Treg Suppression Research Articles

PTEN is required for human Treg suppression of costimulation in vitro.

Regulatory T-cell (Treg) therapy is under clinical investigation for the treatment of transplant rejection, autoimmune disease, and graft-versus-host disease. With the advent of genome editing, attention has turned to reinforcing Treg function for therapeutic benefit. A hallmark of Tregs is dampened activation of PI3K-AKT signaling, of which PTEN is a major negative regulator. Loss-of-function studies of PTEN, however, have not conclusively shown a requirement for PTEN in upholding Treg function and stability. Using CRISPR-based genome editing in human Tregs, we show that PTEN ablation does not cause a global defect in Treg function and stability; rather, it selectively blocks their ability to suppress antigen-presenting cells. PTEN-KO Tregs exhibit elevated glycolytic activity, upregulate FOXP3, maintain a Treg phenotype, and have no discernible defects in lineage stability. Functionally, PTEN is dispensable for human Treg-mediated inhibition of T-cell activity in vitro and in vivo but is required for suppression of costimulatory molecule expression by antigen-presenting cells. These data are the first to define a role for a signaling pathway in controlling a subset of human Treg activity. Moreover, they point to the functional necessity of PTEN-regulated PI3K-AKT activity for optimal human Treg function.

An OGT-STAT5 Axis in Regulatory T Cells Controls Energy and Iron Metabolism.

The immunosuppressive regulatory T (Treg) cells exert emerging effects on adipose tissue homeostasis and systemic metabolism. However, the metabolic regulation and effector mechanisms of Treg cells in coping with obesogenic insults are not fully understood. We have previously established an indispensable role of the O-linked N-Acetylglucosamine (O-GlcNAc) signaling in maintaining Treg cell identity and promoting Treg suppressor function, via STAT5 O-GlcNAcylation and activation. Here, we investigate the O-GlcNAc transferase (OGT)-STAT5 axis in driving the immunomodulatory function of Treg cells for metabolic homeostasis. Treg cell-specific OGT deficiency renders mice more vulnerable to high-fat diet (HFD)-induced adiposity and insulin resistance. Conversely, constitutive STAT5 activation in Treg cells confers protection against adipose tissue expansion and impaired glucose and insulin metabolism upon HFD feeding, in part by suppressing adipose lipid uptake and redistributing systemic iron storage. Treg cell function can be augmented by targeting the OGT-STAT5 axis to combat obesity and related metabolic disorders.

GARP as a Therapeutic Target for the Modulation of Regulatory T Cells in Cancer and Autoimmunity

Regulatory T cells (Treg) play a critical role in immune homeostasis by suppressing several aspects of the immune response. Herein, Glycoprotein A repetitions predominant (GARP), the docking receptor for latent transforming growth factor (LTGF-β), which promotes its activation, plays a crucial role in maintaining Treg mediated immune tolerance. After activation, Treg uniquely express GARP on their surfaces. Due to its location and function, GARP may represent an important target for immunotherapeutic approaches, including the inhibition of Treg suppression in cancer or the enhancement of suppression in autoimmunity. In the present review, we will clarify the cellular and molecular regulation of GARP expression not only in human Treg but also in other cells present in the tumor microenvironment. We will also examine the overall roles of GARP in the regulation of the immune system. Furthermore, we will explore potential applications of GARP as a predictive and therapeutic biomarker as well as the targeting of GARP itself in immunotherapeutic approaches.

Preferential Expansion of Foxp3+ T Regulatory Cells in CTLA-4-Deficient and CTLA-4-Haploinsufficient C57BL/6 Mice.

Foxp3+ cells and CTLA-4 have been ascribed major roles in downregulating immune responses. To address the relationship between CTLA-4 expression and Foxp3+ cells, we generated littermate CTLA-4-sufficient (Ctla4 +/+), CTLA-4-haploinsufficient (Ctla4 +/-), and CTLA-4-deficient (Ctla4 -/-) Foxp3-gfp knock-in C57BL/6 mice, permitting us to characterize the phenotype of Foxp3+ cells and to test their ex vivo T regulatory (Treg) suppressor activity. CD3+, CD4+, and CD8+ cells, but not CD19+ cells, were markedly expanded in Ctla4 -/- mice compared with Ctla4 +/+ or Ctla4 +/- mice. In Ctla4 -/- mice, the relative expansion of the Foxp3+ population was greater than that of the CD3+, CD4+, or CD8+ populations because of increased survival of Foxp3+ cells. Foxp3+ Treg cells from Ctla4 -/- mice and Foxp3+ Treg cells from Ctla4 +/+ mice exerted identical ex vivo suppressor function. This may be related to differential expression of GITR, CD73, and CD39 on Foxp3+ Treg cells from Ctla4 -/- mice versus that on corresponding cells from littermate Ctla4 +/+ or Ctla4 +/- mice, with GITR and CD39 being upregulated and CD73 being downregulated on Foxp3+ Treg cells from Ctla4 -/- mice. Moreover, CTLA-4 expression in Ctla4 +/+, Ctla4 +/-, and Ctla4 -/- mice correlated with their percentages of Foxp3+ cells, suggesting an important role for CTLA-4 expression in Treg cell homeostasis. This may have vital ramifications for the treatment of patients for whom augmentation of suppressor function would be beneficial (e.g., patients with autoimmune diseases) and for whom diminution of suppressor function would be beneficial (e.g., patients with cancer).

