Tregs In Tumor Microenvironment Research Articles

Loss of Y in regulatory T lymphocytes in the tumor micro-environment of primary colorectal cancers and liver metastases

Male sex is a risk factor for colorectal cancer (CRC) with higher illness burden and earlier onset. Thus, we hypothesized that loss of chromosome Y (LOY) in the tumor micro-environment (TME) might be involved in oncogenesis. Previous studies show that LOY in circulating leukocytes of aging men was associated with shorter survival and non-hematological cancer, as well as higher LOY in CD4 + T-lymphocytes in men with prostate cancer vs. controls. However, nothing is known about LOY in leukocytes infiltrating TME and we address this aspect here. We studied frequency and functional effects of LOY in blood, TME and non-tumorous tissue. Regulatory T-lymphocytes (Tregs) in TME had the highest frequency of LOY (22%) in comparison to CD4 + T-lymphocytes and cytotoxic CD8 + T-lymphocytes. LOY score using scRNA-seq was also linked to higher expression of PDCD1, TIGIT and IKZF2 in Tregs. PDCD1 and TIGIT encode immune checkpoint receptors involved in the regulation of Tregs function. Our study sets the direction for further functional research regarding a probable role of LOY in intensifying features related to the suppressive phenotype of Tregs in TME and consequently a possible influence on immunotherapy response in CRC patients.

Strengthening the Combinational Immunotherapy from Modulating the Tumor Inflammatory Environment via Hypoxia-Responsive Nanogels.

Despite the success of immuno-oncology in clinical settings, the therapeutic efficacy is lower than the expectation due to the immunosuppressive inflammatory tumor microenvironment (TME) and the lack of functional lymphocytes caused by exhaustion. To enhance the efficacy of immuno-oncotherapy, a synergistic strategy should be used that can effectively improve the inflammatory TME and increase the tumor infiltration of cytotoxic T lymphocytes (CTLs). Herein, a TME hypoxia-responsive nanogel (NG) is developed to enhance the delivery and penetration of diacerein and (-)-epigallocatechin gallate (EGCG) in tumors. After systemic administration, diacerein effectively improves the tumor immunosuppressive condition through a reduction of MDSCs and Tregs in TME, and induces tumor cell apoptosis via the inhibition of IL-6/STAT3 signal pathway, realizing a strong antitumor effect. Additionally, EGCG can effectively inhibit the expression of PD-L1, restoring the tumor-killing function of CTLs. The infiltration of CTLs increases at the tumor site with activation of systemic immunity after the combination of TIM3 blockade therapy, ultimately resulting in a strong antitumor immune response. This study provides valuable insights for future research on eliciting effective antitumor immunity by suppressing adverse tumor inflammation. The feasible strategy proposed in this work may solve the urgent clinical concerns of the dissatisfactory checkpoint-based immuno-oncotherapy.

Deciphering the immune landscape of head and neck squamous cell carcinoma: A single-cell transcriptomic analysis of regulatory T cell responses to PD-1 blockade therapy

Immunotherapy is changing the Head and Neck Squamous Cell Carcinoma (HNSCC) landscape and improving outcomes for patients with recurrent or metastatic HNSCC. A deeper understanding of the tumor microenvironment (TME) is required in light of the limitations of patients’ responses to immunotherapy. Here, we aimed to examine how Nivolumab affects infiltrating Tregs in the HNSCC TME. We used single-cell RNA sequencing data from eight tissues isolated from four HNSCC donors before and after Nivolumab treatment. Interestingly, the study found that Treg counts and suppressive activity increased following Nivolumab therapy. We also discovered that changes in the CD44-SSP1 axis, NKG2C/D-HLA-E axis, and KRAS signaling may have contributed to the increase in Treg numbers. Furthermore, our study suggests that decreasing the activity of the KRAS and Notch signaling pathways, and increasing FOXP3, CTLA-4, LAG-3, and GZMA expression, may be mechanisms that enhance the killing and suppressive capacity of Tregs. Additionally, the result of pseudo-temporal analysis of the HNSCC TME indicated that after Nivolumab therapy, the expression of certain inhibitory immune checkpoints including TIGIT, ENTPD1, and CD276 and LY9, were decreased in Tregs, while LAG-3 showed an increased expression level. The study also found that Tregs had a dense communication network with cluster two, and that certain ligand-receptor pairs, including SPP1/CD44, HLA-E/KLRC2, HLA-E/KLRK1, ANXA1/FPR3, and CXCL9/FCGR2A, had notable changes after the therapy. These changes in gene expression and cell interactions may have implications for the role of Tregs in the TME and in response to Nivolumab therapy.

Targeting post-translational modifications of Foxp3: a new paradigm for regulatory T cell-specific therapy.

A healthy immune system is pivotal for the hosts to resist external pathogens andmaintain homeostasis; however, the immunosuppressive tumor microenvironment (TME) damages the anti-tumor immunity and promotes tumor progression, invasion, and metastasis. Recently, many studies have found that Foxp3+ regulatory T (Treg) cells are the major immunosuppressive cells that facilitate the formation of TME by promoting the development of various tumor-associated cells and suppressing the activity of effector immune cells. Considering the role of Tregs in tumor progression, it is pivotal to identify new therapeutic drugs to target and deplete Tregs in tumors. Although several studies have developed strategies for targeted deletion of Treg to reduce the TME and support the accumulation of effector T cells in tumors, Treg-targeted therapy systematically affects the Treg population and may lead to the progression of autoimmune diseases. It has been understood that, nevertheless, in disease conditions, Foxp3 undergoes several definite post-translational modifications (PTMs), including acetylation, glycosylation, phosphorylation, ubiquitylation, and methylation. These PTMs not only elevate or mitigate the transcriptional activity of Foxp3 but also affect the stability and immunosuppressive function of Tregs. Various studies have shown that pharmacological targeting of enzymes involved in PTMs can significantly influence the PTMs of Foxp3; thus, it may influence the progression of cancers and/or autoimmune diseases. Overall, this review will help researchers to understand the advances in the immune-suppressive mechanisms of Tregs, the post-translational regulations of Foxp3, and the potential therapeutic targets and strategies to target the Tregs in TME to improve anti-tumor immunity.

