**3. Approaches to enhancing ICB therapy efficiency**

#### **3.1 Improve targeted delivery of ICIs by employing nanotechnology**

The ICP therapeutic effects depends on successful interaction of ICIs with the protein of interest. Nevertheless, the "off-target" effects of ICT therapeutics following systemic administration brings some side effects and limits the maximum allowable doses. Thus, it is significant to achieve targeted delivery and controlled release of ICIs in the desired cell types. To this end, several nanoparticle (NP) systems, such as liposome, polymeric NPs and inorganic NPs, have been used to achieve targeted delivery of ICIs to maximize the therapeutic effects while minimizing the unwanted side effects [15]. The nanotechnology-mediated ICT showed several advantages over traditional method and can improve therapeutic efficacy of ICT, as described below.

#### *3.1.1 Passive targeting*

Employing nanotechnology can improve the tumor accumulation of therapeutic ICIs via enhanced permeability and retention (EPR) effect, which refers to the higher permeability of tumor vessels to NPs than normal vessels and the increased retention of NPs in tumors due to the poor lymphatic clearance. For example, Nikpoor et al. developed PEGylated liposomes for the delivery of αCTLA-4 monoclonal antibodies [16]. They found that tumor accumulation of PEGylated liposomes encapsulated with anti-CTLA-4 antibodies was significantly greater than that of free antibodies in the CT26 colorectal tumor-bearing mice 18 h postinjection. Accordingly, the tumor-bearing mice receiving treatment of PEGylated liposomes loaded with antibody showed obviously extended survival time compared with free antibody group, suggesting that improved tumor accumulation led to greater therapeutic efficacy. It should be noted that the tumor accumulation effects of NPs via EPR effect are closely related with tumor type, heterogeneity, and perfusion.

The size and charges of NPs playing critical roles in passive targeting pattern by affecting the half-life and biodistribution. Such structure–activity relationships guide the rational design of targeted delivery nanoplatform. The size of the NPs should not be too small or too large. The NPs smaller than 7 nm tend to be cleared by renal filtration and urinary excretion [17, 18], while those larger than 200 nm are more likely to be cleared by the reticuloendothelial system (RES) [19]. As for the surface charge, the positively charged NPs show higher cellular uptake efficiency, while those slightly negatively charged and neutral NPs exhibit longer persistence during circulation. Besides, strongly positively or negatively charged NPs tend to be cleared by RES [17].

The surface properties of NPs also have impact on *in vivo* fate and performance by affecting their interaction with endogenous macromolecules. Poly(ethylene)glycol (PEG) is the most widely applied coatings to adjust the NPs surface properties. In a study, PEGylated and non-PEGylated liposomes in similar diameter of 140nm were both applied to deliver anti-CTLA-4 mAb into C26 colon tumor-bearing mice to study antitumor therapeutic effects. PEGylated CTLA-4-liposomes were shown to prolong blood half-lives and induce higher intratumoral accumulation than free antibodies and non-PEGylated groups [16].

#### *3.1.2 Active targeting*

In addition to passive targeting via EPR effects, achieving active targeting by introducing targeting moieties into NPs also can facilitate target site accumulation. Active targeting approaches can promote targeted delivery by directing NPs to action sites, either a specific location or a specific cellular type, and reduce off-target side effects. This strategy often leads to better therapeutic effects compared with those without targeting moieties through passive targeting. For example, LinTT1 is an active targeting peptide which can promote cellular uptake and tumor tissue penetration by intervening low-affinity binding with p32 cell surface receptors on tumor cells, and tumor-associated macrophages [20]. Li et al. incorporated an active targeting peptide LinTT1 into self-assembled micelles for the co-delivery of siRNA for PD-L1 and an IDO inhibitor [21]. The results showed that intravenous administration of LinTT-1-targeted NPs significantly enhanced tumor delivery of the therapeutic cargos than free therapeutics.

