Why cancer immunotherapies targeting regulatory T-cells require translational humanized models

Regulatory T cells (Tregs) are essential guardians of immune homeostasis. Characterized by the expression of the transcription factor FOXP3, these specialized CD4⁺ T cells prevent excessive immune activation, maintain self-tolerance, and protect tissues from immune-mediated damage (Sakaguchi et al., 2008; Josefowicz et al., 2012). In healthy individuals, Tregs suppress the activity of conventional T cells, dendritic cells, NK cells, and other immune effectors through multiple mechanisms, including the secretion of IL-10 and TGF-β, expression of inhibitory receptors such as CTLA-4, metabolic disruption, and direct modulation of antigen-presenting cells (Vignali et al., 2008; Wing & Sakaguchi, 2010). The critical physiological role of Tregs is illustrated by the severe autoimmune syndrome that develops in individuals and mice lacking functional FOXP3 (Sakaguchi et al., 2008).

While indispensable for maintaining immune tolerance, Tregs can become detrimental in cancer. Tumors frequently exploit physiological tolerance mechanisms to evade immune surveillance, leading to the accumulation of highly suppressive Tregs within the tumor microenvironment (TME) (Nishikawa & Sakaguchi, 2010). In many solid malignancies, including melanoma, non-small cell lung cancer, ovarian cancer, gastrointestinal cancers, and glioblastoma, increased intratumoral Treg infiltration is associated with impaired anti-tumor immunity, resistance to immunotherapy, and poor clinical outcomes (Shang et al., 2015; Plitas & Rudensky, 2020). These tumor-infiltrating Tregs suppress cytotoxic CD8⁺ T-cell responses, limit antigen presentation, inhibit NK-cell activity, and contribute to the establishment of an immunosuppressive niche that supports tumor progression (Tanaka & Sakaguchi, 2017).

Treg-targeted therapies for immunotherapy

This strong mechanistic rationale has made Treg depletion or functional inhibition one of the most actively pursued strategies in cancer immunotherapy. Preclinical studies demonstrated that targeting CD25, a known marker of Tregs, for inducing Treg depletion, can restore anti-tumor immunity and promote tumor rejection, particularly when combined with vaccination strategies or immune checkpoint blockade (Shimizu et al., 1999). Clinically, anti-CTLA-4 antibodies such as ipilimumab may exert part of their efficacy through Fc-mediated depletion of intratumoral Tregs in addition to checkpoint inhibition (Simpson et al., 2013). More recently, attention has shifted towards markers preferentially expressed by tumor-resident Tregs, including CCR8, GITR, TIGIT, LAG-3, and CTLA-4, with the goal of achieving selective depletion of suppressive Tregs while preserving systemic immune tolerance. Among these targets, CCR8 has emerged as a particularly promising candidate because of its preferential expression on highly suppressive tumor-infiltrating Tregs in both mice and humans, making CCR8-directed therapies attractive next-generation strategies for selective Treg depletion (Wen et al., 2025).

Importantly, Treg-targeting approaches rarely function as stand-alone therapies. Instead, they are increasingly being developed as combination strategies with PD-1/PD-L1 inhibitors, cancer vaccines, oncolytic viruses, radiotherapy, or adoptive cell therapies. By removing a major immunosuppressive barrier, Treg depletion can enhance T-cell priming, improve effector-cell infiltration into tumors, and increase the durability of anti-tumor responses (Tanaka & Sakaguchi, 2017; Plitas & Rudensky, 2020). However, the therapeutic window remains narrow. Excessive systemic Treg depletion may trigger immune-related adverse events, including colitis, dermatitis, and endocrinopathies, underscoring the need for highly selective approaches that preferentially target tumor-associated Tregs (Wing et al., 2019).

Why translational humanized models are essential

The development of Treg-depleting therapeutics presents unique translational challenges. Many molecules pursued as immunotherapy targets exhibit species-specific expression patterns, receptor biology, Fc interactions, or immune functions. Consequently, conventional mouse models often fail to predict clinical efficacy and safety accurately. Human-specific antibodies designed to deplete Tregs through antibody-dependent cellular cytotoxicity (ADCC), Fc receptor engagement, or checkpoint modulation require experimental systems capable of reproducing both human immune responses and human target biology.

