A Cross-Species Reactive TIGIT-Blocking Antibody Fc Dependently Confers Potent Antitumor Effects

This information is current as Fang Yang, Linlin Zhao, Zhizhong Wei, Yajing Yang, Juan of October 1, 2021. Liu, Yulu Li, Xinxin Tian, Ximing Liu, Xueyuan Lü and Jianhua Sui J Immunol published online 4 September 2020 http://www.jimmunol.org/content/early/2020/09/03/jimmun ol.1901413 Downloaded from

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2020 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. Published September 4, 2020, doi:10.4049/jimmunol.1901413 The Journal of Immunology

A Cross-Species Reactive TIGIT-Blocking Antibody Fc Dependently Confers Potent Antitumor Effects

Fang Yang,*,† Linlin Zhao,†,‡ Zhizhong Wei,†,‡ Yajing Yang,†,x Juan Liu,† Yulu Li,†,{ Xinxin Tian,† Ximing Liu,† Xueyuan Lu¨,†,‡ and Jianhua Sui†,‖

The immunoreceptor with Ig and ITIM domains (TIGIT) has been shown to exert inhibitory roles in antitumor immune responses. In this study, we report the development of a human mAb, T4, which recognizes both human and mouse TIGITand blocks the interaction of TIGIT with its ligand CD155 in both species. The T4 Ab targets the segment connecting F and G strands of TIGIT’s extracellular IgV domain, and we show in studies with mouse tumor models that the T4 Ab exerts strong antitumor activity and induces durable immune memory against various tumor types. Mechanistically, we demonstrate that the T4 Ab’s antitumor effects are mediated via multiple immunological impacts, including a CD8+ T immune response and Fc-mediated effector functions, through NK cells that cause significant reduction in the frequency of intratumoral T regulatory cells (Tregs). Downloaded from Notably, this Treg reduction apparently activates additional antitumor CD8+ T cell responses, targeting tumor-shared Ags that are normally cryptic or suppressed by Tregs, thus conferring cross-tumor immune memory. Subsequent engineering for Fc variants of the T4 Ab with enhanced Fc-mediated effector functions yielded yet further improvements in antitumor efficacy. Thus, beyond demonstrating the T4 Ab as a promising candidate for the development of cancer immunotherapies, our study illustrates how the therapeutic efficacy of an anti-TIGIT Ab can be improved by enhancing Fc-mediated immune effector functions. Our insights about the multiple mechanisms of action of the T4 Ab and its Fc variants should help in developing new strategies that can realize http://www.jimmunol.org/ the full clinical potential of anti-TIGIT Ab therapies. The Journal of Immunology, 2020, 205: 000–000.

mmunotherapies have emerged as extremely potent modali- PD-1 and CTLA-4 combination blockade further improves ties for treating various cancers. One of the most impactful clinical outcomes in patients (1–3). However, there are still a large I approaches is based on the blockade of immune checkpoint number of patients who do not respond optimally to such treat- receptors or ligands. In particular, Abs directed against the pro- ments, suggesting that there are other mechanism(s) that need to grammed cell death protein 1 (PD-1)/programmed cell death ligand be exploited to achieve better antitumor immune response (4). (PD-L1) axis have provided persistent clinical benefits for TIGIT, a member of the poliovirus receptor/nectin family, is an

10–40% of patients with a variety of cancers (1). In addition, immunoreceptor protein mainly expressed on activated T cells, by guest on October 1, 2021 memory T cells, NK cells, and a subset of T regulatory cells (Tregs) (5–8). Studies have shown that TIGIT2/2 mice were more sus- *College of Biological Sciences, China Agricultural University, Beijing 100193, ceptible to immunization-induced autoimmunity (8, 9), supporting China; †National Institute of Biological Sciences, Beijing 102206, China; ‡Peking University-Tsinghua University-National Institute of Biological Sciences that TIGIT functions as an inhibitory receptor in maintaining Joint Graduate Program, School of Life Sciences, Tsinghua University, Beijing immune homeostasis. TIGIT binds its high-affinity cognate ligand 100084, China; xGraduate School of Peking Union Medical College and Chinese Academy of Medical Sciences, Beijing 100730, China; {Peking University- CD155 (also known as poliovirus receptor), which is expressed on Tsinghua University-National Institute of Biological Sciences Joint Graduate Pro- APCs, and this binding inhibits immune responses via T cells and gram, College of Life Sciences, Peking University, Beijing 100871, China; and ‖ APCs as well as through NK cells (9, 10). TIGIT has also been Tsinghua Institute of Multidisciplinary Biomedical Research, Tsinghua University, Beijing 102206, China shown to compete with CD226 for binding to CD155 (7), thus ORCIDs: 0000-0002-9840-1878 (Z.W.); 0000-0002-1272-9662 (J.S.). counterbalancing CD226-mediated costimulatory T cell signaling Received for publication November 26, 2019. Accepted for publication August 8, (5), which is reminiscent of the function of CTLA-4 in counter- 2020. balancing CD28’s costimulatory function (11). This work was supported by grants from the Ministry of Science and Technology of In cancer contexts, TIGIT is known to be upregulated in tumor- the People’s Republic of China (973 Program, Grant 2012CB837600 to J.S.), the infiltrated T cells (12). Given that CD155 is highly expressed by Beijing Municipal Science and Technology Commission, and the Beijing Key Lab- oratory of Pathogen Invasion and Immune Defense (Z171100002217064 to J.S.). both human and mouse tumors and tumor-infiltrating myeloid Address correspondence and reprint requests to Dr. Jianhua Sui, National Institute of cells (12–16), it has been proposed that TIGIT might inhibit an- Biological Sciences, Tsinghua Institute of Multidisciplinary Biomedical Research, titumor immune responses via multiple, sequential steps (4): first, Tsinghua University, 7 Science Park Road, Changping, Beijing 102206, China. inhibiting NK cell–mediated tumor cell killing as well as tumor E-mail address: [email protected] Ag release, then inducing tolerogenic dendritic cells and sup- The online version of this article contains supplemental material. pressing CD8+ T cell function via TIGIT+ Tregs, and finally di- Abbreviations used in this article: ADCC, Ab-dependent cell-mediated cytotoxicity; + ADCP, Ab-dependent cellular phagocytosis; BMDM, bone marrow–derived macro- rectly inhibiting CD8 T cell effector functions. This sequence of phage; CDC, complement-dependent cytotoxicity; DE, S239D/I332E (DE); ECD, steps may ultimately prevent elimination of cancer cells, so TIGIT extracellular domain; FG loop, segment connecting F and G strands; hCD155, human CD155; hIgG1, human IgG1; hTIGIT, human TIGIT; LDH, lactate dehydrogenase; is considered to be a key inhibitor in cancer immunity (4, 17), and mCD155, mouse CD155; mFc, mouse Fc tag; mFcgR, murine FcgR; mIgG2a, mouse targeting TIGIT is viewed as a promising approach for developing IgG2a; mTIGIT, murine TIGIT; PD-1, programmed cell death protein 1; PD-L1, cancer immunotherapies. programmed cell death ligand; PK, pharmacokinetic; Treg, T regulatory cell. Abs targeting immune checkpoint proteins were initially as- Copyright Ó 2020 by The American Association of Immunologists, Inc. 0022-1767/20/$37.50 sumed to modulate the immune activity of effector T cells by

