Assays for Drug Development

Exhausted T cells are a central readout in immuno-oncology in vitro assays, characterized by reduced effector function, altered cytokine production and upregulation of inhibitory receptors.

T cell exhaustion is a distinct differentiation state of T cells observed during chronic infections and cancer; in which chronic antigen exposure leads to T cell dysfunction. Exhausted T cells (T_EX) are characterized by reduced proliferative responses to stimuli, loss of effector function failing to control tumor growth, high expression of inhibitory receptors such as PD-1, LAG-3 and TIM-3 and reduced cytokine secretion in comparison to their conventional, non-exhausted counterparts. 

T_EX undergo extensive transcriptional and epigenetic reprogramming, leading to upregulation of exhaustion-associated factors like TOX and display metabolic disruption due to nutrient-deprived and hypoxic conditions of the TME. These cells become addicted to antigen in what is described as ‘antigen-dependent maintenance’. When chronic antigenic stimulation is withdrawn, T_EX remain dysfunctional, suggesting a commitment to the exhausted T cell state.

At RoukenBio, we have extensive expertise in T cell biology including T cell exhaustion. We were the first CRO to develop a human in vitro T cell exhaustion model that recapitulates the dysfunctional behaviour of T cells in the TME.

Macrophage state and function are critical in shaping the TME playing a key role in creating an immune suppressive environment. Originating from bone-marrow monocytes or tissue-resident precursors, Tumor-associated macrophages (TAMs) are remarkably adaptable, constantly reshaping their function in response to environmental cues. Macrophages are traditionally categorized into M1 (classically activated) and M2 (alternatively activated) subsets. 

In the context of cancer, M1-like TAMs, triggered by type 1 cytokines such as IFN-γ, are associated with pro-inflammatory responses and tumouricidal activity. In contrast, cytokines like IL-4, IL-10, IL-13, TGF-β and PGE2, drive TAMs towards M2-like phenotypes that suppress anti-tumor immunity, support tumor growth, and contribute to remodeling of the TME. This impacts the success of certain therapeutics in treating the disease. 

In most established tumors, TAMs adopt an M2-like phenotype, - marked by high levels of ARG1, CD163, and CD206 - and secrete immunosuppressive cytokines like IL-10 and TGF-β. These cells actively support tumor progression by promoting angiogenesis, remodeling tissue structure, and dampening anti-tumor immune responses via PD-L1, IDO, prostaglandins and the recruitment of regulatory T cells. TAM phenotypes vary widely across tumor types, and even within different lesions or stages of the same tumor. While the M1/M2 classification is a simplification, it remains a valuable framework for understanding macrophage function; especially when applied in the context of evaluating therapeutic candidates.

Key strategies for therapeutic targeting of macrophages in the TME include; CSF1(R) or CCL2/CCR2 inhibition resulting in TAM depletion or prevention of TAM recruitment, VEGF inhibition limiting their tumor-promoting activity, and using CD47/SIRPa inhibitors or TLR agonists to subvert their immunosuppressive function, aiming to re-educate M2-like TAMs to M1-like phenotypes.

At RoukenBio, we understand the complexity of TAM biology. Our advanced macrophage polarization, phagocytosis, and T cell suppression assays are designed to help you evaluate how your therapeutic candidates modulate TAM function, suppress tumor cell growth and drive anti-tumor immunity.

Regulatory T cells (Tregs), typically identified by the CD4⁺CD25^highFOXP3^high phenotype, represent a critical immunosuppressive component within the TME across a broad spectrum of cancers. Their enrichment in tumor tissues frequently correlates with poor cancer prognosis, highlighting their relevance as biomarkers and therapeutic targets in immuno-oncology assays.

Tumor-infiltrating Tregs originate from diverse sources, including thymus-derived Tregs and peripherally induced Tregs derived from conventional T cells. In tumors, these cells exhibit a highly activated phenotype, marked by elevated expression of key immunoregulatory molecules such as CTLA-4, GITR, ICOS, OX40, and TIGIT, along with heightened levels of immune checkpoint receptors like PD-1, LAG-3, and TIM-3.

Functionally, Tregs suppress anti-tumor immune responses via multiple mechanisms: inhibition of effector T cell proliferation (IL-2 sequestration, ATP conversion, glucose deprivation), secretion of immunosuppressive cytokines (IL-10, TGF-β), and modulation of dendritic cell maturation and function (CD80/86 engagement via CTLA-4).

