Affinity IL-2 Receptor Research Articles

Abstract 4225: IL-2Rβ/IL-2R γsynthetic cytokines induce activation of human T and NK cells

Abstract Introduction: Heterodimerization of the intermediate affinity Interleukin-2 Receptors (IL-2R), IL-2Rβ and IL-2Rγ, initiates a signaling cascade in T and NK cells that ultimately results in proliferation and production of Interferon-gamma (IFN-γ). Binding of IL-2 in a dimeric complex with intermediate affinity or in the high affinity trimeric complex, which also includes IL-2Rα and is predominantly expressed on antigen activated T cells and Tregs leads to trans-phosphorylation of signaling motifs on the IL-2Rβ and IL-2Rγ intracellular domains by the associated JAK kinases. The function of the wild type IL-2 is to bring the receptor chains in close proximity to produce an optimal level of phosphorylation and activation of associated STAT transcription factors. Here we established a way to bring IL-2Rβ and IL-2Rγ chains together in a cytokine-independent manner that results in functional activation. Experimental Procedures: Human IL-2Rβ and IL-2Rγ specific heavy chain single domain antibodies (VHH) were generated by camel immunization and screening of VHH libraries prepared from peripheral blood cells for binding. Ten IL-2Rβ VHH segments and six IL-2Rγ VHH segments, all with low nanomolar affinity, were identified and coupled as IL-2Rβ/IL-2Rγ VHH dimers in all possible combinations in both amino-carboxy and carboxy-amino orientations yielding 120 different proteins that were tested in human T and NK cell functional assays. Results: IL-2Rβ/IL-2Rγ VHH dimers were biologically active and induced pSTAT-5 phosphorylation in the IL-2 dependent NK cell line NKL at various levels. The biological activity of these IL-2Rβ/IL-2Rγ VHH dimers was further confirmed on primary cells. IL-2Rβ/IL-2Rγ VHH dimers induced pSTAT5 phosphorylation on NK cells isolated from human peripheral blood and culture with IL-2Rβ/IL-2Rγ VHH dimers resulted in proliferation and production of high but varied levels of IFN-γ. The activity of IL-2Rβ/IL-2Rγ VHH dimers on primary T cells was also examined. Similar to NK cells, IL-2Rβ/IL-2Rγ VHH dimers induced pSTAT5 phosphorylation, proliferation and IFN-γ production by CD4 positive and CD8 positive T cell blasts generated after CD3/CD28 activation of human PBMC. Conclusions: In conclusion, we have generated a series of functional IL-2Rβ/IL-2Rγ VHH dimers representing surrogate cytokine agonists each with unique signaling strengths that will now be further analyzed for potential therapeutic application in tumor immunology and auto-immunity Citation Format: Rene de Waal Malefyt, PJ Aspuria, Sandro Vivona, Priyanka Balasubrahmanyam, Cindy Buffone, Mahalakshmi Ramadass, Romina Riener, Martin Oft, Patrick Lupardus, Robert Kastelein. IL-2Rβ/IL-2R γsynthetic cytokines induce activation of human T and NK cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 4225.

Abstract 2202: The combination of a mouse ortholog of ALKS 4230, a selective agonist of the intermediate affinity IL-2 receptor, and the angiogenesis inhibitor lucitanib enhances antitumor activity