Regulatory T Cells Require CCR6 for Skin Migration and Local Suppression of Vitiligo

Vitiligo is an autoimmune skin disease caused by melanocyte-targeting autoreactive CD8+ T cells. Regulatory T cells (Tregs) have been implicated in restraining vitiligo severity in both mouse models and human patients; however, whether they must be present in the skin for their suppressive function is still unclear. We observed uneven distribution of Tregs within different anatomical locations of mouse skin, which correlated with reduced depigmentation after vitiligo induction. We specifically depleted Tregs in our mouse model of vitiligo and observed increased disease. Next, we found that Tregs contact CD8+ T effector cells in vitiligo lesional skin and that Treg recruitment to the skin inversely correlated with disease severity, suggesting a critical role for Treg suppression within the skin. When we investigated the signals facilitating Treg migration to the skin, we found that although CXCR3 was dispensable for Treg migration and function in vitiligo, Tregs lacking CCR6 exhibited a reduced capacity to migrate to the skin and suppress depigmentation, despite normal systemic numbers in the skin-draining lymph nodes. Our observations highlight a key role for cutaneous Tregs in disease suppression during vitiligo and identify CCR6 as a chemokine receptor that contributes to Treg migration to the skin.

Development of a fully human anti-GITR antibody with potent antitumor activity using H2L2 mice.

Glucocorticoid‐induced TNF receptor‐related (GITR) can act as a co‐stimulatory receptor, representing a potential target for safely enhancing immunotherapy efficacy. GITR is triggered by a GITR ligand or an agonist antibody and activates CD8+ and CD4+ effector T cells, reducing tumor‐infiltrating Treg numbers and resulting in activation of immune responses and tumor cell destruction by effector T cells. GITR is an attractive target for immunotherapy, especially in combination therapy with immune checkpoint inhibitors, as is being explored in clinical trials. Using H2L2 transgenic mice encoding the human immunoglobulin variable region and hybridoma technology, we generated a panel of fully human antibodies that showed excellent specific affinity and strong activation of human T cells. After conversion to fully human antibodies and engineering modification, we obtained an anti‐GITR antibody hab019e2 with enhanced antitumor activity in a B‐hGITR MC38 mouse model compared to Tab9H6V3, an anti‐GITR antibody that activates T cells and inhibits Treg suppression from XenoMouse. As a fully human antibody with its posttranslational modification hot spot removed, the hab019e2 antibody exerted more potent therapeutic effects, and may have potential as a novel and developable antibody targeting GITR for follow‐up drug studies.

Development and Characterization of an Anti-Cancer Monoclonal Antibody for Treatment of Human Carcinomas

Simple SummaryCancers can grow and spread to different parts of the body. The aim of immunotherapy is to stimulate the immune system to eliminate cancer cells in a selective manner. Tumor-targeting monoclonal antibodies (mAb) can be used as immunotherapy to stimulate innate antitumor immunity. In this review, we have described the development and characterization of an anti-cancers mAb NEO-201. NEO-201 is an IgG1 humanized mAb that binds specifically to tumor-associated variants of CEACAM-5 and CEACAM-6 expressed by colon, ovarian, pancreatic, non-small cell lung, head and neck, cervical, uterine and breast cancers, but is not reactive against most normal tissues. The peculiarity of NEO-201 is its ability to counteract tumor growth through different mechanisms. NEO-201 engages components of immune system to kill tumor cells expressing its target via antibody-dependent cell-mediated cytotoxicity and complement dependent cytotoxicity. NEO-201 can also indirectly enhance anti-cancer activity through the blockade of the interaction between CEACAM-5 expressed on tumor cells and CEACAM-1 expressed on natural killer cells to reverse CEACAM-1-dependent inhibition of NK cytotoxicity or through its binding to human regulatory T cells. The specificity of NEO-201 in recognizing suppressive regulatory T cells provides the basis for combination cancer immunotherapy with checkpoint inhibitors targeting the PD-1/PD-L1 pathway.NEO-201 is an IgG1 humanized monoclonal antibody (mAb) that binds to tumor-associated variants of carcinoembryonic antigen-related cell adhesion molecule (CEACAM)-5 and CEACAM-6. NEO-201 reacts to colon, ovarian, pancreatic, non-small cell lung, head and neck, cervical, uterine and breast cancers, but is not reactive against most normal tissues. NEO-201 can kill tumor cells via antibody-dependent cell-mediated cytotoxicity (ADCC) and complement dependent cytotoxicity (CDC) to directly kill tumor cells expressing its target. We explored indirect mechanisms of its action that may enhance immune tumor killing. NEO-201 can block the interaction between CEACAM-5 expressed on tumor cells and CEACAM-1 expressed on natural killer (NK) cells to reverse CEACAM-1-dependent inhibition of NK cytotoxicity. Previous studies have demonstrated safety/tolerability in non-human primates, and in a first in human phase 1 clinical trial at the National Cancer Institute (NCI). In addition, preclinical studies have demonstrated that NEO-201 can bind to human regulatory T (Treg) cells. The specificity of NEO-201 in recognizing suppressive Treg cells provides the basis for combination cancer immunotherapy with checkpoint inhibitors targeting the PD-1/PD-L1 pathway.

Recombinant multimeric dog allergen prevents airway hyperresponsiveness in a model of asthma marked by vigorous TH 2 and TH 17 cell responses.

