A CD137/OX40 Bispecific Antibody Induces Potent Antitumor ... · The LALA (L234A-5 L235A, Eu...

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CD137/OX40 Bispecific Antibody Induces Potent Antitumor Activity 2

That Is Dependent on Target Co-Engagement 3

Anti-Tumour Activity of a CD137/OX40 Bispecific Antibody 4

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Authors/Affiliations 6

Miguel Gaspar 1, John Pravin 1, Leonor N. Rodrigues 1, Sandra Uhlenbroich 1, Katy L. Everett 1, 7

Francisca Wollerton 1, Michelle Morrow 1, Mihriban Tuna 1, and Neil Brewis 1,* 8

1 F-star Therapeutics Ltd., Cambridge, CB22 3AT, UK 9 *Correspondence: [email protected] 10 11

Conflict of Interest Statement 12

All authors are current or former employees of F-star Therapeutics Limited. 13

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on May 13, 2020. © 2020 American Association for Cancer Research. cancerimmunolres.aacrjournals.org Downloaded from

Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on April 9, 2020; DOI: 10.1158/2326-6066.CIR-19-0798

http://cancerimmunolres.aacrjournals.org/

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Abstract 1

Following the success of immune checkpoint blockade (ICB) therapy against cancer, agonistic 2

antibodies targeting T cell co-stimulatory pathways, are in clinical trials. The tumor necrosis factor 3

superfamily of receptors (TNFRSF) members CD137 and OX40 are co-stimulatory receptors that 4

stimulate T cell proliferation and activation upon interaction with their cognate ligands. Activating 5

CD137 and OX40 with agonistic monoclonal antibodies stimulates the immune system due to their 6

broad expression on CD4+ and CD8+ T cells and NK cells and has antitumor effects in pre-clinical 7

models. Most TNFRSF agonist antibodies require crosslinking via Fc receptors (FcRs), which can 8

limit their clinical activity. FS120 mAb2 ™, a dual agonist bispecific antibody targeting CD137 and 9

OX40, activated both CD4+ and CD8+ T cells in a FcR-independent mechanism, dependent on 10

concurrent binding. A mouse surrogate version of the bispecific antibody displayed antitumor 11

activity in syngeneic tumor models, independent of T regulatory cell (Treg) depletion and of FcR-12

interaction, but associated with peripheral T cell activation and proliferation. When compared to a 13

crosslink-independent CD137 agonist monoclonal antibody, the FS120 surrogate induced lower liver 14

T cell infiltration. These data support initiation of clinical development of FS120, a first-in-class dual 15

agonist bispecific antibody for the treatment of human cancer. 16

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Keywords 18

OX40, CD137, Bispecific, Crosslinking, Agonism, Co-stimulation, TNFRSF, FcR; mAb2, Treg. 19 20 21

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Introduction 1

OX40 and CD137 co-stimulatory receptors belong to the tumor necrosis factor receptor superfamily 2 (TNFRSF) (1). Both are expressed on activated T cells and NK cells, and are attractive targets for 3 cancer immunotherapy as stimulation of these receptors results in increased T cell activation, 4 proliferation, and survival in vitro and in vivo (1). The expression patterns of OX40 and CD137 are 5 overlapping but distinct with expression of OX40 higher on CD4+ T cells and that of CD137 higher on 6 CD8+ T cells (2). CD137 stimulation preferentially stimulates CD8+ T cells when compared to CD4+ T 7 cells and OX40 stimulation preferentially stimulates CD4+ T cells when compared to CD8+ T cells (3). 8 However, co-expression of these receptors is demonstrated in both CD4+ and CD8+ T cells and both 9 are expressed in tumor infiltrating lymphocytes (TILs) (4,5). Antibodies stimulating these targets 10 show activity in a variety of murine tumor models by both depleting regulatory T cells (Tregs) and 11 activating CD8+ and CD4+ T cells (6,7). The combination of OX40 and CD137 agonist antibodies 12 stimulate both CD4+ and CD8+ T cells and to induce the cytotoxic function of both antigen 13 experienced and antigen-inexperienced bystander CD4+ T cells (8,9). 14 15 Several clinical trials are underway to test agonist antibodies to OX40 or CD137 either as 16 monotherapies or in combination with other agents to treat various cancers (10). Clinical trials with 17 OX40 agonist antibodies demonstrate peripheral T cell activation and proliferation without 18 associated toxicity (11) but show limited clinical efficacy (12). Two CD137 agonist antibodies have 19 different clinical outcomes. Urelumab (BMS-663513, clone 20H4.9) induces severe transaminitis at 20 doses higher than 1 mg/kg resulting in two hepatotoxicity-related deaths (13) and utomilumab (PF-21 05082566, clone MOR7480.1), which does not induce severe adverse events, has modest clinical 22 activity (14). A combination trial with utomilumab and PF-04518600 (an OX40 agonist antibody) is 23 underway (NCT02315066). 24 25 TNFRSF antibodies typically have no or low intrinsic agonist activity and require secondary 26 crosslinking of antibody-receptor complexes in order to induce sufficient receptor clustering and 27 activation, thereby mimicking the TNFSF ligand superclusters (15). In vivo, this secondary crosslinking 28

requires the interaction with FcRs (16). The availability of FcR expressing cells in the tumor 29

microenvironment and the low affinity-interaction between FcRs and the Fc-region of IgG 30 antibodies may limit the agonist activity of TNFRSF antibodies and, consequently, their antitumor 31

activity (17). Additionally, interaction with FcRs mediates antibody effector functions such as ADCC 32 and ADCP and could lead to the depletion of the tumor-specific T cells that would be activated by 33 these antibodies (18). Consequently, the clinical activity seen with OX40 and CD137 antibodies may 34 not represent the full potential of activating these receptors. 35 36

An alternative to FcR-mediated crosslinking of TNFRSF agonist antibodies is the use of bispecific 37 antibodies, the dual binding of which results in the clustering of the TNFRSF target, independent of 38

