Regulatory T Cells Restrain Interleukin-2- and Blimp-1-Dependent Acquisition of Cytotoxic Function by CD4+ T Cells.

In addition to helper and regulatory potential, CD4+ T cells also acquire cytotoxic activity marked by granzyme B (GzmB) expression and the ability to promote rejection of established tumors. Here, we examined the molecular and cellular mechanisms underpinning the differentiation of cytotoxic CD4+ T cells following immunotherapy. CD4+ transfer into lymphodepleted animals or regulatory T (Treg) cell depletion promoted GzmB expression by tumor-infiltrating CD4+, and this was prevented by interleukin-2 (IL-2) neutralization. Transcriptional analysis revealed a polyfunctional helper and cytotoxic phenotype characterized by the expression of the transcription factors T-bet and Blimp-1. While T-bet ablation restricted interferon-γ (IFN-γ) production, loss of Blimp-1 prevented GzmB expression in response to IL-2, suggesting two independent programs required for polyfunctionality of tumor-reactive CD4+ T cells. Our findings underscore the role of Treg cells, IL-2, and Blimp-1 in controlling the differentiation of cytotoxic CD4+ T cells and offer a pathway to enhancement of anti-tumor activity through their manipulation.

Fc-Optimized Anti-CD25 Depletes Tumor-Infiltrating Regulatory T Cells and Synergizes with PD-1 Blockade to Eradicate Established Tumors.

CD25 is expressed at high levels on regulatory T (Treg) cells and was initially proposed as a target for cancer immunotherapy. However, anti-CD25 antibodies have displayed limited activity against established tumors. We demonstrated that CD25 expression is largely restricted to tumor-infiltrating Treg cells in mice and humans. While existing anti-CD25 antibodies were observed to deplete Treg cells in the periphery, upregulation of the inhibitory Fc gamma receptor (FcγR) IIb at the tumor site prevented intra-tumoral Treg cell depletion, which may underlie the lack of anti-tumor activity previously observed in pre-clinical models. Use of an anti-CD25 antibody with enhanced binding to activating FcγRs led to effective depletion of tumor-infiltrating Treg cells, increased effector to Treg cell ratios, and improved control of established tumors. Combination with anti-programmed cell death protein-1 antibodies promoted complete tumor rejection, demonstrating the relevance of CD25 as a therapeutic target and promising substrate for future combination approaches in immune-oncology.

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

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

An anti-CTLA-4 heavy chain-only antibody with enhanced Treg depletion shows excellent preclinical efficacy and safety profile.

The value of anti-CTLA-4 antibodies in cancer therapy is well established. However, the broad application of currently available anti-CTLA-4 therapeutic antibodies is hampered by their narrow therapeutic index. It is therefore challenging and attractive to develop the next generation of anti-CTLA-4 therapeutics with improved safety and efficacy. To this end, we generated fully human heavy chain-only antibodies (HCAbs) against CTLA-4. The hIgG1 Fc domain of the top candidate, HCAb 4003-1, was further engineered to enhance its regulatory T (Treg) cell depletion effect and to decrease its half-life, resulting in HCAb 4003-2. We tested these HCAbs in in vitro and in vivo experiments in comparison with ipilimumab and other anti-CTLA4 antibodies. The results show that human HCAb 4003-2 binds human CTLA-4 with high affinity and potently blocks the binding of B7-1 (CD80) and B7-2 (CD86) to CTLA-4. The results also show efficient tumor penetration. HCAb 4003-2 exhibits enhanced antibody-dependent cellular cytotoxicity function, lower serum exposure, and more potent anti-tumor activity than ipilimumab in murine tumor models, which is partly driven by a substantial depletion of intratumoral Tregs. Importantly, the enhanced efficacy combined with the shorter serum half-life and less systemic drug exposure in vivo potentially provides an improved therapeutic window in cynomolgus monkeys and preliminary clinical applications. With its augmented efficacy via Treg depletion and improved safety profile, HCAb 4003-2 is a promising candidate for the development of next generation anti-CTLA-4 therapy.

Preclinical development of a novel CCR8/CTLA-4 bispecific antibody for cancer treatment by disrupting CTLA-4 signaling on CD8 T cells and specifically depleting tumor-resident Tregs.

