Combination Cancer Immunotherapy with Dendritic Cell Vaccine and Nanoparticles Loaded with Interleukin-15 and Anti-beta-catenin siRNA Significantly Inhibits Cancer Growth and Induces Anti-Tumor Immune Response.

PURPOSE: The invention and application of new immunotherapeutic methods can compensate for the inefficiency of conventional cancer treatment approaches, partly due to the inhibitory microenvironment of the tumor. In this study, we tried to inhibit the growth of cancer cells and induce anti-tumor immune responses by silencing the expression of the ß-catenin in the tumor microenvironment and transmitting interleukin (IL)-15 cytokine to provide optimal conditions for the dendritic cell (DC) vaccine. METHODS: For this purpose, we used folic acid (FA)-conjugated SPION-carboxymethyl dextran (CMD) chitosan (C) nanoparticles (NPs) to deliver anti-ß-catenin siRNA and IL-15 to cancer cells. RESULTS: The results showed that the codelivery of ß-catenin siRNA and IL-15 significantly reduced the growth of cancer cells and increased the immune response. The treatment also considerably stimulated the performance of the DC vaccine in triggering anti-tumor immunity, which inhibited tumor development and increased survival in mice in two different cancer models. CONCLUSIONS: These findings suggest that the use of new nanocarriers such as SPION-C-CMD-FA could be an effective way to use as a novel combination therapy consisting of ß-catenin siRNA, IL-15, and DC vaccine to treat cancer.

A Chimeric IL-15/IL-15Rα Molecule Expressed on NFκB-Activated Dendritic Cells Supports Their Capability to Activate Natural Killer Cells.

Natural killer (NK) cells, members of the innate immune system, play an important role in the rejection of HLA class I negative tumor cells. Hence, a therapeutic vaccine, which can activate NK cells in addition to cells of the adaptive immune system might induce a more comprehensive cellular response, which could lead to increased tumor elimination. Dendritic cells (DCs) are capable of activating and expanding NK cells, especially when the NFκB pathway is activated in the DCs thereby leading to the secretion of the cytokine IL-12. Another prominent NK cell activator is IL-15, which can be bound by the IL-15 receptor alpha-chain (IL-15Rα) to be transpresented to the NK cells. However, monocyte-derived DCs do neither secrete IL-15, nor express the IL-15Rα. Hence, we designed a chimeric protein consisting of IL-15 and the IL-15Rα. Upon mRNA electroporation, the fusion protein was detectable on the surface of the DCs, and increased the potential of NFκB-activated, IL-12-producing DC to activate NK cells in an autologous cell culture system with ex vivo-generated cells from healthy donors. These data show that a chimeric IL-15/IL-15Rα molecule can be expressed by monocyte-derived DCs, is trafficked to the cell surface, and is functional regarding the activation of NK cells. These data represent an initial proof-of-concept for an additional possibility of further improving cellular DC-based immunotherapies of cancer.

NKTR-255 is a polymer-conjugated IL-15 with unique mechanisms of action on T and natural killer cells.

NKTR-255 is a PEG conjugate of recombinant human IL-15 (rhIL-15) being examined as a potential cancer immunotherapeutic. Since IL-15 responses can be mediated by trans or cis presentation via IL-15Rα or soluble IL-15/IL-15Rα complexes, we investigated the role of IL-15Rα in driving NKTR-255 responses using defined naive and memory OVA-specific CD8+ T cells (OT-I) and NK cells in mice. NKTR-255 induced a 2.5- and 2.0-fold expansion of CD8+ T and NK cells, respectively, in WT mice. In adoptive transfer studies, proliferation of naive and memory WT OT-I T cells in response to NKTR-255 was not impaired in IL-15Rα-/- mice, suggesting trans presentation was not utilized by NKTR-255. Interestingly, naive IL-15Rα-/- OT-I cells had deficient responses to NKTR-255, while memory IL-15Rα-/- OT-I cell responses were partially impaired, suggesting that naive CD8+ T cells are more dependent on cis presentation of NKTR-255 than memory CD8+ T cells. In bone marrow chimera studies, IL-15Rα-/- and WT NK cells present in WT recipients had similar responses to NKTR-255, suggesting that cis presentation is not utilized by NK cells. NKTR-255 could form soluble complexes with IL-15Rα; binding to murine IL-15Rα generated superagonists that preferentially stimulated NK cells, showing that conversion to IL-15Rß agonist biases the response toward NK cells. These findings highlight the ability of NKTR-255 to utilize IL-15Rα for cis presentation and act as an IL-15Rαß agonist on CD8+ T cells.

