Molecular and nanoengineering approaches towards activatable cancer immunotherapy

Chi Zhang and Kanyi Pu *
School of Chemical and Biomedical Engineering, Nanyang Technological University, 70 Nanyang Drive, 637457, Singapore. E-mail:

Received 13th March 2020

First published on 26th May 2020

Cancer immunotherapy is an emerging treatment strategy that modulates the immune system to fight against cancer. Although several immunotherapeutic agents have been utilized in the clinic for cancer treatment, low patient response rates and potential immune-related adverse events remain two major challenges. With the merits of delivery controllability and modular flexibility, nanomedicines provide opportunities to facilitate immunotherapies for clinical translation in a safe and effective manner. In this review, we discuss the convergence of nanomedicine with immunotherapy with a focus on molecular and nanoengineering approaches towards activatable cancer immunotherapy. These activatable nanoagents exert immunotherapeutic action only in response to internal or external stimuli. This allows them to locally reprogram the tumor microenvironment and activate antitumor immunity while reducing the incidence of immune-related adverse events. The category of activatable immunotherapeutic nanoagents are discussed along with an overview of their clinical translation prospects and challenges.

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Chi Zhang

Chi Zhang received his PhD degree from the College of Chemistry and Molecular Sciences, Wuhan University in 2019. Then, he worked as a Postdoctoral Research Fellow in Prof. Kanyi Pu's group at the School of Chemical and Biomedical Engineering (SCBE), Nanyang Technological University (NTU). His current research focuses on the development of phototheranostic agents for cancer therapy.

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Kanyi Pu

Prof. Kanyi Pu received his PhD from the National University of Singapore in 2011 followed by a postdoctoral study at the Stanford University School of Medicine. He joined the School of Chemical and Biomedical Engineering (SCBE) at Nanyang Technological University (NTU) as an Associate Professor in 2015. His research focuses on the development of molecular optical reporters and semiconducting polymer biomaterials for early diagnosis, nanomedicine and photoregulation.

1. Introduction

Cancer has been one of the leading causes of death worldwide and is still difficult to cure although substantial efforts have been devoted.1 The past decade has witnessed a revolution in cancer treatments shifting from traditional therapies (for example, chemotherapy, surgery or radiotherapy) to immunotherapies that modulate antitumor immune responses for cancer regression and elimination.2 The 2018 Nobel Prize in Physiology or Medicine was awarded to pioneers in the field of cancer immunotherapy for enabling the innate and adaptive immune systems to fight against cancer in the clinic. With the increased understanding of cancer and immunity both at cellular and molecular levels, cancer immunotherapy has been rapidly progressing and has been clinically tested. Up to now, the major successes in cancer immunotherapy have been presented by immune checkpoint blockades,3 chimeric antigen receptor (CAR) T cell therapy,4 cytokine therapy,5 and cancer vaccines.6 These emerging immunotherapeutic strategies have been demonstrated to show encouraging results in patients with various types of cancers.7 Besides, several immune drugs (e.g., ipilimumab, nivolumab, imiquimod, blinatumomab, etc.) with remarkable clinical effects and improved cancer prognosis have already been approved by the US Food and Drug Administration (FDA) against some definite solid cancers.8

Despite these advances, the clinical translation of these immunotherapies still faces some challenges. Although certain tumors (including melanoma and microsatellite unstable cancers) exhibit moderate response to immunotherapy, the response rates of the majority of cancer types remain low.9 This is probably associated with many factors including the high mutability, genetic heterogeneity, and poor immunogenicity of cancer, which also make the prediction of patient responses to immunotherapy difficult.10 Moreover, immunotherapy sometimes is accompanied by immune-related adverse events (such as endocrinopathy, pneumonitis, hepatitis, nephritis, and colitis), autoimmune side effects, and cytokine release syndrome with life-threatening lethality.11 Owing to these challenges, development of immunotherapies with high efficacy and increased safety is desired.

Nanomedicines have great potential for the treatment of cancer with enhanced therapeutic efficacy and reduced side effects.12 This mainly results from their ability to modulate the systemic biodistribution and targeted accumulation of administered therapeutic agents.13 Numerous preclinical results have further proved the advantages of nanomedicines such as increased tumor inhibition and prolonged survival of patients compared to free drug treatments due to their specific tumor targeting ability and enhanced tumor accumulation via the enhanced permeability and retention (EPR) effect.14 Recent studies on the design and application of nanomedicines in immune drug delivery have also attracted growing interest.15

Nanotechnology and bioengineering methods play important roles in nanomedicine research to shape the tumor targeting capability and to minimize the off-target toxicity of nanomedicines for cancer immunotherapy.16 Nanomedicine-based immune drug delivery systems have already shown promising results in clinical trials.17 However, traditional drug delivery strategies still suffer from the low bioavailability and inevitable leakage of immune drugs, causing them to encounter the issue of immune-related adverse events.18 Thus, advancement and optimization of nano-immunotherapy lie in the development of innovative approaches to enhance the specificity and controllability of immunotherapeutic action on desired cell types at designated locations.

In this review, we summarize recent progress in nanomedicines for activatable cancer immunotherapy. Particular focus is given to molecular and nanoengineering approaches that lead to the controlled functionality and immune effects of activatable cancer immunotherapeutics (Fig. 1). Such immunotherapeutic nanoagents have spatiotemporal controllability and show on-demand immunoactivation in response to various internal or external stimuli, exhibiting great potential for addressing some of the limitations of traditional immunotherapies and for promoting the translation of cancer immunotherapy.

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Fig. 1 (a) Schematic illustration of the mechanism of activatable cancer immunotherapeutic agents. (b) Summary of four major strategies to induce antitumor immunity via targeting and modulating various immune cells. MDSCs, myeloid-derived suppressor cells; TAM, tumor-associated macrophage; Treg cells, regulatory T cells; Teff cells, T effector cells; TH cells, T helper cells; DCs, dendritic cells; ICD, immunogenic cell death; DAMP, damage-associated molecular patterns; PAMP, pathogen-associated molecular patterns; aCD47, anti-CD47 antibody; IDO, indoleamine-2,3-dioxygenase; TLR, Toll-like receptor; ARG1, arginase 1; SIRPα, signal regulatory protein alpha; aSIRPα, anti-SIRPα antibody; GM-CSF, granulocyte-macrophage-colony-stimulating factor; GM-CSFR, receptors for GM-CSF; APCs, antigen-presenting cells; PD-1, programmed death-1; aPD-1, anti-PD-1 antibody; PD-L1, programmed death ligand 1; aPD-L1, anti-PD-L1 antibody; aCD28, anti-CD28 antibody; MHC, histocompatibility complex class; TCR, T cell receptor; CTLA-4, cytotoxic T lymphocyte antigen 4; aCTLA-4, anti-CTLA-4 antibody; and STING, stimulator of interferon genes.

2. Activatable cancer immunotherapy

Generally, the design of activatable cancer immunotherapeutics relies on chemical or conformational changes in response to various stimuli, followed by the release of immunotherapeutic agents (e.g., immunological adjuvants, drugs, antigens, antibodies, checkpoint inhibitors, etc.) and activation of antitumor immune responses. Such stimulus sources can be internal and external: internal stimuli belong to specific biomarkers in tumor microenvironments such as acidic pH, redox potential, hypoxia, and overexpressed enzymes and external stimuli can be light, magnetic field and ultrasound with artificial manipulation.

2.1. Internal stimuli

Compared to normal tissues, tumor tissues generally show a more acidic microenvironment, slightly higher oxidation and reduction levels, higher hypoxic status, and overexpressed enzymes, owing to fast proliferation, metabolism, maturation, migration, and metastasis.19,20 Tumor cells are also found to possess six major hallmarks distinct from normal cells, consisting of sustaining proliferative signaling, evading growth suppressors, activating invasion and metastasis, enabling replicative immortality, inducing angiogenesis, and resisting cell death.21 As a result, the internal stimuli related to the hallmarks of tumor microenvironments including acidic pH, redox potential, hypoxia, and overexpressed enzymes have been applied for activatable cancer immunotherapy.

2.2. External stimuli

External stimuli generally originate from the outside manipulative energy sources, for example, light, magnetic field, ultrasound, etc. The advancement of materials science allows us to devise biomaterials that respond to these external stimuli through the cleavage of chemical bonds or the transformation of molecular conformations.22 Thus, such external stimuli-responsive biomaterials have been used to control the spatiotemporal resolution of drug release,23 possessing great potential for the development of activatable cancer immunotherapeutics. Moreover, external stimuli can convert light and other forms of electromagnetic energy into cytotoxic signals such as heat and reactive oxygen species (ROS), leading to photothermal, photodynamic, sonodynamic, and radiation therapies.24,25 These therapeutic modalities can induce immunogenic cell death and thus potentiate cancer immunotherapy.

