Depletion Of Treg Cells Research Articles

A phase 1 study of interleukin-15 in combination with mogamulizumab in relapsed and refractory T-cell malignancies

IntroductionRecombinant human interleukin-15 (rhIL-15) is an immunotherapeutic which enhances NK-cells to augment the antibody directed cellular cytotoxicity (ADCC) of monoclonal antibodies. Mogamulizumab is a CCR4 directed monoclonal antibodies that exerts cytotoxicity through ADCC and depletes regulatory T-cells within the tumor microenvironment. MethodsWe conducted a phase 1 clinical trial of rhIL-15 combined with mogamulizumab. Patients with relapsed or refractory adult T-cell leukemia/lymphoma (ATLL), mycosis fungoides (MF) and Sezary syndrome (SS) received fixed dose mogamulizumab combined with escalating doses of rhIL-15 to identify the maximum tolerated dose (MTD). ResultsSix patients were enrolled, 4 with ATLL and 2 with MF/SS. The most common adverse events were rash, infection and fever (67%, in all). Two patients (33%) had grade 4 acute kidney injury and 25% of cycles had grade ≥3 anemia. The MTD was dose level 1. One patient with ATLL had a partial response despite receiving only 4 cycles due to grade 4 myositis. Circulating NK cells were increased in all patients during the first cycle and a rapid reduction in tumor cells within the peripheral circulation was noted. Ex vivo assessment demonstrated increased NK cell activation and increased cell lysis in the presence of monoclonal antibodies after only 5 days. DiscussionOur small study suggests that rhIL-15 combined with mogamulizumab leads to effector NK cell activation and regulatory T-cell depletion but has an unfavorable safety profile. Future development of combinations of immunotherapy that target the microenvironment in relapsed or refractory T-cell lymphomas remain rational. NCT04185220

Development of a humanized mouse model with functional human materno-fetal interface immunity.

Materno-fetal immunity possesses specialized characteristics to ensure pathogen clearance while maintaining tolerance to the semiallogeneic fetus. Most of our understanding on human materno-fetal immunity is based on conventional rodent models that may not precisely represent human immunological processes owing to the huge evolutionary divergence. Herein, we developed a pregnant human immune system (HIS) mouse model through busulfan preconditioning, which hosts multilineage human immune subset reconstitution at the materno-fetal interface. Human materno-fetal immunity exhibits a tolerogenic feature at the midgestation stage (embryonic day [E] 14.5), and human immune regulatory subsets were detected in the decidua. However, the immune system switches to an inflammatory profile at the late gestation stage (E19). A cell-cell interaction network contributing to the alternations in the human materno-fetal immune atmosphere was revealed based on single-cell RNA-Seq analysis, wherein human macrophages played crucial roles by secreting several immune regulatory mediators. Furthermore, depletion of Treg cells at E2.5 and E5.5 resulted in severe inflammation and fetus rejection. Collectively, these results demonstrate that the pregnant HIS mouse model permits the development of functional human materno-fetal immunity and offers a tool for human materno-fetal immunity investigation to facilitate drug discovery for reproductive disorders.

Preclinical investigations and a first-in-human phase 1a trial of JS007, a novel anti-CTLA-4 antibody, in patients with advanced solid tumors

Abstract Background Blocking cytotoxic T lymphocyte-associated antigen-4 (CTLA-4) shows substantial antitumor efficacy. Here, we report the preclinical data and outcomes of a first-in-human phase 1a trial of JS007, a novel anti-CTLA-4 antibody, in advanced solid tumors. Methods In preclinical studies, both in vitro characteristics and in vivo characteristics of JS007 were investigated. The clinical trial included a dose escalation phase and a dose expansion phase. Eligible patients with previously treated advanced solid tumors were enrolled. In the dose escalation phase, JS007 was administered intravenously every 3 weeks at doses of 0.03, 0.3, 1, 3, and 10 mg/kg. Then, 3 and 10 mg/kg were chosen for the dose expansion phase. The primary endpoints included the maximum tolerated dose (MTD) of JS007 based on dose-limiting toxicities (DLTs) and safety. Results JS007 could effectively bind to CTLA-4 and induce an immune response in vitro. Potent in vivo antitumor activity of JS007 was observed. Increased T cell infiltration and T regulatory (Treg) cell depletion in tumor microenvironment of cancer cell xenografts were detected after treated with JS007. Pharmacological analysis in experimental animals showed a dose-proportional increase in exposure. In the clinical trial, a total of 28 patients were treated with JS007 across 5 dose levels. No DLTs occurred. The MTD did not reach at the highest dose tested (10 mg/kg). Twenty-three (82.1%) patients experienced at least one treatment-related adverse event (TRAE). The incidence of Grade ≥ 3 TRAEs was 28.6% (8/28) with alanine aminotransferase increase (7.1%, 2/28) being the most frequently reported TRAE. No severe immune-related adverse event (irAE) occurred. Pharmacological profiles of JS007 in patients were similar to those in animal models. Serum concentration of JS007 showed a dose-dependent escalation, and the half-life of JS007 was 9.4 ~ 12.2 days. Treatment-induced anti-drug antibody was detected in 2 patients. The disease control rate was 50% (14/28), and the median overall survival was 14.7 months. Conclusions JS007 preliminarily demonstrates good tolerance and encouraging antitumor activity in patients with previously treated advanced solid tumors. Trial registration ClinicalTrials.gov identifier: NCT05049265 (Sep 20, 2021).