FcR engagement (19). This study described FS120 mAb2, a dual agonist bispecific antibody targeting 39 CD137 and OX40 that activated both CD4+ and CD8+ T cells, whereas OX40 or CD137 monospecific 40

antibodies only activated CD4+ or CD8+ T cells respectively. FcR disabling mutations (LALA mutations 41 (20)) were introduced to enable antibody crosslinking from the co-engagement of the two different 42 receptors when co-expressed and to potentially avoid depletion of OX40 or CD137 expressing cells. 43

A mouse specific surrogate version of FS120 showed antitumor activity in the absence of FcR-44 interaction or after Treg depletion. When compared to a crosslink-independent CD137 agonist 45 monoclonal antibody, FS120 surrogate showed reduced liver T cell infiltration, which decreased over 46 time. 47

These results indicate that targeting co-expressed receptors with bispecific antibodies may be a 48 potent and safe mechanism to cluster and activate TNFRSF co-stimulatory receptors and induce 49 antitumor immunity 50




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Methods 1

Antibody Reagents 2

Antibodies were cloned by replacing the VH or V domain sequences in human IgG1 with identified 3 sequences from patents or literature using methods described previously (21). The LALA (L234A-4 L235A, Eu numbering (22)) mutations were introduced via site-directed mutagenesis when 5 indicated. Absence of LALA mutations is denoted by the suffix WT after the clone name. For antibody 6 production pTT5 expression vectors (National Research Council of Canada) containing the mAb or 7 mAb2 sequences were transfected into Expi293F cells (Thermo A14528) using PEIpro transfection 8 reagent (Polyplus PPLU115) according to manufacturer’s instructions. Antibodies were purified using 9 a 5 ml MabSelect SuRe column (GE Healthcare, 11003494) on an AKTA Explorer (GE Healthcare) 10 according to manufacturer’s instructions. 11

The following antibodies were used in the experiments described in this manuscript: anti-FITC 12 (Ctrl(4420) WT mAb; clone 4420 used as isotype control (23)), anti-human OX40 (OX40(11D4) mAb; 13 clone 11D4 from patent EP2242771B1), anti-human OX40 Fcab (OX40/Ctrl(4420) mAb2; clone FS20, 14 F-star); anti-human CD137 (CD137(MOR7480.1) mAb; clone MOR7480.1 from patent 15 US8,337,850B2), anti-human CD137 (CD137(20H4.9) mAb; clone 20H4.9 from patent 16 US8,137,667B2), anti-human CD137 (CD137(FS30) mAb; clone FS30, F-star), anti-mouse OX40 17 (mOX40(OX86) WT mAb; clone OX86 from patent US9,738,723B2), anti-mouse CD137 18 (mCD137(Lob12.3) WT mAb; clone Lob12.3 (3)) and anti-mouse CD137 (mCD137(3H3) WT mAb; 19 clone 3H3 (24)) all in human IgG1 format, with (mAb) or without (WT mAb) LALA mutations; anti-20 human IgG CH2 domain (clone MK1A6 (25)) in mouse IgG1 format; anti-human CD28 21 (CD28(TGN1412) mAb; clone TGN1412 from patent US 8,709,414 B2) in human IgG4 format with 22 S228P mutation (Eu numbering); anti-human CD3 antibody (clone UHCT1, R&D Systems); anti-mouse 23 CD3 antibody (clone 17A2, Thermo); anti-mouse CD3 antibody (clone 145-2C11, Thermo). 24

SPR analysis 25

Data were acquired using a BIAcore 3000 or BIAcore T200. Dilution mixtures prepared in HBS-P or 26 HBS-EP buffer (GE Healthcare). For KD determination, FS120 was captured either via the Fc-region 27 using a Human Antibody Capture Kit (GE Healthcare) and human or cynomolgus CD137 was flowed 28 over at a range of concentrations; or via the Fab region using a Human Fab Capture Kit (GE 29 Healthcare) and human or cynomolgus OX40 was flowed over at a range of concentrations. For dual 30 binding determination of FS120, biotinylated human CD137 or OX40 was immobilised on a 31 Streptavidin (SA) chip (GE Healthcare) and antibodies (100 nM) were co-injected with either human 32 OX40 or CD137 (100 nM) respectively or HBS-EP buffer. For dual binding determination of FS120 33 surrogate, mouse CD137 was immobilised on a CM5 chip (GE Healthcare) to a surface density of 34 approximately 1000 RU and antibodies (100 nM) were co-injected with either mouse OX40 or CD137 35

(100 nM) or HBS-EP buffer. Affinity for Fc receptors (R&D Systems hFcR1 (1257-CF-050), hFcR2a 36

(1330-CF-050), hFcR2b (1875-CF-050) and hFcR3a (4325-CF-050)) was tested by coating 37 biotinylated human OX40 (BPS Bioscience 71310-1) or CD137 (in-house) his-tagged antigens onto an 38

SA chip (GE Healthcare) and co-injecting antibodies (100nM) and human FcRs (500nM) at 20 µl/min 39 flow rate and the dissociation was followed for 5 min. For specificity assessment human TNFRSF 40 members (R&D Systems TNFRI (372-RI-050/CF), TNFRII (726-R2-050), GITR (689-GR-100), NGFRI 41 (367-NR-050/CF), CD40 (1493-CD-050) and DR6 (144-DR-100)) were immobilised on CM5 chips (GE 42 Healthcare) to approx. 1000 RU and FS120 (1 µM), isotype control (anti-FITC (Ctrl(4420) WT mAb; 43 clone 4420 in human IgG1 backbone (23)) or positive control antibodies (R&D Systems anti-TNFRI 44 (MAB225R-100); anti-TNFRII (MAB726-100); anti-GITR (MAB689-100); anti-NGFR (MAB367); anti-45 CD40 (MAB6321-100); anti-DR6 (AF144)) were flowed over at a flow rate of 30 µl/min. Data was 46 analysed using BIA evaluation software (GE). 47

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Cell line creation and reporter T cell assay 1