3. Internal stimuli-mediated immunotherapy

3.1. pH-Activated immunotherapy

One of the most representative internal stimuli is the acidic environment in tumor tissues and cell endosomes, which can be harnessed for pH-activated cancer immunotherapy. The extracellular pH of most tumor tissues is reported to be about 6.5–6.8, much lower than those of normal tissues (7.2–7.4).26 This mildly acidic tumor microenvironment is attributed to the high glycolysis rate with overproduction and accumulation of lactic acid by tumor cells. Thus, pH-sensitive biomaterials can be utilized to induce the specific activation of immune drugs at tumor sites. This localized drug activation would greatly promote the initiation of antitumor immune responses and maximize the efficiency of immunotherapy. Up to now, a series of pH-activated nanosystems have been proposed for activatable cancer immunotherapy using immunological adjuvants, antigens, antibodies or immunotherapeutic genes.
3.1.1. pH-Mediated activation of immunological adjuvants and antigens. Generally, immunological adjuvants and antigens play important roles in immune systems for the activation of adaptive immune responses. Adjuvants mainly include immunotherapeutic agents (e.g., various Toll-like receptors (TLRs) modulators,27 indoleamine-2,3-dioxygenase 1 (IDO-1) inhibitors,28 immunomodulatory cytokines,29 stimulator of interferon genes (STING) agonists,30 PD-1/PD-L1 pathway inhibitors,31etc.) which can activate various immune cells and boost immune responses, while antigens can be taken up by antigen-presenting cells (APCs) for antigen presentation and antitumor immunity activation. Both adjuvants and antigens can be easily conjugated to pH-activated nanosystems. Precise and selective modulation of these immunotherapeutic agents will be beneficial for the activation of antitumor immune responses.

Chang et al. constructed pH-responsive poly(ε-caprolactone)–hydrazone–poly(ethylene glycol) (PCL–Hyd–PEG) nanovesicles to encapsulate immunological adjuvants unmethylated cytosine–phosphate–guanine oligodeoxynucleotides (CpG ODNs) and endogenous tumor antigens heat shock protein 70-chaperoned polypeptides (HCP) for activatable cancer immunotherapy (Fig. 2a and b).32 These PCL–Hyd–PEG nanovesicles can be disassociated in the mildly acidic tumor microenvironment and can release encapsulated drugs via pH-mediated hydrolyzation of the hydrazone bond. Dynamic light scattering (DLS) results showed that the nanovesicles rapidly and remarkably swelled at pH 5.0 and pH 6.0, finally resulting in the degradation of nanovesicles (Fig. 2c). Subsequently, the drug release profile demonstrated the stronger release of adjuvants and antigens from the nanovesicle at a lower pH (Fig. 2d). Once accumulated at tumor sites, these nanovesicles could release CpG ODN and HCP effectively. CpG ODN is a TLR9 ligand which involves the TLR signaling for the activation of APCs and initiation of adaptive immune responses. HCP can act as endogenous tumor antigens represented by major histocompatibility complex class I and II (MHC I and II) molecules and can elicit CD8+ T cell responses. Therefore, these mildly acidic tumor microenvironment-responsive systems could activate APCs (including macrophages and dendritic cells (DCs)) at tumor sites and enhance antitumor immune responses for activatable cancer immunotherapy (Fig. 2e and f). In addition to the pH-responsive polymers, many other pH-responsive biomaterials could be utilized to construct drug delivery systems for pH-activated cancer immunotherapy. For instance, Duan et al. developed pH-responsive metal–organic frameworks (MOFs) to realize the release of an immunological adjuvant (CpG) and a model antigen ovalbumin (OVA) for the activation of antitumor immune responses.33 Yuba et al. reported bioactive polysaccharide-based pH-sensitive biomaterials for cytoplasmic delivery of antigens and activation of DCs.34 Curdlan and mannan, microorganism-derived bioactive polysaccharides which can recognize and activate macrophages and DCs, were chosen for the encapsulation of the model antigen OVA. Biodegradable pH-sensitive moieties, such as 3-methyl glutarylated dextran (MGlu-Dex), were introduced into these polysaccharides for OVA release in acidic tumor microenvironments. These released antigens could be efficiently presented to DCs, finally leading to the activation of antigen-specific antitumor immune responses.

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Fig. 2 (a) Schematic illustration of the preparation of HCP + CpG@NPs-CD80 Ab vesicles (EAC-NPs). (b) Schematic illustration of the mechanism of the vesicle-induced cancer immunotherapy. (c) Size distribution of EAC-NPs in PBS at pH 5.0, 6.0, and 7.4 after 10 h. (d) The cumulative release of HCP from EAC-NPs at different time points in the supernatant was measured using the Bradford assay in PBS at pH 5.0, 6.0, and 7.4. (e and f) In vivo flow cytometry analysis of (e) F4/80+ macrophages and (f) CD11c+ DCs with modified NPs (Cy7) in spleens at 24 h after different treatments. Reproduced from ref. 32 with permission from John Wiley & Sons Ltd, copyright 2019.

Apart from TLR modulators and tumor antigens, Chen and co-workers designed pH-sensitive nanovesicles (pRNVs) to deliver an indoleamine 2,3-dioxygenase (IDO) inhibitor, indoximod (1-methyl tryptophan, IND) for cancer immunotherapy.35 IDO is a tryptophan catabolic enzyme overexpressed in tumor tissues and tumor-draining lymph nodes (TDLNs) and can convert tryptophan to kynurenine and additional metabolites. The overexpression of IDO can induce the depletion of tryptophan and accumulation of kynurenine, further leading to the differentiation and hyper-activation of Treg cells, the suppression of Teff cells, and dysfunction of DCs. Thus, IDO plays an important role in the generation of an immunosuppressive microenvironment and IDO inhibitors can re-establish the antitumor immune responses.36 The pH-sensitive nanovesicles were fabricated by the self-assembly of a block copolymer polyethylene glycol-b-cationic polypeptide (PEG-b-cPPT). The IDO inhibitor IND was further encapsulated in the nanovesicles via hydrophobic interactions. After systemic administration, the nanovesicles could release IND in the acidic tumor microenvironment. As a result, IND could inhibit IDO effectively and relieve tryptophan overconsumption and kynurenine accumulation in the tumor microenvironment, finally leading to the reversion of immune suppression and activation of antitumor immune responses. Moreover, Song et al. developed a pH-responsive nanogel for the controllable release of immunotherapeutic cytokine interleukin-2 (IL-2) in the tumor microenvironment.37

IL-2 is an FDA-approved cytokine used to treat metastatic melanoma and renal cell carcinoma and plays important roles in regulating the survival, proliferation, and differentiation of tumor-infiltrating lymphocytes (TILs). In the acidic tumor microenvironment, the nanogels could release IL-2 to activate TILs and reverse immunosuppressive tumor microenvironments for enhanced antitumor immunotherapy.

3.1.2. pH-Mediated activation of immunotherapeutic antibodies. In addition to immunological adjuvants and antigens, immunotherapeutic antibodies with specific target affinity and strong immunomodulatory capability exhibit great potential for the activation of antitumor immunity as well.38 Since the high therapeutic efficacy of a specific antibody requires a relatively high expression of the tumor targets or adequate antibody accumulation at the target sites, developing efficient antibody delivery and release systems to enhance the targeting affinity is urgently needed. The clinically used antibodies mainly include the CD47 blockade antibody,39 checkpoint blockade antibodies (including the anti-PD-1 (programmed death-1) antibody, anti-PD-L1 (programmed death-ligand 1) antibody, and anti-CTLA4 (cytotoxic T lymphocyte antigen 4) antibody),40 T cell-activating antibodies (for example, anti-CD3 and anti-CD28 antibodies),41 and NK cell-activating antibodies (anti-CD16 antibody)42 and can achieve enhanced antitumor immunity via various pathways and mechanisms. Thus, the efficient delivery and activity recovery of a specific antibody at tumor sites by pH-responsive biomaterials are important for activatable cancer immunotherapy.

