FS222, a CD137/PD-L1 Tetravalent Bispecific Antibody, Exhibits Low Toxicity and Antitumor Activity in Colorectal Cancer Models

Published OnlineFirst April 28, 2020; DOI: 10.1158/1078-0432.CCR-19-2958

CLINICAL CANCER RESEARCH | TRANSLATIONAL CANCER MECHANISMS AND THERAPY

FS222, a CD137/PD-L1 Tetravalent Bispeciﬁc Antibody, Exhibits Low Toxicity and Antitumor Activity in Colorectal 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, and Neil Brewis

ABSTRACT ◥ Purpose: With the increased prevalence in checkpoint therapy Results: We demonstrated simultaneous binding of CD137 þ resistance, there remains a significant unmet need for additional andPD-L1andshowedpotentT-cell activation across CD8 therapies for patients with relapsing or refractory cancer. We have T-cell activation assays in a PD-L1–dependent manner with a developed FS222, a bispecific tetravalent antibody targeting CD137 CD137/PD-L1 bispecific antibody, FS222. FS222 also activated and PD-L1, to induce T-cell activation to eradicate tumors without T cells in a human primary mixed lymphocyte reaction assay, with the current toxicity and efficacy limitations seen in the clinic. greater potency than the monospecific mAb combination. FS222 Experimental Design: A bispecific antibody (FS222) was devel- showed no signs of liver toxicity up to 30 mg/kg in a non-human oped by engineering CD137 antigen–binding sites into the Fc region primate dose-range finding study. A surrogate molecule caused of a PD-L1 IgG1 mAb. T-cell activation by FS222 was investigated significant tumor growth inhibition and survival benefit, concom- þ using multiple in vitro assays. The antitumor efficacy, survival itant with CD8 T-cell activation, in CT26 and MC38 syngeneic benefit, pharmacodynamics, and liver pharmacology of a murine mouse tumor models. surrogate molecule were assessed in syngeneic mouse tumor mod- Conclusions: By targeting CD137 agonism to areas of PD-L1 els. Toxicology and the pharmacokinetic/pharmacodynamic profile expression, predominantly found in the tumor microenvironment, of FS222 were investigated in a non-human primate dose-range FS222 has the potential to leverage a focused, potent, and safe finding study. immune response augmenting the PD-(L)1 axis blockade.

Introduction T cells, and dendritic cells (DC; ref. 4). Engagement of CD137 by its ligand CD137L results in receptor trimer formation, and subsequent Immunomodulatory mAbs are a promising approach for patients clustering leads to CD137 signaling cascade activation. This provides a with cancer. Immune checkpoint inhibitors targeting programmed cell survival signal to T cells, thereby sustaining effective T-cell activation death (PD)-1, PD-L1, and cytotoxic T-lymphocyte–associated protein and generation of immunologic memory. The primary functional role 4 are the most advanced immunotherapy agents for oncology. How- of CD137 in enhancing T-cell cytotoxicity was first described in ever, only a subset of patients benefit from long-term survival, and 1997 (5), and soon thereafter CD137 mAbs were proposed as anti- there remains an unmet clinical need (1). Although bispecific T-cell cancer therapeutics. engagers, such as blinatumomab (BLINCYTO) targeting CD3 and Clinical development of CD137 mAbs has been hampered by dose- CD19, are the most advanced next-generation immuno-oncology limiting high-grade liver inflammation associated with CD137 agonist modalities, their use is limited to hematologic malignancies and antibody treatment. Urelumab (Bristol-Myers Squibb, BMS-663513), further limited by acute safety concerns (2). We believe agonist a human IgG4 isotype antibody, was the first CD137 mAb to enter antibodies against specific costimulatory receptors from the tumor clinical trials, but these were halted after significant, on target, dose- necrosis factor receptor superfamily may represent the next stage in dependent liver toxicity was observed (6–8). This outcome was not solid cancer treatment. predicted because urelumab failed to preclinically identify liver inflam- CD137 (4-1BB) is a costimulatory molecule and widely known to þ mation due to its low affinity for the cynomolgus monkey target be upregulated on CD8 T cells following activation (3). CD137 þ molecule (9). More recently, clinical trials of urelumab in the treatment can also be expressed on activated CD4 helper T cells, B cells, of solid cancers were recommended; however, urelumab dosing in regulatory T cells (Treg), natural killer (NK) cells, natural killer these trials had to be limited and efficacy results were disappointing with no objective response reported in the 64 patients with solid F-star Therapeutics Ltd., Cambridge, United Kingdom. tumors treated with monotherapy (6). No dose-limiting toxicity has been observed with CD137 mAb Note: Supplementary data for this article are available at Clinical Cancer fi Research Online (http://clincancerres.aacrjournals.org/). utomilumab (PF-05082566, P zer), a human IgG2 isotype antibody, in dose-escalation phase I clinical trials dosing up to 10 mg/kg in phase I Corresponding Author: Matthew A. Lakins, F-star Therapeutics Ltd., Eddeva clinical trials of advanced cancer (6, 8). However, the overall objective B920, Babraham Research Campus, Cambridge CB22 3AT, United Kingdom. Phone: 01223948159; Fax: 01223410493; E-mail: [email protected] response rate with this antibody was only 3.8% in patients with solid tumors, potentially indicating that utomilumab has a weaker potency Clin Cancer Res 2020;XX:XX–XX and clinical efficacy than urelumab, while having a more favorable doi: 10.1158/1078-0432.CCR-19-2958 safety profile (6, 8). Trials of utomilumab in combination with 2020 American Association for Cancer Research. radiotherapy or chemotherapy, as well as in combination with other

