Antibodies Targeting Human OX40 Expand Effector T Cells and Block Inducible and Natural Regulatory Function

This information is current as Kui S. Voo, Laura Bover, Megan L. Harline, Long T. Vien, of September 30, 2021. Valeria Facchinetti, Kazuhiko Arima, Larry W. Kwak and Yong J. Liu J Immunol 2013; 191:3641-3650; Prepublished online 6 September 2013;

doi: 10.4049/jimmunol.1202752 Downloaded from http://www.jimmunol.org/content/191/7/3641

Supplementary http://www.jimmunol.org/content/suppl/2013/09/06/jimmunol.120275 Material 2.DC1 http://www.jimmunol.org/ References This article cites 39 articles, 16 of which you can access for free at: http://www.jimmunol.org/content/191/7/3641.full#ref-list-1

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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 © 2013 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

Antibodies Targeting Human OX40 Expand Effector T Cells and Block Inducible and Natural Function

Kui S. Voo,* Laura Bover,* Megan L. Harline,* Long T. Vien,* Valeria Facchinetti,* Kazuhiko Arima,† Larry W. Kwak,‡ and Yong J. Liu*,x

Current cancer vaccines induce tumor-specific T cell responses without sustained tumor regression because immunosuppressive elements within the tumor induce exhaustion of effector T cells and infiltration of immune-suppressive regulatory T cells (Tregs). Therefore, much effort has been made to generate agonistic Abs targeting members of the TNFR superfamily, such as OX40, 4- 1BB, and GITR, expressed on effector T cells and Tregs, to reinvigorate T cell effector function and block Treg-suppressive function. In this article, we describe the development of a panel of anti-human OX40 agonistic mouse mAbs that could promote effector CD4+ and CD8+ T cell proliferation, inhibit the induction of CD4+ IL-10 -producing type 1 regulatory T cells, inhibit the expansion of ICOS+IL-10+ Tregs, inhibit TGF-b–induced FOXP3 expression on naive CD4+ T cells, and block natural Downloaded from Treg–suppressive function. We humanized two anti–human OX40 mAb clones, and they retained the potency of their parental clones. These Abs should provide broad opportunities for potential combination therapy to treat a wide realm of cancers and preventative vaccines against infectious diseases. The Journal of Immunology, 2013, 191: 3641–3650.

umerous therapeutic cancer vaccines have been de- cells and macrophages that actively recruit Tregs expressing CCR4

veloped that induce tumor-specific T cell responses in (14, 15). Furthermore, TGF-b and IL-10 secreted by cancer cells http://www.jimmunol.org/ N patients (1–4); however, patient clinical response rates can induce TGF-b–producing Tregs or IL-10–producing Tregs, following vaccination have been low. This low response rate has which actively suppress effector T cell (Teff) function and ex- been attributed largely to the presence of immunosuppressive pansion (10, 16), either directly or indirectly through the induc- elements at the tumor sites that induce exhaustion of tumor- tion of regulatory dendritic cells (10). These two cytokines not only infiltrating lymphocytes (TILs), influx of immune-suppressive can induce iTregs, they also were shown to maintain the expression + CD4 regulatory T cells (Tregs), and secretion of the anti- of the transcription factor FOXP3 and the suppressive function of inflammatory cytokines TGF-b and IL-10 that induce the genera- Tregs (17, 18). As a consequence of these negative factors in the tion of regulatory dendritic cells and maintain CD4+ naturally

