An Antibody Targeting ICOS Increases Intratumoral Cytotoxic to Regulatory T-Cell Ratio and Induces Tumor Regression a C Richard C.A

Published OnlineFirst September 30, 2020; DOI: 10.1158/2326-6066.CIR-20-0034

CANCER IMMUNOLOGY RESEARCH | RESEARCH ARTICLE

An Antibody Targeting ICOS Increases Intratumoral Cytotoxic to Regulatory T-cell Ratio and Induces Tumor Regression A C Richard C.A. Sainson, Anil K. Thotakura, Miha Kosmac, Gwenoline Borhis, Nahida Parveen, Rachael Kimber, Joana Carvalho, Simon J. Henderson, Kerstin L. Pryke, Tracey Okell, Siobhan O’Leary, Stuart Ball, Cassie Van Krinks, Lauriane Gamand, Emma Taggart, Eleanor J. Pring, Hanif Ali, Hannah Craig, Vivian W.Y. Wong, Qi Liang, Robert J. Rowlands, Morgane Lecointre, Jamie Campbell, Ian Kirby, David Melvin, Volker Germaschewski, Elisabeth Oelmann, Sonia Quaratino, and Matthew McCourt

ABSTRACT ◥ The immunosuppressive tumor microenvironment constitutes a antibodies with human variable domains, we selected a fully human significant hurdle to immune checkpoint inhibitor responses. Both IgG1 antibody called KY1044 that bound ICOS from different soluble factors and specialized immune cells, such as regulatory T species. We showed that KY1044 induced sustained depletion of cells (Treg), are key components of active intratumoral immuno- ICOShigh T cells but was also associated with increased secretion of fl low suppression. Inducible costimulatory receptor (ICOS) can be highly proin ammatory cytokines from ICOS effector T cells (Teff). In expressed in the tumor microenvironment, especially on immuno- syngeneic mouse tumor models, KY1044 depleted ICOShigh Treg suppressive Treg, suggesting that it represents a relevant target for and increased the intratumoral TEff:Treg ratio, resulting in preferential depletion of these cells. Here, we performed immune increased secretion of IFNg and TNFa by TEff cells. KY1044 profiling of samples from tumor-bearing mice and patients with demonstrated monotherapy antitumor efficacy and improved cancer to demonstrate differential expression of ICOS in immune anti–PD-L1 efficacy. In summary, we demonstrated that using T-cell subsets in different tissues. ICOS expression was higher on KY1044, one can exploit the differential expression of ICOS on intratumoral Treg than on effector CD8 T cells. In addition, by T-cell subtypes to improve the intratumoral immune contexture immunizing an Icos knockout transgenic mouse line expressing and restore an antitumor immune response.

Introduction through various mechanisms (3, 4). Thus, high numbers of intratumoral Tregs negatively correlates with survival and response to treat- The last decade has seen a paradigm shift in cancer therapies with ments (5, 6). In fact, Treg depletion modifies the tumor microenvi- the approval of antibodies targeting immune checkpoints. These ronment (TME) and favors an antitumor response in preclinical immune checkpoint inhibitors (ICI) trigger a durable response in models (7–9). For these reasons, Treg cells have been investigated as malignancies, including metastatic melanoma, non–small cell lung a prognostic cell type and as therapeutic targets. cancer (NSCLC), head and neck cancer, renal, and bladder cancer (1). The use of therapeutic antibodies for the preferential depletion of However, there is still a high proportion of patients exhibiting resis- intratumoral Treg cells relies on the identification of a marker pref- tance to ICIs that may benefit from novel combinatory approaches. erably expressed on these cells. One such potential target is ICOS, Multiple molecular and cellular mechanisms associate with resis- which belongs to the CD28/CTLA-4 family (10). Unlike CD28, ICOS is tance to ICIs (2). For example, low incidence of cytotoxic T cells and not expressed on na€ve T cells but is induced upon T-cell receptor the presence of immunosuppressive cells prevent an antitumor Eff (TCR) engagement (11, 12). Following activation, ICOS is expressed at response. One class of immunosuppressive cells are regulatory T cells different levels on different T-cell subtypes where it can engage with its (Treg), which block the cytotoxic potential of effector T cells (T ) Eff ligand (ICOS-LG, CD275) expressed on antigen-presenting cells. ICOS/ICOS-LG interaction initiates a costimulatory signal that results in production of either pro- or anti-inflammatory cytokines (IFNg and Kymab Ltd, Babraham Research Campus, Cambridge, United Kingdom. TNFa by TEff cells; IL10 expression by Treg; ref. 13), thus regulating the immune cell homeostasis (14, 15). Of relevance, the accumulation of Note: Supplementary data for this article are available at Cancer Immunology þ Research Online (http://cancerimmunolres.aacrjournals.org/). ICOS Treg cells in the TME is associated with disease progression (16, 17). In marked contrast, the upregulation of ICOS on R.C.A. Sainson and A.K. Thotakura contributed equally to this article. CD4 TEff cells associates with better prognosis in patients treated with Current address for J. Carvalho, SNIPR BIOME, Copenhagen, Denmark; current anti–CTLA-4 (18–21). The relative expression of ICOS varies between address for J. Campbell, Abcam plc, Cambridge, United Kingdom; and current T-cell subtypes, with intratumoral Treg exhibiting higher ICOS expres- address for I. Kirby, ADC Therapeutics, London, United Kingdom. þ þ sion followed by CD4 and CD8 TEff cells (12, 17, 19, 22). This Corresponding Author: Richard C.A. Sainson, Kymab Ltd, Babraham Research differential expression suggests that ICOS represents a relevant target Institute, Bennet Building, Cambridge, Cambridgeshire CB22 3AT, UK. Phone: for a Treg depletion strategy. In fact, ICOS antibodies with depleting 44 (0)1223 833301; E-mail: [email protected] þ capability reduce the numbers of ICOS Treg cells and induce an – Cancer Immunol Res 2020;8:1568 82 antitumor response when combined with a vaccine strategy (7). doi: 10.1158/2326-6066.CIR-20-0034 In this study, using a transgenic mouse platform (23), we identified a 2020 American Association for Cancer Research. fully human ICOS IgG1 antibody called KY1044 that bound to mouse

