Therapeutic potential of co-signaling receptor modulation in hepatitis B.

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.

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.

Chimeric Antigen Receptor Designed to Prevent Ubiquitination and Downregulation Showed Durable Antitumor Efficacy.

Clinical evidence suggests that poor persistence of chimeric antigen receptor-T cells (CAR-T) in patients limits therapeutic efficacy. Here, we designed a CAR with recyclable capability to promote in vivo persistence and to sustain antitumor activity. We showed that the engagement of tumor antigens induced rapid ubiquitination of CARs, causing CAR downmodulation followed by lysosomal degradation. Blocking CAR ubiquitination by mutating all lysines in the CAR cytoplasmic domain (CARKR) markedly repressed CAR downmodulation by inhibiting lysosomal degradation while enhancing recycling of internalized CARs back to the cell surface. Upon encountering tumor antigens, CARKR-T cells ameliorated the loss of surface CARs, which promoted their long-term killing capacity. Moreover, CARKR-T cells containing 4-1BB signaling domains displayed elevated endosomal 4-1BB signaling that enhanced oxidative phosphorylation and promoted memory T cell differentiation, leading to superior persistence in vivo. Collectively, our study provides a straightforward strategy to optimize CAR-T antitumor efficacy by redirecting CAR trafficking.

Antibodies to Costimulatory Receptor 4-1BB Enhance Anti-tumor Immunity via T Regulatory Cell Depletion and Promotion of CD8 T Cell Effector Function.

The costimulatory receptor 4-1BB is expressed on activated immune cells, including activated T cells. Antibodies targeting 4-1BB enhance the proliferation and survival of antigen-stimulated T cells in vitro and promote CD8 T cell-dependent anti-tumor immunity in pre-clinical cancer models. We found that T regulatory (Treg) cells infiltrating human or murine tumors expressed high amounts of 4-1BB. Intra-tumoral Treg cells were preferentially depleted by anti-4-1BB mAbs in vivo. Anti-4-1BB mAbs also promoted effector T cell agonism to promote tumor rejection. These distinct mechanisms were competitive and dependent on antibody isotype and FcγR availability. Administration of anti-4-1BB IgG2a, which preferentially depletes Treg cells, followed by either agonistic anti-4-1BB IgG1 or anti-PD-1 mAb augmented anti-tumor responses in multiple solid tumor models. An antibody engineered to optimize both FcγR-dependent Treg cell depleting capacity and FcγR-independent agonism delivered enhanced anti-tumor therapy. These insights into the effector mechanisms of anti-4-1BB mAbs lay the groundwork for translation into the clinic.

CAR T cells and T cells phenotype and function are impacted by glucocorticoid exposure with different magnitude.

BACKGROUND: Chimeric antigen receptor (CAR) T cell therapy is associated with high risk of adverse events. Glucocorticoids (GCs) are cornerstone in the management of high-grade cytokine release syndrome (CRS) and immune effector cell-associated neurotoxicity syndrome (ICANS). Given the potentially deleterious effects of GCs on CAR T cells anti-tumor activity, increasing our understanding of GCs impact on CAR T cells is crucial. METHODS: Using several CAR T cells i.e., CD19, mesothelin (MSLN)-CD28 and MSLN-41BB CAR T cells (M28z and MBBz), we compared phenotypical, functional, changes and anti-tumor activity between i) transduced CD19 CAR T cells with untransduced T cells, ii) M28z with MBBz CAR T cells induced by Dexamethasone (Dx) or Methylprednisolone (MP) exposures. RESULTS: Higher levels of GC receptor were found in less differentiated CAR T cells. Overall, Dx and MP showed a similar impact on CAR T cells. Compared to untreated condition, GCs exposure increased the expression of PD-1 and TIM-3 and reduced the expression of LAG3 and function of T cells and CAR T cells. GC exposures induced more exhausted (LAG3 + PD1 + TIM3 +) and dysfunctional (CD107a-INFγ-TNF-IL2-) untransduced T cells in comparison to CD19 CAR T cells. GC exposure impaired more CD4 + than CD8 + CD19 CAR T cells. GC exposures increased more PD-1 expression associated with reduced proliferative capacity and function of M28z as compared to MBBz CAR T cells. CAR T cells anti-tumor activity was greatly affected by repeated GC exposure but partly recovered within 48h after GCs withdrawal. CONCLUSIONS: In summary, GCs impacted phenotype and function of untransduced and CAR T cell with different magnitude. The nature of the CAR costimulatory domain influenced the magnitude of CAR T cell response to GCs.