Anti-CTLA-4 antibodies faced challenges due to frequent adverse events and limited efficacy, which spurred the exploration of next-generation CTLA-4 therapeutics to balance regulatory T cells (Tregs) depletion and CD8 T cells activation. CCR8, identified primarily on tumor-infiltrating Tregs, has become a target of interest due to the anti-tumor effects demonstrated by CCR8 antibody-mediated Tregs depletion. Single-cell RNA sequencing analysis reveals that CCR8-positive Tregs constitute a small subset, with concurrent expression of CCR8 and CTLA-4. Consequently, we proposed a novel bispecific antibody targeting CCR8 and CTLA-4 that had the potential to enhance T cell activation while selectively depleting intratumor Tregs. The candidate molecule 2MW4691 was developed in a tetravalent symmetric format, maintaining a strong binding affinity for CCR8 while exhibiting relatively weaker CTLA-4 binding. This selective binding ability allowed 2MW4691 to target and deplete tumor-infiltrating Tregs with higher specificity. In vitro assays verified the antibody's capacity for antibody-dependent cellular cytotoxicity (ADCC) to Tregs with high level of CTLA-4 expression, but not CD8 T cells with relatively low level of CTLA-4 on cell surface. Also, 2MW4691 inhibited the CTLA-4 pathway and enhanced T cell activation. The in vivo therapeutic efficacy of 2MW4691 was further demonstrated using hCCR8 or hCTLA-4 humanized mouse models and hCCR8/hCTLA-4 double knock-in mouse models. In cynomolgus monkeys, 2MW4691 was well-tolerated, exhibited the anticipated pharmacokinetic profile, and had a minimal impact on the peripheral T cell population. The promising preclinical results supported the further evaluation of 2MW4691 as a next-generation Treg-based therapeutics in clinical trials.

High-grade glioma associated immunosuppression does not prevent immune responses induced by therapeutic vaccines in combination with Treg depletion.

High-grade gliomas (HGG) exert systemic immunosuppression, which is of particular importance as immunotherapeutic strategies such as therapeutic vaccines are increasingly used to treat HGGs. In a first cohort of 61 HGG patients we evaluated a panel of 30 hematological and 34 plasma biomarkers. Then, we investigated in a second cohort of 11 relapsed HGG patients receiving immunomodulation with metronomic cyclophosphamide upfront to a DC-based vaccine whether immune abnormalities persisted and whether they hampered induction of IFNγ+ T-cell responses. HGG patients from the first cohort showed increased numbers of leukocytes, neutrophils and MDSCs and in parallel reduced numbers of CD4+/CD8+ T-cells, plasmacytoid and conventional DC2s. MDSCs and T-cell alterations were more profound in WHO IV° glioma patients. Moreover, levels of MDSCs and epidermal growth factor were negatively associated with survival. Serum levels of IL-2, IL-4, IL-5 and IL-10 were altered in HGG patients, however, without any impact on clinical outcome. In the immunotherapy cohort, 6-month overall survival was 100%. Metronomic cyclophosphamide led to > 40% reduction of regulatory T cells (Treg). In parallel to Treg-depletion, MDSCs and DC subsets became indistinguishable from healthy controls, whereas T-lymphopenia persisted. Despite low T-cells, IFNγ-responses could be induced in 9/10 analyzed cases. Importantly, frequency of CD8+VLA-4+ T-cells with CNS-homing properties, but not of CD4+ VLA-4+ T-cells, increased during vaccination. Our study identifies several features of systemic immunosuppression in HGGs. Metronomic cyclophosphamide in combination with an active immunization alleviates the latter and the combined treatment allows induction of a high rate of anti-glioma immune responses.

CTLA-4 antibody-drug conjugate reveals autologous destruction of B-lymphocytes associated with regulatory T cell impairment.

Germline CTLA-4 deficiency causes severe autoimmune diseases characterized by dysregulation of Foxp3+ Tregs, hyper-activation of effector memory T cells, and variable forms autoimmune cytopenia including gradual loss of B cells. Cancer patients with severe immune-related adverse events (irAE) after receiving anti-CTLA-4/PD-1 combination immunotherapy also have markedly reduced peripheral B cells. The immunological basis for B cell loss remains unexplained. Here, we probe the decline of B cells in human CTLA-4 knock-in mice by using anti-human CTLA-4 antibody Ipilimumab conjugated to a drug payload emtansine (Anti-CTLA-4 ADC). The anti-CTLA-4 ADC-treated mice have T cell hyper-proliferation and their differentiation into effector cells which results in B cell depletion. B cell depletion is mediated by both CD4 and CD8 T cells and at least partially rescued by anti-TNF-alpha antibody. These data revealed an unexpected antagonism between T and B cells and the importance of regulatory T cells in preserving B cells.