A CD25×TIGIT bispecific antibody induces anti-tumor activity through selective intratumoral Treg cell depletion

Intratumoral regulatory T cells (Tregs) express high levels of CD25 and TIGIT, which are also recognized as markers of effector T cell (Teff) activation. Targeting these molecules each alone with monoclonal antibodies (mAbs) poses a risk of concurrently depleting both Teffs and peripheral Tregs, thereby compromising the effectiveness and selectivity of intratumoral Treg depletion. Here, leveraging the increased abundance of CD25+ TIGIT+ double positive Tregs in the solid tumor microenvironment (but not in peripheral tissues), we explored the feasibility of using a CD25×TIGIT bispecific antibody (bsAb) to selectively deplete intratumoral Tregs. We initially constructed a bsAb co-targeting mouse CD25 and TIGIT, NSWm7210, and found that NSWm7210 conferred enhanced intratumoral Treg depletion, Teff activation, and tumor suppression as compared to the parental monotherapies in mouse models. We subsequently constructed a bsAb co-targeting human CD25 and TIGIT (NSWh7216), which preferentially eliminated CD25+ TIGIT+ double positive cells over single positive cells in vitro. NSWh7216 exhibited enhanced anti-tumor activity without toxicity of peripheral Tregs in CD25 humanized mice compared to the parental monotherapies. Our study illustrates the use of CD25×TIGIT bsAbs as effective agents against solid tumors based on selective depletion of intratumoral Tregs.

Irreversible electroporation of localised prostate cancerdownregulates immune suppression andinduces systemic anti-tumour T-cell activation - IRE-IMMUNO study.

To prospectively compare systemic anti-tumour immune responses induced by irreversible electroporation (IRE) and robot-assisted radical prostatectomy (RARP) in patients with localised intermediate-risk prostate cancer (PCa). Between February 2021 and June 2022, before and after treatment (at 5, 14 and 30 days) peripheral blood samples of 30 patients with localised PCa were prospectively collected. Patient inclusion criteria were: International Society of Urological Pathologists Grade 2-3, clinical cancer stage ≤T2c, prostate-specific antigen level <20 ng/mL). Patients were treated with IRE (n = 20) or RARP (n = 10). Frequency and activation status of lymphocytic and myeloid immune cell subsets were determined using flow cytometry. PCa-specific T-cell responses to prostatic acid phosphatase(PSAP) and cancer testis antigen (New York oesophageal squamous cell carcinoma 1 [NY-ESO-1]) were determined by interferon-γ enzyme-linked immunospot assay (ELISpot). Repeated-measures analysis of variance and two-sided Student's t-tests were used to compare immune responses over time and between treatment cohorts. Patient and tumour characteristics were similar between the cohorts except for age (median 68 years [IRE] and 62 years [RARP], P = 0.01). IRE induced depletion of systemic regulatory Tcells (P = 0.0001) and a simultaneous increase in activated cytotoxic T-lymphocyte antigen 4 (CTLA-4)+ cluster of differentiation (CD)4+ (P < 0.001) and CD8+ (P = 0.032) Tcells, consistent with reduction of systemic immune suppression allowing for effector T-cell activation, peaking 14 days after IRE. Effects were positively correlated with tumour volume/ablation size. Accordingly, IRE induced expansion of PSAP and/or NY-ESO-1 specific T-cell responses in four of the eight immune competent patients. Temporarily increased activated myeloid derived suppressor cell frequencies (P = 0.047) were consistent with transient immunosuppression after RARP. Irreversible electroporation induces a PCa-specific systemic immune response in patients with localised PCa, aiding conversion of the tumour microenvironment into a more immune permissive state. Therapeutic efficacy might be further enhanced by combination with CTLA-4 checkpoint inhibition, potentially opening up a new synergistic treatment paradigm for high-risk localised or (oligo)metastatic disease.

Low-Dose Interleukin-2 Reverses Traumatic Brain Injury-Induced Cognitive Deficit and Pain in a Murine Model.

Despite the high prevalence, mild traumatic brain injury (mTBI)-induced chronic headache and cognitive deficits are poorly understood and lack effective treatments. Low-dose interleukin-2 (LD-IL-2) treatment soon after mTBI or overexpressing IL-2 in brain astrocytes prior to injury protects mice from developing post-traumatic headache (PTH)-related behaviors and cognitive decline. The present study addresses a clinically relevant knowledge gap: whether LD-IL-2 treatment long after the initial injury is still effective for chronic PTH and cognitive deficits. mTBI was induced by a noninvasive closed-head weight drop method. LD-IL-2 was administered 4-6 weeks post-mTBI to assess its effects on chronic PTH-related facial mechanical hypersensitivity as well as mTBI-induced impairment in novel object recognition and object location tests. Endogenous regulatory T (Treg) cells were depleted to investigate the mechanism of action of LD-IL-2. Delayed LD-IL-2 treatment abolished chronic PTH-related behaviors. It also completely reversed mTBI-induced cognitive impairment in both male and female mice. Treg cell depletion not only prolonged PTH-related behaviors but also abolished the effects of LD-IL-2. Interestingly, LD-IL-2 treatment significantly increased the number of Treg cells in dura but not in brain tissues. These results suggest that the beneficial effects of LD-IL-2 treatment are mediated through the expansion of meningeal Treg cells. Collectively, our study identifies Treg as a cellular target and LD-IL-2 as a promising therapy for both chronic PTH and mTBI-induced cognitive impairment for both males and females, with a wide therapeutic time window and the potential of reducing polypharmacy in mTBI treatment. ANN NEUROL 2024;96:508-525.

