Colorectal cancer, a malignancy occurring in the colon or rectum, accounts for approximately 150,000 new cases annually. Current treatment strategies, including surgery, chemotherapy, radiotherapy, and immunotherapy, face challenges such as cancer recurrence, drug resistance, and significant toxicity. Therefore, more effective treatments are urgently needed for these patients. Ferroptosis, a novel form of regulated cell death characterized by iron-dependent lipid peroxidation, has emerged as a promising new approach for treating colorectal cancer. Inhibition of glutathione peroxidase 4 (GPX4) or the cystine/glutamate antiporter SLC7A11 leads to intracellular glutathione (GSH) depletion, triggering lipid peroxidation and subsequent ferroptosis. Here, Zhang et al. [1] discovered that CDH17 and GUCY2C are co-overexpressed in colorectal cancer cells and developed a bispecific antibody-drug conjugate (BsADC) targeting CDH17 and GUCY2C, conjugated with the ferroptosis inducer RSL3 (a GPX4 inhibitor). Experimental results demonstrated that the BsADC exhibited superior binding and internalization activity compared to monoclonal antibody ADCs and more effectively inhibited tumor cell proliferation. These findings suggest that a CDH17-GUCY2C BsADC capable of inducing ferroptosis in tumor cells may represent a promising novel approach for treating colorectal cancer, also providing new insights for future targeted drug development in difficult-to-treat oncologic diseases.

Main Research Content

The study performed a comprehensive analysis of the Gene Expression Profiling Interactive Analysis (GEPIA) database to identify antigens potentially suitable as tumor-associated targets for developing CDH17-based bispecific antibody-drug conjugates (BsADCs), which should also be highly expressed in colorectal cancer. Across 33 human cancer types, GUCY2C exhibited an expression pattern similar to CDH17 (Figure 1A). CDH17 and GUCY2C were co-highly expressed in most colorectal cancer and rectal adenocarcinoma patients. The study further analyzed CDH17 and GUCY2C expression in the GEPIA database. As shown in Figure 1B, both CDH17 and GUCY2C were highly expressed in colorectal cancer. The study also examined CDH17 and GUCY2C expression in 13 tumor cell lines by RT-PCR and found clear co-expression of CDH17 and GUCY2C across various tumor cells (Figure 1C). These results indicate a strong correlation between the expression levels of CDH17 and GUCY2C in colorectal tumor cells, supporting the rationale for constructing BsADCs targeting CDH17 and GUCY2C.

Figure 1: Validation of the correlation between CDH17 and GUCY2C expression levels in colorectal tumor cells.

The study constructed bispecific antibodies (BsAbs) using CDH17 and GUCY2C antibody sequences derived from two clinical products, BI-905711 and PF07062119. To obtain BsAbs with high binding and internalization capacity, six bispecific antibodies with different formats were constructed (Figure 2A). BsAb1 and BsAb2 adopted a "2+2" tetravalent format, where the C-terminus of one monoclonal antibody was linked to the scFv form of the other antibody. BsAb3 and BsAb4 adopted a "1+1" bivalent format. BsAb5 and BsAb6 also adopted a tetravalent format, but with the variable region sequences of both antibodies located at the N-terminus. The study examined the binding and internalization capacity of these six CDH17 and GUCY2C BsAbs in SW1463 and LS1034 tumor cells. As shown in Figure 2B, BsAb1 demonstrated superior binding capacity in both cell lines compared to its parental monoclonal antibodies. Meanwhile, the CDH17 monoclonal antibody exhibited better binding activity than the GUCY2C monoclonal antibody, possibly due to higher expression levels of CDH17 on tumor cells compared to GUCY2C. However, the internalization activity of the CDH17 monoclonal antibody was significantly lower than that of the GUCY2C monoclonal antibody, potentially reducing the antitumor activity of a CDH17 monoclonal antibody ADC. Nevertheless, when combined with GUCY2C to form BsAbs, several bispecific formats showed enhanced internalization activity, with BsAb1 exhibiting the best internalization activity (Figure 2C). These data suggest that BsAb1, with its "2+2" tetravalent format, enhances binding and internalization activity towards tumor cells, making it suitable for constructing BsADCs.

Figure 2: Development and characterization of CDH17 and GUCY2C bispecific antibodies.

