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CANCER IMMUNOLOGY RESEARCH | RESEARCH ARTICLE

An Antibody Targeting ICOS Increases IntratumoralCytotoxic to Regulatory T-cell Ratio and Induces TumorRegression 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, LaurianeGamand, 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 asignificant hurdle to immune checkpoint inhibitor responses. Bothsoluble factors and specialized immune cells, such as regulatory Tcells (Treg), are key components of active intratumoral immuno-suppression. Inducible costimulatory receptor (ICOS) can be highlyexpressed in the tumor microenvironment, especially on immuno-suppressive Treg, suggesting that it represents a relevant target forpreferential depletion of these cells. Here, we performed immuneprofiling of samples from tumor-bearing mice and patients withcancer to demonstrate differential expression of ICOS in immuneT-cell subsets in different tissues. ICOS expression was higher onintratumoral Treg than on effector CD8 T cells. In addition, byimmunizing an Icos knockout transgenic mouse line expressing

antibodies with human variable domains, we selected a fully humanIgG1 antibody called KY1044 that bound ICOS from differentspecies. We showed that KY1044 induced sustained depletion ofICOShigh T cells but was also associated with increased secretion ofproinflammatory cytokines from ICOSlow effector T cells (Teff). Insyngeneic mouse tumor models, KY1044 depleted ICOShigh Tregand increased the intratumoral TEff:Treg ratio, resulting inincreased secretion of IFNg and TNFa by TEff cells. KY1044demonstrated monotherapy antitumor efficacy and improvedanti–PD-L1 efficacy. In summary, we demonstrated that usingKY1044, one can exploit the differential expression of ICOS onT-cell subtypes to improve the intratumoral immune contextureand restore an antitumor immune response.

IntroductionThe last decade has seen a paradigm shift in cancer therapies with

the approval of antibodies targeting immune checkpoints. Theseimmune checkpoint inhibitors (ICI) trigger a durable response inmalignancies, including metastatic melanoma, non–small cell lungcancer (NSCLC), head and neck cancer, renal, and bladder cancer (1).However, there is still a high proportion of patients exhibiting resis-tance to ICIs that may benefit from novel combinatory approaches.

Multiple molecular and cellular mechanisms associate with resis-tance to ICIs (2). For example, low incidence of cytotoxic T cells andthe presence of immunosuppressive cells prevent an antitumorresponse. One class of immunosuppressive cells are regulatory T cells(Treg), which block the cytotoxic potential of effector T cells (TEff)

through various mechanisms (3, 4). Thus, high numbers of intratu-moral Tregs negatively correlates with survival and response to treat-ments (5, 6). In fact, Treg depletion modifies the tumor microenvi-ronment (TME) and favors an antitumor response in preclinicalmodels (7–9). For these reasons, Treg cells have been investigated asa prognostic cell type and as therapeutic targets.

The use of therapeutic antibodies for the preferential depletion ofintratumoral Treg cells relies on the identification of a marker pref-erably expressed on these cells. One such potential target is ICOS,which belongs to theCD28/CTLA-4 family (10). UnlikeCD28, ICOS isnot expressed on na€�ve TEff cells but is induced upon T-cell receptor(TCR) engagement (11, 12). Following activation, ICOS is expressed atdifferent levels on different T-cell subtypes where it can engage with itsligand (ICOS-LG, CD275) expressed on antigen-presenting cells.ICOS/ICOS-LG interaction initiates a costimulatory signal that resultsin production of either pro- or anti-inflammatory cytokines (IFNg andTNFabyTEff cells; IL10 expressionbyTreg; ref. 13), thus regulating theimmune cell homeostasis (14, 15). Of relevance, the accumulation ofICOSþ Treg cells in the TME is associated with disease progres-sion (16, 17). In marked contrast, the upregulation of ICOS onCD4 TEff cells associates with better prognosis in patients treated withanti–CTLA-4 (18–21). The relative expression of ICOS varies betweenT-cell subtypes,with intratumoral Treg exhibiting higher ICOS expres-sion followed by CD4þ and CD8þ TEff cells (12, 17, 19, 22). Thisdifferential expression suggests that ICOS represents a relevant targetfor a Treg depletion strategy. In fact, ICOS antibodies with depletingcapability reduce the numbers of ICOSþ Treg cells and induce anantitumor response when combined with a vaccine strategy (7).

In this study, using a transgenicmouse platform (23), we identified afully human ICOS IgG1 antibody called KY1044 that bound to mouse

Kymab Ltd, Babraham Research Campus, Cambridge, United Kingdom.

Note: Supplementary data for this article are available at Cancer ImmunologyResearch Online (http://cancerimmunolres.aacrjournals.org/).

R.C.A. Sainson and A.K. Thotakura contributed equally to this article.

Current address for J. Carvalho, SNIPR BIOME, Copenhagen, Denmark; currentaddress for J. Campbell, Abcam plc, Cambridge, United Kingdom; and currentaddress for I. Kirby, ADC Therapeutics, London, United Kingdom.

Corresponding Author: Richard C.A. Sainson, Kymab Ltd, Babraham ResearchInstitute, Bennet Building, Cambridge, Cambridgeshire CB22 3AT, UK. Phone:44 (0)1223 833301; E-mail: [email protected]

Cancer Immunol Res 2020;8:1568–82

doi: 10.1158/2326-6066.CIR-20-0034

�2020 American Association for Cancer Research.

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and human ICOS. In vitro, we established that KY1044 has costimu-latory effect on ICOSLow TEff cells and a depleting effect on ICOSHigh

cells. In vivo, KY1044 induced preferential depletion of ICOSHigh cells,improved the TEff to Treg ratio in the TME and increased secretion ofproinflammatory cytokines, resulting in antitumor efficacy.

Materials and MethodsCell lines used in the study

All cell lines (except MC38) were obtained from LGC standardsATCC. CT26.WT (mouse colon carcinoma, ATCC catalog no. CRL-2638), B16-F10 (mouse melanoma, ATCC catalog no. CRL-6475) andMJ (G11; Human T-cell lymphoma, ATCC catalog no. CRL-8294)cells were acquired between July and November 2015, J558 (mouseplasmacytoma, ATCC catalog no. TIB-6) cell line was obtained inMay2016. A20 (A-20; mouse B-cell lymphoma, ATCC catalog no. TIB-208), and EL4 (mouse lymphoma, ATCC catalog no. TIB-39) cellswere obtained in May 2017. MC38 cells (mouse colon adenocarcino-ma) were obtained in August 2017 from NCI under a license agree-ment. The specific pathogen-free status of these cells was confirmed byPCR screening for mouse/rat comprehensive panel (Charles River).MC38, J558, and B16-F10 cells were cultured in antibiotic-free DMEM(Gibco, catalog no. 41966-029)þ 10% FBS (Gibco, catalog no. 10270)complete cell culture media. CT26.WT cells were cultured in antibi-otic-free RPMI (Gibco, catalog no. 2187) þ 10% FBS complete cellculture media. A20 cells were cultured in antibiotic-free RPMIþ 10%FBS þ 0.05 mmol/L 2-mercaptoethanol (Gibco, catalog no. 21985-023) complete cell culture media. MJ cells were culture in IMDM(Gibco, catalog no. 12440053) þ 20% FBS (I20 media). The passagenumber of cells were kept below 10 generations.

Gene expression analysisThe Cancer Genome Atlas data analysis

The RNA-sequencing data from The Cancer Genome Atlas(TCGA) consortium was downloaded from the UCSC Xena platform(TCGAPan-Cancer, 10,460 samples in total; ref. 24). Samples classifiedas nontumor tissue (727 samples) were excluded as were leukemias,lymphomas, and thymomas (combined 341 samples). Single-samplegene-set enrichment analysis (ssGSEA; refs. 25, 26) was performed forICOS and FOXP3. Samples were grouped by primary disease and thessGSEA scores for each group were compared across the primarydisease groups.

ScRNA sequencingPeripheral blood mononuclear cell (PBMC) and tumors from 5

NSCLC donors were processed using the BD Rhapsody system.Sequencing libraries were generated using the Immune ResponseHuman targeted panel and sequenced using a 2 � 75 bp paired-end run on the Illumina HiSeq 4000 System. Reads were processed byapplying the BD Rhapsody processing pipeline to generate cell countmatrices. The counts were filtered, normalized, and visualized using Rand Bioconductor packages for scRNA-seq data (27–30). Cell type–specific gene sets were constructed by performing a literature searchand cells were classified into one of 27 cell types (SupplementaryTable S1) using the R package AUCell (31). The sequences have beensubmitted to ArrayExpress (accession E-MTAB-9451).

Flow cytometryMouse studies

Tumors and spleens were harvested fromCT26.WT tumor–bearingmice. Tumors were dissociated into single-cell suspensions usingMiltenyi Tumor Dissociation Kit (catalog no. 130-096-730). Spleens

were placed into C-tubes (Miltenyi Biotec, catalog no. 130-096-334)dissociated into single-cell suspensions using gentle MACS dissocia-tor. Tumor samples were filtered through 70-mm nylon filters. Spleenssamples were filtered through 40-mmnylon filters. Red blood cells werelysed using RBC lysis buffer (Sigma, catalog no. R7757). For flowcytometry profiling, 2� 106 tumor samples or 1� 106 spleen sampleswere plated in 96-well plate (Sigma, catalog no. CLS3957). Cellsuspensions were preincubated with Live/Dead fixable YellowDead Cell Stain Kit (Invitrogen, catalog no. L34959). Prior to antibodylabeling, cells were incubated with Fc receptor blocking solution (anti-CD16/CD32 BioLegend, catalog no. 101320) at 4�C for 10 minutes.The staining was performed at 4�C for 30 minutes using fluoro-chrome-conjugated anti-mouse antibodies. Intracellular and intra-nuclear staining was performed using Foxp3 staining buffers (ThermoFisher Scientific, catalog no. 00-5523-00). All flow cytometry anti-bodies or isotype controls were purchased from Thermo FisherScientific. Antibodies used include: anti-CD45 (30-F11), anti-CD3(17A2), anti-CD4 (RM4-5), anti-CD8 (53-6.7), anti-CD25 (PC61.5),anti-ICOS (7E.17G9), anti-Foxp3 (FJK-16s), anti-CD69 (H1.2F3).For the T-cell cytokine staining, single-cell suspensions wereplated at 1� 106 cells per well in RPMIþ 10% FBS cell culture mediawith 1� Brefeldin A solution (eBioscience, catalog no. 00-4506-51) for4 hours at 37�C 5% CO2. Cells were surface stained as above andsubsequently fixed/permeabilized for intracellular staining with anti-IFNg (XMG1.2) and anti-TNFa (MP6-XT22). All flow cytometry datawas acquired using Attune FxT flow cytometer and data were analyzedusing FlowJo software V10.