+ + tumor environment, the majority of TILs are either functionally by guest on September 30, 2021 occurring FOXP3 Tregs (nTregs) or convert CD4 Tcellsinto impaired CD4+/CD8+ T cells (5, 12) or have been converted into IL- inducible IL-10+/TGF-b+ Tregs (iTregs) (5–11). Indeed, recent 10–producing (19, 20) or TGF-b–producing (21) Tregs that prevent reports showed that tumor-specific CD8+ T cells from melanoma antitumor immune responses. patients were functionally impaired and expressed high levels of the Evidence from the literature suggests that these negative ele- inhibitory receptors PD-1, TIM-3, CTLA4, and LAG3 (5, 12). In ments within the tumor microenvironment can be modulated by addition to impaired CD8+ T cells, a large number of CD25+CD4+ Tregs were found in the tumors and draining lymph nodes of many triggering members of the TNFR superfamily, such as OX40, 4- cancer patients (13). The accumulation of Tregs at tumor sites has 1BB, and GITR, which are highly expressed on Teffs and Tregs, to been attributed to the secretion of the chemokine CCL22 by cancer reinvigorate T cell effector function and block Treg-suppressive function (13, 22–27). Therefore, intense research over the last decade focused on generating reagents that trigger these mol- *Department of Immunology, Center for Cancer Immunology Research, The Univer- sity of Texas M.D. Anderson Cancer Center, Houston, TX 77030; †Department of ecules. We recently showed that triggering of human OX40 Medical Biochemistry, Saga Medical School, Saga 849-0937, Japan; ‡Department of (hOX40) with OX40 ligand shuts down the generation and func- Lymphoma and Myeloma, Center for Cancer Immunology Research, The University of Texas M.D. Anderson Cancer Center, Houston, TX 77030; and xBaylor Institute tion of IL-10–producing type 1 Tregs (Tr1), whereas agonists of for Immunology Research, Dallas, TX 75204 4-1BB and GITR were ineffective (28). Recent studies further Received for publication October 4, 2012. Accepted for publication May 21, 2013. showed that triggering of OX40 turns off FOXP3+ Tregs and This work was supported by National Institutes of Health Grants U19 AI071130 and inhibits TGF-b– and Ag-driven conversion of naive CD4+ T cells R01 AI061645 (to Y.J.L.), the University of Texas M.D. Anderson Cancer Founda- + + tion (UTMDACC) (to Y.J.L.), and UTMDACC Specialized Programs of Research into CD25 FOXP3 T cells (29, 30). Studies from mice demon- Excellence (SPORE) (to K.S.V.). L.W.K. was supported by the Leukemia and strated that targeting OX40 using agonistic mAbs (31, 32) could Lymphoma Society (Specialized Center of Research Grant 7262-08). promote Teff function and memory by promoting T cell survival Address correspondence and reprint requests to Dr. Yong-Jun Liu, Baylor Institute for and clonal expansion (33, 34), inhibit the function or survival Immunology Research, 3434 Live Oak Street, Dallas, TX 75204. E-mail address: + [email protected] of normal and tumor-derived FOXP3 nTregs (23, 31), and in- Abbreviations used in this article: CD32-L cell, mouse fibroblast L cell expressing duce changes in the tumor stroma, including a decrease in the CD32; FL, follicular lymphoma; hOX40, human OX40; hOX40-L cell, mouse fibro- number of macrophages and myeloid-derived suppressor cells (35). blast L cell expressing hOX40; ION, ionomycin; iTreg, inducible IL-10+/TGF-b+ regulatory T cell; nTreg, naturally occurring FOXP3+ Tregs; OX40L, OX40 ligand; Abs against hOX40, generated using phage Abs (U.S. Pat. No. PI, propidium iodide; Teff, effector T cell; TIL, tumor-infiltrating lymphocyte; Tr1 7,550,140) or mice immunized with either hOX40 DNA (36) or cell, IL-10–producing type 1 regulatory T cell; Treg, CD4+ regulatory T cell. OX40-transfected L929 cells (37), are capable of promoting Teff Copyright Ó 2013 by The American Association of Immunologists, Inc. 0022-1767/13/$16.00 function. In particular, the Abs generated by Weinberg et al. (36) www.jimmunol.org/cgi/doi/10.4049/jimmunol.1202752 3642 hOX40 Abs INHIBIT REGULATORY T CELLS have in vivo efficacy in prolonging T cell survival in nonhuman Single cells were sorted into two fractions: CD4+CD127lowCD25high (Tregs) + + low primates. and CD4 CD127 CD25 (Teffs). In this article, we describe the successful generation of a new Generation of Tr1 cells from CD4+ T cells and IL-10 assays panel of agonistic anti-hOX40 mAbs that can potently enhance 5 + + + A total of 2 3 10 freshly isolated CD4 T cells was cultured with irra- CD4 and CD8 Teff function, inhibit induction of Tr1 cells and 3 4 + diated (60 Gy) ICOS ligand–expressing CD32-L cells (8 10 ), which FOXP3 Tregs, and completely block the suppressive function of were precoated with anti-CD3 (0.2 mg/ml) in the presence of dexameth- nTregs. We also show that these Abs can block the function of asone (5 3 1028 M; Life Technologies) and 1a,25-dihydroxyvitamin D3 27 Tregs derived from follicular lymphoma (FL) tumors. Further (1 3 10 M; Life Technologies) in T cell culture medium containing 10% characterization involving mechanistic studies suggest that our FCS, RPMI, GlutaMAX (Life Technologies), 1% penicillin-streptomycin, IL-2 (50 IU/ml), and soluble anti-CD28 (0.2 mg/ml), for 7 d in a 48-well anti–hOX40 mAbs block Treg suppression directly by inhibiting tissue culture plate. Expanded T cells were restimulated with 50 ng/ml the function of Tregs and indirectly by making Teffs resistant to PMA and 2 mg/ml ionomycin (ION) for 6 h; 10 mg/ml brefeldin A (all suppression by Tregs. In summary, our Abs can simultaneously from Sigma) was added during the last 4 h. The cells were stained with promote human Teff function and block human Treg-suppressive Alexa Fluor 647–IL-10 Ab (clone JES3-9D7; eBioscience) using a Caltag FIX and PERM kit. To evaluate the effects of OX40 signaling on IL-10+ function, both key targets for developing effective immunotherapy nTregs, freshly sorted CD4+CD127lowCD25highICOS+ Tregs were stimu- strategies to cure cancers. lated with anti-CD3 (0.2 mg/ml) and anti-CD28 (1 mg/ml) in the presence of IL-2 (300 IU IL-2/ml) and mouse fibroblast L cells expressing the OX40 and CD32 ligands or in the presence of CD32-L cells plus anti-hOX40 Materials and Methods mAbs or control Ab for 5 d. Cells were then restimulated with plate-bound Reagents and cell lines anti-CD3 (2 mg/ml) plus soluble anti-CD28 (1 mg/ml) for 24 h, and supernatants were assayed for IL-10 by ELISA or stimulated with PMA/ Downloaded from The following reagents were purchased from the indicated manufacturers: ION and stained with IL-10 Ab, as described above. TLR ligands Pam3CSK4 and flagellin (FLA-ST ultrapure; InvivoGen), IL-2 and IL-10 ELISA kits (R&D Systems), FCS and human serum (Gemini), Proliferation assays CFSE (Invitrogen), and PHA (Sigma-Aldrich). Mouse fibroblast L cells expressing hOX40 (hOX40-L cells), CD32 (CD32-L cells), or CD32 plus Two systems were used to evaluate T cell proliferation in the presence or the ICOS ligand or OX40 ligand were generated in our laboratory and absence of Tregs. Plate-bound proliferation system in the absence of accessory cells: anti-

