InVivoMAb anti-mouse 4-1BB (CD137)
Product Details
The 3H3 monoclonal antibody reacts with mouse 4-1BB, a TNF receptor superfamily member also known as CD137. 4-1BB is a 39 kDa transmembrane protein expressed by T lymphocytes, NK cells, dendritic cells, granulocytes, and mast cells. Upon binding its ligand 4-1BBL, 4-1BB provides costimulatory signals to both CD4 and CD8 T cells through the activation of NF-ĪŗB, c-Jun and p38 downstream pathways. The importance of the 4-1BB pathway has been underscored in a number of diseases, including cancer. Agonistic anti-4-1BB antibodies have been reported to induce T cell mediated antitumor immunity. The 3H3 antibody is an agonistic antibody that has been shown to stimulate 4-1BB signaling in vivo.Specifications
| Isotype | Rat IgG2a |
|---|---|
| Recommended Isotype Control(s) | InVivoMAb rat IgG2a isotype control, anti-trinitrophenol |
| Recommended Dilution Buffer | InVivoPure pH 7.0 Dilution Buffer |
| Conjugation | This product is unconjugated. Conjugation is available via our Antibody Conjugation Services. |
| Immunogen | Mouse CD137 human Fc fusion protein |
| Reported Applications |
in vivo 4-1BB stimulation in vitro 4-1BB stimulation |
| Formulation |
PBS, pH 7.0 Contains no stabilizers or preservatives |
| Endotoxin |
<2EU/mg (<0.002EU/Ī¼g) Determined by LAL gel clotting assay |
| Purity |
>95% Determined by SDS-PAGE |
| Sterility | 0.2 Āµm filtration |
| Production | Purified from cell culture supernatant in an animal-free facility |
| Purification | Protein G |
| RRID | AB_2687721 |
| Molecular Weight | 150 kDa |
| Storage | The antibody solution should be stored at the stock concentration at 4Ā°C. Do not freeze. |
Additional Formats
Recommended Products
-
Recommended Isotype Control(s)
InVivoMAb rat IgG2a isotype control, anti-trinitrophenol
-
Recommended Dilution Buffer
InVivoPure pH 7.0 Dilution Buffer
in vivo activation of 4-1BB
Qi, X., et al. (2019). "Optimization of 4-1BB antibody for cancer immunotherapy by balancing agonistic strength with FcgammaR affinity" Nat Commun 10(1): 2141. PubMed
Costimulation of T cell responses with monoclonal antibody agonists (mAb-AG) targeting 4-1BB showed robust anti-tumor activity in preclinical models, but their clinical development was hampered by low efficacy (Utomilumab) or severe liver toxicity (Urelumab). Here we show that isotype and intrinsic agonistic strength co-determine the efficacy and toxicity of anti-4-1BB mAb-AG. While intrinsically strong agonistic anti-4-1BB can activate 4-1BB in the absence of FcgammaRs, weak agonistic antibodies rely on FcgammaRs to activate 4-1BB. All FcgammaRs can crosslink anti-41BB antibodies to strengthen co-stimulation, but activating FcgammaR-induced antibody-dependent cell-mediated cytotoxicity compromises anti-tumor immunity by deleting 4-1BB(+) cells. This suggests balancing agonistic activity with the strength of FcgammaR interaction as a strategy to engineer 4-1BB mAb-AG with optimal therapeutic performance. As a proof of this concept, we have developed LVGN6051, a humanized 4-1BB mAb-AG that shows high anti-tumor efficacy in the absence of liver toxicity in a mouse model of cancer immunotherapy.
