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OX40, a member of the tumor necrosis factor (TNF) receptor superfamily, is expressed on the surface of activated T cells. Upon interaction with its cognate ligand, OX40L, OX40 transmits costimulatory signals to antigen-primed T cells, promoting their activation, differentiation, and survival - processes essential for the establishment of adaptive immunity. Although the OX40-OX40L interaction has been extensively studied in the context of disease treatment, developing a substitute for the naturally expressed membrane-bound OX40L, particularly a multimerized OX40L trimers, that effectively regulates OX40-driven T cell responses remains a significant challenge. In this study, we successfully engineered soluble OX40L-fusion proteins capable of robustly activating OX40 on T cells. This was achieved by incorporating functional multimerization domains into the TNF homology domain of OX40L. These OX40L proteins bound to OX40, subsequently activated NF-κB signaling, and induced cytokine production by T cells in vitro . In vivo , mice treated with one of the OX40L-fusion proteins - comprising a single-chain OX40L trimer linked to the C-terminus of the human IgG1 Fc domain, forming a dimer of trimers - exhibited significantly enhanced clonal expansion of antigen-specific CD4 + T cells during the primary phase of the immune response. A comparable antibody-fusion single-chain TNF protein incorporating 4-1BBL, CD70 (CD27L), or GITRL in place of OX40L elicited similar in vivo T cell responses. Thus, we propose that optimizing the multimerization of OX40L proteins through innovative design strategies may facilitate the development of more effective agonists for targeted immunotherapies., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be consulted as a potential conflict of interest. The author(s) declared that they were an editorial board member of Frontiers, at the time of submission. This had no impact on the peer review process and the final decision., (Copyright © 2025 Sato, Nagai, Suzuki, Ito, Matsuyama, Shibui, Morita, Hikosaka-Kuniishi, Ishii and So.)

Anti-CTLA-4 and anti-PD-1/PD-L1 antibodies have significantly revolutionized cancer immunotherapy. However, the persistent challenge of low patient response rates necessitates novel approaches to overcome immune tolerance. Targeting immunostimulatory signaling may have a better chance of success for its ability to enhance effector T cell (Teff) function and expansion for antitumor immunity. Among various immunostimulatory pathways, the evidence underscores the potential of activating OX40-OX40L signaling to enhance CD8 + T cell generation and maintenance while suppressing regulatory T cells (Tregs) within the tumor microenvironment (TME). In this study, we introduce a potent agonistic anti-OX40 antibody, SHR-1806, designed to target OX40 receptors on activated T cells and amplify antitumor immune responses. SHR-1806 demonstrates a high affinity and specificity for human OX40 protein, eliciting FcγR-mediated agonistic effects, T cell activation, antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC) activities in vitro . In human OX40 knock-in mice bearing MC38 tumor, SHR-1806 shows a trend toward a higher potency than the reference anti-OX40 antibody produced in-house, GPX4, an analog of pogalizumab, the most advanced drug candidate developed by Roche. Furthermore, SHR-1806 displays promising anti-tumor activity alone or in combination with toll-like receptor 7 (TLR7) agonist or PD-L1 inhibitor in mouse models. Evaluation of SHR-1806 in rhesus monkeys indicates a favorable safety profile and typical pharmacokinetic characteristics. Thus, SHR-1806 emerges as a robust OX40 agonist with promising therapeutic potential.

mRNA vaccines have demonstrated efficacy during the COVID-19 pandemic and are now being investigated for multiple diseases. However, concerns linger about the durability of immune responses, and the high incidence of breakthrough infections among vaccinated individuals highlights the need for improved mRNA vaccines. In this study, we investigated the effects of reinforcing costimulation via 4-1BB, a member of the TNF receptor superfamily, on immune responses elicited by mRNA vaccines. We first immunized mice with mRNA vaccines, followed by treatment with 4-1BB costimulatory antibodies to reinforce the 4-1BB pathway at different time points after vaccination. Consistent with prior studies, reinforcing 4-1BB costimulation on the day of vaccination did not result in a substantial improvement in vaccine responses. However, reinforcing 4-1BB costimulation on day 4 after vaccination, when 4-1BB expression levels were highest, resulted in a profound improvement in CD8+ T cell responses associated with enhanced protection against pathogen challenges. A similar clinical benefit was observed in a therapeutic cancer vaccine model. We also report time-dependent effects with OX40, another costimulatory molecule of the TNF receptor superfamily. These findings demonstrate that delayed reinforcement of costimulation may exert an immunologic benefit, providing insights for the development of more effective mRNA vaccines for infectious diseases and cancer.

Purpose: Human OX40 (hOX40/CD134), a member of the TNF receptor superfamily, is mainly expressed on activated T lymphocytes. Triggered by its ligand OX40L (CD252), it provides costimulatory signals that support the differentiation, proliferation and long-term survival of T cells. Besides being a relevant therapeutic target, hOX40 is also an important biomarker for monitoring the presence or infiltration of activated T cells within the tumor microenvironment (TME), the inflammatory microenvironment (IME) in immune-mediated diseases (IMIDs) and the lymphatic organs. Here, we developed novel single domain antibodies (nanobodies, Nbs) targeting hOX40 to monitor the activation status of T cells by in vivo molecular imaging., Methods: Nbs against hOX40 (hOX40-Nbs) were selected from an immunized Nb-library by phage display. The identified hOX40-Nbs were characterized in vitro , including determination of their specificity, affinity, stability, epitope recognition and their impact on OX40 signaling and T cell function. A lead candidate was site-specifically conjugated with a fluorophore via sortagging and applied for noninvasive in vivo optical imaging (OI) of hOX40-expressing cells in a xenograft mouse model., Results: Our selection campaign revealed four unique Nbs that exhibit strong binding affinities and high stabilities under physiological conditions. Epitope binning and domain mapping indicated the targeting of at least two different epitopes on hOX40. When analyzing their impact on OX40 signaling, an agonistic effect was excluded for all validated Nbs. Incubation of activated T cells with hOX40-Nbs did not affect cell viability or proliferation patterns, whereas differences in cytokine release were observed. In vivo OI with a fluorophore-conjugated lead candidate in experimental mice with hOX40-expressing xenografts demonstrated its specificity and functionality as an imaging probe., Conclusion: Considering the need for advanced probes for noninvasive in vivo monitoring of T cell activation dynamics, we propose, that our hOX40-Nbs have a great potential as imaging probes for noninvasive and longitudinal in vivo diagnostics. Quantification of OX40 + T cells in TME or IME will provide crucial insights into the activation state of infiltrating T cells, offering a valuable biomarker for assessing immune responses, predicting treatment efficacy, and guiding personalized immunotherapy strategies in patients with cancer or IMIDs., Competing Interests: DF, DS, MK, BP, TW, BT, PK, and UR are named as inventors on a patent application claiming the use of the described nanobodies for diagnosis and therapeutics filed by the Natural and Medical Sciences Institute, the Werner Siemens Imaging Center and the University of Tuebingen. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Frecot, Blaess, Wagner, Kaiser, Traenkle, Fandrich, Jakobi, Scholz, Nueske, Schneiderhan-Marra, Gouttefangeas, Kneilling, Pichler, Sonanini and Rothbauer.)

Regulatory T cells (Treg) are highly enriched within many tumors and suppress immune responses to cancer. There is intense interest in reprogramming Tregs to contribute to antitumor immunity. OX40 and CD137 are expressed highly on Tregs, activated and memory T cells, and NK cells. In this study, using a novel bispecific antibody targeting mouse OX40 and CD137 (FS120m), we show that OX40/CD137 bispecific agonism induces potent antitumor immunity partially dependent upon IFNγ production by functionally reprogrammed Tregs. Treatment of tumor-bearing animals with OX40/CD137 bispecific agonists reprograms Tregs into both fragile Foxp3+ IFNγ+ Tregs with decreased suppressive function and lineage-instable Foxp3- IFNγ+ ex-Tregs. Treg fragility is partially driven by IFNγ signaling, whereas Treg instability is associated with reduced IL2 responsiveness upon treatment with OX40/CD137 bispecific agonists. Importantly, conditional deletion of Ifng in Foxp3+ Tregs and their progeny partially reverses the antitumor efficacy of OX40/CD137 bispecific agonist therapy, revealing that reprogramming of Tregs into IFNγ-producing cells contributes to the anti-tumor efficacy of OX40/CD137 bispecific agonists. These findings provide insights into mechanisms by which bispecific agonist therapies targeting costimulatory receptors highly expressed by Tregs potentiate antitumor immunity in mouse models., Significance: The bispecific antibody FS120, an immunotherapy currently being tested in the clinic, partially functions by inducing anti-tumor activity of Tregs, which results in tumor rejection., (©2024 The Authors; Published by the American Association for Cancer Research.)