Epitope Mapping of an Anti-Mouse CCR8 Monoclonal Antibody C8Mab-2 Using Flow Cytometry.

The C-C motif chemokine receptor 8 (CCR8) is highly and selectively expressed in regulatory T (Treg) cells and is associated with tumor progression. The massive accumulation of Treg cells into tumors suppresses the effector function of CD8+ cells against tumor cells. Therefore, selective depletion of Treg cells using anti-CCR8 monoclonal antibodies (mAbs) reinvigorates antitumor immune responses and improves responses to cancer immunotherapy. Previously, we developed an anti-mouse CCR8 (mCCR8) mAb, C8Mab-2, using the Cell-Based Immunization and Screening method. In this study, the binding epitope of C8Mab-2 was investigated using flow cytometry. The mCCR8 extracellular domain-substituted mutant analysis showed that C8Mab-2 recognizes the N-terminal region (1-33 amino acids) of mCCR8. Next, 1×alanine (or glycine) scanning and 2×alanine (or glycine) scanning were conducted in the N-terminal region. The results revealed that the 17-DFFTAP-22 sequence is important for the recognition by C8Mab-2, and Thr20 is a central amino acid of the epitope. These results revealed the involvement of the N-terminus of mCCR8 in the recognition by C8Mab-2.

Phase I/II safety and preliminary efficacy of PM1022, a bispecific antibody targeting PD-L1 and TIGIT, in patients with advanced solid tumors.

e14694 Background: PM1022 is an anti-PD-L1 × TIGIT bispecific antibody that is engineered with a fully human IgG targeting TIGIT in an IgG1 wild type Fc backbone, fused to a VHH at the c-terminus targeting PD-L1. The potential MOA includes releasing T cell inhibition by blocking both the PD-L1/PD-1 and CD155/CD112/TIGIT pathways, as well as depleting Treg cells in the tumor microenvironment and engaging FcγRs to activate myeloid cells. PM1022 showed robust in vitro and in vivo efficacy in preclinical studies, and this is the first in human study. Methods: This phase Ⅰ/Ⅱ study consists of a standard 3+3 dose escalation and dose expansion part. PM1022 was administered intravenously as monotherapy on Day 1 of 21-day cycle. The primary objectives were to assess safety, tolerability and pharmacokinetics in patients with advanced solid tumors. Results: As of January 28, 2024, a total of 15 patients in dose escalation part have received at least 1 dose of PM1022, with 3 patients in each of the 100, 200, 400, 800 and 1200 mg dose levels respectively. No DLT was observed up to 1200 mg. Of the 15 patients, TRAEs occurred in 8 patients (53.3%), ≥ Grade 3 TRAEs occurred in 1 patient with platelet count decreased. No patients were discontinued from PM1022 due to TRAE. The most common TRAEs were rash (20%) and aspartate transaminase increased (20%). 14 patients completed at least one tumor evaluation. The median duration of PM1022 exposure was 6.1 weeks (range, 5.0-48.0 weeks). The ORR per RECIST 1.1 was 7.1% with a DCR of 35.7%. Preliminary antitumor activity has been observed in 1 patient with NSCLC (1200 mg, still on treatment) with previous PD-L1 TPS 100% who was enrolled after previous 3 lines therapy, including chemotherapy and anti-PD-1 treatment, and had a partial response in target lesion with 56.8% reduction. Another patient with esophageal cancer had been on PM1022 for total of 48 weeks until initiation of new anti-cancer therapy. Pharmacokinetics were dose-proportional with a terminal half-life of 7-13 days across the dose range of 100-1200 mg. Conclusions: PM1022 was safe and well-tolerated up to 1200 mg Q3W with preliminary anti-tumor activity. These findings could support further exploration of PM1022 in advanced solid tumors. Clinical trial information: NCT05867771 .