Allergy to dogs affects around 10% of the population in developed countries. Immune therapy of allergic patients with dog allergen extracts has shown limited therapeutic benefit. We established a mouse model of dog allergy by repeatedly administering dog dander and epithelium extracts via the intranasal route. We also assessed the efficacy of a recombinant multimeric protein containing Can f 1, f 2, f 4 and f 6 in preventing inflammatory responses to dog extracts. Repeated inhalation of dog extracts induced infiltration of the airways by TH 2 cells, eosinophils and goblet cells, reminiscent of the house dust mite (HDM) model of asthma. Dog extracts also induced robust airway hyperresponsiveness and promoted TH 17 cell responses, which was associated with a high neutrophilic infiltration of the airways. scRNA-Seq analysis of T helper cells in the airways pinpointed a unique gene signature for TH 17 cells. Analysis of T-cell receptors depicted a high frequency of clones that were shared between TH 17, TH 2 and suppressive Treg cells, indicative of a common differentiation trajectory for these subsets. Importantly, sublingual administration of multimeric Can f 1-2-4-6 protein prior to sensitization reduced airway hyperresponsiveness and type 2-mediated inflammation in this model. Dog allergen extracts induce robust TH 2 and TH 17 cell-mediated responses in mice. Recombinant Can f 1-2-4-6 can induce tolerance to complex dogallergen extracts.

Abstract 6127: Single-cell spatial transcriptome sequencing characterizes CD70+ immune-resilient nasopharyngeal carcinoma cells as a vital mediator of Treg-induced suppression

Abstract Background: The microenvironment of nasopharyngeal carcinoma (NPC) has an intense and heterogenous immune infiltration, due to chronic EBV infection and locoregional lymphoid structures. Phase II clinical trials have shown that the response rate of NPC patients to PD-1 inhibitors is only 20%, indicating the current immunotherapies are not optimal due to insufficient understanding of the tumor-immune interaction. In this study, we have applied an integrated transcriptome sequencing analysis to decipher immune dynamics, and have characterized a new target that can inhibit Treg-mediated suppression in NPC, and provided critical translational evidence to the precision immunotherapy. Methods: We established an integrated NPC single-cell sequencing cohort containing 189,750 T cells from 50 patient samples. Spatial transcriptome sequencing was performed on frozen samples from eight NPC patients. The whole blood was collected from healthy donors and PBMCs were isolated using density gradient centrifugation. T cells were magnetically isolated from PBMCs. The humanized mouse model was established by tail-vein injection of CD3/CD28 activated PBMCs. Results: Single-cell sequencing revealed the enrichment of suppressive Tregs in the NPC microenvironment. We found that the C666-1 cell line facilitated Treg differentiation and activation. Spatial sequencing identified CD70-CD27 interaction was upregulated between NPC cells and suppressive Tregs, indicating its role in regulating Treg function. CD70 was predominantly expressed on NPC cells and correlated to worse prognosis. CD70-knockout (KO) in C666-1 inhibited Treg differentiation. Decreased Treg activities further enhanced the cytotoxicity in CD8+ effector T cells, leading to higher tumor apoptosis. Cusatuzumab also generated a comparable inhibitory effect in Treg-mediated suppression. In the humanized mouse model, CD70-KO and inhibition shrank the size of subcutaneous tumors by enhancing T cell immunity. Conclusion: The enrichment and activation Tregs makes the NPC microenvironment highly immunosuppressive, which might jeopardize the clinical efficacy of anti-PD1 therapies in NPC patients. In this study, we revealed that CD70+ NPC cells were highly resilient to immune surveillance since they were capable of modulating Treg differentiation and activation via the CD70-CD27 interaction to counteract CD8+ T cells. Blocking CD70 has been shown to effectively induce tumor apoptosis by alleviating Treg-mediated suppression. The therapeutic efficacy and safety of the anti-CD70 therapy were tested in the humanized mouse model. These findings suggested that the anti-CD70 therapy exhibited a promising efficacy to kill NPC cells by deteriorating the immunosuppressive microenvironment, and it might synergistically enhance the efficacy of anti-PD1 therapies. Citation Format: Lanqi Gong, Jie Luo, Yuma Yang, Yu Zhang, Xin-Yuan Guan. Single-cell spatial transcriptome sequencing characterizes CD70+ immune-resilient nasopharyngeal carcinoma cells as a vital mediator of Treg-induced suppression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 6127.

Abstract 514: Correlation of sTNFR2 elevation and disease progression in cutaneous T cell lymphomas

Abstract Tumor necrosis factor receptor 2 (TNFR2), a membrane receptor that is a part of the tumor necrosis factor (TNF) superfamily and one of two receptors that can bind to TNF-α, has been emerging in recent years as a promising cancer immunotherapy target. TNFR2 is commonly overexpressed in cancer cells and in the tumor microenvironment. It is also a key receptor in immune evasion, due to its abundance on the most immunosuppressive subset of Treg cells (CD4+Foxp3+), as well as its role in driving proliferation of regulatory T cells (Tregs) and tumor cells via activation of the nuclear factor kappa B (NF-κB) pathway. In environments with high cell proliferation and high surface densities, TNFR2 can be cleaved and shed as serum soluble TNFR2 (sTNFR2). In human cancers, high sTNFR2 has recently been associated with poor prognostic outcomes in colon cancer, esophageal cancer, endometrial cancer and non-Hodgkin’s lymphomas. Elevated sTNFR2 is also potentially associated with poor prognostic outcomes in cutaneous T cell lymphomas (CTCLs), a diverse group of lymphomas primarily composed of mycosis fungoides (MF) and Sézary syndrome (SS), with symptoms that primarily manifest in the skin. Given sTNFR2’s accessibility in serum and association with cancer prognosis, we investigated its role as a potential biomarker for MF and SS prognosis. We also evaluated whether the CTCL tumor type is mechanistically being driven by very suppressive Tregs and tumor cells with this oncogene. In our study, we measured baseline sTNFR2 levels from 31 serum samples across different cancer stages (Stage I-IV) of MF and SS in either treatment-naïve subjects or subjects free of any treatments for ≥3 months. Our initial data shows that sTNFR2 values correlate with levels of blood disease and increase as the cancer advances. These findings strengthen the possibility of using sTNFR2 as a biomarker in MF and SS and also support the possible mechanistic role of TNFR2 in disease pathogenesis. Analysis of the same serum samples for sTNFR1, a non-specific marker of inflammation and renal failure, confirm that the increases are exclusive to sTNFR2 and can indeed be attributed to MF and SS, rather than to non-specific inflammation. Further studies following these patients’ responses to their treatment plans (i.e., mogamulizumab, romidepsin) will help elucidate the potency of sTNFR2 in predicting response to treatment and cancer burden. Citation Format: Amanda Lee, Youn Kim, Michael Khodadoust, Denise L. Faustman. Correlation of sTNFR2 elevation and disease progression in cutaneous T cell lymphomas [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 514.

