Augmented efficacy with the combination of blockade of the Notch-1 pathway, bortezomib and romidepsin in a murine MT-1 adult T-cell leukemia model.

Adult T-cell leukemia (ATL) is an aggressive malignancy caused by human T-cell lymphotropic virus-1. There is no accepted curative therapy for ATL. We have reported that certain ATL patients have increased Notch-1 signaling along with constitutive activation of the nuclear factor-κB pathway. Physical and functional interaction between these two pathways provides the rationale to combine the γ-secretase inhibitor compound E with the proteasome inhibitor bortezomib. Moreover, romidepsin, a histone deacetylase inhibitor, has demonstrated major antitumor action in leukemia/lymphoma. In this study, we investigated the therapeutic efficacy of the single agents and the combination of these agents in a murine model of human ATL, the MT-1 model. Single and double agents inhibited tumor growth as monitored by tumor size (P<0.05), and prolonged survival of leukemia-bearing mice (P<0.05) compared with the control group. The combination of three agents significantly enhanced the antitumor efficacy as assessed by tumor size, tumor markers in the serum (human soluble interleukin-2 receptor-α and ß2-microglobulin) and survival of the MT-1 tumor-bearing mice, compared with all other treatment groups (P<0.05). Improved therapeutic efficacy obtained by combining compound E, bortezomib and romidepsin supports a clinical trial of this combination in the treatment of ATL.

Vaccination with tumor cells expressing IL-15 and IL-15Rα inhibits murine breast and prostate cancer.

A number of antitumor vaccines have recently shown promise in upregulating immune responses against tumor antigens and improving patient survival. In this study, we examine the effectiveness of vaccination using interleukin (IL)-15-expressing tumor cells and also examine their ability to upregulate immune responses to tumor antigens. We demonstrated that the coexpression of IL-15 with its receptor, IL-15Rα, increased the cell-surface expression and secretion of IL-15. We show that a gene transfer approach using recombinant adenovirus to express IL-15 and IL-15Rα in murine TRAMP-C2 prostate or TS/A breast tumors induced antitumor immune responses. From this, we developed a vaccine platform, consisting of TRAMP-C2 prostate cancer cells or TS/A breast cancer cells coexpressing IL-15 and IL-15Rα that inhibited tumor formation when mice were challenged with tumor. Inhibition of tumor growth led to improved survival when compared with animals receiving cells expressing IL-15 alone or unmodified tumor cells. Animals vaccinated with tumor cells coexpressing IL-15 and IL-15Rα showed greater tumor infiltration with CD8(+) T and natural killer (NK) cells, as well as increased antitumor CD8(+) T-cell responses. Vaccination with IL-15/IL-15Rα-modified TS/A breast cancer cells provided a survival advantage to mice challenged with unrelated murine TUBO breast cancer cells, indicating the potential for allogeneic IL-15/IL-15Rα-expressing vaccines.

Intrathecal effects of daclizumab treatment of multiple sclerosis.

OBJECTIVES: We previously reported that daclizumab, a humanized monoclonal antibody against CD25, reduced contrast-enhancing lesions (CEL) in patients with multiple sclerosis (MS) who were suboptimal responders to interferon-ß and that this response correlated with expansion of CD56(bright) NK cells. These data have been reproduced in a placebo-controlled multicenter trial (CHOICE study). The current study investigates whether daclizumab monotherapy reduces CEL in untreated patients with relapsing-remitting MS (RRMS) and the effects of daclizumab on the intrathecal immune system. METHODS: Sixteen patients with RRMS with high inflammatory activity were enrolled in an open-label, baseline-vs-treatment, phase II trial of daclizumab monotherapy for 54 weeks and followed by serial clinical and MRI examinations and immunologic biomarkers measured in the whole blood and CSF. RESULTS: The trial achieved predefined outcomes. There was an 87.7% reduction in brain CEL (primary) and improvements in Multiple Sclerosis Functional Composite (secondary), Scripps Neurologic Rating Scale, and Expanded Disability Status Scale (tertiary) outcomes. There was significant expansion of CD56(bright) NK cells in peripheral blood and CSF, with resultant decrease in T cells/NK cells and B cells/NK cells ratios and IL-12p40 in the CSF. Surprisingly, CD25 Tac epitope was equally blocked on the immune cells in the CSF and in peripheral blood. CONCLUSIONS: Daclizumab monotherapy inhibits formation of MS plaques in patients with RRMS and immunoregulatory NK cells may suppress activation of pathogenic immune responses directly in the CNS compartment. CLASSIFICATION OF EVIDENCE: The study provides Class III evidence that daclizumab reduces the number of contrast-enhancing lesions in treatment-naive patients with RRMS over a 54-week period.

