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. 2016 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- 2016 American Association for Cancer Research. genic efficacy of IFN1 may be optimized by increasing cell-surface

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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 173C. 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 pegyla- the carboxylated dextran surface of the chip via amino groups in tion of IFN1 (33). Furthermore, given that many of antitumori- protein, a CM5 sensor chip was incubated with the IFNAR1-EC genic effects of IFN1 are mediated by the stromal cells, generation subunit and IFNAR2-EC subunit, at 20 and 50 mg/mL, respec- of an elegant transgenic mouse model that expresses human tively. The two tested IFNs were then injected perpendicularly to IFNAR1 and IFNAR2 subunits, and can be used for transplanta- ligands at different concentrations within the range of 100 to tion of human tumors, resulted in improved ability to test the 3,000 nmol/L for IFNa-2b/IFNAR1 binding, 50 to 1,000 nmol/L antitumorigenic effects of IFN1 (34). for sIFN-I/IFNAR1 binding, and 3.125 to 80 nmol/L for both of Here, we characterized antitumorigenic properties of a novel them on IFNAR2 binding. During IFNs/IFNAR2 binding, a 5- recombinant IFN1 derived from human IFNa-2b and other IFN1 second regeneration procedure with 2 mol/L NaCl was added subtypes by mutagenesis and termed "super-compound interfer- between each step of concentration. Data were analyzed by using on-I" (sIFN-I). Compared with IFNa-2b, sIFN-I exhibited higher Biacore T100 software. Dissociation constants KD were deter- anti-HIV activity in SCID mice reconstituted with human periph- mined from the rate constants according to the Equation KD ¼ eral blood leukocytes (35). Current studies revealed that sIFN-I kd/ka (d, dissociation; a, association). exhibits increased affinity for IFNAR1 and has greatly improved pharmacokinetics and signaling in human and mouse cells. sIFN-I robustly inhibits intratumoral angiogenesis and suppresses growth Cells, cell culture, and reagents of transplantable and genetically engineered tumors in immuno- Human amnion epithelium WISH cells, all human (A549, deficient and immunocompetent mice. We discuss the direct and HeLa, HT-29, and SMMC-7721) and murine (MC38, LLC, and indirect mechanisms of potent antitumorigenic effects of sIFN-I B16F10) cancer cell lines were cultured in their complete condi- and potential perspectives of its use in human cancer treatment. tional medium; primary murine melanoma cell line YUMM was cultured as reported previously (36). Lentiviral shRNA targeting Methods and Materials sequences were used for knocking down expression of IFNAR1 in WISH cells. For the construction of A549-IFNAR1-KO cells, the Cytokines IFNAR1 gRNA targeting sequences were inserted into the Cas9/ The novel recombinant super-compound IFN (sIFN-I) and gRNA target vector LentiCRISPR (37). Lentivirus was packaged interferon IFNa-2b were provided by Sichuan Huiyang Life Sci- and used to infect parental A549 cells. The IFNAR1-negative cell ence & Technology Corporation and Shanghai Huaxin Biotech- clones were selected with 0.2 mg/mL puromycin and then con- nology, respectively. Human IFNb (#: 10704-HNAS) and murine firmed by FACS assay and immunoblot. Detailed information

