Associate Professor University of Michigan, United States
Introduction:: Antibody-drug conjugates (ADCs) are a class of targeted therapeutics that combine the antibody backbone and cytotoxic payloads conjugated by a chemical linker. They are known to have multiple mechanisms of action (MoA) including Fc-mediated effector function, receptor signal blockade, and direct payload delivery. However, the contribution of each component is not fully understood, particularly in the context of an intact immune system. While many ADCs focus on payload delivery, studies suggest that ADCs can modulate and synergize with an immune response. To investigate anti-HER2 ADCs for these effects, we first developed a HER2 syngeneic model and compared 1) trastuzumab-based ADCs in immunodeficient and immunocompetent model and 2) WT versus Fc-null ADCs.
Materials and Methods:: E0771 cells were transfected with human HER2 lentiviral particles and sorted into single clones using Fluorescence-activated Cell Sorting (FACS). Clones with stable HER2 expressions (E0771-HER2) were selected using flow cytometry and cultured in growth medium. A cytotoxicity assay was performed to determine the IC50 of different types of payloads (i.e., DNA alkylators, microtubule inhibitors, topoisomerase inhibitors) and the corresponding anti-HER2 ADCs on E0771-HER2. To understand the efficacy of direct payload delivery, an efficacy study was conducted in both immunodeficient and immunocompetent mice. E0771-HER2 cells were injected in the hind limb and mammary fat pad for nude mice and HER2 transgenic mice, respectively. Once the tumor volume reached 250 mm3, 1 mg/kg of trastuzumab-pyrrolobenzodiazepine (T-PBD) was administered through tail vein injection. To compare the effect of the Fc-effector function, Fc-null T-PBD (1.1 mg/kg was given to match the total payload dose) was administered in another set of mice. The tumor sizes were measured every other day until the endpoint.
Results, Conclusions, and Discussions:: Multiple payloads for their potency on E0771-HER2 in vitro, and DNA damaging agents (e.g., PBD, PNU-159682, Calicheamicin D) showed high potency with picomolar IC50s. However, FDA-approved Kadcyla and Enhertu had IC50 greater than 37 nM, indicating they have low potency in this mouse cell line. We then investigated the payload efficacy in vivo by administering 1 mg/kg of T-PBD and T-PNU to immunodeficient nude mice. T-PBD resulted in moderate growth inhibition but not in tumor reduction. Interestingly, T-PNU given at the same dose showed a significant improvement with tumor reduction after initial tumor growth. This demonstrated that although T-PBD and T-PNU have similar IC50 values in vitro, the payload efficacy may differ in vivo. To test the efficacy in the presence of the immune system, the same dose of T-PBD was given to transgenic mice bearing E0771-HER2 tumor. Compared to the efficacy in the immunodeficient mice, T-PBD in the immunocompetent model resulted in tumor decrease with 4 out of 10 complete responses, indicating a synergistic effect of the ADC with the immune system. Finally, we investigated the effect of the Fc-effector function in antitumor activity with Fc-null T-PBD. While Fc-null T-PBD showed similar tumor reduction compared to WT T-PBD, there was only one complete responder. This comparison between T-PBD and Fc-null T-PBD suggests that the Fc-effector function may contribute in E0771-HER2 tumor model and drive durable responses.
In summary, we utilized a HER2 syngeneic model with E0771-HER2 and HER2 transgenic mice to test the efficacy of trastuzumab-based ADCs in an immunocompetent setting. We compared the efficacy of T-PBD in nude mice and transgenic mice and observed greater efficacy with the presence of the immune system. While WT T-PBD showed a range of responses (no, partial, and complete responses), Fc-null T-PBD reduced tumor size but resulted in one complete responder. Using the syngeneic model, we can further test ADCs in the presence of the immune system and different combinations of immunotherapeutic agents for durable responses.
.jpg "Hyeyoung Kwon, MSE (she/her/hers) photo")