All cells used in the experiments described in this manuscript were kept in culture for a maximum of 2 2 months before starting new cultures from master vials. CT26 (CRL-2638), B16-F10 (CRL-6475) cell 3 lines were purchased from ATCC in 2015. DO11.10 cells were purchased from Public Health England 4 (85082301) in 2014 and used under license from National Jewish Health. Expi293F cells were 5 purchased from Thermo (A14528) in 2015. No re-authentication tests were performed. Mycoplasma 6 testing was performed on all cell lines monthly using R&D MycoProbe Mycoplasma detection kit 7 (R&D systems 895285). Human and mouse OX40 and CD137 cDNA constructs were synthesised 8 (Genscript) with flanking 5’ EcoRI and 3’ NotI restriction sites and cloned into the lentivirus vector 9

pLVX-EF1-IRES-puro (Clontech 631988). Lentiviruses were produced using the LentiX Expression 10

system EF1 version (Clontech 631253) and used to transduce DO11.10 cells according to 11 manufacturer’s instruction. Cell lines were selected by incubation with 5 ug/ml Puromycin (Invivogen 12 ant-pr-1) and individual cell lines were cloned by serial dilution. DO11.10 cells expressing human or 13 mouse CD137 were stimulated with coated anti-mouse CD3 antibody (Biolegend 100202 clone 17A2 14 at 0.1 μg/ml) and mouse IL2 concentration in the supernatants was measured by ELISA (Thermo 88-15 7024-88). 16

Flow cytometry 17

For cell binding assays cells were incubated with primary antibodies or mAb2 followed by detection 18 with an anti-human Fc-488 secondary antibody (Jackson Immunoresearch). Excised tissues were 19 dissociated using relevant Miltenyi dissociation kits (tumors – 130-096-730; livers – 130-105-807; 20 spleens – 130-095-926) using a Miltenyi gentleMACS Octo dissociator and C-tubes according to 21

manufacturer’s instructions. Resulting cell suspensions were strained (70 m cell strainer (Corning 22 CLS431751)), washed and resuspended in PBS. Collected blood samples were treated twice with red 23 blood cell lysis buffer (eBioscience 00-430054) according to manufacturer’s instruction. Cells isolated 24 from tissues and blood were stained for flow cytometry with fluorochrome conjugated antibodies 25 including those against CD4, Ki67, FoxP3, CD69, CD3, CD8 (Thermo), CD45 (BD) and fixable live/dead 26 dye (Thermo) in the presence of Fc block (Thermo) according to manufacturer’s recommendations. 27 Cells were analysed in a BD FACS CantoII cytometer and data was analysed with FlowJoX. OX40 and 28 CD137 receptors quantified with Quantum Simply Cellular anti-mouse IgG (Bangslabs 815) according 29 to manufacturer’s recommendations. 30

Peripheral Blood Mononuclear Cells (PBMCs) and T cell stimulation assays 31

Ficoll-purified human PBMCs were stimulated with 100 ng/ml staphylococcal enterotoxin A (Sigma) 32 in the presence of FS120 or control antibodies for 5 days at 37oC, 5% CO2 in T cell media (RPMI1640 33 (Thermo) with 10% FBS (Thermo), 1x Penicillin Streptomycin (Thermo), 1 mM Sodium Pyruvate 34 (Gibco), 10 mM Hepes (Gibco), 2 mM L-Glutamine (Gibco) and 50 µM 2-mercaptoethanol (Gibco)). T 35 cells were isolated from PBMCs using Miltenyi enrichment kits (Human CD3+ - 130-096-535; Human 36 CD4+ - 130-096-533; Human CD8+ - 130-096-495; Mouse CD3+ - 130-095-130) according to 37 manufacturer’s instructions and activated overnight using CD3/CD28 Dynabeads (Life Technologies 38 catalogue numbers: Human – 11131D; Mouse – 11453D) at a 1:1 cell to bead ratio. CD3/CD28 beads 39 were removed using a DynaMag-15 magnet and activated T cells were washed with T cell media and 40

stimulated with coated CD3 antibody (Human: R&D Systems (MAB100) clone UHCT1 at 2.5 g/ml 41

[total and CD4+ T cells] or 5 g/ml [CD8+ T cells] or Mouse: Biolegend (100302) clone 145-2C11 at 2.5 42

g/ml) in the presence of FS120 or control antibodies for 3 days at 37oC, 5% CO2. Anti-human Fc 43 (clone MK1A6, produced in house) was used as crosslinking agent at a 1:1 molar ratio with test 44 antibodies. FITC dextran (70 kDa, Sigma 46945) was used as crosslinking agent at a 1:1 molar ratio 45 with OX40 Fcabs paired with FITC-binding Fab (clone 4420). IL2 concentration in the supernatants 46 was measured by ELISA or electrochemiluminescence (Thermo 88-7025-88 or MSD K151QQD-1). For 47

the cytokine release assay, antibodies were diluted to 10 g/ml in PBS, coated onto 96-well flat-48 bottomed plates and allowed to air dry overnight, washed twice with PBS, and incubated with 2x105 49




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PBMCs for 3 days. Multiple cytokine concentrations in supernatants were measured by Pro-1 inflammatory V-plex kit (Meso Scale Discovery catalogue number K15049D-2) according to 2 manufacturer’s instructions. 3

Mice and Tumor Challenge 4

Balb/c female mice were from Charles River Laboratories, UK. Animals were housed in a local animal 5 facility and were used at approximately 8-10 weeks of age. Antibodies were diluted in PBS before 6 intraperitoneal injection (IP) at the indicated dose and schedule. For tumor trials, mice were 7 anaesthetised by inhalation of isoflurane, and each animal received 106 or 105 of CT26 tumor cells 8 (depending on experiment) or 105 of B16-F10 tumor cells diluted in PBS. Mice were injected 9

subcutaneously with a maximum volume of 100 l in the left flank to generate tumors. Mice were 10 randomised into study cohorts based on tumor volume and any mice which did not have tumors 11 were not assigned into treatment groups and were removed from the study. Tumor measurements 12 were taken under anaesthesia using callipers to determine the longest axis and the shortest axis of 13 the tumor. The following formula was used to calculate the tumor volume: L x (S2) / 2 (Where L = 14 longest axis; S= shortest axis). For peripheral pharmacodynamic analysis blood was collected into 15 EDTA-containing tubes from either the tail vein or by cardiac puncture. Tumors, spleens and livers 16 were collected by dissection when indicated. All procedures involving animals were approved and 17 performed according to UK Home Office (license number 70/7991) and local Ethical Review 18 Committee guidelines 19

