TCR-independent CD137 (4-1BB) signaling promotes CD8+-exhausted T cell proliferation and terminal differentiation.

CD137 (4-1BB)-activating receptor represents a promising cancer immunotherapeutic target. Yet, the cellular program driven by CD137 and its role in cancer immune surveillance remain unresolved. Using T cell-specific deletion and agonist antibodies, we found that CD137 modulates tumor infiltration of CD8+-exhausted T (Tex) cells expressing PD1, Lag-3, and Tim-3 inhibitory receptors. T cell-intrinsic, TCR-independent CD137 signaling stimulated the proliferation and the terminal differentiation of Tex precursor cells through a mechanism involving the RelA and cRel canonical NF-κB subunits and Tox-dependent chromatin remodeling. While Tex cell accumulation induced by prophylactic CD137 agonists favored tumor growth, anti-PD1 efficacy was improved with subsequent CD137 stimulation in pre-clinical mouse models. Better understanding of T cell exhaustion has crucial implications for the treatment of cancer and infectious diseases. Our results identify CD137 as a critical regulator of Tex cell expansion and differentiation that holds potential for broad therapeutic applications.

Regulatory T Cell Specificity Directs Tolerance versus Allergy against Aeroantigens in Humans.

FOXP3+ regulatory T cells (Tregs) maintain tolerance against self-antigens and innocuous environmental antigens. However, it is still unknown whether Treg-mediated tolerance is antigen specific and how Treg specificity contributes to the selective loss of tolerance, as observed in human immunopathologies such as allergies. Here, we used antigen-reactive T cell enrichment to identify antigen-specific human Tregs. We demonstrate dominant Treg-mediated tolerance against particulate aeroallergens, such as pollen, house dust mites, and fungal spores. Surprisingly, we found no evidence of functional impairment of Treg responses in allergic donors. Rather, major allergenic proteins, known to rapidly dissociate from inhaled allergenic particles, have a generally reduced capability to generate Treg responses. Most strikingly, in individual allergic donors, Th2 cells and Tregs always target disparate proteins. Thus, our data highlight the importance of Treg antigen-specificity for tolerance in humans and identify antigen-specific escape from Treg control as an important mechanism enabling antigen-specific loss of tolerance in human allergy.

Costimulatory capacity of CD137 mAbs on T cells depends on elaborate CRD structures but not on blocking ligand-receptor binding.

CD137 is mainly a costimulatory receptor of CD8+ T cells. Two representative CD137 antibodies, utomilumab, and urelumab, show different costimulatory capacities in clinical trials. Balancing the antitumor effect and systemic toxicity of T cells activated by CD137 signaling is a challenge that requires clinical consideration. In this study, a panel of specific anti-human CD137 monoclonal antibodies (mAbs) were prepared and their affinities, isotypes, CD137-CRD (cysteine-rich domain) binding regions, cross-reactivity to mouse and rhesus CD137, inhibition of ligand-receptor binding and costimulatory activities were analyzed. The results showed that anti-human CD137 mAbs had high cross-reactivity with rhesus CD137. MAbs fell into three clusters according to their different binding regions of the CD137 extracellular domain. They bound to CRDI+CRDII, CRDIII or CRDIV+STP. CRDIII-binding mAbs had the strongest blocking activity. Highly costimulatory CD137 mAbs showed stronger abilities to promote CD8+ T-cell proliferation. However, the costimulatory capacity of mAbs on T cells was not closely related to their ability to block CD137L-CD137 binding and may be controlled by more elaborate CRD conformational structures. This study provides additional information for the development of next-generation CD137 mAbs to meet clinical needs.

CD137+ and regulatory T cells as independent prognostic factors of survival in advanced non-oncogene addicted NSCLC patients treated with immunotherapy as first-line.

