Combined agonist stimulation of the TNFR costimulatory receptors 4-1BB (CD137) and OX40(CD134) has been shown to generate supereffector CD8 T cells that clonally expand to greater levels, survive longer, and produce a greater quantity of cytokines compared to T cells stimulated with an agonist of either costimulatory receptor individually. In order to understand the mechanisms for this effect, we have created a mathematical model for the activation of the CD8 T cell intracellular signaling network by mono- or dual-costimulation. We show that supereffector status is generated via downstream interacting pathways that are activated upon engagement of both receptors, and in silico simulations of the model are supported by published experimental results. The model can thus be used to identify critical molecular targets of T cell dual-costimulation in the context of cancer immunotherapy.

The Tumour Necrosis Factor superfamilies of receptors and ligands play a crucial role in the regulation of effective immune responses against pathogens and malignant cells. In chickens, only few members have been identified. Here, we characterise the chicken homologues for mammalian costimulatory molecules OX40 and OX40L, which are involved in sustaining T cell responses. Both genes were identified by virtue of their genomic localisation close to highly conserved genes and their structural relationship to their mammalian homologues. Following cloning and expression of soluble and cell-associated chicken OX40 and OX40L, we confirmed their mutual interaction via ELISA and flow cytometric analyses. In addition, we showed the application of soluble OX40-Fc in staining of chicken cells. Whereas non-activated cells did not express OX40L, activation by IL-2 and IL-12 resulted in upregulation of OX40L on αβ and γδ T cell populations. Our results demonstrate the existence of the costimulatory OX40-OX40L system in the chicken and provide the basis for further investigations of chicken T cell responses.

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.

Our previous studies have implicated signaling through the tumor necrosis family receptors OX40 and CD30 as critical for maintaining CD4 memory responses. We show that signals through both molecules are also required for CD4 effector-mediated autoimmune tissue damage. Under normal circumstances, male mice deficient in the forkhead transcription factor FoxP3, which lack regulatory CD4 T cells, develop lethal autoimmune disease in the first few weeks of life. However, in the combined absence of OX40 and CD30, FoxP3-deficient mice develop normally and breed successfully. The extensive tissue infiltration and organ destruction characteristic of FoxP3 disease does not appear in these mice, and their mortality is not associated with autoimmunity. Although the absence of OX40 plays the dominant role, FoxP3-deficient mice sufficient in CD30 but deficient in OX40 signals still eventually develop lethal disease. This result was supported by the observation that blocking antibodies to OX40 and CD30 ligands also abrogated disease mediated by FoxP3-deficient T cells. These observations identify OX40 and CD30 signals as essential for the development of clinically relevant CD4-dependent autoimmunity and suggest that combination therapies that abrogate these signals might be used to treat established human autoimmune diseases.

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 (CD11ahiCD44hi) 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.

The RNA-binding protein Roquin is required to prevent autoimmunity. Roquin controls T-helper cell activation and differentiation by limiting the induced expression of costimulatory receptors such as tumor necrosis factor receptor superfamily 4 (Tnfrs4 or Ox40). A constitutive decay element (CDE) with a characteristic triloop hairpin was previously shown to be recognized by Roquin. Here we use SELEX assays to identify a novel U-rich hexaloop motif, representing an alternative decay element (ADE). Crystal structures and NMR data show that the Roquin-1 ROQ domain recognizes hexaloops in the SELEX-derived ADE and in an ADE-like variant present in the Ox40 3'-UTR with identical binding modes. In cells, ADE-like and CDE-like motifs cooperate in the repression of Ox40 by Roquin. Our data reveal an unexpected recognition of hexaloop cis elements for the posttranscriptional regulation of target messenger RNAs by Roquin.