in vitro 4-1BB stimulation
Giardino Torchia, M. L., et al. (2015). "c-IAP ubiquitin protein ligase activity is required for 4-1BB signaling and CD8(+) memory T-cell survival" Eur J Immunol 45(9): 2672-2682. PubMed
Cellular inhibitor of apoptosis proteins (c-IAP) 1 and 2 are widely expressed ubiquitin protein ligases that regulate a variety of cellular functions, including the sensitivity of T cells to costimulation. 4-1BB is a TNF receptor family member that signals via a complex that includes TRAF family members and the c-IAPs to upregulate NF-kappaB and ERK, and has been implicated in memory T-cell survival. Here, we show that effector and memory T cells from mice expressing a dominant negative E3-inactive c-IAP2 (c-IAP2(H570A) ) have impaired signaling downstream of 4-1BB. When infected with lymphocytic choriomeningitis virus, unlike mice in which c-IAPs were acutely downregulated by c-IAP antagonists, the primary response of c-IAP2(H570A) mice was normal. However, the number of antigen-specific CD8(+) but not CD4(+) T cells declined more rapidly and to a greater extent in c-IAP2(H570A) mice than in WT controls. Studies with T-cell adoptive transfer demonstrated that the enhanced decay of memory cells was T-cell intrinsic. Thus, c-IAP E3 activity is required for 4-1BB coreceptor signaling and maintenance of CD8(+) T-cell memory.
in vivo 4-1BB stimulation
Guillerey, C., et al. (2015). "Immunosurveillance and therapy of multiple myeloma are CD226 dependent" J Clin Invest 125(5): 2077-2089. PubMed
Multiple myeloma (MM) is an age-dependent hematological malignancy. Evaluation of immune interactions that drive MM relies on in vitro experiments that do not reflect the complex cellular stroma involved in MM pathogenesis. Here we used Vk*MYC transgenic mice, which spontaneously develop MM, and demonstrated that the immune system plays a critical role in the control of MM progression and the response to treatment. We monitored Vk*MYC mice that had been crossed with Cd226 mutant mice over a period of 3 years and found that CD226 limits spontaneous MM development. The CD226-dependent anti-myeloma immune response against transplanted Vk*MYC MM cells was mediated both by NK and CD8+ T cells through perforin and IFN-gamma pathways. Moreover, CD226 expression was required for optimal antimyeloma efficacy of cyclophosphamide (CTX) and bortezomib (Btz), which are both standardly used to manage MM in patients. Activation of costimulatory receptor CD137 with mAb (4-1BB) exerted strong antimyeloma activity, while inhibition of coinhibitory receptors PD-1 and CTLA-4 had no effect. Taken together, the results of this study provide in vivo evidence that CD226 is important for MM immunosurveillance and indicate that specific immune components should be targeted for optimal MM treatment efficacy. As progressive immunosuppression associates with MM development, strategies aimed to increase immune functions may have important therapeutic implications in MM.
in vivo 4-1BB stimulation
Kobayashi, T., et al. (2015). "NKT cell-targeted vaccination plus anti-4-1BB antibody generates persistent CD8 T cell immunity against B cell lymphoma" Oncoimmunology 4(3): e990793. PubMed
Harnessing the immune adjuvant properties of natural killer T (NKT) cells is an effective strategy to generate anticancer immunity. The objective of this study was to increase the potency and durability of vaccine-induced immunity against B cell lymphoma by combining alpha-galactosylceramide (alpha-GalCer)-loaded tumor cell vaccination with an agonistic antibody targeting the immune checkpoint molecule 4-1BB (CD137). We observed potent synergy when combining vaccination and anti-4-1BB antibody treatment resulting in significantly enhanced survival of mice harboring Emu-myc tumors, including complete eradication of lymphoma in over 50% of mice. Tumor-free survival required interferon gamma (IFNgamma)-dependent expansion of CD8+ T cells and was associated with 4-1BB-mediated differentiation of KLRG1+ effector CD8+ T cells. āCuredā mice were also resistant to lymphoma re-challenge 80 days later indicating successful generation of immunological memory. Overall, our results demonstrate that therapeutic anticancer vaccination against B cell lymphoma using an NKT cell ligand can be boosted by subsequent co-stimulation through 4-1BB leading to a sustainable immune response that may enhance outcomes to conventional treatment.
