PD-1-cis IL-2R agonism yields better effectors from stem-like CD8+ T cells.

Expansion and differentiation of antigen-experienced PD-1+TCF-1+ stem-like CD8+ T cells into effector cells is critical for the success of immunotherapies based on PD-1 blockade1-4. Hashimoto et al. have shown that, in chronic infections, administration of the cytokine interleukin (IL)-2 triggers an alternative differentiation path of stem-like T cells towards a distinct population of 'better effector' CD8+ T cells similar to those generated in an acute infection5. IL-2 binding to the IL-2 receptor α-chain (CD25) was essential in triggering this alternative differentiation path and expanding better effectors with distinct transcriptional and epigenetic profiles. However, constitutive expression of CD25 on regulatory T cells and some endothelial cells also contributes to unwanted systemic effects from IL-2 therapy. Therefore, engineered IL-2 receptor ß- and γ-chain (IL-2Rßγ)-biased agonists are currently being developed6-10. Here we show that IL-2Rßγ-biased agonists are unable to preferentially expand better effector T cells in cancer models and describe PD1-IL2v, a new immunocytokine that overcomes the need for CD25 binding by docking in cis to PD-1. Cis binding of PD1-IL2v to PD-1 and IL-2Rßγ on the same cell recovers the ability to differentiate stem-like CD8+ T cells into better effectors in the absence of CD25 binding in both chronic infection and cancer models and provides superior efficacy. By contrast, PD-1- or PD-L1-blocking antibodies alone, or their combination with clinically relevant doses of non-PD-1-targeted IL2v, cannot expand this unique subset of better effector T cells and instead lead to the accumulation of terminally differentiated, exhausted T cells. These findings provide the basis for the development of a new generation of PD-1 cis-targeted IL-2R agonists with enhanced therapeutic potential for the treatment of cancer and chronic infections.

Pharmacokinetics and Pharmacodynamic Effects of Nemvaleukin Alfa, a Selective Agonist of the Intermediate-Affinity IL-2 Receptor, in Cynomolgus Monkeys.

Nemvaleukin alfa (nemvaleukin, ALKS 4230) is a novel cytokine created by the fusion of circularly permuted interleukin-2 (IL-2) to the IL-2Rα subunit of the IL-2 receptor (IL-2R) complex that confers selectivity for the intermediate-affinity IL-2R expressed on CD8+ T cells and natural killer (NK) cells. The pharmacokinetics and selective pharmacodynamic properties of nemvaleukin have been demonstrated using in vitro and in vivo mouse models. The pharmacokinetic/pharmacodynamic effects of nemvaleukin on immune cell subtypes were evaluated in cynomolgus monkeys after intravenous and subcutaneous administration to inform dose selection and predict pharmacodynamic effects in humans. Male drug-naïve cynomolgus monkeys (N = 15) were administered either single-dose (0.3 mg/kg i.v.; 0.3 mg/kg or 1.0 mg/kg s.c.) or repeated doses (0.1 mg/kg i.v. on days 1-5 or 0.5 mg/kg s.c. on days 1 and 4) of nemvaleukin. Serial blood samples were collected for pharmacokinetic assessment, immunophenotyping by flow cytometry, and profiling of serum cytokines. Repeat-dose subcutaneous administration of nemvaleukin with less frequent dosing resulted in total systemic exposure and trough serum concentrations comparable to those seen with intravenous administration, with lower peak serum concentrations. Transient elevation of interferon-γ and IL-6 peaked at 2 and 8 hours after intravenous and subcutaneous administration, respectively. Selective expansion of immunoprotective central memory, effector memory, terminal effector CD8+ T cells, and CD56+ NK cells, and minimal expansion of immunosuppressive CD4+CD25+FoxP3+ regulatory T cells was observed after both intravenous and subcutaneous administration. These data support the ongoing clinical evaluation of intravenous and subcutaneous nemvaleukin. SIGNIFICANCE STATEMENT: Administration of the novel interleukin-2 receptor agonist nemvaleukin alfa to cynomolgus monkeys resulted in selective expansion of immune effector cells, including CD8+ T and natural killer cells with minimal effects on immunosuppressive CD4+ regulatory T cells, confirming the design of nemvaleukin and highlighting its potential as a cancer immunotherapy. Subcutaneous administration of nemvaleukin achieved systemic exposure and immunostimulatory effects similar to those observed after more frequent intravenous dosing and may represent a practical alternative in a clinical setting.

De novo design of potent and selective mimics of IL-2 and IL-15.

