The landscape of cancer immunotherapy is rapidly evolving with the emergence of several cutting-edge modalities. Some examples include genetically engineered chimeric antigen receptor (CAR) immune cell subsets, neoantigen vaccines and multi-specific antibodies.
CAR-Natural Killer (NK) cells and CAR-macrophage cells express synthetic receptors targeting tumor-specific antigens, enabling them to directly recognize and kill cancer cells. Neoantigen vaccines represent a personalized strategy, using tumor-specific mutated peptides unique to each patient to prime the immune system—especially cytotoxic T-cells—to recognize and eliminate cancer cells, with early trials showing durable responses and synergy with immune checkpoint inhibitors. Multi-specific antibodies, such as bispecific T-cell engagers (BiTE), are engineered to simultaneously bind tumor antigens and immune effector cells (like T cells or NK cells), thereby redirecting and activating the immune system to attack cancer with high specificity. Collectively, these approaches harness and precisely direct the immune system to overcome cancer progression.
In vitro immune cell assays are indispensable tools in drug development, enabling precise evaluation of candidate therapies before advancing to clinical trials. By harnessing the unique functions of immune cells, these assays accelerate the discovery and optimization of next-generation cancer immunotherapies.
T-cell assays: are pivotal for assessing the activation, proliferation, and cytotoxicity in response to immunotherapeutic agents. T-cell assays typically measure cytokine production, target cell lysis, and T-cell expansion, providing critical insights into the potential of a therapy to stimulate a robust anti-tumor immune response.
NK cell assays: are essential for characterizing the function of engineered products like CAR-NK cells. These in vitro tests evaluate NK cell-mediated cytotoxicity against tumor targets, degranulation, and cytokine secretion, thereby determining the ability of NK cell therapies to recognize and kill cancer cells with high specificity and safety.
In vitro Macrophage assays: assess phagocytosis, and cytokine/chemokine release, enabling researchers to screen compounds that modulate macrophage activity to enhance anti-tumor immunity or overcome immunosuppression.
In vitro immune cell assays are critical in the early stages of cancer immunotherapy development, allowing precise functional evaluation of candidate therapies. These assays provide essential data on immune activation, cytotoxicity, and modulation, thereby accelerating the optimization and selection of promising treatments before clinical testing.
In this report we focus on cutting edge immunotherapies and in vitro assays that help move them into the clinic.
In Vitro Assays for Cutting-Edge Cancer Immunotherapies
Below is a list of cell-based assays used to move development of drugs across multiple stages of discovery and preclinical research. The assays listed meet requirements for each mechanistically distinct drug modality.
Immune Cell (Non T-Cell) Activation Assays
Macrophages and dendritic cells respond to some stimulants that leads to the production of chemokines and pro-inflammatory cytokines (e.g., IL-1b, TNFa and IL-6) which can be measured in cell culture supernatants by ELISA. Stimulation can also induce the expression of adhesion molecules such as VCAM-1, ICAM-1 on endothelial and stromal cells.
- Bi-specific antibody. e.g. Roche (RO7567132)
- CAR-Macrophages. e.g. Carisma Therapeutics (CT-0508)
Cytotoxic T-Cell Tumor Killing Assay
A cytotoxic T lymphocyte (CTL) tumor killing assay is designed to measure the ability of CTLs to recognize and kill tumor (target) cells in vitro. The basic workflow involves co-culturing effector T-cells with target tumor cells at varying effector-to-target (E:T) ratios, followed by quantification of tumor cell death. Examples of detection assays include Flow cytometry-based assays using Annexin V, 7-AAD, or PI to detect apoptosis/necrosis, or luminescence based assays such as the CytoTox-Glo Cytotoxicity Assay, designed to measure the relative number of dead cells in a population.