Phase I safety and preliminary efficacy of PM1009, a bispecific antibody targeting TIGIT and PVRIG, in patients with advanced solid tumors.

e14695 Background: PM1009 is an anti-TIGIT x PVRIG bispecific antibody containing a fully human anti-TIGIT IgG1 fused with a fully human anti-PVRIG scFv at the C terminus. PM1009 can potently block the TIGIT/CD155/CD112 and PVRIG/CD112 signaling pathways, and supports the depletion of TIGIT-positive Treg cells in the tumor microenvironment through FcγR engagement. This leads to the enhancement of T cell and NK cell activity towards the killing of tumor cells. PM1009 showed robust in vitro and in vivo efficacy with good safety in preclinical studies. Methods: This is the first-in-human study of PM1009, which includes a dose escalation stage (accelerated titration &amp; 3+3 design) followed by dose expansion. In the dose escalation stage, PM1009 was administered as flat doses of 120, 300, 600 and 1200 mg for DLT evaluation after 3 weeks followed by administration Q2W. Primary endpoints were safety and tolerability, and the secondary endpoints were PK, ORR and DCR (per RECIST 1.1). Results: As of February 1, 2024, a total of 12 patients were enrolled for dose escalation and received at least one dose of PM1009, with 3 patients in each of the 120, 300, 600 and 1200 mg dose-levels. Median number of prior therapies was 5 lines (range 1- 9). No DLTs were observed up to 1200 mg. Any-grade TRAEs occurred in 11 patients (91.7%), and ≥ Grade 3 TRAEs occurred in 2 patients (16.7%). Any-Grade irAEs occurred in 7 patients (58.3%), and ≥ Grade 3 irAEs occurred in 1 patient (8.3%). Treatment interruption due to TRAE occurred in 2 patients (16.7%), with infusion related reaction (the main symptom was nausea, and the single infusion was eventually completed), and neutrophil count decrease respectively. Treatment discontinuation due to TRAE occurred in 2 patients (16.7%), with acute myocardial infarction, and neutrophil count decrease respectively. The most common TRAEs were ALP elevation (33.3%) and AST elevation (25.0%). 12 patients completed at least one tumor evaluation. The DCR per RECIST 1.1 by investigator was 33.3% (4/12; 95% CI: 9.92 - 65.11), best overall response was SD. Pharmacokinetics were dose-proportional with a terminal half-life of 5.2-7.4 days across the dose range of 120-1200 mg. Conclusions: PM1009 was well-tolerated up to 1200 mg with signs of anti-tumor activity. These findings support further clinical investigation of PM1009 especially for the combination with chemotherapy or PD-1/L1-based checkpoint inhibitors. Clinical trial information: NCT05607563 .

Quercetin exerts anti-tumor immune mechanism by regulating IL-6/JAK2/STAT3 signaling pathway to deplete Treg cells

Breast cancer is still the leading cause of death among women worldwide. Due to the lack of effective drug targets, triple-negative breast cancer has a worse prognosis and higher mortality compared with other types of breast cancer, and chemotherapy is still the main treatment for triple-negative breast cancer at present. Quercetin (QUE) is a flavonoid compound found in a variety of fruits and vegetables. The mechanism of QUE has been extensively studied, such as prostate cancer, colon cancer, ovarian cancer, etc. However, the anti-tumor immune mechanism of QUE in triple-negative breast cancer remains unclear. Therefore, we assessed the anti-tumor immune effects of QUE on triple-negative breast cancer using both 4T1 cells and a xenograft mouse model of 4T1 cells. In vitro, we examined the inhibitory effects of QUE on 4T1 cells and its molecular mechanisms through MTT, Transwell, ELISA, and Western blotting. In vivo, by establishing a xenograft mouse model, we utilized flow cytometry, immunohistochemistry, ELISA, and Western blotting to evaluate the anti-tumor immune effects of QUE on triple-negative breast cancer. The results indicate that QUE inhibits the proliferation, migration, and invasion of 4T1 cells, concurrently significantly suppressing the IL-6/JAK2/STAT3 signaling pathway. Furthermore, it depletes Treg cell content in 4T1 xenograft mice, thereby improving the tumor immune microenvironment and promoting the cytotoxicity of relevant tumor immune cells. These findings suggest that QUE may serve as a potential adjuvant for immune therapy in triple-negative breast cancer.

CD39 expression by regulatory T cells participates in CD8+ T cell suppression during experimental Trypanosoma cruzi infection.

An imbalance between suppressor and effector immune responses may preclude cure in chronic parasitic diseases. In the case of Trypanosoma cruzi infection, specialized regulatory Foxp3+ T (Treg) cells suppress protective type-1 effector responses. Herein, we investigated the kinetics and underlying mechanisms behind the regulation of protective parasite-specific CD8+ T cell immunity during acute T. cruzi infection. Using the DEREG mouse model, we found that Treg cells play a role during the initial stages after T. cruzi infection, restraining the magnitude of CD8+ T cell responses and parasite control. Early Treg cell depletion increased the frequencies of polyfunctional short-lived, effector T cell subsets, without affecting memory precursor cell formation or the expression of activation, exhaustion and functional markers. In addition, Treg cell depletion during early infection minimally affected the antigen-presenting cell response but it boosted CD4+ T cell responses before the development of anti-parasite effector CD8+ T cell immunity. Crucially, the absence of CD39 expression on Treg cells significantly bolstered effector parasite-specific CD8+ T cell responses, preventing increased parasite replication in T. cruzi infected mice adoptively transferred with Treg cells. Our work underscores the crucial role of Treg cells in regulating protective anti-parasite immunity and provides evidence that CD39 expression by Treg cells represents a key immunomodulatory mechanism in this infection model.