Background: Researches have confirmed that the abnormal signals of OX40 and PD-1 lead to the changes of T cell biological behavior, thus participating the immunopathological process of RA. However, the pathogenesis of RA immunopathological process has not been clarified yet., Methods: 30 DBA/1 mice were randomly divided into 5 groups (6 mice per group): control group, collagen-induced arthritis (CIA) group, PD-1-Fc/CIA group, OX40-Fc/CIA group, and PD-1-Fc + OX40-Fc/CIA group. The pathological changes in mice joints were observed by H&E staining. The proportion of CD4+ T, CD8+ T, CD28+, and CD19+ cells in peripheral blood mononuclear cells (PBMCs) was detected by flow cytometry. Serum inflammatory factors (CRP, IL-2, IL-4, IL-1 β , INF- γ ) and bone metabolism-related genes (CTX-I, TRACP-5b, BALP) were detected by ELISA assay. Western blotting was applied to measure the NF- κ B signaling pathway-related protein (p-IKK β , p-I κ B α , p50) expression in synovial tissue of mice joint., Results: Compared with the control group, CIA mice showed significant increases in arthritis score and pathological score. In the CIA group, a marked decrease was identified in the proportion of CD8+ T, CD19+, and CD68+ cells. Additionally, the CIA group was associated with upregulation of secretion of inflammatory factors in serum and expression of bone metabolism-related genes and NF- κ B pathway-related proteins. Compared with the CIA group, the same indexes above showed a further aggravation in the PD-1-Fc group while all indexes improved in the OX40-Fc group. Besides, OX40-Fc fusion protein slowed down significantly the further deterioration of CIA mouse pathological process caused by PD-1-Fc fusion protein., Conclusion: OX40-Fc fusion protein alleviates PD-1-Fc-aggravated RA by inhibiting inflammatory response. This research provides biological markers with clinical significance for diagnosis and prognosis of RA, as well as offers theoretical and experimental foundation to the new targets for immune intervention., Competing Interests: All authors have no conflicts of interest to declare., (Copyright © 2022 Yanyan Huang et al.)

Antibodies targeting costimulatory receptors of T cells have been developed for the activation of T cell immunity in cancer immunotherapy. However, costimulatory molecule expression is often lacking in tumor-infiltrating immune cells, which can impede antibody-mediated immunotherapy. Here, we hypothesize that delivery of costimulatory receptor mRNA to tumor-infiltrating T cells will enhance the antitumor effects of antibodies. We first design a library of biomimetic nanoparticles and find that phospholipid nanoparticles (PL1) effectively deliver costimulatory receptor mRNA (CD137 or OX40) to T cells. Then, we demonstrate that the combination of PL1-OX40 mRNA and anti-OX40 antibody exhibits significantly improved antitumor activity compared to anti-OX40 antibody alone in multiple tumor models. This treatment regimen results in a 60% complete response rate in the A20 tumor model, with these mice being resistant to rechallenge by A20 tumor cells. Additionally, the combination of PL1-OX40 mRNA and anti-OX40 antibody significantly boosts the antitumor immune response to anti-PD-1 + anti-CTLA-4 antibodies in the B16F10 tumor model. This study supports the concept of delivering mRNA encoding costimulatory receptors in combination with the corresponding agonistic antibody as a strategy to enhance cancer immunotherapy., (© 2021. The Author(s).)

Introduction: Combining CpG oligodeoxynucleotides with anti-OX40 agonist antibody (CpG+OX40) is able to generate an effective in situ vaccine in some tumor models, including the A20 lymphoma model. Immunologically "cold" tumors, which are typically less responsive to immunotherapy, are characterized by few tumor infiltrating lymphocytes (TILs), low mutation burden, and limited neoantigen expression. Radiation therapy (RT) can change the tumor microenvironment (TME) of an immunologically "cold" tumor. This study investigated the effect of combining RT with the in situ vaccine CpG+OX40 in immunologically "cold" tumor models., Methods: Mice bearing flank tumors (A20 lymphoma, B78 melanoma or 4T1 breast cancer) were treated with combinations of local RT, CpG, and/or OX40, and response to treatment was monitored. Flow cytometry and quantitative polymerase chain reaction (qPCR) experiments were conducted to study differences in the TME, secondary lymphoid organs, and immune activation after treatment., Results: An in situ vaccine regimen of CpG+OX40, which was effective in the A20 model, did not significantly improve tumor response or survival in the "cold" B78 and 4T1 models, as tested here. In both models, treatment with RT prior to CpG+OX40 enabled a local response to this in situ vaccine, significantly improving the anti-tumor response and survival compared to RT alone or CpG+OX40 alone. RT increased OX40 expression on tumor infiltrating CD4+ non-regulatory T cells. RT+CpG+OX40 increased the ratio of tumor-infiltrating effector T cells to T regulatory cells and significantly increased CD4+ and CD8+ T cell activation in the tumor draining lymph node (TDLN) and spleen., Conclusion: RT significantly improves the local anti-tumor effect of the in situ vaccine CpG+OX40 in immunologically "cold", solid, murine tumor models where RT or CpG+OX40 alone fail to stimulate tumor regression., Competing Interests: ZM is a member of the scientific advisory board for Archeus Technologies and Seneca Therapeutics and received equity options for these companies. ZM is an inventor on patents or filed patents managed by the Wisconsin Alumni Research Foundation relating to the interaction of targeted radionuclide therapies and immunotherapies, nanoparticles designed to augment the anti-tumor immune response following radiation therapy, and the development of a brachytherapy catheter capable of delivering intra-tumor injectables. PS is an inventor on patents or filed patents managed by the Wisconsin Alumni Research Foundation relating to mAb-related immunotherapies and the interaction of targeted radionuclide therapies and immunotherapies. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The reviewer WS declared a shared affiliation with one of the authors, RP, to the handling editor at the time of the review., (Copyright © 2021 Pieper, Zangl, Speigelman, Feils, Hoefges, Jagodinsky, Felder, Tsarovsky, Arthur, Brown, Birstler, Le, Carlson, Bates, Hank, Rakhmilevich, Erbe, Sondel, Patel and Morris.)

Regulatory T cells (Tregs) are often enriched in tumors, where their immunosuppressive function has a key role in tumor persistence and progression. In colorectal cancer (CRC), however, Tregs are frequently associated with an improved clinical outcome. Tumor-infiltrating Tregs have been shown to exhibit a distinct signature comprising the co-stimulatory molecules (OX40, 4-1BB), cytokine receptors (IL1R2, IL21R, CCR8, CD30), and co-inhibitory molecules (PD-L1, TIGIT). Here, we showed by flow cytometry that circulating CD45RO + Tregs from patients with CRC (n = 25) have elevated CD30 and OX40 expression compared to healthy subjects (n = 14). We identified co-expression of CD30 and OX40 on circulating CD45RO + Tregs using single-cell images captured by the DEPArray ™ system. The frequency of CD30 + OX40 + CD45RO + Tregs was significantly higher in CRC patients than in healthy subjects (P < 0.001). Importantly, receiver operating characteristic analysis confirmed that this CD30 + OX40 + Treg subset could strongly discriminate between CRC patients and healthy subjects with the highest accuracy of 92.3%, an AUC of 0.92, a sensitivity of 88%, a specificity of 100%, a positive predictive value of 100%, a negative predictive value of 82.35%, and a trade-off value of 3.44%, compared to other Treg subsets. Consistently, multiplex-IHC/IF of tumor-infiltrating Tregs revealed a significant association between high densities of CD30 + OX40 + Tregs and improved overall survival; no such association was found for other subsets. These data suggest a potential role for CD30 + OX40 + Tregs as a diagnostic or prognostic biomarker in CRC., (© 2021. The Author(s).)