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about the cell lines and cell culture, shRNA, and sgRNA sequences Untreated mice (n ¼ 3) served as negative control. To determine are provided in Supplementary Materials and Methods and Sup- half-life of the two IFNs in serum, the concentration values, plementary Table S1. determined from ELISA measurements (VeriKine Human IFN Alpha ELISA Kit, #: 41100, PBL Assay Science Inc.), were plotted Preparation of cell suspensions from murine spleen, lymph against time postinjection and numerically fitted using WinNon- node, liver, and small intestinal epithelial tissues lin version 6.2 software (Pharsight) as described elsewhere (39). Spleen, lymph nodes (including inguinal, brachial, axillary, Noncompartmental models were assumed. Data (including SDs) bilateral superficial cervical, and mesenteric lymph nodes), liver, and curve fits were finally plotted with GraphPad Prism 5. and small intestinal epithelial tissue were isolated from C57BL/6 mice. Briefly after organs were mechanically disaggregated, pri- FACS assays mary splenocytes and liver cells were obtained and resuspended in A549 IFNAR1 / cells (3 105) were seeded into 6-well plates. PBS after depletion of red blood cells. For isolation of small After 24 hours, the cells were dissociated with cell dissociation intestinal epithelial tissue cells, the intestinal tube of 3-cm length buffer (#: 13151-014, Life Technologies), centrifuged at 1,500 distant from the connection with stomach was cut out, and the rpm for 5 minutes in FACS tube, and washed with 1 PBS once. interior side was washed from one end by using syringe and sterile Then, cells were stained with the self-made mouse anti-human PBS. Cells were scraped off with the edge of a cover glass, counted, IFNAR1 antibody (1:1,000 diluted in 1% BSA-PBS) for 30 min- and collected for further cell culture or mRNA extraction by utes at room temperature. After washing with PBS, cells were TRIzol. stained with AF488-conjugated goat anti-mouse IgG (1:1,000 diluted in 1% BSA-PBS) for 30 minutes. Cells stained with IgG Preparation of murine bone marrow–derived macrophages isotype and secondary Ab only were used as negative control and Bone marrow cells were flushed from the femurs and tibias of were then analyzed. sacrificed C57BL/6 mice and then depleted for red blood cells For detection of cell populations in spleen from tumor-bearing þ using red cell lysing solution. The cells (1 107 cells/well) were mice (BrafV600E/ , PtenD/D), splenocytes were suspended after red cultured in 6-well plates in medium supplemented with 20 ng/mL cell lysis. Then, cells were incubated with Fc blocker antibody for macrophage colony–stimulating factor (M-CSF). Nonadherent 15 minutes at room temperature. Subsequently, specific antibo- cells were carefully removed, and fresh conditional medium was dies (listed in Supplementary Table S2) were added and staining added every 2 days. On day 5, the adherent murine bone marrow– was continued for 20 minutes on ice. After a washing step, cells derived macrophage cells were collected for further treatment. were stained with 0.5 mg/mL DAPI and were then analyzed immediately. Flow cytometry data acquisition was performed Mice by LSRFortessa machine (BD Biosciences), and analysis was Female nude mice (6–8 weeks old) and female C57BL/6 performed using FlowJo software. mice (8 weeks old) were purchased from Shanghai SLAC Com- þ þ pany. C57BL/6 Ifnar1 / or Ifnar1 / mouse (strain: B6.129S2- Immunologic and other techniques Ifnar1tm1Agt/Mmjax) was purchased from The Jackson Laboratory. Immunoblots, immunofluorescent analysis, and other immu- More detailed information for nude mice models and syngenic nologic techniques using antibodies are listed in the Supplemen- transplantable model is provided in Supplementary Materials and tary Information and have been described in our previous pub- Methods. The experiments and animal procedures conducted at lications (15–17). For details on the methods for RNA extraction, Shanghai Institute of Biochemistry and Cell Biology (Shanghai, cDNA synthesis, quantitative PCR, the information of the syn- China) were approved by the Institution Animal Care and Use thesized primers, H&E staining, cellular senescence detection of Committee (IACUC, protocol recording code: IBCB0029REV1). paraffin sections, immunofluorescent analysis of frozen sections, Experiments and all animal procedures conducted at the University for cell viability assay on human and mouse cells and illustrator of Pennsylvania (Philadelphia, PA) were approved by the IACUC image processing, data analyzing, and statistics are described in (protocols # 803995). Female C57BL/6 mice harboring Tyr:: Supplementary Materials and Methods and our previous publica- þ CreERT2; Braf CA/ ; Ptenf/f alleles (which, upon tamoxifen treatment, tions (22). þ were converted into BrafV600E/ ,PtenD/D specifically in melanocytes) were kindly provided by Drs. McMahon (University of California, Statistical analysis San Francisco, San Francisco, CA) and Bosenberg (Yale University, Comparisons between experimental groups were performed New Haven, CT). Induction of malignant melanoma by tamoxifen using the Student t test and GraphPad Prism 5 software (Graph- treatment was carried out as described previously (22, 38). Pad Prism software Inc.). All data were shown as Mean SEM. Statistically siginificant differences are indicated in figures by Pharmacokinetic animal experiments single (P < 0.05), double (P < 0.01) or triple (P < 0.001) symbols For pharmacokinetics studies, female C57BL/6 mice (8 weeks (such as or #). old; Shanghai SLAC Co.) were injected intraperitoneally with sIFN-I or IFNa-2b. All mice in each IFN-treated group (n ¼ 9, further divided into three subgroups) simultaneously received a Results dose of 50 mg/kg in PBS. Blood samples from each group were sIFN-I differs from IFNa-2b in spatial structure and receptor- collected after 5, 15, 30 minutes, 1, 1.5, 2, 4, 6, 12, and 24 hours binding affinity from the retro-orbital sinus (subgroup I: 5 minutes, 1, 4, and 24 Primary structural analysis showed that sIFN-I has 89% amino hours; subgroup II: 15 minutes, 1.5 and 6 hours; subgroup III: 30 acid sequence homology with IFNa-2b (Fig. 1A). The crystal minutes, 2 and 12 hours). Serum was obtained by centrifugation structure of sIFN-I was solved at 2.6 Å resolution; the resulting at 10,000 rpm for 10 minutes at 4C and was stored at 80C. structure showed that sIFN-I is mainly composed of six helixes