- CAR-T. e.g. Leucid Bio (LEU011)
- Tumor Infiltrating Lymphocyte (TIL) therapy. e.g. Iovance Biotherapeutics (Amtagvi)
- TCR T-Cell Therapy. e.g. Adaptimmune (Tecelra)
- Bispecific T-cell Engager. e.g. Amgen (BiTE-2025)
T-Cell Cytokine Release Assay
A CAR T-cell cytokine release assay involves co-culturing CAR T-cells with target tumor cells that express the relevant antigen. A BiTE cytokine release assay involves co-culturing the BiTE with T cells with target tumor cells that express the relevant antigen. In both cases cytokine expression is the readout. Cytokines such as IL-6, IFN-γ, TNF-α, and IL-2 are quantified using ELISA, Luminex, or similar multiplex assays to assess the magnitude of cytokine release.
- CAR-T. e.g. Leucid Bio LEU011
- Bispecific T-cell Engager. e.g. Amgen (BiTE-2025)
Complement-Dependent Cytotoxicity (CDC) Assay
A common CDC assay example involves testing the cytotoxic effect of the therapeutic monoclonal antibody rituximab on CD20-positive B-cell lymphoma cell lines such as Daudi or Raji. In this setup, rituximab binds to the CD20 antigen on the surface of the cancer cells, and the addition of human serum provides the complement proteins necessary for the cytotoxic cascade. The extent of cell lysis of cancer cells is then measured, often using a dye-release or viability assay.
- Complement Activator. e.g. Commit Biologics (CB-101)
Tumor Infiltrating Lymphocyte Expansion Assay
A tumor infiltrating lymphocyte (TIL) in vitro expansion assay uses the culture of primary tumor tissue, containing high concentrations of interleukin-2 (IL-2), then co-culture with irradiated feeder cells, anti-CD3 antibody. Expanded TILs are then characterized by flow cytometry for CD4+ (T helper) and CD8+ (cytotoxic T cell), and their tumor-killing capacity is evaluated using cytotoxicity assays.
- Tumor Infiltrating Lymphocyte therapy. e.g. Iovance Biotherapeutics (Amtagvi)
Natural Killer Cell Cytotoxicity Assay
Natural killer (NK) cell cytotoxicity assays are designed to measure the ability of NK cells to kill target cells. Primary NK cells are isolated and co-cultured with a target cell line that is sensitive to NK cell-mediated lysis. Flow cytometry, or lactate dehydrogenase release from lysed target cells approaches are common assays to measure dead cells.
- CAR-NK cells. e.g. Fate Therapeutics (FT522)
Natural Killer Cell Degranulation Assay
A widely used method to measure natural killer (NK) cell degranulation in vitro is the CD107a degranulation assay, which uses flow cytometry to detect surface expression of CD107a on NK cells. NK cells are co-incubated with target cells and triggers NK cell degranulation. CD107a is transiently exposed on the NK cell surface as cytotoxic granules are released.
- CAR-NK cells. e.g. Fate Therapeutics (FT522)
Phagocytosis Assay
A standard in vitro assay for measuring macrophage-mediated phagocytosis involves co-culturing macrophages with labeled target cells, followed by quantification of phagocytosis using flow cytometry or microscopy. Generally, macrophages and target cells are labeled with different colored fluorophores. After co-culture, the percentage of macrophages that have internalized target cells and express both fluorophores are considered positive.
- CAR-Macrophages. e.g. Carisma Therapeutics (CT-0508)
Peripheral Blood Mononuclear Cell ELISPOT
The ELISPOT (Enzyme-Linked ImmunoSpot) assay is a sensitive technique used to quantify antigen-specific memory T cell responses at the single-cell level in peripheral blood mononuclear cells (PBMCs) by detecting cytokine secretion, most commonly interferon-gamma (IFN-γ). PBMCs are cultured in the presence of a specific antigen overnight. The secreted cytokines are captured by immobilized antibodies on a membrane, and each spot visualized corresponds to a single cytokine-secreting, antigen-specific T cell. Cytokine spots are developed by adding detection antibodies and substrate. The number of spots is counted and reported as spot-forming cells (SFC) per million PBMCs, reflecting the frequency of antigen-specific memory T cells.
- Neoantigen Vaccine. e.g. Moderna (NEO-PV-01)
Antigen Specific T-cell Activation Assay
Antigen specific T-cell activation assays commonly use peripheral blood mononuclear cells (PBMCs) stimulated in vitro with an antigen, such as tumor specific antigens. If antigen-specific T cells are present, they will become activated. T-cell activation can be detected using Flow Cytometry with the following read-outs:
- Intracellular cytokine staining.
