CAR T cell trogocytosis and cooperative killing regulate tumour antigen escape.

Chimeric antigen receptors (CARs) are synthetic antigen receptors that reprogram T cell specificity, function and persistence1. Patient-derived CAR T cells have demonstrated remarkable efficacy against a range of B-cell malignancies1-3, and the results of early clinical trials suggest activity in multiple myeloma4. Despite high complete response rates, relapses occur in a large fraction of patients; some of these are antigen-negative and others are antigen-low1,2,4-9. Unlike the mechanisms that result in complete and permanent antigen loss6,8,9, those that lead to escape of antigen-low tumours remain unclear. Here, using mouse models of leukaemia, we show that CARs provoke reversible antigen loss through trogocytosis, an active process in which the target antigen is transferred to T cells, thereby decreasing target density on tumour cells and abating T cell activity by promoting fratricide T cell killing and T cell exhaustion. These mechanisms affect both CD28- and 4-1BB-based CARs, albeit differentially, depending on antigen density. These dynamic features can be offset by cooperative killing and combinatorial targeting to augment tumour responses to immunotherapy.

Protective Effect of CD137 Deficiency Against Postinfarction Cardiac Fibrosis and Adverse Cardiac Remodeling by ERK1/2 Signaling Pathways.

ABSTRACT: Myocardial fibrosis, a common complication of myocardial infarction (MI), is characterized by excessive collagen deposition and can result in impaired cardiac function. The specific role of CD137 in the development of post-MI myocardial fibrosis remains unclear. Thus, this study aimed to elucidate the effects of CD137 signaling using CD137 knockout mice and in vitro experiments. CD137 expression levels progressively increased in the heart after MI, particularly in myofibroblast, which play a key role in fibrosis. Remarkably, CD137 knockout mice exhibited improved cardiac function and reduced fibrosis compared with wild-type mice at day 28 post-MI. The use of Masson's trichrome and picrosirius red staining demonstrated a reduction in the infarct area and collagen volume fraction in CD137 knockout mice. Furthermore, the expression of alpha-smooth muscle actin and collagen I, key markers of fibrosis, was decreased in heart tissues lacking CD137. In vitro experiments supported these findings because CD137 depletion attenuated cardiac fibroblast differentiation, and migration, and collagen I synthesis. In addition, the administration of CD137L recombinant protein further promoted alpha-smooth muscle actin expression and collagen I synthesis, suggesting a profibrotic effect. Notably, the application of an inhibitor targeting the extracellular signal-regulated kinase 1/2 (ERK1/2) signaling pathway attenuated the profibrotic effects of CD137L. To conclude, this study provides evidence that CD137 plays a significant role in promoting myocardial fibrosis after MI. Inhibition of CD137 signaling pathways may hold therapeutic potential for mitigating pathological cardiac remodeling and improving post-MI cardiac function.

Long-term antigen-specific immune response by an oncolytic adenovirus encoding SP-SA-E7-4-1BBL in HPV-16 cancer model.

BACKGROUND: To describe an oncolytic adenovirus (OAd) encoding SP-SA-E7-4-1BBL that is capable of inducing tumor regression in therapeutic assays. Herein, we tested whether the antitumor effect is given by the induction of a tumor-specific immune response, as well as the minimum dose needed to elicit antitumor protection and monitor the OAd biodistribution over time. METHODS AND RESULTS: C57BL/6 mice (n = 5) per group were immunized twice with OAds encoding SP-SA-E7-4-1BBL, SA-E7-4-1BBL, or SP-SA-4-1BBL and challenged with TC-1 cancer cells. The DNA construct SP-SA-E7-4-1BBL was employed as a control via biolistic or PBS injection. Groups without tumor development at 47 days were rechallenged with TC-1 cells, and follow-up lasted until day 90. The minimum dose of OAd to induce the antitumor effect was established by immunization using serial dilution doses. The cytometry bead assay and the ELISpot assay were used to evaluate cytokine release in response to ex vivo antigenic stimulation. The distribution profile of the OAd vaccine was evaluated in the different organs by histological, immunohistochemical and qPCR analyses. The OAd SP-SA-E7-4-1BBL-immunized mice did not develop tumors even in a rechallenge. A protective antitumor effect was observed from a dose that is one hundredth of most reports of adenoviral vaccines. Immunization with OAd increases Interferon-gamma-producing cells in response to antigen stimulation. OAd was detected in tumors over time, with significant morphological changes, contrary to nontumor tissues. CONCLUSIONS: The OAd SP-SA-E7-4-1BBL vaccine confers a prophylactic, safe, long-lasting, and antigen-dependent antitumor effect mediated by a Th1 antitumor immune response.

