BGB-A445, a novel non-ligand-blocking agonistic anti-OX40 antibody, exhibits superior immune activation and antitumor effects in preclinical models

Abstract

OX40 is a costimulatory receptor that is expressed primarily on activated CD4⁺, CD8⁺, and regulatory T cells. The ligation of OX40 to its sole ligand OX40L potentiates T cell expansion, differentiation, and activation and also promotes dendritic cells to mature to enhance their cytokine production. Therefore, the use of agonistic anti-OX40 antibodies for cancer immunotherapy has gained great interest. However, most of the agonistic anti-OX40 antibodies in the clinic are OX40L-competitive and show limited efficacy. Here, we discovered that BGB-A445, a non-ligand-competitive agonistic anti-OX40 antibody currently under clinical investigation, induced optimal T cell activation without impairing dendritic cell function. In addition, BGB-A445 dose-dependently and significantly depleted regulatory T cells in vitro and in vivo via antibody-dependent cellular cytotoxicity. In the MC38 syngeneic model established in humanized OX40 knock-in mice, BGB-A445 demonstrated robust and dose-dependent antitumor efficacy, whereas the ligand-competitive anti-OX40 antibody showed antitumor efficacy characterized by a hook effect. Furthermore, BGB-A445 demonstrated a strong combination antitumor effect with an anti-PD-1 antibody. Taken together, our findings show that BGB-A445, which does not block OX40–OX40L interaction in contrast to clinical-stage anti-OX40 antibodies, shows superior immune-stimulating effects and antitumor efficacy and thus warrants further clinical investigation.

Keywords

BGB-A445 / OX40 / agonistic antibody / OX40L noncompetitive

1 Introduction

Blocking immune checkpoints with monoclonal antibodies (mAbs), such as anti-PD-1 and anti-CTLA-4, has achieved unprecedented success across a broad spectrum of cancer types [1]. However, primary and acquired resistance to anti-PD-1 treatment have been observed and raised unmet medical needs for alternative or complementary therapeutic approaches [1]. Notably, the direct stimulation of effector T cells with agonistic OX40 antibodies has emerged as a promising approach and has shown therapeutic potential in preclinical studies [2]. However, the promising antitumor efficacy of agonistic anti-OX40 observed in preclinical models has not been reproduced in clinical trials [3], and conceptual gaps remain to be filled.

OX40, an immune costimulatory receptor belonging to the TNFRSF family, is expressed primarily on activated effector T cells (Teffs) and T regulatory cells (Tregs) to a great extent in the tumor microenvironment [4,5]. Meanwhile, its sole ligand, OX40L, is expressed mainly on activated antigen-presenting cells (APCs), including activated B cells, mature conventional dendritic cells (DCs), plasmacytoid DCs, macrophages, and Langerhans cells [6–9]. OX40–OX40L interaction leads to the activation of the canonical NF-κB1 and noncanonical NF-κB2 pathways, which play key roles in the regulation of the survival, differentiation, expansion, cytokine production, and effector functions of T cells [10–17]. In addition, the ligation of OX40L on DCs can promote DC maturation and enhance DC cytokine production [18].

Treatment with the agonistic anti-OX40 surrogate antibody mAb OX86 has exhibited antitumor efficacy in various preclinical mouse models [19,20]. The mechanisms of action of agonistic anti-OX40 antibodies for cancer treatment involve triggering the costimulatory signaling pathway to improve the effector functions of T cells directly as well as inhibiting the suppressive function of Tregs and inducing the maturation and migration of DCs [20–25]. Moreover, intratumoral Treg depletion in an FcγR-dependent manner plays an important role in the antitumor efficacy of OX40 antibodies [26]. Therefore, OX40 has become an attractive target for cancer immunotherapy using agonistic anti-OX40 mAbs in recent years [27,28].

Although agonistic anti-OX40 has been validated in preclinical studies, its clinical performance is not encouraging. Agonistic anti-OX40 antibodies (e.g., MEDI0562, MOXR0916, PF-04518600, ABBV-368, GSK3174998, and BMS-986178), either as a single agent or in combination with other therapies, have also been tested in phase I/II clinical studies but have only shown a few partial responses at low doses thus far.

A gap between preclinical and clinical studies is the fact that OX86, the widely used OX40 agonist antibody, is a non-OX40L-competitive antibody [29]. By contrast, all anti-OX40 antibodies that have been tested in clinical trials are OX40L-competitive antibodies that block OX40–OX40L interaction. OX40–OX40L interaction is essential for enhancing the effector function of T cells [17]. In addition, this interaction can promote DC maturation through OX40L [30]. Thus, we hypothesized that the blockade of OX40–OX40L restricts the efficacy of ligand-competitive antibodies, whereas a non-ligand-competitive anti-OX40 antibody could potentially activate optimal T cell functions without impairing the positive contributions of OX40L and DCs in the tumor microenvironment. We screened out and engineered a humanized mAb named BGB-A445, which is currently under clinical development for the treatment of human malignancies, to test our hypothesis. BGB-A445 binds to OX40 with high affinity without blocking OX40–OX40L interaction and exerts optimal effects on the activation and proliferation of Teffs and the maturation of DCs. It also presents a better effect on Treg depletion than other anti-OX40 antibodies. In addition, in tumor models, BGB-A445 exhibits significant antitumor efficacy either as a single agent or in combination with anti-PD-1 therapy.

2 Materials and methods

2.1 ELISA

OX40 protein was coated onto 96-well plates at 4 °C overnight. After being washed with PBS/0.05% Tween-20, the plates were blocked with PBS/3% BSA for 2 h at room temperature. Subsequently, the plates were washed with PBS/0.05% Tween-20 and incubated with supernatants of hybridoma clones at room temperature for 1 h. An HRP-linked antimouse immunoglobulin G (IgG) antibody (Cat.: 115035-008, Jackson ImmunoResearch Inc.) and substrate (Cat.: 00-4201-56, eBioscience, USA) were used to develop the color signal, the absorbance of which was measured at a wavelength of 450 nm with a plate reader (SpectraMax Paradigm, Molecular Devices/PHERAstar, BMG LABTECH).