Background/aim: We compared the therapeutic efficacy of two recently developed experimental anticancer technologies: 1) in situ vaccination based on local immunotherapy with CpG oligonucleotides and anti-OX40 antibodies to activate antitumor immune response and 2) "Karanahan" technology [from the Sanskrit kāraṇa ('source') + han ('to kill')] based on the combined injection of cyclophosphamide and double-stranded DNA to eradicate cancer stem cells., Materials and Methods: The anticancer approaches were compared on three types of mouse malignant tumors with different grades of immunogenicity: weakly immunogenic carcinoma Krebs-2, moderately immunogenic Lewis carcinoma, and highly immunogenic A20 В-cellular lymphoma., Results: Our results indicated that in situ vaccination was the most effective against the highly immunogenic tumor А20. In addition, "Karanahan" demonstrated high efficiency in all types of tumors, regardless of their immunogenicity or size., Conclusion: "Karanahan" therapy showed higher efficacy relative to in situ vaccination with CpG oligonucleotides and anti-OX40 antibodies., (Copyright © 2021 International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.)

OX40 is a costimulatory molecule that belongs to the tumor necrosis factor receptor (TNFR) superfamily. OX40 agonist-based combinations are emerging as promising candidates for novel cancer immunotherapy. Clinical trials have shown that OX40 agonist antibodies could lead to better results in cancer patients. Using a hybridoma platform and three different types of immunization strategies, namely recombinant protein, DNA, and overexpressing cells, we identified a chimeric anti-OX40 antibody (mAb035-hIgG1 from DNA immunization) that shows excellent binding specificity, and slightly stronger activation of human memory CD4 + T cells and similar potent antitumor activity compared with BMS 986178, an anti-OX40 antibody currently being evaluated for the treatment of solid tumors. This paper further systematically investigates the antigen-specific immune response, the number of binders, epitope bins, and functional activities of antibodies among different immunization strategies. Interestingly, we found that different immunization strategies affect the biological activity of monoclonal antibodies., (© 2021 Federation of European Biochemical Societies.)

Background: Previous data suggests that anti-OX40 mAb can elicit anti-tumor effects in mice through deletion of Tregs. However, OX40 also has powerful costimulatory effects on T cells which could evoke therapeutic responses. Human trials with anti-OX40 antibodies have shown that these entities are well tolerated but to date have delivered disappointing clinical responses, indicating that the rules for the optimal use of anti-human OX40 (hOX40) antibodies is not yet fully understood. Changes to timing and dosages may lead to improved outcomes; however, here we focus on addressing the role of agonism versus depleting activity in determining therapeutic outcomes. We investigated a novel panel of anti-hOX40 mAb to understand how these reagents and mechanisms may be optimized for therapeutic benefit., Methods: This study examines the binding activity and in vitro activity of a panel of anti-hOX40 antibodies. They were further evaluated in several in vivo models to address how isotype and epitope determine mechanism of action and efficacy of anti-hOX40 mAb., Results: Binding analysis revealed the antibodies to be high affinity, with epitopes spanning all four cysteine-rich domains of the OX40 extracellular domain. In vivo analysis showed that their activities relate directly to two key properties: (1) isotype-with mIgG1 mAb evoking receptor agonism and CD8+ T-cell expansion and mIgG2a mAb evoking deletion of Treg and (2) epitope-with membrane-proximal mAb delivering more powerful agonism. Intriguingly, both isotypes acted therapeutically in tumor models by engaging these different mechanisms., Conclusion: These findings highlight the significant impact of isotype and epitope on the modulation of anti-hOX40 mAb therapy, and indicate that CD8+ T-cell expansion or Treg depletion might be preferred according to the composition of different tumors. As many of the current clinical trials using OX40 antibodies are now using combination therapies, this understanding of how to manipulate therapeutic activity will be vital in directing new combinations that are more likely to improve efficacy and clinical outcomes., Competing Interests: Competing interests: MSC is a retained consultant for BioInvent International and has performed educational and advisory roles for Baxalta and Boehringer Ingleheim. He has received research funding from Roche, Gilead, Bioinvent International and GSK. BF, IT, LM and MS are employees of Bioinvent International., (© Author(s) (or their employer(s)) 2020. Re-use permitted under CC BY. Published by BMJ.)

Background: OX40 (CD134) is a costimulatory molecule of the tumor necrosis factor receptor superfamily that is currently being investigated as a target for cancer immunotherapy. However, despite promising results in murine tumor models, the clinical efficacy of agonistic αOX40 antibodies in the treatment of patients with cancer has fallen short of the high expectation in earlier-stage trials., Methods: Using lymphocytes from resected tumor, tumor-free (TF) tissue and peripheral blood mononuclear cells (PBMC) of 96 patients with hepatocellular and colorectal cancers, we determined OX40 expression and the in vitro T-cell agonistic activity of OX40-targeting compounds. RNA-Seq was used to evaluate OX40-mediated transcriptional changes in CD4+ and CD8+ human tumor-infiltrating lymphocytes (TILs)., Results: Here, we show that OX40 was overexpressed on tumor-infiltrating CD4+ T cells compared with blood and TF tissue-derived T cells. In contrast to a clinical candidate αOX40 antibody, treatment with an Fc-engineered αOX40 antibody (αOX40&#95;v12) with selectively enhanced FcγRIIB affinity, stimulated in vitro CD4+ and CD8+ TIL expansion, as well as cytokine and chemokine secretions. The activity of αOX40&#95;v12 was dependent on FcγRIIB engagement and intrinsic CD3/CD28 signals. The transcriptional landscape of CD4+ and CD8+ TILs shifted toward a prosurvival, inflammatory and chemotactic profile on treatment with αOX40&#95;v12., Conclusions: OX40 is overexpressed on CD4+ TILs and thus represents a promising target for immunotherapy. Targeting OX40 with currently used agonistic antibodies may be inefficient due to lack of OX40 multimerization. Thus, Fc engineering is a powerful tool in enhancing the agonistic activity of αOX40 antibody and may shape the future design of antibody-mediated αOX40 immunotherapy., Competing Interests: Competing interests: JW, TSF, KC, WY, IN, YAY and SS-A are employees of Pfizer and have stocks and ownership interests (including patents) at Pfizer., (© Author(s) (or their employer(s)) 2020. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.)

Plasmacytoid DCs (pDCs), the major producers of type I interferon, are principally recognized as key mediators of antiviral immunity. However, their role in tumor immunity is less clear. Depending on the context, pDCs can promote or suppress antitumor immune responses. In this study, we identified a naturally occurring pDC subset expressing high levels of OX40 (OX40+ pDC) enriched in the tumor microenvironment (TME) of head and neck squamous cell carcinoma. OX40+ pDCs were distinguished by a distinct immunostimulatory phenotype, cytolytic function, and ability to synergize with conventional DCs (cDCs) in generating potent tumor antigen-specific CD8+ T cell responses. Transcriptomically, we found that they selectively utilized EIF2 signaling and oxidative phosphorylation pathways. Moreover, depletion of pDCs in the murine OX40+ pDC-rich tumor model accelerated tumor growth. Collectively, we present evidence of a pDC subset in the TME that favors antitumor immunity.

With the great success of anti-CTLA-4 and anti-PD-1 therapeutics in cancer immunotherapy, tumor necrosis factor receptor superfamily members have been recognized as ideal targets to provide co-stimulatory signals in combination with immune checkpoint blocking antibodies. Among these is OX40 (CD134), a co-stimulatory molecule expressed by activated immune cells. Recently, several anti-OX40 agonistic monoclonal antibodies, pogalizumab as the most advanced, have entered early phase clinical trials. Using a yeast platform and multiple screening methods, we identified a fully human anti-OX40 antibody (IBI101) with distinct modes of action. Unlike pogalizumab, IBI101 partially blocks the binding of OX40 to its ligand OX40L and exhibits both FcγR-dependent and independent agonistic activities in NF-κB luciferase reporter assays. IBI101 also promotes T cell activation and proliferation in vitro. These unique properties partially explain the more potent anti-tumor activity of IBI101 than that of pogalizumab in humanized NOG mice bearing LoVo tumors. In addition, IBI101 shows efficacious anti-tumor activity in mice when administrated alone or in combination with anti-PD-1 antibodies. In human OX40 knock-in mice bearing MC38 colon carcinoma, IBI101 treatment induces tumor antigen-specific CD8+ T-cell responses, decreases immunosuppressive regulatory T cells in tumor, and enhances the immune response to PD-1 inhibition. Preclinical studies of IBI101 in non-human primates demonstrate typical pharmacokinetic characteristics of an IgG antibody and no drug-related toxicity. Collectively, IBI101 has desirable preclinical attributes which support its clinical development for cancer treatment.