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.

T cells engineered with full-length NKG2D linked to signaling domains of 4-1BB and CD3ζ show enhanced antitumor activity.

Since NKG2D ligands (NKG2DLs) are primarily overexpressed on multiple types of solid tumors but absent on most normal tissues, NKG2DLs could be optimal antigens for CAR-T cells. To date, there have been two types of NKG2DL CARs: (i) the extracellular domain of NKG2D fused to the CD8a transmembrane domain, signaling domains of 4-1BB and CD3ζ (NKBz) and (ii) full-length NKG2D fused to the CD3ζ signaling domain (chNKz). Although NKBz- and chNKz-engineered T cells both showed antitumor activities, a comparison of their functions has not been reported. In addition, use of the 4-1BB signaling domain into the CAR construct could prolong the persistence and resistance to antitumor activities of CAR-T cells, we designed a new NKG2DL CAR, full-length NKG2D fused to the signaling domains of 4-1BB and CD3ζ (chNKBz). Among the two types of NKG2DL CAR-T cells reported in previous studies, we found that chNKz T cells had stronger antitumor ability than NKBz T cells in vitro, but their antitumor activity in vivo is similar. The chNKBz T cells showed antitumor activity superior to that of chNKz T cells and NKBz T cells in vitro and in vivo, providing a new option for the immunotherapy of NKG2DL-positive tumor patients.

Development of an antibody-ligand fusion protein scFvCD16A-sc4-1BBL in Komagataella phaffii with stimulatory activity for Natural Killer cells.

BACKGROUND: Natural killer (NK) cell-based immunotherapies have demonstrated substantial potential for the treatment of hematologic malignancies. However, its application is limited due to the difficulty in the production of a large number of NK cells in vitro and the insufficient therapeutic efficacy against solid tumors in vivo. Engineered antibodies or fusion proteins targeting activating receptors and costimulatory molecules of NK cells have been developed to encounter these problems. They are mostly produced in mammalian cells with high cost and long processing times. Yeast systems, such as Komagataella phaffii, present a convenient manipulation of microbial systems with the key advantages of improved folding machinery and low cost. RESULTS: In this study, we designed an antibody fusion protein scFvCD16A-sc4-1BBL, composed of the single chain variant fragment (scFv) of anti-CD16A antibody and the three extracellular domains (ECDs) of human 4-1BBL in a single-chain format (sc) with the GS linker, aiming to boost NK cell proliferation and activation. This protein complex was produced in the K. phaffii X33 system and purified by affinity chromatography and size exclusion chromatography. The scFvCD16A-sc4-1BBL complex showed comparable binding abilities to its two targets human CD16A and 4-1BB as its two parental moieties (scFvCD16A and monomer ECD (mn)4-1BBL). scFvCD16A-sc4-1BBL specifically stimulated the expansion of peripheral blood mononuclear cell (PBMC)-derived NK cells in vitro. Furthermore, in the ovarian cancer xenograft mouse model, adoptive NK cell infusion combined with intraperitoneal (i.p) injection of scFvCD16A-sc4-1BBL further reduced the tumor burden and prolonged the survival time of mice. CONCLUSION: Our studies demonstrate the feasibility of the expression of the antibody fusion protein scFvCD16A-sc4-1BBL in K. phaffii with favourable properties. scFvCD16A-sc4-1BBL stimulates PBMC-derived NK cell expansion in vitro and improves the antitumor activity of adoptively transferred NK cells in a murine model of ovarian cancer and may serve as a synergistic drug for NK immunotherapy in future research and applications.

