CD137/OX40 Bispeciﬁc Antibody Induces Potent Antitumor … · receptorsuperfamily(TNFRSF;ref.1).Bothareexpressedonactivated T cells and natural killer cells, and are attractive

CANCER IMMUNOLOGY RESEARCH | RESEARCH ARTICLE

CD137/OX40 Bispecific Antibody Induces PotentAntitumor Activity that Is Dependent on TargetCoengagement A C

Miguel Gaspar, John Pravin, Leonor Rodrigues, Sandra Uhlenbroich, Katy L. Everett, FranciscaWollerton,Michelle Morrow, Mihriban Tuna, and Neil Brewis

ABSTRACT◥

Following the success of immune checkpoint blockade therapyagainst cancer, agonistic antibodies targeting T-cell costimulatorypathways are in clinical trials. The TNF superfamily of receptors(TNFRSF) members CD137 and OX40 are costimulatory receptorsthat stimulate T-cell proliferation and activation upon interactionwith their cognate ligands. Activating CD137 and OX40 withagonistic mAbs stimulates the immune system due to their broadexpression on CD4þ and CD8þ T cells and natural killer cells andhas antitumor effects in preclinical models. Most TNFRSF agonistantibodies require crosslinking via Fcg receptors (FcgR), which canlimit their clinical activity. FS120 mAb2, a dual agonist bispecific

antibody targeting CD137 and OX40, activated both CD4þ andCD8þ T cells in an FcgR-independent mechanism, dependent onconcurrent binding. A mouse surrogate version of the bispecificantibody displayed antitumor activity in syngeneic tumor models,independent of T regulatory cell depletion and of FcgR interaction,but associated with peripheral T-cell activation and proliferation.When compared with a crosslink-independent CD137 agonistmAb, the FS120 surrogate induced lower liver T-cell infiltration.These data support initiation of clinical development of FS120, afirst-in-class dual agonist bispecific antibody for the treatment ofhuman cancer.

IntroductionOX40 and CD137 costimulatory receptors belong to the TNF

receptor superfamily (TNFRSF; ref. 1). Both are expressed on activatedT cells and natural killer cells, and are attractive targets for cancerimmunotherapy as stimulation of these receptors results in increasedT-cell activation, proliferation, and survival in vitro and in vivo (1). Theexpression patterns of OX40 and CD137 are overlapping, but distinctwith expression of OX40 higher on CD4þ T cells and that of CD137higher on CD8þ T cells (2). CD137 stimulation preferentially stimu-lates CD8þ T cells when compared with CD4þ T cells and OX40stimulation preferentially stimulates CD4þ T cells when comparedwith CD8þ T cells (3). However, coexpression of these receptors isdemonstrated in both CD4þ and CD8þ T cells and both are expressedin tumor-infiltrating lymphocytes (TIL; refs. 4, 5). Antibodies stim-ulating these targets show activity in a variety of murine tumormodelsby both depleting regulatory T cells (Treg) and activating CD8þ andCD4þ T cells (6, 7). The combination of OX40 and CD137 agonistantibodies stimulate both CD4þ and CD8þ T cells and inducethe cytotoxic function of both antigen-experienced and antigen-inexperienced bystander CD4þ T cells (8, 9).

Several clinical trials are underway to test agonist antibodies toOX40 or CD137 either asmonotherapies or in combination with otheragents to treat various cancers (10). Clinical trials with OX40 agonistantibodies demonstrate peripheral T-cell activation and proliferation

without associated toxicity (11) but show limited clinical efficacy (12).Two CD137 agonist antibodies have different clinical outcomes.Urelumab (BMS-663513, clone 20H4.9) induces severe transaminitisat doses higher than 1 mg/kg resulting in two hepatotoxicity-relateddeaths (13) and utomilumab (PF-05082566, cloneMOR7480.1), whichdoes not induce severe adverse events, hasmodest clinical activity (14).A combination trial with utomilumab and PF-04518600 (an OX40agonist antibody) is underway (NCT02315066).

TNFRSF antibodies typically have no or low intrinsic agonistactivity and require secondary crosslinking of antibody–receptorcomplexes to induce sufficient receptor clustering and activation,thereby mimicking the TNFSF ligand superclusters (15). In vivo, thissecondary crosslinking requires the interaction with Fcg receptors(FcgR; ref. 16). The availability of FcgR-expressing cells in the tumormicroenvironment and the low affinity interaction between FcgRs andthe Fc-region of IgG antibodies may limit the agonist activity ofTNFRSF antibodies and, consequently, their antitumor activity (17).In addition, interaction with FcgRs mediates antibody effector func-tions such as antibody-dependent cell-mediated cytotoxicity (ADCC)and antibody-dependent cell-mediated phagocytosis (ADCP) andcould lead to the depletion of the tumor-specific T cells that wouldbe activated by these antibodies (18). Consequently, the clinical activityseen with OX40 and CD137 antibodies may not represent the fullpotential of activating these receptors.

An alternative to FcgR-mediated crosslinking of TNFRSF agonistantibodies is the use of bispecific antibodies, the dual binding of whichresults in the clustering of the TNFRSF target, independent of FcgRengagement (19). This study described FS120 mAb2, a dual agonistbispecific antibody targeting CD137 and OX40 that activated bothCD4þ and CD8þ T cells, whereas OX40 or CD137 monospecificantibodies only activated CD4þ or CD8þ T cells, respectively. FcgR-disabling mutations (LALA mutations; ref. 20) were introduced toenable antibody crosslinking from the coengagement of the twodifferent receptors when coexpressed and to potentially avoid deple-tion of OX40- or CD137-expressing cells. A mouse-specific surrogateversion of FS120 showed antitumor activity in the absence of FcgR

F-star Therapeutics Ltd., Cambridge, United Kingdom.

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

Corresponding Author: Neil Brewis, F-star Biotechnology, Babraham Campus,Cambridge, CB22 3AT, UK. Phone: 44-1223-497400; Fax: 44-1223-497461;E-mail: [email protected]

Cancer Immunol Res 2020;8:781–93

doi: 10.1158/2326-6066.CIR-19-0798

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interaction or after Treg depletion. When compared with a crosslink-independent CD137 agonist mAb, FS120 surrogate showed reducedliver T-cell infiltration, which decreased over time.

These results indicate that targeting coexpressed receptors withbispecific antibodies may be a potent and safe mechanism to clusterand activate TNFRSF costimulatory receptors and induce antitumorimmunity

Materials and MethodsAntibody reagents

Antibodies were cloned by replacing the VH or Vk domainsequences in human IgG1 with identified sequences from patents orliterature using methods described previously (21). The LALA(L234A-L235A, Eu numbering; ref. 22) mutations were introducedvia site-directed mutagenesis when indicated. Absence of LALAmutations is denoted by the suffix WT (wild-type) after the clonename. For antibody production, pTT5 expression vectors (NationalResearch Council of Canada) containing the mAb or mAb2 sequenceswere transfected into Expi293F cells (Thermo Fisher Scientific,A14528) using PEIpro Transfection Reagent (Polyplus, PPLU115)according to themanufacturer's instructions. Antibodies were purifiedusing a 5 mL MabSelect SuRe Column (GE Healthcare, 11003494) onan AKTA Explorer (GE Healthcare) according to the manufacturer'sinstructions.

