Epitope and Fc-Mediated Cross-Linking, but Not High Affinity, Are Critical for Antitumor Activity of CD137 Agonist Antibody with Reduced Liver Toxicity

Published OnlineFirst January 23, 2020; DOI: 10.1158/1535-7163.MCT-19-0608

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Epitope and Fc-Mediated Cross-linking, but Not High Afﬁnity, Are Critical for Antitumor Activity of CD137 Agonist Antibody with Reduced Liver Toxicity Sun K. Ho1, Zhenghai Xu1, Archana Thakur1, Melvin Fox1, Siu Sze Tan1, Enrico DiGiammarino2, Li Zhou3, Mien Sho1, Belinda Cairns1, Vivian Zhao1, Mengli Xiong1, Josue Samayoa1, Charles M. Forsyth1, David B. Powers1, Debra T. Chao1, Diane Hollenbaugh4, Hamsell M. Alvarez1, and Yoshiko Akamatsu1

ABSTRACT ◥ CD137 (TNFRSF9, 4-1BB) agonist antibodies (mAb) have mor efficacy was maintained in Fc gamma receptor (FcgR) III– demonstrated potent antitumor activity with memory response deficient mice but diminished in FcgRIIB-deficient mice, sug- while causing hepatotoxicity in mouse models. In clinical trials, gesting the critical role for FcgRIIB to provide cross-linking the degrees of liver toxicity of anti-CD137 vary from grade 4 in vivo. Interestingly, a single dose of an affinity-reduced variant transaminitis (urelumab) to nonexistent (utomilumab). To was sufficient to control tumor growth, but a higher affinity exploit the antitumor potential of CD137 signaling, we identified variant did not improve efficacy. These observations suggest a new class of CD137 agonist mAbs with strong antitumor that binding epitope and FcgR interaction, but not necessarily potency without significant transaminitis in vivo compared with high affinity, are important for antitumor efficacy and reduced CD137 agonists previously reported. These mAbs are cross- liver toxicity of CD137 mAb. Our study suggests the possibility reactive to mouse and cynomolgus monkey and showed cross- of CD137 agonist therapy with improved safety profile in linking–dependent T-cell costimulation activity in vitro.Antitu- humans.

Introduction T cells (13, 14). In addition, it has been shown that a cytoplasmic domain of CD137 engineered into chimeric antigen receptor T cells CD137 (TNFRSF9, 4-1BB) is a type I transmembrane receptor, a (CAR-T) enhances the survival and induces development of a member of the tumor necrosis factor receptor superfamily. It is a T-cell central-memory phenotype (15). costimulatory molecule which is highly expressed on activated T cells Agonistic CD137 antibodies (mAb) have demonstrated strong and natural killer (NK) cells, while constitutively expressed at a lower antitumor activity in many mouse tumor syngeneic models. For level on monocytes, neutrophils, and dendritic cells (1, 2). CD137 is examples, 1D8 regressed tumor growth in sarcoma (16), glioma (17), also expressed on lung and liver endothelial cells (3), tumor capillar- and lymphoma models (18). 2A demonstrated efficacy in colon ies (4), and activated articular chondrocytes (5). In comparison, carcinoma, lymphoma (19), and myeloma models (20). 3H3 and CD137 ligand (CD137L, TNFSF9) is expressed on activated B cells, MAB9371 showed efficacy in both CT26 and MC-38 models (21). dendritic cells, and macrophages (6). Binding of CD137 with its ligand Most of these mAbs have been used in combination studies and or with an agonistic monoclonal antibody (mAb) induces clustering of demonstrated synergistic effect with checkpoint inhibitors such as the receptors and recruitment of specific TNF receptor–associated anti-CTLA4 (22) and anti–PD-1 (21, 23), chemotherapeutics (24, 25), factors (TRAF1, TRAF2, and TRAF3; ref. 7). Association of these radiotherapy (26), and vaccines (27). However, all these CD137 adaptor proteins in the cytoplasmic domain of CD137 induces mAbs are known to increase transaminases in mice when dosed activation of different T-cell signaling pathways (NF-kB, ERK, repeatedly (28, 29). JNK, and p38 MAPK), upregulation of Bcl-xL, and inhibition of Interestingly, 2 anti-CD137 in clinical trials, urelumab (BMS- proapoptotic protein BIM (8–10). CD137 signaling results in T-cell þ 663513), a fully human IgG4 antibody, and utomilumab (PF- proliferation, cytokine release, enhancement of CD8 T-cell cyto- 05082566), a fully human IgG2 antibody, have shown distinct liver toxicity, and protection from activation-induced cell death. CD137 þ toxicity profiles. The maximum tolerable dose of urelumab is low signaling also enhances antigen-specificmemoryCD8 T-cell þ (0.1 mg/kg every 3 weeks) due to the tendency to cause transamini- accumulation (11, 12) and rescues nonresponsive, anergic CD8 tis (30), which may result in a suboptimal therapeutic window. In contrast, utomilumab can be dosed up to 10 mg/kg every 4 weeks without hepatotoxicity, but it had limited clinical activity in solid 1AbbVie Biotherapeutics Inc., Redwood City, California. 2AbbVie Inc., North Chicago, Illinois. 3AbbVie Bioresearch Center, Worcester, Massachusetts. tumors [3.8% objective response rate (ORR)] and Merkel cell carci- 4Former AbbVie Employee. Good Therapeutics, Inc. Seattle, Washington. noma (13.3% ORR) as a single agent (31). Given the limitations of the current CD137 mAbs in clinical trials, Note: Supplementary data for this article are available at Molecular Cancer Therapeutics Online (http://mct.aacrjournals.org/). we have pursued opportunities to identify CD137 mAbs with potent antitumor efficacy and reduced liver toxicity. Under the assumption Corresponding Author: Yoshiko Akamatsu, AbbVie Biotherapeutics Inc., 1500 that the mechanism of liver toxicity is conserved between human and Seaport Boulevard, Redwood City, CA 94063. Phone: 650-454-2007; E-mail: [email protected] mouse, CD137 agonist mAbs with human and mouse cross-reactivity were selected based on their antitumor efficacy and reduced liver Mol Cancer Ther 2020;XX:XX–XX toxicity in mouse syngeneic models. In this article, we report the doi: 10.1158/1535-7163.MCT-19-0608 identification of a novel class of CD137 mAbs and characterization of 2020 American Association for Cancer Research. their functions in vitro and in vivo.