Abstract ALKS 4230, an engineered cytokine designed for selective binding to the intermediate affinity interleukin-2 receptor (IL-2R), is being evaluated as a monotherapy and in combination with pembrolizumab in patients with advanced solid tumors. The selectivity of ALKS 4230 is achieved through stable fusion of circularly permuted IL-2 to IL-2Rα, with the goal of driving antitumor responses via selective activation of CD8+ T cells and NK cells, while avoiding activation of regulatory T cells (Tregs), which express the high affinity IL-2R. High levels of vascular endothelial growth factor (VEGF) are associated with an immunosuppressive tumor microenvironment and diminished response to high-dose IL-2. Lucitanib is an anti-angiogenic, multi-tyrosine kinase inhibitor targeting VEGFR1-3, PDGFRα/β, and FGFR1-3. We evaluated the antitumor efficacy and mechanism of the combination of RDB 1462, the mouse ortholog of ALKS 4230, and lucitanib in the MC38 mouse syngeneic tumor model. C57BL/6 mice were subcutaneously implanted with MC38 cells and treated with RDB 1462 (1.5 mg/kg and 3 mg/kg once every 3 days for 3 weeks), lucitanib (10 mg/kg daily for 28 days), or the combination of both molecules. After 11 days of treatment, lucitanib monotherapy resulted in 100% tumor growth inhibition (TGI), while 1.5 mg/kg and 3 mg/kg RDB 1462 monotherapy resulted in 25% and 51% TGI, respectively. Lucitanib monotherapy extended survival, with a mean survival time (MST) of 39 days compared to 15 days for vehicle, with no complete tumor regressions. MSTs were 18-22 days for RDB 1462 monotherapy groups, 60 days for the lower dose combination arm, and >60 days for the higher dose combination. Durable complete tumor regressions were observed in 1/20 mice treated with either dose of RDB 1462 monotherapy and 5/10 mice treated with the lower dose of RDB 1462 in combination with lucitanib. 10/10 mice treated with the higher dose of RDB 1462 in combination with lucitanib exhibited complete responses on Day 60. Flow cytometric analyses revealed that RDB 1462 treatment induced significant peripheral CD8+ T cell expansion compared to vehicle (p<0.05) without an increase in Foxp3+ Tregs. This effect was enhanced by the combination of RDB 1462 with lucitanib. The combination led to a significant (p<0.05) increase in intratumoral CD8+ T cells and dendritic cells (CD11c+F4/80−), together with a reduction in tumor-associated macrophages (CD11b+F4/80+). The combination treatment elicited a distinct gene expression profile in tumors by RNA-Seq, merging the immunostimulatory effects of RDB 1462 with the anti-angiogenic effects of lucitanib. The combination of an intermediate affinity IL-2R-selective cytokine and an angiogenesis inhibitor resulted in durable dose-dependent antitumor efficacy in the MC38 mouse tumor model. Citation Format: Jared E. Lopes, Rachel L. Dusek, Liliane Robillard, Minh Nguyen, Bruce Roth, Bryan Vought, Arthur Liberzon, Raymond J. Winquist, Heather C. Losey. The combination of a mouse ortholog of ALKS 4230, a selective agonist of the intermediate affinity IL-2 receptor, and the angiogenesis inhibitor lucitanib enhances antitumor activity [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2202.

Daclizumab for the treatment of adults with relapsing forms of multiple sclerosis

ABSTRACTIntroduction: The goal of the article is to review the mechanism of action and the use of daclizumab, a humanized monoclonal antibody (mAb) against the alpha subunit of the high affinity interleukin-2 (IL-2) receptor, in the treatment of Multiple Sclerosis (MS).Areas covered: PubMed was searched for the terms ‘daclizumab’ and ‘multiple sclerosis’. The mechanisms of action, pharmacokinetics and pharmacodynamics, major studies, side effects and drug interactions of daclizumab in MS are discussed.Expert commentary: Monthly daclizumab-beta [DAC-beta, formerly daclizumab high yield process (DAC HYP), approved as ZINBRYTA®, which has a different form and structure than an earlier form of daclizumab], is an effective and convenient treatment option for patients with relapsing forms of MS who have failed other treatment, or as a first-line option in highly active MS patients. IL-2 signaling modulation by daclizumab constitutes a novel mechanism of action which may also underlie the adverse and serious adverse events and risk profile of the drug that requires appropriate patient selection, monitoring and risk-mitigation programs.

Diphtheria toxin-based bivalent human IL-2 fusion toxin with improved efficacy for targeting human CD25+ cells

Regulatory T cells (Treg) constitute a major inhibitory cell population which suppresses immune responses. Thus, Treg have proven to be key players in the induction of transplantation tolerance, protection from autoimmune disease and prevention of the development of effective anti-tumor immune reactions. Treg express high levels of the high affinity interleukin-2 receptor (IL-2R) consisting of IL-2Rα (CD25) together with IL-2Rβ (CD122) and the common γ-chain (CD132). An effective reagent capable of depleting Treg in vivo would facilitate better cancer treatment and allow mechanistic studies of the role of Treg in transplantation tolerance and the development of autoimmune disease. In this study, we have developed a novel bivalent human IL-2 fusion toxin along with an Ontak®-like monovalent human IL-2 fusion toxin and compared the functional ability of these reagents in vitro. Here we show that genetically linking two human IL-2 domains in tandem, thereby generating a bivalent fusion toxin, results in significantly improved capacity in targeting human CD25+ cells in vitro. Binding analysis by flow cytometry showed that the bivalent human IL-2 fusion toxin has notably increased affinity for human CD25+ cells. In vitro functional analysis demonstrated that the bivalent isoform has an increased potency of approximately 2 logs in inhibiting cellular proliferation and protein synthesis in human CD25+ cells compared to the monovalent human IL-2 fusion toxin. Additionally, we performed two inhibition assays in order to verify that the fusion toxins target the cells specifically through binding of the human IL-2 domain of the fusion toxin to the human IL-2 receptor on the cell surface. These results demonstrated that 1) both monovalent and bivalent human IL-2 fusion toxins are capable of blocking the binding of biotinylated human IL-2 to human CD25 by flow cytometry; and 2) human IL-2 blocked the fusion toxins from inhibiting protein synthesis and cellular proliferation in vitro, thus confirming that the human IL-2 fusion toxins target the cells specifically through binding to the human IL-2 receptor. We believe that the bivalent human IL-2 fusion toxin will be a more potent, and therefore, more optimal agent than the current clinically-used monovalent fusion toxin (denileukin diftitox, Ontak®) for in vivo depletion of Treg.