Co-adjuvant effects of retinoic acid and IL-15 induce inflammatory immunity to dietary antigens.

Under physiological conditions the gut-associated lymphoid tissues not only prevent the induction of a local inflammatory immune response, but also induce systemic tolerance to fed antigens. A notable exception is coeliac disease, where genetically susceptible individuals expressing human leukocyte antigen (HLA) HLA-DQ2 or HLA-DQ8 molecules develop inflammatory T-cell and antibody responses against dietary gluten, a protein present in wheat. The mechanisms underlying this dysregulated mucosal immune response to a soluble antigen have not been identified. Retinoic acid, a metabolite of vitamin A, has been shown to have a critical role in the induction of intestinal regulatory responses. Here we find in mice that in conjunction with IL-15, a cytokine greatly upregulated in the gut of coeliac disease patients, retinoic acid rapidly activates dendritic cells to induce JNK (also known as MAPK8) phosphorylation and release the proinflammatory cytokines IL-12p70 and IL-23. As a result, in a stressed intestinal environment, retinoic acid acted as an adjuvant that promoted rather than prevented inflammatory cellular and humoral responses to fed antigen. Altogether, these findings reveal an unexpected role for retinoic acid and IL-15 in the abrogation of tolerance to dietary antigens.

Daclizumab (anti-Tac, Zenapax) in the treatment of leukemia/lymphoma.

Daclizumab (Zenapax) identifies the alpha subunit of the interleukin-2 (IL-2) receptor and blocks the interaction of this cytokine with its growth factor receptor. The scientific basis for the choice of the IL-2 receptor alpha subunit as a target for monoclonal antibody-mediated therapy of leukemia/lymphoma is that very few normal cells express IL-2R alpha, whereas the abnormal T cells in patients with an array of lymphoid malignancies express this receptor. In 1997, daclizumab was approved by the FDA for use in the prevention of renal allograft rejection. In addition, anti-Tac provided effective therapy for select patients with T-cell malignancies and an array of inflammatory autoimmune disorders. Finally, therapy with this antibody armed with (90)Y has led to clinical responses in the majority of patients with adult T-cell leukemia. These insights concerning the IL-2/IL-2 receptor system facilitated the development of effective daclizumab antibody therapy for select patients with leukemia/lymphoma.

Antileukemic effect of daclizumab in CD25 high-expressing leukemias and impact of tumor burden on antibody dosing.

Humanized anti-CD25 antibody, daclizumab, was applied in a pilot study of 10 patients with CD25(+) leukemias and pharmacokinetic/pharmacodynamic properties were characterized. Two widely held concepts - tumor sink accelerating pharmacokinetics and higher antigen expression correlating with target cell clearance - were supported by this first systematic evaluation of these questions with actual human clinical data. A flexi-dosing regimen was validated for maintaining target drug levels in vivo with a wide range of tumor burdens. Daclizumab induced clearance of peripheral leukemic cells when highly positive for CD25, but durable responses were not obtained. If daclizumab will have a role in antileukemic therapy, it may be in minimal disease settings or as a component of a combination regimen, but only when CD25 expression is high.

Dynamic, yet structured: The cell membrane three decades after the Singer-Nicolson model.