The following antibodies were used in the experiments described inthis article: anti-FITC [Ctrl(4420)WTmAb; clone 4420 used as isotypecontrol; ref. 23], anti-human OX40 [OX40(11D4) mAb; clone 11D4from patent EP2242771B1], anti-human OX40 Fcab [OX40/Ctrl(4420) mAb2; clone FS20, F-star]; anti-human CD137 [CD137(MOR7480.1) mAb; clone MOR7480.1 from patent US8,337,850B2],anti-human CD137 [CD137(20H4.9) mAb; clone 20H4.9 from patentUS8,137,667B2], anti-humanCD137 [CD137(FS30)mAb; clone FS30,F-star], anti-mouse OX40 [mOX40(OX86) WT mAb; clone OX86from patent US9,738,723B2], anti-mouse CD137 [mCD137(Lob12.3)WT mAb; clone Lob12.3; ref. 3], and anti-mouse CD137 [mCD137(3H3) WT mAb; clone 3H3; ref. 24] all in human IgG1 format, with(mAb) or without (WTmAb) LALAmutations; anti-human IgG CH2domain (clone MK1A6; ref. 25) in mouse IgG1 format; anti-humanCD28 [CD28(TGN1412) mAb; clone TGN1412 from patent US8,709,414 B2] in human IgG4 format with S228P mutation (Eunumbering); anti-human CD3 antibody (clone UHCT1, R&D Sys-tems); anti-mouse CD3 antibody (clone 17A2, Thermo Fisher Scien-tific); and anti-mouse CD3 antibody (clone 145-2C11, Thermo FisherScientific).

Surface plasmon resonance analysisDatawere acquired using a BIAcore 3000 or BIAcore T200.Dilution

mixtures prepared in HBS-P or HBS-EP Buffer (GE Healthcare). ForKD determination, FS120 was captured either via the Fc region usinga Human Antibody Capture Kit (GE Healthcare) and human orcynomolgus CD137 was flowed over at a range of concentrations; orvia the Fab region using a Human Fab Capture Kit (GE Healthcare)and human or cynomolgus OX40 was flowed over at a range ofconcentrations. For dual binding determination of FS120, biotinylatedhuman CD137 or OX40 was immobilized on a Streptavidin Chip (GEHealthcare) and antibodies (100 nmol/L) were coinjected with eitherhumanOX40 or CD137 (100 nmol/L), respectively, or HBS-EP buffer.For dual binding determination of FS120 surrogate, mouse CD137wasimmobilized on a CM5 Chip (GE Healthcare) to a surface density ofapproximately 1,000 RU and antibodies (100 nmol/L) were coinjected

with either mouse OX40 or CD137 (100 nmol/L) or HBS-EP buffer.Affinity for FcgRs [R&D Systems, hFcgR1 (1257-CF-050), hFcgR2a(1330-CF-050), hFcgR2b (1875-CF-050), and hFcgR3a (4325-CF-050)] was tested by coating biotinylated humanOX40 (BPS Bioscience71310-1) or CD137 (in-house) his-tagged antigens onto an Strepta-vidin Chip (GE Healthcare) and coinjecting antibodies (100 nmol/L)and human FcgRs (500 nmol/L) at 20 mL/minute flow rate and thedissociation was followed for 5 minutes. For specificity assessment,human TNFRSF members [R&D Systems, TNFRI (372-RI-050/CF),TNFRII (726-R2-050), GITR (689-GR-100), NGFRI (367-NR-050/CF), CD40 (1493-CD-050), and DR6 (144-DR-100)] were immobi-lized on CM5 Chips (GE Healthcare) to approximately 1,000 RU andFS120 (1 mmol/L), isotype control [anti-FITC (Ctrl(4420) WT mAb;clone 4420 in human IgG1 backbone; ref. 23] or positive controlantibodies [R&D Systems, anti-TNFRI (MAB225R-100); anti-TNFRII(MAB726-100); anti-GITR (MAB689-100); anti-NGFR (MAB367);anti-CD40 (MAB6321-100); and anti-DR6 (AF144)] were flowed overat a flow rate of 30 mL/minutes. Data were analyzed using BIAEvaluation Software (GE Healthcare).

Cell line creation and reporter T-cell assayAll cells used in the experiments described in this article were kept in

culture for a maximum of 2 months before starting new cultures frommaster vials. CT26 (CRL-2638) and B16-F10 (CRL-6475) cell lineswere purchased from ATCC in 2015. DO11.10 cells were purchasedfrom Public Health England (85082301) in 2014 and used underlicense from National Jewish Health. Expi293F cells were purchasedfrom Thermo Fisher Scientific (A14528) in 2015. No reauthenticationtests were performed. Mycoplasma testing was performed on all celllines monthly using R&D MycoProbe Mycoplasma Detection Kit(R&D Systems, 895285). Human and mouse OX40 and CD137 cDNAconstructs were synthesized (GenScript) with flanking 50 EcoRI and 30

NotI restriction sites and cloned into the lentivirus vector pLVX-EF1a-IRES-puro (Clontech 631988). Lentiviruses were produced using theLentiX Expression system EF1a Version (Clontech 631253) and usedto transduce DO11.10 cells according to the manufacturer's instruc-tion. Cell lines were selected by incubation with 5 mg/mL Puromycin(InvivoGen, ant-pr-1) and individual cell lines were cloned by serialdilution. DO11.10 cells expressing human or mouse CD137 werestimulated with coated anti-mouse CD3 antibody (BioLegend,100202 clone 17A2 at 0.1 mg/mL) and mouse IL2 concentration inthe supernatants was measured by ELISA (Thermo Fisher Scientific,88-7024-88).

Flow cytometryFor cell binding assays, cells were incubatedwith primary antibodies

or mAb2 followed by detection with an anti-human Fc-488 secondaryantibody (Jackson ImmunoResearch). Excised tissues were dissociatedusing relevantMiltenyi BiotecDissociationKits (tumors, 130-096-730;livers, 130-105-807; and spleens, 130-095-926) using a Miltenyi gen-tleMACS Octo Dissociator and C-tubes according to the manufac-turer's instructions. Resulting cell suspensions were strained [70 mmcell strainer (Corning CLS431751)], washed, and resuspended in PBS.Collected blood samples were treated twice with Red Blood Cell LysisBuffer (eBioscience 00-430054) according to the manufacturer'sinstruction. Cells isolated from tissues and blood were stained forflow cytometry with fluorochrome-conjugated antibodies includingthose against CD4, Ki67, FoxP3, CD69, CD3, andCD8 (Thermo FisherScientific), CD45 (BD Biosciences), and fixable live/dead dye (ThermoFisher Scientific) in the presence of Fc Block (Thermo Fisher Scientific)according to the manufacturer's recommendations. Cells were

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analyzed in a BD FACS CantoII Cytometer and data were analyzedwith FlowJoX. OX40 and CD137 receptors quantified with QuantumSimply Cellular anti-mouse IgG (Bangs Laboratories 815) according tothe manufacturer's recommendations.