www.jimmunol.org/cgi/doi/10.4049/jimmunol.1901413 2 TIGIT-BLOCKING Ab Fc DEPENDENTLY CONFER ANTITUMOR EFFECTS blocking their inhibitory function; however, a growing number of of the V regions of H chain and L chain were subcloned into an IgG (human studies have reported any such therapeutic effects may actually IgG or mouse IgG) H chain expression vector and L chain expression vector, result from the Fc-mediated effector functions of certain immune respectively. 293F cells were cotransfected with the two IgG expression plasmids (HC plus LC plasmids) at a 1:1 ratio. After 3–5 d of transfection, modulatory Abs (such as anti–CTLA-4) (18–23). Abs with the cell culture supernatants were collected for purification in a manner different blockade activities could result in similar antitumor similar to that described previously (26). efficacy, solely depending on FcgR engagement capability (24). Ab library panning and screening of anti-TIGIT Abs Anti-TIGIT Abs have shown therapeutic effects in several murine cancer models, and there several Abs now under assessment in For anti-TIGIT Ab panning, the ECD of hTIGIT and mTIGIT were fused human clinical trials (25). Despite these promising medical re- with His6-Avi tag and biotinylated by BirA ligase. These two proteins were used as Ags in the panning experiments with a human nonimmune Ab sults, our scientific understanding of the mechanism(s) through library (26). To get cross-reactive Abs, we used hTIGIT in the first round which these anti-TIGIT Abs confer their antitumor actions re- of panning and hTIGIT or mTIGIT in the second round of panning as Ags. mains unclear, which limits efforts to develop more efficacious A total of ∼3000 single clones were randomly picked and screened for Abs or combination therapies and limits clinical efforts to identify binding to both hTIGIT and mTIGIT and for competition with CD155 by which patient populations may benefit from such treatments. ELISA. Clones selected out were produced as purified phage-scFv parti- cles or converted into a full-length human IgG1 (hIgG1) format for further In this study, we initially developed Abs with cross-reactivity characterizations. against both human TIGIT (hTIGIT) and mouse TIGIT (mTIGIT), using a large nonimmune phage display human Ab library. The best ELISA-based binding and competition assays candidate, T4 Ab, showed high binding affinity (picomolar level) and For the ELISA-based binding assay, biotinylated protein Ags were captured strong blocking activity against TIGIT–CD155 interactions. Epitope with streptavidin (Sigma-Aldrich)-coated 96-well plates (MaxiSorp; Nunc). mapping studies identified that the TIGIT extracellular IgV domain For phage-scFv–based ELISA, serially diluted phage-scFvs were added Downloaded from segment connecting F and G strands (FG loop), which is known to and then detected by adding mouse anti–M13-HRP Ab (GE Healthcare). For full-length human IgG–based ELISA, the method was similar to the participate in the TIGIT–CD155 interaction, impacts T4 Ab binding. phage-scFvs. The bound Abs were detected using a mouse anti-human IgG The T4 Ab exerts Fc-mediated effector functions in vitro and confers Fc-HRP Ab (Thermo Fisher Scientific). both potent antitumor activity and a capacity to elicit durable sys- The ELISA-based competition assays were performed in a manner temic immune memory in mouse syngeneic tumor models. We similar to ELISA-based binding assays, except that the tested Abs were subsequently used a set of T4 Ab Fc variants with either abolished or incubated with captured Ags in the presence of competitive ligands. Briefly, http://www.jimmunol.org/ different hIgG1 Abs at serially diluted concentrations were mixed with enhanced Fc-mediated effector functions and found that Fc-mediated 2 mg/ml ECD of human CD155 (hCD155) or mouse CD155 (mCD155) effector functions contribute to the therapeutic effect of T4 Ab; we fused with mouse Fc tag (mFc) (hCD155–mFc or mCD155–mFc) and also observed that enhancement of the T4 Ab’s Fc effector function added to the ELISA plates to compete for the binding between TIGIT and further improves its antitumor effects. Immune cell depletion exper- CD155. The signal was measured via ligand detection using HRP–anti- iments using Abs or reagents that efficiently deplete specific immune mouse IgG secondary Ab (Thermo Fisher Scientific). cell populations revealed that both CD8+ T and NK cells are indis- Binding kinetic analysis by surface plasmon resonance pensable for T4 Ab–mediated tumor regression. Analysis of tumor- Kinetic analyses of the bindings of T4 and T4’s Fc variants to the ECD of infiltrating lymphocytes showed thattheT4Ableadstosignificant hTIGIT or mTIGIT and the ECDs of mFcgRs were performed on a Biacore by guest on October 1, 2021 decreases in the proportion of Treg within tumors, doing so in an Fc- T200 instrument (Biacore; GE Healthcare). Anti-human Fc Ab or protein dependent manner and requiring NK cells. Finally, tumor rechallenge A/G (Thermo Fisher Scientific) was covalently attached to surfaces of a experiments demonstrate that T4 Ab treatment can induce cross- CM5 sensor chip using an amine coupling kit (GE Healthcare). Abs at tumor immune memory for mice bearing CT26 and A20 tumors, optimal concentrations were captured on the chip, and the analytes (hTI- GIT, mTIGIT, or mFcgRs) were then injected at determined concentrations and the GSW11-specific T cell response apparently contributes to the or at 2-fold serially diluted concentrations. Binding kinetics were evaluated cross-protective immunity. Together, these results demonstrate that using a 1:1 Langmuir binding model. The ka, kd, and KD were calculated our newly discovered T4 Ab employs multiple mechanisms of action, using Biacore T200 Evaluation Software. including blockade of ligand binding, Fc-mediated effector functions Flow cytometry analyses of cell lines that likely result in depletion of intratumoral Tregs via NK cells, and enhancement of CD8+ T cell responses, which collectively contribute CHO cell lines stably expressing full-length hTIGIT or mTIGIT (CHO- to the potent antitumor activity and immune memory effects of this hTIGIT or CHO-mTIGIT) were constructed and used in FACS analyses for testing anti-TIGIT Ab binding or competition activity against ligand cross-reactive therapeutic Ab. CD155 binding. For examining mCD155 expression on tumor cells, A20 cells were pretreated with 2.4G2 (an Ab that blocks Fc binding to mFcgRII Materials and Methods and mFcgRIII), and subsequently stained with a rat anti-mCD155 mAb, 4.24.1 (BioLegend); CT26 and 4T1 cells were directly stained with 4.24.1 Cell lines Ab. Donkey anti-rat IgG–Alexa Fluor 488 Ab was used as a secondary Ab Raji, CHO, CT26, 4T1, A20, and Jurkat were from the Cell Bank of Type (Thermo Fisher Scientific). Culture Collection, Chinese Academy of Sciences or American Type For T4 Ab epitope mapping, chimeras of hTIGIT IgV domain and Culture Collection; the FreeStyle 293F were from Life Technologies; cell N-terminal IgV domain D1 of hCD155 were constructed by replacing line YTS and the MHC class I–negative mutant B lymphoblastoid cell line residues in the extracellular regions of hTIGIT with the corresponding 721.221 were generously provided by Dr. Zusen Fan (Institute of Bio- residues from hCD155 D1; three hTIGIT variants were generated, as physics, Chinese Academy of Sciences, Beijing, China). CHO or CHO- shown in Supplemental Fig. 2A. These chimeras and variants were derived cell lines were cultured in DMEM supplemented with 10% FBS. expressedbytransienttransfection of CHO cells; their cell surface Raji, CT26, 4T1, A20, YTS, 721.221, and Jurkat cells were cultured with expression levels were assessed by using a mAb, GC33, recognizing RPMI 1640 medium supplemented with 10% FBS. These cells were cul- the N-terminal tag fused to each of the chimeras and variants (27). The tured at 37˚C in a humidified incubator with a 5% CO2 atmosphere. 293F binding of testing Abs in hIgG1 format or hCD155–human Fc to the cells were cultured following the manufacturer’s instructions. CHO transfectants were detected by using goat anti-human IgG–FITC Ab (Thermo Fisher Scientific). Expression and purification of proteins NK cell cytotoxicity assay The extracellular domains (ECD) of TIGIT, CD155, or murine FcgRs (mFcgRs) were produced as His6-, His6-Avi–, or Fc-tagged fusion proteins To test the blockage function of anti-TIGITAbs, YTS cells stably expressing by transient transfection of the FreeStyle 293F cell line and were purified hTIGIT (YTS-hTIGIT) and 721.221 cells stably expressing hCD155 by affinity chromatography. For full-length IgG Abs, the coding sequences (721.221-hCD155) were established and used in the following cytotoxicity The Journal of Immunology 3 assays: 721.221-hCD155 cells (target cells, 5000 cells per well) were tumor cells (1–3 3 105 CT26, A20, or 4T1) into the left flank. Tumors incubated with YTS-hTIGIT (NK effector cells) at the indicated E:T were measured two times per week as described above. ratios of 2:1 in the presence of 5 mg/well Abs for 6 h. Lactate dehy- drogenase (LDH) release by cells was then detected following the in- Tissue processing and flow cytometry analyses of intratumoral structions of a CytoTox 96 Non-Radioactive Cytotoxicity Assay Kit immune cells and tumor cells (Promega). Cytotoxicity percentages were calculated following the manufacturer’s instructions. Spleens, lymph nodes, and tumors from mice were harvested, and single-cell suspensions were used for FACS analyses. Briefly, spleens Ab-dependent cell-mediated cytotoxicity, Ab-dependent were dissociated and treated with RBC Lysis Buffer. Lymph nodes were cellular phagocytosis, and complement-dependent prepared by dissociating them with the back of a syringe into a six-well plate for single-cell suspensions. CT26 tumors were dissected into ∼2- cytotoxicity assays mm fragments and incubated in 2 ml of dissociation solution (PBS, For Ab-dependent cell-mediated cytotoxicity (ADCC) assay, a Raji cell line collagenase type I [200 U/ml], and DNase I [60 mg/ml]) for 30 min at ∼ stably expressing full-length hTIGIT (Raji-hTIGIT) was established and 37˚C on a gentle shaker. A20 tumors were dissected into 2-mm used as target cells in this assay. A Jurkat cell line stably expressing fragments and incubated in 2 ml of PBS for 30 min at 37˚C with gentle mFcgRIV receptor and an NFAT response element–driven firefly luciferase shaking. Cell suspensions were passed through a 40-mm cell strainer to reporter (Jurkat-NFAT-Luc2p/mFcgRIV) was created and used as effector obtain single-cell suspensions. cells. Target cells (15,000 cells per well) were seeded into the wells of a For surface marker staining, cells were incubated with 2.4G2 Ab to U-bottom 96-well cell culture plate and incubated briefly with various block FcgRs binding for 30 min and subsequently stained with Abs concentrations of different Abs. The effector cells were then added (90,000 purchased from BioLegend or specified below. The Abs used were as cells per well) into the wells containing the target cells and Abs at indi- follows: anti-CD8 (clone 53-6.7, PerCp–Cy5.5 labeled), anti-CD4 cated concentrations and incubated for 5 h at 37˚C in RPMI 1640 medium (clone GK1.5, FITC labeled), anti-CD3 (clone 17A2, PE–Cy7 la- supplemented with 1% heat-inactivated FBS. ADCC activity was deter- beled), anti-mTIGIT (T4 Ab), followed by PE-labeled secondary Ab, mined by luciferase expression according to the instruction of Bright-Glo anti-CD45 (clone 30-F1, FITC labeled), CD19 (clone 6D5, BV421 Downloaded from Luciferase Assay reagents (Promega). labeled), or mCD155 (clone TX15, PE labeled). A LIVE/DEAD Fix- For the Ab-dependent cellular phagocytosis (ADCP) assay, mouse bone able Near-IR Stain Kit (Thermo Fisher Scientific) was used to differ- marrow–derived (BMDMs) cells were used as effector cells entiate live and dead cells. For Foxp3 transcription factor staining, in this assay. To prepare BMDMs, mouse bone marrow cells were collected cells were fixed and permeabilized with Foxp3 Transcription Factor from the tibia and femurs of BALB/c mice and induced by GM-CSF in Staining Buffer Set (eBioscience) and then stained with anti-Foxp3 (clone FJK-16s; eBioscience). The gating strategy used for analyzing L929 supernatants for 3 d. The Raji-hTIGIT stable cell line was labeled CD8+,CD4+, and Foxp3+ Tregs was as follows: live cells were first with CFSE and used as target cells. The BMDMs were labeled with anti- http://www.jimmunol.org/ differentiated by live/dead staining and singlet cells were selected mouse F4/80–Alex Fluor 647 (Thermo Fisher Scientific) prior to incuba- basedonforwardandsidescatters.CD4+ and CD8+ T cells were tion with target cells. The CFSE-labeled target cells were incubated with further identified as subpopulations within CD3+ cells. CD4+ Tregs different Abs at room temperature for 15 min and then added to the labeled were further identified as a Foxp3+ subpopulation within CD4+ cells BMDMs in an E:T ratio of 1:2 for 2 h at 37˚C in DMEM supplemented (Supplemental Fig. 4A). with 10% heat-inactivated FBS. Phagocytosis of CFSE-labeled target cells For assessing the level of cytokine production using intracellular cy- by anti-mouse F4/80 Ab–labeled macrophages was recorded using a Nikon tokine staining, single-cell suspensions were isolated from mice 1 d after A1R Confocal Microscope. one dose of T4-mIgG2a treatment in the CT26 tumor model. The isolated For the complement-dependent cytotoxicity (CDC) assay, the Raji- cells were activated for 3 h with a cell activation mixture containing hTIGIT stable cells were used as target cells and seeded in a 96-well brefeldin A (BioLegend). Activated cells were harvested for surface and U-bottom plate at 4 3 105 cells per well and incubated with various Abs in