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and human ICOS. In vitro, we established that KY1044 has costimu- were placed into C-tubes (Miltenyi Biotec, catalog no. 130-096-334) Low High latory effect on ICOS TEff cells and a depleting effect on ICOS dissociated into single-cell suspensions using gentle MACS dissocia- cells. In vivo, KY1044 induced preferential depletion of ICOSHigh cells, tor. Tumor samples were filtered through 70-mm nylon filters. Spleens fi fi improved the TEff to Treg ratio in the TME and increased secretion of samples were ltered through 40-mm nylon lters. Red blood cells were proinflammatory cytokines, resulting in antitumor efficacy. lysed using RBC lysis buffer (Sigma, catalog no. R7757). For flow cytometry profiling, 2 106 tumor samples or 1 106 spleen samples Materials and Methods were plated in 96-well plate (Sigma, catalog no. CLS3957). Cell suspensions were preincubated with Live/Dead fixable Yellow Cell lines used in the study Dead Cell Stain Kit (Invitrogen, catalog no. L34959). Prior to antibody All cell lines (except MC38) were obtained from LGC standards labeling, cells were incubated with Fc receptor blocking solution (anti- ATCC. CT26.WT (mouse colon carcinoma, ATCC catalog no. CRL- CD16/CD32 BioLegend, catalog no. 101320) at 4C for 10 minutes. 2638), B16-F10 (mouse melanoma, ATCC catalog no. CRL-6475) and The staining was performed at 4C for 30 minutes using fluoro- MJ (G11; Human T-cell lymphoma, ATCC catalog no. CRL-8294) chrome-conjugated anti-mouse antibodies. Intracellular and intra- cells were acquired between July and November 2015, J558 (mouse nuclear staining was performed using Foxp3 staining buffers (Thermo plasmacytoma, ATCC catalog no. TIB-6) cell line was obtained in May Fisher Scientific, catalog no. 00-5523-00). All flow cytometry anti- 2016. A20 (A-20; mouse B-cell lymphoma, ATCC catalog no. TIB- bodies or isotype controls were purchased from Thermo Fisher 208), and EL4 (mouse lymphoma, ATCC catalog no. TIB-39) cells Scientific. Antibodies used include: anti-CD45 (30-F11), anti-CD3 were obtained in May 2017. MC38 cells (mouse colon adenocarcino- (17A2), anti-CD4 (RM4-5), anti-CD8 (53-6.7), anti-CD25 (PC61.5), ma) were obtained in August 2017 from NCI under a license agree- anti-ICOS (7E.17G9), anti-Foxp3 (FJK-16s), anti-CD69 (H1.2F3). fi fi ment. The speci c pathogen-free status of these cells was con rmed by For the T-cell cytokine staining, single-cell suspensions were PCR screening for mouse/rat comprehensive panel (Charles River). plated at 1 106 cells per well in RPMI þ 10% FBS cell culture media MC38, J558, and B16-F10 cells were cultured in antibiotic-free DMEM with 1 Brefeldin A solution (eBioscience, catalog no. 00-4506-51) for (Gibco, catalog no. 41966-029) þ 10% FBS (Gibco, catalog no. 10270) 4 hours at 37 C5%CO2. Cells were surface stained as above and complete cell culture media. CT26.WT cells were cultured in antibi- subsequently fixed/permeabilized for intracellular staining with anti- þ otic-free RPMI (Gibco, catalog no. 2187) 10% FBS complete cell IFNg (XMG1.2) and anti-TNFa (MP6-XT22). All flow cytometry data þ culture media. A20 cells were cultured in antibiotic-free RPMI 10% was acquired using Attune FxT flow cytometer and data were analyzed þ FBS 0.05 mmol/L 2-mercaptoethanol (Gibco, catalog no. 21985- using FlowJo software V10. 023) complete cell culture media. MJ cells were culture in IMDM þ (Gibco, catalog no. 12440053) 20% FBS (I20 media). The passage Monkey study number of cells were kept below 10 generations. Blood samples (0.1 mL) were collected into sodium EDTA or Gene expression analysis lithium heparin tubes, mixed, and red blood cells (RBC) were lysed. The leukocytes were washed with FACS buffer (PBS with 2% FBS), The Cancer Genome Atlas data analysis stained with antibody cocktail by incubation in the dark for 45 minutes The RNA-sequencing data from The Cancer Genome Atlas at room temperature. Tissue samples (lymph node), taken from (TCGA) consortium was downloaded from the UCSC Xena platform animals at termination were mechanically disrupted (Medimachine, (TCGA Pan-Cancer, 10,460 samples in total; ref. 24). Samples classified Becton Dickinson GmbH), single-cell filtered, and stained as for blood. as nontumor tissue (727 samples) were excluded as were leukemias, Intracellular FoxP3 staining was carried out by permeabilization with lymphomas, and thymomas (combined 341 samples). Single-sample 10 FACS lysing solution (1:5 with Aqua dest. þ0.1% Tween-20) prior gene-set enrichment analysis (ssGSEA; refs. 25, 26) was performed for to incubation in the dark, 45 minutes at room temperature. All ICOS and FOXP3. Samples were grouped by primary disease and the antibodies or isotype controls were purchased from BD Biosciences, ssGSEA scores for each group were compared across the primary eBioscience (via Fisher Scientific GmbH) or BioLegend. Antibodies disease groups. used included: anti-CD3 (SP34), anti-CD20 (L27 or 2H7), anti- ScRNA sequencing CD14 (M5E2), anti-CD4 (M-T477), anti-CD8 (SK1), anti-CD28 Peripheral blood mononuclear cell (PBMC) and tumors from 5 (CD28.2), anti-CD25 (M-A251), anti-Foxp3 (PCH101), anti-CD95 NSCLC donors were processed using the BD Rhapsody system. (DX2), and anti-CD185/CXCR5 (MU5UBEE). After staining, cells Sequencing libraries were generated using the Immune Response were washed and fixed in 1 BD Stabilizing Fixative. Lymphocytes Human targeted panel and sequenced using a 2 75 bp paired- were gated by forward scatter (FSC), sideward scatter (SSC), and CD45. Multicolour flow cytometric analysis was performed end run on the Illumina HiSeq 4000 System. Reads were processed by þ using the following leukocyte phenotypic characteristics: CD4 Th applying the BD Rhapsody processing pipeline to generate cell count þ þ þ fi cells: CD3 /CD4 /CD8 /CD14 /CD20 ;CD8 cytotoxic T cells: matrices. The counts were ltered, normalized, and visualized using R þ þ – – CD3 /CD4 /CD8 /CD14 /CD20 ; Total memory CD4 T cells: and Bioconductor packages for scRNA-seq data (27 30). Cell type þ þ þ þ fi CD28 /DIM/ CD95 /high; Follicular Th cells: (CD4 /CD185 ), and speci c gene sets were constructed by performing a literature search þ þ and cells were classified into one of 27 cell types (Supplementary Regulatory CD4 T cells: CD25 /FoxP3 . Acquisition of flow data Table S1) using the R package AUCell (31). The sequences have been was performed on a FACSVerse flow cytometer (BD Biosciences) submitted to ArrayExpress (accession E-MTAB-9451). and relative percentages of each of these subpopulations were determined using FlowJo software. Fifty thousand events were Flow cytometry counted for all analyses. Mouse studies Tumors and spleens were harvested from CT26.WT tumor–bearing Human samples studies mice. Tumors were dissociated into single-cell suspensions using In accordance to the declaration of Helsinki and upon approval by Miltenyi Tumor Dissociation Kit (catalog no. 130-096-730). Spleens the Hamburg Medical Association's ethic commission (PV5035),