Eliciting protective titers of HIV-1 broadly neutralizing antibodies (bnAbs) is a goal of HIV-1 vaccine development, but current vaccine strategies have yet to induce bnAbs in humans. Many bnAbs isolated from HIV-1-infected individuals are encoded by immunoglobulin gene rearrangments with infrequent naive B cell precursors and with unusual genetic features that may be subject to host regulatory control. Here, we administer antibodies targeting immune cell regulatory receptors CTLA-4, PD-1 or OX40 along with HIV envelope (Env) vaccines to rhesus macaques and bnAb immunoglobulin knock-in (KI) mice expressing diverse precursors of CD4 binding site HIV-1 bnAbs. CTLA-4 blockade augments HIV-1 Env antibody responses in macaques, and in a bnAb-precursor mouse model, CTLA-4 blocking or OX40 agonist antibodies increase germinal center B and T follicular helper cells and plasma neutralizing antibodies. Thus, modulation of CTLA-4 or OX40 immune checkpoints during vaccination can promote germinal center activity and enhance HIV-1 Env antibody responses.

Background: Immunotherapy, especially immune checkpoint inhibition, has provided powerful tools against cancer. We aimed to detect the expression of common immune checkpoints and evaluate their prognostic values in nasopharyngeal carcinoma (NPC)., Methods: The expression of 9 immune checkpoints consistent with 13 features was detected in the training cohort (n = 208) by immunohistochemistry and quantified by computational pathology. Then, the LASSO cox regression model was used to construct an immune checkpoint-based signature (ICS), which was validated in a validation cohort containing 125 patients., Results: High positive expression of PD-L1 and B7-H4 was observed in tumour cells (TCs), whereas PD-L1, B7-H3, B7-H4, IDO-1, VISTA, ICOS and OX40 were highly expressed in tumour-associated immune cells (TAICs). Eight of the 13 immune features were associated with patient overall survival, and an ICS classifier consisting of 5 features (B7-H3TAIC, IDO-1TAIC, VISTATAIC, ICOSTAIC, and LAG3TAIC) was established. Patients with high-risk scores in the training cohort had shorter overall (P < 0.001), disease-free (P = 0.002), and distant metastasis-free survival (P = 0.004), which were confirmed in the validation cohort. Multivariate analysis revealed that the ICS classifier was an independent prognostic factor. A combination of the ICS classifier and TNM stage had better prognostic value than the TNM stage alone. In addition, the ICS classifier was significantly associated with survivals in patients with high EBV-DNA load., Conclusions: We determined the expression status of nine immune checkpoints consistent with 13 features in NPC and further constructed an ICS prognostic model, which might add prognostic value to the TNM staging system.

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., (©2019 American Association for Cancer Research.)

Hyperinflammatory syndromes are life-threatening disorders caused by overzealous immune cell activation and cytokine release, often resulting from defects in negative feedback mechanisms. In the quintessential hyperinflammatory syndrome familial hemophagocytic lymphohistiocytosis (HLH), inborn errors of cytotoxicity result in effector cell accumulation, immune dysregulation and, if untreated, tissue damage and death. Here, we describe a human case with a homozygous nonsense R688* RC3H1 mutation suffering from hyperinflammation, presenting as relapsing HLH. RC3H1 encodes Roquin-1, a posttranscriptional repressor of immune-regulatory proteins such as ICOS, OX40 and TNF. Comparing the R688* variant with the murine M199R variant reveals a phenotypic resemblance, both in immune cell activation, hypercytokinemia and disease development. Mechanistically, R688* Roquin-1 fails to localize to P-bodies and interact with the CCR4-NOT deadenylation complex, impeding mRNA decay and dysregulating cytokine production. The results from this unique case suggest that impaired Roquin-1 function provokes hyperinflammation by a failure to quench immune activation.

Agonistic antibodies targeting the tumor necrosis factor (TNF) superfamily of co-stimulatory receptors (TNFRSF) are progressing through various stages of clinical development for cancer treatment, but the desired and defining features of these agents for optimal biological activity remain controversial. One idea, based on recent studies with CD40, is that non-ligand-blocking antibodies targeting membrane-distal cysteine-rich domain 1 (CRD1) have superior agonistic activities compared with ligand-blocking antibodies targeting more membrane-proximal CRDs. Here, we determined the binding and functional characteristics of a panel of antibodies targeting CRDs 1-4 of OX40 (also known as TNFRSF4 or CD134). In striking contrast to CD40, we found that ligand-blocking CRD2-binding and membrane-proximal CRD4-binding anti-OX40 antibodies have the strongest agonistic and anti-tumor activities. These findings have important translational implications and further highlight that the relationship between epitope specificity and agonistic activity will be an important issue to resolve on a case-by-case basis when optimizing antibodies targeting different co-stimulatory tumor necrosis factor receptors (TNFRs)., (Copyright © 2019 The Author(s). Published by Elsevier Inc. All rights reserved.)

Background: The CTLA-4 blocking antibody ipilimumab has demonstrated substantial and durable effects in patients with melanoma. While CTLA-4 therapy, both as monotherapy and in combination with PD-1 targeting therapies, has great potential in many indications, the toxicities of the current treatment regimens may limit their use. Thus, there is a medical need for new CTLA-4 targeting therapies with improved benefit-risk profile., Methods: ATOR-1015 is a human CTLA-4 x OX40 targeting IgG1 bispecific antibody generated by linking an optimized version of the Ig-like V-type domain of human CD86, a natural CTLA-4 ligand, to an agonistic OX40 antibody. In vitro evaluation of T-cell activation and T regulatory cell (Treg) depletion was performed using purified cells from healthy human donors or cell lines. In vivo anti-tumor responses were studied using human OX40 transgenic (knock-in) mice with established syngeneic tumors. Tumors and spleens from treated mice were analyzed for CD8 + T cell and Treg frequencies, T-cell activation markers and tumor localization using flow cytometry., Results: ATOR-1015 induces T-cell activation and Treg depletion in vitro. Treatment with ATOR-1015 reduces tumor growth and improves survival in several syngeneic tumor models, including bladder, colon and pancreas cancer models. It is further demonstrated that ATOR-1015 induces tumor-specific and long-term immunological memory and enhances the response to PD-1 inhibition. Moreover, ATOR-1015 localizes to the tumor area where it reduces the frequency of Tregs and increases the number and activation of CD8 + T cells., Conclusions: By targeting CTLA-4 and OX40 simultaneously, ATOR-1015 is directed to the tumor area where it induces enhanced immune activation, and thus has the potential to be a next generation CTLA-4 targeting therapy with improved clinical efficacy and reduced toxicity. ATOR-1015 is also expected to act synergistically with anti-PD-1/PD-L1 therapy. The pre-clinical data support clinical development of ATOR-1015, and a first-in-human trial has started (NCT03782467).

Background: OX40, which is also known as tumor necrosis factor receptor superfamily member 4 (TNFRSF4), and its ligand (OX40L) play a critical role in the pathogenesis of autoimmune diseases. Immune thrombocytopenia (ITP), a hemorrhagic autoimmune disorder, is characterized by low platelet counts that are predominantly caused by antiplatelet autoantibodies. In this study, we firstly investigated the clinical significance of OX40 and OX40L expression in the pathogenesis of ITP in patients., Methods: Fifty-four newly diagnosed ITP patients and 24 healthy controls (HCs) were enrolled in this study. The percentage of OX40 + CD4 + T cells among CD4 + T cells was analyzed by flow cytometry, and the expression levels of OX40 and OX40L mRNA were analyzed by quantitative real-time PCR. Plasma soluble OX40L (sOX40L) levels were analyzed by ELISA, and plasma levels of antiplatelet autoantibodies were analyzed by a solid-phase technique., Results: Compared with HCs, the frequencies of OX40 + CD4 + T cells were significantly increased in ITP patients, particularly in patients with positive antiplatelet autoantibodies compared to those with negative antiplatelet autoantibodies. The elevated frequencies of OX40 + CD4 + T cells were negatively correlated with low platelet counts in patients with positive antiplatelet autoantibodies. Plasma sOX40L levels in ITP patients were significantly greater than those in HCs and increased in patients with positive antiplatelet autoantibodies compared to those with negative antiplatelet autoantibodies. Plasma sOX40L levels were negatively correlated with low platelet counts in patients with positive antiplatelet autoantibodies. Additionally, the mRNA expression levels of OX40 and OX40L in PBMCs from ITP patients were also notably greater than those from HCs, and the expression levels of OX40 and OX40L were significantly different in ITP patients with positive and negative antiplatelet autoantibodies., Conclusion: These data indicated that increased expression levels of OX40 and OX40L were involved in the pathogenesis of ITP, and OX40 and OX40L may be valuable therapeutic targets for ITP.

