fs222, a cd137/pd-l1 tetravalent bispeciﬁc antibody ......simultaneous binding of fs222 to human...

CLINICAL CANCER RESEARCH | TRANSLATIONAL CANCER MECHANISMS AND THERAPY

FS222, a CD137/PD-L1 Tetravalent Bispecific Antibody,Exhibits Low Toxicity and Antitumor Activity inColorectal Cancer Models A C

Matthew A. Lakins, Alexander Koers, Raffaella Giambalvo, Jose Munoz-Olaya, Robert Hughes,Emma Goodman, Sylwia Marshall, Francisca Wollerton, Sarah Batey, Daniel Gliddon, Mihriban Tuna, andNeil Brewis

ABSTRACT◥

Purpose: With the increased prevalence in checkpoint therapyresistance, there remains a significant unmet need for additionaltherapies for patients with relapsing or refractory cancer. We havedeveloped FS222, a bispecific tetravalent antibody targeting CD137and PD-L1, to induce T-cell activation to eradicate tumors withoutthe current toxicity and efficacy limitations seen in the clinic.

Experimental Design: A bispecific antibody (FS222) was devel-oped by engineering CD137 antigen–binding sites into the Fc regionof a PD-L1 IgG1 mAb. T-cell activation by FS222 was investigatedusing multiple in vitro assays. The antitumor efficacy, survivalbenefit, pharmacodynamics, and liver pharmacology of a murinesurrogate molecule were assessed in syngeneic mouse tumor mod-els. Toxicology and the pharmacokinetic/pharmacodynamic profileof FS222 were investigated in a non-human primate dose-rangefinding study.

Results: We demonstrated simultaneous binding of CD137and PD-L1 and showed potent T-cell activation across CD8þ

T-cell activation assays in a PD-L1–dependent manner with aCD137/PD-L1 bispecific antibody, FS222. FS222 also activatedT cells in a human primary mixed lymphocyte reaction assay, withgreater potency than the monospecific mAb combination. FS222showed no signs of liver toxicity up to 30 mg/kg in a non-humanprimate dose-range finding study. A surrogate molecule causedsignificant tumor growth inhibition and survival benefit, concom-itant with CD8þ T-cell activation, in CT26 and MC38 syngeneicmouse tumor models.

Conclusions: By targeting CD137 agonism to areas of PD-L1expression, predominantly found in the tumor microenvironment,FS222 has the potential to leverage a focused, potent, and safeimmune response augmenting the PD-(L)1 axis blockade.

IntroductionImmunomodulatory mAbs are a promising approach for patients

with cancer. Immune checkpoint inhibitors targeting programmed celldeath (PD)-1, PD-L1, and cytotoxic T-lymphocyte–associated protein4 are the most advanced immunotherapy agents for oncology. How-ever, only a subset of patients benefit from long-term survival, andthere remains an unmet clinical need (1). Although bispecific T-cellengagers, such as blinatumomab (BLINCYTO) targeting CD3 andCD19, are the most advanced next-generation immuno-oncologymodalities, their use is limited to hematologic malignancies andfurther limited by acute safety concerns (2). We believe agonistantibodies against specific costimulatory receptors from the tumornecrosis factor receptor superfamily may represent the next stage insolid cancer treatment.

CD137 (4-1BB) is a costimulatory molecule and widely known tobe upregulated on CD8þ T cells following activation (3). CD137can also be expressed on activated CD4þ helper T cells, B cells,regulatory T cells (Treg), natural killer (NK) cells, natural killer

T cells, and dendritic cells (DC; ref. 4). Engagement of CD137 by itsligand CD137L results in receptor trimer formation, and subsequentclustering leads to CD137 signaling cascade activation. This provides asurvival signal to T cells, thereby sustaining effective T-cell activationand generation of immunologic memory. The primary functional roleof CD137 in enhancing T-cell cytotoxicity was first described in1997 (5), and soon thereafter CD137 mAbs were proposed as anti-cancer therapeutics.

Clinical development of CD137 mAbs has been hampered by dose-limiting high-grade liver inflammation associated with CD137 agonistantibody treatment. Urelumab (Bristol-Myers Squibb, BMS-663513),a human IgG4 isotype antibody, was the first CD137 mAb to enterclinical trials, but these were halted after significant, on target, dose-dependent liver toxicity was observed (6–8). This outcome was notpredicted because urelumab failed to preclinically identify liver inflam-mation due to its low affinity for the cynomolgus monkey targetmolecule (9).More recently, clinical trials of urelumab in the treatmentof solid cancers were recommended; however, urelumab dosing inthese trials had to be limited and efficacy results were disappointingwith no objective response reported in the 64 patients with solidtumors treated with monotherapy (6).

No dose-limiting toxicity has been observed with CD137 mAbutomilumab (PF-05082566, Pfizer), a human IgG2 isotype antibody, indose-escalation phase I clinical trials dosing up to 10 mg/kg in phase Iclinical trials of advanced cancer (6, 8). However, the overall objectiveresponse rate with this antibody was only 3.8% in patients with solidtumors, potentially indicating that utomilumab has a weaker potencyand clinical efficacy than urelumab, while having a more favorablesafety profile (6, 8). Trials of utomilumab in combination withradiotherapy or chemotherapy, as well as in combination with other

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

Note: Supplementary data for this article are available at Clinical CancerResearch Online (http://clincancerres.aacrjournals.org/).

Corresponding Author: Matthew A. Lakins, F-star Therapeutics Ltd., EddevaB920, Babraham Research Campus, Cambridge CB22 3AT, United Kingdom.Phone: 01223948159; Fax: 01223410493; E-mail: [email protected]

Clin Cancer Res 2020;26:4154–67

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antibody therapies, are ongoing with early results showing no dose-limiting toxicities for doses up to 5 mg/kg and a 26% patient responserate for the combination of utomilumab and pembrolizumab (10).

PD-1 and its ligands PD-L1 (CD274, B7-H1) and PD-L2 (B7-DC)deliver inhibitory signals that regulate the balance between T-cellactivation, tolerance, and immunopathology. Consequently, PD-L1expression by cells can mediate protection against cytotoxic T lym-phocyte killing. Cancer, as a chronic and proinflammatory disease,subverts this immune-protective pathway through upregulation ofPD-L1 expression to evade the host immune response. PD-L1 expres-sion has been shown in a wide variety of solid tumors (11), and clinicaltrials have shown the benefit of targeting PD-L1 in patients leading tothe approval of three PD-L1–targeting mAbs to date: atezolizumab(MPDL3280A, Tecentriq, Hoffmann-La Roche, Genentech), ahumanized IgG1 antibody; avelumab (MSB0010718C, Bavencio,Merck KGaA, Pfizer), a fully human IgG1 antibody; and durvalumab(MEDI4736, Imfinzi, AstraZeneca) a fully human IgG1 antibody. ThePD-L1/PD-1 immune checkpoint is also being targeted by threeapproved PD-1 mAbs, namely pembrolizumab (Keytruda, Merck),nivolumab (Opdivo, BMS), and cemiplimab (Libtayo, RegeneronPharmaceuticals).

In mouse models resistant to single-agent treatment with eitherCD137 agonists or PD-1/L1 blockade, significant synergistic effectshave been observed when antibodies targeting both pathways arecombined. The mechanistic basis for this synergy, even in poorlyimmunogenic mouse tumor models, is that tumor-infiltrating lym-phocytes coexpress PD-1 and CD137, and following combinationtreatment CD8þ T cells can now effectively respond to tumor-associated neoantigens (12). The mechanistic basis for CD137 agonistantibodies alone can be two-fold. Firstly, their ability to induce effectorcell function can result in antitumor activity in some preclinicalmodels. Secondly, their alternative function to deplete Tregs has beendescribed as the most effective mechanism of action for CD137mAbs (13). However, an alternative approach is to direct potentCD137 agonist activity to tumor-reactive T cells while not relying onTreg depletion. Avoiding Treg depletion can be achieved by removingFcgR binding by L234A and L235A (LALA) mutation while retainingagonist activity via an alternative mechanism of cross-linking asdescribed in this article.

