Background OX40 has been widely studied as a target for immunotherapy with agonist antibodies taken forward into clinical trials for cancer where they are yet to show substantial efficacy. Here, we investigated potential mechanisms of action of anti-mouse (m) OX40 and anti-human (h) OX40 antibodies, including a clinically relevant monoclonal antibody (mAb) (GSK3174998) and evaluated how isotype can alter those mechanisms with the aim to develop improved antibodies for use in rational combination treatments for cancer.Methods Anti-mOX40 and anti-hOX40 mAbs were evaluated in a number of in vivo models, including an OT-I adoptive transfer immunization model in hOX40 knock-in (KI) mice and syngeneic tumor models. The impact of FcγR engagement was evaluated in hOX40 KI mice deficient for Fc gamma receptors (FcγR). Additionally, combination studies using anti-mouse programmed cell death protein-1 (mPD-1) were assessed. In vitro experiments using peripheral blood mononuclear cells (PBMCs) examining possible anti-hOX40 mAb mechanisms of action were also performed.Results Isotype variants of the clinically relevant mAb GSK3174998 showed immunomodulatory effects that differed in mechanism; mIgG1 mediated direct T-cell agonism while mIgG2a acted indirectly, likely through depletion of regulatory T cells (Tregs) via activating FcγRs. In both the OT-I and EG.7-OVA models, hIgG1 was the most effective human isotype, capable of acting both directly and through Treg depletion. The anti-hOX40 hIgG1 synergized with anti-mPD-1 to improve therapeutic outcomes in the EG.7-OVA model. Finally, in vitro assays with human peripheral blood mononuclear cells (hPBMCs), anti-hOX40 hIgG1 also showed the potential for T-cell stimulation and Treg depletion.Conclusions These findings underline the importance of understanding the role of isotype in the mechanism of action of therapeutic mAbs. As an hIgG1, the anti-hOX40 mAb can elicit multiple mechanisms of action that could aid or hinder therapeutic outcomes, dependent on the microenvironment. This should be considered when designing potential combinatorial partners and their FcγR requirements to achieve maximal benefit and improvement of patient outcomes.

In recent years, there has been considerable interest in mAb-based induction of costimulatory receptor signaling as an approach to combat cancer. However, promising nonclinical data have yet to translate to a meaningful clinical benefit. Inducible T-cell costimulator (ICOS) is a costimulatory receptor important for immune responses. Using a novel clinical-stage anti-ICOS immunoglobulin G4 mAb (feladilimab), which induces but does not deplete ICOS+ T cells and their rodent analogs, we provide an end-to-end evaluation of the antitumor potential of antibody-mediated ICOS costimulation alone and in combination with programmed cell death protein 1 (PD-1) blockade. We demonstrate, consistently, that ICOS is expressed in a range of cancers, and its induction can stimulate growth of antitumor reactive T cells. Furthermore, feladilimab, alone and with a PD-1 inhibitor, induced antitumor activity in mouse and humanized tumor models. In addition to nonclinical evaluation, we present three patient case studies from a first-time-in-human, phase I, open-label, dose-escalation and dose-expansion clinical trial (INDUCE-1; ClinicalTrials.gov: NCT02723955), evaluating feladilimab alone and in combination with pembrolizumab in patients with advanced solid tumors. Preliminary data showing clinical benefit in patients with cancer treated with feladilimab alone or in combination with pembrolizumab was reported previously; with example cases described here. Additional work is needed to further validate the translation to the clinic, which includes identifying select patient populations that will benefit from this therapeutic approach, and randomized data with survival endpoints to illustrate its potential, similar to that shown with CTLA-4 and PD-1 blocking antibodies.Significance:Stimulation of the T-cell activation marker ICOS with the anti-ICOS agonist mAb feladilimab, alone and in combination with PD-1 inhibition, induces antitumor activity across nonclinical models as well as select patients with advanced solid tumors.

The cytokine changes in mice receiving PI3K inhibition in combination with anti-OX40 treatment.

The expression and function of OX40 on tumor-reactive T cells

Positively interaction of OX40 agonist antibody and PI3K inhibition in mice bearing PTEN-loss tumors

Clinical patient case studies of feladilimab monotherapy and in combination with pembrolizumab. A, Clinical history and treatment details for a patient with melanoma who received feladilimab monotherapy and a patient with oropharyngeal squamous cell carcinoma who received feladilimab in combination with pembrolizumab. Tumor sample immunohistochemistry and expression of markers for TIL activation, cytotoxic function and proliferation for the monotherapy case study (B) and the combination therapy case study (C). See Supplementary Fig. S11B for CT imaging of tumor lesions for the patient with oropharyngeal squamous cell carcinoma. CPS, combined positive score; CT, computed tomography; HLA-DR, human leukocyte antigen-DR; HPV, human papillomavirus; PD-L1, programmed death ligand-1; TIL, tumor-infiltrating lymphocyte.

Exemplar flow cytometry gating strategy.

Antibodies used for in vitro and in vivo assays, and experimental model

Expression dynamics and cellular localization of ICOS.

Affinity and kinetics

Gene expression in human T-cell

Flow cytometry gating strategy for ICOS on individual T cell populations.

