Juyoung Hwang ^a,b,c,1, Eun-Koung An ^b,c,1, Wei Zhang ^a, Hae-Bin Park ^a,b,c, So-Jung Kim ^b, Dhananjay Yadav ^b, Jihoe Kim ^b,c, Inho Choi ^b,c, Minseok Kwak ^d, Peter CW. Lee ^e, Xiaoyan Zhang ^f, Jianqing Xu ^f, Jun-O Jin ^a,b,c,*

^a Shanghai Public Health Clinical Center & Institutes of Biomedical Sciences, Shanghai Medical College, Fudan University, Shanghai, 201508, China ^b Department of Medical Biotechnology, Yeungnam University, Gyeongsan, 38541, South Korea ^c Research Institute of Cell Culture, Yeungnam University, Gyeongsan, 38541, South Korea ^d Department of Chemistry & Department of Industry 4.0 Convergence Bionics Engineering, Pukyong National University, Busan, 48513, South Korea ^e Department of Biomedical Sciences, University of Ulsan College of Medicine, ASAN Medical Center, Seoul, 05505, South Korea ^f The Laboratory for Immunotherapy, Clinical Center for BioTherapy, Zhongshan Hospital, Fudan University, Shanghai 200032, China

^* Corresponding author.
¹ These authors contributed equally.

Effective cancer therapy aims to treat not only primary tumors but also metastatic and recurrent cancer. Immune check point blockade-mediated immunotherapy showed promising effect against tumors; however, it still has a limited effect in metastatic or recurrent cancer. Here, we extracted recombinant murine programmed death-1 (rmPD-1) proteins. The extracted rmPD-1 effectively bound to CT-26 and 4T1 cells expressing PD-L1 and PD-L2. The rmPD-1 did not alter the activation of dendritic cells (DCs); however, rmPD-1 promoted T cell-mediated anti-cancer immunity against CT-26 tumors in mice. Moreover, rmPD-1 decorated thermal responsive hybrid nanoparticles (piHNPs) promoted apoptotic and necrotic cell death of CT-26 cells in response to laser irradiation at 808 nm consequently, it promoted anti-tumor effects against the 1st challenged CT-26 tumors in mice. In addition, piHNP-mediated cured mice from 1st challenged CT-26 was also prevented the 2nd challenged lung metastatic tumor growth, which was dependent of cancer antigen-specific memory T cell immunity. It was also confirmed that the lung metastatic growth of 2nd challenged 4T1 breast cancer was also prevented in cured mice from 1st challenged 4T1 by piHNP. Thus, these data demonstrate that rmPD-1 functions as an immune checkpoint blockade for the treatment of tumors, and piHNPs could be a novel therapeutic agent for preventing cancer metastasis and recurrence.