Monkey studyBlood samples (0.1 mL) were collected into sodium EDTA or

lithium heparin tubes, mixed, and red blood cells (RBC) were lysed.The leukocytes were washed with FACS buffer (PBS with 2% FBS),stained with antibody cocktail by incubation in the dark for 45minutesat room temperature. Tissue samples (lymph node), taken fromanimals at termination were mechanically disrupted (Medimachine,BectonDickinsonGmbH), single-cell filtered, and stained as for blood.Intracellular FoxP3 staining was carried out by permeabilization with10� FACS lysing solution (1:5withAqua dest.þ0.1%Tween-20) priorto incubation in the dark, 45 minutes at room temperature. Allantibodies or isotype controls were purchased from BD Biosciences,eBioscience (via Fisher Scientific GmbH) or BioLegend. Antibodiesused included: anti-CD3 (SP34), anti-CD20 (L27 or 2H7), anti-CD14 (M5E2), anti-CD4 (M-T477), anti-CD8 (SK1), anti-CD28(CD28.2), anti-CD25 (M-A251), anti-Foxp3 (PCH101), anti-CD95(DX2), and anti-CD185/CXCR5 (MU5UBEE). After staining, cellswere washed and fixed in 1� BD Stabilizing Fixative. Lymphocyteswere gated by forward scatter (FSC), sideward scatter (SSC),and CD45. Multicolour flow cytometric analysis was performedusing the following leukocyte phenotypic characteristics: CD4þ Thcells: CD3þ/CD4þ/CD8�/CD14�/CD20�; CD8þ cytotoxic T cells:CD3þ/CD4�/CD8þ/CD14�/CD20�; Total memory CD4 T cells:CD28þ/DIM/� CD95þ/high; Follicular Th cells: (CD4þ/CD185þ), andRegulatory CD4 T cells: CD25þ/FoxP3þ. Acquisition of flow datawas performed on a FACSVerse flow cytometer (BD Biosciences)and relative percentages of each of these subpopulations weredetermined using FlowJo software. Fifty thousand events werecounted for all analyses.

Human samples studiesIn accordance to the declaration of Helsinki and upon approval by

the Hamburg Medical Association's ethic commission (PV5035),

Antitumor Activity Triggered by an Anti-ICOS Antibody

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NSCLC tissue and whole blood from the same patients were obtainedfrom consented subjects, with ethical approval for analysis of protein,RNA, and DNA content. The NSCLC tumor samples were dissociatedinto single-cell suspensions using enzymatic digestion, while wholeblood was processed into PBMCs using density centrifugation, allspecimens were cryopreserved in Gibco Recovery cell culture freezingmedium before used. Alongside NSCLC specimens, PBMCs from 5healthy individuals were used. Flow cytometry was performed aftercells were thawed and incubated in RPMI1640 þ 10% FBS supple-mented with 100 U of RNase free DNase at 37�C 5% CO2 for 30minutes. Cell suspensions were pre-incubated with Live/Dead fixableYellow Dead Cell Stain Kit (Invitrogen, catalog no. L34959) andFc receptor blocking solution (BioLegend, catalog no. 422302),before staining with fluorochrome-conjugated anti-human antibodiesspecific to anti-CD3 (UCHT1)/anti-CD45 (2D1), anti-CD4 (2A3),anti-CD8 (RPA-T8), anti-CD25 (MA251), anti-CD45RA (H100),anti-ICOS (C398.4A). Cells were incubated for 1 hour at 4�C, washed,and fixed overnight at 4�C with eBioscience intracellular fixationand permeabilization buffer (catalog no. 00-5523-00). Anti-FoxP3(236A-E7) staining was then performed in permeabilization bufferfor 1 hour at 4�C before washing and resuspending cells in DPBS(Gibco). All flow cytometry data was acquired using Attune NxT flowcytometer and data was analyzed using FlowJo software V10.

Immunofluorescence and digital pathologyAn IHC protocol using the Ventana Discovery Ultra platform

(Roche) was developed for costaining of ICOS and FoxP3. Themethodwas optimized in FFPE tonsil tissue, using clone D1K2T for ICOS(Cell Signaling Technology) and clone 236A/E7 (Abcam) for FoxP3.Briefly, the FFPE tissue sections underwent deparaffinization and apH 9-based antigen retrieval step for 64 minutes. The slides wereincubated with anti-FOXP3 antibody or the isotype control at 40 mg/mL for 60 minutes. An anti-mouse HRP (Roche catalog no. 760-4310)labeled detection antibody was applied for 16 minutes and an auto-mated DAB detection (Roche catalog no. 760-159) was carried out asper manufacturer's recommendations. The slides were then incubatedwith the anti-ICOS antibody or the isotype control at 1 mg/mL for60 minutes. An anti-rabbit HRP (Roche, catalog no. 760-4311) labeleddetection antibody was applied for 28 minutes and an automatedpurple detection was carried out as per manufacturer's recommenda-tions. Finally, the slides were counterstained with hematoxylin for12minutes and bluing reagent for 8minutes.Methodswere applied forthe staining of tumor microarrays (TMA) from patients with cervical(CR2088), esophageal (ES2082), lung (LUC2281), head and neck(HN802a), gastric (STC2281), and bladder (BL2081a) cancer (all USBiomax, Inc). The slides were scanned (20� equivalent magnification)and quantified using the Indica Labs Halo platform to determine thenumber of ICOS/FoxP3 single and double positive cells per mm2.

In vitro antibody-dependent cellular cytotoxicity assaysAntibody-dependent cellular cytotoxicity (ADCC) was first tested

in vitro using a reporter bioassay (Promega, catalog no. G7102). Inbrief, the assay uses a Jurkat reporter cell line expressing humanFcgRIIIa V158 and NFAT-induced luciferase. Following engagementwith the Fc region of a relevant antibody bound to a target cell, theFcgRIIIa receptors can activate an intracellular signals resulting inNFAT-mediated luciferase activity that can be quantified via a lumi-nescence readout. CHO cells expressing either human, mouse, rat, orcynomolgus ICOS were used as target cells and coincubated withJurkat reporter cells at a 5:1 ratio. Serial dilutions of KY1044 or isotypeIgG1 control were added to the culture plates, incubated at 37�C

overnight and the luciferase activity was measured using the Bio-GloLuciferase Assay System (Promega, catalog no. G7940) on the EnVi-sion Multilabel Plate Reader (Perkin Elmer). Graph data were nor-malized to background and plotted versus log10 antibodyconcentration.

For the primary ADCC cell assay, human natural killer (NK) cellswere purified using the NK Cell Isolation Kit (StemCell Technologies,catalog no.17955). ICOS-transfected CCRF-CEM (ICOS CEM, targetcells) were preloaded with the fluorescence-enhancing ligand(BATDA) for 30 minutes in the dark at 37�C. The cells were loadedand washed in the presence of an inhibitor of organic anion trans-porters (1 mmol/L Probenecid) to avoid spontaneous dye release fromcells. KY1044 was diluted (1:4 dilutions, 10 points, starting from 33.3nmol/L) in assay buffer. The target ICOS CEM cells (50 mL/well),effector cell (50 mL/well), and reagent dilutions (50 mL/well) werecocultured with 50 mL of the diluted antibody at 37�C, 5% CO2 for 2–4 hours (NK cells to target ratio was 5:1). A digitonin-based lysis buffer(Perkin Elmer) was used to determine complete target cell lysis (100%).

In vitro costimulation assaysFor the MJ cell assays, KY1044 human IgG1 was presented in

three different formats: plate-bound, soluble, or soluble with or withoutF(ab')2 Fragments (Fc linker, catalog no. 109-006-170, Jackson Immu-noresearch). Plate-bound KY1044 and the hIgG1 isotype control werediluted 1:2 in PBS (concentrations ranging from 10 mg/mL to 40 ng/mL,10points).One-hundredmicroliters of diluted antibodieswere coated intriplicate into a 96-well, flat-bottom plate (Corning EIA/RIA plate)overnight at 4�C and then washed. MJ cells (15,000 cells/well) wereadded to precoated wells. For the soluble/cross-linked experiment,KY1044 and the isotype control were serially diluted 1:2 in I20 media(soluble Ab) or in I20 media containing 30 mg/mL of F(ab')2 Fragments(cross-linked Ab) to give an 2� Ab stock concentrations ranging from20mg/mL to 80 ng/mL (10 points). Fiftymicroliters of diluted antibodieswere added to96-wellwith 50mLofMJ cells (3� 105/mL). For the assaysthe cells were cultured for 72 hours at 37�C/5% CO2 and cell-freesupernatants were then collected and used to perform IFNg ELISAusingthe Human IFNg DuoSet assay (R&D Systems, DY285).

