Open access Original research J Immunother Cancer: first published as 10.1136/jitc-2020-000764 on 26 November 2020. Downloaded from Tumors establish resistance to immunotherapy by regulating Treg recruitment via CCR4 Lisa A Marshall,1 Sachie Marubayashi,2 Aparna Jorapur,1 Scott Jacobson,1 Mikhail Zibinsky,1 Omar Robles,1 Dennis Xiaozhou Hu,3 Jeffrey J Jackson,1 Deepa Pookot,1 Jerick Sanchez,1 Martin Brovarney,1 Angela Wadsworth,1 David Chian,4 David Wustrow,1 Paul D Kassner,1 Gene Cutler,1 Brian Wong,1 1 1 Dirk G Brockstedt, Oezcan Talay To cite: Marshall LA, ABSTRACT Statement of significance CPI upregulates CCL17 and Marubayashi S, Jorapur A, et al. Background Checkpoint inhibitors (CPIs) such as anti- CCL22 expression in tumors and increases Treg migration Tumors establish resistance to PD(L)-1 and anti- CTLA-4 antibodies have resulted in into the TME. Pharmacological antagonism of the CCR4 immunotherapy by regulating unprecedented rates of antitumor responses and extension receptor effectively inhibits T recruitment and results T recruitment via CCR4. reg reg of survival of patients with a variety of cancers. But in enhanced antitumor efficacy either as single agent in Journal for ImmunoTherapy some patients fail to respond or initially respond but later CCR4 ligandhigh tumors or in combination with CPIs in of Cancer 2020;8:e000764. low doi:10.1136/jitc-2020-000764 relapse as they develop resistance to immune therapy. CCR4 ligand tumors. One of the tumor- extrinsic mechanisms for resistance to immune therapy is the accumulation of regulatory T cells ► Additional material is published online only. To view (Treg) in tumors. In preclinical and clinical studies, it has INTRODUCTION been suggested that tumor trafficking of T is mediated please visit the journal online reg In recent years, it has become clear that (http:// dx. doi. org/ 10. 1136/ jitc- by CC chemokine receptor 4 (CCR4). Over 90% of human tumors use various immunomodulatory 2020- 000764). T express CCR4 and migrate toward CCL17 and CCL22, reg pathways and recruit suppressive cells as a two major CCR4 ligands that are either high at baseline LAM and SM contributed or upregulated in tumors on CPI treatment. Hence, CCR4 major mechanism of immune resistance, equally. antagonism has the potential to be an effective antitumor particularly against tumor- specific effector T cells (T ).1 2 Cancer immunotherapy with treatment by reducing the accumulation of Treg into the eff Accepted 25 August 2020 tumor microenvironment (TME). monoclonal antibodies that effectively block Methods We developed in vitro and in vivo models PD(L)-1 and CTLA-4 are designed to enhance to assess Treg migration and antitumor efficacy using a antitumor immunity and have shown promise http://jitc.bmj.com/ potent and selective CCR4 antagonist, CCR4-351. We in the treatment of patients with solid tumors used two separate tumor models, Pan02 and CT26 mouse and hematologic malignancies.3–7 However, tumors, that have high and low CCR4 ligand expression, only a subset of patients responds to these respectively. Tumor growth inhibition as well as the checkpoint inhibitor treatments with durable frequency of tumor- infiltrating T and effector T cells was reg clinical benefit, and most patients with cancer assessed following the treatment with CCR4 antagonist fail to respond to the treatment at all or on September 29, 2021 by guest. Protected copyright. alone or in combination with CPI. 3 5 Results Using a selective and highly potent, novel small relapse. The tumor- extrinsic mechanisms of molecule inhibitor of CCR4, we demonstrate that migration either the primary or the acquired resistance © Author(s) (or their + are not fully understood, but evidence points of CCR4 Treg into the tumor drives tumor progression and employer(s)) 2020. Re- use to the accumulation of suppressor cells, such permitted under CC BY-NC. No resistance to CPI treatment. In tumor models with high commercial re- use. See rights baseline levels of CCR4 ligands, blockade of CCR4 reduced as regulatory T cells (Treg), in the tumor as 8 and permissions. Published by the number of Treg and enhanced antitumor immune one such mechanism. BMJ. activity. Notably, in tumor models with low baseline level + + Naturally suppressive CD4 Foxp3 Treg are 1RAPT Therapeutics, South San of CCR4 ligands, treatment with immune CPIs resulted in essential for immune tolerance. Although Francisco, California, USA significant increases of CCR4 ligands and regT numbers. 