Phase I study protocol: NKTR-255 as monotherapy or combined with daratumumab or rituximab in hematologic malignancies.

NKTR-255 is an investigational polyethylene glycol-modified recombinant human IL-15 (rhIL-15) receptor agonist, designed to improve the immunotherapeutic and anti-cancer benefit observed with rhIL-15 while circumventing the toxicities associated with this therapy. In preclinical studies, NKTR-255 has demonstrated enhanced proliferation and function of CD8+ T cells and natural killer cells, as well as enhanced anti-tumor activity and survival both as monotherapy and in combination with monoclonal antibodies in multiple cancer models. Here, we describe the rationale and design of the first-in-human Phase I, dose-escalation and dose-expansion study of NKTR-255 alone and in combination with daratumumab or rituximab in adults with relapsed/refractory multiple myeloma or non-Hodgkin's lymphoma that will determine the maximum tolerated dose and recommended Phase II dose for NKTR-255.

Lay abstract Interleukin-15 (IL-15) is a protein that helps the body's natural immune system to defend itself against infections and diseases like cancer. This article discusses a clinical trial in patients with multiple myeloma or non-Hodgkin's lymphoma that evaluates a new investigational medicine, NKTR-255, a polymer-modified form of IL-15 that has been engineered to improve its ability to provide a sustained anti-tumor immune response. The trial will explore different doses of NKTR-255 to determine patient side effects and to find the highest acceptable dose that patients can tolerate. Based on this, a dose will be chosen that offers an optimal balance between having a positive anti-cancer effect and minimizing side effects. This dose will be tested further in patients who have had different treatments in the past. If the side effects are acceptable, this dose will be tested in a new trial in a large number of patients. Clinical Trial Registration: NCT04136756 (ClinicalTrials.gov).

Development of Stable Chimeric IL-15 for Trans-Presentation by the Antigen Presenting Cells.

IL-15 is one of the important biologics considered for vaccine adjuvant and treatment of cancer. However, a short half-life and poor bioavailability limit its therapeutic potential. Herein, we have structured IL-15 into a chimeric protein to improve its half-life enabling greater bioavailability for longer periods. We have covalently linked IL-15 with IgG2 base to make the IL-15 a stable chimeric protein, which also increased its serum half-life by 40 fold. The dimeric structure of this kind of IgG based biologics has greater stability, resistance to proteolytic cleavage, and less frequent dosing schedule with minimum dosage for achieving the desired response compared to that of their monomeric forms. The structured chimeric IL-15 naturally forms a dimer, and retains its affinity for binding to its receptor, IL-15Rß. Moreover, with the focused action of the structured chimeric IL-15, antigen-presenting cells (APC) would transpresent chimeric IL-15 along with antigen to the T cell, that will help the generation of quantitatively and qualitatively better antigen-specific memory T cells. In vitro and in vivo studies demonstrate the biological activity of chimeric IL-15 with respect to its ability to induce IL-15 signaling and modulating CD8+ T cell response in favor of memory generation. Thus, a longer half-life, dimeric nature, and anticipated focused transpresentation by APCs to the T cells will make chimeric IL-15 a super-agonist for memory CD8+ T cell responses.

Injectable Porous Microchips with Oxygen Reservoirs and an Immune-Niche Enhance the Efficacy of CAR T Cell Therapy in Solid Tumors.