For the primary T-cell assays, leukocyte cones were obtained (HTAIRAS project number 100345). PBMCs were isolated by density-gradient centrifugation. T lymphocytes were them purified using theStemcell EasySep Isolation kit (catalog no. 17951). For ICOS induc-tion, isolated T cells were cultured at 2� 106/mL in R10media (RPMI10% heat-inactivated FBS) in the presence of 20 mL/ml of DynabeadsHuman T-Activator CD3/CD28 (Life Technologies, catalog no.111.31D). These activated primary T cells were tested as for the MJcell assays in three different formats: plate-bound (5 mg/mL), soluble(15 mg/mL), or soluble plus F(ab')2 Fragments cross-linker. T-cellsuspensionwere added to antibody-containing plates to give a final cellconcentration of 1� 106 cells/ml and cultured for 72 hours at 37�Cand5% CO2 until IFNg ELISA quantification. For the three-step culture(stimulation-rest-costimulation assay), the T cells were prestimulatedby Dynabeads for 3 days to induce ICOS before being rested for 3 daysto reduce their activation levels. These stimulated/rested T cells werethen cultured with KY1044 in the presence or absence of an anti-CD3antibody (clone UCHT1, eBioscience) to assess the requirement ofTCR engagement. The effect of ICOS costimulation was assessed after72 hours by measuring the levels of IFNg and TNFa present in theculture (MSD multiplex assay).

For the gene expression analysis, T cells were harvested from the3-step culture (stimulation–rest–costimulation assay) after 6-hourplate-bound antibody stimulation. Total RNA was extracted from the

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cell pellets with the RNeasy Micro Kit (Qiagen), quality controlled onthe Agilent 2100 Bioanalyzer (Agilent Technologies) and subjected toSE50 sequencing following mRNA enrichment (BGI). The sequencereads were aligned using kallisto (32) and further processed usinglimma (33) and metascape (34) and GSVA (26). The sequencing datahas been deposited into ArrayExpress (E-MTAB-9500).

NFAT Luciferase assaysLuciferase reporter assays

Jurkat-Lucia NFAT cells (Invivogen, catalog no. jktl-nfat) stablyexpressing luciferase under the control of NFAT response elementswere further transfected with either the human Icos gene sequenceor a chimeric construct of Icos fused with CD247 (CD3z). Followingselection of stable transgene integration, a luciferase activity bio-assay was performed. High binding 96-well assay plates were coatedovernight with either anti-CD3 (UCHT-1, eBioscience, catalog no.16-0038-85; 10 mg/mL), anti-ICOS (C398.4A, BioLegend catalog no.313512; 10 mg/mL), isotype control (HTK888, BioLegend, catalogno. 400902; 10 mg/mL), anti-CD3 þ anti-ICOS (5 mg/mL each) oranti-CD3 þ isotype control (5 mg/mL each). Transgene expressingcells or control cells (untransfected Jurkat-Lucia NFAT cells) wereseeded at 50,000 cells/well. Following overnight incubation lucif-erase activity was measured by adding BioGlo reagent (Promega,catalog no. G7940) and reading luminescence on a plate reader.Luciferase activity was normalized and scaled to 100% by compar-ing to cells stimulated using Cell Stimulation Cocktail (eBioscience,catalog no. 00-4970-93).

PD-1/PD-L1 blockade luciferase reporter bioassayThe thaw-and-use format of the PD-1/PD-L1 Blockade Bioassay

(Promega, catalog no. CS187111) was performed in a 96-well plateformat according to the manufacturer's instructions. Briefly, PD-L1–expressing APC/CHO-K1 cells were plated on day –1 in cell recoverymedium and incubated overnight at 37�C. The following day, assaymedia was replaced with 10-point serial dilutions of AbW IgG1 orisotype control, prior to addition of PD-1 expressing NFAT Jurkateffector cells. Cells were incubated at 37�C for 6 hours, followed by theaddition of the BioGlo reagent and luminescence signal quantified on aplate reader.

Ligand neutralization assaysFull-length human PD-L1 sequence (Uniprot sequence ID:

Q9NZQ7-1) was codon optimized for mammalian expression andcloned into an expression vector under the CMV promoter flanked by30 and 50 piggyBac-specific terminal repeat sequences, facilitatingstable integration (puromycin selection) into the genome of trans-fected CHO cells (35). Stable PD-L1–expressing CHO cells werecoincubated with a 30 nmol/L concentration of biotinylated ligand(either PD-1 or CD80) and serial dilutions of antibody (150 nmol/L to0.0076 nmol/L of either AbW hIgG1 or control hIgG1) for 1 hour at4�C. The cells were then washed, ligand binding was detected usingAlexa Fluor 647–labeled streptavidin and the samples were acquiredon a BD FACSArray Bioanalyzer.

Mouse T-cell activation assayA T-cell activation assay using the OVA-specific DO11.10 mouse

T-cell hybridoma was used as described previously (36). In brief,human PD-L1–transfected LK35.2 cells (antigen-presenting cells)were incubated with mouse PD-1–expressing DO11.10 cells in thepresence of OVA peptide. Serial dilutions of AbW IgG1 or isotypecontrol were added and incubated at 37�C overnight. The following

day mouse IL2 release was quantified using the Mouse IL2 DuoSetELISA (R&D Systems, catalog no. DY-402).

Antibody internalization assayThe ability of KY1044 to be internalized was assessed by capturing

time course images on the IncuCyte ZOOM Live Cell ImagingPlatform (Sartorius). Briefly, MJ cells endogenously expressing ICOSwere first labeled with the Incucyte CytoLight Rapid Green Reagent(Sartorius) to enable detection of the cytoplasm. Serial dilutions ofeither KY1044, a CD71 antibody (positive control; Sigma, catalog no.SAB4700520-100UG) or a human IgG1 isotype control (negativecontrol) were preincubated with secondary anti-human or anti-mouse antibodies covalently labeled with a pH-sensitive dye (pHrodoRed; Thermo Fisher Scientific, catalog no. P36600) and then added tothe target cells. Antibody internalizationwas tracked for up to 48 hoursand the areas under the time-course curves plotted as a function ofantibody concentration.

MiceAll mice in vivo work was performed in the UK under Home Office

licence (70/08759). All procedures were conducted in accordancewith the United Kingdom Animal (Scientific Procedures) Act 1986and associated guidelines, approved by institutional ethical reviewcommittees (Babraham Institute AWERB). Eight- to 10-week-oldwild-type female Balb/C or C57BL/6J mice were sourced from CharlesRiver UK Ltd and housed in transparent plastic cages with wire covers(391W� 199 L� 160 Hmm, floor area: 500 cm2) containing Grade 6Wood Chip that can be replaced with Lignocell (IPS Product SuppliesLtd, BCM IPS Ltd.; WC1N 3XX) and bale shredded nesting material(IPS Product Supplies Ltd, BCM IPS Ltd.; WC1N 3XX). Four to fivemice were housed per cage in a room with a constant temperature(19�C–23�C) andhumidity (40%–70%) and a 12-hour light–dark cycle(lighting from 7 am to 7 pm). Mice were provided with pellet food(CRM(P), Specialist Diet Services) and RO water ad libitum using anautomatic watering system.

Tumor cell implantation and tumor measurementEarly passage (belowP10)MC38 (3� 106 cells), J558 (1� 106 cells),

CT26.WT (1� 105 cells), A20 (5� 106 cells), EL4 (1� 104 cells), andB16-F10 (1 � 105 cells) tumor cells were prepared in either PBS orMatrigel (Corning, 354230) and the resulting cell suspension wereinjected subcutaneously into the flank of the mice (study day 0). Priorto tumor cell implantation, mice were anesthetised with isoflurane andthe right flank of the mice was shaved. For the implantation, 100 mL ofthe cell suspension were injected using 25 G needles (BD MicrolanceTM 3. VWR 613-4952). Cell numbers and viability (required to beabove 90%)were determined preimplantation by the trypan blue assay.Tumor growth was measured using digital calipers three times a weekuntil end of the study. The tumor volumes (mm3) was estimated usinga standard formula: (L�W2)/2 (with L being the larger diameter, andW the smaller diameter of the tumor). All data were plotted usingGraphPad Prism V10.

Antibody dosingBoth KY1044 mIgG2/mIgG1 and anti–PD-L1 (AbW) mIgG2a/

mIgG1 were produced by Kymab Ltd. The PD-1 (clone RMP1-14)mAbwas purchased fromBioXcell. Antibodies were dosed between 0.1and 10mg/kg or byflat dose of 20 to 200mg/dose via the intraperitonealroute. For the efficacy and pharmacodynamic studies, the treatmentgroups were not blinded. The in vivo depletion of CD8þ and CD4þ Tcells were conducted using a flat dose 200 mg of anti-CD8a (53-6.7)






and/or anti-CD4 (GK1.5). Dosing was performed twice a week for3 weeks starting from day 3 following tumor cell implantation. TheCD8þ andCD4þT-cell depletionwas determined byflow cytometry oftumor, spleen, tumor draining lymphnode (inguinal lymphnode), andblood cells using an anti-CD3 antibody (17A2).