An engineered 4-1BBL fusion protein with "activity on demand".

Engineered cytokines are gaining importance in cancer therapy, but these products are often limited by toxicity, especially at early time points after intravenous administration. 4-1BB is a member of the tumor necrosis factor receptor superfamily, which has been considered as a target for therapeutic strategies with agonistic antibodies or using its cognate cytokine ligand, 4-1BBL. Here we describe the engineering of an antibody fusion protein, termed F8-4-1BBL, that does not exhibit cytokine activity in solution but regains biological activity on antigen binding. F8-4-1BBL bound specifically to its cognate antigen, the alternatively spliced EDA domain of fibronectin, and selectively localized to tumors in vivo, as evidenced by quantitative biodistribution experiments. The product promoted a potent antitumor activity in various mouse models of cancer without apparent toxicity at the doses used. F8-4-1BBL represents a prototype for antibody-cytokine fusion proteins, which conditionally display "activity on demand" properties at the site of disease on antigen binding and reduce toxicity to normal tissues.

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.

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.

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.

Adenovirus vaccine therapy with CD137L promotes CD8+ DCs-mediated multifunctional CD8+ T cell immunity and elicits potent anti-tumor activity.

Renal carcinoma progresses aggressively in patients with metastatic disease while curative strategies are limited. Here, we constructed a recombinant non-replicating adenovirus (Ad) vaccine encoding an immune activator, CD137L, and a tumor antigen, CAIX, for treating renal carcinoma. In a subcutaneous tumor model, tumor growth was significantly suppressed in the Ad-CD137L/CAIX vaccine group compared with the single vaccine group. The induction and maturity of CD11C+ and CD8+CD11C+ dendritic cell (DC) subsets were promoted in Ad-CD137L/CAIX co-immunized mice. Furthermore, the Ad-CD137L/CAIX vaccine elicited stronger tumor-specific multifunctional CD8+ T cell immune responses as demonstrated by increased proliferation and cytolytic function of CD8+ T cells. Notably, depletion of CD8+ T cells greatly compromised the effective protection provided by Ad-CD137L/CAIX vaccine, suggesting an irreplaceable role of CD8+ T cells for the immunopotency of the vaccine. In both lung metastatic and orthotopic models, Ad-CD137L/CAIX vaccine treatment significantly decreased tumor metastasis and progression and increased the induction of tumor-specific multifunctional CD8+ T cells, in contrast to treatment with the Ad-CAIX vaccine alone. The Ad-CD137L/CAIX vaccine also augmented the tumor-specific multifunctional CD8+ T cell immune response in both orthotopic and metastatic models. These results indicated that Ad-CD137L/CAIX vaccine elicited a potent anti-tumor activity by inducing CD8+DC-mediated multifunctional CD8+ T cell immune responses. The potential strategy of CD137L-based vaccine might be served as a novel treatment for renal carcinoma or other malignant tumors.

The CD137 Ligand Is Important for Type 1 Diabetes Development but Dispensable for the Homeostasis of Disease-Suppressive CD137+ FOXP3+ Regulatory CD4 T Cells.