Chimeric antigen receptor (CAR) T cell therapy is a promising new class of hematological malignancy treatment. However, CAR T cells are rarely effective in solid tumor therapy mainly because of the poor trafficking of injected CAR T cells to the tumor site and their limited infiltration and survival in the immunosuppressive and hypoxic tumor microenvironment (TME). Here, we built an injectable immune-microchip (i-G/MC) system to intratumorally deliver CAR T cells and enhance their therapeutic efficacy in solid tumors. In the i-G/MC, oxygen carriers (Hemo) are released to disrupt the TME, and then, CAR T cells migrate from IL-15-laden i-G/MCs into the tumor stroma. The results indicate that Hemo and IL-15 synergistically enhanced CAR T cell survival and expansion under hypoxic conditions, promoting the potency and memory of CAR T cells. This i-G/MC not only serves as a cell carrier but also builds an immune-niche, enhancing the efficacy of CAR T cells.

The advent of de novo proteins for cancer immunotherapy.

Engineered proteins are revolutionizing immunotherapy, but advances are still needed to harness their full potential. Traditional protein engineering methods use naturally existing proteins as a starting point, and therefore, are intrinsically limited to small alterations of a protein's natural structure and function. Conversely, computational de novo protein design is free of such limitation, and can produce a virtually infinite number of novel protein sequences, folds, and functions. Recently, we used de novo protein engineering to create Neoleukin-2/15 (Neo-2/15), a protein mimetic of the function of both interleukin-2 (IL-2) and interleukin-15 (IL-15). To our knowledge, Neo-2/15 is the first de novo protein with immunotherapeutic activity, and in murine cancer models, it has demonstrated enhanced therapeutic potency and reduced toxicity compared to IL-2. De novo protein design is already showcasing its tremendous potential for driving the next wave of protein-based therapeutics that are explicitly engineered to treat disease.

Discovery of a novel IL-15 based protein with improved developability and efficacy for cancer immunotherapy.

Interleukin-15 (IL-15) can promote both innate and adaptive immune reactions by stimulating CD8+/CD4+ T cells and natural killer cells (NK) while showing no effect in activating T-regulatory (Treg) cells or inducing activation-associated death among effector T cells and NK cells. Thus, IL-15 is considered as one of the most promising molecules for antitumor immune therapy. To improve the drug-like properties of natural IL-15, we create an IL-15-based molecule, named P22339, with the following characteristics: 1) building a complex of IL-15 and the Sushi domain of IL-15 receptor α chain to enhance the agonist activity of IL-15 via transpresentation; 2) through a rational structure-based design, creating a disulfide bond linking the IL-15/Sushi domain complex with an IgG1 Fc to augment its half-life. P22339 demonstrates excellent developability, pharmacokinetic and pharmacodynamic properties as well as antitumor efficacy in both in vitro assessments and in vivo studies. It significantly suppresses tumor growth and metastasis in rodent models, and activates T effector cells and NK cells in cynomolgus monkey. Overall, these data suggest that P22339 has a great potential for cancer immunotherapy.

Interleukin-15 and cisplatin co-encapsulated thermosensitive polypeptide hydrogels for combined immuno-chemotherapy.

In situ-forming thermosensitive hydrogels based on poly(ethylene glycol)-poly(γ-ethyl-l-glutamate) diblock copolymers (mPEG-b-PELG) were prepared for the co-delivery of interleukin-15 (IL-15) and cisplatin (CDDP). The polypeptide-based hydrogels as local drug delivery carriers could reduce the systemic toxicity, degrade thoroughly within 3weeks after subcutaneous injection into rats and display an acceptable biocompatibility. When incubated with mouse melanoma B16 cells, only the CDDP-treated groups had significant effects on the S phase cell-cycle arrest in melanoma cells. After a single peritumoral injection of the hydrogel containing IL-15/CDDP in C57BL/6 mice inoculated with B16F0-RFP melanoma cells, the dual drug-loaded hydrogels displayed synergistic anticancer efficacy, which was resulted from a combination of CDDP-mediated S arrest and IL-15/CDDP-induced recovery of CD8+ T cell and NK cell populations to reduce immunosuppression and enhance antitumor immunity. Hence, the as-prepared thermosensitive polypeptide hydrogels for localized and sustained co-delivery of IL-15 and CDDP may have potential for efficient treatment of melanoma.

The level of FoxO1 and IL-15 in skeletal muscle, serum and synovial fluid in people with knee osteoarthritis: a case control study.