Our group developed pH-responsive exosome nano-bioconjugates for activatable cancer immunotherapy.43 Exosome nanoconjugates are composed of M1 macrophage exosomes conjugated with immune-stimulatory antibodies (including the anti-CD47 antibody (aCD47) and anti-signal regulatory protein alpha (SIRPα) antibody (aSIRPα)) via pH-sensitive benzoic-imine bonds (Fig. 3a and b). Flow cytometry analysis demonstrated the successful conjugation of these two antibodies on M1 macrophage exosomes (Fig. 3c). Meanwhile, the release profile of total antibodies further confirmed the acid-mediated release of these antibodies in vitro (Fig. 3d). After systemic administration, the nanoconjugates could release both aCD47 and aSIRPα in the acidic tumor microenvironment, which would individually block the inhibitory receptor CD47 on tumor cells and SIRPα on macrophages, resulting in the abolished “don’t eat me” signaling and improved phagocytosis of macrophages (Fig. 3e and f).

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Fig. 3 (a) Schematic illustration of the fabrication of the aCD47 and aSIRPα-engineered M1 Exo. (b) Mechanisms of Mn2+-induced M1 macrophage polarization and the synergistic anticancer effect of engineered M1 Exo. (c) Flow cytometry analysis of the anti-CD47 and anti-SIRPα antibodies conjugated on M1 Exo (left: pristine M1 Exo; right: M1 Exo-Ab). (d) Release profiles of total Ab from M1 Exo-Ab at different pH values (6.5 and 7.4). (e) Flow cytometry analysis of M1 markers (CD80 and CD86) on M2 before and after treatment. (f) Confocal microscopy images of the phagocytosis of 4T1 by M2 before and after treatment (scale bar = 10 μm). I = pristine M2, II = M2 treated with M1 Exo, III = M2 treated with M1 Exo-Ab. Reproduced from ref. 43 with permission from John Wiley & Sons Ltd, copyright 2020.

Apart from CD47 blockade, PD-1/PD-L1 blockade has attracted great interest owing to its remarkable clinical success. Gu's group designed a self-degradable and pH-sensitive microneedle (MN) patch for the sustained and controllable delivery of the anti-PD-1 antibody for enhanced antitumor immunotherapy.44 The microneedle consists of biocompatible hyaluronic acid (HA) integrated with pH-sensitive dextran NPs which encapsulate the anti-PD-1 antibody, glucose oxidase (GOx) and catalase (CAT). The mildly acidic tumor microenvironment and GOx/CAT-mediated enzymatic generation of gluconic acid could promote the gradual self-dissociation of NPs, resulting in the sustained release of the anti-PD-1 antibody. This pH-sensitive system provided an efficient delivery strategy for the anti-PD-1 antibody and achieved improved antitumor immunity for activatable cancer immunotherapy. In addition, Gu's group reported an in situ formed immunotherapeutic pH-responsive gel encapsulated with aCD47 to promote effective antigen presentation and initiate T cell-mediated immune responses for activatable cancer immunotherapy.45

3.1.3. pH-Mediated activation of immunotherapeutic genes. Gene-based therapy has long fascinated scientists and the general public due to its great potential to treat diseases at the genetic roots.46 The efficient delivery of therapeutic genes into related cells is of vital importance for achieving good therapeutic effects. Recently, several gene-delivery strategies have already been proposed for activatable cancer immunotherapy. Owing to the negatively charged properties of genes and their requirement for endosomal escape, gene-delivery vectors are generally composed of cationic materials that can release genes into the cytosol via pH-activated cleavage.

Wang and co-workers developed cationic lipid-assisted nanoparticles (CLANs) for the delivery of small interfering RNA (siRNA) targeting indoleamine 2,3-dioxygenase-1 (siIDO) as shown in Fig. 4a.47 This siIDO could be efficiently released in acidic endosomes via the “proton sponge” effect. Afterward, the released siRNA could downregulate the target gene and inhibit the expression of IDO-1, finally leading to the interference of the immunosuppressive IDO-1 pathway in TDLNs and tumor tissues. Both the real-time PCR and western blot analysis demonstrated that CLANs encapsulating siIDO could downregulate the IDO-1 mRNA and protein expression levels in CT26 colon cancer cells (Fig. 4b and c). Moreover, the tumor tissues and TDLNs exhibited obvious accumulation of siIDO and downregulation of IDO-1 in CT26 tumor-bearing mice after systemic administration (Fig. 4d and e). This siIDO-delivery system with IDO-1 pathway interference was demonstrated to achieve strong antitumor immune responses for cancer immunotherapy. Moreover, Li's group designed an acid-activatable versatile micelleplex to deliver PD-L1 siRNA for silencing PD-L1 expression and reversing the immunosuppressive tumor microenvironment.48 This siRNA-mediated PD-L1 blockade strategy could achieve pH-activatable cancer immunotherapy.

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Fig. 4 (a) Schematic illustration of the mechanism of CLANsiIDO1-mediated IDO1 activity inhibition in tumor-draining lymph nodes (TDLNs) and tumor tissues. (b and c) The (b) real-time PCR and (c) western blot analysis of IDO1 at 24 and 48 h post-transfection, respectively. (d) Fluorescence imaging of Cy5 siRNA in TDLNs and tumor tissues after administration of CLANCy5-siRNA by confocal microscopy. (e) In vivo IDO1 knockdown efficacy of CLANsiIDO1 in TDLNs and tumor tissues after intravenous injection. Reproduced from ref. 47 with permission from the American Chemical Society, copyright 2019.

3.2. Redox-activated immunotherapy

In general, tumor cells exhibit higher redox status than their normal counterparts.49 This is owing to the upregulation of redox factors including reactive oxygen species (ROS), superoxide dismutase (SOD), nicotinamide adenine dinucleotide phosphate (NADPH) oxidase, free glutathione (GSH)/glutathione disulfide (GSSG), and thioredoxin (TRX-SH2)/oxidized thioredoxin (TRX-SS).50 These redox factors can be used to construct various redox-sensitive biomaterials with controllable drug activation for the treatment of cancers. With the fast development of cancer immunotherapy, utilizing these redox-sensitive biomaterials to encapsulate immunotherapeutic agents and activate specific antitumor immunity is particularly significant.
3.2.1. Reduction-activated immunotherapy. Glutathione (GSH), one of the most abundant cellular metabolites with highly reductive capability, plays an essential role in the regulation of intracellular redox homeostasis.51 Meanwhile, GSH is involved in controlling protein folding by mediating the formation and cleavage of disulfide bonds. The concentration of GSH in tumor cells is reported to be 5–10 mM, which is at least two-fold higher than that in normal cells.52 Thus, a series of GSH-responsive systems have been established to deliver immunotherapeutic agents for activatable cancer immunotherapy. GSH-mediated activation of immunological adjuvants. As mentioned above, the highly efficient delivery of immunological adjuvants to tumor cells can be helpful for the activation of specific antitumor immunity. Due to the high level of GSH in tumor cells, it is convenient to develop GSH-sensitive biomaterials to realize controllable drug release for activatable cancer immunotherapy.

Li's group reported a series of nanosystems with GSH-responsive modulation of IDO-1 activity for activatable cancer immunotherapy. For example, they designed a GSH-responsive heterodimer of the photosensitizer pheophorbide A (PPa) and IDO-1 inhibitor NLG919 and further conjugated it with a light-activatable prodrug of oxaliplatin (OXA) for enhanced cancer immunotherapy (Fig. 5a).53 These self-assembled nanoparticles could be disintegrated and could realize drug release under the stimulation of external light and internal GSH. Both DLS and TEM results demonstrated the morphological transformation of the nanoparticles upon light and GSH stimulation (Fig. 5b–d). Meanwhile, the drug release profile further confirmed the light- and GSH-responsive release of NLG919 in vitro (Fig. 5e). NLG919 can reverse the immunosuppressive tumor microenvironment by inhibiting IDO-1-mediated tryptophan degradation and cytotoxic T-cell lymphocyte (CTL) exhaustion. After systemic administration and tumor accumulation, the released NLG919 could effectively inhibit the activity of IDO-1 and decrease kynurenine accumulation for enhanced T cell activation (Fig. 5f and g). With the combination of photodynamic therapy (PDT) and chemotherapy, this nanosystem could elicit strong antitumor immune responses and reverse immunosuppressive tumor microenvironments for enhanced immunotherapy. In addition, Li et al. developed a GSH-activatable homodimer of NLG919 to self-assemble into nanoparticles for inactivating IDO and enhancing antitumor immunotherapy.54

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Fig. 5 (a) Schematic illustration of the fabrication of light-inducible prodrug nanocargoes LINC. (b and c) Hydrodynamic diameter and representative TEM images of LINC (b) without and (c) with 671 nm laser irradiation (a photodensity of 16 mW cm−2, 5 min). (d) Hydrodynamic diameter and representative TEM images of LINC in 10 × 10−3 M GSH solution. (e) The NLG919 release profiles of LINC under different conditions. (f) The intratumoral Kyn to Trp weight ratio examined at the end of the antitumor study. (g) The CD8+ T cells to Tregs ratios in the tumor mass examined at the end of the antitumor study. Reproduced from ref. 53 with permission from John Wiley & Sons Ltd, copyright 2019.

