Poly-IC Enhances the Effectiveness of Cancer Immunotherapy By
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Open access Original research J Immunother Cancer: first published as 10.1136/jitc-2020-001224 on 21 September 2020. Downloaded from Poly- IC enhances the effectiveness of cancer immunotherapy by promoting T cell tumor infiltration Hussein Sultan,1 Juan Wu,1 Valentyna I Fesenkova,1 Aaron E Fan,1 Diane Addis,1 2 1 Andres M Salazar, Esteban Celis To cite: Sultan H, Wu J, ABSTRACT cell therapy (ACT).1 2 However, there remain Fesenkova VI, et al. Poly- IC Background Immunotherapies, such as immune several caveats that limit the applicability of enhances the effectiveness checkpoint inhibitors and adoptive cell therapies, have these forms of cancer immunotherapy to most of cancer immunotherapy revolutionized cancer treatment and resulted in complete by promoting T cell tumor patients. The efficacy of the most promising and durable responses in some patients. Unfortunately, infiltration. Journal for ICI, programmed cell death 1 (PD-1)/pro- ImmunoTherapy of Cancer most immunotherapy treated patients still fail to respond. Absence of T cell infiltration to the tumor site is one of grammed cell death ligand 1 (PD- L1), relies 2020;8:e001224. doi:10.1136/ on an existing pool of tumor-reactive/tumor - jitc-2020-001224 the major obstacles limiting immunotherapy efficacy against solid tumors. Thus, the development of strategies infiltrating T cells (TILs), which is not a frequent occurrence. ACT requires either ► Additional material is that enhance T cell infiltration and broaden the antitumor published online only. To view, efficacy of immunotherapies is greatly needed. the isolation and expansion in tissue culture please visit the journal online Methods We used mouse tumor models, genetically of TILs, which is only feasible and successful (http:// dx. doi. org/ 10. 1136/ jitc- deficient mice and vascular endothelial cells (VECs) to in rare occasions or in the generation of 2020- 001224). study the requirements for T cell infiltration into tumors. genetically modified T cells expressing T cell Results A specific formulation of poly-IC, containing antigen receptors or chimeric antigen recep- HS and JW contributed equally. poly-lysine and carboxymethylcellulose (PICLC) facilitated tors a task that is technically challenging. The the traffic and infiltration of effector CD8 T cells into Accepted 18 August 2020 the tumors that reduced tumor growth. Surprisingly, expansion of TILs and genetically modified intratumoral injection of PICLC was significantly less T cells to large cell numbers is also laborious effective in inducing tumor T cell infiltration and and expensive. Thus, there is an urgent need controlling growth of tumors as compared with systemic to develop alternative, cost-effective and (intravenous or intramuscular) administration. Systemically broadly applicable cancer immunotherapies. administered PICLC, but not poly-IC stimulated tumor VECs Our laboratory has worked for many years via the double- stranded RNA cytoplasmic sensor MDA5, in the identification of CD8 and CD4 T cell http://jitc.bmj.com/ resulting in enhanced adhesion molecule expression epitopes from tumor antigens (TAgs) and and the production of type I interferon (IFN-I) and T cell the development of synthetic peptide-based recruiting chemokines. Expression of IFNαβ receptor 3 4 in VECs was necessary to obtain the antitumor effects vaccines comprising these epitopes. Using by PICLC and IFN- I was found to directly stimulate mouse tumor models, we developed peptide the secretion of T cell recruiting chemokines by VECs vaccination strategies capable of rapidly on September 26, 2021 by guest. Protected copyright. indicating that this cytokine- chemokine regulatory axis generating vast numbers of tumor- reactive is crucial for recruiting effector T cells into the tumor CD8 T cells, similar to those observed during parenchyma. Unexpectedly, these effects of PICLC were viral infections, where >10% of all CD8 T cells mostly observed in tumors and not in normal tissues. are specific for the immunogen.5 6 However, Conclusions These findings have strong implications as with ICIs and TILs, these vaccines in many © Author(s) (or their for the improvement of all types of T cell-based instances have limited success in eradicating employer(s)) 2020. Re- use immunotherapies for solid cancers. We predict that permitted under CC BY-NC. No systemic administration of PICLC will improve immune large established tumors. Paradoxically, subop- commercial re- use. See rights checkpoint inhibitor therapy, adoptive cell therapies and timal therapeutic effects of these vaccines in and permissions. Published by advanced tumor models are observed even BMJ. therapeutic cancer vaccines. though high numbers of functional TAg- 