maintained with RPMI 1640 culture medium containing 10% FCS, 1% http://www.jimmunol.org/ glutamine, and 1% penicillin-streptomycin. CD3 (3 mg/ml) and anti-hOX40 mAbs (2 mg/ml) in PBS were coated to- gether on a nontissue culture–treated 96-well flat-bottom plate for 1 h at Generation and screening of anti-hOX40 mAbs 37˚C in a CO2 incubator. To determine the proliferation of naive T cells, 105 naive T cells were added per well containing T cell complete medium Anti-hOX40 mAbs were generated using BALB/c female mice immunized (RPMI, GlutaMAX), 10% human AB serum, 1% penicillin-streptomycin). with hOX40-L cells at the M.D. Anderson Monoclonal Antibody Core On the third day, 1.0 mCi/well methyl-[3H]thymidine was added, and Facility following established protocols. Hybridomas secreting mAbs proliferation was assessed by thymidine incorporation 15 h later. To de- recognizing hOX40 were identified by ELISA and flow cytometry, and termine the proliferation of Teffs in the presence of Tregs, CFSE (4 mM)- agonistic function was identified by screening clones for the ability to shut labeled Teffs and nTregs, each at 8 3 104, were added per well. down IL-10+ Tr1 cell induction and block the suppressive function of CD14+ monocyte–based proliferation system: CFSE-labeled Teffs (8 3 4

nTregs. For functional assays, Abs were purified using fast protein liquid 10 ) and Tregs, at a 1:1 or 2:1 ratio, were cultured in T cell medium in the by guest on September 30, 2021 chromatography–protein A FF HiTrap (GE Healthcare) and eluted with presence of irradiated (60 Gy) monocytes at a 1:1 monocyte/Teff ratio for Gentle Ag/Ab elution buffer (Pierce). The generation of humanized anti- 3.5 d in the presence of 0.3 mg/ml anti-CD3 Ab (1, 2). Teff proliferation in hOX40 mAbs was performed by JN Biosciences. the presence of Tregs was determined after 3.5 d of stimulation by CFSE dilution assessed by flow cytometry. Abs, FACS analysis, and cell sorting Direct stimulation of Teffs and nTregs with anti-hOX40 mAbs The following Abs were used for flow cytometry analysis: IL-2–PE, CD4– allophycocyanin–Cy7, CD127-PE, CD25–PE–Cy7, CD14-FITC, CD16- To directly treat nTregs, they were prestimulated with plate-bound anti-CD3 FITC, CD20-FITC, CD56-FITC, CD11C-FITC, and TCRgd-FITC (BD (2 mg/ml) in T cell culture medium plus IL-2 (300 IU/ml) for 12 h, to upregulate OX40 on all Tregs, in a 24-well plate. Activated nTregs were Pharmingen). FOXP3-allophycocyanin was from BioLegend (clone 259D). 6 Functional-grade Abs were anti-CD3 (OKT3; Centocor Ortho Biotech), then pulsed with the anti-hOX40 Abs (20 mg/0.5 3 10 cells) in T cell rhesus monkey cross-reactive anti-CD3 (clone SP34; BD Biosciences), and culture medium for 4 h at 37˚C in a CO2 incubator. Cells were then washed and cultured with CFSE-Teffs in the presence of monocytes and soluble anti-ICOS and anti-CD28 (eBioscience). F(ab9)2 goat anti-human IgG, Fcg anti-CD3 (0.3 mg/ml). To directly treat Teffs, they were stimulated with fragment–specific secondary Ab and ChromPure goat IgG, F(ab9)2 frag- ment control Ab were from Jackson ImmunoResearch Laboratories. Hu- plate-bound anti-CD3 (0.8 mg/ml) in T cell culture medium containing 30 IU/ml IL-2 for 12 h to upregulate OX40; pulsed with anti-hOX40 Abs man IgG1 isotype control was purchased from Sigma. Anti-human CD134 6 (clone ACT35) was purchased from BD Biosciences. Flow cytometry was (20 mg/0.5 3 10 cells) in T cell culture medium for 4 h, as above; washed 4 performed on a flow cytometer (FACSCalibur; Becton Dickinson). FACS three times; and labeled with CFSE, as described. Next, 8 3 10 CFSE- sorting was conducted on a cell sorter (FACSAria IIU; Becton Dickinson). Teffs were cultured with nTregs in the presence of monocytes and soluble All flow cytometry analyses were gated on live T cells. anti-CD3. Isolation of CD4+ T cell subsets from healthy donors and FL Induction of FOXP3 expression from naive T cells patient CFSE-labeled freshly sorted naive CD4+ T cells (105) were stimulated with plate-bound anti-CD3 (3 mg/ml) and an anti-hOX40 mAb clone (2 mg/ml; Buffy coat samples prepared from the peripheral blood of healthy, adult 119-122, 106-222, or 120-270) in the presence of soluble anti-CD28 (1 mg/ml) donors were obtained from the Gulf Coast Regional Blood Center (Houston, and increasing concentrations of TGF-b in serum-free AIM V lymphocyte TX) (IRB LAB03-0390 “Isolation of human dendritic cells, T cells and culture medium (Life Technologies). Three days after stimulation, expanded hematopoietic progenitor cells from human blood and tissue samples”). + cells were stained with FOXP3 Ab using a FOXP3 stain buffer system CD4 naive T cells, Teffs, and Tregs (purity 99% for all) were isolated (eBioscience). from PBMCs using a CD4+ T cell enrichment mixture (STEMCELL Technologies), followed by gating out lineage-negative markers (CD14, Generation of activated human PBMCs and rhesus T cells + low CD16, CD20, CD56, CD11C, TCRgd) and cell sorting the CD4 CD127 for OX40 mAb-binding assays CD25high (top 4–6%) fraction as nTregs, the CD4+CD127+CD25low 2 CD45RA CD45RO+ fraction as Teffs, and the CD4+CD127+CD25low Human PBMCs were stimulated with soluble PHA (10 mg/ml) for 2 d in 2 CD45RO CD45RA+ fraction as naive T cells. To isolate T cells from FL 10% FCS/RPMI 1640. Staining of CD3+OX40+ T cells was performed patients, single-cell suspensions were prepared from FL patient tumor tissue using primary anti-hOX40 mAbs, followed by a secondary Ab against PE- samples obtained from the National Cancer Institute (IRB LAB04-0717). conjugated mouse Ig. CD3+ T cells were detected using anti-CD3–Pacific The Journal of Immunology 3643