Deciphering the heterogeneity and immunosuppressive function of regulatory T cells in osteosarcoma using single-cell RNA transcriptome

Osteosarcoma (OS) is a highly invasive malignant neoplasm with poor prognosis. The tumor microenvironment (TME) plays an essential role in the occurrence and development of OS. Regulatory T cells (Tregs) are known to facilitate immunosuppression, tumor progression, invasion, and metastasis. However, the effect of Tregs in the TME of OS remains unclear. In this study, single-cell RNA sequencing (scRNA-seq) data was used to identify Tregs and various other cell clusters in the TME of OS. Gene set variation analysis (GSVA) was used to investigate the signaling pathways in Tregs from OS and adjacent tissues. The CellChat and iTALK packages were used to analyze cellular communication. In addition, a prognostic model was established based on the Tregs-specific genes using bulk RNA-seq from the TARGET database, and it was verified using a Gene Expression Omnibus dataset. The pRRophetic package was used to predict drug sensitivity. Immunohistochemistry was used to verify the expression of candidate genes in OS. Based on the above methods, we showed that the OS samples were highly infiltrated with Tregs. GSVA revealed that oxidative phosphorylation, angiogenesis and mammalian target of rapamycin complex 1 (mTORC1) were highly activated in Tregs from OS compared with those from adjacent tissues. Using cellular communication analysis, we found that Tregs interacted with osteoblastic, endothelial, and myeloid cells via C-X-C motif chemokine ligand (CXCL) signaling; particularly, they strongly affected the expression of C-X-C motif chemokine receptor 4 (CXCR4) and interacted with other cell clusters through CXCL12/transforming growth factor β1 (TGFB1) to collectively enable tumor growth and progression. Subsequently, two Tregs-specific genes—CD320 and MAF—were screened through univariate, least absolute shrinkage and selection operator regression (LASSO) and multivariate analysis to construct a prognostic model, which showed excellent prognostic accuracy in two independent cohorts. In addition, drug sensitivity analysis demonstrated that OS patients at high Tregs risk were sensitive to sunitinib, sorafenib, and axitinib. We also used immunohistochemistry to validate that CD320 and MAF were significantly upregulated in OS tissues compared with adjacent tissues. Overall, this study reveals the heterogeneity of Tregs in the OS TME, providing new insights into the invasion and treatment of this cancer.

Abstract 1810: Enhanced anti-breast tumor cancer activity using nano-formulated TLR 7/8 agonist and PI3K inhibitor

Abstract Triple-negative breast cancer (TNBC) is characterized by high cell proliferation, abundant heterogeneity, frequent incidence of lung and brain metastases, repeated disease recurrence, and the worst prognosis among all breast cancer subtypes. Extensive evidence from translational studies indicates immunosuppressive cell infiltration into the tumor microenvironment (TME) promotes tumor immune escape and inhibits the efficient antitumor immune response. Previously, we have shown that a large proportion of tumor-infiltrating lymphocytes in claudin-low subtype of TNBC are CD4+FoxP3+ Tregs. Infiltration of Treg in TME is associated with suppressing effector T cell responses, accelerated tumor growth, and poor clinical outcomes. Herein, to convert immunosuppressive TME of TNBC to immune-inflamed immunophenotype we used polymeric micelles nanoparticles for co-delivering immunomodulatory small molecule drugs. We used a novel Full Spectral Flow Cytometry approach for immunophenotyping tumors. We identified significant infiltration of immunosuppressive myeloid-derived suppressor cells (MDSCs) and regulatory T-cells (Tregs) in the orthotopic T11 TNBC tumor. The small molecule resiquimod, an imidazoquinoline Toll-like receptor (TLR) 7/8 agonist, and idelalisib, an inhibitor of phosphatidylinositol 3-kinase (PI3K) p110δ were incorporated in poly(2-oxazoline) block copolymer polymeric micelle NPs. Drug exposure to tumor cells was increased by the small-size nanoparticles associated with the preferential accumulation of NPs in tumors. The resiquimod and idelalisib were used to differentiate MDSC and selectively decrease the number and function of Tregs in TME, respectively. Following targeted radiation therapy in combination with iv administration of nanoformulation to animals bearing orthotopic T11 TNBC tumors, we observed significant suppression of tumor growth and enhanced overall survival compared to targeted radiotherapy alone. Radiation therapy in combination with both nanoformulated resiquimod and idelalisib induced optimal tumor control, increasing median survival with over 50% complete response rate, while all mice in the nanoformulation groups or radiation alone group succumbed to tumor growth. Enhanced adaptive T cell immunity against TNBCs and effective therapeutic improvement in immunocompetent mice following radiotherapy were only observed with simultaneous depletion of Tregs and MDSC. These data suggest radiotherapy in combination with nanoparticle co-delivery of immunomodulators inhibiting MDSCs and Treg, has promising potential for future clinical trials and translation for the treatment of TNBC patients. Citation Format: Mostafa Yazdimamaghani, Oleg Kolupaev, Chaemin Lim, Duhyeong Hwang, Alexander Kabanov, Jonathan Serody. Enhanced anti-breast tumor cancer activity using nano-formulated TLR 7/8 agonist and PI3K inhibitor [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1810.