Peripheral blood mononuclear cells and T-cell stimulationassays

Ficoll-purified human peripheral bloodmononuclear cells (PBMC)were stimulated with 100 ng/mL staphylococcal enterotoxin A (SEA,Sigma) in the presence of FS120 or control antibodies for 5 days at37�C, 5% CO2 in T-cell media [RPMI1640 (Thermo Fisher Scientific)with 10% FBS (Thermo Fisher Scientific), 1� penicillin–streptomycin(Thermo Fisher Scientific), 1 mmol/L Sodium Pyruvate (Gibco), 10mmol/LHepes (Gibco), 2mmol/L L-Glutamine (Gibco), and 50 mmol/L 2-mercaptoethanol (Gibco)]. T cells were isolated from PBMCsusingMiltenyi Enrichment Kits (HumanCD3þ, 130-096-535; HumanCD4þ, 130-096-533; Human CD8þ, 130-096-495; and Mouse CD3þ,130-095-130) according to the manufacturer's instructions and acti-vated overnight using CD3/CD28 Dynabeads (Life Technologiescatalog numbers: Human, 11131D and Mouse, 11453D) at a 1:1 cellto bead ratio. CD3/CD28 beads were removed using a DynaMag-15magnet and activated T cells were washed with T-cell media andstimulated with coated CD3 antibody [Human: R&D Systems(MAB100) clone UHCT1 at 2.5 mg/mL (total and CD4þ T cells) or5 mg/mL (CD8þ T cells) or Mouse: BioLegend (100302) clone 145-2C11 at 2.5 mg/mL] in the presence of FS120 or control antibodies for3 days at 37�C, 5% CO2. Anti-human Fc (clone MK1A6, produced in-house) was used as crosslinking agent at a 1:1 molar ratio with testantibodies. FITC dextran (70 kDa, Sigma 46945) was used as cross-linking agent at a 1:1 molar ratio with OX40 Fcabs paired with FITC-binding Fab (clone 4420). IL2 concentration in the supernatants wasmeasured by ELISA or electrochemiluminescence (Thermo FisherScientific, 88-7025-88 or MSD K151QQD-1). For the cytokine releaseassay, antibodies were diluted to 10 mg/mL in PBS, coated onto 96-wellflat-bottomed plates and allowed to air dry overnight, washed twicewith PBS, and incubated with 2 � 105 PBMCs for 3 days. Multiplecytokine concentrations in supernatants were measured by Pro-inflammatory V-plex Kit (Meso Scale Discovery catalog numberK15049D-2) according to the manufacturer's instructions.

Mice and tumor challengeBalb/c female mice were from Charles River Laboratories. Animals

were housed in a local animal facility and were used at approximately8–10 weeks of age. Antibodies were diluted in PBS before intraper-itoneal injection at the indicated dose and schedule. For tumor trials,mice were anaesthetized by inhalation of isoflurane, and each animalreceived 106 or 105 of CT26 tumor cells (depending on experiment) or105 of B16-F10 tumor cells diluted in PBS. Mice were injectedsubcutaneously with a maximum volume of 100 mL in the left flankto generate tumors.Micewere randomized into study cohorts based ontumor volume and any mice which did not have tumors were notassigned into treatment groups and were removed from the study.Tumor measurements were taken under anesthesia using calipers todetermine the longest axis and the shortest axis of the tumor. Thefollowing formula was used to calculate the tumor volume: L� (S2)/2(where L ¼ longest axis; S ¼ shortest axis). For peripheral pharma-codynamic analysis, blood was collected into EDTA-containing tubesfrom either the tail vein or by cardiac puncture. Tumors, spleens, andlivers were collected by dissection when indicated. All proceduresinvolving animals were approved and performed according to UK

Home Office (license number 70/7991) and local ethical reviewcommittee guidelines.

Statistical analysisOne- and two-way ANOVA with Tukey or Dunnett multiple

comparisons test, and survival analysis (log-rank Mantel–Cox test)was performed using Prism Software (GraphPad). For comparison oftreatment responses and EC50 determinations, data were log trans-formed before analysis and fit using the log agonist versus responseusing Prism Software (GraphPad). Where indicated, statistical testingof tumor volume over time was analyzed using a mixed model. Aseparate model was fitted to each pair of treatments of interest. Themodel is

log10 volumeð Þ ¼ Aþ B� day � start dayð Þ þ "

A and B are the intercept and slope, respectively; they are differentfor each mouse, and include a fixed effect for the group and a randomeffect for the animal:

A ¼ A0 þ A1T þ "A

B ¼ B0 þ B1T þ "B

T is a dummy variable representing the treatment groupwith value 0in one group and 1 in the other. The random effects are distributedwith a normal distribution:

"A �N 0; sAð Þ; "B �N 0; sBð Þ

where sA and sB are the SDs of the interanimal variability in theintercept and slope, respectively. The intra-animal variability is alsonormally distributed with SD s:

" �N 0; sð Þ

For each pair of treatments, the model above was fitted to the data.For A1 and B1, the (two-sided) P value for a difference from zero wascalculated; a P value below 0.05 is statistically significant evidence for adifference between the treatment groups.

ResultsFS120 simultaneously bound to CD137 and OX40

Phage and yeast libraries were usedwith directed evolutionmethodsdescribed previously (21, 26) to identify and improveOX40-binding Fcantigen binders, termed Fcabs (Fc-region with antigen binding), aswell as CD137-binding Fab regions. The Fcab (OX40 Fcab, clone FS20)and Fab (CD137 Fab, clone FS30) with the overall highest activity in T-cell stimulation assays and affinity in cell binding assays were com-bined to generate the bispecific FS120 mAb2 (or FS120; Fig. 1A).Affinity determination by surface plasmon resonance (SPR) showedthat FS120 has subnanomolar binding to both human and cynomolgusmonkey OX40 (KD: Human, 0.2 nmol/L and Cyno, 0.9 nmol/L) andCD137 (KD: Human, 0.2 nmol/L and Cyno, 0.2 nmol/L; Fig. 1B) andFS120 bound both OX40 and CD137 simultaneously (Fig. 1C). FS120did not bind to other related members of the TNFR superfamily(CD40, GITR, NGFR, DR6, TNFRI, and TNFRII; SupplementaryFig. S1). FS120, which contains the LALA mutations, had reducedFcgR binding as compared with WT IgG1 (non-LALA containing)OX40 and CD137 antibodies (Supplementary Fig. S2). FS120 bound tocell surface expressed human and cynomolgus OX40 and CD137receptors on engineered DO11.10 T cell lines but not to the non-transduced parental DO11.10 cell line (Fig. 1D).

Antitumor Activity of a CD137/OX40 Bispecific Antibody

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FS120 agonistic activity was dependent on the dual binding ofCD137 and OX40

To test the agonist activity of FS120, PBMCs were stimulated withSEA superantigen, which crosslinks MHC class II molecules at thesurface of antigen-presenting cells and the T-cell receptor (TCR) of Tcells (27), in the presence or absence of secondary crosslinking agentsto mimic the effect of FcgR-mediated crosslinking. The amount ofT-cell activation resulting from OX40 or CD137 stimulation wasthen measured by the production of IL2. All antibodies weretested in the same isotype background as FS120, human IgG1 withthe LALA mutations, to minimize interference from FcgR-mediatedcrosslinking.