The fluid mosaic membrane model proved to be a very useful hypothesis in explaining many, but certainly not all, phenomena taking place in biological membranes. New experimental data show that the compartmentalization of membrane components can be as important for effective signal transduction as is the fluidity of the membrane. In this work, we pay tribute to the Singer-Nicolson model, which is near its 30th anniversary, honoring its basic features, "mosaicism" and "diffusion," which predict the interspersion of proteins and lipids and their ability to undergo dynamic rearrangement via Brownian motion. At the same time, modifications based on quantitative data are proposed, highlighting the often genetically predestined, yet flexible, multilevel structure implementing a vast complexity of cellular functions. This new "dynamically structured mosaic model" bears the following characteristics: emphasis is shifted from fluidity to mosaicism, which, in our interpretation, means nonrandom codistribution patterns of specific kinds of membrane proteins forming small-scale clusters at the molecular level and large-scale clusters (groups of clusters, islands) at the submicrometer level. The cohesive forces, which maintain these assemblies as principal elements of the membranes, originate from within a microdomain structure, where lipid-lipid, protein-protein, and protein-lipid interactions, as well as sub- and supramembrane (cytoskeletal, extracellular matrix, other cell) effectors, many of them genetically predestined, play equally important roles. The concept of fluidity in the original model now is interpreted as permissiveness of the architecture to continuous, dynamic restructuring of the molecular- and higher-level clusters according to the needs of the cell and as evoked by the environment.

Colocalization and nonrandom distribution of Kv1.3 potassium channels and CD3 molecules in the plasma membrane of human T lymphocytes.

Distribution and lateral organization of Kv1.3 potassium channels and CD3 molecules were studied by using electron microscopy, confocal laser scanning microscopy, and fluorescence resonance energy transfer. Immunogold labeling and electron microscopy showed that the distribution of FLAG epitope-tagged Kv1.3 channels (Kv1.3/FLAG) significantly differs from the stochastic Poisson distribution in the plasma membrane of human T lymphoma cells. Confocal laser scanning microscopy images showed that Kv1.3/FLAG channels and CD3 molecules accumulated in largely overlapping membrane areas. The numerical analysis of crosscorrelation of the spatial intensity distributions yielded a high correlation coefficient (C = 0.64). A different hierarchical level of molecular proximity between Kv1.3/FLAG and CD3 proteins was reported by a high fluorescence resonance energy transfer efficiency (E = 51%). These findings implicate that reciprocal regulation of ion-channel activity, membrane potential, and the function of receptor complexes may contribute to the proper functioning of the immunological synapse.

Vascular-targeted radioimmunotherapy with the alpha-particle emitter 211At.

Astatine-211, an alpha-particle emitter, was employed in a model system for vascular-targeted radioimmunotherapy of small tumors in mouse lung to compare its performance relative to other radioisotopes in the same system. Astatine-211 was coupled to the lung blood vessel-targeting monoclonal antibody 201B with N-succinimidyl N-(4-[211At]astatophenethyl) succinamate linker. Biodistribution data showed that the conjugate delivered 211At to the lung (260-418% ID/g), where it remained with a biological half-time of about 30 h. BALB/c mice bearing about 100 lung tumor colonies of EMT-6 cells, each about 2000 cells in size, were treated with 211At-labeled monoclonal antibody 201B. The administered activity of 185 kBq per animal extended the life span of treated mice over untreated controls. Injections of 370 kBq, corresponding to an absorbed dose of 25-40 Gy, were necessary to eradicate all of the lung tumors. Mice receiving 740 kBq of 211At-labeled monoclonal antibody 201B developed pulmonary fibrosis 3-4 months after treatment, as did mice treated with 3700 kBq of the alpha-particle emitter 213Bi-labeled monoclonal antibody 201B in previous work. Animals that were injected with 211At bound to untargeted IgG or to glycine, as control agents, also demonstrated therapeutic effects relative to untreated controls. Control groups that received untargeted 211At required about twice as much administered activity for effective therapy as did groups with lung-targeted radioisotope. These results were not consistent with radioisotope biodistribution and dosimetry calculations that indicated that lung-targeted 211At should be at least 10-fold more efficient for lung colony therapy than 211At bound to nontargeting controls. The data showed that 211At is useful for vascular-targeted radioimmunotherapy because lung tumor colonies were eradicated in the mice. Work in this model system demonstrates that vascular targeting of alpha-particle emitters is an efficient therapy for small perivascular tumors and may be applicable to human disease when specific targeting agents are identified.