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Materials and Methods volumes reached a maximum of 2,500 mm3 or if animal health was Mice compromised, per IACUC-approved protocol. Female, 5- to 6-week-old BALB/c and C57BL/6 mice were pur- Liver immunohistochemistry (IHC) chased from Charles River Laboratories. FcgRIIB knockout mice Mouse livers were perfused with PBS prior to harvesting and (C.129S4(B6)-Fcgr2btm1TtK/cAnNTac N12) were purchased from processed as formalin-fixed paraffin-embedded blocks. The sections Taconic, and FcgRIII knockout mice (B6.129P2-Fcgr3tm1Sjv/J) were were stained using rat anti-mouse CD45 (BD), rabbit anti-CD8 (Cell purchased from The Jackson Laboratory. Mice were housed under Signaling Technology), and rabbit anti-Iba-1 (Wako) after the antigen specific pathogen-free conditions in an AAALAC accredited facility. retrieval process. The sections were then stained using a standard IHC All mouse procedures were performed in accordance with protocols processing protocol with secondary mAbs; Envision-HRP (Dako) for approved by the internal Institutional Animal Care and Use Com- CD8 and Iba-1 and a rat anti-mouse-HRP for CD45. Images were mittee (IACUC). captured by Aperio AT2 digital scanner (Leica) and analyzed by HALO imaging analysis system. Hybridoma generation and screening Rat monoclonal mAbs were generated according to standard Human T-cell costimulation assay hybridoma technology. Recombinant mouse and human CD137- A 96-well plate was coated with 0.5 mg/mL anti-CD3 (OKT3, human Fc fusion (Creative Biomart) and mouse CD137-His proteins eBioscience) in PBS. The plate was washed with assay medium were generated in-house and used as immunogens. Hybridoma were (AIM-V, Life Technologies) and blocked dry before adding 50 mLof fi screened for antigen-speci c binding as well as for the ability to CD137 mAb at 30 mg/mL and 50 mL of medium with or without Fc activate NFkB by a reporter assay using HEK-293 cells transduced cross-linker (F(ab')2 fragment of human Fcg-specific goat polyclonal þ with pLenti-NFkB-Luciferase vector and a plasmid expressing human mAbs, Jackson ImmunoResearch Labs) at 120 mg/mL. CD8 T cells CD137. isolated by negative selection (Stem Cell Technologies) from periph- eral blood mononuclear cells of healthy donors were then added (105 Cell lines and antibodies cells/well). In some experiments, 4 104 CHO-K1 cells expressing þ CT26, B16-F10, CHO-K1, and HEK-293 were purchased from the FcgRs were seeded overnight as cross-linkers and 105 CD8 T cells, ATCC, and MC-38 cell line was obtained from the University of CD137 mAb, and OKT3 (0.5 mg/mL) were added for a final volume of Chicago under a license agreement with the NIH (Rockville, MD). Cell 200 mL. Plates were incubated for 3 days, and supernatants were lines overexpressing FcgRIIB and human or murine CD137 were collected for cytokine analysis. Cells were pulsed with 0.5 mCi/well generated by stably transfecting CHO-K1 and HEK-293, respectively, methyl-[3H] thymidine for 6 hours and proliferation was assessed by and maintained in the media containing 0.5 mg/mL G418. All cell lines thymidine incorporation (Wallac 1450 MicroBeta Liquid Scintillation were tested negative for mycoplasmas and banked after purchasing or Counter). genetic modifications. They were maintained in culture for no more than 10 passages from master cell banking. Mouse T-cell costimulation assay CD137 mAbs 106, 107, and their isotype variants, murine IgG1 A 96-well plate was coated with 1 mg/mL of anti-CD3 (145-2C11, þ isotype control, and human IgG1 isotype control were produced in- BD) in PBS. CD8 T cells were isolated from the splenocytes using a þ house as described in PCT/US2017/034687 (106 and 107 were stated as mouse CD8 T-cell isolation kit (STEMCELL Technologies) from TABBY106 and 107, respectively). Sequence of the 1D8 was from US spleens of na€ve BALB/c mice and resuspended in media to 5 105 þ patent#7,754,209 B2 (SEQ ID Nos.: 101 and 103). 3H3 and rat IgG1 cells/mL. CD8 T cells (100 mL) with 1 mg/mL of anti-CD28 (37.51, were purchased from Bio X Cell. MAB9371 was purchased from R&D BD) were added to each well. CD137 mAbs were mixed with F(ab')2 Systems. Anti-human CD137 control reagents, Mab A (20H4.9) and fragment of rat or mouse Fcg-specific goat polyclonal mAbs used as Mab B (MOR.7480.1), were expressed using published sequences in US cross-linkers (Jackson ImmunoResearch Labs) at a molar ratio of 1:4 patent#7,288,638 and PCT/US2012/032704, respectively. Rat 106 and incubated at room temperature for 1 hour. The mixture was mAb was humanized using VH1-69 and Vk6-57 frameworks as serially diluted in 100 mL and then added to the cells and incubated for acceptors with rodent complementarity-determining regions (CDR) 66 hours before supernatants were collected for cytokine measure- fi along with back mutations (106M). 106L is a low-af nity variant that ment. The methyl-[3H] thymidine incorporation measurement was contains an amino acid substitution in a CDR, whereas 106H was carried out as previously described. isolated from yeast display library and contains several amino acid substitutions in CDRs. All constructs were subcloned into in-house Flow cytometry fi expression vectors and puri ed by protein A from the supernatant of Single-cell suspensions from tumors were prepared by digesting HEK293-6E cells that had been transiently transfected with heavy and tumors with a mouse Tumor Dissociation Kit in combination with the light chain vectors as previously described (32). gentle MACS Octo Dissociator system (Miltenyi Biotec). Fluorescent- conjugated mAbs used in flow cytometry analysis were purchased Syngeneic tumor models from BD: CD45 (30–11); CD3 (145-2C11); CD4 (cRM4-5); CD8 (53- Log-phase cells of CT26 (2.5 105), MC-38 (5 105), and B16-F10 6.7); CD11b (M1/70), CD25 (PC61); CD49b (DX5), CD62L (MEL-14); (2.5 105) were implanted by subcutaneous (s.c.) injection in the right B220 (RA3-6B2), Ly6G (1A8), Ly6C (AL-21), F4/80 (T45-2342), and flank of BALB/c or C57BL/6 mice. Tumor-bearing mice were ran- FoxP3 (MF23). Dead cells were excluded from analysis using the domized into treatment groups based on mean tumor volume of 100 Fixable Viability Dye eFluor 780 (Thermo Fisher Scientific). Binding of mm3 and were treated with CD137 mAb or isotype control (murine or mouse anti-human CD137 mAbs were detected by PE-conjugated goat d rat) by intraperitoneal (i.p.) injections. Tumor volume was monitored F(ab')2 anti-mouse IgG (Jackson ImmunoResearch). H-2L MuLV using electronic calipers and calculated according to the formula: V ¼ gp70 Tetramer-SPSYVYHQF was purchased from MBL. Flow data 1 /2 length width height. Mice were euthanized when tumor were analyzed by FlowJo V10.