Blue–conjugated Ab. Ag-binding competition assays were performed by Statistical analysis preincubating 0.5 mg/ml allophycocyanin-conjugated 106-222 Ab (Ab labeling kit; A20186; Life Technologies) with increasing concentrations of Statistical differences between experimental groups were determined by recombinant hOX40 protein (TP311253; OriGene) in FACS buffer (1% either paired or unpaired t tests or two-way ANOVA using Prism software FCS/2 mM EDTA) for 20 min. The Ab–Ag complex formed was added to (GraphPad). 5 3 105 unstimulated or activated human T cells and incubated for another 20 min. The binding was then analyzed on the CD4+ cell subpopulation. Results The indicated mean fluorescence was plotted for 0, 0.078, 0.625, 2.5, and Generation and identification of potent agonistic anti-hOX40 10 mg/ml the recombinant protein. Rhesus CD4+ T cells were isolated from rhesus PBMCs using anti-CD4 MicroBeads (clone M-T466; Miltenyi mAbs + Biotec). Enriched CD4 T cells were stimulated with 8 mg/ml plate-bound Although OX40 signaling can break , the com- anti-CD3 (clone SP34) for 72 h in the presence of IL-2 (50 IU/ml). mercially available anti-hOX40 mAb ACT35 is ineffective in Staining of OX40-expressing T cells was performed with allophycocyanin- conjugated 106-222 and compared with isotype control mouse IgG1- blocking the function of nTregs. Therefore, we decided to immunize allophycocyanin (BD Biosciences). mice with mouse fibroblast L cells stably expressing recombinant Downloaded from http://www.jimmunol.org/ by guest on September 30, 2021

FIGURE 1. Identification of anti-hOX40 mAbs that inhibit induction of IL-10–producing T cells. (A) Flow cytometry analysis of L cells and human OX40 (hOX40-L) cells mixed (1:1) in FACS buffer and incubated with 0.5 mg of fast protein liquid chromatography–purified, anti-hOX40 mAb. Anti- hOX40 mAbs were derived from three hybridoma fusions: 106, 119, and 120. Numbers following the fusion number denote a specific Ab. Two peaks indicate positive and negative staining by the mAb. A single peak indicates no binding or nonspecific binding of Abs. Shown is a representative of two independent experiments. (B) Screening procedure to identify anti-hOX40 mAbs that inhibit the generation of Tr1 cells from CD4+ T cells stimulated by anti-CD3/CD28 in the presence of vitamin D3/dexamethasone. An OX40 mAb was added on day 0 of cell culture. After 7 d of stimulation, IL-10 in- tracellular staining, followed by flow cytometry analysis, was performed on the CD4+ T cells. Representative FACS data (left panel). Percentage of Tr1 cells after treatment with the indicated anti-hOX40 mAb (right panel). (C) Titration of anti-hOX40 mAbs for their ability to inhibit the induction of Tr1 cells from CD4+ T cells. Experiment was the same as in (B) but with decreasing amounts of mAb. Representative FACS data (left panel). Percentage of Tr1 cells after treatment with decreasing concentrations of indicated anti-hOX40 mAb (right panel). (D) OX40 triggering induces TNF-a– and IFN-g–pro- ducing T cells from the IL-10–induction assay. Experiments were performed as in (B), and cells were stained with TNF-a and IFN-g Abs, followed by flow cytometry. Two representative FACS analyses from six Abs with similar results are shown. (E) The OX40 mAbs that inhibit the induction of Tr1 cells also inhibit the expansion of IL-10+ nTregs. Freshly sorted CD4+CD127lowCD25highICOS+ Tregs were stimulated with anti-CD3 and anti-CD28 in the presence of the indicated anti-hOX40 mAb or control isotype for 5 d. (EI) Cells were then restimulated with anti-CD3/CD28 for 24 h, and supernatants were assayed for IL-10 by ELISA. (EII) Cells were stimulated with PMA/ION and stained with IL-10 and IL-2 Abs, followed by flow cytometry. Data in (D) and (E) are representative of two independent experiments. (F) OX40 Abs shut down TGF-b1–mediated induction of FOXP3+ expression. Freshly sorted CFSE-labeled naive CD4+ T cells were stimulated with plate-bound anti-CD3 in the presence of the indicated OX40 mAb and increasing concentrations of TGF-b1in serum-free lymphocyte culture medium for 3 d. Expanded cells were stained with FOXP3 Ab. Results are from two independent experiments, with SD of the mean shown as error bars. The p values were calculated by two-way ANOVA. 3644 hOX40 Abs INHIBIT REGULATORY T CELLS hOX40 on the cell surface. We performed three fusions and induction of Tr1 cells was inhibited by anti-hOX40 mAbs, the screened the generated hybridoma clones by ELISA for binding percentages of TNF-a+ and IFN-g+ cells were increased. To eval- to plate-bound hOX40-L cells. We found that .500 hybridoma uate the ability of our anti-hOX40 mAbs to also inhibit the ex- clones bound to hOX40-L cells but not the control L cells (data pansion of IL-10+FOXP3+ Tregs, we stimulated freshly sorted not shown). We performed flow cytometry analysis to demonstrate CD4+CD127lowCD25highICOS+ Tregs with anti-CD3 Ab-coated that these clones bound specifically to hOX40 expressed on the human CD32-L cells in the presence of anti-CD28 and an anti- cell surface. Supernatants from hybridomas were screened by hOX40 mAb or control IgG1 isotype for 5 d. Then cells were staining a mixture of hOX40-L cells and parental L cells at a 1:1 either restimulated with anti-CD3/CD28 Abs for IL-10 cytokine ratio. We found that only 20 of 500 of the anti-hOX40 mAbs stained secretion or PMA/ION for staining of IL-10+IL-22 T cells. Fig. OX40-expressing L cells but not the parental L cells (Fig. 1A), 1EI shows that all nine anti-hOX40 mAbs tested blocked the indicating that they bind specifically to surface hOX40. secretion of IL-10 from expanded IL-10+FOXP3+ Tregs. This OX40 signaling was shown to block the induction of Tr1 cells, reduction in IL-10 release was reflected by the 80% decrease in TGF-b–mediated conversion of naive CD4+ T cells to FOXP3+ thenumberofIL-10+IL-22 Tregs (Fig. 1EII). To further eval- Tregs, and suppressive function of nTregs, but it augmented the uate the ability of our anti-hOX40 mAbs to inhibit TGF-b– proliferation of CD4+ and CD8+ T cells (28, 29, 34, 38). To mediated conversion of naive CD4+ T cells to FOXP3+ Tregs, we evaluate the agonistic activity of the 20 anti-hOX40 Abs, we pu- stimulated naive CD4+ T cells with plate-bound anti-CD3 and an rified the Abs by protein A/G–mediated affinity chromatography anti-hOX40 mAb with increasing concentrations of TGF-b in and then tested them for their ability to inhibit the induction of serum-free medium. Fig. 1F shows that an anti-hOX40 mAb (three

