Immunogenic Cell Death Inducing Fluorinated Mitochondria‐Disrupting Helical Polypeptide Synergizes with PD‐L1 Immune Checkpoint Blockade

Abstract Immunogenic cell death (ICD) is distinguished by the release of tumor‐associated antigens (TAAs) and danger‐associated molecular patterns (DAMPs). This cell death has been studied in the field of cancer immunotherapy due to the ability of ICD to induce antitumor immunity. Herein, endoplasmic reticulum (ER) stress‐mediated ICD inducing fluorinated mitochondria‐disrupting helical polypeptides (MDHPs) are reported. The fluorination of the polypeptide provides a high helical structure and potent anticancer ability. This helical polypeptide destabilizes the mitochondrial outer membrane, leading to the overproduction of intracellular reactive oxygen species (ROS) and apoptosis. In addition, this oxidative stress triggers ER stress‐mediated ICD. The in vivo results show that cotreatment of fluorinated MDHP and antiprogrammed death‐ligand 1 antibodies (αPD‐L1) significantly regresses tumor growth and prevents metastasis to the lungs by activating the cytotoxic T cell response and alleviating the immunosuppressive tumor microenvironment. These results indicate that fluorinated MDHP synergizes with the immune checkpoint blockade therapy to eliminate established tumors and to elicit antitumor immune responses.

CuCO 3 /Cu(OH) 2 (3.63 g, 16.424 mmol) was added into 80 mL of an aqueous solution of Llysine hydrochloride (3 g, 16.424 mmol). The solution was refluxed for 3 h and filtered to remove any unreacted CuCO 3 . The filtered solution was cooled to 0 ℃, and NaHCO 3 (5.52 g, 65.7 mmol) was added. 4-(chloromethyl)benzoyl chloride (6.21 g, 32.85 mmol) dissolved in tetrahydrofuran (THF) was slowly added into the filtered solution under stirring. After 30 min, a precipitate started to form, and the reaction was conducted overnight at room temperature (RT). The precipitated product was filtered and washed with THF and deionized (DI) water to remove any unreacted 4-(chloromethyl)benzoyl chloride and ( L -lysine) 2 •Cu(II).
Finally, the product was obtained as a violet solid powder after lyophilization (Yield : 80%).

Lys-NCA)
In a glove box, [N ε -(4-(chloromethyl)benzoyl)-L -lysine] 2 •Cu(II) (4.34 g, 6.585 mmol) was dissolved in THF and triphosgene (1.95 g, 6.585 mmol) was added. The reaction mixture was refluxed, and the color of the mixture turned brown. During the reaction, the precipitation of CuCl 2 occurred. After 3 h, the reaction mixture was cooled to 0 ℃, and it was poured into a separatory funnel with ethyl acetate (100 mL). Subsequently, this mixture was washed with cold saturated EDTA-2Na dihydrate/NaHCO 3 /H 2 O solution three times. The separated organic layer was dried over MgSO 4 and concentrated under vacuum to approximately 1/3 of its initial volume. The concentrated organic layer was poured into excessive n-hexane for recrystallization. The precipitated product was filtered and kept in a vacuum to remove nhexane. A yellowish solid powder was obtained (Yield : 51%).

Synthesis of N ε -Carbobenzoxy-L -lysine based N-carboxyanhydride (CBZ-L -Lys-NCA)
In a glove box, N ε -Carbobenzoxy-L -lysine (1.5 g, 5.35 mmol) was dissolved in THF. Then, α-Pinene (1.7 mL, 10.7 mmol) and triphosgene (0.635 g, 2.14 mmol) were added. The reaction proceeded under continuous stirring at 45 ℃ for 3 h. The reaction mixture was precipitated with excessive n-hexane. The precipitated product was filtered and kept in a vacuum to remove the n-hexane. A white solid powder was obtained (Yield : 85%).

Carbobenzoxy-L -lysine) (PDAC)
PCBC was dissolved in DMF (10 mL), followed by the addition of sodium iodide (3 equivalents of chloro groups) dissolved in acetonitrile (10 mL) and N-Butyldimethylamine (3 equivalents of chloro groups). The reaction was conducted at 80 ℃ for 48 h under continuous stirring. The resulting product was dialyzed against DI water for 48 h in a dialysis bag with a cutoff molecular weight of 3.5 kDa. The product was obtained by lyophilization (Yield : PDAC-1 : 77%, PDAC-2 : 91%).

Acidolysis of CBZ groups
PDAC was dissolved in trifluoroacetic acid (TFA) (6 mL). After complete dissolution of the polypeptide, 33 wt% Hydrogen bromide solution in acetic acid (4 equivalents of CBZ groups) was added. The reaction was conducted at RT for 1 h, and the resulting product was poured into excessive ethyl ether. The precipitated polypeptide was filtered and dried under vacuum (Yield : PDA-1 : 93%, PDA-2 : 98%).

Fluorination of the polypeptides
CBZ deprotected polypeptides were dissolved in methanol (5 mL). Heptafluorobutyric anhydride (5 equivalents of deprotected amine groups) and triethylamine (6 equivalents of deprotected amine groups) were added into the above solution under continuous stirring. The reaction was conducted at RT for 24 h. The polypeptides were purified against 1 M NaCl aqueous solution for 1 day and DI water for 1 day in a dialysis bag with a cutoff molecular weight of 3.5 kDa. The polypeptides were obtained by lyophilization as a white solid powder (Yield : FHP-1 : 85%, FHP-2 : 83%).

IRDye 800CW conjugation
CBZ deprotected polypeptides were dissolved in PBS (5 mL), and subsequently, IRDye 800CW NHS ester (5 mg mL -1 in DMSO, LI-COR, USA) (1 equivalent of CBZ deprotected polypeptides) was added to the solution. The reaction was conducted at RT for 24 h and dialyzed against DI water for 2 days in a dialysis bag with a molecular cutoff weight of 3.5 kDa. IRDye 800CW conjugated polypeptides were obtained by lyophilization.

Characterization
The molecular weights of the synthesized polypeptides were determined by gel permeation chromatography (GPC) with a Younglin YL9100  were treated at 37 ℃ for 3 h. After the treatment, the cells were washed with PBS containing heparin (40 U mL -1 ) and 0.1% trypan blue was added for 2 min to quench extracellular fluorescence. Then, the cells were washed with PBS and fixed with 4% paraformaldehyde at 37 ℃ for 10 min. After fixation, the cells were stained with DAPI at RT for 10 min and mounted onto slides. Stained cells were visualized with confocal laser scanning microscopy (LSM 800 META, ZEISS, Germany) to observe endo/lysosomal escape of polypeptides.

Nuclear condensation
CT26 cells seeded on coverslips in a 24-well plate were treated with M of HP, FHP-1, and FHP-2 for 24 h. Then, the cells were fixed with 4% paraformaldehyde at 37 ℃ for 10 min. After fixation, the cells were stained with DAPI at RT for 10 min and mounted onto slides. Stained cells were visualized with confocal laser scanning microscopy (LSM 800 META, ZEISS, Germany) to observe nuclear morphological changes.
Fluorescent images were obtained from the anesthetized mice using the IVIS imaging system.                  Table S1. Measurement of bilirubin level in blood for liver toxicity.
Cell type Ab