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cancer without toxicity (7, 8), we believe there remains a need to Translational Relevance develop treatments that combine PD-L1 blockade and provoke We developed an mAb2 bispecific antibody for targeting CD137 strong CD137 agonism in safe and efficacious therapies that do not þ and PD-L1 in solid tumors which potently activated CD8 T cells rely on a combination approach. An alternative to combining CD137 in vitro only in the presence of PD-L1–expressing cells. Our and PD-L1 monotherapies is the development of a bispecific antibody þ surrogate molecule activated intratumoral CD8 T cells and that encompasses the two modalities. It is anticipated that such a effectively controlled tumor growth in syngeneic mouse tumor bispecific mAb could deliver superior antitumor efficacy over com- þ models without toxicity. We found that FS222 activates CD4 and bining monotherapies. There are existing preclinical approaches com- þ CD8 T cells in vitro with activity superior to the combination of bining CD137 mAb activity with PD-L1 mAb activity into bispecific monospecific, mAbs representative of those used in the clinic therapies. These can be subdivided into two broad range categories, currently, providing evidence that our tetravalent bispecific clinical non–IgG- and IgG-like molecules, both of which can be further candidate will provide greater benefit to patients than a combina- divided by their binding valency for each target. tion approach against both targets in solid tumors. Considering the Here, we describe a fully human, tetravalent, IgG bispecific antibody broad expression of PD-L1 in many solid tumors, FS222 may (mAb2, FS222) comprising a PD-L1–specific mAb with 5 amino acid provide therapeutic opportunities for patients with cancer who insertions and 7 amino acid substitutions in the CH3 region of the Fc remain challenging to treat. domain to create two binding sites forming an Fc fragment antigen- binding (Fcab) for CD137 (15). FS222 blocked PD-L1 and activated þ CD137 tumor-reactive T cells in a PD-L1–dependent manner. It demonstrated similar potency in primates, and preliminary toxicity antibody therapies, are ongoing with early results showing no dose- studies in this species showed significant pharmacodynamic (PD) limiting toxicities for doses up to 5 mg/kg and a 26% patient response responses and a lack of toxicity. A surrogate mouse cross-reactive rate for the combination of utomilumab and pembrolizumab (10). CD137/PD-L1 mAb2 with homologous mechanisms of action to PD-1 and its ligands PD-L1 (CD274, B7-H1) and PD-L2 (B7-DC) FS222 was observed to provide a substantial survival benefit in multiple deliver inhibitory signals that regulate the balance between T-cell mouse tumor models with no toxicity and showed potent in vivo PD activation, tolerance, and immunopathology. Consequently, PD-L1 changes related to antitumor immune responses. expression by cells can mediate protection against cytotoxic T lymphocyte killing. Cancer, as a chronic and proinflammatory disease, subverts this immune-protective pathway through upregulation of Materials and Methods PD-L1 expression to evade the host immune response. PD-L1 expres- Production and characterization of a CD137/PD-L1 mAb2, sion has been shown in a wide variety of solid tumors (11), and clinical FS222 trials have shown the benefit of targeting PD-L1 in patients leading to The CD137/PD-L1 mAb2 molecule named FS222 consisting of the approval of three PD-L1–targeting mAbs to date: atezolizumab an IgG1 molecule comprising the human CD137 Fcab was prepared (MPDL3280A, Tecentriq, Hoffmann-La Roche, Genentech), a by substituting part of the CH3 domain comprising the AB, CD, humanized IgG1 antibody; avelumab (MSB0010718C, Bavencio, and EF loops (15), for the corresponding region of the CH3 domain of Merck KGaA, Pfizer), a fully human IgG1 antibody; and durvalumab a PD-L1 mAb (E12v2). Fcab generation has been previously (MEDI4736, Imfinzi, AstraZeneca) a fully human IgG1 antibody. The described (16). FS222 incorporates a LALA mutation (leucine to PD-L1/PD-1 immune checkpoint is also being targeted by three alanine at positions 234 and 235 according to Eu numbering) in the approved PD-1 mAbs, namely pembrolizumab (Keytruda, Merck), CH2 domain (AA) to reduce Fcg receptor binding (17, 18). FS222 was nivolumab (Opdivo, BMS), and cemiplimab (Libtayo, Regeneron expressed transiently using HEK293 6E (National Research Council Pharmaceuticals). Canada, Canada) cells. Supernatants were purified on MabSelect In mouse models resistant to single-agent treatment with either SuRe LX Protein-A prepacked columns using AKTAxpress€ instrument CD137 agonists or PD-1/L1 blockade, significant synergistic effects (both GE Healthcare Life Sciences). IgG protein content was have been observed when antibodies targeting both pathways are quantified by BioLayer Interferometry (BLI) using the Octet QKe combined. The mechanistic basis for this synergy, even in poorly System (ForteBio) platform with Protein A quantitation biosensors immunogenic mouse tumor models, is that tumor-infiltrating lym- (ForteBio; 18-5013). FS222 was purified by Protein A affinity phocytes coexpress PD-1 and CD137, and following combination chromatography using mAb SelectSure columns. þ treatment CD8 T cells can now effectively respond to tumor- 2 associated neoantigens (12). The mechanistic basis for CD137 agonist Biophysical characterization of mAb by size exclusion antibodies alone can be two-fold. Firstly, their ability to induce effector chromatography and SDS-PAGE cell function can result in antitumor activity in some preclinical After purification, size exclusion–high-performance liquid chro- models. Secondly, their alternative function to deplete Tregs has been matography (SE-HPLC) was performed on an Agilent 1100 series described as the most effective mechanism of action for CD137 HPLC Value System (Agilent Technologies, Inc.), fitted with a TSKgel mAbs (13). However, an alternative approach is to direct potent SUPERSW3000 HPLC 4.6 mm ID 30 cm column (Tosoh Bioscience, CD137 agonist activity to tumor-reactive T cells while not relying on LLC) using 20 mmol/L sodium phosphate and 200 mmol/L sodium Treg depletion. Avoiding Treg depletion can be achieved by removing chloride, pH 6.8, as a mobile phase. Quantification of percentage FcgR binding by L234A and L235A (LALA) mutation while retaining monomer was performed using ChemStation software (Agilent Tech- agonist activity via an alternative mechanism of cross-linking as nologies, Inc.). Capillary electrophoresis–sodium dodecyl sulfate (CE- described in this article. SDS) analysis was performed on a 2100 Bioanalyzer Capillary Elec- As only a fraction of patients respond to monotherapies that block trophoresis System (Agilent Technologies, Inc.), according to manu- the PD-1/PD-L1 pathway (14), and CD137-targeting agonistic mole- facturer's instructions. For reducing conditions, dithiothreitol (DTT) cules have yet to demonstrate significant responses in patients with was added and samples were denatured at 70 C for 5 minutes.

