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Purpose: Albuleukin fusion protein is a recombinant human interleukin-2 (rIL-2) genetically fused to recombinant human serum albumin (rHSA). The pharmacokinetics and pharmacologic activity of Albuleukin were examined in mice to determine whether the fusion protein had the immunomodulatory and anti-tumor properties of rIL-2 as well as a prolonged serum half-life due to the rHSA., Methods: The effect of Albuleukin on lymphocyte proliferation, IL-2 receptor binding, and release of IFN-gamma from human NK cells were examined in vitro. For the pharmacokinetic analysis, Albuleukin and rIL-2 were administered intravenously (i.v.) and subcutaneously (s.c.) to BALB/c mice, both at a single dose of 500 microg/kg. The anti-tumor properties of Albuleukin were evaluated in a Renca tumor model in BALB/c mice and in a metastatic liver model of B16F10 melanoma in C57B1/6 mice. In the Renca tumor model, BALB/c mice were dosed intraperitoneally (i.p.) and s.c. with Albuleukin on days 12, 14, 16, 19, 21, and 23 and i.p. with rIL-2 daily for two periods of 5 days (days 10-14 and 17-21). In the B16 melanoma model, C57B1/6 mice were dosed s.c. with rIL-2 twice daily or Albuleukin every 48 h for 14 days., Results: In vitro, Albuleukin induced the proliferation of primary human and mouse T cells and B cells and primary human NK cells, competed with rIL-2 for binding to the IL-2 receptors, and induced the production of IFN-gamma from primary human NK cells. The s.c. bioavailability of Albuleukin was about 45% relative to the i.v. dose. Plasma half-life was prolonged and ranged from 6 to 8 h with Albuleukin, compared to 19-57 min with rIL-2. Total clearance of Albuleukin was about 50-fold slower than that of rIL-2 after i.v. dosing. In vivo, Albuleukin suppressed the growth of Renca tumors and induced a dense infiltration of CD4+ and CD8+ T cells. Both Albuleukin and rIL-2 significantly reduced the tumor burden in mice with hepatic B16F10 metastases. Albuleukin significantly reduced the incidence of residual macroscopic hepatic tumors, resulting in improved survival relative to controls and rIL-2., Conclusion: Results from these studies suggest that the therapeutic efficacy of rIL-2 is improved in mice by prolonging its in vivo half-life through genetic fusion to albumin. Albuleukin, the fusion protein, had pronounced anti-tumor effects in Renca and hepatic melanoma tumor models without an increase in mortality. On the basis of its preclinical effects, Albuleukin was brought to the clinic to assess its therapeutic benefit in a variety of cancers.

Cytokines, such as interleukin-2 (IL-2), activate intracellular signaling pathways via rapid tyrosine phosphorylation of their receptors, resulting in the activation of many genes involved in cell growth and survival. The deubiquitinating enzyme DUB-2 is induced in response to IL-2 but as yet its function has not been determined. The results of this study show that DUB-2 is expressed in human T-cell lymphotropic virus-I (HTLV-1)-transformed T cells that exhibit constitutive activation of the IL-2 JAK/STAT (signal transducers and activators of transcription) pathway, and when expressed in Ba/F3 cells DUB-2 markedly prolonged IL-2-induced STAT5 phosphorylation. Although DUB-2 did not enhance IL-2-mediated proliferation, when withdrawn from growth factor, cells expressing DUB-2 had sustained STAT5 phosphorylation and enhanced expression of IL-2-induced genes cis and c-myc. Moreover, DUB-2 expression markedly inhibited apoptosis induced by cytokine withdrawal allowing cells to survive. Taken together these data suggest that DUB-2 can enhance signaling through the JAK/STAT pathway, prolong lymphocyte survival, and, when constitutively expressed, may contribute to the activation of the JAK/STAT pathway observed in some transformed cells.

CIS is a cytokine-induced SH2-containing protein that was originally cloned as an interleukin (IL)-3-inducible gene. CIS is known to associate with the IL-3 receptor beta chain and erythropoietin receptor and to inhibit signaling mediated by IL-3 and erythropoietin. We now demonstrate that CIS also interacts with the IL-2 receptor beta chain (IL-2Rbeta). This interaction requires the A region of IL-2Rbeta (residues 313-382), which also mediates the association of IL-2Rbeta with Lck and Jak3. Correspondingly, CIS inhibits functions associated with both of these kinases: Lck-mediated phosphorylation of IL-2Rbeta and IL-2-mediated activation of Stat5. Thus, we demonstrate that CIS can negatively control at least two independent IL-2 signaling pathways. Although a functional SH2 binding domain of CIS was not required for its interaction with IL-2Rbeta in vitro, its phosphotyrosine binding capability was essential for the inhibitory action of CIS. On this basis, we have generated a mutant form of CIS protein with an altered SH2 domain that acts as a dominant negative and should prove useful in further understanding CIS action.