As only a fraction of patients respond to monotherapies that blockthe PD-1/PD-L1 pathway (14), and CD137-targeting agonistic mole-cules have yet to demonstrate significant responses in patients with

cancer without toxicity (7, 8), we believe there remains a need todevelop treatments that combine PD-L1 blockade and provokestrong CD137 agonism in safe and efficacious therapies that do notrely on a combination approach. An alternative to combining CD137and PD-L1 monotherapies is the development of a bispecific antibodythat encompasses the two modalities. It is anticipated that such abispecific mAb could deliver superior antitumor efficacy over com-bining monotherapies. There are existing preclinical approaches com-bining CD137 mAb activity with PD-L1 mAb activity into bispecifictherapies. These can be subdivided into two broad range categories,non–IgG- and IgG-like molecules, both of which can be furtherdivided by their binding valency for each target.

Here, we describe a fully human, tetravalent, IgG bispecific antibody(mAb2, FS222) comprising a PD-L1–specific mAb with 5 amino acidinsertions and 7 amino acid substitutions in the CH3 region of the Fcdomain to create two binding sites forming an Fc fragment antigen-binding (Fcab) for CD137 (15). FS222 blocked PD-L1 and activatedCD137þ tumor-reactive T cells in a PD-L1–dependent manner. Itdemonstrated similar potency in primates, and preliminary toxicitystudies in this species showed significant pharmacodynamic (PD)responses and a lack of toxicity. A surrogate mouse cross-reactiveCD137/PD-L1 mAb2 with homologous mechanisms of action toFS222was observed to provide a substantial survival benefit inmultiplemouse tumor models with no toxicity and showed potent in vivo PDchanges related to antitumor immune responses.

Materials and MethodsProduction and characterization of a CD137/PD-L1 mAb2,FS222

The CD137/PD-L1 mAb2 molecule named FS222 consisting ofan IgG1 molecule comprising the human CD137 Fcab was preparedby substituting part of the CH3 domain comprising the AB, CD,and EF loops (15), for the corresponding region of the CH3 domain ofa PD-L1 mAb (E12v2). Fcab generation has been previouslydescribed (16). FS222 incorporates a LALA mutation (leucine toalanine at positions 234 and 235 according to Eu numbering) in theCH2 domain (AA) to reduce Fcg receptor binding (17, 18). FS222 wasexpressed transiently using HEK293 6E (National Research CouncilCanada, Canada) cells. Supernatants were purified on MabSelectSuRe LXProtein-Aprepacked columns using €AKTAxpress instrument(both GE Healthcare Life Sciences). IgG protein content wasquantified by BioLayer Interferometry (BLI) using the Octet QKe

System (Fort�eBio) platform with Protein A quantitation biosensors(Fort�eBio; 18-5013). FS222 was purified by Protein A affinitychromatography using mAb SelectSure columns.

Biophysical characterization of mAb2 by size exclusionchromatography and SDS-PAGE

After purification, size exclusion–high-performance liquid chro-matography (SE-HPLC) was performed on an Agilent 1100 seriesHPLC Value System (Agilent Technologies, Inc.), fitted with a TSKgelSUPERSW3000HPLC4.6mmID� 30 cm column (TosohBioscience,LLC) using 20 mmol/L sodium phosphate and 200 mmol/L sodiumchloride, pH 6.8, as a mobile phase. Quantification of percentagemonomer was performed using ChemStation software (Agilent Tech-nologies, Inc.). Capillary electrophoresis–sodium dodecyl sulfate (CE-SDS) analysis was performed on a 2100 Bioanalyzer Capillary Elec-trophoresis System (Agilent Technologies, Inc.), according to manu-facturer's instructions. For reducing conditions, dithiothreitol (DTT)was added and samples were denatured at 70�C for 5 minutes.

Translational Relevance

We developed an mAb2 bispecific antibody for targeting CD137and PD-L1 in solid tumors which potently activated CD8þ T cellsin vitro only in the presence of PD-L1–expressing cells. Oursurrogate molecule activated intratumoral CD8þ T cells andeffectively controlled tumor growth in syngeneic mouse tumormodels without toxicity. We found that FS222 activates CD4þ andCD8þ T cells in vitro with activity superior to the combination ofmonospecific, mAbs representative of those used in the cliniccurrently, providing evidence that our tetravalent bispecific clinicalcandidate will provide greater benefit to patients than a combina-tion approach against both targets in solid tumors. Considering thebroad expression of PD-L1 in many solid tumors, FS222 mayprovide therapeutic opportunities for patients with cancer whoremain challenging to treat.

Characterization of a CD137/PD-L1 Bispecific Antibody

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Simultaneous binding of FS222 to human PD-L1 and humanCD137 by surface plasmon resonance (SPR)

His-tagged human PD-L1 antigen was coated on to a CM5 chip toapproximately 1,100 RU and was used to immobilize FS222 wheninjected at 100 nmol/L which resulted in approximately 300 RU ofFS222 being captured. Fc-tagged human CD137 antigen was theninjected at a single concentration (100 nmol/L), using the Biacore T200(GE Healthcare Life Sciences), to observe dual binding.

FS222 binding to cell-expressed receptors by flow cytometryDO11.10 T cells overexpressing human CD137 were cultured in

DMEM containing 10% heat-inactivated FBS, 1 mmol/L sodiumpyruvate, and 50 mg/mL puromycin. HEK cells overexpressing humanPD-L1 were cultured in DMEM containing 10% FBS, 100 mg/mLhygromycin B, 15 mg/mL blasticidin, and 1 mg/mL doxycycline. Cellswere resuspended in 40 mL of FS222, CD137/Ctrl(HelD1.3) mAb2,CD137(MOR7480.1) mAb, Ctrl(4420) mAb, or PD-L1(E12v2) mAb(Supplementary Table S1) titrations prepared in DPBS and thenwashed and resuspended in a secondary human IgG detection anti-body (A-21445, Thermo Fisher Scientific) which had been diluted inDPBS. Cells were then resuspended in the viability dye 7-AAD (A1310,Thermo Fisher Scientific) and examined with either a BD FACSCantoII or BD LSRFortessa II (BD Biosciences) before being analyzed usingFlowJo V10 (TreeStar, Inc.).

FS222 binding in human primary T-cell assayHuman primary T-cell isolation and activation

Peripheral blood mononuclear cells (PBMC) were isolated fromleukocyte cones, obtained fromplatelet donors, by Ficoll (GE17-1440-02,Sigma-Aldrich) gradient separation. Pan T cells were isolated from thePBMCs present in the eluent using the Pan T Cell Isolation kit IIaccording to the manufacturer's instructions (130-096-535, MiltenyiBiotec). Pan T cells were incubated overnight at 37�C and 5% CO2

in RPMI supplemented with 10% heat-inactivated FBS, 1 mmol/L2-mercaptoethanol, penicillin (100 U/mL)/streptomycin (100 U/mL),and 1 mmol/L sodium pyruvate. Dynabeads Human T-Activator CD3/CD28 beads (11132D, Thermo Fisher Scientific) were used to activateT cells and upregulate CD137 and PD-L1 surface expression. Beads werewashed from the T cells using a DynaMag-15 Magnet (12301, ThermoFisher Scientific) following the manufacturer's instructions.