UNLABELLED: The molecular regulation of muscle function in knee osteoarthritis is unclear. Elevated muscle atrophy regulation marker expression was associated with reduced muscle strength in knee osteoarthritis. The level of protein expression appears to be different between muscle, knee joint and serum, suggesting that inflammation is regulated differently within these tissues. INTRODUCTION: Impaired muscle function is common in knee osteoarthritis (OA). Numerous biochemical molecules have been implicated in the development of OA; however, these have only been identified in the joint and serum. We compared the expression of interleukin-15 (IL-15) and Forkhead box protein-O1 (FoxO1) in muscle of patients with knee OA and asymptomatic individuals and examined whether IL-15 was also present in the joint and serum. METHODS: Muscle and blood samples were collected from 19 patients with knee OA and 10 age-matched asymptomatic individuals. Synovial fluid and muscle biopsies were collected from the OA group during knee replacement surgery. IL-15 and FoxO1 were measured in the skeletal muscle. IL-15 abundance was also analysed in the serum of both groups and synovial fluid from the OA group. Knee extensor strength was measured and correlated with IL-15 and FoxO1 in the muscle. RESULTS: FoxO1 protein expression was higher (p = 0.04), whereas IL-15 expression was lower (p = 0.02) in the muscle of the OA group. Strength was also lower in the OA group and was inversely correlated with FoxO1 expression. No correlation was found between IL-15 in the joint, muscle or serum. CONCLUSION: Skeletal muscle, particularly the quadriceps, is affected in people with knee OA where elevated FoxO1 protein expression was associated with reduced muscle strength. While IL-15 protein expression in the muscle was lower in the knee OA group, no correlation was found between the expression of IL-15 protein in the muscle, joint and serum, which suggests that inflammation is regulated differently within these tissues. Australian Clinical Trials Registry (ACTR) number: ACTRN12613000467730 ( http://www.anzctr.org.au/TrialSearch.aspx?searchTxt=ACTRN12613000467730&isBasic=True ).

Molecular characterization of interleukin 15 mRNA from rohu, Labeo rohita (Hamilton): Its prominent role during parasitic infection as indicated from infection studies.

Interleukin 15 (IL-15) is an important cytokine of fish immune system. Sequence characterization of IL-15 from rohu, Labeo rohita revealed a mRNA sequence of 1064 bp with coding sequence of 567 bp and signal peptide of 16 amino acids. There are four characteristic sequence features viz., presence of four out-of-frame AUG initiation codons, four highly conserved cysteine residues, constitutive expression in all tissues and evolutionary similarity. The ontogeny study revealed maternal transfer of this molecule and higher expression up to 3 h post-fertilization in fertilized embryos. Its expression was down-regulated in anterior and posterior kidneys, intestine and liver tissues of rohu infected with Aeromonas hydrophila. Mild up-regulation in liver and higher expression in spleen was noticed in rohu stimulated with poly I:C (poly ionosinic:cytidylic), whereas down-regulation was observed in intestine and kidney tissues. However, a consistent higher expression was noticed in kidney and skin tissues during Argulus siamensis infection. Therefore, rohu IL-15 might possess more defensive role during early development and parasitic infection.

Production of bioactive soluble interleukin-15 in complex with interleukin-15 receptor alpha from a conditionally-replicating oncolytic HSV-1.