IL-15 plays a major role in the persistence of Tax-specific CD8 cells in HAM/TSP patients.

IL-15 is a critical cytokine for the maintenance of memory-phenotype CD8 cells in mice. Here, we investigated the role of IL-15 in the neurological disease termed human T cell lymphotropic virus I-associated myelopathy/tropical spastic paraparesis (HAM/TSP). The high number of viral-specific CD8 cells in these patients is associated with inflammatory responses in the central nervous system. Because IL-15 is overexpressed in these patients, we asked whether IL-15 contributes to the persistence of human T cell lymphotropic virus I viral-specific CD8 cells. Using ex vivo cultures of HAM/TSP peripheral blood mononuclear cells, we demonstrated that in the majority of patients examined here blocking IL-15 action resulted in a decrease in the number of viral-specific CD8 cells. This decrease was caused by both inhibition of proliferation and induction of apoptosis in these cells. The data indicate that IL-15 plays a major role in the maintenance of viral-specific CD8 cells in HAM/TSP.

Preparation and in vivo evaluation of linkers for 211At labeling of humanized anti-Tac.

The syntheses, radiolabeling, antibody conjugation, and in vivo evaluation of new linkers for 211At labeling of humanized anti-Tac (Hu-anti-Tac), an antibody to the alpha-chain of the IL-2 receptor (IL-2Ralpha) shown to be a useful target for radioimmunotherapy are described. Synthesis of the organometallic linker precursors is accomplished by reaction of the corresponding bromo- or iodoaryl esters with bis(tributyltin) in the presence of a palladium catalyst. Subsequent conversion to the corresponding N-succinimidyl ester and labeling with 211At of two new linkers, N-succinimidyl 4-[211At]astato-3-methylbenzoate and N-succinimidyl N-(4-[211At]astatophenethyl)succinamate (SAPS), together with the previously reported N-succinimidyl 4-[211At]astatobenzoate and N-succinimidyl 3-[211At]astato-4-methylbenzoate, are each conjugated to Hu-anti-Tac. The plasma survival times of these conjugates are compared to those of directly iodinated (125I) Hu-anti-Tac. The N-succinimidyl N-(4-[211At]astatophenethyl)succinamate compound (SAPS) emerged from this assay as the most viable candidate for 211At-labeling of Hu-anti-Tac. SAPS, along with the directly analogous radio-iodinated reagent, N-succinimidyl N-(4-[125I]astatophenethyl)succinamate (SIPS), are evaluated in a biodistribution study along with directly iodinated (125I) Hu-anti-Tac. Blood clearance and biological accretion results indicate that SAPS is a viable candidate for further evaluation for radioimmunotherapy of cancer.

Comparative cellular catabolism and retention of astatine-, bismuth-, and lead-radiolabeled internalizing monoclonal antibody.

UNLABELLED: Monoclonal antibodies (mAbs) labeled with alpha-emitting radionuclides such as (211)At, (212)Bi, (213)Bi, and (212)Pb (which decays by beta-emission to its alpha-emitting daughter, (212)Bi) are being evaluated for their potential applications for cancer therapy. The fate of these radionuclides after cells are targeted with mAbs is important in terms of dosimetry and tumor detection. METHODS: In this study, we attached various radionuclides that result in alpha-emissions to T101, a rapidly internalizing anti-CD5 mAb. We then evaluated the catabolism and cellular retention and compared them with those of (125)I- and (111)In-labeled T101. T101 was labeled with (211)At, (125)I, (205,6)Bi, (111)In, and (203)Pb. CD5 antigen-positive cells, peripheral blood mononuclear cells (PBMNC), and MOLT-4 leukemia cells were used. The labeled T101 was incubated with the cells for 1 h at 4 degrees C for surface labeling. Unbound activity was removed and 1 mL medium added. The cells were then incubated at 37 degrees C for 0, 1, 2, 4, 8, and 24 h. The activity on the cell surface that internalized and the activity on the cell surface remaining in the supernatant were determined. The protein in the supernatant was further precipitated by methanol for determining protein-bound and non-protein-bound radioactivity. Sites of internal cellular localization of radioactivity were determined by Percoll gradient centrifugation. RESULTS: All radiolabeled antibodies bound to the cells were internalized rapidly. After internalization, (205,6)Bi, (203)Pb, and (111)In radiolabels were retained in the cell, with little decrease of cell-associated radioactivity. However, (211)At and (125)I were released from cells rapidly ((211)At < (125)I) and most of the radioactivity in the supernatant was in a non-protein-bound form. Intracellular distribution of radioactivity revealed a transit of the radiolabel from the cell surface to the lysosome. The catabolism patterns of MOLT-4 cells and PBMNC were similar. CONCLUSION: (211)At catabolism and release from cells were somewhat similar to that of (125)I, whereas (205,6)Bi and (203)Pb showed prolonged cell retention similar to that of (111)In. These catabolism differences may be important in the selection of alpha-radionuclides for radioimmunotherapy.

