antimurine interleukin-2 receptor (IL-2+) beta chain monoclonal antibody (MoAb)(TM- beta 1), followed by daily injection of human recombinant IL-2 until 60 days after cell injection. SCID mice injected with ATL cells from 6 of 8 ATL patients were found to have the tumor or leukemia 5 to 7 weeks after the inoculation of cells. Serum levels of soluble form of human IL-2R alpha chain (Tac) were markedly elevated in such mice. The cells recovered from the mice injected with leukemic cells from four different ATL patients had the same cell surface phenotype as that of original leukemic cells which were CD4+Tac+. Furthermore, we detected the same integration site of human T-cell leukemia virus type I (HTLV- I) provirus and the same rearrangement pattern of human T-cell receptor (TCR) beta chain gene as those of ATL cells by Southern blot hybridization, indicating that the cells proliferating in SCID mice were derived from the original ATL cell clone. Histologic examination showed that the pattern of the infiltration of ATL cells into various organs in SCID mice was similar to that of an ATL patient. Such a model of in vivo cell proliferation of ATL cells will be useful for the study of the mechanism of neoplastic cell proliferation and for the development of a new and effective treatment of ATL.     

Leukemic cells from some adult T-cell leukemia patients proliferate in response to interleukin-4

The proliferative response of fresh peripheral blood leukemic cells from eight adult T cell leukemia (ATL) patients to interleukin-4 (IL-4) was studied to determine the possibility that the IL-4-mediated T-cell growth pathway is involved in the cell growth of leukemic cells in ATL. Resting lymphocytes from ten normal individuals did not proliferate in response to IL-4. Leukemic cells from two ATL patients did not respond to interleukin-2 (IL-2) or IL-4. Leukemic cells from two patients did respond to IL-2, but not to IL-4. In contrast, a strong proliferative response was observed in the IL-4 culture, but not in the IL-2 culture in the remaining four patients. Chromosome analysis of mitotic cells, performed in one of four patients, confirmed that the cells dividing in response to IL-4 were leukemic cells, but not activated normal lymphocytes. These results indicate the activation of IL-4/IL-4 receptor system in leukemic cells from some ATL patients and suggest the possible involvement of the system in the proliferation of leukemic cells and the leukemogenesis in ATL.     

Interleukin-4 induces proliferation of adult T-cell leukemia cells

Abstract: To evaluate the effect of IL-4 on the growth of leukemic cells from adult T-cell leukemia (ATL) patients (ATL cells) and determine whether the IL-4 autocrine mechanism is involved in the growth of ATL cells, we studied the proliferative response of ATL cells, from 11 patients, cultured in the presence or absence of IL-4 in vitro. Leukemic cells from 10 of the 11 patients examined proliferated in response to both IL-2 and IL-4 in a dose-dependent manner. The proliferative response to IL-4 was higher than that obtained with IL-2 in 8 patients. The expression of the IL-2 receptor (IL-2R) αα-chain in leukemic cells from some patients was also enhanced by IL-4. The IL-4 receptor was demonstrated by flow cytometry on the surface of ATL cells. Neither IL-4-induced proliferation of ATL cells nor IL-4-induced IL-2R expression on ATL cells was inhibited by anti-Tac or anti-IL-2 antibody and, therefore, these effects of IL-4 are considered independent of endogenous IL-2 activity. However, IL-2 and IL-4 were undetectable in the culture supernatants of ATL cells from any patient by enzyme-linked immunosorbent assay. Interferon-γ (IFN-γ) partially inhibited IL-2- or IL-4-induced proliferation of ATL cells. These results suggest that leukemic cells from ATL patients proliferate by an IL-2 or IL-4 paracrine mechanism in lymphoid tissue in vivo and that IFN-γ inhibits IL-2- or IL-4-induced proliferation of ATL cells.     

E-selectin and vascular cell adhesion molecule-1 mediate adult T-cell leukemia cell adhesion to endothelial cells

We studied the adhesion properties of peripheral blood leukemic cells from 10 patients with adult T-cell leukemia (ATL) to endothelial cells to better understand the mechanism of leukemic cell infiltration. ATL cells expressed lymphocyte function-associated antigen-1 (LFA-1), but the expression of very late antigen-4 (VLA-4) and sialyl-Lewisx (SLex) was variable. They did not express sialyl-Lewisa (SLea). Cell adhesion assays, which were performed in nine patients, showed marked adhesion of ATL cells to interleukin [IL]-1-activated human umbilical vein endothelial cells (HUVEC). A monoclonal antibody (MoAb) against E- selectin consistently inhibited ATL cell adhesion, and an MoAb against vascular cell adhesion molecule-1 (VCAM-1) or an MoAb against VLA-4 sometimes diminished it. In contrast, an MoAb against LFA-1 had a minor effect on freshly isolated ATL cell adhesion to HUVEC. The percentage of SLex+ cells in the cell population adherent to IL-1-activated HUVEC was slightly higher than that in unseparated cells. These results, together with the detection of E-selectin expression on the endothelium at ATL skin lesions, indicate that E-selectin-mediated adhesion is the major pathway for the adherence of ATL cells to endothelial cells. In addition, the ligand for E-selectin on ATL cells appears to differ from that on neutrophils.     

