CD137/OX40 Bispecific Antibody Induces Potent Antitumor Activity That Is Dependent on Target Coengagement

Published OnlineFirst April 9, 2020; DOI: 10.1158/2326-6066.CIR-19-0798

CANCER IMMUNOLOGY RESEARCH | RESEARCH ARTICLE

CD137/OX40 Bispeciﬁc Antibody Induces Potent Antitumor Activity that Is Dependent on Target Coengagement A C Miguel Gaspar, John Pravin, Leonor Rodrigues, Sandra Uhlenbroich, Katy L. Everett, Francisca Wollerton, Michelle Morrow, Mihriban Tuna, and Neil Brewis

ABSTRACT ◥ þ Following the success of immune checkpoint blockade therapy antibody targeting CD137 and OX40, activated both CD4 and þ against cancer, agonistic antibodies targeting T-cell costimulatory CD8 T cells in an FcgR-independent mechanism, dependent on pathways are in clinical trials. The TNF superfamily of receptors concurrent binding. A mouse surrogate version of the bispecific (TNFRSF) members CD137 and OX40 are costimulatory receptors antibody displayed antitumor activity in syngeneic tumor models, that stimulate T-cell proliferation and activation upon interaction independent of T regulatory cell depletion and of FcgR interaction, with their cognate ligands. Activating CD137 and OX40 with but associated with peripheral T-cell activation and proliferation. agonistic mAbs stimulates the immune system due to their broad When compared with a crosslink-independent CD137 agonist þ þ expression on CD4 and CD8 T cells and natural killer cells and mAb, the FS120 surrogate induced lower liver T-cell infiltration. has antitumor effects in preclinical models. Most TNFRSF agonist These data support initiation of clinical development of FS120, a antibodies require crosslinking via Fcg receptors (FcgR), which can first-in-class dual agonist bispecific antibody for the treatment of limit their clinical activity. FS120 mAb2, a dual agonist bispecific human cancer.

Introduction without associated toxicity (11) but show limited clinical efficacy (12). Two CD137 agonist antibodies have different clinical outcomes. OX40 and CD137 costimulatory receptors belong to the TNF Urelumab (BMS-663513, clone 20H4.9) induces severe transaminitis receptor superfamily (TNFRSF; ref. 1). Both are expressed on activated at doses higher than 1 mg/kg resulting in two hepatotoxicity-related T cells and natural killer cells, and are attractive targets for cancer deaths (13) and utomilumab (PF-05082566, clone MOR7480.1), which immunotherapy as stimulation of these receptors results in increased does not induce severe adverse events, has modest clinical activity (14). T-cell activation, proliferation, and survival in vitro and in vivo (1). The A combination trial with utomilumab and PF-04518600 (an OX40 expression patterns of OX40 and CD137 are overlapping, but distinct þ agonist antibody) is underway (NCT02315066). with expression of OX40 higher on CD4 T cells and that of CD137 þ TNFRSF antibodies typically have no or low intrinsic agonist higher on CD8 T cells (2). CD137 stimulation preferentially stimu- þ þ activity and require secondary crosslinking of antibody–receptor lates CD8 T cells when compared with CD4 T cells and OX40 þ complexes to induce sufficient receptor clustering and activation, stimulation preferentially stimulates CD4 T cells when compared þ thereby mimicking the TNFSF ligand superclusters (15). In vivo, this with CD8 T cells (3). However, coexpression of these receptors is þ þ secondary crosslinking requires the interaction with Fcg receptors demonstrated in both CD4 and CD8 T cells and both are expressed (FcgR; ref. 16). The availability of FcgR-expressing cells in the tumor in tumor-infiltrating lymphocytes (TIL; refs. 4, 5). Antibodies stim- microenvironment and the low affinity interaction between FcgRs and ulating these targets show activity in a variety of murine tumor models þ the Fc-region of IgG antibodies may limit the agonist activity of by both depleting regulatory T cells (Treg) and activating CD8 and þ TNFRSF antibodies and, consequently, their antitumor activity (17). CD4 T cells (6, 7). The combination of OX40 and CD137 agonist þ þ In addition, interaction with FcgRs mediates antibody effector func- antibodies stimulate both CD4 and CD8 T cells and induce tions such as antibody-dependent cell-mediated cytotoxicity (ADCC) the cytotoxic function of both antigen-experienced and antigen- þ and antibody-dependent cell-mediated phagocytosis (ADCP) and inexperienced bystander CD4 T cells (8, 9). could lead to the depletion of the tumor-specific T cells that would Several clinical trials are underway to test agonist antibodies to be activated by these antibodies (18). Consequently, the clinical activity OX40 or CD137 either as monotherapies or in combination with other seen with OX40 and CD137 antibodies may not represent the full agents to treat various cancers (10). Clinical trials with OX40 agonist potential of activating these receptors. antibodies demonstrate peripheral T-cell activation and proliferation An alternative to FcgR-mediated crosslinking of TNFRSF agonist antibodies is the use of bispecific antibodies, the dual binding of which F-star Therapeutics Ltd., Cambridge, United Kingdom. results in the clustering of the TNFRSF target, independent of FcgR engagement (19). This study described FS120 mAb2, a dual agonist Note: Supplementary data for this article are available at Cancer Immunology bispecific antibody targeting CD137 and OX40 that activated both Research Online (http://cancerimmunolres.aacrjournals.org/). þ þ CD4 and CD8 T cells, whereas OX40 or CD137 monospecific Corresponding Author: Neil Brewis, F-star Biotechnology, Babraham Campus, þ þ g Cambridge, CB22 3AT, UK. Phone: 44-1223-497400; Fax: 44-1223-497461; antibodies only activated CD4 or CD8 T cells, respectively. Fc R- E-mail: [email protected] disabling mutations (LALA mutations; ref. 20) were introduced to enable antibody crosslinking from the coengagement of the two Cancer Immunol Res 2020;XX:XX–XX different receptors when coexpressed and to potentially avoid deple- doi: 10.1158/2326-6066.CIR-19-0798 tion of OX40- or CD137-expressing cells. A mouse-specific surrogate 2020 American Association for Cancer Research. version of FS120 showed antitumor activity in the absence of FcgR