The traditional chinese medicine monomer Ailanthone improves the therapeutic efficacy of anti-PD-L1 in melanoma cells by targeting c-Jun

BackgroundC-Jun, a critical component of AP-1, exerts essential functions in various tumors, including melanoma, and is believed to be a druggable target for cancer therapy. Unfortunately, no effective c-Jun inhibitors are currently approved for clinical use. The advent of immune checkpoint inhibitor (ICI) has brought a paradigm shift in melanoma therapy, but more than half of patients fail to exhibit clinical responses. The exploration of new combination therapies has become the current pursuit of melanoma treatment strategy. This study aims to screen out Chinese herbal monomers that can target c-Jun, explore the combined effect of c–Jun inhibitor and ICI, and further clarify the related molecular mechanism. MethodsWe adopted a combinatorial screening strategy, including molecular docking, ligand-based online approaches and consensus quantitative structure-activity relationship (QSAR) model, to filter out c-Jun inhibitors from a traditional Chinese medicine (TCM) library. A mouse melanoma model was used to evaluate the therapeutic efficacy of monotherapy and combination therapy. Multicolor flow cytometry was employed to assess the tumor microenvironment (TME). Multiple in vitro assays were performed to verify down-streaming signaling pathway. CD4 + T-cell differentiation assay was applied to investigate Treg differentiation in vitro.ResultsAilanthone (AIL) was screened out as a c-Jun inhibitor, and inhibited melanoma cell growth by directly targeting c-Jun and promoting its degradation. Surprisingly, AIL also facilitated the therapeutic efficacy of anti-programmed death ligand-1 (PD-L1) in melanoma cells by reducing the infiltration of Tregs in TME. Additionally, AIL treatment inhibited c-Jun-induced PD-L1 expression and secretion. As a consequence, Treg differentiation was attenuated after treatment with AIL through the c-Jun/PD-L1 axis.ConclusionOur findings identified AIL as a novel c-Jun inhibitor, and revealed its previously unrecognized anti-melanoma effects and the vital role in regulating TME by Treg suppression, which provides a novel combination therapeutic strategy of c-Jun inhibition by AIL with ICI.Graphical AIL down-regulates c-Jun by reducing its stability, and inhibits the function of Tregs via AIL-c-Jun-PD-L1 pathway, ultimately suppressing melanoma progression and enhancing the efficacy of anti-PD-L1.

704TiP SAR445256 (KY1044) in combination with atezolizumab in patients (pts) with recurrent/metastatic head and neck squamous cell carcinoma (HNSCC)

SAR445256 (KY1044) is a fully human IgG1 targeting ICOS with a dual mechanism of action: dose dependent depletion of ICOShigh Tregs in tumor microenvironment and activation of ICOSlow Teff. NCT03829501 is the first in human study testing safety, efficacy, and pharmacological properties of SAR445256 with/without atezolizumab in PD-L1 naïve or pretreated pts with advanced malignancies. Dose escalation part was guided by an adaptive dose-finding method and Ph 1 enrichment part was included for a better understanding of safety, tolerability, preliminary anti-tumor efficacy and PK/PD relationships of SAR445256 in combination with atezolizumab. Selected Ph2 dose was 8mg with atezolizumab 1200mg, both given IV, Q3W. In Ph1, a 59-year-old male pt with HPV+ HNSCC who progressed on 5 prior lines of therapy (including nivolumab), experienced a PR (42% tumor shrinkage), still holding as of C26D1, and on treatment for >20 months as of 10Feb2022. Strong expression of ICOS on intratumoral Tregs in head and neck cancer as well as the promising clinical activity previously reported suggest HNSCC to be favorable indication for SAR445256. Ph2 cohorts (2 cohorts, PD-L1 naïve and pretreated) are being initiated in HNSCC pts. Approximately 40 pts will be enrolled in each cohort. Key inclusion criteria: anti -PD-L1 therapy naïve and pretreated, 1-2 prior lines of systemic therapy for advanced disease, histologically documented advanced/metastatic malignancies, measurable disease by RECIST 1.1, site of disease amenable to biopsy. Key exclusion criteria: CNS metastases, active autoimmune disease, significant heart disease and/or QT prolongation, steroid therapy, or any immunosuppressive therapy. Efficacy measures will be performed as per RECIST 1.1 Q8W for the first 16 weeks and then Q12W, while AEs will be classified according to CTCAE v5. SAR445256 was well tolerated and showed initial signs of activity, in a HNSCC pt who received multiple prior lines of therapy. Ph2 assessment is planned in 2 cohorts: HNSCC pts PD-L1 naïve and pretreated. Note: Atezolizumab will continue to be provided by Roche for the RP2D part of this study as well. 1. Sainson R et al 2020. 2. Patel MR et al 2021. NCT03829501. Mukul Rastogi, PhD of Sanofi. Sanofi.

Localized Chemotherapy Based on Injectable Hydrogel Boosts the Antitumor Activity of Adoptively Transferred T Lymphocytes In Vivo.

The adoptive transfer of antigen-specific T cells has been successfully applied in the treatment of hematological malignancies. However, its application in the treatment of solid tumors has been overshadowed by the immunosuppressive tumor microenvironment. In this context, a preprocessing strategy is developed to reprogram the immunosuppressive tumor microenvironment using a thermoresponsive hydrogel loaded with doxorubicin (DOX@Gel). Compared with hydrogel-based chemotherapy alone or adoptive T cell therapy alone, this combination exhibits enhanced anti-tumor efficacy. In addition to the direct killing of tumor cells, the local chemotherapy releases tumor-associated antigens which enhance the proliferation and effector function of endogenous and adoptively transferred T cells. Moreover, DOX@Gel significantly reduces the numbers of both myeloid derived suppressor cells and Tregs in tumor microenvironment. It is suggested that DOX@Gel promotes the efficacy of adoptively transferred T cells against solid tumors, overcoming the key limitations of adoptive T cell therapy.