DAB389IL-2 suppresses autoimmune inflammation in the CNS and inhibits T cell-mediated lysis of glial target cells

In multiple sclerosis (MS) and its rodent model, experimental autoimmune encephalomyelitis (EAE), activated CD4+ T cells with upregulated IL-2R mediate inflammation and demyelination in the central nervous system (CNS). DAB389IL-2, a chimeric fusion protein of IL-2 and diphtheria toxin, inhibits human and rodent IL-2 activated T cells that express the high affinity interleukin-2 receptor. In the present study, DAB389IL-2 was used to treat rats with EAE. We wanted to investigate the possibility that DAB389IL-2 could prevent tissue destruction within the CNS. We used a suboptimal dose of DAB389IL-2 that allowed substantial transmigration of inflammatory cells across the blood–brain barrier. DAB389IL-2 inhibited infiltration of CD4+, CD8+, CD25+ and TCR αβ+ associated mononuclear cells and inflammatory macrophages in the spinal cord on day 13 post-immunization, at the peak of disease. Gene expression study showed that DAB389IL-2 treatment suppressed TNF-α and IFN-γ as well as IL-10 cytokine gene expression in the spinal cord of rats with EAE on day 13. DAB389IL-2 in vitro treatment suppressed cytotoxicity of MBP-activated T cells from rats with EAE against oligodendrocytes in culture by 66%. Astrocytes were less targeted by MBP activated T cells in vitro. This study suggests that DAB389IL-2 directly targets CD4+ and CD25+ (IL-2R) T cells and effector T cell function and also indirectly suppresses the activation of macrophage CD169+ (ED3+) and microglia CD11b/c (OX42+) populations in the CNS.

Expression of Heme Oxygenase‐1 in Neural Stem/Progenitor Cells as a Potential Mechanism to Evade Host Immune Response

Besides their therapeutic benefit as cell source, neural stem/progenitor cells (NSPCs) exhibit immunosuppressive properties of great interest for modulating immune response in the central nervous system. To decipher the mechanisms of NSPC-mediated immunosuppression, activated T cells were exposed to NSPCs isolated from fetal rat brains. Analyses revealed that NSPCs inhibited T-cell proliferation and interferon-gamma production in a dose-dependent manner. A higher proportion of helper T cells (CD4+ T cells) was found in the presence of NSPCs, but analyses of FoxP3 population indicated that T-cell suppression was not secondary to an induction of suppressive regulatory T cells (FoxP3+ CD4+ CD25+). Conversely, induction of the high affinity interleukin-2 (IL-2) receptor (CD25) and the inability of IL-2 to rescue T-cell proliferation suggest that NSPCs display immunosuppressive activity without affecting T-cell activation. Cultures in Transwell chambers or addition of NSPC-conditioned medium to activated T cells indicated that part of the suppressive activity was not contact dependent. We therefore searched for soluble factors that mediate NSPC immunosuppression. We found that NSPCs express several immunosuppressive molecules, but the ability of these cells to inhibit T-cell proliferation was only counteracted by heme oxygenase (HO) inhibitors in association or not with nitric oxide synthase inhibitors. Taken together, our findings highlight a dynamic crosstalk between NSPCs and T lymphocytes and provide the first evidence of an implication of HO-1 in mediating the immunosuppressive effects of the NSPCs.

