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A Potent in Vivo Antitumor Efficacy of Novel Recombinant Type I Interferon

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A Potent in Vivo Antitumor Efficacy of Novel Recombinant Type I Interferon Published OnlineFirst September 28, 2016; DOI: 10.1158/1078-0432.CCR-16-1386 Cancer Therapy: Preclinical Clinical Cancer Research A Potent In Vivo Antitumor Efficacy of Novel Recombinant Type I Interferon Kang-Jian Zhang1,2,3, Xiao-Fei Yin1, Yuan-Qin Yang1,4, Hui-Ling Li1, Yan-Ni Xu1, Lie-Yang Chen1, Xi-Jun Liu1, Su-Jing Yuan1, Xian-Long Fang1, Jing Xiao1, Shuai Wu1, Hai-Neng Xu1,5, Liang Chu1, Kanstantsin V. Katlinski2, Yuliya V. Katlinskaya2, Rong-Bing Guo3, Guang-Wen Wei3, Da-Cheng Wang6, Xin-Yuan Liu1,4, and Serge Y.Fuchs2 Abstract Purpose: Antiproliferative, antiviral, and immunomodulatory Results: sIFN-I displayed greater affinity for IFNAR1 (over activities of endogenous type I IFNs (IFN1) prompt the design of IFNAR2) chain of the IFN1 receptor and elicited a greater recombinant IFN1 for therapeutic purposes. However, most of the extent of IFN1 signaling and expression of IFN-inducible designed IFNs exhibited suboptimal therapeutic efficacies against genes in human cells. Unlike IFNa-2b, sIFN-I induced solid tumors. Here, we report evaluation of the in vitro and in vivo JAK–STAT signaling in mouse cells and exhibited an extend- antitumorigenic activities of a novel recombinant IFN termed sIFN-I. ed half-life in mice. Treatment with sIFN-I inhibited intra- þ Experimental Design: We compared primary and tertiary tumoral angiogenesis, increased CD8 T-cell infiltration, and structures of sIFN-I with its parental human IFNa-2b, as well as robustly suppressed growth of transplantable and genetically affinities of these ligands for IFN1 receptor chains and pharma- engineered tumors in immunodeficient and immunocompe- cokinetics. These IFN1 species were also compared for their ability tent mice. to induce JAK–STAT signaling and expression of the IFN1-stim- Conclusions: These findings define sIFN-I as a novel recom- ulated genes and to elicit antitumorigenic effects. Effects of sIFN-I binant IFN1 with potent preclinical antitumorigenic effects on tumor angiogenesis and immune infiltration were also tested against solid tumor, thereby prompting the assessment of in transplanted and genetically engineered immunocompetent sIFN-I clinical efficacy in humans. Clin Cancer Res; 23(8); 2038–49. mouse models. Ó2016 AACR. Introduction 40 years of trials, the use of IFN1 against tumors is limited by the suboptimal ratio between clinical efficacy and the severity of its Type I IFN (IFN1) family of antiviral cytokines comprises 13 side effects (6), as well as limited response rate, which is often different subtypes of IFNa, as well as IFNb, IFNe, IFNk, IFNw, etc attributed to the downregulation of IFN1 receptor (7). This (1–3). Potent antiproliferative, proapoptotic, antiangiogenic, and heterodimeric receptor complex encompassing the IFNAR1 and immunomodulatory effects of IFN1 prompted their use for anti- IFNAR2 chains mediates all effects of IFN1 on cells (8–10). Levels cancer treatment (reviewed in refs. 4, 5). However, after more than of IFN1 receptor were indeed shown to correlate with IFN1- induced growth arrest (11) and apoptosis in the tumor samples (12, 13). 1State Key Laboratory of Cell Biology, Shanghai Institute of Biochemistry and The levels of IFN1 receptor on cell surface are largely regulated Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of 2 by the ubiquitin-mediated internalization and degradation of Sciences, Shanghai, China. Department of Biomedical Sciences, University of – 3 IFNAR1 (10, 14 18). Downregulation of IFNAR1 can be accel- Pennsylvania, Philadelphia, Pennsylvania. Sichuan Huiyang Life Science and – Technology Corp., Chengdu, Sichuan, China. 4Xinyuan Institute of Medicine and erated in some cancers (19 22), thereby limiting the antitumori- Biotechnology, Zhejiang Sci-Tech University, Hangzhou, China. 5Department of genic effects of IFN1. Remarkably, although activation of the JAK– Radiation Oncology, University of Pennsylvania Perelman School of Medicine, STAT pathway is required for both antiviral and antitumor effects Philadelphia, Pennsylvania. 6National Laboratory of Biomacromolecules, Insti- of IFN1, lower receptor density still allows efficient antiviral tute of Biophysics, Chinese Academy of Sciences, Beijing, China. responses while impeding ability of IFN1 to suppress cell prolif- Note: Supplementary data for this article are available at Clinical Cancer eration (23). Schreiber and colleagues have proposed that Research Online (http://clincancerres.aacrjournals.org/). responses to IFN1 could be classified as "robust" (such as antiviral K.-J. Zhang, X.-F. Yin, and Y.