Abstract
Background: Heterodimeric interleukin-15 (hetIL-15) is a cytokine that sustains the growth and activation of NK and cytotoxic T cells. It consists of a membrane-anchored polypeptide (IL-15 receptor α), which interacts and stabilizes the bioactive polypeptide (IL-15). This plasma membrane-anchored complex is cleaved and liberated as a bioactive heterodimeric cytokine (hetIL-15). Recombinant single-chain IL-15 and other forms of the cytokine have been shown to lead to expansion of T cells with an effector-memory, cytotoxic phenotype and enhanced cytotoxic function. hetIL-15 and other forms of IL-15 have also been shown to have anti-tumor effects in mouse models of cancer; hetIL-15 is currently being tested in humans for treatment of metastatic cancer, delivered as fixed-dose subcutaneous injections. In Rhesus macaques, high- dose hetIL-15 led to early toxicity, while immune activation decreased towards the end of treatment cycles, suggesting a need to optimize the treatment regimen. The ...
Background: Heterodimeric interleukin-15 (hetIL-15) is a cytokine that sustains the growth and activation of NK and cytotoxic T cells. It consists of a membrane-anchored polypeptide (IL-15 receptor α), which interacts and stabilizes the bioactive polypeptide (IL-15). This plasma membrane-anchored complex is cleaved and liberated as a bioactive heterodimeric cytokine (hetIL-15). Recombinant single-chain IL-15 and other forms of the cytokine have been shown to lead to expansion of T cells with an effector-memory, cytotoxic phenotype and enhanced cytotoxic function. hetIL-15 and other forms of IL-15 have also been shown to have anti-tumor effects in mouse models of cancer; hetIL-15 is currently being tested in humans for treatment of metastatic cancer, delivered as fixed-dose subcutaneous injections. In Rhesus macaques, high- dose hetIL-15 led to early toxicity, while immune activation decreased towards the end of treatment cycles, suggesting a need to optimize the treatment regimen. The ability of hetIL-15 to increase the abundance of cytotoxic cells makes it an appealing drug for use in HIV-1 infection, especially if activated cells infiltrate tissues known to comprise the viral reservoir, such as in lymph node follicles and the gastrointestinal tract-associated lymphoid tissues (GALT). For cancer therapy, the efficacy of IL-15 and other drugs has been shown to be significant when they are targeted to the sites of tumors. Eukaryotic cells secrete ~100 nm vesicles (extracellular vesicles; EV), which can be engineered to carry therapeutic molecules. In the emerging field of EV therapeutics, these vesicles are being designed as biological nanoparticles to deliver multiple therapeutics to tumors and other tissues. Aims: This study sought to: (a) develop an optimized dosing regimen for hetIL-15 based on its mechanism of action, in order to minimize toxicity and maximize immunological activation, (b) explore the use of hetIL-15 as part of an immunotherapeutic approach to treatment of HIV-1 infection, and (c) develop the technology and tools necessary for further development of EV as a platform to deliver hetIL-15 and other therapeutics to tumors. Methods: Healthy and SHIV-infected Rhesus macaques were treated with a 2-week cycle of increasing hetIL-15 dosages (2-64 μg/kg; “step-dose”), either subcutaneously or intraperitoneally. At the end of treatment, animals were sacrificed. Blood was collected throughout the study, lymph nodes (LN) were biopsied before/after treatment, and multiple organs (including the gastrointestinal tract) were sampled at necropsy. Vital signs, blood chemistry, clinical observation, and hetIL-15 drug level measurements were carried out throughout the study. Sampled tissues were analyzed for immune cell frequencies and phenotype by flow cytometry and/or multiplex confocal imaging with Histocytometry. Plasma viral load and cell associated viral burden was measured by qPCR and qRT-PCR using primers for SIV gag. For EV studies, a new form of the cytokine was generated by recombinant technology, consisting of a human hetIL-15/Lactadherin fusion protein. HEK293 cell clones stably expressing wildtype cytokine or hetIL-15/Lactadherin were generated and grown in tissue culture flasks or in a hollow-fiber bioreactor with serum-free media. EV were purified from conditioned media using combinations of (ultra)centrifugation, filtration, dialysis, tangential flow-filtration, and size exclusion chromatography. EV preparations were characterized biophysically (nanoparticle tracking