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Simultaneous binding of FS222 to human PD-L1 and human 100 mL T-cell culture medium [RPMI medium with 10% FBS, 1X CD137 by surface plasmon resonance (SPR) penicillin–streptomycin, 1 mmol/L sodium pyruvate, 10 mmol/L His-tagged human PD-L1 antigen was coated on to a CM5 chip to Hepes (Sigma-Aldrich, H0887), and 50 mmol/L 2-mercaptoethanol þ approximately 1,100 RU and was used to immobilize FS222 when (Gibco, M6250)]. CD8 T cells were added. Cells were treated with a injected at 100 nmol/L which resulted in approximately 300 RU of titration of FS222, CD137(20H4.9) mAb, or Ctrl(4420) mAb (Sup- FS222 being captured. Fc-tagged human CD137 antigen was then plementary Table S1). Supernatants were assayed with human IL2 injected at a single concentration (100 nmol/L), using the Biacore T200 ELISA Ready-SET-Go! kit (88-7025-88, Fisher Scientific) following the (GE Healthcare Life Sciences), to observe dual binding. manufacturer's instructions. Plates were read at 450 nm using the plate reader with the Gen5 Software. The concentration of human IL2 FS222 binding to cell-expressed receptors by flow cytometry (hIL2) was plotted versus the log concentration of antibody, and the DO11.10 T cells overexpressing human CD137 were cultured in resulting curves were fitted using the log (agonist) versus response DMEM containing 10% heat-inactivated FBS, 1 mmol/L sodium equation in GraphPad Prism. pyruvate, and 50 mg/mL puromycin. HEK cells overexpressing human PD-L1 were cultured in DMEM containing 10% FBS, 100 mg/mL Human primary mixed lymphocyte reaction þ hygromycin B, 15 mg/mL blasticidin, and 1 mg/mL doxycycline. Cells Generation of expanded CD4 T cells þ were resuspended in 40 mL of FS222, CD137/Ctrl(HelD1.3) mAb2, Human primary CD4 T cells were isolated from leukocyte cones þ CD137(MOR7480.1) mAb, Ctrl(4420) mAb, or PD-L1(E12v2) mAb using a Human CD4 T Cell Isolation Kit (130-096-533, Miltenyi (Supplementary Table S1) titrations prepared in DPBS and then Biotec Ltd.) according to the manufacturer's instructions. Dynabeads washed and resuspended in a secondary human IgG detection anti- Human T-Activator CD3/CD28 (11131D, Thermo Fisher Scientific) body (A-21445, Thermo Fisher Scientific) which had been diluted in were used in the presence of 50 IU/mL recombinant human IL2 DPBS. Cells were then resuspended in the viability dye 7-AAD (A1310, (PeproTech, 200-02) with 3:1 bead to cell ratio to expand cells for þ Thermo Fisher Scientific) and examined with either a BD FACSCanto 7 days. Dynabeads were removed, and CD4 T cells were rested II or BD LSRFortessa II (BD Biosciences) before being analyzed using overnight with reduced 10 IU/mL recombinant human IL2. FlowJo V10 (TreeStar, Inc.). Differentiation of iDCs FS222 binding in human primary T-cell assay Monocytes were isolated from human PBMCs using a Human Pan Human primary T-cell isolation and activation Monocyte Isolation Kit (130-096-537, Miltenyi Biotec Ltd.) following Peripheral blood mononuclear cells (PBMC) were isolated from the manufacturer's instructions. Monocytes were differentiated to leukocyte cones, obtained from platelet donors, by Ficoll (GE17-1440-02, iDCs using Human Mo-DC Differentiation Medium (130-094-812, Sigma-Aldrich) gradient separation. Pan T cells were isolated from the Miltenyi Biotec Ltd.) following the manufacturer's instructions. PBMCs present in the eluent using the Pan T Cell Isolation kit II according to the manufacturer's instructions (130-096-535, Miltenyi Mixed lymphocyte reaction: Expanded T cells were cultured in AIM fi Biotec). Pan T cells were incubated overnight at 37 Cand5%CO2 V Medium (12055091, Thermo Fisher Scienti c) and incubated in RPMI supplemented with 10% heat-inactivated FBS, 1 mmol/L overnight. Titrations of FS222, PD-L1(E12v2) mAb and CD137 2-mercaptoethanol, penicillin (100 U/mL)/streptomycin (100 U/mL), (20H4.9) mAb, PD-L1(E12v2) mAb, CD137/Ctrl(HelD1.3) mAb2 and and 1 mmol/L sodium pyruvate. Dynabeads Human T-Activator CD3/ PD-L1(E12v2) mAb, CD137(20H4.9) mAb, CD137/Ctrl(HelD1.3) CD28 beads (11132D, Thermo Fisher Scientific) were used to activate mAb2, or Ctrl(4420) mAb (Supplementary Table S1) were used to þ T cells and upregulate CD137 and PD-L1 surface expression. Beads were treat a 1:10 mix of iDC cells and expanded CD4 T cells in AIM V washed from the T cells using a DynaMag-15 Magnet (12301, Thermo Medium for 5 days. Supernatants were analyzed for IFNg using Fisher Scientific) following the manufacturer's instructions. Human IFN gamma ELISA Ready-SET-Go! Kit (88-7316-86, Thermo Fisher Scientific). Plates were read at 450 nm using the plate reader Human primary T-cell binding assay with the Gen5 Software. The concentration of human IFNg was plotted Stimulated pan human primary T-cell suspensions were resus- versus the log concentration of antibody, and the resulting curves were pended in 40 mL FS222, CD137/Ctrl(HelD1.3) mAb2, PD-L1 mAb, fitted using the log (agonist) versus response equation in GraphPad CD137(MOR7480.1) mAb, or Ctrl(4420) mAb (Supplementary Prism. Table S1) titrations prepared in DPBS and treated with AF647 goat þ anti-human IgG (H þ L; 1:500; A-21445, Thermo Fisher Scientific), Murine primary OT-1 CD8 T-cell activation assay þ anti-hCD4 FITC (1:200; 550628, BD Biosciences), and anti-hCD8 CD8 T-cell activation was achieved by antigen stimulation of eF450 (1:200; 48-0087-42, Thermo Fisher Scientific) prepared in genetically modified OT-1 T cells, isolated from C57BL/6 OT-1 DPBS. 7-AAD (A1310, Thermo Fisher Scientific) was used as a mice (003831, The Jackson Laboratory) having a T-cell receptor viability dye, and samples were then examined with a BD FACSCanto specific for ovalbumin peptide 257–264, and was determined by the II before being analyzed using FlowJo V10 Prism software. release of IFNg. OT-1 T cells were incubated with B16-F10 melanoma cells, which had previously been cultured in the presence of þ Human primary CD8 T-cell assay with HEK.hPD-L1 cross-linking 20 ng/mL IFNg (AF-315-05-100UG, PeproTech) to induce PD-L1 þ Human primary CD8 T cells were isolated from PBMCs obtained expression, and that were then pulsed with 500 nmol/L SIINFEKL þ from leucocyte depletion cones using the CD8 T-cell isolation kit II peptide for 1 hour at 37C, to drive T-cell activation. The efficacy of (130-096-495, Miltenyi Biotec Ltd.) according to the manufacturer's surrogate FS222 was subsequently assessed by ELISA for secreted instructions. For cell-based cross-linking, HEK293 cells overexpres- mIFNg (88-7314-88, Thermo Fisher Scientific) after 3 days. This sing hPD-L1 (HEK.hPD-L1) or HEK wild-type cells or a mixture of the assay was also carried out utilizing MC38.OVA cells that express two populations were plated on to CD3 mAb–coated (8 mg/mL, Clone ovalbumin, in an identical protocol, except for peptide pulsing UCHT1, R&D Systems, MAB100-SP) 96-well flat-bottom plates in which was not necessary.