Tr1 cells. We found that 9 of the 20 hOX40-specific Abs could represented in figure) potently inhibited the induction of FOXP3+ Downloaded from block vitamin D3/dexamethasone-mediated generation of Tr1 T cells. Together, these results suggest that our anti-hOX40 mAbs cells from total CD4+ T cells (Fig. 1B). We next titrated the Abs inhibit the induction and expansion of IL-10+ Tregs and FOXP3+ to determine their potency. As shown in Fig. 1C, five of the nine Tregs. Abs potently suppressed the generation of Tr1 cells at a concen- We further determined which of the 20 originally selected Abs tration as low as 4 ng/ml. Fig. 1D further shows that, although the could augment TCR-triggered naive CD4+ or CD8+ T cell pro- http://www.jimmunol.org/ by guest on September 30, 2021

FIGURE 2. Anti-hOX40 Abs enhance CD4+ and CD8+ T cell proliferation. (A) A total of 1 3 105 CFSE-labeled freshly sorted CD4+CD25lowCD45RO2 CD45RA+ naive T cells were stimulated with plate-bound anti-CD3 (3 mg/ml) and the indicated anti-hOX40 mAb (2 mg/ml). T cell proliferation was evaluated 4 d after stimulation. Mouse IgG1 and 119-42 served as negative controls. Results from the commercially available anti-hOX40 mAb ACT35 are shown to the far right. Fifteen anti-hOX40 mAbs (under the bracket; 119-122 to 119-220C) significantly enhanced T cell proliferation compared with control mouse IgG1 (*p , 0.05). Error bars represent mean 6 SD. The p values were calculated by paired t tests. (B) CD3+CD45RA+CD27+CD8+ T cells were stimulated as in (A); [3H]thymidine was added on the third day of culture, and cells were harvested after another 15 h of incubation (left panel). Proliferation of T cells was evaluated by thymidine incorporation. CFSE-labeled CD8+ T cells were stimulated with plate-bound anti-CD3 (1.5 mg/ml) and an anti-hOX40 mAb (0.5 mg/ml) (right panel). T cell proliferation was evaluated 3.5 d after stimulation. Mouse IgG1 and anti-CD28 served as negative and positive controls, respectively. Data are representative of two donors. Error bars represent means 6 SD. The p values were calculated by unpaired t tests. The Journal of Immunology 3645 liferation. Plate-bound anti-hOX40 and anti-CD3 mAbs were used Together, these results indicate that several of our anti-hOX40 mAbs to stimulate CFSE-labeled T cells. We found that 11 of the anti- are potent agonists that can potently inhibit the generation and sup- hOX40 mAbs potently enhanced (5–9-fold) the proliferation of pressive function of iTregs and nTregs. naive CD4+ T cells, whereas the only commercially available anti- hOX40 mAb, ACT35, had no such effect (Fig. 2A). In addition to Anti-hOX40 mAbs block normal donor– and cancer this stimulation of CD4+ T cells, one of the clones tested, 119-122, patient–derived Treg function in the presence of accessory cells also stimulated CD8+ T cell proliferation (Fig. 2B). We next in- Because Abs are likely to interact with accessory cells invivo to block vestigated whether these anti-hOX40 mAbs could inhibit nTreg- Treg function, we evaluated the ability of our anti-hOX40 mAbs to suppressive function. We performed similar proliferation assays block Treg suppression in the presence of CD14+ monocytes, at a 1:0 or 1:2 ratio of CFSE-labeled CD4+CD252CD45RA2 CFSE-labeled Teffs, nTregs, and soluble anti-CD3 Abs. We found CD45RO+ Teffs to CD4+CD127lowCD25high Tregs (nTregs). We that 20 mg/ml of clone 106-222, 119-122, 119-43, or 120-270 blocked found that the 11 Abs that were strong activators of naive CD4+ the suppressive function of nTregs (Fig. 3C). Surprisingly, clones T cell proliferation were also strong activators of memory Teff 119-173B and 119-8B, which blocked nTreg function in the plate- proliferation (Fig. 3A) and could also completely block the bound proliferation system, could not block nTreg function in this suppression of nTregs (Fig. 3B). We note that the commercial system. We next wondered whether the anti-hOX40 mAbs could anti-hOX40 mAb ACT35 is a relatively weak activator of Teff also block the function of allogeneic CD4+CD127lowCD25high proliferation and a weak blocker of nTreg-suppressive function. Tregs isolated from a FL tumor sample in the presence of monocytes. Downloaded from http://www.jimmunol.org/ by guest on September 30, 2021