Abstract 2043: The role of CCL20 in response to radiation in head and neck squamous cell carcinomas

Abstract Background: Radioresistance is responsible for most cases of tumor progression and recurrence in head and neck squamous cell carcinomas (HNSCC). Combined radiotherapy (RT) and immunotherapy (IT) treatment has improved survival for a minority of patients with inflamed tumors or tumors susceptible to RT-induced inflammation. To enhance the response of radioresistant tumors to radiation, an understanding of the factors that suppress anti-tumor immunity is necessary. In this regard, regulatory T cells (Tregs) are critical mediators of immune suppression in HNSCCs and their depletion improves tumor response to RT. In this study, we investigated how radiation modulates Treg infiltration through the chemokine CCL20. We hypothesized that radiation induces CCL20 secretion resulting in Treg infiltration and suppression of anti-tumor immunity and that blocking CCL20 could lead to improved response to RT. Methods: Human (Cal27, SCC9) and mouse (MOC1, MOC2) cell lines with different immune phenotypes were irradiated at doses of 2Gy or 10Gy. Conditioned media (CM), RNA and protein were collected for the assessment of CCL20. qPCR was used to determine CCL20 gene expression. In vivo, MOC2 cell were were implanted into the right buccal cavity of mice and the effect of neutralizing CCL20 antibody was determined alone and in combination with RT. Blood samples were collected before and after RT for the analysis of serum levels of CCL20. Tumor samples were analyzed by flow cytometry to determine immune infiltrates, including CD8 T cells and Tregs. Results: Cal27 and MOC2 tumors had a gene signature associated with immunosuppression and Treg infiltration whereas SCC9 and MOC1 tumors displayed a gene signature associated with an inflamed microenvironment. In vitro, high-dose radiation significantly induced CCL20 in Cal27 and MOC2 cells relative to control. Multiplex analysis of chemokines from conditioned media samples showed that induction of CCL20 was associated with increased secretion of GM-CSF by MOC2 and Cal27 cells. In vivo, inhibition of CCL20 alone resulted in a significant decrease in tumor growth compared to control in MOC2 tumors. The combination of RT and anti-CCL20 showed a significant decrease in tumor volume compared to RT alone. Conclusion: Our data suggests that high-dose radiation promotes the induction of CCL20 secretion by tumor cells which is responsible for attraction of Tregs. Inhibition of CCL20 secretion using anti-CCL20 antibodies can slow down tumor growth and enhance the response to radiation. Citation Format: Cleopatra Rutihinda, Ayman J. Oweida, Patrick Delage, Léanie Moreau, Safia Chelighem, Nour Elhouda Saidi. The role of CCL20 in response to radiation in head and neck squamous cell carcinomas [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2043.

Abstract 2866: BAY 3375968: An afucosylated anti-CCR8 antibody depleting activated intratumoral regulatory T cells as a cancer immunotherapy

Abstract Regulatory T cells (Tregs) play an indispensable role in mediating peripheral tolerance to self-antigens. They can also promote tumor growth by suppressing the function of effector CD8+ and CD4+ T cells in the tumor microenvironment (TME). Furthermore, Tregs are identified as one of the key resistance mechanisms hampering the efficacy of immune checkpoint inhibitors (ICIs) across many tumor types. Therefore, there is a high need for safe and effective agents that would specifically deplete tumor-infiltrating Tregs while sparing both peripheral Tregs and effector T cells. Chemokine receptor 8 (CCR8) is predominantly expressed on activated tumor-infiltrating Tregs and marks the most suppressive and proliferative Treg population. CCR8+ Tregs are associated with high tumor grade and poor overall survival across many tumor types such as lung, breast, or head-neck cancer. Thus, unlike other approaches directed against Tregs, targeting CCR8 offers the opportunity to specifically deplete intra-tumoral Tregs without impacting peripheral Tregs or other immune cells. BAY 3375968 is a non-internalizing fully human glycoengineered (afucosylated) monoclonal IgG1 antibody, which in vitro selectively depleted human CCR8+ Tregs via antibody dependent cellular cytotoxicity (ADCC) and antibody dependent cellular phagocytosis (ADCP). In vivo efficacy studies using mouse surrogate antibodies showed strong monotherapeutic efficacy across a variety of murine tumor models with clear correlation of intratumoral CCR8+ Treg depletion and CD8+ T cell increase. The monotherapeutic efficacy of CCR8 depleting antibodies was further enhanced by combinations with ICIs. BAY 3375968 also showed a good safety profile in cynomolgus monkeys. In conclusion, CCR8 is a novel Treg depleting immunotherapy target, and due to its highly tumor-restricted expression profile, BAY 3375968 may provide superior clinical safety and efficacy profile comparing to other less specific Treg targeting approaches. Based on the promising pre-clinical data, preparations for a phase I clinical trial investigating the safety, tolerability, pharmacokinetics, pharmacodynamics, and preliminary anti-tumor activity of BAY 3375968 are underway. Citation Format: Helge Roider, Su-Yi Tseng, Sabine Hoff, Sandra Berndt, Katharina Filarski, Uwe Gritzan, Beatrix Stelte-Ludwig, Wiebke M. Nadler, Joanna Grudzinska-Goebel, Philipp Ellinger, Mark Trautwein, Matyas Gorjanacz. BAY 3375968: An afucosylated anti-CCR8 antibody depleting activated intratumoral regulatory T cells as a cancer immunotherapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2866.

