InVivoMAb anti-mouse 4-1BB (CD137)

Early pregnancy loss (EPL) is closely associated with imbalances in the maternal-foetal immune microenvironment. Here we identify CD11c + CD8 + T cells, an unconventional cytotoxic T cell subset, as significantly enriched and activated in EPL cases. These cells contribute to immune dysregulation and inhibit trophoblast invasion through secreting granzyme B, perforin, CD107a, TNF-α, and IFN-γ. Furthermore, we present an effective early prediction model for EPL, based on cytokine and cytotoxic molecule profiles of CD11c + CD8 + T cells in maternal serum, collected 12-16 days post-embryo transfer. Functional assays reveal that IFN-γ triggers trophoblast pyroptosis via the NLRP3/Caspase-1/GSDMD pathway, impairing trophoblast invasion. In vivo validation using abortion-prone mice and an anti-4-1BB antibody-induced model of CD11c + CD8 + T cell activation confirms increased embryo resorption and reduced trophoblast infiltration. These findings highlight the role of dysregulated CD11c + CD8 + T cells at the maternal-foetal interface in EPL, and suggest their potential as biomarkers and therapeutic targets for EPL-management.

T-dependent germinal center (GC) output, comprising plasma cells and memory B cells (MBCs), is crucial for clearance of Plasmodium infection and protection against reinfection. In this study, we examine the effect of an agonistic antibody targeting 4-1BB (CD137) during experimental malaria. We show that exogenous 4-1BB stimulation, despite delaying the effector GC response, surprisingly enhances humoral memory recall and protection from reinfection. Single-cell RNA and assay for transposase-accessible chromatin (ATAC) sequencing of MBCs from mice receiving 4-1BB stimulation reveal populations with distinct transcriptional signatures and a chromatin landscape indicative of superior recall and proliferative potential. Importantly, our results indicate that the effects of 4-1BB stimulation are dependent on interleukin (IL)-9R signaling in B cells but independent of parasite load during primary infection. Our study proposes an immunomodulatory approach to enhance the quality of the MBC pool, providing superior protection during infection and vaccination, particularly in the context of malaria.

Lung pathogenic T helper type 2 (pTh2) cells are important in mediating allergic asthma, but fundamental questions remain regarding their heterogeneity and epigenetic regulation. Here we investigate immune regulation in allergic asthma by single-cell RNA sequencing in mice challenged with house dust mite, in the presence and absence of histone deacetylase 1 (HDAC1) function. Our analyses indicate two distinct highly proinflammatory subsets of lung pTh2 cells and pinpoint thymic stromal lymphopoietin (TSLP) and Tumour Necrosis Factor Receptor Superfamily (TNFRSF) members as important drivers to generate pTh2 cells in vitro. Using our in vitro model, we uncover how signalling via TSLP and a TNFRSF member shapes chromatin accessibility at the type 2 cytokine gene loci by modulating HDAC1 repressive function. In summary, we have generated insights into pTh2 cell biology and establish an in vitro model for investigating pTh2 cells that proves useful for discovering molecular mechanisms involved in pTh2-mediated allergic asthma.

Immune checkpoint inhibitors have revolutionized cancer therapy, but many patients fail to respond or develop resistance, often due to reduced T cell activity. Costimulation via 4-1BB has emerged as a promising approach to enhance the effector function of antigen-primed T cells. Bispecific T cell-engaging (TCE) antibodies are an effective way to provide tumor-specific T cell receptor-mediated signaling to tumor-infiltrating lymphocytes. mRNA-based delivery of bispecific antibodies, offer a novel approach to enhance tumor-specific immune responses while minimizing adverse effects.

Human papillomavirus (HPV) is a prevalent infection affecting both men and women, leading to various cytological lesions. Therapeutic vaccines mount a HPV-specific CD8+ cytotoxic T lymphocyte response, thus clearing HPV-infected cells. However, no therapeutic vaccines targeting HPV are currently approved for clinical treatment due to limited efficacy. Our goal is to develop a vaccine that can effectively eliminate tumors caused by HPV.