Oncolytic type-1 herpes simplex viruses (oHSVs) lacking the γ134.5 neurovirulence gene are being evaluated for treatment of a variety of malignancies. oHSVs replicate within and directly kill permissive cancer cells. To augment their anti-tumor activity, oHSVs have been engineered to express immunostimulatory molecules, including cytokines, to elicit tumor-specific immune responses. Interleukin-15 (IL-15) holds potential as an immunotherapeutic cytokine because it has been demonstrated to promote both natural killer (NK) cell-mediated and CD8(+) T cell-mediated cytotoxicity against cancer cells. The purpose of these studies was to engineer an oHSV producing bioactive IL-15. Two oHSVs were constructed encoding murine (m)IL-15 alone (J100) or with the mIL-15 receptor α (mIL-15Rα, J100D) to determine whether co-expression of these proteins is required for production of bioactive mIL-15 from oHSV. The following were demonstrated: i) both oHSVs retain replication competence and cytotoxicity in permissive tumor cell lines. ii) Enhanced production of mIL-15 was detected in cell lysates of neuro-2a cells following J100D infection as compared to J100 infection, suggesting that mIL-15Rα improved mIL-15 production. iii) Soluble mIL-15 in complex with mIL-15Rα was detected in supernates from J100D-infected, but not J100-infected, neuro-2a, GL261, and CT-2A cells. These cell lines vary in permissiveness to oHSV replication and cytotoxicity, demonstrating soluble mIL-15/IL-15Rα complex production from J100D was independent of direct oHSV effects. iv) The soluble mIL-15/IL-15Rα complex produced by J100D was bioactive, stimulating NK cells to proliferate and reduce the viability of syngeneic GL261 and CT-2A cells. v) J100 and J100D were aneurovirulent inasmuch as no neuropathologic effects were documented following direct inoculation into brains of CBA/J mice at up to 1x10(7) plaque forming units. The production of mIL-15/mIL-15Rα from multiple tumor lines, as well as the lack of neurovirulence, renders J100D suitable for investigating the combined effects of oHSV and mIL-15/IL-15Rα in various cancer models.

Characterization and favorable in vivo properties of heterodimeric soluble IL-15·IL-15Rα cytokine compared to IL-15 monomer.

Interleukin-15 (IL-15), a 114-amino acid cytokine related to IL-2, regulates immune homeostasis and the fate of many lymphocyte subsets. We reported that, in the blood of mice and humans, IL-15 is present as a heterodimer associated with soluble IL-15 receptor α (sIL-15Rα). Here, we show efficient production of this noncovalently linked but stable heterodimer in clonal human HEK293 cells and release of the processed IL-15·sIL-15Rα heterodimer in the medium. Purification of the IL-15 and sIL-15Rα polypeptides allowed identification of the proteolytic cleavage site of IL-15Rα and characterization of multiple glycosylation sites. Administration of the IL-15·sIL-15Rα heterodimer reconstituted from purified subunits resulted in sustained plasma IL-15 levels and in robust expansion of NK and T cells in mice, demonstrating pharmacokinetics and in vivo bioactivity superior to single chain IL-15. These identified properties of heterodimeric IL-15 provide a strong rationale for the evaluation of this molecule for clinical applications.

Artificial antigen-presenting cells plus IL-15 and IL-21 efficiently induce melanoma-specific cytotoxic CD8+ CD28+ T lymphocyte responses.

OBJECTIVE: To develop a novel artificial antigen-presenting system for efficiently inducing melanoma-specific CD8(+) CD28(+) cytotoxic T lymphocyte (CTL) responses. METHODS: Cell-sized Dynabeads® M-450 Epoxy beads coated with H-2K(b): Ig-TRP2180-188 and anti-CD28 antibody were used as artificial antigen-presenting cells (aAPCs) to induce melanoma-specific CD8(+)CD28(+) CTL responses with the help of IL-21 and IL-15. Dimer staining, proliferation, ELISPOT, and cytotoxicity experiments were conducted to evaluate the frequency and activity of induced CTLs. RESULTS: Dimer staining demonstrated that the new artificial antigen-presenting system efficiently induced melanoma TRP2-specific CD8(+)CD28(+)CTLs. Proliferation and ELISPOT assays indicated that the induced CTLs rapidly proliferate and produce increased IFN- γ under the stimulation of H-2K(b): Ig-TRP2-aAPCs, IL-15, and IL-21. In addition, cytotoxicity experiments showed that induced CTLs have specific killing activity of target cells. CONCLUSIONS: The new artificial antigen-presenting system including aAPCs plus IL-21 and IL-15 can induce a large number of antigen-specific CD8(+) CD28(+) CTLs against the melanoma. Our study provides evidence for a novel adoptive immunotherapy against tumors.

Mechanistic and structural insight into the functional dichotomy between IL-2 and IL-15.