IL-10 derived from Hepatocarcinoma cells improves human induced regulatory T cells function via JAK1/STAT5 pathway in tumor microenvironment

Forkhead box P3 (Foxp3) expressing CD4+CD25+ regulatory T cells (Tregs), an essential subset of immune T cells for maintaining immune homeostasis is implicated as a negative regulator in an anti-tumor immune response. Current researches suggest that reducing tumor-infiltrating Tregs contribute to enhanced anti-cancer effect. However, the mechanism of infiltration of a large number of Tregs into tumor tissues is still unclear. In this study, human induced Tregs (iTregs) were co-cultured with human hepatocytes and various types of cancer cells (HepG2, NSCLC, and AsPC-1) supernatants. Foxp3, multiple cytokines, levels of apoptosis and suppressive ability of iTregs were detected by FACS. Western blot was employed to test of proteins. Impact of HepG2 supernatants on T cell subpopulations differentiation, cytokines in supernatants were examed by FACS and ELISA respectively. Anti-IL-10R antibody and JAK1 inhibitor were used to reconfirm the role of tumor-derived IL-10 play in the regulation on iTregs. Hepatocarcinoma cells (HCC) supernatants treatment increases Foxp3 stability and reduces apoptosis level in human iTregs without influencing its differentiation trend. Furthermore, IL-10 was found to be extremely higher in HCC supernatants than other groups, IL-10R blockade neutralize the effect of HCC supernatants on iTregs in vitro obviously. HCC supernatants also reversed IL-1β/6 triggered decline on Foxp3 which may be related to higher expression of JAK1 and elevated phosphorylation level of STAT5 induced by IL-10. Our results suggest that improved stability and abnormal accumulation of Tregs in tumor microenvironment is IL-10/JAK1/STAT5 signal pathway-dependent and provide a novel approach for improving the efficiency of anti-tumor immunotherapy.

Abstract CT140: Proof-of-concept evidence of immune modulation by blockade of the phosphatidylinositol 3-kinase (PI3K)-AKT signaling pathway in the phase I dose escalation study of Ipatasertib (Ipa) in combination with atezolizumab (A) in patients (pts) with advanced solid tumors (Ice-CAP)

Abstract Background: Hyperactivation of the PI3K/AKT pathway correlates with impaired anti-tumor responses, including reduced T cell infiltration into tumor, and reduced efficacy of immune checkpoint inhibitors. Blockade of this pathway synergizes with PD-L1/PD-1 axis blockade preclinically. Methods: This Phase I clinical trial (NCT03673787) assessed the safety, pharmacodynamic, and preliminary clinical activity of Ipa (200mg or 400mg OD) given in combination with A 1200mg q3 wk in refractory pts. Serial paired blood and tumor samples were analysed to interrogate the effect of Ipa on the tumor micro-environment and host immune system prior to the addition of the immune check point inhibitor, A. Results: 18 adult pts were treated in dose escalation. Median age 49 yrs. All pts had ECOG PS 0-1 and median 7 prior therapies. Most common TRAEs (>15%) were mild Gr1-2 diarrhea (56%), rash (50%), fatigue (33%), nausea (33%), raised ALT/AST (33%), headache (28%) and arthralgia (22%). 1 pt had G2 systemic immune activation; 2 pts had G3 rash, both rapidly reversible. 1 DLT of G3 raised ALT seen at 200mg (1 DLT/9 evaluable pts) but none at 400mg (0 DLT/6). Of 14 RECIST evaluable patients, there were 2 confirmed PRs, and 5 SD (clinical benefit rate 50%). Reductions of CD4+FOXP3+ Tregs in tumor microenvironment were seen after 2wks of single agent Ipa, regardless of PIK3/AKT somatic mutation status (Table 1). Responding pts had a >400% median increase in intra-tumoral CD8+ Teff cell infiltration, effectively switching from a desert phenotype to an inflamed phenotype. Paired changes in FACS, transcriptome and cytokine will also be presented.Conclusions: The RP2D of Ipa 400mg OD combination with A was well tolerated with early efficacy signals. Further biomarker work is ongoing and will be evaluated in expansion cohorts. Table 1:Changes in immune cell populations as assessed by multicolour Immunofluorescence in paired biopsies of breast/gynae patients, % change in cell number/mm2 from baseline (median [min,max$])&Post 2 weeks single agent Ipatasertib(n=9)Post 1 cycle of combination Ipatasertib and Atezolizumab(n=7)CD4+FOXP3+Tregs cellsCD 8+ Teff cellsCD4+FOXP3+Tregs cellsCD 8+ Teff cellsIntra-tumourstromaIntra-tumourstromaIntra-tumourstromaIntra-tumourstromaAll patients-23.9*[-89.7, BL0]-30.0*[-91.6, BL0]-37.7*[-84.4, -24.5]-28.4[-92.4, 259.8]335.9[-44.0,BL0]45.4[-51.0, BL0]59.6[-60.6,493.3]64.7[-51.7,293.3]Stratified by somatic PI3K/AKT/PTEN mutational statusPathogenic mutations (mt)11.1[-82.2, BL0]#-10.7[-91.6, BL0]Φnsnsnsns-30.5[-60.6,-0.5]11.3[-51.7,50.0]Wildtype (wt)-63.1[-89.7,19.0]#-47.5[-77.0,11.1]Φnsnsnsns426.5[59.6,493.3]126.7[79.4,293.3]Stratified by responseResponders (PR + SD>4 cycles). 1 ER+ HER2+ breast cancer (wt), 1 ER+ HER2- breast cancer (wt)459.9[426.5,493.3]@103.1[79.4,126.7]Non-responders (PD at 4 cycles) 1 cervical cancer, 4 ER+ breast cancer-0.5[-60.6, 59.6]@30.6[-51.7,293.3]*significant change (p≤0.05; Wilcoxon sign-rank test) from baseline, $maximum values denoted by BL0indicate that the baseline value was zero, and so percentage change from baseline is not defined. For the analysis, the baseline value has been replaced by a nominal value of 0.1 so that a large percentage increase is associated with these cases. Note that these large percentage increases do not affect the non-parametric statistical tests used.#no significant difference in distribution of reduction in intra-tumoural CD4+ FOXP3+Tregsbetween pts with pathogenic mutations in PI3K/AKT and those without (p=0.30; Wilcoxon rank-sum test)Φno significant difference in distribution of reduction in stromal CD4+FOXP3+Tregsbetween pts with pathogenic mutations in PI3K/AKT and those without (p=0.44; Wilcoxon rank-sum test) @ difference between responders and non-responders p=0.083; Wilcoxon rank-sum test)mt pathogenic mutations in PI3K/AKT and PTEN as per COSMIC database present in tumour or PTEN loss by IHC. wt no pathogenic mutations in PI3K/AKT and PTEN as per COSMIC database detected in tumour and intact PTEN expression by IHC. &exploratory analyses with no adjustment for multiple testing Citation Format: Juanita S. Lopez, Andrea Biondo, Crescens Tiu, Mariana Scaranti, Malaka Ameratunga, Anna Zachariou, Alison Turner, Nina Tunariu, Toby Prout, Mona Parmar, Hannah Badham, Karen Swales, Wei Yuan, Ricardo Morilla, Mateus Crespo, Rob Daly, Ines Figueiredo, Bora Gurel, Rita Pereira, Ruth Riisnaes, Igor Vivanco, Anna Minchom, Ben Jenkins, Christina Yap, Udai Banerji, Johann De Bono. Proof-of-concept evidence of immune modulation by blockade of the phosphatidylinositol 3-kinase (PI3K)-AKT signaling pathway in the phase I dose escalation study of Ipatasertib (Ipa) in combination with atezolizumab (A) in patients (pts) with advanced solid tumors (Ice-CAP) [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr CT140.