A Pilot Study of IL-2Rα Blockade during Lymphopenia Depletes Regulatory T-cells and Correlates with Enhanced Immunity in Patients with Glioblastoma

BackgroundPreclinical studies in mice have demonstrated that the prophylactic depletion of immunosuppressive regulatory T-cells (TRegs) through targeting the high affinity interleukin-2 (IL-2) receptor (IL-2Rα/CD25) can enhance anti-tumor immunotherapy. However, therapeutic approaches are complicated by the inadvertent inhibition of IL-2Rα expressing anti-tumor effector T-cells.ObjectiveTo determine if changes in the cytokine milieu during lymphopenia may engender differential signaling requirements that would enable unarmed anti-IL-2Rα monoclonal antibody (MAbs) to selectively deplete TRegs while permitting vaccine-stimulated immune responses.MethodologyA randomized placebo-controlled pilot study was undertaken to examine the ability of the anti-IL-2Rα MAb daclizumab, given at the time of epidermal growth factor receptor variant III (EGFRvIII) targeted peptide vaccination, to safely and selectively deplete TRegs in patients with glioblastoma (GBM) treated with lymphodepleting temozolomide (TMZ).Results and ConclusionsDaclizumab treatment (n = 3) was well-tolerated with no symptoms of autoimmune toxicity and resulted in a significant reduction in the frequency of circulating CD4+Foxp3+ TRegs in comparison to saline controls (n = 3)( p = 0.0464). A significant (p<0.0001) inverse correlation between the frequency of TRegs and the level of EGFRvIII specific humoral responses suggests the depletion of TRegs may be linked to increased vaccine-stimulated humoral immunity. These data suggest this approach deserves further study.Trial RegistrationClinicalTrials.gov NCT00626015

Natural killer T cells constitutively expressing the interleukin‐2 receptor α chain early in life are primed to respond to lower antigenic stimulation

Invariant natural killer T (iNKT) cells are known to constitutively express the high affinity interleukin-2 receptor α chain (CD25) in neonates, but the functional consequence of this phenotype is unknown. Here, we show that high numbers of CD25-expressing iNKT cells are present early in gestation and represent a significant proportion of the developing immune system. Despite their activated phenotype, neonatal iNKT cells express high levels of the Krüppel-like factor-2, a transcription factor associated with quiescent T cells, and require de novo T-cell receptor and CD28 co-stimulation to proliferate. In contrast to bona fide CD4/CD25-expressing regulatory T cells, neonatal iNKT cells do not suppress T-cell responses, indicating that they do not represent an immunosuppressive cell subset. Evidence that neonatal iNKT cells respond to dramatically reduced amounts of CD1d-restricted antigen compared with adult iNKT cells or T cells, and that their proliferation can be induced in the absence of early interleukin-2 suggest that constitutive expression of CD25 'primes' neonatal iNKT cells to respond rapidly to low amounts of antigen. This unique phenotype, which is distinct from adult iNKT cells, as well as other CD25-expressing activated T or regulatory T cells, may be important to ensure stability of a structurally limited peripheral iNKT-cell repertoire early in life.

The effect of selenium supplementation on DTH skin responses in healthy North American Men

The trace element selenium (Se) is essential for immune system development and function in animals. However, the exact functions of Se in the human immune system and the achievable health benefits from Se supplementation remain unclear. To test whether an increased intake of dietary Se affects immune function, we conducted a randomized, controlled trial of Se supplementation in healthy free-living men. Forty-two men were administered 300 μg of Se a day as high-Se Baker's yeast, or low-Se yeast for 48 weeks. Serum immunoglobulins, differential complete blood counts and lymphocyte sub-populations were measured every 6 weeks. Tests of delayed-type hypersensitivity (DTH) skin responses to mumps, candida, trychophyton, tuberculin-purified protein, and tetanus were performed at baseline and at the end of 48 weeks of treatment. Supplementation increased blood Se concentration by 50%. Surprisingly, consumption of the low-Se yeast induced anergy in DTH skin responses and increased counts of natural killer (NK) cells and T lymphocytes expressing both subunits of the high affinity interleukin-2 receptor (IL2R). DTH skin responses and IL2R+ cells did not change in the high-Se group, suggesting Se supplementation blocked induction of DTH anergy. There were no differences between groups in quality of life indicators, number of days sick, other leukocyte phenotypes, serum immunoglobulins, or complement factors. These results suggest that Se plays a role in immunotolerization, a cell-mediated process involved in many aspects of immune function.