FIGURE 3. Anti-hOX40 mAbs block the activity of CD4+CD25high nTregs. (A and B) Blocking of nTreg function in the absence of accessory cells. Syngeneic CFSE-labeled CD4+CD25lowCD127+CD45RA2CD45RO+ Teffs and CD4+CD25highCD1272 nTregs were cultured at a 1:2 ratio with soluble anti-CD28 (0.4 mg/ml), plate-bound anti-CD3 (3 mg/ml), and the indicated anti-hOX40 mAb or isotype control (2 mg/ml). Representative FACS data showing the proliferation of Teffs in the presence of naive CD4+ T cells, nTregs, or nTregs plus the anti-hOX40 mAb 119-33A (left panels). Percentage of Teff proliferation from six to nine donors in the absence (A) or presence (B) of nTregs after treatment with the indicated anti-hOX40 mAb. Error bars represent means 6 SD. Anti-hOX40 mAbs under the bracket significantly enhanced T cell proliferation and blocked nTreg-suppressive function. (C) Anti- hOX40 mAbs inhibit normal donor–derived nTreg function in the presence of accessory cells. Freshly sorted healthy donor–derived nTregs (3.5 3 104) were cultured for 3–4 d with CFSE-Teff (7 3 104), irradiated CD14+ monocytes (7 3 104; 60 Gy), anti-CD3 (0.3 mg/ml), and increasing concentrations of anti-hOX40 mAb. (D) Inhibition of FL-derived Treg function. CD4+CD127lowCD25high Tregs were sorted from FL-infiltrating lymphocytes. CFSE-labeled CD4+CD127+CD252 Teffs and monocytes were derived from a healthy donor. In (A–C), representative FACS analyses are shown in left panels, unlabeled CD4+ T cells replace nTregs as control, and cell proliferation was assessed by flow cytometry for CFSE dilution. Percentages of Teff proliferation after treatment with the indicated Abs are shown in the right panels. All experiments were performed in duplicate. Data are representative from two or three donors. *p , 0.05, compared with control mouse IgG1, paired t test. 3646 hOX40 Abs INHIBIT REGULATORY T CELLS

We found that 119-43 and 119-122 Abs completely restored the evaluated by gating out live cells on a forward light scatter and proliferation of Teffs at the three Treg:Teff ratios tested, whereas side scatter plot decreased significantly (Fig. 4A, 4B). With an Ab the control Abs IgG1 and 106-118 (does not bind to hOX40) did concentration of 30 mg/ml, the viability of nTregs was reduced not (Fig. 3D). These results demonstrate that some of the anti- from 60% to ,20%, whereas the viability of memory T cells was hOX40 mAbs are capable of blocking Tregs isolated from healthy only reduced 10–15% and was unaffected in naive T cells. To donors, as well as cancer patients. further monitor the precise apoptotic effect of OX40 triggering, we stained nTregs with annexin V and propidium iodide (PI). Anti-hOX40 mAbs act directly on T cells to block Treg Consistently, we found that, at high doses of anti-hOX40 mAb, suppression a significant number of nTregs stained positive for annexin V or Evidence from the literature suggests that a strong OX40-triggering annexin V and PI (Fig. 4C). These results suggest that a stronger signal can block Treg suppression either directly or indirectly (23, OX40 triggering can preferentially induce apoptosis on nTregs, 39). To test these possibilities, we first determined the sensitivity leading to the loss of suppressive function. We next asked whether of a T cell subset to strong OX40 triggering. Each T cell subset, a brief pulse of preactivated nTregs with anti-hOX40 mAb was including naive, memory, and nTregs, was stimulated with soluble sufficient to block suppression activity. We pulsed preactivated anti-CD3 Abs in the presence of CD14+ monocytes and increasing nTregs with anti-hOX40 mAbs for 4 h and then cocultured them concentrations of the anti-hOX40 mAb 106-222, which blocks with CFSE-labeled Teffs in the presence of monocytes. We found nTreg-suppression function. We found that the anti-hOX40 that the brief pulse was sufficient to completely block nTreg mAb stimulated the proliferation of naive T cells and nTregs in a suppression, whereas control Abs had no such effect (Fig. 4D). dose-dependent manner (data not shown). However, when the Ab Because Teffs are resistant to killing by OX40 triggering, we next Downloaded from concentration reached 20 mg/ml, the percentage of viable nTregs determined whether OX40 triggering could instead render Teffs http://www.jimmunol.org/ by guest on September 30, 2021