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Surrogate FS222 in vivo characterization in CT26.WT and MC38 cynomolgus macaques at Charles River Laboratories in line with syngeneic mouse tumor models Institutional Animal Care and Use Committee guidelines and in The CT26.WT colon carcinoma cell line (ATCC) was initially accordance with the “Guide for the Care and Use of Laboratory expanded, stored, and then prescreened by IDEXX Bioresearch for Animals” (1996) by the Institute of Laboratory Animals Research pathogens using the IMPACT I protocol and shown to be pathogen- Commission on Life Sciences (National Research Council, Washing- free. BALB/c female mice (Charles River) aged 8 to 10 weeks and ton, DC). weighing 18 to 22 g each received 1 105 CT26.WT cells injected Briefly, FS222 was administered to cynomolgus monkeys (1/sex/ subcutaneously in the left flank in 100 mL DMEM serum-free culture group) via intravenous infusion at 3 mg/kg as a single dose on day 1 or medium. at 0.1, 1, 10, or 30 mg/kg as repeat doses on days 1, 8, 15, and 22. For the The MC38 colon carcinoma cell line (ATCC) was initially expand- 3 mg/kg dose group, serial serum samples were collected for PK ed, stored, and then prescreened by IDEXX Bioresearch for pathogens assessment on day 1 (predose, 0.083, 0.5, 2, 6, and 12 hours postdose) using the IMPACT I protocol and shown to be pathogen free. C57BL/6 and days 2, 3, 4, 6, 8, 11, 15, 22, 29, 36, and 43. For the remaining female mice (The Jackson Laboratory) aged 9 to 10 weeks and weighing groups, PK serum samples were collected on day 1 (predose, 0.083, 0.5, 18 to 24 g each received 1 106 MC38 cells injected subcutaneously in 2, 6, and 12 hours postdose) and days 2, 3, 7, 8 (predose and 0.083 hours the right flank in 100 mL DMEM serum-free culture medium. postdose); day 15 (predose, 0.083, 0.5, 2, 6, and 12 hours postdose); The surrogate FS222 and control antibodies were injected intraper- days 16, 17, 21, and 22 (predose and 0.083 hours postdose); and day 25. itoneally into mice at appropriate mg per mouse in DPBS, 100 mmol/L Serum levels of FS222 were measured using a qualified Gyros-based arginine, and 0.05% Tween 80 on days 7, 9, and 11 following tumor immunoassay developed in-house to specifically detect free drug inoculation. Tumor volume measurements were taken with callipers to (human biotinylated PD-L1 was used as a capture reagent and human determine the longest axis and the shortest axis of the tumor, and the Alexa Fluor labeled CD137 as a detection reagent). following formula was used to calculate the tumor volume: For the evaluation of tolerability, standard toxicology parameters ÀÁ LX S2 =2 such as body weight, food consumption, clinical observations, hematology, and blood chemistry were evaluated over the duration of the Where L ¼ longest axis; S ¼ shortest axis. study. The study was terminated 25 days after administration of first dose in repeat dose animals and 43 days after administration of single PD assessment and receptor occupancy in a CT26.WT syngeneic dose (PK group of animals). mouse tumor model For evaluation of the PD response to FS222, immunophenotyping of A single-dose PD study was run in the same CT26.WT syngeneic peripheral blood was performed to assess peripheral lymphocyte tumor model as described above. This study comprised three dosing populations (monocytes, T cells, B cells, and NK cells) as well as the groups, receiving either control antibody or surrogate FS222 at one of induction of proliferation and activation of central memory and þ þ two doses. Samples from tumor tissue and blood were analyzed over 8 effector memory CD4 and CD8 T-cell subpopulations. Serial blood time points (2, 6, 24, 48, 72, 96, 120, and 192 hours). Each dosing cohort samples (on EDTA) were collected prior to first dosing of FS222 had 64 mice (8 mice per timepoint). Each animal received 1 105 (predose) and on the indicated study days over the course of the study; CT26.WT cells injected subcutaneously in the left flank in 100 mL stained with antibodies against CD45, CD3, CD4, CD8, CD16, CD28, DMEM. Eleven days following tumor cell inoculation, each mouse CD25, FoxP3, CD95, CD69, and Ki67 (Supplementary Table S3); and received the test sample via a 100 mL i.v. injection. analyzed using a FACSCanto II flow cytometer (BD Biosciences) and Tumor tissue and blood were tested for drug-bound–positive T FACSDiva (BD Biosciences), Excel, and GraphPad Prism software. cells, T-cell proliferation, and free PD-L1. Blood (100 mL) was collected into EDTA-coated capillaries by tail vein bleeding and was lysed twice in red blood cell lysis buffer (Thermo Fisher Scientific, 00-4333-57) Results 2 according to manufacturer's instructions. Tumor tissue was collected Creation of a CD137/PD-L1 bispecific antibody (mAb , FS222) by dissection and was disaggregated to single-cell suspension by FS222 was created by incorporating into a proprietary PD-L1 mAb, standard mechanical and enzymatic methods. Red blood cells were an engineered IgG1 Fc region termed Fcab (Fc-region with antigen lysed in red blood cell lysis buffer according to the manufacturer's binding), where high-affinity antigen binding for CD137 was intro- instructions. duced in the C-terminal region (Fig. 1A). Binding to FcgR was Cells were stained with Fixable Viability Dye eFluor 780 (65-0865- removed by the introduction of L234A and L235A (LALA) mutations 14, Thermo Fisher Scientific) following the manufacturer's instruc- (Supplementary Fig. S1A), while binding to human FcRn was main- tions. Cells were stained with an antibody staining panel (Supple- tained (Supplementary Fig. S1B). The mAb2 is tetravalent, with two mentary Table S4, all but Ki67 and FoxP3 antibodies) in the presence of binding sites for PD-L1 (one in each Fab region) and two binding sites CD16/CD32 mAb Fc block (1:50, 14-0161-86, Thermo Fisher Scien- for CD137 (one in each CH3, due to the homodimeric nature of the Fc tific) and then fixed and permeabilized with the eBioscience Foxp3 region), and maintains the IgG1 structure. staining Kit (00-5523-00, Thermo Fisher Scientific) according to the manufacturer's instructions. Cells were stained with Ki67 and Foxp3 Binding affinities of FS222 are equivalent to individual antibodies in the presence of Fc block and then examined in a BD component antibodies Fortessa flow cytometer. Data were analyzed with FlowJo, Excel, and The simultaneous binding of FS222 to human CD137 and human GraphPad Prism. PD-L1 was tested via SPR and then individually to cells expressing either human CD137 or human PD-L1. Preliminary toxicology study in cynomolgus monkeys FS222 simultaneously bound to both targets as observed by SPR A preliminary dose-range finding study was conducted to evaluate (Fig. 1B) with affinities of 0.66 nmol/L to dimeric human CD137 and the pharmacokinetic (PK)/PD response to and tolerability of FS222 in 0.19 nmol/L to monomeric human PD-L1 (Supplementary Fig. S1C– cynomolgus monkeys. The study was performed using Mauritian S1E). The cell-binding assays demonstrated that FS222 bound human

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Figure 1. mAb2 structure and concurrent high-affinity binding of CD137/PD-L1 mAb2 equivalent to individual component antibodies. A, Representation of the bispecific CD137/ PD-L1 mAb2, FS222, on a human IgG1 backbone with FcgR binding removed by L234A and 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, FS222 simultaneous 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 determined by 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 primary CD4þ and CD8þ T cells as determined by flow cytometry.

CD137 to an equivalent level as the component CD137 Fcab (CD137/ a DO11.10 human PD-L1 T-cell activation assay (data not shown). As 2 þ þ Ctrl(HelD1.3) mAb ) alone (EC50 6.2 nmol/L; Fig. 1C). FS222 shown in Fig. 1E, FS222 bound to activated CD4 and CD8 primary also bound human PD-L1 to an equivalent level as the component T cells with an EC50 of 0.8 and 0.9 nmol/L, respectively. This was – Fab (PD-L1(E12v2) mAb) alone (EC50 3.7 nmol/L; Fig. 1D). PD-L1 equivalent to the PD-L1(E12v2) mAb binding characteristics but not (E12v2) mAb was found to be just as effective as the PD-L1(S70) mAb equivalent to the CD137/Ctrl(HelD1.3) mAb2, showing that this cell (YW243.55.S70) at blocking PD-L1 binding to PD-1 as investigated in binding was driven primarily through PD-L1 rather than CD137. The