intracellular cytokine staining. Surface staining of the cells was performed by guest on October 1, 2021 the presence of 5% rabbit sera (Sigma-Aldrich). After 2 h of incubation, by incubating with PerCp–Cy5.5–conjugated anti-CD8 Ab (clone 53-6.7; the supernatants in each well were analyzed for LDH release using a BioLegend) and BV605-conjugated anti-NKp46 Ab (clone 29A1.4; Bio- CytoTox 96 Non-Radioactive Cytotoxicity Assay Kit (Promega). Legend) to identify the cellular origin of cytokines (i.e., CD8+ T cell or Animal studies NK cell origin). After surface staining, the cells were fixed and per- meabilized with a Cytofix/Cytoperm Kit (BD Biosciences). Intracellular All animal experiments were conducted following the National Guidelines cytokine staining was performed by incubating with PE-labeled Abs for Housing and Care of Laboratory Animals in China and performed under against IFN-g (clone XMG1.2; BioLegend), BV421-labeled TNF-a (clone the approved Institutional Animal Care and Use Committee protocols at the MP6-XT22; BioLegend), and allophycocyanin-labeled IL-2 (clone JES6- National Institute of Biological Sciences, Beijing, China. 5H4; BioLegend). All the FACS data were acquired using a BD FACSAria For mouse tumor models, 6–8-wk-old female BALB/c or BALB/c nude III cytometer. 3 5 mice were inoculated s.c. with 1–3 10 CT26, A20, or 4T1 cells in the + right flank. Based on similar mean tumor volumes (50–100 mm3, except as Tumor Ag-specific CD8 T cell analyses otherwise specifically indicated), mice were randomized into groups (n = Age-matched naive BALB/c mice and mice with complete tumor regression 3–6 per group) and received an i.p. injection of T4 Ab (10 mg/kg) or its after T4 Ab treatment were inoculated with tumor cells (1–3 3 105 CT26 variants two times per week for a total of five or six injections. Tumor or A20) into the left flank. Spleen cells were collected after 7 d and volume was measured with an electronic caliper and calculated using the restimulated in vitro as follows: 5 3 106/ml spleen cells were cultured with modified ellipsoid equation 1/2 3 (length 3 width2). + + 50 IU/ml rIL-2 (PeproTech) and 5 mg/ml synthetic peptides (AH1 or For depletion of CD4 or CD8 T cells, tumor-bearing BALB/c mice GSW11) for 7 d. Cells were then stained with particular synthetic MHC were injected with 200 mg of CD4-depleting Abs (clone GK1.5; Bio X class I tetramers (MBL International) as follows: AH1, PE-labeled H-2Ld Cell) or CD8-depleting Abs (clone 2.43; Bio X Cell) 2 d before T4 Ab MuLV tetramer-SPSYVYHQF; GSW11, PE-labeled H-2Dd MuLV treatment and two times per week. For depletion of NK cells, mice were tetramer-GGPESFYCASW), and an FITC-labeled Ab against CD8 (clone injected with 50 mg of anti–Asialo-GM1 polyclonal Ab (Poly21460; KT15; MBL International), followed by FACS analysis. BioLegend) 1 d before T4 Ab treatment and once every 5 d. For depletion of neutrophils, mice were injected with 400 mg of anti–Ly-6G Ab (1A8 Pharmacokinetic analysis clone; Bio X Cell) 2 d before T4 Ab treatment and two times per week. For depletion of , mice were injected with 100 ml of clodronate Six- to eight-week-old female BALB/c mice were used in this experiment. liposomes (FormuMax Scientific) 1 d before T4 Ab treatment and then Blood was collected at different time points after a single i.p. injection of once a week. Depletion Abs or reagents were given until the end of T4 Ab testing Abs. Ab concentrations in serum were measured by an mTIGIT- treatment for all of the immune cell depletion experiments. For depletion binding ELISA. of Tregs, mice were injected with 200 mg of anti–CD25–mouse IgG2a Statistical analyses (mIgG2a)–S239D/I332E (DE) Ab (PC61 clone, an Fc variant DE with enhanced effector functions) on days 27, 24, and 0 (before tumor im- Two-way ANOVAwas used to determine statistically significant differences plantation). The efficiencies of immune cell depletion methods described in inhibition of tumor growth between groups in animal studies, and two- above were confirmed by using tumor-naive BALB/c mice. tailed unpaired Student t tests were used to compare two groups in the For tumor rechallenge studies, age-matched naive BALB/c mice and other experiments. Data were analyzed using Prism 5.0 (GraphPad Soft- mice with tumor regression after T4 Ab treatment were inoculated with ware); *p , 0.05, **p , 0.01, ***p , 0.001, and ****p , 0.0001. 4 TIGIT-BLOCKING Ab Fc DEPENDENTLY CONFER ANTITUMOR EFFECTS

Results evaluated its antitumor function using immune-competent murine Identification of a blocking anti-TIGIT human mAb with cross- syngeneic tumor models. BALB/c mice were s.c. inoculated with reactivity to both hTIGIT and mTIGIT CT26 tumor cells, a mCD155+ mouse colon carcinoma cell line (Fig. 1C). When tumors were ∼40–80 mm3 in volume, they were Abs with species cross-reactivity for a given Ag facilitate the treated via i.p. injection two times per week for 3 wk with T4 Ab preclinical evaluation of their therapeutic potential and mecha- in one of two forms: the hIgG1 (T4-hIgG1) or the mIgG2a nisms of action in animal models (28). To identify cross-reactive (T4-mIgG2a) isoform. Monitoring tumor growth over time showed human mAbs recognizing both hTIGIT and mTIGIT, we used our that both T4-hIgG1 and T4-mIgG2a significantly inhibited tu- large naive human phage-displayed scFv Ab library (26) and mor growth, eventually resulting in complete tumor regression performed two rounds of library selection by using the ECD of (Fig. 1D). hTIGIT and mTIGIT as the targets, respectively, in the first and Recall that the mCD155 ligand is expressed by multiple cell second rounds of selection. After the second round of selection, types within tumors (e.g., tumor cells but also tumor-infiltrating ∼3000 phage–Ab clones were screened for cross-binding activity immune cells, in particular, myeloid cells) (16). We examined to both hTIGIT-ECD and mTIGIT-ECD using ELISA, and ∼100 whether the performance of T4 Ab in tumors is impacted by ex- clones with cross-binding activity were eventually obtained. These pression of mCD155 on tumor cells. Specifically, we treated clones were subsequently converted into the hIgG1 format and BALB/c mice bearing two other syngeneic tumors with T4- were analyzed for binding to both hTIGIT- and mTIGIT- mIgG2a: 4T1 (an mCD155+ mouse breast cancer cell line) or expressing CHO cells using FACS. Five clones (T4, number 18, A20 (an mCD1552 mouse B lymphoma cell line) (Fig. 1C). In- number 43, number 45, and hm7) showed high binding affinity to triguingly, we observed that T4-mIgG2a treatment resulted in Downloaded from both CHO-hTIGIT and CHO-mTIGIT cells (Supplemental Fig. complete tumor regression for the mCD1552 A20 tumors but had 1A). Owing to its top-ranking binding kinetics for both hTIGIT no obvious effect on the mCD155+ 4T1 tumors (Fig. 1E). To and mTIGIT, the T4 Ab was selected as the lead candidate exclude any regulatory effect on the expression of mCD155 during (Supplemental Fig. 1B, 1C). The affinity of T4 binding to hTIGIT the course of tumor formation, we analyzed the cells isolated from was further improved by k-chain shuffling and mutagenesis. The the tumors using FACS and confirmed that implanted tumor cells engineered T4 Ab exhibited similarly high binding affinity for exhibited the same mCD155 expression status as the cultured both hTIGIT and mTIGIT (KD: 8.75 and 2.74 nM, respectively) tumor cells (i.e., 4T1 and CT26 as mCD155-positive, A20 as http://www.jimmunol.org/ (Supplemental Fig. 1C). Competition ELISA assays testing in- mCD155-negative) (Fig. 1C). These results highlight that the teractions of TIGIT’s ligand CD155 with TIGIT-ECD showed that antitumor efficacy of the T4 Ab varies among different tumor T4 Ab potently outcompetes CD155 (Fig. 1A). models and also suggest that expression of mCD155 directly on To explore the basis of the T4 Ab’s cross-reactivity with hTIGIT tumor cells is not a prerequisite for the T4 Ab’s antitumor effects. and mTIGIT and to map the binding epitope of the T4 Ab, we That is, beyond the T4 Ab’s capacity to interrupt the TIGIT– generated full-length hTIGIT/hCD155 chimeras, each of which CD155 interaction on tumor cells, the T4 Ab is apparently able to replaced an extracellular fragment of hTIGIT with the corre- exert its effects via some alternative mechanism(s). sponding fragment from its cognate ligand hCD155; hTIGIT variants, each containing four to six residue changes, were also The Fc-mediated effector functions of T4 Ab contribute to its by guest on October 1, 2021 constructed (Supplemental Fig. 2A). By transfecting CHO cells potent antitumor effects in mice with these hTIGIT/hCD155 chimeras or hTIGIT variants and Seeking how the T4 Ab exerts its antitumor effects independently subsequently analyzing T4 Ab or hCD155 binding to these of the interaction between TIGIT and CD155 on tumors, we first transfected CHO cells by FACS, we identified that the FG loop examined whether T4 Ab possesses Fc-mediated effector functions of the hTIGIT’s extracellular IgV domain participates in T4 Ab using in vitro assays, including ADCC, ADCP, and CDC. To test T4 binding. Note that, consistent with previous findings (29), this Ab’s ADCC, we established a reporter system in which Jurkat experiment confirmed that the FG loop participates in the binding T lymphocyte cells expressing mFcgRIV (32, 33) and an NFAT of hTIGIT to the hCD155 ligand (Supplemental Fig. 2B). Thus, response element driving the expression of firefly luciferase were the binding of the T4 Ab to this FG loop blocks TIGIT from used as effector cells, and Raji-hTIGIT were used as target cells. binding with its ligand CD155. Moreover, owing to the high This ADCC assay revealed that T4 Ab in its mIgG2a form, a amino acid for this FG loop, T4 Ab exhibits murine Fc isotype capable of binding to Fc receptors and com- cross-reactivity with both hTIGIT and mTIGIT orthologs. plements (Supplemental Fig. 3B), induced a potent and dose- We next evaluated whether T4 Ab’s binding with TIGIT can dependent cytotoxic effect as executed by the effector cells interrupt TIGIT–CD155 interaction-mediated inhibitory functions, against the target cells (Fig. 2A). For testing T4 Ab’s ADCP ac- based on a previously reported YTS and 721.221 cell assay tivity, mouse BMDMs and Raji-hTIGIT were used as effector and strategy (10, 30, 31). Briefly, previous studies showed that YTS target cells, respectively. T4-mIgG2a was able to induce phago- cells (an NK cell line) achieve restricted killing of 721.221 target cytosis of BMDMs against Raji-hTIGIT cells (Fig. 2B). Similarly, cells (an MHC class I–negative human B cell line) (10, 30, 31) and T4-mIgG2a also induced CDC activity against Raji-hTIGIT target also showed that this killing can be effectively inhibited by cells in a CDC assay using LDH release as readout of complement- expressing hTIGIT in YTS cells (YTS-hTIGIT) and by expressing mediated target cell lysis (Fig. 2C). Collectively, these in vitro hCD155 in 721.221 cells (721.221–hCD155) (10, 30). Using this studies demonstrate that, in addition to its ability to block the assay system, we found that the T4 Ab completely restored the TIGIT–CD155 interaction, T4 Ab also exerts Fc-mediated effector ability of YTS-hTIGIT cells to kill 721.221-hCD155 cells functions. (Fig. 1B), thus demonstrating that the T4 Ab efficiently blocks To test whether the effector functions of T4 Ab contribute to its TIGIT–CD155 interaction-mediated inhibitory functions. antitumor effects in vivo, we constructed a set of T4 Ab Fc variants with either enhanced or abolished effector functions and compared T4 Ab exerts potent antitumor activity in mouse models their antitumor activities in the two aforementioned immune- Given our demonstrations that the T4 Ab cross-reacts with mTIGIT competent tumor-bearing mouse models. Specifically, we tested and blocks the interaction between mTIGIT and mCD155, we next two mIgG2a variants S239D/I332E and S239D/A330L/I332E The Journal of Immunology 5 Downloaded from http://www.jimmunol.org/ by guest on October 1, 2021