Aim: We investigate combining Prussian Blue nanoparticles (PBNPs), as photothermal therapy (PTT) agents, with agonistic CD137 antibodies (αCD137) on a single nanoparticle platform to deliver non-toxic, anti-tumor efficacy in SM1 murine melanoma.Methods: We electrostatically coated PBNPs with αCD137 (αCD137-PBNPs) and quantified their physicochemical characteristics, photothermal and co-stimulatory capabilities. Next, we tested the efficacy and hepatotoxicity of PTT using αCD137-PBNPs (αCD137-PBNP-PTT) in SM1 tumor-bearing mice.Results: The αCD137-PBNPs retained both the photothermal and agonistic properties of the PBNPs and αCD137, respectively. In vivo, SM1 tumor-bearing mice treated with αCD137-PBNP-PTT exhibited a significantly higher survival rate (50%) without hepatotoxicity, compared with control treatments.Conclusion: These data suggest the potential utility of co-localizing PBNP-PTT with αCD137-based agonism as a novel combination nanomedicine.

Agonistic antibodies against CD137 have been demonstrated to completely regress established tumors through activating T cell immunity. Unfortunately, current CD137 antibodies failed to benefit patients with cancer. Moreover, their antitumor mechanisms in vivo remain to be determined. Here, we report the development of a small molecular CD137 agonist, JNU-0921. JNU-0921 effectively activates both human and mouse CD137 through direct binding their extracellular domains to induce oligomerization and signaling and effectively shrinks tumors in vivo. Mechanistically, JNU-0921 enhances effector and memory function of cytotoxic CD8+ T cells (CTLs) and alleviates their exhaustion. JNU-0921 also skews polarization of helper T cells toward T helper 1 type and enhances their activity to boost CTL function. Meanwhile, JNU-0921 attenuates the inhibitory function of regulatory T cells on CTLs. Our current work shows that JNU-0921 shrinks tumors by enhancing the cytotoxicity of CTLs in cis and in trans and sheds light on strategy for developing CD137 small molecular agonists.

Reversing CD8+ T cell dysfunction is crucial in treating chronic hepatitis B virus (HBV) infection, yet specific molecular targets remain unclear. Our study analyzed co-signaling receptors during hepatocellular priming and traced the trajectory and fate of dysfunctional HBV-specific CD8+ T cells. Early on, these cells upregulate PD-1, CTLA-4, LAG-3, OX40, 4-1BB, and ICOS. While blocking co-inhibitory receptors had minimal effect, activating 4-1BB and OX40 converted them into antiviral effectors. Prolonged stimulation led to a self-renewing, long-lived, heterogeneous population with a unique transcriptional profile. This includes dysfunctional progenitor/stem-like (TSL) cells and two distinct dysfunctional tissue-resident memory (TRM) populations. While 4-1BB expression is ubiquitously maintained, OX40 expression is limited to TSL. In chronic settings, only 4-1BB stimulation conferred antiviral activity. In HBeAg+ chronic patients, 4-1BB activation showed the highest potential to rejuvenate dysfunctional CD8+ T cells. Targeting all dysfunctional T cells, rather than only stem-like precursors, holds promise for treating chronic HBV infection.