Apart from IDO inhibitors, some other immunological adjuvants could be utilized for activatable cancer immunotherapy. Lam and co-workers encapsulated imiquimod, a potent small molecule TLR7/8 agonist, into the structurally defined telodendrimers for GSH-responsive drug release and enhanced activation of immune cells.55 Li and co-workers developed a photothermal CpG nanotherapeutic by integrating Au nanorods with CpG via GSH-sensitive Au–thiol bonds for photothermal and activatable immunotherapy.56 These drug delivery nanosystems with GSH-responsive activation of immunological adjuvants would be promising for activatable cancer immunotherapy. GSH-mediated activation of antigens. Tumor-associated antigens can be used to construct tumor vaccines for immunoactivation. Due to the specific tumor microenvironment, GSH-responsive biomaterials can also be utilized for the delivery and activation of antigens at tumor sites. Moon and co-workers synthesized uniform and biodegradable mesoporous silica nanoparticles (bMSN) for neoantigen-based cancer vaccination.57 This bMSN nanoplatform could load multiple neoantigen peptides, CpG oligodeoxynucleotide adjuvants, and the photosensitizer chlorin e6 (Ce6) for synergistic cancer therapy. These neoantigen peptides were conjugated on the surface of bMSN via disulfide bonds for specific tumor targeting, which could further be cleaved by the highly abundant GSH in the tumor intracellular environment. With the release of these neoantigens and PDT-mediated recruitment of DCs, strong neoantigen-specific CD8+ CTL responses could be elicited for personalized cancer immunotherapy. GSH-mediated activation of immunotherapeutic antibodies. Apart from the immunological adjuvants and tumor-associated antigens, immunotherapeutic antibodies can achieve robust antitumor immunity via GSH-mediated release. Irvine and co-workers designed cell surface-conjugated protein nanogels (NGs) that responded to an increase in T cell surface reduction potential after antigen recognition and limited drug release to sites of antigen encounter.58 The NGs were loaded with an interleukin-15 super-agonist (IL-15Sa) complex and further conjugated on the surface of T cells. Once T cells were activated, the T cell-surface reduction potential would be increased, resulting in drug release and stronger activation of these T cells. This GSH-mediated drug activation exhibited eight-fold more IL-15Sa release, enabling substantially improved therapeutic efficacy for enhanced activatable cancer immunotherapy.
3.2.2. Oxidation-activated immunotherapy. Owing to the highly oxidative tumor microenvironment, oxidation-sensitive biomaterials can be utilized to modulate the activation of immunotherapeutic agents for cancer immunotherapy. Gu's group reported a series of ROS-responsive biomaterials for activatable cancer immunotherapy. For example, they developed a ROS-responsive scaffold for the local release of gemcitabine (GEM) and the anti-PD-L1 blocking antibody (aPD-L1).59 This scaffold was composed of an injectable ROS-responsive hydrogel encapsulated with GEM and aPD-L1 (Fig. 6a). The release profile of GEM and aPD-L1 showed that the scaffold could induce the drug release under high ROS conditions in vitro (Fig. 6b and c). After local administration of the aPD-L1–GEM scaffold, GEM and aPD-L1 could be efficiently released with the disassociation of the hydrogel by the highly abundant ROS in the tumor microenvironment, leading to immune-mediated tumor regression. Immunofluorescence staining and flow cytometry analysis of tumor tissues further demonstrated that the tumor-infiltrating CD8+ and CD4+ T cells exhibited remarkable upregulation, and the intratumoral ratios of effector T cells to regulatory T cells (Treg) were increased after treatment (Fig. 6d and e). This tumor microenvironment ROS-responsive drug delivery system is promising for the local activation of immunotherapeutic agents and antitumor immunity.
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Fig. 6 (a) Schematic illustration of combined chemoimmunotherapy using a ROS-degradable hydrogel scaffold to deliver GEM and aPDL1 into the tumor microenvironment. (b and c) Cumulative release profiles of (b) GEM and (c) aPDL1 from hydrogels incubated with PBS with or without H2O2 (1 mM). (d) Immunofluorescence staining of CD4+ and CD8+ T cells in tumor tissues of B16F10 tumor-bearing mice. (e) Absolute numbers of the CD8+ and CD4+ T cells per gram of the tumor, and ratios of the tumor-infiltrating CD8+ T cells and CD4+ T cells to Tregs in the tumors upon various treatments. Teff, effector T cells. Reproduced from ref. 59 with permission from the American Association for the Advancement of Science, copyright 2018.

Afterward, they further utilized aPD-1 and aCD47 to construct the aPD-1@aCD47 complexes for enhanced immunotherapy.60 Such aPD-1@aCD47 complexes were engineered by conjugating albumin with aPD-1 and aCD47 using ROS responsive linkers. At high ROS levels in the tumor microenvironment, these complexes could be degraded and could release aPD-1 and aCD47 to activate the recognition of cancer cells by the innate immune system and boost T cell responses. In addition, an injectable ROS-responsive polypeptide gel was developed for sustained release of aPD-L1 and an IDO inhibitor dextro-1-methyl tryptophan (D-1MT) for synergistic immunotherapy.61 The hydrogel was fabricated by conjugating PEG with two polypeptides including ROS-responsive L-methionine (Me) and D-1MT to form a functional triblock copolymer. After local injection, the copolymer could transfer to the hydrogel upon rising the temperature and release aPD-L1 and D-1MT at high ROS levels to improve T cell activity and relieve the immunosuppressive tumor microenvironment.

3.3. Hypoxia-activated immunotherapy

Hypoxia is one of the major biological features of tumor microenvironments owing to the restricted oxygen delivery by abnormally formed blood vessels and excessive oxygen consumption by rapidly proliferating tumor cells.62 Based on the hypoxic characteristics of the tumor microenvironment, a growing number of hypoxia-sensitive biomaterials have been developed for specific drug activation at tumor sites. Meanwhile, hypoxia-activated immunotherapy has attracted a lot of interest in the treatment of cancers.

Kim and co-workers designed a hypoxia-responsive mesoporous silica nanocarrier to encapsulate the photosensitizer Ce6 and the immunological adjuvant CpG for PDT and enhanced cancer immunotherapy (Fig. 7a and b).63 This hypoxia-responsive nanosystem (CAGE) was fabricated by decorating glycol chitosan (GC) on the surface of Ce6-doped mesoporous silica nanoparticles via a hypoxia-responsive labile linker, azobenzene linker. Negatively charged CpG was further loaded on the nanoparticle via electrostatic interactions with GC. Under hypoxia conditions, CpG could be released from CAGE upon the cleavage of the azobenzene linker (Fig. 7c). After systemic administration and laser irradiation, CAGE-mediated PDT could eradicate tumor cells and induce DC recruitment to tumor sites. The flow cytometry results showed an obvious increase of CD11c+ MHC II+ DC population in tumor tissues (Fig. 7d). Afterward, the hypoxia-responsive release of CpG could enhance the maturation of DCs with an elevated CD80+ CD86+ DC population (Fig. 7e). The recruitment and maturation of DCs would improve the antigen-presenting process of DCs, which was further demonstrated by the increased OVA-presenting DC population in tumor tissues (Fig. 7f). The hypoxia-responsive immunological adjuvant delivery strategy combined with PDT could greatly induce the maturation and activation of DCs for antigen presentation and enhanced cancer immunotherapy. In addition, Chen et al. developed a synergistic therapeutic strategy by the combination of vascular disrupting agents (VDAs) with hypoxia-sensitive imiquimod (hs-IMQ).64 VDAs could exacerbate the hypoxic status of the tumor microenvironment, which would induce the fast release of active imiquimod (IMQ). IMQ, a small molecule TLR7/8 agonist, can transform immature plasmacytoid dendritic cells (pDCs) into their active form. This hypoxia-sensitive strategy with IMQ activation in tumor tissues offers new approaches to reverse the immunosuppressive microenvironment for activatable cancer immunotherapy.