FIGURE 1. Functional analysis of a human anti-TIGIT Ab, T4, with cross-reactivity to both hTIGIT and mTIGIT. (A) T4 competes with CD155 for binding to TIGIT. T4-hIgG1 at serially diluted concentrations was tested for competing of hCD155-mFc or mCD155-mFc (2 mg/ml) to biotinylated hTIGIT or mTIGIT captured by immobilized streptavidin on an ELISA plate. The competition activity of T4 Ab is shown as the percentage of inhibition for CD155 binding to TIGIT. (B) T4 Ab effectively reverses NK cell cytotoxicity suppressed by TIGIT–CD155 interactions. The expression of hTIGIT on YTS- hTIGIT or hCD155 on 721.221-hCD155 stable cell line was assessed using anti-TIGIT Ab (10A7) or hTIGIT-Fc fusion protein by flow cytometry. For killing assays of 721.221 target cells by NK effector YTS cells and killing of 721.221-hCD155 cells by YTS-hTIGIT cells, the E:T ratios are indicated on the x-axis. For killing assays in the presence of T4 Ab, the E:T ratio was 2:1. 10A7 was included as an anti-TIGIT reference Ab control. Both T4 and 10A7 were full-length hIgG1 Abs. (C) The expression of mCD155 on tumor cells. Tumor cells from cell cultures (left) or cells reisolated from implanted tumors (right) were analyzed for expression of mCD155 using an mCD155-specific Ab. CT26 and 4T1 cells from tumors were defined as CD452 cells; A20 cells from tumors were defined as CD19+ cells. (D) Antitumor activity of T4 Ab in syngeneic mouse models. BALB/c mice bearing CT26 tumors were ran- domized into two groups (n = 3–5 per group) with similar tumor volumes (50–200 mm3): no-treatment control group or T4 Ab treatment group (10 mg/kg). The time points for Ab treatment are marked by arrows. Tumor volumes are shown as mean 6 SEM. (E)Asin(D), but for A20 and 4T1 tumor models. The results shown are representative data for at least two to three experiments.

(DLE) with enhanced FcgR-mediated effector functions (34–41) T4-mIgG2a-DANA did not induce any ADCC (Fig. 2A). As for and one mIgG2a 265A/N297A (DANA) variant with abolished ADCP, T4-mIgG2a, T4-mIgG2a-DE, and T4-mIgG2a-DLE FcgR-mediated effector functions (42–46). Prior to mouse exhibited similar levels of ADCP activities, whereas T4- studies, we first tested these T4 Ab Fc variants’ binding to mIgG2a-DANA did not induce any ADCP (Fig. 2B). For mFcgRs, and found that each elicited the expected differential CDC, T4-mIgG2a, T4-mIgG2a-DE, and T4-mIgG2a-DANA trends in relative binding activity for mFcgRs, all while maintaining showed similar CDC activities, but T4-mIgG2a-DLE did not similar binding affinity with hTIGIT and mTIGIT (Supplemental elicit CDC at all (Fig. 2C). These findings support three major Fig. 3A, 3B). conclusions. T4-mIgG2a induces ADCC, ADCP, and CDC T4-mIgG2a-DE and T4-mIgG2a-DLE variants both exhibited in vitro. T4-mIgG2a-DE outperforms T4-mIgG2a for its ADCC significantly higher ADCC activities than unmodified T4-mIgG2a; function but has similarly strong ADCP and CDC functions 6 TIGIT-BLOCKING Ab Fc DEPENDENTLY CONFER ANTITUMOR EFFECTS Downloaded from http://www.jimmunol.org/ by guest on October 1, 2021

FIGURE 2. T4 Ab’s antitumor activity depends on Fc-mediated effector functions. (A) ADCC effector functions induced by T4 Abs. ADCC activity was measured using a reporter assay system. Jurkat-NFAT-Luc2p/mFcgRIV and Raji-hTIGIT cells were used as effector cells and target cells, respectively. Abs were tested at the indicated concentrations with three replicates. The E:T ratio was 6:1. (B) ADCP effector functions induced by T4 Abs via macrophages. Raji-hTIGIT target cells were labeled with CFSE fluorescent dye prior to mixing with mouse BMDMs stained with F4/80–Alexa Fluor 633 Ab. The E:T ratio was 1:2. ADCP activity was monitored via fluorescence microscopy. The phagocytosis index was determined as the number of CFSE-positive target cells per 100 macrophages. (C) CDC effector functions induced by T4 Abs. Raji-hTIGIT target cells were incubated with T4 Abs in the presence of 5% rabbit complement sera. CDC activity was measured using LDH-releasing assays. Abs were tested at 15–20 mg/ml (B and C). (D) A comparison table of Fc- mediated effector functions inducted by T4 Ab and its Fc variants; positive (+), negative (2), and enhanced (++) activities are shown. (E) Antitumor activities of T4 Ab and its Fc variants in CT26 and A20 tumor models. The animal studies were performed in a manner similar to those in Fig. 1D and 1E. *p , 0.05 for the T4-mIgG2a group compared with the control group, **p , 0.01 for the T4-mIgG2a-DLE group or the T4-mIgG2a-DE group compared with the control group.