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(A–F as shown in Fig. 1A) and two distinct loops (AB and BC). demonstrated that these cells do not harbor defects in JAK This structure was generally comparable with the one previ- signaling. Importantly, STAT1 phosphorylation in IFNAR1-defi- ously reported for IFNa-2b (40). Nevertheless, a difference cient clones was not induced by sIFN-I (Fig. 3E). These data between these proteins was noted in the structure of AB loop suggest that sIFN-I signals through the IFNAR1/IFNAR2–JAK (residues 25–33: SPFSCLKDR) and BC loop (residues 44–52: pathway in human cells. DGNQFQKAQ; Fig. 1A and Supplementary Fig. S1A). Given previously published data regarding putative role of these loops sIFN-I can act on mouse cells and exhibits distinct in the interaction with the ligands (30), we next sought to pharmacokinetics and tissue responses in vivo determine relative affinities of sIFN-I for the receptor chains Poor sensitivity of mouse IFN1 receptor to human IFN1 species IFNAR1 and IFNAR2. and suboptimal pharmacokinetics of IFN1-based agents pose a Surface plasmon resonance assay indeed demonstrated differ- challenge for efficient testing of biological effects of human IFN1 ent receptor-binding affinities for sIFN-I and IFNa-2b. Under (34). Notably, treatment of primary mouse cells with sIFN-I the condition of steady-analysis model used in this experiment, revealed that activity of this ligand in induction of ISGs (Irf7 and sIFN-I exhibited greater affinity for the extracellular domain of Isg15) is superior to that of human IFNa-2b. All these effects were 7 IFNAR1 [KD 6.003 10 mol/L (0.6 mmol/L)] than IFNa-2b [KD dependent on IFN1 receptor status as evident from the lack of 2.835 10 6 mol/L (2.8 mmol/L); Fig. 1B and D]. Affinity sIFN-I–induced gene expression increase in Ifnar1 knockout mice constant of the extracellular domain of IFNAR2 chain analyzed (Fig. 3F). by the dynamic-analysis model exhibited KD for sIFN-I of 2.192 We further compared pharmacokinetics of sIFN-I and IFNa-2b 8 9 10 mol/L (21.9 nmol/L) and KD for IFNa-2b of 1.843 10 in mice after intraperitoneal injection of these agents. To this end, mol/L (1.84 nmol/L). Compared with IFNa-2b, sIFN-I displayed blood samples were taken at fixed time points after IFN admin- a higher affinity to IFNAR1 (4.72-fold) but lower affinity for istration, and IFN concentrations in serum were assessed by IFNAR2 (11.9-fold; Fig. 1C and D). These properties distinguish ELISA, followed by numerical analysis using WinNonlin6.2 soft- sIFN-I from other IFN1 variants, such as IFN-YNS and IFN-YNS- ware (Fig. 4A and B). The pharmacokinetic parameters of sIFN-I a8tail, which exhibit increased affinities to both IFNAR1 and and IFNa-2b after administration at the same dose are summa- IFNAR2 (24). In fact, with weaker binding toward IFNAR2 but rized in Supplementary Table S3. At 15 minutes after injection, the stronger binding to IFNAR1, sIFN-I is mostly reminiscent of mean serum peak concentration (Cmax) for IFNa-2b was 16,730 properties reported for IFNa-21 (29) that shared 95 % homology pg/mL. However, the Cmax of sIFN-I with 9,915 pg/mL was with sIFN-I (Supplementary Fig. S1B). delayed to 1 hour after administration. Despite the Cmax differences between sIFN-I and IFNa-2b, the area under concentration sIFN-I requires IFNAR1/IFNAR2 for activating the JAK–STAT versus time curve [AUC (0i)] for sIFN-I and IFNa-2b exhibited pathway comparable value (27,425 and 24,648 pg per hour/mL, respec- We next compared signaling elicited by sIFN-I and IFNa-2b tively) at the same dosage. Such pharmacokinetics data suggested in human A549 or HeLa cells. A similar extent of STAT1, STAT2, volume distribution (Vz-F) of sIFN-I (4,384 mL/kg) is more and STAT3 tyrosine phosphorylation was detected after admin- extensive than that of IFNa-2b (2,055 mL/kg) at steady state. In istering both IFN1 types. However, IFNa-2b–induced signaling other words, the tissue concentrations of sIFN-I were higher than was more sensitive to inhibition by the vaccinia virus–derived that of IFNa-2b. Consistent with these data, the induction of B18R protein mimicking soluble IFN1 receptor and known to expression of IFN-induced genes Irf7 and Isg15 in mouse lymph inhibit IFN1 pathway via ligand squelching (41) in both cell nodes, spleen, liver, and intestinal epithelial cells was notably lines (Fig. 2A and B). This result suggests that sIFN-I may greater after treatment of mice with sIFN-I compared with IFNa- exhibit an enhanced signaling capacity under signaling limiting 2b (Fig. 4C–F) treatment. In all, these data suggest that compared conditions. with IFNa-2b, sIFN-I exhibits a greater distribution in mouse Recombinant IFN1 proteins were shown to opportunistically tissues and accordingly elicits a greater IFN-stimulated genes bind other receptors besides IFNAR1/2, such as the opioid induction in these tissues. receptors (42–44). Thus, we sought to determine whether signaling by sIFN-I depends on canonical IFNAR1/2-JAK-STAT sIFN-I inhibits growth of solid tumors pathway. Experiments in human fibrosarcoma 2fTGH cells We next compared the antitumorigenic properties of sIFN-I and (sensitive to IFN1) and derivative clones lacking IFNAR2 of IFNa-2b. These agents administered at the doses of 50 to 150 mg (U5A) or JAK1 (U4A) revealed that both IFNAR2 and JAK1 per mice were reasonably well tolerated by the A549 or HT-29 are required for sIFN-I–induced phosphorylation of STAT1 and tumor-bearing immunocompromised mice; these mice did not STAT3 (Fig. 3A). exhibit body weight loss during the course of treatment (Supple- Consistent with these results, sIFN-I did not induce the expres- mentary Fig. S4). Whereas a modest inhibition of tumor growth sion of IFN-stimulated genes (ISG), such as ISG15 or CCL5 in was elicited by IFNa-2b, administration of sIFN-I robustly either U4A or U5A cells (Fig. 3B). Furthermore, RNAi-mediated suppressed this growth and led to a stable disease (Fig. 5A knockdown of IFNAR1 attenuated sIFN-I–induced phosphoryla- and Supplementary Fig. S4). Analysis of tumor tissues revealed tion of STAT1/STAT3 and expression of TRAIL in WISH cells that sIFN-I treatment increased cell senescence markers (senes- (Fig. 3C and D and Supplementary Fig. S2), suggesting an impor- cence-associated b-galactosidase) and dramatically decreased tant role of IFNAR1 in sIFN-I signaling. To corroborate these data, the rate of cell proliferation (assessed by Ki67 staining). Accord- we used CRISPR/Cas9 approach to knock out IFNAR1 in human ingly, an increased expression of p53 tumor suppressor protein A549 cells (Supplementary Fig. S3). A robust phosphorylation of as well as cyclin-dependent kinase inhibitors p21 and p27 was STAT1 observed in response to IFNg (which utilizes type II IFN found in tumor tissues from mice treated with sIFN-I (Supple- þ receptor; ref. 45) in selected IFNAR1 / or IFNAR1 / clones mentary Fig. S5).