Interleukin 15 (IL-15) and IL-2 have distinct immunological functions even though both signal through the receptor subunit IL-2Rß and the common γ-chain (γ(c)). Here we found that in the structure of the IL-15-IL-15Rα-IL-2Rß-γ(c) quaternary complex, IL-15 binds to IL-2Rß and γ(c) in a heterodimer nearly indistinguishable from that of the IL-2-IL-2Rα-IL-2Rß-γ(c) complex, despite their different receptor-binding chemistries. IL-15Rα substantially increased the affinity of IL-15 for IL-2Rß, and this allostery was required for IL-15 trans signaling. Consistent with their identical IL-2Rß-γ(c) dimer geometries, IL-2 and IL-15 showed similar signaling properties in lymphocytes, with any differences resulting from disparate receptor affinities. Thus, IL-15 and IL-2 induced similar signals, and the cytokine specificity of IL-2Rα versus IL-15Rα determined cellular responsiveness. Our results provide new insights for the development of specific immunotherapeutics based on IL-15 or IL-2.

Design and characterization of a protein superagonist of IL-15 fused with IL-15Rα and a high-affinity T cell receptor.

To avoid high systemic doses, strategies involving antigen-specific delivery of cytokine via linked antibodies or antibody fragments have been used. Targeting cancer-associated peptides presented by major histocompatibility complex (MHC) molecules (pepMHC) increases the number of potential target antigens and takes advantage of cross-presentation on tumor stroma and in draining lymph nodes. Here, we use a soluble, high-affinity single-chain T cell receptor Vα-Vß (scTv), to deliver cytokines to intracellular tumor-associated antigens presented as pepMHC. As typical wild-type T cell receptors (TCRs) exhibit low affinity (K(d) = 1-100 µM or more), we used an engineered TCR, m33, that binds its antigenic peptide SIYRYYGL (SIY) bound to the murine class I major histocompatability complex protein H2-K(b) (SIY/K(b) ) with nanomolar affinity (K(d) = 30 nM). We generated constructs consisting of m33 scTv fused to murine interleukin 2 (IL-2), interleukin 15 (IL-15), or IL-15/IL-15Rα (IL-15 linked to IL-15Rα sushi domain, called "superfusion"). The fusions were purified with good yields and bound specifically to SIY/K(b) with high affinity. Proper cytokine folding and binding were confirmed, and the fusions were capable of stimulating proliferation of cytokine-dependent cells, both when added directly and when presented in trans, bound to cells with the target pepMHC. The m33 superfusion was particularly potent and stable and represents a promising design for targeted antitumor immunomodulation.

Circulating IL-15 exists as heterodimeric complex with soluble IL-15Rα in human and mouse serum.

IL-15 is an important cytokine for the function of the immune system, but the form(s) of IL-15 produced in the human body are not fully characterized. Coexpression of the single-chain IL-15 and the IL-15 receptor alpha (IL-15Rα) in the same cell allows for efficient production, surface display, and eventual cleavage and secretion of the bioactive IL-15/IL-15Rα heterodimer in vivo, whereas the single-chain IL-15 is poorly secreted and unstable. This observation led to the hypothesis that IL-15 is produced and secreted only as a heterodimer with IL-15Rα. We purified human IL-15/IL-15Rα complexes from overproducing human cell lines and developed an ELISA specifically measuring the heterodimeric form of IL-15. Analysis of sera from melanoma patients after lymphodepletion revealed the presence of circulating IL-15/IL-15Rα complexes in amounts similar to the total IL-15 quantified by a commercial IL-15 ELISA that detects both the single-chain and the heterodimeric forms of the cytokine. Therefore, in lymphodepleted cancer patients, the serum IL-15 is exclusively present in its heterodimeric form. Analysis of the form of IL-15 present in either normal or lymphodepleted mice agrees with the human data. These results have important implications for development of assays and materials for clinical applications of IL-15.

Characterization of recombinant human IL-15 deamidation and its practical elimination through substitution of asparagine 77.

