Nanoparticles for tumor immunotherapy

European Journal of Pharmaceutics and Biopharmaceutics 115 (2017) 243–256

Contents lists available at ScienceDirect

European Journal of Pharmaceutics and Biopharmaceutics journal homepage: www.elsevier.com/locate/ejpb

Review article

Nanoparticles for tumor immunotherapy Xinlong Zang, Xiuli Zhao, Haiyang Hu, Mingxi Qiao, Yihui Deng ⇑, Dawei Chen ⇑ Department of Pharmaceutics, School of Pharmacy, Shenyang Pharmaceutical University, No. 103, Wenhua Road, Shenyang 110016, China

a r t i c l e

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Article history: Received 13 November 2016 Revised 1 March 2017 Accepted in revised form 17 March 2017 Available online 18 March 2017 Keywords: Tumor immunotherapy Antitumor immune response Immunosuppression Nanoparticles Nanovaccines Artificial antigen presenting cells Targeting to immunosuppressive cells

a b s t r a c t Although most researches and therapies have been focused on the tumor itself, it is becoming clear that immune cells can not only suppress tumor development but support and maintain their malignant type. Promising recent developments in immunology will provide opportunities for tumor-specific immunotherapy, which can orchestrate the patients immune system to target, fight and eradicate cancer cells without destroying healthy cells. However, antitumor immunity driven by self-immune system alone may be therapeutically insufficient. Developments in nanoparticle based drug delivery system can promote immunotherapy and re-educate immunosuppressive tumor microenvironment (TME), which provide promising strategies for cancer therapy. In this review, we will focus on nanoparticlebased immunotherapeutic approaches against cancer, ranging from nanovaccines, artificial antigen presenting cells (aAPCs) to nanoparticles reversing tumor immunosuppressive microenvironment. Ó 2017 Published by Elsevier B.V.

Contents 1.

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Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.1. Innate and adaptive immune cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.2. Tumor immunoediting model. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.3. Nanoparticulate drug delivery system . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Tumor and immune system. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.1. Tumor surveillance and elimination . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.2. Tumor promotion and immune suppression . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.2.1. Tumor associated macrophages . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.2.2. Myeloid derived suppressive cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.2.3. Regulatory T cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Current immunotherapies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.1. DCs vaccinations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.2. T cell therapies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Nanoparticle for immunotherapy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.1. Targeting delivery TAAs to APCs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.1.1. Liposomes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.1.2. Poly(propylene) sulfide (PPS) nanoparticles . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.1.3. Gold nanoparticles . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.1.4. PLGA nanoparticles. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.1.5. Artificial exosomes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.1.6. Micelles . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.1.7. Dendrimers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.2. Artificial antigen presenting cells (aAPCs) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.2.1. Liposomes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.2.2. PLGA nanoparticles. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

⇑ Corresponding authors. E-mail addresses: [email protected] (Y. Deng), [email protected] (D. Chen). http://dx.doi.org/10.1016/j.ejpb.2017.03.013 0939-6411/Ó 2017 Published by Elsevier B.V.

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4.2.3. Carbon nanotubes. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.2.4. Magnetic nanoparticles . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.2.5. Nanoworms. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.2.6. Janus particles. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.2.7. Artificial exosomes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.3. Targeting to immunosuppressive microenvironment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.3.1. Targeting to TAMs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.3.2. Targeting to MDSCs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.3.3. Targeting to Tregs. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Conclusion and further perspectives . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

1. Introduction 1.1. Innate and adaptive immune cells The innate and adaptive immune cells are the central components of the defense system of fighting against pathogens infections in mammalians, which are respectively responsible for immediate and long-term protection. The innate immune cells, such as macrophages, dendritic cells (DCs) and natural killer cells (NK), contribute to the first-line protection by recognizing conserved pathogen-associated molecular patters (PAMPs) via pattern-recognition receptors (PRRs), including membrane-bound toll-like receptors (TLRs), C-type receptor (e.g. mannose receptors and asialoglycoprotein receptors), cytoplasmic NOD-like receptors, RIG-I-like receptors and DNA sensors [1]. Pathogens engulfed by antigen presenting cells (APCs), such as macrophages and DCs, are degraded into peptide fragments that are then present on major histocompatibility complexes (MHC) followed by T cells recognize using T cell receptors (TCRs) [2]. Besides, NK cells can destroy compromised host cells rather than invaded pathogens for low expression of MHC I on infectious cells. In general, adaptive immunity proceed innate immune response and possess activated pathogen-specific T and B lymphocytes. Unlike invariant PRRs in innate immune cells, the pathogenspecific T and B cells are enabled by randomized rearrangement of extensive arrays of gene cassettes encoding the pathogen receptor molecular complexes, which allow generating a vast number repertoire of antigen receptors [3]. T cells can be defined into various subsets dependent on functions and surface markers. One subset is cytotoxic T lymphocyte (CTL, also called CD8+ T cells), which can induce the death of invaded or damaged and dysfunctional cells. To activate CTLs, it’s essential to stimulate T cells with strong MHC signals or additional signals produced by ‘‘helper” T (TH) cells. TH cells (CD4+ T cells) play crucial roles in adaptive immune regulation whereas they do not directly involve in pathogen phagocytosis or destruction. There are other important adaptive immune cells, B cells, which are the major cells releasing antibodies involved in pathogen inactivation.

1.2. Tumor immunoediting model Growing evidences have supported that the immune system play crucial roles in initiation, progression, invasion and metastasis throughout tumor development [4–6]. It has also been unveiled a critical role for immunological parameters in predicting tumor prognosis and clinical response to anticancer therapeutics. Immune system can either inhibit or promote tumor growth, which has become a hallmark of tumor. A tumor immunoediting model has been developed to demonstrate the paradoxical roles of the immune system in tumor development, whereby the immune system could alter or ‘edit’ the process of tumor growth

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[7]. The diverse functions of immune cells and their roles that confer protumor or antitumor activities will be discussed in next section. 1.3. Nanoparticulate drug delivery system In the past decades, a variety of nanoparticles (NPs) based drug delivery system has been developed and some of these have been employed in clinical diagnosis and therapy. These NPs include liposomes, dendrimers, micelles, polymer nanoparticles, nanotube and inorganic nanoparticles, which can deliver therapeutic and/or contrast agents to targeting sites [8]. NPs allow cancer specific drug delivery by inherent passive targeting and adopted active targeting strategies in addition to improving pharmaceutics of the loaded drugs [9]. Drug loaded NPs have the capacity of improving its safety and prolonging its circulation time and bioavailability, resulting in enhanced therapeutic outcomes [10]. Therefore, NPs have gained a growing interest in the field of biology and medicine. Cancer immunotherapy has become an intensely researched filed given the increased understanding of tumorigenesis as having an important immunological component. Current strategies for immunomodulation have addressed the efficacy of immunotherapy, however, there are still many fundamental challenges. Nonspecific modulation may have an unfavorable system cytotoxicity whereas specific methods may generate subtherapeutic responses through work against some targets [11]. The apparent dichotomy provides opportunities for nanoparticulate platforms with unique properties to overcome limitations of current treatments and boost efficacious immune response against cancer [12]. 2. Tumor and immune system 2.1. Tumor surveillance and elimination Tumor acquires mutations in cancer suppressor or oncogenes that lead to morphological transformation, inhibition of apoptosis, uncontrolled proliferation, angiogenesis, invasion and metastasis [13,14]. Rapid tumor growth is usually associated with numerous tumor cells death and damage due to hypoxia or mutations of essential gene for survivorship. Necrotic cancer cells release a large number of intracellular components, such as calreticulin, filamentous actin, high mobility group box 1 protein and nucleotide [15], which will activate APCs as tumor associated antigens (TAAs). Furthermore, the addition of adjuvant has generated a favorable immune environment with viable and motile cells available to initiate a successful APCs activation rather ineffective activation with antigen alone [16]. After ‘‘clone selection” process, CD8+ T cells proliferate and differentiate into effector T cells that will search for tumor cells bearing MHC I through the body and then bind directly and destroy them by releasing perforin and granzyme. Activated CD4+ T cells can release pro-inflammatory cytokines,