CD137 modulates type 1 diabetes (T1D) progression in NOD mice. We previously showed that CD137 expression in CD4 T cells inhibits T1D, but its expression in CD8 T cells promotes disease development by intrinsically enhancing the accumulation of ß-cell-autoreactive CD8 T cells. CD137 is expressed on a subset of FOXP3+ regulatory CD4 T cells (Tregs), and CD137+ Tregs are the main source of soluble CD137. Soluble CD137 suppresses T cells in vitro by binding to the CD137 ligand (CD137L) upregulated on activated T cells. To further study how the opposing functions of CD137 are regulated, we successfully targeted Tnfsf9 (encoding CD137L) in NOD mice using the CRISPR/Cas9 system (designated NOD.Tnfsf9 -/-). Relative to wild-type NOD mice, T1D development in the NOD.Tnfsf9 -/- strain was significantly delayed, and mice developed less insulitis and had reduced frequencies of ß-cell-autoreactive CD8 T cells. Bone marrow chimera experiments showed that CD137L-deficient hematopoietic cells were able to confer T1D resistance. Adoptive T cell transfer experiments showed that CD137L deficiency on myeloid APCs was associated with T1D suppression. Conversely, lack of CD137L on T cells enhanced their diabetogenic activity. Furthermore, neither CD137 nor CD137L was required for the development and homeostasis of FOXP3+ Tregs. However, CD137 was critical for the in vivo T1D-suppressive activity of FOXP3+ Tregs, suggesting that the interaction between CD137 and CD137L regulates their function. Collectively, our results provide new insights into the complex roles of CD137-CD137L interaction in T1D.

Intratumoral Activation of 41BB Costimulatory Signals Enhances CD8 T Cell Expansion and Modulates Tumor-Infiltrating Myeloid Cells.

The activation of 41BB costimulatory signals by agonistic Abs enhances the expansion and function of tumor-infiltrating lymphocytes (TILs) for treating cancer patients with adoptive cell therapy. However, the impact of 41BB agonism is not limited to enhancing the activity of T cells, and the mechanism by which additional activation of this costimulatory axis in tumor-associated myeloid cells is poorly understood. In this study, we describe that the intratumoral administration of 41BB agonistic Abs led to increases in CD8 T cell infiltration followed by tumor regression in murine models. We found that granulocytes and monocytes rapidly replaced macrophages and dendritic cells in tumors following administration of anti-41BB Abs. Overall, myeloid cells from anti-41BB-treated tumors had an improved capacity to stimulate T cells in comparison with control-treated tumors. In human coculture systems, we demonstrated that the agonism of the 41BB-41BBL axis enhanced costimulatory signals and effector functions among APC and autologous TILs. Overall, these findings suggest that the effect of 41BB agonistic Abs are supported by additional costimulatory signals from tumor-associated myeloid cells,v leading to enhanced TIL expansion and function.

4-1BBL Regulates the Polarization of Macrophages, and Inhibition of 4-1BBL Signaling Alleviates Imiquimod-Induced Psoriasis.

4-1BBL, a member of the TNF superfamily, regulates the sustained production of inflammatory cytokines in macrophages triggered by TLR signaling. In this study, we have investigated the role of 4-1BBL in macrophage metabolism and polarization and in skin inflammation using a model of imiquimod-induced psoriasis in mice. Genetic ablation or blocking of 4-1BBL signaling by Ab or 4-1BB-Fc alleviated the pathology of psoriasis by regulating the expression of inflammatory cytokines associated with macrophage activation and regulated the polarization of macrophages in vitro. We further linked this result with macrophage by finding that 4-1BBL expression during the immediate TLR response was dependent on glycolysis, mitochondrial oxidative phosphorylation, and fatty acid metabolism, whereas the late-phase 4-1BBL-mediated sustained inflammatory response was dependent on glycolysis and fatty acid synthesis. Correlating with this, administration of a fatty acid synthase inhibitor, cerulenin, also alleviated the pathology of psoriasis. We further found that 4-1BBL-mediated psoriasis development is independent of its receptor 4-1BB, as a deficiency of 4-1BB augmented the severity of psoriasis linked to a reduced regulatory T cell population and increased IL-17A expression in γδ T cells. Additionally, coblocking of 4-1BBL signaling and IL-17A activity additively ameliorated psoriasis. Taken together, 4-1BBL signaling regulates macrophage polarization and contributes to imiquimod-induced psoriasis by sustaining inflammation, providing a possible avenue for psoriasis treatment in patients.

