DT3C Recombinant Protein

The efficiency of antibody internalization is key to designing effective antibody-drug conjugates (ADCs). For the cytotoxic drug to reach and destroy tumor cells, the antibody must first be internalized by the target cell. Without this step, the drug cannot be released where it's needed.

DT3C protein is a reliable tool for measuring how well antibodies are internalized. It helps researchers evaluate the internalization efficiency of antibodies in target cells, making it easier to identify those with strong binding and efficient internalization. These high-performing antibodies can be used in ADCs to improve drug targeting and safety.

To see why DT3C works, we need to explore its structural and functional features.

DT3C protein is a recombinant fusion protein that consists of a diphtheria toxin (DT) protein lacking the receptor-binding domain (only catalytic and translocation domains) but containing the C1, C2, and C3 domains (IgG-binding domain) of Streptococcus protein G (3C). It is characterized by low toxicity and high affinity with antibodies.

Figure 1. Mechanism of antibody: DT3C-induced cytotoxicity

DT3C's unique structure enables its role in mAb-DT3C conjugates, which mimic ADC behavior in vitro.

A mAb-DT3C conjugate, which functions in vitro similarly to an antibody-drug conjugate (ADC), can kill cancer cells or reduce the viability of cancer cells only when the mAb being tested is internalized by the target cells through antibody-antigen interactions.

After the mAb-DT3C conjugate recognizes and binds to cell surface antigens, the mAb-DT3C-antigen complex is internalized into the cell via endocytosis. In the cell, the translocated terminus of DT3C is cleaved by the cellular furin protease, and the catalytic domain of DT3C is released into the cytoplasm, leading to ADP ribosylation of EF-2, subsequent blockade of the protein translation machinery, and ultimate cytotoxicity.

Therefore, the internalization efficiency of antibodies can be evaluated according to the cell-killing situation.

Figure 2. DT3C-Induced Cell Killing Mechanism

Generally speaking, antibody internalization assays primarily include pH probe-based assays, live-cell imaging-based assays, temperature shift-based assays, and toxin-conjugated cytotoxicity assays.

Among these methods, temperature shift-based assays have room for improvement in accuracy and specificity. Live-cell imaging-based assay, on the other hand, require advanced equipment, which limits its accessibility. Toxin-conjugated cytotoxicity assays have had relatively few well-established products in the past.

As a result, pH probe-based assays remain the most commonly used method for antibody internalization today.

pH probe-based assays may be affected by other intracellular factors that influence fluorescent signals. However, they are affordable, simple, and efficient. These advantages make them highly favored by researchers. As a result, they often overshadow newer methods for antibody internalization detection—for example, the DT3C protein, as mentioned earlier.

Similar to the well-known mAb-ZAP, DT3C is also a toxin-conjugated cytotoxicity assay. It matches the pH probe in performance for naked antibody screening and offers a key advantage—it can be used during the early-stage supernatant screening of hybridomas, significantly accelerating the initial stages of ADC development. This is something neither pH probe-based methods nor mAb-ZAP can achieve.

Compared to mAb-ZAP, DT3C protein offers more distinct advantages for antibody internalization assays. Research by M. Yamaguchi et al. has shown that DT3C performs significantly better than mAb-ZAP in antibody internalization efficiency. Let’s take a closer look at the comparison between these two toxin-conjugated cytotoxicity methods.