BACKGROUND: Immune checkpoint inhibitors (ICIs), administered alone or combined with chemotherapy, are the standard of care in advanced non-oncogene addicted Non-Small Cell Lung Cancer (NSCLC). Despite these treatments' success, most long-term survival benefit is restricted to approximately 20% of patients, highlighting the need to identify novel biomarkers to optimize treatment strategies. In several solid tumors, immune soluble factors, the activatory CD137+ Tcells, and the immunosuppressive cell subsets Tregs and MDSCs (PMN(Lox1+)-MDSC and M-MDSCs) correlated with responses to ICIs and clinical outcomes thus becoming appealing predictive and prognostic factors. This study investigated the role of distinct CD137+ Tcell subsets, Tregs, MDSCs, and immune-soluble factors in NSCLC patients as possible biomarkers. METHODS: The levels of T cells, MDSCs and soluble factors were evaluated in 89 metastatic NSCLC patients who underwent ICIs as first- or second-line treatment. T cell analysis was performed by cytoflurimetry evaluating Tregs and different CD137+ Tcell subsets also combined with CD3+, CD8+, PD1+, and Ki67+ markers. Circulating cytokines and immune checkpoints were also evaluated by Luminex analysis. All these parameters were correlated with several clinical factors (age, sex, smoking status, PS and TPS), response to therapy, PFS , and OS . The analyses were conducted in the overall population and in patients treated with ICIs as first-line (naïve patients). RESULTS: In both groups of patients, high levels of circulating CD137+ and CD137+PD1+ T cells (total, CD4 and CD8) and the soluble factor LAG3 positively correlated with response to therapy. In naïve patients, PMN(Lox1+)-MDSCs negatively correlated with clinical response, and a high percentage of Tregs was associated with favorable survival. Moreover, the balance between Treg/CD137+ Tcells or PMN(Lox1+)-MDSC/CD137+ Tcells was higher in non-responding patients and was associated with poor survival. CD137+ Tcells and Tregs resulted as two positive independent prognostic factors. CONCLUSION: High levels of CD137+, CD137+PD1+ Tcells and sLAG3 could predict the response to ICIs in NSCLC patients independently by previous therapy. Combining the evaluation of CD137+ Tcells and Tregs also as Treg/CD137+ T cells ratio it is possible to identify naive patients with longer survival.

Pharmacokinetics and safety of LEAD-452, an EGFR-specific 4-1BB-agonistic trimerbody in non-human primates.

LEAD-452 is a humanized bispecific EGFR-targeted 4-1BB-agonistic trimerbody with a unique trimeric configuration compared to other 4-1BB-specific antibodies that are currently in development. Indeed, enhanced tumor-specific costimulation and very remarkable safety and efficacy profiles have been observed in mouse models. Here, we conducted for the first time a preclinical pharmacokinetic and toxicity study in non-human primates (NHP) (Macaca fascicularis). LEAD-452 exhibits comparable binding affinity for human and macaque targets, indicating its pharmacological significance for safety testing across species. The NHP were administered LEAD-452 in a series of ascending doses, ranging from 0.1 mg/kg to 10 mg/kg, and repeated doses up to 20 mg/kg. The administration of LEAD-452 was found to be clinically well tolerated, with no major related adverse effects observed. Furthermore, there have been no reported cases of liver toxicity, thrombocytopenia, and neutropenia, which are commonly associated with treatments using conventional anti-4-1BB IgG-based antibodies. In addition, neither IgM nor IgG-based anti-drug antibodies were detected in serum samples from NHP during the study, regardless of the dose of LEAD-452 administered. These results support the clinical development of LEAD-452 for the treatment of solid tumors.

CD137+ tumor infiltrating lymphocytes predicts ovarian cancer survival.