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Fig. 1. Immunomodulatory factors in tumor microenvironment. Antigens derived from tumor cells stimulate APCs, which then activate CD4+ , CD8+ T cells to induce immune responses against cancer. Besides, NK cells will destroy tumor cells that are associated with low expression of MHC I complexes for immune escape. However, tumor promotion by various mechanisms that operates in parallel with anti-tumor immunity. anti-tumor immunity can be suppressed by various immune cells, such as TAMs, MDSCs and Tregs, which can produce versatile cytokines and chemokines not only inhibit activity of T cells and NK but recruit immunosuppressive cells infiltrating into tumor. In addition to VEGF, MMP promote angiogenesis whereas TGF-b enhances tumor cells invasion and metastasis via epithelial-to-mesenchymal transition (EMT). In conclusion, immune system supports tumor growth by various mechanisms, including immune suppression and enhanced vascularization, invasion and metastasis.

including IFN-c, IL-12 and TNF-a, that can suppress tumor cells and drive their MHC I expression facilitating specific recognition by CD8+ T cells. NK cells can mediate cellular cytotoxicity in an antigen-dependent manner when the antibody binding to Fc receptors more than lysis tumor cells in absence of antigen presentation [17]. Despite the potential antitumor activity, increased NK cells infiltration into peritoneum and pleura in metastatic ovarian carcinoma is associated with poor prognosis. Natural killer T (NKT) cells that express semi-invariant TCRs recognizing lipid antigens can mediate immune responses against tumor cells through proinflammatory cytokines. They also have NK-like cytotoxicity to tumor cells via perforin, Fas-FasL and IFN-c. In addition, cdT cells typically provide large amounts of proinflammatory cytokines to inhibit tumor cells growth in situ, however, they preferentially produce IL-17 rather than IFN-c, which mobilizes unconventional small peritoneal macrophages that directly and uniquely promote ovarian cancer proliferation [18].

2.2. Tumor promotion and immune suppression In contrast to immune surveillance and elimination, there are some types of immune cells that play major roles in ‘‘selftolerance”, promote tumor cells growth and protect them from discovery and elimination (Fig. 1). The immunosuppressive cells are

mainly tumor associated macrophages (TAMs), myeloid derived suppressor cells (MDSCs) and regulatory T cells (Tregs).

2.2.1. Tumor associated macrophages Contrast with conventional concepts that TAMs might display cytotoxicity to tumor cells, more and more recent studies have proven their protumor functions [19]. TAMs that are mainly from M2-type significantly foster tumor progression, which drives from regulation of angiogenesis, invasion and metastasis of malignant cells as well as direct and/or indirect immunosuppressive activity [20]. The first way through which TAMs support tumor is the induction of vascularization that play a major role in oxygenation and nutrition supply as well as waste disposal when tumor above a certain size. Important for this activity is M2-derived vascular endothelial growth factor (VEGF) that has a dominant effect in angiogenesis. Matrix metalloproteinase (MMP) derived from TAMs can also regulate VEGF bioavailability, thus providing an alternative VEGF-dependent pathway for angiogenesis [20]. CCL18+ TAMs infiltration was positively associated with MVD (a well-known marker of tumor vascularization) in breast cancer samples and blocking CCL18 or VEGF with neutralizing antibodies significantly inhibited endothelial migration and angiogenesis [21]. Tie2+ macrophages, a subtype expressing Tie2 that binds to ANG2 on vessels, are important actors in angiogenesis. Targeting Tie2 or

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ANG restrained tumor growth by impacting the later stages of remodeling and vascular maturation whereas VEGF primarily acts on immature vessels [22]. TAMs can improve tumor cells invasion and metastasis into ectopic tissue through the paracrine loop consisted of tumor released colony-stimulating factor 1 (CSF-1) and macrophagederived epidermal growth factor (EGF) as well as family ligands [23]. Besides promoting angiogenesis, VEGF has been demonstrated to increase intratumoral lymphangiogenesis, resulting in significantly enhanced metastasis to regional lymph nodes and to lungs [24]. TAMs also regulate the composition and structure of extracellular matrix (ECM) through collagens deposition and protease release [20]. Epithelial-myofibroblast transdifferentiation (EMT) is a well-described pathogenetic feature in tumor metastasis since it acquires highly aggressive capability supporting cancer cells spreading [25]. TAM derived TGF-b can promote EMT and TGF-b1 neutralizing antibodies blocked the effect of TAM-induced EMT on promotion of CSC-like properties in Hepa1-6 cells [26]. TAMs also develop immune suppressive microenvironment that facilitates tumor cells escape from immune surveillance by inhibiting antitumor immune response. To achieve this goal, TAMs pathologically exaggerate the normal regulatory circuits that control self-tolerance, homeostasis of myeloid cells, wound-healing and response to dying cells while not novel mechanisms generation [27]. They can make significant contribution to protumor immunosuppression by expressing immune suppression factors, such as IL10, PGE2, TGF-b and arginase 1 (Arg1) [28]. TGF-b, IL-10 and PGE2 can suppress the expression of MHC Ⅱ molecules on TAMs to inhibit antigen presentation. IL-10 can inhibit antigen presentation through interference in TLR or IFN-c-mediated APCs activation and induction expression of genes abolishing APCs functions [29]. Besides, IL-10 suppresses IFN-c production that play a major role in naïve T cell differentiation [30]. IL-10 also enhances IL-4 mediated expression of Arg1 that can transform L-arginine into Lornithine (a precursor of proline and polyamines), which causes dysfunction of TCR signal and subsequent CD8+ T cells response [31]. Proline is a key component of collagen and polyamines involve tumor cell proliferation, differentiation and division, and resistance to chemotherapeutic drugs [32,33]. Arg1 can also reduce L-arginine available to inducible nitric oxide synthase (iNOS) to decrease nitric oxide (NO) production, which influences proapoptotic effects on NO-sensitive cancer cells and T cell activation [34]. TAMs also secrete an array of chemokines that can suppress CD4+ and CD8+ T cells, recruit Tregs infiltration and induce CD4+ T differentiation to Tregs and sustain their survival [13]. In addition, the expression of B7 ligands specific for cytotoxic T lymphocyte-associated antigen 4 (CTLA-4) on TAMs suppress cytotoxic functions of CD8+ T cells and induce DCs inhibitory phenotype. Comparison of T cells activities between pre- and posttreatment using CTLA-4 blockade demonstrated a significant increase in the frequency of T cells specific for melanoma as a component of the clinical mode of action [35]. TAMs also express programmed cell death-ligand 1 (PD-L1) inducing T cell dysfunction, as defined by the presence of low cytotoxicity to tumor cells and a reduction in T cell proliferation [36]. 2.2.2. Myeloid derived suppressive cells MDSCs, a heterogeneous population of immature cells, are important obstacles for natural antitumor immunity and immunotherapy. The appearance of MDSCs has been documented in many different cancers such as breast cancer, head and neck squamous cell carcinoma, non-small lung cancer, colon an colorectal cancer, renal cell carcinoma, bladder cancer, gastrointestinal cancer, prostate cancer and hepatocellular carcinoma [37]. MDSCs directly inhibit T cells in a cell-cell manner, which includes amino acid starvation, apoptosis induction and intracellular signaling