1Georgia Cancer Center, Augusta University Medical College of specific CD8 T cells are present in lymphoid Georgia, Augusta, Georgia, USA INTRODUCTION tissues (spleen, bone marrow (BM), blood), 2Oncovir, Inc, Washington, Over the past decade, there has been a suggesting that the lack of tumor control/ District Columbia, USA remarkable resurgence in the field of cancer rejection could be due to a lack of trafficking Correspondence to immunotherapy sparked by great clinical and infiltration of the T cells to the tumor Dr Esteban Celis; results obtained with immune checkpoint parenchyma. In addition, various sources of ecelis@ augusta. edu inhibitors (ICIs) and with T cell adoptive immunosuppressive activities abound in the Sultan H, et al. J Immunother Cancer 2020;8:e001224. doi:10.1136/jitc-2020-001224 1 Open access J Immunother Cancer: first published as 10.1136/jitc-2020-001224 on 21 September 2020. Downloaded from TME, which neutralize the effector function of the few Human umbilical vein endothelial cells (HUVECs) were T cells that manage to infiltrate the tumor parenchyma. obtained from Lonza. The mouse endothelial cell lines Indeed, absence of T cell infiltration to the tumor site from brain (bEnd.3) and heart (H5V) were from Amer- has been proposed as one of the major obstacles that ican Type Culture Collection. limits ICI and TIL immunotherapy efficacy against solid tumors.7–10 Reagents and antibodies T cell trafficking and infiltration to tissues where PICLC (Hiltonol) is a clinical grade formulation of poly- IC they are needed such as tumors is a complex multistep stabilized with poly- L- lysine and carboxymethylcellulose process, which involves the expression of adhesion mole- manufactured by Oncovir. High molecular weight (hmw) cules and corresponding ligands by vascular endothelial poly- IC and poly- AU were purchased from InvivoGen. cells (VECs) and activated T cells and the production of Poly- IC/PEI was prepared by mixing hmw poly-IC with T cell recruiting chemokines by the tissue cells in need in vivo- JetPEI (Polyplus) at a nucleotide:PEI ratio of 6 of effector cells.11–14 Although this process readily occurs in a 5% glucose solution. Antimouse PD- L1 monoclonal during most acute infections due to the interaction of antibody (mAb) (clone, 10F.9G2) was from BioXcell. pathogen- associated molecular patterns (PAMPs) with Mouse recombinant Interferon-beta and human recom- pattern recognition receptors (PRRs), clearly it is not binant Interferon-α2 were purchased from BioLegend. effective in the case of cold tumors, where T cell infil- CXCL10 ELISA kits were purchased from R&D Systems. trates are absent and a relevant PAMP may not be present. EliSpot plates were from eBioSciences. Dispase I (neutral We report here that systemic administration of a synthetic protease, grade I) was purchased from Sigma-Aldrich. PAMP mimic consisting of polyinosinic:polycytidylic acid Fluorescence- conjugated Abs for flow cytometry were (poly- IC) formulated with poly-lysine and carboxymethyl- from Biolegend or eBioscience. cellulose (PICLC), resulted in the enhancement of T cell infiltrates into mouse solid tumors, which correlated with Therapeutic protocols and evaluation of antitumor effects a substantial therapeutic effect. In addition, we describe Mice were injected subcutaneously with 5×105 LLC, or some of cellular and molecular mechanisms involved in 3×105 B16F10 cells in a shaved rear flank. Seven days the antitumor effects of PICLC. (B16F10 tumor model), or 9 days (LLC tumor model) later, poly- IC, PICLC or JETPEI/poly- IC were adminis- tered intravenously, intramuscularly or intratumorally at MATERIALS AND METHODS 50 µg/dose. The administration of PICLC was repeated 3 Mice and cell lines times, 5 days apart. Anti- PD- L1 mAb (200 µg/dose) was Female C57BL/6 (B6) mice aged 6–8 weeks were from administered intraperitoneally on days 1 and 3 after each the National Cancer Institute/Charles River Program PICLC administration. Tumor growth was monitored (Wilmington, Massachusetts, USA). MDA5 KO, TLR3 every 2–3 days in individually tagged mice by measuring KO and mice expressing diphtheria toxin receptor two opposing diameters with a set of calipers. Mice were 2 (DTR) under CD11c promoter (CD11cDTR) were euthanized when the tumor area reached 400 mm . http://jitc.bmj.com/ from Jackson Laboratory. Interferon (IFN)αβ receptor- Results are presented as the mean tumor size (area in deficient (IFNαβR KO) mice were originally provided by mm2±SD for every treatment group at various time points P. Marrack (National Jewish Health, Denver, Colorado, until the termination of the experiment. USA) and bred in our facility. Mice deficient of IFNαβR in endothelial cells were produced by crossing Tie2- Cre Tissue digestions and cell composition analysis (Tek- Cre, Jackson Stock No: 004128)