2.2 Cell binding assay

The HuT78/OX40 cell line was established through the transfection of an expression plasmid containing the human OX40 gene into HuT78 cells (ATCC) and selection with 1 mg/mL G418 (Sigma) in culture medium. The cells were washed with fluorescence-activated cell sorting (FACS) buffer (1% FBS in DPBS), seeded at 5 × 10⁴ per well in 200 µL of FACS buffer in a 96-well V-bottomed FACS plate (Corning), and blocked for at least 10 min. The cells were then incubated with hybridoma supernatants (for antibody screening) and gradient concentrations of BGB-A445 (for BGB-A445 characterization) on ice for 1 h. Antimouse IgG eFluor® 660 antibodies (Cat.: 50-4010-82, eBioscience, USA, for antibody screening) or fluorescence-labeled antihuman (F(abʹ)2) (Jackson ImmunoResearch, for BGB-A445 characterization) were used to detect antibody binding to the cell surface. Finally, the cells were fixed with 1% paraformaldehyde (PFA) in DPBS, and cell-bound fluorescence was measured by using a flow cytometer.

2.3 OX40 receptor occupancy of BGB-A445

T cells were isolated from the whole blood of healthy donors by using a T cell enrichment kit (stem cell) and blocked with 10% AB-human serum for 60 min. Different concentrations of biotin-labeled BGB-A445 were then added to the T cells, and the cells were incubated for an additional 30 min. The percentage of biotin-positive cells among human CD4⁺ T cells was analyzed by FACS. Data were presented as the percentage of OX40 receptor occupancy (RO) by normalizing the actual binding rate to the max binding rate.

2.4 OX40L competition assay

The OX40L-expressing cell line HEK293/OX40L was established to determine the effects of different agonistic anti-OX40 mAbs on the binding of OX40 to OX40L. For a competition assay, HEK293/OX40L cells (10⁵/well) were seeded in 100 µL of staining buffer (DPBS + 1% FBS) in 96-well V-bottomed plates. The fusion protein of human OX40 with murine IgG2a Fc (hOX40–mIgG2a) was preincubated with anti-OX40 mAbs or human IgG (huIgG) at a molar ratio of 1:1 at room temperature for 10 min. The protein mixture was then added to the cell suspension, and the cells were incubated at 4 °C for 30 min before being stained with Alexa Fluor® 647 antimouse IgG. The OX40–OX40L binding signal (mean fluorescence intensity, MFI) was determined and analyzed through flow cytometry by using a flow cytometer (Guava easyCyte 12HT, Merck-Millipore, USA) and guavaSoft 3.1.1.

2.5 OX40 costimulation assay with HuT78/OX40 cells

OX40-expressing HuT78 cells (ATCC, HuT78/OX40) were generated via stable transfection. Similarly, target cells (HEK293/FcγRI/anti-CD3) expressing the T cell engager anti-CD3 [31] and Fc gamma receptor I (FcγRI) were established. HuT78/OX40 cells (3 × 10⁴/well) were cocultured overnight with HEK293/ FcγRI/anti-CD3 cells (2 × 10⁴/well) in the presence of anti-OX40 mAbs in 96-well plates (Costar 3599) to determine the costimulation activity of agonistic anti-OX40 mAbs. IL-2 levels in the culture supernatants were determined by ELISA by using a kit from Thermo Fisher (88-7025-88) in accordance with the manufacturer’s instructions.

2.6 Costimulation assay with autologous CD4⁺ T cells and DCs

Human CD4⁺ T cells and CD14⁺ monocytes were isolated from the peripheral blood mononuclear cells (PBMCs) of healthy donors by using magnetic beads in accordance with the manufacturer’s instructions (Miltenyi, Cat.: 130-096-533 and Cat.: 130-050-201). Purified CD4⁺ T cells were cryopreserved in liquid nitrogen before the assay. CD14⁺ monocytes were then cultured with GM-CSF (Sino Biological, Cat.: 10015-H07H 100 ng/mL) and IL-4 (Sino Biological, Cat.: GMP-11846-HNAE, 100 ng/mL) for 5 days. Loosely adherent cells (monocyte-derived immature DCs) were collected by gentle pipetting. In the coculture, purified CD4⁺ T cells (freshly thawed, 2 × 10⁴/well) were mixed with DCs (10⁴/well) in the presence of SEB (in-house produced, 0.1 μg/mL) and anti-OX40 mAbs in 96-well plates for 3 days. In some cases, an anti-OX40L blocking polyclonal antibody (R&D Systems, Cat.: AF1054) was added to the coculture to determine the effects of endogenous OX40–OX40L interaction on CD4⁺ T cells and DCs. IL-2 production in the supernatant was determined through ELISA (Invitrogen, Cat.: 887025). The expression of CD83 and CD86 on DCs was examined with APC/Cyanine7-conjugated anti-CD83 (BioLegend, Cat.: 305330) and APC-conjugated anti-CD86 (BioLegend, Cat.: 374208), respectively. The samples were subjected to flow cytometry with a NovoCyte 3005 system and NovoExpress software (version 1.4.1).