OBJECTIVE: Ovarian cancer (OC) is the leading cause of death from gynecologic malignancy in the United States, and biomarkers of patient outcomes are limited. Data using immunohistochemical (IHC) analysis are mixed regarding whether and which tumor infiltrating lymphocytes (TILs) impact survival, and IHC does not adequately quantify rare cell populations, including CD137+ (4-1BB) tumor-reactive TILs. Our study investigates if a higher percentage of CD3+ CD137+ TILs is associated with improved overall survival (OS) in OC. METHODS: Flow cytometry was performed on viably banked OC digests. Chart review and statistical analysis were performed. Forty-seven patients were included, 40 of whom were diagnosed with high-grade serous ovarian carcinoma (HGSOC), papillary serous carcinoma, or undifferentiated histology. RESULTS: A high percentage of CD3+ CD137+ TILs correlated with improved OS (n = 40, r = 0.48, P = 0.0016). Subjects were divided into CD3+ CD137+ TIL high and low groups by the median. Subjects with high CD3+CD137+ TIL frequencies (>9.6%) had longer OS (Wilcoxon rank-sum test; P = 0.0032) and improved OS (logrank test; P = 0.007). Differences in CD3+ or CD3+ CD8+ TILs did not impact survival. CD3+ CD137+ TILs were predictive of OS regardless of germline mutation or debulking status. Analysis of subgroups including late stage HGSOC and late stage HGSOC with primary optimal cytoreduction indicated CD3+ CD137+ TILs correlated with improved OS after adjusting for age and PARP inhibitor use (P = 0.034 and P = 0.016, respectively). CONCLUSIONS: Prevalence of CD3+ CD137+ TILs in digested OC specimens is associated with improved OS, while general TIL markers are not. CD137 has the potential to be a novel biomarker for survival in OC.

Antitumor efficacy and reduced toxicity using an anti-CD137 Probody therapeutic.

Costimulation via CD137 (4-1BB) enhances antitumor immunity mediated by cytotoxic T lymphocytes. Anti-CD137 agonist antibodies elicit mild liver inflammation in mice, and the maximum tolerated dose of Urelumab, an anti-human CD137 agonist monoclonal antibody, in the clinic was defined by liver inflammation-related side effects. A protease-activated prodrug form of the anti-mouse CD137 agonist antibody 1D8 (1D8 Probody therapeutic, Pb-Tx) was constructed and found to be selectively activated in the tumor microenvironment. This construct, which encompasses a protease-cleavable linker holding in place a peptide that masks the antigen binding site, exerted antitumor effects comparable to the unmodified antibody but did not result in liver inflammation. Moreover, it efficaciously synergized with both PD-1 blockade and adoptive T-cell therapy. Surprisingly, minimal active Pb-Tx reached tumor-draining lymph nodes, and regional lymphadenectomy did not abrogate antitumor efficacy. By contrast, S1P receptor-dependent recirculation of T cells was absolutely required for efficacy. The preferential cleavage of the anti-CD137 Pb-Tx by tumor proteases offers multiple therapeutic opportunities, including neoadjuvant therapy, as shown by experiments in which the Pb-Tx is given prior to surgery to avoid spontaneous metastases.

Benralizumab affects NK cell maturation and proliferation in severe asthmatic patients.

INTRODUCTION: The mechanism of action of benralizumab is determined by its afucosylated constant fragment that binds CD16a receptors on the membrane of natural killer cells. Here we analysed changes in Natural Killer and T-cells in Severe asthmatic patients, before and after benralizumab. METHODS: Natural Killer and T-cell subsets were detected through multiparametric flow cytometry. The concentrations of serum cytokines levels were detected through multiplex assay. Functional proliferation assay was performed in follow-up samples in severe asthmatic patients. RESULTS: At baseline, severe asthmatic patients showed higher percentages of immature Natural Killer cells when compared with healthy controls. We demonstrate the proliferative capacity of these cells and their activation after benralizumab administration. Benralizumab shifted Natural Killer cell phenotypes towards maturity. Correlation between the Natural Killer cells and functional parameters and with steroid-sparing was observed. CONCLUSION: Together this data contributes to our understanding of the mechanisms of action of benralizumab in the resolution of inflammation in severe asthma patients.

Antigen-specific T cell balance reveals Why patients with atopic dermatitis fail to achieve immune tolerance.

The number of regulatory T cells (Tregs) and how they behave in the pathogenesis of atopic dermatitis (AD) are still controversial. We identified and quantified Tregs, mite-specific Tregs, and mite-specific effector T cells (Teffs) in patients with AD and healthy controls (HCs). We collected peripheral blood and analyzed the cells using flow cytometry after stimulation with mite antigens. Mite-specific Tregs and mite-specific Teffs were recognized by the expression of CD137 and CD154, respectively. Patients with AD had more Tregs than HCs; however, when focusing on a single antigen, the ratio of mite-specific Tregs/Teffs was lower in patients with AD than in HCs. Furthermore, the mite-specific Teffs in patients with AD were more likely to produce proinflammatory cytokines interleukin (IL)-4 and IL-13. This Teff-dominant imbalance is thought to be the cause of development of atopic status in patients with AD without immune tolerance.