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Epitope grouping CD137, Mab A, suggesting they bind to a unique epitope. Interestingly, Epitope grouping of anti-CD137 was determined by competition 106 and 107 mAbs compete with a ligand blocking anti-human assays using Biacore T200 instrument (GE Healthcare) at 12C to slow CD137, Mab B. These results suggest the epitopes of 106 and 107 the off-rate of mAb: antigen interactions. Recombinant human and mAbs are different from that of Mab B even though they partially mouse CD137 and CD137 ligand proteins (His-tags) were produced overlap. in-house (HEK-293). Chip preparation and binding kinetic measure- To further investigate the efficacy and liver toxicity of 106 mAb, we ments were made in the assay buffer HBS-EPþ (10 mmol/L HEPES, performed a dose titration study in CT26 tumor model using 3H3 as a pH 7.4, 150 mmol/L NaCl, 3 mmol/L EDTA, 0.05% Tween 20). control CD137 mAb. Compared with 3H3, 106 mAb showed a similar Approximately 2,000 RU of goat anti-mouse or anti-rat IgG Fc dose-dependent antitumor activity (Fig. 2A) while induced lower polyclonal mAb (Thermo Fisher Scientific), diluted to 25 mg/mL in serum ALT levels (Fig. 2B). It has been previously suggested that 10 mmol/L sodium acetate (pH 4.5), was directly immobilized across a CD137 mAbs caused liver damage by increasing the infiltration of þ CM5 biosensor chip using a standard amine coupling kit according to CD8 T cells and macrophages (28, 33), so we examined liver tissues the manufacturer's instructions. Each assay cycle consisted of the from the treated animals to evaluate the level of immune cell infil- following steps: (i) capture of first test mAb at 10 mg/mL on test tration. Livers from 3H3 treated mice showed higher number of þ þ surface only; (ii) blocking injection of isotype control cocktail at infiltrated leukocytes including CD8 T cells and Iba1 macrophages 100 mg/mL over both reference and test surface; (iii) analyte injection; in comparison with 106 mAb (Fig. 2C). Plasma concentrations of 106 (iv) second test mAb injection at 10 mg/mL; (v) regeneration of and 3H3 mAbs 24 hours after last dose were comparable (Supple- capture surface by 10 mmol/L Glycine-HCL, pH1.5 injections. mentary Fig. S2), suggesting that the drug exposure is not a factor for the differences in liver toxicity. These data demonstrated the antitumor Measurement of liver enzymes and cytokines/chemokines in activity and liver toxicity with anti-CD137 agonist treatment can be serum segregated in mice. Blood chemistry was measured by VetScan VS2 analyzer (ABAXIS) using Mammalian Liver Profile rotors. Human or mouse cytokines/ In vivo antitumor activity is Fc and Fc gamma receptor (FcgR) chemokines in culture or serum samples were analyzed using the IIB dependent MILLIPLEX MAP Human or Mouse Cytokine/Chemokine kit (Milli- Fc-mediated biology is known to have an important role in driving pore) and measured by FLEXMAP 3D (Luminex). antitumor efficacy by immunomodulatory mAbs. To assess if the in vivo activity of our CD137 mAbs is Fc dependent, we first compared Statistical analysis the antitumor potency induced by a murine 107 IgG2a and 107 IgG2a Statistical significance between multiple groups was determined by DANA (D265A-N297A, mutant that eliminates FcgR binding) in one-way ANOVA (single variable) or two-way ANOVA (two vari- CT26 and MC-38 syngeneic tumor models. As shown in Fig. 3A ables). Student t test (unpaired, two-tailed, 95% confidence interval) and B, an IgG2a has stronger antitumor activity than the IgG2a DANA was used to compare 2 groups with one variable. For tumor survival variant of 107, which indicates that FcgR-mediated activity is impor- analysis, Kaplan–Meier test was used. Error bars indicate SEM. All tant for in vivo antitumor activity. Murine IgG1 consistently showed graphs and statistical analysis were generated using Prism 7 software strong efficacy in both models; however, no statistical difference was (GraphPad Software). observed between IgG1 and IgG2a under the condition tested. A similar result was observed in 106 mAb (Supplementary Fig. S3C). Interestingly, 107 with a murine IgG1 resulted in more tumor-free Results mice than IgG2a-treated mice (5/8 vs. 0/8 in CT26 and 2/8 vs. 0/8 in Novel CD137 mAbs 106 and 107 demonstrated strong antitumor MC-38 for IgG1 and IgG2a, respectively). It is also important to note activity with low alanine aminotransferase (ALT) level that the serum ALT remained low in both BALB/c or C57BL/6 mice Because CD137 mAbs are known to induce transaminitis in both treated with 106 and 107 (Supplementary Fig. S3A, S3B, and S3D), human and mouse, we hypothesized that the biological pathway indicating that they induce less liver toxicity. We observed a slight causing the liver cell damage may be conserved between human and increase of ALT levels when MC-38 tumors in C57BL/6 were treated mouse. Therefore, it is critical to identify mAbs that cross-react to with either 106 or 107 in IgG1 but not with IgG2a (Supplementary mouse in order to screen in vivo for potential clinical CD137 agonists Fig. S3B and S3D). Similarly, a small increase in ALT levels compared with strong antitumor efficacy and low liver toxicity using mouse with control group was observed when CT26 and MC-38 tumors syngeneic tumor models. Three selected anti-CD137 were tested in the were treated with 106 mouse G1 (Fig. 2B and Supplementary CT26 syngeneic model for their antitumor efficacy and liver function Fig. S3D). It is important to note that these increases in liver enzyme measured by the level of liver enzyme ALT in serum (Fig. 1A and B). levels are 10-fold lower than those observed in serum treated with Even though all 3 clones were similarly effective in controlling the 105 (mean 529 U/L) or 3H3 mAbs (mean 388 U/L), Fig. 1B tumor growth, mAb 105 treatment showed increased ALT level but not and Fig. 2B, respectively. Thus, we conclude that these CD137 mAbs mAbs 106 or 107 treatment under the condition tested (10 mg/kg, q7d )106and 107) induce significantly reduced liver toxicity compared 3). Both 106 and 107 mAbs showed cross-reactivity to human, with typical CD137 agonist mAbs reported elsewhere, regardless of cynomolgus monkey, and mouse CD137 (Supplementary Table S1). mouse genetic background. Furthermore, the epitopes of 106 and 107 clones were characterized by Because both murine IgG1 and IgG2a engage FcgRIIB and III, we competition to other mAbs and CD137 ligand (Fig. 1C and Supple- evaluated whether FcgR interactions are critical for the bioactivity of mentary Fig. S1). CD137 mAbs 106 and 107 compete with each other CD137 mAbs using FcgR knockout mice. Efficacy was completely lost but not with either human or mouse CD137 ligand, suggesting these 2 for both IgG1 and IgG2a when CT26 tumor was grown and treated in mAbs share similar epitopes that do not overlap with the ligand FcgRIIB / background (Fig. 3C). In MC-38, the difference between binding site. 106 and 107 mAbs also did not compete with other 107 IgG1 and IgG2a was insignificant (Fig. 3B) while they remained anti-mouse CD137 (1D8, 3H3, and MAB9371) or an anti-human active in FcgRIII / mice (Fig. 3D). Because murine IgG1 engages