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Statistical analysis 21

One and two-way ANOVA with Tukey’s or Dunnett’s multiple comparisons test, and survival analysis 22

(log rank Mantel-Cox test) were performed using Prism software (GraphPad). For comparison of 23

treatment responses and EC50 determinations, data were log transformed before analysis and fit 24

using the log agonist vs response using Prism software (GraphPad). Where indicated, statistical 25

testing of tumor volume over time was analysed using a mixed model. A separate model was fitted 26

to each pair of treatments of interest. The model is 27

log10(𝑣𝑜𝑙𝑢𝑚𝑒) = 𝐴 + 𝐵 × (𝑑𝑎𝑦 − 𝑠𝑡𝑎𝑟𝑡 𝑑𝑎𝑦) + 𝜀

A and B are the intercept and slope respectively; they are different for each mouse, and include a 28

fixed effect for the group and a random effect for the animal: 29

𝐴 = 𝐴0 + 𝐴1𝑇 + 𝜀𝐴

𝐵 = 𝐵0 + 𝐵1𝑇 + 𝜀𝐵

T is a dummy variable representing the treatment group with value 0 in one group and 1 in the 30

other. The random effects are distributed with a normal distribution: 31

𝜀A~𝑁(0, 𝜎A), 𝜀B~𝑁(0, 𝜎B) 32

where 𝜎A and 𝜎B are the standard deviations of the inter-animal variability in the intercept and 33

slope respectively. The intra-animal variability is also normally distributed with standard deviation 𝜎: 34

ε~𝑁(0, σ)

For each pair of treatments, the model above was fitted to the data. For 𝐴1 and 𝐵1, the (two-sided) 35

p-value for a difference from zero was calculated; a p-value below 0.05 is statistically significant 36

evidence for a difference between the treatment groups. 37




7

Results 1

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FS120 simultaneously bound to CD137 and OX40 3

4 Phage and yeast libraries were used with directed evolution methods described previously (21,26) to 5 identify and improve OX40-binding Fc antigen binders, termed Fcabs (Fc-region with antigen 6 binding), as well as CD137-binding Fab regions. The Fcab (OX40 Fcab, clone FS20) and Fab (CD137 7 Fab, clone FS30) with the overall highest activity in T cell stimulation assays and affinity in cell 8 binding assays were combined to generate the bispecific FS120 mAb2 (or FS120) (Fig. 1A). Affinity 9 determination by SPR showed that FS120 has sub-nanomolar binding to both human and 10 cynomolgus monkey OX40 (KD : Human - 0.2 nM; Cyno - 0.9 nM) and CD137 (KD : Human - 0.2 nM; 11 Cyno - 0.2 nM) (Fig. 1B) and FS120 bound both OX40 and CD137 simultaneously (Fig. 1C). FS120 did 12 not bind to other related members of the TNFR superfamily (CD40, GITR, NGFR, DR6, TNFRI and 13

TNFRII) (Supplemental Fig. S1). FS120, which contains the LALA mutations, had reduced FcR binding 14 as compared to WT IgG1 (non-LALA containing) OX40 and CD137 antibodies (Supplemental Fig. S2). 15 FS120 bound to cell surface expressed human and cynomolgus OX40 and CD137 receptors on 16 engineered DO11.10 T cell lines but not to the non-transduced parental DO11.10 cell line (Fig. 1D). 17 18

FS120 agonistic activity was dependent on the dual binding of CD137 and OX40 19

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To test the agonist activity of FS120, PBMCs were stimulated with staphylococcal enterotoxin A (SEA) 21 superantigen, which crosslinks MHC class II (MHC II) molecules at the surface of antigen-presenting 22 cells (APCs) and the TCR of T cells (27), in the presence or absence of secondary crosslinking agents 23

to mimic the effect of FcR-mediated crosslinking. The amount of T cell activation resulting from 24 OX40 or CD137 stimulation was then measured by the production of IL2. All antibodies were tested 25 in the same isotype background as FS120, human IgG1 with the LALA mutations, to minimise 26

interference from FcR-mediated crosslinking. 27

Agonistic activity of OX40 or CD137 monospecific antibodies was only observed in the presence of 28 crosslinking (Fig. 2A). In contrast, FS120 mAb2 showed activity in the absence of secondary 29 crosslinking agent suggesting the co-engagement of OX40 and CD137 resulted in effective receptor 30 clustering and activation (Fig. 2A). Similar results were observed when isolated T cells were 31 stimulated with plate-bound CD3 antibody and co-stimulated with OX40 or CD137-specific 32 antibodies or FS120 mAb2 (Fig. 2B). The activity of FS120 was not increased by the secondary 33 crosslinking agent, either in maximum response or in a decrease in EC50 (Fig. 2C and 2D), indicating 34 that the dual binding to OX40 and CD137 resulted in the maximum stimulation induced by FS120. 35

When CD137 agonist antibodies were crosslinked, the 20H4.9 Fab clone (Fab present in urelumab) 36 was observed to have a higher activity as compared to clones MOR7480.1 (Fab present in 37 utomilumab) or FS30 (Fab present in FS120) (Fig. 2A and 2B). The crosslinked OX40-targeting 38 antibodies induced higher IL2 production than the crosslinked CD137-targeting antibodies, and the 39 combination of the OX40 Fcab and the CD137 Fab components of FS120 did not show a synergistic 40 effect as compared to the OX40 Fcab alone (Fig. 2A and 2B). This result indicated that these assays 41 were more sensitive to OX40 stimulation and that only potent CD137 stimulation resulted in 42 substantial T cell activation. The higher response to OX40 agonism could have been explained by the 43 higher proportion of CD4+ T cells in human PBMCs and the higher expression of OX40 on activated 44 CD4+ T cells (Supplemental Fig. S3). 45

Titrations of these antibodies in both the PBMC SEA stimulation and T cell CD3 stimulation assays 46 were performed and the concentration at which these antibodies induced the highest IL2 production 47




