# An Innovative In Vivo Model for CAR-T-Cell Therapy Development: Efficacy Evaluation of CD19-Targeting CAR-T Cells on Human Lymphoma, Using the Chicken CAM Assay

## TL;DR

This paper introduces a new chicken-based model to test CAR-T-cell therapy for lymphoma, offering a faster and more ethical alternative to rodent models.

## Contribution

The study introduces the chicken CAM assay as a novel in vivo platform for evaluating CAR-T-cell therapy efficacy.

## Key findings

- CD19-targeting CAR-T cells effectively reduced tumor growth, metastasis, and angiogenesis in the chicken CAM model.
- The chicken CAM assay supports interactions between CAR-T cells and the host immune system during tumor challenge.
- The chicken model is a 3Rs-compliant alternative to traditional rodent models for CAR-T-cell preclinical studies.

## Abstract

Chimeric antigen receptor (CAR)-T-cell therapy is a revolutionary approach in immunotherapy that has shown remarkable success in the treatment of blood cancers. Many preclinical studies are currently underway worldwide to extend the CAR-T-cell therapy benefits to a broad spectrum of cancers, using rodent models. Alternative in vivo platforms are essential for overcoming the drawbacks associated with rodent models, including immunodeficiency in humanized models, ethical concerns, extended time requirements, and cost. In this work, we used the chicken chorioallantoic membrane (CAM) assay to evaluate the in vivo efficacy of cluster-of-differentiation 19 (CD19)-targeting CAR-T cells expressing a second-generation CAR construct against human lymphoma derived from the Raji cell line. Our results confirm the efficacy of selected CAR-T cells on tumor growth, metastasis, and angiogenesis. Further, the chicken embryo has an intrinsic active immune system. Therefore, the dialog between CAR-T cells and endogenous immune cells, as well as their participation in the tumor challenge, has also been studied. In conclusion, our study demonstrates that the chicken CAM assay provides a relevant in vivo, 3Rs (Replacement, Reduction and Refinement)-compliant new approach methodology (NAM), which is well-suited for the current needs of preclinical research on CAR-T-cell therapy.