The study aims to explore the active molecules of traditional Chinese medicine that specifically bind to interleukin-15 receptor α (IL-15Rα) using molecular docking and surface plasmon resonance (SPR) technology. AutoDock molecular docking software was used to perform simulated docking of more than 3 000 compounds from 48 traditional Chinese medicines at IL-15Rα and screen the specific binding compounds. Then Biocore T200 biomolecular interaction analysis system of SPR was used to confirm the binding specificity of the selected target compounds. Finally, the biological effects of the target compounds on IL-15Rα were verified by cell biological experiments. The results showed that neoprzewaquinone A (Neo) possessed the highest specific binding affinity among the active molecules from traditional Chinese medicine, and the dissociation constant (KD) value was (0.62 ± 0.20) µmol/L. The results of cell experiment showed that Neo significantly inhibited the proliferation of Mo7e cells induced by IL-15, and the IC50 was 1.075 µmol/L, approximately 1/120 of the IC50 of Cefazolin (IL-15 specific antagonist). These results suggest that Neo is a specific inhibitor of IL-15Rα and may be a potential active drug for the treatment of diseases related to the dysfunction of the IL-15Rα signaling.

Chimeric antigen receptor (CAR)-T cell therapy is a powerful adoptive immunotherapy against both B-cell malignancies and some types of solid tumors. Interleukin (IL) -15 is an important immune stimulator that may provide ideal long-term persistent CAR-T cells. However, higher base line or peak serum IL-15 levels are also related to severe toxicity, such as cytokine release syndrome (CRS), graft-versus-host disease (GVHD), and neurotoxicity.
We successfully constructed CD19 specific armored CAR-T cells overexpressing IL-I5 and IL-15 receptor alpha (IL-15Ra). In vitro cell differentiation and viability were monitored by flow cytometry, and an in vivo xenograft mouse models was used to evaluate the anti-tumor efficiency and liver damage of CAR-T cells.
CAR-T cells overexpressing IL-15 alone demonstrated enhanced viability, retarded exhaustion in vitro and superior tumor-inhibitory effects in vivo. However, these tumor-free mice had lower survival rates, with serious liver injuries, as a possible result of toxicity. As expected, CAR-T cells overexpressing IL-15 combined with IL-15Ra had reduced CD132 expression and released fewer cytokines (IFNγ, IL-2 and IL-15) in vitro, as well as had the tendency to improve mouse survival via repressing the growth of tumor cells and keeping livers healthier compared to CAR-IL-15 T cells.
These results indicated the importance of IL-15 in enhancing T cells persistence and IL-15Ra in reducing the adverse effects of IL-15, with superior tumor retardation during CAR-T therapy. This study paves the way for the rapid exploitation of IL-15 in adoptive cell therapy in the future.

BACKGROUND: Interleukin-15 (IL-15) is a critical cytokine for the development, proliferation, and function of natural killer (NK) cells, NKT cells, and CD8+ memory T cells and has become one of the most promising protein molecules for the treatment of cancer and viral diseases. However, there are several limitations in applying IL-15 in therapy, such as its low yield in vitro, limited potency, and short half-life in vivo. To date, there are several recombinant IL-15 agonists based on configurational modifications that are being pursued in the treatment of cancer, such as ALT-803, which are mainly produced from mammalian cells.
RESULTS: In this study, we designed two different forms of the IL-15 complex, which were formed by the noncovalent assembly of IL-15 with dimeric or monomeric sushi domain of IL-15 receptor α (SuIL-15Rα)-IgG4 Fc fusion protein and designated IL-15/SuIL-15Rα-dFc and IL-15/SuIL-15Rα-mFc, respectively. The two IL-15 complexes were expressed in Pichia pastoris (P. pastoris), and their activities and half-lives were evaluated and compared. Pharmacokinetic analysis showed that IL-15/SuIL-15Rα-dFc had a half-life of 14.26 h while IL-15/SuIL-15Rα-mFc had a half-life of 9.16 h in mice, which were much longer than the 0.7-h half-life of commercial recombinant human IL-15 (rhIL-15). Treatment of mice with intravenous injection of the two IL-15 complexes resulted in significant increases in NK cells, NKT cells, and memory CD8+ T cells, which were not observed after rhIL-15 treatment. Treatment of human peripheral blood mononuclear cells (PBMCs) from healthy donors with the two IL-15 complexes yielded enhanced NK and CD8+ T cell activation and proliferation, which was comparable to the effect of rhIL-15.
CONCLUSIONS: These findings indicate that the IL-15/SuIL-15Rα-dFc and IL-15/SuIL-15Rα-mFc produced in P. pastoris exhibit potent activities and prolonged half-lives and may serve as superagonists for immunotherapy in further research and applications.