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Figure 1. Characterization of novel anti-CD137 mAbs in vivo and in vitro. A, Efﬁcacy study of anti-CD137 in CT26 mouse syngeneic tumor model. BALB/c mice were inoculated s. c. with CT26 cells, and when mean tumor volume reached 100 mm3, mice were treated i.p. with 10 mg/kg of rat IgG1 control and rat CD137 mAbs once per week for 3 weeks (arrows). N ¼ 6 mice per group. B, Serum ALT level in treated animals at the end of the study on day 40. C, Simultaneous binding assay to bin epitopes via Biacore. Assay schematic is shown (left) and resulting binding matrix (middle) for human (top) and mouse (bottom) CD137; (þ) indicates simultaneous binding occurs, whereas () does not. Results are also shown as Venn diagrams (right) to illustrate separate and overlapping epitopes. 106 and 107 mAbs do not compete with Mab A, 1D8, 3H3, 9371, mouse or human CD137L, but do compete with Mab B. Actual sensorgrams are shown in Supplementary Fig. S1. Rat chimeric 106 and 107 with human IgG1 constant region were used for epitope binning. See Materials and Methods about tool mAbs used for comparison.

only FcgRIIB and FcgRIII, FcgRIIB that remained in the FcgRIII / independent activity, T-cell costimulation of Mab A, Mab B, 106, and mice can still provide sufficient activity to control MC-38 tumor 107 was also compared with IgG4 (Supplementary Fig. S4A). Only growth. These results demonstrated that FcgRIIB is crucial for induc- Mab A showed activity without cross-linking, indicating the activity is ing antitumor activity in mice in vivo. due to its intrinsic epitope instead of Fc isotype being G4. To further confirm the effect of Fc-mediated cross-linking, different IgG variants mAbs 106 and 107 require Fc cross-linking for T-cell activation of 106 and 107 mAbs were compared in a T-cell costimulation assay in in vitro the presence of FcgRIIB expressed on CHO-K1. The human IgG4 To investigate the intrinsic costimulation activity of 106 and 107 isotype induced higher IFNg secretion than the IgG1 and IgG2 isotypes þ mAbs, a mouse CD8 T-cell costimulation assay was performed with for both mAbs (Fig. 4C). In the absence of FcgRIIB-expressing CHO- 3H3 mAb as a positive control. In the absence of Fc cross-linking, only K1 cells, no activity was observed for all variants. These data indicate 3H3 mAb has significant bioactivity, indicating 106 and 107 mAbs are that 106 and 107 mAbs require Fc cross-linking to induce T-cell not strong agonists intrinsically; however, in the presence of anti-rat costimulation activity, and human IgG4 is much more efficient than IgG as cross-linkers, 106 and 107 mAbs induced GM-CSF cytokine IgG2 in mediating the activity in the presence of FcgIIB. þ release (Fig. 4A). Similarly, in a human CD8 T-cell proliferation assay, Mab B-IgG2, 106-hIgG1AA (rat mAb chimeric with human In vivo pharmacodynamics analysis of anti-CD137 agonists in IgG1 Fc with L234A/L235A mutations for reduced FcgR binding), and mouse syngeneic models 107-hIgG1AA induced T-cell proliferation following the effect of To understand the mechanism of action of 106 and 107 mAbs cross-linking with a secondary mAb, except for Mab A (IgG4 or in vivo, we used an MC-38 tumor model and included 1D8 mAb as the IgG1AA), which is active even in the absence of cross-linking (Fig. 4B). positive control to allow comparison as mouse IgG1. 1D8 mAb is also These data suggested that, similar to Mab B, 106 and 107 mAbs require known to induce liver toxicity similar to 3H3 mAb (29). Consistent Fc cross-linking to function as T-cell costimulators, whereas Mab A is a with previous results (Fig. 3B), i.p. injection of IgG1 isotypes of 106, strong agonist that is capable of inducing signal by itself. In order to 107, and 1D8 mAbs at 10 mg/kg on days 7, 14, and 21 resulted in exclude the possibility that IgG4 isotype induces cross-linking– significant regression of established MC-38 tumors (Fig. 5A). Systemic

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Figure 2. Novel CD137 mAb demonstrated potent antitumor efficacy with reduced liver toxicity. A, In vivo efficacy of 106 (murine IgG1) was compared with 3H3 (rat IgG2a) at 10 mg/kg, 1 mg/kg, or 0.1 mg/kg, dosed i.p. once per week for 3 weeks, in CT26 syngeneic tumor model. N ¼ 8 mice per group. B, Serum ALT level in treated animals at the end of the study on day 35. C, Liver IHC showing infiltration of CD45þ, CD8þ, and Ibaþ cells on day 35. Liver biopsies of animals treated with 10 mg/kg mAbs were shown. Percent stain-positive tissue area were quantitated by HALO imaging analysis. Data represent mean SEM; , P < 0.05; , P < 0.01; , P < 0.0001 using one- way ANOVA.

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Figure 3. Antitumor activity of CD137 mAb is FcgRIIB-dependent. A and B, Antitumor activity of 107 in mouse IgG1, IgG2a, and IgG2a DANA was compared in CT26-BALB/c (A) and MC-38-C57BL/6 (B) syngeneic tumor models. When mean tumor volume reached 100 mm3, mice were treated i.p. at 10 mg/kg once per week for 3 weeks. Tumor volumes of individual animal of each group were shown on the right. Each line represented a single animal. Numbers of tumor-free mice at the end of the study are shown in parenthesis. C and D, Efficacy of 107 mAb was lost when FcgRIIB-deficient mice were used as a host (C); however, it was not affected in FcgRIII-deficient mice (D). Mean tumor volume was shown. Number of mice for each group: 10; ns ¼ not significant.

administration of 106 and 107 mAbs resulted in significant decrease of the circulation 24 hours after the second and third doses (days 15 and þ tumor-infiltrating CD4 T cells at early time point (day 15; Fig. 5B). day 22, respectively); while in the tumor-draining lymph nodes, All 3 CD137 mAbs induced a significant increase of tumor-infiltrating changes in immune cell populations were usually small and transient þ CD8 T cells at later time point (day 22). There was a slight decrease in between the control group– and CD137 mAb–treated groups (Sup- þ T regulatory cells (Treg) (106 mAb at day 15) and an increase of CD8 / plementary Fig. S5A). Treg ratio, although they are not statistically significant at day 22. We Serum cytokine and chemokine levels were measured 24 hours after also observed that total T cells, B cells, and NK cells were decreased in the second (day 15) and third doses (day 21). No significant changes