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NSCLC tissue and whole blood from the same patients were obtained overnight and the luciferase activity was measured using the Bio-Glo from consented subjects, with ethical approval for analysis of protein, Luciferase Assay System (Promega, catalog no. G7940) on the EnVi- RNA, and DNA content. The NSCLC tumor samples were dissociated sion Multilabel Plate Reader (Perkin Elmer). Graph data were nor- into single-cell suspensions using enzymatic digestion, while whole malized to background and plotted versus log10 antibody blood was processed into PBMCs using density centrifugation, all concentration. specimens were cryopreserved in Gibco Recovery cell culture freezing For the primary ADCC cell assay, human natural killer (NK) cells medium before used. Alongside NSCLC specimens, PBMCs from 5 were purified using the NK Cell Isolation Kit (StemCell Technologies, healthy individuals were used. Flow cytometry was performed after catalog no.17955). ICOS-transfected CCRF-CEM (ICOS CEM, target cells were thawed and incubated in RPMI1640 þ 10% FBS supple- cells) were preloaded with the fluorescence-enhancing ligand mented with 100 U of RNase free DNase at 37 C5%CO2 for 30 (BATDA) for 30 minutes in the dark at 37 C. The cells were loaded minutes. Cell suspensions were pre-incubated with Live/Dead fixable and washed in the presence of an inhibitor of organic anion trans- Yellow Dead Cell Stain Kit (Invitrogen, catalog no. L34959) and porters (1 mmol/L Probenecid) to avoid spontaneous dye release from Fc receptor blocking solution (BioLegend, catalog no. 422302), cells. KY1044 was diluted (1:4 dilutions, 10 points, starting from 33.3 before staining with fluorochrome-conjugated anti-human antibodies nmol/L) in assay buffer. The target ICOS CEM cells (50 mL/well), specific to anti-CD3 (UCHT1)/anti-CD45 (2D1), anti-CD4 (2A3), effector cell (50 mL/well), and reagent dilutions (50 mL/well) were – anti-CD8 (RPA-T8), anti-CD25 (MA251), anti-CD45RA (H100), cocultured with 50 mL of the diluted antibody at 37 C, 5% CO2 for 2 anti-ICOS (C398.4A). Cells were incubated for 1 hour at 4C, washed, 4 hours (NK cells to target ratio was 5:1). A digitonin-based lysis buffer and fixed overnight at 4C with eBioscience intracellular fixation (Perkin Elmer) was used to determine complete target cell lysis (100%). and permeabilization buffer (catalog no. 00-5523-00). Anti-FoxP3 (236A-E7) staining was then performed in permeabilization buffer In vitro costimulation assays for 1 hour at 4C before washing and resuspending cells in DPBS For the MJ cell assays, KY1044 human IgG1 was presented in (Gibco). All flow cytometry data was acquired using Attune NxT flow three different formats: plate-bound, soluble, or soluble with or without cytometer and data was analyzed using FlowJo software V10. F(ab')2 Fragments (Fc linker, catalog no. 109-006-170, Jackson Immu- noresearch). Plate-bound KY1044 and the hIgG1 isotype control were Immunofluorescence and digital pathology diluted 1:2 in PBS (concentrations ranging from 10 mg/mL to 40 ng/mL, An IHC protocol using the Ventana Discovery Ultra platform 10 points). One-hundred microliters of diluted antibodies were coated in (Roche) was developed for costaining of ICOS and FoxP3. The method triplicate into a 96-well, flat-bottom plate (Corning EIA/RIA plate) was optimized in FFPE tonsil tissue, using clone D1K2T for ICOS overnight at 4C and then washed. MJ cells (15,000 cells/well) were (Cell Signaling Technology) and clone 236A/E7 (Abcam) for FoxP3. added to precoated wells. For the soluble/cross-linked experiment, Briefly, the FFPE tissue sections underwent deparaffinization and a KY1044 and the isotype control were serially diluted 1:2 in I20 media pH 9-based antigen retrieval step for 64 minutes. The slides were (soluble Ab) or in I20 media containing 30 mg/mL of F(ab')2 Fragments incubated with anti-FOXP3 antibody or the isotype control at 40 mg/ (cross-linked Ab) to give an 2 Ab stock concentrations ranging from mL for 60 minutes. An anti-mouse HRP (Roche catalog no. 760-4310) 20 mg/mL to 80 ng/mL (10 points). Fifty microliters of diluted antibodies labeled detection antibody was applied for 16 minutes and an auto- were added to 96-well with 50 mL of MJ cells (3 105/mL). For the assays mated DAB detection (Roche catalog no. 760-159) was carried out as the cells were cultured for 72 hours at 37 C/5% CO2 and cell-free per manufacturer's recommendations. The slides were then incubated supernatants were then collected and used to perform IFNg ELISA using with the anti-ICOS antibody or the isotype control at 1 mg/mL for the Human IFNg DuoSet assay (R&D Systems, DY285). 60 minutes. An anti-rabbit HRP (Roche, catalog no. 760-4311) labeled For the primary T-cell assays, leukocyte cones were obtained (HTA detection antibody was applied for 28 minutes and an automated IRAS project number 100345). PBMCs were isolated by density- purple detection was carried out as per manufacturer's recommenda- gradient centrifugation. T lymphocytes were them purified using the tions. Finally, the slides were counterstained with hematoxylin for Stemcell EasySep Isolation kit (catalog no. 17951). For ICOS induc- 12 minutes and bluing reagent for 8 minutes. Methods were applied for tion, isolated T cells were cultured at 2 106/mL in R10 media (RPMI the staining of tumor microarrays (TMA) from patients with cervical 10% heat-inactivated FBS) in the presence of 20 mL/ml of Dynabeads (CR2088), esophageal (ES2082), lung (LUC2281), head and neck Human T-Activator CD3/CD28 (Life Technologies, catalog no. (HN802a), gastric (STC2281), and bladder (BL2081a) cancer (all US 111.31D). These activated primary T cells were tested as for the MJ Biomax, Inc). The slides were scanned (20 equivalent magnification) cell assays in three different formats: plate-bound (5 mg/mL), soluble fi and quanti ed using the Indica Labs Halo platform to determine the (15 mg/mL), or soluble plus F(ab')2 Fragments cross-linker. T-cell number of ICOS/FoxP3 single and double positive cells per mm2. suspension were added to antibody-containing plates to give a final cell concentration of 1 106 cells/ml and cultured for 72 hours at 37C and In vitro fi antibody-dependent cellular cytotoxicity assays 5% CO2 until IFNg ELISA quanti cation. For the three-step culture Antibody-dependent cellular cytotoxicity (ADCC) was first tested (stimulation-rest-costimulation assay), the T cells were prestimulated in vitro using a reporter bioassay (Promega, catalog no. G7102). In by Dynabeads for 3 days to induce ICOS before being rested for 3 days brief, the assay uses a Jurkat reporter cell line expressing human to reduce their activation levels. These stimulated/rested T cells were FcgRIIIa V158 and NFAT-induced luciferase. Following engagement then cultured with KY1044 in the presence or absence of an anti-CD3 with the Fc region of a relevant antibody bound to a target cell, the antibody (clone UCHT1, eBioscience) to assess the requirement of FcgRIIIa receptors can activate an intracellular signals resulting in TCR engagement. The effect of ICOS costimulation was assessed after NFAT-mediated luciferase activity that can be quantified via a lumi- 72 hours by measuring the levels of IFNg and TNFa present in the nescence readout. CHO cells expressing either human, mouse, rat, or culture (MSD multiplex assay). cynomolgus ICOS were used as target cells and coincubated with For the gene expression analysis, T cells were harvested from the Jurkat reporter cells at a 5:1 ratio. Serial dilutions of KY1044 or isotype 3-step culture (stimulation–rest–costimulation assay) after 6-hour IgG1 control were added to the culture plates, incubated at 37C plate-bound antibody stimulation. Total RNA was extracted from the

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cell pellets with the RNeasy Micro Kit (Qiagen), quality controlled on day mouse IL2 release was quantified using the Mouse IL2 DuoSet the Agilent 2100 Bioanalyzer (Agilent Technologies) and subjected to ELISA (R&D Systems, catalog no. DY-402). SE50 sequencing following mRNA enrichment (BGI). The sequence reads were aligned using kallisto (32) and further processed using Antibody internalization assay limma (33) and metascape (34) and GSVA (26). The sequencing data The ability of KY1044 to be internalized was assessed by capturing has been deposited into ArrayExpress (E-MTAB-9500). time course images on the IncuCyte ZOOM Live Cell Imaging Platform (Sartorius). Briefly, MJ cells endogenously expressing ICOS NFAT Luciferase assays were first labeled with the Incucyte CytoLight Rapid Green Reagent Luciferase reporter assays (Sartorius) to enable detection of the cytoplasm. Serial dilutions of Jurkat-Lucia NFAT cells (Invivogen, catalog no. jktl-nfat) stably either KY1044, a CD71 antibody (positive control; Sigma, catalog no. expressing luciferase under the control of NFAT response elements SAB4700520-100UG) or a human IgG1 isotype control (negative were further transfected with either the human Icos gene sequence control) were preincubated with secondary anti-human or anti- or a chimeric construct of Icos fused with CD247 (CD3z). Following mouse antibodies covalently labeled with a pH-sensitive dye (pHrodo selection of stable transgene integration, a luciferase activity bio- Red; Thermo Fisher Scientific, catalog no. P36600) and then added to assaywasperformed.Highbinding96-wellassayplateswerecoated the target cells. Antibody internalization was tracked for up to 48 hours overnight with either anti-CD3 (UCHT-1, eBioscience, catalog no. and the areas under the time-course curves plotted as a function of 16-0038-85; 10 mg/mL), anti-ICOS (C398.4A, BioLegend catalog no. antibody concentration. 313512; 10 mg/mL), isotype control (HTK888, BioLegend, catalog no. 400902; 10 mg/mL), anti-CD3 þ anti-ICOS (5 mg/mL each) or Mice anti-CD3 þ isotype control (5 mg/mL each). Transgene expressing All mice in vivo work was performed in the UK under Home Office cells or control cells (untransfected Jurkat-Lucia NFAT cells) were licence (70/08759). All procedures were conducted in accordance seeded at 50,000 cells/well. Following overnight incubation lucif- with the United Kingdom Animal (Scientific Procedures) Act 1986 erase activity was measured by adding BioGlo reagent (Promega, and associated guidelines, approved by institutional ethical review catalog no. G7940) and reading luminescence on a plate reader. committees (Babraham Institute AWERB). Eight- to 10-week-old Luciferase activity was normalized and scaled to 100% by compar- wild-type female Balb/C or C57BL/6J mice were sourced from Charles ing to cells stimulated using Cell Stimulation Cocktail (eBioscience, River UK Ltd and housed in transparent plastic cages with wire covers catalog no. 00-4970-93). (391 W 199 L 160 H mm, floor area: 500 cm2) containing Grade 6 Wood Chip that can be replaced with Lignocell (IPS Product Supplies PD-1/PD-L1 blockade luciferase reporter bioassay Ltd, BCM IPS Ltd.; WC1N 3XX) and bale shredded nesting material The thaw-and-use format of the PD-1/PD-L1 Blockade Bioassay (IPS Product Supplies Ltd, BCM IPS Ltd.; WC1N 3XX). Four to five (Promega, catalog no. CS187111) was performed in a 96-well plate mice were housed per cage in a room with a constant temperature format according to the manufacturer's instructions. Briefly, PD-L1– (19 C–23 C) and humidity (40%–70%) and a 12-hour light–dark cycle expressing APC/CHO-K1 cells were plated on day –1 in cell recovery (lighting from 7 am to 7 pm). Mice were provided with pellet food medium and incubated overnight at 37C. The following day, assay (CRM(P), Specialist Diet Services) and RO water ad libitum using an media was replaced with 10-point serial dilutions of AbW IgG1 or automatic watering system. isotype control, prior to addition of PD-1 expressing NFAT Jurkat effector cells. Cells were incubated at 37C for 6 hours, followed by the Tumor cell implantation and tumor measurement addition of the BioGlo reagent and luminescence signal quantified on a Early passage (below P10) MC38 (3 106 cells), J558 (1 106 cells), plate reader. CT26.WT (1 105 cells), A20 (5 106 cells), EL4 (1 104 cells), and B16-F10 (1 105 cells) tumor cells were prepared in either PBS or Ligand neutralization assays Matrigel (Corning, 354230) and the resulting cell suspension were Full-length human PD-L1 sequence (Uniprot sequence ID: injected subcutaneously into the flank of the mice (study day 0). Prior Q9NZQ7-1) was codon optimized for mammalian expression and to tumor cell implantation, mice were anesthetised with isoflurane and cloned into an expression vector under the CMV promoter flanked by the right flank of the mice was shaved. For the implantation, 100 mLof 30 and 50 piggyBac-specific terminal repeat sequences, facilitating the cell suspension were injected using 25 G needles (BD Microlance stable integration (puromycin selection) into the genome of trans- TM 3. VWR 613-4952). Cell numbers and viability (required to be fected CHO cells (35). Stable PD-L1–expressing CHO cells were above 90%) were determined preimplantation by the trypan blue assay. coincubated with a 30 nmol/L concentration of biotinylated ligand Tumor growth was measured using digital calipers three times a week (either PD-1 or CD80) and serial dilutions of antibody (150 nmol/L to until end of the study. The tumor volumes (mm3) was estimated using 0.0076 nmol/L of either AbW hIgG1 or control hIgG1) for 1 hour at a standard formula: (L W2)/2 (with L being the larger diameter, and 4C. The cells were then washed, ligand binding was detected using W the smaller diameter of the tumor). All data were plotted using Alexa Fluor 647–labeled streptavidin and the samples were acquired GraphPad Prism V10. on a BD FACSArray Bioanalyzer. Antibody dosing Mouse T-cell activation assay Both KY1044 mIgG2/mIgG1 and anti–PD-L1 (AbW) mIgG2a/ A T-cell activation assay using the OVA-specific DO11.10 mouse mIgG1 were produced by Kymab Ltd. The PD-1 (clone RMP1-14) T-cell hybridoma was used as described previously (36). In brief, mAb was purchased from BioXcell. Antibodies were dosed between 0.1 human PD-L1–transfected LK35.2 cells (antigen-presenting cells) and 10 mg/kg or by flat dose of 20 to 200 mg/dose via the intraperitoneal were incubated with mouse PD-1–expressing DO11.10 cells in the route. For the efficacy and pharmacodynamic studies, the treatment þ þ presence of OVA peptide. Serial dilutions of AbW IgG1 or isotype groups were not blinded. The in vivo depletion of CD8 and CD4 T control were added and incubated at 37C overnight. The following cells were conducted using a flat dose 200 mg of anti-CD8a (53-6.7)