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positive control CD137 mAb, CD137(MOR7480.1) mAb, bound to allowed the investigation of varying ratios of human PD-L1–expres- activated human primary T cells as expected, whereas the CD137/ sing cells to wild-type cells to model the likely heterogeneity of Ctrl(HelD1.3) mAb2 had minimal binding to the same cells. There- expression present within different human tumors. fore, FS222 bound to PD-L1 with high affinity and, through design, FS222 showed maximum activity, measured by human IL2 release, þ bound CD137 with high avidity, which are two features of the from activated human primary CD8 T cells, when 100% of HEK 293 molecule critical for the cross-linking–dependent activity as cells expressed PD-L1 (Fig. 2A). The maximum IL2 release (Emax) described below. reduced in proportion to the reduction in the percentage of cells expressing human PD-L1 present; however, the EC50 value remained T-cell activation through CD137 agonism was dependent upon broadly the same at 0.05 nmol/L (Fig. 2A). cross-linking via PD-L1 þ Subsequent investigations were aimed to dissect the nature of FS222 elicited superior activity in a CD4 mixed lymphocyte FS222-mediated CD137 agonism. To demonstrate that FS222 can be reaction by FS222 compared with a combination of cross-linked to mediate human CD137 signaling only in the presence monospecific antibodies þ of cells expressing human PD-L1, human primary CD8 T cells The activity of FS222 was tested in a mixed lymphocyte reaction þ stimulated by plate-bound CD3 mAb were cocultured with wild- (MLR) which utilizes human primary CD4 T cells and immature type HEK 293 cells, HEK 293 cells engineered to overexpress human monocyte-derived DCs (iDC) expressing endogenous levels of both PD-L1, or mixtures of the two cell lines in different proportions. This targets. 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 complete FS222 mAb2 either alone or in combination with each other. Significance determined by extra sum-of-squares F test. , P < 0.001.

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potent activity in the MLR assay (EC50 0.08 nmol/L). The human 11 after tumor implantation with approximately 10 mg/kg surrogate CD137 mAb, CD137(20H4.9) mAb, even when cross-linked with FS222, PD-L1(S70) mAb, mCD137(Lob12.3) mAb, or Ctrl(HelD.13) hCH2 mAb, did not elicit activity. This suggested that CD137 signaling isotype control mAb. Surrogate FS222 was shown to substantially alone is ineffective in this assay. However, a combination of PD-L1 reduce tumor growth (Fig. 3B), and 42% of mice remained tumor free (E12v2) and CD137(20H4.9) mAbs cross-linked with hCH2 mAb up to 68 days after treatment (Fig. 3C and Table 1). In contrast, showed potent activity (EC50 0.14 nmol/L) with a higher maximum treatment with PD-L1(S70) mAb or mCD137(Lob12.3) mAb failed to IFNg release compared with PD-L1(E12v2) mAb alone, which show significant survival advantage (Fig. 3C and Table 1; Supple- indicated a synergisticeffectofthetwomAbs(Fig. 2B). FS222 mentary Tables S8 and S9). showed similarly potent activity to the combination of the two In the MC38 tumor model, surrogate FS222 was able to eradicate separate monospecific antibodies with an EC50 of 0.07 nmol/L and all tumors at a lower dose of 1 mg/kg. Mice were injected intra- comparable maximum IFNg release level (Fig. 2B). FS222 was also peritoneally 7, 9, and 11 days after tumor cell inoculation with a tested against each component part of the mAb2 that makes up dose of approximately 1 mg/kg of either surrogate FS222, PD-L1 FS222, the CD137/Ctrl(HelD1.3) mAb2,andthePD-L1(E12v2) (S70) mAb, mCD137(Lob12.3) mAb, a combination of both, or Ctrl mAb, alone and in combination. CD137(HelD1.3) mAb2 showed (4420) isotype control mAb. Surrogate FS222–treated mice showed no activity in this assay, indicating that the CD137 Fcab component full tumor regression in all mice which remained tumor free until had an inability to activate T cells in an MLR. There was no day 49 when the study was ended. In contrast, single and combined additional effect on activation by treating with a CD137/Ctrl treatment of PD-L1(S70) mAb and mCD137(Lob12.3) mAb (HelD1.3) mAb2 plus PD-L1(E12v2) mAb combination above that resulted in durable tumor regression in only a fraction (4/12 or already seen with PD-L1(E12v2) mAb alone. FS222 showed a less) of animals (Fig. 3D and E, Table 1; Supplementary Tables S10 similar potency to the combination of its component parts and S11). 2 CD137/Ctrl(HelD1.3) mAb and PD-L1(E12v2) mAb (EC50 of 0.07 and 0.06 nmol/L, respectively) with superior efficacy as indi- Surrogate FS222 produced dose-dependent survival benefitin cated by higher IFNg production (Emax; Fig. 2B). CT26.WT syngeneic mouse tumor model In the syngeneic CT26.WT tumor model, we assessed surrogate Surrogate FS222, a mouse CD137/PD-L1 tetravalent bispecific FS222 dose-dependency in vivo andshoweddose-dependentsur- antibody, demonstrated potent in vitro activity greater than an vival benefit between doses of approximately 0.1 mg/kg and approx- mAb combination approach imately 10 mg/kg. To evaluate dose-dependent efficacy, surrogate FS222 did not bind to mouse CD137 (data not shown) so it was not FS222 dose levels equivalent to approximately 0.1, 0.3, 1, and 10 mg/kg, possible to evaluate FS222 in mouse syngeneic tumor model systems and Ctrl(4420) mAb isotype control dosed at 10 mg/kg, were in vivo. Therefore, a mouse surrogate of FS222 was created using an administered intraperitoneally usingthesamestudydesigndes- Fcab targeting mouse CD137 selected using yeast display. The Fcab cribed above. Surrogate FS222 showed an antitumor efficacy from against mouse CD137 was selected based on affinity measurements 0.3 mg/kg and durable tumor regression in 21% of treated animals and cross-link–dependent activation of CD137 and was tested in at 1 mg/kg and 40% at 10 mg/kg (Supplementary Fig. S2B). Using similar mouse systems to those used to determine the function of the log-rank (Mantel–Cox) test, a significant survival dose depen- FS222 in human systems. Surrogate FS222 was tested for cell binding dency was shown for surrogate FS222 at 0.3 mg/kg up to 1 mg/kg fi similar to FS222 and was found to have an EC50 of 2.7 and 2.6 nmol/L compared with Ctrl(4420) mAb treatment, but no signi cant to cells engineered to overexpress mouse CD137 and mouse PD-L1, benefit raising from 1 mg/kg to 10 mg/kg surrogate FS222 despite respectively. the median survival extending from 29 to 39 days, respectively The functional activity of surrogate FS222 was determined in a (Fig. 4 and Table 2). primary assay where CD137 and PD-L1 are endogenously expressed on activated T cells and B16-F10 tumor cells, respec- Surrogate FS222–regulated dose-dependent PD changes in tively. B16-F10 mouse melanoma cells that had previously been tumor and blood þ pulsed with OVA peptide (SIINFEKL) were cocultured with CD8 CT26.WT tumor–bearing mice were treated with a single intrave- þ antigen–specific OT-1 T cells. CD8 T-cell activation (IFNg nous dose of surrogate FS222 (1 mg/kg and 10 mg/kg) 11 days after release) was increased after treatment with either mCD137 subcutaneous inoculation of CT26.WT. Tumor tissue and blood (Lob12.3) mAb, PD-L1(S70) mAb, or a combination approach. were tested for T-cell–bound surrogate FS222, T-cell proliferation, However, the greatest potency was seen upon treatment with the and PD-L1 receptor occupancy over time (between 2 and 192 hours). surrogate FS222 (EC50 0.003 nmol/L; Fig. 3A). The same result was Total CD137 receptor expression was also assessed. achieved using a similar assay described above but utilizing MC38 A high percentage of peripheral and tumor-resident T cells showed cells expressing ovalbumin as the source of both OVA peptide and bound surrogate FS222 as early as 2 hours after intravenous admin- PD-L1 (Supplementary Fig. S2A). As with FS222, no activation was istration (Fig. 5A). There was a dose-dependent correlation in the detected in the absence of PD-L1 cross-linking which indicated a longevity of binding, with surrogate FS222 no longer detected after similar mode of action between the surrogate and human-specific 96 hours on T cells isolated from mice administered with 1 mg/kg, molecules. whereas surrogate FS222 was still detected between 120 and 192 hours after administration of 10 mg/kg. Surrogate FS222 controlled tumor growth in syngeneic mouse Ki67 expression was used as a marker for T-cell proliferation on þ þ tumor models CD4 and CD8 T cells. T cells isolated from tumor tissue exhibited a þ To evaluate the antitumor effects of surrogate FS222 in vivo, higher frequency of Ki67 T cells as expected in an inflammatory CT26.WT or MC38 tumor cell lines were injected subcutaneously into tumor microenvironment. At both dose levels, surrogate FS222 þ the flank of BALB/c and C57BL/6 mice, respectively. In the CT26.WT resulted in increases in the frequency of Ki67 peripheral blood tumor model, mice were injected intraperitoneally on days 7, 9, and T 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 assay with cell-based cross-linking provided by B16-F10 tumor cells expressing mouse PD-L1. Signiﬁcance determined by extra sum-of-squares F 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/kg Ctrl(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) þ CD137(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 with 1 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 5/12 Percent 0% 0% 8% N/A 42% MC38 tumor–free animals Number 0/12 2/12 2/12 4/12 12/12 Percent 0% 17% 17% 33% 100%

