Recently, immunotherapy has rapidly become the "fifth pillar" of cancer treatment, along with surgery, radiotherapy, chemotherapy, and other targeted treatments. Various categories of immuno-oncology (IO) treatments have been developed, including monoclonal antibodies, Antibody Drug Conjugates (ADCs), Immune Checkpoint Inhibitors (ICIs), vaccines, adoptive cell therapies, immune system modulators. During pre-clinical phase, rodents still represent the most commonly-used in vivo model. However, these models have several drawbacks, such as the immunodeficiency of humanized models, ethical constraints, time requirements and a high cost. Therefore, an alternative, pertinent, 3Rs-compliant in vivo model is strongly needed for accelerating cancer immunotherapy research. In this work, the chicken ChorioAllantoic Membrane (CAM) assay is used to investigate different types of IO treatments.

On embryonic development day (EDD) 9, different human tumor cell lines are grafted on the upper CAM. A treatment with different immunotherapy reagents is performed between EDD10 and EDD18: monoclonal Abs (bevacizumab, trastuzumab, rituximab) in 4 doses; ADC (trastuzumab emtansine) in 1 dose; ICIs (pembrolizumab, nivolumab, atezolizumab, avelumab) in 5 doses; tumor cell based vaccine (STC1010) in 3 doses; CAR-T cells & CAR-NK cells in 1 dose. IO reagents are evaluated in monotherapy or in combination with other drugs. The in ovo part ends at EDD18. Anti-cancer efficacy is evaluated on tumor growth through tumor mass weight, on metastatic invasion through Alu sequence detection in the lower CAM based on the qPCR, on angiogenesis development by counting the vessels surrounding the tumor, on immune response activation through tumor infiltrating immune biomarker quantification based on the RT-qPCR.

The efficacy of different IO reagents is revealed in ovo: all treatments lead to a significant tumor growth regression (24.79%-49.53%), on different tumor models. Additional analyses show more conclusive evidence, including metastasis regression, the inhibition of angiogenesis development, as well as immune activation and immune cell infiltration in the tumor. Furthermore, the combination regimens show increased anti-tumoral potential when compared to the monotherapy.

Our work demonstrates that the CAM assay is suitable for testing different IO anticancer treatments. The presence of an active immune system allows a suitable characterization of the effects of immunotherapy reagents on tumor cells, the microenvironment, and the entire organism. As one part of New Approach Methodologies (NAMs), the CAM assay provides a relevant alternative, 3Rs-compliant, in vivo model for testing novel immunotherapies on a large spectrum of cancer types, as a monotherapy or as an in combination approach.