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was chosen for this analysis. FS120, and crosslinked OX40 Fcab, induced the production of additional 1

pro-inflammatory cytokines (IL-6, IL-12p70, IL-13 and TNF) by T cells and reduced the levels of IL-2 10, a typical anti-inflammatory cytokine (Supplemental Fig. S4). 3

To test if the activity of FS120 required simultaneous binding to the two receptors, the ability of 4 FS120 to co-engage OX40 and CD137 was blocked using 100-fold molar excess of either the OX40 5 Fcab or the CD137 Fab components of FS120 or both. The results showed that the FS120 induced T 6 cell activation was reduced when the mAb2 component parts were present either individually or in 7 combination (Fig. 2E), indicating that FS120 required dual binding to OX40 and CD137 to induce the 8 clustering and activation of these receptors. 9

FS120 did not induce T cell activation in a cytokine release assay (28) in the absence of TCR 10 stimulation, unlike the two positive control antibodies used, a CD28 antibody shown to induce 11 cytokine storm in the clinic (TGN1412) (29) and a CD3 antibody (Supplemental Fig. S5). 12

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FS120 stimulated both CD4+ and CD8+ T cells 14

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OX40 and CD137 receptors were co-expressed on CD4+ and CD8+ T cells, with OX40 expressed in 16 higher percentages and at a higher receptor number in CD4+ T cells than CD8+ T cells and, 17 conversely, more CD137 receptors were expressed on CD8+ T cells (Supplemental Fig. S1). The 18 differential expressions correlated with the activity of OX40 or CD137 targeting antibodies. On CD4+ 19 T cells stimulated with plate-coated CD3 antibody, both the crosslinked OX40 mAb and the OX40 20 Fcab induced IL2 production, but CD137 antibodies did not show activity (Fig. 3A and 3B). On CD8+ T 21 cells the crosslinked CD137 antibodies (Fab clones 20H4.9 and FS30) induced IL2 production and 22 OX40 antibodies did not (Fig. 3A and 3B). When tested in the absence of secondary crosslinking 23 agent, FS120 increased IL2 production on both CD4+ and CD8+ T cells (Fig. 3A). The crosslinked 24 activity of clone FS30 (CD137(FS30) mAb) demonstrated that the mAb2 could activate the CD137 25 receptor when crosslinked by binding of the Fcab to OX40 on CD8+ T cells. The activity of the 26 crosslinked OX40 Fcab (OX40/Ctrl(4420) mAb2) showed that the mAb2 could activate the OX40 27 receptor when crosslinked by binding of the Fab arms to CD137 on CD4+ T cells. 28

The different CD137 antibodies tested showed varying activity on CD8+ T cells. Fab clone MOR7480.1 29 did not show activity in the absence of crosslinking and only a moderate, non-significant, increase 30 when crosslinked (Fig. 3A). Clone FS30 displayed higher activity when crosslinked but no activity in 31 the absence of crosslinking (Fig. 3A). However, Fab clone 20H4.9 induced higher levels of IL2 32 production in the absence of crosslinking, which was increased with crosslinking (Fig. 3A). When 33 these antibodies were tested in a model DO11.10 T cell line expressing human CD137 and stimulated 34 with coated anti-mouse CD3 antibody, Fab clone 20H4.9 also showed crosslink-independent activity, 35 whereas Fab clones FS30 and MOR7480.1 did not (Supplemental Fig. S6A). 36

37

FS120 surrogate bound mouse OX40 and CD137 and activates T cells 38

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As the FS120 mAb2 does not bind to mouse OX40 or CD137, a surrogate molecule was generated for 40 in vivo testing by pairing a mouse-specific OX40 Fcab with a CD137 mAb (clone Lob12.3). The Fc-41 modifying technology used to create Fcabs is based on human IgG1 therefore the FS120 surrogate 42 has a human IgG1 domain. All in vivo experiments were performed using molecules with the same 43 human IgG1 backbone. The FS120 surrogate bound to cell surface expressed mouse OX40 and 44




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CD137 receptors on engineered DO11.10 T cell lines (Supplemental Fig. S7A) and concurrent binding 1 to mouse OX40 and CD137 was demonstrated by SPR (Supplemental Fig. S7B). 2

FS120 surrogate was functionally characterised by testing its ability to co-stimulate primary mouse T 3 cells in the presence of coated anti-mouse CD3. FS120 surrogate induced the production of IL2 in the 4 absence of secondary crosslinking agent, unlike the OX40 monospecific antibody which required 5 anti-Fc antibody for crosslinking (Supplemental Fig. S8A). No activity of the CD137 antibody was 6 detected in this assay, but in DO11.10 T cell line expressing mouse CD137 and stimulated with 7 coated anti-mouse CD3 antibody, clone Lob12.3 increased IL2 production in the presence of 8 crosslinking (Supplemental Fig. S6B). A different anti-mouse CD137 (clone 3H3) was also tested in 9 this assay and showed crosslink-independent activity, similar to that seen with Fab clone 20H4.9 (Fig. 10 3A and Supplemental Fig. S6A and S6B). This result indicated that these two molecules could be 11 surrogates for each other, whereas clone Lob12.3 only showed activity when crosslinked and was 12 therefore a surrogate for Fab clones MOR7480.1 and FS30 mAb. 13

The activity of FS120 surrogate in the absence of secondary crosslinking agent was also dependent 14 on dual binding to OX40 and CD137; when either of these receptors was blocked with excess mouse 15 OX40 Fcab or mouse CD137 mAb (clone Lob12.3), the activity of FS120 surrogate was reduced, 16 similar to that observed with FS120 in human T cells (Supplemental Fig. S8B). 17

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Antitumor activity of the FS120 surrogate 19

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OX40 and CD137 antibodies demonstrate antitumor activity in a variety of syngeneic models with 21 responses depending on dose, time of treatment initiation, antibody isotype and clones used 22 (3,25,26,27,28). Intra-tumoral Treg depletion is part of the mechanism of antitumor activity of OX40 23 (clone OX86) (6) and CD137 antibodies (clone Lob12.0) (7). 24