in vivo 4-1BB stimulation
Tewalt, E. F., et al. (2012). "Lymphatic endothelial cells induce tolerance via PD-L1 and lack of costimulation leading to high-level PD-1 expression on CD8 T cells" Blood 120(24): 4772-4782. PubMed
Lymphatic endothelial cells (LECs) induce peripheral tolerance by direct presentation to CD8 T cells (T(CD8)). We demonstrate that LECs mediate deletion only via programmed cell death-1 (PD-1) ligand 1, despite expressing ligands for the CD160, B- and T-lymphocyte attenuator, and lymphocyte activation gene-3 inhibitory pathways. LECs induce activation and proliferation of T(CD8), but lack of costimulation through 4-1BB leads to rapid high-level expression of PD-1, which in turn inhibits up-regulation of the high-affinity IL-2 receptor that is necessary for T(CD8) survival. Rescue of tyrosinase-specific T(CD8) by interference with PD-1 or provision of costimulation results in autoimmune vitiligo, demonstrating that LECs are significant, albeit suboptimal, antigen-presenting cells. Because LECs express numerous peripheral tissue antigens, lack of costimulation coupled to rapid high-level up-regulation of inhibitory receptors may be generally important in systemic peripheral tolerance.
in vivo 4-1BB stimulation
Verbrugge, I., et al. (2012). "Radiotherapy increases the permissiveness of established mammary tumors to rejection by immunomodulatory antibodies" Cancer Res 72(13): 3163-3174. PubMed
It is becoming increasingly evident that radiotherapy may benefit from coincident or subsequent immunotherapy. In this study, we examined whether the antitumor effects of radiotherapy, in established triple-negative breast tumors could be enhanced with combinations of clinically relevant monoclonal antibodies (mAb), designed to stimulate immunity [anti-(alpha)-CD137, alpha-CD40] or relieve immunosuppression [alpha-programmed death (PD)-1]. While the concomitant targeting of the costimulatory molecules CD137 and CD40 enhanced the antitumor effects of radiotherapy and promoted the rejection of subcutaneous BALB/c-derived 4T1.2 tumors, this novel combination was noncurative in mice bearing established C57BL/6-derived AT-3 tumors. We identified PD-1 signaling within the AT-3 tumors as a critical limiting factor to the therapeutic efficacy of alpha-CD137 therapy, alone and in combination with radiotherapy. Strikingly, all mice bearing established orthotopic AT-3 mammary tumors were cured when alpha-CD137 and alpha-PD-1 mAbs were combined with single- or low-dose fractionated radiotherapy. CD8+ T cells were essential for curative responses to this combinatorial regime. Interestingly, CD137 expression on tumor-associated CD8+ T cells was largely restricted to a subset that highly expressed PD-1. These CD137+PD-1High CD8+ T cells, persisted in irradiated AT-3 tumors, expressed Tim-3, granzyme B and Ki67 and produced IFN-gamma ex vivo in response to phorbol 12-myristate 13-acetate (PMA) and ionomycin stimulation. Notably, radiotherapy did not deplete, but enriched tumors of functionally active, tumor-specific effector cells. Collectively, these data show that concomitant targeting of immunostimulatory and inhibitory checkpoints with immunomodulatory mAbs can enhance the curative capacity of radiotherapy in established breast malignancy.
in vivo 4-1BB stimulation
Vezys, V., et al. (2011). "4-1BB signaling synergizes with programmed death ligand 1 blockade to augment CD8 T cell responses during chronic viral infection" J Immunol 187(4): 1634-1642. PubMed
Previous studies have identified the inhibitory role that the programmed death 1 (PD-1) pathway plays during chronic infection. Blockade of this pathway results in rescue of viral-specific CD8 T cells, as well as reduction of viral loads in mice chronically infected with lymphocytic choriomeningitis virus (LCMV). We tested the effect of combining PD ligand 1 (PD-L1) blockade with an agonistic regimen that induces 4-1BB costimulation during chronic LCMV infection. There is a boosting effect in the rescue of LCMV-specific CD8 T cell responses after dual treatment with PD-L1 blockade and 4-1BB agonistic Abs when the amount and timing of 4-1BB costimulation are carefully controlled. When PD-L1-blocking Abs are given together with a single low dose of anti-4-1BB agonistic Abs, there is an enhanced and stable expansion of viral-specific CD8 T cells. Conversely, when blocking Abs to PD-L1 are given with a repetitive high dose of anti-4-1BB, there is an initial synergistic expansion of viral-specific CD8 T cells by day 7, followed by dramatic apoptosis by day 14. Viral control paralleled CD8 T cell kinetics after dual treatment. By day 7 posttreatment, viral titers were lower in both of the combined regimens (compared with PD-L1 blockade alone). However, whereas the high dose of anti-4-1BB plus PD-L1 blockade resulted in rebound of viral titers to original levels, the low dose of anti-4-1BB plus PD-L1 blockade resulted in a stable reduction of viral loads. These findings demonstrate the importance of carefully manipulating the balance between activating and inhibitory signals to enhance T cell responses during chronic infection.