pathway abrogation. Peroxynitrite, a reactive nitrogen species derived from MDSCs, retards recognition of MHC I molecules by CD8+ T cells as it has been shown to covalently bind to proteins expressed in TCR-CD80 complexes [38]. MDSCs also inhibit functional antigen presentation of DCs and drive macrophage differentiation to M2 type. MDSCs that are inversely correlated with NK cells functions in liver and spleen, have been demonstrated to suppress the latter killing functions by membrane-bound TGF-b1 [39]. Not surprisingly, MDSCs also impact other immune cells involving immunosuppression. Macrophage production of IL-12 decreasing depends on MDSCs production of IL-10, and Tregs immunosuppression is in coordination with CD80 over-expressing MDSCs [39]. 2.2.3. Regulatory T cells Antitumor immunity was also suppressed by CD4+ CD25+ Foxp3+ T cells (Tregs), which have been identified as important negative regulatory cells to suppress adaptive immune responses and limit excessive immunity such as autoimmune diseases [40]. The increased density of Tregs has been associated with multiple cancers including liver, lung, ovary, melanoma and head and neck cancer, which usually predicts poor prognosis and recurrence [41–45]. Increased Tregs infiltration could be attributed to recruitment by cytokines and chemokines in tumor microenvironment as well as upregulation of anti-apoptotic and downregulation of proapoptotic genes [46]. Knockdown of TGF-b using RNA interference reduced Tregs infiltration, revealing depressed immunosuppression in tumor environment and metastatic nodules in mouse xenograft hepatocellular carcinoma tumor [47]. Tregs have high expression of CLTA-4 that can interact with CD80/86 on APCs to induce the production of IDO, which may play an important role in immune suppression [39]. Blocking CTLA4-B7 interactions reduced times of Tregs-T cells interactions followed by enhanced T cells proliferation in vivo [48]. Although the potential protumor mechanisms of Tregs remain a challenge, it is well-established that infiltration of Tregs correlates with poor prognosis. 3. Current immunotherapies Cancer immunotherapy is a rapidly moving field and have achieved endurable antitumor responses clinically. This section will discuss recent immunotherapies that endogenous APCs or T cells specific for antigen after ex vivo activation are directly readministrated to the patients. 3.1. DCs vaccinations Immunotherapy based on tumor antigen-loaded DCs represents a promising strategy in the multimodal treatment for different types of cancer [49]. In this method, tumor antigen-loaded DCs are re-injected into cancer patients to stimulate T cells and initiate tumor eradication, which have achieved effective immune responses and favorable clinical outcomes. In a clinical trial, 14 stage IV melanoma patients without previous systemic treatment received autologous CD1+ myeloid DCs activated with tyrosinase and gp100 ex vivo [50]. The results demonstrated de novo immune and objective responses against cancer even at numbers of 3– 10  106 DCs per vaccine, resulting in prolonged progression-free survival. Interestingly, the most common side effects associated with DCs vaccination are flu-like symptoms. The potential impact of prior therapies on DCs phenotype and function is key to the success or failure of DCs vaccination. Nair et al. found that fewer DCs after induction chemotherapy and G-CSF mobilization met the phenotypic and functional requirements for DC-based cancer immunotherapy, suggesting limited application of DCs vaccination [51].

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X. Zang et al. / European Journal of Pharmaceutics and Biopharmaceutics 115 (2017) 243–256 Table 1 Nanoparticulate drug delivery system for immunotherapy. Strategy

Carriers

Advantages

Payload

Tumor model

Reference

Targeting to DCs

Liposomes

Easy modification, production and pH sensitive Passive targeting Lymph node targeting Cross-presentation Induction of both CD4+ and CD8+ T cell response Tunable surface chemistry, easy controlled sizes and shapes, adjuvants Controlled release of antigen, endosomal/lysosomal escape

OVA Cyclic-di-GMP CpG ODN – OVA peptides OVA

E.G7-OVA

[2,59] [61] [63] [64] [65] [66]

OVA and CpG ODN conjugation WNVE protein OVA, Pam3Csk4 and/or Poly (I:C) SPIO particles or gold nanoparticles and OVA Tumor lysates

Protection from viral infection – OVA-expressing melanoma

[67]

–

[70]

Head and neck squamous cancer Protection from lewis lung carcinoma cells OVA-expressing melanoma challenge

[74]

OVA

–

[76]

hTERT-HSP70 OVA conjugation, CpG ODN complexation

S180/H22 –

[79] [81]

OVA

–

[84]

MHC II peptide Anti-CD3, anti-CD28 and anti-LFA-1 Anti-CD3, anti-CD28 and IL2 MHC IgG dimer, anti-CD28 Gp100-MHC-IgG dimer and anti-CD28 Anti-CD3 aCD3 and aCD28 MHC-IgG dimer and antiCD28 Anti-CD3

– –

[87,88] [89]

–

[90]

B16 melanoma tumor –

[91] [92]

– – Melanoma tumor

[94] [95] [86,96]

–

[97]

Anti-CD3 MHC I peptide

– –

[98] [100]

CpG ODN anti-IL-10 ODN and anti-IL-10 receptor ODN Poy(L-arginine)

Hepatoma heap 1–6

[103]

C26 tumor

[104]

Alendronate

S180

[107]

Cross blood barrier and infiltrate into tumor

Liposomal doxorubicin

A549

[108]

Functional modification Passive targeting ability

Baccation III 6-Thioguanine

[112] [113]

Poly(propylene) sulfide nanoparticles

Gold nanoparticles

PLGA nanoparticles

Artificial Exosomes Micelles

Dendrimers Artificial antigen presenting cells

Liposomes

Activate T cells in a more natural manner

PLGA nanoparticles

Incorporate multi-signals in one platform Tunable ellipsoidal shape

Carbon nanotubes Magnetic nanoparticles Nanoworms Janus particles Artificial exosomes Targeting to TAMs

Targeting to Tregs

Large surface and high protein loading efficiency Enhanced clusters formation by magnet application Flexible structures, multiple interactions with T cells Signals spatial organization Stimulate T expansion in a similar manner as natural exosomes

PEG detachable active targeting nanoparticles

pH sensitive, TAM targeting,

Polyion micelles

L-arginine supply enhanced NO production TAM targeting, TAM depletion

Polysaccharide-alendronate conjugation Macrophage Targeting to MDSCs

Efficacious antigen delivery to Langerhans cells through microneedle Enhanced stability of nanoparticle and loaded proteins Easy production Small particle sizes, controlled release, endosomal/lysosomal escape Controlled sizes and surface properties, functional groups

Liposomes PEG-PPS micelles

GM-CSF and IL-2 stimulated Tumor lysates OVA

Melanoma – – –

[68] [69]

[75] [73]