Cynomolgus monkey studyThe effects of KY1044 in nonhuman primates (NHP) were studied

as part of a repeat dose toxicity study. Na€�ve male cynomolgusmonkeys (Macaca fascicularis) were obtained from a certified supplier,group housed, allowed access to water ad libitum and fed on a pelleteddiet for monkeys supplemented with fresh fruit and biscuits. Animalsranged from 4 to 7 years old andweighed 3–6 kg at time of dosing. Fourgroups of three cynomolgus monkeys received weekly intravenousdoses of KY1044 (slow bolus over approximately 1 minute) for amonth (5 doses in total) at doses of 0 (vehicle control; PBS pH 7.4), 10,30, or 100 mg/kg at a dose volume of 2 mL/kg. Blood samples weretaken at 1 or 2 timepoints prior to dosing and atmultiple timepoints upto 29 days after the first dose for measurement of serum KY1044 usinga qualified ELISA assay, ICOS occupancy on CD4þ cells in blooddetermined using a validated flow cytometry method and/or immu-nophenotyping of whole blood (described above). For the receptoroccupancy, free ICOS was measured by using a competing anti-ICOS that does not displace KY1044 while an anti-human IgGwas used to detect KY1044 directly or after saturating the cells withan excess of KY1044 to quantify bound and total ICOS, respectively.Scheduled necropsies were conducted 1 day after the final dose(day 30) and spleen and mesenteric lymph nodes were taken forimmunophenotyping.

NHP ethics statementThe cynomolgus monkey study was conducted at Covance preclin-

ical ServicesGmbH in strict accordancewith a study plan reviewed andapproved by the local Institutional Animal Care and Use Committee(IACUC) of the testing facility and the German Animal Welfare Act.The study was performed according to DIRECTIVE 2010/63/EU OFTHE EUROPEAN PARLIAMENT AND OF THE COUNCIL ofSeptember 22, 2010 on the protection of animals used for scientificpurposes and the Commission Recommendation 2007/526/EC onguidelines for the accommodation and care of animals used forexperimental and other scientific purposes (Appendix A of Conven-tion ETS 123). The study was in compliance with the KymabWorkingPractice on Experiments involving Animals and was approved by theKymab Ethics Committee.

Statistical analysesUnless stated otherwise in the figure legends, the efficacies observed

for the different treatment groups were compared using either t test orone-way ANOVA and Tukey multiple comparison post hoc test.Differences between groups were significant at a P < 0.05. On thegraphs, �, P < 0.05; ��, P < 0.01; ��, P ≤ 0.001; ��, P ≤ 0.0001.Statistical analyses were performed with GraphPad Prism 10.0(GraphPad Software, Inc.).

ResultsHigh ICOS expression on intratumoral Treg

Ovarian, gastric, and liver cancers, are highly infiltrated with ICOSþ

Treg cells (16, 17, 37) and this results in poor prognosis (17, 38). Toassess ICOS expression on intratumoral T cells and to identify

additional tumor types containing high ICOSþ Treg cells, we analyzedthe content of T cells in tumors using different approaches.

Using the TCGA datasets, we identified tumors originating fromthe head and neck, stomach, cervix, thymus, testis, skin, and thelungs as indications with high mRNA expression of ICOS andFOXP3 (a marker of Treg; Fig. 1A). To establish which types ofcells expressed ICOS and/or FOXP3, we assessed these markers atthe mRNA and protein levels by single-cell transcriptomics andFACS analysis, respectively. We generated paired PBMCs andtumor scRNA-seq data for 79,544 cells from 5 patients with NSCLC(Supplementary Table S2), which we subsequently stratified into27 cell subtypes based on published immune gene expressionsignatures. As expected, genes such as foxp3, ccr8, il2ra, tnfrsf4were highly expressed in intratumoral Tregs (Fig. 1B). ICOSmRNAexpression was higher in Tregs than in other T-cell subsets. Finally,ICOS expression was higher in the TME than in the periphery,whereas the expression pattern in both compartments followed thegeneral trend of: Treg > CD4non-Treg > CD8 > Other (Fig. 1B). Byflow cytometry analysis (Fig. 1C; Supplementary Fig. S1A), ICOSexpression was higher on Treg (CD4þ/FOXP3þ) than on the otherT-cell subsets (CD8þ and CD4þ/FOXP3�). ICOS protein was alsomore expressed in the TME than on PBMCs (P < 0.001 for Tregs).No differences in ICOS expression between PBMCs from healthydonors and patients with NSCLC were observed. Finally, T-cellimmunophenotyping of CT26.WT tumor–bearing mice revealedthat ICOS was also more expressed in the TME than in the spleen.This analysis also confirmed higher ICOS expression on Treg thanon CD4/CD8 TEff cells (Supplementary Fig. S1B–S1D).

ICOS immunostaining on tumor tissue microarrays (from esoph-ageal, head and neck, gastric, lung, bladder, and cervical cancerbiopsies) confirmed the upregulation of ICOS in the TME(Fig. 1D). The costaining for ICOS and FOXP3 (Fig. 1E), alsohighlighted a high number of ICOSþ Treg cells for the six selectedindications, with only bladder cancer demonstrating a lower density ofICOS/FOXP3-positive cells (Fig. 1F).

Altogether, our data confirmed that ICOS was not homogenouslyexpressed on the T-cell subsets andwas induced in the TME, especiallyon the surface of Treg. In addition, we identified indications with highlevels of intratumoral ICOSþ Treg cells including esophageal, headand neck, gastric, lung, and cervical cancers.

KY1044: a human ICOS IgG1 antibody with both depleting andagonistic functions

The strong expression of ICOS on intratumoral Tregs suggests thatthe targeting of these cells with an effector enabled ICOS antibodycould lead to their preferential depletion (e.g., via ADCC and/orADCP). The level of target expression and the amount of antibodybound to a target are key parameters influencing cell killing byADCC (39). Therefore, it is expected that ICOSLow cells (e.g., CD8þ

TEff cells) will be less sensitive to depletion than ICOSHigh Treg, butcould be sensitive to costimulation if the antibody also harborsagonistic properties (via target clustering through FcgR receptors;refs. 40, 41). With this in mind, we identified a fully human mono-clonal IgG1 antibody called KY1044. KY1044 was generated in atransgenic mouse line for the human immunoglobulin genes in whichthe endogenous Icos gene was knocked-out. Lack of endogenous ICOSexpression was aimed to facilitate the generation of a mouse crossreactive antibodies. KY1044 binds ICOS from different species(human, Cynomolgus monkey, rat, and mouse) with similar affinity(a Kd below 3 nmol/L for the Fab, Supplementary Fig. S2A).

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KY1044-dependent ADCC was assessed using a luciferase report-er assay. As shown in Fig. 2A, KY1044 significantly induced theluciferase signal (EC50 of 0.15 nmol/L, n ¼ 3). A similar EC50 wasobtained against mouse ICOS (0.53 nmol/L, n ¼ 3), rat ICOS (0.48nmol/L, n ¼ 3), or cynomolgus monkey ICOS (0.22 nmol/L, n ¼ 3).We validated the data using human NK cells as effector cells.When incubated with KY1044 and CEM cells expressing hICOS(5:1 effector:target cell ratio), these NK cells induced potent ADCC-

mediated killing (EC50 of 5.6 pmol/L, n ¼ 8, Fig. 2B). Altogether,these data confirmed that KY1044 triggers ADCC of ICOSHigh-expressing cells.

The agonistic potential of KY1044 was assessed using the MJ [G11]CD4þ cells that we demonstrated express ICOS and do not require aprimary stimulatory signal (e.g., TCR engagement) for cytokineproduction. KY1044 was either precoated on culture plates (mimick-ing cross-presentation/clustering) or used in solution with or without

Figure 1.

ICOS expression in cancer Tregs. A, Density plots showing the combined Icos and Foxp3 expression in solid tumors (TCGA datasets). Combined expression wasscored using ssGSEA. The median ssGSEA score (marked by vertical ticks) is shown. The TCGA standard cancer type abbreviations were used. B, Immune responsegenes 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 (n¼ 5). Each gene was scaled individually across all cells of thedataset, and themean scaled expression for eachof 17 T-cell subtypes is presented. Right, scatter plots showing ICOSmRNAexpression in Treg, CD4non-Treg, CD8, andall other T cells. Eachdot corresponds to a single cell. Full lines indicate themean ICOSgene expression of the total cell compartment, and the dashed lines indicate themode (density peak) of ICOSþ cells.C,Relative ICOS expression (flow cytometry) on CD4þ, CD8þ, and Treg (CD4þ/FOXP3þ) in healthy donor PBMCs (n¼ 5), NSCLCtumor samples (n¼ 5), and matched NSCLC patient PBMCs (n¼ 4; two-way ANOVAwith Tukeymultiple comparison). D, Graph showing the density of ICOSþ cellsper mm2 in tumor cores (n¼ 995) from six indications and matching healthy tissues (n¼ 48; Mann–Whitney unpaired t test). E, Example of ICOS/FOXP3 staining ofgastric tumor core showing (i) original whole core image with ICOS staining in purple and FOXP3 in brown; (ii) classifier analysis overlays showing tissue region ofinterest (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 permm2 from the tumor cores (n¼ 995) from the six indications.






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 withFcgRIIIA Jurkat reporter cells and ICOSþCHO cells as target cells. B, KY1044 induced ADCC in a human primary NK cell assay. KY1044 or isotype control wascoincubatedwith dye-loaded ICOSþCEM target cells andNKcells for 4 hours. ADCCactivitywas quantifiedbymeasuring SpecificDyeRelease upon target killing andnormalized to percentage of maximum lysis compared with the positive control, a digitonin-based lysis buffer (mean of triplicates� SEM). For A and B, the verticaldotted line indicates 100 ng/mL. Plate-bound (C) and cross-linked soluble (D) KY1044 induced the release of IFNg fromMJ cells. For C andD, the vertical dotted lineindicates 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 primaryT 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; n¼ 10) or with either15 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, Cytokineproduction byKY1044 requires anti-CD3. Levels of TNFa in T-cell culturewith 5mg/mLof KY1044or isotype control IgG1 (plate bound; n¼ 8) in the presence or not ofanti-CD3 (Wilcoxon statistic test for E, F, andG).H,RNA-sequencing analysis comparing T-cell stimulationwith either KY1044þ 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)Metascapepathway enrichment analysis of the differentially expressedgenes. Enrichedgene setswere rankedon the basis of the significance of theirPvalues. (iii) Heatmap showing unsupervised clustering of samples based on the expression levels of the top gene ontology terms from the Metascape enrichmentanalysis. IC, isotype control.

