Butyrophilin 2A2 promotes T cell immunoregulation via CD45 phosphatase activation and protects against murine autoimmune glomerulonephritis and pregnancy loss

Nature Communications · 2026-01-08 · 1 citations

B7 costimulatory family member Butyrophilin 2A2 (BTN2A2) is predominantly expressed by antigen presenting cells and regulates T cell immunity, but molecular mechanisms are unclear. Using immunoblots analyzing TCR-initiated signaling intermediaries, co-immunoprecipitation studies, confocal microscopy, structural modeling-guided mutational analyses, and microscale thermophoresis, we demonstrate that BTN2A2 directly interacts with CD45RO, resulting in CD45 retention within the immune synapse during TCR activation. Recombinant BTN2A2 increases murine CD4+Foxp3+ regulatory T cells (Treg) and reduces T helper 17 (Th17) cells in vitro through mechanisms dependent on CD45 phosphatase activity. BTN2A2 therapy alleviates disease severity in murine nephrotoxic glomerulonephritis and autoimmune miscarriage while increasing Treg/Th17 ratios. Analyses of BTN2A2-deficient animals show exacerbation of disease associated with reduced Treg/Th17 ratios. BTN2A2 functions analogously on human T cells suppressing Th17, Th1 and Th2 responses while inducing Tregs. Together, our studies identify BTN2A2 as a modulator of CD45RO signaling in T cells, providing insight into how BTN2A2 regulates T cell-dependent immune responses including those mediating autoimmunity and transplant rejection. Butyrophilin 2A2 is a member of the B7 costimulatory family that is expressed on antigen presenting cells and is linked to the regulation of T cells. Here the authors implicate butyrophilin 2A2 in enhancement of CD45 phosphatase activity within the immunological synapse during T cell activation, leading to expansion of regulatory T cells and reduction of proinflammatory Th17 CD4 T cells.

ONECUT2: a validated drug target and lineage plasticity driver in prostate cancer and other malignancies

Endocrine Related Cancer · 2025-11-01

The CUT/Hox transcription factor ONECUT2 (OC2) promotes lineage plasticity and is a confirmed therapeutic target in prostate cancer and several other malignancies where cell phenotype plays a substantial role in treatment resistance. OC2 governs a broad growth and lineage identity process in prostate cancer that promotes neuroendocrine (NE) differentiation, androgen receptor (AR) suppression, and the emergence of a wide range of treatment-resistant pathways. The mode of action of OC2 includes incorporation of the protein into transcription complexes at gene promoters as an activator and repressor, alteration of chromatin accessibility and epigenetic marks, and extensive alteration of large-scale chromatin modifications, such as super-enhancers and chromatin loops. Notably, OC2 may be unique among NE drivers in that it can promote AR indifference in adenocarcinoma as a direct upstream activator of the glucocorticoid receptor, thus assuming indirect control of a portion of the AR cistrome. OC2 expression and activity increase substantially following hormone therapy in association with aggressive disease in prostate and breast cancer. Experiments in model systems have shown that OC2 has a survival function in both human castration-sensitive and castration-resistant prostate cancer cells. OC2 can be targeted directly with a family of novel small-molecule inhibitors that show therapeutic efficacy in vivo in prostate, breast, and gastric cancer models, including regression of established distant metastases in mice. These findings suggest that inhibition of OC2 clinically may confer substantial therapeutic benefit in some aggressive malignancies, including in localized hormone-sensitive disease.

Effect of KROS 101, a small molecule GITR ligand agonist, on T effector cells, T reg cells and intratumoral CD8 T cell cytotoxicity.