Differential effects of IL-2 and IL-15 on expression of IL-2 receptor alpha.

IL-2 and IL-15 have overlapping functions since they share the IL-2Rbetagamma receptor complex. However, each cytokine has a private alpha receptor namely IL-2Ralpha for IL-2 and IL-15Ralpha for IL-15. As a consequence the effects of the two cytokines may differ. We describe the differential effects of the two cytokines regarding the induction of cell surface expression of the IL-2Ralpha subunit on YT-l cells. Both cytokines induced transcription of the IL-2Ralpha gene. Furthermore translation of IL-2Ralpha leading to intracellular expression of the receptor was observed following either IL-2 or IL-15 addition. However, only IL-15 was associated with the induction of cell surface expression of IL-2Ralpha. With IL-2 there appears to be an impediment to the translocation of IL-2Ralpha to the cell membrane. Since surface expression of IL-2Ralpha is a key element in the formation of the high affinity IL-2 receptor, translocation of IL-2Ralpha to the membrane represents another level of control of the immune response in addition to regulation of IL-2Ralpha transcription and translation.

Onset of natural killer cell lymphomas in transgenic mice carrying a truncated HMGI-C gene by the chronic stimulation of the IL-2 and IL-15 pathway.

Rearrangements of the high mobility group protein I-C (HMGI-C) gene, consisting in the loss of the carboxyl-terminal tail, have been frequently detected in benign human tumors of mesenchymal origin. We have previously demonstrated that transgenic (TG) mice carrying a truncated HMGI-C construct (HMGI-C/T) exhibit a giant phenotype together with a predominantly abdominal/pelvic lipomatosis. Here, we report that HMGI-C/T TG mice develop natural killer (NK)-T/NK cell lymphomas starting from 12 months of age. We found an increased expression of IL-2 and IL-15 proteins and their receptors in these lymphomas, and we demonstrate that HMGI-C/T protein positively regulates their expression in vitro. Therefore, the HMGI-C/T-mediated chronic stimulation of the IL-2/IL-15 pathway could be responsible for the onset of NK-T/NK cell lymphomas in HMGI-C/T TG mice.

The combination of zidovudine and interferon alpha-2B in the treatment of adult T-cell leukemia/lymphoma.

Adult T-cell leukemia/lymphoma (ATL) is frequently a very aggressive malignancy with a poor survival despite aggressive multiagent chemotherapy. The combination of the antiretroviral drug zidovudine (AZT) and interferon alpha (IFNalpha) has been reported to induce remissions in patients with ATL. The purpose of this study was to evaluate the clinical response and toxicity following administration of a combination of IFNalpha-2b and AZT in patients with human T-cell lymphotropic virus type I (HTLV-I)-associated ATL. Eighteen patients with ATL (chronic. crisis, acute or lymphoma type) were treated with the combination of AZT (50 - 200 mg orally 5 times a day) and IFNalpha-2b (2.5 - 10 million units subcutaneously daily). Three patients had objective responses lasting more than one month. One patient had a clinical complete remission, lasting 21.6 months and two patients had partial remissions lasting 3.7 and 26.5 months. Six patients were not considered evaluable for response due to short and/or interrupted periods of treatment. Seventeen patients have died with a median survival time after initiation of therapy of 6 months. Neutropenia and thrombocytopenia were the dose limiting toxicities. In conclusion, the response rate in this study was lower than noted in the two previous published series. This may be due to the amount and type of prior treatment our patients had received.

