On the basis of the results of preclinical studies in animal models, and following obtaining support from the Director NCI, Dr. Waldmann oversaw an interinstitute collaboration (NCI-NIAID) directed toward the production under current good manufacturing practices (cGMP) of rhIL-15 for clinical trials in patients with metastatic malignancy and those with HIV AIDS. In a modification of an expression system and purification method developed in the Waldmann Laboratory, the Biopharmaceutical Development Program (BDP) of the National Cancer Institute produced recombinant human IL-15 (rhIL-15). In this work an Escherichia coli (E. coli) based fermentation and purification process was developed for the production of clinical grade recombinant human IL-15. DNA sequences from human IL-15 were inserted into pET-28b plasmid and were expressed in the E. coli host BL2I-AL. In a toxicity study directed by the Waldmann Laboratory rhIL-15 was evaluated for safety (toxicity), pharmacokinetics, immunogenicity, autoimmunity and impact on the elements of the normal immune system of rhIL-15 in rhesus macaques. The only biologically meaningful laboratory abnormality was a grade 3/4 neutropenia that was secondary to a redistribution of neutrophils from the circulation to the tissues. Twelve day bolus intravenous administration of 20 mcg/kg/day of IL-15 was associated with a four to eightfold increase in the number of circulating NK and CD3, CD8 T- cells especially stem, central and effector memory CD8 T-cells. More recently in conjunction with collaborators from NIAID in rhesus macaques an evaluation of alternative routes of administration of IL-15 was completed, that involved subcutaneous administration (s.c.) and continuous intravenous infusion (c.i.v.) of IL-15. The administration of IL-15 by c.i.v. at 20 mcg/kg/day for 10 days led to a massive eighty to one-hundredfold increase in the number of effector memory CD8 T cells. When compared to c.i.v., subcutaneous infusion at 20 mcg/kg/day for 10 days led to a more modest tenfold expansion in the number of circulating memory CD8 T-cells. CDER FDA and IRB NCI approval was granted to perform a clinical phase I trial of bolus intravenous recombinant IL-15 (rhIL-15) in adults with refractory metastatic malignant melanoma and metastatic renal cell cancer, with the Metabolism Branch CCR, NCI as sponsor, Dr. Kevin Conlon as the Principal Investigator (PI), and Dr. Thomas Waldmann as study Chairman and IND holder. This study has been completed with five patients having completed their course of therapy at 3.0 mcg/kg/day, four patients having entered at 1.0 mcg/kg/day dose and five patients at 0.3 mcg/kg/day dose for 12 daily infusions. Following the bolus i.v. infusion of IL-15 the serum concentrations of IL-15 at 10 minutes following infusion ranged from 20,000 to 90,000 picograms/mL levels--sufficient to signal through the IL-2/IL-15R beta and common gamma receptors shared with IL-2. The serum IL-15 concentrations declined with an alpha phase of approximately 30 minutes for the first two hours and then with a beta phase that had a survival T 1/2 of 2-3 hours. At 24 hours IL-15 was still detectable in the serum but at the very low level of 25 to 100 picograms/mL--levels that are below those required to effectively signal through by the heterotrimeric receptor despite its high affinity for IL-15. With a bolus infusion of 3.0 mcg/kg/day there was a pattern of fever beginning 2 to 2.5 hours following the start of the rhIL-15 infusions, peaking reliably at the 3-hour timepoint. Rigors occurred at the 4-hour timepoint. Rigors and fever were also observed at the 1.0 mcg/kg/day dose but were less severe. There was minimal fever observed at the 0.3 mcg/kg/day dose. These toxicities were associated with maximum elevations at 4 to 8 hours post-bolus infusion of IL-15 in the serum concentrations of inflammatory cytokines (e.g. IL-6, IL-1, IL-8, IL-10 and IFN-gamma). Two patients manifested dose-limiting toxicity (DLT) at both the 3.0 mcg/kg/day (hypotension) and at the 1.0 mcg/kg/day (abnormalities of liver function). However the 0.3 mcg/kg/day dose has proven to be without DLT in the 5 patients examined. In the patients with the 3.0 and 1.0 mcg/kg doses of IL-15 the total lymphocyte count fell from its normal 1,000 to 2,000 mm3 level to 200-600 mm3 virtually immediately after the bolus IL-15 infusion presumably by redistribution and this lymphopenia extended through day 3. Following that timepoint there was a rise in the number of lymphocytes from the period days 8 through 12 to approximately 4,000 mm3. The circulating NK-cell numbers during the pretherapy evaluation period ranged from absolute numbers of 200 to 300mm3. By day 8 the absolute NK-cell numbers rose to 1,000 ? 