2.7 Preparation of BGB-A445 Fab and OX40 proteins

For crystallography, mutations were introduced at residues T148 and N160 of OX40 to block the glycosylation of OX40 and to improve the homogeneity of the protein. DNA encoding the mutant human OX40 (residues M1–D170 with the two mutated sites T148A and N160A) was cloned into an expression vector with the inclusion of a hexa-His tag. This construct was transiently transfected into 293F cells (Thermo Fisher) for protein expression at 37 °C for 7 days. The cells were harvested, and the supernatant was collected and incubated with His tag affinity resin at 4 °C for 1 h. The resin was rinsed three times with a buffer containing 20 mmol/L Tris (pH 8.0), 300 mmol/L NaCl, and 30 mmol/L imidazole. The OX40 protein was then eluted with a buffer containing 20 mmol/L Tris (pH 8.0), 300 mmol/L NaCl, and 250 mmol/L imidazole. Subsequently, further purification was performed with Superdex 200 (GE Healthcare) in a buffer containing 20 mmol/L Tris (pH 8.0) and 100 mmol/L NaCl. Paired plasmids containing the heavy and light chains of BGB-A445 Fab were transiently cotransfected into HEK 293F cells (Thermo Fisher) for protein expression. The supernatant was collected, and BGB-A445 Fab was purified with the His tag affinity resin before further purification with Superdex 200 (GE Healthcare) in a buffer containing 20 mmol/L Tris and 150 mmol/L NaCl (pH 8.0). The purified OX40 and BGB-A445 Fab were mixed at a molar ratio of 1:1, incubated for 30 min on ice, then further purified with Superdex 200 (GE Healthcare) in a buffer containing 20 mmol/L Tris (pH 8.0) and 100 mmol/L NaCl. The complex peak was collected and concentrated to approximately 30 mg/mL.

The extracellular domain of OX40 with a C-terminal human Fc tag was constructed and transiently transfected into the HEK 293F cell line for protein expression at 37 °C for 7 days to prepare the proteins used for surface plasmon resonance (SPR) analysis. The cells were harvested, and the supernatant was collected and incubated with MabSelect SuRe resin (GE Healthcare) at 4 °C for 1 h. The resin was rinsed three times with DPBS buffer. The OX40 protein was eluted with 50 mmol/L acetic acid (pH 3.5) then further purified with Superdex 200 (GE Healthcare) in DPBS buffer. OX40 mutants were created with a mutagenesis system from TransGen then subjected to expression and purification in accordance with the same method used for the expression and purification of the wild-type (WT) OX40.

2.8 Crystallography study

Cocrystal screening was performed by mixing the protein complex with a reservoir solution at a volume ratio of 1:1. Cocrystals were obtained from hanging drop culture at 20 °C through vapor diffusion with a reservoir solution containing 0.1 mol/L HEPES (pH 7.0), 1% PEG 2000 MME, and 0.95 mol/L sodium succinate. Nylon loops were used to harvest the cocrystals, and the crystals were immersed in reservoir solution supplemented with 20% glycerol for 10 s. Diffraction data were collected at BL17U1 at the Shanghai Synchrotron Radiation Facility and processed with the XDS program [32]. Phases were solved with the program PHASER [33] by using the structures of IgG Fab (chains C and D of PDB 5CZX) and OX40 (chain R of PDB 2HEV) as the molecular replacement searching models [34,35]. The Phenix.refine graphical interface was used to perform rigid body, TLS, and restrained refinement against X-ray data; the results were adjusted with the COOT program and further refined with the Phenix.refine program [36,37]. The X-ray data collection and refinement statistics are summarized in Table S1. The atomic coordinates and structural factors of the complex structure have been deposited in the RCSB Protein Data Bank with the accession number PDB:7EUH.

2.9 Antibody preparation

A Chinese hamster ovary (CHO) stable cell line expressing BGB-A445 was established by following a standard protocol [38]. The CHO stable cell line was cultured in a shaker at 37 °C under 5% CO₂ for 7–14 days to express BGB-A445, and the culture was harvested until cell viability dropped below 80%. The coding sequences of the antibodies against MOXR0916 (Genentech, patent WO2015153513), INCAGN1949 (AGENUS, patent WO2016179517), BMS-986178 (Bristol–Myers–Squibb, patent WO2019089921), GSK3174998 (GlaxoSmithKline, patent WO2017021910), Hu3738 (AbbVie, patent WO2018112346), and muCh15mt (homolog of an antihuman PD-1 antibody BGB-A317 (patent US8735553) with the constant region substituted with a murine source) were synthesized and cloned into an expression plasmid. Paired plasmids (containing the heavy and light chains) for each antibody were transiently cotransfected into HEK 293F cells (Thermo Fisher) for antibody expression at 37 °C under 5% CO₂ for 7 days. For the purification of all the antibodies above, the supernatants of the cell cultures were collected, and each antibody was purified with a protein A column then with a Superdex 200 column (GE Healthcare) in PBS buffer. The purified antibodies were concentrated and stored in aliquots in a −80 °C freezer.

2.10 SPR analysis

The binding affinity of the OX40 point mutants to BGB-A445 Fab was characterized through SPR assays using a BIAcore 8K system (GE Life Sciences). WT or mutant OX40 was immobilized on a CM5 biosensor chip (Cat.: BR100530, GE Life Sciences) by using EDC and NHS. Then, a serial dilution of BGB-A445 Fab in HBS-EP + buffer (Cat.: BR-1008-26, GE Life Sciences) was flowed over the chip surface with a contact time of 180 s and a dissociation time of 600 s at 30 µL/min.

For the binding of mouse/human OX40 to the mouse OX40 ligand, 50 nM mouse OX40 ligand was immobilized on a chip. A series of dilutions of the OX40 extracellular domain of specific species (human and mouse) were injected into the BIAcore system.

Changes in SPR signals were analyzed to calculate association rates (ka) and dissociation rates (kd) by using the one-to-one Langmuir binding model (BIA Evaluation Software, GE Life Sciences). The equilibrium dissociation constant (K_D) was calculated as the ratio of kd/ka. The K_D fold shift of the mutant was calculated as the ratio of mutant K_D/WT K_D.