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

Surrogate FS222 immunopharmacology did not result in hepatotoxicity In 2008, clinical trials investigating urelumab in solid tumors were halted due to severe treatment-related immune events which mani- fested in the liver as severe hepatotoxicity resulting in patient deaths (7). More recently, urelumab has been administered at far reduced dose levels to mitigate this toxicity. Preclinical mechanistic work was undertaken in mice wherein animals dosed with CD137 Figure 4. agonist mAbs showed similar hepatotoxicity. These studies showed a Surrogate FS222 was tested for efficacy and survival in a CT26.WT syngeneic requirement for T cells and CD137 expression in the resultant mouse tumor model dose-range finding study. Kaplan–Meier survival plot of hepatotoxicity (9, 19). Therefore, these animal models have some dose-range finding study in CT26.WT of surrogate FS222 dosing in the range 0.1 translational relevance for predicting the risk of hepatotoxicity in the to 10 mg/kg showing significant survival benefit of increasing doses of surrogate clinic in human patients following administration of other CD137 FS222 above 0.1 mg/kg compared with IgG control. agonists such as FS222. Mice from our CT26.WT syngeneic tumor studies showed no overt signs of toxicity following repeated dosing indicating a PD response (Fig. 5B). The effect appeared stronger for with surrogate FS222 and maintained normal bodyweight throughout. þ þ CD8 T cells which is in line with CD8 T cells expressing higher To determine whether immune activation and antitumor activity þ CD137 levels than CD4 T cells. observed as a result of treatment with 1 mg/kg surrogate FS222 A proportion of cells isolated from the blood and tissue of correlated with hepatotoxicity, liver samples were taken at necropsy mice dosed with Ctrl(4420) mAb isotype control were saturated with for histologic assessment. Surrogate FS222–treated and control mice 100 nmol/L surrogate FS222 ex vivo which acted as control for 100% were necropsied 4, 7, and 14 days after the last administration whereby PD-L1 receptor engagement and was confirmed by fully blocking liver samples were excised and examined. binding with a competing mouse PD-L1 mAb (clone 10F.9G2). Each liver section was scored for pathology, and the frequencies of Cells isolated from tumor tissue and the blood of mice treated with mice showing zero, minimal, slight, and moderate effects within each surrogate FS222 at the 10 mg/kg dose showed near-complete PD-L1 group are shown in Supplementary Table S2 (0 ¼ zero, 1 ¼ minimal, blockade for 8 days, as shown in Fig. 5C represented by 100% PD-L1 2 ¼ slight, 3 ¼ moderate). Surrogate FS222–treated animals showed receptor occupancy. At 1 mg/kg, surrogate FS222 achieved near- minimal liver pathology (Supplementary Fig. S3B and Supplementary complete PD-L1 receptor occupancy for approximately 72 hours on Table S2). Specifically, the livers showed minimal to slight hepatocel- peripheral T cells. PD-L1 receptor engagement on T cells present in lular necrosis with mixed lymphocyte infiltrate in the parenchyma, blood showed an accelerated decrease compared with T cells present in minimal to slight mixed inflammatory cells in periportal tracts, no the tumor tissue which retained a greater PD-L1 receptor occupancy degenerative hepatocytes, and minimal to slight increased mitoses with greater longevity. (Supplementary Table S2). These findings are not deemed to represent Serum cytokines were analyzed by multiplex electrochemilumines- adverse hepatotoxicity, as observed with other examples of CD137 cent immunoassay (Meso Scale Discovery, MSD) to assess cytokine agonist mAbs. production after surrogate FS222 treatment in the same model. In a similar liver pharmacology mouse study, which included dosing Surrogate FS222, when dosed at 10 mg/kg, resulted in increased serum CT26.WT tumor–bearing mice with 10 mg/kg of a CD137 targeting proinflammatory cytokines IFNg, TNFa, and IL6. The immunosup- mAb clone 3H3 known to induce liver toxicity (20), both surrogate pressive cytokine IL10 likewise shows increase in serum after dosing, FS222–treated (also at 10 mg/kg) and CD137(3H3) mAb–treated þ presumably to counter the proinflammatory response. This effect was animals showed CD3 T-cell liver infiltration from day 13 onward dose-dependent, and serum cytokine levels remained similar in mice to similarly high levels above control mice (Fig. 5E). However, þ treated with 1 mg/kg surrogate FS222 compared with Ctrl(4420) activated CD8 T cells remained at significantly higher levels with mAb–treated mice (Fig. 5D). Antitumor activity observed via tumor greater longevity in CD137(3H3) mAb–treated animals compared þ growth inhibition, tracked by measuring excised tumors at indicated with animals treated with surrogate FS222 (Fig. 5F). The CD8 T-cell response for both CD137(3H3) mAb and surrogate FS222 peaks at fi Table 2. Increased dose of surrogate FS222 correlated with approximately 95% positive for Ki67 across days 8 and 13 after rst increased survival. dose. However, for surrogate FS222, this returned to baseline by day 16, whereas for CD137(3H3) mAb, this did not happen by the end of the Median P values Log-rank study at day 28 after first dose (Fig. 5F). This indicated a difference in þ survival (group-wise comparison the mode of action of these two CD137 targeting agents; CD8 T cells Surrogate FS222 (days) with lower dosed group) activated by surrogate FS222 proliferated less so and for a shorter þ period compared with CD8 T cells activated by CD137(3H3) mAb. 10 mg/kg 39 0.2 CD137(3H3) mAb has been shown to lead to hepatic degeneration 1 mg/kg 29.5 0.02 þ þ 0.3 mg/kg 24 0.007 previously (20). CD4 FoxP3 Tregs were present at higher levels in – – 0.1 mg/kg 21 0.6 surrogate FS222 treated livers compared with CD137(3H3) mAb 0 mg/kg (IgG Control) 21 treated livers at day 8 and day 13 after first dose (Supplementary þ Fig. S3C). Approximately 30% of CD4 T cells were positive for FoxP3

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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 in the 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 1 dose of surrogate FS222. B, The frequency of Ki67þ CD8þ T cells or Ki67þ CD4þ T cells in the tumor or blood as determined by flow cytometry similarly to A. C, PD-L1 receptor 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–treated mice 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 then saturated 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 a percentage of total CD45þ immune cells) in the liver of treated mice as determined by flow cytometry. F, Proliferating CD8þ T cells present in the liver, using Ki67 expression as a marker of proliferation, as determined by flow cytometry.