Abstract 718: CCR8 antibody drug conjugates: Targeting regulatory T cells for tumor sensitization

Abstract Background: Regulatory T (Treg) cells in the tumor microenvironment have an immune suppressive function, and their presence is associated with poor prognosis. Depletion of Treg cells may be a mechanism to sensitize tumors and represents a novel strategy for cancer immunotherapy. High expression of chemokine receptor CCR8 has been found in Treg cells in microenvironments of numerous tumors but not on Treg cells in systemic lymphoid tissues. The ability to selectively kill Treg cells in tumor environments may provide an opportunity for therapy that leaves Treg function intact elsewhere in the body where they normally mediate tolerance to self-antigen. Methods: We sought to identify CCR8 antibodies for therapeutics and designed an antibody discovery strategy to address the challenges of this target. CCR8 is a member of the G protein-coupled receptor (GPCR) family. Like many GPCRs, CCR8 is a valuable therapeutic antibody target but challenging to access due to its poor expression, membrane-dependent structure, and poor immunogenicity due to sequence conservation. We developed an antibody discovery platform (MPS) that specifically addresses each of these challenges through use of advanced immunization techniques and evolutionarily divergent host species (chickens) for robust immune responses against conserved targets. We extensively engineered CCR8 antigen to enable high expression for immunization. When sufficient antibody diversity was not obtained using our standard immunization techniques, we developed mRNA immunization techniques for this program. Antibody binding was measured using flow cytometry on transfected cells and human Treg cells. The ability of antibodies to inhibit CCR8 function was measured using intracellular calcium influx assays. Antibody internalization was measured using dye uptake assays and flow cytometry. Antibodies were conjugated with cytotoxic payloads and tested for the ability to mediate killing. Results: Using these approaches, we identified a panel of high-affinity antibodies that bound in the nanomolar or subnanomolar range with high specificity, and we selected a lead molecule to progress. We will present data from our antibody discovery campaign including mRNA immunization, binding profiles, GPCR functional data, receptor-mediated internalization, and the ability of antibody-drug conjugates (ADC) to kill CCR8+ cells, including human Treg cells, in vitro. Conclusion: Treg cells are a primary driver of the immunosuppressive microenvironment in many solid tumors. Targeting immune-suppressive CCR8+ Treg cells using ADC has potential to expand the treatment of solid tumors Citation Format: Lewis J. Stafford, Hayley Roth, Sarah Campbell, Samantha Brady, Valerie Fiers, FeiFan Yu, Andrew Tsourkas, Riley Payne, Meghan Pitts, Kyle Guldner, Tim Phillips, Breanna Tyrell, Colleen Ulke, Allison Snyder, Marianne Assogba, Holden Ohl, Ileine Sanchez, Benjamin Doranz, Joseph Rucker, Ross Chambers. CCR8 antibody drug conjugates: Targeting regulatory T cells for tumor sensitization [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 718.

Abstract 6659: Oncolytic herpes simplex virus expressing XCL1 and FLT3L modulate the intratumoral immune response and improve the anti-tumor efficacy in a metastatic breast tumor model

Abstract Patients with metastatic breast cancer (MBC) have a dismal prognosis, with a 5-year survival rate of only 26%. The inadequate infiltration of immune cells targeting the tumor microenvironment (TME) is a key factor contributing to this low survival rate. Immune effector cells, including CD8+ cytotoxic T cells, NKT cells, and NK cells, play a crucial role in targeting tumor cells. The activation of immune effector cells relies on the presence of conventional type I dendritic cells (cDC1), which present tumor-specific antigenic peptides on their surface. Recent studies have identified the chemokine XCL1 and growth factor FLT3L as critical factors involved in the intratumoral recruitment and expansion of dendritic cells. To enhance the activity of cDC1 in the TME we designed an oncolytic herpes simplex virus (oHSV) expressing XCL1 and FLT3L. The recombinant oHSV, rQ1-XF, was generated by incorporating LoxP sites into the viral genome through recombination with a carrier plasmid carrying the polycistronic cassette. To enhance the intratumoral penetration of oHSV injected intravenously in primary tumors and MBC lesions in the lung, we employed the cancer drug ganetespib, which can increase the permeability of tumor blood vessels. In comparison to untreated mice, ganetespib combined with rQ1-XF reduced the size of primary tumors and induced a short but significant increase in the survival of mice. Additionally, compared to the ganetespib alone treatment, ganetespib combined with rQ1-XF reduced MBC burden in the lung. The flow cytometry analysis of immune cells revealed an increased infiltration of CD11c+CD11b- dendritic cells in primary tumors treated with oHSV combination therapy compared to the ganetespib only control. A similar trend was also observed in CD4 T cells and CD8 T cells in primary tumors. As expected, there was a negative correlation between tumor burden and the infiltration of CD4+ T cells and CD8+ T cells in primary tumors. Ganetespib combined with oHSV also reduced the infiltration of CD4+ regulatory T cells (Tregs) in primary tumors, however the number of Tregs increased with primary tumor size as well as tumor burden in the lung. This could potentially lead to reduced efficacy of tumor-infiltrating dendritic cells in activating effector T cells in tumors treated with ganetespib and rQ1-XF. Moreover, the combination therapy did not yield significant alterations in the populations of DCs, CD4+ T cells, and CD8+ T cells in lungs with metastatic lesions. Further studies are underway to investigate whether the depletion of Treg cells in combination with rQ1-XF enhances effector T cell activation against metastatic breast tumor cells. The findings of this study will provide valuable insights into the effectiveness of using XCL1 and FLT3L-armed oncolytic viruses in patients with MBC. Citation Format: Himanshu Soni, Liqun Gu, Sylvie Roberge, Heena Kumra, Hiroshi Nakashima, E. Antonio Chiocca, Yves Boucher. Oncolytic herpes simplex virus expressing XCL1 and FLT3L modulate the intratumoral immune response and improve the anti-tumor efficacy in a metastatic breast tumor model [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6659.