Effect of daclizumab on TReg counts and EGFRvIII-specific immune responses in GBM

2034 Background: TRegs are increased in patients with GBM and constitutively express the high affinity interleukin-2 receptor (IL-2Rα). Treatment with an antibody that blocks IL-2Rα signaling functionally inactivates and eliminates TRegs without inducing autoimmune toxicity in murine models. We hypothesized that daclizumab, a commercially-available, IL-2Rα-specific antibody would function identically.Methods: A randomized phase II clinical trial assessed the effects of daclizumab in the context of the cancer vaccine, CDX-110, which is comprised of an EGFRvIII-specific peptide sequence linked to KLH. EGFRvIII is a constitutively activated and immunogenic mutation not expressed in normal tissues, but widely expressed in GBMs and other neoplasms. In patients with newly-diagnosed, EGFRvIII+ GBM, after resection and radiation/TMZ, patients received CDX-110 vaccinations biweekly x 3, then monthly until tumor progression in combination with TMZ (200 mg/m2 x 5/28 days). Half the patients were randomized to receive daclizumab (1mg/Kg x1) at the first vaccine. The others received saline in a double-blinded fashion.Results: There were no drug related SAEs. EGFRvIII-specific immune responses were generated in all patients, and all immune responses were sustained or enhanced during subsequent TMZ cycles. Preliminary analysis (n = 4) suggests that daclizumab reduces Treg (CD4+CD25+CD45RO+FOXP3+) numbers [change 82.4 ± 7.1% from baseline (p = 0.011; t-test)] without reducing overall CD8+ or CD4+ T-cell counts. Tregs decreased only 3.7 + 11.0% after vaccination in the saline treated group during the same interval. Preliminary analysis (n = 4) also suggest that daclizumab enhanced EGFRvIII-specific immune responses (p = 0.01; t-test) and enhanced the titer of cytotoxic EGFRvIII-specific IgG1 isotype antibodies compared to the saline treated group (p = 0.003; t-test) and compared to previously vaccinated patients who did not receive daclizumab (p = 0.0015; t-test). TTP and OS survival in both arms has not been reached. Conclusions: Daclizumab may reduce Treg counts in patients with GBM. TMZ and daclizumab may enhance EGFRvIII-targeted immune responses despite lymphodepletion. These combinations are currently under further investigation. [Table: see text]

Dendritic cells as controllers of antigen-specific Foxp3+ regulatory T cells

Regulatory T cells (Treg) are a subpopulation of CD4(+) lymphocytes that maintain immunological self-tolerance in the periphery. Treg also regulate or suppress other classes of immune response such as allograft rejection, allergy, tumor immunity, and responses to microbes. Treg express the Foxp3 transcription factor and CD25, the high affinity interleukin-2 receptor (IL-2R). Treg are divided into two types: naturally occurring Treg derived from thymus (natural Treg) and Treg induced from Foxp3(-) CD4(+) T cells in the periphery (induced Treg). It would be valuable to understand how to control the generation of antigen-specific Treg, which could also provide a new approach to treat autoimmunity, allergy or allograft rejection without suppressing immune responses to tumor and microbes. In this review, we will discuss the role of dendritic cells (DCs) in controlling antigen-specific natural Treg and induced Treg. Natural Treg are anergic upon T cell receptor stimulation generally, however, we found that the antigen-specific natural Treg can be expanded by antigen-presenting mature bone marrow-derived dendritic cells (BM-DCs). Furthermore, recent studies showed that antigen-specific Treg can be induced from Foxp3(-) CD25(-) CD4(+) T cells by antigen-presenting DCs, particularly select subsets of DCs in the periphery. These findings need to be pursued to develop novel immune suppressive therapies using antigen-specific Treg educated by DCs.

Changes in natural killer cell phenotype in patients with post-viral fatigue syndrome

We analysed peripheral blood CD56+ natural killer (NK) cell subsets in 23 carefully characterized patients with post-viral fatigue syndrome (PFS), compared with 19 healthy controls, using fluorochrome-conjugated, specific monoclonal antibodies and the FACScan. We found significantly increased percentages of CD56+, and especially CD56bright+ NK cells in PFS patients. We also found significantly increased percentages of CD56+ high affinity interleukin-2 (IL-2) receptor (CD25)+ and CD56+ transferrin receptor (CD71+) subsets of cells, most of which also stained brightly for CD56. Also, we found an increased percentage of CD56+ CD3+ cells, many of which stained brightly for CD56, although there was no increase in the percentage of CD56- CD3+ T cells in these patients. These observations, in conjunction with very low percentage of CD56- CD25+ cells, suggest that there is a preferential involvement of this minor subset of CD56+ CD3+ T cells in PFS. Finally, a decreased percentage of CD56+ Fc gamma receptor (CD16)+ NK cells was identified, which suggests a reduced capacity of antibody-dependent cellular cytotoxicity in PFS patients. Subsets of CD56+ NK cells co-expressing CD2, CD4 or CD8 did not show any significant difference between PFS patients and healthy controls. These phenotypic changes provide laboratory evidence of immunological abnormalities in this syndrome, and, we suggest, may be consistent with persistent viral infection.