Journal of Clinical Oncology · 2025-05-28

2587 Background: A small molecule was identified that stabilizes the trimerization of the glucacorticoid-induced tumor necrosis factor receptor (GITR) ligand which then leads to the trimerization of GITR and magnified signaling of GITR. GITR signaling of T cells results in T effector cell expansion and T reg reduction. An antagonist to the GITR ligand was also indentified which stabilizes the GITR ligand dimer formation preventing trimerization. Methods: Binding of KROS 101 to the GITR ligand was assessed and the binding region of GITR ligand to KROS was determined with targeted deletion of GITR. T cell suppression studies were performed with T cell proliferation assays and T cell cytotoxicity assays with glioblastoma target cells using patient derived PBMCs. Double humanized GITR/GITRL mice bearing B16-F10-LUC2 tumors were treated with KROS 101 or controls. Tumor-infiltrating lymphocytes were analyzed by flow cytometry and tumors assessed. Results: KROS 101 agonist binds to GITRL with high affinity with a K D of 340nM by Surface Plasmon resonance. T cell suppression assay showed KROS 101 had peak proliferative induction of T effector cells at 25 uM concentration to 52% increase in proliferation, and peak proliferation induction of T effector cells with 1:1 ratio of T reg cells at 50uM concentration to 80% increase in proliferation of T effectors. KROS 101 treated T cell show enhanced effetor function and selectively target glioblastoma and cancer stem cells in vitro. KROS 101 enhances tumor immune infiltration by Increasing CD3+ T Cells, CD8+ T Cells, and M1 Macrophages While Reducing Tregs and Myeloid Cells in vivo. KROS 101 enhances cytotoxicity by increasing IFNγ and TNFα While Reducing TIGIT and TIM3 in CD4+ and CD8+ T Cells I n Vivo . Conclusions: KROS 101 is a GITR ligand agonist that increases T cell proliferation and increased cytotoxicity and reduces the T reg population more effectively than TRX 518 which is a therapeutic GITR antibody that was in clinical trial.

HDAC4/MybL1/YAP novel signaling axis is required for pancreatic cancer metastasis to the liver

International Journal of Biological Sciences · 2025-10-24 · 1 citations

Pancreatic ductal adenocarcinoma (PDAC) is one of the deadliest forms of human malignancy, and there is an urgency to develop more effective therapy. We previously showed that Metavert, a dual inhibitor of glycogen synthase kinase 3-beta (GSK-3β) and histone deacetylases (HDACs) prevents pancreatic ductal adenocarcinoma (PDAC) metastasis. In this study, we investigated the mechanisms that mediate metastasis and the roles of GSK-3β, HDACs, and Yes-associated protein (YAP) in this process. We found that HDAC4 and YAP are highly expressed in PDAC from patients with rapid disease progression and metastasis compared to those with prolonged recurrence-free survival. Pan-HDAC inhibition decreases metastasis in the splenic PDAC metastatic mouse model. Inhibition of HDAC4 reduces migration of cancer cells and decreases the mRNA and protein levels of transcription factor MYB Proto-Oncogene Like 1 (MybL1) and YAP. Mechanistic studies show that HDAC4 regulates transcription of YAP through up-regulating MybL1 expression. Comparable results were observed in colon and prostate cancers. ATAC-seq studies show that inhibition of HDAC4 causes chromatin remodeling in the YAP promoter region and reduces accessibility to the binding sites of multiple transcription factors, including those of MybL1. Pharmacological or molecular inhibition of YAP significantly decreases PDAC metastasis in vivo. Imaging Mass Cytometry (IMC) reveals no significant changes in immune cells, but a notable shift in the distribution patterns of cancer-associated hepatic stellate cells in the metastatic niche, when YAP is ablated in the cancer cells. The results demonstrate a novel metastasis-driving cell signaling pathway mediated by the functional interaction between HDAC4 and MybL1, which regulates YAP expression and metastasis.