Abstract 6529: GI101, A novel CD80-IgG4-IL2 variant bispecific protein, inhibits tumor growth and induces anti-tumor immune response in multiple preclinical models

Abstract Introduction: Immune checkpoint blockades (ICBs) have revolutionized cancer treatment and broadened clinical applicability. However, the majority of patients still fail to respond to standard ICBs. To overcome such unmet needs in a clinical study, we designed GI-101, combining the extracellular domain of CD80 serve as a CTLA-4 blockade and an IL-2 variant that preferentially binds the IL-2 receptor β subunit (IL-2Rβ) together. The harmonizing mechanisms of action are projected to translate into improved clinical benefits for this first-in-class immune checkpoint inhibitor fusion protein, even in non-inflamed “cold” tumors. Methods: Binding affinity of GI101 to IL2Rs, CTLA4, and CD28 was determined by SPR. Immune cell proliferation was analyzed by CFSE assay. In vivo anti-tumor efficacy was tested by single or combination treatment on CT26, MC38 and B16F10 syngeneic tumor models. To elucidate the involvement of GI101 on tumor microenvironment (TME), immune cell population was analyzed by flow cytometry from tumor. Tumor specific T cells (surrogate marker, gp70) were measured by splenocyte proliferation assay and IFN-γ ELISPOT assay. RNA sequencing was performed to elucidate immune mechanism of GI-101. Results: GI101 highly binds to CTLA-4 (Kd, 2.9 nM) which leads to the reinforcement of endogenous CD80 and CD28 interaction resulting in the activation of T cells. Bivalent IL-2 variant of GI101 triggers both CD8+ T and NK cells proliferation in vitro and in vivo without Tregs proliferation. GI101 has no evidence for toxicity associated with IL-2 activity including vascular leakage syndrome and cytokine storm in non-GLP monkey studies whereas isolated mortality was observed in the anti-PD-1 and anti-CTLA4 combination treatment group. GI101 elicits restoration of immune functions in vitro settings using mouse splenocytes co-cultured with different PDL-1 and CTLA-4 expression level tumor cells. A dose-dependent (3 to 12 mg/kg) inhibition of tumor growth was observed in CT26 syngeneic models without toxicity. Immune profiling of tumor samples also revealed that a robust increment of M1 macrophages, CD8+ central memory T cells (Tcm) and Ki-67+ proliferating T cells but not Tregs in TME (p < 0.05). Tumor specific T cells were strongly proliferated when stimulated with CT26 neoantigens (gp70, RSPWFTTLI and MGPLIVLLL) in splenocyte. IFN-γ+ cells were significantly increased in draining lymph nodes from GI101 treated mice. Furthermore, drastic tumor regression was observed in MC38 tumor-bearing mice treated with GI101 and anti-PD-1 combination. Conclusion: GI101 facilitates the dual function of checkpoint blockade and IL2 activity that enhances the proliferation and activation of T and NK cells. This novel target drug is expected to be interpreted as superior clinical efficacy and safety as indicated even in ‘cold tumor' models. GI101 is the promising immune-oncology drug to replace the first-generation ICBs by single or combining with other immunotherapies. Our findings provide a rationale for further clinical investigations. Keywords: CD80, IL-2 variant, GI101, Bispecific fusion protein, immunotherapy Citation Format: Kyoung-Ho Pyo, Young Jun Koh, Chun-Bong Synn, Jae Chan Park, Jae-Hwan Kim, Yeongseon Byeon, Sung Eun Kim, Ji Min Lee, Ha Ni Jo, Wongeun Lee, Do Hee Kim, Sungwon Park, Yoo Jeong Song, Won Jae Lee, Ji Young Kim, Hyung Nam Ji, Sang Su Park, Kyung Wha Lee, Young Gyu Cho, Young Min Oh, Bo Gie Yang, Su Youn Nam, Myoung Ho Jang, Byoung Chul Cho. GI101, A novel CD80-IgG4-IL2 variant bispecific protein, inhibits tumor growth and induces anti-tumor immune response in multiple preclinical models [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 6529.