Antibodies used for in vitro and in vivo assays, and experimental model

Healthy human donor CD4+ T cells were pre-activated with antiCD3/CD28 for 48 hours and then re-stimulated with plate-bound anti-CD3 in the presence of increasing concentrations of soluble or plate-bound feladilimab or isotype control for 72 hours. IFNg levels were then assessed from cell-free supernatants using Meso Scale Discovery (MSD)-based detection.

List of detailed information for antibodies used for CytoF and Flow Cytometry

In vivo effect of PI3K inhibition in combination with anti-OX40 treatment on T cell proliferation

Feladilimab induces phospho-AKT in ICOS-expressing Ba/F3 cells and cytokine production in PBMC from healthy donors and cancer patients.

B-cell maturation antigen (BCMA) is an attractive therapeutic target highly expressed on differentiated plasma cells in multiple myeloma and other B-cell malignancies. GSK2857916 (belantamab mafodotin, BLENREP) is a BCMA-targeting antibody-drug conjugate approved for the treatment of relapsed/refractory multiple myeloma. We report that GSK2857916 induces immunogenic cell death in BCMA-expressing cancer cells and promotes dendritic cell activation in vitro and in vivo GSK2857916 treatment enhances intratumor immune cell infiltration and activation, delays tumor growth, and promotes durable complete regressions in immune-competent mice bearing EL4 lymphoma tumors expressing human BCMA (EL4-hBCMA). Responding mice are immune to rechallenge with EL4 parental and EL4-hBCMA cells, suggesting engagement of an adaptive immune response, immunologic memory, and tumor antigen spreading, which are abrogated upon depletion of endogenous CD8+ T cells. Combinations with OX40/OX86, an immune agonist antibody, significantly enhance antitumor activity and increase durable complete responses, providing a strong rationale for clinical evaluation of GSK2857916 combinations with immunotherapies targeting adaptive immune responses, including T-cell-directed checkpoint modulators.

Sepsis is characterized as life-threatening organ dysfunction caused by a dysregulated host immune response to infection. The purpose of this investigation was to determine the differential effect of sepsis on innate versus adaptive immunity, in humans, by examining RNA expression in specific immune cell subsets, including monocytes/macrophages and CD4 and CD8 T cells. A second aim was to determine immunosuppressive mechanisms operative in sepsis that might be amenable to immunotherapy. Finally, we examined RNA expression in peripheral cells from critically ill nonseptic patients and from cancer patients to compare the unique immune response in these disorders with that occurring in sepsis. Monocytes, CD4 T cells, and CD8 T cells from septic patients, critically ill nonseptic patients, patients with metastatic colon cancer, and healthy controls were analyzed by RNA sequencing. Sepsis induced a marked phenotypic shift toward downregulation of multiple immune response pathways in monocytes suggesting that impaired innate immunity may be fundamental to the immunosuppression that characterizes the disorder. In the sepsis cohort, there was a much more pronounced effect on gene transcription in CD4 T cells than in CD8 T cells. Potential mediators of sepsis-induced immunosuppression included Arg-1, SOCS-1, and SOCS-3, which were highly upregulated in multiple cell types. Multiple negative costimulatory molecules, including TIGIT, Lag-3, PD-1, and CTLA-4, were also highly upregulated in sepsis. Although cancer had much more profound effects on gene transcription in CD8 T cells, common immunosuppressive mechanisms were present in all disorders, suggesting that immunoadjuvant therapies that are effective in one disease may also be efficacious in the others.

Abstract Purpose: OX40 agonist–based combinations are emerging as a novel avenue to improve the effectiveness of cancer immunotherapy. To better guide its clinical development, we characterized the role of the OX40 pathway in tumor-reactive immune cells. We also evaluated combining OX40 agonists with targeted therapy to combat resistance to cancer immunotherapy. Experimental Design: We utilized patient-derived tumor-infiltrating lymphocytes (TILs) and multiple preclinical models to determine the direct effect of anti-OX40 agonistic antibodies on tumor-reactive CD8+ T cells. We also evaluated the antitumor activity of an anti-OX40 antibody plus PI3Kβ inhibition in a transgenic murine melanoma model (Braf mutant, PTEN null), which spontaneously develops immunotherapy-resistant melanomas. Results: We observed elevated expression of OX40 in tumor-reactive CD8+ TILs upon encountering tumors; activation of OX40 signaling enhanced their cytotoxic function. OX40 agonist antibody improved the antitumor activity of CD8+ T cells and the generation of tumor-specific T-cell memory in vivo. Furthermore, combining anti-OX40 with GSK2636771, a PI3Kβ-selective inhibitor, delayed tumor growth and extended the survival of mice with PTEN-null melanomas. This combination treatment did not increase the number of TILs, but it instead significantly enhanced proliferation of CD8+ TILs and elevated the serum levels of CCL4, CXCL10, and IFNγ, which are mainly produced by memory and/or effector T cells. Conclusions: These results highlight a critical role of OX40 activation in potentiating the effector function of tumor-reactive CD8+ T cells and suggest further evaluation of OX40 agonist–based combinations in patients with immune-resistant tumors.