Biomarkers for cancer immunotherapy are an unmet medical need. The group of Daniela Thommen at the NKI recently reported on novel methodologies based on short-term cultures of patient-derived tumor fragments whose cytokine concentrations in the supernatants and activation markers on infiltrating T cells were associated with clinical response to PD-1 blockade. We set up a similar culture technology with tumor-derived fragments using mouse tumors transplanted into syngeneic immunocompetent mice to test an agonist anti-CD137 mAb and its combinations with anti-PD-1 and/or anti-TGF-β. Increases in IFNγ concentrations in the tissue culture supernatants were detected upon in-culture activation with the anti-CD137 and anti-PD-1 mAb combinations or concanavalin A as a positive control. No other cytokine from a wide array was informative of stimulation with these mAbs. Interestingly, increases in Ki67 and other activation markers were substantiated in lymphocytes from cell suspensions gathered at the end of 72 h cultures. In mice bearing bilateral tumors in which one was excised prior to in vivo anti-CD137 + anti-PD-1 treatment to perform the fragment culture evaluation, no association was found between IFNγ production from the fragments and the in vivo therapeutic outcome in the non-resected contralateral tumors. The experimental system permitted freezing and thawing of the fragments with similar functional outcomes. Using a series of patient-derived tumor fragments from excised solid malignancies, we showed IFNγ production in a fraction of the studied cases, that was conserved in frozen/thawed fragments. The small tumor fragment culture technique seems suitable to preclinically explore immunotherapy combinations.

Immunosuppressive macrophages restrict anti-cancer immunity in glioblastoma (GBM). Here, we studied the contribution of microglia (MGs) and monocyte-derived macrophages (MDMs) to immunosuppression and mechanisms underlying their regulatory function. MDMs outnumbered MGs at late tumor stages and suppressed T cell activity. Molecular and functional analysis identified a population of glycolytic MDM expressing GLUT1 with potent immunosuppressive activity. GBM-derived factors promoted high glycolysis, lactate, and interleukin-10 (IL-10) production in MDMs. Inhibition of glycolysis or lactate production in MDMs impaired IL-10 expression and T cell suppression. Mechanistically, intracellular lactate-driven histone lactylation promoted IL-10 expression, which was required to suppress T cell activity. GLUT1 expression on MDMs was induced downstream of tumor-derived factors that activated the PERK-ATF4 axis. PERK deletion in MDM abrogated histone lactylation, led to the accumulation of intratumoral T cells and tumor growth delay, and, in combination with immunotherapy, blocked GBM progression. Thus, PERK-driven glucose metabolism promotes MDM immunosuppressive activity via histone lactylation.

ADG106, a ligand-blocking agonistic antibody targeting CD137 (4-1BB), exhibits promising results in preclinical studies, demonstrating tumor suppression in various animal models and showing a balanced profile between safety and efficacy. This phase 1 study enrolls 62 patients with advanced malignancies, revealing favorable tolerability up to the 5.0 mg/kg dose level. Dose-limiting toxicity occurs in only one patient (6.3%) at 10.0 mg/kg, resulting in grade 4 neutropenia. The most frequent treatment-related adverse events include leukopenia (22.6%), neutropenia (22.6%), elevated alanine aminotransferase (22.6%), rash (21.0%), itching (17.7%), and elevated aspartate aminotransferase (17.7%). The overall disease control rates are 47.1% for advanced solid tumors and 54.5% for non-Hodgkin's lymphoma. Circulating biomarkers suggest target engagement by ADG106 and immune modulation of circulating T, B, and natural killer cells and cytokines interferon γ and interleukin-6, which may affect the probability of clinical efficacy. ADG106 has a manageable safety profile and preliminary anti-tumor efficacy in patients with advanced cancers (this study was registered at ClinicalTrials.gov: NCT03802955).

Human metapneumovirus (HMPV) is a leading cause of respiratory infection in adults >65 y. Nearly all children worldwide are seropositive for HMPV by age 5 y, but reinfections occur throughout life, and there is no licensed vaccine. Recurrent HMPV infection is mild and self-resolving in immunocompetent individuals. However, elderly individuals develop severe respiratory disease on HMPV reinfection that leads to a high risk for morbidity and mortality. In this study, we developed a mouse model to mirror HMPV reinfection in elderly humans. C57BL/6J mice were infected with HMPV at 6-7 wk old, aged in-house, and rechallenged with high-dose virus at 70 wk. Aged rechallenged mice had profound weight loss similar to primary infected mice, increased lung histopathology, and accumulated cytotoxic CD8+CD44+CD62L-CD69+CD103+ memory cells despite having undetectable lung virus titer. When aged mice 14 mo postinfection (p.i.) or young mice 5 wk p.i. were restimulated with HMPV cognate Ag to mimic epitope vaccination, aged mice had an impaired CD8+ memory response. Convalescent serum transfer from young naive or 5 wk p.i. mice into aged mice on day of infection did not protect. Aged mice vaccinated with UV-inactivated HMPV also exhibited diminished protection and poor CD8+ memory response compared with young mice. These results suggest aged individuals with HMPV reinfection have a dysregulated CD8+ memory T cell response that fails to protect and exacerbates disease. Moreover, aged mice exhibited a poor memory response to either epitope peptide or UV-inactivated vaccination, suggesting that aged CD8+ T cell dysfunction presents a barrier to effective vaccination strategies.