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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 after repeat dosing with FS222. D, Kinetic changes in peripheral CD4þ central memory cell frequency expressing Ki67 after repeat dosing with FS222. E, Kinetic changes in peripheral CD8þ central memory cell frequency after repeat dosing with FS222.

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after surrogate FS222 treatment, whereas the level after CD137(3H3) Discussion mAb remained nearer baseline at 10% (Supplementary Fig. S3C). This FS222, a CD137/PD-L1 tetravalent bispecific antibody, exhibited indicated a potentially more immunosuppressive environment which þ potent in vitro CD137-mediated T-cell activation upon engagement of could dampen the damaging effect of activated CD8 T-cell accumu- PD-L1. No cross-reactivity was observed to mouse CD137; therefore, a lation, shown to otherwise lead to hepatocyte death (21). mouse surrogate molecule was developed. Surrogate FS222 outper- Due to the potential preclinical limitations of surrogate molecules formed CD137 and PD-L1 monospecific mAbs as monotherapies or in and mouse models for predicting CD137-induced liver toxicity, we ran combination in multiple syngeneic mouse tumor models. a non-GLP PK/PD toxicity study of single and repeat dosing of FS222 No liver pharmacology or toxicity, previously reported with other in cynomolgus monkeys. CD137 agonist mAbs, was observed with FS222 or the mouse surro- FS222 elicited immune activation with no liver toxicity in a gate. Contrasting observations of liver toxicity in the clinic, with preliminary toxicity study in cynomolgus monkeys CD137 mAbs urelumab and utomilumab, suggest that targeting of CD137 is not an intrinsically toxic pathway for therapy, but the way it is FS222 was shown to be fully cross-reactive in cell binding assays and targeted is crucial. Urelumab is a potent fully human IgG4 antibody primary immune cell functional assays using PBMCs from human or but causes dose-dependent and on-target liver toxicity, whereas cynomolgus blood (Supplementary Fig. S4A–S4D). Therefore, the PK utomilumab demonstrates no dose-limiting toxicity but weaker poten- behavior of FS222 was characterized in cynomolgus monkeys after cy on a human IgG2 backbone. FS222, although a human IgG1, had no intravenous administration of FS222 in a non-GLP dose-range finding Fc-mediated effector function, and its potent CD137 activity was study (Fig. 6A). FS222 displayed a dose proportional increase in C max dependent upon PD-L1 expression. This resulted in a highly active and AUC (0–168 hours; Supplementary Table S5) and linear plasma molecule as seen in vitro and in vivo in multiple syngeneic tumor clearance (at doses ≥ 1 mg/kg; Fig. 6A). FS222 had a mean terminal models, with no liver toxicity. Furthermore, the results from our half-life of approximately 148 hours, which is in line with human preliminary toxicology study indicated that FS222, which is cross- antibodies in monkeys targeting PD-L1 (atezolizumab BLA #761034 reactive with cynomolgus monkeys and has the same in vitro potency Pharmacology Review). In general, FS222 PK followed a linear dose in this species and human, had potent in vivo pharmacologic activity in response (at dose levels ≥ 1 mg/kg) and clearance rates (CLp), and the the cynomolgus monkey and is well tolerated up to 30 mg/kg. volumes of distribution were similar between animals (Supplementary Despite being able to bind cell-expressed human CD137, the Fcab Tables S6 and S7). FS222 was generally well tolerated up to 30 mg/kg component of FS222 was unable to cluster and activate CD137 in the dosed weekly as determined by clinical chemistry and histopathology absence of PD-L1–mediated cross-linking, a significant safety feature results (Table 3). of the molecule. Coupled with directing CD137 activity to areas of As shown in Fig. 6B, the levels of serum soluble PD-L1 (sPD-L1) PD-L1 expression, for example tumor microenvironments, FS222 is were quantified as a measure of direct target engagement and indic- designed to overcome the adverse side effects associated with CD137 ative of downstream cell activation (22). Increased serum sPD-L1 agonists currently in the clinic. This is further strengthened by levels were observed in all animals on day 1, with an apparent peak at reducing FcgR binding and allows FS222 to not rely on FcgR- 168 hours after end of infusion, following which the levels declined in expressing cells to provide the cross-linking necessary for CD137 line with the decline in the systemic levels of FS222. Repeat admin- clustering in current mAb therapies. The combination approach istration of FS222 resulted in prolonged increase in serum sPD-L1 in adopted for in vitro experiments of using two separate monospecific animals that were shown to have no or low levels of antidrug anti- antibodies relied not only on releasing the PD-1/PD-L1 blockade via bodies. Consistent with the findings of the study to assess the PD one antibody, but also on maximum cross-linking of CD137(20H4.9) response of the surrogate FS222 in a syngeneic mouse tumor model mAb by a hCH2-specific mAb. For this combination in the clinic, the described previously, a drug-related increase in cell proliferation and þ natural cross-linking mechanism would be via FcgR cross-linking via activation was also observed in NK cells (Fig. 6C) and CD4 and þ the Fc region of IgG4-based urelumab (20H4.9). Not only are FcgR- CD8 central memory T cells (Fig. 6D and E). In many animals, Ki67 expressing cells found throughout the body, therefore bringing anoth- expression reached plateau at day 11, remained high at day 15, and er significant safety concern for aberrant IgG cross-linking and CD137 deceased progressively to reach baseline expression between days 18 agonism, they are also varied in prevalence with diverse FcgR expres- and 22 with a maximum response being observed between 3 and sion levels, making them an unreliable source of cross-linking– 10 mg/kg. A moderate but transient increase in the relative percentage þ þ dependent activation within a tumor (23). Therefore, FS222 mitigated and absolute counts of CD4 FoxP3 Tregs was also seen (Supple- high systemic toxicity and variable antitumor activity by not relying on mentary Fig. S4E). FcgR cross-linking for potent site-specific activity. FS222 also did not Table 3. Changes in clinical chemistry parameters relating to liver rely on Fc-mediated cell killing as a mechanism of action on account of fi function of cynomolgus monkeys in FS222 repeat dose phase. signi cantly reduced FcgR binding. This resulted in highly potent activity which did not come at the loss of important CD137- or PD-L1– Lower limit Upper limit expressing immune cells that can then potentiate the cell-mediated of normal of normal tumor killing action. range FS222 range FS222 showed potent activity in human primary T-cell assays, but only when PD-L1–expressing cells were present. There was also AST (U/L) 20 23–69 94 – no activity when the CD137 Fcab was paired with an irrelevant, ALT (U/L) 21 19 111 112 – ALP (U/L) 140 485–1,310 1,350 non PD-L1 binding Fab domain, HelD1.3. Surrogate FS222 had in vitro TBIL (mg/dL) 0.06 0.07–0.38 0.43 comparable activity to FS222, therefore justifying its use in syngeneic mouse tumor models. We believe the tumor control activity Abbreviations: ALP, alkaline phosphatase; ALT, alanine aminotransferase; AST, shown by surrogate FS222 addresses one of the hurdles of treating a aspartate aminotransferase; TBIL, total bilirubin. PD-L1–insensitive tumor, or one that has become refractory to PD-L1