We describe a de novo computational approach for designing proteins that recapitulate the binding sites of natural cytokines, but are otherwise unrelated in topology or amino acid sequence. We use this strategy to design mimics of the central immune cytokine interleukin-2 (IL-2) that bind to the IL-2 receptor ßγc heterodimer (IL-2Rßγc) but have no binding site for IL-2Rα (also called CD25) or IL-15Rα (also known as CD215). The designs are hyper-stable, bind human and mouse IL-2Rßγc with higher affinity than the natural cytokines, and elicit downstream cell signalling independently of IL-2Rα and IL-15Rα. Crystal structures of the optimized design neoleukin-2/15 (Neo-2/15), both alone and in complex with IL-2Rßγc, are very similar to the designed model. Neo-2/15 has superior therapeutic activity to IL-2 in mouse models of melanoma and colon cancer, with reduced toxicity and undetectable immunogenicity. Our strategy for building hyper-stable de novo mimetics could be applied generally to signalling proteins, enabling the creation of superior therapeutic candidates.

Modeling the receptor pharmacology, pharmacokinetics, and pharmacodynamics of NKTR-214, a kinetically-controlled interleukin-2 (IL2) receptor agonist for cancer immunotherapy.

Cytokines are potent immune modulating agents but are not ideal medicines in their natural form due to their short half-life and pleiotropic systemic effects. NKTR-214 is a clinical-stage biologic that comprises interleukin-2 (IL2) protein bound by multiple releasable polyethylene glycol (PEG) chains. In this highly PEG-bound form, the IL2 is inactive; therefore, NKTR-214 is a biologic prodrug. When administered in vivo, the PEG chains slowly release, creating a cascade of increasingly active IL2 protein conjugates bound by fewer PEG chains. The 1-PEG-IL2 and 2-PEG-IL2 species derived from NKTR-214 are the most active conjugated-IL2 species. Free-IL2 protein is undetectable in vivo as it is eliminated faster than formed. The PEG chains on NKTR-214 are located at the region of IL2 that contacts the alpha (α) subunit of the heterotrimeric IL2 receptor complex, IL2Rαßγ, reducing its ability to bind and activate the heterotrimer. The IL2Rαßγ complex is constitutively expressed on regulatory T cells (Tregs). Therefore, without the use of mutations, PEGylation reduces the affinity for IL2Rαßγ to a greater extent than for IL2Rßγ, the receptor complex predominant on CD8 T cells. NKTR-214 treatment in vivo favors activation of CD8 T cells over Tregs in the tumor microenvironment to provide anti-tumor efficacy in multiple syngeneic models. Mechanistic modeling based on in vitro and in vivo kinetic data provides insight into the mechanism of NKTR-214 pharmacology. The model reveals that conjugated-IL2 protein derived from NKTR-214 occupy IL-2Rßγ to a greater extent compared to free-IL2 protein. The model accurately describes the sustained in vivo signaling observed after a single dose of NKTR-214 and explains how the properties of NKTR-214 impart a unique kinetically-controlled immunological mechanism of action.

The biology of interleukin-2.

Much data support an essential role for interleukin (IL)-2 in immune tolerance. This idea is much different from the early paradigm in which IL-2 is central for protective immune responses. This change in thinking occurred when a T regulatory cell defect was shown to be responsible for the lethal autoimmunity associated with IL-2/IL-2R deficiency. This realization allowed investigators to explore immune responses in IL-2-nonresponsive mice rendered autoimmune-free. Such studies established that IL-2 sometimes contributes to optimal primary immune responses, but it is not mandatory. Emerging findings, however, suggest an essential role for IL-2 in immune memory. Here, the current understanding of the dual role of IL-2 in maintaining tolerance and contributing to immunity in vivo is reviewed with some emphasis on T regulatory cell production and homeostasis. Also discussed are implications of this new appreciation concerning the immunobiology of IL-2 with respect to targeting IL-2 or its receptor in immunotherapy.

Transient ischemia-induced changes of interleukin-2 and its receptor beta immunoreactivity and levels in the gerbil hippocampal CA1 region.