-Q. Yang contributed equally to this article. effects) or "tunable" (such as antiproliferative or proinflamma- Corresponding Authors: Xin-Yuan Liu, Institute of Biochemistry and Cell Bio- tory), the latter being much more sensitive to receptor density logy, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, (24). Indeed, high cell-surface receptor density and maximal 320 Yue-Yang Road, Shanghai, China 200031. Phone: 8621-5492-1127; Fax: receptor occupancy by relatively high doses of ligands are required 8621-5492-1256; E-mail: [email protected]; and Serge Y. Fuchs, University of to mount an efficient antiproliferative effect (24, 25). Pennsylvania, 380 South University Avenue, Room 316 Hill, Philadelphia, PA Furthermore, the affinity of IFN1 subtypes for the extracellular 19104. Phone: 215-573-6949; Fax: 215-573-5188; E-mail: [email protected] domain of IFNAR1 correlates with the ability of these subtypes to doi: 10.1158/1078-0432.CCR-16-1386 elicit specific antiproliferative effect (26–29). Thus, antitumori- Ó2016 American Association for Cancer Research. genic efficacy of IFN1 may be optimized by increasing cell-surface 2038 Clin Cancer Res; 23(8) April 15, 2017 Downloaded from clincancerres.aacrjournals.org on September 23, 2021. © 2017 American Association for Cancer Research. Published OnlineFirst September 28, 2016; DOI: 10.1158/1078-0432.CCR-16-1386 Antitumor Effects of Recombinant Interferon sIFN-I IFNb (#: 50708-M02H), M-CSF (#: 11792-H08H), recombinant Translational Relevance type I IFN receptor subunit extracellular domain IFNAR1-EC Despite potent antitumorigenic properties of natural and (#: 13222-H08H) and IFNAR2-EC (#: 10359-H08H) were pur- pharmacologic type I IFNs (IFN1), these agents achieved only a chased from Sino Biological Inc. Recombinant B18R protein limited success in cancer therapy. This article describes the (vaccinia virus-encoded neutralizing type I interferon receptor) molecular and biological characterization of de novo engi- was purchased from eBioscience (#: 14-8185). neered and highly potent recombinant IFN (sIFN-I), which has evoked massive clinical interest and is currently undergo- Protein crystallization, data collection, and structure ing clinical trials in patients with solid tumors in Singapore determination (CTC1300056) and the United States (NCT02464007), as Crystals of super interferon (sIFN-I) were grown by the hang- well as patients with HBV in China (2009L04155). Here, we ing-drop vapor diffusion method (3 mg/mL protein concentra- present data obtained in both in vitro and in vivo settings; these tion) at 20 C with, in the buffer of 1.2 mol/L Li2SO4, 0.1 mol/L data demonstrate that sIFN-I exhibits superior pharmacody- 3-(cyclohexylamino)-1-propanesulfonic acid, pH 11.1, 0.02 namic and pharmacokinetic characteristics compared with its mol/L MgCL2. Before data collection, the crystals were equilibrat- parental human IFNa-2b species. Furthermore, studies con- ed in a solution containing paraffin oil for a few seconds, and then ducted in cells and in animals harboring transplantable and flash cooled in a liquid nitrogen stream at À173C. Original data genetically engineered tumor models reveal that sIFN-I evokes collection to 2.6 Å resolutions was conducted by using the potent antitumorigenic effects at least in part by inhibiting synchrotron radiation from beamline BL5A at a photon factory stromal angiogenesis and by stimulating antitumor immunity. in Tsukuba, Japan. Primary structural determination was achieved by a combination of molecular replacement method. The posi- tion of the sIFN-I was found by molecular replacement using PHASER with the crystal structure of IFNa (Protein Data Bank receptor density and/or by designing novel recombinant IFN1 name: IB5L) used as the search model. The final sIFN-I structure species that display a greater affinity for IFNAR1. A number of was refined by using molecular modeling techniques and a IFN1 variants were generated and shown to be effective against computerized optimization program, CNS1.1. tumor cells. For example, a mutant derivative of IFNa-2, IFNa- YNS exhibited tight binding to IFNAR1 and elicited potent proa- Surface plasmon resonance assay poptotic activity and antiproliferative/antiangiogenesis effects in On the basis of surface plasmon resonance technology, binding vivo; this mutant surpassed IFNa-2 in antitumorigenic activity in a affinities of both IFNa-2b and sIFN-I toward recombinant extra- breast cancer xenograft (28, 30). cellular (EC) domain of type I interferon receptor subunit fi Yet, another approach to increase ef cacy of IFN1 treatment is IFNAR1-EC or IFNAR2-EC were measured using the Biacore to improve its pharmacokinetics and biological activities. Various T100 Protein Interaction Array system (General Electric Health- efforts in this direction include the use of IFNa-2b-albumin Care Co.). For immobilization of the receptor subunit via binding fusion protein (31), antibody armed with IFN1 (32), and
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