Deplete Tumors with Dual OX40 and IL-2 Targeting

Following T-cell receptor stimulation, the tumor necrosis factor receptor family member OX40 is transiently expressed on CD4+ T and CD8+ T cells, playing critical roles in cell activation, effector function, and long-term survival.1 OX40 ligand (OX40L), expressed on activated antigen-presenting cells, facilitates signaling transduction to activate antigen-specific T cells.2 However, Tregs greatly upregulate OX40 expression inside the tumor microenvironment (TME).3 4 Despite several being under clinical investigation, no anti-OX40 antibodies have been approved, most of which do not achieve clear antitumor effects.5 6 The Fc region of antibodies plays a critical role in engaging immune effector mechanisms. Both human IgG1 (human immunoglobulin G subclass 1) and mouse IgG2a (mouse immunoglobulin G subclass 2a) exhibit a preferential affinity for activating Fcγ receptors, thereby promoting the clearance of target cells via antibody-dependent cellular cytotoxicity and antibody-dependent cellular phagocytosis. Recent studies have shown that the hIgG1 backbone of aOX40 antibodies is essential for effectively depleting Treg cells within the TME through Fc-Fcγ receptor interactions.7 8 However, despite effective regulatory T cell (Treg) depletion, these antibodies have demonstrated limited capacity to enhance the expansion of tumor-infiltrating lymphocytes (TILs), which restricts their overall therapeutic efficacy.9 10 We hypothesize that Treg depletion alone is insufficient to rejuvenate TILs. Therefore, the addition of exogenous T cell-stimulatory cytokines may be necessary to expand and activate TILs for effective tumor control.

Interleukin-2 (IL-2), a pleiotropic cytokine that can proliferate and activate cytotoxic T cells or natural killer (NK) cells, has been approved for treating metastatic melanoma and renal cell cancer.11 IL-2 preferentially binds to the high-affinity IL-2Rαβγ receptor complex, then peripheral or intratumoral Treg expressed trimer IL-2 receptor complex could absorb a large proportion of IL-2, potentially impairing the therapeutic efficacy.12 13 High-dose IL-2 expands CD8+ T and NK cells for tumor control but causes severe peripheral toxicity.14 15 To address these limitations, no-alpha IL-2 muteins and anti-programmed cell death protein-1 (PD-1) fusion proteins have been developed to selectively target and activate PD-1+ tumor-specific T cells, reducing CD25 binding to prevent Treg activation.16–18 Despite these advances, such approaches may still induce peripheral NK cell expansion and associated toxicity. Other IL-2 muteins, designed to reduce binding to CD122 or CD132, demonstrate significantly lower peripheral toxicity by limiting NK activation and proliferation.19–21 However, these CD25-biased IL-2 muteins may still bind Tregs and could weaken antitumor activity.22 23 To improve IL-2-based cancer therapy, the innovative design of IL-2 needs to have a distinct binding affinity to these target cells, aiming to expand tumor-specific CD8+ T cells and decrease Tregs within the TME while preventing peripheral NK and endothelial cell activation.

We found that IL-2 signaling is essential during anti-OX40 (aOX40) antibody-mediated tumor control. To achieve efficient Treg depletion while enhancing TIL functionality within the TME, we engineered a novel anti-OX40 bispecific fusion protein with attenuated IL-2, using a combination of Fab physical blocking and the Rβ binding reducing IL-2 N88D mutation to alter the IL-2’s binding pattern. This design is intended to minimize NK cell proliferation in the peripheral circulation, thereby decreasing cytokine consumption and systemic toxicity, while effectively depleting Treg within the TME and rejuvenating TIL for enhanced tumor control.

BALB/c and C57BL/6 mice, aged 6–8 weeks, were purchased from Vital River Laboratories (Beijing, China). C57BL Rag1 KO mice were purchased from the model animal research center of Nanjing University. All mice were maintained under specific pathogen-free conditions in the Institute of Biophysics and Tsinghua University animal facilities. All studies were approved by the Animal Care and Use Committee of the Institute of Biophysics and Tsinghua University.

Cell lines and reagents

Invitrogen FreeStyle 293-F cells (R79007) were cultured in SMM 293-TII medium (M293TII, Sino Biological) or EX-CELL 293 serum-free medium (14571C, Sigma-Aldrich). MC38 and CT26 cell lines were purchased from the American Type Culture Collection. MC38-OVA cells were selected from single-cell clones transduced with a lentivirus expressing OVA. Both MC38 and MC38-OVA cells were cultured and maintained in Dulbecco’s Modified Eagle’s Medium supplemented with 10% heat-inactivated fetal bovine serum, 2 mM l-glutamine, 0.1 mM minimum essential medium non-essential amino acids, 100 U/mL penicillin, and 100 mg/mL streptomycin. Purified mouse splenocytes and CTLL2 cells were maintained in complete Roswell Park Memorial Institute (RPMI) 1640 medium, supplemented with 10% heat-inactivated fetal bovine serum. All cell lines were cultured at 37°C in a 5% CO2 atmosphere and routinely tested for Mycoplasma contamination using a Mycoplasma detection kit (R&D Systems). Antibodies, including anti-CD8 antibody (TIB210), FcγRII/III blocking antibody (2.4G2), anti-NK1.1 antibody (PK136), anti-interferon (IFN)-γ antibody (XMG1.2), anti-IL-2 antibody (JES6-1A12), and anti-programmed death-ligand 1 (PD-L1) antibody (10F. 9G2), were purchased from Bio X Cell (USA). FTY720 was purchased from Sigma-Aldrich.