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addition of a secondary cross-linking antibody. While soluble KY1044did not induce IFNg production, plate-bound, and cross-linkedKY1044 effectively induced IFNg secretion (EC50 of 10.5 nmol/L �2.7 nmol/L; n ¼ 2 and 0.50 nmol/L � 0.18 nmol/L, n ¼ 3,respectively; Fig. 2C and D). This agonism potential was confirmedusing primary T cells preactivated with anti-CD3/CD28 (to induceICOS expression; Supplementary Fig. S2B) and cultured with KY1044.Plate-bound (Fig. 2E) and cross-linked (Fig. 2F) KY1044 inducedIFNg production in primary cells. KY1044-dependent IFNg secretionwas significantly higher than for the isotype-treated cells in both plate-bound (2.4 � 0.5-fold at 5 mg/mL mAb, P < 0.01) and cross-linkedassays (2.5 � 0.2-fold at 15 mg/mL mAb, P < 0.01). Importantly, athree-step stimulation–resting–costimulation experiment confirmedthat TCR engagement was required for KY1044-dependent upregula-tion of cytokines (such as TNFa; Fig. 2G).

Finally, a transcriptomic analysis of CD3þ T cells was performedfollowing 6 hours of combined anti-CD3 and KY1044 costimulation.In line with previous reports (14), we observed cytokine upregulation,most notably IFNG, IL10, and IL4 (Fig. 2H). Gene set enrichmentanalysis confirmed that KY1044 induced genes involved in cytokine–cytokine receptor interactions and and lymphocyte activation(Fig. 2H). Of relevance, no significant change in genes associatedwith proliferation and cell-cycle progression were observed. However,in agreement with a previous report (42), the analysis showed anenrichment of genes involved in cell locomotion, adhesion, anddifferentiation. This finding also reflected our observations of MJ[G11] CD4þmorphology changes in response to anti-ICOS (C398.4Aor KY1044; Supplementary Fig. S2C; Supplementary Movies S1 andS2). Even though ICOS signaling depends on the PI3K/AKT/mTORaxis (43), following KY1044 agonism, we noticed an overrepresenta-tion of genes downstream of the nuclear factor of activated T cells

(NFAT; Fig. 2H), as seen previously (44). NFAT-dependent ICOSagonism was confirmed using reporter cell lines (SupplementaryFig. S2D). Target-mediated internalization of KY1044 was limited(Supplementary Fig. S2E).

Altogether the data presented here demonstrated that KY1044 has adual mechanism of action with both depleting and costimulatoryproperties.

KY1044 (mIgG2a) monotherapy blocks tumor growth inlymphoma/myeloma tumor models

KY1044 cross-reactivity tomouse ICOS facilitated the in vivomousepharmacology work for which the antibody was reformatted as amouse IgG2a (the effector enabled format in mouse). Within theTCGA datasets, ICOS expression was high in diffuse large B-celllymphoma (Supplementary Fig. S3). On the basis of the role ofICOS/ICOS-LG signaling in the generation and maintenance ofgerminal centres (10, 45) and on the antitumor efficacy of ICOSantibodies in lymphoma (46), we assessed the effect of KY1044mIgG2a in the ICOS-LGþ A20 tumor lymphoblast B-cell model (47).Mice were dosed (2qW for 3 weeks at 10 mg/kg) starting from 6 daysfollowing tumor cell implantation with saline and KY1044 mIgG2a.KY1044 mIgG2a treatment triggered an antitumor response, withmore than 90% of the mice being free from measurable disease at theend of the study (day 42; Fig. 3A). This efficacy was confirmed inanother B-cell–derived syngeneic model, the J558 plasmacytomamodel in which around 70% of the KY1044 mIgG2a–treated micewere tumor free at the end of the study (Fig. 3B).

Monotherapy efficacy was also assessed in models of hematologicmalignancies (T-cell lymphoma EL4) and models of solid tumors(CT26.WT,MC-38, and B16.F10). Overall, themonotherapy responsewas absent or low in all these models. Mice harboring CT26.WT or

Figure 3.

Effect of KY1044mIgG2 in the A20 and J558 tumormodels.A, Spider plots of a representative experiment showing individual A20 tumor volumes (n¼ 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, Spiderplots 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 salinestarting from day 9 following tumor cell implantation. The dosing days are indicated by vertical dotted lines. The survival curves depicting the control and KY1044mIgG2a groups are shown. Statistics were calculated using log-rank (Mantel–Cox) test. Data are representative of at least two experiments.






MC-38 tumors showed only tumor growth delay (SupplementaryFig. S4).

Altogether, our in vivo studies demonstrated that KY1044 mIgG2awas highly effective as amonotherapy in B-cell–derived tumormodels.

KY1044 (mIgG2a) improved anti–PD-L1 efficacyCT26.WT tumors respond poorly to anti–PD-L1 (48). Because

anti-ICOS and the anti–PD-L1 act on complementary immunepathways, we combined KY1044 mIgG2a with anti–PD-L1 (AbW).AbW binds to mouse PD-L1 with high affinity (Kd ¼ �2 nmol/L)and blocks PD-L1/PD-1 and PD-L1/CD80 interactions (Supple-mentary Fig. S5A). These studies (Fig. 4A) demonstrated a com-plete antitumor response in the majority (ranging from 50% to 90%)of mice treated with the KY1044 mIgG2a (equivalent of 3 mg/kg)and anti–PD-L1 (equivalent of 10 mg/kg) combination. Of rele-vance, we observed a trend (albeit not significant) showing higherantitumor efficacy when anti–PD-L1 was used as mIgG2a than asmIgG1 (Supplementary Fig. S6A). Finally, when KY1044 wasreformatted as a mouse IgG1 (low depleting potential in mouse)and combined with anti–PD-L1, the resulting antitumor efficacywas weaker than the one observed with the corresponding KY1044mIgG2a/anti–PD-L1 combination, thus arguing for the contribu-tion of ICOS-mediated depletion to the stronger antitumor efficacyseen in the CT26.WT model (Supplementary Fig. S6B).

Mice that survived the first CT26.WT challenge in response to theKY1044 mIgG2a/anti–PD-L1 treatment were resistant to a CT26.WTrechallenge but sensitive to the growth of EMT-6 cells (Fig. 4A)suggesting a tumor antigen–specific memory response. Finally, wedemonstrated that the response to the combination was primarily

dependent onCD8þT cells, with the improved survival fully abrogatedwhen CD8þ T cells were depleted (Fig. 4B; Supplementary Fig. S5).Conversely, addition of a CD4-depleting antibody (which depletesboth CD4 Tregs and CD4 non-Tregs) to the combination was stillassociated with tumor growth delay but CD4 depletion eliminated anylong-term survival benefit (Fig. 4B). Altogether, the coadministrationof KY1044 mIgG2a and anti–PD-L1 triggered a durable antitumorimmune response in the CT26.WT tumor model. The combination ofanti–PD-1 (clone RMP1-14) with KY1044 was poorly effective in theCT26.WTmodel (Supplementary Fig. S6C), whereas it was effective inanother model (MC38; Supplementary Fig. S6D).

The KY1044 mIgG2a and anti–PD-L1 combination was also testedin other models (Supplementary Fig. S7A). Although the J558 modelalready responded well to either the anti-ICOS or the anti–PD-L1monotherapy, a 100% response was achieved with the combination inthis model. Similarly, the combination was effective in the MC38model (Supplementary Fig. S7B). The B16F10, 4T1, and EL4 tumormodels did not respond to the combination (Supplementary Fig. S7A).

Collectively, our efficacy studies demonstrated that the cotargetingof ICOS and PD-L1 resulted in a strong combinatorial effect in selectedtumor models, including those in which both monotherapies werepoorly effective.

KY1044 depleted ICOSHigh cells in vivo in mice and NHPsTo assess the mechanism of action of KY1044 in vivo, we first

conducted pharmacodynamic studies in the CT26.WTmodel. Tumor-bearing mice were dosed with KY1044 mIgG2a (ranging from 0.3 to10 mg/kg) on day 13 and day 15 following tumor cell implantation(Fig. 5A). The T-cell content in the tumors and the spleens were

Figure 4.

KY1044mIgG2a improved the efficacy of anti–PD-L1.A, Survival curves ofmice harboringCT26.WT tumors and treatedwith control, KY1044mIgG2amonotherapy at60 mg/dose, anti–PD-L1 mIgG2amonotherapy at 200 mg/dose, and the combination of KY1044mIgG2a 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 sixindependent experiments. Tumor-bearing mice that cleared CT26.WT tumors were randomly allocated to two groups and rechallenged with either CT26.WTor EMT6 cells (right).B,Effect of CD4þ andCD8þT-cell depletion on antitumor efficacy. Mice implantedwith CT26.WT cells (n¼ 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 twoindependent experiments.