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interaction or after Treg depletion. When compared with a crosslink- with either mouse OX40 or CD137 (100 nmol/L) or HBS-EP buffer. independent CD137 agonist mAb, FS120 surrogate showed reduced Affinity for FcgRs [R&D Systems, hFcgR1 (1257-CF-050), hFcgR2a liver T-cell infiltration, which decreased over time. (1330-CF-050), hFcgR2b (1875-CF-050), and hFcgR3a (4325-CF- These results indicate that targeting coexpressed receptors with 050)] was tested by coating biotinylated human OX40 (BPS Bioscience bispecific antibodies may be a potent and safe mechanism to cluster 71310-1) or CD137 (in-house) his-tagged antigens onto an Strepta- and activate TNFRSF costimulatory receptors and induce antitumor vidin Chip (GE Healthcare) and coinjecting antibodies (100 nmol/L) immunity and human FcgRs (500 nmol/L) at 20 mL/minute flow rate and the dissociation was followed for 5 minutes. For specificity assessment, human TNFRSF members [R&D Systems, TNFRI (372-RI-050/CF), Materials and Methods TNFRII (726-R2-050), GITR (689-GR-100), NGFRI (367-NR-050/ Antibody reagents CF), CD40 (1493-CD-050), and DR6 (144-DR-100)] were immobi- Antibodies were cloned by replacing the VH or Vk domain lized on CM5 Chips (GE Healthcare) to approximately 1,000 RU and sequences in human IgG1 with identified sequences from patents or FS120 (1 mmol/L), isotype control [anti-FITC (Ctrl(4420) WT mAb; literature using methods described previously (21). The LALA clone 4420 in human IgG1 backbone; ref. 23] or positive control (L234A-L235A, Eu numbering; ref. 22) mutations were introduced antibodies [R&D Systems, anti-TNFRI (MAB225R-100); anti-TNFRII via site-directed mutagenesis when indicated. Absence of LALA (MAB726-100); anti-GITR (MAB689-100); anti-NGFR (MAB367); mutations is denoted by the suffix WT (wild-type) after the clone anti-CD40 (MAB6321-100); and anti-DR6 (AF144)] were flowed over name. For antibody production, pTT5 expression vectors (National at a flow rate of 30 mL/minutes. Data were analyzed using BIA Research Council of Canada) containing the mAb or mAb2 sequences Evaluation Software (GE Healthcare). were transfected into Expi293F cells (Thermo Fisher Scientific, A14528) using PEIpro Transfection Reagent (Polyplus, PPLU115) Cell line creation and reporter T-cell assay according to the manufacturer's instructions. Antibodies were purified All cells used in the experiments described in this article were kept in using a 5 mL MabSelect SuRe Column (GE Healthcare, 11003494) on culture for a maximum of 2 months before starting new cultures from an AKTA Explorer (GE Healthcare) according to the manufacturer's master vials. CT26 (CRL-2638) and B16-F10 (CRL-6475) cell lines instructions. were purchased from ATCC in 2015. DO11.10 cells were purchased The following antibodies were used in the experiments described in from Public Health England (85082301) in 2014 and used under this article: anti-FITC [Ctrl(4420) WT mAb; clone 4420 used as isotype license from National Jewish Health. Expi293F cells were purchased control; ref. 23], anti-human OX40 [OX40(11D4) mAb; clone 11D4 from Thermo Fisher Scientific (A14528) in 2015. No reauthentication from patent EP2242771B1], anti-human OX40 Fcab [OX40/Ctrl tests were performed. Mycoplasma testing was performed on all cell (4420) mAb2; clone FS20, F-star]; anti-human CD137 [CD137 lines monthly using R&D MycoProbe Mycoplasma Detection Kit (MOR7480.1) mAb; clone MOR7480.1 from patent US8,337,850B2], (R&D Systems, 895285). Human and mouse OX40 and CD137 cDNA anti-human CD137 [CD137(20H4.9) mAb; clone 20H4.9 from patent constructs were synthesized (GenScript) with flanking 50 EcoRI and 30 US8,137,667B2], anti-human CD137 [CD137(FS30) mAb; clone FS30, NotI restriction sites and cloned into the lentivirus vector pLVX-EF1a- F-star], anti-mouse OX40 [mOX40(OX86) WT mAb; clone OX86 IRES-puro (Clontech 631988). Lentiviruses were produced using the from patent US9,738,723B2], anti-mouse CD137 [mCD137(Lob12.3) LentiX Expression system EF1a Version (Clontech 631253) and used WT mAb; clone Lob12.3; ref. 3], and anti-mouse CD137 [mCD137 to transduce DO11.10 cells according to the manufacturer's instruc- (3H3) WT mAb; clone 3H3; ref. 24] all in human IgG1 format, with tion. Cell lines were selected by incubation with 5 mg/mL Puromycin (mAb) or without (WT mAb) LALA mutations; anti-human IgG CH2 (InvivoGen, ant-pr-1) and individual cell lines were cloned by serial domain (clone MK1A6; ref. 25) in mouse IgG1 format; anti-human dilution. DO11.10 cells expressing human or mouse CD137 were CD28 [CD28(TGN1412) mAb; clone TGN1412 from patent US stimulated with coated anti-mouse CD3 antibody (BioLegend, 8,709,414 B2] in human IgG4 format with S228P mutation (Eu 100202 clone 17A2 at 0.1 mg/mL) and mouse IL2 concentration in numbering); anti-human CD3 antibody (clone UHCT1, R&D Sys- the supernatants was measured by ELISA (Thermo Fisher Scientific, tems); anti-mouse CD3 antibody (clone 17A2, Thermo Fisher Scien- 88-7024-88). tific); and anti-mouse CD3 antibody (clone 145-2C11, Thermo Fisher Scientific). Flow cytometry For cell binding assays, cells were incubated with primary antibodies Surface plasmon resonance analysis or mAb2 followed by detection with an anti-human Fc-488 secondary Data were acquired using a BIAcore 3000 or BIAcore T200. Dilution antibody (Jackson ImmunoResearch). Excised tissues were dissociated mixtures prepared in HBS-P or HBS-EP Buffer (GE Healthcare). For using relevant Miltenyi Biotec Dissociation Kits (tumors, 130-096-730; K D determination, FS120 was captured either via the Fc region using livers, 130-105-807; and spleens, 130-095-926) using a Miltenyi gen- a Human Antibody Capture Kit (GE Healthcare) and human or tleMACS Octo Dissociator and C-tubes according to the manufac- cynomolgus CD137 was flowed over at a range of concentrations; or turer's instructions. Resulting cell suspensions were strained [70 mm via the Fab region using a Human Fab Capture Kit (GE Healthcare) cell strainer (Corning CLS431751)], washed, and resuspended in PBS. and human or cynomolgus OX40 was flowed over at a range of Collected blood samples were treated twice with Red Blood Cell Lysis concentrations. For dual binding determination of FS120, biotinylated Buffer (eBioscience 00-430054) according to the manufacturer's human CD137 or OX40 was immobilized on a Streptavidin Chip (GE instruction. Cells isolated from tissues and blood were stained for Healthcare) and antibodies (100 nmol/L) were coinjected with either flow cytometry with fluorochrome-conjugated antibodies including human OX40 or CD137 (100 nmol/L), respectively, or HBS-EP buffer. those against CD4, Ki67, FoxP3, CD69, CD3, and CD8 (Thermo Fisher For dual binding determination of FS120 surrogate, mouse CD137 was Scientific), CD45 (BD Biosciences), and fixable live/dead dye (Thermo immobilized on a CM5 Chip (GE Healthcare) to a surface density of Fisher Scientific) in the presence of Fc Block (Thermo Fisher Scientific) approximately 1,000 RU and antibodies (100 nmol/L) were coinjected according to the manufacturer's recommendations. Cells were

