**1. Introduction**

Malignant tumor is a global threat to the health and life of humans [1]. The commonly used clinical treatment approaches include surgical resection, chemotherapy, radiotherapy, and immunotherapy [2,3]. Immunotherapy is a rapidly developing cancer treatment technology recently, including immune checkpoint blocking agents, immune adjuvants, chimeric antigen receptor T (CAR-T) cells, and cancer vaccines [3]. Immune checkpoints are key regulators for immunological tolerance, which can protect tumors from the recognition and attack of the immune system. In the past decade, immune checkpoint blockade (ICB) therapy has witnessed great progress in cancer therapy [4–6]. Cytotoxic T-lymphocyteassociated protein 4 (CTLA-4) and programmed cell death protein 1 (PD-1) are typical immune checkpoint transmembrane proteins that play key roles in different immunosuppressive processes. CTLA-4, a transmembrane protein expressed on T cells, is able to inhibit antigen presentation from antigen-presenting cells (APCs) to T cells and, therefore, restrain successive activation of T cells, through competitively binding with B7 molecules on APCs [7]. In the effector stage, the tumor recognition and killing process by effector T cells can be inhibited by the binding of PD-1 on T cells to ligands (PD-L1 and PD-L2) on tumor cells. Thus, the ICB therapy based on antibodies that block the CTLA-4 and/or PD-1 pathways has achieved remarkable clinical outcomes recently in the treatment of several types of cancers [8].

It is worth mentioning that the anti-cancer efficiency of ICB therapy is still limited by some major shortcomings, especially the insufficient objective response rates (ORRs) and

**Citation:** Chen, Z.; Rong, Y.; Ding, J.; Cheng, X.; Chen, X.; He, C. Injectable Polypeptide Hydrogel Depots Containing Dual Immune Checkpoint Inhibitors and Doxorubicin for Improved Tumor Immunotherapy and Post-Surgical Tumor Treatment. *Pharmaceutics* **2023**, *15*, 428. https://doi.org/10.3390/ pharmaceutics15020428

Academic Editors: João Paulo Figueiró Longo and Lúis Alexandre Muehlmann

Received: 26 December 2022 Revised: 14 January 2023 Accepted: 19 January 2023 Published: 28 January 2023

**Copyright:** © 2023 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

serious immune-related adverse events (irAEs) [8]. It has been found that the cooperative inhibition of the CTLA-4 and PD-1/PD-L1 pathways with anti-CTLA-4 (aCTLA-4) and anti-PD-1 (aPD-1) antibodies lead to significantly enhanced ORRs [9–19]. In addition, it has been shown that the anti-tumor immune response can be improved through the combination with other therapy approaches, such as chemotherapy and radiation therapy [20,21]. For instance, several chemotherapeutics such as anthracyclines, oxaliplatin and cyclophosphamide can induce immunogenic cell death (ICD) of tumor cells, leading to the generation of tumor-associated antigens (TAAs) and damage-associated molecular patterns (DAMPs) [22]. Subsequently, the release of TAAs and DAMPs can stimulate an antigen-specific immune response against tumors.

On the other hand, to reduce systemic irAEs of ICB therapy, localized delivery systems based on injectable hydrogels have attracted considerable attention in recent years [23–26]. Injectable, biodegradable hydrogels have shown several advantages as topical delivery systems, including facile encapsulation of both small-molecule drugs and biomacromolecules, minimally invasive drug administration, good biocompatibility, as well as prolonged drug release behavior at targeted sites [27,28]. After the injection of drug-loaded hydrogels to the tumor sites, the ICB antibodies and combined drugs can be released locally and sustainedly, which may lead to persistently elevated drug concentration at the tumor sites while reducing systemic side effects [29,30]. Thus, several hydrogel systems loaded with an ICB antibody and a chemotherapy drug have been developed recently for improved antitumor chemo-immunotherapy [31–36]. Nevertheless, a study on the localized co-delivery of dual or multiple ICB antibodies and chemotherapeutics with injectable hydrogels has not been reported yet.

In this work, an injectable drug depot based on a thermosensitive polypeptide hydrogel was developed for the topical co-delivery of aCTLA-4, aPD-1 and an anthracycline chemotherapy drug, doxorubicin (Dox), for antitumor chemo-immunotherapy (Scheme 1). As the hydrogel precursor, the methoxy poly(ethylene glycol)-*block*-poly (γ-ethyl-L-glutamate) (mPEG-PELG) aqueous solutions presented sol–gel phase transitions with the temperature rising from room temperature to 37 ◦C. This property facilitated the mixing of antitumor agents with the solutions at a low temperature, and spontaneous formation of drug-loaded hydrogels at the physiological temperature. The sustained release of Dox and a model antibody from the hydrogel was investigated in PBS with or without proteinase K. The capacity of the Dox-loaded hydrogel to cause ICD in B16F10 melanoma cells was revealed by testing the expression of calreticulin (CRT). The antitumor efficacy and systemic side effects of the aCTLA-4/aPD-1/Dox co-loaded hydrogel in vivo were evaluated by peritumoral injection of the hydrogel into C57BL/6 mice bearing B16F10 melanoma. The immune response of the treatment was investigated by analyzing immune cells and pro-inflammatory cytokines. Additionally, the inhibition of tumor reoccurrence after tumor resection surgery by the treatment of the hydrogel depot was further studied in a tumor resection model of melanoma-bearing mice.