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analysed on day 16. Although KY1044 monotherapy did not inducemuch antitumor efficacy in this model (Fig. 4A), KY1044mIgG2a wasassociatedwith intratumoral Treg cell depletion, even at doses as low as0.3mg/kg (Fig. 5B). The antibody format was critical to decrease Tregsas shown with partial and nonsignificant Treg depletion when using amIgG1 (poor depleter; Supplementary Fig. S8A). In addition, anincrease in the CD8þ TEff to Treg cell ratio (known to be associatedwith improved response to ICIs) in the TME was observed at all dosestested (Fig. 5B).However, the highest dose of 10mg/kg showed a lowerCD8þ TEff to Treg cell ratio than the one resulting from the 1 and

3 mg/kg doses. This decrease in the ratio at the highest dose may havebeen caused by the depletory effect of KY1044 (albeit not significant)on CD8þ T cells (Supplementary Fig. S8B). These effects were notobserved in the spleen (Fig. 5C; Supplementary Fig. S8C) potentiallydue to lower ICOS expression in this tissue (Supplementary Fig. S1).We repeated similar experiment by dosing mice once with KY1044mIgG2a. The tumors were then immunophenotyped up to 7 daysposttreatment (Supplementary Fig. S8D). Treg depletion was observedafter a single dose of 0.3 mg/kg; however, at this dose, the incidence ofTregs in the TME recovered quickly back to the level observed in the

Figure 5.

KY1044 depleted ICOShigh T cells in vivo.A, In vivo experimental protocol of the pharmacodynamic study in the CT26.WT tumor–bearingmice. Micewere dosed twicewith saline or KY1044mIgG2a (ranging from 0.3–10 mg/kg) on days 13 and 15, and the tumor and spleenwere harvested on day 16 and the immune cells analyzed byFACS. Effect of KY1044 on Treg depletion (plotted as a percentage of live cells) and on the CD8þ TEff to Treg cell ratio in the TME (B) and in the spleen (C) of treatedmice (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 indicatedin 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-doseof KY1044 human IgG1 at 0, 10, 30, and 100 mg/kg (n ¼ 3 NHP per groups).






control group. A long-lasting Treg depletion and an increase of theCD8 to Treg ratio were observed at higher dose (3 and 10 mg/kg;Supplementary Fig. S8D). Altogether, these data suggest that KY1044mIgG2a preferentially depleted ICOShigh Treg cells and improvedCD8þ TEff to Treg cell ratio in the TME.

ICOS was expressed on cynomolgus monkey CD4þmemory T cells(TM: CD3

þ/CD4þ/CD95high/CD28�/Dim/þ), CD4þ follicular Th cells(TFH: CD3þ/CD4þ/CD185þ) and on Treg cells (CD3þ/CD4þ/CD25þFoxP3þ; Fig. 5D). Similarly, circulating NHP CD8þ T cellsshowed the lowest ICOS expression. We assessed the pharmacody-namic effects of KY1044 hIgG1 in NHP after repeated weekly intra-venous administration at doses of 0, 10, 30, and 100 mg/kg for 4 weeks(5 doses). Exposure and full occupancy of ICOS on circulating CD4þTcells was maintained at all doses (Supplementary Fig. S9A and S9B).Immunophenotyping indicated a decrease in TM and TFH cells inperipheral blood (Fig. 5E and F). KY1044 did not affect circulatingTreg (Supplementary Fig. S9C), which represented less than 3% of

circulating CD4þ cells in monkeys (Fig. 5D). KY1044 did not alterCD8þ T cells in blood (Supplementary Fig. S9D). Finally, we did notobserve a significant decrease in ICOShigh cells such as TM or Treg or inICOSlow CD8þ T cells in the in the lymph node (or spleen) of treatedmonkeys (Supplementary Fig. S9E–S9G). In summary, these datademonstrated that high dose of KY1044 elicited some depletion ofICOShigh cells in peripheral blood but not in lymphoid tissues ofNHPs.

Increased cytokine expression in response to KY1044 in vivoTo demonstrate that KY1044 activates and increases proinflamma-

tory cytokine production in intratumoral TEff cells, we assessed theexpression of the activationmarkers CD69 andCD44 onCD8þT cells.T cells from CT26.WT tumors were analyzed 24 hours after a seconddose of KY1044 mIgG2a. KY1044 mIgG2a induced a significantincrease in CD69 expression (Fig. 6A; P < 0.05 vs. control) at dosesof 1 and 3mg/kg. Similarly, the analysis of CD69/CD44 double positivecells confirmed a significant increase (P < 0.05) of CD8 activation in

A

B

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 pergroups). Measurement of CD69 and CD69/CD44 double-positive cells was performed 24 hours after a second dose of KY1044 mIgG2a (see Fig. 5A). B, Graphsshowing theflowcytometry analysis of intracellular IFNg and/or TNFa expression in intratumoral CD8þ andCD4þT cells fromCT26.WT tumor collected 7days after asingle 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 separateexperiment, we examined KY1044-dependent induction of IFNg andTNFa by CD4þ and CD8þ T cells. Using an intracellular stainingapproach on intratumoral CD4þ and CD8þ T cells collected 7 daysposttreatment (3 mg/kg of KY1044 mIgG2a), we demonstrated asignificant increase in cytokine production (Fig. 6B). Although, onecannot differentiate between direct activation (through direct ICOScostimulation on TEff cells) or indirect activation (via Treg depletion),this analysis of intratumoral T cells revealed that KY1044 was associatedwith some activation of both ICOSLow CD8þ and CD4þ TEff cells.

Increased efficacy of KY1044 at an intermediate dose incombination with anti–PD-L1

The pharmacodynamic studies confirmed that KY1044 mIgG2aeffectively depleted ICOShigh Treg and resulted in activation ofICOSLow TEff cells (Fig. 4A). However, we noticed that when used athigh doses, KY1044 mIgG2a also affected the numbers of intratu-moral CD8þ T cells (Supplementary Fig. S8), resulting in a lowerCD8þ TEff to Treg cell ratio. Because a higher baseline CD8þ TEff toTreg cell ratio positively correlates with a response to ICIs such asatezolizumab (49), we aimed to assess whether the antitumorefficacy would be superior at an intermediate dose. For this, werepeated the CT26.WT efficacy study using a range of differentdoses of KY1044 mIgG2a (20, 60, and 200 mg/dose) combined witha fixed dose of anti–PD-L1 (200 mg/dose). As shown in Fig. 7, all thecombination treatments were associated with an improved response(vs. control or the anti–PD-L1 group). However, an improvedresponse at 60 mg/dose of KY1044 mIgG2a was observed, with90% of the mice being tumor free on day 54.

DiscussionThe approval of ICIs provides a novel approach that adds to existing

cancer therapies. However, responses to ICIs are not universal (2). Animmunosuppressive TME can underlie the lack of response. Notably,intratumoral Tregs maintain an immunosuppressive environment.Strategies reducing Treg numbers and improving the ratio of effectorT cells to Treg cells have thus emerged. Blocking the recruitment,function, expansion, and/or the survival of Treg, may achieve this goaland a number of molecules, targeting CTLA-4, CD25, GITR, OX40,CCR8, CD137 and CCR2, are under investigation (50). However, thedifferential expression of these targets (i.e., within immune and othercell subtypes) is crucial to preferentially target TReg (49). Here, wedemonstrated that ICOS expression differs between T-cell subsets,with the highest expression observed onTreg and confirmed that ICOSexpression is higher in the TME than in the periphery (blood orspleen). In addition, we identified head and neck, gastric, esophageal,lung, bladder, skin, and cervical cancers as tumors with a high contentof ICOSþ cells. However, costaining of ICOSwith FOXP3 showed thatthese tumors differ by their incidence of ICOSþ Treg with cervical,esophageal, and head and neck cancers being more infiltrated byICOSþ Treg. Because the ICOS expression on different cell subsetsshould affect the response to anti-ICOS therapies, this work suggeststhat the specific expression of ICOS on Treg may support a patientselection strategy.

High ICOS expression on intratumoral Tregs highlights the poten-tial of this target for a depletion strategy. Using a transgenic mouseplatform (23), we selected a fully human ICOS antibody called KY1044and demonstrated that KY1044 depletes ICOShigh cells via ADCC(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 showingindividual mouse tumor volumes from BALB/c mice (n ¼ 9 per group) harboring subcutaneous CT26.WT tumors and treated with different doses of KY1044mIgG2a [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 miceinjected with CT26.WT tumor and treated with different doses of the KY1044 mIgG2a/anti–PD-L1 combination. The dosing days are indicated by verticaldotted lines.






molecule on cells expressing lower ICOS levels, such as CD8þ TEff

cells (through FcgR-dependent clustering). KY1044 has monotherapyefficacy in lymphoma models and combination efficacy (with anti–PD-L1) in models of solid tumors that are resistant to PD-1/PD-L1blockade. In the CT26.WT tumor model, we showed better efficacywhen combining anti–PD-L1 with KY1044 mIgG2a (effector enable)than with a poorly depleting mIgG1 format. Similarly, in this model,we showed better efficacywhen combiningKY1044mIgG2awith anti–PD-L1 than with anti–PD-1. Although it was not clear why such adifference was observed in this particular model, one could postulatethat anti–PD-1, which blocks binding to both PD-L1 and PD-L2, maybe associated with a different phenotype to anti–PD-L1, which affectsPD-1 and CD80 biology (51). Speculatively, there is also the possibilitythat anti-ICOS and anti–PD-1 may interfere with each other's func-tions within the immunologic synapse, as both ICOS and PD-1 areoften expressed on the same cells (52–54).

There is an ongoing debate regarding the value of mouse modelswhen predicting the depletion potential of effector function enabledantibodies in human (8, 9, 22, 55, 56). Experiments in mouse dem-onstrated complete depletion of intratumoral CTLA-4þ Treg, whereasdepletion of these cells in patients treated with ipilimumab remainsdisputed. The differences in FcgR expression and Fc/FcgR interactionbetween rodents and primates may contribute to these discrepan-cies (57). Here, we performed pharmacodynamic studies in both mice(using mIgG2a) and NHPs (using hIgG1) to confirm that KY1044decreased the frequency of ICOShigh cells in vivo in both species. Withthe mouse work, we demonstrated that KY1044 reduced Treg andimproved the CD8þ TEff to Treg cell ratio, which led the tumors torespond to anti–PD-L1. Combined with the decrease in ICOShigh cellsobserved in NHP, these data suggested that KY1044 could also depleteICOShigh cells in human tumors.