Agonistic activity of OX40 or CD137 monospecific antibodies wasonly observed in the presence of crosslinking (Fig. 2A). In contrast,FS120 mAb2 showed activity in the absence of secondary crosslinkingagent suggesting the coengagement of OX40 and CD137 resulted in

effective receptor clustering and activation (Fig. 2A). Similar resultswere observed when isolated T cells were stimulated with plate-boundCD3 antibody and costimulated with OX40- or CD137-specific anti-bodies or FS120 mAb2 (Fig. 2B). The activity of FS120 was notincreased by the secondary crosslinking agent, either in maximumresponse or in a decrease in EC50 (Fig. 2C and D), indicating that thedual binding to OX40 and CD137 resulted in the maximum stimu-lation induced by FS120.

When CD137 agonist antibodies were crosslinked, the 20H4.9 Fabclone (Fab present in urelumab) was observed to have a higher activityas compared with clones MOR7480.1 (Fab present in utomilumab) orFS30 (Fab present in FS120; Fig. 2A and B). The crosslinked OX40-targeting antibodies induced higher IL2 production than the cross-linked CD137-targeting antibodies, and the combination of the OX40Fcab and the CD137 Fab components of FS120 did not show asynergistic effect as compared with the OX40 Fcab alone (Fig. 2A

Figure 1.

FS120 bound to OX40 and CD137 with high affinity simultaneously. A, Schematic representation of FS120 mAb2, a bispecific antibody binding to OX40 viathe Fc region and to CD137 via the Fab region, containing LALA mutations. B, SPR of FS120 binding to human OX40 or human CD137. KD determinationperformed using BIA evaluation software (representative results of three independent experiments). C, SPR of FS120 simultaneous binding to immobilizedOX40 and CD137 in solution and to immobilized CD137 and OX40 in solution (representative results of two independent experiments). D, Geometric meanfluorescence intensity (GMFI) of FS120 binding to cell surface–expressed human OX40 and CD137 determined by flow cytometry. Data of triplicates presentedas mean � SD. For EC50 determinations, antibody concentration and GMFI data were log transformed before analysis and fit using the log agonist versusresponse using Prism Software (GraphPad; representative results of three independent experiments).

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and B). This result indicated that these assays were more sensitive toOX40 stimulation and that only potent CD137 stimulation resulted insubstantial T-cell activation. The higher response to OX40 agonismcould have been explained by the higher proportion of CD4þ T cells inhuman PBMCs and the higher expression of OX40 on activated CD4þ

T cells (Supplementary Fig. S3).Titrations of these antibodies in both the PBMC SEA stimulation

and T-cell CD3 stimulation assays were performed and the con-centration at which these antibodies induced the highest IL2production was chosen for this analysis. FS120, and crosslinkedOX40 Fcab, induced the production of additional proinflammatorycytokines (IL6, IL12p70, IL13, and TNFa) by T cells and reduced

the levels of IL10, a typical anti-inflammatory cytokine (Supple-mentary Fig. S4).

To test whether the activity of FS120 required simultaneous bindingto the two receptors, the ability of FS120 to coengageOX40 andCD137was blocked using 100-foldmolar excess of either theOX40 Fcab or theCD137 Fab components of FS120 or both. The results showed that theFS120-induced T-cell activation was reduced when the mAb2 com-ponent parts were present either individually or in combination(Fig. 2E), indicating that FS120 required dual binding to OX40 andCD137 to induce the clustering and activation of these receptors.

FS120 did not induce T-cell activation in a cytokine releaseassay (28) in the absence of TCR stimulation, unlike the two positive

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 coatedCD3-antibody (cloneUCHT-1 at 2.5mg/mL;B) and costimulatedwith FS120or control antibodies (3.7 nmol/L) in the presence or absence of crosslinking reagents at 1:1molar 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 thepresence or absence of anti-Fc crosslinking in SEA-stimulated PBMCs (C) or CD3-stimulated T cells (D). E,CD3-stimulated T cells costimulatedwith 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 (representativeresults of three independent experiments). Statistical testing by two-way ANOVA and Tukey multiple comparison test (A and B) or one-way ANOVAand 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 fit using thelog agonist versus response using Prism Software (GraphPad; C and D). See also Supplementary Figs. S2 and S3.






control antibodies used, a CD28 antibody shown to induce cytokinestorm in the clinic (TGN1412; ref. 29) and a CD3 antibody (Supple-mentary Fig. S5).

FS120 stimulated both CD4þ and CD8þ T cellsOX40 and CD137 receptors were coexpressed on CD4þ and CD8þ

T cells, with OX40 expressed in higher percentages and at a higherreceptor number in CD4þ T cells than CD8þ T cells and, conversely,more CD137 receptors were expressed on CD8þ T cells (Supplemen-tary Fig. S1). The differential expressions correlated with the activity ofOX40- or CD137-targeting antibodies. On CD4þ T cells stimulatedwith plate-coated CD3 antibody, both the crosslinked OX40 mAb andthe OX40 Fcab induced IL2 production, but CD137 antibodies did notshow activity (Fig. 3A and B). On CD8þ T cells, the crosslinkedCD137 antibodies (Fab clones 20H4.9 and FS30) induced IL2 pro-duction and OX40 antibodies did not (Fig. 3A and B). When tested inthe absence of secondary crosslinking agent, FS120 increased IL2production on both CD4þ and CD8þ T cells (Fig. 3A). The cross-linked activity of clone FS30 [CD137(FS30) mAb] demonstrated thatthe mAb2 could activate the CD137 receptor when crosslinked bybinding of the Fcab toOX40 on CD8þT cells. The activity of the cross-linked OX40 Fcab [OX40/Ctrl(4420) mAb2] showed that the mAb2

could activate the OX40 receptor when crosslinked by binding of theFab arms to CD137 on CD4þ T cells.

The different CD137 antibodies tested showed varying activity onCD8þ T cells. Fab clone MOR7480.1 did not show activity in theabsence of crosslinking and only a moderate, nonsignificant, increasewhen crosslinked (Fig. 3A). Clone FS30 displayed higher activitywhen crosslinked, but no activity in the absence of crosslinking(Fig. 3A). However, Fab clone 20H4.9 induced higher levels of IL2production in the absence of crosslinking, which was increased withcrosslinking (Fig. 3A). When these antibodies were tested in a modelDO11.10 T cell line expressing human CD137 and stimulated withcoated anti-mouse CD3 antibody, Fab clone 20H4.9 also showedcrosslink-independent activity, whereas Fab clones FS30 andMOR7480.1 did not (Supplementary Fig. S6A).

FS120 surrogate bound mouse OX40 and CD137 and activatesT cells

As the FS120 mAb2 does not bind to mouse OX40 or CD137, asurrogate molecule was generated for in vivo testing by pairinga mouse-specific OX40 Fcab with a CD137 mAb (clone Lob12.3).The Fc-modifying technology used to create Fcabs is based onhuman IgG1 therefore the FS120 surrogate has a human IgG1domain. All in vivo experiments were performed using moleculeswith the same human IgG1 backbone. The FS120 surrogate boundto cell surface expressed mouse OX40 and CD137 receptors onengineered DO11.10 T cell lines (Supplementary Fig. S7A) and

Figure 3.