2.11 Antibody-dependent cellular cytotoxicity against phytohemagglutinin-stimulated PBMCs

CD16a-expressing NK92MI/CD16V cells were generated from NK92MI cells (ATCC, CRL-2408) via the stable transfection of expression constructs containing CD16a (V158 allele) and FcRγ genes. NK92MI/CD16V cells were cultured in MEM alpha (Gibco, 12561-056) supplemented with 0.2 mM inositol (Sigma, I7508), 0.1 mmol/L 2-mercaptoethanol (Sigma, 97622), 0.02 mmol/L folic acid (Sigma, F8758), and 20% heat-inactivated FBS (Corning, 35-079-CV). Whether BGB-A445 triggers antibody-dependent cellular cytotoxicity (ADCC) against human T cells was evaluated by using phytohemagglutinin (PHA)-stimulated PBMCs as target cells. In brief, PBMCs from healthy donors were preactivated with PHA (1 μg/mL, Sigma, L2769) in RPMI 1640 (Corning, 10-040-CVR) supplemented with 10% heat-inactivated FBS and 50 μmol/L β-mercaptoethanol for 3 days. PHA-preactivated PBMCs (5 × 10⁴/well) or OX40^Hi target BW5417.3/OX40 cells were then cocultured with an equal number of NK92MI/CD16V cells in 96-well V-bottomed plates in the presence of BGB-A445 (0.01–10 μg/mL), BGB-A445/huIgG1mf (mutant Fc without FcγR binding, negative control), or MOXR0916 for 18 h. The percentages of CD8⁺ cells and Tregs (CD3⁺CD4⁺Foxp3⁺) among CD3⁺ cells were determined through flow cytometry with a Guava flow cytometry analyzer (Merck). The results were analyzed by using guavaSoft (3.1.1).

2.12 OX40 expression evaluation

Human PBMCs derived from healthy donors were cultured in 2 mL of RPMI-1640 complete culture medium (2 × 10⁶/well) and added with 1 μg/mL OKT3 (anti-CD3 functional antibody) to stimulate OX40 expression. On day 1 post-stimulation, the cells were collected and OX40 expression on the CD8⁺ cells, CD4⁺ cells, and Tregs (CD3⁺CD4⁺Foxp3⁺) among CD3⁺ cells were determined through flow cytometry.

2.13 Mixed lymphocyte reaction assay

A mixed lymphocyte reaction (MLR) assay was performed as previously described [39]. In brief, mature DCs were induced from human PBMC-derived CD14⁺ myeloid cells through culture with GM-CSF and IL-4 followed by lipopolysaccharide (LPS) stimulation. Next, mitomycin C-treated DCs were cocultured with allogenic CD4⁺ T cells in the presence of BGB-A445 or MOXR0916 (0.1–10 μg/mL) for 2 days with or without the anti-PD-1 antibody BGB-A317. IL-2 production in the coculture was detected by ELISA as the readout of the MLR.

2.14 Antitumor efficacy in syngeneic mouse models

All animal studies were performed with the approval of the Institutional Animal Care and Use Committee of BeiGene. Six- to eight-week-old female humanized OX40 knock-in (hOX40) mice against a C57BL/6 background (Jiangsu Biocytogen Co., China) were used for efficacy and pharmacodynamics studies. For single-agent efficacy studies, 1 × 10⁶ MC38 tumor cells (a gift from the laboratory of Chen Dong, PhD, Institute for Immunology, Tsinghua University, Beijing, China) suspended in 100 µL of PBS were implanted subcutaneously (s.c.) into the right flanks of the hOX40 mice. On day 7 after implantation, the mice were randomly allocated into groups in accordance with tumor volume and treated with the vehicle (PBS or placebo buffer), BGB-A445, or MOXR0916 at various doses as indicated once a week for 3 weeks. For the combination study in the MC38 (Kerafast, Inc., Boston, MA, USA) model, the mice were randomized and intraperitoneally (i.p.) treated with the vehicle, BGB-A445, an antimouse PD-1 antibody (muCh15mt) as monotherapy [31], or a combination of agents at indicated doses once a week for 2 weeks.

Bodyweight and tumor volume were measured twice a week. Tumor volumes are shown as mm³ and were calculated by using the formula V = 0.5(a × b²), where a and b are the long and short diameters of the tumor, respectively. The mice were euthanized by using carbon dioxide once their tumor volumes reached ≥2000 mm³, their tumors became ulcerated, or their bodyweight loss exceeded 20%. Tumor growth inhibition (TGI) was calculated by using the following formula:

\mathrm{\%}\mathrm{g}\mathrm{r}\mathrm{o}\mathrm{w}\mathrm{t}\mathrm{h}\mathrm{i}\mathrm{n}\mathrm{h}\mathrm{i}\mathrm{b}\mathrm{i}\mathrm{t}\mathrm{i}\mathrm{o}\mathrm{n}=100\times \left(1-\left({\displaystyle \frac{(\mathrm{t}\mathrm{r}\mathrm{e}\mathrm{a}\mathrm{t}\mathrm{e}\mathrm{d}t)-(\mathrm{t}\mathrm{r}\mathrm{e}\mathrm{a}\mathrm{t}\mathrm{e}\mathrm{d}\mathrm{t}0)}{(\mathrm{p}\mathrm{l}\mathrm{a}\mathrm{c}\mathrm{e}\mathrm{b}\mathrm{o}t)-(\mathrm{p}\mathrm{l}\mathrm{a}\mathrm{c}\mathrm{e}\mathrm{b}\mathrm{o}\mathrm{t}0)}}\right)\right)

where treated t = the treated tumor volume at time t, treated t0 = the treated tumor volume at time 0, placebo t = the placebo tumor volume at time t, and placebo t0 = the placebo tumor volume at time 0.