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analyzed in a BD FACS CantoII Cytometer and data were analyzed Home Office (license number 70/7991) and local ethical review with FlowJoX. OX40 and CD137 receptors quantified with Quantum committee guidelines. Simply Cellular anti-mouse IgG (Bangs Laboratories 815) according to the manufacturer's recommendations. Statistical analysis One- and two-way ANOVA with Tukey or Dunnett multiple Peripheral blood mononuclear cells and T-cell stimulation comparisons test, and survival analysis (log-rank Mantel–Cox test) assays was performed using Prism Software (GraphPad). For comparison of Ficoll-purified human peripheral blood mononuclear cells (PBMC) treatment responses and EC50 determinations, data were log trans- were stimulated with 100 ng/mL staphylococcal enterotoxin A (SEA, formed before analysis and fit using the log agonist versus response Sigma) in the presence of FS120 or control antibodies for 5 days at using Prism Software (GraphPad). Where indicated, statistical testing fi 37 C, 5% CO2 in T-cell media [RPMI1640 (Thermo Fisher Scienti c) of tumor volume over time was analyzed using a mixed model. A with 10% FBS (Thermo Fisher Scientific), 1 penicillin–streptomycin separate model was fitted to each pair of treatments of interest. The (Thermo Fisher Scientific), 1 mmol/L Sodium Pyruvate (Gibco), 10 model is L mmol/L Hepes (Gibco), 2 mmol/L -Glutamine (Gibco), and 50 mmol/ log10ðÞ¼volume A þ B ðÞþday start day " L 2-mercaptoethanol (Gibco)]. T cells were isolated from PBMCs þ using Miltenyi Enrichment Kits (Human CD3 , 130-096-535; Human A and B are the intercept and slope, respectively; they are different þ þ þ CD4 , 130-096-533; Human CD8 , 130-096-495; and Mouse CD3 , for each mouse, and include a fixed effect for the group and a random 130-095-130) according to the manufacturer's instructions and acti- effect for the animal: vated overnight using CD3/CD28 Dynabeads (Life Technologies A ¼ A0 þ A1T þ "A catalog numbers: Human, 11131D and Mouse, 11453D) at a 1:1 cell to bead ratio. CD3/CD28 beads were removed using a DynaMag-15 B ¼ B0 þ B1T þ "B magnet and activated T cells were washed with T-cell media and T stimulated with coated CD3 antibody [Human: R&D Systems is a dummy variable representing the treatment group with value 0 þ (MAB100) clone UHCT1 at 2.5 mg/mL (total and CD4 T cells) or in one group and 1 in the other. The random effects are distributed þ 5 mg/mL (CD8 T cells) or Mouse: BioLegend (100302) clone 145- with a normal distribution: 2C11 at 2.5 mg/mL] in the presence of FS120 or control antibodies for "A NðÞ0; sA ;"B NðÞ0; sB 3 days at 37C, 5% CO . Anti-human Fc (clone MK1A6, produced in- 2 s s house) was used as crosslinking agent at a 1:1 molar ratio with test where A and B are the SDs of the interanimal variability in the antibodies. FITC dextran (70 kDa, Sigma 46945) was used as cross- intercept and slope, respectively. The intra-animal variability is also s linking agent at a 1:1 molar ratio with OX40 Fcabs paired with FITC- normally distributed with SD : binding Fab (clone 4420). IL2 concentration in the supernatants was " NðÞ0; s measured by ELISA or electrochemiluminescence (Thermo Fisher fi Scientific, 88-7025-88 or MSD K151QQD-1). For the cytokine release For each pair of treatments, the model above was tted to the data. A B P assay, antibodies were diluted to 10 mg/mL in PBS, coated onto 96-well For 1 and 1, the (two-sided) value for a difference from zero was P fi flat-bottomed plates and allowed to air dry overnight, washed twice calculated; a value below 0.05 is statistically signi cant evidence for a with PBS, and incubated with 2 105 PBMCs for 3 days. Multiple difference between the treatment groups. cytokine concentrations in supernatants were measured by Pro- inflammatory V-plex Kit (Meso Scale Discovery catalog number Results K15049D-2) according to the manufacturer's instructions. FS120 simultaneously bound to CD137 and OX40 Mice and tumor challenge Phage and yeast libraries were used with directed evolution methods Balb/c female mice were from Charles River Laboratories. Animals described previously (21, 26) to identify and improve OX40-binding Fc were housed in a local animal facility and were used at approximately antigen binders, termed Fcabs (Fc-region with antigen binding), as 8–10 weeks of age. Antibodies were diluted in PBS before intraper- well as CD137-binding Fab regions. The Fcab (OX40 Fcab, clone FS20) itoneal injection at the indicated dose and schedule. For tumor trials, and Fab (CD137 Fab, clone FS30) with the overall highest activity in T- fi mice were anaesthetized by inhalation of isoflurane, and each animal cell stimulation assays and af nity in cell binding assays were com- 6 5 fi 2 received 10 or 10 of CT26 tumor cells (depending on experiment) or bined to generate the bispeci c FS120 mAb (or FS120; Fig. 1A). 5 fi 10 of B16-F10 tumor cells diluted in PBS. Mice were injected Af nity determination by surface plasmon resonance (SPR) showed subcutaneously with a maximum volume of 100 mL in the left flank that FS120 has subnanomolar binding to both human and cynomolgus K to generate tumors. Mice were randomized into study cohorts based on monkey OX40 ( D: Human, 0.2 nmol/L and Cyno, 0.9 nmol/L) and K tumor volume and any mice which did not have tumors were not CD137 ( D: Human, 0.2 nmol/L and Cyno, 0.2 nmol/L; Fig. 1B) and assigned into treatment groups and were removed from the study. FS120 bound both OX40 and CD137 simultaneously (Fig. 1C). FS120 Tumor measurements were taken under anesthesia using calipers to did not bind to other related members of the TNFR superfamily determine the longest axis and the shortest axis of the tumor. The (CD40, GITR, NGFR, DR6, TNFRI, and TNFRII; Supplementary following formula was used to calculate the tumor volume: L (S2)/2 Fig. S1). FS120, which contains the LALA mutations, had reduced (where L ¼ longest axis; S ¼ shortest axis). For peripheral pharma- FcgR binding as compared with WT IgG1 (non-LALA containing) codynamic analysis, blood was collected into EDTA-containing tubes OX40 and CD137 antibodies (Supplementary Fig. S2). FS120 bound to from either the tail vein or by cardiac puncture. Tumors, spleens, and cell surface expressed human and cynomolgus OX40 and CD137 livers were collected by dissection when indicated. All procedures receptors on engineered DO11.10 T cell lines but not to the non- involving animals were approved and performed according to UK transduced parental DO11.10 cell line (Fig. 1D).

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Figure 1. FS120 bound to OX40 and CD137 with high afﬁnity simultaneously. A, Schematic representation of FS120 mAb2,abispeciﬁc antibody binding to OX40 via

the Fc region and to CD137 via the Fab region, containing LALA mutations. B, SPR of FS120 binding to human OX40 or human CD137. KD determination performed using BIA evaluation software (representative results of three independent experiments). C, SPR of FS120 simultaneous binding to immobilized OX40 and CD137 in solution and to immobilized CD137 and OX40 in solution (representative results of two independent experiments). D, Geometric mean ﬂuorescence intensity (GMFI) of FS120 binding to cell surface–expressed human OX40 and CD137 determined by ﬂow cytometry. Data of triplicates presented

as mean SD. For EC50 determinations, antibody concentration and GMFI data were log transformed before analysis and ﬁt using the log agonist versus response using Prism Software (GraphPad; representative results of three independent experiments).

