2.1 Different therapeutic agents for promoted CAR-T cell activity

Since the cytokines have been frequently applied to enhance the function of CAR-T cells and can be easily incorporated into hydrogels,¹⁴ codelivery hydrogel platforms comprising CAR-T cells and cytokines have been explored as a universal strategy for achieving promoted anticancer efficacy. Recently, Grosskopf et al.¹⁵ developed a simple-to-implement injectable hydrogel for the locoregional delivery and controlled release of CAR-T cells and the stimulatory cytokine IL-15 to enhance the therapeutic efficacy against solid tumor (Figure 2A). Dodecyl-modified hydroxypropyl methylcellulose (HPMC-C₁₂) polymers were mixed with biodegradable poly(ethylene glycol)-b-poly(lactic acid) nanoparticles (PEG-PLA NPs) to form the robust polymer–nanoparticle (PNP) hydrogels via physical crosslinking. Leveraging its unique encapsulation property, the PNP hydrogel could serve as an injectable depot of both CAR-T cells and IL-15 that simultaneously inhibited the passive diffusion of the entrapped IL-15 and allowed the active movement of encapsulated CAR-T cells, to enable the long-term retention, sustained exposure, and promoted expansion and activation of CAR-T cells. The codelivery of CAR-T cells and IL-15 via the peritumoral administration of PNP hydrogel in human medulloblastoma solid tumor-bearing non–obese diabetic (NOD) scid gamma (NSG) mice (Figure 2B) successfully created a transient inflammatory niche where CAR-T cells could exhibit a more tumor-reactive CAR-T phenotype and effectively expand. Results demonstrated that the hydrogel-based codelivery strategy realized enhanced T cell expansion and promoted anticancer efficacy without any adverse immune response. Interestingly, the transient inflammatory niche could also be formed in the tumor-bearing NSG mouse who received a distal subcutaneous administration of the PNP hydrogel depot (Figure 2C). The formation of the transient inflammatory niche within the PNP hydrogel realized the promoted expansion and maintained antitumor activity of CAR-T cells, thereby eliciting a potent abscopal effect. Utilizing a similar strategy, Zhou et al.¹⁶ constructed a gelatin methacryloyl (GelMA)-based injectable and photocurable hydrogel depot of CAR-T cells and cytokines (IL-2, IL-7, and IL-15) to build a suitable living environment for the activation, proliferation, and infiltration of CAR-T cells at the tumor site, achieving promoted efficacy in a melanoma-bearing mouse model (Figure 2D). Besides cytokines, Chao et al.¹⁷ discovered that metformin (Met), a drug prescribed for patients with type 2 diabetes, displayed dual modulation ability of cancer cells and CAR-T cells. Met could suppress the oxidative and glycolytic metabolism of cancer cells, leading to the relief of tumor hypoxia. Meanwhile, Met could upregulate the oxidative metabolism of CAR-T cells and make them transform toward a long-lived memory-like phenotype with enhanced anticancer responses. To realize a potent yet safe anticancer therapy, the authors constructed a sodium alginate (ALG)-based hydrogel scaffold (termed CAR-T@Met/gel) for the regional codelivery of CAR-T cells and Met (Figure 2E). In several postsurgical tumor models, the CAR-T@Met/gel implanted in the tumor resection cavity remarkably increased the tumor infiltration and proliferation of CAR-T cells and displayed enhanced efficacy against both local tumors and distant abscopal tumors with reduced systemic immune-related adverse effects. Furthermore, Luo et al.¹⁸ incorporated IL-15 and oxygen carriers (HEMOXCell, a marine extracellular hemoglobin with a strong capacity of oxygen storage) into an ALG-based CAR-T cell delivery hydrogel to elevate the oxygen tension throughout the solid tumor, contributing to the survival of infiltrated CAR-T cells and the downregulation of the expression of hypoxia-inducible factor-1α (HIF-1α) in the tumor site.

Besides the incorporation of extra immunostimulatory molecules, “armor” proteins such as cytokines and antibodies can be secreted by modified CAR-T cells. In a recent study, Wang et al.¹⁹ encapsulated ganglioside 2 (GD2)-specific CAR-T cells (termed GD2.CAR-Ts) with the ability to secret IL-15 (termed GD2.CAR.15-Ts) within a chitosan-PEG injectable hydrogel. In a retinoblastoma (RB) tumor-bearing nude mouse model, GD2.CAR.15-Ts intratumorally delivered by hydrogels enabled complete tumor eradication with minimized inflammation and retinal detachment. The authors suggested that the increased persistence of CAR-T cells and/or the enhanced retention of IL-15 within the tumor site might underlie the robust anticancer effect of this local delivery strategy.