analysis, transmission electron microscopy), biochemically (Western blot, ELISA, flow cytometry, immuno-transmission electron microscopy, mass spectrometry) and with regard to in vitro bioactivity. Uptake of EV was tested in vitro by culturing cell lines or PBMC with fluorescently labelled EV, in the presence or absence of uptake receptor blockers, and monitored by flow cytometry. In vivo biodistribution of fluorescently labelled EV injected i.v. was tested in healthy FVB and 4T1 tumor-bearing Balb/c mice, and monitored by live fluorescence imaging and ex vivo imaging of excised organs. Results: The subcutaneous step-dose regimen was associated with less toxicity than a fixed-dose administration of 50 μg/kg hetIL-15. Toxicity was limited to transient fever and a decrease in serum albumin. Furthermore, proliferation and immune activation of cytotoxic cells was maintained throughout the treatment cycle. The regimen was equally well tolerated in SHIV-infected macaques, and led to significant infiltration of lymph nodes (including follicles) with proliferating cytotoxic cells. Intraperitoneal administration in a small cohort was not associated with increased toxicity, and led to improved immune activation within the mesenteric LN and GALT. Animals that received s.c hetIL-15 were also tested for virological outcome. Plasma viral load decreased in 11 of 13 treated animals (range 3-176 fold reduction), as did cell- associated viral RNA in LN (10-103 fold reduction) and peripheral blood cells (3-100 fold reduction) in 4 of 4 tested animals. We found that human hetIL-15/Lactadherin increased cytokine loading of EV by 100 fold over wildtype hetIL-15, and that EV-associated cytokine was bioactive in vitro, leading to dose- dependent proliferation of the NK92 cell line. Bioreactor-conditioned medium contained 40-60 fold more EV per mL than conventional cell culture, and lacked serum contaminants found in conventional cell culture. Combining tangential flow filtration with SEC proved to be a scalable methodology for producing highly purified, bioactive EV . EV administered to mice intravenously were rapidly cleared by liver macrophages. We found Scavenger Receptor A to be an important mediator of this uptake, and showed that blocking it with dextran sulfate led to decreased liver clearance and significantly increased plasma levels and tumor accumulation of administered EV. Conclusions: Administration of hetIL-15 using a step-dose regimen in Rhesus macaques is a safe way to administer high-dose cytokine, leading to significant increases in cytotoxic cell infiltration of tissues throughout the body, including at sites relevant to cancer and HIV-1 infection. In SHIV-infected animals, subcutaneous hetIL-15 therapy led to a decrease in viral burden associated with blood and peripheral LN, but did not eradicate the virus. Augmenting hetIL-15 effects in other reservoir sites (such as mesenteric LN and GALT), and incorporating hetIL-15 into a protocol that combines other agents (e.g. latency reversal agents, checkpoint inhibitors) may increase the likelihood of a functional cure of HIV-1 infection. Similar approaches, capitalizing on the potent effects of hetIL-15 in combination protocols may also be beneficial in the treatment of cancer. Targeted tumor delivery of the cytokine on EV expressing high amounts of the novel hetIL-15/Lactadherin fusion protein may increase efficacy. Use of our human lactadherin construct could be employed to increase EV loading of other protein therapeutics. Bioreactor production and purification with tangential flow filtration + SEC were not only efficient in lab-scale production of EV for development, but are scalable, cGMP-compatible technologies that could be readily adapted to industrial manufacturing of EV. Thus, these methods may be more broadly applicable to the further translational development of EV therapeutics. While delivery of EV to tumors can be limited by liver macrophage clearance upon i.v. administration (especially by interaction with Scavenger Receptor A), blocking this uptake is possible, and facilitates tumor accumulation of EV. Development of new uptake blocking molecules, EV targeting/stealth modification, as well as a deeper understanding of what determines the fate of EV in vivo may allow for more effective delivery. As hetIL-15 is already tested in humans, the findings of our studies may direct the design of future clinical trials of this cytokine for immunotherapy of HIV-1 infection and cancer.
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