FS120 agonistic activity was dependent on the dual binding of effective receptor clustering and activation (Fig. 2A). Similar results CD137 and OX40 were observed when isolated T cells were stimulated with plate-bound To test the agonist activity of FS120, PBMCs were stimulated with CD3 antibody and costimulated with OX40- or CD137-speciﬁc anti- SEA superantigen, which crosslinks MHC class II molecules at the bodies or FS120 mAb2 (Fig. 2B). The activity of FS120 was not surface of antigen-presenting cells and the T-cell receptor (TCR) of T increased by the secondary crosslinking agent, either in maximum cells (27), in the presence or absence of secondary crosslinking agents response or in a decrease in EC50 (Fig. 2C and D), indicating that the to mimic the effect of FcgR-mediated crosslinking. The amount of dual binding to OX40 and CD137 resulted in the maximum stimu- T-cell activation resulting from OX40 or CD137 stimulation was lation induced by FS120. then measured by the production of IL2. All antibodies were When CD137 agonist antibodies were crosslinked, the 20H4.9 Fab tested in the same isotype background as FS120, human IgG1 with clone (Fab present in urelumab) was observed to have a higher activity the LALA mutations, to minimize interference from FcgR-mediated as compared with clones MOR7480.1 (Fab present in utomilumab) or crosslinking. FS30 (Fab present in FS120; Fig. 2A and B). The crosslinked OX40- Agonistic activity of OX40 or CD137 monospeciﬁc antibodies was targeting antibodies induced higher IL2 production than the cross- only observed in the presence of crosslinking (Fig. 2A). In contrast, linked CD137-targeting antibodies, and the combination of the OX40 FS120 mAb2 showed activity in the absence of secondary crosslinking Fcab and the CD137 Fab components of FS120 did not show a agent suggesting the coengagement of OX40 and CD137 resulted in synergistic effect as compared with the OX40 Fcab alone (Fig. 2A

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Figure 2. FS120 stimulated T cells in the absence of anti-Fc crosslinking. A and B, Human PBMCs stimulated with SEA (100 ng/mL; A) or human T cells stimulated with coated CD3-antibody (clone UCHT-1 at 2.5 mg/mL; B) and costimulated with FS120 or control antibodies (3.7 nmol/L) in the presence or absence of crosslinking reagents at 1:1 molar ratio [FITC dextran for OX40/Ctrl(4420) mAb2 and anti-Fc antibody (clone MK1A6 in mouse IgG1 format) for other antibodies]. C and D, FS120 titration in the presence or absence of anti-Fc crosslinking in SEA-stimulated PBMCs (C) or CD3-stimulated T cells (D). E, CD3-stimulated T cells costimulated with FS120 (1 nmol/L) and isotype control antibody or component parts of FS120 and their combination (100 nmol/L). A–E, Data from duplicates is shown as mean SD (representative results of three independent experiments). Statistical testing by two-way ANOVA and Tukey multiple comparison test (A and B) or one-way ANOVA and Dunnett multiple comparisons test (E). Asterisks on top of error bars represent the significant difference to Ctrl(4420) mAb–treated samples (, P < 0.032; , P < 0.0021; , P < 0.0002; , P < 0.0001). For EC50 determinations, antibody concentration data were log transformed before analysis and fitusingthe log agonist versus response using Prism Software (GraphPad; C and D). See also Supplementary Figs. S2 and S3. and B). This result indicated that these assays were more sensitive to the levels of IL10, a typical anti-inflammatory cytokine (Supple- OX40 stimulation and that only potent CD137 stimulation resulted in mentary Fig. S4). substantial T-cell activation. The higher response to OX40 agonism To test whether the activity of FS120 required simultaneous binding þ could have been explained by the higher proportion of CD4 T cells in to the two receptors, the ability of FS120 to coengage OX40 and CD137 þ human PBMCs and the higher expression of OX40 on activated CD4 was blocked using 100-fold molar excess of either the OX40 Fcab or the T cells (Supplementary Fig. S3). CD137 Fab components of FS120 or both. The results showed that the Titrations of these antibodies in both the PBMC SEA stimulation FS120-induced T-cell activation was reduced when the mAb2 com- and T-cell CD3 stimulation assays were performed and the con- ponent parts were present either individually or in combination centration at which these antibodies induced the highest IL2 (Fig. 2E), indicating that FS120 required dual binding to OX40 and production was chosen for this analysis. FS120, and crosslinked CD137 to induce the clustering and activation of these receptors. OX40 Fcab, induced the production of additional proinflammatory FS120 did not induce T-cell activation in a cytokine release cytokines (IL6, IL12p70, IL13, and TNFa) by T cells and reduced assay (28) in the absence of TCR stimulation, unlike the two positive

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Figure 3. þ þ þ þ þ FS120 mAb2 stimulated both CD4 and CD8 T cells. A, Human CD4 and CD8 T cells stimulated with coated CD3 antibody (clone UCHT-1 at 2.5 mg/mL for CD4 and þ 5 mg/mL for CD8 T cells) and costimulated with FS120 or OX40 and CD137 antibody controls (3.7 nmol/L) in the presence or absence of crosslinking reagents at 1:1 molar ratio [FITC dextran for OX40/Ctrl(4420) mAb2 and anti-Fc (clone MK1A6 in mouse IgG1 format) for other antibodies]. Data from duplicates are shown as mean SD (representative results of three independent experiments). Statistical testing by two-way ANOVA and Dunnett multiple comparison test. Asterisks on top of error bars represent the significant difference to Ctrl(4420) mAb–treated samples (, P < 0.032; , P < 0.0021; , P < 0.0002; , P < 0.0001). B, OX40 and CD137 receptor quantification in CD4þand CD8þ T cells from 3 PBMC donors, activated overnight with CD3/CD28 beads (1:1 ratio), by flow cytometric fluorescence quantification with beads (Bangs Laboratories). See also Supplementary Figs. S1 and S4.

control antibodies used, a CD28 antibody shown to induce cytokine The different CD137 antibodies tested showed varying activity on þ storm in the clinic (TGN1412; ref. 29) and a CD3 antibody (Supple- CD8 T cells. Fab clone MOR7480.1 did not show activity in the mentary Fig. S5). absence of crosslinking and only a moderate, nonsigniﬁcant, increase when crosslinked (Fig. 3A). Clone FS30 displayed higher activity þ þ FS120 stimulated both CD4 and CD8 T cells when crosslinked, but no activity in the absence of crosslinking þ þ OX40 and CD137 receptors were coexpressed on CD4 and CD8 (Fig. 3A). However, Fab clone 20H4.9 induced higher levels of IL2 T cells, with OX40 expressed in higher percentages and at a higher production in the absence of crosslinking, which was increased with þ þ receptor number in CD4 T cells than CD8 T cells and, conversely, crosslinking (Fig. 3A). When these antibodies were tested in a model þ more CD137 receptors were expressed on CD8 T cells (Supplemen- DO11.10 T cell line expressing human CD137 and stimulated with tary Fig. S1). The differential expressions correlated with the activity of coated anti-mouse CD3 antibody, Fab clone 20H4.9 also showed þ OX40- or CD137-targeting antibodies. On CD4 T cells stimulated crosslink-independent activity, whereas Fab clones FS30 and with plate-coated CD3 antibody, both the crosslinked OX40 mAb and MOR7480.1 did not (Supplementary Fig. S6A). the OX40 Fcab induced IL2 production, but CD137 antibodies did not þ show activity (Fig. 3A and B). On CD8 T cells, the crosslinked FS120 surrogate bound mouse OX40 and CD137 and activates CD137 antibodies (Fab clones 20H4.9 and FS30) induced IL2 pro- T cells duction and OX40 antibodies did not (Fig. 3A and B). When tested in As the FS120 mAb2 doesnotbindtomouseOX40orCD137,a the absence of secondary crosslinking agent, FS120 increased IL2 surrogate molecule was generated for in vivo testingbypairing þ þ production on both CD4 and CD8 T cells (Fig. 3A). The cross- a mouse-speciﬁc OX40 Fcab with a CD137 mAb (clone Lob12.3). linked activity of clone FS30 [CD137(FS30) mAb] demonstrated that The Fc-modifying technology used to create Fcabs is based on the mAb2 could activate the CD137 receptor when crosslinked by human IgG1 therefore the FS120 surrogate has a human IgG1 þ binding of the Fcab to OX40 on CD8 T cells. The activity of the cross- domain. All in vivo experiments were performed using molecules linked OX40 Fcab [OX40/Ctrl(4420) mAb2] showed that the mAb2 withthesamehumanIgG1backbone.TheFS120surrogatebound could activate the OX40 receptor when crosslinked by binding of the to cell surface expressed mouse OX40 and CD137 receptors on þ Fab arms to CD137 on CD4 T cells. engineered DO11.10 T cell lines (Supplementary Fig. S7A) and