Mouse models with a specific humanized target or with a human immune system (HIS mice) address this critical gap by enabling the evaluation of therapeutic antibodies in the context of a functional immune system. These models enable the assessment of Treg biology, target engagement, immune-cell depletion, cytokine responses, anti-tumor activity, and safety risks within a more clinically relevant setting.

genOway's humanized platform for Treg research

genOway has developed a portfolio of humanized models, both target-specific or with a human immune system, that enables the study of Treg biology, target engagement, and anti-tumor immune responses in vivo.

At the foundation of this portfolio is the genO-BRGSF-HIS model, reconstituted with human CD34+ hematopoietic stem cells, which develops functional human myeloid and lymphoid cell compartments, including monocytes, DCs, NK cells, γδT cells and regulatory T cells. This model enables evaluation of human immune responses, monitoring of Treg dynamics, and testing of depletion strategies in vivo, as well as testing the safety of these strategies (Martin et al., AACR 2026; Martin et al., 2024; Li et al., 2017). Importantly, the model is particularly valuable for the safety assessment of CCR8-targeting therapies, as CCR8 displays species-dependent expression patterns. In the genO-BRGSF-HIS mice, CCR8 is expressed by naïve Tregs in the blood and spleen as observed in humans, while in the hCCR8/hCCL1 knockin mice, CCR8 expression is not observed in the spleen. Consequently, knockin mice might underestimate the safety effects of CCR8-targeted therapies, and a far more translational assessment can be achieved using the genO-BRGSF-HIS model.

Complementing this platform, genOway's CCR8/CCL1, CTLA-4, GITR, LAG-3, and TIGIT humanized models support the development of therapies targeting pathways involved in Treg recruitment, suppressive function, and selective depletion. These models are particularly relevant for investigating novel checkpoint inhibitors and next-generation antibodies designed to overcome Treg-mediated immunosuppression while preserving systemic immune tolerance. In particular, the genO-hCCR8/hCCL1 double-humanized model was designed to evaluate therapies targeting the CCR8-CCL1 axis, a pathway strongly associated with tumor-infiltrating suppressive Tregs and increasingly recognized as a promising mechanism for selective Treg depletion (Wen et al., 2025). Additionally, hCCR8-targetting antibodies have been shown to lead to efficient tumor growth inhibition in this model, validating it for studying the efficacy of Treg-targeted therapies (Sônego et al., AACR 2025). To support the development of combination immunotherapies, genOway has also developed a genO-hCCR8/hCCL1/hPD-1 triple-humanized model, which enables the simultaneous evaluation of CCR8-mediated Treg depletion and PD-1 checkpoint blockade. This model provides a translationally relevant platform for assessing potential synergistic mechanisms between selective Treg targeting and immune checkpoint inhibition.

Since Fc-mediated mechanisms are critical for the activity of many Treg-depleting antibodies, genOway's FcγR-humanized models (genO-hFcγR and genO-hCCR8/hCCL1/hFcγR) enable the evaluation of antibody-dependent cellular cytotoxicity and phagocytosis (ADCP), and other Fc-mediated effector functions in a clinically relevant setting. The genO-hFcγR model supports the assessment of antibody effector functions and Fc-engineering strategies using a human Fcγ-receptor repertoire, while the genO-hCCR8/hCCL1/hFcγR model combines human target biology with human FcγRs, enabling comprehensive evaluation of anti-CCR8 antibodies. This includes assessment of target engagement, Fc-mediated Treg depletion, and anti-tumor efficacy, making the model particularly valuable for the development of next-generation CCR8-targeting therapeutics.

Whereas these models are primarily designed to evaluate antibody-mediated cell depletion, another strategy for eliminating specific cell populations relies on T-cell engagers, which redirect and activate T cells against target-expressing cells. To support the development of these therapeutics, genOway developed the genO-panhCD3 model. This model is particularly valuable for assessing the efficacy and safety of T-cell engager approaches, including those designed to deplete immunosuppressive cell populations such as Tregs. Importantly, the model has already been validated for selective cell-depletion strategies in autoimmune disease settings, demonstrating its utility for investigating CD3-mediated therapeutic mechanisms across multiple indications (Perico et al., 2024).

Together, these models provide complementary tools that enable researchers to investigate Treg biology across multiple therapeutic modalities, including monoclonal antibodies, bispecific antibodies, T-cell engagers, and combination immunotherapies. Importantly, they allow mechanistic exploration of how selective Treg depletion can enhance anti-tumor immunity while minimizing systemic toxicity, one of the central challenges facing next-generation immuno-oncology programs. By combining human immune system reconstitution with target-specific genetic humanization, genOway's platform provides a powerful framework for advancing the next generation of Treg-directed immunotherapies.

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References

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