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Fig. 7 (a) Schematic illustration of the synthetic scheme of the CAGE complex. (b) Schematic illustration of the mechanism of CAGE for in vivo hypoxia-triggered transformation, tumor protein release, DC recruitment and maturation, enhanced antigen presentation, and improved cancer immunotherapy. (c) Hypoxia-responsive CpG ODN release from CAGE. (d–f) Flow cytometry analysis of (d) recruited (CD11c+ MHC II+), (e) mature (CD80+ CD86+), and (f) OVA-presenting DC population in tumor tissues. Reproduced from ref. 63 with permission from the American Chemical Society, copyright 2019.

3.4. Enzyme-activated immunotherapy

A wide variety of cancers are reported to abnormally express various enzymes, which play crucial roles in tumor progression. These enzymes generally have distinctive activities and functions and can catalyze the degradation of related substrates with high selectivity and efficacy.65 Thus, lots of enzyme-sensitive biomaterials have been developed for specific cancer diagnosis and therapy.66,67 Recently, a series of enzyme-sensitive systems have been reported for activatable cancer immunotherapy. There are three frequently used enzymes including matrix metalloprotease (MMP), caspase and HAase for tumor-specific drug delivery and enhanced antitumor immunity.
3.4.1. MMP-activated immunotherapy. MMPs, a family of extracellular zinc-dependent endopeptidases, can regulate physiological tumor progression processes including proliferation, invasion, migration, and metastasis.68 MMPs are overexpressed in a variety of tumors and can recognize and cleave specific peptide substrates. As a result, plenty of research studies have focused on the development of MMP-sensitive drug delivery systems.69 Recently, MMP-activated immunotherapy has also been proposed and studied.

Li and co-workers reported a combinational nanosystem by integrating the checkpoint blockade inhibitor aPD-L1 with the photosensitizer indocyanine green (ICG) for MMP-sensitive drug activation and antitumor immunotherapy (Fig. 8a and b).70 This nanoparticle was fabricated by the self-assembly of aPD-L1, ICG, the (−)-epigallocatechin-3-O-gallate dimer (dEGCG) and an MMP-2-liable PEGylated dEGCG (PEG-PLGLAG-dEGCG). The activity of aPD-L1 could be efficiently shielded in the nanoparticles and aPD-L1 could be reactivated upon MMP-2 or Triton X-100-mediated disassociation of the nanoparticles (Fig. 8c). The aPD-L1 release profile further demonstrated that the nanoparticle could respond to MMP-2 and induce aPD-L1 release (Fig. 8d). After the passive accumulation of this nanoparticle at tumor sites through the EPR effect, the highly expressed MMP-2 in the tumor microenvironment could induce the activation of aPD-L1 for PD-L1 blockade, finally resulting in the remarkable activation of CD8+ T cells and an increased ratio of CD8+ T cells to Treg for checkpoint inhibition and immune stimulation (Fig. 8e and f). This MMP-responsive nanoparticle with the controllable activation of aPD-L1 could overcome immunological tolerance for checkpoint blockade immunotherapy.

image file: c9cs00773c-f8.tif
Fig. 8 (a) Schematic illustration of the fabrication of MMP-2-liable S-aPDL1/ICG@NP. (b) Schematic illustration of S-aPDL1/ICG@NP-mediated combined checkpoint blockade immunotherapy and PDT. (c) Activity assay of aPDL1, proteinase K, RNase, and the HRP antibody released from the nanoparticles after different treatments. (d) aPDL1 release profiles of S-aPDL1/ICG@NP in the presence or absence of MMP-2. (e and f) Flow cytometry analysis of (e) intratumoral infiltration of CD8+ T cells and (f) the ratios of CD8+ T cells to Treg in tumor tissues (1, PBS; 2, aPDL1; 3, S-ICG@NP; 4, S-ICG@NP + laser; 5, aPDL1/ICG@NP; 6, aPDL1/ICG@NP + laser; 7, S-aPDL1/ICG@NP; 8, S-aPDL1/ICG@NP + laser). Reproduced from ref. 70 with permission from the American Association for the Advancement of Science, copyright 2019.

Moreover, Nie and co-workers reported an MMP-2-responsive therapeutic peptide assembling nanoparticle to deliver a short D-peptide antagonist of PD-L1 (DPPA-1) and an IDO inhibitor (NLG919) for activatable cancer immunotherapy.71 This nanoparticle was composed of an amphiphilic peptide, an MMP-2-responsive peptide linker and DPPA-1 and further co-assembled with NLG919. DPPA-1 can block PD-L1 for checkpoint blockade therapy and NLG919 could reverse the immunosuppressive tumor microenvironment for enhanced antitumor immune responses. In vivo results further demonstrated that the localized release of DPPA-1 and NLG919 upon the overexpression of MMP-2 in tumor tissues could promote the survival and activation of cytotoxic T lymphocytes, leading to tumor regression and systemic antitumor immunity. Zhang and co-workers designed an MMP-2-responsive peptide-based prodrug platform with structure-transformable properties for codelivery of antitumor agents (cisplatin and adjudin) and an immunomodulatory peptide (formyl peptide receptor 1 (FPR-1)).72 Cisplatin and adjudin could inhibit tumor progression, while FPR-1 played an important role in the activation of chemotherapy-mediated antitumor immunity by promoting DC recruitment and antigen processing. The tumor overexpressed MMP-2 could induce the localized activation of chemotherapeutic drugs and immunological agents to eradicate tumor cells and elicit robust antitumor immunity for enhanced chemotherapy and immunotherapy.

3.4.2. Caspase-activated immunotherapy. As one of the cysteine aspartic-specific proteases, caspase is highly conversed and plays a central role in the regulation of cell death and inflammation.73 Nevertheless, cancer and other diseases generally involve dysregulation of caspases. Hence, many caspase-responsive substrates have been developed to construct functional biomaterials for caspase-related imaging and drug delivery. Some immune drugs have also been introduced in the drug delivery systems for caspase-activated cancer immunotherapy.

Zhang's group developed a caspase-responsive chimeric peptide for tumor photodynamic and immunotherapy (Fig. 9a).74 This chimeric peptide was synthesized by integrating the photosensitizer protoporphyrin IX (PpIX) with an IDO inhibitor 1-methyltryptophan (1MT) by a caspase-3-cleavage peptide linker Asp-Glu-Val-Asp (DEVD). This peptide could self-assemble into PpIX-1MT nanoparticles and passively accumulate at tumor sites. Upon 630 nm light irradiation, the nanoparticles generated ROS to induce the apoptosis of tumor cells and thus facilitated the expression of caspase-3, which further induced the release of 1MT to reverse immunosuppressive tumor microenvironments and activate antitumor immune responses. The in vitro drug release profile demonstrated that this peptide could achieve about 80% release of 1MT in 48 h in the presence of caspase-3 (Fig. 9b). After co-incubation with CT26 tumor cells and lymphocytes isolated from BALB/c mice, this peptide could generate ROS and induce the apoptosis of tumor cells under laser irradiation and further recruit CD8+ T cells via blocking the IDO pathway upon the release of 1MT (Fig. 9c). After systemic administration, the peptide could also eradicate tumor cells with a remarkable increase of caspase-3 (Fig. 9d). Meanwhile, the immune-facilitated indexes including necrosis factor-alpha (TNF-α), interferon-gamma (IFN-γ), interleukin-17 (IL-17), CD-8, CD-86, and granzyme-B exhibited an upward tendency in both tumor and lung tissues owing to the release of 1MT (Fig. 9e). This cascade synergistic antitumor strategy by combining PDT with the caspase-3-responsive activation of an immune drug 1MT provides a practical way for activatable cancer immunotherapy. In addition, they utilized this caspase-3-activated cancer immunotherapy strategy to combine with chemotherapy for enhanced antitumor therapy.75 The iRGD-modified mesoporous silica nanoparticles were encapsulated with doxorubicin (DOX) and decorated with 1MT by the caspase-3-cleavage peptide linker DEVD. Once taken up by tumor cells, DOX could be released to induce the apoptosis of cells and expression of caspase-3. Afterward, the cascade release of 1MT via the cleavage of the DEVD peptide sequence could activate antitumor immunity with enhanced antitumor therapeutic effects.

image file: c9cs00773c-f9.tif
Fig. 9 (a) Schematic illustration of the structure of the chimeric peptide PpIX-1MT and the mechanism of 1MT release and enhanced antitumor immune responses. (b) 1MT release profile of PpIX-1MT nanoparticles in the presence of caspase-3. (c) Flow cytometry analysis of the ratio of CD8+ T cells to CD4+ T cells in CT26 cells cocultured with splenocytes after different treatments. (d) Western blot analysis of the expression of cleaved caspase-3 in tumor tissues in different groups after the 21-day treatment. (e) Western blot analysis of the expression of TNF-α, IFN-γ, IL-17, CD-8, CD-86, and granzyme-B in tumor and lung tissues after different treatments. Reproduced from ref. 74 with permission from the American Chemical Society, copyright 2018.
3.4.3. HAase-activated immunotherapy. The tumor extracellular matrix (ECM) is known to be a hydrated gel-like matrix composed of many ECM-associated proteoglycans and proteins including elastin, collagen, MMPs, hyaluronic acid (HA) and hyaluronidase (HAase).76 HA has been widely used as a biodegradable material for cancer therapy. In addition, HAase is a hydrolytic enzyme that can specifically degrade HA. Several HAase-sensitive nanosystems have been established to deliver immunotherapeutic agents into tumor sites for HAase-activated immunotherapy.