with T4-mIgG2a. T4-mIgG2a-DLE exerts no CDC function yet normal CDC function in vitro, it is conceivable that the observed induces a substantial enhancement of ADCC function over T4- antitumor effects of the T4 variants do not require CDC function. mIgG2a, whereas it induces a similar extent of ADCP activity We also examined the pharmacokinetic (PK) properties of the (Fig. 2D). It is therefore clear that the T4 Ab Fc variants we T4 Ab Fc variants in BALB/c mice and found that T4-mIgG2a, developed represent a useful tool set for in vivo studies to T4-mIgG2a-DANA, as well as T4-hIgG1 all had comparable dissect the specific impacts of the T4 Ab’s effector functions PK profiles, whereas T4-mIgG2a-DE and T4-mIgG2a-DLE and its ability to block TIGIT–CD155 interactions. had markedly faster clearance and shorter serum half-lives In BALB/c mice bearing CT26 or in A20 tumor models, (Supplemental Fig. 3C), presumably because of the relatively treatment with the T4-mIgG2a-DLE or T4-mIgG2a-DE variants lower stability of these Abs in vivo. Despite these apparently resulted in improved antitumor effects as compared with wild type inferior PK profiles, our data showed that T4-mIgG2a-DE and T4-mIgG2a, with especially pronounced improvements against the T4-mIgG2a-DLE exert stronger antitumor activity (Fig. 2E), so A20 tumors, findings consistent with their enhanced ADCC ef- we tentatively conclude that the improved antitumor activities fector functions (Fig. 2E). The T4-mIgG2a-DANA variant, which observed for these two Ab variants can be attributed to their su- exerts no FcgR-mediated effector functions in vitro, exhibited perior effector functions. Taken together, our results demonstrate minimal or no antitumor activity against either the CT26 or A20 that the blockade function of T4 Abs alone is insufficient to confer tumors (Fig. 2E). Recalling our finding that T4-mIgG2a-DLE does antitumor effects in vivo, that FcgR engagement-mediated effector not exert any CDC function and that T4-mIgG2a-DANA exerts functions are required for antitumor activity, and that Fc variants The Journal of Immunology 7 with enhanced effector functions exert improved antitumor effi- status, highlighting the function of CD8 T cells in mediating tumor cacy in vivo. regression. Moreover, the depletion of CD8+ T cells completely ab- rogated the therapeutic effect of T4 Ab (Fig. 4B), supporting that CD8+ T4 Ab mediates depletion of Tregs in tumors T cells are essential for the observed antitumor effects of the T4 Ab. Tregs, a potent type of immunosuppressive immune cell, are able to We next assessed potential roles of NK cells in the antitumor infiltrate into a vast array of different tumor types, in which they activity of the T4 Ab with NK cell depletion experiments using an promote the early stages of tumor development (47–50). Of par- anti–Asialo-GM1 Ab. NK cell depletion began 1 d before T4 Ab ticular note, Tregs with high TIGIT expression are known to be treatment in mice bearing established CT26 tumors and continued highly active and to confer immunosuppressive effects (12). Pre- until the end of T4 Ab treatment (Supplemental Fig. 4B). Similar vious studies have reported that the FcgR engagement-dependent to our observations with CD8+ cells, we found that NKs in this antitumor activities of Abs that target CTLA-4, OX40, or GITR study function as antitumor effector cells; tumors grew much are accompanied by reductions in Treg frequency within the tumor faster in NK-depleted mice than in nondepleted mice. Moreover, microenvironment (18–21, 51–53). the depletion of NK cells greatly impaired the therapeutic effect of To test whether T4 Ab treatment also causes reduced Treg T4 Ab treatment (Fig. 4B), indicating that the antitumor effects + frequency in tumors, we analyzed the frequency of Foxp3 Tregs of the T4 Ab involve NK cells. We also assessed potential roles of + + as well as total CD4 and CD8 T cells in tumors, spleens, and neutrophils and macrophages in the antitumor effects of the T4 Ab lymph nodes in both CT26 and A20 tumor model mice, either with by depleting these cell types, specifically using an anti–Ly-6G Ab T4 Ab treatment, an isotype control, or an Fc-silent T4 variant. On to deplete neutrophils and using clodronate liposomes to deplete the one hand, upon treatment with the mIgG2a isotype control, we macrophages (Supplemental Fig. 4B). Depletion of neutrophils or + + Downloaded from detected little difference in the proportion of CD4 and CD8 macrophages had no significant effect on the antitumor activity of + T cells among CD3 T cells in tumors, spleens, and lymph nodes, the T4 Ab, indicating they are apparently uninvolved in the anti- + + whereas the proportion of Foxp3 Tregs among CD4 T cells in tumor effects of T4 Ab (Fig. 4B). tumors was much higher compared with spleens or lymph nodes Similar immune cell depletion studies in syngeneic BALB/c (Fig. 3A, Supplemental Fig. 4A). Likewise, both types of tumor mice bearing A20 tumors were conducted, and we observed + exhibited significantly higher proportions of TIGIT Tregs relative similar results to the CT26 tumor model; the depletion of CD8+ Tor to spleens or lymph nodes (Fig. 3B). NK cells blocked or substantially reduced the antitumor effects of http://www.jimmunol.org/ On the other hand, T4-mIgG2a treatment significantly reduced the T4 Ab. However, depletion of CD4+ T cells, Tregs, neutro- + + the proportion of Foxp3 Tregs among CD4 T cells in both types phils, or macrophages had little or no effect on tumor outcomes of tumor, but no such treatment-induced change occurred in following T4 Ab treatment (Supplemental Fig. 4C). spleens or lymph nodes. Moreover, T4 Ab treatment did not Given our finding that T4 Ab treatment causes a reduction in the + + affect total CD4 or CD8 T cell proportions in tumors, spleens, proportion of intratumoral Treg in an Fc-dependent manner and or lymph nodes. Treatment with an Fc-silent T4-mIgG2a-DANA considering our demonstration that NK cells are required for the T4 + + variant did not have any effect on the proportion of CD4 Foxp3 Ab’s efficacy, we surmised that NK cells are the immune cells Tregs in tumors (Fig. 3A, Supplemental Fig. 4A). These results

responsible for the effector function that drives the observed by guest on October 1, 2021 demonstrate that the T4 Ab targets Tregs specifically in tumors, intratumoral Treg reduction. To test this, we depleted NK cells in which it decreases the proportion of Foxp3+ Tregs in an Fc- before T4-mIgG2a treatment and monitored the frequency of dependent manner. intratumoral CD4+Foxp3+ Tregs after T4-mIgG2a treatment. We + found that depletion of NK cells indeed rendered T4 Ab unable to Both CD8 T and NK cells are required for T4 Ab’s + + antitumor activity reduce the frequency of CD4 Foxp3 Tregs in tumors (Fig. 4D). We further evaluated the effects of T4 Ab treatment on the We next sought to identify the immune cell types that contribute to proinflammatory cytokines of CD8+ T and NK cells in the tumor the antitumor effect of the T4 Ab. We first evaluated the contri- microenvironment. One day after one dose of T4-mIgG2a treat- bution of T cells using the aforementioned CT26 tumor model. T4 ment in the CT26 tumor model, single-cell suspensions containing Ab treatment induced effective CT26 tumor regression in WT immune cells were isolated from mice. and intracellular cytokine BALB/c mice but induced much less antitumor activity in T cell– staining (IFN-g, TNF-a, and IL-2) was performed to assess the deficient nude mice (Fig. 4A), clearly suggesting that T cells are level of cytokine production in the CD8+ T and NK cells. We required for the full therapeutic effect of T4 Ab. found that T4 treatment caused significantly elevated production To identify which T cell subset(s) are involved in T4 Ab– of these cytokines in the CD8+ T and NK cells from tumors but not + + mediated tumor regression, CD4 or CD8 T cells were depleted the cells from spleens or lymph nodes (Fig. 4C), indicating that T4 by treating mice with an anti-CD4 or an anti-CD8 Ab, respectively Ab treatment specifically increased antitumor immune responses in BALB/c mice bearing established CT26 tumors (Supplemental in tumors without affecting immune cell function in lymph organs. + Fig. 4B). The depletion of CD4 cells alone greatly repressed Taken together, our results demonstrate that CD8+ T and NK tumor growth, regardless of T4 Ab treatment status, demonstrating immune cells contribute to the therapeutic effect of T4 Ab in both + that CD4 cells are apparently not required for the antitumor CT26 and A20 tumor-bearing mouse models. Further, our results activity of the T4 Ab and suggesting that anti-CD4 Ab treatment support that the mechanisms of T4 Ab’s antitumor activity involve + resulted in removal of immunosuppressive CD4 Tregs (Fig. CD8+ T cells as well as promotion of Fc-dependent Treg clearance + 4B). To confirm that CD4 depletion confers depletion of im- via NK-mediated effector functions (i.e., ADCC). munosuppressive Tregs, we used a Treg-specific ablating Ab (anti-CD25, clone PC61) to deplete Tregs in CT26 tumors. We T4 Ab treatment induces durable and cross-protective observed the same results as upon depletion of all CD4+ Tcells; antitumor immune memory similar to that induced by that is, Treg depletion alone greatly repressed tumor growth, Treg depletion regardless of T4 Ab treatment status. In contrast, tumors grew We next investigated whether T4 Ab treatment induces immune faster in mice from which CD8+ T cells were depleted as com- memory. Mice with complete regressions of CT26 or A20 tumors pared with nondepleted mice, regardless of T4 Ab treatment after T4 Ab treatment were rechallenged with CT26, A20, or 4T1 8 TIGIT-BLOCKING Ab Fc DEPENDENTLY CONFER ANTITUMOR EFFECTS Downloaded from http://www.jimmunol.org/ by guest on October 1, 2021

FIGURE 3. T4 Ab induces Treg depletion in the tumor microenvironment. (A) FACS analysis of T cells isolated from tumor model mice with or without T4 Ab treatment. When CT26 or A20 tumors reached ∼50–200 mm3 in size, mice were randomized into groups (n = 3–5 per group) and treated with a single dose (5 mg/kg) of T4-mIgG2a, T4-mIgG2a-DANA, or an isotype control Ab. Top panel, The frequency of CD8+ or CD4+ cells in T cells (CD3+) and the frequency of Foxp3+ Tregs as a percentage of CD4+ T cells from CT26 or A20 tumor-bearing mice were analyzed after 2 d of Ab treatment. Bottom panel, Representative FACS plots of the data shown in the top panel. Each FACS plot represents one mouse in each group. (B) Intratumoral Tregs express high levels of mTIGIT in both CT26 and A20 tumors. Left panel, Frequency 6SEM of TIGIT+ Tregs. The gating strategy is shown in Supplemental Fig. 4A. Right panel, Representative flow cytometry data showing TIGIT expression on Tregs in tumors. tumors on day 80–100 after the initial tumor inoculation and in the (54, 55). Specifically, a T cell epitope (GSW11) derived from en- absence of any further treatment. Mouse sera were collected prior dogenous murine leukemia virus envelope protein GP90 was the to tumor cell rechallenge and confirmed to have no detectable T4 target of the cross-protective immunity that was selectively sup- Ab before the rechallenge. In contrast to age-matched naive mice, pressed by the presence of Tregs. Consistent with the cross-reactivity the T4 Ab–cured mice were resistant to rechallenge with the same pattern we observed for T4 Ab, GP90 has not been detected in 4T1 tumor (i.e., mice that exhibited full CT26 regression were sub- cells but is present in both CT26 and A20 cells (54, 56). sequently resistant to rechallenge with CT26) (Fig. 5A), and the We next determined whether the cross-protective GSW11- same was true for A20 tumor mice (Fig. 5B). specific CD8+ T cells were induced in these T4 Ab–cured mice Surprisingly, mice with complete regressions of CT26 or A20 after tumor cell rechallenge. These experiments used two syn- tumors actually developed cross-tumor immunity. That is, both groups thetic MHC class I tetramers to enable FACS-based analysis of of mice successfully rejected both tumor types; we did not observe distinct T cell populations. The H-2Dd-GSW11 tetramer is com- such cross-tumor immunity upon rechallenge with cells from 4T1 mon to both CT26 and A20 because of the shared MHC class I tumors (Fig. 5A, 5B). Helping to contextualize this, previous studies allele (H-2Dd) and the GP90 Ag. The other tetramer is a CT26 reported a specific type of cross-protective CD8 T cell immunity tumor-specific H-2Ld-AH1 tetramer [H-2Ld is CT26-specific; against different tumors that was only induced upon Treg ablation AH1 is also a GP90-derived T cell epitope (57)]. The Journal of Immunology 9 Downloaded from http://www.jimmunol.org/ by guest on October 1, 2021

