ABSTRACT
The clinical applications of immunocytokines are severely restricted by dose-limiting toxicities. To address this challenge, here we propose a next-generation immunocytokine concept involving the design of LH05, a tumor-conditional anti-PD-L1/interleukin-15 (IL-15) prodrug. LH05 innovatively masks IL-15 with steric hindrance, mitigating the "cytokine sink" effect of IL-15 and reducing systemic toxicities associated with wild-type anti-PD-L1/IL-15. Moreover, upon specific proteolytic cleavage within the tumor microenvironment, LH05 releases an active IL-15 superagonist, exerting potent antitumor effects. Mechanistically, the antitumor efficacy of LH05 depends on the increased infiltration of CD8+ T and natural killer cells by stimulating the chemokines CXCL9 and CXCL10, thereby converting cold tumors into hot tumors. Additionally, the tumor-conditional anti-PD-L1/IL-15 can synergize with an oncolytic virus or checkpoint blockade in advanced and metastatic tumor models. Our findings provide a compelling proof of concept for the development of next-generation immunocytokines, contributing significantly to current knowledge and strategies of immunotherapy.
Subject(s)
B7-H1 Antigen , Interleukin-15 , Tumor Microenvironment , Interleukin-15/immunology , B7-H1 Antigen/metabolism , B7-H1 Antigen/immunology , B7-H1 Antigen/genetics , Animals , Humans , Mice , Tumor Microenvironment/immunology , Tumor Microenvironment/drug effects , Cell Line, Tumor , CD8-Positive T-Lymphocytes/immunology , CD8-Positive T-Lymphocytes/drug effects , Neoplasms/immunology , Neoplasms/drug therapy , Neoplasms/pathology , Immunotherapy/methods , Mice, Inbred C57BL , Female , Killer Cells, Natural/immunology , Killer Cells, Natural/drug effects , Immune Checkpoint Inhibitors/pharmacologyABSTRACT
Being an important immune stimulant of T lymphocytes and NK cells, the recombinant human interleukin-15 (rhIL-15) has been extensively researched in tumor immunotherapy or as a vaccine adjuvant. However, the rhIL-15 manufacturing level lags far behind its growing clinical demand due to the lack of efficient and exact analysis methodologies to characterize the trace by-products, typically redox and deamidation. In order to improve the production and quality control of rhIL-15, here we developed an expanded resolution reverse-phase high-performance liquid chromatography (ExRP-HPLC) approach to quickly and accurately analyze the oxidation and reduction by-products of rhIL-15, which may appear during the purification processes. Firstly, we developed RP-HPLC methods which can separate rhIL-15 fractions with different levels of oxidization or reduction, respectively, and the redox status of each peak was then determined by measuring the intact mass with a high-resolution mass spectrometer (UPLC-MS). To further clarify the complex pattern of oxidization of specific residues, the peaks with various oxidation levels were digested into pieces for peptide mapping to pinpoint the exact changes of oxygen and hydrogen atoms in the rhIL-15 by-products. In addition, we performed the ExRP-HPLC and UPLC-MS analysis of partially deamidated rhIL-15 to characterize their oxidation and reduction. Our work is the first in-depth characterization of the redox by-products of rhIL-15, even for deamidated impurities. The ExRP-HPLC method we reported can facilitate the rapid and accurate quality analysis of rhIL-15, which is substantially helpful for streamlining the industrial manufacturing of rhIL-15 to better meet the demands of clinical applications. KEYPOINTS: ⢠The oxidization and reduction rhIL-15 by-products were characterized for the first time. ⢠The changes of oxygen and hydrogen atoms in rhIL-15 redox by-products were accurately determined by UPLC-MS. ⢠Oxidation and reduction by-products of deamidated rhIL-15 were further analyzed.