Abstract A048: Targeting M2 macrophage polarization: The anti-tumor effects of IL-4/IL-13 blocking peptide (KROS-401) and iron oxide nanoparticles

Cancer Immunology Research · 2025-02-23 · 1 citations

Abstract Glioblastoma (GBM) is the most aggressive primary brain tumor, with a five-year survival rate of only 5-7%. Current therapies include surgery, chemotherapy, and radiation, but there is an urgent need for alternative strategies to reduce the side effects of these treatments. The paradigm of cancer treatment is shifting toward immunotherapy, which offers tumor target specificity through mechanisms that target specific antigen and receptors. Macrophages play a major role in innate immune system, primarily through phagocytosis and the destruction of harmful organisms. In cancer, they comprise the highest cell concentration among immune cells in the tumor microenvironment (TME). However, most of these macrophages possess pro-tumoral and immunosuppressive (M2-like) phenotype which promotes angiogenesis, tumor invasion and aid in metastasis. Tumor associated macrophages (TAMs) represent the predominant infiltrating immune cell population in gliomas, accounting for 30–50% of the total tumor cell population, and are associated with poor prognosis and higher tumor grades. M1-like TAMs produce proinflammatory cytokines that inhibit tumor progression whereas M2-like TAMs contribute to an immunosuppressive microenvironment, facilitating tumor growth. Hence, inhibition of M2 polarization could be a promising strategy to create a proinflammatory state (M1). We developed a peptide inhibitor, KROS-401, targeting the shared IL-4Ra subunit of the IL-4 and IL-13 receptor complex, aiming to inhibit macrophage polarization to the M2 phenotype and reprogram the immunosuppressive TME. Initially, we derived and differentiated macrophages from human peripheral blood mononuclear cell (PBMC) and characterized for M2 surface markers. These differentiated macrophages demonstrated excellent phagocytotic activity using red zymosan. Subsequently we assessed the effect of KROS-401 on M2 macrophage polarization. Our preliminary results showed decrease in the M2 macrophage population in a dose-dependent manner in the presence of IL-4 and IL-13. Furthermore, our in vivo studies indicated that KROS-401 had an anti-tumor effect in glioma bearing mice, with treated mice showed significantly extended overall survival compared to control group. It is reported that iron oxide nanoparticles can skew macrophages towards the M1 phenotype but no studies have explored their potential for reprogramming macrophages in glioblastoma treatment. To enhance the efficacy of KROS-401 and further reprogram the macrophage/microglia population, we developed an iron oxide nanoparticle conjugated to KROS-401. Preliminary in vitro and in vivo studies shows that the iron oxide nanoparticle conjugated to KROS-401 is safe and may effectively reprogram macrophages to inhibit tumor growth. Citation Format: Dnyaneshwar Kalyane, John Yu, Tesfahun Admasu, Michelot Michel, Hongqiang Wang, Ramachandran Murali. Targeting M2 macrophage polarization: The anti-tumor effects of IL-4/IL-13 blocking peptide (KROS-401) and iron oxide nanoparticles [abstract]. In: Proceedings of the AACR IO Conference: Discovery and Innovation in Cancer Immunology: Revolutionizing Treatment through Immunotherapy; 2025 Feb 23-26; Los Angeles, CA. Philadelphia (PA): AACR; Cancer Immunol Res 2025;13(2 Suppl):Abstract nr A048.

Modulation of the PTPRS proteoglycan switch by antibodies binding to the membrane-proximal fibronectin-type III domain

Journal of Biological Chemistry · 2025-07-15 · 1 citations

Protein tyrosine phosphatases (PTPs) receptor type II A (R2A) are negatively regulated through oligomerization upon binding of their extracellular domains to glycosaminoglycans (GAGs) on heparan sulfate proteoglycans (HSPGs). Inactivation of receptor PTP sigma (PTPRS) by HSPGs promotes the aggressive behavior of fibroblast-like synoviocytes (FLS) in rheumatoid arthritis (RA). Blocking the binding of its N-terminal, membrane-distal immunoglobulin-like 1 and 2 (Ig1&2) domains to its GAG ligands on the HSPG syndecan-4 (SDC4) promotes PTPRS activity and reverses the pathogenic phenotype of FLS. The potential for therapeutically leveraging other PTPRS ectodomain regions is, however, unknown. We show targeting the membrane-proximal fibronectin type III-like 9 (Fn9) domain offers a novel avenue to activate PTPRS. We mapped PTPRS Fn9 as the binding site of three antibodies (Abs) (13G5, 22H8, 49F2) and characterized their effects on cells. Despite sharing similar epitopes, we found large differences in the ability of these Abs to regulate PTPRS activity. One of these, 13G5, reduced PTPRS-dependent cell migration, PTPRS co-localization with SDC4, and PTPRS oligomerization. Single-chain variable fragment Abs of 13G5 and 22H8 were similarly effective at activating cellular PTPRS as 13G5. Replacing the entire 13G5 constant region enhanced its binding and cellular activity, indicating the Ab's potency can be optimized via isotype engineering. Treatment of cells with recombinant Fn9 protein acted as a decoy, disrupting PTPRS colocalization with SDC4 and oligomerization, and inhibiting FLS migration. Finally, significant disease mitigation in mice using 13G5-derived Abs suggests a viable strategy for the generation of novel drugs for RA therapy.