FS120mAb2 stimulatedboth CD4þandCD8þT cells.A,HumanCD4þ andCD8þT cells stimulatedwith coatedCD3 antibody (cloneUCHT-1 at 2.5mg/mL for CD4þ and5 mg/mL for CD8þ 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 1:1molar ratio [FITC dextran for OX40/Ctrl(4420)mAb2 and anti-Fc (cloneMK1A6 inmouse IgG1 format) for other antibodies]. Data fromduplicates are shown asmean� SD (representative results of three independent experiments). Statistical testing by two-way ANOVA and Dunnett multiple comparison test. Asterisks on top oferror 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 CD137receptor quantification in CD4þand CD8þ T cells from 3 PBMC donors, activated overnight with CD3/CD28 beads (1:1 ratio), by flow cytometric fluorescencequantification with beads (Bangs Laboratories). See also Supplementary Figs. S1 and S4.

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concurrent binding to mouse OX40 and CD137 was demonstratedby SPR (Supplementary Fig. S7B).

FS120 surrogate was functionally characterized by testing its abilityto costimulate primary mouse T cells in the presence of coated anti-mouse CD3. FS120 surrogate induced the production of IL2 in theabsence of secondary crosslinking agent, unlike the OX40 monospe-cific antibody, which required anti-Fc antibody for crosslinking (Sup-plementary Fig. S8A). No activity of the CD137 antibody was detectedin this assay, but in DO11.10 T cell line expressing mouse CD137 andstimulated with coated anti-mouse CD3 antibody, clone Lob12.3increased IL2 production in the presence of crosslinking (Supplemen-tary Fig. S6B). A different anti-mouse CD137 (clone 3H3) was alsotested in this assay and showed crosslink-independent activity, similarto that seen with Fab clone 20H4.9 (Fig. 3A; Supplementary Fig. S6Aand S6B). This result indicated that these two molecules could besurrogates for each other, whereas clone Lob12.3 only showed activitywhen crosslinked and was therefore a surrogate for Fab clonesMOR7480.1 and FS30 mAb.

The activity of FS120 surrogate in the absence of secondary cross-linking agent was also dependent on dual binding toOX40 andCD137;when either of these receptors was blocked with excess mouse OX40Fcab or mouse CD137 mAb (clone Lob12.3), the activity of FS120surrogate was reduced, similar to that observedwith FS120 in humanTcells (Supplementary Fig. S8B).

Antitumor activity of the FS120 surrogateOX40 and CD137 antibodies demonstrate antitumor activity in a

variety of syngeneic models with responses depending on dose, time oftreatment initiation, antibody isotype, and clones used (3, 30–32).Intratumoral Treg depletion is part of the mechanism of antitumoractivity of OX40 (clone OX86; ref. 6) and CD137 antibodies (cloneLob12.0; ref. 7).

To test the antitumor activity of FS120 and to understand the in vivomechanism of action, FS120 surrogate or control antibodies wereinjected intraperitoneally at 1 mg/kg on days 11, 13, and 15 post-CT26tumor cells inoculation (Fig. 4A). The OX40 (clone OX86) or CD137(clone Lob12.3) control antibodies with human IgG1 isotype or theircombination showed no antitumor activity (Fig. 4B), unlike previouslypublished results using the original rat versions of these antibodies (3).This could be explained by the later start time of antibody treatment,the lower dose, or the human IgG1 isotype used. FS120 surrogateshowed significant antitumor activity as compared with the isotypecontrol antibody and the antitumor activity observed for FS120surrogate was similar to a WT IgG1 variant of FS120 surrogate inwhich FcgR interaction was not reduced by the LALA mutations[FS120 surrogate (WT mAb2); Fig. 4B]. This indicated themechanism of antitumor activity of FS120 surrogate was independentof FcgR interaction, either FcgR-mediated crosslinking or FcgR-mediated effector functions such as ADCC or ADCP. FS120 surro-gate–treated mice with no tumors at day 60 were rechallenged withCT26 tumors and did not show tumor growth (SupplementaryFig. S9).

When intratumoral Tregs were analyzed, CT26 tumors treated withCD137 antibody or the combination of OX40 and CD137 antibodieshad fewer Tregs compared with the isotype control–treated tumors(Fig. 4C). CT26 tumors treated with the FS120 surrogate (WTmAb2)also had fewer intratumoral Tregs, similar to the percentages observedwith the combination of OX40 and CD137 antibodies (Fig. 4C).However, due to the presence of the LALA mutations, the FS120surrogate–treated tumors did not have fewer intratumoral Tregscompared with the isotype control–treated tumors (Fig. 4C). None

of the treatments induced any significant changes in the frequency ofCD4þ or CD8þ T cells or T-cell proliferation (Fig 4C). The antitumoractivity of the FS120 surrogate was therefore not associated withintratumoral Treg depletion.

When the FS120 surrogate was tested in the B16-F10 model, apoorly immunogenic model and thus harder to treat with immu-notherapies (33), FS120 surrogate also had antitumor activity ascompared with an isotype control antibody (Supplementary Fig. S10).

Peripheral T-cell activation andproliferationmediatedbyFS120surrogate

The induction of T-cell proliferation by OX40 and CD137 agonistantibodies in vitro and in vivo is described in both preclinical modelsand in the clinic (11, 34). Comparing T-cell proliferation and activa-tion in the blood of CT26 tumor–bearing mice treated with FS120surrogate or control antibodies overtime, it was observed that FS120surrogate induced more CD4þ and CD8þ T-cell proliferation, asmeasured by the expression of Ki67 (Fig. 5A and B) than the OX40and CD137 antibodies or their combination. The effect of FS120surrogate on T-cell activation, as measured by the expression of CD69,was delayed as compared with the proliferative effect with the highestfrequencies observed 3 days after the third dose (Fig. 5B). Similareffects on T-cell activation were observed with the combination ofOX40 and CD137 antibodies (Fig. 5B).

FS120 surrogate did not induce liver inflammationIncreased liver T-cell infiltration was observed with various CD137

agonist antibodies in mice, suggesting a similar mechanism of livertoxicity for CD137 agonism in mice and humans (35, 36). To under-stand the potential hepatotoxicity risk of FS120, T cells and theirproliferation and activation in the liver, spleen, and blood induced byFS120 surrogate was comparedwith that induced byOX40 andCD137agonist antibodies or their combination (Fig. 6A). The results showeda clear difference between the two CD137 agonist antibodies tested,clone 3H3 induced a sustained increase in T-cell infiltration, prolif-eration, and activation in the liver and spleen, whereas clone Lob12.3did not (Fig. 6B). OX40 stimulation did not show an increase in T cellsin the liver, spleen, or blood, but induced transient T-cell proliferationin all tissues studied and T-cell activation in the liver at 14 days post-last dose (Fig. 6B). Combination of OX40 and CD137 (clone Lob12.3)agonism induced a transient increase in T cells in the liver, which wasassociated with increased T-cell proliferation. FS120 surrogate alsoshowed amoderate, but not statistically significant, increase in liver T-cell infiltration, proliferation, and activation at 7 days post-last dose,which returned to normal at 14 days post-last dose (Fig. 6B). Thistransient increase in T cells and proliferation was also observed in theblood of these na€�ve mice, as expected from other studies in CT26tumor–bearing mice (Fig. 5B). In the spleen, FS120 surrogate alsoinduced transient T-cell proliferation (Fig. 6A and B).