Abstract 3590: ZL-1218, a novel anti-CCR8 antibody, exerts potent antitumor effect by depleting intratumoral regulatory T cells

Abstract Despite the promise of immunotherapy for cancer treatment, nearly 80% of patients fail to respond to checkpoint inhibitor (CPI) therapy. Regulatory T cells (Tregs), which inhibit immune responses in the tumor microenvironment via multiple suppressive mechanisms, have been proposed to play a key role in those patients who are not responding to CPI therapy. Therefore, targeted depletion of Tregs should promote more effective antitumor immunity. CCR8 is a chemokine receptor that is selectively expressed on highly activated human tumor-resident Tregs, and these intratumoral CCR8+ Tregs have been shown to drive immunosuppression that leads to poor prognosis for cancer patients. Here, we demonstrate that CCR8 is highly expressed on intratumoral FoxP3+ Treg cells in multiple cancers and is absent on other major immune cell populations in tumor microenvironment including effector T cells, conventional CD4 T cells, B cells, NK cells, some FoxP3+ cells, and myeloid cells. Importantly, no CCR8 protein expression was observed on any peripheral human leukocyte subset examined. These results provide strong rationale for targeting CCR8 as a cancer immunotherapy by selectively depleting the most suppressive intratumoral Treg cells. We have developed a humanized therapeutic antibody, ZL-1218, that binds to human CCR8 with high affinity and specificity and can induce potent ADCC activity enabling strong NK cell-mediated killing of CCR8-expressing Tregs. We show that in human CCR8 knock-in mouse models bearing syngeneic tumors, ZL-1218 reduces intratumoral Treg cells and thus elicits significant tumor growth inhibition in a dose-dependent manner. We have recently explored the potential for ZL-1218 in combination immunotherapy, examining the enhanced antitumor activity when ZL-1218 is combined with anti-PD-1. Using human dissociated tumor samples, we further observed that different tumor types may induce different CCR8 expression levels on intratumoral Tregs leading to multiple, distinct CCR8+ subsets in various indications. We are currently exploring the significance of these distinct populations and the impact of ZL-1218-mediated depletion of both CCR8 high- and CCR8 low-expressing subsets in multiple indications. Together, these data support the advancement of ZL-1218 into clinical evaluation as a novel therapeutic candidate to treat human solid tumors. Citation Format: Jing Zhang, Wenhua Shi, Min Chen, Xinchuan Dai, Hongshui Liu, Shou Li, Lina Wang, Bee-Chun Sun, Monica You, Vivian Morton, Qiuping Ye, Lishan Kang, Bing Wan, Peter Brams, David I. Bellovin. ZL-1218, a novel anti-CCR8 antibody, exerts potent antitumor effect by depleting intratumoral regulatory T cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3590.

Abstract 1111: Identification of selective IKZF2 degraders that reprogram suppressive regulatory T cells in solid tumors

Abstract Targeted protein degradation (TPD) using the endogenous Ubiquitin Proteasome System (UPS) is a rapidly growing drug discovery strategy to eliminate pathogenic proteins. Molecular glues are small molecules that promote a novel interaction between a protein of interest with an E3 ubiquitin ligase leading to proximity induced protein degradation. This has enabled targeting undruggable proteins, such as the zinc-finger transcription factor Helios (IKZF2), that have no known small molecule binding pocket. Despite recent clinical breakthroughs in checkpoint blockade in treating solid tumors, suppression of the antitumor immune response in the tumor microenvironment (TME) is a major obstacle to tumor regression. Regulatory T cells (Tregs) in the TME are potent immunosuppressive cells that promote progression of cancer. IKZF2 has been shown to be a marker of highly suppressive Treg cells and is critical for maintaining the anergic and suppressive phenotype in the highly inflammatory tumor microenvironment. Genetic depletion of IKZF2 in Treg cells results in both loss of suppressive activity and conversion of Tregs into T effector cells, leading to enhanced anti-tumor immunity. Collectively, these findings support that an IKZF2-specific degrader could be beneficial in enhancing the efficacy of current immunotherapies. Here we report our efforts leading to the development of a series of potent and selective IKZF2 degraders for the treatment of cancer. Our lead compound, PLX-4107, is a novel molecular glue that was optimized to be a highly selective, deep, and rapid IKZF2 degrader via the redirection of the E3 substrate receptor, cereblon. Proteome-wide analysis demonstrated that PLX-4107 depletes IKZF2 protein levels without degrading other known cereblon neo-substrates. PLX-4107 mediated degradation of IKZF2 resulted in conversion of highly suppressive Tregs into T effector cells, coupled with an increased expression of the effector cytokines IL2 and IFNγ. Oral administration of PLX-4107 to cynomolgus monkeys caused rapid, complete, and prolonged degradation of IKZF2 in Tregs, indicative of the catalytic nature of degraders where sustained pharmacodynamic response is observed well beyond plasma drug exposure levels. These preliminary data provide a strong rationale for developing small molecule therapeutics that target the undruggable IKZF2 transcription factor with the potential to enhance the efficacy of immune checkpoint therapy. Citation Format: Peggy A. Thompson, Pengyu Yang, Xiaoming Li, Stephen Chien, Mary E. Spalding, Alejandro Dearie, Elizabeth Daniele, Linette Yang, E Adam Kallel, Aleksandar Jamborcic, Julia Toth, Gregory Parker, Simon Bailey. Identification of selective IKZF2 degraders that reprogram suppressive regulatory T cells in solid tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1111.