Luan et al. reported a three-in-one immunotherapy nanoplatform to boost antitumor immune responses (Fig. 10a).77 This nanoplatform (designated as aPD-L1@HC/PM NPs) was synthesized by the assembly of Ce6-conjugated HA, 1MT-conjugated polylysine and aPD-L1. The TEM images exhibited the uniform particle size of aPD-L1@HC/PM NPs. After incubation with HAase for 4 h, the particle size was decreased owing to the degradation of HC by HAase and the subsequent release of aPD-L1 (Fig. 10b). When the HAase-incubated aPD-L1@HC/PM NPs were further cultured with papain to simulate the enzyme microenvironment of the tumor cell lysosome, the structure of the nanoparticles was completely collapsed due to the degradation of polylysine via the cleavage of amide bonds. Afterward, the IDO inhibition effects were evaluated by measuring the concentration of kynurenine, which is an immunosuppressive product of IDO metabolism. After incubation with B16F10 cells, the aPD-L1@HC/PM NPs could release 1MT and inhibit the activity of IDO with an obvious decrease of kynurenine (Fig. 10c). After systemic administration, the nanoparticles could be degraded by tumor ECM overexpressed HAase, leading to the release of aPD-L1, subsequent charge reversal for enhanced tumor uptake, and IDO inhibition by 1MT for enhanced immunoactivation. In vivo flow cytometry results also demonstrated the DC maturation and T cell proliferation for enhanced cancer immunotherapy (Fig. 10d). Moreover, Gu's group designed a microneedle-based transcutaneous delivery approach to deliver 1MT and aPD-1 for cancer immunotherapy.78 HA covalently conjugated with 1MT could form an amphiphilic structure and could further self-assemble into the nanoparticles by encapsulating with aPD-1. After being integrated into the microneedle system, the nanoparticles could be transported across the stratum corneum and could release 1MT and aPD-1 in the presence of the tumor overexpressed HAase to activate tumor-infiltrating lymphocytes. These HAase-activatable nanoplatforms were demonstrated to be excellent immunotherapy systems for the treatment of tumors.

image file: c9cs00773c-f10.tif
Fig. 10 (a) Schematic illustration of the step-by-step detached release behavior of aPD-L1, Ce6, and 1-mt and the immunotherapy capability via the cascade-amplifying cancer-immunity cycle. (b) TEM images of (i) the aPD-L1@HC/PM NPs and the (ii) aPD-L1@HC/PM NPs after incubation with HAase for 4 h without papain and (iii) with papain. (c) The effect of IDO inhibition on the level of kynurenine in B16F10 cells. (d) Flow cytometry analysis of DC maturation in mouse bone marrow dendritic cells (BMDCs) and T cell proliferation in mouse peripheral blood mononuclear cells (PBMCs) with photodynamically treated B16F10 cells. Reproduced from ref. 77 with permission from the American Chemical Society, copyright 2019.

4. External stimuli-mediated immunotherapy

4.1. Photoactivated immunotherapy

Light, one of the most frequently used external stimulus sources, has been widely applied in scientific studies and pre-clinical research for the treatment of cancer, for example, fluorescence imaging, photodynamic therapy, photothermal imaging, and photothermal therapy.79 Owing to its excellent controllability and high spatiotemporal resolution, light can also be used as a convenient tool for remotely triggering drug release in an on/off switching manner.80 In addition, a broad range of parameters including wavelength, light intensity, duration of irradiation and beam diameter can be regulated to meet various requirements for light-triggered drug release.81–83 This light-triggered drug activation strategy can also be used to activate immune drugs. The photo-activated immunotherapy can be mainly divided into four classes, including photo-mediated activation, photo-mediated ROS generation for indirect activation, photothermal transformation-mediated activation, and photogenetic control.
4.1.1. Photo-mediated direct activation. Several small molecules and chemical bonds are reported to specifically respond to NIR or high-energy ultraviolet (UV) light, which can be used to construct photo-sensitive systems for drug activation.84 Immunotherapy can be efficiently combined with photo-sensitive systems to realize highly efficient immunoactivation. This photo-activated immunotherapy has the potential for enhancing antitumor efficiency and reducing off-target toxicity and immune-related adverse events.

Li and co-workers reported an activatable engineered immunodevice that could remotely control the antitumor immunity both in vitro and in vivo with near-infrared (NIR) light (Fig. 11a and b).85 This immunodevice was composed of a rationally designed UV light-activatable immunostimulatory agent and upconversion nanoparticles, which could shift the light sensitivity of the device to the NIR window. Upon NIR light irradiation, the immunodevice could transfer NIR to UV light, leading to the cleavage of the photocleavable bonds and the subsequent activation of the immunological adjuvant CpG oligonucleotides (ONDs). The photo-induced activation of CpG ONDs could be examined using Förster resonance energy transfer (FRET). CpG ONDs were labeled with Cy3 (Cy3-CpG), and the complementary ssDNA containing photocleavable (PC) bonds were conjugated to the quencher BHQ-2 (Q-PcDNA). After the Q-PcDNA was hybridized to Cy3-CpG, the fluorescence intensity was decreased due to the energy transfer from Cy3 to BHQ-2 (Fig. 11c). When irradiated with UV light, the fluorescence intensity was gradually increased, indicating the release of Cy3-CpG from the hybrid due to the photolysis of Q-PcDNA (Fig. 11d). The inflammatory cytokine detection of RAW264.7 cells with different treatments further demonstrated that CpG could be efficiently released upon laser irradiation to stimulate RAW264.7 cells for immunoactivation (Fig. 11e–h). In vivo antitumor results also indicated that the locally activated CpG ONDs could elicit effective immune responses at tumor sites without disturbing immunity elsewhere in the body.

image file: c9cs00773c-f11.tif
Fig. 11 (a) Schematic illustration of the design of the photoactivatable immunodevice through the integration of UCNPs with the UV light-responsive PCpG. (b) Schematic illustration of the photoactivatable immunodevice for spatially selective triggering of immunoactivity through NIR light irradiation. (c) Fluorescence spectra of Cy3-CpG before and after the formation of PCpG. (d) Fluorescence spectra of FRET pair-labeled PCpG with increased dose of 365 nm light irradiation (5 mW cm−2). (e–h) Cytokine secretion by RAW264.7 cells upon indicated treatments. The concentration of cytokines in the culture medium was measured (e and f) immediately and (g and h) subsequently at 24 h after the initial 8 h treatment. Reproduced from ref. 85 with permission from Springer Nature, copyright 2019.
4.1.2. Photo-mediated indirect activation. PDT is widely reported to kill cancer cells by the generation of ROS under light irradiation.86 ROS-sensitive immune drug delivery systems can also be efficiently integrated with PDT for activatable cancer immunotherapy.87 The photo-mediated cleavage of ROS-sensitive bonds for drug activation can be more controllable and precise than that of the oxidative tumor microenvironment.88 Thus, the photo-mediated ROS generation can be applied for activatable cancer immunotherapy.