FIGURE 4. High concentrations of anti-hOX40 mAb kill nTregs. (A and B) Naive T cell subsets (CD4+CD25lowCD127+CD45RO2CD45RA+), Teffs (CD4+CD25lowCD127+CD45RA2CD45RO+), or nTregs (CD4+CD25highCD127low) were cultured with CD14+ monocytes (1:1) plus soluble anti-CD3 (0.3 mg/ml) and increasing concentrations of the 106-222 anti-hOX40 mAb. Cell viability was determined after 3 d of culture by flow cytometry by gating out live cells on a forward light scatter and side scatter plot (two donors). (C) nTregs were stimulated as in (A), except that, after 3 d of culture, the nTregs were stained with annexin V and PI. (D) Anti-hOX40–treated Tregs are unable to suppress Teff proliferation. nTregs were prestimulated with plate-bound anti-CD3 (2 mg/ml) for 12 h and then pulsed with anti-hOX40 mAb (119-122 or 106-222) or a control Ab (anti-ICOS or IgG1), washed, and cultured with CFSE-labeled Teffs plus monocytes and soluble anti-CD3 (0.3 mg/ml). Proliferation was evaluated by CFSE dilution. (E) Anti-hOX40 mAb confers Teff resistance to suppression by nTregs. Teffs were stimulated with plate-bound anti-CD3 (0.8 mg/ml) for 12 h and pulsed with the anti-hOX40 mAb 119-122 (20 mg/0.5 M cells) for 4 h, and 8 3 104 CFSE-Teffs were cultured with decreasing ratios of nTregs in the presence of monocytes and soluble anti-CD3. Proliferation of Teffs was evaluated by CFSE dilution (two experiments/two donors). The Journal of Immunology 3647 resistant to suppression by nTregs. We found that preactivated to unstimulated or activated human T cells, and the binding was Teffs became resistant to suppression by nTregs when they were analyzed on the CD4+ cell subpopulation. We found that the binding subjected to a brief pulse with the anti-hOX40 mAb clone 119- of 106-222 to activated CD4+ T cells could be blocked by re- 122, whereas a control Ab had no such effect (Fig. 4E). Together, combinant hOX40 protein in a dose-dependent manner, whereas these results suggest that several of our anti-hOX40 mAbs act on control protein had no such effect (Fig. 5B, 5C). Mean fluorescence both Teffs and nTregs to block nTreg function. was plotted for only four of the concentrations used of the re- combinant protein (0.078, 0.625, 2.5, and 10 mg/ml), showing that, Anti-hOX40 mAbs bind specifically to human and rhesus OX40 even if the percentage of CD4+ proliferating cells at lower con- For the purpose of future preclinical testing of the efficacy and toxicity centrations is similar (.40%), the mean fluorescence was reduced of our anti-hOX40 mAbs, we asked whether anti-hOX40 mAbs could (Fig. 5C). Taken together, these results demonstrate that our anti- bind specifically to activated CD3+ T cells in human PBMCs and hOX40 mAb binds specifically to hOX40 expressed on activated rhesus OX40 expressed on rhesus-activated CD4+ cells. We obtained CD3+ T cells. To determine whether our anti-hOX40 mAbs could fresh human PBMCs from healthy donors and performed Ab-binding bind specifically to rhesus OX40, we performed similar binding assays. We found that two of our best Abs (119-122 and 106-222) assays using fresh and anti-CD3 polyclonally stimulated CD4+ specifically bound to 10–25% of activated CD3+ T cells but not T cells. We found that the allophycocyanin-conjugated anti-hOX40 CD32 cells, whereas the same Abs bound to only 2–5% of unstimu- mAb 106-222 bound only to activated CD4+ T cells (Fig. 5D), lated CD3+ T cells (Fig. 5A). In contrast, no such binding was ob- whereas no such binding was observed using mouse IgG1 isotype served with control Abs, mouse IgG1 and 319. Surprisingly, we found control, suggesting that our OX40 mAb also binds specifically to that the commercially available anti-hOX40 mAb ACT35 bound to rhesus OX40. We next extended our study to all of the anti-hOX40 Downloaded from both CD32 and CD3+ T cells in unstimulated PBMCs (Fig. 5A), mAbs. We purified CD4+ T cells from rhesus PBMCs and stimulated suggesting that the Ab is less specific for OX40 binding. To further them with PHA for 2 d. Then, anti-hOX40 mAbs were tested for assess the specificity of OX40 mAbs, we performed a competition binding to the activated CD4+ T cells. We found that 10 Abs (107, 108, assay in which a fixed amount of fluorochrome-conjugated 106-222 148, 8B, 58, 69A, 122, 140A, 222, and 270) could bind to rhesus OX40 Ab was preincubated with increasing concentrations of re- CD69+CD4+ T cells (Fig. 5E), whereas control Ab IgG1 did not bind,

combinant hOX40 protein. The Ab–Ag complex formed was added suggesting that these Abs recognize rhesus OX40 on activated T cells. http://www.jimmunol.org/ by guest on September 30, 2021

FIGURE 5. Anti-hOX40 mAbs bind specifically to human and rhesus OX40. (A) Anti-hOX40 mAbs bind specifically to activated human CD3+ cells. Fresh and PHA-activated PBMCs were tested with two agonistic anti-hOX40 mAbs (1 mg/106 cells) and negative controls (mouse IgG1 and mAb 106-319, which do not bind to hOX40). Bound cells were determined by flow cytometry. Data are representative from three donors. (B and C) Ag-binding com- petition assay. Allophycocyanin-conjugated 106-222 Ab (0.5 mg/ml) was preincubated with increasing concentrations of recombinant hOX40 protein. The Ab–Ag complex formed was added to unstimulated or activated human T cells, and the binding was analyzed on the CD4+ cell subpopulation. Bound cells were expressed as the percentage of CD4+OX40+ cells (B) or mean fluorescence intensity (MFI) (C). (D) 106-222 mAb binds specifically to activated rhesus CD4+ T cells. CD4+ T cells were activated using plate-bound anti-CD3, and binding was detected using increasing concentrations of allophycocyanin–106- 222 or allophycocyanin-IgG1. Bound cells were determined by flow cytometry and shown as the percentage of CD4+OX40+ cells. (E) Ten anti-hOX40 mAbs bind to activated rhesus CD4+ cells. CD4+ T cells were stimulated with 10 mg/ml PHA. After 2 d, cells were stained with the indicated anti-hOX40 mAb, followed by goat anti-mouse Ig-allophycocyanin and CD69-PE. Mouse IgG1 and 106-317 served as negative controls. Bound cells were determined by flow cytometry. Data are representative from two donors. 3648 hOX40 Abs INHIBIT REGULATORY T CELLS