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Figure 2. sIFN-I is capable of an increased signaling under limiting conditions. A, IFN signaling in A549 cells: 10-fold serial dilutions of recombinant B18R protein (1 to 100 ng/mL, final assay concentration) were prepared in media and combined with a constant amount (1 ng/mL, final assay concentration) of each IFN protein (sIFN-I or IFNa-2b) for 1 hour at room temperature. The B18R/IFN complexes were transferred to cells and then incubated for 30 minutes. The phosphorylation and total signal of STAT1, STAT2, and STAT3 were detected by immunoblot, and the p-STAT levels were quantified compared with their corresponding total STAT proteins. þ, treatment with 100 ng/mL B18R protein. B, IFN signaling in HeLa cells was analyzed as in A.

When tested for growth inhibition in vitro, both IFNa-2b knockoutanimalswerechosenashosts,tumorsgrewmore and sIFN-I exhibited robust effects on human WISH cells at the aggressively and did not respond to treatment with sIFN-I (Fig. dose of 1 mg/mL (Fig. 5B). A greater dose (50 mg/mL) was 6A). Given this Ifnar1-dependent difference in responses to required to detect modest inhibitory effect of either of these sIFN-I and the fact that YUMM cells were poorly sensitive to IFN1 agents on growth of A549, HT-29 human cancer cells, growth inhibition by sIFN-I in vitro (Fig. 5B and C), these results and MC38 mouse cancer cell line. Under these conditions, suggest that sIFN-I can suppress tumor growth through affect- sIFN-I was slightly more efficient than IFNa-2b, while growth ing tumor stromal compartment. of some of human (SPC-A4) or mouse (YUMM) cancer cell Consistent with this possibility, compared with untreated lines in vitro was not inhibited by IFN1 even at 50 mg/mL (Fig. animals or treated Ifnar1 / mice, tumors from sIFN-I–treated þ þ 5C). Given that IFN1 can act on tumor vascularization and Ifnar1 / mice contained fewer blood vessels and were less antitumor immunity (45), it is plausible that these indirect positive for endothelial marker CD31 (Fig. 6B and C). Further- þ þ mechanisms may contribute to potent antitumorigenic effects more, these tumors contained a greater number of CD3 CD8 of sIFN-I observed in vivo. cytotoxic lymphocytes (Fig. 6B and C). These results support a notion that sIFN acts on tumor stromal compartment and may impede tumor growth via inhibiting tumor angiogenesis and sIFN-I suppresses angiogenesis and stimulates antitumor þ þ immunity increasing tumor infiltration by CD3 CD8 cytotoxic lympho- Treatment of C57BL/6 mice bearing a syngeneic B16F10 mel- cytes (indicative of reversing tumor immunosuppression) in an anoma with sIFN-I but not IFNa-2b resulted in suppression of IFNAR1-dependent manner. tumor growth (Supplementary Fig. S6A). sIFN-I also suppressed Having observed a robust therapeutic effect of sIFN-I in trans- tumor growth in mice burdened with syngeneic colorectal planted tumors, we sought to determine whether this agent can (MC38) or lung (LLC) adenocarcinomas (Supplementary Fig. also be active in genetically engineered models. To this end, we V600E/þ D/D S6B and S6C). These results suggest that sIFN-I can elicit its induced melanoma tumors in Braf ; Pten mice and antitumorigenic activities in immunocompetent hosts. started the treatment after establishing tumors with the average 3 To further understand the antitumor effects of sIFN-I on tumor size of 51 mm in both groups. Administration of sIFN-I notably host, we tested its action in immunocompetent C57BL/6 mice suppressed growth of these tumors (Fig. 6D). When all control inoculated with syngeneic murine melanoma cell line YUMM mice receiving vehicle had to be sacrificed for humane reasons þ (BrafV600E/ /PtenD/D/Cdkn2aD/D). Administration of sIFN-I into (i.e., tumor size reaching the limit required by IACUC), animals þ þ tumor-bearing Ifnar1 / mice led to a dramatic suppression receiving sIFN-I exhibited either stable disease or partial/complete of growth of transplanted tumor. Importantly, when Ifnar1 tumor regression (Fig. 6D and E).

Figure 1. sIFN-I exibits altered binding affinities toward the receptor subunits compared with IFNa-2b. A, Protein sequence and structure comparison between sIFN-I (red) and IFNa-2b (green). Top, amino acid sequence alignment between IFNa-2b and sIFN-I; bottom, the secondary structures on monomer including side view (left) and vertical view (right). Each monomer consists of 6 main segments of the helices (A–F) and the connecting peptide segments. Broken ellipses represent the AB or BC loop. B, Comparison of the dissociation constants for sIFN-I (black) and IFNa-2b (blue) to immobilized IFNAR1-EC. The constants were determined by steady-analysis model. C, Binding curves of IFNa-2b or sIFN-I to immobilized IFNAR2-EC. The constants were determined by dynamic-analysis model. D, Quantification of the binding affinities toward the two receptor subunits between sIFN-I and IFNa-2b.

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Figure 3. sIFN-I elicits its signaling in an IFNAR1/2-dependent manner. A, Human fibrosarcoma 2fTGH cells (sensitive to IFN1) and derivative clones deficient in either JAK1 (U4A) or IFNAR2 (U5A) were treated with human IFNb, IFNa-2b, or sIFN-I (10 ng/mL). The phosphorylation and total signal of STATs were detected by Western blot analysis after 15-minute treatment. B, The induction of indicated IFN-stimulated genes in cells described in A was detected by qPCR after 24-hour treatment. n.s., not significant. C, WISH cells with stable IFNAR1 knockdown expression were treated with human IFNa-2b or sIFN-I (10 ng/mL) or mouse IFNb (negative control) for 15 minutes. The phosphorylation of STAT proteins was detected by immunoblot. D, Cells described in C were treated with indicated IFN for 24 hours, and the induction of TRAIL mRNA was detected by qPCR. E, A549 cells harboring various IFNAR1 status expression were treated with indicated IFNs (10 ng/mL) for 30 minutes. The phosphorylation of STAT1 and ERK was detected by immunoblot. F, Lymphocytes (lym), splenocytes (spl), and bone marrow– derived macrophages (BMM) were obtained from Ifnar1 knockout (Ifnar1/) or wild-type (Ifnar1þ/þ) mice. Similar numbers (6–10 106) of these primary cells were cultured and treated with murine IFNb (10 ng/mL), hIFNa-2b (1 mg/mL), or sIFN-I (1 mg/mL) for 24 hours, and then the induction of Irf7 and Isg15 was quantified by qPCR. , P < 0.05; , P < 0.01; , P < 0.001, versus mock group; #, P < 0.05; ##, P < 0.01; ###, P < 0.001, versus IFNa-2b group.