Systematic analysis of CD39, CD103, CD137, and PD-1 as biomarkers for naturally occurring tumor antigen-specific TILs.

The detection of tumor-specific T cells in solid tumors is integral to interrogate endogenous antitumor responses and to advance downstream therapeutic applications. Multiple biomarkers are reported to identify endogenous tumor-specific tumor-infiltrating lymphocytes (TILs), namely CD137, PD-1, CD103, and CD39; however, a direct comparison of these molecules has yet to be performed. We evaluated these biomarkers in primary human ovarian tumor samples using single-cell mass cytometry to compare their relative phenotypic profiles, and examined their response to autologous tumor cells ex vivo. PD-1+ , CD103+ , and CD39+ TILs all contain a CD137+ cell subset, while CD137+ TILs highly co-express the aforementioned markers. CD137+ TILs exhibit the highest expression of cytotoxic effector molecules compared to PD-1+ , CD103+ , or CD39+ TILs. Removal of CD137+ cells from PD-1+ , CD103+ , or CD39+ TILs diminish their IFN-γ secretion in response to autologous tumor cell stimulation, while CD137+ TILs maintain high HLA-dependent IFN-γ secretion. CD137+ TILs exhibited an exhausted phenotype but with CD28 co-expression, suggesting possible receptiveness to reinvigoration via immune checkpoint blockade. Together, our findings demonstrate that the antitumor abilities of PD-1+ , CD103+ , and CD39+ TILs are mainly derived from a subset of CD137-expressing TILs, implicating CD137 as a more selective biomarker for naturally occurring tumor-specific TILs.

ATOR-1017 (evunzekibart), an Fc-gamma receptor conditional 4-1BB agonist designed for optimal safety and efficacy, activates exhausted T cells in combination with anti-PD-1.

BACKGROUND: 4-1BB (CD137) is a co-stimulatory receptor highly expressed on tumor reactive effector T cells and NK cells, which upon stimulation prolongs persistence of tumor reactive effector T and NK cells within the tumor and induces long-lived memory T cells. 4-1BB agonistic antibodies have been shown to induce strong anti-tumor effects that synergize with immune checkpoint inhibitors. The first generation of 4-1BB agonists was, however, hampered by dose-limiting toxicities resulting in suboptimal dose levels or poor agonistic activity. METHODS: ATOR-1017 (evunzekibart), a second-generation Fc-gamma receptor conditional 4-1BB agonist in IgG4 format, was designed to overcome the limitations of the first generation of 4-1BB agonists, providing strong agonistic effect while minimizing systemic immune activation and risk of hepatoxicity. The epitope of ATOR-1017 was determined by X-ray crystallography, and the functional activity was assessed in vitro and in vivo as monotherapy or in combination with anti-PD1. RESULTS: ATOR-1017 binds to a unique epitope on 4-1BB enabling ATOR-1017 to activate T cells, including cells with an exhausted phenotype, and NK cells, in a cross-linking dependent, FcγR-conditional, manner. This translated into a tumor-directed and potent anti-tumor therapeutic effect in vivo, which was further enhanced with anti-PD-1 treatment. CONCLUSIONS: These preclinical data demonstrate a strong safety profile of ATOR-1017, together with its potent therapeutic effect as monotherapy and in combination with anti-PD1, supporting further clinical development of ATOR-1017.

Polyfunctional donor-reactive T cells are associated with acute T-cell-mediated rejection of the kidney transplant.

