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PD-1 Blockade and CD27 Stimulation Activate Distinct Transcriptional Programs That Synergize for Cd8þ T-Cell–Driven Antitumor Immunity Sarah L

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PD-1 Blockade and CD27 Stimulation Activate Distinct Transcriptional Programs That Synergize for Cd8þ T-Cell–Driven Antitumor Immunity Sarah L Published OnlineFirst March 7, 2018; DOI: 10.1158/1078-0432.CCR-17-3057 Cancer Therapy: Preclinical Clinical Cancer Research PD-1 Blockade and CD27 Stimulation Activate Distinct Transcriptional Programs That Synergize for CD8þ T-Cell–Driven Antitumor Immunity Sarah L. Buchan1, Mohannad Fallatah1, Stephen M. Thirdborough2, Vadim Y. Taraban1, Anne Rogel1, Lawrence J.Thomas3, Christine A. Penfold1, Li-Zhen He3, Michael A. Curran4, Tibor Keler3, and Aymen Al-Shamkhani1 Abstract Purpose: PD-1 checkpoint blockade has revolutionized the bination therapy triggered a convergent program largely driven by field of cancer immunotherapy, yet the frequency of responding IL2 and Myc. However, division of labor was also apparent such patients is limited by inadequate T-cell priming secondary to a that anti–PD-1/L1 activates a cytotoxicity–gene expression pro- paucity of activatory dendritic cells (DC). DC signals can be gram whereas anti-CD27 preferentially augments proliferation. In þ bypassed by CD27 agonists, and we therefore investigated if the tumor models, either dependent on endogenous CD8 T cells or effectiveness of anti–PD-1/L1 could be improved by combining adoptive transfer of transgenic T cells, anti-CD27 mAb synergized with agonist anti-CD27 monoclonal antibodies (mAb). with PD-1/L1 blockade for antitumor immunity. Finally, we show Experimental Design: The efficacy of PD-1/L1 blockade or that a clinically relevant anti-human CD27 mAb, varlilumab, agonist anti-CD27 mAb was compared with a dual-therapy similarly synergizes with PD-L1 blockade for protection against approach in multiple tumor models. Global transcriptional pro- lymphoma in human–CD27 transgenic mice. filing and flow cytometry analysis were used to delineate mechan- Conclusions: Our findings suggest that suboptimal T-cell isms underpinning the observed synergy. invigoration in cancer patients undergoing treatment with PD-1 Results: PD-1/PD-L1 blockade and agonist anti-CD27 mAb checkpoint blockers will be improved by dual PD-1 blockade þ synergize for increased CD8 T-cell expansion and effector func- and CD27 agonism and provide mechanistic insight into how þ tion, exemplified by enhanced IFNg, TNFa, granzyme B, and T- these approaches cooperate for CD8 T-cell activation. Clin þ bet. Transcriptome analysis of CD8 T cells revealed that com- Cancer Res; 24(10); 2383–94. Ó2018 AACR. Introduction well-studied inhibitory receptor triggered by binding to PD-L1 þ or PD-L2, and which counters T-cell activation through a While it is clear that activated CD8 T cells can be harnessed mechanism dependent on recruitment of the protein tyrosine to kill tumor cells, T-cell activation is a complex, multifaceted phosphatase SHP-2 to an immunoreceptor-based tyrosine process, tightly controlled by activatory and inhibitory recep- switch motif (ITSM) in its cytoplasmic tail. Subsequent signal- tors. To effectively utilize T cells for immunotherapy, it is ingeventsacttorepressTCR-driven signals, inhibit the PI3K, therefore necessary to understand how these signals can be Ras, and ERK signaling pathways, limit T-cell/dendritic cell exploited to support appropriate T-cell activation. PD-1 is a (DC)-dwell time, and promote expression of the inhibitory transcription factor BATF (1). More recently, Hui and collea- gues (2) showed that CD28 is the primary target of PD-1– 1Cancer Sciences Unit, Faculty of Medicine, University of Southampton, South- recruited SHP-2, suggesting that PD-1 inhibits T-cell function ampton, United Kingdom. 2Cancer Research UK Centre, University of South- 3 through inactivation of CD28 signaling. The transient upregu- ampton, Southampton, United Kingdom. Celldex Therapeutics Inc., Hampton, lation of PD-1 during T-cell activation and its maintenance at New Jersey. 4Department of Immunology, The University of Texas MD Anderson Cancer Center, Houston, Texas. high levels on chronically stimulated (exhausted) T cells enable PD-1 to negatively regulate T-cell function during priming, Note: Supplementary data for this article are available at Clinical Cancer secondary activation, and in conditions of prolonged antigen Research Online (http://clincancerres.aacrjournals.org/). exposure (1, 3, 4). Furthermore, PD-1 is expressed on a large S.L. Buchan and M. Fallatah contributed equally to this article. proportion of tumor-infiltrating T cells, yet even within this Current address for V.Y. Taraban: School of Sport, Health and Social Science, population, PD-1 expression is skewed toward the tumor- Southampton Solent University, Southampton, United Kingdom. reactive, and presumably chronically stimulated, subpopula- Corresponding Author: Aymen Al-Shamkhani, University of Southampton, tion (1, 5). Southampton General Hospital, Tremona Road, Southampton SO16 6YD, United Encouragingly, countering PD-1 with PD-1– or PD-L1– Kingdom. Phone: 44-23-80796285; Fax: 44-23-80704061; specific monoclonal antibodies (mAb) enables functional