Therapeutic strategies aim to; deplete Treg (anti-CTLA-4), prevent their infiltration (CCR4 antagonists), or impair their immunosuppressive function in the TME (CD39/CD73 blockade) without compromising peripheral immune tolerance.

At RoukenBio, we offer comprehensive capabilities to evaluate how your therapeutic candidate influences Treg biology, providing in-depth analysis into the phenotype and functional activity of both natural and induced Treg populations.

Immune cell-based killing assays are powerful tools for driving innovation in cancer immunotherapy, offering mechanistic insights and therapeutic evaluation of immune-mediated tumour cell destruction. By mimicking real-time tumour–immune cell interactions, these assays enable precise assessment of potency, specificity, and safety for cutting-edge modalities such as immune cell engagers and engineered cell therapies.

Beyond efficacy testing, they illuminate mechanisms of resistance and inform smarter therapeutic design. For researchers aiming to accelerate innovation in immuno-oncology, these assays are essential for translating breakthrough science into next-generation cancer treatments.

Our off-the-shelf and bespoke assay design allows us to test the cytotoxic potential of small molecules, biologics (BiTEs or ADCs) and cell-based therapies (CAR-T, CAR-NK or γδ T cells) against cancer cell lines or custom-built target expressing cells. 

Available formats include:

T cell mediated: 

• T cell dependent cellular cytotoxicity (TDCC): Assess ability of BiTEs to engage primary human T cells to kill tumour cells
• CAR-T cells: Determine cytotoxic potential of your CAR-T cell candidates 
• TCR-engineered T cells: Assess therapeutic molecules in an antigen specific system

NK cell mediated: 

• Antibody dependent cellular cytotoxicity (ADCC): Assess ability of antibodies to engage primary NK cells to kill tumour cells 
• CAR-NK cells: Determine cytotoxic potential of your CAR-NK cell candidates 

γδ T cell-mediated:

• Screen γδ T cell-engaging therapeutics using our ex vivo expanded γδ T cells (CD3+ Vγ9+Vδ2+) 

Standard readouts include:

• Flow cytometry- or impedance-based target cytolysis
• Effector cell phenotyping, proliferation and activation via flow cytometry
• Cytokine analysis by ELISA or multiplex bead-based array

RoukenBio leverages extensive experience in T cell biology, having pioneered the development of a robust in vitro model of T cell exhaustion in-house. Using isolated pan CD3+ T cells that are repeatedly stimulated, our system drives cells into a hyporesponsive, dysfunctional state, characterized by upregulation of multiple immune checkpoint molecules and a marked reduction in IL-2 production.

Combining in vitro generated exhausted T cells with a functional assay such as a mixed lymphocyte reaction (MLR), this medium-throughput, flexible assay format is ideally suited for mechanism-of-action (MoA) studies of checkpoint inhibitors, co-stimulatory agents, T cell engagers, and modulators of T cell signaling, tailored to support both early discovery and translational research.

Functional Readouts Include:

1. Cytokine secretion (e.g. IFNγ via ELISA or cytokine multiplex assays)
2. Proliferation (Ki-67 expression by flow cytometry)
3. Co-inhibitory and co-stimulatory marker profiling via flow cytometry

An example of data from an MLR functional readout is shown below. In this assay, proliferation as indicated by %Ki67 expression was compared in conventional T (TCON) cells and exhausted T (TEX) cells treated with nivolumab or an IgG4 control antibody. 

T cell fitness is often compromised by the complex and highly immunosuppressive nature of the TME. Within this dynamic ecosystem, diverse cellular populations - including regulatory T cells (Tregs) and tumor-associated macrophages (TAMs) - act through direct contact and soluble mediators to suppress T cell activation and proliferation, as well as limit their trafficking.

At RoukenBio, we specialize in T cell suppression assays using a variety of immunosuppressive cell types, including M2-polarized macrophages and both natural and induced Tregs. Our flexible platforms support evaluation across therapeutic modalities, helping you assess how your candidates modulate immune suppression.

We also offer test article integration during macrophage polarization or Treg induction to evaluate effects on their differentiation. 

Key readouts include:

1. T cell proliferation and T cell phenotyping via flow cytometry
2. Cytokine profiling through ELISA or multiplex array

Explore our collaboration with Miltenyi showcasing how Cbl-b antagonism reverses Treg-mediated T cell suppression.