**Scheme 1.** Schematic illustration of sustained co-delivery of Dox, aCTLA-4 and aPD-1 by an injectable hydrogel for antitumor chemo-immunotherapy. After peritumoral injection of the multiple agent co-loaded hydrogel into tumor-bearing mice, the Dox release can induce ICD of tumor cells to release DAMPs and TAAs, promoting the maturation of dendritic cells (DCs). The subsequent antigen presentation by DCs to T cells can be enhanced by inhibiting the binding between CTLA-4 and CD80/86, with aCTLA-4. Additionally, the continuous release of aPD-1 can block the PD-1/PD-L1 pathway, thereby inhibiting the immune escape of tumor cells. **Scheme 1.** Schematic illustration of sustained co-delivery of Dox, aCTLA-4 and aPD-1 by an injectable hydrogel for antitumor chemo-immunotherapy. After peritumoral injection of the multiple agent co-loaded hydrogel into tumor-bearing mice, the Dox release can induce ICD of tumor cells to release DAMPs and TAAs, promoting the maturation of dendritic cells (DCs). The subsequent antigen presentation by DCs to T cells can be enhanced by inhibiting the binding between CTLA-4 and CD80/86, with aCTLA-4. Additionally, the continuous release of aPD-1 can block the PD-1/PD-L1 pathway, thereby inhibiting the immune escape of tumor cells.

### **2. Materials and Methods 2. Materials and Methods**

#### *2.1. Materials 2.1. Materials*

Amino-terminated monomethyl poly(ethylene glycol) (mPEG-NH2, *M*<sup>n</sup> = 2000) was bought from Jemkem Inc., China. Tetrahydrofuran (THF), N,N-dimethylformamide (DMF) and toluene were refluxed with CaH2 and distilled under N2 before use. γ-Ethyl-Lglutamate N-carboxyanhydride (ELG NCA) was prepared according to our previous method [37]. All the other chemical reagents were bought from Sinopharm Chemical Reagent Co., Ltd., China and used as obtained. Amino-terminated monomethyl poly(ethylene glycol) (mPEG-NH2, *M*<sup>n</sup> = 2000) was bought from Jemkem Inc., China. Tetrahydrofuran (THF), N,N-dimethylformamide (DMF) and toluene were refluxed with CaH<sup>2</sup> and distilled under N<sup>2</sup> before use. γ-Ethyl-Lglutamate N-carboxyanhydride (ELG NCA) was prepared according to our previous method [37]. All the other chemical reagents were bought from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China), China and used as obtained.

InvivoMab anti-mouse PD-1 (CD279) and invivoMab anti-mouse CTLA-4 (CD152) were purchased from Bioxcell Inc. (West Lebanon, NH, USA). Dox was purchased from Beijing HVSF United Chemical Materials Co., Ltd. (Beijing, China). CD3-FITC, CD4-PE and CD8-APC antibodies were bought from Biolegend Inc (San Diego, CA, USA). Treg cell test kit was bought from Ebioscience Inc. (San Diego, CA, USA). ELISA kits for the detection of IL-2, TNF-α and IFN-γ were obtained from Shanghai Lengton Bioscience Co., Ltd. (Shanghai, China). InvivoMab anti-mouse PD-1 (CD279) and invivoMab anti-mouse CTLA-4 (CD152) were purchased from Bioxcell Inc. (West Lebanon, NH, USA). Dox was purchased from Beijing HVSF United Chemical Materials Co., Ltd. (Beijing, China). CD3-FITC, CD4-PE and CD8-APC antibodies were bought from Biolegend Inc (San Diego, CA, USA). Treg cell test kit was bought from Ebioscience Inc. (San Diego, CA, USA). ELISA kits for the detection of IL-2, TNF-α and IFN-γ were obtained from Shanghai Lengton Bioscience Co., Ltd. (Shanghai, China).

#### *2.2. Characterization 2.2. Characterization*

1H NMR spectra of mPEG-PELG solutions in deuterated trifluoroacetic acid (CF3COOD) were recorded on a Bruker AV 500 NMR spectrometer (Bruker Optik GmbH, Ettlingen, Germany). Number-average molecular weight (*M*n) and polydispersity index (PDI) were examined by gel permeation chromatography (GPC), which was equipped with 2 Styragel® HMW 6E columns (7.8 mm \* 300 mm) and Waters 515 HPLC pump with a Waters 2414 refractive index detector (Waters, Milford, MA, USA). The eluant was DMF containing 0.05 M LiBr at a flow rate of 1.0 mL/min at 50 °C. Monodispersed poly(methyl <sup>1</sup>H NMR spectra of mPEG-PELG solutions in deuterated trifluoroacetic acid (CF3COOD) were recorded on a Bruker AV 500 NMR spectrometer (Bruker Optik GmbH, Ettlingen, Germany). Number-average molecular weight (*M*n) and polydispersity index (PDI) were examined by gel permeation chromatography (GPC), which was equipped with 2 Styragel® HMW 6E columns (7.8 mm \* 300 mm) and Waters 515 HPLC pump with a Waters 2414 refractive index detector (Waters, Milford, MA, USA). The eluant was DMF containing 0.05 M LiBr at a flow rate of 1.0 mL/min at 50 ◦C. Monodispersed poly(methyl methacrylate)

methacrylate) standards were used to generate the calibration curve. The ellipticity of

standards were used to generate the calibration curve. The ellipticity of polymer aqueous solution (0.05 wt%) was obtained on a Chirascan CD spectrometer (Applied Photophysics, Leatherhead, UK) as a function of temperature in the range of 10–60 ◦C. The microstructure of freeze-dried hydrogel sample was observed by field emission scanning electron microscopy (SEM, Gemini 2, Carl Zeiss, Oberkochen, Germany).