Denileukin diftitox: a biotherapeutic paradigm shift in the treatment of lymphoid-derived disorders

Denileukin diftitox (Ontak®) represents an example of a fused molecule that targets cells bearing high affinity interleukin-2 receptors internalized via receptor-mediated endocytosis in an acidified vesicle. Denileukin diftitox is proteolytically cleaved within the endosome liberating the enzymatically active portion of the diphtheria toxin, the A fragment. Diphtheria toxin fragment A is released into the cytosol inhibiting the protein synthesis through the ADP-ribosylation of the elongation factor-2, and leading to cell death. This review focuses on the clinical trials that led to the FDA approval of the drug for cutaneous T cell lymphoma in the US, and investigational studies demonstrating drug-activity against B and T-cell non-Hodgkin’s lymphoma, chronic lymphocytic lymphoma and acute graft versus disease within allogeneic hematopoietic stem cell transplant.

A Novel Human CD34(+) Subset That Constitutively Expresses the High Affinity Interleukin-2 Receptor Traffics to Lymph Nodes and Differentiates into CD56Bright Natural Killer Cells.

In adult humans, T cells differentiate in the thymus and B cells develop in the bone marrow, but the site(s) of natural killer (NK) cell differentiation are unclear. Here we describe, for the first time, a unique CD34(+) population found in human lymph nodes (LN) that differentiates into NK cells. CD56bright NK cells represent <10% of NK cells in peripheral blood (PB) yet predominate in LN where they can compete for endogenous T cell-derived IL-2 during immune activation due to their unique expression of functional high affinity (HA) interleukin (IL)-2 receptors (IL-2R). We hypothesized that a subset of CD34(+) hematopoietic precursor cells (HPC) might also express functional HA IL-2R and potentially differentiate into CD56bright NK cells via activation with low dose IL-2. We first identified a novel human CD34dimCD45RA(+) HPC in PB with constitutive expression of the HA IL-2R. When cultured in picomolar concentrations of IL-2 that selectively saturate the HA IL-2R, these cells give rise to CD56bright NK cells, and this effect is blocked when IL-2 cannot bind to its HA receptor. This unique CD34(+) population expresses IL-2Rα, CD2, CD7, c-kit, L-selectin, and NKR-P1A, all of which are also expressed by CD56bright NK cells. Unique among total PB CD34(+) cells, this novel population displays high integrin α4β7 expression. This attribute, in addition to its high L-selectin expression, suggested that these cells may traffic to LN where their progeny, CD56bright NK cells, represent the major NK subset. Indeed we found a distinct CD34dimCD45RA(+)α4β7bright population that resides in the T cell rich regions of human LN, and when stimulated in vitro with 10 pM IL-2, this cell gives rise to CD56bright NK cells. This novel population represents only ~6% of all PB CD34(+) HPC yet is the major if not exclusive CD34(+) subset in LN. While murine studies strongly support the notion that most if not all NK cells require IL-15 for their development, these new human data suggest a model for development of a minor human NK subset, the CD56bright NK cells, whereby CD34dimCD45RA(+)α4β7bright HPC constitutively expressing the HA IL-2R traffic to peripheral LN where endogenous T cell-derived IL-2 can drive CD56bright NK cell differentiation in vivo.