Recent studies also reveal the possibility of constructing hydrogel-based multifunctional CAR-T cell delivery platforms to compatibly incorporate more than one type of therapeutic agents and/or functional cells by utilizing the flexibility of hydrogels, thus realizing combined cancer therapy. For instance, Hu et al.²⁰ constructed a hyaluronic acid (HA)-based hydrogel reservoir to encapsulate CAR-T cells, IL-15-loaded polymeric NPs (IL-15 NPs), and aPDL1-conjugated platelets (P–aPDL1) to fight against cancer recurrence after surgery via combination immunotherapy (Figure 3A). The encapsulated platelets could be activated by the inflammation occurring after tumor surgery and these activated platelets could not only form microplatelets to promote the local release of aPDL1 but also be considered as a source of ligands and chemokines. Cooperating with the IL-15 NPs, the P–aPDL1 could further boost CAR-T cell immunity, preserve the T cells from exhaustion, and recruit other immune cells, thus creating a favorable milieu for CAR-T cells (Figure 3B). Results confirmed that this HA hydrogel reservoir could promote the persistence and expansion of CAR-T cells, and therefore prevent the recurrence of local tumors as well as inhibit the growth of distant tumors by the abscopal effect in WM115 melanoma tumor resection models.

2.2 Diverse hydrogel formulations utilized in the construction of codelivery platforms

In addition to hydrogels based on polymer chains (e.g., chitosan and ALG), an increasing number of biomolecular hydrogels that contain peptides or proteins have been designed and applied as delivery platforms.^{21, 22} Peptide molecules can form secondary structural motifs such as β-sheet and α-helix and the resultant secondary structures can further self-assemble into nanofibers.²³ Then, the elongation of these nanofibers can generate thicker and longer fibers to spontaneously assemble into a fibrous hydrogel network. The peptide-based hydrogels possess nanofiber structures that are similar to the extracellular matrix (ECM) for the growth and differentiation of cells, as demonstrated by Jie et al.²⁴ In this study, the authors engineered CAR-T cells to secrete antiprogrammed death-1 single-chain variable fragments (anti-PD-1 scFv) and loaded these engineered CAR-T cells and cytokines within a peptide-based hydrogel scaffold for effective treatment against breast cancer. The anti-PD-1 scFv could realize intrinsic checkpoint blockade of CAR-T cells in the hydrogel scaffold to reverse the depletion of CAR-T cells and promote T cell proliferation and cytotoxicity. Furthermore, as an important protein from ECM, fibrin has also been considered as a promising candidate for the construction of protein-based hydrogels due to its good biocompatibility and cell adhesion ability.²⁵ The formation of the fibrin hydrogel is based on the natural polymerization process of fibrin after vascular injury. With the help of thrombin, the polymerization sites of the fibrin precursor fibrinogen can be exposed to make fibrinogen molecules be polymerized into a three-dimensional fibrin network. In 2021, Ogunnaike et al.²⁶ utilized a fibrin hydrogel to regionally deliver CAR-T cells in a subtotal glioblastoma resection model and demonstrated that the hydrogel-based delivery of CAR-T cells exhibited better control over the growth of partially resected tumor compared with the direct injection of CAR-T cells in the resection cavity. The authors also proposed the possibility of loading cytokines and biological agents in fibrin gels to sustain CAR-T cell growth and reprogram the immunosuppressive TME in solid tumors. Recently, Uslu et al.²⁷ constructed an in-situ forming fibrin hydrogel as a cell delivery carrier by simply mixing the mesothelin-specific CAR-T cells, cytokines, fibrinogen, and thrombin in the tumor resection cavity and realized effective clearance of residual cancer cells in adenocarcinoma and triple-negative breast cancer xenograft models.

Overall, diverse types of hydrogels based on hydroxypropyl methylcellulose,¹⁵ GelMA,¹⁶ ALG,^{17, 19} chitosan,¹⁸ HA,²⁰ peptide,²⁴ and fibrin^{27, 26} have been introduced in this perspective. Similar to ECM, these hydrogels have a porous structure, which endows them with good encapsulation ability of cells (e.g., around 1–2 million CAR-T cells per 100 μL of hydrogel). Also, the biochemical and biophysical properties of hydrogels can be easily tailored and optimized to mimic ECM, further supporting the proliferation and activity of the loaded cells.²⁸ Furthermore, the shear-thinning property of injectable hydrogels can realize in vivo implantation of cell-laden hydrogels without invasive procedures.