In this model, sIFN-I did not noticeably affect infiltration of Discussion tumors with CD31-positive cells. However, consistent with Endogenous IFN1 plays an important role in protection against tumor regression, we observed significant increase of infiltrat- tumors due to their antiproliferative, antiangiogenic, and immu- ing cytotoxic lymphocytes in tumors treated with sIFN-I in nostimulating activities (2). The response rate and therapeutic genetically engineered mouse melanomas (Fig. 6F and Supple- efficacy of IFN1-based pharmaceutical agents is limited, especially mentaryFig.S7).Furthermore,sIFN-Inotablysuppressed in solid tumors (4, 6) because oncogene signaling, tumor micro- metastases of genetically engineered melanoma into the lymph environment stress, unfolded protein response, and inflamma- nodes (Fig. 6G). These results strongly suggest that sIFN-I tion can decrease the levels of IFNAR1 available for ligand exhibits a potent antitumorigenic effect against primary tumors interaction (19–21, 46, 47). Besides developing means to reverse and metastatic disease.

2044 Clin Cancer Res; 23(8) April 15, 2017 Clinical Cancer Research

Antitumor Effects of Recombinant Interferon sIFN-I

Figure 4. sIFN-I displays different pharmacokinetics and distinct tissue responsiveness in vivo. A, Calibration curve of human IFNa ELISA used for pharmacokinetic assay. B, C57BL/6 wild-type mice were subjected to intraperitoneal injection in each group (n ¼ 3) with hIFNa-2b or sIFN-I. Serum was obtained at the indicated time point, and the concentrations of serum IFNs were detected by ELISA assay. Comparison of the pharmacokinetic curves of hIFNa-2b or sIFN-I administered as in A. Additional information is provided in Supplementary Table S3. C, C57BL/6 wild-type mice were intraperitoneally injected with murine IFNb (1 mg/mL), human IFNa-2b (1 mg/mL), or sIFN-I (1 mg/mL), respectively. n.s., not significant. Primary tissues were collected for gene expression detection after 6-hour treatment. The induction of Irf7 and Isg15 mRNA in lymph nodes was quantified by qPCR. D, Analysis of Irf7 and Isg15 mRNA was quantified by qPCR in spleen tissues of mice described in C. E, Analysis of Irf7 and Isg15 mRNA was quantified by qPCR in intestinal epithelial tissues of mice described in C. F, Analysis of Irf7 and Isg15 mRNA was quantified by qPCR in liver tissues of mice described in C. , P < 0.05; , P < 0.01; , P < 0.001, versus mock group; #, P < 0.05; ##, P < 0.01; ###, P < 0.001, versus IFNa-2b group.

downregulation of IFNAR1, additional solutions for optimizing sIFN-I elicits notable activation of STAT proteins and ensuing IFN1 therapy can be based on the observation that antitumori- inductionofISGs(Figs.2and3F);importantly,alltheseeffects genic efficacy of diverse IFN1 subtypes parallels affinity of these of sIFN-I depend on integrity of the IFNAR1/IFNAR2–JAK types for IFNAR1 (48, 49). Here, we describe sIFN-I, a novel pathway (Fig. 3). Furthermore, tumor-bearing mice lacking recombinant IFN1 exhibiting increased affinity for IFNAR1 and Ifnar1 are poorly responsive to antitumorigenic activities of potent antitumorigenic properties. sIFN-I (Fig. 6). These results suggest that despite (or because of) Intriguingly, although it tightly binds to IFNAR1, sIFN-I exhi- potentially altered ligand–IFNAR1–IFNAR2 complex, sIFN-I bits a lesser affinity for IFNAR2 (normally a chain with greater robustly activates this receptor and downstream IFN1 signaling affinity for endogenous ligands; ref. 48) compared with its pathway. "parental" molecule IFNa-2b (Fig. 1), which is different from Remarkably, compared with human IFNa-2b, the effects of the other reported IFN variants, such as IFN-YNS and IFN-YNS- sIFN-I appear to transcend the species differences. Data presented a8tail (24). The latter variants displayed enhanced ligand binding here reveal that sIFN-I elicits the IFN1-stimulated gene induction affinity to both IFNAR1/2, and also showed enhanced anti-pro- responses in primary mouse cells and mice in vivo (Figs. 4 and 5). liferation activity for cancer cells in vitro (28). Whereas in vitro Furthermore, in terms of pharmacokinetics in mouse, sIFN-I activities of sIFN-I are relatively underwhelming, sIFN-I exerts its exhibited longer half-life and lower peak drug concentration in potent antitumor effect in vivo (Figs. 5A and 6 and Supplementary serum compared with IFNa-2b (Fig. 4). Intriguingly, there was a Fig. S4A). two-step serum increase for sIFN-I; this phenomenon was not