Humanized mouse anti-hOX40 mAbs retain the functional Discussion activities of their parental mAbs A key question in the development of effective immunotherapy In view of a future clinical trial, we selected the anti-hOX40 mAbs against cancer is how to reinvigorate Teff function and eliminate or 119-122 and 106-222 for humanization based on their ability to block the suppressive function of Tregs. Recent studies focused on inhibit the induction of Tr1 cells and FOXP3+ Tregs, enhance the targeting OX40 because it is highly expressed on the surface of + + proliferation of naive CD4+ T cells, block the suppressive func- Tregs and activated CD4 and CD8 T cells, and OX40 activation + + tion of nTregs in the absence or presence of accessory cells, and on effector CD4 and CD8 T cells was shown to promote their bind to rhesus OX40. The humanized anti-hOX40 mAbs Hu106- survival and expansion (33). Importantly, triggering of mouse 222 and Hu119-122 bound to OX40 with similar binding affinity OX40 on Tregs using agonistic anti-mouse OX40 Abs was shown (Supplemental Fig. 1). We next tested the ability of the humanized to block Treg-suppressive function and induce Treg-specific apo- Abs to enhance naive CD4+ T cell proliferation and block nTreg ptosis and tumor rejection in mice previously subjected to che- function. As shown in Fig. 6A, plate-bound Hu106-222 and motherapy (23, 24, 27, 30, 33). Therefore, OX40 represents an Hu119-122 mAbs have similar potency in enhancing CD4+ T cell attractive target molecule to develop immunotherapy for a wide proliferation. They both stimulated naive CD4+ T cell prolifera- realm of cancers. In this study, we performed an exhaustive im- tion to a level similar to that induced in the presence of anti-CD28 munization strategy using hOX40-L cells and innovative multitask Ab (Fig. 6A). However, when the humanized or mouse anti-hOX40 screening procedures to identify a set of robust agonistic anti- + + mAbs were tested as soluble Abs, they failed to stimulate T cell hOX40 mAbs that enhance naive CD4 and CD8 T cell prolif- + + proliferation (Fig. 6A), suggesting that cross-linking is required eration, inhibit expansion of IL-10 FOXP3 nTregs, shut down the + + Downloaded from for Ab activity. To demonstrate that cross-linking was required induction of Tr1 cells and FOXP3 Tregs from CD4 T cells, and for Ab activity, naive CD4+ T cells were stimulated with plate- act directly on Tregs to inhibit their function. We found that 11 bound anti-CD3 Abs plus soluble mouse anti-hOX40 mAb 119- anti-hOX40 mAbs (132, 222, 8B, 33A, 43, 58, 122, 173B, 157A, 122 in the presence or absence of a secondary Ab against Fcg.We 140A, 270) enhanced the levels of naive CD4+ T cell proliferation, found that the addition of a secondary Ab against Fcg to the cell each .5–10-fold, compared with the control Ab. Although less culture restored the Ab’s ability to enhance T cell proliferation, robust, the anti-hOX40 mAb 119-122 also enhanced CD3+CD8+ whereas control Abs were ineffective (Fig. 6B), thus demonstrating T cell proliferation. This effect might explain why some of our http://www.jimmunol.org/ that cross-linking is required for anti-hOX40 mAb function. We anti-hOX40 mAbs could completely inhibit nTreg suppression further determined whether Hu119-122 and Hu106-222 could of Teff proliferation. Importantly, we found that activated nTregs block nTreg function. Fig. 6C shows that both plate-bound Abs were highly susceptible to anti-hOX40 mAb– or OX40 ligand potently stimulated Teff proliferation and blocked nTreg-suppressive (OX40L)-triggered cell death, whereas other T cell subtypes, such function. Interestingly, in the presence of Hu106-222 mAb and as naive and memory CD4+ T cells, were less susceptible. These nTregs, the percentage of Teff proliferation exceeded that in the results are in agreement with two previous reports showing that absence of nTregs, suggesting that, in these conditions, OX40 stimulation with OX40L or OX40L plus TNF induced apoptosis signaling blocks nTreg function by the combined action of re- of an OX40-expressing CD4+ T cell line and that pretreatment by guest on September 30, 2021 versing nTreg function and inducing Teff resistance to suppres- of mice with the alkylating agent cyclophosphamide, which sion. These results suggest that the Hu106-222 and Hu119-122 upregulates the expression of OX40 on CD4+ FOXP3+ Tregs, mAbs retained the functional activities of their parental mAbs. combined with OX40 triggering could induce apoptosis in vivo

FIGURE 6. Anti-hOX40 mAbs require cross-linking to enhance T cell proliferation and block Treg-suppressive function. (A) Only plate-bound anti- hOX40 mAbs enhance CD4+ T cell proliferation. Freshly sorted naive CD4+ T cells were stimulated with plate-bound anti-CD3 (3 mg/ml) plus the in- dicated humanized or mouse anti-hOX40 mAb (2 mg/ml), plate-bound or soluble, in the absence of accessory cells. After 3 d, [3H]thymidine was added to the culture, and cell proliferation was assessed after 15 h by thymidine incorporation. Human and mouse IgG1 and anti-CD28 served as negative and positive controls, respectively. (B) Cross-linked soluble anti-hOX40 mAbs augment CD4+ T cell proliferation. Naive CD4+ T cells were stimulated with plate-bound anti-CD3 (3 mg/ml). Soluble anti-hOX40 mAb clone 119-122 (2 mg/ml) was added alone or in combination with an equal amount of a sec- ondary Ab against Fc. Mouse IgG1 and ChromPure goat IgG, F(ab9)2 fragment, served as negative controls. Cell proliferation was evaluated as described in (A). Each treatment in (A) and (B) was performed in triplicate, and data are representative from two donors. (C) Plate-bound humanized anti-hOX40 mAbs Hu106-222 and Hu119-122 block nTreg-suppressive function. Teffs and nTregs cells were prepared and cultured as described in Fig. 3A and 3B. The percentage of Teff proliferation taken from seven donors in the absence (d) or presence (s) of nTregs after treatment with Hu106-222 or Hu119-122. Human IgG1 served as a negative control. Error bars represent mean 6 SD. Statistical significance between treatment groups (*p , 0.05 compared with control Hu IgG1, paired t test) is indicated by a top bracket. The Journal of Immunology 3649

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