Members of the tumour necrosis factor (TNF) superfamily OX40L and CD70 and their receptors are costimulating signalling axes critical for adequate T cell activation in humans and mice but characterisation of these molecules in other species including ruminants is lacking. Here we cloned and expressed the predicted ovine orthologues of the receptors OX40 and CD27, as well as soluble recombinant forms of their potential ovine ligands, OaOX40L and OaCD70. Using biochemical and immunofluorescence analyses, we show that both signalling axes are functional in sheep. We show that oligomeric recombinant ligand constructs are able to induce signalling through their receptors on transfected cells. Recombinant defective human adenoviruses were constructed to express the soluble forms of OaOX40L and OaCD70. Both proteins were detected in the supernatant of adenovirus-infected cells and shown to activate NF-κB signalling pathway through their cognate receptor. These adenovirus-secreted OaOX40L and OaCD70 forms could also activate ovine T cell proliferation and enhance IFN-γ production in CD4+ and CD8+ T cells. Altogether, this study provides the first characterisation of the ovine costimulatory OX40L-OX40 and CD70-CD27 signalling axes, and indicates that their activation in vivo may be useful to enhance vaccination-induced immune responses in sheep and other ruminants.

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.

Immune checkpoint therapy (ICT) has the power to eradicate cancer, but the mechanisms that determine effective therapy-induced immune responses are not fully understood. Here, using high-dimensional single-cell profiling, we interrogate whether the landscape of T cell states in the peripheral blood predict responses to combinatorial targeting of the OX40 costimulatory and PD-1 inhibitory pathways. Single-cell RNA sequencing and mass cytometry expose systemic and dynamic activation states of therapy-responsive CD4+ and CD8+ T cells in tumor-bearing mice with expression of distinct natural killer (NK) cell receptors, granzymes, and chemokines/chemokine receptors. Moreover, similar NK cell receptor-expressing CD8+ T cells are also detected in the blood of immunotherapy-responsive cancer patients. Targeting the NK cell and chemokine receptors in tumor-bearing mice shows the functional importance of these receptors for therapy-induced anti-tumor immunity. These findings provide a better understanding of ICT and highlight the use and targeting of dynamic biomarkers on T cells to improve cancer immunotherapy.

Immunostimulatory receptors belonging to the tumor necrosis factor receptor (TNFR) superfamily are emerging as promising targets for cancer immunotherapies. To optimize the agonism of therapeutic antibodies to these receptors, Fc engineering of antibodies was applied to facilitate the clustering of cell surface TNFRs to activate downstream signaling pathways. One engineering strategy is to identify Fc mutations that facilitate antibody multimerization on the cell surface directly. From the analyses of the crystal packing of IgG1 structures, we identified a novel set of Fc mutations, T437R and K248E, that facilitated antibody multimerization upon binding to antigens on cell surface. In a NF-κB reporter assay, the engineered T437R/K248E mutations could facilitate enhanced agonism of an anti-OX40 antibody without the dependence on FcγRIIB crosslinking. Nonetheless, the presence of cells expressing FcγRIIB could facilitate a boost of the agonism of the engineered antibody with mutations on IgG1 Fc, but not on the silent IgG2σ Fc. The Fc engineered antibody also showed enhanced effector functions, including antibody-dependent cell-meditated cytotoxicity, antibody-dependent cellular phagocytosis, and complement-dependent cytotoxicity, depending on the IgG subtypes. Also, the engineered antibodies showed normal FcRn binding and pharmacokinetic profiles in mice. In summary, this study elucidated a novel Fc engineering approach to promote antibody multimerization on a cell surface, which could enhance agonism and improve effector function for anti-TNFR antibodies as well as other therapeutic antibodies.

Background: Natural Killer (NK) cell-based immunotherapy used to treat cancer requires the adoptive transfer of a large number of activated NK cells. Here, we report a new effective method to expand human NK cells ex vivo using K562 cells genetically engineered (GE) to express OX40 ligand (K562-OX40L) in combination with a short exposure to soluble IL-21. In addition, we describe a possible mechanism of the NK cell expansion through the OX40 receptor-OX40 ligand axis which is dependent on NK cell homotypic interaction. Methods: K562-OX40L cells were generated by lentiviral transduction and were used as feeder cells to expand and activate NK cells from PBMCs in the presence of IL-2/IL-15. Soluble IL-21 was also added in various concentrations only once at the beginning of the culture. NK cells were expanded for 4-5 weeks, and the purity, expansion rate, phenotype and function (cytotoxicity, antibody-dependent cell-mediated cytotoxicity (ADCC), cytokine production, CD107a degranulation) of these expanded NK cells were compared to those generated by using K562 feeder cells. Results: The culture of NK cells with K562-OX40L cells in combination with the transient exposure to IL-21 highly enhanced NK cell expansion to approximately 2,000-fold after 4 weeks of culture, compared to a 303-fold expansion using the conventional K562 cells. Mechanistically, the OX40-OX40L axis between the feeder cells and NK cells as well as the homotypic interaction between NK cells through the OX40-OX40L axis were both necessary for NK cell expansion. The short exposure of NK cells to IL-21 had a synergistic effect with OX40 signaling for NK cell expansion. Apart from their enhanced expansion, NK cells grown with K562-OX40L feeder cells were similar to those grown with conventional K562 cells in regard to the surface expression of various receptors, cytotoxicity, ADCC, cytokine secretion, and CD107 degranulation. Conclusion: Our data suggest that OX40 ligand is a potent co-stimulant for the robust expansion of human NK cells and the homotypic NK cell interactions through the OX40-OX40L axis is a mechanism of NK cell expansion.