A comprehensive profiling of soluble immune checkpoints from the sera of patients with non-small cell lung cancer.

BACKGROUND: Immunotherapy was widely used for the treatment of non-small cell lung cancer (NSCLC). However, whether inhibition of immune checkpoints individually or simultaneously could improve the therapeutic efficacy of NSCLC remains to be investigated. Here, we explored the aberrant levels of several checkpoints and evaluated their potential diagnostic values for NSCLC. METHODS: Serum samples of 89 NSCLC patients and 57 healthy donors were collected from Nanjing Drum Tower Hospital between November 2019 and July 2020. Fourteen human immune checkpoints were quantified by Procarta-Plex Human Immuno-Oncology Checkpoint Panel. RESULTS: The expression levels of sTIM-3, sCD137, sCD27, sLAG-3, sIDO, sPD-L2, sCD152, sCD80, and sPD-1 were all significantly increased in serum of NSCLC patients. Especially, sLAG-3 was significantly elevated in serum of NSCLC patients at early-stage (stages I and II), TIM-3, CD137, and CD27 were significantly higher in the advanced NSCLC patients (stages III and IV) than in the early-stage groups. Receiver operating characteristics (ROC) results showed that except for PD-1, all the other immune checkpoint proteins had potential diagnostic values for NSCLC. sTIM-3 had the highest diagnostic accuracy, followed by sLAG-3. Combining sTIM-3, sLAG-3, and sCD137 could increase the accuracy to a higher level. Moreover, sCD27 was correlated with NSCLC cancer type, age, sex, and disease stage, while sCD137 was correlated with age and disease stage. sTIM-3 and sIDO were correlated with stage and age, respectively. CONCLUSIONS: TIM-3 and LAG-3 were independent biomarkers for the early diagnosis of NSCLC. The combination of TIM-3, LAG-3, and CD137 could increase the diagnostic accuracy.

A Novel L-Phenylalanine Dipeptide Inhibits the Growth and Metastasis of Prostate Cancer Cells via Targeting DUSP1 and TNFSF9.

Prostate cancer (PCa) is a common malignant cancer of the urinary system. Drug therapy, chemotherapy, and radical prostatectomy are the primary treatment methods, but drug resistance and postoperative recurrence often occur. Therefore, seeking novel anti-tumor compounds with high efficiency and low toxicity from natural products can produce a new tumor treatment method. Matijin-Su [N-(N-benzoyl-L-phenylalanyl)-O-acetyl-L-phenylalanol, MTS] is a phenylalanine dipeptide monomer compound that is isolated from the Chinese ethnic medicine Matijin (Dichondra repens Forst.). Its derivatives exhibit various pharmacological activities, especially anti-tumor. Among them, the novel MTS derivative HXL131 has a significant inhibitory effect against prostate tumor growth and metastasis. This study is designed to investigate the effects of HXL131 on the growth and metastasis of human PCa cell lines PC3 and its molecular mechanism through in vitro experiments combined with proteomics, molecular docking, and gene silencing. The in vitro results showed that HXL131 concentration dependently inhibited PC3 cell proliferation, induced apoptosis, arrested cell cycle at the G2/M phase, and inhibited cell migration capacity. A proteomic analysis and a Western blot showed that HXL131 up-regulated the expression of proliferation, apoptosis, cell cycle, and migration-related proteins CYR61, TIMP1, SOD2, IL6, SERPINE2, DUSP1, TNFSF9, OSMR, TNFRSF10D, and TNFRSF12A. Molecular docking, a cellular thermal shift assay (CETSA), and gene silencing showed that HXL131 had a strong binding affinity with DUSP1 and TNFSF9, which are important target genes for inhibiting the growth and metastasis of PC3 cells. This study demonstrates that HXL131 exhibited excellent anti-prostate cancer activity and inhibited the growth and metastasis of prostate cancer cells by regulating the expression of DUSP1 and TNFSF9.