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Figure 4. In vitro activity of 106 and 107 mAbs requires Fc cross-linking. A, Mouse CD8 T-cell costimulation (mGM-CSF secretion) of 3H3 (rat IgG2a), 106 and 107 mAbs (rat IgG1) in the absence and presence of anti-rat Fc cross-linkers. B, Human CD8þ T-cell proliferation of Mab A, Mab B, 106, and 107 mAbs with human constant regions in the absence and presence of anti-human Fc cross-linkers. C, 106/107 mAbs with different human IgG isotypes displays different capability of inducing T-cell costimulation (measured by IFNg release) in the presence of FcgRIIB transfected CHO cells. , P < 0.0001 using one-way ANOVA. Data are representative of 2 independent experiments.

fi were observed in the levels of IL2, TNFa, and IL6 cytokines in sera after af nity mutant 106H with a KD (dissociation constant) of 0.54 nmol/L treatment (Supplementary Fig. S5B). Detectable levels of IFNg, IL10, and a low-affinity variant 106L with a KD of 220 nmol/L (KD values MIG, MCP-1, MIP-1b, and RANTES were identified in groups treated were determined by Biacore; see Supplementary Table S1) generated with CD137 mAbs (Fig. 5C). The level of these cytokines/chemokines by amino acid substitutions in CDRs. In comparison, the parental 106 fi seems to increase from day 15 to day 22. mAb (106M) has an intermediate KD of 17 nmol/L. Binding af nities An antigen-specific memory T-cell response has been demonstrated were also confirmed by flow cytometry using CD137 stable transfec- in CD137 mAb–treated CT26 mice based on tumor rechallenge studies tant (Fig. 6A). T-cell proliferation assay showed high-affinity 106H following treatment with mIgG1 isoforms of 106 and 1D8 mAbs at induces a strong T-cell activation, whereas low-affinity 106L has very 10 mg/kg on days 7, 14, and 21 (Supplementary Fig. S5C). Accumu- limited bioactivity, which indicates a direct correlation between affin- þ lation of antigen-specific CD8 T cells in the spleen has also been ity and in vitro activity (Fig. 6B). In vivo activity of these variants observed by gp70–peptide–MHC tetramer staining (34) following was tested using the CT26 tumor syngeneic model. Interestingly, treatment with the 106 mAb (Fig. 5D). the efficacy of 106L was comparable with parental and high-affinity mAbs following a single-dose treatment at high concentration (3 mg/- CD137 mAb binding affinity and in vitro activity may not kg; Fig. 6C). Surprisingly, at low dose (0.3 mg/kg), parental (106M) correlate with in vivo efficacy and hepatotoxicity and low-affinity mAb (106L) controlled tumor growth better than the Although binding epitope is critical to its liver toxicity, it is possible high-affinity 106H without causing significant increase in ALT. All of that affinity difference plays a role for the activity of CD137 agonist these mAbs slightly increased the level of serum ALT at 3 mg/kg, less mAbs. To investigate whether the affinity of a CD137 mAb would also than 2-fold of the control (Fig. 6D), which is consistent with the results affect liver toxicity and affect its functional activity, two 106 variant in Fig. 2B at weekly dose. These results suggest the affinity of CD137 mAbs with different CD137 binding affinity were created: a high- mAbs may not play a major role in hepatotoxicity, and a low-affinity

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Figure 5. Efficacy and pharmacodynamics analysis of CD137 agonistic mAbs in MC-38 mouse syngeneic model. A, Invivo efficacy of 106, 107, and 1D8 mAbs in MC-38. n ¼ 7 mice per group. B, Frequency of intratumoral CD4þ, CD8þ, Treg (CD4þCD25þFoxP3þ), and CD8þ/Treg ratio 24 hours after the second (day 14) and third dose (day 21), in relative to live CD45þ cells in each tumor. Dotted line represents the mean value of isotype control. C, Serum cytokine and chemokine levels (pg/mL) measured at 24 hours after the second (day 14) and third dose (day 21). N ¼ 4 mice per group. D, Accumulation of gp70þ antigen-specific CD8þ T cells following CT26 rechallenge in anti-CD137-treated mice. Splenocytes from mice that showed a total tumor regression following treatment with 106 and 1D8 mAbs and repelled CT26 rechallenge were stimulated with gp70 peptide in vitro for 4 days and then stained using a gp70–MHC tetramer. Statistics were generated with Student t test. , P < 0.05; , P < 0.01; , P < 0.001.