Differential expression of CCR8 in tumors versus normal tissue allows specific depletion of tumor-infiltrating T regulatory cells by GS-1811, a novel Fc-optimized anti-CCR8 antibody.

The presence of T regulatory (Treg) cells in the tumor microenvironment is associated with poor prognosis and resistance to therapies aimed at reactivating anti-tumor immune responses. Therefore, depletion of tumor-infiltrating Tregs is a potential approach to overcome resistance to immunotherapy. However, identifying Treg-specific targets to drive such selective depletion is challenging. CCR8 has recently emerged as one of these potential targets. Here, we describe GS-1811, a novel therapeutic monoclonal antibody that specifically binds to human CCR8 and is designed to selectively deplete tumor-infiltrating Tregs. We validate previous findings showing restricted expression of CCR8 on tumor Tregs, and precisely quantify CCR8 receptor densities on tumor and normal tissue T cell subsets, demonstrating a window for selective depletion of Tregs in the tumor. Importantly, we show that GS-1811 depleting activity is limited to cells expressing CCR8 at levels comparable to tumor-infiltrating Tregs. Targeting CCR8 in mouse tumor models results in robust anti-tumor efficacy, which is dependent on Treg depleting activity, and synergizes with PD-1 inhibition to promote anti-tumor responses in PD-1 resistant models. Our data support clinical development of GS-1811 to target CCR8 in cancer and drive tumor Treg depletion in order to promote anti-tumor immunity.

IL-2 availability regulates the tissue specific phenotype of murine intra-hepatic Tregs.

CD4+CD25+Foxp3+ Tregs are known to acquire tissue-specific features and exert cytoprotective and regenerative functions. The extent to which this applies to liver-resident Tregs is unknown. In this study, we aimed to explore the phenotypic and functional characteristics of adult murine liver resident Tregs during homeostasis. Additionally, we investigated their role in ameliorating liver inflammation and tissue damage. Quantification of Foxp3+CD4+CD25+ cells comparing different tissues showed that the liver contained significantly fewer resident Tregs. A combination of flow cytometry phenotyping and microarray analysis of intra-hepatic and splenic Tregs under homeostatic conditions revealed that, although intra-hepatic Tregs exhibited the core transcriptional Treg signature, they expressed a distinct transcriptional profile. This was characterized by reduced CD25 expression and increased levels of pro-inflammatory Th1 transcripts Il1b and Ifng. In vivo ablation of Tregs in the Foxp3-DTR mouse model showed that Tregs had a role in reducing the magnitude of systemic and intra-hepatic inflammatory responses following acute carbon tetrachloride (CCl4) injury, but their absence did not impact the development of hepatocyte necrosis. Conversely, the specific expansion of Tregs by administration of IL-2 complexes increased the number of intra-hepatic Tregs and significantly ameliorated tissue damage following CCl4 administration in C57BL/6 mice. The cytoprotective effect observed in response to IL-2c was associated with the increased expression of markers known to regulate Treg suppressive function. Our results offer insight into the transcriptome and complex immune network of intra-hepatic Tregs and suggest that strategies capable of selectively increasing the pool of intra-hepatic Tregs could constitute effective therapies in inflammatory liver diseases.

Lack of Specific Regulatory T Cell Depletion and Cytoreduction Associated with Extensive Toxicity After Administration of Low and High Doses of Cyclophosphamide.

Up to 93% of the human immunodeficiency virus (HIV) latent reservoir comprised defective proviruses, suggesting that a functional cure is possible through the elimination of a small population of cells containing intact virus, instead of the entire reservoir. Cyclophosphamide (Cy) is an established chemotherapeutic agent for immune cell cancers. In high doses, Cy is a nonselective cytoreductor, used in allogeneic stem-cell transplantation, while in a low dose, metronomic schedule, Cy selectively depletes regulatory T cells (Tregs). We administered low and high doses to simian immunodeficiency virus (SIV)-infected rhesus macaques (RM) to assess their effects on the SIV reservoirs. As a Treg-depleting agent, Cy unselectively depleted Treg and total lymphocytes, resulting in minimal immune activation and no viral reactivation. As a cytoreductive agent, Cy induced massive viral reactivation in elite controller RMs without ART. However, when administered with antiretroviral therapy (ART), Cy had substantial adverse effects, including mortality. Our study thus dissuades further investigation of Cy as an HIV cure agent.