2.15 In vivo pharmacodynamics studies

The MC38 (a gift from the laboratory of Chen Dong, PhD, Institute for Immunology, Tsinghua University, Beijing, China) model was used for in vivo pharmacodynamics studies. When their tumor sizes reached approximately 200 mm³, the mice were randomly grouped and i.p. injected weekly with BGB-A445 or MOXR0916 twice. At 24 h after the second injection, their spleens and tumors were isolated. The isolated spleens were placed into a cell strainer (Falcon, Cat.: 352340) in a tissue culture dish (Corning, Cat.: 430166). Then, the spleens were pressed with the plunger of a 1 mL syringe in ice-cold DMEM. After centrifugation, the cell pellets were lysed in ACK lysis buffer then washed twice with FACS buffer (1% FBS in DPBS). The cell pellets were resuspended in FACS buffer. Splenocytes were counted with Guava ViaCount Reagent (Millipore, Cat.: 400-0041) and prepared for staining. Tumors were dissociated into single cells following the protocol of a Tumor Dissociation Kit (Miltenyi Biotec, Cat.: 130-096-730). Tumor-infiltrating lymphocytes (TILs, CD45⁺) were isolated by using mouse CD45 MicroBeads (Miltenyi Biotec, Cat.: 130-110-618).

The following antibodies were used for the flow cytometry of the single-cell suspensions of spleen and tumor cells: antimouse-CD45-BV421 (BD Bioscience, Cat.: 563890), antimouse CD3-AF532 (eBioscience, Cat.: 58-5961-82), antimouse-CD4-AF660 (BD Bioscience, Cat.: 100520), antimouse-FOXP3-eF450 (eBioscience, Cat.: 48-5773-82), antimouse-CD8a-Pacific Orange (Invitrogen, Cat.: MCD0830), antimouse-Ki67-FITC (BD Bioscience, Cat.: 556026), antimouse-CD44-Percp/Cy5.5 (BD Bioscience, Cat.: 560570), and antihuman-OX40-PE/Cy5 (BD Bioscience, Cat.: 551500). Staining for surface antibodies, fixation/permeabilization, and staining for intracellular FOXP3 were performed by following the recommended protocol of a FOXP3 buffer kit (eBioscience, Cat.: 48-5773-82). All samples were analyzed by using a Cytek® Aurora apparatus (Cytek Biosciences, Fremont, California, USA) with SpectroFlo® software (Cytek Biosciences, Fremont, California, USA) and FlowJo™ software (TreeStar, Ashland, Oregon, USA).

2.16 Statistics

T effector cell counts, Treg (%), CD8⁺ (%) and CD8⁺/Treg ratios, and tumor volume in the MC38 model treated with single agents were analyzed on logarithmic scale by performing one-way ANOVA with Holm–Sidak’s multiple comparison test. Tumor volume in the MC38 model treated with single agents was analyzed on logarithmic scale by conducting one-way ANOVA with Holm–Sidak’s multiple comparison test. PAN02 and MC38 models under combination therapy were analyzed by using nonparametric Kruskal–Wallis test with Dunn’s multiple comparison test due to the violation of normality. All analyses were performed by using GraphPad Prism 8. P < 0.05 was considered as statistically significant.

3 Results

3.1 BGB-A445 is an anti-OX40 antibody that does not compete with OX40L for binding

On the basis of the hypothesis that an OX40L-noncompetitive agonistic antibody of OX40 will have better efficacy than a competitive antibody, we aim to discover an agonistic anti-OX40 antibody that does not compete with OX40L for binding. A schematic overview of the whole antibody screening workflow is presented in Fig.1. We generated anti-OX40 mAbs by fusing mouse hybridoma SP2/0 cells with splenocytes from OX40 extracellular domain-immunized BALB/c mice. Supernatants of hybridoma clones were initially screened by ELISA on the basis of OX40 binding. ELISA-positive clones were further verified through a cell-based binding assay and SPR analysis to exclude antibodies with fast off-rates. Subsequently, binding-positive clones were tested for agonistic activities with a cell-based OX40 costimulation assay using IL-2 secretion as a readout. We then established an OX40L competition assay to determine whether the top agonistic clones interfered with OX40–OX40L interaction to obtain non-OX40L-competitive antibodies. Clone 445, the sole non-ligand-competitive antibody with the desired properties, was then used for humanization and optimization. Here, we refer to the resulting humanized 445 antibody as BGB-A445.

First, we performed flow cytometry to test the cellular binding activity of BGB-A445 to OX40-overexpressing HuT78 cells and the primary human CD4⁺ T cell line HuT78 derived from healthy donors. BGB-A445 bound potently to cell-surface-overexpressed OX40 in a dose-dependent manner with an EC₅₀ of 0.219 µg/mL (1.46 nmol/L) in HuT78 cells (Fig.1) and an EC₅₀ of 0.157 µg/mL (1.05 nmol/L) in primary human CD4⁺ T cells from six healthy donors (Fig.1).

Then, we conducted a cell-based FACS assay to confirm that BGB-A445 is an OX40-noncompetitive agonistic antibody of OX40L. Briefly, OX40L-expressing HEK293 cells were incubated with BGB-A445 and hOX40–mIgG2a (human OX40 fusion protein with murine IgG2a Fc). Alexa Fluor® 647 antimouse IgG was used to detect the OX40 binding signal to OX40L. In this assay, the reference antibodies MOXR0916 (Genentech), INCAGN1949 (AGENUS), BMS-986178 (Bristol–Myers–Squibb), GSK3174998 (GlaxoSmithKline), and Hu3738 (AbbVie) were also tested for comparison with BGB-A445. All the antibodies tested in the assay and following studies are in IgG1 format. As expected, BGB-A445 did not reduce the binding signal of OX40 to OX40L, indicating that BGB-A445 does not interfere with OX40–OX40L interaction. By contrast, all the other tested clinical-stage anti-OX40 antibodies significantly blocked OX40 binding to OX40L (Fig.1 and S1).