Human primary T-cell binding assayStimulated pan human primary T-cell suspensions were resus-

pended in 40 mL FS222, CD137/Ctrl(HelD1.3) mAb2, PD-L1 mAb,CD137(MOR7480.1) mAb, or Ctrl(4420) mAb (SupplementaryTable S1) titrations prepared in DPBS and treated with AF647 goatanti-human IgG (H þ L; 1:500; A-21445, Thermo Fisher Scientific),anti-hCD4 FITC (1:200; 550628, BD Biosciences), and anti-hCD8eF450 (1:200; 48-0087-42, Thermo Fisher Scientific) prepared inDPBS. 7-AAD (A1310, Thermo Fisher Scientific) was used as aviability dye, and samples were then examined with a BD FACSCantoII before being analyzed using FlowJo V10 Prism software.

Humanprimary CD8þT-cell assaywithHEK.hPD-L1 cross-linkingHuman primary CD8þ T cells were isolated from PBMCs obtained

from leucocyte depletion cones using the CD8þ T-cell isolation kit II(130-096-495, Miltenyi Biotec Ltd.) according to the manufacturer'sinstructions. For cell-based cross-linking, HEK293 cells overexpres-sing hPD-L1 (HEK.hPD-L1) orHEKwild-type cells or amixture of thetwo populations were plated on to CD3mAb–coated (8 mg/mL, CloneUCHT1, R&D Systems, MAB100-SP) 96-well flat-bottom plates in

100 mL T-cell culture medium [RPMI medium with 10% FBS, 1Xpenicillin–streptomycin, 1 mmol/L sodium pyruvate, 10 mmol/LHepes (Sigma-Aldrich, H0887), and 50 mmol/L 2-mercaptoethanol(Gibco, M6250)]. CD8þ T cells were added. Cells were treated with atitration of FS222, CD137(20H4.9) mAb, or Ctrl(4420) mAb (Sup-plementary Table S1). Supernatants were assayed with human IL2ELISAReady-SET-Go! kit (88-7025-88, Fisher Scientific) following themanufacturer's instructions. Plates were read at 450 nm using the platereader with the Gen5 Software. The concentration of human IL2(hIL2) was plotted versus the log concentration of antibody, and theresulting curves were fitted using the log (agonist) versus responseequation in GraphPad Prism.

Human primary mixed lymphocyte reactionGeneration of expanded CD4þ T cells

Human primary CD4þ T cells were isolated from leukocyte conesusing a Human CD4þ T Cell Isolation Kit (130-096-533, MiltenyiBiotec Ltd.) according to the manufacturer's instructions. DynabeadsHuman T-Activator CD3/CD28 (11131D, Thermo Fisher Scientific)were used in the presence of 50 IU/mL recombinant human IL2(PeproTech, 200-02) with 3:1 bead to cell ratio to expand cells for7 days. Dynabeads were removed, and CD4þ T cells were restedovernight with reduced 10 IU/mL recombinant human IL2.

Differentiation of iDCsMonocytes were isolated from human PBMCs using a Human Pan

Monocyte Isolation Kit (130-096-537, Miltenyi Biotec Ltd.) followingthe manufacturer's instructions. Monocytes were differentiated toiDCs using Human Mo-DC Differentiation Medium (130-094-812,Miltenyi Biotec Ltd.) following the manufacturer's instructions.

Mixed lymphocyte reaction: Expanded T cells were cultured in AIMV Medium (12055091, Thermo Fisher Scientific) and incubatedovernight. Titrations of FS222, PD-L1(E12v2) mAb and CD137(20H4.9) mAb, PD-L1(E12v2)mAb, CD137/Ctrl(HelD1.3) mAb2 andPD-L1(E12v2) mAb, CD137(20H4.9) mAb, CD137/Ctrl(HelD1.3)mAb2, or Ctrl(4420) mAb (Supplementary Table S1) were used totreat a 1:10 mix of iDC cells and expanded CD4þ T cells in AIM VMedium for 5 days. Supernatants were analyzed for IFNg usingHuman IFN gamma ELISA Ready-SET-Go! Kit (88-7316-86, ThermoFisher Scientific). Plates were read at 450 nm using the plate readerwith theGen5 Software. The concentration of human IFNg was plottedversus the log concentration of antibody, and the resulting curves werefitted using the log (agonist) versus response equation in GraphPadPrism.

Murine primary OT-1 CD8þ T-cell activation assayCD8þ T-cell activation was achieved by antigen stimulation of

genetically modified OT-1 T cells, isolated from C57BL/6 OT-1mice (003831, The Jackson Laboratory) having a T-cell receptorspecific for ovalbumin peptide 257–264, and was determined by therelease of IFNg . OT-1 T cells were incubated with B16-F10 mel-anoma cells, which had previously been cultured in the presence of20 ng/mL IFNg (AF-315-05-100UG, PeproTech) to induce PD-L1expression, and that were then pulsed with 500 nmol/L SIINFEKLpeptide for 1 hour at 37�C, to drive T-cell activation. The efficacy ofsurrogate FS222 was subsequently assessed by ELISA for secretedmIFNg (88-7314-88, Thermo Fisher Scientific) after 3 days. Thisassay was also carried out utilizing MC38.OVA cells that expressovalbumin, in an identical protocol, except for peptide pulsingwhich was not necessary.

Lakins et al.

Clin Cancer Res; 26(15) August 1, 2020 CLINICAL CANCER RESEARCH4156




Surrogate FS222 in vivo characterization in CT26.WT and MC38syngeneic mouse tumor models

The CT26.WT colon carcinoma cell line (ATCC) was initiallyexpanded, stored, and then prescreened by IDEXX Bioresearch forpathogens using the IMPACT I protocol and shown to be pathogen-free. BALB/c female mice (Charles River) aged 8 to 10 weeks andweighing 18 to 22 g each received 1 � 105 CT26.WT cells injectedsubcutaneously in the left flank in 100 mL DMEM serum-free culturemedium.

The MC38 colon carcinoma cell line (ATCC) was initially expand-ed, stored, and then prescreened by IDEXX Bioresearch for pathogensusing the IMPACT I protocol and shown to be pathogen free. C57BL/6femalemice (The Jackson Laboratory) aged 9 to 10weeks andweighing18 to 24 g each received 1� 106MC38 cells injected subcutaneously inthe right flank in 100 mL DMEM serum-free culture medium.

The surrogate FS222 and control antibodies were injected intraper-itoneally into mice at appropriate mg per mouse in DPBS, 100 mmol/Larginine, and 0.05% Tween 80 on days 7, 9, and 11 following tumorinoculation. Tumor volumemeasurements were takenwith callipers todetermine the longest axis and the shortest axis of the tumor, and thefollowing formula was used to calculate the tumor volume:

L X S2� �

=2

Where L ¼ longest axis; S ¼ shortest axis.

PDassessment and receptor occupancy in aCT26.WT syngeneicmouse tumor model

A single-dose PD study was run in the same CT26.WT syngeneictumor model as described above. This study comprised three dosinggroups, receiving either control antibody or surrogate FS222 at one oftwo doses. Samples from tumor tissue and blood were analyzed over 8timepoints (2, 6, 24, 48, 72, 96, 120, and 192hours). Each dosing cohorthad 64 mice (8 mice per timepoint). Each animal received 1 � 105

CT26.WT cells injected subcutaneously in the left flank in 100 mLDMEM. Eleven days following tumor cell inoculation, each mousereceived the test sample via a 100 mL i.v. injection.

Tumor tissue and blood were tested for drug-bound–positive Tcells, T-cell proliferation, and free PD-L1. Blood (100mL)was collectedinto EDTA-coated capillaries by tail vein bleeding and was lysed twicein red blood cell lysis buffer (Thermo Fisher Scientific, 00-4333-57)according to manufacturer's instructions. Tumor tissue was collectedby dissection and was disaggregated to single-cell suspension bystandard mechanical and enzymatic methods. Red blood cells werelysed in red blood cell lysis buffer according to the manufacturer'sinstructions.