www.aacrjournals.org Clin Cancer Res; 23(8) April 15, 2017 2045

Zhang et al.

Figure 5. sIFN-I exhibits potent antisolid tumor effects in xenotransplanted tumor models. A, A549 or HT-29 xenograft tumors were treated with intratumoral injection of sIFN-I or IFNa-2b (5 mg/kg) every other day for the indicated days; in the HT-29 model, 5 mg/kg mitomycin (MMC) treatment as a positive control. n.s., not signiﬁcant. The tumor volume was measured and calculated as follows: tumor volume (mm3) ¼ (length width2)/2. B and C, Indicated cancer cell lines (human lung cancer cells SPC-A4, A549, human colon adenocarcinoma cell HT-29, murine colorectal cancer cell line MC38, and murine primary melanoma cell line YUMM) and WISH cells were treated with 1 mg/mL (B)or50mg/mL (C) IFNs for 4 days. Cell viability and proliferation were assessed by WST1 assay. , P < 0.05; , P < 0.01; , P < 0.001, versus mock group; #, P < 0.05; ###, P < 0.001, versus IFNa-2b group.

observed for IFNa-2b injected into mice. These differences could the pharmacodynamic activity of IFN) observed in blood after be attributed to the different binding model for sIFN-I toward sIFN-I subcutaneous injection for the healthy volunteers (51). plasma protein or lipoprotein in blood, which lead to re-release of Altered pharmacokinetic characteristics of sIFN-I may contribute sIFN-I from the sIFN-I/plasma protein or sIFN-I/lipoprotein to greater ISG induction and improved antitumorigenic activities dynamic binding complex (50). Such possibility would be con- in vivo (Fig. 5) and, furthermore, may potentially cause lesser side sistent with two peaks in concentration–time curve for serum effects. These possibilities in humans will be revealed by clinical concentration of 205-OAS (a well-known downstream marker of trials of sIFN-I in Singapore (CTC1300056) and United States

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Antitumor Effects of Recombinant Interferon sIFN-I

Figure 6. Antitumorigenic, antiangiogenic, and immunostimulating effects of sIFN-I in immunocompetent mouse models. A, YUMM (BrafV600E/þ; PtenD/D; CDKN2A/) cells þ þ were injected subcutaneously into Ifnar1 / and Ifnar1 / mice to establish transplantable tumor model. sIFN-I or IFNa-2b (5 mg/kg) was injected intraperitoneally every other day for the indicated days. The tumor volume was measured and calculated. B, H&E and immunofluorescence staining of YUMM tumors isolated þ from mice after sIFN-I treatment. Arrows, vessels in tumor tissue. Scale bar, 100 mm. C, Quantification of the positive CD31 vessel number in the fields (n ¼ 7) and CD3þCD8þ T cells infiltrated in YUMM allograft tumor microenvironment (n ¼ 10). D, Melanocyte-specific Cre activity was induced in adult mice (BrafCA/þPtenf/f) by topical application of 4-HT to shaved back skin. Melanoma growth was measured after intraperitoneal injection with sIFN-I every other day. E, Volume of melanoma tumors that grew in BrafV600E/þ; PtenD/D mice to initial volume ("," blue circles). After that, mice were randomly assigned to two groups treated with PBS (black squares, left mouse in the inset) or sIFN-I (red triangles, right mouse at inset) for 32 days. P < 0.001 between PBS and sIFN-I group. F, Immunofluorescence staining of the tumor isolated from BrafV600E/þ; PtenD/D mice after sIFN-I treatment. Bottom, quantification of the average positive CD31þ vessel number in the fields (n ¼ 10) and the double positive CD3þCD8þ cells in the fields (n ¼ 10) presenting infiltrated effector T cells in tumors from BrafV600E; PtenD/D mice. þ Scale bar, 100 mm. G, H&E staining of the tumors and superficial lymph nodes (n ¼ 12) isolated from BrafV600E/ ; PtenD/D mice after sIFN-I treatment. Bottom, quantification on the number of metastatic tumors in lymph node (LN). Scale bar, 100 mm. www.aacrjournals.org Clin Cancer Res; 23(8) April 15, 2017 2047

Zhang et al.