Acute T-cell-mediated rejection (aTCMR) still remains a clinical problem after kidney transplantation despite significant improvements in immunosuppressive regimens. Polyfunctional T cells, i.e. T cells producing multiple pro-inflammatory cytokines, are believed to be the most relevant T cells in an immune response. The aim of this study was to determine whether polyfunctional donor-reactive T cells are associated with aTCMR. In a case-control study, 49 kidney transplant recipients with a biopsy-proven aTCMR in the first year after transplantation were included, as well as 51 controls without aTCMR. Circulating donor-reactive T cells were identified by the expression of CD137 after short-term co-culture with donor antigen-presenting cells. Polyfunctional donor-reactive T cells were further characterized by dissection into different T-cell subsets encompassing the spectrum of naïve to terminally differentiated effector T cells. Prior to kidney transplantation, proportions of donor-reactive CD4+ (0.03% versus 0.02%; P < 0.01) and CD8+ (0.18% versus 0.10%; P < 0.01) CD137++ T cells were significantly higher in recipients with a biopsy-proven aTCMR versus non-rejectors. Polyfunctionality was higher (P = 0.03) in this subset of CD137-expressing T cells. These cells were predominantly of the EM/EMRA-phenotype, with polyfunctional donor-reactive CD137++CD4+ T cells predominantly co-expressing CD28 whereas approximately half of the polyfunctional CD137++CD8+ T cells co-expressed CD28. In addition, at the time of aTCMR, polyfunctional donor-reactive CD137++ CD4+, but not CD8+, T cells, were specifically decreased by 75% compared to before transplantation in recipients with as well as those without an aTCMR. Prior to transplantation, the proportion of polyfunctional donor-reactive CD137++ T cells is associated with the occurrence of a biopsy-proven aTCMR within the first year after transplantation.

A bispecific antibody AP203 targeting PD-L1 and CD137 exerts potent antitumor activity without toxicity.

BACKGROUND: Bispecific antibody has garnered considerable attention in the recent years due to its impressive preliminary efficacy in hematological malignancies. For solid tumors, however, the main hindrance is the suppressive tumor microenvironment, which effectively impedes the activation of infiltrating T cells. Herein, we designed a bispecific antibody AP203 with high binding affinity to PD-L1 and CD137 and assessed its safety and anti-tumor efficacy, as well as explored the mechanism of action. METHODS: The optimal antibody binders against PD-L1 and CD137 were screened from the OmniMab phagemid library. The binding affinity of the constructed AP203 were evaluated using enzyme-linked immunosorbent assay (ELISA) and biolayer interferometry (BLI). T-cell stimulatory capacity was assessed using the allogeneic mixed lymphocyte reaction (MLR), antigen-specific recall response, and coculture with PD-L1-expressing cells. In vivo antitumor efficacy was evaluated using two models of tumor-xenografted humanized mice with profiling of tumor infiltrating lymphocytes (TILs). The possible toxicity of AP203 was examined using in vitro cytokine release assay by human PBMCs. RESULTS: AP203, which simultaneously targeted PD-L1 and costimulatory CD137, elicit superior agonistic effects over parental antibodies alone or in combination in terms of T cell activation, enhanced memory recall responses, and overcoming Treg-mediated immunosuppression (P < 0.05). The agonistic activity of AP203 was further demonstrated PD-L1-dependent by coculturing T cells with PD-L1-expressing cells. In vivo animal studies using immunodeficient or immunocompetent mice both showed a dose-related antitumor efficacy superior to parental antibodies in combination (P < 0.05). Correspondingly, AP203 significantly increased tumor infiltrating CD8 + T cells, while decreased CD4 + T cells, as well as Treg cells (P < 0.05), resulting in a dose-dependent increase in the CD8 + /CD4 + ratio. Moreover, either soluble or immobilized AP203 did not induce the production of inflammatory cytokines by human PBMCs. CONCLUSIONS: AP203 exerts potent antitumor activity not only by blocking PD-1/PD-L1 inhibitory signaling, but also by activating CD137 costimulatory signaling in effector T cells that consequently counteracts Treg-mediated immunosuppression. Based on promising preclinical results, AP203 should be a suitable candidate for clinical treatment of solid tumors.

Phase 1 trial of 4-1BB-based adoptive T-cell therapy targeting human telomerase reverse transcriptase in patients with advanced refractory solid tumors.