While depletion of Treg is an attractive strategy, it is crucial notto deplete all Treg in all tissues to avoid autoimmune dis-eases (58, 59). KY1044 did not deplete Treg in the spleen andlymph node. Although the reasons behind this selective depletion isnot fully understood, this response has been shown for otherdepleting antibodies and, in our case, could be explained by thelower expression of ICOS on the surface of T cells in lymphoidtissues (Supplementary Fig. S1) or to lower expression of FcgRIIIain the periphery. In the tumor rechallenge experiment, a long-termimmune memory response was observed after treatment withKY1044. Because some memory T cells expressed ICOS, these datasuggested that KY1044 has the potential to deplete ICOShigh cellswithout removing all ICOS cells. In cynomolgus monkeys, noadverse toxic effects were associated with the depletion of someof the ICOShigh cells. Finally, we demonstrated that KY1044 hascostimulatory properties, as shown by the activation and secretionof cytokines such as IFNg and TNFa. Although no effect on T-cellproliferation was observed, we noted a strong effect on cell mor-phology in response to KY1044-dependent ICOS stimulation. Thiscostimulatory agonistic property was shown to require targetclustering and concomitant engagement of the TCR.

Finally, while assessing the effect of KY1044 on the CD8þ TEff toTreg intratumoral ratio, we noticed that although the treatmentimproved the ratio at all doses tested, a bell-shaped response patternwas observed. Although all combinations of anti–PD-L1 with different

doses of KY1044mIgG2a were shown to trigger an antitumor immuneresponse, the intermediate dose of 60 mg of KY1044 resulted in thestrongest response.

Altogether, this study demonstrated that KY1044 was pharmaco-logically active, modified the intratumoral immune contexture, andinduced a strong and long-lasting antitumor response. These findings,therefore, warrant the further assessment of KY1044 as amonotherapyor a combination therapy with anti–PD-L1 as a potential treatment forsolid tumors.

Disclosure of Potential Conflicts of InterestR.C.A. Sainson reports other from Kymab Ltd (full-time employee) during the

conduct of the study, as well as a patent for US9957323 issued (author/coinventor onthe 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 US16/323980pending. G. Borhis reports a patent for US9957323 issued and a patent forUS16/323980 pending. N. Parveen reports a patent for US9957323 issued and apatent for US16/323980 pending. C. Van Krinks reports a patent for US9957323issued and a patent for US16/323980 pending. H. Ali reports a patent for US9957323issued to Kymab Ltd and a patent for US16/323980 pending to Kymab Ltd. H. Craigreports other from Kymab Ltd (full-time employee) during the conduct of the study.V. Germaschewski reports Kymab Ltd employment and share options. S. Quaratinoreports other from Kymab Ltd (full-time employee) during the conduct of the study.M. McCourt reports personal fees from Kymab Ltd (full-time employee) during theconduct of the study, as well as a patent for US9957323B2 issued to Kymab Ltd and apatent forUS20190330345A1pending toKymabLtd.Nopotential conflicts of interestwere disclosed by the other authors.

Authors’ ContributionsR.C.A. Sainson: Conceptualization, data curation, formal analysis, supervision,

investigation, writing–original draft, writing–review and editing. A.K. Thotakura:Resources, data curation, writing–original draft. M. Kosmac: Resources, datacuration, writing–original draft, writing–review and editing. G. Borhis: Resources,data curation, writing–original draft, writing–review and editing. N. Parveen:Resources, data curation. R. Kimber: Resources, data curation. J. Carvalho:Resources, data curation. S.J. Henderson: Resources, data curation. K.L. Pryke:Resources. T. Okell: Resources. S. O'Leary: Resources, data curation. S. Ball:Resources, data curation. C. Van Krinks: Data curation. L. Gamand: Resources,data curation. E. Taggart: Resources. E.J. Pring: Resources. H. Ali: Resources, datacuration. H. Craig: Resources, data curation. V.W.Y. Wong: Resources, datacuration. Q. Liang: Resources, supervision. R.J. Rowlands: Resources, datacuration. M. Lecointre: Resources, data curation. J. Campbell: Resources, datacuration, supervision. I. Kirby: Resources, data curation. D. Melvin: Software.V. Germaschewski: Resources, supervision. E. Oelmann: Writing–review andediting. S. Quaratino: Supervision, writing–review and editing. M. McCourt:Conceptualization, supervision, writing–review and editing.

AcknowledgmentsThe authors are extremely grateful to Kymab's molecular biology and antibody

expression team as well as the Kymab BSU team. The authors also thank PropathUK and OracleBio for assistance with IHC staining and digital image analysis aswell as Stephanie Grote-Wessels (Covance Preclinical Services GmbH) for theirsupport with this study. They also thank the NCI Division of Cancer Treatmentand Diagnosis, Developmental Therapeutics Program, Biological Testing Branch,Tumor Repository for providing the MC38 syngeneic tumor cell line.

The costs of publication of this article were defrayed in part by the payment of pagecharges. This article must therefore be hereby marked advertisement in accordancewith 18 U.S.C. Section 1734 solely to indicate this fact.

Received January 14, 2020; revised June 1, 2020; accepted September 18, 2020;published first September 30, 2020.

References1. PostowMA, CallahanMK,Wolchok JD. Immune checkpoint blockade in cancer

therapy. J Clin Oncol 2015;33:1974–82.2. Sharma P, Hu-Lieskovan S, Wargo JA, Ribas A. Primary, adaptive, and acquired

resistance to cancer immunotherapy. Cell 2017;168:707–23.

Sainson et al.





3. Takeuchi Y, Nishikawa H. Roles of regulatory T cells in cancer immunity.Int Immunol 2016;28:401–9.

4. Wang H, Franco F, Ho P-C. Metabolic regulation of Tregs in cancer: oppor-tunities for immunotherapy. Trends Cancer 2017;3:583–92.

5. Shang B, Liu Y, Jiang S, Liu Y. Prognostic value of tumor-infiltrating FoxP3þregulatoryT cells in cancers: a systematic review andmeta-analysis. Sci Rep 2015;5:15179.

6. Fridman WH, Zitvogel L, Saut�es-Fridman C, Kroemer G. The immune contex-ture in cancer prognosis and treatment. Nat Rev Clin Oncol 2017;14:717–34.

7. Mo L, Chen Q, Zhang X, Shi X, Wei L, Zheng D, et al. Depletion of regulatory Tcells by anti-ICOS antibody enhances anti-tumor immunity of tumor cellvaccine in prostate cancer. Vaccine 2017;35:5932–8.

8. Arce Vargas F, Furness AJS, Solomon I, Joshi K, Mekkaoui L, Lesko MH, et al.Fc-optimized anti-CD25 depletes tumor-infiltrating regulatory T cells andsynergizes with PD-1 blockade to eradicate established tumors. Immunity2017;46:577–86.

9. Selby MJ, Engelhardt JJ, Quigley M, Henning KA, Chen T, Srinivasan M, et al.Anti-CTLA-4 antibodies of IgG2a isotype enhance antitumor activitythrough reduction of intratumoral regulatory T cells. Cancer Immunol Res2013;1:32–42.

10. Amatore F, Gorvel L, Olive D. Inducible co-stimulator (ICOS) as a potentialtherapeutic target for anti-cancer therapy. Expert Opin Ther Targets 2018;22:343–51.

11. McAdam AJ, Greenwald RJ, Levin MA, Chernova T, Malenkovich N, Ling V,et al. ICOS is critical for CD40-mediated antibody class switching. Nature 2001;409:102–5.

12. Hutloff A, Dittrich AM, Beier KC, Eljaschewitsch B, Kraft R, Anagnostopoulos I,et al. ICOS is an inducible T-cell co-stimulator structurally and functionallyrelated to CD28. Nature 1999;397:263–6.

13. Burmeister Y, Lischke T, Dahler AC, Mages HW, Lam KP, Coyle AJ, et al. ICOScontrols the pool size of effector-memory and regulatoryT cells. J Immunol 2008;180:774–82.

14. Wikenheiser DJ, Stumhofer JS. ICOS co-stimulation: friend or foe? FrontImmunol 2016;7:304.

15. Leconte J, Bagherzadeh Yazdchi S, Panneton V, SuhWK. Inducible costimulator(ICOS) potentiates TCR-induced calcium flux by augmenting PLCg1 activationand actin remodeling. Mol Immunol 2016;79:38–46.

16. Nagase H, Takeoka T, Urakawa S, Morimoto-Okazawa A, Kawashima A,Iwahori K, et al. ICOS(þ) Foxp3(þ) TILs in gastric cancer are prognosticmarkers and effector regulatory T cells associated with Helicobacter pylori.Int J Cancer 2016;138:1328–36.

17. Tu JF, Ding YH, Ying XH,Wu FZ, Zhou XM, ZhangDK, et al. Regulatory T cells,especially ICOSþ FOXP3þ regulatory T cells, are increased in the hepatocellularcarcinoma microenvironment and predict reduced survival. Sci Rep 2016;6:35056.

18. Ng TangD, Shen Y, Sun J,Wen S,Wolchok JD, Yuan J, et al. Increased frequencyof ICOSþ CD4 T cells as a pharmacodynamic biomarker for anti-CTLA-4therapy. Cancer Immunol Res 2013;1:229–34.

19. Fan X, Quezada SA, Sepulveda MA, Sharma P, Allison JP. Engagement of theICOS pathway markedly enhances efficacy of CTLA-4 blockade in cancerimmunotherapy. J Exp Med 2014;211:715–25.