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.

Radiolabelling and preclinical characterisation of [89Zr]Zr-Df-ATG-101 bispecific to PD-L1/4-1BB.

PURPOSE: ATG-101, a bispecific antibody that simultaneously targets the immune checkpoint PD-L1 and the costimulatory receptor 4-1BB, activates exhausted T cells upon PD-L1 crosslinking. Previous studies demonstrated promising anti-tumour efficacy of ATG-101 in preclinical models. Here, we labelled ATG-101 with 89Zr to confirm its tumour targeting effect and tissue biodistribution in a preclinical model. We also evaluated the use of immuno-PET to study tumour uptake of ATG-101 in vivo. METHODS: ATG-101, anti-PD-L1, and an isotype control were conjugated with p-SCN-Deferoxamine (Df). The Df-conjugated antibodies were radiolabelled with 89Zr, and their radiochemical purity, immunoreactivity, and serum stability were assessed. We conducted PET/MRI and biodistribution studies on [89Zr]Zr-Df-ATG-101 in BALB/c nude mice bearing PD-L1-expressing MDA-MB-231 breast cancer xenografts for up to 10 days after intravenous administration of [89Zr]Zr-labelled antibodies. The specificity of [89Zr]Zr-Df-ATG-101 was evaluated through a competition study with unlabelled ATG-101 and anti-PD-L1 antibodies. RESULTS: The Df-conjugation and [89Zr]Zr -radiolabelling did not affect the target binding of ATG-101. Biodistribution and imaging studies demonstrated biological similarity of [89Zr]Zr-Df-ATG-101 and [89Zr]Zr-Df-anti-PD-L1. Tumour uptake of [89Zr]Zr-Df-ATG-101 was clearly visualised using small-animal PET imaging up to 7 days post-injection. Competition studies confirmed the specificity of PD-L1 targeting in vivo. CONCLUSION: [89Zr]Zr-Df-ATG-101 in vivo distribution is dependent on PD-L1 expression in the MDA-MB-231 xenograft model. Immuno-PET with [89Zr]Zr-Df-ATG-101 provides real-time information about ATG-101 distribution and tumour uptake in vivo. Our data support the use of [89Zr]Zr-Df-ATG-101 to assess tumour and tissue uptake of ATG-101.

Characterization of tumoricidal activities mediated by a novel immune cell regimen composing interferon-producing killer dendritic cells and tumor-specific cytotoxic T lymphocytes.