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þ and/or anti-CD4 (GK1.5). Dosing was performed twice a week for additional tumor types containing high ICOS Treg cells, we analyzed 3 weeks starting from day 3 following tumor cell implantation. The the content of T cells in tumors using different approaches. þ þ CD8 and CD4 T-cell depletion was determined by flow cytometry of Using the TCGA datasets, we identified tumors originating from tumor, spleen, tumor draining lymph node (inguinal lymph node), and the head and neck, stomach, cervix, thymus, testis, skin, and the blood cells using an anti-CD3 antibody (17A2). lungs as indications with high mRNA expression of ICOS and FOXP3 (a marker of Treg; Fig. 1A). To establish which types of Cynomolgus monkey study cells expressed ICOS and/or FOXP3, we assessed these markers at The effects of KY1044 in nonhuman primates (NHP) were studied the mRNA and protein levels by single-cell transcriptomics and as part of a repeat dose toxicity study. Na€ve male cynomolgus FACS analysis, respectively. We generated paired PBMCs and monkeys (Macaca fascicularis) were obtained from a certified supplier, tumor scRNA-seq data for 79,544 cells from 5 patients with NSCLC group housed, allowed access to water ad libitum and fed on a pelleted (Supplementary Table S2), which we subsequently stratified into diet for monkeys supplemented with fresh fruit and biscuits. Animals 27 cell subtypes based on published immune gene expression ranged from 4 to 7 years old and weighed 3–6 kg at time of dosing. Four signatures. As expected, genes such as foxp3, ccr8, il2ra, tnfrsf4 groups of three cynomolgus monkeys received weekly intravenous were highly expressed in intratumoral Tregs (Fig. 1B). ICOS mRNA doses of KY1044 (slow bolus over approximately 1 minute) for a expression was higher in Tregs than in other T-cell subsets. Finally, month (5 doses in total) at doses of 0 (vehicle control; PBS pH 7.4), 10, ICOS expression was higher in the TME than in the periphery, 30, or 100 mg/kg at a dose volume of 2 mL/kg. Blood samples were whereas the expression pattern in both compartments followed the taken at 1 or 2 timepoints prior to dosing and at multiple timepoints up general trend of: Treg > CD4non-Treg > CD8 > Other (Fig. 1B). By to 29 days after the first dose for measurement of serum KY1044 using flow cytometry analysis (Fig. 1C; Supplementary Fig. S1A), ICOS þ þ þ a qualified ELISA assay, ICOS occupancy on CD4 cells in blood expression was higher on Treg (CD4 /FOXP3 )thanontheother þ þ determined using a validated flow cytometry method and/or immu- T-cell subsets (CD8 and CD4 /FOXP3 ). ICOS protein was also nophenotyping of whole blood (described above). For the receptor moreexpressedintheTMEthanonPBMCs(P < 0.001 for Tregs). occupancy, free ICOS was measured by using a competing anti- No differences in ICOS expression between PBMCs from healthy ICOS that does not displace KY1044 while an anti-human IgG donors and patients with NSCLC were observed. Finally, T-cell was used to detect KY1044 directly or after saturating the cells with immunophenotyping of CT26.WT tumor–bearing mice revealed an excess of KY1044 to quantify bound and total ICOS, respectively. that ICOS was also more expressed in the TME than in the spleen. Scheduled necropsies were conducted 1 day after the final dose This analysis also confirmed higher ICOS expression on Treg than – (day 30) and spleen and mesenteric lymph nodes were taken for on CD4/CD8 TEff cells (Supplementary Fig. S1B S1D). immunophenotyping. ICOS immunostaining on tumor tissue microarrays (from esophageal, head and neck, gastric, lung, bladder, and cervical cancer NHP ethics statement biopsies) confirmed the upregulation of ICOS in the TME The cynomolgus monkey study was conducted at Covance preclin- (Fig. 1D). The costaining for ICOS and FOXP3 (Fig. 1E), also þ ical Services GmbH in strict accordance with a study plan reviewed and highlighted a high number of ICOS Treg cells for the six selected approved by the local Institutional Animal Care and Use Committee indications, with only bladder cancer demonstrating a lower density of (IACUC) of the testing facility and the German Animal Welfare Act. ICOS/FOXP3-positive cells (Fig. 1F). The study was performed according to DIRECTIVE 2010/63/EU OF Altogether, our data confirmed that ICOS was not homogenously THE EUROPEAN PARLIAMENT AND OF THE COUNCIL of expressed on the T-cell subsets and was induced in the TME, especially September 22, 2010 on the protection of animals used for scientific on the surface of Treg. In addition, we identified indications with high þ purposes and the Commission Recommendation 2007/526/EC on levels of intratumoral ICOS Treg cells including esophageal, head guidelines for the accommodation and care of animals used for and neck, gastric, lung, and cervical cancers. experimental and other scientific purposes (Appendix A of Conven- tion ETS 123). The study was in compliance with the Kymab Working KY1044: a human ICOS IgG1 antibody with both depleting and Practice on Experiments involving Animals and was approved by the agonistic functions Kymab Ethics Committee. The strong expression of ICOS on intratumoral Tregs suggests that the targeting of these cells with an effector enabled ICOS antibody Statistical analyses could lead to their preferential depletion (e.g., via ADCC and/or Unless stated otherwise in the figure legends, the efficacies observed ADCP). The level of target expression and the amount of antibody for the different treatment groups were compared using either t test or bound to a target are key parameters influencing cell killing by þ one-way ANOVA and Tukey multiple comparison post hoc test. ADCC (39). Therefore, it is expected that ICOSLow cells (e.g., CD8 fi P High Differences between groups were signi cant at a < 0.05. On the TEff cells) will be less sensitive to depletion than ICOS Treg, but graphs, , P < 0.05; , P < 0.01; , P ≤ 0.001; , P ≤ 0.0001. could be sensitive to costimulation if the antibody also harbors Statistical analyses were performed with GraphPad Prism 10.0 agonistic properties (via target clustering through FcgR receptors; (GraphPad Software, Inc.). refs. 40, 41). With this in mind, we identified a fully human mono- clonal IgG1 antibody called KY1044. KY1044 was generated in a transgenic mouse line for the human immunoglobulin genes in which Results the endogenous Icos gene was knocked-out. Lack of endogenous ICOS High ICOS expression on intratumoral Treg expression was aimed to facilitate the generation of a mouse cross þ Ovarian, gastric, and liver cancers, are highly infiltrated with ICOS reactive antibodies. KY1044 binds ICOS from different species Treg cells (16, 17, 37) and this results in poor prognosis (17, 38). To (human, Cynomolgus monkey, rat, and mouse) with similar affinity K assess ICOS expression on intratumoral T cells and to identify (a d below 3 nmol/L for the Fab, Supplementary Fig. S2A).