Nano-Polymers as Cas9 Inhibitors

Polymers · 2025-02-05

Despite wide applications of CRISPR/Cas9 technology, effective approaches for CRISPR delivery with functional control are limited. In an attempt to develop a nanoscale CRSIPR/Cas9 delivery platform, we discovered that several biocompatible polymers, including polymalic acid (PMLA), polyglutamic acid (PGA), and polyaspartic acid (PLD), when conjugated with a trileucine (LLL) moiety, can effectively inhibit Cas9 nuclease function. The Cas9 inhibition by those polymers is dose-dependent, with varying efficiency to achieve 100% inhibition. Further biophysical studies revealed that PMLA-LLL directly binds the Cas9 protein, resulting in a substantial decrease in Cas9/sgRNA binding affinity. Transmission electron microscopy and molecular docking were performed to provide a possible binding mechanism for PMLA-LLL to interact with Cas9. This work identified a new class of Cas9 inhibitor in nano-polymer form. These biodegradable polymers may serve as novel Cas9 delivery vehicles with a potential to enhance the precision of Cas9-mediated gene editing.

IMMU-05. KROS 101: A next-generation GITR ligand agonist boosting anti-tumor T cell responses to glioblastoma and reprogramming the tumor microenvironment

Neuro-Oncology · 2025-11-01

Abstract Glucocorticoid-induced Tumor Necrosis Factor Receptor (GITR) and its ligand GITRL, members of the TNFR superfamily, play critical roles in T cell activation and modulation. GITR is expressed on activated T cells and Tregs, while GITRL is found on APCs, including dendritic cells and macrophages. GITR-GITRL interaction induces trimerization, essential for signaling and T cell activation. In humans, trimerization enhances T cell proliferation and suppresses Tregs. However, clinical trials with GITR agonist antibodies in humans have shown limited efficacy, likely due to insufficient GITRL trimerization. To address this, we analyzed soluble human GITRL and found an equilibrium between dimers and trimers. We developed KROS 101, a novel small molecule GITR agonist, to stabilize GITRL trimerization and enhance signaling. Surface plasmon resonance assays confirmed high-affinity binding of KROS 101 to hGITRL. Functional assays demonstrated that KROS 101 significantly increased CD3+ T cell proliferation, particularly CD4+ and CD8+ subsets, while suppressing Tregs. Treated T cells exhibited enhanced cytotoxicity against glioblastoma (GBM) cell lines and patient-derived glioma cancer stem cells (CSCs), showing superior anti-tumor activity compared to controls. To evaluate in vivo effects, we used a humanized GITR/GITRL double knock-in mouse model. In a melanoma model (B16-F10-Luc2), KROS 101 significantly inhibited tumor growth, with reduced luminescence intensity and smaller tumors compared to those treated with the clinical-stage GITR antibody TRX518. Immune profiling revealed increased CD3+ and CD8+ T cell infiltration and reduced Tregs within the tumor microenvironment (TME). KROS 101 also enhanced effector T cell populations, shifting the TME towards an effective anti-tumor response. These effects surpassed those observed with TRX518. In summary, KROS 101 is a potent GITR agonist that promotes T cell infiltration, proliferation, and cytotoxicity while reducing Treg-mediated suppression. These findings support further development of KROS 101 for clinical trials in cancer immunotherapy, particularly for melanoma and glioblastoma.