BACKGROUND AIMS: Human telomerase reverse transcriptase (hTERT) is an attractive target for anti-cancer therapies. We developed an effective method for generating hTERT-specific CD8+ T cells (hTERT-induced natural T cells [TERTiNTs]) using peripheral blood mononuclear cells (PBMCs) from patients with solid cancers and investigated their feasibility and safety. METHODS: This was a single-center phase 1 trial using a 3 + 3 dose escalation design to evaluate six dose levels of TERTiNTs. PBMCs from each patient were screened using an hTERT peptide panel to select those that stimulated CD8+ T cells. The four most stimulatory peptides were used to produce autologous CD8+ T cells from patients refractory or intolerant to standard therapies. Eligible patients received a single intravenous infusion of TERTiNTs at different dose levels (4 × 108 cells/m2, 8 × 108 cells/m2 and 16 × 108 cells/m2). Pre-conditioning chemotherapy, including cyclophosphamide alone or in combination with fludarabine, was administered to induce lymphodepletion. RESULTS: From January 2014 to October 2019, a total of 24 patients with a median of three prior lines of therapy were enrolled. The most common adverse events were lymphopenia (79.2%), nausea (58.3%) and neutropenia (54.2%), mostly caused by pre-conditioning chemotherapy. The TERTiNT infusion was well tolerated, and dose-limiting toxicities were not observed. None of the patients showed objective responses. Seven patients (30.4%) achieved stable disease with a median progression-free survival of 3.9 months (range, 3.2-11.3). At the highest dose level (16 × 108 cells/m2), four of five patients showed disease stabilization. CONCLUSIONS: The generation of TERTiNTs was feasible and safe and provided an interesting disease control rate in heavily pre-treated cancer patients.

An Fc-muted bispecific antibody targeting PD-L1 and 4-1BB induces antitumor immune activity in colorectal cancer without systemic toxicity.

BACKGROUND: Resistance to immune checkpoint inhibitor (ICI) therapy narrows the efficacy of cancer immunotherapy. Although 4-1BB is a promising drug target as a costimulatory molecule of immune cells, no 4-1BB agonist has been given clinical approval because of severe liver toxicity or limited efficacy. Therefore, a safe and efficient immunostimulatory molecule is urgently needed for cancer immunotherapy. METHODS: HK010 was generated by antibody engineering, and the Fab/antigen complex structure was analyzed using crystallography. The affinity and activity of HK010 were detected by multiple in vitro bioassays, including enzyme-linked immunosorbent assay (ELISA), surface plasmon resonance (SPR), flow cytometry, and luciferase-reporter assays. Humanized mice bearing human PD-L1-expressing MC38 (MC38/hPDL1) or CT26 (CT26/hPDL1) tumor transplants were established to assess the in vivo antitumor activity of HK010. The pharmacokinetics (PK) and toxicity of HK010 were evaluated in cynomolgus monkeys. RESULTS: HK010 was generated as an Fc-muted immunoglobulin (Ig)G4 PD-L1x4-1BB bispecific antibody (BsAb) with a distinguished Fab/antigen complex structure, and maintained a high affinity for human PD-L1 (KD: 2.27 nM) and low affinity for human 4-1BB (KD: 493 nM) to achieve potent PD-1/PD-L1 blockade and appropriate 4-1BB agonism. HK010 exhibited synergistic antitumor activity by blocking the PD-1/PD-L1 signaling pathway and stimulating the 4-1BB signaling pathway simultaneously, and being strictly dependent on the PD-L1 receptor in vitro and in vivo. In particular, when the dose was decreased to 0.3 mg/kg, HK010 still showed a strong antitumor effect in a humanized mouse model bearing MC38/hPDL1 tumors. Strikingly, HK010 treatment enhanced antitumor immunity and induced durable antigen-specific immune memory to prevent rechallenged tumor growth by recruiting CD8+ T cells and other lymphocytes into tumor tissue and activating tumor-infiltrating lymphocytes. Moreover, HK010 not only did not induce nonspecific production of proinflammatory cytokines but was also observed to be well tolerated in cynomolgus monkeys in 5 week repeated-dose (5, 15, or 50 mg/kg) and single-dose (75 or 150 mg/kg) toxicity studies. CONCLUSION: We generated an Fc-muted anti-PD-L1x4-1BB BsAb, HK010, with a distinguished structural interaction with PD-L1 and 4-1BB that exhibits a synergistic antitumor effect by blocking the PD-1/PD-L1 signaling pathway and stimulating the 4-1BB signaling pathway simultaneously. It is strictly dependent on the PD-L1 receptor with no systemic toxicity, which may offer a new option for cancer immunotherapy.