Our group developed an organic semiconducting pro-nanostimulant (OSPS) with NIR photoactivatable immunotherapeutic action for synergetic cancer therapy (Fig. 12a).89 Organic semiconducting materials are an emerging class of organic optical agents, which have been proved to be effective in the area of biophotonics.90 Our group has designed a series of organic semiconducting materials for optical imaging,91 cancer phototherapy,92 and biological photoactivation.93 The OSPS was composed of a semiconducting polymer nanoparticle (SPN) core, an IDO inhibitor (NLG919), and a singlet oxygen (1O2) cleavable linker. Under NIR light irradiation, The OSPS could generate 1O2 for both killing tumor cells through PDT and inducing NLG919 release via the cleavage of the 1O2-sensitive linker (Fig. 12b). In vitro HPLC analysis demonstrated the release of NLG919 upon NIR irradiation (Fig. 12c). After incubation with 4T1 cells, the OSPS could greatly decrease the concentration of kynurenine and reverse the immunosuppressive tumor microenvironment (Fig. 12d). In vivo results also showed that the OSPS with laser irradiation group could decrease the concentration of the immunosuppressive kynurenine and activate cytotoxic T cells (CD3+CD8+ T cells and IFN-γ-producing T cells) for enhanced antitumor immunity (Fig. 12e–g). The combination of phototherapy with photoactivatable remote-controlled immunotherapy could show an amplified therapeutic efficacy for both primary/distant tumors and lung metastasis.

image file: c9cs00773c-f12.tif
Fig. 12 (a) Schematic illustration of the photoactivation of OSPS for synergistic phototherapy and checkpoint blockade immunotherapy. (b) Structure and NIR photoactivation mechanism of OSPS. (c) HPLC profiles of NLG919 (30 μg mL−1) and OSPS ([PCB] = 40 μg mL−1) with or without 808 nm NIR laser irradiation (0.3 W cm−2) for 15 min. (d) Relative Kyn content in the cell culture medium after 24 h treatment with OSPS or CSPN ([PCB] = 20 μg mL−1) with or without 808 nm laser irradiation (0.3 W cm−2) for 6 min. (e) The Kyn/Trp ratio in the primary tumors of living mice after different treatments. (f and g) Flow cytometry analysis of the population of (f) CD3+ CD8+ T cells and (g) IFN-γ producing T-cells in distant tumors. Reproduced from ref. 89 with permission from John Wiley & Sons Ltd, copyright 2019.
4.1.3. Photothermal-mediated activation. Photothermal therapy (PTT) is a recently developed tumor therapeutic strategy that can transform light into heat for eradicating tumor cells.94 It is reported that the photo-mediated generation of heat can also enhance drug accumulation and promote drug release at tumor sites by improving blood circulation and accelerating molecule diffusion.95 Thus, the photothermal transformation strategy can be used for both PTT and activatable cancer immunotherapy.

Ran and co-workers designed a photothermally triggered immunotherapeutic nanosystem for synergistic PTT and immunotherapy (Fig. 13a).96 This nanosystem (MINPs) was fabricated by loading superparamagnetic iron oxide (SPIO) nanoparticles and CpG ODNs in the amphiphilic polymer nanoparticles. Under NIR laser irradiation, SPIO could generate heat with high photothermal conversion efficiency for photothermal destruction of the primary tumors, releasing immunological adjuvants CpG ODNs and activating antitumor immune responses. After co-culturing the MINP-treated 4T1 cells with the DCs, DC maturation was observed with remarkable upregulation of typical co-stimulatory molecules (CD11c+, CD80+, and CD86+) as shown in Fig. 13b. Upon DC maturation, different cytokines TNF-α and IL-6 were increased to regulate and activate specific immune cells (Fig. 13c and d). In vivo results also demonstrated the enhanced expression of CD8+ T cells and upregulation of IFN-γ in tumor tissues (Fig. 13e). This combinational PTT and photothermal-activated immunotherapy would be potentially applied for precise and personalized cancer immunotherapy.

image file: c9cs00773c-f13.tif
Fig. 13 (a) Schematic illustration of imaging-guided photothermally triggered immunotherapy based on magnetic-responsive immunostimulatory nanoagents (MINPs) for both primary treated and distant untreated tumors. (b) Flow cytometry analysis of the mature DCs (CD11c+, CD86+, and CD80+) after co-incubation of different treated 4T1 cells with DCs. (c and d) The secretion levels of (c) TNF-α and (d) IL-6 in DC suspensions after different treatments. (e) Immunofluorescence images of effector CD8+ T cells and IFN-γ in the distant tumors on day 7 after different treatments of the primary tumors. Reproduced from ref. 96 with permission from Elsevier, copyright 2019.
4.1.4. Optogenetic control. With the rapid development of optics and genetic engineering, optogenetic control has attracted increasing attention based on the spatiotemporally precise control of molecular processes, cellular signals, and animal behavior by the genetically encoded light-dependent receptors.97 Besides, optogenetic control is used for the treatment of cancers by regulating the expression of corresponding therapeutic proteins or immunostimulatory cytokines.

Kim et al. developed a strategy for optically controlling T-cell trafficking using a photoactivatable (PA) chemokine receptor (Fig. 14a).98 This photoactivatable-chemokine C-X-C motif receptor 4 (PA-CXCR4) was efficiently transfected into gene engineered T cells for adoptive T-cell transfer immunotherapy. The rhodopsin-chemokine receptor chimera was expressed in T cells for the photoactivated release of CXCR4. After transfecting PA-CXCR4-mCherry into T cells, the fluorescence of mCherry and anti-rhodopsin demonstrated the successful expression of the construct in the plasma membrane (Fig. 14b). Generally, the concentration of the intracellular Ca2+ increased transiently after chemokine stimulation. Therefore, the increased fluorescence of Ca2+ in the PA-CXCR4-expressing cells indicated the release of CXCR4 after 488 nm light irradiation (Fig. 14c and d). Upon light irradiation, PA-CXCR4 could transmit intracellular CXCR4 signals, resulting in T-cell polarization and directional migration to tumor sites. However, WT CXCR4-expressing cells could only respond to CXCL12, which was specifically bound to CXCR4 for driving Ca2+ signals. This strategy demonstrated that remotely controlled T cell trafficking and activation with outstanding spatial resolution would be feasible and potential for activatable cancer immunotherapy.

image file: c9cs00773c-f14.tif
Fig. 14 (a) Schematic illustration of the design of photoactivatable CXCR4 (PA-CXCR4). (b) Fluorescence images of the expression of PA-CXCR4-mCherry in human primary T cells. (c) Ca2+ fluorescence images and (d) the intensity traces of mouse T cells after different treatments. Reproduced from ref. 98 with permission from National Academy of Sciences, copyright 2014.

4.2. Magnet-activated immunotherapy

Apart from phototherapy, the magnetic field based on its excellent controllability and the magnetic driving force has been widely utilized for magnetic resonance imaging (MRI), magnetic targeting, and magnet-responsive drug delivery.99 Besides, the magnetic field has been reported to manipulate the activation of antitumor immunity.

Schneck et al. developed a reductionist T cell activation platform via magnetic field manipulation (Fig. 15a and b).100 This platform is based on paramagnetic nanoparticles decorated with different types of signaling molecules. Each of these signaling molecules has their own distinct functions to stimulate the corresponding signaling pathway. However, T cell activation generally requires the coordination of a variety of signaling molecules including T cell receptor-specific signals and costimulatory signals. Thus, these single-signal nanoparticles could recognize their relevant receptors and drive the aggregation and clustering of these surface-bonded antigens or costimulus by a magnetic field, leading to enhanced T cell activation for immunotherapy (Fig. 15c).

image file: c9cs00773c-f15.tif
Fig. 15 (a) Schematic illustration of T cell activation by nanoparticles separately expressing signal 1 and signal 2 when particles are clustered within a magnetic field. (b) Schematic comparing standard artificial antigen-presenting cells (aAPC) co-expressing stimulatory signals on the same nanoparticle, and a separate signal 1 + signal 2 particle platform with each stimulatory signal conjugated to distinct nanoparticles. (c) TCR transgenic PMEL CD8+ T cells were stimulated with signal 1 only Db-gp100 dimer particles in the presence (black) or absence (gray) of signal 2 only anti-CD28 particles. CFSE dilution was measured after 3 days. (d) Schematic illustration of ultrasound-induced cell activation and gene expression. (e) Diagram of an integrated system of ultrasound stimulation and FRET imaging. (f) The time courses of the normalized FRET/ECFP (enhanced cyan fluorescent protein) ratio (mean ± SEM) of a calcium biosensor in HEK293T cells before and after 5 s of ultrasound stimulation with different treatments. (g) Representative FRET/ECFP ratio images of the calcium biosensor in HEK293T cells with different treatments. (a)–(c) were reproduced from ref. 100 with permission from the American Chemical Society, copyright 2018. (d)–(g) were reproduced from ref. 102 with permission from National Academy of Sciences, copyright 2018.

