Abstract 5194: Humanized PD-1 knock-in mice as a model system for combination therapies with human specific PD-1 therapeutics
Authors: Metzger Philipp , Sônega Fabiane , Berthomme Hervé , Obodozie Cynthia , Weber Holger 
Published: April 2023

Abstract
Checkpoint inhibitor treatment has become a common therapy for various cancer types. As an escape mechanism, tumor cells express PD-L1 on their surface, as a ligand for PD-1 with players of the immune system (such as T cells), aimed at preventing the immune system from exerting its anti-tumour activities. Antibodies blocking the PD-L1/PD-1 interaction have emerged as front-line treatments for various oncological indications. Several anti-PD-L1 and anti-PD-1 therapeutics are already approved and in clinical use. For preclinical testing of such human specific antibodies, syngeneic tumour models are of limited use due to the limited cross-specificity of such antibodies, giving rise to the development of humanized mouse models in which combination therapies testing new drugs with already clinically approved human checkpoint inhibitors addressing human specific targets can be evaluated.
The anti-tumor efficacies of pembrolizumab (Keytruda) a human specific anti-hPD-1 checkpoint inhibitor and the mouse specific anti-mPD-1 counterpart (clone RPMI1-14) were tested in C57BL/6 wildtype and humanized hPD-1 C57BL/6 mice engrafted with syngeneic MC38-CEA colorectal carcinoma cells. The MC38-CEA tumor cells were implanted into the mammary fat pad (subQperiorTM), a procedure that prevents tumor ulceration and leads to a more homogenous tumor growth compared to subcutaneous implantation. Treatment with pembrolizumab extended the survival of tumor-bearing human PD-1 C57BL/6 mice significantly, whereas no effect in C57BL/6 wildtype mice was observed, in line with the reported specificity of Keytruda towards the human version of PD-1. Further supporting this finding, the application of the anti-mPD-1 antibody led to a prolonged survival of C57BL/6 mice with no life extension of hPD-1 C57BL/6 mice.
The validation of the humanized subQperior™ platform was further complemented by conducting flow cytometry analysis of intra-tumoral immune populations present in tumor tissues at 7 days post-treatment. Two staining panels were executed: a well-established and validated 17 marker staining allowing to quantify the frequency of all major immune cell populations and a staining panel addressing the activity of T and NK cells after ex vivo stimulation with PMA/Ionomycin. An increased activity of CD8+ T cells and NK cells was observed as major players in the response to treatment with these anti-PD-1 antibodies.
In summary, the humanized subQperior™ platform using hPD-1 C57BL/6 mice is a suitable tool to evaluate novel cancer therapies in combination with human specific anti-PD-1 therapeutics and tumor ulceration was completely prevented by subQperior implantation of the tumor cells.
Citation Format: Philipp Metzger, Fabiane Sônega, Hervé Berthomme, Cynthia Obodozie, Holger Weber. Humanized PD-1 knock-in mice as a model system for combination therapies with human specific PD-1 therapeutics. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5194.