Abstract 4123: Attenuating breast cancer metastasis with BCL-XL-targeting PROTACs

Abstract B-cell lymphoma X-large (BCL-XL) emerges as a significant player in cancer progression. In our recent findings, we unveil the efficacy of proteolysis-targeting chimeras (PROTACs) directed against BCL-XL, demonstrating their capacity to not only eliminate cancer cells but also target tumor-induced regulatory T cells (TI-Tregs). Notably, BCL-XL's role in promoting breast cancer metastasis extends beyond its anti-apoptotic function. We posit that BCL-XL-targeting PROTACs (BCL-XL-Ps) hold promise for treating metastatic breast cancer. Their potential lies in directly eliminating circulating tumor cells during vulnerable stages and/or triggering anti-cancer immunity through Treg cell depletion. Our experiments underscore the potency of IAP-based PROTACs in facilitating BCL-XL degradation via the ubiquitin-proteasome system (UPS) while mitigating on-target platelet toxicity. Treatment of breast cancer-bearing mice with these compounds demonstrates a tangible reduction in breast cancer metastasis to the lungs. Additionally, our studies reveal a significant depletion of tumor-infiltrating Tregs in mice treated with BCL-XL-Ps. While affirming BCL-XL as a viable therapeutic target for breast cancer metastasis, further exploration into the mechanisms behind BCL-XL-P targeting is imperative. Our future endeavors will be devoted to unraveling whether our BCL-XL-Ps effectively target both tumor-intrinsic BCL-XL, potentially inducing tumor cell death, and tumor-infiltrating Tregs, thereby fostering an adaptive immune response against the tumor cells. Citation Format: Madison E. Carelock, Peiyi Zhang, Mo Jiao, Umasankar De, Zeng Jin, Rachel M. Stump, Sarah G. Williams, Guangrong Zheng, Weizhou Zhang. Attenuating breast cancer metastasis with BCL-XL-targeting PROTACs [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4123.

Abstract 2358: YH41723 (IMC-202), a Novel TIGITxPD-L1 bispecific antibody, leads to potent anti-tumor immunity through T cell engaging and T/NK cell activation

Abstract Immune checkpoint inhibitors (ICIs) have been making innovative advances in current cancer therapy. In particular, the PD-L1/PD-1 blockades changed the paradigm of anti-cancer treatment, showing high efficiency compared to conventional therapy. However, drug resistance has been observed in many patients receiving PD-1/PD-L1 monoclonal antibodies (mAbs), and there are still many limitations in restoring T cell. To overcome this, various ICI targets including T cell immunoreceptor with Ig and ITIM domain (TIGIT) are being studied. TIGIT is a T cell co-inhibitory receptor, highly expressed on regulatory T (Treg) cells and memory T cells and natural killer (NK) cells. In combination with PD-1/PD-L1 inhibition, the activation of CD226 through the blocking of TIGIT/PVR ligation is important for T cell activation. In this study, we demonstrated the synergistic effect that can be obtained by simultaneously blocking of PD-L1 and TIGIT and effect of T cell engager function using TIGITxPD-L1 bispecific antibody (BsAb). We generated an anti-TIGITxPD-L1 BsAb, YH41723 (IMC-202), with Fc-engineering to enhance the effector function. In order to confirm superior efficacy of YH41723 compared to combination of two monoclonal antibodies, PD-L1 mAb+TIGIT mAb, we verified the CD8+ T cell-mediated tumor killing effect by T cell engager function of YH41723. We also observed strong antibody-dependent cell-mediated cytotoxicity (ADCC) effect of YH41723 against various cancer cell lines. In addition, YH41723 showed superior anti-tumor efficacy than combination treatment in NK cell-mediated tumor killing assay. We further confirmed the significant Treg cell depletion efficacy of YH41723 in vitro compared to combination. We tested in vivo anti-cancer efficacy of YH41723 in hPD-L1 KI CT26 with BALB/c-H/K-dKO mouse models (B cell-deficient mice). As a result, YH41723 showed superior tumor growth inhibition effect compared with anti-PD-L1 mAb and anti-TIGIT mAb combination. Even, 7 of 8 mice showed complete regression in 30 mg/kg dosing group. 60 days after the last administration, we further performed tumor re-challenge test. The tumor-free state of tumor re-challenged mice was sustained, indicating that the immune memory was generated by YH41723. Taken together, YH41723 showed strong efficacy in NK/T cell activation and Treg depletion in vitro and excellent anti-tumor efficacy in vivo, supporting that YH41723 as an effective anti-cancer immunotherapy and combination candidate with other ICIs. Citation Format: Min Hyeok Jee, Young Bong Park, Byeong-Yun An, Hee Ra Jung, Eunjung Lee, Jun Hwan Kim, Ji Eun Park, Ji Young Yoon, Seung Mi Jeong, Yang Kyun Ryu, Sung Ho Kim, Heung Tae Kim, Se-Woong Oh, Yeol Hong Kim. YH41723 (IMC-202), a Novel TIGITxPD-L1 bispecific antibody, leads to potent anti-tumor immunity through T cell engaging and T/NK cell activation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2358.