Moreover, introducing multiple targeting molecules into one nanoplatform can further enhance the active targeting ability. For instance, Chiang et al. fabricated anti-CD3 antibodies modified magnetic NPs for anti-PD-1 mAb delivery [22]. In addition to facilitating T cells delivery mediated by anti-CD3 antibodies bounding to the CD3 T-cell surface marker, ferromagnetic properties also facilitated tumor targeting under an external magnetic field. This dual-targeting strategy improved tumor accumulation of anti-PD-1 mAb drugs and antitumor therapeutic effects compared with the anti-CD3 single targeting group. Multivalent active targeting strategies not only can promote the NPs transportation to targeting sites, but also enable to attract specific immune cells to the site of interest. For example, Au et al. established a PEG-PLGA based trispecific NK cell engager platform for combining targeted chemoimmunotherapy and co-stimulatory 4-1BB molecule-based ICT [23]. The NPs were functionalized with tumor targeting anti-epidermal growth factor receptor (α-EGFR) antibody and two NK-activating components, anti-CD16 (α-CD16) and anti–4-1BB (α-4-1BB) antibodies, and encapsulated chemotherapeutics epirubicin (EPI). This trispecific α-EGFR/α-CD16/α-4-1BB NPs not only can achieve targeted delivery of EPI to EGFR-overexpressed tumor cells and NK cells, but also can recruit and activate circulating NK cells to the TME following systemic delivery. This multifunctional and multivalent active targeting strategy led to the greatest therapeutic efficacy and extended survival in EGFR-overexpressing murine tumor model compared with other treatment groups. These finding demonstrated that multiple targeting strategy can be applied to improve targeting specificity or drive two different targets together into spatial proximity to improve treatment outcomes.

#### *3.1.3 Controlled release*

In addition to passive and active targeting strategy, the NPs also can be engineered to achieve selective and controlled release of ICI cargos at the action sites so as to maximize the therapeutic effects. Several NPs have been reported to utilize the characteristics of TME as triggers to realize controlled release of ICT drugs, such as acidic pH and matrix metalloproteinases (MMPs) in TME [24, 25]. For instance, Lang et al. encapsulated chemotherapeutic drug paclitaxel (PTX), anti- cancer stem cells (CSC) agent thioridazine (THZ), and the PD-1/PD-L1 inhibitor HY19991 (HY) into an

MMPs enzymes as well as pH dual-responsive double-layer structured NPs [25]. The MMPs in TME triggered outer layer degradation and achieved release of HY, THZ, and PTX-loaded. Subsequently, the micelles internalized into cells and disrupted under endosomes/lysosomes acidic, leading to the PTX release and cancer cell death. This controlled release strategy controlled spatial and temporal delivery to showed powerful synergy among different therapeutic effects.

#### *3.1.4 Codelivery of different therapeutics*

Utilizing nanotechnology enable co-delivery of different therapeutics simultaneously. Mi et al. explored the dual immunotherapy nanoparticles (DINP) for the co-delivery of αPD-1 monoclonal antibodies and agonistic antibodies for the co-stimulatory receptor αOX40, to prevent T-cell inhibition and elicit T-cell activation simultaneously [26]. They proved that using DINP induced higher levels of T-cell activation compared with free immunotherapeutic antibodies or single therapeutic NPs. This NP-based co-delivery strategy enabled to increase T-cell activation, improve therapeutic efficacy and enhance immunological memory. Cheng et al. developed amphiphilic peptides containing NPs for the codelivery of PD-1/PD-L1 peptide ICI, DPPA-1, and an IDO inhibitor, NLG919 [27]. At neutral conditions, the hydrophobic segments of amphiphilic peptides formed a tight shell to protect hydrophobic cargos. At the weak acidic pH at TME, the NP swelled and MMPs diffusing into the internal hydrophobic domain, leading to the disassembly of NP and release of DPPA-1 and NLG919. This co-delivery of DPPA-1 and NLG919 enhanced tumor inhibition effects and survival in tumor-bearing mice compared to the delivery of either therapeutic alone. These finding confirmed the superiorities of nanotechnology in terms of integrating different therapeutic into a single platform.

#### *3.1.5 Other superiorities of nanotechnology*

The application of nanotechnology allows for real-time delivery monitoring. For instance, Meir et al. developed an integrated diagnostic and therapeutic nanoplatform by conjugating α-PD-L1 antibodies to gold nanoparticles (αPDL1-GNPs) to stratify patient response to ICIs [28]. αPDL1-GNPs were intravenously injected into subcutaneous MC38 colon tumors bearing mice and accumulated in tumor, which generated CT signal contrast and could be used to predict response to ICT. A strong correlation was observed between αPD-L1-GNPs related CT signal and tumor growth, leading to the facile precise prediction of the ICT response via CT signal levels. Although more validation in other tumor models is required, this proof-ofconcept study suggested that nanotechnology may promote non-invasive monitoring of ICT response.

Additionally, the combination of nanotechnology can promote development of novel delivery approaches. As an example, Wang et al. established a microneedle patch coated with pH-sensitive dextran nanoparticles for the sustained delivery of αPD1 [28]. The αPD1 was encapsulated into the NPs, which can dissociate at acidic pH and achieve controlled and sustained released αPD-1 antibodies over 3 days. This sustained release of αPD-1 antibodies improved tumor retention of antibodies and prolonged the survival time of subcutaneous B16F10 melanomas bearing mice. Nevertheless, this delivery approach seems be limited to superficial tumors, such as melanomas, and need more investigation to confirm strategies.