Members of the recently discovered SOCS/CIS/SSI family have been proposed as regulators of cytokine signaling, and while targets and mechanisms have been suggested for some family members, the precise role of these proteins remains to be defined. To date no SOCS proteins have been specifically implicated in interleukin-2 (IL-2) signaling in T cells. Here we report SOCS-3 expression in response to IL-2 in both T-cell lines and human peripheral blood lymphocytes. SOCS-3 protein was detectable as early as 30 min following IL-2 stimulation, while CIS was seen only at low levels after 2 h. Unlike CIS, SOCS-3 was rapidly tyrosine phosphorylated in response to IL-2. Tyrosine phosphorylation of SOCS-3 was observed upon coexpression with Jak1 and Jak2 but only weakly with Jak3. In these experiments, SOCS-3 associated with Jak1 and inhibited Jak1 phosphorylation, and this inhibition was markedly enhanced by the presence of IL-2 receptor beta chain (IL-2Rbeta). Moreover, following IL-2 stimulation of T cells, SOCS-3 was able to interact with the IL-2 receptor complex, and in particular tyrosine phosphorylated Jak1 and IL-2Rbeta. Additionally, in lymphocytes expressing SOCS-3 but not CIS, IL-2-induced tyrosine phosphorylation of STAT5b was markedly reduced, while there was only a weak effect on IL-3-mediated STAT5b tyrosine phosphorylation. Finally, proliferation induced by both IL-2- and IL-3 was significantly inhibited in the presence of SOCS-3. The findings suggest that when SOCS-3 is rapidly induced by IL-2 in T cells, it acts to inhibit IL-2 responses in a classical negative feedback loop.

Interleukin 2 (IL-2) rapidly induces tyrosine phosphorylation of intracellular substrates, including the IL-2 receptor beta chain (IL-2Rbeta), Janus kinase 1 (Jak1), Jak3, signal transducer/activator of transcription proteins, and Shc, but the mechanism underlying dephosphorylation of these proteins is not known. The src homology 2 (SH2) containing tyrosine phosphatase 1 (SHP-1) is recruited by several hematopoietic surface receptors indicating that this phosphatase plays an important role as a regulator of signaling. We have found that IL-2 induces association of SHP-1 with the IL-2 receptor complex, and that once SHP-1 is recruited to the activated receptor it is able to decrease tyrosine phosphorylation of IL-2Rbeta and the associated tyrosine kinases Jak1 and Jak3. This dephosphorylation is specific as expression of a catalytically inactive form of SHP-1, or expression of the related phosphatase SHP-2 did not result in dephosphorylation of the IL-2 receptor components. Furthermore, we have found that SHP-1 expression is greatly decreased or undetectable in a number of IL-2 independent HTLV-I transformed T cell lines that exhibit constitutive Jak/signal transducer/activator of transcription activation. In HTLV-I infected T cells, down-regulation of SHP-1 expression was also found to correlate with the acquisition of IL-2 independence. These observations suggest that SHP-1 normally functions to antagonize the IL-2 signal transduction pathway and that HTLV-I infection and oncogenic transformation can lead to loss of SHP-1 expression resulting in constitutive activation of IL-2 regulated T cell responses.

To understand the molecular bases for cytokine redundancy and pleiotropy, we have compared the Stat proteins activated in peripheral blood lymphocytes (PBLs) by cytokines with shared and distinct actions. Interleukin-2 (IL-2) rapidly activated Stat5 in fresh PBL, and Stat3 and Stat5 in preactivated PBL. IL-7 and IL-15 induced the same complexes as IL-2, a feature explained by the existence of similar tyrosine-phosphorylated motifs in the cytoplasmic domains of IL-2R beta and IL-7R that can serve as docking sites for Stat proteins. IL-13 Induced the same complexes as IL-4, a finding explained by our studies implicating IL-4R as a shared component of the receptors. These studies demonstrate that a single cytokine can activate different combinations of Stat proteins under different physiological conditions, and also indicate two mechanisms by which distinct cytokines can activate the same Stat protein.