Role of CD4+ T-cells for regulating splenic myelopoiesis and monocyte differentiation after experimental myocardial infarction

Myocardial infarction (MI) induces the generation of proinflammatory Ly6Chigh monocytes in the spleen and the recruitment of these cells to the myocardium. CD4+ Foxp3+ CD25+ T-cells (Tregs) promote the healing process after myocardial infarction by engendering a pro-healing differentiation state in myocardial monocyte-derived macrophages. We aimed to study the effects of CD4+ T-cells on splenic myelopoiesis and monocyte differentiation. We instigated MI in mice and found that MI-induced splenic myelopoiesis is abrogated in CD4+ T-cell deficient animals. Conventional CD4+ T-cells promoted myelopoiesis in vitro by cell–cell-contact and paracrine mechanisms, including interferon-gamma (IFN-γ) signalling. Depletion of regulatory T-cells enhanced myelopoiesis in vivo, as evidenced by increases in progenitor cell numbers and proliferative activity in the spleen 5 days after MI. The frequency of CD4+ T-cells-producing factors that promote myelopoiesis increased within the spleen of Treg-depleted mice. Moreover, depletion of Tregs caused a proinflammatory bias in splenic Ly6Chigh monocytes, which showed predominantly upregulated expression of IFN-γ responsive genes after MI. Our results indicate that conventional CD4+ T-cells promote and Tregs attenuate splenic myelopoiesis and proinflammatory differentiation of monocytes.

Targeting JMJD1C to selectively disrupt tumor Treg cell fitness enhances antitumor immunity.

Regulatory T (Treg) cells are critical for immune tolerance but also form a barrier to antitumor immunity. As therapeutic strategies involving Treg cell depletion are limited by concurrent autoimmune disorders, identification of intratumoral Treg cell-specific regulatory mechanisms is needed for selective targeting. Epigenetic modulators can be targeted with small compounds, but intratumoral Treg cell-specific epigenetic regulators have been unexplored. Here, we show that JMJD1C, a histone demethylase upregulated by cytokines in the tumor microenvironment, is essential for tumor Treg cell fitness but dispensable for systemic immune homeostasis. JMJD1C deletion enhanced AKT signals in a manner dependent on histone H3 lysine 9 dimethylation (H3K9me2) demethylase and STAT3 signals independently of H3K9me2 demethylase, leading to robust interferon-γ production and tumor Treg cell fragility. We have also developed an oral JMJD1C inhibitor that suppresses tumor growth by targeting intratumoral Treg cells. Overall, this study identifies JMJD1C as an epigenetic hub that can integrate signals to establish tumor Treg cell fitness, and we present a specific JMJD1C inhibitor that can target tumor Treg cells without affecting systemic immune homeostasis.

Exendin-4-enriched exosomes from hUCMSCs alleviate diabetic nephropathy via gut microbiota and immune modulation.

Diabetic nephropathy (DN) presents a significant therapeutic challenge, compounded by complex pathophysiological mechanisms. Recent studies suggest Exendin-4 (Ex-4) as a potential ameliorative agent for DN, albeit with unclear mechanisms. This research investigates the effects and underlying mechanisms of Ex-4-enriched exosomes derived from human umbilical cord mesenchymal stem cells (hUCMSCs) on DN, focusing on their renoprotective properties and interactions with gut microbiota. Exosomes from hUCMSCs (hUCMSCs-Exo) were loaded with Ex-4 via electroporation. A streptozotocin (STZ) -induced DN mouse model was employed to assess the therapeutic impact of these engineered exosomes. The study further explored immune cell dynamics, mainly CD4+ regulatory T (Treg) cells, using bioinformatics, flow cytometry, and the influence of gut microbiota through antibiotic treatment and specific bacterial reintroduction. Treatment with hUCMSCs-Exo@Ex-4 significantly improved key DN markers, including blood glucose and proteinuria, alleviating kidney damage. A notable decrease in natural Treg cell infiltration in DN was observed, while Ex-4-loaded exosomes promoted CD4+ Treg cell induction. The therapeutic benefits of hUCMSCs-Exo@Ex-4 were diminished upon CD4+ Treg cell depletion, underscoring their role in this context. Notably, CD4+ Treg cell induction correlated with the presence of Prevotella species, and disruption of gut microbiota adversely affected these cells and the therapeutic efficacy of the treatment. However, the reintroduction of Prevotella strains counteracted these adverse effects. This study elucidates a novel therapeutic mechanism of Ex-4-loaded hUCMSCs exosomes in DN, highlighting the induction of CD4+ Treg cells mediated by specific gut microbiota components. These findings underscore the potential of leveraging gut microbiota and immune cell interplay in developing effective DN treatments.