BACKGROUND: Although immune cell therapy has long been used for treating solid cancer, its efficacy remains limited. Interferon (IFN)-producing killer dendritic cells (IKDCs) exhibit cytotoxicity and present antigens to relevant cells; thus, they can selectively induce tumor-associated antigen (TAA)-specific CD8 T cells and may be useful in cancer treatment. Various protocols have been used to amplify human IKDCs from peripheral sources, but the complexity of the process has prevented their widespread clinical application. Additionally, the induction of TAA-specific CD8 T cells through the adoptive transfer of IKDCs to immunocompromised patients with cancer may be insufficient. Therefore, we developed a method for generating an immune cell-based regimen, Phyduxon-T, comprising a human IKDC counterpart (Phyduxon) and expanded TAA-specific CD8 T cells. METHODS: Peripheral blood mononuclear cells from ovarian cancer patients were cultured with human interleukin (hIL)-15, hIL-12, and hIL-18 to generate Phyduxon-T. Then, its phenotype, cytotoxicity, and antigen-presenting function were evaluated through flow cytometry using specific monoclonal antibodies. RESULTS: Phyduxon exhibited the characteristics of both natural killer and dendritic cells. This regimen also exhibited cytotoxicity against primary ovarian cancer cells and presented TAAs, thereby inducing TAA-specific CD8 T cells, as evidenced by the expression of 4-1BB and IFN-γ. Notably, the Phyduxon-T manufacturing protocol effectively expanded IFN-γ-producing 4-1BB+ TAA-specific CD8 T cells from peripheral sources; these cells exhibited cytotoxic activities against ovarian cancer cells. CONCLUSIONS: Phyduxon-T, which is a combination of natural killer cells, dendritic cells, and TAA-specific CD8 T cells, may enhance the efficacy of cancer immunotherapy.

CD19 CAR T cells for B cell malignancies: a systematic review and meta-analysis focused on clinical impacts of CAR structural domains, manufacturing conditions, cellular product, doses, patient's age, and tumor types.

CD19-targeted chimeric antigen receptors (CAR) T cells are one of the most remarkable cellular therapies for managing B cell malignancies. However, long-term disease-free survival is still a challenge to overcome. Here, we evaluated the influence of different hinge, transmembrane (TM), and costimulatory CAR domains, as well as manufacturing conditions, cellular product type, doses, patient's age, and tumor types on the clinical outcomes of patients with B cell cancers treated with CD19 CAR T cells. The primary outcome was defined as the best complete response (BCR), and the secondary outcomes were the best objective response (BOR) and 12-month overall survival (OS). The covariates considered were the type of hinge, TM, and costimulatory domains in the CAR, CAR T cell manufacturing conditions, cell population transduced with the CAR, the number of CAR T cell infusions, amount of CAR T cells injected/Kg, CD19 CAR type (name), tumor type, and age. Fifty-six studies (3493 patients) were included in the systematic review and 46 (3421 patients) in the meta-analysis. The overall BCR rate was 56%, with 60% OS and 75% BOR. Younger patients displayed remarkably higher BCR prevalence without differences in OS. The presence of CD28 in the CAR's hinge, TM, and costimulatory domains improved all outcomes evaluated. Doses from one to 4.9 million cells/kg resulted in better clinical outcomes. Our data also suggest that regardless of whether patients have had high objective responses, they might have survival benefits from CD19 CAR T therapy. This meta-analysis is a critical hypothesis-generating instrument, capturing effects in the CD19 CAR T cells literature lacking randomized clinical trials and large observational studies.

The enhanced antitumor activity of bispecific antibody targeting PD-1/PD-L1 signaling.

The programmed cell death 1 (PD-1) signaling pathway, a key player in immune checkpoint regulation, has become a focal point in cancer immunotherapy. In the context of cancer, upregulated PD-L1 on tumor cells can result in T cell exhaustion and immune evasion, fostering tumor progression. The advent of PD-1/PD-L1 inhibitor has demonstrated clinical success by unleashing T cells from exhaustion. Nevertheless, challenges such as resistance and adverse effects have spurred the exploration of innovative strategies, with bispecific antibodies (BsAbs) emerging as a promising frontier. BsAbs offer a multifaceted approach to cancer immunotherapy by simultaneously targeting PD-L1 and other immune regulatory molecules. We focus on recent advancements in PD-1/PD-L1 therapy with a particular emphasis on the development and potential of BsAbs, especially in the context of solid tumors. Various BsAb products targeting PD-1 signaling are discussed, highlighting their unique mechanisms of action and therapeutic potential. Noteworthy examples include anti-TGFß × PD-L1, anti-CD47 × PD-L1, anti-VEGF × PD-L1, anti-4-1BB × PD-L1, anti-LAG-3 × PD-L1, and anti-PD-1 × CTLA-4 BsAbs. Besides, we summarize ongoing clinical studies evaluating the efficacy and safety of these innovative BsAb agents. By unraveling the intricacies of the tumor microenvironment and harnessing the synergistic effects of anti-PD-1/PD-L1 BsAbs, there exists the potential to elevate the precision and efficacy of cancer immunotherapy, ultimately enabling the development of personalized treatment strategies tailored to individual patient profiles.