The increased liver T-cell infiltration observed with the crosslink-independent CD137 agonist antibody (clone 3H3) as comparedwith the crosslink-dependent CD137 agonist antibody (cloneLob12.3; Fig. 6B) correlated with observations that urelumab (cross-link-independent clone 20H4.9) induces hepatotoxicity at doses above1 mg/kg and utomilumab (crosslink-dependent clone MOR7480.1) iswell-tolerated up to 10 mg/kg (13, 14). Both FS120 and the FS120surrogatemolecules had crosslink-dependent CD137 agonist Fab armsand were only able to induce CD137 agonism via binding to OX40 asshown by the competition experiments in Fig. 2E and SupplementaryFig. S8B. This dependency on OX40 binding for CD137 stimulationresulted in decreased liver T-cell infiltration in this preclinical study






Figure 4.

Antitumor activity of the FS120 surrogate.A andB,Balb/cmice (n¼ 15) inoculatedwith 106CT26 cells subcutaneously (s.c.) and treatedwith 1mg/kg FS120 surrogateor controls every 2days (Q2D) startingonday 13 post-tumor inoculation for threedoses injected intraperitoneally (IP). Tumor volumemeasured every other day.Datashown 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/kgFS120 surrogate or controls Q2D starting on day 10 for three doses injected intraperitoneally by flow cytometry. Individual sample data are shown aswell as mean�SD (representative data from two independent experiments). Statistical testing by one-way ANOVA and Tukey multiple comparisons test. Asterisks on top of errorbars 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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Figure 5.

Peripheral T-cell activation and proliferation induced by the FS120 surrogate. A, Schematic representation of experimental design. B, Balb/c mice (n¼ 5) inoculatedwith 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 threedoses injected intraperitoneally (IP). Tail vein blood collected ondays 10 (predose), 11, 15, 17, and 24 forflowcytometric analysis. Data shownaremean�SD. Statisticaltesting by two-wayANOVA and Tukeymultiple comparisons test. Asterisks on top of error bars represent the significant difference to Ctrl(4420)mAb–treatedmice(�� , P < 0.0001).






Figure 6.

Increased inflammation induced by the crosslink-independent CD137 agonist antibody. A, Schematic representation of experimental design. B, Balb/c mice (n¼ 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 3mice 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 aswell as mean� SD. Statistical testing by two-way ANOVA and Tukeymultiple comparisons test. Asterisks on top of error bars represent the significant difference toCtrl(4420) mAb–treated mice (� , P < 0.032; �� , P < 0.0021; �� , P < 0.0002; �� , P < 0.0001).

Gaspar et al.





and suggested that FS120 may have a lower hepatotoxicity risk thancrosslink-independent CD137 agonist antibodies.

DiscussionTILs express various checkpoint receptors and costimulatory recep-

tors, includingOX40 andCD137 (4, 5). These receptors, in the absenceof ligand interaction, are likely to contribute to the dysfunctionalphenotype of tumor reactive TILs (37, 38). Activating TILs withagonist antibodies against OX40 and CD137 has the potential tounleash existing antitumor immune responses and reduces tumorgrowth and increases survival in several syngeneic tumor mod-els (39, 40). In clinical trials, however, despite inducing peripheralT-cell activation, neither OX40 antibodies nor CD137 antibodiesinduce complete responses unlike the results observed in preclinicalstudies (12).

The lack of translation between the preclinical models and theclinical results is likely due to various factors. Limited availability ofFcgR-expressing cells in the tumor microenvironment of humancancers and low affinity interaction between FcgRs and the Fc regionof IgG antibodies (41) could result in suboptimal crosslinking of theseagonist antibodies (42). The depletion of intratumoral Tregs, describedas the mechanism of action of OX40 and CD137 antibodies in mousesyngeneic tumor models, may not be as effective in human can-cers (6, 7, 43) and may also result in the depletion of the very samecells the OX40 and CD137 antibodies aim to stimulate (15).

CD137 agonist antibodies are associated with liver inflammation inpreclinical models and urelumab induces lethal hepatic inflammationin clinical trials at doses above 1 mg/kg (13). Although the mechanismof toxicity in the clinic is unclear, in preclinicalmodels this is associatedwith activation of liver myeloid cells, which express CD137 and theproduction of IL27, which then recruits CD8þ T cells that mediate theinflammation damage (35). Whereas these results explain the mech-anism of liver inflammation inmousemodels, they do not explain whyin the clinic, a different CD137 antibody, utomilumab, showed nosigns of liver inflammation andwas well-tolerated up to 10mg/kg (14).Several differences could account for this disparity because urelumab isreported as being a human IgG4 non-ligand blocking antibody andutomilumab as being a human IgG2 ligand blocking antibody (44). Inaddition, the epitopes of urelumab and utomilumab are different andcould also account for their different activities (45). In this study, theincreased in vitro potency of the CD137 antibody clone present inurelumabwas observed and this clone (20H4.9) was also able to inducecrosslink-independent activation of CD8þ T cells, and T cell linesengineered to express CD137. In a separate study, the clone present inurelumab also induces increased IFNg production from CD3-stimu-lated PBMCs and purified T cells in the absence of crosslinking (46).

When two anti-mouse CD137 antibodies, clones Lob12.3 and 3H3were compared, a corresponding difference was observed, with clone3H3 able to induce crosslink-independent activation of CD137-expressing cells and clone Lob12.3 requiring crosslinking. The liverinflammation observed in mice treated with these antibodies wasmarkedly different, with clone 3H3 showing sustained increase ofT cells in the liver. Increased alanine aminotransferase and liver T-cellinfiltration is observed in mice treated with rat versions of the sameclones (46). Because urelumab and clone 3H3 are associated withincreased liver inflammation and both clones (20H4.9 and 3H3) havethe ability to stimulate CD137 in the absence of crosslinking, it ispossible that this may contribute to the hepatotoxicity risk presentedby CD137-targeting antibodies.

In conclusion, FS120 is a bispecific antibody targeting OX40 andCD137 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 wasdependent on coengagement with both receptors, suggested that thedual binding resulted in efficient receptor clustering and activation.The existence of two OX40-binding sites in the Fcab and two CD137-binding sites in the Fab region of FS120 raised the possibility oftetravalent binding, which was likely to be involved in the clusteringmechanism. In addition, the binding to these two separate TNFRSFmembers with a single molecule could potentially create receptorsuperclusters resulting in increased signaling via these receptors asthey share intracellular signaling intermediates such as TRAF2 but alsohave unique adapters (TRAF1 for CD137 and TRAF5 for OX40) andstimulate distinct pathways (47). The crosslinking ofOX40 andCD137receptors by FS120 could also lead to increased internalization, as isrequired for CD137 signaling (48). The increased T-cell activation byFS120 surrogate which resulted in FcgR-independent antitumor activ-ity was independent of Treg depletion. Furthermore, due to itscrosslink-dependent CD137-targeting Fab, which required Fcab bind-ing to OX40 for activity, FS120 may potentially provide a potent andsafe way of stimulating CD137. These data support initiation of clinicaldevelopment of FS120, a first-in-class dual agonist bispecific antibodyfor the treatment of human cancer.