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concurrent binding to mouse OX40 and CD137 was demonstrated of the treatments induced any signiﬁcant changes in the frequency of þ þ by SPR (Supplementary Fig. S7B). CD4 or CD8 T cells or T-cell proliferation (Fig 4C). The antitumor FS120 surrogate was functionally characterized by testing its ability activity of the FS120 surrogate was therefore not associated with to costimulate primary mouse T cells in the presence of coated anti- intratumoral Treg depletion. mouse CD3. FS120 surrogate induced the production of IL2 in the When the FS120 surrogate was tested in the B16-F10 model, a absence of secondary crosslinking agent, unlike the OX40 monospe- poorly immunogenic model and thus harder to treat with immu- ciﬁc antibody, which required anti-Fc antibody for crosslinking (Sup- notherapies (33), FS120 surrogate also had antitumor activity as plementary Fig. S8A). No activity of the CD137 antibody was detected compared with an isotype control antibody (Supplementary Fig. S10). in this assay, but in DO11.10 T cell line expressing mouse CD137 and stimulated with coated anti-mouse CD3 antibody, clone Lob12.3 Peripheral T-cell activation and proliferation mediated by FS120 increased IL2 production in the presence of crosslinking (Supplemen- surrogate tary Fig. S6B). A different anti-mouse CD137 (clone 3H3) was also The induction of T-cell proliferation by OX40 and CD137 agonist tested in this assay and showed crosslink-independent activity, similar antibodies in vitro and in vivo is described in both preclinical models to that seen with Fab clone 20H4.9 (Fig. 3A; Supplementary Fig. S6A and in the clinic (11, 34). Comparing T-cell proliferation and activa- and S6B). This result indicated that these two molecules could be tion in the blood of CT26 tumor–bearing mice treated with FS120 surrogates for each other, whereas clone Lob12.3 only showed activity surrogate or control antibodies overtime, it was observed that FS120 þ þ when crosslinked and was therefore a surrogate for Fab clones surrogate induced more CD4 and CD8 T-cell proliferation, as MOR7480.1 and FS30 mAb. measured by the expression of Ki67 (Fig. 5A and B) than the OX40 The activity of FS120 surrogate in the absence of secondary cross- and CD137 antibodies or their combination. The effect of FS120 linking agent was also dependent on dual binding to OX40 and CD137; surrogate on T-cell activation, as measured by the expression of CD69, when either of these receptors was blocked with excess mouse OX40 was delayed as compared with the proliferative effect with the highest Fcab or mouse CD137 mAb (clone Lob12.3), the activity of FS120 frequencies observed 3 days after the third dose (Fig. 5B). Similar surrogate was reduced, similar to that observed with FS120 in human T effects on T-cell activation were observed with the combination of cells (Supplementary Fig. S8B). OX40 and CD137 antibodies (Fig. 5B).

Antitumor activity of the FS120 surrogate FS120 surrogate did not induce liver inflammation OX40 and CD137 antibodies demonstrate antitumor activity in a Increased liver T-cell infiltration was observed with various CD137 variety of syngeneic models with responses depending on dose, time of agonist antibodies in mice, suggesting a similar mechanism of liver treatment initiation, antibody isotype, and clones used (3, 30–32). toxicity for CD137 agonism in mice and humans (35, 36). To under- Intratumoral Treg depletion is part of the mechanism of antitumor stand the potential hepatotoxicity risk of FS120, T cells and their activity of OX40 (clone OX86; ref. 6) and CD137 antibodies (clone proliferation and activation in the liver, spleen, and blood induced by Lob12.0; ref. 7). FS120 surrogate was compared with that induced by OX40 and CD137 To test the antitumor activity of FS120 and to understand the in vivo agonist antibodies or their combination (Fig. 6A). The results showed mechanism of action, FS120 surrogate or control antibodies were a clear difference between the two CD137 agonist antibodies tested, injected intraperitoneally at 1 mg/kg on days 11, 13, and 15 post-CT26 clone 3H3 induced a sustained increase in T-cell infiltration, prolif- tumor cells inoculation (Fig. 4A). The OX40 (clone OX86) or CD137 eration, and activation in the liver and spleen, whereas clone Lob12.3 (clone Lob12.3) control antibodies with human IgG1 isotype or their did not (Fig. 6B). OX40 stimulation did not show an increase in T cells combination showed no antitumor activity (Fig. 4B), unlike previously in the liver, spleen, or blood, but induced transient T-cell proliferation published results using the original rat versions of these antibodies (3). in all tissues studied and T-cell activation in the liver at 14 days post- This could be explained by the later start time of antibody treatment, last dose (Fig. 6B). Combination of OX40 and CD137 (clone Lob12.3) the lower dose, or the human IgG1 isotype used. FS120 surrogate agonism induced a transient increase in T cells in the liver, which was showed significant antitumor activity as compared with the isotype associated with increased T-cell proliferation. FS120 surrogate also control antibody and the antitumor activity observed for FS120 showed a moderate, but not statistically significant, increase in liver T- surrogate was similar to a WT IgG1 variant of FS120 surrogate in cell infiltration, proliferation, and activation at 7 days post-last dose, which FcgR interaction was not reduced by the LALA mutations which returned to normal at 14 days post-last dose (Fig. 6B). This [FS120 surrogate (WT mAb2); Fig. 4B]. This indicated the transient increase in T cells and proliferation was also observed in the mechanism of antitumor activity of FS120 surrogate was independent blood of these na€ve mice, as expected from other studies in CT26 of FcgR interaction, either FcgR-mediated crosslinking or FcgR- tumor–bearing mice (Fig. 5B). In the spleen, FS120 surrogate also mediated effector functions such as ADCC or ADCP. FS120 surro- induced transient T-cell proliferation (Fig. 6A and B). gate–treated mice with no tumors at day 60 were rechallenged with The increased liver T-cell infiltration observed with the crosslink- CT26 tumors and did not show tumor growth (Supplementary independent CD137 agonist antibody (clone 3H3) as compared Fig. S9). with the crosslink-dependent CD137 agonist antibody (clone When intratumoral Tregs were analyzed, CT26 tumors treated with Lob12.3; Fig. 6B) correlated with observations that urelumab (cross- CD137 antibody or the combination of OX40 and CD137 antibodies link-independent clone 20H4.9) induces hepatotoxicity at doses above had fewer Tregs compared with the isotype control–treated tumors 1 mg/kg and utomilumab (crosslink-dependent clone MOR7480.1) is (Fig. 4C). CT26 tumors treated with the FS120 surrogate (WT mAb2) well-tolerated up to 10 mg/kg (13, 14). Both FS120 and the FS120 also had fewer intratumoral Tregs, similar to the percentages observed surrogate molecules had crosslink-dependent CD137 agonist Fab arms with the combination of OX40 and CD137 antibodies (Fig. 4C). and were only able to induce CD137 agonism via binding to OX40 as However, due to the presence of the LALA mutations, the FS120 shown by the competition experiments in Fig. 2E and Supplementary surrogate–treated tumors did not have fewer intratumoral Tregs Fig. S8B. This dependency on OX40 binding for CD137 stimulation compared with the isotype control–treated tumors (Fig. 4C). None resulted in decreased liver T-cell infiltration in this preclinical study