3.2 Cocrystal structure of the OX40/BGB-A445 Fab complex reveals the binding mechanism and confirms the noncompetitiveness of BGB-A445 binding to OX40L

The extracellular domain of OX40 consists of four cysteine-rich domains (CRDs) [34,40]. The cocrystal structure of OX40 in complex with BGB-A445 Fab was solved at a resolution of 2.55 Å to further investigate the mechanism of BGB-A445 binding to OX40 (Table S1). The structure revealed that BGB-A445 utilized five out of six complementarity-determining region (CDR) loops, including all three heavy-chain CDRs (HCDRs) and light-chain CDRs (LCDRs) 1 and 3, to interact with OX40 with a total buried surface of 1578 Ų (Fig.2). Under the guidance of this structure, we performed mutagenesis on several residues of the OX40 protein in the OX40/BGB-A445 interface then evaluated the binding affinity of the mutants to BGB-A445 by SPR analysis. The SPR results show that the H153A mutation of OX40 almost abolished the binding to BGB-A445, while the mutations T154A, I165A, E167A, and D170A significantly reduced the binding affinity of BGB-A445 (Fig. S2). The detailed interactions between BGB-A445 and the five residues of OX40 discussed above are shown in Fig.2. The side chain of H153 on OX40 was surrounded by a small pocket of BGB-A445 on the interaction interface, forming hydrogen bonds with residue S31 of the heavy chain of BGB-A445 (_heavyS31) and _heavyG102 and engaging in pi–pi stacking with _heavyY101. The side chain of E167 formed hydrogen bonds with _heavyY50 and _heavyN52. Meanwhile, D170 formed a hydrogen bond and a salt bridge with _heavyS31 and _heavyK28, respectively. Van der Waals interactions between T154 and _heavyY105 and between I165 and _heavyR59 also contributed to the binding of BGB-A445 to OX40. In conclusion, residues H153, T154, I165A, E167A, and D170A of OX40 were identified as key epitopes of BGB-A445 on the basis of structural biology and experimentally confirmed through SPR analysis.

The OX40L trimer has been reported to interact with one face of OX40 primarily through the CRD1, CRD2, and partial CRD3 regions of OX40 [34]. However, Yang et al. [41] reported that the antibody MOXR0916 shares a partially overlapped binding face of OX40 similar to OX40L and interacts mainly with CRD3 of OX40. The structural superposition of OX40 complexed with OX40L (PDB: 2HEV) or MOXR0916 (PDB: 6OKN) and binding surface analyses showed that MOXR0916 overlapped with part of the OX40L–OX40 binding area and directly occupied the position of the OX40L trimer harboring OX40 (Fig.2 and 2D). This finding is consistent with our in vitro ligand competition data suggesting that MOXR0916 competes with OX40L when binding to OX40 (Fig.1). By contrast, BGB-A445 bound to the opposite face of OX40 as OX40L and interacted with OX40 only through the CRD4 region (Fig.2 and 2D), wherein all epitopes of this antibody were far away from the OX40–OX40L interface, indicating that BGB-A445 can bind to OX40 without interfering with OX40L–OX40 binding. These results are consistent with the noncompetitiveness of BGB-A445’s interaction with OX40L (Fig.1).

3.3 BGB-A445 induces superior T cell activation and DC maturation

We first performed a T cell costimulation assay to evaluate the in vitro costimulation activity of BGB-A445 in augmenting T cell responses (Fig.3). Upon the coculture of HEK293/anti-CD3/FcγRI cells with OX40-expressing HuT78/OX40 T cells in the presence of a Fc-competent agonistic anti-OX40 mAb, HuT78/OX40 cells were activated and secreted IL-2. As shown in Fig.3, BGB-A445 could dose-dependently enhance IL-2 production (EC₅₀: 0.059 μg/mL) with a potency comparable with the potency of MOXR0916 (EC₅₀: 0.110 μg/mL). Given that BGB-A445 did not block OX40–OX40L interaction in the binding assay and structural analysis (Fig.1 and Fig.2), how this attribute affects T cells in the presence of DCs remains unknown. We established a T cell/DC coculture system to determine whether non-OX40L blocking anti-OX40 mAbs demonstrate any advantages over ligand-blocking mAbs (Fig.3). DCs express high levels of OX40L, which can provide endogenous OX40 costimulation via interaction with OX40L. We added the superantigen SEB to the coculture to mimic T cell receptor signaling. Similar to the HuT78/OX40 assay, this analysis showed that BGB-A445 significantly induced primary CD4⁺ T cells to secrete IL-2 in a dose-dependent manner (Fig.3). By contrast, the OX40L-competitive mAb MOXR0916 induced only modest increments in IL-2 at low concentrations and demonstrated a hook effect when the antibody concentration reached 10 μg/mL. The different IL-2 release patterns induced by BGB-A445 and MOXR0916 in the T cell/DC coculture system cannot be explained by their binding activity to OX40 (Fig.1 and 1C) or their direct agonistic function on T cells (Fig.3). The addition of saturated polyclonal OX40L-blocking antibodies with BGB-A445 (10 μg/mL) to the T/DC coculture significantly reduced IL-2 production, further suggesting that endogenous OX40L and exogenous agonistic anti-OX40 mAbs play critical roles in OX40 costimulation (Fig.3).

In addition to enhancing the T cell response, BGB-A445 induced DC maturation, as indicated by the upregulation of the DC maturation/activation markers CD83 and CD86. Compared with those of BGB-A445, the effects of MOXR0916 on DC maturation were marginal (CD83 upregulation) or weaker (CD86 upregulation). In contrast to OX40L-competitive agonistic anti-OX40 mAbs, the noncompetitive mAb BGB-A445 induced optimal T cell costimulation and DC maturation/activation.