2 Treg- mediated suppression of effector cells is Arcus Biosciences Inc, Inhibition of CCR4 reduced T frequency and potentiated 9 reg important to maintain host self-tolerance, Hayward, California, USA the antitumor effects of CPIs. 3 the presence of T in the tumor microenvi- Genentech Inc, South San Conclusion Taken together, we demonstrate that CCR4- reg Francisco, California, USA dependent T recruitment into the tumor is an important ronment (TME) has been shown to dampen 4Lyell Immunopharma, South reg 10 11 tumor- extrinsic mechanism for immune resistance. antitumor immune responses. In fact, San Francisco, California, USA studies revealed that elevated frequencies Blockade of CCR4 led to reduced frequency of Treg and Correspondence to resulted in increased antitumor activity, supporting the of Treg at the tumor site correlate with poor Dr Dirk G Brockstedt; clinical development of CCR4 inhibitors in combination clinical outcome, raising the need for a treat- dbrockstedt@ rapt. com with CPI for the treatment of cancer. ment to reduce the number of Treg in the Marshall LA, et al. J Immunother Cancer 2020;8:e000764. doi:10.1136/jitc-2020-000764 1 Open access J Immunother Cancer: first published as 10.1136/jitc-2020-000764 on 26 November 2020. Downloaded from 12–14 tumor. The observed increased frequency of Treg cells of Medicine, Stanford University). Pan02- OVA cells were has been hypothesized to be attributed to the expansion engineered by stable transduction of Pan02 cells with of tumor- resident Treg, conversion from conventional pChac- puro plasmid expressing full-length chicken oval- + CD4 T cells, or migration of Treg from the periphery bumin (OVA; LakePharma). All cells were confirmed into the TME. Evidence indicates that Treg are actively mycoplasma negative and identity of all cell lines was vali- recruited to the TME by surface receptors that recognize dated by short- tandem repeat profiling. chemokines that are highly expressed within the TME.15 The CC chemokine receptor 4 (CCR4) is expressed on CCR4 antagonist 16 most (>90%) human Treg. In patients with various types The discovery of the selective and potent small molecule of cancers, such as ovarian, lung and breast cancer, high CCR4 inhibitor (referenced as CCR4-351 in this manu- levels of CCR4 ligands (CCL17 and CCL22) are produced script) is described elsewhere.22 This novel compound by tumor cells, tumor- associated macrophages17 and/or was designed, synthesized and characterized at RAPT dendritic cells (DCs).18 Importantly, recent preclinical Therapeutics. In several experiments, an independent and clinical studies demonstrated that the number of CCR4 antagonist highly related to CCR4-351 was used. intratumoral Treg increased in subjects after treatment The potency, selectivity and pharmacokinetic properties with immunotherapies.19 20 However, whether immuno- of this antagonist are similar to CCR4-351. therapies increase levels of CCR4 ligands in the tumor and result in the increased migration of Treg is not well Antibodies and reagents understood. Antibodies and dyes used for surface staining: unless In this study, we demonstrated that immune resistance specified, antibodies were purchased from BioLegend, in several mouse tumor models, at least in part, is estab- San Diego. Anti- CD45 BV510 (clone 30-F11), anti- CD4 lished by producing high levels of the two CCR4 ligands, APC- Cy7 (clone RM4-5), anti-CD8 FITC (clone 53-6.7), CCL17 and CCL22, leading to the recruitment of CCR4+ anti- CD69 PE (clone H1.2F3), anti- PD-1 PE- Cy7 (clone Treg into the TME. This finding is supported by gene 29F.1A12, BD Biosciences), anti-CCR4 APC (clone 2G12), expression data from various human tumors, suggesting anti- CCR4 PerCP/CY5.5 (clone L291H4), anti- CCR5 that high CCL17 and CCL22 gene expression strongly PE (clone HEK/1/85), anti- CCR6 PE (clone G034E3), correlates with increased Treg presence as assessed by anti- CCR7 PE (clone G034H7), anti- CXCR3 PE (clone FOXP3 expression.21 In contrast, there was poor correla- G025H7), anti- CD45RA BV510 (clone HI100), anti- tion between FOXP3 and other chemokine ligands or CD45RO Pacific Blue (clone UCHL1), anti-CD25 APC- transforming growth factor beta (TGF-β) expression, CY7 (clone BC96), anti-CD127 APC (clone A019D5), suggesting that Treg accumulation occurs predominantly anti- CD4 FITC (clone GK1.5) and 7AAD. Reagents and through CCR4- mediated recruitment. A selective and antibodies used for intracellular staining: cells were fixed potent small molecule CCR4 inhibitor (CCR4-351) and permeabilized following the protocol for the FoxP3/ effectively blocked migration of Treg into the tumor and Transcription Factor Staining Buffer (eBiosciences). increased single-agent antitumor efficacy in CCR4 ligand- Fixable viability dye eFluor780 (eBiosciences) and anti- http://jitc.bmj.com/ high mouse tumors. Interestingly, treatment with check- FoxP3 APC (clone FJK- 16s, eBiosciences). Sample acqui- point inhibitors such as anti- CTLA-4 antibody or other sition was performed on either a FACSCanto II or