Successful hematopoietic stem cell transplantation in a 4-1BB deficient patient with EBV-induced lymphoproliferation.

Complete remission from recurrent EBV-positive lymphoma is not mandatory before HSCT to achieve long-term cure in a patient suffering from a recently described immunodeficiency affecting the T-cell coactivation molecule 4-1BB.

Expanded clinical-grade membrane-bound IL-21/4-1BBL NK cell products exhibit activity against acute myeloid leukemia in vivo.

BACKGROUND: Adoptive NK cell infusion is a promising immunotherapy for acute myeloid leukemia (AML) patients. The aim of this study was to test the activity of clinical-grade membrane-bound IL-21/4-1BBL-expanded NK cell products against AML in vivo. METHODS: Fresh peripheral blood mononuclear cells (PBMCs) were incubated with equal numbers of irradiated membrane-bound IL-21/4-1BBL-expressing K562 cells for 2-3 weeks to induce clinical-grade NK cell expansion. RESULTS: Expansion for 2 and 3 weeks produced ∼4 and 8 × 109 NK cells from 2 × 107 PBMCs. The production of CD107a and TNF-α in NK cell products in response to AML cell lines and primary blasts was higher than that observed in resting NK cells. The 2-week expanded NK cell products were xenografted into immunodeficient mice with leukemia and were persistently found in the BM, spleen, liver, lung, and peripheral blood for at least 13 days; furthermore, these expanded products reduced the AML burden in vivo. Compared with matched AML patients with persistent or relapsed minimal residual disease (MRD+ ) who underwent regular consolidation therapy, MRD+ patients who underwent NK treatment had better overall survival and showed no major adverse events. CONCLUSIONS: Clinical-grade mbIL-21/4-1BBL-expanded NK cells exhibited antileukemic activity against AML in vitro and in vivo.

Anti-tumor effects of RTX-240: an engineered red blood cell expressing 4-1BB ligand and interleukin-15.

Recombinant agonists that activate co-stimulatory and cytokine receptors have shown limited clinical anticancer utility, potentially due to narrow therapeutic windows, the need for coordinated activation of co-stimulatory and cytokine pathways and the failure of agonistic antibodies to recapitulate signaling by endogenous ligands. RTX-240 is a genetically engineered red blood cell expressing 4-1BBL and IL-15/IL-15Rα fusion (IL-15TP). RTX-240 is designed to potently and simultaneously stimulate the 4-1BB and IL-15 pathways, thereby activating and expanding T cells and NK cells, while potentially offering an improved safety profile through restricted biodistribution. We assessed the ability of RTX-240 to expand and activate T cells and NK cells and evaluated the in vivo efficacy, pharmacodynamics and tolerability using murine models. Treatment of PBMCs with RTX-240 induced T cell and NK cell activation and proliferation. In vivo studies using mRBC-240, a mouse surrogate for RTX-240, revealed biodistribution predominantly to the red pulp of the spleen, leading to CD8 + T cell and NK cell expansion. mRBC-240 was efficacious in a B16-F10 melanoma model and led to increased NK cell infiltration into the lungs. mRBC-240 significantly inhibited CT26 tumor growth, in association with an increase in tumor-infiltrating proliferating and cytotoxic CD8 + T cells. mRBC-240 was tolerated and showed no evidence of hepatic injury at the highest feasible dose, compared with a 4-1BB agonistic antibody. RTX-240 promotes T cell and NK cell activity in preclinical models and shows efficacy and an improved safety profile. Based on these data, RTX-240 is now being evaluated in a clinical trial.