- Activation-induced marker (AIM) assays, which detect upregulation of surface markers (such as CD69, CD137, OX40) on T-cells.
- Proliferation assays using dyes like CFSE to track T-cell division in response to antigen.
- TCR T-Cell Therapy. e.g. Adaptimmune (Tecelra)
- Neoantigen Vaccine. e.g. Moderna (NEO-PV-01)
CONCLUSION
The field of cancer immunotherapy is undergoing a remarkable transformation, driven by innovative approaches. Cutting-edge therapies are redefining how we harness and direct the immune system to target and eliminate cancer with unprecedented precision and efficacy.
However, the successful translation of these novel modalities from the laboratory to the clinic relies heavily on robust in vitro immune cell assays. These assays are indispensable for evaluating the functional activity, specificity, and safety of candidate therapies—providing critical insights into immune cell activation, cytotoxicity, and modulation. By enabling rapid and precise assessment of therapeutic potential, in vitro assays play a pivotal role in accelerating the discovery, optimization, and clinical advancement of next-generation immunotherapies. Ultimately, the synergy between innovative immunotherapeutic strategies and sophisticated in vitro testing platforms is paving the way for more effective, personalized, and safer cancer treatments.
The table below highlights nine cutting edge cancer immunotherapies, each with unique mechanisms, and both standard and specific immune-cell based assays used for research and development. The information in the table is based on the latest 2025 oncology trends and conference disclosures.
Cutting Edge Cancer Immunotherapies and Essential Cell-Based Assays
| Drug Name | Main Mechanism | Company | Cell-Based Assay |
| Bi-specific Antibody (RO7567132) | Bispecific antibody targeting FAP and LTBR in tumor stroma/vasculature | Roche | Immune cell (non- T cell) activation assays |
| CAR-T (LEU011) | Chimeric antigen receptor T-cell therapy for solid tumors | Leucid Bio | Cytotoxic T-cell tumor killing assay and T-cell cytokine release assay using patient-derived tumor cells |
| Complement Activator (CB-101) | Harnesses complement system for tumor cell lysis | Commit Biologics | Complement-dependent cytotoxicity (CDC) assays in cancer cell lines |
| TIL Therapy (Amtagvi) | Tumor-infiltrating lymphocyte therapy with enhanced persistence | Iovance Biotherapeutics | Cytotoxic T-cell tumor killing assay and TIL expansion assays using patient tumor samples |
| CAR-NK Cells | Chimeric antigen receptor-modified NK cells for tumor targeting | Fate Therapeutics | NK cell cytotoxicity and degranulation assays with cancer cell lines |
| CAR-Macrophages | Chimeric antigen receptor-modified macrophages for tumor phagocytosis | Carisma Therapeutics | Phagocytosis and immune cell (non-T cell) activation assays in tumor cell co-culture |
| Neoantigen Vaccine (NEO-PV-01) | Personalized vaccine induces T-cell response to tumor neoantigens | Moderna/partner | T-cell cytokine release assay and ELISPOT with patient PBMCs |
| TCR-T Cell Therapy | T-cell receptor engineered T cells target tumor-specific antigens | Adaptimmune | Cytotoxic T-cell tumor killing assay and antigen specific T-cell activation assay |
| Bispecific T-cell Engager (BiTE-2025) | Links T-cells to tumor antigens for direct killing | Amgen | Cytotoxic T-cell tumor killing assay and T-cell cytokine release assay in tumor co-culture |
**Cell-based assays listed are standard for the modality and target described. Where possible, specific assay types are included.
Disclaimer: This blog post is intended solely for educational and scientific informational purposes. Any mention of therapeutic drug names, including FDA-approved medications, is for the purpose of accurate reporting and discussion of biomedical research and does not constitute medical advice, endorsement, or promotion. Readers should not interpret the content as a recommendation for any specific treatment. Always consult a qualified healthcare professional for medical advice or treatment decisions.
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