Cells were stained with Fixable Viability Dye eFluor 780 (65-0865-14, Thermo Fisher Scientific) following the manufacturer's instruc-tions. Cells were stained with an antibody staining panel (Supple-mentary Table S4, all but Ki67 and FoxP3 antibodies) in the presence ofCD16/CD32 mAb Fc block (1:50, 14-0161-86, Thermo Fisher Scien-tific) and then fixed and permeabilized with the eBioscience Foxp3staining Kit (00-5523-00, Thermo Fisher Scientific) according to themanufacturer's instructions. Cells were stained with Ki67 and Foxp3antibodies in the presence of Fc block and then examined in a BDFortessa flow cytometer. Data were analyzed with FlowJo, Excel, andGraphPad Prism.

Preliminary toxicology study in cynomolgus monkeysA preliminary dose-range finding study was conducted to evaluate

the pharmacokinetic (PK)/PD response to and tolerability of FS222 incynomolgus monkeys. The study was performed using Mauritian

cynomolgus macaques at Charles River Laboratories in line withInstitutional Animal Care and Use Committee guidelines and inaccordance with the “Guide for the Care and Use of LaboratoryAnimals” (1996) by the Institute of Laboratory Animals ResearchCommission on Life Sciences (National Research Council, Washing-ton, DC).

Briefly, FS222 was administered to cynomolgus monkeys (1/sex/group) via intravenous infusion at 3 mg/kg as a single dose on day 1 orat 0.1, 1, 10, or 30mg/kg as repeat doses on days 1, 8, 15, and 22. For the3 mg/kg dose group, serial serum samples were collected for PKassessment on day 1 (predose, 0.083, 0.5, 2, 6, and 12 hours postdose)and days 2, 3, 4, 6, 8, 11, 15, 22, 29, 36, and 43. For the remaininggroups, PK serum samples were collected on day 1 (predose, 0.083, 0.5,2, 6, and 12 hours postdose) and days 2, 3, 7, 8 (predose and 0.083 hourspostdose); day 15 (predose, 0.083, 0.5, 2, 6, and 12 hours postdose);days 16, 17, 21, and 22 (predose and 0.083 hours postdose); and day 25.Serum levels of FS222 were measured using a qualified Gyros-basedimmunoassay developed in-house to specifically detect free drug(human biotinylated PD-L1 was used as a capture reagent and humanAlexa Fluor labeled CD137 as a detection reagent).

For the evaluation of tolerability, standard toxicology parameterssuch as body weight, food consumption, clinical observations, hema-tology, and blood chemistry were evaluated over the duration of thestudy. The study was terminated 25 days after administration of firstdose in repeat dose animals and 43 days after administration of singledose (PK group of animals).

For evaluation of the PD response to FS222, immunophenotyping ofperipheral blood was performed to assess peripheral lymphocytepopulations (monocytes, T cells, B cells, and NK cells) as well as theinduction of proliferation and activation of central memory andeffector memory CD4þ and CD8þ T-cell subpopulations. Serial bloodsamples (on EDTA) were collected prior to first dosing of FS222(predose) and on the indicated study days over the course of the study;stained with antibodies against CD45, CD3, CD4, CD8, CD16, CD28,CD25, FoxP3, CD95, CD69, and Ki67 (Supplementary Table S3); andanalyzed using a FACSCanto II flow cytometer (BD Biosciences) andFACSDiva (BD Biosciences), Excel, and GraphPad Prism software.

ResultsCreation of a CD137/PD-L1 bispecific antibody (mAb2, FS222)

FS222 was created by incorporating into a proprietary PD-L1 mAb,an engineered IgG1 Fc region termed Fcab (Fc-region with antigenbinding), where high-affinity antigen binding for CD137 was intro-duced in the C-terminal region (Fig. 1A). Binding to FcgR wasremoved by the introduction of L234A and L235A (LALA) mutations(Supplementary Fig. S1A), while binding to human FcRn was main-tained (Supplementary Fig. S1B). The mAb2 is tetravalent, with twobinding sites for PD-L1 (one in each Fab region) and two binding sitesfor CD137 (one in each CH3, due to the homodimeric nature of the Fcregion), and maintains the IgG1 structure.

Binding affinities of FS222 are equivalent to individualcomponent antibodies

The simultaneous binding of FS222 to human CD137 and humanPD-L1 was tested via SPR and then individually to cells expressingeither human CD137 or human PD-L1.

FS222 simultaneously bound to both targets as observed by SPR(Fig. 1B) with affinities of 0.66 nmol/L to dimeric human CD137 and0.19 nmol/L to monomeric human PD-L1 (Supplementary Fig. S1C–S1E). The cell-binding assays demonstrated that FS222 bound human






CD137 to an equivalent level as the component CD137 Fcab (CD137/Ctrl(HelD1.3) mAb2) alone (EC50 6.2 nmol/L; Fig. 1C). FS222also bound human PD-L1 to an equivalent level as the componentFab (PD-L1(E12v2) mAb) alone (EC50 3.7 nmol/L; Fig. 1D). PD-L1(E12v2) mAb was found to be just as effective as the PD-L1(S70) mAb(YW243.55.S70) at blocking PD-L1 binding to PD-1 as investigated in

a DO11.10 human PD-L1 T-cell activation assay (data not shown). Asshown in Fig. 1E, FS222 bound to activated CD4þ and CD8þ primaryT cells with an EC50 of 0.8 and 0.9 nmol/L, respectively. This wasequivalent to the PD-L1(E12v2) mAb–binding characteristics but notequivalent to the CD137/Ctrl(HelD1.3) mAb2, showing that this cellbinding was driven primarily through PD-L1 rather than CD137. The

Figure 1.

mAb2 structure and concurrent high-affinity binding of CD137/PD-L1mAb2 equivalent to individual component antibodies.A,Representation of the bispecific CD137/PD-L1mAb2, FS222, on a human IgG1 backbonewith FcgRbinding removedby L234Aand L235A (LALA)mutations highlighted in green. The PD-L1 complementarity-determining regions (CDR) of heavy and light chains are highlighted in orange. The CD137 CH3 domain AB- and EF-binding loops are highlighted in cyan. B, FS222simultaneous binding to both human PD-L1 and human CD137 as determined by SPR. C, FS222 binding to DO11.10 T cells expressing human CD137 as determinedby flow cytometry. D, FS222 binding to HEK cells expressing human PD-L1 as determined by flow cytometry. E, FS222 binding to in vitro–activated human primaryCD4þ and CD8þ T cells as determined by flow cytometry.

Lakins et al.





positive control CD137 mAb, CD137(MOR7480.1) mAb, bound toactivated human primary T cells as expected, whereas the CD137/Ctrl(HelD1.3) mAb2 had minimal binding to the same cells. There-fore, FS222 bound to PD-L1 with high affinity and, through design,bound CD137 with high avidity, which are two features of themolecule critical for the cross-linking–dependent activity asdescribed below.

T-cell activation through CD137 agonism was dependent uponcross-linking via PD-L1

Subsequent investigations were aimed to dissect the nature ofFS222-mediated CD137 agonism. To demonstrate that FS222 can becross-linked to mediate human CD137 signaling only in the presenceof cells expressing human PD-L1, human primary CD8þ T cellsstimulated by plate-bound CD3 mAb were cocultured with wild-type HEK 293 cells, HEK 293 cells engineered to overexpress humanPD-L1, or mixtures of the two cell lines in different proportions. This

allowed the investigation of varying ratios of human PD-L1–expres-sing cells to wild-type cells to model the likely heterogeneity ofexpression present within different human tumors.

FS222 showed maximum activity, measured by human IL2 release,from activated human primary CD8þ T cells, when 100% of HEK 293cells expressed PD-L1 (Fig. 2A). The maximum IL2 release (Emax)reduced in proportion to the reduction in the percentage of cellsexpressing human PD-L1 present; however, the EC50 value remainedbroadly the same at 0.05 nmol/L (Fig. 2A).