Single-cell and chromatin accessibility profiling reveals regulatory programs of pathogenic Th2 cells in allergic asthma.
In Nat Commun on 15 March 2025 by Khan, M., Alteneder, M., et al.
PubMed
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 generatedĀ 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.
- Cancer Research,
- Immunology and Microbiology
Neoadjuvant anti-4-1BB confers protection against spontaneous metastasis through low-affinity intratumor CD8+T cells in triple-negative breast cancer
In bioRxiv on 2 February 2025 by Lim, B. J. W., Liu, M., et al.
- Flow cytometry/Cell sorting,
- Cancer Research,
- Genetics,
- Immunology and Microbiology
Effective cancer immunotherapy combining mRNA-encoded bispecific antibodies that induce polyclonal T cell engagement and PD-L1-dependent 4-1BB costimulation.
In Front Immunol on 21 January 2025 by Hangiu, O., Navarro, R., et al.
PubMed
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.
- Cancer Research,
- Immunology and Microbiology
Developing an Effective Therapeutic HPV Vaccine to Eradicate Large Tumors by Genetically Fusing Xcl1 and Incorporating IL-9 as Molecular Adjuvants.
In Vaccines (Basel) on 9 January 2025 by Sun, Z., Wu, Z., et al.
PubMed
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.
- Cancer Research
Photothermal therapy co-localized with CD137 agonism improves survival in an SM1 melanoma model without hepatotoxicity.
In Nanomedicine (Lond) on 3 September 2024 by Medina, J. A., Ledezma, D. K., et al.
PubMed
Aim: We investigate combining Prussian Blue nanoparticles (PBNPs), as photothermal therapy (PTT) agents, with agonistic CD137 antibodies (Ī±CD137) on a single nanoparticle platform to deliver non-toxic, anti-tumor efficacy in SM1 murine melanoma.Methods: We electrostatically coated PBNPs with Ī±CD137 (Ī±CD137-PBNPs) and quantified their physicochemical characteristics, photothermal and co-stimulatory capabilities. Next, we tested the efficacy and hepatotoxicity of PTT using Ī±CD137-PBNPs (Ī±CD137-PBNP-PTT) in SM1 tumor-bearing mice.Results: The Ī±CD137-PBNPs retained both the photothermal and agonistic properties of the PBNPs and Ī±CD137, respectively. In vivo, SM1 tumor-bearing mice treated with Ī±CD137-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 Ī±CD137-based agonism as a novel combination nanomedicine.
- In vivo experiments,
- In vivo experiments,
- Mus musculus (Mouse)
Therapeutic potential of co-signaling receptor modulation in hepatitis B.
In Cell on 25 July 2024 by Andreata, F., Laura, C., et al.
PubMed
Reversing CD8+ TĀ 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+ TĀ 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+ TĀ cells. Targeting all dysfunctional TĀ cells, rather than only stem-like precursors, holds promise for treating chronic HBV infection.
- Mus musculus (Mouse),
- Cancer Research,
- Immunology and Microbiology
Short-term cultured tumor fragments to study immunotherapy combinations based on CD137 (4-1BB) agonism.
In Oncoimmunology on 11 July 2024 by Eguren-Santamaria, I., Rodriguez, I., et al.
PubMed
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-Ī². Increases in IFNĪ³ 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 72āh cultures. In mice bearing bilateral tumors in which one was excised prior to in vivo anti-CD137ā+āanti-PD-1 treatment to perform the fragment culture evaluation, no association was found between IFNĪ³ 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 IFNĪ³ 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.