Abstract LB189: Pharmacodynamic assessment of a-TIGIT mAb EOS-448 highlights multiple FcγR-mediated mode-of-actions in blood and tumor of patients with advanced solid tumors

Abstract T cell immunoglobulin and ITIM domain (TIGIT) is an immune checkpoint inhibitor expressed mainly on NK and T cell populations which has demonstrated clinical proof of concept in NSCLC for its inhibition in combination with a-PD(L)-1 agents1. EOS-448 (also named GSK4428859A) is an antagonistic anti-TIGIT human immunoglobulin G1 (hIgG1) antibody showing picomolar activity both to prevent ligand binding and to engage Fc gamma receptors (FcγR). EOS-448 was selected based on its potent affinity to TIGIT, translating into strong antagonist and functional activity to restore T cell functions and potent antitumor activity in murine models2. Building EOS-448 on an isotype allowing to engage FcγR added potential additional mechanisms of action (MoAs), including the FcγR-induced activation of myeloid/NK populations and antibody-mediated depletion of TIGIT high cells. To characterize and experimentally validate the multiple MoA triggered by EOS-448, we tested, both in preclinical and clinical settings, its potential to (i) activate effector T cells (ii) modulate antigen-presenting through FcγR engagement, and (iii) deplete suppressive regulatory T cells and terminally exhausted CD8 T cells that express the highest levels of TIGIT. Our results obtained in murine models confirm the strong potential for the a-TIGIT + a-PD1 combination as well as major differences in activity depending on isotype, with only the Fc-engaging format inducing strong antitumor effect correlating with Treg depletion and CD8 T cell activation within the tumor. Similarly, activation markers on DC and antigen-mediated activation were only observed with the Fc-engaging version but totally absent with the Fc-dead format. In addition, we demonstrated, ex-vivo on human samples, that EOS-448 could preferentially deplete Tregs over effector CD8 T cells and progenitor-like exhausted T cells (Tpex) with low effector function over stem-like memory T cells (Tstem) with stronger effector potential3. In a first-in-human trial, EOS-448 demonstrated a good tolerability profile with early signs of efficacy4. Pharmacodynamic assessment made by flow cytometry in the blood of patients treated across multiple dose levels of EOS-448 confirmed the multiple MoAs triggered by the drug with (i) increased Ki67 expression in memory CD8 T cells during the first treatment cycle, (ii) sustained depletion of suppressive Tregs, and (iii) of TIGIT high CD8+ T cells, known to be terminally exhausted5, overall resulting in an increased effector CD8/Treg ratio. This activity also translated into a significant decrease in the detection of TIGIT+ cells by immunohistochemistry (IHC), including FoxP3+ double stained cells, in 21 out of 22 tested paired biopsies, with EOS-448 being the first reported a-TIGIT mAb to engage TIGIT within patient tumor. Overall, the preclinical and clinical evaluation demonstrate the multiple MoAs of EOS-448 supporting its clinical development as a differentiated a-TIGIT mAb to restore anti-tumor immune response. Multiple randomized studies are planned to be initiated in 2022. 1.Cho BC, et al. Abstract #LBA2 ESMO-IO; 2021 2.Preillon J, et al. Mol Cancer Ther ; 2021. 3.Galletti G, et al. Nat Immunol ; 2020. 4.Van den Mooter TF et al. Abstract #CT118 AACR; 2021. 5.Guo X et al. Nat Medicine ; 2018. Citation Format: Julia Cuende, Julie Preillon, Noemie Wald, Marjorie Mercier, Paola Tieppo, Iain Welsby, Veronique Bodo, Yvonne McGrath, Tom F. Van Den Mooter, Jean-Pascal Machiels, Clarisse Truong, Olivier De Henau, Gregory Driessens, Marion Libouban. Pharmacodynamic assessment of a-TIGIT mAb EOS-448 highlights multiple FcγR-mediated mode-of-actions in blood and tumor of patients with advanced solid tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr LB189.

