Jul 2026· Journal of Immunology· Vol 215· 0 citations
TL;DR
The first FoxP3-specific small molecule degrader which promotes proteasomal degradation of FoxP3 and partially reduces Treg suppressive function is reported, demonstrating proof-of-concept and feasibility of targeting FoxP3, which has been known to be “undruggable”, in a chemical manner by small molecules.
Abstract
T regulatory cell (Treg) infiltration and accumulation within solid tumors leads to tumor immune evasion and is a major barrier to immunotherapy efficacy. Thus, immunotherapeutic strategies aimed at reducing Treg abundance and/or suppressive function hold significant promise for cancer treatment. Efforts to modulate Tregs have largely relied on non-specific molecular or cellular approaches; however, targeting Forkhead box protein 3 (FoxP3), the master transcription factor for Tregs holds significant promise. Here, we report the first FoxP3-specific small molecule degrader which promotes proteasomal degradation of FoxP3 and partially reduces Treg suppressive function.
To identify potential hits, we used high content imaging to screen a 640 small, electrophilic compound library. The top hits were resynthesized and validated by flow cytometry in human Treg-like MT-2 cells, leading to identification of our lead compound, termed FD03. To explore the mechanism by which FD03 mediated FoxP3 reduction, we employed co-immunoprecipitation and immunoblotting techniques. We then evaluated therapeutic efficacy of our compound in ex vivo and in vivo settings using flow cytometry as a readout.
After identifying FD03 as the lead compound, we further characterized its efficacy finding that FD03 had a EC50 of ∼5-10 uM in primary murine and human Tregs. Mechanistically, FD03 degrades FoxP3 in part through facilitating FoxP3 interaction with its E3 ligase, STUB1, to promote FoxP3 ubiquitination-mediated proteasomal degradation. Finally, we characterized the therapeutic potential of FD03: we illustrated that FD03 can reduce Treg suppressive function. Importantly, we demonstrated that FD03 treatment in tumor-bearing mice can decrease tumor burden and skew the immune cell landscape towards an inflammatory phenotype.
Overall, we demonstrated proof-of-concept and feasibility of targeting FoxP3, which has been known to be “undruggable”, in a chemical manner by small molecules.
1F31CA287701-01, T32GM15538-10, 1T32GM149439-01
Tumor Immunology: Checkpoints, Prevention, and Treatment (TIPT)
The immunosuppressive tumor microenvironment (TME), orchestrated largely by tumor -infiltrating regulatory T cells (TI -Tregs), represents a critical barrier to effective cancer immunotherapy. This review focuses on the chemokine receptor CCR8, which has recently emerged as a highly promising and specific target for therapeutic intervention due to its preferential and stable expression on highly suppressive TI -Treg subsets. We synthesize current evidence demonstrating that CCR8 is selectively upregulated on TI -Tregs across multiple solid tumor types, where its expression correlates with poor patient prognosis and resistance to immune checkpoint inhibitors (ICIs). This expression pattern —enriched within the tumor but minimal in peripheral blood and normal tissues —provides a compelling rationale for tumor -selective targeting. We critically examine the primary therapeutic strategies under development, including ADCC -enhanced monoclonal antibodies, which have shown the most clinical promise, alongside bispecific antibodies and small molecule antagonists. While early -phase clinical trials report encouraging safety and on -target pharmacodynamic activity, the field faces significant challenges. These include unresolved questions regarding CCR8's functional role versus its value as a targeting biomarker, a lack of validated predictive biomarkers for patient stratification, and the physical barriers to effective drug delivery in solid tumors. This review aims to provide a comprehensive and nuanced understanding of CCR8 biology and to outline the key steps necessary for the successful clinical translation of CCR8-targeted therapies.
Underperformance and dysfunction of CD4+ regulatory T cells (Tregs) has been implicated in the pathogenesis of many autoimmune diseases. As such, multiple approaches toward Treg immunotherapy are being developed, most of which require ex vivo expansion and/or manipulation of Tregs, and in vivo strategies remain challenging to accomplish. Here, we hypothesized that constitutively high expression of the high-affinity IL2 receptor on Tregs could be exploited to target selective uptake of mRNA-LNPs by Tregs, enabling the potential development of an in vivo immunotherapy platform.
IL2 or αCD25 was conjugated to LNPs containing N1-Methylpseudouridine-substituted mRNA for eGFP using a SATA-maleimide based strategy. In vivo experiments were done using 6-week-old female C57BL/6 mice given 5 µg of LNP intravenously. In vitro experiments were performed using normal donor human splenocytes or PBMCs. Targeting was assessed using eGFP expression and flow cytometry.
To define the optimal method to target Tregs with mRNA-LNPs, we first compared αCD25 vs IL-2 conjugated LNPs. IL2-LNPs demonstrated superior targeting in vivo of splenic Tregs compared to αCD25-LNPs in both frequency (69.7% vs. 41.9% eGFP+ respectively, p = 0.03) and expression (1399 vs. 424 eGFP MFI, p = 0.01) 24 hours after delivery. IL2-LNP targeted Tregs expressing eGFP were present in spleen, lymph node, and blood 24 hours post-treatment (40-70% eGFP+) and remained detectable for at least 7 days. Little background uptake of IL2-LNPs was observed in other immune cell subsets. Finally, IL2-LNPs also delivered mRNA effectively to FoxP3+Tregs in vitro in human splenocytes in a dose-dependent manner with little uptake by other cell subsets.
Altogether, these data demonstrate that IL2-LNPs are an efficient and effective method of targeting Tregs in situ. Future studies will apply this powerful tool to transiently enhance and alter Treg function in vivo for interventional and therapeutic strategies in autoimmune disease models.
