*Article* **Injectable Polypeptide Hydrogel Depots Containing Dual Immune Checkpoint Inhibitors and Doxorubicin for Improved Tumor Immunotherapy and Post-Surgical Tumor Treatment**

**Zhixiong Chen † , Yan Rong † , Junfeng Ding, Xueliang Cheng, Xuesi Chen and Chaoliang He \***

> CAS Key Laboratory of Polymer Ecomaterials, Changchun Institute of Applied Chemistry, Chinese Academy of Sciences, Changchun 130022, China

**\*** Correspondence: clhe@ciac.ac.cn; Tel.: +86-431-8526-2901

† These authors contributed equally to this work.

**Abstract:** In this work, we developed a strategy for local chemo-immunotherapy through simultaneous incorporation of dual immune checkpoint blockade (ICB) antibodies, anti-cytotoxic T-lymphocyteassociated protein 4 (aCTLA-4) and anti-programmed cell death protein 1 (aPD-1), and a chemotherapy drug, doxorubicin (Dox), into a thermo-gelling polypeptide hydrogel. The hydrogel encapsulating Dox or IgG model antibody showed sustained release profiles for more than 12 days in vitro, and the drug release and hydrogel degradation were accelerated in the presence of enzymes. In comparison to free drug solutions or hydrogels containing Dox or antibodies only, the Dox/aCTLA-4/aPD-1 co-loaded hydrogel achieved improved tumor suppression efficiency, strengthened antitumor immune response, and prolonged animal survival time after peritumoral injection into mice bearing B16F10 melanoma. Additionally, after injection of Dox/aCTLA-4/aPD-1 co-loaded hydrogel into the surgical site following tumor resection, a significantly enhanced inhibition on tumor reoccurrence was demonstrated. Thus, the polypeptide hydrogel-based chemo-immunotherapy strategy has potential in anti-tumor therapy and the prevention of tumor reoccurrence.

**Keywords:** hydrogel; combination therapy; immunotherapy; polypeptide; ICB therapy