IL15 is a pleiotropic cytokine with multiple roles that improve immune responses to tumor cells. Oncolytic viruses (OV) specifically lyse tumors and activate immune responses. Systemic administration of IL15 or its complex with the IL15Rα and chimeric antigen receptor (CAR) natural killer (NK) cells are currently being tested in the clinic. Here, we generated a herpes simplex 1-based OV-expressing human IL15/IL15Rα sushi domain fusion protein (named OV-IL15C), as well as off-the-shelf EGFR-CAR NK cells, and studied their monotherapy and combination efficacy in vitro and in multiple glioblastoma (GBM) mouse models. In vitro, soluble IL15/IL15Rα complex was secreted from OV-IL15C-infected GBM cells, which promoted GBM cytotoxicity and improved survival of NK and CD8+ T cells. Frozen, readily available off-the-shelf EGFR-CAR NK cells showed enhanced killing of tumor cells compared with empty vector-transduced NK cells. In vivo, OV-IL15C significantly inhibited tumor growth and prolonged survival of GBM-bearing mice in the presence of CD8+ T cells compared with parental OV. OV-IL15C plus EGFR-CAR NK cells synergistically suppressed tumor growth and significantly improved survival compared with either monotherapy, correlating with increased intracranial infiltration and activation of NK and CD8+ T cells and elevated persistence of CAR NK cells in an immunocompetent model. Collectively, OV-IL15C and off-the-shelf EGFR-CAR NK cells represent promising therapeutic strategies for GBM treatment to improve the clinical management of this devastating disease. SIGNIFICANCE: The combination of an oncolytic virus expressing the IL15/IL15Rα complex and frozen, ready-to-use EGFR-CAR NK cells elicits strong antitumor responses in glioblastoma.

OBJECTIVE: This study aimed to find polymorphic loci associated with OPLL in Mongolian and Han population, the relationship of 9 polymorphic loci in Runx2 and IL-15RA with OPLL were identified in Mongolian and Han populations in Inner Mongolia.
METHODS: Gene polymorphism of two candidate genes Runx2 and IL-15RA were detected by sequencing in 99 OPLL patients of Han population and 98 patients of Mongolian people. Controls included 102 healthy Han people and 104 healthy Mongolian people. The result of sequencing of patients were compared with control subjects to screen loci with significant difference.
RESULTS: In Han population, results of genotyping showed rs1321075 and rs12333172 in Runx2 and rs2296139 in IL-15RA differed between patients and healthy people (P<0.05); Genotype of rs1321075 and rs16873379 and rs2296139 in IL-15RA have significant difference between patients and controls in Mongolian people (P<0.05); There was no significant difference found in genotype and frequency of other loci (P>0.05).
CONCLUSIONS: Polymorphism of rs1321075 and rs2296139 in Runx2 and IL-15RA may be responsible for OPLL in Mongolian and Han population patients. rs12333172 was related to OPLL in Han population and rs16873379 was responsible for OPLL in Mongolian people in Inner Mongolia.

Schizophrenia is a complex and debilitating mental disorder with strong heritability. Its pathogenesis involves immune dysregulation. Interleukin 15 and interleukin 15 receptor alpha(IL-15Rα) are classical immune molecules. They also help maintain normal brain function, leading to our hypothesis that IL-15Rα gene(IL- 15RA) variants contribute to the pathogenesis of schizophrenia.
We determine whether the genetic variants of IL-15RA are associated with the development and progression of schizophrenia and whether IL-15RA single nucleotide polymorphism(SNP) plays a key role in downstream signaling transduction.
We sequenced IL-15RA exon from 132 Chinese schizophrenic patients and identified a rare variant(rs528238821) in a patient diagnosed with catatonic schizophrenia and ankylosing spondylitis(AS). We overexpressed this missense variant in cells driven by pBI-CMV vector. The cells showed attenuated STAT3 phosphorylation in response to interleukin15.
IL-15RA mutation is rare in schizophrenic patients but interfered with IL- 15Rα intracellular signal transduction. Given the similarity of symptoms of catatonic schizophrenia and the known phenotype of IL-15Rα knockout mice, gene variation might offer diagnostic value for sub-types of schizophrenia.