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Figure 4. Antitumor activity of the FS120 surrogate. A and B, Balb/c mice (n ¼ 15) inoculated with 106 CT26 cells subcutaneously (s.c.) and treated with 1 mg/kg FS120 surrogate or controls every 2 days (Q2D) starting on day 13 post-tumor inoculation for three doses injected intraperitoneally (IP). Tumor volume measured every other day. Data shown are mean SEM. Statistical testing of tumor volume over time by mixed model analysis. C, TIL analysis of day 21 CT26 tumors (n ¼ 5) treated with 1 mg/kg FS120 surrogate or controls Q2D starting on day 10 for three doses injected intraperitoneally by ﬂow cytometry. Individual sample data are shown as well as mean SD (representative data from two independent experiments). Statistical testing by one-way ANOVA and Tukey multiple comparisons test. Asterisks on top of error bars represent the signiﬁcant difference to Ctrl(4420) mAb–treated mice (, P < 0.032; , P < 0.0002). See also Supplementary Figs. S5, S6, and S7.

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Antitumor Activity of a CD137/OX40 Bispeciﬁc Antibody

Figure 5. Peripheral T-cell activation and proliferation induced by the FS120 surrogate. A, Schematic representation of experimental design. B, Balb/c mice (n ¼ 5) inoculated with 106 CT26 cells subcutaneously (s.c.) and treated with 1 mg/kg FS120 surrogate or controls every 2 days starting on day 10 post-tumor inoculation for three doses injected intraperitoneally (IP). Tail vein blood collected on days 10 (predose), 11, 15, 17, and 24 for ﬂow cytometric analysis. Data shown are mean SD. Statistical testing by two-way ANOVA and Tukey multiple comparisons test. Asterisks on top of error bars represent the signiﬁcant difference to Ctrl(4420) mAb–treated mice (, P < 0.0001).

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Figure 6. Increased inflammation induced by the crosslink-independent CD137 agonist antibody. A, Schematic representation of experimental design. B, Balb/c mice (n ¼ 6) treated with 10 mg/kg FS120 surrogate or controls every 2 days starting on day 1 for three doses injected intraperitoneally (IP). Livers, spleens, and blood from 3 mice collected on days 7 and 14 post-last dose (experiment days 11 and 18) and processed for flow cytometric analysis. Individual sample data are shown as well as mean SD. Statistical testing by two-way ANOVA and Tukey multiple comparisons test. Asterisks on top of error bars represent the significant difference to Ctrl(4420) mAb–treated mice (, P < 0.032; , P < 0.0021; , P < 0.0002; , P < 0.0001).

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Antitumor Activity of a CD137/OX40 Bispeciﬁc Antibody

and suggested that FS120 may have a lower hepatotoxicity risk than In conclusion, FS120 is a bispecific antibody targeting OX40 and crosslink-independent CD137 agonist antibodies. CD137 that simultaneously bound these receptors and induced FcgR- independent T-cell activation in vitro; the FS120 surrogate induced T- cell activation in vivo. This in vitro and in vivo activity, which was Discussion dependent on coengagement with both receptors, suggested that the TILs express various checkpoint receptors and costimulatory recep- dual binding resulted in efficient receptor clustering and activation. tors, including OX40 and CD137 (4, 5). These receptors, in the absence The existence of two OX40-binding sites in the Fcab and two CD137- of ligand interaction, are likely to contribute to the dysfunctional binding sites in the Fab region of FS120 raised the possibility of phenotype of tumor reactive TILs (37, 38). Activating TILs with tetravalent binding, which was likely to be involved in the clustering agonist antibodies against OX40 and CD137 has the potential to mechanism. In addition, the binding to these two separate TNFRSF unleash existing antitumor immune responses and reduces tumor members with a single molecule could potentially create receptor growth and increases survival in several syngeneic tumor mod- superclusters resulting in increased signaling via these receptors as els (39, 40). In clinical trials, however, despite inducing peripheral they share intracellular signaling intermediates such as TRAF2 but also T-cell activation, neither OX40 antibodies nor CD137 antibodies have unique adapters (TRAF1 for CD137 and TRAF5 for OX40) and induce complete responses unlike the results observed in preclinical stimulate distinct pathways (47). The crosslinking of OX40 and CD137 studies (12). receptors by FS120 could also lead to increased internalization, as is The lack of translation between the preclinical models and the required for CD137 signaling (48). The increased T-cell activation by clinical results is likely due to various factors. Limited availability of FS120 surrogate which resulted in FcgR-independent antitumor activ- FcgR-expressing cells in the tumor microenvironment of human ity was independent of Treg depletion. Furthermore, due to its cancers and low affinity interaction between FcgRs and the Fc region crosslink-dependent CD137-targeting Fab, which required Fcab bind- of IgG antibodies (41) could result in suboptimal crosslinking of these ing to OX40 for activity, FS120 may potentially provide a potent and agonist antibodies (42). The depletion of intratumoral Tregs, described safe way of stimulating CD137. These data support initiation of clinical as the mechanism of action of OX40 and CD137 antibodies in mouse development of FS120, a first-in-class dual agonist bispecific antibody syngeneic tumor models, may not be as effective in human can- for the treatment of human cancer. cers (6, 7, 43) and may also result in the depletion of the very same cells the OX40 and CD137 antibodies aim to stimulate (15). Disclosure of Potential Conflicts of Interest CD137 agonist antibodies are associated with liver inflammation in M. Gaspar is a principal scientist at and has ownership interest (including patents) preclinical models and urelumab induces lethal hepatic inflammation in F-star Therapeutics Ltd. S. Uhlenbroich is a senior scientist at and has ownership in clinical trials at doses above 1 mg/kg (13). Although the mechanism interest (including patents) in F-star Therapeutics Ltd. K.L. Everett is a senior scientist at F-star Therapeutics Ltd. N. Brewis is Chief ScientificOfficer at, reports receiving a of toxicity in the clinic is unclear, in preclinical models this is associated commercial research grant from, and has ownership interest (including patents) in with activation of liver myeloid cells, which express CD137 and the F-star Therapeutics Ltd. No potential conflicts of interest were disclosed by the other þ production of IL27, which then recruits CD8 T cells that mediate the authors. inflammation damage (35). Whereas these results explain the mechanism of liver inflammation in mouse models, they do not explain why Authors’ Contributions in the clinic, a different CD137 antibody, utomilumab, showed no Conception and design: M. Gaspar, M. Morrow, M. Tuna, N. Brewis signs of liver inflammation and was well-tolerated up to 10 mg/kg (14). Development of methodology: M. Gaspar, J. Pravin, L. Rodrigues, S. Uhlenbroich, Several differences could account for this disparity because urelumab is K.L. Everett Acquisition of data (provided animals, acquired and managed patients, provided reported as being a human IgG4 non-ligand blocking antibody and facilities, etc.): M. Gaspar, J. Pravin, L. Rodrigues, S. Uhlenbroich, F. Wollerton utomilumab as being a human IgG2 ligand blocking antibody (44). In Analysis and interpretation of data (e.g., statistical analysis, biostatistics, addition, the epitopes of urelumab and utomilumab are different and computational analysis): M. Gaspar, J. Pravin, L. Rodrigues, F. Wollerton, could also account for their different activities (45). In this study, the M. Morrow, N. Brewis increased in vitro potency of the CD137 antibody clone present in Writing, review, and/or revision of the manuscript: M. Gaspar, J. Pravin, urelumab was observed and this clone (20H4.9) was also able to induce F. Wollerton, M. Morrow, M. Tuna, N. Brewis þ Administrative, technical, or material support (i.e., reporting or organizing data, crosslink-independent activation of CD8 T cells, and T cell lines constructing databases): M. Gaspar, J. Pravin engineered to express CD137. In a separate study, the clone present in Study supervision: M. Gaspar, M. Tuna urelumab also induces increased IFNg production from CD3-stimulated PBMCs and purified T cells in the absence of crosslinking (46). Acknowledgments When two anti-mouse CD137 antibodies, clones Lob12.3 and 3H3 The authors wish to thank Delphine Buffet, Marine Houee, and Cyril were compared, a corresponding difference was observed, with clone Privezentzev for contributions to OX40 Fcab discovery, to Melanie Medcalf 3H3 able to induce crosslink-independent activation of CD137- and Edouard Souteyrand for contributions to CD137 Fab discovery and FS120 fi in vivo expressing cells and clone Lob12.3 requiring crosslinking. The liver af nity determination, to Drug Discovery, Protein Sciences and team fl members for assistance with experimental procedures, and to Jacqueline Doody in ammation observed in mice treated with these antibodies was for contributions to project strategy and supervision. markedly different, with clone 3H3 showing sustained increase of T cells in the liver. Increased alanine aminotransferase and liver T-cell infiltration is observed in mice treated with rat versions of the same The costs of publication of this article were defrayed in part by the payment of page advertisement clones (46). Because urelumab and clone 3H3 are associated with charges. This article must therefore be hereby marked in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. increased liver inflammation and both clones (20H4.9 and 3H3) have the ability to stimulate CD137 in the absence of crosslinking, it is possible that this may contribute to the hepatotoxicity risk presented Received October 15, 2019; revised January 31, 2020; accepted March 31, 2020; by CD137-targeting antibodies. published first April 9, 2020.

