Published on 06 Dec 24 | Updated on 10 July 2025

Immunotherapy represents a transformative advancement in cancer treatment, offering significant success in managing advanced cancers while causing fewer side effects. To date, the FDA has approved over 20 immunotherapy agents, with more than 70 additional molecules undergoing clinical trials for various oncology indications. This approach includes a wide range of innovative techniques, such as immune checkpoint inhibitors, bispecific antibodies, ADCs, and CAR-T cell therapies.

Learn how o2h discovery supports immuno-oncology research:

  • o2h scientists can perform immune phenotyping of whole blood-derived PBMCs and quantify the expression of surface receptors and intracellular proteins
  • Develop co-culture systems of various cancer cell lines with PBMCs, T cells and NK cells; characterize paracrine signalling events; activation, proliferation, and differentiation using fluorochrome-conjugated antibodies towards cell-surface receptors
  • Therapeutic small molecules and antibodies can be screened for their cytotoxic potential against cancer cell lines using CFSE/7-AAD dyes to quantify the percentage of cellular apoptosis and necrosis
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Figure 1: o2h scientist working on CytoFLEX S.

PBMC Immunophenotyping using T-cell Antibody Panel

Figure 2: Human PBMCs stained with a T cell antibody panel. The gating strategy involved first identifying the lymphocyte population by forward scatter (FSC) and side scatter (SSC). Live T cells (CD3+) were gated and then the two main types of T cells were defined by CD4+ (T helper cells) and CD8+ (cytotoxic T cells).


Understanding Lymphocyte-Mediated Cytotoxicity and Its Role in Immunotherapy

Lymphocyte-mediated cytotoxicity is an essential part of the immune system, allowing immune cells such as T cells to identify and destroy cells infected by viruses or transformed into cancer cells. Studying this process is crucial for developing new cancer treatments, including small-molecule drugs and antibody-based immunotherapies. Two key strategies in cancer immunotherapy are checkpoint blockade inhibitors and bispecific antibodies.

Checkpoint Blockade Inhibitors
Cancer cells often exploit “checkpoint” proteins to avoid immune attack. For example, PD-1 is a protein on T cells that, when bound to its partner PD-L1 on cancer cells, sends a signal that dampens the immune response. This allows tumours to hide from the immune system. Checkpoint inhibitors are drugs designed to block this interaction, preventing PD-1 and PD-L1 from binding. This effectively “releases the brakes” on T cells, allowing them to attack cancer cells more strongly.

Bispecific Antibodies
Bispecific antibodies are engineered molecules that can bind two different targets at the same time. One arm of the antibody attaches to a T cell (for example, via the CD3 receptor), while the other arm binds a tumour-specific antigen. This dual binding brings T cells close to cancer cells, triggering the T cells to directly kill the tumor cells.


How Lymphocyte-Mediated Cytotoxicity Is Measured

Several lab-based assays help determine how effectively immune cells kill cancer cells. Two common methods are:

  • ADCC (Antibody-Dependent Cellular Cytotoxicity): measures how antibodies help immune cells kill target cells.
  • TDCC (T Cell-Dependent Cellular Cytotoxicity): measures how T cells directly kill target cells.

A widely used method for assessing cytotoxicity is the CFSE/7-AAD Cytotoxicity Assay.

  • CFSE (Carboxyfluorescein succinimidyl ester) is a fluorescent dye used to stain target cells before they are mixed with immune cells. It labels live target cells, allowing them to be distinguished from other cells in the mixture during analysis. As the cells remain viable, CFSE stays inside them.
  • 7-AAD (7-aminoactinomycin D) is a dye that can enter only cells with compromised membranes. i.e. dead or dying cells.

In the following assay, peripheral blood mononuclear cells (PBMCs), which include immune cells, are combined with CFSE-labelled target cells. After incubation, 7-AAD is added to identify dead cells. Using flow cytometry, it’s possible to measure the proportion of CFSE-labelled target cells that have taken up 7-AAD, indicating they’ve been killed. This provides insights into how well immune cells or therapeutic agents are eliminating cancer cells.

Understanding these mechanisms, along with the design of these assays, is critical for developing effective immunotherapies for cancer.

At o2h, we have used the cytotoxicity assay described above to show that the small-molecule PD-1/PD-L1 checkpoint inhibitor BMS-1166 can restore PBMC-mediated killing of MDA-MB-231 breast cancer cells in a PBMC : cancer cell co-culture system. We have also demonstrated that a BCMA × CD3 bispecific antibody induces dose-dependent killing of RPMI 8226 multiple myeloma cells using a similar co-culture approach.

Figure 3: Cell surface expression of the checkpoint ligand PD-L1 on the MDA-MB-231 breast cancer cell line was assessed by flow cytometry. Cells were stained with LIVE/DEAD Fixable Violet Cell Stain, followed by incubation with PE-conjugated anti-PD-L1 antibody (clone 29E.2A3, 1:100 dilution) for 30 minutes on ice. The analysis showed (A) approximately 90% of cells expressing PD-L1 and (B) a four-fold increase in PD-L1 median fluorescence intensity (MFI) compared to the isotype control.
Figure 4: Cell surface expression of PD-1 was assessed by flow cytometry on human PBMC effector cells co-cultured with MDA-MB-231 target cells at an effector-to-target (E:T) ratio of 5:1 for 48 hours, (A) without or (B) with BMS-1166 treatment. Histogram plots depict PD-1 expression in pan T cells (CD3⁺). Cells were pre-treated with Human TruStain FcX to block Fc receptors, then stained with PE-conjugated anti-PD-1 antibody (clone EH12.2H7, 1:100 dilution) and BV605-conjugated anti-CD3 antibody (clone UCHT1, 1:100 dilution) for 30 minutes on ice. (C) Treatment with BMS-1166 resulted in a modest increase in the percentage of PD-1–expressing T cells after 48 hours.
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Figure 5: Cytolytic activity of the small-molecule PD-1/PD-L1 checkpoint inhibitor BMS-1166 assessed using the CFSE/7-AAD cytotoxicity assay. CFSE-labelled MDA-MB-231 target cells were co-cultured with human PBMC effector cells at a 5:1 effector-to-target (E:T) ratio for 48 hours in the presence of IL-2 (218 IU/ml) and anti-CD28 antibody (1 µg/ml), either (A) without or (B) with BMS-1166 at 15 µM. Cell lysis was determined by measuring the percentage of CFSE⁺/7-AAD⁺ MDA-MB-231 cells via flow cytometry. (C) BMS-1166 induced dose-dependent lysis of target cells, with an IC₅₀ of 6 µM, demonstrating its ability to restore cytotoxicity through PD-1/PD-L1 checkpoint blockade.
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Figure 6: Cytolytic activity of the BCMA × CD3 bispecific antibody assessed using the CFSE/7-AAD cytotoxicity assay. CFSE-labeled RPMI 8226 target cells were co-cultured with human PBMC effector cells at an effector-to-target (E:T) ratio of 5:1 in the presence of a 10-point, 3-fold serial dilution of the BCMA × CD3 bispecific antibody for either (A) 24 hours or (B) 48 hours. Cell lysis was determined by measuring the percentage of CFSE⁺/7-AAD⁺ target cells via flow cytometry. The BCMA × CD3 bispecific antibody induced dose-dependent lysis of target cells, with an IC₅₀ of 0.22 nM.

We are dedicated to advancing immuno-oncology research, demonstrating our commitment to innovation in drug discovery services and improving human health. For more information or to explore collaboration opportunities, contact us at discovery@o2h.com.

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