Denileukin diftitox (ONTAK) as targeted therapy against steroid refractory graft-versus-host disease (GVHD) after allogeneic hematopoietic stem cell transplantation (HSCT)

Denileukin diftitox (ONTAK®) is a recombinant fusion protein composed of human interleukin-2 linked to the membrane translocation and catalytic domains of diphtheria toxin. ONTAK has potent selective killing activity against activated lymphocytes that express the high or intermediate affinity interleukin-2 receptor (IL-2R). Monoclonal antibodies that target the IL-2R have demonstrated clinical activity against acute GVHD. ONTAK may be superior to these agents because it does not rely on secondary effector mechanisms for cell kill, and entry of a few molecules into the cytoplasm may be sufficient to induce apoptosis. Furthermore, because ONTAK selectively destroys activated T-cells and spares resting lymphoid populations, valuable immune function and reconstitution potential may be preserved. We have completed a phase I study to assess the dosing of ONTAK in patients with steroid-refractory GVHD after allogeneic HSCT. Thirty patients (3 related donor, 27 unrelated donor) were treated for grade II (n = 10), grade III (n = 15), or grade IV (n = 5) steroid-refractory acute GVHD. Seven received ONTAK at dose level I (9 μg/kg IV on day 1 and 15), eighteen were treated on dose level II (9 μg/kg IV days 1,3,5,15,17,19), five were treated on dose level III (9 μg/kg IV days 1–5, 15–19). At dose level III, 4 of 5 subjects developed dose-limiting toxicity (1 acute renal failure, 3 hepatic transaminase elevation). Therefore, dose level II was deemed the maximum tolerated dose (MTD). In all patients, elevated transaminases resolved promptly upon withdrawal of ONTAK. Only 2 (7%) developed infusional reactions with transient hypotension. Twenty-four patients were evaluable for GVHD response. After 4 weeks, 8 (33%) had complete GVHD resolution (CR) of, 9(38%) had partial response (PR), for an overall response rate of 71%. Among the 9 PRs, 4 subsequently entered CR without additional GVHD therapy, resulting in an overall CR of 50%. Response was highest at the MTD, where 6 (46%) achieved CR, and 3 (23%) had PR, all of whom eventually converted to CR (overall CR = 69%). Of the 24 patients evaluable for GVHD response, 9(38%) are alive, with the longest survivor being GVHD free at 20 months. Our results show that ONTAK at a dose schedule of 9 μg/kg IV on days 1,3,5,15,17,19 is tolerable and has promising activity for steroid-refractory GVHD. Future studies using the MTD are warranted to investigate the potential role of ONTAK as primary treatment of acute GVHD.

DAB389IL2 (ONTAK®) fusion protein therapy of chronic lymphocytic leukaemia

Despite multiple new therapeutic options for patients with chronic lymphocytic leukaemia (CLL), the prognosis of patients with relapsed disease is poor. After first-line therapy with fludarabine, alkylating agents, rituximab or combinations of these agents, most patients relapse within a few years. While second-line therapy with alemtuzumab, or other combinations of the above agents, have produced remissions, most of these are partial responses lasting months rather than years. Patients commonly die from progressive disease or infections related to the underlying disease or treatment. The authors sought to develop a novel therapeutic with the capacity to kill chemotherapy-resistant CLL cells and with reduced toxicities to normal tissues. Based on the presence of high affinity interleukin-2 receptor (IL-2R) on CLL cells, therapy of relapsed CLL patients with a diphtheria fusion protein targeting IL-2R – DAB389IL2 (ONTAK®,Seragen, Inc., Hopkinton, MA, USA) – was tested in a pilot Phase II study. Biological activity and a partial remission were observed with modest drug-related side effects. Based on these encouraging findings, alternative schedules and combinations with agents that may enhance CLL cell expression of IL-2R are being tested. Hopefully, the use of these targeted therapeutic approaches will provide additional therapeutic options with fewer side effects for this common and incurable condition.

Receptor upregulation enhances cell surface receptor targeted therapies

A number of new therapeutic agents for hematologic malignancies have been approved in the last 5 years that target cell surface receptors on the malignant cell. These include a mouse–human chimeric monoclonal antibody to CD20 (rituximab), a 90Y-labeled mouse monoclonal antibody to CD20 (ibritumomab tiuxetan), a humanized antibody to CD52 (alemtuzumab), an immunoconjugate consisting of a humanized antibody to CD33 coupled to calicheamicin (gemtuzumab ozogamicin), and a diphtheria fusion protein (denileukin diftitox) directed at the high affinity interleukin-2 receptor (IL2R) complex [1–5]. In addition, there are a number of targeted protein molecules in various stages of clinical development reactive with various hematopoietic cell surface receptors including CD20, CD22, CD33, HLA-DR, the granulocyte–macrophage colonystimulating factor receptor, among others (see Table 1). Many of these biologics show impressive clinical activity, but most treated patients relapse. To improve the remission rate and duration, one approach has been to combine the biologic compound with cytotoxic drugs [6]. This has yielded outstanding results with rituximab. Another approach is to selectively enhance the potency of the biologic on the target malignant stem cell. Based on both preclinical and clinical data, tumor cell kill with toxin immunoconjugates is proportional to receptor density [7,8]. Foss and coworkers have studied methods to increase the expression of high affinity IL2R on malignant hematopoietic cells to enhance killing by denileukin diftitox [9–11]. In this issue, her group documents that arginine butyrate increases the expression of the subunit of the IL2R on leukemia cells and that this leads to increased sensitivity to denileukin diftitox [12]. Upregulation of receptors on malignant cells is an attractive strategy. This may involve enhanced gene transcription, improved protein stability, or increased protein trafficking to the cell surface. Compounds have been found which modify each of these pathways for a number of receptors (see Table 2). The molecular mechanisms for receptor upregulation by most of these compounds are poorly characterized.