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.

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).

It is well-known that vaccines comprising of irradiated whole tumor cells or tumor-derived heat shock proteins can generate tumor-specific immune responses. In contrast, we showed recently that vaccines composed of autophagosomes (DRibbles) derived from syngeneic sarcomas could induce cross-reactive T-cell responses and cross-protection against the tumor. This unusual property of DRibbles was related to the selective recruitment of defective ribosomal products (DRiPs) and other short-lived proteins (SLiPs) into autophagosomes via sequestosome (SQSTM1, p62) mediated association of ubiquitinated SLiPs to the autophagy gene product LC3. Here, we extend our observations to mammary carcinomas from mice of different genetic background. We demonstrated that combined of intranodal administration of autologous or allogeneic DRibbles together with anti-OX40 antibody led to robust proliferation, expansion, and differentiation of memory and effector T cells. We also showed that SLiPs is an excellent source of antigen for cross-priming of CD8+ T-cells that recognize shared tumor antigens in the context of host MHC class I molecules. Thus, our results provide a strong basis for novel clinical trials that combine allogeneic "off-the-shelf" DRibble vaccines together with antibodies against co-stimulatory molecules.

Mast cells are major effector cells of allergic diseases related to IgE. This study was undertaken to determine whether IgE or IgA, produced by CD40-CD40L or OX40-OX40L interactions between B cells and mast cells, re-activate FcεRI or FcαRI on mast cell surface. C57BL mice were sensitized and subjected to OVA challenge to induce asthma. Bone marrow-derived mast cells (BMMCs) and primary B cells were co-cultured. Mast cell recruitment into airways was stained by May-Grünwald Giemsa, the expression of markers or signaling molecules were determined by immunohistochemistry or Western blotting, and co-localization of B cells and mast cells by immunofluorescence. Anti-CD40 plus anti-OX40L Abs synergistically reduced IgE and IgA production, and mediators (histamine, LTs and cytokines) released in mast cells, and additively reduced other responses, such as, numbers of mast cells, the expression of markers (tryptase, mMCP5, B220 and CD19), surface molecules (CD40, CD40L, OX40 and OX40L), FcεRI or FcαRI and the co-localization of BMMCs and B cells, and IgE- or IgA-producing cells, as compared with individual blocking Ab treatment which reducedresponses in BAL cells or lung tissues of OVA-challenged mice or in co-culture of B and mast cells. The data suggest that IgE and IgA, produced by OX40-OX40L or CD40-CD40L interaction between B cells and mast cells, may re-activate receptors of FCεRI and FcαRI on mast cell surfaces, followed by more mediator release, and furthermore, that treatment with anti-CD40 plus anti-OX40L Abs offers a potential treatment for allergic asthma.

The clinical approval of immune checkpoint inhibitors is an important advancement in the field of cancer immunotherapy. However, the percentage of beneficiaries is still limited and it is becoming clear that combination therapies are required to further enhance the treatment efficacy. The potential of strategies targeting the immunoregulatory network by "hitting the gas pedal" as opposed to "blocking the brakes" is being recognized and intensively investigated. Hence, next to immune checkpoint inhibitors, agonists of co-stimulatory receptors of the tumor necrosis factor superfamily (TNF-SF) are emerging as promising options to expand the immunomodulatory toolbox. In this review the development of different categories of recombinant antibody and ligand-based agonists of 4-1BB, OX40, and GITR is summarized and discussed in the context of the challenges presented by the structural and mechanistical features of the TNFR-SF. An overview of current formats, trends, and clinical studies is provided.