Soluble levels of 4-1BB (CD137) and OX40 (CD134) are associated with cancer progression in gastric adenocarcinoma.

BACKGROUND AND OBJECTIVES: Previous studies have demonstrated that soluble forms of T-cell costimulatory molecules 4-1BB (s4-1BB) and OX40 (sOX40) interact with immune cells and may constitute a mechanism of immune evasion by tumors in various cancers. The role of the soluble forms of 4-1BB and OX40 in GC remains unclear. We aimed to examine the association between serum levels of s4-1BB and sOX40 and tumor progression in patients with GC. METHODS: Between 2017 and 2018, a cross-sectional study was performed with serum samples of 83 GC patients and 20 healthy controls. RESULTS: Patients with stage IV metastatic gastric cancer had significantly higher levels of soluble OX40 in comparison with stage III patients with lymph nodes metastasis (p = 0.0003) and stages I and II patients (p = 0.005), whereas the opposite was found for soluble 4-1BB levels, with lower levels being found in advanced stage III (p = 0.003) compared with initial stages I/II. CONCLUSIONS: The sOX40 and s4-1BB-mediated T cell interactions may be involved in antitumor immune responses in GC, possibly favoring tumor escape and progression. Serum levels of sOX40 and s4-1BB are associated with staging in GC and may constitute biomarkers for prognosis, as well as potential targets for immunotherapy.

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.

Safety and Pharmacokinetic Assessment of the FIC CLDN18.2/4-1BB Bispecific Antibody in Rhesus Monkeys.

Gastric cancer is one of the most common cancers worldwide, particularly in China, with over half a million new cases and over 400 thousand deaths in 2022. Zolbetuximab, a first-in-class investigational monoclonal antibody (mAb) targeting tumor-associated antigen CLDN18.2 which is highly expressed on gastric cancer cells, was recently reported to meet the primary endpoint in Phase III trial as first-line treatment in CLDN18.2 positive and HER2-negative gastric cancers. In the present study, we developed a humanized bispecific antibody (bsAb) CLDN18.2/4-1BB named PM1032. PM1032 activates immune cells via CLDN18.2 mediated crosslinking of 4-1BB, a potent stimulator of T/NK cells. It induced strong immunological memory in multiple tumor-bearing animal models, indicating significant potential as an effective treatment for CLDN18.2 positive cancers such as gastric cancer. Since liver and gastrointestinal (GI) related toxicities were reported in 4-1BB and CLDN18.2 targeting programs during the clinical development, respectively, extensive pharmacokinetics (PK) and safety profile characterization of PM1032 was performed in rhesus monkeys. PM1032 had a half-life comparable to a conventional IgG1 mAb, and serum drug concentration increased in a dose-dependent pattern. Furthermore, PM1032 was generally well tolerated, with no significant abnormalities observed in toxicity studies, including the liver and stomach. In summary, PM1032 demonstrated good PK and an exceptional safety profile in rhesus monkeys supporting further investigation in clinical studies.

Uncovering the Expression Pattern of the Costimulatory Receptors ICOS, 4-1BB, and OX-40 in Exhausted Peripheral and Tumor-Infiltrating Natural Killer Cells from Patients with Cervical Cancer.