In order to test the antitumor activity of FS120 and to understand the in vivo mechanism of action, 25 FS120 surrogate or control antibodies were injected intraperitoneally (IP) at 1 mg/kg on days 11, 13 26 and 15 post CT26 tumor cells inoculation (Fig. 4A). The OX40 (clone OX86) or CD137 (clone Lob12.3) 27 control antibodies with human IgG1 isotype or their combination showed no antitumor activity (Fig. 28 4B), unlike previously published results using the original rat versions of these antibodies (3). This 29 could be explained by the later start time of antibody treatment, the lower dose or the human IgG1 30 isotype used. FS120 surrogate showed significant antitumor activity as compared to the isotype 31 control antibody and the antitumor activity observed for FS120 surrogate was similar to a WT IgG1 32

variant of FS120 surrogate in which FcR-interaction was not reduced by the LALA mutations (FS120 33 surrogate (WT mAb2)) (Fig. 4B). This indicated the mechanism of antitumor activity of FS120 34

surrogate was independent of FcR interaction, either FcR-mediated crosslinking or FcR-mediated 35 effector functions such as ADCC or ADCP. FS120 surrogate-treated mice with no tumors at day 60 36 were re-challenged with CT26 tumors and did not show tumor growth (Supplemental Fig. S9). 37

When intra-tumoral Tregs were analysed, CT26 tumors treated with CD137 antibody or the 38 combination of OX40 and CD137 antibodies had fewer Tregs compared to the isotype control 39 treated tumors (Fig. 4C). CT26 tumors treated with the FS120 surrogate (WT mAb2) also had fewer 40 intra-tumoral Tregs, similar to the percentages observed with the combination of OX40 and CD137 41 antibodies (Fig. 4C). However, due to the presence of the LALA mutations, the FS120 surrogate 42 treated tumors did not have fewer intra-tumoral Tregs compared to the isotype control treated 43 tumors (Fig. 4C). None of the treatments induced any significant changes in the frequency of CD4+ or 44 CD8+ T cells or T cell proliferation (Fig 4C). The antitumor activity of the FS120 surrogate was 45 therefore not associated with intra-tumoral Treg depletion. 46




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When the FS120 surrogate was tested in the B16-F10 model, a poorly immunogenic model and thus 1 harder to treat with immunotherapies (33), FS120 surrogate also had antitumor activity as compared 2 to an isotype control antibody (Supplemental Fig. S10). 3

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Peripheral T cell activation and proliferation mediated by FS120 surrogate 5

6

The induction of T cell proliferation by OX40 and CD137 agonist antibodies in vitro and in vivo is 7 described in both preclinical models and in the clinic (11,34). Comparing T cell proliferation and 8 activation in the blood of CT26 tumor-bearing mice treated with FS120 surrogate or control 9 antibodies overtime, it was observed that FS120 surrogate induced more CD4+ and CD8+ T cell 10 proliferation, as measured by the expression of Ki67 (Fig. 5A and 5B) than the OX40 and CD137 11 antibodies or their combination. The effect of FS120 surrogate on T cell activation, as measured by 12 the expression of CD69, was delayed as compared to the proliferative effect with the highest 13 frequencies observed 3 days after the third dose (Fig. 5B). Similar effects on T cell activation were 14 observed with the combination of OX40 and CD137 antibodies (Fig. 5B). 15

16

FS120 surrogate did not induce liver inflammation 17

18

Increased liver T cell infiltration is observed with various CD137 agonist antibodies in mice, 19

suggesting a similar mechanism of liver toxicity for CD137 agonism in mice and humans (35,36). To 20

understand the potential hepatotoxicity risk of FS120, T cells and their proliferation and activation in 21

the liver, spleen and blood induced by FS120 surrogate was compared to that induced by OX40 and 22

CD137 agonist antibodies or their combination (Fig. 6A). The results showed a clear difference 23

between the two CD137 agonist antibodies tested, clone 3H3 induced a sustained increase in T cell 24

infiltration, proliferation and activation in the liver and spleen, whereas clone Lob12.3 did not (Fig. 25

6B). OX40 stimulation did not show an increase in T cells in the liver, spleen or blood, but induced 26

transient T cell proliferation in all tissues studied and T cell activation in the liver at 14 days post-last 27

dose (Fig. 6B). Combination of OX40 and CD137 (clone Lob12.3) agonism induced a transient 28

increase in T cells in the liver, which was associated with increased T cell proliferation. FS120 29

surrogate also showed a moderate, but not statistically significant, increase in liver T cell infiltration, 30

proliferation and activation at 7 days post last dose, which returned to normal at 14 days post-last 31

dose (Fig. 6B). This transient increase in T cells and proliferation was also observed in the blood of 32

these naïve mice, as expected from other studies in CT26-tumor bearing mice (Fig. 5B). In the spleen 33

FS120 surrogate also induced transient T cell proliferation (Fig. 6A and 6B). 34

The increased liver T cell infiltration observed with the crosslink-independent CD137 agonist 35

antibody (clone 3H3) as compared to the crosslink-dependent CD137 agonist antibody (clone 36

Lob12.3) (Fig. 6B) correlated with observations that urelumab (crosslink-independent clone 20H4.9) 37

induces hepatotoxicity at doses above 1 mg/kg and utomilumab (crosslink-dependent clone 38

MOR7480.1) is well tolerated up to 10 mg/kg (13,14). Both FS120 and the FS120 surrogate molecules 39

had crosslink-dependent CD137 agonist Fab arms and were only able to induce CD137 agonism via 40

binding to OX40 as shown by the competition experiments in Fig. 2E and Supplemental Fig. S8B. This 41

dependency on OX40 binding for CD137 stimulation resulted in decreased liver T cell infiltration in 42

this preclinical study and suggested that FS120 may have a lower hepatotoxicity risk than crosslink-43

independent CD137 agonist antibodies. 44




11

Discussion 1

TILs express various checkpoint receptors and co-stimulatory receptors, including OX40 and CD137 2 (4,5). These receptors, in the absence of ligand interaction, are likely to contribute to the 3 dysfunctional phenotype of tumor reactive TILs (37,38). Activating TILs with agonist antibodies 4 against OX40 and CD137 has the potential to unleash existing antitumor immune responses and 5 reduces tumor growth and increases survival in several syngeneic tumor models (39,40). In clinical 6 trials however, despite inducing peripheral T cell activation, neither OX40 antibodies nor CD137 7 antibodies induce complete responses (CRs) unlike the results observed in preclinical studies (12). 8 9 The lack of translation between the preclinical models and the clinical results is likely due to various 10

factors. Limited availability of FcR-expressing cells in the tumor microenvironment of human 11

cancers and low affinity interaction between FcRs and the Fc region of IgG antibodies (41) could 12 result in sub-optimal crosslinking of these agonist antibodies (42). The depletion of intra-tumoral 13 Tregs, described as the mechanism of action of OX40 and CD137 antibodies in mouse syngeneic 14 tumor models, may not be as effective in human cancers (6,7,43) and may also result in the 15 depletion of the very same cells the OX40 and CD137 antibodies aim to stimulate (15). 16