- Mus musculus (Mouse),
- Cancer Research
Preclinical characterization and phase 1 results of ADG106 in patients with advanced solid tumors and non-Hodgkin's lymphoma.
In Cell Rep Med on 20 February 2024 by Ma, Y., Luo, F., et al.
PubMed
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 5.0Ā mg/kg dose level. Dose-limiting toxicity occurs in only one patient (6.3%) at 10.0Ā 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 Ī³ 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).
- Mus musculus (Mouse)
Human Metapneumovirus Reinfection in Aged Mice Recapitulates Increased Disease Severity in Elderly Humans Infected with Human Metapneumovirus.
In Immunohorizons on 1 June 2023 by Parks, O. B., Eddens, T., et al.
PubMed
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.
- Mus musculus (Mouse),
- Immunology and Microbiology
Dendritic cell therapy augments antitumor immunity triggered by CDK4/6 inhibition and immune checkpoint blockade by unleashing systemic CD4 T-cell responses.
In J Immunother Cancer on 1 May 2023 by Kumar, A., Ramani, V., et al.
PubMed
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.
- Genetics,
- Immunology and Microbiology
mRNAs encoding IL-12 and a decoy-resistant variant of IL-18 synergize to engineer T cells for efficacious intratumoral adoptive immunotherapy.
In Cell Rep Med on 17 March 2023 by Olivera, I., BolaƱos, E., et al.
PubMed
Interleukin-12 (IL-12) gene transfer enhances the therapeutic potency of adoptive TĀ cell therapies. We previously reported that transient engineering of tumor-specific CD8 TĀ cells with IL-12 mRNA enhanced their systemic therapeutic efficacy when delivered intratumorally. Here, we mix TĀ 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 TĀ cell mixtures are repeatedly injected into mouse tumors. Pmel-1 TĀ cell receptor (TCR)-transgenic TĀ cells electroporated with scIL-12 or DRIL18 mRNAs exert powerful therapeutic effects in local and distant melanoma lesions. These effects are associated with TĀ 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) TĀ cells on IL-12 and DRIL18 mRNA electroporation.
- Cancer Research,
- Immunology and Microbiology
Tumor targeted 4-1BB agonist antibody-albumin fusions with high affinity to FcRn induce anti-tumor immunity without toxicity.
In iScience on 16 September 2022 by Hangiu, O., Compte, M., et al.
PubMed
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 FcĪ³R 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 inĀ 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 inĀ 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.
- In vivo experiments,
- Mus musculus (Mouse),
- Genetics,
- Immunology and Microbiology
Optimal CD8+ T cell effector function requires costimulation-induced RNA-binding proteins that reprogram the transcript isoform landscape.
In Nat Commun on 20 June 2022 by Karginov, T. A., Menoret, A., et al.
PubMed
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.
- Immunology and Microbiology
Evaluating the glycolytic potential of mouse costimulated effector CD8+ T cells ex vivo.
In STAR Protoc on 17 June 2022 by Agliano, F., Menoret, A., et al.
PubMed
Studying the metabolic fitness of TĀ cells is fundamental to understand how immune responses are regulated. Here, we describe a step-by-step protocol optimized to efficiently generate and isolate effector antigen-specific CD8+ TĀ cells ex vivo using costimulation. We also detail steps to evaluate their metabolic activity using Seahorse technology. This protocol can be used to measure the glycolytic potential of effector murine TĀ cells in response to different manipulations, such as infections, adjuvant studies, gene editing, or metabolite supplementation. For complete details on the use and execution of this protocol, please refer to Agliano etĀ al. (2022).
- Immunology and Microbiology,
- Mus musculus (Mouse)
Nicotinamide breaks effector CD8 T cell responses by targeting mTOR signaling.
In iScience on 18 March 2022 by Agliano, F., Karginov, T. A., et al.