RSL3 was confirmed as a ferroptosis activator functioning through inhibition of glutathione peroxidase 4 (GPX4). CDH17 and GUCY2C bispecific antibody-drug conjugates (BsADCs) were generated by bioconjugating the RSL3-NH2 payload to endogenous cysteine residues within the BsAbs. Hydrophobic interaction chromatography revealed uniform drug distribution, and the synthesized ADC had a drug-to-antibody ratio (DAR) of approximately 4 (Figure 3B). The study performed cytotoxicity assays of BsADCs in SW1463 and LS1034 cells, which highly express CDH17 and GUCY2C. The BsADC was found to inhibit tumor cell proliferation in a dose-dependent manner, and this activity was significantly higher than that of monospecific ADCs targeting CDH17 or GUCY2C alone (IC50 of 4.97 μg/mL vs. 27.72 μg/mL and 11.59 μg/mL in SW1463; IC50 of 2.249 μg/mL vs. 39.15 μg/mL and 10.02 μg/mL in LS1034) (Figure 3C). In contrast, an antibody-drug conjugate containing the same linker and payload (IgG-ADC) exhibited no cell growth inhibitory activity (Figure 3C). These results indicate that the BsADC can specifically inhibit tumor cells with high CDH17 and GUCY2C expression. Compared to traditional immunooncology therapies, a significant advantage of ADCs is their ability to induce bystander killing effects. To test whether the CDH17 and GUCY2C BsADC possesses bystander cytotoxic effects, the study assessed its ability to induce bystander cytotoxicity on CDH17/GUCY2C-negative cells (HT-29) after 72 hours of co-culture with CDH17/GUCY2C-positive cells (LS1034, SW1463). As shown in Figure 3D, when SW1463 or LS1034 cells were co-cultured with HT-29 cells, the killing of SW1463 or LS1034 cells did not increase; however, co-culture with SW1463/HT-29 or LS1034/HT-29 increased bystander killing of CDH17/GUCY2C-negative HT-29 cells compared to controls. In contrast, SW1463/HT-29 or LS1034/HT-29 co-cultures incubated with IgG1-ADC for 72 hours showed no significant change in cytotoxicity (Figure 3E).

Figure 3: In vitro antitumor efficacy validation of CDH17 and GUCY2C bispecific antibody-drug conjugates.

Subsequently, the project investigated whether the BsADC induces cell death through ferroptosis. SW1463 and LS1034 cells were treated with 5 μg/mL of hIgG1-ADC, CDH17-ADC, GUCY2C-ADC, or BsADC for 24 hours, and relevant ferroptosis markers were assessed. Ferroptosis is typically accompanied by intracellular accumulation of malondialdehyde (MDA). Detection of MDA levels in cell samples revealed that cells treated with the BsADC exhibited significantly elevated MDA levels compared to those treated with hIgG1-ADC, CDH17-ADC, and GUCY2C-ADC (Figure 4A). Furthermore, changes in lipid peroxidation levels were further investigated by examining reactive oxygen species (ROS) changes in cell samples using BODIPY-C11 staining. As shown in Figure 4B, tumor cells treated with the BsADC showed a significant increase in lipid peroxidation products, markedly higher than in cells treated with CDH17-ADC or GUCY2C-ADC. Taken together, these results indicate that the BsADC induces ferroptosis by delivering RSL3 into tumor cells.

Figure 4: The CDH17 and GUCY2C BsADC induces ferroptosis in colon cancer cells.

This study developed a CDH17 and GUCY2C bispecific antibody-drug conjugate (BsADC). CDH17 and GUCY2C exhibit low expression levels in normal tissues, and the BsADC does not increase binding and internalization to normal tissues, thereby avoiding toxicity. Currently, this BsADC has entered clinical stages and demonstrated promising therapeutic efficacy. It is hoped that, in the future, BsADCs will also bring new treatment options for more colorectal cancer patients.

Tek Biotech (Tianjin) Co., Ltd. specializes in phage display and yeast display antibody development services as its core business. We are committed to providing high-quality bispecific antibody-drug conjugate (BsADC) development services to scientists worldwide. We also offer comprehensive supporting services including downstream in vitro affinity validation (comprising EC50 validation, BLI validation, SPR validation), cell killing validation, and in vivo animal imaging validation, providing robust technical support for our partners' drug development programs.

References 

[1] Zhang, Y., Du, J., Cui, X. et al. Development of a bispecific CDH17-GUCY2C ADC bearing the ferroptosis inducer RSL3 for the treatment of colorectal cancer. Cell Death Discov. 11, 347 (2025).