Clustering of class I HLA oligomers with CD8 and TCR: three-dimensional models based on fluorescence resonance energy transfer and crystallographic data.

Fluorescence resonance energy transfer (FRET) data, in accordance with lateral mobility measurements, suggested the existence of class I HLA dimers and oligomers at the surface of live human cells, including the B lymphoblast cell line (JY) used in the present study. Intra- and intermolecular class I HLA epitope distances were measured on JY B cells by FRET using fluorophore-conjugated Ag-binding fragments of mAbs W6/32 and L368 directed against structurally well-characterized heavy and light chain epitopes, respectively. Out-of-plane location of these epitopes relative to the membrane-bound BODIPY-PC (2-(4,4-difluoro-5-(4-phenyl-1,3-butadienyl)-4-bora-3a,4a-diaza-s-indacene-3-pentanoyl)-1-hexadecanoyl-sn-glycero-3-phosphocholine) was also determined by FRET. Computer-simulated docking of crystallographic structures of class I HLA and epitope-specific Ag-binding fragments, with experimentally determined interepitope and epitope to cell surface distances as constraints, revealed several sterically allowed and FRET-compatible class I HLA dimeric and tetrameric arrangements. Extension of the tetrameric class I HLA model with interacting TCR and CD8 resulted in a model of a supramolecular cluster that may exist physiologically and serve as a functionally significant unit for a network of CD8-HLA-I complexes providing enhanced signaling efficiency even at low MHC-peptide concentrations at the interface of effector and APCs.

Comparative assessment of virulence of recombinant vaccinia viruses expressing IL-2 and IL-15 in immunodeficient mice.

IL-2 and -15 belong to the four alpha-helix bundle family of cytokines and display a spectrum of overlapping immune functions because of shared signal transducing receptor components of the IL-2 receptor complex. However, recent evidence suggests a nonredundant unique role for IL-15 in the establishment and perhaps maintenance of peripheral natural killer (NK) cell populations in vivo. To explore the contribution of locally released IL-15 on peripheral NK-cell-mediated innate immune responses, we generated a recombinant vaccinia virus that expresses IL-15 and evaluated the course of vaccinial disease in athymic nude mice. Coexpression of IL-15 resulted in the attenuation of virulence of vaccinia virus, and mice inoculated with 10(5) plaque-forming units or less resolved the infection successfully. In contrast, mice inoculated with a similar dose of the control vaccinia virus failed to eliminate the virus and died of generalized vaccinial disease. Enhanced expression of IL-12 and IFN-gamma as well as induction of chemokines were evident in the mice inoculated with IL-15-expressing vaccinia virus in addition to an increase in NK cells in the spleen. However, in this model system, the degree of attenuation in viral virulence attained with coexpression of IL-15 was much less than that achieved with coexpression of IL-2, suggesting that the peripheral NK-cell-mediated events are more responsive to IL-2 than to IL-15.

Human T cell lymphotropic virus type I Tax activates IL-15R alpha gene expression through an NF-kappa B site.

IL-15 mRNA levels are increased in diseases caused by human T cell lymphotropic virus type I (HTLV-I). In this study, we demonstrated that IL-15Ralpha, the IL-15-specific binding receptor, mRNA and protein levels were also elevated in HTLV-I-infected cells. We showed that transient HTLV-I Tax expression lead to increased IL-15Ralpha mRNA levels. In addition, by using a reporter construct that bears the human IL-15Ralpha promoter, we demonstrated that Tax expression increased promoter activity by at least 4-fold. Furthermore, using promoter deletion constructs and gel shift analysis, we defined a functional NF-kappaB-binding motif in the human IL-15Ralpha promoter, suggesting that Tax activation of IL-15Ralpha is due, in part, to the induction of NF-kappaB. These data indicate that IL-15Ralpha is transcriptionally regulated by the HTLV-I Tax protein through the action of NF-kappaB. These findings suggest a role for IL-15Ralpha in aberrant T cell proliferation observed in HTLV-I-associated diseases.