3.4 BGB-A445 increases the proliferation of Teffs in a dose-dependent manner in vivo

We investigated the proliferation and activation of splenic T cells in an MC38 tumor model by using hOX40 knock-in mice to further study the mechanism underlying the differential activities of BGB-A445 against MOXR0916 in T cell augmentation in vivo. First, we confirmed that similar to mouse OX40 in the SPR assay, the mouse OX40 ligand can bind to human OX40 (Fig. S3A). Then, we conducted a cell-based competition FACS assay to confirm that BGB-A445 did not interfere with human OX40–mouse OX40L interaction, whereas MOXR0916 can significantly block the binding of human OX40 to mouse OX40L (Fig. S3B). Taken together, our data suggest that human OX40 knock-in mice can serve as an appropriate model for further evaluating the difference between OX40-ligand-competitive and -noncompetitive antibodies in vivo. BGB-A445 dose-dependently increased the number of proliferating (Ki67⁺) Teffs, including CD4 T effector cells (CD3⁺CD4⁺FOXP3⁻) and CD8 T cells (CD3⁺CD8⁺) (Fig.4 and 4B). The numbers of proliferating memory phenotype (CD44^hi) CD4 Teffs and CD8 T cells also significantly increased in a dose-dependent manner (Fig.4 and 4D). Although MOXR0916 also increased the proliferation of Teffs at low doses (0.3, 1, and 3 mg/kg [mpk]), increasing its dose to 10 mpk did not further increase the number of proliferating Teffs (Fig.4–4D). Consistent with the differential T cell activation effects of BGB-A445 and MOXR0916 observed in vitro (Fig.3), BGB-A445 elicited a dose-dependent increase in proliferating T cells without showing the hook effect presented by MOXR0916.

In addition, at low doses, BGB-A445 demonstrated significantly more potent activation in promoting the proliferation of CD8 T cells and memory phenotype CD8 T cells (1 mpk) than MOXR0916. It also exhibited a similar trend in activating CD4 Teffs and memory phenotype CD4 Teffs, albeit without any statistically significant difference.

3.5 BGB-A445 preferentially depletes Tregs and increases the CD8⁺/Treg ratio

PHA-stimulated PBMCs from healthy donors were used as target cells and cocultured with NK92MI/CD16V cells in the presence of BGB-A445 (0.001–10 μg/mL) to explore BGB-A445-mediated ADCC in primary human T cells in vitro. Compared with the medium (blank) control or BGB-A445/huIgG1mf (mutant Fc without the FcγR-binding function [42], i.e., effectorless), BGB-A445 increased the percentage of CD8⁺ cells while reducing the percentage of Tregs among T cells (Fig.5–5D, S5A and S5B). The differential ADCC activities observed on CD8⁺ and Tregs may be attributed to the higher OX40 expression on human Treg cells than on CD8⁺ T cells (Fig. S5C).

Treg depletion was further evaluated in an MC38 tumor model in vivo. Interestingly, our analysis of the percentage and absolute number of Tregs revealed an obvious reduction in TILs but not in splenic Tregs (Fig.5 and S4A–S4B). These different observations may be ascribed to the different OX40 expression percentages and densities. BGB-A445 induced ADCC against tumor Tregs with high OX40 levels and high percentages of OX40-expressing cells (Fig. S4D–S4F). Compared with MOXR0916, BGB-A445 showed better effects on tumor Treg depletion (Fig.5 and S4B) and comparable CD8⁺ T cell infiltration (Fig.5 and S4C). The CD8⁺/Treg ratios in tumors significantly increased in a dose-dependent manner after BGB-A445 treatment but not after MOXR0916 treatment (Fig.5). These findings together indicate that BGB-A445 induces more potent Treg depletion and more strongly increases the CD8⁺/Treg ratio in vitro and in vivo than MOXR0916.

3.6 BGB-A445 exerts dose-dependent antitumor activity in the MC38 syngeneic tumor model and shows superior efficacy in the PD-1 resistant model PAN02

Next, we tested the antitumor effect of BGB-A445 in the MC38 syngeneic model.

BGB-A445 dosed at 0.4, 2, and 10 mpk exhibited dose-dependent antitumor efficacy (Fig.6) with the TGI rates of 14%, 80%, and 78%, respectively (Fig.6). By contrast, MOXR0916 achieved the maximum antitumor effect at 2 mpk, but its TGI decreased at high doses (Fig.6). Specifically, it obtained TGI rates of < 0%, 60%, and < 0% at various doses (Fig.6). Consistent with the observations in the in vitro T/DC coculture assay (Fig.3), the in vivo efficacy data of the MC38 model suggest that MOXR0916 presents a hook effect. We found no significant effect on animal bodyweight in any treatment group throughout the study (Fig.6). Taken together, our results indicate that compared with MOXR0916, BGB-A445 showed better dose-dependent antitumor efficacy likely due to its non-ligand-blocking property.

We further tested whether BGB-A445 functions in the anti-PD1-resistant PAN02 syngeneic model. Fig.6 shows that the surrogate antimouse PD-1 antibody muCh15mt did not show any antitumor efficacy as expected. However, BGB-A445 monotherapy achieved a TGI rate of 75% by day 32, whereas MOXR0916 at the same dose inhibited tumor growth only by 41%, which was not statistically significant.

3.7 BGB-A445 exerts a combined effect with the anti-PD-1 antibody and shows antitumor effects in an anti-PD-1-resistant model

We further studied the antitumor efficacy of BGB-A445 when BGB-A445 was used in combination with an anti-PD1 antibody in vitro and in vivo. In the MLR assay, BGB-A445 alone significantly promoted IL-2 secretion by primary T cells (Fig.7), suggesting that it can be used as a single agent to activate CD4⁺ T cells. Strong MLR was observed when BGB-A445 was used in combination with a human anti-PD-1 antibody, BGB-A317 [43] (tislelizumab, an approved anti-PD-1 antibody developed by BeiGene). By contrast, MOXR0916 showed significantly weakened activity even when combined with the anti-PD-1 antibody and, similar to BGB-A445, did not elicit a dose-dependent increase in MLR (Fig.7).