Tregs are impaired in human systemic lupus erythematosus (SLE) and contribute to effector T cell activation. However, the mechanisms responsible for the Treg deficiency in SLE remain unclear. We hypothesized that the OX40L/OX40 axis is implicated in Treg and regulatory follicular helper T (Tfr) cell dysfunction in human SLE. OX40L/OX40 axis engagement on Tregs and Tfr cells not only specifically impaired their ability to regulate effector T cell proliferation, but also their ability to suppress T follicular helper (Tfh) cell-dependent B cell activation and immunoglobulin secretion. Antigen-presenting cells from patients with active SLE mediated Treg dysfunction in an OX40L-dependent manner, and OX40L-expressing cells colocalized with Foxp3+ cells in active SLE skin lesions. Engagement of the OX40L/OX40 axis resulted in Foxp3 downregulation in Tregs, and expression in SLE Tregs correlated with the proportion of circulating OX40L-expressing myeloid DCs. These data support that OX40L/OX40 signals are implicated in Treg dysfunction in human SLE. Thus, blocking the OX40L/OX40 axis appears to be a promising therapeutic strategy.

Tumor neoantigens arising from somatic mutations in the cancer genome are less likely to be subject to central immune tolerance and are therefore attractive targets for vaccine immunotherapy. We utilized whole-exome sequencing, RNA sequencing (RNASeq), and an in silico immunogenicity prediction algorithm, NetMHC, to generate a neoantigen-targeted vaccine, PancVAX, which was administered together with the STING adjuvant ADU-V16 to mice bearing pancreatic adenocarcinoma (Panc02) cells. PancVAX activated a neoepitope-specific T cell repertoire within the tumor and caused transient tumor regression. When given in combination with two checkpoint modulators, namely anti-PD-1 and agonist OX40 antibodies, PancVAX resulted in enhanced and more durable tumor regression and a survival benefit. The addition of OX40 to vaccine reduced the coexpression of T cell exhaustion markers, Lag3 and PD-1, and resulted in rejection of tumors upon contralateral rechallenge, suggesting the induction of T cell memory. Together, these data provide the framework for testing personalized neoantigen-based combinatorial vaccine strategies in patients with pancreatic and other nonimmunogenic cancers.

Malaria is a disease with diverse symptoms depending on host immune status and pathogenicity of Plasmodium parasites. The continuous parasite growth within a host suggests mechanisms of immune evasion by the parasite and/or immune inhibition in response to infection. To identify pathways commonly inhibited after malaria infection, we infected C57BL/6 mice with four Plasmodium yoelii strains causing different disease phenotypes and 24 progeny of a genetic cross. mRNAs from mouse spleens day 1 and/or day 4 post infection (p.i.) were hybridized to a mouse microarray to identify activated or inhibited pathways, upstream regulators, and host genes playing an important role in malaria infection. Strong interferon responses were observed after infection with the N67 strain, whereas initial inhibition and later activation of hematopoietic pathways were found after infection with 17XNL parasite, showing unique responses to individual parasite strains. Inhibitions of pathways such as Th1 activation, dendritic cell (DC) maturation, and NFAT immune regulation were observed in mice infected with all the parasite strains day 4 p.i., suggesting universally inhibited immune pathways. As a proof of principle, treatment of N67-infected mice with antibodies against T cell receptors OX40 or CD28 to activate the inhibited pathways enhanced host survival. Controlled activation of these pathways may provide important strategies for better disease management and for developing an effective vaccine.

Regulatory T cells (Treg) are negative regulators of the immune response; however, it is poorly understood whether and how Foxp3 transcription is induced and regulated in the periphery during T-cell responses. Using Foxp3 -Timer of cell kinetics and activity (Tocky) mice, which report real-time Foxp3 expression , we show that the flux of new Foxp3 expressors and the rate of Foxp3 transcription are increased during inflammation. These persistent dynamics of Foxp3 transcription determine the effector Treg programme and are dependent on a Foxp3 autoregulatory transcriptional circuit. Persistent Foxp3 transcriptional activity controls the expression of coinhibitory molecules, including CTLA-4 and effector Treg signature genes. Using RNA-seq, we identify two groups of surface proteins based on their relationship to the temporal dynamics of Foxp3 transcription, and we show proof of principle for the manipulation of Foxp3 dynamics by immunotherapy: new Foxp3 flux is promoted by anti-TNFRII antibody, and high-frequency Foxp3 expressors are targeted by anti-OX40 antibody. Collectively, our study dissects time-dependent mechanisms behind Foxp3-driven T-cell regulation and establishes the Foxp3 -Tocky system as a tool to investigate the mechanisms behind T-cell immunotherapies., (© 2018 The Authors. Published under the terms of the CC BY 4.0 license.)

Protection against a malaria infection can be achieved by immunization with live-attenuated Plasmodium sporozoites and while the precise mechanisms of protection remain unknown, T cell responses are thought to be critical in the elimination of infected liver cells. In cancer immunotherapies, agonistic antibodies that target T cell surface proteins, such as CD27, OX40 (CD134), and 4-1BB (CD137), have been used to enhance T cell function by increasing co-stimulation. In this study, we have analyzed the effect of agonistic OX40 monoclonal antibody treatment on protective immunity induced in mice immunized with genetically attenuated parasites (GAPs). OX40 stimulation enhanced protective immunity after vaccination as shown by an increase in the number of protected mice and delay to blood-stage infection after challenge with wild-type sporozoites. Consistent with the enhanced protective immunity enforced OX40 stimulation resulted in an increased expansion of antigen-experienced effector (CD11a hi CD44 hi ) CD8 + and CD4 + T cells in the liver and spleen and also increased IFN-γ and TNF producing CD4 + T cells in the liver and spleen. In addition, GAP immunization plus α-OX40 treatment significantly increased sporozoite-specific IgG responses. Thus, we demonstrate that targeting T cell costimulatory receptors can improve sporozoite-based vaccine efficacy.

In situ cancer vaccines are under active clinical investigation, given their reported ability to eradicate both local and disseminated malignancies. Intratumoral vaccine administration is thought to activate a T cell-mediated immune response, which begins in the treated tumor and cascades systemically. In this study, we describe a PET tracer (64Cu-DOTA-AbOX40) that enabled noninvasive and longitudinal imaging of OX40, a cell-surface marker of T cell activation. We report the spatiotemporal dynamics of T cell activation following in situ vaccination with CpG oligodeoxynucleotide in a dual tumor-bearing mouse model. We demonstrate that OX40 imaging was able to predict tumor responses on day 9 after treatment on the basis of tumor tracer uptake on day 2, with greater accuracy than both anatomical and blood-based measurements. These studies provide key insights into global T cell activation following local CpG treatment and indicate that 64Cu-DOTA-AbOX40 is a promising candidate for monitoring clinical cancer immunotherapy strategies.

Ligation of OX40 (CD134, TNFRSF4) on activated T cells by its natural ligand (OX40L, CD252, TNFSF4) enhances cellular survival, proliferation, and effector functions such as cytokine release and cellular cytotoxicity. We engineered a recombinant human OX40L IgG4P Fc fusion protein termed MEDI6383 that assembles into a hexameric structure and exerts potent agonist activity following engagement of OX40. MEDI6383 displayed solution-phase agonist activity that was enhanced when the fusion protein was clustered by Fc gamma receptors (FcγRs) on the surface of adjacent cells. The resulting costimulation of OX40 on T cells induced NFκB promoter activity in OX40-expressing T cells and induced Th1-type cytokine production, proliferation, and resistance to regulatory T cell (Treg)-mediated suppression. MEDI6383 enhanced the cytolytic activity of tumor-reactive T cells and reduced tumor growth in the context of an alloreactive human T cell:tumor cell admix model in immunocompromised mice. Consistent with the role of OX40 costimulation in the expansion of memory T cells, MEDI6383 administered to healthy nonhuman primates elicited peripheral blood CD4 and CD8 central and effector memory T-cell proliferation as well as B-cell proliferation. Together, these results suggest that OX40 agonism has the potential to enhance antitumor immunity in human malignancies. Mol Cancer Ther; 17(5); 1024-38. ©2018 AACR ., (©2018 American Association for Cancer Research.)