E-mail: [email protected] þ CD8 T cells to accumulate, leading to tumor rejection in doi: 10.1158/1078-0432.CCR-17-3057 preclinical models and dramatically improved outcomes Ó2018 American Association for Cancer Research. across a range of cancer types in patients. Despite these www.aacrjournals.org 2383 Downloaded from clincancerres.aacrjournals.org on October 1, 2021. © 2018 American Association for Cancer Research. Published OnlineFirst March 7, 2018; DOI: 10.1158/1078-0432.CCR-17-3057 Buchan et al. tors in regulating T-cell activation and provide new impetus for Translational Relevance evaluating combined agonist anti-CD27 and PD-1/L1 blockade in þ Antitumor effector CD8 T cells are often suppressed by patients. PD-1 signaling. Although blocking antibodies to PD-1 and PD-L1 have shown impressive results in multiple tumor Materials and Methods types, only a minority of patients respond, and recent Cell lines and reagents findings suggest that stimulatory signals for T-cell activation The cell lines B16-OVA-GFP, B16-BL6, FVAX, and C1498 may be lacking in some patients. We here show that agonist have all been described previously (18–20) and were main- antibodies targeting mouse or human CD27, a key costi- tained in Dulbecco's modified Eagles Medium containing 10% mulatory receptor, act synergistically with anti–PD-1/L1 to þ FCS, pyruvate, L-glutamine, and antibiotics. Stocks of cells were increase CD8 T-cell proliferation, effector function, and maintained in liquid nitrogen. Defrosted cells were discarded tumor clearance in wild-type and human–CD27 transgenic within 3 months and regularly visualized for any changes in mice. Detailed mechanistic analysis indicates cooperative, morphology or growth rate. Cell lines were routinely tested for yet also independent, influences of these antibodies on mycoplasma contamination using endosafe-PTS cartridges aspects of T-cell activation. Thus, our data reveal synergy (Charles River Laboratories). BCL1 tumor was maintained by between anti–PD-1/L1 and anti-CD27 for immunotherapy passage in BALB/c mice as described (21). Endotoxin-low and provide renewed impetus for clinical evaluation of peptides hgp100 – (KVPRNQDWL), OVA – (SIIN- combined anti-CD27 and anti–PD-1/L1. 25 33 257 264 FEKL),andthealteredpeptideligandSIIQFEKLwereobtained from Peptide Protein Research Ltd. Cyclophosphamide and FTY720 were obtained from Sigma-Aldrich, and Cell Prolifer- ation Dye eFluor450 and Fixable Viability Dye eFluor506 were from eBioscience. PE-labeled SIINFEKL peptide/H-2Kb tetra- unprecedented clinical successes, response rates remain mod- mers were manufactured in house (Protein Core Facility). est and rarely exceed 40% (6). Reasons for the observed Antibodies used in vivo were rat anti-mouse CD27 (AT124; rat patient variability to PD-1/L1 blockade are doubtless multi- þ IgG2a), anti-OX40 (OX-86), anti-GITR (DTA-1), anti-4-1BB factorial, but the extent of CD8 T-cell infiltration is likely to (LOB12.3), anti-CD4 (GK1.5 and YTA3.1.2), anti-CD8 be an important factor in the resistance to immunotherapy (YTS169), rat IgG2a isotype control antibodies Mc106A5 or (7). Recent findings demonstrate that the lack of T-cell infil- Mc39-16 (all prepared <10 EU/mg endotoxin in house); anti- tration is the result of defective recruitment and activation of þ mouse PD-1 (RMP1-14), anti-mouse PD-L1 (10F.9G2), and rat DCs, which leads to reduced cross-priming of CD8 T cells IgG2b isotype control mAbs were obtained from Bio X Cell. (8). Based on these findings, a number of studies have shown Anti-human CD27 mAb, varlilumab, has been described pre- that the intratumoral administration of inflammatory med- viously (21). iators alone or together with DCs can result in T-cell priming and improved antitumor immunity (8). However, the broad applicability of this approach, particularly for the treatment of Mice and in vivo protocols metastatic disease, is likely to be limited. We and others have C57BL/6, BALB/c, OT-I, pmel1, and hCD27-Tg mice have previously demonstrated that DC expression of CD70, the been described previously (21, 22). Experiments were con- þ ligand for CD27, is required for efficient priming of CD8 T ducted according to UK Home Office Regulations or according cells and effective antitumor immunity in murine models (9– to the guidelines established by the Institutional Animal Care 14). Furthermore, we have shown that the requirement for DC and Use Committee (IACUC) at Celldex Therapeutics, Inc. For þ activation can be largely replaced by systemic administration adoptive CD8 T-cell transfer, single-cell suspensions of sple- of soluble recombinant CD70 or agonist anti-CD27 mAb (12, nocytes were prepared in phosphate-buffered saline (PBS) and þ þ þ 14, 15). We reasoned that the ability of CD27 agonists to 1 106 to  106 CD8 Vb13 Thy1.1 cells (pmel1) or 1  104 þ þ þ enhance CD8 T-cell priming could combine with PD-1 CD8 SIINFEKL/H-2Kb tetramer (OT-I) cells were injected þ blockade to further improve CD8 T-cell responses and anti- intravenously (i.v.). For tumor challenge, adherent cell lines tumor immunity. Indeed we have shown previously in a CD4- were treated with trypsin-EDTA, washed, and viable cells were independent model of donor lymphocyte infusion in which counted and injected into mice.
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