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Figure 1. ICOS expression in cancer Tregs. A, Density plots showing the combined Icos and Foxp3 expression in solid tumors (TCGA datasets). Combined expression was scored using ssGSEA. The median ssGSEA score (marked by vertical ticks) is shown. The TCGA standard cancer type abbreviations were used. B, Immune response genes were sequenced in PBMCs and tumors from patients with NSCLC. Cells were classiﬁed into one of 27 immunologic subtypes (Supplementary Table S1). Left, heatmap of scaled gene expression in T cells from PBMCs and tumors from NSCLC patient samples (n ¼ 5). Each gene was scaled individually across all cells of the

dataset, and the mean scaled expression for each of 17 T-cell subtypes is presented. Right, scatter plots showing ICOS mRNA expression in Treg, CD4non-Treg, CD8, and all other T cells. Each dot corresponds to a single cell. Full lines indicate the mean ICOS gene expression of the total cell compartment, and the dashed lines indicate the mode (density peak) of ICOSþ cells. C, Relative ICOS expression (flow cytometry) on CD4þ, CD8þ, and Treg (CD4þ/FOXP3þ) in healthy donor PBMCs (n ¼ 5), NSCLC tumor samples (n ¼ 5), and matched NSCLC patient PBMCs (n ¼ 4; two-way ANOVA with Tukey multiple comparison). D, Graph showing the density of ICOSþ cells per mm2 in tumor cores (n ¼ 995) from six indications and matching healthy tissues (n ¼ 48; Mann–Whitney unpaired t test). E, Example of ICOS/FOXP3 staining of gastric tumor core showing (i) original whole core image with ICOS staining in purple and FOXP3 in brown; (ii) classifier analysis overlays showing tissue region of interest (lilac) and white space (white); (iii) cellular analysis overlay showing single FOXP3 positive (green overlay), single ICOS positive (red overlay), and ICOS/ FOXP3 dual positive (cyan overlay); and (iv–vi) 20 magnified detailed area. F, Quantification of ICOS/FOXP3 double-positive cells per mm2 from the tumor cores (n ¼ 995) from the six indications.

n ¼ KY1044-dependent ADCC was assessed using a luciferase report- mediated killing (EC50 of 5.6 pmol/L, 8, Fig. 2B). Altogether, er assay. As shown in Fig. 2A, KY1044 signiﬁcantly induced the these data conﬁrmed that KY1044 triggers ADCC of ICOSHigh- n ¼ luciferase signal (EC50 of 0.15 nmol/L, 3). A similar EC50 was expressing cells. obtained against mouse ICOS (0.53 nmol/L, n ¼ 3), rat ICOS (0.48 The agonistic potential of KY1044 was assessed using the MJ [G11] þ nmol/L, n ¼ 3), or cynomolgus monkey ICOS (0.22 nmol/L, n ¼ 3). CD4 cells that we demonstrated express ICOS and do not require a We validated the data using human NK cells as effector cells. primary stimulatory signal (e.g., TCR engagement) for cytokine When incubated with KY1044 and CEM cells expressing hICOS production. KY1044 was either precoated on culture plates (mimick- (5:1 effector:target cell ratio), these NK cells induced potent ADCC- ing cross-presentation/clustering) or used in solution with or without

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Figure 2. KY1044 hIgG1 triggered ADCC and had costimulatory properties. A, ADCC cell reporter assay with KY1044. KY1044 hIgG1 or the isotype control was incubated with FcgRIIIA Jurkat reporter cells and ICOSþCHO cells as target cells. B, KY1044 induced ADCC in a human primary NK cell assay. KY1044 or isotype control was coincubated with dye-loaded ICOSþ CEM target cells and NK cells for 4 hours. ADCC activity was quantified by measuring Specific Dye Release upon target killing and normalized to percentage of maximum lysis compared with the positive control, a digitonin-based lysis buffer (mean of triplicates SEM). For A and B, the vertical dotted line indicates 100 ng/mL. Plate-bound (C) and cross-linked soluble (D) KY1044 induced the release of IFNg from MJ cells. For C and D, the vertical dotted line indicates 10 mg/mL. The concentration of IFNg was assessed in the supernatant of MJ cells cultured for 72 hours. E and F, Levels of IFNg induced in human primary T cells activated with anti-CD3/CD28 (to induce ICOS) and cultured with either 5 mg/mL of plate-bound KY1044 or the isotype control IgG1 (E; n ¼ 10) or with either 15 mg/mL of soluble KY1044 or soluble isotype control with or without the addition of Fc cross-linking anti-human F(ab')2 Fragments (F; n ¼ 10). G, Cytokine production by KY1044 requires anti-CD3. Levels of TNFa in T-cell culture with 5 mg/mL of KY1044 or isotype control IgG1 (plate bound; n ¼ 8) in the presence or not of anti-CD3 (Wilcoxon statistic test for E, F,andG). H, RNA-sequencing analysis comparing T-cell stimulation with either KY1044 þ anti-CD3 or control (anti-CD3 only). (i) Genes reported to be downstream of ICOS signaling showed a pattern of upregulation following KY1044 costimulation, with significant P values obtained for Ifng, Il10 and Il4. (ii) Metascape pathway enrichment analysis of the differentially expressed genes. Enriched gene sets were ranked on the basis of the significance of their P values. (iii) Heatmap showing unsupervised clustering of samples based on the expression levels of the top gene ontology terms from the Metascape enrichment analysis. IC, isotype control.