17 CD137 agonist antibodies are associated with liver inflammation in preclinical models and urelumab 18 induces lethal hepatic inflammation in clinical trials at doses above 1 mg/kg (13). Although the 19 mechanism of toxicity in the clinic is unclear, in preclinical models this is associated with activation 20 of liver myeloid cells which express CD137 and the production of IL27 which then recruits CD8+ T 21 cells that mediate the inflammation damage (35). Whereas these results explain the mechanism of 22 liver inflammation in mouse models, they do not explain why in the clinic, a different CD137 23 antibody, utomilumab, showed no signs of liver inflammation and was well tolerated up to 10 mg/kg 24 (14). Several differences could account for this disparity since urelumab is reported as being a 25 human IgG4 non-ligand-blocking antibody and utomilumab as being a human IgG2 ligand-blocking 26 antibody (44). Additionally, the epitopes of urelumab and utomilumab are different and could also 27 account for their different activities (45). In this study, the increased in vitro potency of the CD137 28 antibody clone present in urelumab was observed and this clone (20H4.9) was also able to induce 29 crosslink-independent activation of CD8+ T cells, and T cell lines engineered to express CD137. In a 30

separate study, the clone present in urelumab also induces increased IFN production from CD3-31 stimulated PBMCs and purified T cells in the absence of crosslinking (46). 32 33 When two anti-mouse CD137 antibodies, clones Lob12.3 and 3H3 were compared, a corresponding 34 difference was observed, with clone 3H3 able to induce crosslink-independent activation of CD137 35 expressing cells and clone Lob12.3 requiring crosslinking. The liver inflammation observed in mice 36 treated with these antibodies was markedly different, with clone 3H3 showing sustained increase of 37 T cells in the liver. Increased ALT and liver T cell infiltration is observed in mice treated with rat 38 versions of the same clones (46). Since urelumab and clone 3H3 are associated with increased liver 39 inflammation and both clones (20H4.9 and 3H3) have the ability to stimulate CD137 in the absence 40 of crosslinking, it is possible that this may contribute to the hepatotoxicity risk presented by CD137 41 targeting antibodies. 42 43 In conclusion, FS120 is a bispecific antibody targeting OX40 and CD137 that simultaneously bound 44

these receptors and induced FcR-independent T cell activation in vitro; the FS120 surrogate induced 45 T cell activation in vivo. This in vitro and in vivo activity, which was dependent on co-engagement 46 with both receptors, suggested that the dual binding resulted in efficient receptor clustering and 47 activation. The existence of two OX40-binding sites in the Fcab and two CD137 binding sites in the 48 Fab region of FS120 raised the possibility of tetravalent binding, which was likely to be involved in 49 the clustering mechanism. Additionally, the binding to these two separate TNFRSF members with a 50




12

single molecule could potentially create receptor superclusters resulting in increased signalling via 1 these receptors as they share intracellular signalling intermediates such as TRAF2 but also have 2 unique adapters (TRAF1 for CD137 and TRAF5 for OX40) and stimulate distinct pathways (47). The 3 crosslinking of OX40 and CD137 receptors by FS120 could also lead to increased internalization, as is 4 required for CD137 signalling (48). The increased T cell activation by FS120 surrogate which resulted 5

in FcR-independent antitumor activity was independent of Treg depletion. Further, due to its 6 crosslink-dependent CD137-targeting Fab, which required Fcab-binding to OX40 for activity, FS120 7 may potentially provide a potent and safe way of stimulating CD137. These data support initiation of 8 clinical development of FS120, a first-in-class dual agonist bispecific antibody for the treatment of 9 human cancer. 10 11 12

Acknowledgements 13

The authors wish to thank Delphine Buffet, Marine Houee and Cyril Privezentzev for contributions to 14

OX40 Fcab discovery, to Melanie Medcalf and Edouard Souteyrand for contributions to CD137 Fab 15

discovery and FS120 affinity determination, to Drug Discovery, Protein Sciences and In vivo team 16

members for assistance with experimental procedures, and to Jacqueline Doody for contributions to 17

project strategy and supervision. All authors are current or former employees of F-star 18

Biotechnology Ltd. 19

Authors Contributions 20

Conceptualisation, M.G.; Methodology, S.U., K.L.E., F.P.G.W. and M.G.; Investigation, J.P., L.N.R., 21

E.P., S.U., K.L.E., F.P.G.W. and M.G.; Formal Analysis, E.P and M.G.; Writing - Original Draft, M.G.; 22

Writing - Review & Editing, M.M., M.T. and N.B.; Project Administration, M.T.; Supervision, N.B. 23

24




13

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8

Figure Legends 9

Figure 1. 10

FS120 bound to OX40 and CD137 with high affinity simultaneously. (A) Schematic representation of 11 FS120 mAb2, bispecific antibody binding to OX40 via the Fc region and to CD137 via the Fab region, 12 containing LALA mutations. (B) SPR of FS120 binding to human OX40 or human CD137. KD 13 determination performed using BIA evaluation software (representative results of 3 independent 14 experiments). (C) SPR of FS120 simultaneous binding to immobilised OX40 and CD137 in solution 15 and to immobilised CD137 and OX40 in solution (representative results of 2 independent 16 experiments). (D) Geometric mean fluorescence intensity (GMFI) of FS120 binding to cell surface 17 expressed human OX40 and CD137 determined by flow cytometry. Data of triplicates presented as 18 mean ± standard deviation (SD). For EC50 determinations, antibody concentration and GMFI data was 19 log transformed before analysis and fit using the log agonist vs response using Prism software 20 (GraphPad) (representative results of 3 independent experiments). 21 22