1,100 and they increased further so that by day 14 their levels were 2,600 mm3. The pharmacokinetics of IL-15 following bolus infusion, discussed above, are clearly not optimal with exceedingly high levels initially that may have caused the toxicity. Therefore permission has been obtained and a trial has been initiated using continuous intravenous infusion of rhIL-15 in a dose escalation trial for a 10-day period. Furthermore, the plan is to evaluate a subcutaneous dosing strategy for IL-15 as well. A second limitation in the present trial of rhIL-15 is that the levels of IL-15R alpha required for optimal IL-15 action observed in the patients prior to and during the IL-15 infusions were exceptionally low. Therefore we propose to translate our preclinical studies in murine tumor models by initiating a combination agent clinical trial that involves the use of an agonistic antibody to CD40 to induce IL-15R alpha expression in conjunction with IL-15 administration. Furthermore, our present and future plans include administration of IL-15 in conjunction with anti-PD1 with anti-CTLA4 monoclonal antibodies to remove these checkpoints on the immune response.Collectively the 2-decade long scientific odyssey that involved the discovery and development of IL-15 by the Waldmann Group has accelerated progress in the field of Immunobiology that focuses on the role of the common gamma cytokines in the normal regulation of NK and CD8T-cell homeostasis, and in disorders in this regulation in disease state. Furthermore the completed initial trial evaluating IL-15 in patients with metastatic malignancy has provided the phase I goal information required for the initiation of a broad range of studies using IL-15 in clinical trials by intramural and extramural NCI supported groups such as the Cancer Immunotherapy Network (CITN).

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Ratner, Lee; Waldmann, Thomas A; Janakiram, Murali et al. (2018) Rapid Progression of Adult T-Cell Leukemia-Lymphoma after PD-1 Inhibitor Therapy. N Engl J Med 378:1947-1948
Perera, Liyanage P; Zhang, Meili; Nakagawa, Masao et al. (2017) Chimeric antigen receptor modified T cells that target chemokine receptor CCR4 as a therapeutic modality for T-cell malignancies. Am J Hematol 92:892-901
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Waldmann, Thomas A (2015) The shared and contrasting roles of IL2 and IL15 in the life and death of normal and neoplastic lymphocytes: implications for cancer therapy. Cancer Immunol Res 3:219-27
Yu, Huifeng; Sui, Yongjun; Wang, Yichuan et al. (2015) Interleukin-15 Constrains Mucosal T Helper 17 Cell Generation: Influence of Mononuclear Phagocytes. PLoS One 10:e0143001
Mitra, Suman; Ring, Aaron M; Amarnath, Shoba et al. (2015) Interleukin-2 activity can be fine tuned with engineered receptor signaling clamps. Immunity 42:826-38
Pilipow, Karolina; Roberto, Alessandra; Roederer, Mario et al. (2015) IL15 and T-cell Stemness in T-cell-Based Cancer Immunotherapy. Cancer Res 75:5187-5193
Conlon, Kevin C; Lugli, Enrico; Welles, Hugh C et al. (2015) Redistribution, hyperproliferation, activation of natural killer cells and CD8 T cells, and cytokine production during first-in-human clinical trial of recombinant human interleukin-15 in patients with cancer. J Clin Oncol 33:74-82
Zhang, Yi; Tian, Shenghe; Liu, Zuqiang et al. (2014) Dendritic cell-derived interleukin-15 is crucial for therapeutic cancer vaccine potency. Oncoimmunology 3:e959321
Valkenburg, Sophie A; Li, Olive T W; Mak, Polly W Y et al. (2014) IL-15 adjuvanted multivalent vaccinia-based universal influenza vaccine requires CD4+ T cells for heterosubtypic protection. Proc Natl Acad Sci U S A 111:5676-81

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