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References 1. Sanmamed MF, Pastor F, Rodriguez A, JL P-G, ME R-R, Jure-Kunkel M, et al. 24. Rickert KW, Grinberg L, Woods RM, Wilson S, Bowen MA, Baca M. Combining Agonists of co-stimulation in cancer immunotherapy directed against CD137, phage display with de novo protein sequencing for reverse engineering of OX40, GITR, CD27, CD28, and ICOS. Semin Oncol 2015;42:640–55. monoclonal antibodies. MAbs 2016;8:501–12. 2. Ma BY, Mikolajczak SA, Danesh A, Hosiawa KA, Cameron CM, 25. Lund J, Takahashi N, Popplewell A, Goodall M, Pound JD, Tyler R, et al. Takaori-Kondo A, et al. The expression and the regulatory role of OX40 and Expression and characterization of truncated forms of humanized L243 IgG1: 4-1BB heterodimer in activated human T cells. Blood 2005;106:2002–10. architectural features can influence synthesis of its oligosaccharide chains and 3. Taraban VY, Rowley TF, O'Brien L, Chan HT, Haswell LE, Green MH, et al. affect superoxide production triggered through human Fcg receptor I. Eur J Expression and costimulatory effects of the TNF receptor superfamily members Biochem 2000;267:7246–56. CD134 (OX40) and CD137 (4-1BB), and their role in the generation of anti- 26. Wozniak-Knopp G, Bartl S, Bauer A, Mostageer M, Woisetschl€ager M, Antes B, tumor immune responses. Eur J Immunol 2002;32:3617–27. et al. Introducing antigen-binding sites in structural loops of immunoglobulin 4. Montler R, Bell RB, Thalhofer C, Leidner R, Feng Z, Fox BA, et al. OX40, PD-1 constant domains: Fc fragments with engineered HER2/neu-binding sites and and CTLA-4 are selectively expressed on tumor-infiltrating T cells in head and antibody properties. Protein Eng Des Sel 2010;23:289–97. neck cancer. Clin Transl Immunology 2016;5:e70–8. 27. Scherer MT, Ignatowicz L, Winslow GM, Kappler JW, Marrack P. Superantigens: 5. Ye Q, De-Gang S, Poussin M, Yamamoto T, Best A, Li C, et al. CD137 accurately bacterial and viral proteins that manipulate the immune system. Annu Rev Cell identifies and enriches for naturally occurring tumor-reactive T cells in tumor. Biol 1993;9:101–28 Clin Cancer Res 2014;20:44–55. 28. Vessillier S, Eastwood D, Fox B, Sathish J, Sethu S, Dougall T, et al. Cytokine 6. Bulliard Y, Jolicoeur R, Zhang J, Dranoff G, Wilson NS, Brogdon JL. OX40 release assays for the prediction of therapeutic mAb safety in first-in man trials - engagement depletes intratumoral Tregs via activating FcgRs, leading to anti- whole blood cytokine release assays are poorly predictive for TGN1412 cytokine tumor efficacy. Immunol Cell Biol 2014;92:475–80. storm. J Immunol Methods 2015;424:43–52. 7. Buchan SL, Dou L, Remer M, Booth SG, Dunn SN, Lai C, et al. Antibodies to 29. Stebbings R, Findlay L, Edwards C, Eastwood D, Bird C, North D, et al. “Cytokine costimulatory receptor 4-1BB enhance anti-tumor immunity via T regulatory storm” in the phase I trial of monoclonal antibody TGN1412: better under- cell depletion and promotion of CD8þ T cell effector function. Immunity 2018; standing the causes to improve preclinical testing of immunotherapeutics. 49:958–70. J Immunol 2007;179:3325–31. 8. Lee S-J, Myers L, Muralimohan G, Dai J, Qiao Y, Li Z, et al. 4-1BB and OX40 dual 30. Weinberg AD, Rivera M-M, Prell R, Morris A, Ramstad T, Vetto JT, et al. costimulation synergistically stimulate primary specific CD8 T cells for robust Engagement of the OX-40 receptor in vivo enhances antitumor immunity. effector function. J Immunol 2004;173:3002–12. J Immunol 2000;164:2160–9. 9. Qui HZ, Hagymasi AT, Bandyopadhyay S, St. Rose M-C, Ramanarasimhaiah R, 31. Linch SN, McNamara MJ, Redmond WL. OX40 agonists and combination Menoret A, et al. CD134 plus CD137 dual costimulation induces eomesodermin immunotherapy: putting the pedal to the metal. Front Oncol 2015;5:1–14. in CD4 T cells to program cytotoxic Th1 differentiation. J Immunol 2011;187: 32. Bartkowiak T, Curran MA. 4-1BB agonists: multi-potent potentiators of tumor 3555–64. immunity. Front Oncol 2015;5:1–16. 10. Cabo M, Offringa R, Zitvogel L, Kroemer G, Galluzzi L, Cabo M, et al. Trial 33. Lechner MG, Karimi SS, Barry-Holson K, Angell TE, Murphy KA, Church watch: immunostimulatory monoclonal antibodies for oncological indications. CH, et al. Immunogenicity of murine solid tumor models as a defining Oncoimmunology 2017;6:1–16. feature of in vivo behavior and response to immunotherapy. J Immunother 11. Curti BD, Kovacsovics-Bankowski M, Morris N, Walker E, Chisholm L, Floyd K, 2013;36:477–89. et al. OX40 is a potent immune-stimulating target in late-stage cancer patients. 34. Segal NH, He AR, Doi T, Levy R, Bhatia S, Pishvaian MJ, et al. Phase I study of Cancer Res 2013;73:7189–98. single-agent utomilumab (PF-05082566), a 4-1BB/CD137 agonist, in patients 12. Marin-Acevedo JA, Dholaria B, Soyano AE, Knutson KL, Chumsri S, Lou Y. Next with advanced cancer. Clin Cancer Res 2018;24:1816–23. generation of immune checkpoint therapy in cancer: new developments and 35. Bartkowiak T, Jaiswal AR, Ager CR, Chin R, Chen C-H, Budhani P, et al. challenges. J Hematol Oncol 2018;11:39. Activation of 4-1BB on liver myeloid cells triggers hepatitis via an interleukin-27 13. Segal NH, Logan TF, Hodi FS, McDermott D, Melero I, Hamid O, et al. Results dependent pathway. Clin Cancer Res 2018;24:1138–51. from an integrated safety analysis of urelumab, an agonist anti-CD137 mono- 36. Niu L, Strahotin S, Hewes B, Zhang B, Zhang Y, Archer D, et al. Cytokine- clonal antibody. Clin Cancer Res 2017;23:1929–36. mediated disruption of lymphocyte trafficking, hemopoiesis, and induction of 14. Segal NH, He AR, Doi T, Levy R, Bhatia S, Pishvaian MJ, et al. Phase I study of lymphopenia, anemia, and thrombocytopenia in anti-CD137-treated mice. single-agent utomilumab (PF-05082566), a 4-1bb/cd137 agonist, in patients with J Immunol 2007;178:4194–213. advanced cancer. Clin Cancer Res 2018;24:1816–23. 37.WilliamsJB,HortonBL,ZhengY,DuanY,PowellJD,GajewskiTF.The 15. Wajant H. Principles of antibody-mediated TNF receptor activation. Cell Death EGR2 targets LAG-3 and 4-1BB describe and regulate dysfunctional antigen- þ Differ 2015;22:1727–41. specificCD8 T cells in the tumor microenvironment. J Exp Med 2017;214: 16. Li F, Ravetch J V. Antitumor activities of agonistic anti-TNFR antibodies require 381–400. differential FcgRIIB coengagement in vivo. Proc Natl Acad Sci U S A 2013;110: 38. Thommen DS, Schumacher TN. T cell dysfunction in cancer. Cancer Cell 2018; 19501–6. 33:547–62. 17. Stewart R, Hammond SA, Oberst M, Wilkinson RW. The role of Fc gamma 39. Aspeslagh S, Postel-Vinay S, Rusakiewicz S, Soria JC, Zitvogel L, Marabelle A. receptors in the activity of immunomodulatory antibodies for cancer. Rationale for anti-OX40 cancer immunotherapy. Eur J Cancer 2016;52: J Immunother Cancer 2014;2:29. 50–66. 18. Mayes PA, Hance KW, Hoos A. The promise and challenges of immune agonist 40. Sanchez-Paulete AR, Labiano S, Rodriguez-Ruiz ME, Azpilikueta A, Etxeberria I, antibody development in cancer. Nat Rev Drug Discov 2018;17:509–27. Bolanos~ E, et al. Deciphering CD137 (4-1BB) signaling in T-cell costimulation 19. Junttila TT, Li J, Johnston J, Hristopoulos M, Clark R, Ellerman D, et al. for translation into successful cancer immunotherapy. Eur J Immunol 2016;46: Antitumor efficacy of a bispecific antibody that targets HER2 and activates T 513–22. cells. Cancer Res 2014;74:5561–71. 41. Bruhns P, Iannascoli B, England P, Mancardi DA, Fernandez N, Jorieux S, et al. 20. Lund J, Winter G, PT J, JD P, Tanaka T, MR W, et al. Human Fc gamma RI and Fc Specificity and affinity of human Fc gamma receptors and their polymorphic gamma RII interact with distinct but overlapping sites on human IgG. J Immunol variants for human IgG subclasses. Blood 2009;113:3716–25. 1991;147:2657–62. 42. Bruhns P. Properties of mouse and human IgG receptors and their contribution 21. Everett KL, Kraman M, Wollerton FPG, Zimarino C, Kmiecik K, Gaspar M, et al. to disease models. Blood 2012;119:5640–9. Generation of Fcabs targeting human and murine LAG-3 as building blocks for 43. Selby MJ, Engelhardt JJ, Quigley M, Henning KA, Chen T, Srinivasan M, et al. novel bispecific antibody therapeutics. Methods 2019;154:60–9. Anti-CTLA-4 antibodies of IgG2a isotype enhance antitumor activity 22. Edelman GM, Cunningham BA, Gall WE, Gottlieb PD, Rutishauser U, Waxdal through reduction of intratumoral regulatory T cells. Cancer Immunol Res MJ. The covalent structure of an entire gammaG immunoglobulin molecule. 2013;1:32–42. Proc Natl Acad Sci U S A 1969;63:78–85. 44. Fisher TS, Kamperschroer C, Oliphant T, Love VA, Lira PD, Doyonnas R, et al. 23. Bedzyk WD, Weidner KM, Denzin LK, Johnson LS, Hardman KD, Pantoliano Targeting of 4-1BB by monoclonal antibody PF-05082566 enhances T-cell MW, et al. Immunological and structural characterization of a high affinity anti- function and promotes anti-tumor activity. Cancer Immunol Immunother fluorescein single-chain antibody. J Biol Chem 1990;265:18615–20. 2012;61:1721–33.