Disclosure of Potential Conflicts of InterestM. Gaspar is a principal scientist at and has ownership interest (including patents)

in F-star Therapeutics Ltd. S. Uhlenbroich is a senior scientist at and has ownershipinterest (including patents) in F-star Therapeutics Ltd. K.L. Everett is a senior scientistat F-star Therapeutics Ltd. N. Brewis is Chief Scientific Officer at, reports receiving acommercial research grant from, and has ownership interest (including patents) inF-star Therapeutics Ltd. No potential conflicts of interest were disclosed by the otherauthors.

Authors’ ContributionsConception and design: M. Gaspar, M. Morrow, M. Tuna, N. BrewisDevelopment of methodology: M. Gaspar, J. Pravin, L. Rodrigues, S. Uhlenbroich,K.L. EverettAcquisition of data (provided animals, acquired and managed patients, providedfacilities, etc.): M. Gaspar, J. Pravin, L. Rodrigues, S. Uhlenbroich, F. WollertonAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): M. Gaspar, J. Pravin, L. Rodrigues, F. Wollerton,M. Morrow, N. BrewisWriting, review, and/or revision of the manuscript: M. Gaspar, J. Pravin,F. Wollerton, M. Morrow, M. Tuna, N. BrewisAdministrative, technical, or material support (i.e., reporting or organizing data,constructing databases): M. Gaspar, J. PravinStudy supervision: M. Gaspar, M. Tuna

AcknowledgmentsThe authors wish to thank Delphine Buffet, Marine Houee, and Cyril

Privezentzev for contributions to OX40 Fcab discovery, to Melanie Medcalfand Edouard Souteyrand for contributions to CD137 Fab discovery and FS120affinity determination, to Drug Discovery, Protein Sciences and in vivo teammembers for assistance with experimental procedures, and to Jacqueline Doodyfor contributions to project strategy and supervision.

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

Received October 15, 2019; revised January 31, 2020; accepted March 31, 2020;published first April 9, 2020.






References1. Sanmamed MF, Pastor F, Rodriguez A, JL P-G, ME R-R, Jure-Kunkel M, et al.

Agonists of co-stimulation in cancer immunotherapy directed against CD137,OX40, GITR, CD27, CD28, and ICOS. Semin Oncol 2015;42:640–55.

2. Ma BY, Mikolajczak SA, Danesh A, Hosiawa KA, Cameron CM,Takaori-Kondo A, et al. The expression and the regulatory role of OX40 and4-1BB heterodimer in activated human T cells. Blood 2005;106:2002–10.

3. Taraban VY, Rowley TF, O'Brien L, Chan HT, Haswell LE, Green MH, et al.Expression and costimulatory effects of the TNF receptor superfamily membersCD134 (OX40) and CD137 (4-1BB), and their role in the generation of anti-tumor immune responses. Eur J Immunol 2002;32:3617–27.

4. Montler R, Bell RB, Thalhofer C, Leidner R, Feng Z, Fox BA, et al. OX40, PD-1and CTLA-4 are selectively expressed on tumor-infiltrating T cells in head andneck cancer. Clin Transl Immunology 2016;5:e70–8.

5. Ye Q, De-Gang S, PoussinM, Yamamoto T, Best A, Li C, et al. CD137 accuratelyidentifies and enriches for naturally occurring tumor-reactive T cells in tumor.Clin Cancer Res 2014;20:44–55.

6. Bulliard Y, Jolicoeur R, Zhang J, Dranoff G, Wilson NS, Brogdon JL. OX40engagement depletes intratumoral Tregs via activating FcgRs, leading to anti-tumor efficacy. Immunol Cell Biol 2014;92:475–80.

7. Buchan SL, Dou L, Remer M, Booth SG, Dunn SN, Lai C, et al. Antibodies tocostimulatory receptor 4-1BB enhance anti-tumor immunity via T regulatorycell depletion and promotion of CD8þ T cell effector function. Immunity 2018;49:958–70.

8. Lee S-J,Myers L,MuralimohanG, Dai J, Qiao Y, Li Z, et al. 4-1BB andOX40 dualcostimulation synergistically stimulate primary specific CD8 T cells for robusteffector function. J Immunol 2004;173:3002–12.

9. Qui HZ, Hagymasi AT, Bandyopadhyay S, St. Rose M-C, Ramanarasimhaiah R,Menoret A, et al. CD134 plus CD137 dual costimulation induces eomesoderminin CD4 T cells to program cytotoxic Th1 differentiation. J Immunol 2011;187:3555–64.

10. Cabo M, Offringa R, Zitvogel L, Kroemer G, Galluzzi L, Cabo M, et al. Trialwatch: immunostimulatory monoclonal antibodies for oncological indications.Oncoimmunology 2017;6:1–16.

11. Curti BD, Kovacsovics-BankowskiM,Morris N,Walker E, Chisholm L, Floyd K,et al. OX40 is a potent immune-stimulating target in late-stage cancer patients.Cancer Res 2013;73:7189–98.

12. Marin-Acevedo JA,Dholaria B, SoyanoAE, KnutsonKL, Chumsri S, LouY.Nextgeneration of immune checkpoint therapy in cancer: new developments andchallenges. J Hematol Oncol 2018;11:39.

13. Segal NH, Logan TF, Hodi FS, McDermott D, Melero I, Hamid O, et al. Resultsfrom an integrated safety analysis of urelumab, an agonist anti-CD137 mono-clonal antibody. Clin Cancer Res 2017;23:1929–36.

14. Segal NH, He AR, Doi T, Levy R, Bhatia S, Pishvaian MJ, et al. Phase I study ofsingle-agent utomilumab (PF-05082566), a 4-1bb/cd137 agonist, in patientswithadvanced cancer. Clin Cancer Res 2018;24:1816–23.

15. Wajant H. Principles of antibody-mediated TNF receptor activation. Cell DeathDiffer 2015;22:1727–41.

16. Li F, Ravetch J V. Antitumor activities of agonistic anti-TNFR antibodies requiredifferential FcgRIIB coengagement in vivo. Proc Natl Acad Sci U S A 2013;110:19501–6.

17. Stewart R, Hammond SA, Oberst M, Wilkinson RW. The role of Fc gammareceptors in the activity of immunomodulatory antibodies for cancer.J Immunother Cancer 2014;2:29.

18. Mayes PA, Hance KW, Hoos A. The promise and challenges of immune agonistantibody development in cancer. Nat Rev Drug Discov 2018;17:509–27.

19. Junttila TT, Li J, Johnston J, Hristopoulos M, Clark R, Ellerman D, et al.Antitumor efficacy of a bispecific antibody that targets HER2 and activates Tcells. Cancer Res 2014;74:5561–71.

20. Lund J,Winter G, PT J, JDP, Tanaka T,MRW, et al. Human Fc gammaRI and FcgammaRII interact with distinct but overlapping sites on human IgG. J Immunol1991;147:2657–62.