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addition of a secondary cross-linking antibody. While soluble KY1044 (NFAT; Fig. 2H), as seen previously (44). NFAT-dependent ICOS did not induce IFNg production, plate-bound, and cross-linked agonism was confirmed using reporter cell lines (Supplementary KY1044 effectively induced IFNg secretion (EC50 of 10.5 nmol/L Fig. S2D). Target-mediated internalization of KY1044 was limited 2.7 nmol/L; n ¼ 2 and 0.50 nmol/L 0.18 nmol/L, n ¼ 3, (Supplementary Fig. S2E). respectively; Fig. 2C and D). This agonism potential was confirmed Altogether the data presented here demonstrated that KY1044 has a using primary T cells preactivated with anti-CD3/CD28 (to induce dual mechanism of action with both depleting and costimulatory ICOS expression; Supplementary Fig. S2B) and cultured with KY1044. properties. Plate-bound (Fig. 2E) and cross-linked (Fig. 2F) KY1044 induced IFNg production in primary cells. KY1044-dependent IFNg secretion KY1044 (mIgG2a) monotherapy blocks tumor growth in was significantly higher than for the isotype-treated cells in both plate- lymphoma/myeloma tumor models bound (2.4 0.5-fold at 5 mg/mL mAb, P < 0.01) and cross-linked KY1044 cross-reactivity to mouse ICOS facilitated the in vivo mouse assays (2.5 0.2-fold at 15 mg/mL mAb, P < 0.01). Importantly, a pharmacology work for which the antibody was reformatted as a three-step stimulation–resting–costimulation experiment confirmed mouse IgG2a (the effector enabled format in mouse). Within the that TCR engagement was required for KY1044-dependent upregula- TCGA datasets, ICOS expression was high in diffuse large B-cell tion of cytokines (such as TNFa; Fig. 2G). lymphoma (Supplementary Fig. S3). On the basis of the role of þ Finally, a transcriptomic analysis of CD3 T cells was performed ICOS/ICOS-LG signaling in the generation and maintenance of following 6 hours of combined anti-CD3 and KY1044 costimulation. germinal centres (10, 45) and on the antitumor efficacy of ICOS In line with previous reports (14), we observed cytokine upregulation, antibodies in lymphoma (46), we assessed the effect of KY1044 þ most notably IFNG, IL10, and IL4 (Fig. 2H). Gene set enrichment mIgG2a in the ICOS-LG A20 tumor lymphoblast B-cell model (47). analysis confirmed that KY1044 induced genes involved in cytokine– Mice were dosed (2qW for 3 weeks at 10 mg/kg) starting from 6 days cytokine receptor interactions and and lymphocyte activation following tumor cell implantation with saline and KY1044 mIgG2a. (Fig. 2H). Of relevance, no significant change in genes associated KY1044 mIgG2a treatment triggered an antitumor response, with with proliferation and cell-cycle progression were observed. However, more than 90% of the mice being free from measurable disease at the in agreement with a previous report (42), the analysis showed an end of the study (day 42; Fig. 3A). This efficacy was confirmed in enrichment of genes involved in cell locomotion, adhesion, and another B-cell–derived syngeneic model, the J558 plasmacytoma differentiation. This finding also reflected our observations of MJ model in which around 70% of the KY1044 mIgG2a–treated mice þ [G11] CD4 morphology changes in response to anti-ICOS (C398.4A were tumor free at the end of the study (Fig. 3B). or KY1044; Supplementary Fig. S2C; Supplementary Movies S1 and Monotherapy efficacy was also assessed in models of hematologic S2). Even though ICOS signaling depends on the PI3K/AKT/mTOR malignancies (T-cell lymphoma EL4) and models of solid tumors axis (43), following KY1044 agonism, we noticed an overrepresenta- (CT26.WT, MC-38, and B16.F10). Overall, the monotherapy response tion of genes downstream of the nuclear factor of activated T cells was absent or low in all these models. Mice harboring CT26.WT or

Figure 3. Effect of KY1044 mIgG2 in the A20 and J558 tumor models. A, Spider plots of a representative experiment showing individual A20 tumor volumes (n ¼ 10 per group). Tumor-bearing mice were dosed intraperitoneally with 200 mg of KY1044 mIgG2a or 200 mL saline starting from day 8 following tumor cell implantation. B, Spider plots showing individual J558 tumor volumes (n ¼ 7 per group). Tumor-bearing mice were dosed intraperitoneally with 60 mg of KY1044 mIgG2a or 200 mL saline starting from day 9 following tumor cell implantation. The dosing days are indicated by vertical dotted lines. The survival curves depicting the control and KY1044 mIgG2a groups are shown. Statistics were calculated using log-rank (Mantel–Cox) test. Data are representative of at least two experiments.

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þ MC-38 tumors showed only tumor growth delay (Supplementary dependent on CD8 T cells, with the improved survival fully abrogated þ Fig. S4). when CD8 T cells were depleted (Fig. 4B; Supplementary Fig. S5). Altogether, our in vivo studies demonstrated that KY1044 mIgG2a Conversely, addition of a CD4-depleting antibody (which depletes was highly effective as a monotherapy in B-cell–derived tumor models. both CD4 Tregs and CD4 non-Tregs) to the combination was still associated with tumor growth delay but CD4 depletion eliminated any KY1044 (mIgG2a) improved anti–PD-L1 efficacy long-term survival benefit(Fig. 4B). Altogether, the coadministration CT26.WT tumors respond poorly to anti–PD-L1 (48). Because of KY1044 mIgG2a and anti–PD-L1 triggered a durable antitumor anti-ICOS and the anti–PD-L1 act on complementary immune immune response in the CT26.WT tumor model. The combination of pathways, we combined KY1044 mIgG2a with anti–PD-L1 (AbW). anti–PD-1 (clone RMP1-14) with KY1044 was poorly effective in the AbW binds to mouse PD-L1 with high affinity (Kd ¼2nmol/L) CT26.WT model (Supplementary Fig. S6C), whereas it was effective in and blocks PD-L1/PD-1 and PD-L1/CD80 interactions (Supple- another model (MC38; Supplementary Fig. S6D). mentary Fig. S5A). These studies (Fig. 4A) demonstrated a com- The KY1044 mIgG2a and anti–PD-L1 combination was also tested plete antitumor response in the majority (ranging from 50% to 90%) in other models (Supplementary Fig. S7A). Although the J558 model of mice treated with the KY1044 mIgG2a (equivalent of 3 mg/kg) already responded well to either the anti-ICOS or the anti–PD-L1 and anti–PD-L1 (equivalent of 10 mg/kg) combination. Of rele- monotherapy, a 100% response was achieved with the combination in vance, we observed a trend (albeit not significant) showing higher this model. Similarly, the combination was effective in the MC38 antitumor efficacy when anti–PD-L1 was used as mIgG2a than as model (Supplementary Fig. S7B). The B16F10, 4T1, and EL4 tumor mIgG1 (Supplementary Fig. S6A). Finally, when KY1044 was models did not respond to the combination (Supplementary Fig. S7A). reformatted as a mouse IgG1 (low depleting potential in mouse) Collectively, our efficacy studies demonstrated that the cotargeting andcombinedwithanti–PD-L1, the resulting antitumor efficacy of ICOS and PD-L1 resulted in a strong combinatorial effect in selected was weaker than the one observed with the corresponding KY1044 tumor models, including those in which both monotherapies were mIgG2a/anti–PD-L1 combination, thus arguing for the contribu- poorly effective. tion of ICOS-mediated depletion to the stronger antitumor efficacy seen in the CT26.WT model (Supplementary Fig. S6B). KY1044 depleted ICOSHigh cells in vivo in mice and NHPs Mice that survived the first CT26.WT challenge in response to the To assess the mechanism of action of KY1044 in vivo,wefirst KY1044 mIgG2a/anti–PD-L1 treatment were resistant to a CT26.WT conducted pharmacodynamic studies in the CT26.WT model. Tumor- rechallenge but sensitive to the growth of EMT-6 cells (Fig. 4A) bearing mice were dosed with KY1044 mIgG2a (ranging from 0.3 to suggesting a tumor antigen–specific memory response. Finally, we 10 mg/kg) on day 13 and day 15 following tumor cell implantation demonstrated that the response to the combination was primarily (Fig. 5A). The T-cell content in the tumors and the spleens were

Figure 4. KY1044 mIgG2a improved the efﬁcacy of anti–PD-L1. A, Survival curves of mice harboring CT26.WT tumors and treated with control, KY1044 mIgG2a monotherapy at 60 mg/dose, anti–PD-L1 mIgG2a monotherapy at 200 mg/dose, and the combination of KY1044 mIgG2a and anti–PD-L1 mIgG2a at 60 and 200 mg/dose, respectively (left). The dosing days are indicated by vertical dotted lines. Statistics were calculated using log-rank (Mantel–Cox) test. The data are representative of six independent experiments. Tumor-bearing mice that cleared CT26.WT tumors were randomly allocated to two groups and rechallenged with either CT26.WT or EMT6 cells (right). B, Effect of CD4þ and CD8þ T-cell depletion on antitumor efﬁcacy. Mice implanted with CT26.WT cells (n ¼ 10 per group) were depleted of CD8þ and/or CD4þ T cells and treated with saline or with KY1044 mIgG2a and anti–PD-L1 mIgG2a combination as described in A. Data are representative of two independent experiments.

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Figure 5. KY1044 depleted ICOShigh T cells in vivo. A, In vivo experimental protocol of the pharmacodynamic study in the CT26.WT tumor–bearing mice. Mice were dosed twice with saline or KY1044 mIgG2a (ranging from 0.3–10 mg/kg) on days 13 and 15, and the tumor and spleen were harvested on day 16 and the immune cells analyzed by þ FACS. Effect of KY1044 on Treg depletion (plotted as a percentage of live cells) and on the CD8 TEff to Treg cell ratio in the TME (B) and in the spleen (C) of treated mice (n ¼ 7/8 mice per groups). D, Relative expression of ICOS and frequency of T-cell subsets in the blood of NHP (the number of datapoints are indicated in brackets). The percentages indicate the frequency of each cell types within CD3/CD4 T cells in NHP PBMCs. geoMFI, geometric mean ﬂuorescence intensity.