FS222 elicited superior activity in a CD4þ mixed lymphocytereaction by FS222 compared with a combination ofmonospecific antibodies

The activity of FS222 was tested in a mixed lymphocyte reaction(MLR) which utilizes human primary CD4þ T cells and immaturemonocyte-derived DCs (iDC) expressing endogenous levels of bothtargets. The PD-L1–specific antibody [PD-L1(E12v2) mAb] showed

Figure 2.

CD137 agonism via FS222 is dependent upon cross-linking via PD-L1 in a human primary T-cell assay and has activity superior to mAb combinations in an MLR.A, FS222 activity in a human primary CD8þ T-cell activation assay with varying ratios of HEK cells that are positive for PD-L1 to HEK cells that are negative for PD-L1.Significance determined by extra sum-of-squares F test. �� , P < 0.001. B, FS222 activity in MLR against monospecific component parts that make up the completeFS222 mAb2 either alone or in combination with each other. Significance determined by extra sum-of-squares F test. �� , P < 0.001.






potent activity in the MLR assay (EC50 0.08 nmol/L). The humanCD137 mAb, CD137(20H4.9) mAb, even when cross-linked withhCH2mAb, did not elicit activity. This suggested that CD137 signalingalone is ineffective in this assay. However, a combination of PD-L1(E12v2) and CD137(20H4.9) mAbs cross-linked with hCH2 mAbshowed potent activity (EC50 0.14 nmol/L) with a higher maximumIFNg release compared with PD-L1(E12v2) mAb alone, whichindicated a synergistic effect of the two mAbs (Fig. 2B). FS222showed similarly potent activity to the combination of the twoseparate monospecific antibodies with an EC50 of 0.07 nmol/L andcomparable maximum IFNg release level (Fig. 2B). FS222 was alsotested against each component part of the mAb2 that makes upFS222, the CD137/Ctrl(HelD1.3) mAb2, and the PD-L1(E12v2)mAb, alone and in combination. CD137(HelD1.3) mAb2 showedno activity in this assay, indicating that the CD137 Fcab componenthad an inability to activate T cells in an MLR. There was noadditional effect on activation by treating with a CD137/Ctrl(HelD1.3) mAb2 plus PD-L1(E12v2) mAb combination above thatalready seen with PD-L1(E12v2) mAb alone. FS222 showed asimilar potency to the combination of its component partsCD137/Ctrl(HelD1.3) mAb2 and PD-L1(E12v2) mAb (EC50 of0.07 and 0.06 nmol/L, respectively) with superior efficacy as indi-cated by higher IFNg production (Emax; Fig. 2B).

Surrogate FS222, a mouse CD137/PD-L1 tetravalent bispecificantibody, demonstrated potent in vitro activity greater than anmAb combination approach

FS222 did not bind to mouse CD137 (data not shown) so it was notpossible to evaluate FS222 in mouse syngeneic tumor model systemsin vivo. Therefore, a mouse surrogate of FS222 was created using anFcab targeting mouse CD137 selected using yeast display. The Fcabagainst mouse CD137 was selected based on affinity measurementsand cross-link–dependent activation of CD137 and was tested insimilar mouse systems to those used to determine the function ofFS222 in human systems. Surrogate FS222 was tested for cell bindingsimilar to FS222 and was found to have an EC50 of 2.7 and 2.6 nmol/Lto cells engineered to overexpress mouse CD137 and mouse PD-L1,respectively.

The functional activity of surrogate FS222 was determined in aprimary assay where CD137 and PD-L1 are endogenouslyexpressed on activated T cells and B16-F10 tumor cells, respec-tively. B16-F10 mouse melanoma cells that had previously beenpulsed with OVA peptide (SIINFEKL) were cocultured with CD8þ

antigen–specific OT-1 T cells. CD8þ T-cell activation (IFNgrelease) was increased after treatment with either mCD137(Lob12.3) mAb, PD-L1(S70) mAb, or a combination approach.However, the greatest potency was seen upon treatment with thesurrogate FS222 (EC50 0.003 nmol/L; Fig. 3A). The same result wasachieved using a similar assay described above but utilizing MC38cells expressing ovalbumin as the source of both OVA peptide andPD-L1 (Supplementary Fig. S2A). As with FS222, no activation wasdetected in the absence of PD-L1 cross-linking which indicated asimilar mode of action between the surrogate and human-specificmolecules.

Surrogate FS222 controlled tumor growth in syngeneic mousetumor models

To evaluate the antitumor effects of surrogate FS222 in vivo,CT26.WT orMC38 tumor cell lines were injected subcutaneously intothe flank of BALB/c and C57BL/6 mice, respectively. In the CT26.WTtumor model, mice were injected intraperitoneally on days 7, 9, and

11 after tumor implantation with approximately 10 mg/kg surrogateFS222, PD-L1(S70) mAb, mCD137(Lob12.3) mAb, or Ctrl(HelD.13)isotype control mAb. Surrogate FS222 was shown to substantiallyreduce tumor growth (Fig. 3B), and 42% of mice remained tumor freeup to 68 days after treatment (Fig. 3C and Table 1). In contrast,treatment with PD-L1(S70) mAb or mCD137(Lob12.3) mAb failed toshow significant survival advantage (Fig. 3C and Table 1; Supple-mentary Tables S8 and S9).

In the MC38 tumor model, surrogate FS222 was able to eradicateall tumors at a lower dose of 1 mg/kg. Mice were injected intra-peritoneally 7, 9, and 11 days after tumor cell inoculation with adose of approximately 1 mg/kg of either surrogate FS222, PD-L1(S70) mAb, mCD137(Lob12.3) mAb, a combination of both, or Ctrl(4420) isotype control mAb. Surrogate FS222–treated mice showedfull tumor regression in all mice which remained tumor free untilday 49 when the study was ended. In contrast, single and combinedtreatment of PD-L1(S70) mAb and mCD137(Lob12.3) mAbresulted in durable tumor regression in only a fraction (4/12 orless) of animals (Fig. 3D and E, Table 1; Supplementary Tables S10and S11).

Surrogate FS222 produced dose-dependent survival benefit inCT26.WT syngeneic mouse tumor model

In the syngeneic CT26.WT tumor model, we assessed surrogateFS222 dose-dependency in vivo and showed dose-dependent sur-vival benefit between doses of approximately 0.1 mg/kg and approx-imately 10 mg/kg. To evaluate dose-dependent efficacy, surrogateFS222 dose levels equivalent to approximately 0.1, 0.3, 1, and 10mg/kg,and Ctrl(4420) mAb isotype control dosed at 10 mg/kg, wereadministered intraperitoneally using the same study design des-cribed above. Surrogate FS222 showed an antitumor efficacy from0.3 mg/kg and durable tumor regression in 21% of treated animalsat 1 mg/kg and 40% at 10 mg/kg (Supplementary Fig. S2B). Usingthe log-rank (Mantel–Cox) test, a significant survival dose depen-dency was shown for surrogate FS222 at 0.3 mg/kg up to 1 mg/kgcompared with Ctrl(4420) mAb treatment, but no significantbenefit raising from 1 mg/kg to 10 mg/kg surrogate FS222 despitethe median survival extending from 29 to 39 days, respectively(Fig. 4 and Table 2).

Surrogate FS222–regulated dose-dependent PD changes intumor and blood

CT26.WT tumor–bearing mice were treated with a single intrave-nous dose of surrogate FS222 (�1mg/kg and�10mg/kg) 11 days aftersubcutaneous inoculation of CT26.WT. Tumor tissue and bloodwere tested for T-cell–bound surrogate FS222, T-cell proliferation,and PD-L1 receptor occupancy over time (between 2 and 192 hours).Total CD137 receptor expression was also assessed.

A high percentage of peripheral and tumor-resident T cells showedbound surrogate FS222 as early as 2 hours after intravenous admin-istration (Fig. 5A). There was a dose-dependent correlation in thelongevity of binding, with surrogate FS222 no longer detected after96 hours on T cells isolated from mice administered with 1 mg/kg,whereas surrogate FS222 was still detected between 120 and 192 hoursafter administration of 10 mg/kg.

