Antibody-drug conjugates (ADCs) represent a rapidly advancing therapeutic class for both solid tumors and hematological malignancies. Unlike conventional monoclonal antibodies, ADCs combine a tumor-targeting antibody with a highly potent cytotoxic payload, enabling selective drug delivery to cancer cells. 

At GemPharmatech, we provide integrated end-to-end preclinical solutions to support ADC development. These solutions center around an advanced in vitro platform as well as in vivo capabilities to support every step of ADC functional assessment, including: 

• Antigen expression and target validation
  

• Binding affinity and specificity

• Internalization efficiency

• Cytotoxicity and dose response

• Cell cycle and apoptosis induction

• Bystander killing effects

• Combination therapy assessment   

Figure 1. GemPharmatech in vitro service for ADC evaluation

Case Study 1: Antigen Expression Analysis

Objective: Assess c-Met and HER2 surface expression across 8 cell lines to determine suitability for ADC targeting

Method: Flow cytometric analysis

Result: Both c-Met and HER2 were widely distributed across tumor cells, highlighting opportunities for dual-targeted ADCs or combination therapies to enhance cancer treatment efficacy.

Figure 2. HER2 and c-Met surface expression across 8 cell lines

Case Study 2: Binding Affinity Assay

Objective: Compare binding of ABBV-399(c-Met ADC Drug) to c-Met with its parental antibody, Telisotuzumab

Method: Flow cytometric analysis

Result: ABBV-399 demonstrated robust binding across all three tumor cell lines, with binding characteristics similar to Telisotuzumab (c-Met antibody).

Figure 3. Binding assay of ABBV-399 in different cancer cell lines 

Case Study 3: Cytotoxicity Screening 

Objective: Identify tumor cell lines most susceptible to ABBV-399-mediated cytotoxicity

Method: CellTiter-Glo (CTG) assay

Result: ABBV-399 induced potent cytotoxic activity against four tumor cell lines, with its activity strongly correlated to the levels of c-Met overexpression. Notably, in SNU-5 cells, ABBV-399 exhibited significantly stronger anti-proliferative effects compared to its free cytotoxic payload (MMAE).

Figure 4. Dose-response curves of ABBV-399 against tumor cell lines 

Table 1. c-Met expression on tumor cells in vitro and sensitivity to ABBV-399

Case Study 4: Internalization Assay

Objective: Evaluate the internalization of ABBV-399 

Method: Flow cytometric analysis with pHrodo™ dye

Result: ABBV-399 demonstrated a time-dependent internalization effect similar to Telisotuzumab. Notably, low internalization signal in c-Met-positive NCI-H1650 cells, accounted for the absence of cytotoxicity despite binding.

Figure 5. The internalization kinetics of ABBV-399

Case Study 5: Cell Cycle and Apoptosis Assay

Objective: Evaluate the effects of DS-8201a on SK-BR-3 cells

Method: Flow cytometric analysis

Result: Following treatment for 48 h, the proportion of S phase cells in the DS-8201a group was significantly increased compared with the control group, suggesting S-phase arrest via topoisomerase I inhibition (Figure 6). The proportion of apoptotic cells in DS-8201a group was also significantly increased compared with the control group, suggesting that DS-8201a induced cell apoptosis in a cell cycle-dependent or-independent manner (Figure 7). 

Figure 6. The effects of different concentrations of DS-8201a on the cell cycle of the target cell SK-BR-3

Figure 7. Apoptosis induced by DS-8201a in target cells SK-BR-3 at different concentrations

Case Study 6: Bystander Killing in Co-culture Conditions in vitro (FACS)

Objective: Assess the bystander effect of DS-8201a on HER2-positive and HER2-low tumor cells

Method: Flow cytometric analysis

Result: DS-8201a exerted direct cytotoxicity against HER2-positive SK-BR-3 cells and demonstrated bystander killing effects on antigen-negative MDA-MB-468 cells in coculture.

Figure 8. In vitro bystander killing ffect of ABBV-399. (a) Population analysis by flow cytometry. (b) Number of viable SK-BR-3 and MDA-MB-468 cells following DS-8201a treatment.

Case Study 7: Bystander Killing in Co-culture Conditions in vitro (Luciferase Reporter Cell)

Objective: Evaluate ABBV-399 bystander effects in mixed cultures of c-Met+ SNU-5 cells c-Met-low cells

Method: Co-culture with luciferase-labeled reporter cell

Result: ABBV-399 reduced the viability of c-Met-low reporter cells in a concentration-dependent manner, demonstrating a robust bystander killing effect.

Figure 9. In Vitro Bystander Killing Effect of ABBV-399

GemPharmatech’s ADC evaluation platform provides a comprehensive toolkit for the mechanistic characterization of ADCs, from antigen profiling and binding affinity to internalization efficiency, cytotoxicity and bystander effects. These insights enable more informed decision-making in ADC design and development, supporting the translation of promising candidates into successful clinical therapies. 

Contact us at sales@gempharmatech.com to learn how we can support your ADC program.

Reference

1. Zhiwen Fu et al.nature.2022

2. Ogitani et al. Cancer Sci.2016