FIGURE 4. CD8+ T cells and NK cells are required for T4 Ab–mediated tumor regression. (A) T cells are required for an optimal therapeutic effect of T4 Ab. The antitumor effect of T4 Ab for treating CT26 tumor was compared in BALB/c (n = 5 per group) and nude mice (n = 6 per group). (B) Effects of selective immune cell depletions on T4 Ab’s therapeutic efficacy in treating CT26 tumor. CD4-depleting Abs (clone GK1.5) or CD8-depleting Abs (clone 2.43) were used to deplete CD4+ or CD8+ T cells. Treg-depleting Ab (ani-CD25, PC61-mIgG2a-DE) was used to deplete Tregs. Anti–Asialo-GM1 polyclonal Ab (Poly21460), anti–Ly-6G Ab (1A8 clone), or clodronate liposomes were used for the depletion of NK cells, neutrophils, or macrophages, respectively. Black arrows indicate the administrations of depletion Abs or clodronate liposomes; red arrows indicate T4 Ab treatment (10 mg/kg). Tumor volumes over time are shown (n = 4–6 per group). In the CD4+ T cell depletion experiment, the DCD4 group was compared with the control group. In the Treg-depletion experiment, the Ab, DTreg, or Ab plus DTreg groups were compared with the control group. In the CD8+ T or NK cell depletion experiment, the DCD8 or DNK groups were compared with the control group (blue stars). Ab plus DCD8 or Ab plus DNK groups were compared with the Ab-alone treatment group (purple stars). (C) Cytokine production analyses of CD8+ T and NK cells. Single-cell suspensions were isolated (Figure legend continues) 10 TIGIT-BLOCKING Ab Fc DEPENDENTLY CONFER ANTITUMOR EFFECTS

Seven days after tumor rechallenge, spleen cells were collected CD155, thereby preventing or reversing NK and CD8+ T cell and activated with GSW11 or AH1 peptides and were subsequently exhaustion and enhancing these cells’ effector functions (5, 59). assessed for the frequency of tetramer-specific CD8+ T cells by Consistently, we found that both NK and CD8+ T cells are es- flow cytometry. Compared with age-matched naive control mice, sential for the antitumor effects of our T4 anti-TIGIT Ab and the abundance of both AH1 and GSW11 tetramer-specific CD8+ additionally determined that mCD155 expression on tumor cells is T cells was increased in T4 Ab–cured mice after CT26 rechal- not a prerequisite for T4’s antitumor effects. Furthermore, our lenge (Fig. 5C). In contrast, only GSW11 tetramer-specific CD8+ study demonstrated that T4’s blockade function alone is clearly T cells were increased in the T4 Ab–cured mice after A20 insufficient and showed that the Fc–FcgR interaction-mediated rechallenge (Fig. 5C), findings indicating that GSW11-specific effector function is required for the T4 Ab’s antitumor activity. T cell immunity apparently contributes to the T4 Ab treatment- Specifically, by using two T4 Ab variants with changes in their Fc induced cross-protective immunity we observed for mice bearing domain with in vivo tumor models, we observed different antitu- CT26 and A20 tumors. mor effects. On the one hand, the FcgR binding-deficient variant Viewed together, these results establish that T4 Ab treatment can with normal complement binding activity (T4-mIgG2a-DANA) induce antitumor immune memory and demonstrate that such showed minimal or no antitumor activity. On the other hand, the memory is capable of providing durable cross-protection against FcgR binding-increased variant with no complement binding ac- tumor types that feature 1) common MHC class I allele(s)-restricted tivity (T4-mIgG2a-DLE) exerted improved antitumor effects as T cell epitope(s) and 2) Treg-mediated immune suppression of any compared with T4-mIgG2a wild type Ab. These results support responses capable of targeting such epitope(s). Recalling that T4 that T4 Ab does not require CDC function; rather, it requires FcgR Ab treatment reduces intratumoral Treg frequency, our results engagement-mediated effector functions for exerting its antitumor support that the antitumor T cell immunity conferred by our anti- activity. Future studies using FcgR knockout or complement- Downloaded from TIGIT Ab is dependent on Treg depletion. deficient mice with in vivo tumor models will help to defini- tively determine the in vivo role of FcgRs or complements Discussion for T4 Ab. Although several anti-TIGITAbs used alone or in combination with T4 Ab treatment caused a significant decrease in the proportion anti–PD-1/PD-L1 mAbs have entered clinical trials (25), it re- of intratumoral CD4+Foxp3+ Tregs, and we showed that NK cells mains unclear if (and how) the efficacy initially observed in mice are the immune cells responsible for the Fc–FcgR interaction- http://www.jimmunol.org/ can be effectively translated into humans. Owing to practical and mediated ADCC against intratumoral Tregs, which express ethical concerns associated with clinical trials, in-depth studies in TIGIT at a high level. It should be noted that a previous study mouse models are still essential for dissecting anti-TIGIT Abs’ showed that tumor-infiltrating macrophages are responsible for the complex antitumor mechanisms. The clinical stage anti-TIGIT Treg depletion caused by anti-CTLA4 Ab treatment (20). Whether Abs all specifically recognize hTIGIT, so their preclinical effi- the depletion of Tregs is one of the mechanisms for anti-TIGIT cacy in mouse models required the use of anti-mouse TIGIT Ab Abs has been controversial (5, 23, 60–62). A functional impact of surrogates (or transgenic mice knock-in for hTIGIT) (58). Thus, a FcgR engagement was reported in a recent study (23). However, in contrast to our work, that study did not report Fc-dependent

cross-species Ab able to bind both hTIGIT and mTIGIT should by guest on October 1, 2021 both simplify and accelerate preclinical development of TIGIT Treg depletion but, rather, concluded that the coengagement of Ab–based therapies and combination therapies. specific FcgRs on APCs might enhance Ag-specific T cell re- 10A7 is a previously reported anti-TIGIT Ab generated by sponses and antitumor activity (23). Another study also reported immunization of hamster (5) that possesses cross-species reac- that an anti-TIGIT Ab used in combination with an anti-PD1 Ab tivity; it has high affinity for mTIGIT yet very low affinity for did not reduce intratumoral Treg frequencies (5). We suspect that hTIGIT (Supplemental Fig. 1B). In the current study, we these discrepancies may reflect some differential influences from employed a cross-panning approach using phage display libraries the particular Abs in the separate investigations; alternatively, and identified the cross-species T4 Ab. To our knowledge, this is these may reflect differences in the experimental procedures or the first report of a functional anti-TIGIT Ab with human and differences in the time points at which intratumoral Tregs were murine cross-species reactivity that features similar binding af- examined. In our study, we examined immune cell distributions finities for both hTIGIT and mTIGIT. We explored the mechanism (including Tregs) within tumors as well as lymph organs after 2 d of its cross-species binding activity and characterized its potent of T4 Ab treatment and found that T4 Ab treatment Fc depen- inhibition against the growth of syngeneic murine tumors without dently caused a significant decrease in the proportion of Foxp3+ observing adverse effects in mice. The species cross-reactivity of Tregs among CD4+ T cells in tumors but not in lymph organs. T4 Ab also allowed us to directly dissect its mechanisms in mouse Studies have shown that tumor-bearing mice (those for which models and to develop a deeper understanding that can be lever- anti-TIGIT Ab treatment conferred full tumor regression) can aged for the design of more efficacious anti-TIGIT Abs or more develop immune memory against rechallenge with the same type of effective clinical deployment for treating cancer patients. tumor cells (5, 59). We found that T4 Ab treatment elicited durable Previous studies reported that anti-TIGITAbs enhance antitumor immune memory against rechallenge with the same tumor cells responses through blockage of the interaction between TIGIT and and also observed that T4 Ab treatment induced cross-immunity

from CT26-bearing mice 1 d after T4 Ab treatment. Intracellular cytokine staining (TNF-a, IFN-g, or IL-2) was performed to assess the level of cytokine production in the CD8+ T and NK cells. Prior to intracellular cytokine staining, cells were activated for 3 h in vitro with a cell activation mixture (with brefeldin A) (n = 3–6 per group). (D) NK is responsible for the decreased proportion of intratumoral Tregs after T4 treatment. When CT26 tumors reached ∼50–200 mm3 in size, mice were randomized into groups (n = 3–5 per group) and were treated with a single dose (5 mg/kg) of T4-mIgG2a Ab or an isotype control Ab. In DNK plus T4-mIgG2a group, anti–Asialo-GM1 polyclonal Ab (Poly21460) was used 1 d before T4-mIgG2a treatment to deplete NK cells in mice. The frequency of Foxp3+ Tregs as a percentage of CD4+ T cells in tumors from CT26 tumor-bearing mice was analyzed after 1 d of T4 Ab treatment. ****p , 0.0001. The Journal of Immunology 11 Downloaded from http://www.jimmunol.org/ by guest on October 1, 2021