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Figure 6. In vitro binding affinity and activity do not predict in vivo efficacy. A, Flow cytometry binding of 106 affinity variant mAbs, high affinity: 106H; medium: 106M; low: 106L. B, Activity of 106 affinity variant mAbs was tested in an in vitro murine CD8 T-cell proliferation assay. C, In vivo efficacy of 106 affinity variant mAbs in CT26 syngeneic mouse model dosing at 3 mg/kg and 0.3 mg/kg, respectively. , P < 0.001 by two-way ANOVA. D, Serum ALT levels in mice treated with 106 affinity variant mAbs. Dotted line represents the average value of control group. , P < 0.05; , P < 0.01 by paired Student t test. Na€ve: samples from mice with no tumor. Data are representative of 2 independent experiments. mAb might be more efficacious in vivo in a suboptimal dose. Finally, found that cross-linking dependency of an agonistic mAb to induce these results clearly indicate that binding affinity and bioactivity CD137 signaling could be independent from the ability to compete in vitro do not predict in vivo antitumor efficacy with CD137 mAbs with ligand binding. Although 106/107 mAb and Mab B compete in in mice. binding to CD137, they likely bind to different epitopes because 106/107 do not compete with ligand binding while Mab B does. This could be potentially explained by the differences in conformational Discussion change induced by mAb binding; further study such as X-ray Based on the observation that anti-CD137 induced transaminitis in cocrystallography will be necessary to understand the molecular both mouse and human, we used mouse syngeneic models to screen for mechanism. lead candidates with low liver toxicity and potent antitumor efficacy. In Bioactivity of CD137 mAb described in this study is dependent on this study, we successfully generated and identified 2 rat mAbs, 106 and FcgRIIB in vivo, which is in agreement with mAbs against CD40 (35) 107, which cross-react to human, cynomolgus monkey, and mouse and DR5 (36). In the literature, 2 other CD137 mAbs have been tested CD137. These mAbs have unique properties that are important for in FcgRIIB / mice. First, clone 2A (rat IgG2a) was shown to be improved safety while maintaining strong antitumor potency in vivo. efficacious in FcgRIIB / mice in an EL4E7 lymphoma model (37). TNFR superfamily signaling depends on receptor clustering and Second, clone LOB12.0 (mIgG1) showed enhanced efficacy in conformation change induced by ligand binding. In this study, we FcgRIIB / mice in a CT26 syngeneic tumor model (38). The

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antitumoractivityofLOB12.0wasreported to dependent primarily part due to a Treg-mediated anti-inflammatory response or may also þ on depletion of intratumoral Tregs, which explains why FcgRIIB- play a critical role in the proliferation of antigen-specific CD8 T mediated cross-linking was not essential for its in vivo efficacy. It is cells (46, 47). MCP-1 may be particularly important for initiating liver likely that 2A mAb also has some Treg-depleting activity because rat toxicity because its receptor CCR2 has been shown to be involved in G2a binds to FcgRIII; however, these observations may simply CD137-mediated hepatotoxicity (33). However, we did not detect reflect differences in tumor microenvironment in the models used TNFa, which has been shown to be essential for CD137-induced liver and/or size of tumor when treatment was initiated. Interestingly, in toxicity (28). mice lacking all activating FcgRs (Fcer1g / ), yet still retaining An inverse correlation between affinity and potency has been FcgRIIB, both 2A and LOB12.0 (mIgG1) achieved 100% long- observed for agonist mAbs against Fas in vitro (48) and erythropoietin term survival in EL4E7 and EG7 models, respectively (37, 38). These receptor (EPOR) in vivo (49). The authors suggested that these low- results indicate that activating FcgRs interfered with FcgRIIB- affinity mAbs have fast off-rates that favored the formation of receptor mediated CD137 signaling and negatively affected the in vivo clusters with active conformation and hence enhanced signaling. What efficacy under certain circumstances. Hence, an anti-CD137 with surprised us is the strong in vivo antitumor activity of the low-affinity Fc engineered to enhance FcgRIIB binding with minimal interaction 106L mAb, which showed the weakest T-cell costimulation activity with activating FcgRs would likely produce an optimal antitumor in vitro. It is known that CD137 can be expressed in a variety of response. We have compared the in vivo efficacy of murine IgG1 and immune cells, including myeloid cells in liver that have been reported IgG2a isoforms of 106, 107, and 1D8 in CT26 or MC-38 models and to play a role in toxicity mediated by CD137 agonist mAbs (33). It is found that the efficacy of murine IgG1 is more consistent than the possible that a low-affinity agonist provides selective binding and murine IgG2a isoform, resulting in higher numbers of tumor-free activation of immune cells with higher CD137 expression such as þ miceattheendofthestudy.SimilartoratIgG2a,murineIgG1binds cytotoxic CD8 T cells, which are directly involved in antitumor FcgRIIB and FcgRIII, whereas murine