The recombinant immunotoxin anti-Tac(Fv)-Pseudomonas exotoxin 40 is cytotoxic toward peripheral blood malignant cells from patients with adult T-cell leukemia.

Anti-Tac(Fv)-PE40 is a recombinant single-chain immunotoxin containing the heavy and light variable regions of the anti-Tac monoclonal antibody fused to a mutant form of Pseudomonas exotoxin (PE). Anti-Tac binds to the p55 subunit of the human interleukin 2 (IL-2) receptor, and anti-Tac(Fv)-PE40 kills human or monkey cell lines that contain either the intact IL-2 receptor or its p55 subunit alone. To assess the usefulness of anti-Tac(Fv)-PE40 in treatment of IL-2 receptor-positive leukemia, we tested peripheral blood mononuclear cells from six patients with adult T-cell leukemia. In each of the six patients, anti-Tac(Fv)-PE40 was extremely cytotoxic to the malignant cells. Metabolic activity and sensitivity of the fresh cells improved when a small amount of IL-2 (10 units per ml) was present during incubation. The toxin concentration necessary to inhibit protein synthesis by 50% after 16-hr incubation of cells with immunotoxin varied from 1.6 to 16 ng/ml (2.5-25 x 10(-11) M). In every case, binding was by means of the Tac antigen because anti-Tac(Fv)-PE40 cytotoxicity was prevented by adding excess anti-Tac antibody. Moreover, anti-Tac alone or an inactive mutant of anti-Tac(Fv)-PE40 without ADP-ribosylation activity had very little cytotoxic activity. Peripheral blood mononuclear cells from normal controls, from a patient with Tac-negative leukemia, and from adult T-cell leukemia patients without significant peripheral blood involvement were not sensitive to anti-Tac(Fv)-PE40. These results indicate that anti-Tac(Fv)-PE40 is a potent cytotoxin against adult T-cell leukemia cells in vitro and warrants clinical testing.     

Frequent lack of antibodies against human T-cell leukemia virus type I gag proteins p24 or p19 in sera of patients with adult T-cell leukemia

Summary Specificities of antibodies against human T-cell leukemia virus type I (HTLV-I) in sera of 27 patients with adult T-cell leukemia (ATL) were studied. All sera were positive for HTLV-I antigens by immunofluorescence assay using cells expressing HTLV-I antigens; sera from five ATL patients, however, did not react or hardly reacted with p19 or p24 gag proteins of HTLV-I upon immunoprecipitation assay. Therefore, the relationships among antibody specificities against HTLV-I, the proviral structures of HTLV-I genomes in leukemic cells, and the expression of viral antigens by leukemic cells after cultivation in vitro for a few days were examined. Analyses of the genomic structures of the proviruses revealed deletions in at least seven cases. However, we could not detect deletions in the proviral genomes of four out of the five ATL patients who lacked antibodies against gag proteins. Furthermore, expression of p19 and p24 was detected in these patients' peripheral blood lymphocytes (PBL) cultured in vitro for a few days. Thus, some ATL patients could not or could hardly raise antibodies against gag proteins, although they harbored complete HTLV-I genomes and their PBL expressed gag proteins in vitro. All patients harboring deleted proviruses, so far tested, raised antibodies not only against viral proteins that should be encoded by the integrated proviruses, but also against viral proteins that should be encoded by the deleted regions. Antibodies against viral proteins were detected also in sera of ATL patients whose PBL did not express viral proteins after in vitro cultivation. Specificities of antibodies against viral proteins in ATL patients could not be predicted by the structures of proviruses in leukemic cells or by expression of viral proteins in vitro. Immune responses to HTLV-I antigens were weak or lost in some ATL patients.Abbreviations used HTLV-I human T-cell leukemia virus type I - ATL adult T-cell leukemia - PBL peripheral blood lymphocytes - PAGE Polyacrylamide gel electrophoresis - kb kilobases Supported in part by a grant-in-aid from the Ministry of Health and Welfare of Japan for a comprehensive 10-year strategy for cancer control, and a grant-in-aid from the Ministry of Education, Science and Culture of Japan     

Characterization and regulation of interleukin-4 receptor in adult T-cell leukemia cells

We studied the expression of the receptor of interleukin (IL)-4, one of the T cell growth factors, on fresh peripheral blood leukemic cells from adult T-cell leukemia (ATL) patients. Flow cytofluorometric analysis with a monoclonal antibody to the IL-4 receptor (IL-4R) were used to investigate whether expression of IL-4R on ATL cells is different from that on normal lymphocytes and other types of leukemic cells. Leukemic cells from acute type ATL patients synthesize IL-4R without stimulation, at levels much higher than normal resting lymphocytes and other types of leukemic cells. Furthermore, leukemic cells from acute type ATL showed higher IL-4R expression than that of chronic type ATL or human T-cell leukemia virus type I carriers. In addition, there was correlation between expression of IL-4R on the cell surface and the proliferative response to IL-4. Both IL-4 and IL-2 induced upregulation of IL-4R on activated normal T cells but not on ATL cells. These results suggest that abnormal expression of IL-4R may display different biological activities in ATL compared with other types of leukemia. Furthermore, the high expression of IL-4R in ATL may be involved in the proliferation of leukemic cells and the leukemogenesis in this disease.     