FIGURE 5. Anti-TIGIT T4 Ab elicits durable systemic immunity. CT26 tumor-bearing mice cured by T4 Ab treatment were rechallenged with CT26, A20, or 4T1. The second tumor inoculation started ∼80–100 d after initial tumor inoculation. Age-matched naive mice were included as controls (n = 3–5 per group). (B) A20 tumor-bearing mice cured by T4 Ab treatment were rechallenged with A20, CT26, or 4T1. The study was performed in a manner similar to (A). (C) Seven days after tumor cell rechallenge, spleen cells from each mouse were isolated and activated using AH1 or GSW11 peptide and IL-2 for 7 d. Subsequently, the frequencies of CD8+ H-2Ld-AH1 tetramer+ and CD8+ H-2Dd-GSW11 tetramer+ cells among total splenocytes were assessed by flow cytometry. The dot plots shown are representative data for one mouse of each group (n = 3); the mean 6 SEM of tetramer+/CD8+ percentage shown is from three mice in each group. Data are representative of two independent experiments. **p , 0.01, ***p , 0.001, ****p , 0.0001 between two histologically distinct tumors (CT26 and A20); these two studies also showed the GSW11-specific CD8+ Tcell such phenomena have not been reported previously for any PD- activity was suppressed by the presence of Tregs (54, 55). In 1/PD-L1, CTLA4, or TIGIT Abs. A recent study showed that a our study, T4 Ab treatment induced the same cross-protective Treg-depleted anti-CD25 immunotoxin facilitated the devel- GSW11-specific T cell activity in both CT26 and A20 tumors. opment of CD8+ T cell–dependent cross-tumor immunity (63). Again, this result strongly supports that the T4 Ab’s effects Two other studies reported thatTregablationinducedcross- apparently rely on depletion of Tregs to exert antitumor T cell protective CD8+ T cell immunity (GSW11-specific) against immunity, at least for certain types of tumor in which immune challenges by different tumors (CT26 and A20) (54, 55), and responses are suppressed by Tregs. 12 TIGIT-BLOCKING Ab Fc DEPENDENTLY CONFER ANTITUMOR EFFECTS

In summary, our study demonstrates that an anti-TIGIT Ab’s 19. Selby, M. J., J. J. Engelhardt, M. Quigley, K. A. Henning, T. Chen, M. Srinivasan, and A. J. Korman. 2013. Anti-CTLA-4 antibodies of IgG2a antitumor effects are mediated via multiple immunological events isotype enhance antitumor activity through reduction of intratumoral regulatory + involving CD8 T cells and NK cells. Specifically, Fc-mediated T cells. Cancer Immunol. Res. 1: 32–42. effector functions mediated by NK cells initiate Treg depletion- 20. Simpson, T. R., F. Li, W. Montalvo-Ortiz, M. A. Sepulveda, K. Bergerhoff, F. Arce, C. Roddie, J. Y. Henry, H. Yagita, J. D. Wolchok, et al. 2013. Fc- mediated immune processes in which an anti-TIGIT Ab can ap- dependent depletion of tumor-infiltrating regulatory T cells co-defines the effi- parently induce CD8+ T cell responses and immune memory cacy of anti-CTLA-4 therapy against melanoma. J. Exp. Med. 210: 1695–1710. against formerly suppressed or cryptic T cell Ags. Fundamentally, 21. Arce Vargas, F., A. J. S. Furness, K. Litchfield, K. Joshi, R. Rosenthal, E. Ghorani, I. Solomon, M. H. Lesko, N. Ruef, C. Roddie, et al.; TRACERx our discovery that multiple complementary mechanisms syner- Melanoma.; TRACERx Renal.; TRACERx Lung Consortia. 2018. Fc effector gistically confer an Ab’s therapeutic effects strongly supports the function contributes to the activity of human anti-CTLA-4 antibodies. Cancer Cell 33: 649–663.e4. conceptual merit of developing combinatory therapeutic strategies 22. Dahan, R., E. Sega, J. Engelhardt, M. Selby, A. J. Korman, and J. V. Ravetch. comprising anti-TIGIT Abs alongside other agents that target 2015. FcgRs modulate the anti-tumor activity of antibodies targeting the PD-1/ checkpoint proteins. PD-L1 axis. [Published erratum appears in 2015 Cancer Cell 28: 543.] Cancer Cell 28: 285–295. 23. Waight, J. D., D. Chand, S. Dietrich, R. Gombos, T. Horn, A. M. Gonzalez, Acknowledgments M. Manrique, L. Swiech, B. Morin, C. Brittsan, et al. 2018. Selective FcgRco- We thank the Animal Facility at the National Institute of Biological Sciences engagement on APCs modulates the activity of therapeutic antibodies targeting T cell . Cancer Cell 33: 1033–1047.e5. for help in handling and care of mice, and the Biological Resource Center at 24. Du, X., F. Tang, M. Liu, J. Su, Y. Zhang, W. Wu, M. Devenport, C. A. Lazarski, the National Institute of Biological Sciences for DNA sequencing. P. Zhang, X. Wang, et al. 2018. A reappraisal of CTLA-4 checkpoint blockade in cancer immunotherapy. Cell Res. 28: 416–432. 25. Burugu, S., A. R. Dancsok, and T. O. Nielsen. 2018. Emerging targets in cancer Disclosures immunotherapy. Semin. Cancer Biol. 52: 39–52. Downloaded from The authors have no financial conflicts of interest. 26. Li, D., W. He, X. Liu, S. Zheng, Y. Qi, H. Li, F. Mao, J. Liu, Y. Sun, L. Pan, et al. 2017. A potent human neutralizing antibody Fc-dependently reduces established HBV infections. eLife 6: e26738. 27. Ishiguro, T., M. Sugimoto, Y. Kinoshita, Y. Miyazaki, K. Nakano, H. Tsunoda, References I. Sugo, I. Ohizumi, H. Aburatani, et al. 2008. Anti-glypican 3 antibody as a 1. Iwai, Y., J. Hamanishi, K. Chamoto, and T. Honjo. 2017. Cancer immunother- potential antitumor agent for human liver cancer. Cancer Res. 68: 9832–9838. apies targeting the PD-1 signaling pathway. J. Biomed. Sci. 24: 26. 28. Pegram, M., and D. Ngo. 2006. Application and potential limitations of animal 2. Ott, P. A., F. S. Hodi, H. L. Kaufman, J. M. Wigginton, and J. D. Wolchok. 2017. models utilized in the development of trastuzumab (herceptin): a case study. Adv.

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costimulatory signaling in agonistic GITR targeting for tumor immunotherapy. tumors with an anti-CD25 immunotoxin induces CD8 T cell-mediated systemic http://www.jimmunol.org/ Cancer Res. 77: 1108–1118. antitumor immunity. Proc. Natl. Acad. Sci. USA 116: 4575–4582. by guest on October 1, 2021 Supplemental Fig.1

A 48 48 48 48 48 48 T4 #18 #43 #45 hm7 10A7 36 36 36 36 36 36 Ctrl. s t

n 24 24 24 24 24 24 CHO-hTIGIT u

o 12 12 12 12 12 12 C CHO-mTIGIT

0 0 0 0 0 0 0 1 2 3 4 100 101 102 103 104 100 101 102 103 104 100 101 102 103 104 100 101 102 103 104 10 10 10 10 10 100 101 102 103 104 TIGIT

B T4 #18 #43 #45 hm7 10A7

hTIGIT

mTIGIT

C hTIGIT mTIGIT 35 18 30 25 ) 13 ) U U 20 R R ( ( e e 15 s 8 s n n

T4 o o 10 p p s s e e 5

R 3 R 0 -2 -5 -100 0 100 200 300 400 -100 0 100 200 300 400 Time (s) Time (s)

25 30 25 20 ) U R ) ( 20

U 15 e R s (

n 15 o e 10 s p s n 10 e T4M o p R

s 5

e 5 R 0 0 -5 -5 -100 0 100 200 300 400 -100 0 100 200 300 400 Time (s) Time (s)

Antigen Ab ka (1/Ms) kd (1/s) KD (M) T4 1.82E+05 4.55E-03 2.50E-08 hTIGIT T4M 6.31E+04 5.52E-04 8.75E-09 T4 1.40E+05 4.55E-04 3.24E-09 mTIGIT T4M 1.34E+05 3.67E-04 2.74E-09

Supplemental Figure 1. Binding analyses of anti-TIGIT Abs to hTIGIT and mTIGIT using FACS and SPR. (A) Binding of anti-TIGIT Abs to CHO-hTIGIT and CHO-mTIGIT cells. The selected Abs in full-length human IgG1 form (hIgG1) were tested for binding to CHO-hTIGIT and CHO-mTIGIT stable cell lines using FACS. (B) Single-cycle kinetic analysis of anti- TIGIT Abs binding to hTGIT or mTIGIT using SPR (Biacore T200). Abs were captured on a CM5 chip via immobilized anti- human IgG1 Ab on the chip. The ECD of hTIGIT or mTIGIT (100 nM) were injected over the chip surface. The result was analyzed using the Biacore T200 evaluation software. 10A7 was used as a reference Ab control. (C) As in B, but for multi- cycle kinetic analysis of the interaction between T4-hIgG1 Ab and hTIGIT or mTIGIT using SPR. Values of ka, kd, and KD were listed in the table. Supplemental Fig.2

A

10 20 30 40 50 60 hTIGIT M T G T I E T T G N I S A E K G G S I I L Q C H L S S - - - T T A Q V T Q V N W E Q Q D Q - - L L A I C N A D L G - W H I S P S F K D R V A mTIGIT - - - . . D . K R . . . . . E . . . V . . . . . F . . - - - D . . E . . . . D . K . . . . - - . . . . Y S V . . . - . . V A S V . S . . . V hCD155 - D V V V Q A P T Q V P G F L . D . V T . P . Y . Q V P N M E V T H . S . L T . A R H G E S G S M . V F H Q T Q . P S Y S E S K R L E F . . Chimera A - ...... V T . P . Y . Q V P N M E V T H . S ...... Chimera B ...... L T . A R H G E S G S M ...... Chimera C ...... C Strand variant ...... A . A A . A ...... C’C’’ Loop variant ...... S . H Q T Q A ...... FG Loop variant ...... C strand C’C’’ loop

70 80 90 100 110 hTIGIT P - - G P G L G - - - L T L Q S L T V N D T G E Y F C I Y H T Y P D G T Y T G R I F L E V L E S S V A E H G A R F Q I P mTIGIT . - - . . S . . - - - . . F . . . . M ...... T . . . . . G . I . K . . . . . K . Q . . . . . Q F Q T A P L G G hCD155 A R L . A E . R N A S . R M F G . R . E . E . N . T . L F V . F . Q . S R S V D . W . R . . Chimera A ...... Chimera B ...... Chimera C ...... N . T . L F V . F . Q . S R S V D . W ...... C Strand variant ...... C’C’’ Loop variant ...... FG Loop variant ...... A . R . A A R ...... FG loop

B Cell only Chimera A Chimera B Chimera C

T4-hIgG1

0 2 4 6 hCD155-hFc 100 102 104 10 6 10 10 10 10 100 102 104 10 6 100 102 104 10 6 10A7-hIgG1 WT C Stard virant C’C’’ Loop variant FG Loop variant GC33-hIgG1 Neg. Ctrl.