Immunostimulatory antibodies against the tumor necrosis factor receptors (TNFR) are emerging as promising cancer immunotherapies. The agonism activity of such antibodies depends on crosslinking to Fc gamma RIIB receptor (FcγRIIB) to enable the antibody multimerization that drives TNFR activation. Previously, Fc engineering was used to enhance the binding of such antibodies to Fcγ receptors. Here, we report the identification of Centyrins as alternative scaffold proteins with binding affinities to homologous FcγRIIB and FcγRIIA, but not to other types of Fcγ receptors. One Centyrin, S29, was engineered at distinct positions of an anti-OX40 SF2 antibody to generate bispecific and tetravalent molecules named as mAbtyrins. Regardless of the position of S29 on the SF2 antibody, SF2-S29 mAbtyrins could bind FcγRIIB and FcγRIIA specifically while maintaining binding to OX40 receptors. In a NFκB reporter assay, attachment of S29 Centyrin molecules at the C-termini, but not the N-termini, resulted in SF2 antibodies with increased agonism owing to FcγRIIB crosslinking. The mAbtyrins also showed agonism in T-cell activation assays with immobilized FcγRIIB and FcγRIIA, but this activity was confined to mAbtyrins with S29 specifically at the C-termini of antibody heavy chains. Furthermore, regardless of the position of the molecule, S29 Centyrin could equip an otherwise Fc-silent antibody with antibody-dependent cellular phagocytosis activity without affecting the antibody's intrinsic antibody-dependent cell-meditated cytotoxicity and complement-dependent cytotoxicity. In summary, the appropriate adoption FcγRII-binding Centyrins as functional modules represents a novel strategy to engineer therapeutic antibodies with improved functionalities.

Combination immunotherapies utilizing complementary modalities that target distinct tumor attributes or immunosuppressive mechanisms, or engage different arms of the antitumor immune response, can elicit greater therapeutic efficacy than the component monotherapies. Increasing the number of agents included in a therapeutic cocktail can further increase efficacy, however, this approach poses numerous challenges for clinical translation. Here, a novel platform to simplify combination immunotherapy by covalently linking immunotherapeutic agonists to the costimulatory receptors CD134 and CD137 into a single heterodimeric drug, "OrthomAb", is shown. This reagent not only retains costimulatory T cell activity, but also elicits unique T cell functions that are not programmed by either individual agonist, and preferentially expands effector T cells over Tregs. Finally, in an aggressive melanoma model OrthomAb elicits better therapeutic efficacy compared to the unlinked agonists. This demonstration that two drugs can be combined into one provides a framework for distilling complex combination drug cocktails into simpler delivery platforms.

The ubiquitously expressed RNA-binding proteins Roquin-1 and Roquin-2 are essential for appropriate immune cell function and postnatal survival of mice. Roquin proteins repress target mRNAs by recognizing secondary structures in their 3'-UTRs and by inducing mRNA decay. However, it is unknown if other cellular proteins contribute to target control. To identify cofactors of Roquin, we used RNA interference to screen ~1500 genes involved in RNA-binding or mRNA degradation, and identified NUFIP2 as a cofactor of Roquin-induced mRNA decay. NUFIP2 binds directly and with high affinity to Roquin, which stabilizes NUFIP2 in cells. Post-transcriptional repression of human ICOS by endogenous Roquin proteins requires two neighboring non-canonical stem-loops in the ICOS 3'-UTR. This unconventional cis-element as well as another tandem loop known to confer Roquin-mediated regulation of the Ox40 3'-UTR, are bound cooperatively by Roquin and NUFIP2. NUFIP2 therefore emerges as a cofactor that contributes to mRNA target recognition by Roquin.

In recent years, monoclonal antibodies (mAbs) able to reinvigorate antitumor T-cell immunity have heralded a paradigm shift in cancer treatment. The most high profile of these mAbs block the inhibitory checkpoint receptors PD-1 and CTLA-4 and have improved life expectancy for patients across a range of tumor types. However, it is becoming increasingly clear that failure of some patients to respond to checkpoint inhibition is attributable to inadequate T-cell priming. For full T-cell activation, 2 signals must be received, and ligands providing the second of these signals, termed costimulation, are often lacking in tumors. Members of the TNF receptor superfamily (TNFRSF) are key costimulators of T cells during infection, and there has been an increasing interest in harnessing these receptors to augment tumor immunity. We here review the immunobiology of 2 particularly promising TNFRSF target receptors, CD27 and OX40, and their respective ligands, CD70 and OX40L, focusing on their role within a tumor setting. We describe the influence of CD27 and OX40 on human T cells based on in vitro studies and on the phenotypes of several recently described individuals exhibiting natural deficiencies in CD27/CD70 and OX40. Finally, we review key literature describing progress in elucidating the efficacy and mode of action of OX40- and CD27-targeting mAbs in preclinical models and provide an overview of current clinical trials targeting these promising receptor/ligand pairings in cancer., (© 2018 by The American Society of Hematology.)

The delivery of immunomodulators directly into the tumor potentially harnesses the existing antigen, tumor-specific infiltrating lymphocytes, and antigen presenting cells. This can confer specificity and generate a potent systemic anti-tumor immune response with lower doses and less toxicity compared to systemic administration, in effect an in situ vaccine. Here, we test this concept using the novel combination of immunomodulators anti-CTLA4, -CD137, and -OX40. The triple combination administered intratumorally at low doses to one tumor of a dual tumor mouse model had dramatic local and systemic anti-tumor efficacy in lymphoma (A20) and solid tumor (MC38) models, consistent with an abscopal effect. The minimal effective dose was 10 μg each. The effect was dependent on CD8 T-cells. Intratumoral administration resulted in superior local and distant tumor control compared to systemic routes, supporting the in situ vaccine concept. In a single tumor A20 model, injection close to the tDLN resulted in similar efficacy as intratumoral and significantly better than targeting a non-tDLN, supporting the role of the tDLN as a viable immunotherapy target in addition to the tumor itself. Distribution studies confirmed expected concentration of antibodies in tumor and tDLN, in keeping with the anti-tumor results. Overall intratumoral or peri-tDLN administration of the novel combination of anti-CTLA4, anti-CD137, and anti-OX40, all agents in the clinic or clinical trials, demonstrates potent systemic anti-tumor effects. This immunotherapeutic combination is promising for future clinical development via both these safe and highly efficacious routes of administration.

Thalidomide (TM) has been reported to have anti-cancer and anti-inflammatory properties, and dexamethasone (DX) is known to reduce inflammation and inhibit production of inflammatory cytokines. Many studies have reported that combinatorial therapy with TM and DX is clinically used to treat multiple myeloma and lupus nephritis, but the mechanism responsible for its effects has not been elucidated. In this study, we determined that TM and DX co-treatment had an enhanced immune-modulatory effect on T cells through regulating the expression of co-stimulatory molecules. Splenic naive T cells from C57BL/6 mice were sort-purified and cultured for CD4 + T cell proliferation and regulatory T (Treg) cell conversion in the presence of TM and/or DX. Following incubation with the drugs, cells were collected and OX40, 4-1BB, and glucocorticoid-induced tumour necrosis factor receptor-related protein (GITR) expression was quantified by flow cytometry. TM (1 or 10 μm) decreased CD4 + T cell proliferation in a dose-dependent manner, whereas TM/DX (0·1 or 1 nm) co-treatment further decreased proliferation. Treg cell populations were preserved following drug treatment. Furthermore, expression of co-stimulatory molecules decreased upon TM/DX co-treatment in effector T (Teff) cells and was preserved in Treg cells. Splenic CD4 + T cells isolated from TM- and DX-treated mice exhibited the same patterns of Teff and Treg cell populations as observed in vitro. Considering the selective effect of TM on different T cell subsets, we suggest that TM may play an immunomodulatory role and that TM/DX combinatorial treatment could further enhance these immunomodulatory effects by regulating GITR, OX40, and 4-1BB expression in CD4 + T cells., (© 2017 John Wiley & Sons Ltd.)

