Abstract 4018: An innovative in vivo model for CAR-T cell therapy development: Tolerability and efficacy evaluation of CD19-targeting CAR-T cells on human lymphoma using the chicken CAM assay
Authors: Wang Yan , Rousset Xavier , Prunier Chloé , Dosda Emilien , Gutierrez-Guerrero Alejandra , Abrial Pauline , Menkova Inna , Viallet Jean 
Published: March 2024

Abstract
BACKGROUND Chimeric antigen receptor (CAR) T cell therapy is a revolutionary approach in cellular immunotherapy, where a patient’s T cells are genetically engineered to recognize and attack cancer cells. In the last decade, several CAR-T cell therapies have been approved by the FDA. However, this encouraging outcome was obtained only with certain blood cancers, like B cell leukemias, lymphomas, etc. Many research/clinical trials are now underway worldwide to extend the CAR T-cell therapy benefits to a large spectrum of cancers, using rodents. This model presents several drawbacks, such as the immunodeficiency of humanized models, ethical constraints, time, and cost. An alternative, pertinent, 3Rs-compliant in vivo model for developing CAR-T therapy is urgently needed. Xenografts on the chicken embryo’s ChorioAllantoic Membrane (CAM) have proven extremely valuable for in vivo cancerology studies. In this work, we evaluated in vivo the anti-tumor efficacy of CD19-targeting CAR-T cells on human lymphoma using Inovotion’s CAM assay.
METHODS Human lymphoma Raji cells (ATCC) were grafted on the CAM on embryonic development day (EDD) 9. A 2nd generation of CD19-targeting CAR-T cells (CAR-T-(CD3/4-1BB)) developed by Allogenica (France) were co-injected at EDD9 at E/T (effector CAR-T cell vs target Raji cell) ratio 1:1. One group was injected with CAR-T (CD3/4-1BB) only once at EDD9 and a second group received a first injection of CAR-T (CD3/4-1BB) at EDD9 and a second injection at EDD13. Embryonic viability was checked daily. On EDD18. The efficacy of CAR-T therapy was evaluated basing on tumor weight, metastatic invasion in the lower CAM and CAR-T infiltration in tumors detected by qPCR. Activation of the chicken embryo immune system was detected as well.
RESULTS The treatment with CAR-T-(CD3/4-1BB) cells was well tolerated in ovo, no significant embryo mortality/abnormality was observed. CAR-T-(CD3/4-1BB) cells prevented tumor formation (tumor weight was 88.67% to 90.62% lower than control, p &lt; 0.001); and prevented metastasis formation in the lower CAM (86.88% to 81.43% lower than control, p &lt; 0.001).
CONCLUSION Different to in vitro models and immunodeficient murine models, chicken embryo possesses a progressively mature immune system. Our results show that CAR-T-(CD3/4-1BB) cells can prevent tumor formation and metastatic invasion when co-injected with tumor cells. Inovotion’s CAM assay is a viable alternative in vivo model for developing CAR-T cell therapy on a large spectrum of cancers. It is fully 3Rs-compliant.
Citation Format: Yan Wang, Xavier Rousset, Chloé Prunier, Emilien Dosda, Alejandra Gutierrez-Guerrero, Pauline Abrial, Inna Menkova, Jean Viallet. An innovative in vivo model for CAR-T cell therapy development: Tolerability and efficacy evaluation of CD19-targeting CAR-T cells on human lymphoma using the chicken CAM assay [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4018.