Poly(amidoamine) dendrimers

Self-assemble, pH sensitive

All-trans retinoic acids

4T1 tumor E.G7-OVA thymoma cells and B16-F10 melanoma cells –

Ligand conjugated to PEGmodified single-walled carbon nanotubes PPS NPs

Efficient and selective targeting ability

–

B16 tumor

[116]

Targeting to TDLN

Paclitaxel

B16-F10 melanoma

[63]

3.2. T cell therapies Adoptive cell therapy (ACT) is the use of extracting and ex vivo expanding tumor-specific T cells, and then reinfusing them into tumor patients [52]. One strategy is that tumor-infiltrating lymphocytes (TILs) after ex vivo expansion are transferred back into the patients, which has achieved impressive clinical results in

[115]

phase Ⅱ trials for metastatic melanoma treatment after classical lymphodepletion and attenuated regimen of IL-2 [53]. This can be alternatively done by genetically-redirected T cells that recognize predefined tumor surface antigens by chimeric antigen receptor (CAR) rather than MHC restriction. Zhao and colleagues investigated the functionality and persistence of T cells using 7 different CAR structures providing CD28 and/or 4-1BB costimulation

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Fig. 2. The nanoparticle-based delivery system to activate DCs or T cells for immunotherapy. TAAs loaded within nanoparticles are released from endosomes/lysosomes and then degraded into peptide fragments by cytoplasmic proteasomes. Combined with TLRs cell signaling pathway activation, antigens-MHC complexes are sequentially formed and presented on the surface of DCs. Artificial antigen presenting cells (aAPCs) that are functionalized with MHC-antigen complexes and T cells co-stimulatory molecules such as anti-CD28 or anti-CD86 antibodies have capability to trigger T cells activation. Recognition of MHC-antigen complexes and costimulatory molecules displayed on DCs or aAPCs can activate T cells, resulting in immune response against tumor cells.

[54]. One configuration with two signaling domains (CD28 & CD3f) and 4-1BB costimulation achieved therapeutic efficiency with both balanced tumoricidal function and increased persistence. Although these therapies are potent strategies against cancer, abundant generation of CAR T cells is expensive and can only be performed by a relatively restricted manipulation [55]. 4. Nanoparticle for immunotherapy Nanoparticulate immunotherapy is a rapidly moving field and represents a novel approach for cancer therapy [56]. Table 1 provides some examples of nanoparticles for immunotherapy. 4.1. Targeting delivery TAAs to APCs Efficient and targeted delivery of immunomodulatory and costimulatory molecules to APCs provide opportunities to develop nanocarriers for immunotherapy [57]. Versatile delivery systems, including liposomes, poly(propylene) sulfide nanoparticles, gold nanoparticles, PLGA nanoparticles, exosomes, micelles and dendrimers, have achieved cytoplasmic delivery of exogenous antigens into DCs and enhanced immune responses (Fig. 2). 4.1.1. Liposomes pH sensitive liposome will be a good candidate for induction of cellular immunity because the loaded antigen can be released into cytoplasm by destabilizing or fusing with endosomal/lysosomal membrane. Hyperbranced poly(glycidol) derivatives exhibited hydrophobicity and intensive interactions with biomembrane with increasing degree of polymerization, which conferred liposomes escape from cellular acidic compartments and cytoplasmic delivery of antigen in DCs [58]. 3-methylglutarylated poly(glycidol) (MGluPG)-modified liposomes entrapping OVA and cationic liposomes-DNA encoding IFN-c complexes (lipoplexes) selfassembled into hybrid complexes via electrostatic interactions [2]. The hybrid complexes could translocate OVA and DNA to cytoplasm from endosome/lysosome after co-delivery of OVA and DNA into murine DC2.4 cells, which engendered efficient IFN-c production. Although the hybrid complexes after subcutaneous administration

demonstrated strong tumor suppression and prolonged survival in mice bearing E.G7-OVA cells, the antitumor effect of the hybrid was almost equal to that of OVA-loaded MGluPG-liposomes alone. The combination of MGluPG-liposomes and lipoplexes without pre-mixing achieved more efficient antitumor immune responses than the hybrid complexes because efficient antigen presentation and IFN-c secretion were achieved concomitantly. Therefore, the combination of pH sensitive liposomes mediated antigen presentation and lipoplexes mediated IFN-c gene delivery is a promising strategy for tumor immunotherapy. To establish peptide vaccinebased cancer immunotherapy, they also investigated the improvement of antigenic peptides by encapsulation with 3methylglutarylated hyperbranched poly(glycidol) (MGlu-HPG)modified liposomes for induction of antigen-specific immunity [59]. The liposomes were loaded with peptides derived from OVA-I or OVA-Ⅱ, which is respectively specific for MHC I and MHC Ⅱ on DCs. In vivo OVA-I peptides loaded MGlu-HPG-modified liposomes achieved CTLs activation more efficiently than free OVA-I did in mice bearing E.G7-OVA tumors. Furthermore, efficient suppression of tumor volume was observed in mice immunized with OVA-I MGlu-HPG liposomes whereas OVA-Ⅱ MGlu-HPG liposomes exhibited much lower tumor-suppressive effects, which is mainly due to that OVA-Ⅱ peptides were designed to engender the induction of OVA-specific CD4+ T cells. YSK05 is a synthetic pH sensitive lipid that has optimal functionality at pH 6.4 and possesses a high fusogenic activity, with endosomal escape ability to promote gene silencing [60]. Miyabe et al. utilized YSK05 as vaccine carriers (c-di-GMP/YSK05 liposomes) for cytosol transport of cyclic-di-GMP, an cyclic dinucleotide that can serve as adjuvants to stimulate innate immune system via STING-TBK1-IRF3 pathway [61]. In vitro and in vivo results demonstrated that c-di-GMP/YSK05 liposomes achieved cytosol cyclic-di-GMP delivery and IFN-b production in APCs, resulting in increased expressions of CD80 and MHC I and higher CTLs activities. These results demonstrated that liposomes are good candidates for vaccines delivery, however, there are several major disadvantages. For example, PLGA nanoparticles demonstrated sustained release of antigens resulting in enhanced CD8+ T cells responses compared to fast-releasing liposomes [62], suggesting that strategies to alter release behaviors of antigens from