Recombinant human interleukin-15 (IL-15) is a potent cancer immunotherapeutic candidate due to its excellent immune stimulating effects. Previous work demonstrated that IL-15 appeared with short half-life in circulation system, while the complex with its receptor can prolong the half-life as well as benefit its activities in vivo. Therefore, IL-15 complex was more favorably considered for clinical development. Herein we developed IL-15·sIL-15Rα/Fc, a complex comprising of IL-15 and the extracellular region of its receptor alpha subunit which fused to Immunoglobulin G (IgG1) Fc to further prolong the half-life in plasma. Through transient gene expression in HEK293 cells, we expressed the superagonist by co-transfection of plasmids encoding IL-15 and sIL-15Rα/Fc respectively, yielding 36 mg/L of product after purification. Pharmacokinetic study demonstrated that the combination profoundly prolonged the half-life of IL-15 to 13.1 h in mice, about 18 folds longer than that of IL-15 monomer which is around 0.7 h. The bioactivity of the superagonist was characterized by CTLL-2 cells proliferation assay in vitro, showing its capability of stimulating the expansion of memory CD8+ T cells (cluster of differentiation) in mouse spleen. Using a HT-29 xenograft NOD-SCID mouse model, we observed tumor growth inhibition in all groups that received the superagonist, indicating its anti-tumor efficacy via stimulating infused human immune cells. In addition, combo cancer treatment by IL-15·sIL-15Rα/Fc and programmed death-1 (PD-1) antibody have shown stronger inhibitory effects as compared with treatment with either single molecule. Therefore, we developed IL-15·sIL-15Rα/Fc to be a long half-life potential cancer immunotherapy candidate that can be applied alone or in synergy with PD-1/PD-L1 blockade.

Recent studies on CD19-specific chimeric antigen receptor (CAR)-modified T cells (CARTs) have demonstrated unprecedented successes in treating refractory and relapsed B cell malignancies. The key to the latest CART therapy advances can be attributed to the improved costimulatory signals in the CAR design.
Here, we established several novel CARs by incorporating T cell signaling domains of CD28 in conjunction with intracellular signaling motif of 4-1BB, CD27, OX40, ICOS, and IL-15Rα. These novel CARs were functionally assessed based on a simple target cell killing assay.
The results showed that the CD28/IL-15Rα co-signaling (153z) CAR demonstrated the fastest T cell expansion potential and cytotoxic activities. IL-15 is a key cytokine that mediates immune effector activities. The 153z CARTs maintained prolonged killing activities after repetitive rounds of target cell engagement. Consistent with the enhanced target killing function, the 153z CARTs produced increased amount of effector cytokines including IFN-γ, TNFα and IL-2 upon interaction with the target cells.
In a follow-up clinical study, an acute lymphoblastic leukemia (ALL) patient, who experienced multiple relapses of central nervous system leukemia (CNSL) and failed all conventional therapies, was enrolled to receive the CD19-specific 153z CART treatment. The patient achieved complete remission after the 153z CART cell infusion. The translational outcome supports further investigation into the safety and enhanced therapeutic efficacy of the IL-15Rα-modified CART cells in cancer patients.

Interleukin 15 (IL15) is a pleiotropic cytokine that participates in innate and adaptive immunity along with its receptor α-chain (IL15Rα). In order to investigate the potential roles of IL15 and IL15Rα in dojo loach (Misgurnus anguillicaudatus), we firstly cloned the cDNA sequence of Ma-IL15 and Ma-IL15Rα, which contain 1096bp and 1236bp and code proteins of 193 amino acids and 210 amino acids, respectively. A short signal peptide and Pfam IL15 domain were found in Ma-IL15, while a highly conserved sushi domain existed in Ma-IL15Rα. Ontogeny analysis indicated that significantly increased expression of Ma-IL15 and Ma- IL15Rα mRNA were detected in larvae from 1d to 7d post hatching, while relative high expression levels were detected in both systematic and mucosal immune-related tissues of adult dojo loach. Then three dojo loach infection models with F. columnare G4, I. multifiliis and Saprolegnia parasitica were constructed, which resulted in increased skin goblet cells and serious lesions in gill. Ma-IL15 and Ma-IL15Rα showed different expression patterns in different tissues during three infection models. Ma-IL15Rα mRNA was found to be more significantly elevated than Ma-IL15 after infection with F. columnare G4 in all examined tissues including kidney, spleen, gill and skin. I. multifiliis infection induced higher expression of Ma-IL15 in mucosal tissues including skin and gill, while it mainly increased Ma-IL15Rα expression in kidney. Moreover, our study firstly evaluated the influence of fungal infection on IL15 and IL15Rα expression in teleost, and it is interesting to find that both Ma-IL15 and Ma-IL15Rα expression showed consistent up-regulation after Saprolegnia parasitica infection compared to two other infection models. Therefore, our results suggest that Ma-IL15 and Ma-IL15Rα possess important defensive roles in systematic and mucosal tissues of dojo loach during bacterial, fungal and parasitic infection.

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.