Cervical cancer (CC) poses a significant health burden, particularly in low- and middle-income countries. NK cells play a crucial role against CC; however, they can become exhausted and lose their cytotoxic capacity. This work explores the expression of costimulatory receptors (ICOS, 4-1BB, OX-40) in exhausted NK cells from CC patients. Peripheral blood and tumor biopsies were collected, and flow cytometry was used to evaluate the expression of costimulatory receptors in exhausted NK cells. There is an increase of peripheral exhausted NK cells (PD-1+TIGIT+) in CC patients; this subpopulation has a selectively increased expression of the costimulatory receptors ICOS and 4-1BB. An exhausted population is also highly increased in tumor-infiltrating NK cells, and it shows a dramatically increased expression of the costimulatory receptors ICOS (>15×) and 4-1BB (>10×) compared to peripheral NK cells. The exhausted cells, both in the periphery and in the tumor infiltrating lymphocytes (TILs), are also more likely than non-exhausted NK cell populations (PD-1-TIGIT-) to express these costimulatory receptors; increases ranging from 2.0× ICOS, 2.4× 4-1BB, and 2.6× OX-40 in CD56dim PBMCs to 1.5× ICOS, 5× 4-1BB, and 10× OX-40 in TILs were found. Our study demonstrates for the first time the increased expression of the costimulatory receptors ICOS, 4-1BB, and OX-40 in peripheral CD56dim, CD56bright, and tumor-infiltrating NK cells in CC. Targeting these receptors for stimulation could reverse exhaustion and be a promising immunotherapy strategy.

Comparative Evaluation of STEAP1 Targeting Chimeric Antigen Receptors with Different Costimulatory Domains and Spacers.

We have developed a chimeric antigen receptor (CAR) against the six-transmembrane epithelial antigen of prostate-1 (STEAP1), which is expressed in prostate cancer, Ewing sarcoma, and other malignancies. In the present study, we investigated the effect of substituting costimulatory domains and spacers in this STEAP1 CAR. We cloned four CAR constructs with either CD28 or 4-1BB costimulatory domains, combined with a CD8a-spacer (sp) or a mutated IgG-spacer. The CAR T-cells were evaluated in short- and long-term in vitro T-cell assays, measuring cytokine production, tumor cell killing, and CAR T-cell expansion and phenotype. A xenograft mouse model of prostate cancer was used for in vivo comparison. All four CAR constructs conferred CD4+ and CD8+ T cells with STEAP1-specific functionality. A CD8sp_41BBz construct and an IgGsp_CD28z construct were selected for a more extensive comparison. The IgGsp_CD28z CAR gave stronger cytokine responses and killing in overnight caspase assays. However, the 41BB-containing CAR mediated more killing (IncuCyte) over one week. Upon six repeated stimulations, the CD8sp_41BBz CAR T cells showed superior expansion and lower expression of exhaustion markers (PD1, LAG3, TIGIT, TIM3, and CD25). In vivo, both the CAR T variants had comparable anti-tumor activity, but persisting CAR T-cells in tumors were only detected for the 41BBz variant. In conclusion, the CD8sp_41BBz STEAP1 CAR T cells had superior expansion and survival in vitro and in vivo, compared to the IgGsp_CD28z counterpart, and a less exhausted phenotype upon repeated antigen exposure. Such persistence may be important for clinical efficacy.

Anti-4-1BB immunotherapy enhances systemic immune effects of radiotherapy to induce B and T cell-dependent anti-tumor immune activation and improve tumor control at unirradiated sites.