The Impact of Tregs on the Anticancer Immunity and the Efficacy of Immune Checkpoint Inhibitor Therapies.

Although cancers arise from genetic mutations enabling cells to proliferate uncontrollably, they cannot thrive without failure of the anticancer immunity due in a large part to the tumor environment's influence on effector and regulatory T cells. The field of immune checkpoint inhibitor (ICI) therapy for cancer was born out of the fact that tumor environments paralyze the immune cells that are supposed to clear them by activating the immune checkpoint molecules such as PD-1. While various subsets of effector T cells work collaboratively to eliminate cancers, Tregs enriched in the tumor environment can suppress not only the native anticancer immunity but also diminish the efficacy of ICI therapies. Because of their essential role in suppressing autoimmunity, various attempts to specifically deplete tumor-associated Tregs are currently underway to boost the efficacy of ICI therapies without causing systemic autoimmune responses. A better understanding the roles of Tregs in the anti-cancer immunity and ICI therapies should provide more specific targets to deplete intratumoral Tregs. Here, we review the current understanding on how Tregs inhibit the anti-cancer immunity and ICI therapies as well as the advances in the targeted depletion of intratumoral Tregs.

Effects of Regulatory T Cell Depletion on NK Cell Responses against Listeria monocytogenes in Feline Immunodeficiency Virus Infected Cats.

Regulatory T cells (Treg) are key players in the maintenance of peripheral tolerance, preventing autoimmune diseases and restraining chronic inflammatory diseases. Evidence suggests Treg cells and NK cells have important roles in feline immunodeficiency virus (FIV) pathogenesis; however, in vivo studies investigating the interplay between these two cell populations are lacking. We previously described innate immune defects in FIV-infected cats characterized by cytokine deficits and impaired natural killer cell (NK) and NK T cell (NKT) functions. In this study, we investigated whether in vivo Treg depletion by treatment with an anti-feline CD25 monoclonal antibody would improve the innate immune response against subcutaneous challenge with Listeria monocytogenes (Lm). Treg depletion resulted in an increased overall number of cells in Lm-draining lymph nodes and increased proliferation of NK and NKT cells in FIV-infected cats. Treg depletion did not normalize expression of perforin or granzyme A by NK and NKT cells, nor did Treg depletion result in improved clearance of Lm. Thus, despite the quantitative improvements in the NK and NKT cell responses to Lm, there was no functional improvement in the early control of Lm. CD1a+ dendritic cell percentages in the lymph nodes of FIV-infected cats were lower than in specific-pathogen-free control cats and failed to upregulate CD80 even when Treg were depleted. Taken together, Treg depletion failed to improve the innate immune response of FIV-infected cats against Lm and this may be due to dendritic cell dysfunction.

Injection site and regulatory T cells influence durable vaccine-induced tumor immunity to an over-expressed self tumor associated antigen.

Tumor protein D52 (D52) is constitutively expressed in healthy tissues and overexpressed in multiple cancers, including (but not limited to) breast, prostate and ovarian carcinomas. Although the normal functions of D52 are unknown, it is clear that increased D52 expression levels not only stimulate cell proliferation and metastasis, but also correlate with poor prognosis in a subset of breast cancer patients. The murine orthologs of D52 (mD52) shares 86% identity with its human counterpart (hD52) and mirrors hD52 expression patterns. The forced overexpression of mD52 induces anchorage-independent growth in vitro and promotes tumor formation as well as spontaneous metastasis in vivo. We have previously reported that the intramuscular administration of recombinant mD52 elicits immune responses capable of rejecting a challenge with tumor cells and preventing spontaneous metastasis only in 50% of mice. We hypothesized that mechanisms of peripheral tolerance dampen immune responses against mD52, thus limiting the protective effects of vaccination. To test this hypothesis, mice were depleted of CD25+ regulatory T cells (Tregs) and subcutaneously immunized with mD52 prior to a tumor challenge. The subcutaneous immunization failed to induce protective antitumor immunity unless accompanied by Treg depletion, which resulted in a rate of protection of 70% as compared with.