The Effects of WW2/WW3 Domains of Smurf2 Molecule on CD4+CD25+/CD4+ Proportion in Spleen of 4T1 Tumor Bearing BALB/c Mice.

Background:TGF-β has long been considered as the main inducer of Tregs in tumor microenvironment and is the reason for the aberrant number of Tregs in tumor-bearing individuals. Recently, it has been suggested that the enzyme arginase I is able to mediate the induction of Tregs in a TGF-β-independent fashion. The recombinant WW2/WW3 domains from Smurf2 molecule was demonstrated to increase TGF-β signaling while reducing arginase I gene expression. In this study, we aimed to examine the effects of this recombinant protein on CD4+CD25+/CD4+ proportion in the spleen of 4T1 mammary carcinoma-bearing BALB/c mice. Methods:Flow cytometry was used to evaluate CD4+CD25+ spleen cell populations of the tumor-bearing mice that received WW2/WW3 protein treatment and those of the control group. Results:The results indicated a significant rise in CD4+CD25+/CD4+ ratio, along with an average increase in tumor mass of the subjects that underwent protein treatment. Conclusion:It can be inferred that the heightened CD4+CD25+/CD4+ proportion in the spleen of protein-treated tumor-bearing mice can be the result of the increased TGF-β signaling despite the reduced arginase I expression.

Deciphering the Role of Regulatory CD4 T Cells in Oral and Oropharyngeal Cancer: A Systematic Review.

Background: Recruiting regulatory CD4 T cells (Tregs) into the tumor microenvironment is an important tumor escape mechanism. Diminishing these suppressive cells is therefore one of the targets of cancer immunotherapy. Selective depletion of Tregs has proven successful in enhancing anti-tumor immunity and therapeutic efficacy in multiple tumor types. However, the role of Tregs in oral/oropharyngeal cancers is unclear with conflicting evidence regarding the effect of these suppressive cells on tumor prognosis. In this study, we sought to review the role of Tregs in oral/oropharyngeal cancer with the aim of deciphering the controversy regarding their effect on tumor progression and prognosis.Methods: A systematic review of the literature pertaining to the role of Tregs in oral/oropharyngeal cancer was performed using Scopus, Embase, and PubMed. Forty-five records were deemed eligible and data describing methodology of Treg detection, tumor type, and association with prognosis were extracted.Results: Of the 45 eligible manuscripts accepted for this systematic review, thirty-nine studies reported data from human subjects while the remaining studies focused on animal models. Sixteen studies were carried out using peripheral blood samples, while samples from the tumor site were analyzed in 18 studies and 11 studies assessed both blood and tumor samples. The transcriptional factor, Foxp3, was the most commonly used marker for Treg identification (38/45). The findings of 25 studies suggested that an increase in Tregs in the tumor microenvironment and/or peripheral blood was associated with poorer prognosis. These conclusions were attributed to the suppression of immune responses and the consequent tumor progression. Conversely, nine studies showed an increase in Tregs in peripheral blood and/or tumor microenvironment was related to a favorable prognosis, particularly in the presence of human papilloma virus (HPV), the status of which was only assessed in 11 studies.Conclusions: This review underlines the importance of host immunity in the behavior of oral/oropharyngeal cancer. Furthermore, we report an apparent lack of clarity regarding the true role Tregs play in oral/oropharyngeal cancer progression which could be attributed to inconsistent detection techniques of Tregs. Our results therefore highlight the need for clearer methodologies and more robust phenotyping when defining Tregs.

Reprogramming the murine colon cancer microenvironment using lentivectors encoding shRNA against IL-10 as a component of a potent DC-based chemoimmunotherapy

BackgroundThe excessive amounts of immunosuppressive factors present in a tumor microenvironment (TME) reduce the effectiveness of cancer vaccines. The main objective of our research was to improve the effectiveness of dendritic cell (DC)-based immunotherapy or chemoimmunotherapy composed of cyclophosphamide (CY) and DCs by application of lentivectors encoding shRNA specific to IL-10 (shIL10 LVs) in murine colon carcinoma MC38 model.MethodsThe efficacy of shIL10 LVs in silencing of IL-10 expression was measured both in vitro and in vivo using Real-Time PCR and ELISA assays. In addition, the influence of intratumorally inoculated lentivectors on MC38 tumor microenvironment was examined using flow cytometry method. The effect of applied therapeutic schemes was determined by measurement of tumor growth inhibition and activation state of local and systemic immune response.ResultsWe observed that intratumorally inoculated shIL10 LVs transduced tumor and TME-infiltrating cells and reduced the secretion of IL-10. Application of shIL10 LVs for three consecutive weeks initiated tumor growth inhibition, whereas treatment with shIL10 LVs and BMDC/TAg did not enhance the antitumor effect. However, when pretreatment with CY was introduced to the proposed scheme, we noticed high MC38 tumor growth inhibition accompanied by reduction of MDSCs and Tregs in TME, as well as activation of potent local and systemic Th1-type antitumor response.ConclusionsThe obtained data shows that remodeling of TME by shIL10 LVs and CY enhances DC activity and supports them during regeneration and actuation of a potent antitumor response. Therefore, therapeutic strategies aimed at local IL-10 elimination using lentiviral vectors should be further investigated in context of combined chemoimmunotherapies.