Radiation therapy (RT) can prime and boost systemic anti-tumor effects via STING activation, resulting in enhanced tumor antigen presentation and antigen recognition by T cells. It is increasingly recognized that optimal anti-tumor immune responses benefit from coordinated cellular (T cell) and humoral (B cell) responses. However, the nature and functional relevance of the RT-induced immune response are controversial, beyond STING signaling, and agonistic interventions are lacking. Here, we show that B and CD4+ T cell accumulation at tumor beds in response to RT precedes the arrival of CD8+ T cells, and both cell types are absolutely required for abrogated tumor growth in non-irradiated tumors. Further, RT induces increased expression of 4-1BB (CD137) in both T and B cells; both in preclinical models and in a cohort of patients with small cell lung cancer treated with thoracic RT. Accordingly, the combination of RT and anti-41BB therapy leads to increased immune cell infiltration in the tumor microenvironment and significant abscopal effects. Thus, 4-1BB therapy enhances radiation-induced tumor-specific immune responses via coordinated B and T cell responses, thereby preventing malignant progression at unirradiated tumor sites. These findings provide a rationale for combining RT and 4-1bb therapy in future clinical trials.

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.

Engineered soluble, trimerized 4-1BBL variants as potent immunomodulatory agents.

Targeting co-stimulatory receptors promotes the activation and effector functions of anti-tumor lymphocytes. 4-1BB (CD137/TNFSF9), a member of the tumor necrosis factor receptor superfamily (TNFR-SF), is a potent co-stimulatory receptor that plays a prominent role in augmenting effector functions of CD8+ T cells, but also CD4+ T cells and NK cells. Agonistic antibodies against 4-1BB have entered clinical trials and shown signs of therapeutic efficacy. Here, we have used a T cell reporter system to evaluate various formats of 4-1BBL regarding their capacity to functionally engage its receptor. We found that a secreted 4-1BBL ectodomain harboring a trimerization domain derived from human collagen (s4-1BBL-TriXVIII) is a strong inducer of 4-1BB co-stimulation. Similar to the 4-1BB agonistic antibody urelumab, s4-1BBL-TriXVIII is very potent in inducing CD8+ and CD4+ T cell proliferation. We provide first evidence that s4-1BBL-TriXVIII can be used as an effective immunomodulatory payload in therapeutic viral vectors. Oncolytic measles viruses encoding s4-1BBL-TriXVIII significantly reduced tumor burden in a CD34+ humanized mouse model, whereas measles viruses lacking s4-1BBL-TriXVIII were not effective. Natural soluble 4-1BB ligand harboring a trimerization domain might have utility in tumor therapy especially when delivered to tumor tissue as systemic administration might induce liver toxicity.

A novel agonist of 4-1BB costimulatory receptor shows therapeutic efficacy against a tobacco carcinogen-induced lung cancer.

Immunotherapy utilizing checkpoint inhibitors has shown remarkable success in the treatment of cancers. In addition to immune checkpoint inhibitors, immune co-stimulation has the potential to enhance immune activation and destabilize the immunosuppressive tumor microenvironment. CD137, also known as 4-1BB, is one of the potent immune costimulatory receptors that could be targeted for effective immune co-stimulation. The interaction of the 4-1BB receptor with its natural ligand (4-1BBL) generates a strong costimulatory signal for T cell proliferation and survival. 4-1BBL lacks costimulatory activity in soluble form. To obtain co-stimulatory activity in soluble form, a recombinant 4-1BBL protein was generated by fusing the extracellular domains of murine 4-1BBL to a modified version of streptavidin (SA-4-1BBL). Treatment with SA-4-1BBL inhibited the development of lung tumors in A/J mice induced by weekly injections of the tobacco carcinogen NNK for eight weeks. The inhibition was dependent on the presence of T cells and NK cells; depletion of these cells diminished the SA-4-1BBL antitumor protective effect. The number of lung tumor nodules was significantly reduced by the administration of SA-4-1BBL to mice during ongoing exposure to NNK. The data presented in this paper suggest that utilizing an immune checkpoint stimulator as a single agent generate a protective immune response against lung cancer in the presence of a carcinogen. More broadly, this study suggests that immune checkpoint stimulation can be extended to a number of other cancer types, including breast and prostate cancers, for which improved diagnostics can detect disease at the preneoplastic stage.