Figure 2. 23

FS120 stimulated T cells in the absence of anti-Fc crosslinking. A-B. Human PBMCs stimulated with 24 SEA (100 ng/ml) (A) or human T cells stimulated with coated CD3-antibody (clone UCHT-1 at 2.5 25

g/ml) (B) and co-stimulated with FS120 or control antibodies (3.7 nM) in the presence or absence 26 of crosslinking reagents at 1:1 molar ratio (FITC dextran for OX40/Ctrl(4420) mAb2 and anti-Fc 27 antibody (clone MK1A6 in mouse IgG1 format ) for other antibodies). C-D FS120 titration in the 28 presence or absence of anti-Fc crosslinking in SEA-stimulated PBMCs (C) or CD3-stimulated T cells 29 (D). (E) CD3-stimulated T cells co-stimulated with FS120 (1nM) and isotype control antibody or 30 component parts of FS120 and their combination (100nM). (A-E) Data from duplicates is shown as 31 mean ± standard deviation (SD) (representative results of 3 independent experiments). Statistical 32 testing by two-way ANOVA and Tukey’s multiple comparison test (A-B) or one-way ANOVA and 33 Dunnett’s multiple comparisons test (E). Asterisks on top of error bars represent the significant 34 difference to Ctrl(4420) mAb treated samples (* p<0.032, ** p<0.0021, *** p<0.0002, **** 35 p<0.0001). For EC50 determinations, antibody concentration data was log transformed before 36 analysis and fit using the log agonist vs response using Prism software (GraphPad) (C-D). See also 37 Figures S2 and S3. 38 39

Figure 3. 40

FS120 mAb2 stimulated both CD4+and CD8+ T cells. (A) Human CD4+ and CD8+ T cells stimulated with 41

coated CD3 antibody (clone UCHT-1 at 2.5 g/ml for CD4+ and 5 g/ml for CD8+ T cells) and co-42 stimulated with FS120 or OX40 and CD137 antibody controls (3.7 nM) in the presence or absence of 43 crosslinking reagents at 1:1 molar ratio (FITC dextran for OX40/Ctrl(4420) mAb2 and anti-Fc (clone 44 MK1A6 in mouse IgG1 format) for other antibodies). Data from duplicates is shown as mean ± 45 standard deviation (SD) (representative results of 3 independent experiments). Statistical testing by 46 two-way ANOVA and Dunnett’s multiple comparison test. Asterisks on top of error bars represent 47




17

the significant difference to Ctrl(4420) mAb treated samples (* p<0.032, ** p<0.0021, *** p<0.0002, 1 **** p<0.0001). (B) OX40 and CD137 receptor quantification in CD4+and CD8+ T cells from 3 PBMC 2 donors, activated overnight with CD3/CD28 beads (1:1 ratio), by flow cytometric fluorescence 3 quantification with beads (BangsLabs). See also Figures S1 and S4. 4 5

Figure 4. 6

Antitumor activity of the FS120 surrogate. (A-B) Balb/c mice (n=15) inoculated with 106 CT26 cells 7 subcutaneously and treated with 1 mg/kg FS120 surrogate or controls Q2D starting on day 13 post-8 tumor inoculation for 3 doses injected intraperitoneally. Tumor volume measured every other day. 9 Data shown is mean ± standard error mean (SEM). Statistical testing of tumor volume over time by 10 mixed model analysis. (C) TIL analysis of day 21 CT26 tumors (n=5) treated with 1 mg/kg FS120 11 surrogate or controls Q2D starting on day 10 for 3 doses injected intraperitoneally by flow 12 cytometry. Individual sample data is shown as well as mean ± standard deviation (SD) 13 (representative data from 2 independent experiments). Statistical testing by one-way ANOVA and 14 Tukey’s multiple comparisons test. Asterisks on top of error bars represent the significant difference 15 to Ctrl(4420) mAb treated mice (* p<0.032, ** p<0.0021, *** p<0.0002, **** p<0.0001). See also 16 Figures S5, S6 and S7. 17 18

Figure 5. 19

Peripheral T cell activation and proliferation induced by the FS120 surrogate. (A) Schematic 20 representation of experimental design. (B) Balb/c mice (n=5) inoculated with 106 CT26 cells 21 subcutaneously and treated with 1 mg/kg FS120 surrogate or controls Q2D starting on day 10 post-22 tumor inoculation for 3 doses injected intraperitoneally. Tail vein blood collected on days 10 (pre-23 dose), 11, 15, 17 and 24 for flow cytometric analysis. Data shown is mean ± standard deviation (SD). 24 Statistical testing by two-way ANOVA and Tukey’s multiple comparisons test. Asterisks on top of 25 error bars represent the significant difference to Ctrl(4420) mAb treated mice (* p<0.032, ** 26 p<0.0021, *** p<0.0002, **** p<0.0001). 27 28

Figure 6. 29

Increased inflammation induced by the crosslink-independent CD137 agonist antibody. (A) 30 Schematic representation of experimental design. (B) Balb/c mice (n=6) treated with 10 mg/kg FS120 31 surrogate or controls Q2D starting on day 1 for 3 doses injected intraperitoneally. Livers, spleens and 32 blood from 3 mice collected on days 7 and 14 post last dose (experiment days 11 and 18) and 33 processed for flow cytometric analysis. Individual sample data is shown as well as mean ± standard 34 deviation (SD). Statistical testing by two-way ANOVA and Tukey’s multiple comparisons test. 35 Asterisks on top of error bars represent the significant difference to Ctrl(4420) mAb treated mice (* 36 p<0.032, ** p<0.0021, *** p<0.0002, **** p<0.0001). 37 38




Published OnlineFirst April 9, 2020.Cancer Immunol Res Miguel Gaspar, John Pravin, Leonor Rodrigues, et al. Activity That Is Dependent on Target Co-EngagementA CD137/OX40 Bispecific Antibody Induces Potent Antitumor

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