Ki67 expression was used as a marker for T-cell proliferation onCD4þ and CD8þ T cells. T cells isolated from tumor tissue exhibited ahigher frequency of Ki67þ T cells as expected in an inflammatorytumor microenvironment. At both dose levels, surrogate FS222resulted in increases in the frequency of Ki67þ peripheral bloodT cells when compared with Ctrl(4420) mAb isotype control,

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Figure 3.

In vitro characterization of surrogate FS222 and in vivo efficacy and survival in two syngeneic mouse tumor models. A, Surrogate FS222 activity in an OT-1 CD8þ

mouse T-cell activity assaywith cell-based cross-linking provided by B16-F10 tumor cells expressingmouse PD-L1. Significance determined by extra sum-of-squaresF test. �� , P < 0.001. B, Individual tumor growth spaghetti plots for CT26.WT tumor–bearing mice treated on days 7, 9, and 11 after tumor inoculation with 10 mg/kgCtrl(HelD.13), PD-L1(S70), CD137(Lob12.3), or surrogate FS222. C, Survival data for CT26.WT tumor–bearing mice treated with 10 mg/kg Ctrl(HelD.13), PD-L1(S70),CD137(Lob12.3), or surrogate FS222. D, Survival data for MC38 tumor–bearing mice treated with 1 mg/kg Ctrl(4420), PD-L1(S70), CD137(Lob12.3), PD-L1(S70) þCD137(Lob12.3), or surrogate FS222. E, Individual tumor growth spider plots for MC38 tumor–bearing mice treated on days 7, 9, and 11 after tumor inoculation with1 mg/kg Ctrl(4420), PD-L1(S70), CD137(Lob12.3), PD-L1(S70) þ CD137(Lob12.3), or surrogate FS222.

Table 1. Summary table of tumor-free animals by end of study.

Compound IgG control PD-L1(S70) CD137(Lob12.3) PD-L1(S70) þ CD137(Lob12.3) Surrogate FS222

CT26 tumor–free animals Number 0/12 0/12 1/12 N/A 5/12Percent 0% 0% 8% N/A 42%

MC38 tumor–free animals Number 0/12 2/12 2/12 4/12 12/12Percent 0% 17% 17% 33% 100%






indicating a PD response (Fig. 5B). The effect appeared stronger forCD8þ T cells which is in line with CD8þ T cells expressing higherCD137 levels than CD4þ T cells.

A proportion of cells isolated from the blood and tissue ofmice dosed with Ctrl(4420) mAb isotype control were saturated with100 nmol/L surrogate FS222 ex vivo which acted as control for 100%PD-L1 receptor engagement and was confirmed by fully blockingbinding with a competing mouse PD-L1 mAb (clone 10F.9G2).Cells isolated from tumor tissue and the blood of mice treated withsurrogate FS222 at the 10 mg/kg dose showed near-complete PD-L1blockade for 8 days, as shown in Fig. 5C represented by 100% PD-L1receptor occupancy. At 1 mg/kg, surrogate FS222 achieved near-complete PD-L1 receptor occupancy for approximately 72 hours onperipheral T cells. PD-L1 receptor engagement on T cells present inblood showed an accelerated decrease comparedwith T cells present inthe tumor tissue which retained a greater PD-L1 receptor occupancywith greater longevity.

Serum cytokines were analyzed by multiplex electrochemilumines-cent immunoassay (Meso Scale Discovery, MSD) to assess cytokineproduction after surrogate FS222 treatment in the same model.Surrogate FS222, when dosed at 10mg/kg, resulted in increased serumproinflammatory cytokines IFNg , TNFa, and IL6. The immunosup-pressive cytokine IL10 likewise shows increase in serum after dosing,presumably to counter the proinflammatory response. This effect wasdose-dependent, and serum cytokine levels remained similar in micetreated with 1 mg/kg surrogate FS222 compared with Ctrl(4420)mAb–treated mice (Fig. 5D). Antitumor activity observed via tumorgrowth inhibition, tracked by measuring excised tumors at indicated

timepoints, still showed highly significant tumor growth inhibition inmice treated with a single dose of 1 mg/kg surrogate FS222 (Supple-mentary Fig. S3A). This indicated localized antitumor cytotoxicactivity without systemic exposure to inflammatory cytokines.

Surrogate FS222 immunopharmacology did not result inhepatotoxicity

In 2008, clinical trials investigating urelumab in solid tumors werehalted due to severe treatment-related immune events which mani-fested in the liver as severe hepatotoxicity resulting in patientdeaths (7). More recently, urelumab has been administered at farreduced dose levels to mitigate this toxicity. Preclinical mechanisticwork was undertaken in mice wherein animals dosed with CD137agonist mAbs showed similar hepatotoxicity. These studies showed arequirement for T cells and CD137 expression in the resultanthepatotoxicity (9, 19). Therefore, these animal models have sometranslational relevance for predicting the risk of hepatotoxicity in theclinic in human patients following administration of other CD137agonists such as FS222. Mice from our CT26.WT syngeneic tumorstudies showed no overt signs of toxicity following repeated dosingwith surrogate FS222 andmaintained normal bodyweight throughout.To determine whether immune activation and antitumor activityobserved as a result of treatment with 1 mg/kg surrogate FS222correlated with hepatotoxicity, liver samples were taken at necropsyfor histologic assessment. Surrogate FS222–treated and control micewere necropsied 4, 7, and 14 days after the last administration wherebyliver samples were excised and examined.

Each liver section was scored for pathology, and the frequencies ofmice showing zero, minimal, slight, and moderate effects within eachgroup are shown in Supplementary Table S2 (0 ¼ zero, 1 ¼ minimal,2 ¼ slight, 3 ¼ moderate). Surrogate FS222–treated animals showedminimal liver pathology (Supplementary Fig. S3B and SupplementaryTable S2). Specifically, the livers showed minimal to slight hepatocel-lular necrosis with mixed lymphocyte infiltrate in the parenchyma,minimal to slight mixed inflammatory cells in periportal tracts, nodegenerative hepatocytes, and minimal to slight increased mitoses(Supplementary Table S2). These findings are not deemed to representadverse hepatotoxicity, as observed with other examples of CD137agonist mAbs.

In a similar liver pharmacologymouse study, which included dosingCT26.WT tumor–bearing mice with 10 mg/kg of a CD137 targetingmAb clone 3H3 known to induce liver toxicity (20), both surrogateFS222–treated (also at 10 mg/kg) and CD137(3H3) mAb–treatedanimals showed CD3þ T-cell liver infiltration from day 13 onwardto similarly high levels above control mice (Fig. 5E). However,activated CD8þ T cells remained at significantly higher levels withgreater longevity in CD137(3H3) mAb–treated animals comparedwith animals treated with surrogate FS222 (Fig. 5F). The CD8þ T-cellresponse for both CD137(3H3) mAb and surrogate FS222 peaks atapproximately 95% positive for Ki67 across days 8 and 13 after firstdose.However, for surrogate FS222, this returned to baseline by day 16,whereas for CD137(3H3) mAb, this did not happen by the end of thestudy at day 28 after first dose (Fig. 5F). This indicated a difference inthe mode of action of these two CD137 targeting agents; CD8þ T cellsactivated by surrogate FS222 proliferated less so and for a shorterperiod compared with CD8þ T cells activated by CD137(3H3) mAb.CD137(3H3) mAb has been shown to lead to hepatic degenerationpreviously (20). CD4þ FoxP3þ Tregs were present at higher levels insurrogate FS222–treated livers compared with CD137(3H3) mAb–treated livers at day 8 and day 13 after first dose (SupplementaryFig. S3C). Approximately 30% of CD4þT cells were positive for FoxP3

Figure 4.