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liposomes are needed. In addition, liposomal vaccines tend to induce CD4+ rather than CD8+ T cells responses, which might be related to their trafficking in cytoplasm. 4.1.2. Poly(propylene) sulfide (PPS) nanoparticles Antigen alone is sometimes difficult to induce efficient DCs activation and addition of adjuvant will be a good choice for enhanced immune response. In patients with melanoma, the maturation and activation of DCs in tumor-draining lymph nodes (TDLNs) are suppressed even in the presence of abundant immunogenic antigens, which inhibits sequential adaptive immune responses. Thomas and his colleagues assumed that delivery adjuvant alone to TDLNs may exploit the crosstalk between TDLNs and tumor, potentially reverse immunosuppressive microenvironment toward inflammatory one and activate DCs [63]. To validate this, pluronic modified poly(propylene) sulfide (PPS)-core nanoparticles were prepared to deliver CpG oligonucleotide for DCs activation in the TDLNs and reverse immune suppression. The NPs could target to TDLNs after ipsilateral administration, activate DCs and reverse immunosuppression and finally increase CD4+ and CD8+ T cells infiltration into tumor. In order to target DCs in TDLNs, it’s critical to design nanocarriers that can be readily internalized into lymphatic vessels and retained in draining lymph nodes. It has been established that particles size plays an important role in lymphatic uptake from interstitial space [64]. Reddy et al. investigated PEG-stabilized PPS (PEG-PPS) nanoparticles with sizes of 20, 45 and 100 nm in diameter to target DCs in lymphatic nodes. They found that 20 nm nanoparticles were most readily taken up into lymphatics whereas 20 and 45 nm particles exhibited a consistent and strong presence even after 120 h injection. Furthermore, approximately half of PEG-PPS particles even without targeting ligands were internalized by residual DCs in TDLNs, suggesting PPS nanoparticles of 20–45 nm have the potential to target DCs in TDLNs. Exogenous soluble antigens uptake by APCs generally accumulate in lysosomes, resulting in MHC Ⅱ presentation but inefficient MHC I presentation, and their particulate forms can promote antigen presentation onto MHC I complexes, termed as crosspresentation, as demonstrated by direct presentation and tumor challenge [65]. Hirosue et al. explore routes of processing and efficiency of MHC I cross-presentation of OVA peptides conjugated to PPS-PEG NPs using both reducible and non-reducible linkages. They showed that antigens conjugated to NPs by disulfide linkage can be presented both MHC I and MHC Ⅱ molecules, translating to TCRs transgenic T cell activation both in vitro and in vivo. However, antigens conjugated to PPS-PEG NPs were more efficient in MHC I but not in MHC Ⅱ presentation pathway. Antigen loaded into PPSbl-PEG polymers (PS) were, in contrast, delivered more efficiently into MHC Ⅱ than MHC I presentation pathway, stimulating CD4+ T cells immunity. The differences observed in ability to promote T cell subtypes immunity with PS or PPS-PEG NPs might due to differences in distribution and targeting to DCs subpopulations, resulting in different translocation to subcellular compartments [66]. Take this advantage, both strong CD4+ and CD8+ T cells responses were concomitantly obtained through the coadministration of PS and PPS-PEG NPs, which has important implications for particulate-based vaccine design and highlights the potential of combination of different antigen delivery system specific for the induction of helper and cytotoxic T cells immune responses. 4.1.3. Gold nanoparticles Gold nanoparticles possess unique properties, such as tunable surface chemistry, low cytotoxicity, and easily controlled sizes and shapes, which are important factors affecting immune response induction [67]. To evaluate the influence of particle sizes on DCs based immunotherapy, a series of gold nanoparticles with

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diameter ranging from 15 to 80 nm were conjugated to OVA peptides or CpG ODN. The nanoparticles of 60 and 80 nm revealed superior efficiency in antigen presentation, which were optimal for OVA peptides and CpG ODN delivery, respectively. Their combination (NanoAu-cocktail) achieved dual targeting of OVA and CpG to DCs subcellular compartments, enhancement in antigen presentation and TH1 cytokines release. NanoAu-cocktail after intravenous injection demonstrated dramatic improvement in DCs homing to secondary lymphoid and settling in T cell locations, and strong antigen specific CD8+ T response. To investigate differences in shapes as affecting factors of immune responses, spherical, rod and cubic gold NPs were prepared as adjuvants and coated with west nile virus (WNV) envelope protein [68]. The spherical nanoparticles with size of 40 nm induced the highest level of WNV-specific antibodies production whereas rod one did only half of that. Interestingly, antibodies production was independent of endocytosis efficiency, which needs further investigation. In addition, cytokines from bone marrow derived DCs revealed that only rod nanoparticle could significantly induce the release of inflammasome-dependent cytokines such as IL-1b and IL-18 whereas spherical and cubic ones significantly led to production of IL-6, TNF-a and GM-CSF. All these results suggested gold nanoparticles as effective adjuvants to enhance immune responses via different cytokines pathway, which is associated with sizes and shapes.

4.1.4. PLGA nanoparticles PLGA nanoparticles can be formulated to incorporate protein or short/long peptide, which facilitates antigen cross-presentation and anti-tumor T cell response in comparison with soluble antigen in vivo [69]. As only a small fraction of conventional vaccine is delivered to DCs after subcutaneous administration while the majority are removed by other immune cells, Rosalia et al. developed anti-CD40 conjugated PLGA nanoparticles (NP-CD40) encapsulating OVA, Pam3Csk4 and poly(I:C). NP-CD40 demonstrated efficient and selective delivery to DCs in vivo upon subcutaneous injection, which achieved enhanced priming of CD8+ T cells and significantly prolonged survival. To investigate DC-targeted nanoparticles in vivo and subcellular distribution, superparamagnetic iron oxide particles (SPIO) or gold nanoparticles and fluorescence labelled antigen were incorporated in PLGA nanoparticles coated with lipid-PEG-antibody conjugation [70]. Quantification of the nanoparticles at different subcellular compartments indicated their successful endosomal/lysosomal escape and cytosol release. Magnetic resonance images showed that the PLGA NPs were detected within lymph nodes as early as one hour after injection. Antibody modification did not significantly affect in vivo distribution compared to unconjugated PLGA, however, it preferred to target to DCs residing in peripheral lymph nodes. PLGA NPs were also conjugated to other ligands specific for DEC205 or CD11c to compare the efficiency of different targeting strategies to activate DCs and elicit a potent CD8+ T response [71]. Although DEC205or CD11c-conjugation showed decreased internalization efficiency in comparison with CD40 modification, it showed an equal capacity to elicit cytotoxic CD8+ T cell response. The administration route is indispensable for vaccination. Microneedle array can cross stratum corneum and transdermally deliver antigen and costimulatory molecules to the langerhans cells, inducing more efficacious immune response with respect to conventional injection route [72]. OVA incorporated PLGA nanoparticles were loaded within soluble microneedle array to increase vaccine immunogenicity [73]. Following in situ uptake, langerhans cells could deliver NPs to cutaneous lymph nodes, resulting in antigen-specific T cells proliferation and efficient immune responses against tumor. Therefore, soluble microneedle