Production of bispecific and monoclonal antibodies

Using the heterodimeric Fc variant KiHss-AkKh technology, the wild-type human IL-2 or IL-2 muteins were fused with the knob variant Fc region, while the aOX40 Fab was fused with the hole variant Fc region. The aOX40-mIL2-Fc antibody was generated by transient co-transfection of three plasmid constructs into FreeStyle 293-F cells. The supernatant containing bispecific antibodies was purified using protein A affinity chromatography according to the manufacturer’s protocol.

Tumor growth and treatment

A total of 5×105 MC38, 7×105 MC38OVA, and 4×105 CT26 cells in 100 µL phosphate-buffered saline (PBS) were inoculated subcutaneously into the right dorsal flanks of 6–8 weeks old mice. Tumor-bearing mice were randomly assigned to treatment groups when tumors reached approximately 100–150 mm3. For the depletion of NK1.1 and CD8+ T cells, 400 µg or 200 µg antibodies were injected intraperitoneally 1 day before the initial treatment, followed by biweekly administration for 2 weeks. To neutralize IL-2, 200 µg of mouse IL-2 blocking antibody was administered on days 12, 15, and 18. FTY720 exhibits agonistic activity at sphingosine 1-phosphate (S1P) receptors, thereby inhibiting S1P/S1P1-dependent lymphocyte egress from secondary lymphoid tissues and the thymus. FTY720 was intraperitoneally administered at a dose of 25 µg 1 day before treatment initiation, followed by 10 µg every other day for 2 weeks. Anti-IFN-γ was intraperitoneally injected (500 µg per mouse) 1 day before aOX40-mIL2-Fc treatment. For anti-PD-L1 and anti-cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) treatment, 200 µg of anti-PD-L1 or 100 µg of anti-CTLA-4 antibody were administered intraperitoneally. Tumor volumes were measured using the formula: volume=(length×width×height)/2.

Flow cytometry analysis

To evaluate the infiltration of immune cells and the ratio of CD8+ T/Treg, tumor-bearing mice were sacrificed and tumors were harvested 2 days after a single or second treatment. Single-cell suspensions from the spleen, tumor, or in vitro co-cultured cells were incubated with anti-FcγIII/II receptor (clone 2.4G2) for 15 min at 4°C to block non-specific binding before staining with the conjugated antibodies. LIVE/DEAD fixable yellow dye (Thermo Fisher Scientific) was employed to exclude dead cells. For intracellular staining of IFN-γ and Foxp3, samples were fixed, permeabilized, and subsequently stained with anti-mouse IFN-γ or anti-mouse Foxp3 antibodies. All staining procedures were conducted in the dark at 4°C. Cytokine levels in the supernatants from mice serum were measured using the BD CBA Mouse Th1/Th2/Th17 Kit. Data were collected on FACS Fortessa flow cytometer (BD) and analyzed using FlowJo (TreeStar) software.

Ex vivo binding assay

To evaluate the binding affinity of fusion protein, single-cell suspension from C57BL6 spleen (1×10e6) or MC38 tumor was incubated with anti-CD16/32 (anti-FcγIII/II receptor, clone 2.4G2) for 30 min at 4°C. IL-2, aOX40 or various forms of IL-2/aOX40 antibodies were added for 15 min on ice, followed by two washes and staining with anti-human IgG Fcγ-PE for 20 min on ice. After additional washes, samples were analyzed.

CTLL-2 proliferation assay

To assess IL-2 biological activity, various fusion proteins were co-cultured with CTLL-2 cells. Purified proteins, including IL-2/aOX40-Fc, aOX40-IL2-Fc, or aOX40-mIL2-Fc, were serially diluted in 100 µL medium and added to a 96-well plate. Each well received 3×103 of CTLL-2 cells in 100 µL medium and was incubated for 72 hours at 37°C in a 5% CO2. Following incubation, 20 µL of CCK-8 reagent (Cell Counting Kit-8) was added, and the plate was incubated for an additional 3–4 hours at 37°C in 5% CO2. Absorbance was read at 450 nm.

CD8+ T-cell binding and expansion in vitro

Splenocytes from C57BL6 mice were cultured with aCD3 and aCD28 (1 µg/mL) and subsequently stimulated with or without IL-2. OX40 and CD25 receptor expression on CD8+ T cells was detected after IL-2 or aOX40-Fc stimulation. For binding affinity assessment, splenocytes were incubated with aOX40 or IL-2/aOX40-Fc antibodies for 15 min on ice, followed by two washes and staining with anti-human IgG Fcγ-PE for 20 min on ice, then analyzed for CD8+ T cells binding throughflow cytometry. For proliferation analysis, CD8+ T cells were sorted from the cultural splenocytes using a negative CD8+ T Isolation Kit (STEMCELL Technologies) following the manufacturer’s instructions, and co-cultured with aOX40 or IL-2/aOX40 fusion proteins for 72 hours, followed by evaluation with the CCK-8 assay.

C57BL/6 mice bearing MC38 tumors were treated with varying doses of fusion proteins, with body weight monitored daily. Serum samples were collected 12 hours after the second antibody injection for cytokine analysis, measuring serum IFN-γ levels via CBA. Peripheral blood was collected 48 hours after the second injection to assess NK cell numbers using flow cytometry.

Single-cell RNA sequencing and analysis