IgG2a binds strongly to FcgRI activity in vivo. and FcgRIV, yet weakly to FcgRIIB. Higher efficacy of IgG1 over The major concern of targeting CD137 for immunotherapy is liver IgG2aislikelyduetoitsincreasedbindingaffinity to FcgRIIB (39). It toxicity. We addressed this problem by using mice as a screening is known that Fc-engineered CD40 mAbs with enhanced binding platform to select for mAbs with high potency and low liver toxicity. specifically to FcgRIIB results in a dramatic enhancement of their These mAbs demonstrated minimum liver enzyme elevation while þ antitumor efficacy (40). With the common mechanism in receptor retaining antitumor efficacy, accompanied by increased CD8 T cells clustering of TNFR superfamily members, similar Fc mutations is in the tumor which resulted in an antigen-specific memory T-cell likely applicable to 106/107 to further enhance activity in vivo. response. This characteristic of inducing efficacy without significant Because the functions of FcgRIIB are conserved in both mouse and liver toxicity is likely attributed by the specific epitope. It is also human, the choice of isotype for a clinical CD137 agonistic mAb that noteworthy that clone 105 showing high ALT in Fig. 1B belonged can engage FcgRIIB becomes critical to its therapeutic potential and to the same epitope group as 1D8. Because hepatic myeloid cells liver toxicity. Our results show that human IgG4 is the most potent express both CD137 and FcgR are thought to drive liver toxicity (33), isotype, whereas IgG2 is the weakest in human T-cell costimulation. one could speculate that certain mAbs in an epitope group may activate The observations were not limited to 106 and 107, but also applied to these liver myeloid cells more than the others by coengagement of Mab A and Mab B (Supplementary Fig. S4B). These results are in FcgR on the same cell. Further investigation will be required to fully agreement with the binding affinity of isotypes to FcgRIIB, IgG4 is understand the mechanism(s) utilized by different epitopes of CD137 about 2-fold higher than IgG1, and 10-fold higher than IgG2 (41). The mAbs resulting in differential liver toxicity. clinical outcomes of 2 current agonistic CD137 mAbs also agree with In conclusion, this study demonstrated the importance of epitope our hypothesis. Urelumab is a strong CD137 agonist like Mab A, and and FcgRIIB-mediated activity to develop a new class of CD137 the human IgG4 isotype will likely boost its in vivo activity, which agonistic mAbs that have an enhanced therapeutic window for effec- explains why it has severe hepatotoxicity and must be dosed at tive cancer immunotherapy. Synergistic antitumor activity by com- 0.1 mg/kg or lower. On the other hand, utomilumab is human IgG2, bination of CD137 mAb with anti–PD-1 or anti–PD-L1 in B16-F10 the weakest isotype for FcgRIIB cross-linking, which may result in a tumor model was confirmed as previously reported (Supplementary weak agonist unless G2 hinge causes the activity to be Fc-independent Fig. S6; refs. 21, 50); however, it is important to address the toxicity as described in the example of a CD40 agonist mAb (42). Assuming the effect because the checkpoint inhibitors may alter lymphocytes infil- activity of utomilumab is independent from this hinge effect, it may tration not only to tumors but also to other organs. Thus, enhanced explain why utomilumab can be dosed at 10 mg/kg without any liver safety monitoring will be critical for any CD137-related human toxicity issues. In addition, utomilumab blocks CD137 ligand whereas therapy development. urelumab does not, which may also explain the difference in activity (43). Urelumab and utomilumab represent two extremes of CD137 Disclosure of Potential Conflicts of Interest agonist and their choice of isotype and subsequent clinical outcomes E. DiGiammarino is a principal scientist at AbbVie, Inc., reports receiving provide valuable lessons for choosing the optimal isotype for future a commercial research grant from the same. L. Zhou is a senior scientist at AbbVie. CD137 mAbs. C.M. Forsyth is a principal scientist at AbbVie. D.B. Powers is a research fellow at Treatment with CD137 agonist induced a variety of cytokines AbbVie. D.T. Chao is a director at AbbVie. H.M. Alvarez is a principal research scientist and chemokines that are detectable in serum, which might be impor- at AbbVie. Y. Akamatsu is a senior principal research scientist at, reports receiving fi commercial research grant from, and has ownership interest (including patents) in tant for CD137-mediated antitumor ef cacy. For example, IFNg AbbVie. No potential conflicts of interest were disclosed by the other authors. has been shown to be essential for antitumor activity of CD137 mAb fi fi þ because it is required for the in ltration of tumor-speci c CD8 Authors’ Contributions cytotoxic T cells into the tumors (44). MIG is induced by IFNg as a Conception and design: D.B. Powers, D.T. Chao, D. Hollenbaugh, Y. Akamatsu chemoattractant for activated T cells, which inhibits angiogenesis and Development of methodology: S.K. Ho, Z. Xu, A. Thakur, E. DiGiammarino, promotes cytotoxic T-cell activity (45). The presence of IL10 may be in L. Zhou, V. Zhao, M. Xiong, J. Samayoa