Surrogate FS222 was tested for efficacy and survival in a CT26.WT syngeneicmouse tumor model dose-range finding study. Kaplan–Meier survival plot ofdose-range finding study in CT26.WT of surrogate FS222 dosing in the range 0.1to 10mg/kg showing significant survival benefit of increasing doses of surrogateFS222 above 0.1 mg/kg compared with IgG control.

Table 2. Increased dose of surrogate FS222 correlated withincreased survival.

Surrogate FS222

Mediansurvival(days)

P values Log-rank(group-wise comparisonwith lower dosed group)

10 mg/kg 39 0.21 mg/kg 29.5 0.020.3 mg/kg 24 0.0070.1 mg/kg 21 0.60 mg/kg (IgG Control) 21

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Figure 5.

Surrogate FS222 was tested for PD and liver pharmacology in a CT26.WT syngeneic mouse tumor model.A, The percentage of CD8þ and CD4þ T-cell populations inthe tumor or blood determined by flow cytometry to be positive for bound surrogate FS222 in a PD study for CT26.WT tumor–bearing mice upon treatment with 1dose of surrogate FS222.B, The frequency of Ki67þCD8þ T cells or Ki67þ CD4þ T cells in the tumor or blood as determined by flow cytometry similarly toA.C, PD-L1receptor occupancy of CD8þ and CD4þ T cells in the tumor or blood as determined by normalizing to a negative control [cells isolated from Ctrl(4420)mAb–treatedmice set to 0% receptor occupancy at each time point, black circles] and a positive control [cells isolated from Ctrl(4420) mAb–treated mice which were thensaturated with surrogate FS222 set to 100% receptor occupancy at each time point, black triangles]. D, Serum cytokine levels as determined by MSD analysis,significance determined by two-way ANOVA and shown for surrogate FS222 10 mg/kg group vs. Ctrl(4420) mAb group. � , P < 0.05; �� , P < 0.001. E, CD3þ T cells(as apercentageof total CD45þ immune cells) in the liver of treatedmice as determined byflowcytometry.F,Proliferating CD8þT cells present in the liver, usingKi67expression as a marker of proliferation, as determined by flow cytometry.






Figure 6.

Non-GLP PK/PD toxicity study of single and repeat dosing of FS222 in cynomolgus monkeys. A, PK profile of FS222 in cynomolgus monkeys (SD, single dose).B, Kinetic changes in serum soluble PD-L1 (sPD-L1) levels after repeat dosing with FS222. C, Kinetic changes in peripheral NK-cell frequency expressing Ki67 afterrepeat dosingwith FS222.D,Kinetic changes in peripheral CD4þ central memory cell frequency expressing Ki67 after repeat dosingwith FS222. E,Kinetic changes inperipheral CD8þ central memory cell frequency after repeat dosing with FS222.

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after surrogate FS222 treatment, whereas the level after CD137(3H3)mAb remained nearer baseline at 10% (Supplementary Fig. S3C). Thisindicated a potentially more immunosuppressive environment whichcould dampen the damaging effect of activated CD8þ T-cell accumu-lation, shown to otherwise lead to hepatocyte death (21).

Due to the potential preclinical limitations of surrogate moleculesandmousemodels for predicting CD137-induced liver toxicity, we rana non-GLP PK/PD toxicity study of single and repeat dosing of FS222in cynomolgus monkeys.

FS222 elicited immune activation with no liver toxicity in apreliminary toxicity study in cynomolgus monkeys

FS222was shown to be fully cross-reactive in cell binding assays andprimary immune cell functional assays using PBMCs from human orcynomolgus blood (Supplementary Fig. S4A–S4D). Therefore, the PKbehavior of FS222 was characterized in cynomolgus monkeys afterintravenous administration of FS222 in a non-GLP dose-range findingstudy (Fig. 6A). FS222 displayed a dose proportional increase in Cmax

and AUC (0–168 hours; Supplementary Table S5) and linear plasmaclearance (at doses ≥ 1 mg/kg; Fig. 6A). FS222 had a mean terminalhalf-life of approximately 148 hours, which is in line with humanantibodies in monkeys targeting PD-L1 (atezolizumab BLA #761034Pharmacology Review). In general, FS222 PK followed a linear doseresponse (at dose levels ≥ 1 mg/kg) and clearance rates (CLp), and thevolumes of distribution were similar between animals (SupplementaryTables S6 and S7). FS222 was generally well tolerated up to 30 mg/kgdosed weekly as determined by clinical chemistry and histopathologyresults (Table 3).

As shown in Fig. 6B, the levels of serum soluble PD-L1 (sPD-L1)were quantified as a measure of direct target engagement and indic-ative of downstream cell activation (22). Increased serum sPD-L1levels were observed in all animals on day 1, with an apparent peak at168 hours after end of infusion, following which the levels declined inline with the decline in the systemic levels of FS222. Repeat admin-istration of FS222 resulted in prolonged increase in serum sPD-L1 inanimals that were shown to have no or low levels of antidrug anti-bodies. Consistent with the findings of the study to assess the PDresponse of the surrogate FS222 in a syngeneic mouse tumor modeldescribed previously, a drug-related increase in cell proliferation andactivation was also observed in NK cells (Fig. 6C) and CD4þ andCD8þ central memory T cells (Fig. 6D and E). In many animals, Ki67expression reached plateau at day 11, remained high at day 15, anddeceased progressively to reach baseline expression between days 18and 22 with a maximum response being observed between 3 and10 mg/kg. Amoderate but transient increase in the relative percentageand absolute counts of CD4þ FoxP3þ Tregs was also seen (Supple-mentary Fig. S4E).

DiscussionFS222, a CD137/PD-L1 tetravalent bispecific antibody, exhibited

potent in vitroCD137-mediated T-cell activation upon engagement ofPD-L1. No cross-reactivity was observed tomouse CD137; therefore, amouse surrogate molecule was developed. Surrogate FS222 outper-formedCD137 and PD-L1monospecificmAbs asmonotherapies or incombination in multiple syngeneic mouse tumor models.

No liver pharmacology or toxicity, previously reported with otherCD137 agonist mAbs, was observed with FS222 or the mouse surro-gate. Contrasting observations of liver toxicity in the clinic, withCD137 mAbs urelumab and utomilumab, suggest that targeting ofCD137 is not an intrinsically toxic pathway for therapy, but theway it istargeted is crucial. Urelumab is a potent fully human IgG4 antibodybut causes dose-dependent and on-target liver toxicity, whereasutomilumab demonstrates no dose-limiting toxicity but weaker poten-cy on a human IgG2 backbone. FS222, although a human IgG1, had noFc-mediated effector function, and its potent CD137 activity wasdependent upon PD-L1 expression. This resulted in a highly activemolecule as seen in vitro and in vivo in multiple syngeneic tumormodels, with no liver toxicity. Furthermore, the results from ourpreliminary toxicology study indicated that FS222, which is cross-reactive with cynomolgus monkeys and has the same in vitro potencyin this species and human, had potent in vivo pharmacologic activity inthe cynomolgus monkey and is well tolerated up to 30 mg/kg.

Despite being able to bind cell-expressed human CD137, the Fcabcomponent of FS222 was unable to cluster and activate CD137 in theabsence of PD-L1–mediated cross-linking, a significant safety featureof the molecule. Coupled with directing CD137 activity to areas ofPD-L1 expression, for example tumor microenvironments, FS222 isdesigned to overcome the adverse side effects associated with CD137agonists currently in the clinic. This is further strengthened byreducing FcgR binding and allows FS222 to not rely on FcgR-expressing cells to provide the cross-linking necessary for CD137clustering in current mAb therapies. The combination approachadopted for in vitro experiments of using two separate monospecificantibodies relied not only on releasing the PD-1/PD-L1 blockade viaone antibody, but also on maximum cross-linking of CD137(20H4.9)mAb by a hCH2-specific mAb. For this combination in the clinic, thenatural cross-linking mechanism would be via FcgR cross-linking viathe Fc region of IgG4-based urelumab (20H4.9). Not only are FcgR-expressing cells found throughout the body, therefore bringing anoth-er significant safety concern for aberrant IgG cross-linking and CD137agonism, they are also varied in prevalence with diverse FcgR expres-sion levels, making them an unreliable source of cross-linking–dependent activation within a tumor (23). Therefore, FS222 mitigatedhigh systemic toxicity and variable antitumor activity by not relying onFcgR cross-linking for potent site-specific activity. FS222 also did notrely on Fc-mediated cell killing as amechanism of action on account ofsignificantly reduced FcgR binding. This resulted in highly potentactivity which did not come at the loss of important CD137- or PD-L1–expressing immune cells that can then potentiate the cell-mediatedtumor killing action.