Circulating CD137+ T Cell Levels Are Correlated with Response to Pembrolizumab Treatment in Advanced Head and Neck Cancer Patients.

Pembrolizumab, an anti-PD-1 antibody, has been approved as first-line treatment for recurrent or metastatic head and neck squamous cell carcinoma ((R/M) HNSCC). However, only a minority of patients benefit from immunotherapy, which highlights the need to identify novel biomarkers to optimize treatment strategies. CD137+ T cells have been identified as tumour-specific T cells correlated with immunotherapy responses in several solid tumours. In this study, we investigated the role of circulating CD137+ T cells in (R/M) HNSCC patients undergoing pembrolizumab treatment. PBMCs obtained from 40 (R/M) HNSCC patients with a PD-L1 combined positive score (CPS) ≥1 were analysed at baseline via cytofluorimetry for the expression of CD137, and it was found that the percentage of CD3+CD137+ cells is correlated with the clinical benefit rate (CBR), PFS, and OS. The results show that levels of circulating CD137+ T cells are significantly higher in responder patients than in non-responders (p = 0.03). Moreover, patients with CD3+CD137+ percentage ≥1.65% had prolonged OS (p = 0.02) and PFS (p = 0.02). Multivariate analysis, on a combination of biological and clinical parameters, showed that high levels of CD3+CD137+ cells (≥1.65%) and performance status (PS) = 0 are independent prognostic factors of PFS (CD137+ T cells, p = 0.007; PS, p = 0.002) and OS (CD137+ T cells, p = 0.006; PS, p = 0.001). Our results suggest that levels of circulating CD137+ T cells could serve as biomarkers for predicting the response of (R/M) HNSCC patients to pembrolizumab treatment, thus contributing to the success of anti-cancer treatment.

CD137 costimulation is associated with reduced herpetic stromal keratitis and with developing normal CD8+ T cells in trigeminal ganglia.

Costimulatory interactions can be critical in developing immune responses to infectious agents. We recently reported that herpes simplex type 1 (HSV-1) infections of the cornea require a functional CD28-CD80/86 interaction to not only reduce the likelihood of encephalitis, but also to mediate herpetic stromal keratitis (HSK) following viral reactivation. In this same spirit we decided to determine the role that CD137 costimulation plays during HSK. Using both B6-CD137L-/- mice, as well as antagonistic and agonistic antibodies to CD137 we characterize the immune response and to what extent CD137 plays an important role during this disease. Immune responses were measured in both the cornea and in the trigeminal ganglia where the virus forms a latent infection. We demonstrate that CD137 costimulation leads to reduced corneal disease. Interestingly, we observed that lack of CD137 costimulation resulted in significantly reduced CD8+ T expansion and function in the trigeminal ganglia. Finally, we showed that viruses that have been genetically altered to express CD137 display significantly reduced corneal disease, though they did present similar levels of trigeminal infection and peripheral virus production following reactivation of a latent infection. CD137 interactions lead to reduced HSK and are necessary to develop robust trigeminal CD8+ T cell responses.

Epstein-Barr virus-induced ectopic CD137 expression helps nasopharyngeal carcinoma to escape immune surveillance and enables targeting by chimeric antigen receptors.