P3.02c-071 Spatially Selective Depletion of Regulatory T Cells with near Infrared Photoimmunotherapy for Syngeneic Lewis Lung Carcinoma: Topic: IT Biomarkers

CD4+CD25+Foxp3+ regulatory T-cells (Tregs) are known to suppress immune responses. Treg-mediated immunosuppression is a key mechanism for tumor immune-evasion, which could lead cancer immunotherapies to failures. Systemic depletion of Tregs has been tried; however, intravenously delivering antibodies against Tregs might not sufficiently deplete Tregs in tumor-microenvironment or could deplete effector cells. Moreover, auto-immune responses could cause potential side effects. To overcome these problems, we exploited near infrared photoimmunotherapy (NIR-PIT) at the tumor to deplete only intratumoral-Tregs. F(ab’)2 fragments of an anti-mouse CD25 antibody, PC61.3, were generated and conjugated with a phthalocyanine dye, IRDye-700DX (αnti-CD25-F(ab’)2-IR700). In vitro NIR-PIT effect was examined against CD25-expressing-T-lymphocytes, HT2-clone-A5E-cells. In vivo CD25-target-NIR-PIT was performed after an intravenous injection of the conjugate to mice bearing subcutaneous, luciferase transfected, LL/2 (Lewis lung carcinoma, LL/2-luc). Tumor-volume, bioluminescence signals (BLI), and Immune responses following the therapy were examined. In vitro NIR-PIT-induced cytotoxicity was light-dose-dependent. Local CD25-target-NIR-PIT selectively depleted intratumoral-Tregs; yet, Tregs in any other organs were not affected. The local CD25-target-NIR-PIT induced a intratumoral rapid activation and cytotoxic action of CD8-T cells and NK-cells. This led to significant reductions of tumor-volume (p < 0.0001) and BLI (p < 0.05) and prolonged survival (p < 0.0001) compared to non-treated controls. Intriguingly, this local CD25-target-NIR-PIT induced a transient systemic cytokine storm and antitumor-effects on distant non-irradiated specific tumors. Effects of local CD25-target-NIR-PIT were significantly (p < 0.0001) inhibited by a CD8-, NK-, or INFg-depletion, suggesting the anti-tumor roles of CD8 T-cells and NK-cells. Depletion of intratumoral-Tregs with a local CD25-target-NIR-PIT rapidly induced CD8 T- and NK-cell activation, thereby restoring local anti-tumor immunity. Consequently, activated immunity led to regression of not only NIR-PIT-treated-tumors but also non-NIR-light-exposed-tumors in separate parts of the body (Fig). These observations suggest that local CD25-target-NIR-PIT may be a promising new strategy for cancer immunotherapy.

Abstract 4945: The cancer-associated fibroblasts-targeted strategycan augment the potency of the dendritic cell-based vaccine immunotherapy.

Abstract The dendritic cell (DC) -based vaccine immunotherapy has been a promising cancer immunotherapy, but has been insufficient to eradicate the tumor in patients with advanced cancer. This can result from the complicated tumor microenvironment (TME) that is implicated in suppression of anti-tumor immune responses. Several immune cell types in TME, such as Tregs, myeloid derived suppressor cells (MDSC) and tumor-associated macrophages (TAMs), have been reported to regulate anti-tumor immune responses negatively. Cancer-associated fibroblasts (CAFs) are also primary stromal cells in TME, and contribute to tumor growth and metastases through the secretion of TGF-β and stromal cell-derived factor-1 (SDF-1). We considered that TME-targeted strategies should be innovated for the development of the potent cancer immunotherapy. On the basis of these viewpoints, we focused on the role of CAFs in TME, and hypothesized that inhibition of CAFs would lead to improvement of systemic anti-tumor immune responses and enhancement of the potency of the DCs-based vaccine immunotherapy. In this study, we applied tranilast in order to inhibit CAFs, the anti-fibrotic and -allergic agent that is used clinically and has been shown to inhibit fibroblast in the scar tissue. In in vitro studies, we examined effects of tranilast on CAFs that were isolated from established EG7 (mouse lymphoma cells) tumors. As results, tranilast was able to suppress the proliferation of CAFs, and decrease the production of SDF-1 as well as TGF-β from CAFs. Regarding the effect on Tregs, tranilast was able to decrease the induction of them from spleen cells of normal mice. Based on these results, we confirmed that tranilast could inhibit the function of CAFs. Next, we examined the association between inhibition of CAFs and anti-tumor immune responses in tumor-bearing mouse model. C57BL/6 mice bearing EG7 were administered tranilast into the established tumor in combination with tumor antigen-loaded DCs vaccination, and were evaluated anti-tumor immune responses. As results, the population of CAFs was decreased by targeting them, leading to lower expressions of TGF-β as well as SDF-1 in TME. Inhibition of CAFs in TME resulted in the decreased distributions of Tregs in TME and tumor-draining lymph nodes (TDLs). On the induction of effector cells, antigen-specific CD8+ cells producing IFN-γ were significantly increased in TDLs and spleen through inhibition of CAFs in TME. In these mice, systemic antigen-specific cytotoxic responses were augmented, leading to suppression of tumor growth as compared with mice in control groups. These results demonstrate that CAFs are associated with immune suppression, and inhibition of CAFs functions in TME can augment systemic anti-tumor immune responses. Our mouse models provide a new rationale with TME-targeted strategies for enhancing the potency of the DCs-based vaccine immunotherapy. Citation Format: Yasuhiko Ohshio, Ryosuke Kaku, Keiko Ishida, Masayuki Hashimoto, Shoji Kitamura, Koji Teramoto, Jun Hanaoka, Noriaki Tezuka. The cancer-associated fibroblasts-targeted strategycan augment the potency of the dendritic cell-based vaccine immunotherapy. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4945. doi:10.1158/1538-7445.AM2013-4945

Increased prevalence of regulatory T cells in the lung cancer microenvironment: a role of thymic stromal lymphopoietin.