FS222 showed potent activity in human primary T-cell assays, butonly when PD-L1–expressing cells were present. There was alsono activity when the CD137 Fcab was paired with an irrelevant,non–PD-L1 binding Fab domain, HelD1.3. Surrogate FS222 hadcomparable in vitro activity to FS222, therefore justifying its use insyngeneic mouse tumor models. We believe the tumor control activityshown by surrogate FS222 addresses one of the hurdles of treating aPD-L1–insensitive tumor, or one that has become refractory to PD-L1

Table 3. Changes in clinical chemistry parameters relating to liverfunction of cynomolgus monkeys in FS222 repeat dose phase.

Lower limitof normalrange FS222

Upper limitof normalrange

AST (U/L) 20 23–69 94ALT (U/L) 21 19–111 112ALP (U/L) 140 485–1,310 1,350TBIL (mg/dL) 0.06 0.07–0.38 0.43

Abbreviations: ALP, alkaline phosphatase; ALT, alanine aminotransferase; AST,aspartate aminotransferase; TBIL, total bilirubin.






therapy. It does this by harnessing PD-L1 target expression in analternative way to exert direct cytotoxic T-cell activation throughCD137 engagement and clustering. The highly immunogenicMC38 tumor model demonstrated insensitivity to PD-L1 mAbtreatment and Fc-disabled CD137 mAb treatment as monothera-pies at the dose levels employed in this in vivo study. However,despite also being Fc-disabled, treatment with surrogate FS222resulted in complete tumor eradication and 100% animal survival.In this model, PD-L1 expression, presumably in the tumor micro-environment, provided a setting in which surrogate FS222 can exertsuperior activity and efficacy to either monotherapy. This is clearlythrough PD-L1–dependent cross-linking of FS222 and CD137receptor clustering on T cells resulting ultimately in enhancedtumor-specific CD8þ T-cell cytotoxic activity. Surrogate FS222 andtherefore FS222 could be bridging a PD-L1–expressing tumor celland tumor-infiltrating T cell, localizing T-cell cytotoxic activationto the tumor cell/T-cell interface. The same is true for the significantactivity and superiority over monotherapy of surrogate FS222 in theless immunogenic CT26.WT model (24), which in our hands isalso insensitive to PD-L1 mAb treatment.

The PD changes after treatment with surrogate FS222 in aCT26.WT tumor–bearing mouse model were investigated afteradministration of a single high (10 mg/kg) and a single low (1 mg/kg)dose. Strikingly, T cells with bound drug were present in the tumor at2 hours after administration for both dose levels. These T cells hadprolonged PD-L1 occupancy specifically in the tumor, the longevity,but notmagnitude, of whichwas correlatedwith dose. By the end of thestudy (8 days after drug administration), intratumoral PD-L1 occu-pancy was still approximately 80% on T cells at 10 mg/kg, whereasperipheral PD-L1 occupancy on T cells had decreased substantially.This highlights the potential of surrogate FS222 to locate to the tumormicroenvironment, in preference to remaining in the periphery.Evidence of cytokine production as a consequence of surrogate FS222treatmentwas also observed in the serumofmice treatedwith 10mg/kg,whereas this was less pronounced with the lower dose level perhapsindicating efficacy without systemic cytokine exposure.

Given the relevance of preclinical studies inmice for risk assessmentof severe hepatotoxicity in human patients treated with CD137 agonistagents, the lack of hepatotoxicity inmice in these studies indicates thata mAb2 agonizing CD137 via PD-L1–mediated cross-linking has asignificantly reduced risk of inducing hepatotoxicity in humanpatientstreated at therapeutic doses. FS222 had a mean terminal half-life ofapproximately 6 days in cynomolgus monkeys, in line with PD-L1–targeting antibodies such as atezolizumab and followed a linear doseresponse at dose levels≥ 1mg/kg. FS222was generally well tolerated upto 30 mg/kg dosed weekly. With comparable potency between thecynomolgus and human immune systems, we believe these findingswill translate successfully to a human setting. Both the surrogate

molecule in mouse and FS222 in cynomolgus monkeys caused adrug-related increase in T-cell proliferation and activation as mea-sured by Ki67 expression. This would indicate that the PD responsesseen in our mouse models translate to the effect of FS222 on cyno-molgus monkey T cells.

In summary, we have developed FS222, a CD137/PD-L1 tetravalentbispecific antibody with a novel mode of action, and potentiallyimproved therapeutic index for the treatment of human cancer. FS222did not cause evident toxicity in cynomolgus monkeys upon repeateddosing which we believe further encourages clinical developmenttargeted at tumors where a significant unmet medical need exists inimmunotherapy. Checkpoint inhibitors are failing or only providingmodest clinical benefit in many tumor settings, and for many of those,we feel there is a mechanistic rationale for improvement in clinicaloutcomes with FS222.

Disclosure of Potential Conflicts of InterestAll authors are current or former employees of F-star Therapeutics Ltd.

Authors’ ContributionsConception anddesign:M.A. Lakins, A.Koers, J.Munoz-Olaya, S. Batey, D.Gliddon,M. Tuna, N. BrewisDevelopment of methodology: M.A. Lakins, A. Koers, R. Giambalvo, M. TunaAcquisition of data (provided animals, acquired and managed patients, providedfacilities, etc.): M.A. Lakins, A. Koers, R. Giambalvo, R. Hughes, E. Goodman,F. WollertonAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): M.A. Lakins, A. Koers, R. Giambalvo, J. Munoz-Olaya,R. Hughes, E. Goodman, S. Marshall, F. Wollerton, S. Batey, D. GliddonWriting, review, and/or revision of the manuscript:M.A. Lakins, J. Munoz-Olaya,R. Hughes, E. Goodman, S. Marshall, F. Wollerton, S. Batey, D. Gliddon, M. Tuna,N. BrewisAdministrative, technical, or material support (i.e., reporting or organizing data,constructing databases): M.A. Lakins, S. MarshallStudy supervision: M.A. Lakins, M. Tuna, N. BrewisOther (Coordinated cynomolgus monkey DRF study and associated data analysisand interpretation): S. Marshall

AcknowledgmentsThe authors would like to thank the F-star Protein Sciences, in vivo, Assay

Development and Drug Discovery team; Cristian Gradinaru for statistical analyses;Jacqueline Doody for scientific contributions; Alison McGhee for critical review;Babraham BSU staff members for animal husbandry and technical assistance;Dr. Sarah Taplin for pathology assessment; and Dr. Sarah Burl and Natalie Allenfor article editing and review.

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 September 11, 2019; revised February 7, 2020; accepted April 13, 2020;published first April 28, 2020.

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2020;26:4154-4167. Published OnlineFirst April 28, 2020.Clin Cancer Res Matthew A. Lakins, Alexander Koers, Raffaella Giambalvo, et al. Low Toxicity and Antitumor Activity in Colorectal Cancer ModelsFS222, a CD137/PD-L1 Tetravalent Bispecific Antibody, Exhibits

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fs222, a cd137/pd-l1 tetravalent bispeciﬁc antibody ......simultaneous binding of fs222 to human...

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