Cyclin-dependent kinase 4/6 inhibitors (CDK4/6i) combined with endocrine therapy are a mainstay treatment for hormone receptor-positive breast cancer. While their principal mechanism is inhibition of cancer cell proliferation, preclinical and clinical evidence suggests that CDK4/6i can also promote antitumor T-cell responses. However, this pro-immunogenic property is yet to be successfully harnessed in the clinic, as combining CDK4/6i with immune checkpoint blockade (ICB) has not shown a definitive benefit in patients.

Immune checkpoint-inhibitor combinations are the best therapeutic option for advanced hepatocellular carcinoma (HCC) patients, but improvements in efficacy are needed to improve response rates. We develop a multifocal HCC model to test immunotherapies by introducing c-myc using hydrodynamic gene transfer along with CRISPR-Cas9-mediated disruption of p53 in mouse hepatocytes. Additionally, induced co-expression of luciferase, EGFP, and the melanosomal antigen gp100 facilitates studies on the underlying immunological mechanisms. We show that treatment of the mice with a combination of anti-CTLA-4 + anti-PD1 mAbs results in partial clearance of the tumor with an improvement in survival. However, the addition of either recombinant IL-2 or an anti-CD137 mAb markedly improves both outcomes in these mice. Combining tumor-specific adoptive T cell therapy to the aCTLA-4/aPD1/rIL2 or aCTLA-4/aPD1/aCD137 regimens enhances efficacy in a synergistic manner. As shown by multiplex tissue immunofluorescence and intravital microscopy, combined immunotherapy treatments enhance T cell infiltration and the intratumoral performance of T lymphocytes.

Immune checkpoint-inhibitor combinations are the best therapeutic option for advanced hepatocellular carcinoma (HCC) patients, but improvements in efficacy are needed to improve response rates. We develop a multifocal HCC model to test immunotherapies by introducing c-myc using hydrodynamic gene transfer along with CRISPR-Cas9-mediated disruption of p53 in mouse hepatocytes. Additionally, induced co-expression of luciferase, EGFP, and the melanosomal antigen gp100 facilitates studies on the underlying immunological mechanisms. We show that treatment of the mice with a combination of anti-CTLA-4 + anti-PD1 mAbs results in partial clearance of the tumor with an improvement in survival. However, the addition of either recombinant IL-2 or an anti-CD137 mAb markedly improves both outcomes in these mice. Combining tumor-specific adoptive T cell therapy to the aCTLA-4/aPD1/rIL2 or aCTLA-4/aPD1/aCD137 regimens enhances efficacy in a synergistic manner. As shown by multiplex tissue immunofluorescence and intravital microscopy, combined immunotherapy treatments enhance T cell infiltration and the intratumoral performance of T lymphocytes.