BGB-A445 and the surrogate antimouse PD-1 antibody muCh15mt were applied in the MC38 syngeneic tumor model in hOX40 knock-in mice to verify the combination effect in vivo. Fig.7 and 7C show that treatment with either muCh15mt or BGB-A445 monotherapies once a week resulted in a negligible TGI effect. By contrast, significantly improved antitumor activity was observed in the combination treatment group, which exhibited a TGI of 78% on day 12 (P < 0.001, combination versus vehicle; P < 0.05, combination versus BGB-A445 monotherapy; and P < 0.001, combination versus muCh15mt monotherapy). We observed no significant effect on animal bodyweight in any treatment group throughout the study (Fig.7). These data demonstrate that the targeting of OX40 and PD-1 results in a synergistic antitumor activity in vivo.

Taken together, our results indicate that BGB-A445 exerts a synergistic effect when used in combination with anti-PD-1.

4 Discussion

Although the agonistic anti-OX40 surrogate antibody OX86 has demonstrated robust antitumor activity in several syngeneic models preclinically, anti-OX40 antibodies have not demonstrated satisfactory efficacy in clinical trials [4,44]. Considering that the anti-OX40 antibodies used in clinical trials are all ligand-competitive, we hypothesized that the blockade of OX40–OX40L interaction might restrict their efficacy. By contrast, a non-ligand-competitive anti-OX40 antibody could potentially activate optimal T cell functions without impairing the positive contributions of OX40–OX40L interaction. Given that OX40L occupies three of the four CRDs of OX40 upon binding [34], generating an agonistic antibody with a non-OX40L-competitive property is difficult. After expending extensive efforts on antibody screening, we successfully generated BGB-A445, a sole humanized IgG1 mAb that targets OX40 without blocking the binding of OX40 to its native ligand OX40L. The produced mAb showed promising and superior efficacy compared with the ligand-blocking antibody MOXR0916.

Cell–cell interaction between T cells and APCs elicits not only T cell proliferation and activation but also APC activation [45]. OX40L is a potent stimulus that upregulates the production of bioactive IL-12 and the expression of ICAM-1, CD80, and CD86 molecules on DCs [18]. The ligation of OX40L can promote DC maturation and increase DC capacity to trigger T cell proliferation and IFN-γ production [30]. Given that a single DC can interact with a cluster of T cells, its increased stimulatory capacity might benefit numerous nearby T cells [7]. The non-OX40L-blocking antibody BGB-A445 potentially has an inherent advantage over the OX40L-blocking antibody in boosting T cells and maintaining APC activity without compromising the benefit of T cell–APC interaction. As we hypothesized and illustrated in Fig.8, the OX40L-blocking antibody can bind to the OX40L-competing OX40 receptors on T cell surfaces and impair the functions of APCs, thus limiting their immune stimulating effects. By contrast, a non-OX40L-blocking antibody (e.g., BGB-A445), in addition to executing an agonistic function, does not block T cell/APC interaction and maintains T cell activation by APCs regardless of antibody concentration. Hence, non-OX40L-blocking antibodies promote T cell proliferation and activation to a higher level than OX40L-blocking antibodies.

Previous studies on ligand-blocking agonistic anti-OX40 antibodies have discovered a hook effect wherein low doses show better efficacy than high doses [46]. An investigation of antibody-mediated OX40 receptor modulation revealed that the maximum T cell activation is achieved at a relatively low dose when the OX40 RO reaches approximately 20%. However, with increasing doses of antibody, the saturated OX40 RO leads to the downregulation of surface OX40, which may occur via the cellular internalization of antibody-bound OX40 and the reduced functional activity of agonistic antibodies [46]. Here, we provide another possible explanation for this phenomenon (Fig.8). At high concentrations, the ligand-blocking antibody competes with OX40–OX40L binding, thus dampening T cell stimulation during APC/T cell interaction. This hypothesis is supported by the findings showing that BGB-A445 dose-dependently enhanced the activation and proliferation of CD4⁺ and CD8⁺ Teffs, whereas the ligand-blocking reference antibody exhibited only a limited effect at a relatively low dose (Fig.3 and Fig.4).

Intratumoral Treg depletion in an FcγR-dependent manner also contributes to the antitumor efficacy of agonistic anti-OX40 antibodies [26,47]. We found that at the percentage and MFI levels, tumor Tregs highly expressed OX40 compared with spleen Tregs (Fig. S4). This finding is consistent with the recent results of animal tumor models demonstrating that tumor-infiltrating Tregs express higher levels of OX40 than Teffs and peripheral Tregs [29]. High OX40 expression on Treg cells has not only been proven in mouse models but also observed in human patient tumor samples. Compared with all other T cell populations analyzed in the tumor or periphery, the Treg TIL population showed the highest OX40 level [48]. Our results indicate that BGB-A445 preferentially depleted Tregs and increased CD8⁺/Treg ratios in vitro and in vivo. The membrane-proximal location of antibody-binding epitopes may facilitate synapse formation between T and tumor cells and induce the optimal T cell engager-mediated killing of tumor cells [49]. Another study showed that in contrast to ADCC, antibody-dependent cellular phagocytosis favors epitopes positioned far away, indicating that the distance of an antibody from the cell membrane might affect the effector mechanism differently [50]. One possibility of superior efficacy of BGB-A445 is that the membrane-proximal location of the BGB-A445-binding epitope may facilitate synapse formation between NK cells and Tregs, thus increasing the NK killing of Tregs.

In summary, we found that BGB-A445 binds to the extracellular proximal domain of human OX40 with high specificity and affinity without blocking the binding of the native ligand OX40L. In an MC38 model of colon adenocarcinoma established in hOX40 knock-in mice, BGB-A445 showed superior antitumor activity to the OX40L-blocking reference antibody MOXR0916. Moreover, compared with either monotherapy, combined treatment with BGB-A445 and an anti-PD-1 antibody significantly inhibited tumor growth. Overall, our results show that BGB-A445 possesses unique biophysical and biological characteristics that make it a promising candidate for the clinical development of treatments for human cancer. A phase I trial to evaluate the safety and preliminary effectiveness of BGB-A445 as a single agent and in combination with tislelizumab in patients with advanced solid tumors is ongoing.

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