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45. Chin SM, Kimberlin CR, Roe-Zurz Z, Zhang P, Xu A, Liao-Chan S, et al. 47. Konstorum A, Vella AT, Adler AJ, Laubenbacher RC. A mathematical model of Structure of the 4-1BB/4-1BBL complex and distinct binding and functional combined CD8 T cell costimulation by receptors. Sci Rep 2019;9:10862–74. properties of utomilumab and urelumab. Nat Commun 2018;9:4679. 48. Martinez-Forero I, Azpilikueta A, Bolanos-Mateo E, Nistal-Villan E, Palazon A, 46. Qi X, Li F, Wu Y, Cheng C, Han P, Wang J, et al. Optimization of 4-1BB antibody Teijeira A, et al. T cell costimulation with anti-CD137 monoclonal antibodies is for cancer immunotherapy by balancing agonistic strength with FcgRafﬁnity. mediated by K63-polyubiquitin-dependent signals from endosomes. J Immunol Nat Commun 2019;10:2141–52. 2013;190:6694–706.

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CD137/OX40 Bispecific Antibody Induces Potent Antitumor Activity that Is Dependent on Target Coengagement

Miguel Gaspar, John Pravin, Leonor Rodrigues, et al.

Cancer Immunol Res Published OnlineFirst April 9, 2020.

Updated version Access the most recent version of this article at: doi:10.1158/2326-6066.CIR-19-0798

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