Recent findings show that immune cells constitute a large fraction of the tumor microenvironment and that they modulate tumor progression. Clinical data indicate that chronic inflammation is present at tumor sites and that IL-4, in particular, is upregulated. Thus, we tested whether IL-4 neutralization would affect tumor immunity. Current results demonstrate that the administration of a neutralizing antibody against IL-4 enhances anti-tumor immunity and delays tumor progression. IL-4 blockade also alters inflammation in the tumor microenvironment, reducing the generation of both immunosuppressive M2 macrophages and myeloid-derived suppressor cells, and enhancing tumor-specific cytotoxic T lymphocytes. In addition, IL-4 blockade improves the response to anti-OX40 Ab or CpG oligodeoxynucleotide immunotherapies. These findings suggest that IL-4 affects anti-tumor immunity and constitutes an attractive therapeutic target to reduce immune suppression in the tumor microenvironment, thus enhancing the efficacy of cancer therapy.

Adaptive immunity plays a critical role in IR and T2DM development; however, the biological mechanisms linking T cell costimulation and glucose metabolism have not been fully elucidated. In this study, we demonstrated that the costimulatory molecule OX40 controls T cell activation and IR development. Inflammatory cell accumulation and enhanced proinflammatory gene expression, as well as high OX40 expression levels on CD4 + T cells, were observed in the adipose tissues of mice with diet-induced obesity. OX40-KO mice exhibited significantly less weight gain and lower fasting glucose levels than those of WT mice, without obvious adipose tissue inflammation. The effects of OX40 on IR are mechanistically linked to the promotion of T cell activation, Th1 cell differentiation and proliferation-as well as the attenuation of Treg suppressive activity and the enhancement of proinflammatory cytokine production-in adipose tissues. Furthermore, OX40 expression on T cells was positively associated with obesity in humans, suggesting that our findings are clinically relevant. In summary, our study revealed that OX40 in CD4 + T cells is crucial for adipose tissue inflammation and IR development. Therefore, the OX40 signaling pathway may be a new target for preventing or treating obesity-related IR and T2DM.

LPS is a powerful adjuvant, and although LPS-mediated TLR4 signaling has been exquisitely delineated, the in vivo mechanism of how TLR4 responses impact T cell priming is far less clear. Besides costimulation, TNF and type 1 IFN are dominant cytokines released after TLR4 activation and can shape T cell responses, but other downstream factors have not been examined extensively. Depending on context, we show that IFNαR1 blockade resulted in minor to major effects on specific CD4 T cell clonal expansion. To help explain these differences, it was hypothesized that IFNαR1 blockade would inhibit specific T cell migration by reducing chemokine receptor signaling, but specific CD4 T cells from IFNαR1-blocked mice were readily able to migrate in response to specific chemokines. Next, we examined downstream factors and found that type 1 IFN signaling was necessary for chemokine production, even when mice were immunized with specific Ag with LPS and CD134 costimulation. IFNαR1 signaling promoted CXCL9 and CXCL10 synthesis, suggesting that these chemokines might be involved in the LPS and CD134 costimulation response. After immunization, we show that CXCL9 blockade inhibited CD4 T cell accumulation in the liver but also in LNs, even in the presence of elevated serum IFN-β levels. Thus, whereas type 1 IFN might have direct effects on primed CD4 T cells, the downstream chemokines that play a role during migration also impact accumulation. In sum, CXCL9 production is a key benchmark for productive CD4 T cell vaccination strategies., (© Society for Leukocyte Biology.)

Patients with advanced melanoma have a compromised anti-tumor immune response leading to tumor immune tolerance and a tumor microenvironment conducive to disease progression. Immunotherapy that successfully overcomes this tumor-mediated immune suppression has made the greatest impact in the management of this disease over the past few years. This progress through immunotherapy builds upon earlier successes that interferon-α had in the treatment of melanoma in the adjuvant setting, as well as that of high-dose interleukin-2 in advanced melanoma. The development of immune checkpoint inhibitors has led to dramatic clinical activity in advanced melanoma. In particular, anti-CTLA4 and anti-PD1 monoclonal antibodies have taken us forward into the realm of longer survival and durable responses with the possibility of cure in a continuously increasing proportion of patients. Combination immunotherapeutic strategies and novel immunotherapeutic agents are being tested at an accelerated pace where the outlook for long-term survival benefits for the majority of patients appears brighter than ever.

Background: CD4 + CD28 - T cells exhibit autoreactive potential in autoimmune disorders, including rheumatoid arthritis (RA). It is not well known which costimulator functions as an alternative second signal in the activation of this subset after CD28 expression is downregulated. Tumor necrosis factor receptor superfamily member OX40 is a key costimulator in the activation of T cells. The aim of this study was to investigate the costimulatory effects of OX40 on CD4 + CD28 - T cells in autoimmune arthritis., Methods: Clinical samples were collected from patients with RA and control subjects. Collagen-induced arthritis (CIA) was induced with collagen type II (CII) in DBA/1 mice. The CD4 + CD28 - OX40 + T-cell subset and its cytokine production were detected by flow cytometry. After T-cell purification, adoptive transfer was performed in CIA mice. The regulatory role of OX40 was determined by blocking experiments in vitro and in vivo., Results: OX40 and OX40L were abnormally expressed in patients with RA and CIA mice. Further analysis showed that CD4 + CD28 - OX40 + T cells accumulated in patients with RA and in animal models. These cells produced higher levels of proinflammatory cytokines and were closely correlated with the clinicopathological features of the affected individuals. Adoptive transfer of CII-specific CD4 + CD28 - OX40 + T cells remarkably aggravated arthritic development and joint pathology in CIA mice. Moreover, OX40 blockade significantly reduced the proinflammatory responses and ameliorated arthritis development., Conclusions: OX40 acts as an alternative costimulator of CD4 + CD28 - T cells and plays a pathogenic role in autoimmune arthritic development, suggesting that it is a potential target for immunomodulatory therapy of RA.

Agonistic antibodies directed against immunostimulatory receptors belonging to the tumor necrosis factor receptor (TNFR) superfamily are emerging as promising cancer immunotherapies. Several Fc engineering approaches discovered recently can augment the anti-tumor activities of TNFR antibodies by enhancing their agonistic activities and/or effector functions. In this study, we compared these approaches for their effects on an anti-OX40 antibody. Both S267E/L328F and V12 mutations facilitated enhanced binding to FcγRIIB and thus increased FcγRIIB cross-linking mediated agonist activity. However, both mutations abrogated the binding to FcγRIIIA and thereby decreasing the antibody-dependent cellular cytotoxicity activities. In contrast, the E345R mutation, which can promote antibody multimerization upon receptor binding, facilitated anti-OX40 antibody to have increased agonism by promoting the clustering of OX40 receptors without the dependence on FcγRIIB cross-linking. Nonetheless, cross-linking to FcγRIIB can lead to a further boost of the agonism of the anti-OX40 antibody with IgG1 Fc but not with the silent IgG2σ Fc. The antibody-dependent cellular cytotoxicity and complement-dependent cytotoxicity activities of the anti-OX40 antibody with the E345R mutation were affected by the choice of IgG subtypes. However, there was little change in the antibody-dependent cellular phagocytosis activity. In summary, different Fc engineering approaches can guide the design of engineered antibodies to OX40 and other TNFR with improved anti-tumor activity., (© 2016 by The American Society for Biochemistry and Molecular Biology, Inc.)