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array loaded with antigen carrier provides a more promising technology for tumor immunotherapy. As a majority of tumor agents have not been identified, inactivated tumor cells lysate containing all relevant TAA may be a good choice to induce a polyvalent immune response. Head and neck squamous carcinoma lysates were incorporated into PLGA nanoparticles for cancer immunotherapy [74]. In four of five patients treated with the NPs, cytotoxic CD8+ T cells significantly increased IFN-c production and decreased immunosuppressive IL-10 excretion. However, immune response generated from inactivated cells alone is insufficient for immunogenicity generation. Liu et al. developed multi-adjuvant whole cell tumor vaccine (WCTV) loaded PLGA nanoparticles decorated with penetrating peptide to facilitate GM-CSF and IL-2 uptake into tumor cells [75]. After inactivation, the lysates enhanced DCs recruitment and antigen presentation followed by effector T cells activation, which are responsible for depressed tumor growth, metastasis and relapse. Although PLGA based nanovaccine is more efficient in inducing CD8+ T mediated immune response than Freud’s vaccine, its main shortcomings are aggregation and denaturation and acylation of the loaded proteins. The functionalized poly esters such as poly(lactic-co-hydroxymethyl glycolic acid) (PLHMGA) and poly(lactic-co-glycolic-hydroxymethyl glycolic acid) (PLGHMGA) have been synthesized to overcome these limitations [76]. PLHMGA nanoparticles have demonstrated capability of antigen delivery to DCs for antigen-specific T cell activation through cross presentation in vitro and in vivo. 4.1.5. Artificial exosomes Exosomes that are small 40–120 nm vesicles generated from nearly all mammalian cells play important roles in intercellular communication. Increasingly, exosomes are being recognized as potential therapeutics since their ability to elicit potent cells response in vitro and in vivo [77]. Tumor derived exosomes carrying antigen have been demonstrated to induce antitumor immune responses [78]. The numbers of natural exosomes relatively limit purification and further application, thus, artificial biomimetic exosomes are attractive for cancer immunotherapy. We have developed biomimetic exosomes (DECE) incorporating hTERTHSP70 complexes using microemulsion-micelle assembling method meanwhile anti-DEC205 monoclonal antibodies were introduced as targeting ligands [79]. DECE have mean particles sizes of 81 nm and encapsulation efficiency of 93%. There is a significant enhancement in DECE uptake due to high expression of DEC205 on DCs. In our unpublished paper, DECE vaccine significantly inhibited tumor growth and prolonged survival in mice bearing H22 and S180 tumor. 4.1.6. Micelles Exogenous antigens prefer MHC Ⅱ presentation pathway due to endosomal/lysosomal degradation whereas endogenous ones can induce robust cytotoxic CD8+ T cell response via MHC I. pHdependent nanoparticles provide a versatile approach for enhanced antigen delivery through MHC I presentation pathway [80]. pH sensitive micelles based on poly[(dimethylaminoethyl methacrylate-co-pyridyl disulfide ethyl methacrylate)-block(dimethylaminoethyl methacrylate-co-butyl methacrylate-copropylacrylic acid)] were designed for co-delivery of OVA and CpG ODN via disulfide exchange reaction and electric complexation, respectively [81]. The polymer assembled in 23 nm micelles and subcutaneous vaccination significantly elicited CD8+ T response compared to other formulations. Furthermore, the micelles not only improved TH1 immune responses but induced a balanced IgG1/IgG2 production. This work showed that the pH responsive micelles actively promote antigen cross presentation and offer a promising platform for efficacious antigen delivery.

4.1.7. Dendrimers Dendrimers are promising chemotherapeutics and gene platforms for controlled sizes and surface properties [82,83]. Additionally, there are multifarious functional groups in dendrimers, which allows for bioactive molecules conjugation. However, it is not suitable for complete protein delivery due to similar molecular weights. Guanidineterminated dendrimers (Gd) that can form salt bridges with oxyanions of proteins may be an eligible carrier for antigen delivery [84]. Complexation of OVA with Gd bearing amyloid-promoting peptide derived from helix B region (HB) in OVA induced conformational changes and sequential digestion of OVA, suggesting HB as a possible anchor to OVA. Cell association assay showed that the NPs were effectively internalized into RAW 264.7 cells. 4.2. Artificial antigen presenting cells (aAPCs) The nanoparticles described above are designed to deliver antigens and co-stimulatory molecules to APCs, where active immune responses are dependent on efficacious antigen presentation [85]. The nanovaccines have demonstrated more immunogenicity compared to traditional vaccine, nevertheless, clinical efficacy has been unsatisfactory to date. The failure may be attributed to that specific and efficient antigen delivery to immunogenic DCs cannot be controlled well. Additionally, DCs also express inhibitory ligands binding to receptors on activated T cells, which can abolish immune responses partly or eventually. To address this problem, one alternative approach is to develop artificial antigen-presenting cells (aAPCs) (Fig. 2). Signals from aAPCs mainly include two parts: a cognate antigenic peptide presented in the context of MHC and co-stimulatory molecules, which can respectively bind to TCRs and co-stimulatory receptors to activate T cells [86]. 4.2.1. Liposomes To facilitate T cell activation in a more natural context similar to the fluid membrane interactions between natural APCs and T cells, Parkken developed liposomes based aAPCs, where MHC Ⅱ peptides was conjugated and allowed for free movement in the membrane [87]. The results showed that the aAPCs induced 5.4% CD4+ T cells activation whereas only 0.7% in mice treated with incomplete Freund’s adjuvant only. However, this aAPCs are limited to some T cell hybridomas and may not be available in other T cells. On this basis, two types of aAPCs were prepared: (1) on one MHC Ⅱ peptides were uniformly distributed while (2) on the other the peptides clustered on the surface [88]. The peptides clustered aAPCs (PCaAPCs) were constructed by addition of GM, a component of lipid rafts, where MHC Ⅱ peptides were anchored through biotinylated CTB. A significantly higher percentage of CD4+ activation was observed when stimulated with PCaAPCs carrying anti-CD3 and anti-CD28 antibodies. No significant differences in expression of CD69 were found in T cells treated with PCaAPCs in comparison with natural APCs, confirming comparable efficiency of PCaAPCs in ex vivo CD4+ T cell activation. This may be attributed to the organization of the relevant MHC Ⅱ peptides on liposomes, which allow manipulation of molarity, density and affinity to achieve efficacious T cell stimulation. Furthermore, aAPCs were generated with anti-CD3, anti-CD28 and anti-LFA-1 (adhesion protein) antibodies preclustered on liposomes, which is expected to facilitate immunological synapse formation [89]. The aAPCs achieved the most efficient expansion of antigen-specific CD8+ T cells that display an immunotype consistent with in vivo potential persistence meanwhile no increase was in the frequency of regulatory T cells. Liposomes with preclustered antibodies represent efficient aAPCs to rapidly obtain a sufficient number of antigen-specific T cells for immunotherapy.

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4.2.2. PLGA nanoparticles Besides MHC and costimulatory molecules, a third signal provided by cytokines can contribute to expansion, survival, effector function and memory of activated T cells. To incorporate three signals in one aAPCs, Steenblock et al. developed PLGA microparticles that present a high density of adaptor elements for attaching CD3 and CD28, and a core for encapsulating IL-2 [90]. The sustaining IL2 release in the vicinity of T cells significantly improved stimulatory capacity in comparison with exogenous addition of cytokine, resulting in a 45-fold promotion in T cells expansion. It’s well known that natural APCs can alter their morphologies to increase the overall surface area, which will facilitate their interactions with naïve T cells. To imitate the property of natural APCs, ellipsoidal PLGA microparticles with different aspect ratios were prepared and functionalized as aAPCs for T cells activation [91]. The PLGA microparticles were stretched into ellipsoids using a film stretching method, which allows a direct comparison in shape and surface area in the equivalent volumes. The ellipsoidal PLGA was more effective in inducing CD8+ T cells activation than the spherical one. It also shows that increased aspect ratios were positively correlated with enhanced CD8+ T cell proliferation, suggesting that T activation depends on ellipsoidal aAPC geometry rather than density or amount of antibody conjugation. In addition, there were no significant differences observed in the quality of CD8+ T cells between spherical and ellipsoidal PLGA microparticles induced INF-c or CD107a expression. Ellipsoidal PLGA microparticles also achieved enhanced T activation in vivo, which significantly prolonged survival of mice bearing B16 melanoma relative to spherical aAPCs. Furthermore, nanoscale aAPCs are preferred to microscale platforms because they possess satisfactory biocompatibility, favorable translocation and biodistribution in vivo. Ellipsoidal PLGA NPs coupled with gp100-MHC dimer and anti-CD28 were then prepared using the same method described above [92]. Besides superior efficiency in T cell activation, ellipsoidal nano aAPCs can reduce non-specific interactions with macrophages and HUVECs, which are all models of non-specific uptake [93]. Therefore, the ellipsoidal PLGA NPs mediated significantly higher CD8+ T cell expansion in vivo, which is consistent with superior pharmacokinetics profiles. 4.2.3. Carbon nanotubes Carbon nanotubes (NT) can be adapted to nanoscale vehicles due to their unique physiochemical properties. Owing to large surface of these bubbles (1560 m2/g), bioactive molecules such as antibodies can be presented at a high concentration for potential therapy in biological systems. NT bundles absorbing anti-CD3 revealed a dramatic effect on T cell activation by determining IL2 release compared to antibodies immobilized on tissue culture plate or free in solution [94]. NT exhibited more efficient T activation than other materials even when normalized antibody presentation on surface, suggesting their unique properties associated with T cell activation. FRET acceptor photobleaching (FRET-AP) technique apparently revealed that aCD3 and aCD28 formed cluster on NT, which is a possible mechanism for the enhanced activation of T cells [95]. 4.2.4. Magnetic nanoparticles Perica et al. developed aAPCs based on paramagnetic irondextran nanoparticles (50–100 nm) to incorporate MHC-IgG dimers and anti-CD28 antibodies [86]. The aAPCs not only induced antigen-specific splenocytes expansion in mice but stimulated human peripheral blood T cells proliferation. The nanoscale aAPCs diffusely spread to draining lymph nodes within 24 h compared to microbeads up to 72 h. In mice bearing melanoma tumor, the NPs significantly revealed tumor rejection. Furthermore, incubation in a magnetic field significantly led to iron-dextran nanoparticles