20. Wei SC, Levine JH, Cogdill AP, Zhao Y, Anang NAAS, Andrews MC, et al.Distinct cellular mechanisms underlie anti-CTLA-4 and anti-PD-1 checkpointblockade. Cell 2017;170:1120–33.

21. Fu T, He Q, Sharma P. The ICOS/ICOSL pathway is required for optimalantitumor responses mediated by anti-CTLA-4 therapy. Cancer Res 2011;71:5445–54.

22. ArceVargas F, Furness AJS, LitchfieldK, Joshi K, Rosenthal R, Solomon I, et al. Fceffector function contributes to the activity of human anti-CTLA-4 antibodies.Cancer Cell 2018;33:649–63.

23. Lee EC, LiangQ,AliH, Bayliss L, BeasleyA, Bloomfield-Gerdes T, et al. Completehumanization of the mouse immunoglobulin loci enables efficient therapeuticantibody discovery. Nat Biotechnol 2014;32:356–63.

24. Goldman M, Craft B, Zhu J, Haussler D. The UCSC Xena system for cancergenomics data visualization and interpretation [abstract]. In: Proceedings of theAmerican Association for Cancer Research AnnualMeeting 2017; 2017Apr 1–5;Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2584.

25. Barbie DA, Tamayo P, Boehm JS, Kim SY, Moody SE, Dunn IF, et al. SystematicRNA interference reveals that oncogenic KRAS-driven cancers require TBK1.Nature 2009;462:108–12.

26. H€anzelmann S, Castelo R, Guinney J. GSVA: gene set variation analysis formicroarray and RNA-Seq data. BMC Bioinformatics 2013;14:7.

27. Lun ATL, McCarthy DJ, Marioni JC. A step-by-step workflow for low-levelanalysis of single-cell RNA-seq data with Bioconductor. F1000Res 2016;5:2122.

28. McCarthy DJ, Campbell KR, Lun ATL, Wills QF. Scater: pre-processing, qualitycontrol, normalization and visualization of single-cell RNA-seq data in R.Bioinformatics 2017;33:1179–86.

29. Young MD, Behjati S. SoupX removes ambient RNA contamination fromdroplet based single cell RNA sequencing data. BioRxiv 303727 [Preprint].2018. Available from: https://doi.org/10.1101/303727.

30. McInnes L, Healy J, Melville J. UMAP: uniform manifold approximation andprojection for dimension reduction. arXiv 1802.03426 [Preprint]. 2018. Avail-able from: https://arxiv.org/abs/1802.03426.

31. Aibar S, Gonz�alez-Blas CB, Moerman T, Huynh-Thu VA, Imrichova H,Hulselmans G, et al. SCENIC: single-cell regulatory network inference andclustering. Nat Methods 2017;14:1083–6.

32. Bray NL, Pimentel H,Melsted P, Pachter L. Near-optimal probabilistic RNA-seqquantification. Nat Biotechnol 2016;34:525–7.

33. Ritchie ME, Phipson B, Wu D, Hu Y, Law CW, Shi W, et al. limma powersdifferential expression analyses for RNA-sequencing and microarray studies.Nucleic Acids Res 2015;43:e47.

34. Zhou Y, Zhou B, Pache L, Chang M, Khodabakhshi AH, Tanaseichuk O, et al.Metascape provides a biologist-oriented resource for the analysis of systems-leveldatasets. Nat Commun 2019;10:1–10.

35. YusaK, ZhouL, LiMA, BradleyA, CraigNL.Ahyperactive piggyBac transposasefor mammalian applications. Proc Natl Acad Sci U S A 2011;108:1531–6.

36. Kraman M, Faroudi M, Allen NL, Kmiecik K, Gliddon D, Seal C, et al.FS118, a bispecific antibody targeting LAG-3 and PD-L1, enhances T cellactivation resulting in potent anti-tumor activity. Clin Cancer Res 2020;26:3333–44.

37. ConradC,Gregorio J,WangYH, Ito T,Meller S,Hanabuchi S, et al. Plasmacytoiddendritic cells promote immunosuppression in ovarian cancer via ICOS costi-mulation of Foxp3þ T-regulatory cells. Cancer Res 2012;72:5240–9.

38. Strauss L, Bergmann C, Szczepanski MJ, Lang S, Kirkwood JM, Whiteside TL.Expression of ICOS on human melanoma-infiltrating CD4þCD25highFoxp3þT regulatory cells: implications and impact on tumor-mediated immune sup-pression. J Immunol 2008;180:2967–80.

39. Hoffman F, Gavaghan D, Osborne J, Barrett IP, You T, Ghadially H, et al. Amathematical model of antibody-dependent cellular cytotoxicity (ADCC).J Theor Biol 2018;436:39–50.

40. Waight JD, Chand D, Dietrich S, Gombos R, Horn T, Gonzalez AM, et al.Selective FcgR co-engagement on APCs modulates the activity of therapeuticantibodies targeting T cell antigens. Cancer Cell 2018;33:1033–47.

41. Barnhart BC,QuigleyM.Role of Fc-FcgR interactions in the antitumor activity oftherapeutic antibodies. Immunol Cell Biol 2017;95:340–6.

42. Franko JL, Levine AD. Antigen-independent adhesion and cell spreadingby inducible costimulator engagement inhibits T cell migration in a PI-3K-dependent manner. J Leukoc Biol 2009;85:526–38.

43. ChenH, Fu T, SuhW-K, Tsavachidou D,Wen S, Gao J, et al. CD4 T cells requireICOS-mediated PI3K signaling to increase T-Bet expression in the setting ofanti-CTLA-4 therapy. Cancer Immunol Res 2014;2:167–76.

44. Chen Q, Mo L, Cai X, Wei L, Xie Z, Li H, et al. ICOS signal facilitates Foxp3transcription to favor suppressive function of regulatory T cells. Int J Med Sci2018;15:666–73.

45. Pratama A, Vinuesa CG. Control of TFH cell numbers: why and how?Immunol Cell Biol 2014;9269:40–8.

46. Le KS, Thibult ML, Just-Landi S, Pastor S, Gondois-Rey F, Granjeaud S, et al.Follicular B lymphomas generate regulatory T cells via the ICOS/ICOSLpathwayand are susceptible to treatment by anti-ICOS/ICOSL therapy. Cancer Res 2016;76:4648–60.

47. Wang B, Ma N, Cheng H, Zhou H, Qiu H, Yang J, et al. Effects of ICOSLGexpressed in mouse hematological neoplasm cell lines in the GVL reaction.Bone Marrow Transplant 2013;48:124–8.

48. Dovedi SJ, Adlard AL, Lipowska-Bhalla G,McKennaC, Jones S, Cheadle EJ, et al.Acquired resistance to fractionated radiotherapy can be overcome by concurrentPD-L1 blockade. Cancer Res 2014;74:5458–68.

49. McDermott DF, Sosman JA, Sznol M, Massard C, Gordon MS, Hamid O, et al.Atezolizumab, an anti–programmed death-ligand 1 antibody, inmetastatic renalcell carcinoma: long-term safety, clinical activity, and immune correlates from aphase Ia study. J Clin Oncol 2016;34:833–42.





https://doi.org/10.1101/303727

https://arxiv.org/abs/1802.03426


50. Shitara K, Nishikawa H. Regulatory T cells: a potential target in cancer immu-notherapy. Ann N Y Acad Sci 2018;1417:104–15.

51. Zak KM, Grudnik P, Magiera K, D€omling A, Dubin G, Holak TA. Structuralbiology of the immune checkpoint receptor PD-1 and its ligands PD-L1/PD-L2.Structure 2017;25:1163–74.

52. Suvas S, Channappanavar R, Twardy B. Inducible costimulator (ICOS) nega-tively regulate programmed death-1 (PD-1) induced apoptosis of effectormemory CD4 T cells under homeostatic condition (47.11). J Immunol 2012;188(1 Suppl):47.11.

53. Zhang Y, Luo Y, Qin SL, Mu YF, Qi Y, YuMH, et al. The clinical impact of ICOSsignal in colorectal cancer patients. Oncoimmunology 2016;5:e1141857.

54. Beyrend G, van der Gracht E, Yilmaz A, van Duikeren S, CampsM, H€ollt T, et al.PD-L1 blockade engages tumor-infiltrating lymphocytes to co-express targetableactivating and inhibitory receptors. J Immunother Cancer 2019;7:217.

55. SharmaA, Subudhi SK, Blando J, Scutti J, Vence L,Wargo JA, et al. Anti-CTLA-4immunotherapy does not deplete FOXP3þ regulatory T cells (Tregs) in humancancers. Clin Cancer Res 2019;25:1233–8.

56. Quezada SA, Peggs KS. Lost in translation: deciphering the mechanism of actionof anti-human CTLA-4. Clin Cancer Res 2019;25:1130–2.

57. Overdijk MB, Verploegen S, Ortiz Buijsse A, Vink T, Leusen JHW, Bleeker WK,et al. Crosstalk between human IgG isotypes andmurine effector cells. J Immunol2012;189:3430–8.

58. Ellis JS, Wan X, Braley-Mullen H. Transient depletion of CD4þ CD25þ

regulatory T cells results in multiple autoimmune diseases in wild-type andB-cell-deficient NOD mice. Immunology 2013;139:179–86.

59. Stephens LA, Gray D, Anderton SM. CD4þ CD25þ regulatory T cells limit therisk of autoimmune disease arising fromT cell receptor crossreactivity. ProcNatlAcad Sci U S A 2005;102:17418–23.


Sainson et al.




2020;8:1568-1582. Published OnlineFirst September 30, 2020.Cancer Immunol Res Richard C.A. Sainson, Anil K. Thotakura, Miha Kosmac, et al. Regulatory T-cell Ratio and Induces Tumor RegressionAn Antibody Targeting ICOS Increases Intratumoral Cytotoxic to

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