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therapy. It does this by harnessing PD-L1 target expression in an molecule in mouse and FS222 in cynomolgus monkeys caused a alternative way to exert direct cytotoxic T-cell activation through drug-related increase in T-cell proliferation and activation as mea- CD137 engagement and clustering. The highly immunogenic sured by Ki67 expression. This would indicate that the PD responses MC38 tumor model demonstrated insensitivity to PD-L1 mAb seen in our mouse models translate to the effect of FS222 on cyno- treatment and Fc-disabled CD137 mAb treatment as monothera- molgus monkey T cells. pies at the dose levels employed in this in vivo study. However, In summary, we have developed FS222, a CD137/PD-L1 tetravalent despite also being Fc-disabled, treatment with surrogate FS222 bispecific antibody with a novel mode of action, and potentially resulted in complete tumor eradication and 100% animal survival. improved therapeutic index for the treatment of human cancer. FS222 In this model, PD-L1 expression, presumably in the tumor micro- did not cause evident toxicity in cynomolgus monkeys upon repeated environment, provided a setting in which surrogate FS222 can exert dosing which we believe further encourages clinical development superior activity and efficacy to either monotherapy. This is clearly targeted at tumors where a significant unmet medical need exists in through PD-L1–dependent cross-linking of FS222 and CD137 immunotherapy. Checkpoint inhibitors are failing or only providing receptor clustering on T cells resulting ultimately in enhanced modest clinical benefit in many tumor settings, and for many of those, þ tumor-specificCD8 T-cell cytotoxic activity. Surrogate FS222 and we feel there is a mechanistic rationale for improvement in clinical therefore FS222 could be bridging a PD-L1–expressing tumor cell outcomes with FS222. and tumor-infiltrating T cell, localizing T-cell cytotoxic activation to the tumor cell/T-cell interface. The same is true for the significant Disclosure of Potential Conflicts of Interest activity and superiority over monotherapy of surrogate FS222 in the All authors are current or former employees of F-star Therapeutics Ltd. less immunogenic CT26.WT model(24),whichinourhandsis also insensitive to PD-L1 mAb treatment. Authors’ Contributions The PD changes after treatment with surrogate FS222 in a Conception and design: M.A. Lakins, A. Koers, J. Munoz-Olaya, S. Batey, D. Gliddon, CT26.WT tumor–bearing mouse model were investigated after M. Tuna, N. Brewis Development of methodology: M.A. Lakins, A. Koers, R. Giambalvo, M. Tuna administration of a single high (10 mg/kg) and a single low (1 mg/kg) Acquisition of data (provided animals, acquired and managed patients, provided dose. Strikingly, T cells with bound drug were present in the tumor at facilities, etc.): M.A. Lakins, A. Koers, R. Giambalvo, R. Hughes, E. Goodman, 2 hours after administration for both dose levels. These T cells had F. Wollerton prolonged PD-L1 occupancy specifically in the tumor, the longevity, Analysis and interpretation of data (e.g., statistical analysis, biostatistics, but not magnitude, of which was correlated with dose. By the end of the computational analysis): M.A. Lakins, A. Koers, R. Giambalvo, J. Munoz-Olaya, study (8 days after drug administration), intratumoral PD-L1 occu- R. Hughes, E. Goodman, S. Marshall, F. Wollerton, S. Batey, D. Gliddon Writing, review, and/or revision of the manuscript: M.A. Lakins, J. Munoz-Olaya, pancy was still approximately 80% on T cells at 10 mg/kg, whereas R. Hughes, E. Goodman, S. Marshall, F. Wollerton, S. Batey, D. Gliddon, M. Tuna, peripheral PD-L1 occupancy on T cells had decreased substantially. N. Brewis This highlights the potential of surrogate FS222 to locate to the tumor Administrative, technical, or material support (i.e., reporting or organizing data, microenvironment, in preference to remaining in the periphery. constructing databases): M.A. Lakins, S. Marshall Evidence of cytokine production as a consequence of surrogate FS222 Study supervision: M.A. Lakins, M. Tuna, N. Brewis treatment was also observed in the serum of mice treated with 10 mg/kg, Other (Coordinated cynomolgus monkey DRF study and associated data analysis and interpretation): S. Marshall whereas this was less pronounced with the lower dose level perhaps fi indicating ef cacy without systemic cytokine exposure. Acknowledgments Given the relevance of preclinical studies in mice for risk assessment The authors would like to thank the F-star Protein Sciences, in vivo, Assay of severe hepatotoxicity in human patients treated with CD137 agonist Development and Drug Discovery team; Cristian Gradinaru for statistical analyses; agents, the lack of hepatotoxicity in mice in these studies indicates that Jacqueline Doody for scientific contributions; Alison McGhee for critical review; a mAb2 agonizing CD137 via PD-L1–mediated cross-linking has a Babraham BSU staff members for animal husbandry and technical assistance; significantly reduced risk of inducing hepatotoxicity in human patients Dr. Sarah Taplin for pathology assessment; and Dr. Sarah Burl and Natalie Allen treated at therapeutic doses. FS222 had a mean terminal half-life of for article editing and review. approximately 6 days in cynomolgus monkeys, in line with PD-L1– The costs of publication of this article were defrayed in part by the payment of page targeting antibodies such as atezolizumab and followed a linear dose charges. This article must therefore be hereby marked advertisement in accordance ≥ response at dose levels 1 mg/kg. FS222 was generally well tolerated up with 18 U.S.C. Section 1734 solely to indicate this fact. to 30 mg/kg dosed weekly. With comparable potency between the cynomolgus and human immune systems, we believe these findings Received September 11, 2019; revised February 7, 2020; accepted April 13, 2020; will translate successfully to a human setting. Both the surrogate published first April 28, 2020.

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OF14 Clin Cancer Res; 2020 CLINICAL CANCER RESEARCH

FS222, a CD137/PD-L1 Tetravalent Bispecific Antibody, Exhibits Low Toxicity and Antitumor Activity in Colorectal Cancer Models

Matthew A. Lakins, Alexander Koers, Raffaella Giambalvo, et al.

Clin Cancer Res Published OnlineFirst April 28, 2020.

Updated version Access the most recent version of this article at: doi:10.1158/1078-0432.CCR-19-2958

Supplementary Access the most recent supplemental material at: Material http://clincancerres.aacrjournals.org/content/suppl/2020/04/28/1078-0432.CCR-19-2958.DC1

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