Non-keratinizing nasopharyngeal carcinoma (NPC) is a malignancy with a poor prognosis for relapsing patients and those with metastatic disease. Here, we identify a novel disease mechanism of NPC which may be its Achilles' heel that makes it susceptible to immunotherapy. CD137 is a potent costimulatory receptor on activated T cells, and CD137 agonists strongly enhance anti-tumor immune responses. A negative feedback mechanism prevents overstimulation by transferring CD137 from T cells to CD137 ligand (CD137L)-expressing antigen presenting cells (APC) during cognate interaction, upon which the CD137-CD137L complex is internalized and degraded. We found ectopic expression of CD137 on 42 of 122 (34.4%) NPC cases, and that CD137 is induced by the Epstein-Barr virus latent membrane protein (LMP) 1. CD137 expression enables NPC to hijack the inbuilt negative feedback mechanism to downregulate the costimulatory CD137L on APC, facilitating its escape from immune surveillance. Further, the ectopically expressed CD137 signals into NPC cells via the p38-MAPK pathway, and induces the expression of IL-6, IL-8 and Laminin γ2. As much as ectopic CD137 expression may support the growth and spread of NPC, it may be a target for its immunotherapeutic elimination. Natural killer cells that express a CD137-specific chimeric antigen receptor induce death in CD137+ NPC cells, in vitro, and in vivo in a murine xenograft model. These data identify a novel immune escape mechanism of NPC, and lay the foundation for an urgently needed immunotherapeutic approach for NPC.

Dendritic cell therapy with CD137L-DC-EBV-VAX in locally recurrent or metastatic nasopharyngeal carcinoma is safe and confers clinical benefit.

INTRODUCTION: Epstein-Barr virus (EBV) is associated with nasopharyngeal carcinoma (NPC), and provides a target for a dendritic cell (DC) vaccine. CD137 ligand (CD137L) expressed on antigen presenting cells, costimulates CD137-expressing T cells, and reverse CD137L signaling differentiates monocytes to CD137L-DC, a type of DC, which is more potent than classical DC in stimulating T cells. METHODS: In this phase I study, patients with locally recurrent or metastatic NPC were administered CD137L-DC pulsed with EBV antigens (CD137L-DC-EBV-VAX). RESULTS: Of the 12 patients treated, 9 received full 7 vaccine doses with a mean administered cell count of 23.9 × 106 per dose. Treatment was well tolerated with only 4 cases of grade 1 related adverse events. A partial response was obtained in 1 patient, and 4 patients are still benefitting from a progression free survival (PFS) of currently 2-3 years. The mean pre-treatment neutrophil: lymphocyte ratio was 3.4 and a value of less than 3 was associated with prolonged median PFS. Progressors were characterized by a high frequency of naïve T cells but a low frequency of CD8+ effector T cells while patients with a clinical benefit (CB) had a high frequency of memory T cells. Patients with CB had lower plasma EBV DNA levels, and a reduction after vaccination. CONCLUSION: CD137L-DC-EBV-VAX was well tolerated. The use of CD137L-DC-EBV-VAX is demonstrated to be safe. Consistent results were obtained from all 12 patients, indicating that CD137L-DC-EBV-VAX induces an anti-EBV and anti-NPC immune response, and warranting further studies in patients post effective chemotherapy. PRECIS: The first clinical testing of CD137L-DC, a new type of monocyte-derived DC, finds that CD137L-DC are safe, and that they can induce an immune response against Epstein-Barr virus-associated nasopharyngeal carcinoma that leads to tumor regression or prevents tumor progression.

CD137L and CD4 T cells limit BCL6-expressing pre-germinal center B cell expansion and BCL6-driven B cell malignancy.

Aberrant expression of the proto-oncogene BCL6 is a driver of tumorigenesis in diffuse large B cell lymphoma (DLBCL). Mice overexpressing BCL6 from the B cell-specific immunoglobulin heavy chain µ intron promoter (Iµ-Bcl6Tg/+ ) develop B cell lymphomas with features typical of human DLBCL. While the development of B cell lymphoma in these mice is tightly controlled by T cells, the mechanisms of this immune surveillance are poorly understood. Here we show that CD4 T cells contribute to the control of lymphoproliferative disease in lymphoma-prone Iµ-Bcl6Tg/+ mice. We reveal that this CD4 T cell immuno-surveillance requires signaling by the co-stimulatory molecule CD137 ligand (CD137L; also known as 4-1BBL), which may promote the transition of pre-malignant B cells with an activated phenotype into the germinal center stage via reverse signaling, preventing their hazardous accumulation. Thus, CD137L-mediated CD4 T cell immuno-surveillance adds another layer of protection against B cell malignancy to that provided by CD8 T cell cytotoxicity.