4.3. Ultrasound-activated immunotherapy

As one of the mechanical forces, ultrasound has long been utilized in clinical diagnosis and ultrasound-assisting cancer therapy.101 Due to its simplicity, security, inexpensiveness, and spatiotemporal controllability, ultrasound has also been increasingly studied to regulate drug activation for cancer chemotherapy and even immunotherapy.

Wang et al. utilized mechanogenetics to develop gene engineered CAR T cells for the remote and non-invasive control of cancer immunotherapy (Fig. 15d and e).102 These specific T cells were engineered with an ultrasound activated Piezo1 ion channel to regulate the CAR expression by ultrasound stimulation. Upon ultrasound stimulation, Piezo1 could be activated to induce the consequent calcium influx, which could further activate a calcium-sensitive phosphatase calcineurin to dephosphorylate a transcription factor, the nuclear factor of activated T-cells (NFAT). This NFAT would translocate to the nucleus for the activation of an NFAT response element to drive the expression of designed target genes. HEK293T cells were co-transfected with Piezo1-tdTomato and the D3cpv FRET calcium biosensor for the examination of calcium influx upon ultrasound stimulation. Then, RGD-modified microbubbles could connect to Piezo1 to enhance calcium influx under low-frequency ultrasound treatment. The fluorescence of Ca2+ indicated that the ultrasound stimulation could effectively induce calcium influx and further activate the expression of designed target genes (Fig. 15f and g). This ultrasound-activated CAR expression could efficiently activate T cells and elicit enhanced antitumor immune responses for cancer immunotherapy.

5. Summary and outlook

Cancer immunotherapy is an emerging therapeutic modality that enables the immune system to fight against cancer. However, traditional cancer immunotherapies still suffer from poor patient response rates and potential immune-related adverse events. Both issues can be mitigated by developing activatable cancer immunotherapeutics as exemplified in Table 1. Integration of stimuli-responsive nanomedicines into immunotherapy has led to these activatable immunotherapeutic nanoagents, which could simultaneously possess the merits of optimized biodistribution, specific cell targeting, and controllable immune activation. With the rapid development and convergence of nanotechnology, biological science, and biomedical engineering, various internal and external stimuli-responsive biomaterials have been exploited for the construction of activatable cancer immunotherapeutics.
Table 1 Summary of nanoagents for activatable cancer immunotherapy
Immunotherapeutic strategies Mechanism of action Targets Immunotherapeutic agents Stimuli sources Sensitive molecules/cargos Delivery platforms Ref.
TLR agonist Activating APCs and inducing proinflammatory immune response TLR9 CpG ONDs pH Hydrazone (Hyd) bond Polymer 32
Metal ions–phosphate coordination bond MOF 33
GSH Disulfide bond MSN 57
Au–thiol bond Au nanorod 56
Hypoxia Azobenzene linker MSN 63
NIR light Photocleavable bonds UCNP 85
Photothermal conversion SPIO 96
TLR7 Imiquimod GSH Disulfide bond Dendrimer 55
Hypoxia 4-Nitrobenzyl bond Polymer 64
Antigen presentation Enhancing the presentation of tumor antigens to APCs APCs HSP70-chaperoned polypeptides pH Hyd bond Polymer 32
Ovalbumin (OVA) pH Metal ions–phosphate coordination bond MOF 33
Carboxylic acid Polymer 34
Neoantigen peptides GSH Au–thiol bond Au nanorod 56
Disulfide bond MSN 57
Cytokine stimulation Regulating the survival, proliferation, and differentiation of activated T and NK cells T and NK cells IL-2 pH Tertiary amines Nanogel 37
IL-15Sa GSH Disulfide bond Nanogel 58
CXCR4 chimera Light Photoactivatable rhodopsin T cells 98
FPR-1 agonist Building stable interactions between DCs and tumor cells FPR-1 WKYMVm MMP-2 PLGVRG peptide Nanoparticle 72
TCR signaling Enhancing T cell activation TCR Specific peptide-MHC Magnet Paramagnetic iron Paramagnetic iron-dextran nanoparticles 100
CD28 anti-CD28 antibody
CAR T cell therapy Targeting tumor antigen and eradicating tumor cells CD19 CAR protein Ultrasound Ultrasound-activatable Piezo1 ion channel CAR-expressing T cells 102
CD47/SIRPα blockade Abolishing the “don’t eat me” signaling and improving the phagocytosis of macrophages CD47 aCD47 pH Benzoic-imine bond M1 macrophage exosomes 43
CaCO3 nanoparticle Hydrogel 45
ROS Thioketal Protein complex 60
SIRPα aSIRPα pH Benzoic–imine bond M1 macrophage exosomes 43
PD-1/PD-L1 blockade Reversing the immunosuppressive tumor microenvironment and activating T cells PD-1 aPD-1 pH Acetal Dextran nanoparticle 44
ROS Thioketal Protein complex 60
HAase Hyaluronic acid Microneedle 78
PD-L1 aPD-L1 ROS Phenylboronic acid pinacol group Hydrogel 59
Sulfoether group Polymer 61
MMP-2 PLGLAG peptide Nanoparticle 70
HAase Hyaluronic acid Nanoparticle 77
aPD-L1 siRNA pH Tertiary amines Polymer 48
D-peptide antagonist of PD-L1 MMP-2 PLGLAG peptide Nanoparticle 71
IDO inhibition Inhibiting IDO activity and reversing of immune suppression IDO 1MT pH Tertiary amines Polymer 35
ROS Sulfoether group Polymer 61
Caspase-3 DEVD peptide Peptide 74
MSN 75
HAase Hyaluronic acid Nanoparticle 77
Microneedle 78
IDO1 siRNA pH Tertiary amines Polymer 47
NLG919 GSH Disulfide bond Nanoparticle 53 and 54
MMP-2 PLGLAG peptide Nanoparticle 70
NIR light Thioketal Nanoparticle 89

Internal stimuli associated with cancer biomarkers including acidic pH, redox potential, hypoxia, and various tumor-overexpressed enzymes (such as MMPs, caspases, HAase, etc.) can be utilized for localized immune stimulation. Due to the difference in these biomarkers between tumor and normal tissues, activatable immunotherapeutic nanoagents that responded to these internal stimuli could show preferable activation of immunotherapeutic action at the tumor site. However, these nanoagents still face a certain level of off-target side effects due to the ubiquity of these biomarkers in normal tissues. In contrast, activatable cancer immunotherapeutics in response to external stimuli (such as light, magnet field, ultrasound, etc.) do not share this issue, and their spatiotemporal resolution and precision of immunotherapeutic action are mainly reliant on the interaction between the nanoagents and external stimuli. However, external-stimuli activatable nanoagents generally require more sophisticated chemistry to synthesize and additional equipment to operate as compared with internal stimuli nanoagents.

Although inspiring progress and results have been achieved in this emerging field, activatable cancer immunotherapeutic nanoagents remain in the proof-of-concept stage and face challenges on the way towards their translation into the clinic. For instance, the general issue in the field of nanomedicine, that is, how to improve their targeting efficacies to intended cells (cancer or immune cells), is also the obstacle for activatable cancer immunotherapeutics. This is because a major fraction of systemically administrated immunotherapeutics can be taken up by nonspecific cells during blood circulation, ended up in and metabolized by the liver, kidneys, and other organs. Although a small portion of immunotherapeutic nanoagents can be accumulated at the tumor site, the exact amount that enters the target cells can be even less. Thus, innovative approaches are desired to overcome this issue so as to decrease the administrated doses while enhancing the drug bioavailability. In this regard, in-situ self-assembly of small molecules into nanoparticles in tumors may be promising as it has the advantages of both small molecules (high tumor permeability and precise cell targeting) and nanoparticles (enhanced retention in tumor cells).103,104

Another challenge is to personalize and adjust the immunotherapeutic intervention at different stages, as the immune microenvironment of tumors is dynamic and changes during the therapy. In addition to using molecular imaging to closely monitor the immune response and therapeutic efficacy,105 one alternative solution is to integrate diagnostic, theragnostic, and even prognostic functions into systems to form activatable cancer immunotheranostics. The ultimate goal is to allow such immunotheranostic systems to act as intelligent nanorobots to activate the appropriate therapeutic agents at the correct dosage at the right time after self-analysis of the tumor microenvironment according to the detection results.

Conflicts of interest

There are no conflicts to declare.


K. P. thanks Nanyang Technological University (Start-up Grant No. NTU-SUG: M4081627.120) and Singapore Ministry of Education, Academic Research Fund Tier 1 (2017-T1-002-134; 2019-T1-002-045) and Academic Research Fund Tier 2 (MOE2018-T2-2-042) for the financial support.

Notes and references

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