Acquisition of suppressive function by conventional T cells limits antitumor immunity upon Treg depletion.

Regulatory T (Treg) cells contribute to immune homeostasis but suppress immune responses to cancer. Strategies to disrupt Treg cell-mediated cancer immunosuppression have been met with limited clinical success, but the underlying mechanisms for treatment failure are poorly understood. By modeling Treg cell-targeted immunotherapy in mice, we find that CD4+ Foxp3- conventional T (Tconv) cells acquire suppressive function upon depletion of Foxp3+ Treg cells, limiting therapeutic efficacy. Foxp3- Tconv cells within tumors adopt a Treg cell-like transcriptional profile upon ablation of Treg cells and acquire the ability to suppress T cell activation and proliferation ex vivo. Suppressive activity is enriched among CD4+ Tconv cells marked by expression of C-C motif receptor 8 (CCR8), which are found in mouse and human tumors. Upon Treg cell depletion, CCR8+ Tconv cells undergo systemic and intratumoral activation and expansion, and mediate IL-10-dependent suppression of antitumor immunity. Consequently, conditional deletion of Il10 within T cells augments antitumor immunity upon Treg cell depletion in mice, and antibody blockade of IL-10 signaling synergizes with Treg cell depletion to overcome treatment resistance. These findings reveal a secondary layer of immunosuppression by Tconv cells released upon therapeutic Treg cell depletion and suggest that broader consideration of suppressive function within the T cell lineage is required for development of effective Treg cell-targeted therapies.

Discovery of Novel, First-in-Class Allosteric Modulators of MALT1 Scaffolding Function with Differentiated Pharmacology for NFκB-Driven Malignancies

Constitutive activation of NFκB is an oncogenic driver for many lymphoid and selected solid tumor malignancies. BTK inhibitors, which affect the upstream NFκB pathway, have been successfully used in some diseases, but their utility is limited. The majority of DLBCL patients, for instance, are insensitive to BTK inhibition, supporting the need for alternative mechanisms to block NFκB-driven tumor progression. Knockdown of MALT1, a key component of the CBMT complex (CARD-BCL10-MALT1-TRAF6) involved in direct signal transduction of NFκB, is able to block NFκB-dependent DLBCL tumor growth in BTKi sensitive or resistant tumors, but pharmacologic modulation has been challenged by the dual-functionality of MALT1 in a complexed signalosome. MALT1 functions as the scaffolding component of the CBMT complex. Activated MALT1 recruits multiple proteins and directly activates NFκB canonical signaling. MALT1 also has protease activity that fine-tunes various signaling pathways including NFκB via the cleavage of many substrates. The scaffolding activity of MALT1 is critical for cancer cell survival. Most MALT1 inhibitors discovered so far only block protease activity, which results in incomplete NFκB inhibition, suboptimal anti-tumor activity, and the induction of severe autoimmunity due to protease inhibition. Using our Smart Allostery platform, we elucidated and then drugged a natural hotspot on MALT1 that selectively impacts scaffolding but not protease function. The series of scaffolding modulators demonstrate potent inhibition of MALT1 scaffolding activity with cellular IC 50 values ranging from 15 to 250 nM, while exerting minimal influence on MALT1 protease function. Unlike protease inhibitors, which only partially affect NFκB activity, scaffolding modulators completely inhibit the NFκB downstream signaling manifested by the suppression of NFκB activity with cellular IC 50 values ranging from 2 to 90 nM. Consequently, scaffolding modulators exhibit a superior and broader anti-proliferation profile compared to MALT1 protease inhibitors and BTK inhibitors in a panel of lymphoma cell lines. Pathway and genetic analyses indicate that B-cell lymphoma lines driven by NFκB are especially sensitive to scaffolding modulators. In vivo studies demonstrate a robust, dose-dependent tumor growth inhibition (TGI) by one representative scaffolding modulator in several B-cell lymphoma xenograft models. In a protease inhibitor sensitive ABC-DLBCL model, OCI-LY10, our scaffolding modulator achieved a maximal TGI of 94%, while a clinical MALT1 protease inhibitor showed 62% maximal TGI (p&amp;lt;0.001). In protease resistant NFκB driven DLBCL models, MALT1 scaffolding modulation resulted in tumor regression or stasis. These findings highlight the differentiated pharmacology of our scaffolding modulators. Distinct from protease inhibitors that suppress T cell activation and Treg population in vivo, scaffolding modulators also did not deplete Treg cells nor suppress T cell activation in a 14-day study. This potentially mitigates the risk of development of autoimmune disease caused by chronic MALT1 protease inhibition. In summary, we have discovered novel, first-in-class allosteric modulators of MALT1 scaffolding activities. These scaffolding modulators drive enhanced and broader anti-tumor effect in models resistant to MALT1 protease or BTK inhibitors and do so without immune dysregulation. Scaffolding modulation represents a potential best-in-class therapeutic opportunity to modulate MALT1 in patients with NFκB-driven malignancies.