Regulatable CAR platforms could circumvent toxicities associated with CAR-T therapy, but existing systems have shortcomings including leakiness and attenuated activity. Here, we present SNIP CARs, a protease-based platform for regulating CAR activity using an FDA-approved small molecule. Design iterations yielded CAR-T cells that manifest full functional capacity with drug and no leaky activity in the absence of drug. In numerous models, SNIP CAR-T cells were more potent than constitutive CAR-T cells and showed diminished T cell exhaustion and greater stemness. In a ROR1-based CAR lethality model, drug cessation following toxicity onset reversed toxicity, thereby credentialing the platform as a safety switch. In the same model, reduced drug dosing opened a therapeutic window that resulted in tumor eradication in the absence of toxicity. SNIP CARs enable remote tuning of CAR activity, which provides solutions to safety and efficacy barriers that are currently limiting progress in using CAR-T cells to treat solid tumors.

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.

Agonist antibodies that activate cellular receptors are being pursued for therapeutic applications ranging from neurodegenerative diseases to cancer. For the tumor necrosis factor (TNF) receptor superfamily, higher-order clustering of three or more receptors is key to their potent activation. This can be achieved using antibodies that recognize two unique epitopes on the same receptor and mediate receptor superclustering. However, identifying compatible pairs of antibodies to generate biepitopic antibodies (also known as biparatopic antibodies) for activating TNF receptors typically requires animal immunization and is a laborious and unpredictable process. Here, we report a simple method for systematically identifying biepitopic antibodies that potently activate TNF receptors without the need for additional animal immunization. Our approach uses off-the-shelf, receptor-specific IgG antibodies, which lack intrinsic (Fc-gamma receptor-independent) agonist activity, to first block their corresponding epitopes. Next, we perform selections for single-chain antibodies from human nonimmune libraries that bind accessible epitopes on the same ectodomains using yeast surface display and fluorescence-activated cell sorting. The selected single-chain antibodies are finally fused to the light chains of IgGs to generate human tetravalent antibodies that engage two different receptor epitopes and mediate potent receptor activation. We highlight the broad utility of this approach by converting several existing clinical-stage antibodies against TNF receptors, including ivuxolimab and pogalizumab against OX40 and utomilumab against CD137, into biepitopic antibodies with highly potent agonist activity. We expect that this widely accessible methodology can be used to systematically generate biepitopic antibodies for activating other receptors in the TNF receptor superfamily and many other receptors whose activation is dependent on strong receptor clustering.

Immunotherapy with T cells expressing chimeric antigen receptors (CARs) is an attractive approach to improve outcomes for patients with glioblastoma (GBM). IL13Rα2 is expressed at a high frequency in GBM but not in normal brain, making it a promising CAR T-cell therapy target. IL13Rα2-specific CARs generated up to date contain mutated forms of IL13 as an antigen-binding domain. While these CARs target IL13Rα2, they also recognize IL13Rα1, which is broadly expressed. To overcome this limitation, we constructed a panel of IL13Rα2-specific CARs that contain the IL13Rα2-specific single-chain variable fragment (scFv) 47 as an antigen binding domain, short or long spacer regions, a transmembrane domain, and endodomains derived from costimulatory molecules and CD3.ζ (IL13Rα2-CARs). IL13Rα2-CAR T cells recognized IL13Rα2-positive target cells in coculture and cytotoxicity assays with no cross-reactivity to IL13Rα1. However, only IL13Rα2-CAR T cells with a short spacer region produced IL2 in an antigen-dependent fashion. In vivo, T cells expressing IL13Rα2-CARs with short spacer regions and CD28.ζ, 41BB.ζ, and CD28.OX40.ζ endodomains had potent anti-glioma activity conferring a significant survival advantage in comparison to mice that received control T cells. Thus, IL13Rα2-CAR T cells hold the promise to improve current IL13Rα2-targeted immunotherapy approaches for GBM and other IL13Rα2-positive malignancies.