Recent studies have revealed various Foxp3(-) regulatory T (Treg) cell subsets effectively protect mice from colitis. In the present study, we demonstrated that B cells induced a particular subset of regulatory T (Treg-of-B) cells, expressing programmed cell death 1 (PD-1), inducible costimulator (ICOS), lymphocyte-activation gene 3 (LAG3), glucocorticoid-induced tumor necrosis factor receptor (GITR), and OX-40, did not express Foxp3. Treg-of-B cells produced abundant levels of IL-10 and low levels of IL-4 and TGF-β. Adoptive transfer of Treg-of-B cells protected mice from CD4(+)CD45RB(hi) T-cell-induced colitis, including infiltration of leukocytes, depletion of goblet cells, epithelial hyperplasia, and inhibition of Th1 and Th17 cytokines. These features were similar to IL-10-producing type 1 regulatory T (Tr1) cells; however, IL-10-deficient Treg-of-B cells maintained their suppressive function in vitro as well as in vivo, while the regulation of Tr1 cells depended on IL-10. In conclusion, Treg-of-B cells protected against experimental colitis through an IL-10-independent mechanism. We reported a novel subpopulation of regulatory T cells was different from conventional Foxp3(+) Treg and IL-10-producing Tr1 cells.

OX40 receptor-expressing regulatory T cells (Tregs) populate tumors and suppress a variety of immune cells, posing a major obstacle for cancer immunotherapy. Different ways to functionally inactivate Tregs by triggering OX40 receptor have been suggested, including anti-OX40 antibodies and Fc:OX40L fusion proteins. To investigate whether the soluble extracellular domain of OX40L (OX40Lexo) is sufficient to enhance antitumor immune response, we generated an OX40Lexo-expressing CT26 colon carcinoma cell line and studied its tumorigenicity in immunocompetent BALB/c and T cell deficient nu/nu mice. We found that soluble OX40L expressed in CT26 colon carcinoma favors the induction of an antitumor response which is not limited just to cells co-expressing EGFP as an antigenic determinant, but also eliminates CT26 cells expressing another fluorescent protein, KillerRed. Tumor rejection required the presence of T lymphocytes, as indicated by the unhampered tumor growth in nu/nu mice. Subsequent re-challenge of tumor-free BALB/c mice with CT26 EGFP cells resulted in no tumor growth, which is indicative of the formation of immunological memory. Adoptive transfer of splenocytes from mice that successfully rejected CT26 OX40Lexo EGFP tumors to naïve mice conferred 100% resistance to subsequent challenge with the CT26 EGFP tumor., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

The anecdotal reports of promising results seen with immunotherapy and radiation in advanced malignancies have prompted several trials combining immunotherapy and radiation. However, the ideal timing of immunotherapy with radiation has not been clarified. Tumor bearing mice were treated with 20Gy radiation delivered only to the tumor combined with either anti-CTLA4 antibody or anti-OX40 agonist antibody. Immunotherapy was delivered at a single timepoint around radiation. Surprisingly, the optimal timing of these therapies varied. Anti-CTLA4 was most effective when given prior to radiation therapy, in part due to regulatory T cell depletion. Administration of anti-OX40 agonist antibody was optimal when delivered one day following radiation during the post-radiation window of increased antigen presentation. Combination treatment of anti-CTLA4, radiation, and anti-OX40 using the ideal timing in a transplanted spontaneous mammary tumor model demonstrated tumor cures. These data demonstrate that the combination of immunotherapy and radiation results in improved therapeutic efficacy, and that the ideal timing of administration with radiation is dependent on the mechanism of action of the immunotherapy utilized.

CD4+CD25+ regulatory T cells (Tregs) are involved in the regulation of physiological and pathological hepatic immune responses, but the roles are not well explored in natural killer (NK) cell-mediated liver diseases. In this study, using the NK cell-mediated oversensitive liver injury model of hepatitis B virus transgenic (HBs-Tg) mice triggered by a low dose of concanavalin A, it was observed that an increased number of CD4+CD25+Foxp3+ Tregs were accumulated in the liver, along with the recovery of liver injury. Adoptive transfer of hepatic Tregs from HBs-Tg mice but not wild B6 mice could significantly attenuate the oversensitive liver injury via inhibiting liver accumulation and decreasing NK cell group 2D-mediated activation of NK cells in the recipient HBs-Tg mice. Furthermore, upregulated expression of membrane-bound TGF-β (mTGF-β) and OX40 on hepatic Tregs were demonstrated to account for inhibiting the NK cell-mediated hepatic injury in HBs-Tg mice through cell-cell contact, confirmed by antibody blockade and cell Transwell experiments in vivo and in vitro. Our findings for the first time indicated that CD4+CD25+ Tregs directly suppressed NK cell-mediated hepatocytotoxicity through mTGF-β and OX40/OX40L interaction in a cell-cell contact manner in HBV-associated liver disease., (© 2015 S. Karger AG, Basel.)

Rationale: Most immunocompetent patients diagnosed with latent tuberculosis infection (LTBI) will not progress to tuberculosis (TB) reactivation. However, current diagnostic tools cannot reliably distinguish nonprogressing from progressing patients a priori, and thus LTBI therapy must be prescribed with suboptimal patient specificity. We hypothesized that LTBI diagnostics could be improved by generating immunomarker profiles capable of categorizing distinct patient subsets by a combinatorial immunoassay approach., Objectives: A combinatorial immunoassay analysis was applied to identify potential immunomarker combinations that distinguish among unexposed subjects, untreated patients with LTBI, and treated patients with LTBI and to differentiate risk of reactivation., Methods: IFN-γ release assay (IGRA) was combined with a flow cytometric assay that detects induction of CD25(+)CD134(+) coexpression on TB antigen-stimulated T cells from peripheral blood. The combinatorial immunoassay analysis was based on receiver operating characteristic curves, technical cut-offs, 95% bivariate normal density ellipse prediction, and statistical analysis. Risk of reactivation was estimated with a prediction formula., Measurements and Main Results: Sixty-five out of 150 subjects were included. The combinatorial immunoassay approach identified at least four different T-cell subsets. The representation of these immune phenotypes was more heterogeneous in untreated patients with LTBI than in treated patients with LTBI or unexposed groups. Patients with IGRA(+) CD4(+)CD25(+)CD134(+) T-cell phenotypes had the highest estimated reactivation risk (4.11 ± 2.11%)., Conclusions: These findings suggest that immune phenotypes defined by combinatorial assays may potentially have a role in identifying those at risk of developing TB; this potential role is supported by risk of reactivation modeling. Prospective studies will be needed to test this novel approach.

Cancer therapy with T cells expressing chimeric antigen receptors (CARs) has produced remarkable clinical responses in recent trials, but also severe side effects. Whereas most protocols use permanently reprogrammed T cells, we have developed a platform for transient CAR expression by mRNA electroporation. This approach may be useful for safe clinical testing of novel receptors, or when a temporary treatment period is desirable. Herein, we investigated therapy with transiently redirected T cells in vitro and in a xenograft mouse model. We constructed a series of CD19-specific CARs with different spacers and co-stimulatory domains (CD28, OX40 or CD28-OX40). The CAR constructs all conferred T cells with potent CD19-specific activity in vitro. Unexpectedly, the constructs incorporating a commonly used IgG1-CH2CH3 spacer showed lack of anti-leukemia activity in vivo and induced severe, partly CD19-independent toxicity. By contrast, identical CAR constructs without the CH2-domain eradicated leukemia in vivo, without notable toxicity. Follow-up studies demonstrated that the CH2CH3-spacer bound soluble mouse Fcγ-receptor I and mediated off-target T-cell activation towards murine macrophages. Our findings highlight the importance of non-signalling CAR elements and of in vivo studies. Finally, the results show that transiently redirected T cells control leukemia in mice and support the rationale for developing an mRNA-CAR platform.

Decline in CD4 T cell immune responses is associated with aging. Although a number of immunological defects have been identified in elderly mice (>18 months old), a key early-onset immune defect at middle age could be a driver or contributor to defective CD4 T cell responses. Our studies demonstrate that age-related alterations in DC subsets within the priming environment of middle-aged mice (12 months old) correlate with and can directly contribute to decreases in antigen-specific CD4 T cell Th1 differentiation, which measured by T-bet and IFN-γ expression, was decreased significantly in T cells following VSV infection or s.c. immunization with a protein antigen in the context of immune stimulation via OX40. The deficient Th1 phenotype, observed following protein antigen challenge, was found to be the result of an age-related decrease in an inflammatory DC subset (CD11b+ Gr-1/Ly6C+) in the dLN that corresponded with T cell dysfunction. In the virus model, we observed significant changes in two DC subsets: mDCs and pDCs. Thus, different, early age-related changes in the DC profile in the priming environment can significantly contribute to impaired Th1 differentiation, depending on the type of immunological challenge., (© 2014 Society for Leukocyte Biology.)