High affinity interleukin-3 receptor expression on blasts from patients with acute myelogenous leukemia correlates with cytotoxicity of a diphtheria toxin/IL-3 fusion protein

Diphtheria fusion proteins are a novel class of agents for the treatment of chemotherapy resistant AML. We prepared DT 388IL3 composed of human interleukin-3 (IL3) fused to the catalytic and translocation domain of diphtheria toxin (DT 388) and assessed its activity on patient AML blasts. The number and affinity of IL3 receptors in circulating blasts was measured using a radiolabeled IL3 agonist (SC-65461). Ninety-two percent of patients’ blasts had both high and low affinity IL3 receptors. DT 388IL3 cytotoxicity (>1 log cell kill) was seen in nine of 25 samples (36%). There was a significant correlation between DT 388IL3 log cell kill and blast high affinity IL3 receptor density ( P=0.0044). These results show that specific high affinity IL3 binding is one factor important in the sensitivity of patients’ leukemic blasts to DT 388IL3.

Solution assembly of the pseudo-high affinity and intermediate affinity interleukin-2 receptor complexes.

The high affinity interleukin-2 receptor is composed of three cell surface subunits, IL-2Ralpha, IL-2Rbeta, and IL-2Rgamma. Functional forms of the IL-2 receptor exist, however, that enlist only two of the three subunits. On activated T-cells, the alpha- and beta-subunits combine as a preformed heterodimer (the pseudo-high affinity receptor) that serves to capture IL-2. On a subpopulation of natural killer cells, the beta- and gamma-subunits interact in a ligand-dependent manner to form the intermediate affinity receptor site. Previously, we have demonstrated the feasibility of employing coiled-coil molecular recognition for the solution assembly of a heteromeric IL-2 receptor complex. In that study, although the receptor was functional, the coiled-coil complex was a trimer rather than the desired heterodimer. We have now redesigned the hydrophobic heptad sequences of the coiled-coils to generate soluble forms of both the pseudo-high affinity and the intermediate affinity heterodimeric IL-2 receptors. The properties of these complexes were examined and their relevance to the physiological IL-2 receptor mechanism is discussed.

Intermediate Affinity Interleukin-2 Receptor Mediates Survival via a Phosphatidylinositol 3-Kinase-dependent Pathway

Peripheral blood T lymphocytes require two signals to enter and progress along the cell cycle from their natural quiescent state. The first activation signal is provided by the stimulation through the T cell receptor, which induces the synthesis of cyclins and the expression of the high affinity interleukin-2 receptor. The second signal, required to enter the S phase, is generated upon binding of interleukin-2 to the high affinity alphabetagamma interleukin-2 receptor. However, resting T cells already express intermediate affinity betagamma interleukin-2 receptors. As shown here, T cell stimulation through intermediate affinity receptors is capable of inducing cell rescue from the apoptosis suffered in the absence of stimulation. Characterization of the signaling pathways utilized by betagamma interleukin-2 receptors in resting T cells, indicated that pp56(lck), but not Jak1 or Jak3, is activated upon receptor triggering. Compelling evidence is presented indicating that phosphatidylinositol 3-kinase associates with the intermediate affinity interleukin-2 receptor and is activated upon interleukin-2 addition. Bcl-xL gene was also found to be induced upon betagamma interleukin-2 receptor stimulation. Finally, pharmacological inhibition of phosphatidylinositol 3-kinase blocked both interleukin-2-mediated bcl-xL induction and cell survival. We conclude that betagamma interleukin-2 receptor mediates T-cell survival via a phosphatidylinositol 3-kinase-dependent pathway, possibly involving pp56(lck) and bcl-xL as upstream and downstream effectors, respectively.