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CD137 Agonist mAb with Reduced Liver Toxicity

Acquisition of data (provided animals, acquired and managed patients, provided Acknowledgments facilities, etc.): S.K. Ho, Z. Xu, A. Thakur, M. Fox, S.S. Tan, E. DiGiammarino, We thank Jieyi Wang (Lyvgen BioPharma) for his earlier works to the study when L. Zhou, M. Sho, M. Xiong, C.M. Forsyth he was an employee of AbbVie. We also thank the Protein Science Core group in Analysis and interpretation of data (e.g., statistical analysis, biostatistics, AbbVie Redwood City for producing reagents. computational analysis): S.K. Ho, Z. Xu, A. Thakur, S.S. Tan, E. DiGiammarino, L. Zhou, B. Cairns, V. Zhao, J. Samayoa, H.M. Alvarez The costs of publication of this article were defrayed in part by the payment of page Writing, review, and/or revision of the manuscript: S.K. Ho, Z. Xu, charges. This article must therefore be hereby marked advertisement in accordance A. Thakur, M. Fox, E. DiGiammarino, L. Zhou, B. Cairns, V. Zhao, with 18 U.S.C. Section 1734 solely to indicate this fact. J. Samayoa, C.M. Forsyth, D.B. Powers, D.T. Chao, D. Hollenbaugh, H.M. Alvarez, Y. Akamatsu Received June 14, 2019; revised November 15, 2019; accepted January 17, 2020; Study supervision: H.M. Alvarez, Y. Akamatsu published ﬁrst January 23, 2020.

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OF12 Mol Cancer Ther; 2020 MOLECULAR CANCER THERAPEUTICS

Epitope and Fc-Mediated Cross-linking, but Not High Affinity, Are Critical for Antitumor Activity of CD137 Agonist Antibody with Reduced Liver Toxicity

Sun K. Ho, Zhenghai Xu, Archana Thakur, et al.

Mol Cancer Ther Published OnlineFirst January 23, 2020.

Updated version Access the most recent version of this article at: doi:10.1158/1535-7163.MCT-19-0608

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