Expansion of CD4+CD25+ regulatory T cells (Tregs) in tumor microenvironment was one of the mechanisms by which cancer cells escaped host defense. Thymic stromal lymphopoietin (TSLP) contributes to the generation of natural Tregs in thymus. Therefore, the purpose of this report was to investigate the role of TSLP in the increasing prevalence of Tregs in lung cancer microenvironment. The expression ratio of TSLP protein in tumor tissues was significantly increased compared with that in benign lesion and non-cancer lung tissue. The prevalence of Tregs in tumor microenvironment was correlated with the expression of TSLP in lung cancer. Dendritic cells (DCs) were induced from peripheral blood mononuclear cells (PBMCs) collected from lung cancer patients and left unstimulated (imDCs) or exposed to hTSLP (TSLP-DCs) or LPS (LPS-DCs). TSLP-DCs expressed intermediate levels of CD83 and high levels of CD86, CD11C, and HLA-DR, which showed a characteristic of less mature DCs. TSLP-DCs secreted low levels of IL-6, IL-12, IL-10, TNF-α and IFN-γ, and high levels of TGF-β and MDC. The percentage of Tregs in CD4+CD25- T cells cocultured with TSLP-DCs group was statistically higher than that of LPS-DCs and imDCs. Transwell assays showed that TSLP-DCs exhibited increased ability to attract the migration of CD4+CD25- Tregs, when compared with imDCs. These results indicated that TSLP proteins were expressed in lung tumor tissue and correlated with the prevalence of Tregs. TSLP-DCs could induce CD4+CD25- T cells to differentiate into CD4+CD25+foxp3+ T cells and the migration of CD4+CD25+ T cells.

Expansion of peripheral and intratumoral regulatory T-cells in hepatocellular carcinoma: A case-control study

Hepatocellular carcinoma (HCC) is notorious for poor prognosis with limited therapeutic options. A better understanding of the role of regulatory T-cells (Tregs) in HCC is important for design of immunotherapy based clinical protocol. The objective of the present study was to evaluate the presence of Tregs in tumor microenvironment in patients with HCC compared to chronic hepatitis (CH). The frequency of CD4 + CD25 + Treg cells was evaluated from peripheral blood (PB) of 28 patients of HCC and 30 controls including CH cases and healthy donors using flowcytometry. Intratumoral Treg were also analyzed in tissue samples from 17 HCC cases and 15 CH cases. In addition the expression of FOXP3 and CTLA-4 was also studied by RT-PCR. Frequency of CD4 + CD25 + cells in the PBMCs of HCC cases was significantly higher than in HC (10.8 ± 7.64 vs 3.05 ± 1.30, P < 0.005) and CH patients (2.88 ± 1.92, P < 0.005). Also Treg population was significantly higher in HCC tumor microenvironment compared to CH biopsies (15.8 ± 5.32 vs 5.51 ± 3.40, P < 0.05). Expression of FOXP3 and CTLA-4 was also significantly higher in HCC patients ( P < 0.05) compared to CH group. We provide evidence of an increased population of Treg not only in the PB but also in tumor microenvironment of HCC patients, suggesting association of enhanced Treg activity with poor immune responses to tumor antigens. These findings may in future play a significant role in designing immunotherapeutic approaches in HCC.

Correlation of dynamic alteration of regulatory T cells with the genesis and development of intrahepatic cholangiocarcinoma in a rat model

Objective To investigate the correlation of dynamic alteration of regulatory T cells(Tregs) with the genesis and development of intrahepatic cholangiocarcinoma in a rat model. Methods A total of 40 male SD rats were administered orally with drinking water containing 300 mg/L thioacetamide, and 40 rats administered normally as control group. Peripheral blood mononuclear cells(PBMCs) ,spleen, tumor and liver were harvested and detected at a two-weeks interval from 10 weeks after initiation of thioacetamide. Tregs in PBMCs and spleen were examined by flow cytometry, and those in tumor microenvironment by immunohistochemistry. Results(1) The main pathologic features in different phases were observed as follows:hyperplasia of duct gland from 12th to 14th week;atypical hyperplasia at 16th week;the visible tumor after 18th week.(2) In PBMCs,an increasing proportion of Tregs was observed from the 14th week [(4.25±0. 64) % vs(3. 76±0. 60) % , P<0.01 ]. The proportion of Tregs reached the peak at 18th week [(6.18±0.64)% ]. Tregs in the spleen began to increase at 18th week,reached the peak at 20th week [(8.54±0.36)% ].(3) A significant increase of Tregs was examined at 18th week in tumor microenvironment(P<0.05). With the progress of tumor,the number of Tregs in tumor microenvironment was increased. Conclusion The increased Tregs in PBMCs and the spleen were related to tumor genesis. But they did not show any correlation with tumor progression. The proportion of Tregs in tumor microenvironment was correlated with tumor progression. Key words: Cholangiocarcinoma; Model,animal; T cells