(NCT02464007) that are currently conducted in patients with Acquisition of data (provided animals, acquired and managed patients, solid tumors. provided facilities, etc.): K.-J. Zhang, X.-F. Yin, Y.-Q. Yang, J. Xiao, Previous published data suggested that sIFN-I can suppress the K.V. Katlinski, Y.V. Katlinskaya, G.-W. Wei, D.-C. Wang, X.-Y. Liu Analysis and interpretation of data (e.g., statistical analysis, biostatistics, tumor growth in some isolated clinical cases in human patients computational analysis): K.-J. Zhang, X.-F. Yin, X.-J. Liu, K.V. Katlinski, (52). Our current data demonstrate greater efficacy of sIFN-I over D.-C. Wang, X.-Y. Liu, S.Y. Fuchs IFNa-2b against human tumors xenotransplanted into immuno- Writing, review, and/or revision of the manuscript: K.-J. Zhang, X.-F. Yin, compromised mice (Fig. 5A). Given a robust response of mouse L. Chu, R.-B. Guo, G.-W. Wei, D.-C. Wang, X.-Y. Liu, S.Y. Fuchs tissues to sIFN-I, this response may at least in part be attributed to Administrative, technical, or material support (i.e., reporting or organizing the effects of sIFN-I on mouse stromal cells. Indeed, in immu- data, constructing databases): K.-J. Zhang, Y.-N. Xu, L.-Y. Chen, X.-J. Liu, S.-J. Yuan, X.-L. Fang, S. Wu, H.-N. Xu, G.-W. Wei, D.-C. Wang, X.-Y. Liu, nocompetent syngeneic transplantation or genetically engineered S.Y. Fuchs mouse melanoma models, sIFN-I notably suppressed angiogen- Study supervision: K.-J. Zhang, X.-Y. Liu, S.Y. Fuchs esis and/or increased tumor infiltration with cytotoxic lymphocytes. These antiangiogenic and immunostimulatory effects of Acknowledgments sIFN-I are likely to contribute to robust antitumorigenic efficacy of We are grateful to Drs. McMahon (UCSF), Bosenberg (Yale University), Jiang sIFN-I that elicit stable disease or/and tumor regression in very (Peking University), Melissa Wong (Oregon Health and Science University), aggressive melanoma tumors (Fig. 6). Detailed studies of the and Stark (Cleveland Clinics) for sharing the reagents and to members of Fuchs and Liu labs for insightful comments. mechanisms underlying immunostimulatory and other effects of sIFN-I are ongoing. These studies will be instrumental in design- Grant Support ing clinical trials in humans that will address clinical efficacy of This work was supported by NIH/NCI grant CA092900(to S.Y. Fuchs), sIFN-I alone or in combination with traditional, molecularly Sichuan Science and Technology project 2013ZZ0004 (to K.-J. Zhang), Shang- targeted or immune-targeted therapies. hai Institutes for Biological Science, Chinese Academy of Sciences, and Sichuan Huiyang Life Science and Technology Corp. research program Y363S21763 (to Disclosure of Potential Conflicts of Interest X.-Y. Liu), National Basic Research Program of China 973 Program, no. 2011CB510104 (to X.-Y. Liu), Zhejiang Sci-Tech University grant 1204807-Y G.-W. Wei holds ownership interest (including patents) in Sichuan Huiyang (to X.-Y. Liu), Chinese Ministry of Science and Technology fund 2014CB964704 Life Science & Technology Corp. X.-J. Liu is a consultant/advisory board member (to X.-Y. Liu), and grant from the Sino-American joint laboratory between for Sichuan Huiyang Life Science & Technology Corp. No potential conflicts of Conba Group and Zhejiang Sci-Tech University(to X.-Y. Liu). interest were disclosed by the other authors. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in Authors' Contributions accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Conception and design: K.-J. Zhang, X.-Y. Liu, S.Y. Fuchs Development of methodology: K.-J. Zhang, H.-L. Li, X.-L. Fang, Y.V. Katlins- Received June 1, 2016; revised August 28, 2016; accepted September 11, 2016; kaya, G.-W. Wei, D.-C. Wang, X.-Y. Liu published OnlineFirst September 28, 2016.

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www.aacrjournals.org Clin Cancer Res; 23(8) April 15, 2017 2049

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

Kang-Jian Zhang, Xiao-Fei Yin, Yuan-Qin Yang, et al.

Clin Cancer Res 2017;23:2038-2049. Published OnlineFirst September 28, 2016.

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