Adult T-cell leukemia

Adult T-cell leukemia (ATL) is a leukemia caused by a monoclonal expansion of HTLV-I-infected T-cells expressing a CD4 antigen. The clinical features of ATL include lymphadenopathy, hepatosplenomegaly, frequent skin lesions, hypercalcemia and a rapidly fatal course. The cell surface phenotype, cytogenetics and functions of leukemic cells are described in association with various clinical manifestations and HTLV-I infection. Leukemic cells constitutively express the p55 (Tac antigen) subunit of the interleukin-2 (IL-2) receptor. Its association with the function of HTLV-I gene products and its possible role in the leukemogenesis of ATL are discussed. Finally, the potential of some therapeutic agents which may selectively eliminate the Tac-expressing leukemic cells in vitro are described, and these may provide an improvement over currently ineffective combination chemotherapy.     

Cell membrane expression and functional role of the p75 subunit of interleukin-2 receptor in lymphoproliferative disease of granular lymphocytes

The cell membrane expression and functional role of the interleukin-2 receptor (IL-2R) was analyzed in nine patients with lymphoproliferative disease of granular lymphocytes (LDGL) using monoclonal antibodies (MoAbs) specific for the p75 (TU27) and the p55 (anti-Tac) subunits of IL-2R. Four patients were characterized by the proliferation of CD3+CD8+ granular lymphocytes (GL) expressing the alpha/beta T-cell receptor (T alpha beta) and one case by the proliferation of CD3+CD4-CD8- GL expressing the gamma/delta T-cell receptor (T gamma delta); in four additional cases proliferating cells were CD3 negative GL. Consistent with data observed on normal GL, phenotypic analysis demonstrated that patients' GL lack the expression of the p55 IL-2R, whereas the p75 subunit is constitutionally expressed by expanding GL of both T-cell (either T alpha beta and T gamma delta) and natural killer (NK) origin in variable proportions (11% to 94% of cells). The analysis of the cytotoxic and proliferative activity demonstrated that the anti-p55 MoAb failed to inhibit IL-2-mediated activation, whereas a marked inhibition of both cytotoxicity and proliferation were obtained using the anti-p75 chain specific MoAb. These data indicate that the p75 chain of IL-2R is responsible for IL-2 signal transduction in both CD3+ and CD3- LDGL patients' GL.     

CD8-positive adult T-cell leukemia cells with an integrated defective HTLV-I genome show a paracrine growth to IL-2

We describe a 50-year-old man with adult T-cell leukemia complicated by laryngeal tuberculosis whose tumor cells proliferate in response to IL-2 in a paracrine manner. On admission, the patient's white blood cell count was 17,900/mm³; 73% were abnormal lymphocytes with convoluted nuclel. FACS analysis showed that the tumor cells were CD4-negative, CD8-positive T cells. Southern blot analysis of tumor cells revealed integration of a defective HTLV-I genome lacking gag and pol genes. He was diagnosed with chronic ATL complicated by laryngeal tuberculosis. The primary leukemic cells expressed IL-2Rα and IL-2Rβ detected by FACS and Northern blot analysis and showed marked growth in response to exogenously added recombinant IL-2 in short-term cultures. Northern blot analysis did not show any IL-2 mRNA. We have previously demonstrated that primary leukemic cells from some ATL patients grow in response to IL-2 in an autocrine or paracrine manner. These results suggest that in CD8 ATL, IL-2 may beinvolved in a paracrine manner. © 1994 Wiley-Liss, Inc.     

Adult T-cell leukemia/lymphoma not associated with human T-cell leukemia virus type I.

We describe five patients with adult T-cell leukemia/lymphoma (ATL) with neither integration of human T-cell leukemia virus type I (HTLV-I) into their leukemia cells nor anti-HTLV-I antibody in their sera. These findings indicate that HTLV-I may not have been involved in leukemogenesis in these patients. The clinicohematological, cytopathological, and immunological features of HTLV-I-negative ATL were exactly the same as those of HTLV-I-associated ATL. Leukemia cells with pleomorphic nuclei, generalized lymphadenopathy, hepatosplenomegaly, skin lesions, hypercalcemia, and elevated lactate dehydrogenase levels, all of which are characteristic features of typical ATL, were also seen in these patients with HTLV-I-negative ATL. Leukemia cells expressed T3, T4, and pan-T-cell antigens in three cases, and T3 and pan-T-cell antigens in two. All five patients had lived in ATL-nonendemic areas. The finding of HTLV-I-negative ATL suggests that factor(s) other than HTLV-I infection may be involved in ATL leukemogenesis.