Immune checkpoint-inhibitor combinations are the best therapeutic option for advanced hepatocellular carcinoma (HCC) patients, but improvements in efficacy are needed to improve response rates. We develop a multifocal HCC model to test immunotherapies by introducing c-myc using hydrodynamic gene transfer along with CRISPR-Cas9-mediated disruption of p53 in mouse hepatocytes. Additionally, induced co-expression of luciferase, EGFP, and the melanosomal antigen gp100 facilitates studies on the underlying immunological mechanisms. We show that treatment of the mice with a combination of anti-CTLA-4 + anti-PD1 mAbs results in partial clearance of the tumor with an improvement in survival. However, the addition of either recombinant IL-2 or an anti-CD137 mAb markedly improves both outcomes in these mice. Combining tumor-specific adoptive T cell therapy to the aCTLA-4/aPD1/rIL2 or aCTLA-4/aPD1/aCD137 regimens enhances efficacy in a synergistic manner. As shown by multiplex tissue immunofluorescence and intravital microscopy, combined immunotherapy treatments enhance T cell infiltration and the intratumoral performance of T lymphocytes.

Interleukin-12 (IL-12) gene transfer enhances the therapeutic potency of adoptive T cell therapies. We previously reported that transient engineering of tumor-specific CD8 T cells with IL-12 mRNA enhanced their systemic therapeutic efficacy when delivered intratumorally. Here, we mix T cells engineered with mRNAs to express either single-chain IL-12 (scIL-12) or an IL-18 decoy-resistant variant (DRIL18) that is not functionally hampered by IL-18 binding protein (IL-18BP). These mRNA-engineered T cell mixtures are repeatedly injected into mouse tumors. Pmel-1 T cell receptor (TCR)-transgenic T cells electroporated with scIL-12 or DRIL18 mRNAs exert powerful therapeutic effects in local and distant melanoma lesions. These effects are associated with T cell metabolic fitness, enhanced miR-155 control on immunosuppressive target genes, enhanced expression of various cytokines, and changes in the glycosylation profile of surface proteins, enabling adhesiveness to E-selectin. Efficacy of this intratumoral immunotherapeutic strategy is recapitulated in cultures of tumor-infiltrating lymphocytes (TILs) and chimeric antigen receptor (CAR) T cells on IL-12 and DRIL18 mRNA electroporation.

Costimulation of tumor-infiltrating T lymphocytes by anti-4-1BB monoclonal antibodies (mAbs) has shown anti-tumor activity in human trials, but can be associated with significant off-tumor toxicities involving FcγR interactions. Here, we introduce albumin-fused mouse and human bispecific antibodies with clinically favorable pharmacokinetics designed to confine 4-1BB costimulation to the tumor microenvironment. These Fc-free 4-1BB agonists consist of an EGFR-specific VHH antibody, a 4-1BB-specific scFv, and a human albumin sequence engineered for high FcRn binding connected in tandem (LiTCo-Albu). We demonstrate in vitro cognate target engagement, EGFR-specific costimulatory activity, and FcRn-driven cellular recycling similar to non-fused FcRn high-binding albumin. The mouse LiTCo-Albu exhibited a prolonged circulatory half-life and in vivo tumor inhibition, with no indication of 4-1BB mAb-associated toxicity. Furthermore, we show a greater therapeutic effect when used in combination with PD-1-blocking mAbs. These findings demonstrate the feasibility of tumor-specific LiTCo-Albu antibodies for safe and effective costimulatory strategies in cancer immunotherapy.

Boosting T cell activation through costimulation directs defense against cancer and viral infections. Despite multiple studies targeting costimulation in clinical trials, the increased potency and reprogramming of T cells endowed by costimulation is poorly understood. Canonical dogma states that transcription mediates T cell activation. Here, we show that the spliceosome, controlling post-transcriptional alternative splicing and alternative polyadenylation, is the most enriched pathway in T cells after CD134/CD137 costimulation. Costimulation of CD8+ T cells significantly increases expression of 29 RNA-binding proteins while RNA-seq uncovers over 1000 differential alternative splicing and polyadenylation events. Using in vivo mouse and in vitro human models, we demonstrate that RNA-binding protein Tardbp is required for effector cytokine production, CD8+ T cell clonal expansion, and isoform regulation after costimulation. The prospect of immune response optimization through reprogramming of mRNA isoform production offered herein opens new avenues for experimentally and therapeutically tuning the activities of T cells.