100 102 104 10 6 100 102 104 10 6 100 102 104 10 6 100 102 104 10 6 FITC

Supplemental Figure 2. Epitope mapping of T4 Ab. (A) Amino acid sequence alignment of N-terminal IgV domains of hTIGIT, mTIGIT, hCD155, hTIGIT/hCD155 chimeras, and hTIGIT variants. Dots denote identical amino acids, dashes indi- cate gaps. The replaced or mutated regions are shaded with gray color. The amino acid numbers are based on hTIGIT. (B) Flow cytometry analysis of different Abs or hCD155-hFc ligand binding to CHO cells transiently expressing full-length hTIGIT (wild type, WT), or its chimeras or variants. The secondary Ab staining only was used as a negative control (Neg. Ctrl.); The cell surface expression levels of WT hTGIT, chimeras, and variants were assessed using a mAb (GC33) recognizing the N- terminal tag. Supplemental Fig.3

A C Binding ELISA (hTIGIT) Binding ELISA (mTIGIT) T4 Ab (10mg/kg) in mice

1.5 150000 1.5 T4-mIgG2a T4-mIgG2a hIgG1 T4-mIgG2a-DANA T4-mIgG2a-DANA mIgG2a 0

3 mIgG2a-DANA 6 1.0 T4-mIgG2a-DE 1.0 T4-mIgG2a-DE l 100000 D

m mIgG2a-DE /

O T4-mIgG2a-DLE T4-mIgG2a-DLE - g

0 mIgG2a-DLE n

5 Ctrl. Ctrl. ] 4

0.5 0.5 b 50000 D A O [

0.0 0.0 0

-4 -3 -2 -1 0 1 -4 -3 -2 -1 0 1 0 100 200 300 400 500 Log [Ab] (µg/ml) Log [Ab] (µg/ml) Time (hours)

B T4-mIgG2a T4-mIgG2a- T4-mIgG2a- T4-mIgG2a- DANA DE DLE n.m.

mFcγRI

n.m.

mFcγRIIb

n.m.

mFcγRIII

n.m.

mFcγRIV

Supplemental Figure 3. Binding analyses of T4 Ab Fc variants to hTIGIT, mTIGIT, or mFcγRs. (A) Binding analyses of T4 Ab and its Fc variants to purified TIGIT-ECD proteins via ELISA assay. Biotinylated hTIGIT-ECD or mTIGIT-ECD was cap- tured by immobilized streptavidin. Abs were examined at indicated concentrations. (B) Characterization of the binding of T4 Ab and its Fc variants to mFcγRs using Biacore T200. The experiments were similarly performed as described in Supplemen- tal Fig. 1B-C. The binding kinetics or affinity was evaluated by the Biacore T200 evaluation software using “Kinetic evalua- tion” model for high affinity interactions, or “Affinity evaluation” model for low affinity interactions. “n.m.” indicates the affinity was not measurable. (C) Pharmacokinetic analyses of T4 Abs in BALB/c mice. Serum concentrations of Abs were detected using ELISA as described in the “Materials and Methods” section and calculated according to a standard curve. Serum Ab concentration vs. time profile of a single administration of T4 Ab (10 mg/kg) is shown. Data are presented as mean values ± SEM. Supplemental Fig.4

A 250K 105 150K 150K 200K 4 Single cells-SSC d 10 Single cells-FSC

a 100K A 100K A 150K 98.5 A - - - e 95.9 CD3 C C C

d 3 / 10 100K 50K 50K 14.7 S S S e S F F

v live

i 0 50K l 71.6 0 0 -103 0 0 50K 100K150K200K250K 0 50K 100K150K200K250K 0 50K 100K 150K -103 0 103 104 105 FSC-A FSC-H SSC-H CD3

100 5 5 10 10 CD4 80 65.7 4 104 10 s CD8 t 60

Ctrl. Ab 4 4 n 28.6 3 3 D D u 40 10 Foxp3 in CD4 10 o C C

C 36.8 Anti-mTIGIT Ab 20 0 0 3 -103 0 -10 0 104 105 -103 0 103 104 10 5 -103 0 103 104 105 mTIGIT Foxp3 CD8 B Ctrl. CD4 depletion Ctrl. Treg depletion Ctrl. CD8 depletion 7 7 7 7 10 10 5 5 10 10 10 10 6 10.6% 4.00% 106 10 106 106 4 4 5 23.9% 5 0.49% 10 10 5 12.4% 5 0.51% 10 10 10 10

4 4 4 4 8 4 4 10 10 3 3 10 10 D D 10 10 D

C 3 3 C C 3 3 10 10 0 0 10 10 2 2 2 2 -10 -10 3 3 -10 -10 3 3 -10 -10 3 3 -10 -10 -10 -10 2 3 4 5 6 7 3 3 4 5 3 3 4 5 2 3 4 5 6 7 2 3 4 5 6 7 10 10 10 10 10 10 102 103 104 105 106 107 -10 0 10 10 10 -10 0 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 CD45 Foxp3 CD45 Ctrl. NK depletion Ctrl. Neutrophil depletion Ctrl. Macrophage depletion 7 7 7 7 7 10 107 10 10 10 10

6 6 6 8.53% 6 0.13% 6 6 10 4.01% 10 0.43% 10 10 10 10 5 5 5 5 5 5

1 10 10 10 10 10 10 b 3.31% 0.20% G 1 M 4 10 104 4 4 4 4 1 6 10 10 10 10 G y

3 3 3 3 D S

10 10 L 10 10 3 3

C 10 10 A 2 2 2 2 10 10 102 102 -10 -10 -102 -102

2 3 4 5 6 7 2 3 4 5 6 7 2 3 4 5 6 7 10 10 10 10 10 10 10 10 10 10 10 10 -103 103 104 105 106 107 -103 103 104 105 106 107 10 10 10 10 10 10 102 103 104 105 106 107 CD45 CD11b F4/80 C CD4 T cell depletion (A20) Treg cell depletion (A20) CD8 T cell depletion (A20)

) 2000 ) 2000 ) 2500 3 Ctrl. 3 Ctrl. 3 Ctrl. * m m m

m Ab m Ab m 2000 Ab ( 1500 ∆ ( 1500 ∆ ( ∆ e CD4 e Treg e CD8 ****

m Ab+∆CD4 m Ab+∆Treg m 1500 Ab+∆CD8 u u u

l 1000 l 1000 l

o o o 1000 v v v

r r 500 r

o 500 o o **** 500 m **** m **** m u n.s. u **** u T 0 T 0 T 0

0 5 10 15 20 25 30 -10 0 10 20 30 0 5 10 15 20 25 30

NK depletion (A20) Neutrophil depletion (A20) Macrophage depletion (A20)

) 2500 ) 2000 ) 2000 3 Ctrl. **** 3 Ctrl. 3 Ctrl. m m m

m 2000 Ab m Ab m Ab ( ∆ ( 1500 ∆ ( 1500 ∆ ϕ e NK e neutrophil e M 1500 **** m Ab+∆NK m Ab+∆neutrophil m Ab+∆Mϕ u u u

l l 1000 l 1000

o 1000 o o v v v r r r

o 500 o 500 o 500 m m m

u u n.s. u n.s. T 0 T 0 T 0

0 5 10 15 20 25 30 0 5 10 15 20 25 30 0 5 10 15 20 25 30

Days post tumor implantation Days post tumor implantation Days post tumor implantation

Supplemental Figure 4. Gating strategy for TIL analysis, the efficiency of in vivo immune cell depletion, and the effects of selec- tive immune-cell depletions on T4 Ab’s therapeutic efficacy in treating A20 tumor. (A) An example of gating strategy for the identifica- tion of CD4+, CD8+, and Foxp3+ Treg cells in TILs of CT26 or A20 tumor models. (B) The immune cell depletion efficiency in tumor-naive BALB/c mice. The Abs or reagents used for selective depleting different immune cell subtypes were the same as that described in Fig. 4, and the detailed methods are described in “Materials and Methods”. The CD4+ or CD8+ T cell depletion efficiency was confirmed to be greater than 95% in splenic T cells; the efficiency of NK cell depletion in spleen was nearly 90%; the depletion efficiency of neutrophils in blood was greater than 98%, and the efficiency of macrophage depletion in spleen was greater than 94%. The Treg depletion efficiency in spleen was about 63%. The controls represent results obtained from mice that were not treated with the depletion Abs or reagents. Each FACS dot plot represents the results obtained from two mice with the same treatment. (C) Effects of selective immune-cell depletions on T4 Ab’s therapeutic efficacy in treating A20 tumor. As shown in Fig.4C for CT26 tumor, this panel is for A20 tumor.