PubMed
Nicotinamide (NAM) shapes TĀ cell responses but its precise molecular mechanism of action remains elusive. Here, we show that NAM impairs naive TĀ cell effector transition but also effector TĀ cells themselves. Although aerobic glycolysis is a hallmark of activated TĀ cells, CD8+ TĀ cells exposed to NAM displayed enhanced glycolysis, yet producing significantly less IFNĪ³. Mechanistically, NAM reduced mTORC1 activity independently of NAD+ metabolism, decreasing IFNĪ³ translation and regulating TĀ cell transcriptional factors critical to effector/memory fate. Finally, the role of NAM in a biomedically relevant model of lung injury was tested. Specifically, a NAM-supplemented diet reduced systemic IL-2, antigen-specific TĀ cell clonal expansion, and effector function after inhalation of Staphylococcus aureus enterotoxin A. These findings identify NAM as a potential therapeutic supplement that uncouples glycolysis from effector cytokine production and may be a powerful treatment for diseases associated with TĀ cell hyperactivation.
- Cancer Research,
- Enzyme-linked immunosorbent assay,
- Mus musculus (Mouse)
The immunotoxicity, but not anti-tumor efficacy, of anti-CD40 and anti-CD137 immunotherapies is dependent on the gut microbiota.
In Cell Rep Med on 21 December 2021 by Blake, S. J., James, J., et al.
PubMed
Immune agonist antibodies (IAAs) are promising immunotherapies that target co-stimulatory receptors to induce potent anti-tumor immune responses, particularly when combined with checkpoint inhibitors. Unfortunately, their clinical translation is hampered by serious dose-limiting, immune-mediated toxicities, including high-grade and sometimes fatal liver damage, cytokine release syndrome (CRS), and colitis. We show that the immunotoxicity, induced by the IAAs anti-CD40 and anti-CD137, is dependent on the gut microbiota. Germ-free or antibiotic-treated mice have significantly reduced colitis, CRS, and liver damage following IAA treatment compared with conventional mice or germ-free mice recolonized via fecal microbiota transplant. MyD88 signaling is required for IAA-induced CRS and for anti-CD137-induced, but not anti-CD40-induced, liver damage. Importantly, antibiotic treatment does not impair IAA anti-tumor efficacy, alone or in combination with anti-PD1. Our results suggest that microbiota-targeted therapies could overcome the toxicity induced by IAAs without impairing their anti-tumor activity.
- Immunology and Microbiology
CD137 (4-1BB) costimulation of CD8+ T cells is more potent when provided in cis than in trans with respect to CD3-TCR stimulation.
In Nat Commun on 15 December 2021 by Otano, I., Azpilikueta, A., et al.
PubMed
CD137 (4-1BB; TNFSR9) is an activation-induced surface receptor that through costimulation effects provide antigen-primed T cells with augmented survival, proliferation and effector functions as well as metabolic advantages. These immunobiological mechanisms are being utilised for cancer immunotherapy with agonist CD137-binding and crosslinking-inducing agents that elicit CD137 intracellular signaling. In this study, side-by-side comparisons show that provision of CD137 costimulation in-cis with regard to the TCR-CD3-ligating cell is superior to that provided in-trans in terms of T cell activation, proliferation, survival, cytokine secretion and mitochondrial fitness in mouse and human. Cis ligation of CD137 relative to the TCR-CD3 complex results in more intense canonical and non-canonical NF-ĪŗB signaling and provides a more robust induction of cell cycle and DNA damage repair gene expression programs. Here we report that the superiority of cis versus trans CD137-costimulation is readily observed in vivo and is relevant for understanding the immunotherapeutic effects of CAR T cells and CD137 agonistic therapies currently undergoing clinical trials, which may provide costimulation either in cis or in trans.
- Cancer Research,
- Immunology and Microbiology,
- Flow cytometry/Cell sorting
ACKR4 in Tumor Cells Regulates Dendritic Cell Migration to Tumor-Draining Lymph Nodes and T-Cell Priming.
In Cancers (Basel) on 7 October 2021 by Wangmo, D., Premsrirut, P. K., et al.