Butyrophilin 2A2 promotes T cell immunoregulation by enhancing CD45 phosphatase activity within the immune synapse

bioRxiv (Cold Spring Harbor Laboratory) · 2024-11-15

ABSTRACT B7 costimulatory family member Butyrophilin 2A2 ( BTN2A2) is predominantly expressed by antigen presenting cells and regulates T cell immunity, but molecular mechanisms are unclear. Using immunoblots analyzing TCR-initiated signaling intermediaries, co-immunoprecipitation studies, confocal microscopy, structural modeling-guided mutational analyses, and microscale thermophoresis, we demonstrate that BTN2A2 directly interacts with CD45RO, resulting in CD45 retention within the immune synapse during TCR activation. Recombinant BTN2A2 increased murine CD4+Foxp3+ regulatory T cells (Treg) and reduced T helper 17 (Th17) cells in vitro through mechanisms dependent on CD45 phosphatase activity. BTN2A2 treatment reduced clinical expression of two murine autoimmune disease models and increased Treg/Th17 ratios. Analyses of BTN2A2-deficient animals showed exacerbated disease associated with reduced Treg/Th17 ratios. Addition of BTN2A2 to human mixed lymphocyte responses similarly enhanced human Treg and suppressed Th17 cells and was CD45 phosphatase dependent. Together, our studies identify BTN2A2 as a physiological CD45RO ligand that enhances CD45 phosphatase activity in murine and human T cells, providing mechanisms for BTN2A2-mediated amelioration of autoimmunity. Summary Butyrophilin 2A2 ameliorates autoimmunity by binding to CD45RO on activated T cell surfaces leading to dampened TCR signaling which in turn leads to expansion of T regulatory cells and reduction of Th17 differentiation.

296 Towards Nose-to-brain Inhibition of NFB: Activity of a Novel p65 Inhibitor in Glioblastoma and Feasibility of Intranasal Delivery With a Near-infrared Fluorescent HMC-Chitosan Nanoparticle

Neurosurgery · 2024-03-15

INTRODUCTION: Constitutive NFκB inflammatory signaling is a driving force of tumor progression, immune evasion, and therapeutic resistance in glioblastoma (GBM). CRL1101 is a novel small molecule inhibitor of RelA (p65), a transcription factor of NFκB-mediated gene expression. Previously, we have shown that heptamethine carbocyanine (HMC) conjugated nanoparticles target GBM tumor sites via organic anion transporter proteins. The activity of CRL1101 in GBM has not been explored. Poor blood-brain barrier (BBB) penetration and theoretical toxicities related to p65 inhibition pose challenges to its implementation in vivo. METHODS: GBM transcriptomic analysis was performed using Gene Profiling Interactive Analysis. In vitro cytotoxicity and migration assays were performed in U87 and GL261. Western blot was used to confirm the mechanism of CRL1101. Quantitative PCR was used to assess the relative expression of NFκB-regulated genes. HMC-chitosan-CRL1101 nanoparticles were synthesized by cross-linking chitosan with HMC and their uptake was visualized using immunocytochemistry. For survival analysis, we implanted GL261 cells into immunocompetent mice and treated intranasally with HMC-chitosan-CRL1101. RESULTS: RelA is overexpressed in GBM relative to normal brain tissue. CRL1101 alone effectively kills U87 cells (IC50 = 16.0 uM) and prevents migration in vitro (p < 0.0001) by disabling the nuclear translocation of RelA. Treatment with CRL1101 was associated with a decrease in IL-8 (p = 0.01) and PD-L1 (p = 0.44) expression. HMC-chitosan-CRL1101 demonstrated an IC50 of 25.23 uM in U87. Near-infrared imaging confirmed intranasal delivery of HMC-chitosan-CRL1101 across the BBB in vivo. CONCLUSIONS: RelA-targeted NFκB inhibition with CRL1101 is a promising immunotherapeutic strategy in GBM. Intranasal nanoparticle delivery is a practical route to bypass the BBB and minimize systemic toxicities in mouse models of GBM, with expected survival benefit in vivo.