21. Everett KL, KramanM,Wollerton FPG, Zimarino C, Kmiecik K, GasparM, et al.Generation of Fcabs targeting human and murine LAG-3 as building blocks fornovel bispecific antibody therapeutics. Methods 2019;154:60–9.

22. Edelman GM, Cunningham BA, Gall WE, Gottlieb PD, Rutishauser U, WaxdalMJ. The covalent structure of an entire gammaG immunoglobulin molecule.Proc Natl Acad Sci U S A 1969;63:78–85.

23. Bedzyk WD, Weidner KM, Denzin LK, Johnson LS, Hardman KD, PantolianoMW, et al. Immunological and structural characterization of a high affinity anti-fluorescein single-chain antibody. J Biol Chem 1990;265:18615–20.

24. Rickert KW, Grinberg L,Woods RM,Wilson S, BowenMA, BacaM. Combiningphage display with de novo protein sequencing for reverse engineering ofmonoclonal antibodies. MAbs 2016;8:501–12.

25. Lund J, Takahashi N, Popplewell A, Goodall M, Pound JD, Tyler R, et al.Expression and characterization of truncated forms of humanized L243 IgG1:architectural features can influence synthesis of its oligosaccharide chains andaffect superoxide production triggered through human Fcg receptor I. Eur JBiochem 2000;267:7246–56.

26. Wozniak-Knopp G, Bartl S, Bauer A, Mostageer M, Woisetschl€ager M, Antes B,et al. Introducing antigen-binding sites in structural loops of immunoglobulinconstant domains: Fc fragments with engineered HER2/neu-binding sites andantibody properties. Protein Eng Des Sel 2010;23:289–97.

27. SchererMT, Ignatowicz L,WinslowGM,Kappler JW,Marrack P. Superantigens:bacterial and viral proteins that manipulate the immune system. Annu Rev CellBiol 1993;9:101–28

28. Vessillier S, Eastwood D, Fox B, Sathish J, Sethu S, Dougall T, et al. Cytokinerelease assays for the prediction of therapeutic mAb safety in first-in man trials -whole blood cytokine release assays are poorly predictive for TGN1412 cytokinestorm. J Immunol Methods 2015;424:43–52.

29. Stebbings R, Findlay L, Edwards C, EastwoodD, Bird C,NorthD, et al. “Cytokinestorm” in the phase I trial of monoclonal antibody TGN1412: better under-standing the causes to improve preclinical testing of immunotherapeutics.J Immunol 2007;179:3325–31.

30. Weinberg AD, Rivera M-M, Prell R, Morris A, Ramstad T, Vetto JT, et al.Engagement of the OX-40 receptor in vivo enhances antitumor immunity.J Immunol 2000;164:2160–9.

31. Linch SN, McNamara MJ, Redmond WL. OX40 agonists and combinationimmunotherapy: putting the pedal to the metal. Front Oncol 2015;5:1–14.

32. Bartkowiak T, Curran MA. 4-1BB agonists: multi-potent potentiators of tumorimmunity. Front Oncol 2015;5:1–16.

33. Lechner MG, Karimi SS, Barry-Holson K, Angell TE, Murphy KA, ChurchCH, et al. Immunogenicity of murine solid tumor models as a definingfeature of in vivo behavior and response to immunotherapy. J Immunother2013;36:477–89.

34. Segal NH, He AR, Doi T, Levy R, Bhatia S, Pishvaian MJ, et al. Phase I study ofsingle-agent utomilumab (PF-05082566), a 4-1BB/CD137 agonist, in patientswith advanced cancer. Clin Cancer Res 2018;24:1816–23.

35. Bartkowiak T, Jaiswal AR, Ager CR, Chin R, Chen C-H, Budhani P, et al.Activation of 4-1BB on liver myeloid cells triggers hepatitis via an interleukin-27dependent pathway. Clin Cancer Res 2018;24:1138–51.

36. Niu L, Strahotin S, Hewes B, Zhang B, Zhang Y, Archer D, et al. Cytokine-mediated disruption of lymphocyte trafficking, hemopoiesis, and induction oflymphopenia, anemia, and thrombocytopenia in anti-CD137-treated mice.J Immunol 2007;178:4194–213.

37. Williams JB, Horton BL, Zheng Y, Duan Y, Powell JD, Gajewski TF. TheEGR2 targets LAG-3 and 4-1BB describe and regulate dysfunctional antigen-specific CD8 þ T cells in the tumor microenvironment. J Exp Med 2017;214:381–400.

38. Thommen DS, Schumacher TN. T cell dysfunction in cancer. Cancer Cell 2018;33:547–62.

39. Aspeslagh S, Postel-Vinay S, Rusakiewicz S, Soria JC, Zitvogel L, Marabelle A.Rationale for anti-OX40 cancer immunotherapy. Eur J Cancer 2016;52:50–66.

40. Sanchez-Paulete AR, Labiano S, Rodriguez-RuizME, Azpilikueta A, Etxeberria I,Bola~nos E, et al. Deciphering CD137 (4-1BB) signaling in T-cell costimulationfor translation into successful cancer immunotherapy. Eur J Immunol 2016;46:513–22.

41. Bruhns P, Iannascoli B, England P, Mancardi DA, Fernandez N, Jorieux S, et al.Specificity and affinity of human Fc gamma receptors and their polymorphicvariants for human IgG subclasses. Blood 2009;113:3716–25.

42. Bruhns P. Properties of mouse and human IgG receptors and their contributionto disease models. Blood 2012;119:5640–9.

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

44. Fisher TS, Kamperschroer C, Oliphant T, Love VA, Lira PD, Doyonnas R, et al.Targeting of 4-1BB by monoclonal antibody PF-05082566 enhances T-cellfunction and promotes anti-tumor activity. Cancer Immunol Immunother2012;61:1721–33.

Gaspar et al.





45. Chin SM, Kimberlin CR, Roe-Zurz Z, Zhang P, Xu A, Liao-Chan S, et al.Structure of the 4-1BB/4-1BBL complex and distinct binding and functionalproperties of utomilumab and urelumab. Nat Commun 2018;9:4679.

46. Qi X, Li F,WuY, Cheng C, Han P,Wang J, et al. Optimization of 4-1BB antibodyfor cancer immunotherapy by balancing agonistic strength with FcgR affinity.Nat Commun 2019;10:2141–52.

47. Konstorum A, Vella AT, Adler AJ, Laubenbacher RC. A mathematical model ofcombined CD8 T cell costimulation by receptors. Sci Rep 2019;9:10862–74.

48. Martinez-Forero I, Azpilikueta A, Bolanos-Mateo E, Nistal-Villan E, Palazon A,Teijeira A, et al. T cell costimulation with anti-CD137 monoclonal antibodies ismediated by K63-polyubiquitin-dependent signals from endosomes. J Immunol2013;190:6694–706.






2020;8:781-793. Published OnlineFirst April 9, 2020.Cancer Immunol Res Miguel Gaspar, John Pravin, Leonor Rodrigues, et al. that Is Dependent on Target CoengagementCD137/OX40 Bispecific Antibody Induces Potent Antitumor Activity

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CD137/OX40 Bispeciﬁc Antibody Induces Potent Antitumor … · receptorsuperfamily(TNFRSF;ref.1).Bothareexpressedonactivated T cells and natural killer cells, and are attractive