@article {Zhoue001925, author = {Shujuan Zhou and Fanyan Meng and Shiyao Du and Hanqing Qian and Naiqing Ding and Huizi Sha and Mei Zhu and Xiaoxiao Yu and Lifeng Wang and Baorui Liu and Jia Wei}, title = {Bifunctional iRGD-anti-CD3 enhances antitumor potency of T cells by facilitating tumor infiltration and T-cell activation}, volume = {9}, number = {5}, elocation-id = {e001925}, year = {2021}, doi = {10.1136/jitc-2020-001925}, publisher = {BMJ Specialist Journals}, abstract = {Background Poor infiltration and limited activation of transferred T cells are fundamental factors impeding the development of adoptive cell immunotherapy in solid tumors. A tumor-penetrating peptide iRGD has been widely used to deliver drugs deep into tumor tissues. CD3-targeting bispecific antibodies represent a promising immunotherapy which recruits and activates T cells.Methods T-cell penetration was demonstrated in tumor spheroids using confocal microscope, and in xenografted tumors by histology and in vivo real-time fluorescence imaging. Activation and cytotoxicity of T cells were assessed by flow cytometry and confocal microscope. Bioluminescence imaging was used to evaluate in vivo antitumor effects, and transmission electron microscopy was used for mechanistic studies.Results We generated a novel bifunctional agent iRGD-anti-CD3 which could immobilize iRGD on the surface of T cells through CD3 engaging. We found that iRGD-anti-CD3 modification not only facilitated T-cell infiltration in 3D tumor spheroids and xenografted tumor nodules but also induced T-cell activation and cytotoxicity against target cancer cells. T cells modified with iRGD-anti-CD3 significantly inhibited tumor growth and prolonged survival in several xenograft mouse models, which was further enhanced by the combination of programmed cell death protein 1 (PD-1) blockade. Mechanistic studies revealed that iRGD-anti-CD3 initiated a transport pathway called vesiculovacuolar organelles in the endothelial cytoplasm to promote T-cell extravasation.Conclusion Altogether, we show that iRGD-anti-CD3 modification is an innovative and bifunctional strategy to overcome major bottlenecks in adoptive cell therapy. Moreover, we demonstrate that combination with PD-1 blockade can further improve antitumor efficacy of iRGD-anti-CD3-modified T cells.Data are available upon reasonable request. All data relevant to the study are included in the article or uploaded as supplementary information. The data that support the findings of this study are available from the corresponding author (weijia01627@hotmail.com) upon reasonable request.}, URL = {https://jitc.bmj.com/content/9/5/e001925}, eprint = {https://jitc.bmj.com/content/9/5/e001925.full.pdf}, journal = {Journal for ImmunoTherapy of Cancer} }