PubMed
Colorectal cancer (CRC) is one of the most common malignancies in both morbidity and mortality. Immune checkpoint blockade (ICB) treatments have been successful in a portion of mismatch repair-deficient (dMMR) CRC patients but have failed in mismatch repair-proficient (pMMR) CRC patients. Atypical Chemokine Receptor 4 (ACKR4) is implicated in regulating dendritic cell (DC) migration. However, the roles of ACKR4 in CRC development and anti-tumor immunoregulation are not known. By analyzing human CRC tissues, transgenic animals, and genetically modified CRC cells lines, our study revealed an important function of ACKR4 in maintaining CRC immune response. Loss of ACKR4 in CRC is associated with poor immune infiltration in the tumor microenvironment. More importantly, loss of ACKR4 in CRC tumor cells, rather than stromal cells, restrains the DC migration and antigen presentation to the tumor-draining lymph nodes (TdLNs). Moreover, tumors with ACKR4 knockdown become less sensitive to immune checkpoint blockade. Finally, we identified that microRNA miR-552 negatively regulates ACKR4 expression in human CRC. Taken together, our studies identified a novel and crucial mechanism for the maintenance of the DC-mediated T-cell priming in the TdLNs. These new findings demonstrate a novel mechanism leading to immunosuppression and ICB treatment resistance in CRC.
- Immunology and Microbiology,
- Mus musculus (Mouse)
Context-Dependent Immunomodulatory Effects of MEK Inhibition Are Enhanced with T-cell Agonist Therapy.
In Cancer Immunol Res on 1 October 2021 by Dennison, L., Ruggieri, A., et al.
PubMed
MEK inhibition (MEKi) is proposed to enhance antitumor immunity but has demonstrated mixed results as an immunomodulatory strategy in human clinical trials. MEKi exerts direct immunomodulatory effects on tumor cells and tumor-infiltrating lymphocytes (TIL), but these effects have not been independently investigated. Here we modeled tumor-specific MEKi through CRISPR/Cas-mediated genome editing of tumor cells [MEK1 knockout (KO)] and pharmacologic MEKi with cobimetinib in a RAS-driven model of colorectal cancer. This approach allowed us to distinguish tumor-mediated and tumor-independent mechanisms of MEKi immunomodulation. MEK1 KO tumors demonstrated upregulation of JAK/STAT signaling, enhanced MHCI expression, CD8+ T-cell infiltration and T-cell activation, and impaired tumor growth that is immune dependent. Pharmacologic MEKi recapitulated tumor-intrinsic effects but simultaneously impaired T-cell activation in the tumor microenvironment. We confirmed a reduction in human peripheral-lymphocyte activation from a clinical trial of anti-PD-L1 (atezolizumab) with or without cobimetinib in biliary tract cancers. Impaired activation of TILs treated with pharmacologic MEKi was reversible and was rescued with the addition of a 4-1BB agonist. Collectively, these data underscore the ability of MEKi to induce context-dependent immunomodulatory effects and suggest that T cell-agonist therapy maximizes the beneficial effects of MEKi on the antitumor immune response.
- Binding experiments,
- Mus musculus (Mouse),
- Cancer Research,
- Immunology and Microbiology
Switchable immune modulator for tumor-specific activation of anticancer immunity.
In Sci Adv on 10 September 2021 by Zhao, Y., Xie, Y. Q., et al.
PubMed
Immune stimulatory antibodies and cytokines elicit potent antitumor immunity. However, the dose-limiting systemic toxicity greatly hinders their clinical applications. Here, we demonstrate a chemical approach, termed āswitchableā immune modulator (Sw-IM), to limit the systemic exposure and therefore ameliorate their toxicities. Sw-IM is a biomacromolecular therapeutic reversibly masked by biocompatible polymers through chemical linkers that are responsive to tumor-specific stimuli, such as high reducing potential and acidic pH. Sw-IMs stay inert (switch off) in the circulation and healthy tissues but get reactivated (switch on) selectively in tumor via responsive removal of the polymer masks, thus focusing the immune boosting activities in the tumor microenvironment. Sw-IMs applied to antiā4-1BB agonistic antibody and IL-15 cytokine led to equivalent antitumor efficacy to the parental IMs with markedly reduced toxicities. Sw-IM provides a highly modular and generic approach to improve the therapeutic window and clinical applicability of potent IMs in mono- and combinational immunotherapies.