E–G, Graphs showing the changes (vs. baseline pretreatment) of total CD4 TM cells and follicular Th cells in the blood of NHP over time following a single-dose of KY1044 human IgG1 at 0, 10, 30, and 100 mg/kg (n ¼ 3 NHP per groups).

analysed on day 16. Although KY1044 monotherapy did not induce 3 mg/kg doses. This decrease in the ratio at the highest dose may have much antitumor efficacy in this model (Fig. 4A), KY1044 mIgG2a was been caused by the depletory effect of KY1044 (albeit not significant) þ associated with intratumoral Treg cell depletion, even at doses as low as on CD8 T cells (Supplementary Fig. S8B). These effects were not 0.3 mg/kg (Fig. 5B). The antibody format was critical to decrease Tregs observed in the spleen (Fig. 5C; Supplementary Fig. S8C) potentially as shown with partial and nonsignificant Treg depletion when using a due to lower ICOS expression in this tissue (Supplementary Fig. S1). mIgG1 (poor depleter; Supplementary Fig. S8A). In addition, an We repeated similar experiment by dosing mice once with KY1044 þ increase in the CD8 TEff to Treg cell ratio (known to be associated mIgG2a. The tumors were then immunophenotyped up to 7 days with improved response to ICIs) in the TME was observed at all doses posttreatment (Supplementary Fig. S8D). Treg depletion was observed tested (Fig. 5B). However, the highest dose of 10 mg/kg showed a lower after a single dose of 0.3 mg/kg; however, at this dose, the incidence of þ CD8 TEff to Treg cell ratio than the one resulting from the 1 and Tregs in the TME recovered quickly back to the level observed in the

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þ control group. A long-lasting Treg depletion and an increase of the circulating CD4 cells in monkeys (Fig. 5D). KY1044 did not alter þ CD8 to Treg ratio were observed at higher dose (3 and 10 mg/kg; CD8 T cells in blood (Supplementary Fig. S9D). Finally, we did not fi high Supplementary Fig. S8D). Altogether, these data suggest that KY1044 observe a signi cant decrease in ICOS cells such as TM or Treg or in þ mIgG2a preferentially depleted ICOShigh Treg cells and improved ICOSlow CD8 T cells in the in the lymph node (or spleen) of treated þ – CD8 TEff to Treg cell ratio in the TME. monkeys (Supplementary Fig. S9E S9G). In summary, these data þ ICOS was expressed on cynomolgus monkey CD4 memory T cells demonstrated that high dose of KY1044 elicited some depletion of þ þ high /Dim/þ þ high (TM: CD3 /CD4 /CD95 /CD28 ), CD4 follicular Th cells ICOS cells in peripheral blood but not in lymphoid tissues of NHPs. þ þ þ þ þ (TFH: CD3 /CD4 /CD185 ) and on Treg cells (CD3 /CD4 / þ þ þ CD25 FoxP3 ; Fig. 5D). Similarly, circulating NHP CD8 T cells Increased cytokine expression in response to KY1044 in vivo showed the lowest ICOS expression. We assessed the pharmacody- To demonstrate that KY1044 activates and increases proinflamma- namic effects of KY1044 hIgG1 in NHP after repeated weekly intra- tory cytokine production in intratumoral TEff cells, we assessed the þ venous administration at doses of 0, 10, 30, and 100 mg/kg for 4 weeks expression of the activation markers CD69 and CD44 on CD8 T cells. þ (5 doses). Exposure and full occupancy of ICOS on circulating CD4 T T cells from CT26.WT tumors were analyzed 24 hours after a second cells was maintained at all doses (Supplementary Fig. S9A and S9B). dose of KY1044 mIgG2a. KY1044 mIgG2a induced a significant P Immunophenotyping indicated a decrease in TM and TFH cells in increase in CD69 expression (Fig. 6A; < 0.05 vs. control) at doses peripheral blood (Fig. 5E and F). KY1044 did not affect circulating of 1 and 3 mg/kg. Similarly, the analysis of CD69/CD44 double positive Treg (Supplementary Fig. S9C), which represented less than 3% of cells confirmed a significant increase (P < 0.05) of CD8 activation in

Figure 6. KY1044 mIgG2a activated intratumoral T cells in vivo. A, Effect of KY1044 mIgG2a on intratumoral CD8þ T-cell expression of CD69 and CD44 (n ¼ 7/8 mice per groups). Measurement of CD69 and CD69/CD44 double-positive cells was performed 24 hours after a second dose of KY1044 mIgG2a (see Fig. 5A). B, Graphs þ þ showing the ﬂow cytometry analysis of intracellular IFNg and/or TNFa expression in intratumoral CD8 and CD4 T cells from CT26.WT tumor collected 7 days after a single intraperitoneal injection of saline or KY1044 mIgG2a at 3 mg/kg. Groups of mice (n ¼ 4 mice/group; Tukey multiple comparisons test).

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response to KY1044 at a dose of 1 mg/kg (Fig. 6A). In a separate Discussion experiment, we examined KY1044-dependent induction of IFNg and The approval of ICIs provides a novel approach that adds to existing þ þ TNFa by CD4 and CD8 T cells. Using an intracellular staining cancer therapies. However, responses to ICIs are not universal (2). An þ þ approach on intratumoral CD4 and CD8 T cells collected 7 days immunosuppressive TME can underlie the lack of response. Notably, posttreatment (3 mg/kg of KY1044 mIgG2a), we demonstrated a intratumoral Tregs maintain an immunosuppressive environment. significant increase in cytokine production (Fig. 6B). Although, one Strategies reducing Treg numbers and improving the ratio of effector cannot differentiate between direct activation (through direct ICOS T cells to Treg cells have thus emerged. Blocking the recruitment, costimulation on TEff cells) or indirect activation (via Treg depletion), function, expansion, and/or the survival of Treg, may achieve this goal this analysis of intratumoral T cells revealed that KY1044 was associated and a number of molecules, targeting CTLA-4, CD25, GITR, OX40, Low þ þ with some activation of both ICOS CD8 and CD4 TEff cells. CCR8, CD137 and CCR2, are under investigation (50). However, the differential expression of these targets (i.e., within immune and other Increased efficacy of KY1044 at an intermediate dose in cell subtypes) is crucial to preferentially target TReg (49). Here, we combination with anti–PD-L1 demonstrated that ICOS expression differs between T-cell subsets, The pharmacodynamic studies confirmed that KY1044 mIgG2a with the highest expression observed on Treg and confirmed that ICOS effectively depleted ICOShigh Treg and resulted in activation of expression is higher in the TME than in the periphery (blood or Low fi ICOS TEff cells (Fig. 4A). However, we noticed that when used at spleen). In addition, we identi ed head and neck, gastric, esophageal, high doses, KY1044 mIgG2a also affected the numbers of intratu- lung, bladder, skin, and cervical cancers as tumors with a high content þ þ moral CD8 T cells (Supplementary Fig. S8), resulting in a lower of ICOS cells. However, costaining of ICOS with FOXP3 showed that þ þ þ CD8 TEff to Treg cell ratio. Because a higher baseline CD8 TEff to these tumors differ by their incidence of ICOS Treg with cervical, Treg cell ratio positively correlates with a response to ICIs such as esophageal, and head and neck cancers being more infiltrated by þ atezolizumab(49),weaimedtoassess whether the antitumor ICOS Treg. Because the ICOS expression on different cell subsets efficacy would be superior at an intermediate dose. For this, we should affect the response to anti-ICOS therapies, this work suggests repeated the CT26.WT efficacy study using a range of different that the specific expression of ICOS on Treg may support a patient doses of KY1044 mIgG2a (20, 60, and 200 mg/dose) combined with selection strategy. a fixed dose of anti–PD-L1 (200 mg/dose). As shown in Fig. 7,allthe High ICOS expression on intratumoral Tregs highlights the poten- combination treatments were associated with an improved response tial of this target for a depletion strategy. Using a transgenic mouse (vs. control or the anti–PD-L1 group). However, an improved platform (23), we selected a fully human ICOS antibody called KY1044 response at 60 mg/dose of KY1044 mIgG2a was observed, with and demonstrated that KY1044 depletes ICOShigh cells via ADCC 90% of the mice being tumor free on day 54. (through the engagement of FcgRIIIa) and act as a costimulatory

Figure 7. Improved combination efﬁcacy with anti–PD-L1 was obtained at intermediate dose of KY1044 mIgG2a. Spider plots from a representative experiment showing individual mouse tumor volumes from BALB/c mice (n ¼ 9 per group) harboring subcutaneous CT26.WT tumors and treated with different doses of KY1044 mIgG2a [20 (A), 60 (B), and 200 (C) mg/dose] with a ﬁxed dose of anti–PD-L1 mIgG2a (200 mg/dose). D, Kaplan–Meier plot depicting the survival of mice injected with CT26.WT tumor and treated with different doses of the KY1044 mIgG2a/anti–PD-L1 combination. The dosing days are indicated by vertical dotted lines.

