Introduction

T Cell Engagers (TCEs) are bispecific antibodies that simultaneously target tumor-associated cell surface antigens (Tumor-Associated Antigens, TAA) and T-cell surface receptors (T-Cell Receptor, TCR). Compared to traditional monoclonal antibodies, a significant advantage of TCE bispecific antibodies is their powerful targeted cytotoxicity. By directly recruiting and activating T cells, TCEs can induce stronger and more sustained immune responses.

In recent years, many companies have accelerated the commercialization of TCE bispecific antibodies, viewing them as a new growth area after ADCs. As of December 2024, there are approximately 400+ TCE bispecific antibody drug pipelines under research globally, with the number of pipelines increasing year by year. Among them, pipelines targeting non-solid tumors (such as multiple myeloma and various types of lymphoma) and hematological diseases account for more than 50% of the total pipelines, with approximately 50% of these entering clinical stages. In addition, TCE bispecific antibodies targeting solid tumors also account for about 30% of the pipelines, with recent breakthroughs. In May 2024, Amgen announced that its TCE antibody, Tarlatamab (brand name IMDELLTRA), specifically targets small cell lung cancer and has received FDA accelerated approval for market release.

Mechanism of Action of TCE Drug

TCEs are a new type of antibody with a unique Fab and Fc structure. Their mechanism of action (MOA) involves one Fab binding to a specific tumor-associated antigen (TAA), while the other Fab binds to the T-cell receptor (TCR), with CD3 being a common target. This dual targeting allows TCEs to precisely locate tumor cells while drawing T cells to the tumor cell surface, activating T cells, and inducing tumor cell killing. Activated T cells form an immunological synapse with the tumor cells, leading to tumor cell lysis and releasing a series of cytokines (such as IFN-γ, TNF-α, IL-2, IL-10, etc.), which further enhance T cell proliferation and antitumor effects (Figure 1). In addition, the Fc region of the TCE design can significantly extend its half-life and reduce metabolism, thus enhancing the persistence of its therapeutic effect.

Figure 1. Schematic of the TCE Bispecific Antibody Mechanism

Compared to traditional IgG monoclonal antibodies, TCE bispecific antibodies exhibit significant advantages. Traditional IgG monoclonal antibodies mainly activate antigen-presenting cells through Fc-mediated mechanisms such as ADCC, CDC, and ADCP, which in turn activate T cells. This indirect mechanism limits the ability of monoclonal antibodies to directly activate T cells and kill tumor cells. On the other hand, TCE bispecific antibodies can directly activate T cells, significantly improving their antitumor effect.

More importantly, the Fab region of TCEs can be custom-designed to target specific TAAs for different tumor types. For example, popular targets for hematological tumors include CD19, CD20, and BCMA, while common targets for solid tumors include DLL3, HER2, and CLDN18.2.

Cytokine Detection in TCE Drug Development

In the early stages of drug development and preclinical research, researchers use in vitro cultured lymphocytes to stimulate cytokine secretion to analyze the stimulatory effect of TCE drugs on immune cells, thereby evaluating their efficacy. For instance, for the TCE bispecific antibody Tarlatamab, which targets CD3 and the small cell lung cancer-specific antigen DLL3, the mechanism of action was established by detecting the secretion of cytokines such as IL-2, IL-4, IL-6, IL-10, IFN-γ, and TNF-α in the cell supernatant under different drug concentrations (Figure 2).

Figure 2. In Vitro Cell Stimulation Experiment of Tarlatamab

In addition, Blinatumomab, a TCE drug approved in 2014, targets T-cell surface CD3 and the large B-cell lymphoma-specific antigen CD19. According to clinical trial data published by the FDA, a series of cytokines (IL-2, IFN-γ, TNF-α, IL-4, IL-6, IL-8, IL-10, IL-12) were established as biomarkers for T-cell activation in clinical-phase pharmacodynamics (Figure 3).

Figure 3. Cytokine Biomarker Detection in Blinatumomab Clinical Trials

Summary

Cytokines serve as key detection indicators in the development of TCE bispecific antibody drugs. By detecting the secretion levels of various cytokines in cell supernatants or serum during early pharmacology studies, preclinical, and clinical trials, they can reflect the activation status of the body's immune system.

To accelerate TCE drug development, ACROBiosystems offers various quantitative analysis ELISA kits for cytokines and biomarkers. These kits have been extensively validated with real samples and experiments to ensure the precision, specificity, accuracy, sensitivity, and consistency of the analysis results.

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Case Study

In Vitro Activity Evaluation of TCE Drug Blinatumomab

Experimental Protocol

1. Use Human PBMC cells as effector cells and GMP ActiveMax® Human T cell Activation/Expansion CD3/CD28 Beads (ACROBiosystems, Cat. No. GMP-MBS001) for T cell expansion.

2. On Day 10, FACS-sort CD3+ T cells and co-culture them with target cells (Raji Cells), then stimulate with different concentrations of TCE bispecific antibody (Blinatumomab).

3. The TCE bispecific antibody binds simultaneously to TCR/CD3 on effector cells and tumor antigens on target cells.

4. TCE binding stimulates the release of IFN-γ, IL-6, and IL-10. Collect cell supernatants at 0h, 24h, and 48h, and use ClinMax™ Human Cytokines ELISA Kit to measure the cytokine content.

Figure 4. Assay Protocol for Measuring CD3 Bispecific Antibody (Blinatumomab) Activity

Results

When effector and target cells are co-cultured at an E:T ratio of 1:10 and 1:5, the release of IFN-γ increases with drug concentration. At 100 ng/mL Blinatumomab, the secretion of IFN-γ was higher than in other experimental groups (Figure 5).

Figure 5. Elevation of IFN-γ under Different Stimulation Conditions

When effector and target cells are co-cultured at E:T ratios of 1:10 and 1:5, IL-6 and IL-10 secretion increase with drug concentration, compared to the control group (Figure 6).

Figure 6. Elevation of IL-6 and IL-10 under Different Stimulation Conditions

Product Validation Data

ClinMax™ Human IL-6 ELISA Kit (Cat. No. CRS-B001)

- Intra-Assay and Inter-Assay Precision: The CV of the measured quality control (QC) samples is all below 10%, as shown in Figure 7.

Figure 7. (A) Intra-Assay Precision Data for CRS-B001; (B) Inter-Assay Precision Data for CRS-B001

- Recovery:  IL-6 was spiked into 3 human serum samples, and then analyzed. The average recovery of IL-6 for serum samples is 92.29% (see Table 1).

Table 1. Recovery of IL-6 in Spiked Samples

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