Abstract 3298: Propagation competence of a self-antigen-targeting arenavirus vector based cancer therapy determines antitumor efficacy in mouse melanoma

Abstract Breaking self-tolerance and inducing a robust immune response targeting tumor cell derived self-antigens by therapeutic vaccines has remained challenging. We have generated a replicating viral vector based on the arenavirus lymphocytic choriomeningitis virus expressing the melanoma tissue-specific self-antigen Trp2 (artLCMV-Trp2) and show that a single immunization of mice bearing established melanoma with this vector can break tolerance against this self antigen and induces high levels of tumor-infiltrating TRP2+ CD8+ T cells. These T cells express the effector cytokines IFNγ and TNFα, lack markers of T cell exhaustion, and their appearance coincides with marked inhibition of tumor growth and significant survival benefit. artLCMV-TRP2 induced a strong type I interferon response that resulted in transient type I-dependent Treg inhibition that was necessary for the generation of optimal antitumoral T cell responses. Moreover, artLCMV-TRP2 vaccination proved able to induce complete tumor remission when combined with infusion of exogenous TRP2-specific T cells into mice. In conclusion, replication competence is a key property of artLCMV-TRP2 that enabled effective anti-cancer immunity and therapeutic effect. Citation Format: Mette-Triin Purde, Fabienne Hartmann, Jovana Cupovic, Sarah Schmidt, David Bomze, Felix Stemeseder, Alexander Lercher, Lenka Besse, Fiamma Berner, Thomas Tüting, Andreas Bergthaler, Andrea Schietinger, Stefan Kochanek, Burkhard Ludewig, Klaus K. Orlinger, Tobias Bald, Sandra S. Ring, Lukas Flatz. Propagation competence of a self-antigen-targeting arenavirus vector based cancer therapy determines antitumor efficacy in mouse melanoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3298.

Selected Articles from This Issue

Intratumoral electroporation of IL12-encoding plasmids (IT-pIL12-EP) enhances antitumor immunity in treated and distant tumors. However, IT-pIL12-EP outcomes are in part dictated by infiltrating T-cell composition, where higher proportions of cytotoxic T cells as opposed to more immunosuppressive immune cells predicts enhanced IT-pIL12-EP responsiveness. In an effort to increase proportions of activated cytotoxic T cells in IT-pIL12-EP-treated tumors, Han and colleagues supplemented IT-pIL12-EP with electroporation of plasmids encoding membrane-anchored anti-CD3 single-chain variable fragments (scFv). The authors found that including anti-CD3 scFv with IL12 enhances proliferation and interferon-γ (IFNγ) secretion in both CD8+ cytotoxic T cells and CD4+ T regulatory (Treg) cells, and that costimulation with membrane-bound anti-CD3 with IL12 reduces Treg suppression. Supplementing IT-pIL12-EP with plasmids encoding anti-CD3 scFv in vivo enhances proliferation and cytotoxicity of tumor antigen-specific T cells, leading to augmented antitumor immunity against B16-F10 melanoma and 4T1 mammary tumor cells. Treating human melanoma-infiltrating T cells with IL12 and anti-CD3 scFv enhances cytotoxic CD8+ expansion, IFNγ secretion, and PD-1 expression in vitro, suggesting IT-pIL12-EP and anti-CD3 scFv could augment PD-1 immune checkpoint inhibition responsiveness in melanoma patients.Tumor necrosis factor alpha (TNFα)-mediated cytotoxicity can be resisted via NF-κB activation and downstream survival signals in head and neck squamous cell carcinoma (HNSCC). Toxicities accompanying NF-κB signaling inhibition necessitate other methods of targeting the pathway. To search for novel targets affecting NF-κB signaling, Hu and colleagues performed a RNAi screen targeting the kinome and druggable genomic targets in a HNSCC cell line expressing NF-κB promoter response elements in tandem with a β-lactamase reporter gene. The screen revealed that G2/M checkpoint kinases WEE1 and cyclin dependent kinase 1 (CDK1) promote NF-κB signaling. WEE1 inhibition using AZD1775 disrupts WEE1 interactions with IκB kinase (IKK)α/β and RELA, reducing IKKα/β and RELA phosphorylation and NF-κB signaling. Correspondingly, AZD1775 augments TNFα-mediated HNSCC cell death, including in HNSCC radiotherapy in vivo. WEE1 and CDK1 expression levels and disease outcome correlations are dependent on human papillomavirus (HPV) infection status, suggesting HPV influences the signaling pathway. In addition to unveiling a novel signaling axis, this study presents a new potential way in which NF-κB can be therapeutically targeted in HNSCC.Decreased SIN3A expression in human breast cancer cell lines enhances tumorigenic phenotypes in vitro and tumor progression in vivo. However, how SIN3A abundance in breast tumors compares to its abundance in normal breast tissue is unknown, as is how SIN3A expression is regulated in breast tumors. To address these questions, Davenport and colleagues assessed SIN3A mRNA and corresponding protein levels in patients and validated these findings using public data sets (TCGA and METABRIC). The authors found that the SIN3A mRNA 3′ untranslated region harbors a sequence complementary to that of oncogenic miR-183, and that miR-183 silences SIN3A expression and enhances expression of genes that SIN3A transcriptionally represses. Accordingly, miR-183 expression negatively correlates with breast cancer patient survival and induces breast cancer cell migration and invasion in vitro and pulmonary metastasis in vivo through SIN3A downregulation. Overall, the novel miR-183-mediated regulation of SIN3A expression uncovered in this study could be used to inform metastatic breast cancer intervention strategies moving forward.Radiation and temozolomide (TMZ), the standard treatments for glioblastoma (GBM), are known to induce a non-proliferative, senescent phenotype in surviving GBM cells. However, GBM cells often eventually escape treatment-induced senescence, leading to residual tumor outgrowth and GBM recurrence. To assess whether recently discovered senolytic drugs can eliminate senescent GBM cells, Rahman and colleagues tested the effects of 10 senolytic drugs on 12 human GBM cell lines treated with radiation or TMZ. The authors found that senolytic drugs targeting BCL-XL, as well as siRNA-mediated BCL-XL abrogation, enhance senescent GBM cell death. The results of the study suggest that pharmacological BCL-XL inhibition could augment antitumor effects of radiation and TMZ treatment, and potentially inhibit residual GBM cell outgrowth and disease recurrence.