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aggregation, resulting in increased TCR-anti-CD3 aggregate sizes and decreased numbers of that per cell [96]. Enhanced T cells activation was also obviously observed in paramagnetic iron-dextran nanoparticle combination with magnetic field since receptor aggregation is a sufficient signal for T cell activation. These studies reveal that magnetic nanoparticles as aAPCs are potentially coupled with magnetic field for enhanced T cells activation. 4.2.5. Nanoworms Nanoworms were synthesized as novel aAPCs due to their high aspect ratio, flexible architecture and multiple interactions with T cells. The structures are semi-flexible filamentous polymers comprising semi-stiff poly(isocyano peptide) with oligo(ethylene oxide) side chains that were then functionalized with anti-CD3 antibodies [97]. Even at very low concentration, nanoworms significantly achieved T cell activation and IFN-c production in accordance to a higher fraction of anti-CD3 on T cells surfaces with respect to microbead based aAPCs and free anti-CD3 antibodies. This can be probably explained by the semi-flexibility and the density of effector molecules of nanoworms, which assists in cluster formation on the surface of T cells. 4.2.6. Janus particles Janus particles that have distinct surface component in one entity offer many promising applications in drug delivery, imaging/sensing and magnetic therapy. Chen and his colleagues reported their application as aAPCs, where the ligand presentation was designed to mimic ‘‘bull’s eye” in the immunological synapse [98]. The results showed that T cells activation was related to both surface coverage of anti-CD3 and ligands spatial organization. It has been reported that TCRs departing from the center of the immunological synapse stimulates T cells activation whereas their accumulation terminates signaling pathway [99]. Janus particles with reverse ‘‘bull’s eye” pattern that anti-CD3 and fibronectin are spatially separated directed the dissociation of membrane receptors and intracellular proteins and activated T cells more efficiently. 4.2.7. Artificial exosomes Besides as vaccine carriers, artificial exosomes can serve as aAPCs for cancer immunotherapy. Peña et al. constructed artificial exosomes by coating liposomes with optimized MHC I peptide and a selected specific range of ligands for adhesion, early and late activation as well as survival T cell receptors [100]. The artificial exosomes resulted in reproducible high T cell expansion in a similar manner as natural exosomes did, though exosomes are not categorized into APCs due to deficient specific T cell stimulation. 4.3. Targeting to immunosuppressive microenvironment TAMs, MDSCs and Tregs are major players that construct immunosuppressive tumor microenvironment. Designing a system to address immunosuppressive cells in the TME presents numerous challenges, specifically with respect to delivering combinations of immune modulating payloads in a bioactive state to tumor site while minimizing systemic toxicity. Nanoparticulate carriers can facilitate the preferential accumulation of immunomodulatory cargos in the TME, by being designed to either passively accumulate in the TME via enhanced permeability and retention (EPR) effect, or actively target to the TME via diverse strategies using antibodies, peptides, aptamers and small molecules specific for immunosuppressive cells or other components in the TME [101]. In carriers design, it is important to consider the spatial and temporal pattern of delivery that would be optimal for the activity of payloads. For targeting to TAMs, MDSCs or Tregs, the carrier should be designed to minimize interactions with reticuloendothelial system (RES),

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Fig. 3. The nanoparticulate drug delivery system targeting to tumor immunosuppressive microenvironment. The cargo loaded NPs conjugated to targeting ligands, such as antibodies, folic acid and saccharide, can deplete immunosuppressive immune cells or repolarize their protumor phenotype in tumor microenvironment, resulting in antitumor immune response and tumor regression.

which can be achieved by some strategies such as PEG modification, and size distribution. For the delivery of immune modulators such as TLRs agonists or siRNA silencing tumor promoting gene, the vehicle should be design to target to the intracellular compartments. For example, cytosolic targeting can be achieved through the use of pH sensitive lipid or polymers that promote endosomal escape following endocytosis. The temporal pattern of cargo release can be tuned by varying system parameters including the method of payload loading, degradability of the vehicle materials and other relative physicochemical properties. Developing immunomodulatory factors loaded nanoparticles against these immunosuppressive mediators and immune cells will improve therapeutic efficiency of vaccine and other immunotherapies (Fig. 3). 4.3.1. Targeting to TAMs TAMs are the main source of intratumoral IL-10 that involves in maintenance of M2 phenotype in an autocrine manner. It has been reported that CpG ODN combined with anti-IL 10 antibodies could repolarize the phenotype of TAMs and then inhibit tumor growth [102]. Therefore, CpG ODN, anti-IL-10 and anti-IL-10 receptor ODN were associated with galactosylated cationic dextran to form stable nanocomplexes (GDO) for targeting delivery to TAMs [103]. PEG-histidine-modified alginate (PHA) as pH sensitive materials shielding cationic charge of dextran was coated on the complexes to develop PDO (PHA + GDO), which could release GDO in the

acidic tumor microenvironment in favor of TAMs endocytosis mediated by galactose receptor. The complexes were accumulated in F4/80+ macrophages after tail vein administration, and then prompted IL-12 production and reduced the expression of IL-10 and IL-10 receptor. IL-12high and IL-10low TAMs were characterized with decreased expression of M2-like genes such as Arg1, Ym1, Msr2, Fizz1, Mgl2, MMP9 and VEGF, indicating successful reversal of TAM phenotype to M1-like macrophages. The antitumor results revealed that PDO significantly suppressed tumor growth due to TAMs phenotype reverse. NO can induce tumor cells apoptosis through condensation with amine or thiol groups of certain protein and damage to DNA related to p53 signaling or not [104,105]. However, NO production is frequently down-regulated in TAMs due to inadequate Larginine available to iNOS. Kudo and Nagasaki developed polyion complex micelles (PEG-b-P(l-Arg)/m) composed of poly(ethylene glycol)-block-poly-(L-arginine) and chondroitin sulfate as NOtriggered immune therapeutics for systemic antitumor therapy [104]. As expected tremendous amount of nitric oxide was generated from RAW 264.7 cells stimulated with LPS, which suggested Larginine liberation from PEG-b-p(L-Arg)/m. Systemic administration of PEG-b-p(L-Arg)/m inhibited tumor growth in a dosedependent manner, which enhanced tumor growth in a low dosage whereas high concentration exhibited antitumor effect. This was probably attributed to angiogenesis promotion of low concentration NO.