Breakthrough T1D (3-SRA-2024-1612-S-B)
Therapeutic Approaches to Autoimmunity (THER)
Erin Maule, Amie Albertus, Vladimir Shuvaev et al.· Journal of Immunology· 1 citation
Deletion of Satb1 specifically in Tregs impaired the function of Satb1+ pro-tumorigenic Tregs, leading to enhanced CD8+ T cell antitumor immune responses, and complete tumor eradication without any systemic autoimmune conditions.
Ephraim A. Ansa-Addo, Parviz Azimnasab-sorkhabi, Musab Bouhajra et al.· Journal of Immunology· 0 citations
Regulatory T cells (Tregs) are key modulators of immune responses and are increasingly targeted for their therapeutic potential in hematologic malignancies, autoimmune diseases, and immune-mediated complications of hematopoietic cell transplantation. Robust human Treg assays are therefore essential to evaluate novel targets and therapeutic modalities. However, due to experimental challenges in isolation, expansion, and optimization of reliable functional readouts, progress has been slow and results remain variable. In this study, we established a human Treg platform enabling high-purity isolation, expansion, functional profiling, and gene modification, using the oncogenic deubiquitinase USP22 as a reference target.
In our hands, Tregs achieved >90% purity (CD4+CD25+FOXP3+) with 92% viability and maintained a stable phenotype through Days 7, 15, and 18, reaching approximately 60–80-fold expansion before plateauing. Expanded Tregs demonstrated potent suppressive activity, with reduced responder T-cell proliferation as Tresp:Treg ratios decreased from 32:1 to 2:1, accompanied by increased anti-inflammatory cytokine production. Genome editing of USP22 was efficient and compatible with downstream proliferation, phenotypic stability, and cytokine analyses. Approximately 60% knockdown of USP22 led to a ∼4-fold increase in IFN-γ mRNA expression and loss of FOXP3 at both mRNA and protein levels, accompanied by reduced IL-2 mRNA expression, while IL-2RA (CD25) and CTLA-4 mRNA levels remained unchanged.
We developed a human Treg workflow that integrates high-purity isolation, robust expansion, and functional characterization with gene modification capability. USP22 perturbation demonstrates the utility of this system in dissecting molecular regulators of Treg function. This flexible platform can be adapted for diverse gene targets, including deubiquitinases such as USP22, to support preclinical immunology and cell therapy research relevant to hematologic and autoimmune diseases.
Shereen Kesserwan, Sarju Patel, Madison Greer, Sukanya Jadhav, Palin Biner, Ajita V. Singh. A gene-editable human regulatory T-cell platform reveals USP22 as a regulator of FOXP3 stability and IFN-γ production [abstract]. In: Proceedings of AACR Drug Discovery and Development (AACR D3) Conference; 2026 Jul 21-24; Boston, MA. Philadelphia (PA): AACR; Clin Cancer Res 2026;32(14_Suppl):Abstract nr B063.
Shereen Kesserwan, Sarju Patel, Madison Greer et al.· Clinical Cancer Research· 0 citations
Background: Successful translation of cancer immunotherapy is underscored by the efficacy of PD-1/PD-L1 and CTLA-4 inhibitors in the treatment of various malignancies. However, their limited efficacy in glioma indicates alternative immune escape mechanisms. We investigated B and T Lymphocyte Attenuator (BTLA), a co-inhibitory receptor structurally and functionally analogous to PD-1, to determine if it constitutes a key, unaddressed mechanism of immune escape and a novel therapeutic target in glioma. Methods: We analyzed BTLA expression and function within the tumor microenvironment of a Moroccan cohort (n = 44). This was complemented by multiparameter flow cytometry on peripheral blood from glioblastoma (GBM) patients (n = 8) to assess circulating T cell profiles. Findings were corroborated using independent transcriptomic datasets from TCGA and CGGA cohorts. Single-cell RNA-seq and citeSeq identified specific BTLA-expressing cell populations. Results: Elevated BTLA expression was significantly associated with aggressive features and poor overall survival in glioma patients. Mechanistically, BTLA levels were positively correlated with pro-tumorigenic factors, immune infiltration, and immunosuppressive checkpoints. Single-cell and citeSeq analyses revealed that BTLA was primarily expressed by exhausted T cells and conventional type 1 dendritic cells (cDC1) within the GBM microenvironment. Crucially, this phenotype was translated systemically; BTLA defined dysfunctional circulating CD8+ and CD4+ T cells characterized by diminished IFN-γ production, alongside reduced granzyme B and perforin in CD8+ T cells. Conclusions: Our findings indicate that BTLA may represent a relevant pathway associated with an immunosuppressive glioma microenvironment. The therapeutic potential of targeting this pathway, particularly in combination with PD-1/PD-L1 blockade, warrants further investigation.
S. Souat, Khadija El Azhary, Sara Bourdoukh et al.· Medical Science· 0 citations
Summary Cancer immunotherapy has made significant advancements, as immune checkpoint inhibitors and cytokines are widely applied in the clinic. Nonetheless, these therapies are often challenged by severe side effects arising from inappropriate activation of the immune system or systemic toxicity. Herein, we constructed a tumor-selective, safe therapeutic gene cassette specifically activated by NF-κB to produce cytokine IL-15 and microRNAs silencing two key immune checkpoints, PD-L1 and CD47. High NF-κB activity in cancer cells drove expression of the cassette to produce IL-15 and suppress PD-L1 and CD47, while low basal NF-κB rendered the cassette silent in most normal cells. This design simultaneously activated and enhanced adaptive and innate immunity. A recombinant adeno-associated virus (AAV2) delivering the cassette showed significant antitumor effects, favorable tumor selectivity, and biosafety across multiple murine solid tumor models. Consequently, this treatment may offer a potentially more effective and safer immunotherapy against cancers in the future.