Interlukin-2 (IL-2) is an important cytokine in the brain: IL-2 and its receptors are involved with inflammatory processes. Chronological changes in IL-2 level in serum, and IL-2 and its receptor (IL-2 receptor beta, IL-2Rbeta) immunoreactivities and levels were examined in the hippocampal CA1 region after transient forebrain ischemia in gerbils. IL-2 level in serum significantly decreased 12 h after ischemia/reperfusion. IL-2 immunoreactivity was detected in the somata of pyramidal cells in sham-operated group. At 15 min after ischemia, IL-2 immunoreactivity was shown in non-pyramidal cells as well as pyramidal cells. One day after ischemia, IL-2 immunoreactivity was lowest, and IL-2 immunoreactivity is shown in non-pyramidal cells from 2 days after ischemia. Four days after ischemia, IL-2 immunoreactivity was shown in dying pyramidal cells. IL-2Rbeta immunoreactivity in the sham-operated and 15 min-3 min post-ischemic groups is detected in the cell membrane of pyramidal cells. From 3 h after ischemia, IL-2Rbeta immunoreactivity is found in cytoplasm and nuclei, but not in cell membrane. IL-2Rbeta immunoreactivity decreases from 6 h after ischemia and is shown mainly in non-pyramidal cells from 3 days after ischemia. The data of Western blot analyses for IL-2 and IL-2Rbeta was similar to the immunohistochemical data. IL-2 infusion into cerebrospinal fluid did not protect hippocampal neurons from ischemic damage. These results suggest that IL-2 and IL-2Rbeta show malfunction from 3 h after ischemia, and exogenous IL-2 does not protect ischemic neuronal damage.

Soluble interleukin-15 receptor alpha (IL-15R alpha)-sushi as a selective and potent agonist of IL-15 action through IL-15R beta/gamma. Hyperagonist IL-15 x IL-15R alpha fusion proteins.

Interleukin-15 (IL-15) is crucial for the generation of multiple lymphocyte subsets (natural killer (NK), NK-T cells, and memory CD8 T cells), and transpresentation of IL-15 by monocytes and dendritic cells has been suggested to be the dominant activating process of these lymphocytes. We have previously shown that a natural soluble form of IL-15R alpha chain corresponding to the entire extracellular domain of IL-15R alpha behaves as a high affinity IL-15 antagonist. In sharp contrast with this finding, we demonstrate in this report that a recombinant, soluble sushi domain of IL-15R alpha, which bears most of the binding affinity for IL-15, behaves as a potent IL-15 agonist by enhancing its binding and biological effects (proliferation and protection from apoptosis) through the IL-15R beta/gamma heterodimer, whereas it does not affect IL-15 binding and function of the tripartite IL-15R alpha/beta/gamma membrane receptor. Our results suggest that, if naturally produced, such soluble sushi domains might be involved in the IL-15 transpresentation mechanism. Fusion proteins (RLI and ILR), in which IL-15 and IL-15R alpha-sushi are attached by a flexible linker, are even more potent than the combination of IL-15 plus sIL-15R alpha-sushi. After binding to IL-15R beta/gamma, RLI is internalized and induces a biological response very similar to the IL-15 high affinity response. Such hyper-IL-15 fusion proteins appear to constitute potent adjuvants for the expansion of lymphocyte subsets.

IL-2R beta agonist P1-30 acts in synergy with IL-2, IL-4, IL-9, and IL-15: biological and molecular effects.

From the sequence of human IL-2 we have recently characterized a peptide (p1-30), which is the first IL-2 mimetic described. P1-30 covers the entire alpha helix A of IL-2 and spontaneously folds into a alpha helical homotetramer mimicking the quaternary structure of a hemopoietin. This neocytokine interacts with a previously undescribed dimeric form of the human IL-2 receptor beta-chain likely to form the p1-30 receptor (p1-30R). P1-30 acts as a specific IL-2Rbeta agonist, selectively inducing activation of CD8 and NK lymphocytes. From human PBMC we have also shown that p1-30 induces the activation of lymphokine-activated killer cells and the production of IFN-gamma. Here we demonstrate the ability of p1-30 to act in synergy with IL-2, -4, -9, and -15. These synergistic effects were analyzed at the functional level by using TS1beta, a murine T cell line endogenously expressing the common cytokine gamma gene and transfected with the human IL-2Rbeta gene. At the receptor level, we show that expression of human IL-2Rbeta is absolutely required to obtain synergistic effects, whereas IL-2Ralpha specifically impedes the synergistic effects obtained with IL-2. The results suggest that overexpression of IL-2Ralpha inhibits p1-30R formation in the presence of IL-2. Finally, concerning the molecular effects, although p1-30 alone induces the antiapoptotic molecule bcl-2, we show that it does not influence mRNA expression of c-myc, c-jun, and c-fos oncogenes. In contrast, p1-30 enhances IL-2-driven expression of these oncogenes. Our data suggest that p1-30R (IL-2Rbeta)(2) and intermediate affinity IL-2R (IL-2Rbetagamma), when simultaneously expressed at the cell surface, may induce complementary signal transduction pathways and act in synergy.

