PD-1/PD-L1 Checkpoint Inhibitors Are Active in the Chicken Embryo Model and Show Antitumor Efficacy In Ovo

Abstract

(1) Purpose: To assess the use of the chicken embryo (in ovo) model as an alternative in vivo model for immuno-oncology (IO) drug development, focusing on programmed cell death protein-1 (PD-1)/programmed cell death-ligand 1 (PD-L1) immune checkpoint inhibitors. (2) Methods: First, the presence of immune cells in the model was detected through the immunophenotyping of chicken peripheral blood mononuclear cells (PBMCs) based on fluorescence activated cell sorting (FACS) analysis and the immunohistochemistry (IHC) analysis of in ovo tumor-infiltrating lymphocytes. Second, the cross-reactivity between one anti-human PD-1 Ab, pembrolizumab (KEYTRUDA®), and chicken PD-1 was verified through the labelling of chicken splenocytes with pembrolizumab by FACS analysis. Third, the blockade effect of pembrolizumab on chicken PBMCs was assessed in vitro through cytotoxicity assay based on MTT. Fourth, the CAM assay was used to estimate the anti-tumor performance of pembrolizumab through the analyses of tumor growth and chicken immune cell infiltration in tumors. Finally, the efficacy of several PD-1 or PD-L1 inhibitors (nivolumab, atezolizumab and avelumab) on tumor growth was further assessed using the CAM assay. (3) Results: The presence of CD3+, CD4+, CD8+ T lymphocytes and monocytes was confirmed by FACS and IHC analyses. During in vitro assays, pembrolizumab cross-reacted with chicken lymphocytes and induced PD-1/PD-L1 blockade, which permitted the restoration of chicken T-cell’s cytotoxicity against human lung cancer H460 tumor cells. All these in vitro results were correlated with in ovo findings based on the CAM assay: pembrolizumab inhibited H460 tumor growth and induced evident chicken immune cell infiltration (with significant chicken CD45, CD3, CD4, CD8 and CD56 markers) in tumors. Furthermore, the potency of the CAM assay was not limited to the application of pembrolizumab. Nivolumab, atezolizumab and avelumab also led to tumor growth inhibition in ovo, on different tumor models. (4) Conclusions: The chicken embryo affords a physiological, immune reactive, in vivo environment for IO research, which allows observation of how the immune system defense against tumor cells, as well as the different immune tolerance mechanisms leading to tumor immune escape. The encouraging results obtained with PD-1/PD-L1 inhibitors in this study reveal the potential use of the chicken embryo model as an alternative, fast, and reliable in vivo model in the different fields of IO drug discovery.

In ovo model in cancer research and tumor immunology

“…Here, human monocyte-derived dendritic cells isolated from healthy donors were engrafted 1:1 with the immortalized tumor cell lines and grown on the CAM for 7 days. Reduction in tumor burden correlated well with results obtained in a model of peritoneal (75). However, appropriate controls and quantification of crucial mechanistic insights, including checkpoint antibody binding assays, were largely missing, questioning the scientific relevance of such findings.…”

“…A widely different approach was employed by Wang and colleagues, who took advantage of homologies in the PD1/PDL1 axis between chickens and humans and proposed the TUM-CAM model as an alternative for immunooncological drug development. Clinically approved checkpoint inhibitors mitigated tumor growth in ovo and partially restored T cell-mediated tumor toxicity of chicken lymphocytes in vitro ( 75 ). However, appropriate controls and quantification of crucial mechanistic insights, including checkpoint antibody binding assays, were largely missing, questioning the scientific relevance of such findings.…”

“…Staining of cleaved caspase 3 or TdT-mediated dUTP-biotin nick end labeling (TUNEL) ( 29 ) has been used to evaluate cell death in tissues combined with Ki-67 as a biomarker reflecting the proliferative state ( 15 , 47 ). Various surface markers of interest, including integrins related to EMT ( view chapter 2.2 ) ( 26 ), the immunogenicity of cell death, myeloid ( 28 ), and classical checkpoints ( 75 ) ( view chapter 3.3 ) as well as diagnostic ( 9 , 15 , 35 , 45 ) and predictive biomarkers ( 14 , 31 , 73 ) can be assessed. Tumor dissociation using adequate dissociation kits can be used to validate previous results at single cell levels using flow cytometry after intra- and extracellular staining with high throughput, including intracellular ROS levels, translocation, and phosphorylation of transcription factors, and an unlimited range of surface markers ( 13 ).…”

The Chorioallantoic Membrane Xenograft Assay as a Reliable Model for Investigating the Biology of Breast Cancer

“…Animal protocol approvals are not required for the use of the CAM model (country-dependent). Moreover, being naturally immunodeficient until embryonic day (ED) 10, transplantations from different tissues and species may be performed with only minor immune responses, whereas at ED16, cytotoxic T cells and monocyte populations are available [ 78 ]. Furthermore, in contrast to the CAM model, murine in vivo models allow for a longer observation time.…”

949 Inovativein vivo(in ovo) cam model to predict efficacy and mode of action of a new antitumor vaccine stc-1010 on human colorectal adenocarcinoma

“…At EDD18, i.e., 9 days post-graft, in ovo anti-tumor efficacy was evaluated by tumor weight, metastatic invasion (qPCR analysis of human Alu sequence in lower CAM) and quantification of tumor-infiltrating by CD8, CD4, IFN-gamma, Perforin and TNF-alpha. 1,2 Results Compared to negative control, STC-1010 vaccine induced: 1) significant increase of IL-12 and IL-2 secretion in peripheric blood during the generation of all three batches of PBMCs, confirming previous results (IL-12: +52%, p=0.0003 ; IL-2: +482%, p=0.0033); 2) a significant expression of IFN-gamma in tumor (+130,83%, p=0.0185); 3) a tendency to increase infiltrating cells: CD4+: +79,2%, CD8+: +29,4% , Perforin: +105,5%, TNFa : +78,63% confirmed by immunohistochemistry and translated into 4) a significant increase of tumor necrosis (p = 0.0267); and 5) a tendency of metastasis regression (-49%); with 6) no embryonic toxicity/mortality (daily evaluation of embryonic viability) induced by STC-1010. Conclusions This in ovo study confirms efficacy of the STC-1010 observed in previous CRC syngeneic models and gives more insight about STC mechanism of action with the activation and maturation of dendritic cells, induction of CD8+ and LTh1 against tumor as the main driver of the response, all without toxicity.…”