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TAMs are known to express abundant mannose receptor, which may be a desirable target mediated drug delivery to macrophages. Glucomannan polysaccharide (GP) has been demonstrated superior affinity to mannose receptor and have capability in mediating therapeutic nucleotide delivery into macrophages in vitro [106]. Zhan et al. employed GP conjugated alendronate (GP-ALN) for specific TAMs depletion in tumor microenvironment [107]. In vitro and in vivo results showed that GP-ALN preferentially accumulated in TAMs and then induced their apoptosis, which significantly inhibited angiogenesis, recovered immune surveillance and finally induced tumor regression in a S180 tumor-bearing mice model. Additionally, GP-ALN elicited no unwanted side effects such as systematic immune response, suggesting its potential depletion of TAMs for cancer immunotherapy. Macrophages have to cross blood barriers in the process of recruitment into tumor tissues, which allow to exploit their intratumoral infiltration to specifically target to the tumor area. Choi et al. used mouse peritoneal macrophage as a biologically active carrier to deliver doxorubicin loaded liposomes (Macrophage-LPDox) [108]. Macrophage-LP-Dox showed plenty amount of doxorubicin infiltration in tumor in comparison with less amount of that in tumor of LP-Dox group in both subcutaneous and metastasis xenograft tumor models. More importantly, M-LP-Dox remarkably reduced tumor growth ratio compared to reference formula after systematic administration. These results proved the feasibility of macrophage-LP-drug as an active biocarrier for efficient cancer therapy and afford new perspectives for active drug delivery. 4.3.2. Targeting to MDSCs Infiltrating MDSCs involve immunosuppressive microenvironment creation and suppress antitumor immune responses, and their depletion can restore effector T cells anti-tumor responses. Baccatin III is the precursors for the semisynthesis of paclitaxel that can decrease MDSCs infiltration [109]. Although its cytotoxicity is much lower than that of paclitaxel, baccatin III reduced immunosuppressive functions and accumulation of MDSCs, resulting in significant tumor regression [110,111]. However, baccatin III suffers from non-specific toxicity and dose-limiting delivery to MDSCs as other small-molecule drugs. Kullberg et al. employed endogenous activated complement C3 to target liposomes (C3Lip) to CD11b+ MDSCs and the results showed that C3-Lip were specifically endocytosed by CD11b+ MDSCs even in complete serum [112]. Jeanbart developed PEG-PPS polymer micelles (MCTG) conjugated to 6-thioguanine to deplete MDSCs and enhance T cell mediated immune response [113]. MC-TG achieved circulating MDSCs and Ly6chi macrophage depletion for up to 7 days even taking a single administration. All-trans retinoic acids (ATRA) could dramatically decrease the number of MDSCs by enhancing myeloid cells differentiation into mature DCs, macrophages and granulocytes [114]. Wang et al. have prepared poly(amidoamine) dendrimers based nanoparticles for ATRA delivery [115]. These dendrimers can self-assembly from 125 to 435 nm nanoparticles, which are relatively stable at pH 7.4 but dissociated in acidic environments. However, their anti-MDSCs effects were not studied in the paper. 4.3.3. Targeting to Tregs Recent evidences regarding the role of intratumor Tregs have suggested that their selective depletion will be a desirable approach for efficacious immunotherapy. In order to distinguish and selectively deplete tumor-infiltrating Tregs while preserving other Tregs critical for suppressing autoimmunity, it is important to select surface molecules expressed specifically or selectively on intratumoral Tregs. Sacchetti et al. designed PEG modified single-walled carbon nanotubes (PEG-SWCNTs) decorated with anti-glucocorticoid-induced TNFR-related receptor (GITR) mAb

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(DTA-1) for targeting to intratumoral Tregs as GITR showed higher overexpression on intratumoral than peripheral Tregs in comparison with other Tregs-specific markers such as folate receptor 4, CD103 and CD39 [116]. DTA-1 conjugation enabled efficient and selective in vitro PEG-SWCNTs uptake into the cytoplasm even in GITR low expression Jurkat T cells. In vivo investigations showed PEG-SWCNTs armed with GITR ligands targeted to residual Tregs in a B16 melanoma more efficiently than non- or splenic-Tregs. Interestingly, this could be translated into more selective targeting ability to intratumor Tregs due to increased Tregs infiltration in tumor microenvironment. Besides targeting to Tregs directly, Thomas developed paclitaxel loaded PPS-NPs to reshaped immunosuppressive lymph node (LN) that is an important site for induction of immune tolerance because Tregs require recirculation into LN for their controlling the priming phase of an immune response, and the results demonstrated decreased frequencies of activated Tregs in TDLN [63]. More therapies targeting Tregs are under active investigation when considering the role of Tregs in tumor immunity. It is worth noting that combination of Tregs attenuation with activation of tumor-specific T cells may mutually enhance each individual treatment [117]. 5. Conclusion and further perspectives Advances in studies of the dynamic and complex interactions between immune system and tumor can guide immunotherapy, which will be helpful to innovative therapeutics for cancer. Blocking the pathway through which tumor cells seek to evade immune surveillance is critical for successful immunotherapy. Although checkpoint blockades combined with other therapies have shown the potential to elicit durable controls of cancers, there remain many challenges for broader achievement of immunotherapy. The manipulation of immune system through nanoparticlebased immune-modulating therapeutics is in its infancy but provides promising strategies for cancer therapy. Numerous studies have harnessed nanoparticles as vehicles for coordinated delivery of antigens and costimulatory molecules to APCs, which demonstrated enhanced CD4+ and CD8+ T responses against tumor. Likewise, nanoparticulate aAPCs aimed at directly activating T cells have also been proven their efficacy. Armies of immune cells against tumors can also be enhanced by utilizing small-molecule inhibitors/gene loaded nanocarriers to deplete/repolarize TAMs, MDSCs and Tregs as well as by inhibiting immunosuppressive factors in tumor. However, the clinical translation of nanoparticulate immunotherapies to safely and potently modulate the immune system in patients with cancer can pose significant challenges. The first challenge is that in vitro assays, although perhaps useful intracellular evaluation, are not equivalent to recapitulate in vivo conditions because of the absence of critical factors such as hostand tumor-derived microenvironmental factors. Next, animal models can serve as an important source of in vivo information, but they are limited in ability to mimic the extremely complex process of human carcinogenesis, physiology and progression [118]. To ensure low systemic cytotoxicity, high specificity, long-term efficacy and bioavailability of payloads can be clinically challenging for delivery systems design and optimization. Optimizing efficacious immunotherapy in patients with cancer will also require targeting ligands specific for immune cells and stable in plasma. Finally, differences in diverse human cancers, such as difference between liquid and solid tumor profiles and therapies, must be taken into consideration in nanoparticle based immunotherapies for clinical applications. Therefore, success in this endeavor will require partnerships between materials scientists, bioengineers, pharmaceutical scientists, chemists, immunologists, vaccinologists and clinicians [119,120].

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