The first alpha helix of interleukin (IL)-2 folds as a homotetramer, acts as an agonist of the IL-2 receptor beta chain, and induces lymphokine-activated killer cells.

Interleukin (IL)-2 interacts with two types of functional receptors (IL-2Ralphabetagamma and IL-2Rbetagamma) and acts on a broad range of target cells involved in inflammatory reactions and immune responses. For the first time, we show that a chemically synthesized fragment of the IL-2 sequence can fold into a molecule mimicking the quaternary structure of a hemopoietin. Indeed, peptide p1-30 (containing amino acids 1-30, covering the entire alpha helix A of IL-2) spontaneously folds into an alpha-helical homotetramer and stimulates the growth of T cell lines expressing human IL-2Rbeta, whereas shorter versions of the peptide lack helical structure and are inactive. We also demonstrate that this neocytokine interacts with a previously undescribed dimeric form of IL-2Rbeta. In agreement with its binding to IL-2Rbeta, p1-30 activates Shc and p56(lck) but unlike IL-2, fails to activate Janus kinase (Jak)1, Jak3, and signal transducer and activator of transcription 5 (STAT5). Unexpectedly, we also show that p1-30 activates Tyk2, thus suggesting that IL-2Rbeta may bind to different Jaks depending on its oligomerization. At the cellular level, p1-30 induces lymphokine-activated killer (LAK) cells and preferentially activates CD8(low) lymphocytes and natural killer cells, which constitutively express IL-2Rbeta. A significant interferon gamma production is also detected after p1-30 stimulation. A mutant form of p1-30 (Asp20-->Lys), which is likely unable to induce vascular leak syndrome, remains capable of generating LAK cells, like the original p1-30 peptide. Altogether, our data suggest that p1-30 has therapeutic potential.

The IL-2 receptor in hemocytes of the sea mussel Mytilus galloprovincialis Lmk.

In the hemolymph of the sea mussel Mytilus galloprovincialis, two different cell types have been found. Rounded (RH) cells display a nucleus that is very large in relation to the cell size; spread (SH) cells have an expanded cytoplasm and multiple granules. We determined by flow cytometry that only the SH cell types express three interleukin-2 receptor (IL-2R) subunits. Mussel IL-2R alpha and IL-2R beta subunits display a molecular weight similar to those in vertebrates tissues, whereas mussel IL-2R gamma is smaller than that in vertebrates. Both lipopolysaccharide and IL-2 induce IL-2R alpha expression, and such induction depends on the concentration of both agonists.

Human neutrophils express the interleukin-15 receptor alpha chain (IL-15Ralpha) but not the IL-9Ralpha component.

The interleukin-15 receptor (IL-15R) is composed of at least three chains, namely gammac, IL-2Rbeta, and the recently identified IL-15Ralpha, while the IL-9R complex consists of gammac and a subunit designated IL-9Ralpha. Our previous work and that of others have shown that human neutrophils express gammac and IL-2Rbeta (two components shared with IL-2R) but not IL-2Ralpha and that IL-15 is a neutrophil agonist, whereas IL-2 is not. In this study, using flow cytometry with a specific anti-human IL-15Ralpha, we show for the first time that human neutrophils express surface IL-15Ralpha. Although we previously found that IL-15 is a neutrophil agonist, our present work shows that IL-15 does not trigger superoxide production nor cell spreading onto glass. In addition, we report that human neutrophils do not respond to IL-9 with respect to the functions/responses studied, namely, superoxide production, spreading onto glass, cell shape changes, phagocytosis, RNA synthesis, and apoptosis. Further, our results show that neutrophils do not express IL-9Ralpha as assessed by flow cytometry with a specific anti-human IL-9Ralpha antibody that stains the transfected cell line BW-h9R used as positive control. Finally, our results indicate that gammac expression was not modulated and remained stable for up to 24 h when neutrophils were stimulated with all currently known "gammac users," namely, IL-2, IL-4, IL-7, IL-9, and IL-15. We conclude that human neutrophils express all IL-15R components on their surface, including IL-15Ralpha, that IL-15 activates human neutrophils (as the IL-4 neutrophil agonist) by a mechanism which does not involve upregulation of gammac cell surface expression, and that IL-9 is not a neutrophil agonist as demonstrated by the inability to modulate the tested functions/responses that correlate with lack of the IL-9R component, namely, IL-9Ralpha.