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þ molecule on cells expressing lower ICOS levels, such as CD8 TEff doses of KY1044 mIgG2a were shown to trigger an antitumor immune cells (through FcgR-dependent clustering). KY1044 has monotherapy response, the intermediate dose of 60 mg of KY1044 resulted in the efficacy in lymphoma models and combination efficacy (with anti– strongest response. PD-L1) in models of solid tumors that are resistant to PD-1/PD-L1 Altogether, this study demonstrated that KY1044 was pharmaco- blockade. In the CT26.WT tumor model, we showed better efficacy logically active, modified the intratumoral immune contexture, and when combining anti–PD-L1 with KY1044 mIgG2a (effector enable) induced a strong and long-lasting antitumor response. These findings, than with a poorly depleting mIgG1 format. Similarly, in this model, therefore, warrant the further assessment of KY1044 as a monotherapy we showed better efficacy when combining KY1044 mIgG2a with anti– or a combination therapy with anti–PD-L1 as a potential treatment for PD-L1 than with anti–PD-1. Although it was not clear why such a solid tumors. difference was observed in this particular model, one could postulate that anti–PD-1, which blocks binding to both PD-L1 and PD-L2, may Disclosure of Potential Conflicts of Interest be associated with a different phenotype to anti–PD-L1, which affects R.C.A. Sainson reports other from Kymab Ltd (full-time employee) during the PD-1 and CD80 biology (51). Speculatively, there is also the possibility conduct of the study, as well as a patent for US9957323 issued (author/coinventor on that anti-ICOS and anti–PD-1 may interfere with each other's func- the patent) and a patent for US16/323980 pending (author/coinventor on the patent). M. Kosmac reports a patent for US9957323 issued and a patent for US16/323980 tions within the immunologic synapse, as both ICOS and PD-1 are pending. G. Borhis reports a patent for US9957323 issued and a patent for – often expressed on the same cells (52 54). US16/323980 pending. N. Parveen reports a patent for US9957323 issued and a There is an ongoing debate regarding the value of mouse models patent for US16/323980 pending. C. Van Krinks reports a patent for US9957323 when predicting the depletion potential of effector function enabled issued and a patent for US16/323980 pending. H. Ali reports a patent for US9957323 antibodies in human (8, 9, 22, 55, 56). Experiments in mouse dem- issued to Kymab Ltd and a patent for US16/323980 pending to Kymab Ltd. H. Craig þ onstrated complete depletion of intratumoral CTLA-4 Treg, whereas reports other from Kymab Ltd (full-time employee) during the conduct of the study. V. Germaschewski reports Kymab Ltd employment and share options. S. Quaratino depletion of these cells in patients treated with ipilimumab remains reports other from Kymab Ltd (full-time employee) during the conduct of the study. disputed. The differences in FcgR expression and Fc/FcgR interaction M. McCourt reports personal fees from Kymab Ltd (full-time employee) during the between rodents and primates may contribute to these discrepan- conduct of the study, as well as a patent for US9957323B2 issued to Kymab Ltd and a cies (57). Here, we performed pharmacodynamic studies in both mice patent for US20190330345A1 pending to Kymab Ltd. No potential conflicts of interest (using mIgG2a) and NHPs (using hIgG1) to confirm that KY1044 were disclosed by the other authors. decreased the frequency of ICOShigh cells in vivo in both species. With ’ the mouse work, we demonstrated that KY1044 reduced Treg and Authors Contributions þ R.C.A. Sainson: Conceptualization, data curation, formal analysis, supervision, improved the CD8 TEff to Treg cell ratio, which led the tumors to investigation, writing–original draft, writing–review and editing. A.K. Thotakura: respond to anti–PD-L1. Combined with the decrease in ICOShigh cells Resources, data curation, writing–original draft. M. Kosmac: Resources, data observed in NHP, these data suggested that KY1044 could also deplete curation, writing–original draft, writing–review and editing. G. Borhis: Resources, high ICOS cells in human tumors. data curation, writing–original draft, writing–review and editing. N. Parveen: While depletion of Treg is an attractive strategy, it is crucial not Resources, data curation. R. Kimber: Resources, data curation. J. Carvalho: to deplete all Treg in all tissues to avoid autoimmune dis- Resources, data curation. S.J. Henderson: Resources, data curation. K.L. Pryke: eases (58, 59). KY1044 did not deplete Treg in the spleen and Resources. T. Okell: Resources. S. O'Leary: Resources, data curation. S. Ball: Resources, data curation. C. Van Krinks: Data curation. L. Gamand: Resources, lymph node. Although the reasons behind this selective depletion is data curation. E. Taggart: Resources. E.J. Pring: Resources. H. Ali: Resources, data not fully understood, this response has been shown for other curation. H. Craig: Resources, data curation. V.W.Y. Wong: Resources, data depleting antibodies and, in our case, could be explained by the curation. Q. Liang: Resources, supervision. R.J. Rowlands: Resources, data lower expression of ICOS on the surface of T cells in lymphoid curation. M. Lecointre: Resources, data curation. J. Campbell: Resources, data tissues (Supplementary Fig. S1) or to lower expression of FcgRIIIa curation, supervision. I. Kirby: Resources, data curation. D. Melvin: Software. – in the periphery. In the tumor rechallenge experiment, a long-term V. Germaschewski: Resources, supervision. E. Oelmann: Writing review and editing. S. Quaratino: Supervision, writing–review and editing. M. McCourt: immune memory response was observed after treatment with Conceptualization, supervision, writing–review and editing. KY1044. Because some memory T cells expressed ICOS, these data high suggested that KY1044 has the potential to deplete ICOS cells Acknowledgments without removing all ICOS cells. In cynomolgus monkeys, no The authors are extremely grateful to Kymab's molecular biology and antibody adverse toxic effects were associated with the depletion of some expression team as well as the Kymab BSU team. The authors also thank Propath high of the ICOS cells. Finally, we demonstrated that KY1044 has UK and OracleBio for assistance with IHC staining and digital image analysis as costimulatory properties, as shown by the activation and secretion well as Stephanie Grote-Wessels (Covance Preclinical Services GmbH) for their ofcytokinessuchasIFNg and TNFa. Although no effect on T-cell support with this study. They also thank the NCI Division of Cancer Treatment proliferation was observed, we noted a strong effect on cell mor- and Diagnosis, Developmental Therapeutics Program, Biological Testing Branch, Tumor Repository for providing the MC38 syngeneic tumor cell line. phology in response to KY1044-dependent ICOS stimulation. This costimulatory agonistic property was shown to require target The costs of publication of this article were defrayed in part by the payment of page clustering and concomitant engagement of the TCR. advertisement þ charges. This article must therefore be hereby marked in accordance Finally, while assessing the effect of KY1044 on the CD8 TEff to with 18 U.S.C. Section 1734 solely to indicate this fact. Treg intratumoral ratio, we noticed that although the treatment improved the ratio at all doses tested, a bell-shaped response pattern Received January 14, 2020; revised June 1, 2020; accepted September 18, 2020; was observed. Although all combinations of anti–PD-L1 with different published first September 30, 2020.

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1582 Cancer Immunol Res; 8(12) December 2020 CANCER IMMUNOLOGY RESEARCH

An Antibody Targeting ICOS Increases Intratumoral Cytotoxic to Regulatory T-cell Ratio and Induces Tumor Regression

Richard C.A. Sainson, Anil K. Thotakura, Miha Kosmac, et al.

Cancer Immunol Res 2020;8:1568-1582. Published OnlineFirst September 30, 2020.

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