PURPOSE: The use of recombinant human interleukin (rhIL)-15 as a potential therapeutic immune modulator and anticancer agent requires pure, stable preparations. However, purified rhIL-15 preparations readily accumulated heterogeneities. We sought to improve rhIL-15 stability through process, formulation, and targeted amino acid changes. METHODS: The solution state of rhIL-15 versus buffer composition and temperature was studied using SEC and IEX methods. rhIL-15 deamidation was confirmed using RP-HPLC/ESI-MS, enzymatic labeling, and peptide mapping. Deamidation kinetics were measured versus buffer composition and pH using RP-HPLC. Deamidation-resistant rhIL-15 variants (N77A, N77S, N77Q, G78A, and [N71S/N72A/N77A]) were produced in E. coli, then assayed for T-cell culture expansion potency and deamidation resistance. RESULTS: Adding 20% ethanol to buffers or heating at ≥32°C dispersed rhIL-15 transient pairs, improving purification efficiencies. Asparagine 77 deamidated rapidly at pH 7.4 with activation energy of 22.9 kcal per mol. Deamidation in citrate buffer was 17-fold slower at pH 5.9 than at pH 7.4. Amino acid substitutions at N77 or G78 slowed deamidation ≥23-fold. rhIL-15 variants N77A and (N71S/N72A/N77A) were active in a CTLL-2 proliferation assay equivalent to unsubstituted rhIL-15. CONCLUSIONS: The N77A and (N71S/N72A/N77A) rhIL-15 variants are resistant to deamidation and remain potent, thus providing enhanced drug substances for clinical evaluation.

Enhancement of the inhibitory effect of an IL-15 antagonist peptide by alanine scanning.

IL-15 is a proinflammatory cytokine that acts early in the inflammatory response and has been associated with several autoimmune diseases including rheumatoid arthritis, where it had been proposed as a therapeutic target. We recently reported an IL-15 antagonist peptide corresponding to sequence 36-45 of IL-15 (KVTAMKCFLL) named P8, which specifically binds to IL-15Rα and inhibits IL-15 biological activity with a half maximal inhibitory concentration (IC50) of 130 µ m in CTLL-2 proliferation assay. In order to improve binding of peptide P8 to the receptor IL-15Rα, we used an Ala scan strategy to study contribution of each individual amino acid to the peptide's antagonist effect. Here, we found that Phe and Cys are important for peptide binding to IL-15Rα. We also investigated other single site mutations and replaced the second Lys in the sequence by the polar non-charged amino acid threonine. The resulting peptide [K6T]P8 exhibited a higher activity than P8 with an IC50 of 24 µm. We also found that this peptide was more active than peptide P8 in the inhibition of TNFα secretion by synovial cells from rheumatoid arthritis patients. The peptide [K6T]P8 described in this work is a new type of IL-15 antagonist and constitutes a potential therapeutic agent for rheumatoid arthritis.

Elevated expression of transmembrane IL-15 in immune cells correlates with the development of murine lupus: a potential target for immunotherapy against SLE.

Presentation in trans by the Interleukin-15 receptor alpha chain (IL-15Ralpha) has been suggested as the main mechanism for IL-15 anchoring to the cell surface, but it is also evident that IL-15 can exist as a transmembrane protein. We herein demonstrate that replacement of the first 41 residues of human IL-15 (hIL-15) with Igkappa chain leader sequence resulted in secretion of most of the recombinant hIL-15 expressed in transfectant cells, thus identifying the transmembrane region of IL-15. A fusion protein (hIL-15Ralpha-Fc) between the extracellular domain of hIL-15Ralpha and the Fc fragment of IgG1 was prepared and shown to be able to bind with transmembrane IL-15 (tmIL-15). The level of tmIL-15 expression in macrophages, activated T cells and B cells from 6-month-old BXSB male mice, an animal model for systemic lupus erythematosus (SLE), was significantly increased compared with that from BXSB females or young males. In addition, hIL-15Ralpha-Fc was able to block the T cell stimulating and anti-apoptotic effect of the tmIL-15-positive BXSB macrophages in vitro. Intravenous administration of hIL-15Ralpha-Fc reduced the titre of autoantibodies against dsDNA and also proteinuria in aged BXSB males, implying that neutralization of IL-15 activity in vivo may be an effective way of treating SLE.