Immune tumor microenvironnement (iTME) post-neoadjuvant chemotherapy, beyond PD-L1: Novel immune targets in ovarian cancer, data from the CHIVA trial, a GINECO/GINEGEPS study.

5554 Background: Antibodies targeting PDL1 or PD1 have been disappointing so far in the treatment of ovarian cancer (OC). A greater understanding of the complex iTME and of the impact of chemotherapy on immune features could uncover promising immune targets. We previously reported that neoadjuvant chemotherapy (NACT) increased CD4+ and CD8+ immune cells (IC) and depleted FOXP3+ suppressive T-regs in OC iTME. Here we aimed to describe the expression of PDL1 as well as other co-regulatory molecules in OC and their changes under NACT. Methods: Tumor samples and clinical data were prospectively collected from patients (pts) in the randomized CHIVA trial of NACT +/- nintedanib. Samples were evaluable for immune profiling for 116-124 pts at diagnosis and 89-107 at surgery after 3 cycles of NACT. IC stained for CD4, CD8 were scored as number of IC+/mm². Expression of immune co-regulatory molecules PDL1, TIM3, LAG3 and IDO was scored as percentage of positive cells, and tumors were classified as PDL1/TIM3/IDO/LAG3 positive if > 1% of IC and/or tumor cells (TC) were positive. Highly sensitive pts, defined as objective response to NACT and prolonged median progression-free survival (mPFS > 24months), were compared to refractory pts (progressing during or within 3mo of platinum). Results: As expected, about one third (36%) of tumors were PDL1+ at diagnosis. In contrast, the prevalence of other co-regulatory molecules was higher with 52%, 54% and 93% of tumors being positive for IDO, LAG3 and TIM3, respectively. There was no significant change in PDL1 expression with NACT. However, in paired samples NACT significantly increased IDO and LAG3 expression (p < 0.05), such that 60% and 66% of tumors post-NACT were positive for IDO and LAG3, respectively. TIM3 expression remained high post-NACT with 92% of positive tumors. Highly sensitive tumors (vs refractory tumors) had significantly higher IC expression of TIM3 after NACT (24% vs 6%, p = 0.005), and were significantly more infiltrated by CD4+ (441 vs 228 cells/mm2, p = 0.04) and CD8+ (460 vs 225 cells/mm2, p = 0.045) T cells. Conclusions: Other immune targets beyond PDL1 are highly expressed in OC. In addition NACT appears to prime the iTME by increasing effector T cell infiltration and the expression of other relevant co-regulatory molecules (LAG3, TIM3 and IDO). Future studies could be performed by priming the iTME with NACT and testing novel immune therapies based on target expression in samples obtained at interval debulking surgery.

Distinct Injury Responsive Regulatory T Cells Identified by Multi-Dimensional Phenotyping.

CD4+ regulatory T cells (Tregs) activate and expand in response to different types of injuries, suggesting that they play a critical role in controlling the immune response to tissue and cell damage. This project used multi-dimensional profiling techniques to comprehensively characterize injury responsive Tregs in mice. We show that CD44high Tregs expand in response to injury and were highly suppressive when compared to CD44low Tregs. T cell receptor (TCR) repertoire analysis revealed that the CD44high Treg population undergo TCRαβ clonal expansion as well as increased TCR CDR3 diversity. Bulk RNA sequencing and single-cell RNA sequencing with paired TCR clonotype analysis identified unique differences between CD44high and CD44low Tregs and specific upregulation of genes in Tregs with expanded TCR clonotypes. Gene ontology analysis for molecular function of RNA sequencing data identified chemokine receptors and cell division as the most enriched functional terms in CD44high Tregs versus CD44low Tregs. Mass cytometry (CyTOF) analysis of Tregs from injured and uninjured mice verified protein expression of these genes on CD44high Tregs, with injury-induced increases in Helios, Galectin-3 and PYCARD expression. Taken together, these data indicate that injury triggers the expansion of a highly suppressive CD44high Treg population that is transcriptionally and phenotypically distinct from CD44low Tregs suggesting that they actively participate in controlling immune responses to injury and tissue damage.

Helminth infection modulates number and function of adipose tissue Tregs in high fat diet-induced obesity.

BackgroundEpidemiological and experimental studies have shown a protective effect of helminth infections in weight gain and against the development of metabolic dysfunctions in the host. However, the mechanisms Treg cells exert in the helminth-obesity interface has been poorly investigated. The present study aimed to verify the influence of Heligmosomoides polygyrus infection in early stages of high fat diet-induced obesity.Principal findingsThe presence of infection was able to prevent exacerbated weight gain in mice fed with high fat diet when compared to non-infected controls. In addition, infected animals displayed improved insulin sensitivity and decreased fat accumulation in the liver. Obesity-associated inflammation was reduced in the presence of infection, demonstrated by lower levels of leptin and resistin, lower infiltration of Th1 and Th17 cells in adipose tissue, higher expression of IL10 and adiponectin, increased infiltration of Th2 and eosinophils in adipose tissue of infected animals. Of note, the parasite infection was associated with increased Treg frequency in adipose tissue which showed higher expression of cell surface markers of function and activation, like LAP and CD134. The infection could also increase adipose Treg suppressor function in animals on high fat diet.ConclusionThese data suggest that H. polygyrus modulates adipose tissue Treg cells with implication for weight gain and metabolic syndrome.