Cancer nanoimmunotherapy using advanced pharmaceutical ...

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Cancer nanoimmunotherapy using advanced pharmaceutical nanotechnology

Immunotherapy is a promising option for cancer treatment that might cure cancer with fewer side effects by primarily activating the host’s immune system. However, the effect of traditional immunotherapy is modest, frequently due to tumor escape and resistance of multiple mechanisms. Pharmaceutical nanotechnology, which is also called cancer nanotechnology or nanomedicine, has provided a practical solution to solve the limitations of traditional immunotherapy. This article reviews the latest developments in immunotherapy and nanomedicine, and illustrates how nanocarriers (including micelles, liposomes, polymer–drug conjugates, solid lipid nanoparticles and biodegradable nanoparticles) could be used for the cellular transfer of immune effectors for active and passive nanoimmunotherapy. The fine engineering of nanocarriers based on the unique features of the tumor microenvironment and extra-/intra-cellular conditions of tumor cells can greatly tip the triangle immunobalance among host, tumor and nanoparticulates in favor of antitumor responses, which shows a promising prospect for nanoimmunotherapy. Keywords: antibody engineering • biodegradable polymers • cancer nanotechnology • cancer vaccine • dendritic cells • nanoimmunotherapy • nanomedicine

Cancer immunotherapy In the clinic setting, a tumor frequently experiences relapse, which results in therapeutic failure and an unfavorable postoperative life. This phenomenon is partly attributed to the micrometastases of disseminated cancer cells. Overcoming such tumor relapse is critical in clinical oncology for curing tumors [1] . Fortunately, the host immune system can recognize, eliminate and protect the body from viral or bacterial infections, as well as prevent the formation of transformed cells (including precancerous cells) [2] . In immune system, all immune cells and factors participate in tumor suppressing by the well-known lineal immunobalance, which is regulated by the host immune system and tumor. Many immunocells such as natural killer (NK) cells, B cells, dendritic cells (DCs) and T cells and cytokines work together to prevent and control tumor initiation [3] .

10.2217/NNM.14.127 © 2014 Future Medicine Ltd

Two main strategies have been developed for cancer immunotherapy: nonspecific immune activation and tumor-specific immune activation. The nonspecific immune activation strategy involves the application of cytokines, interferons and ligands for Toll-like receptors (TLRs) [2] . The cytokines mainly refer to interleukins including IL-2, IL-8 and IL-15. IL-2 not only promotes the proliferation of effector immune cells and enhances their cytotoxicity, but it can also restore immune response by suppressing negative regulatory receptors such as programmed death-1. IL-8 can augment antitumor therapy by inducing antitumor cytokines, activating effector T cells and enhancing NK cell cytotoxicity. The interferons include type I and type II interferons. Type I interferons (IFN-α and -β) exhibit antitumor effects via promoting the activity of NK cells, increasing expression of Fcγ receptors and inhibiting the generation of Treg cells. Type II interferon (IFN-γ)

Nanomedicine (Lond.) (2014) 9(16), 2587–2605

Wei Li*,‡,1,2, Huafeng Wei1, HuafeiLi1,JieGao1,Si-ShenFeng3 & Yajun Guo*,‡,1,2,4 International Joint Cancer Institute, The Second Military Medical University, 800 Xiangyin Road, Shanghai 200433, PR China 2 State Key Laboratory of Antibody Medicine & Targeting Therapy & Shanghai Key Laboratory of Cell Engineering, 399 Libing Road, Shanghai 201203, PR China 3 Department of Chemical & Biomolecular Engineering, National University of Singapore, Block E5, 02–11, Engineering Drive 4, 117576, Singapore 4 PLA General Hospital Cancer Center, PLA Graduate School of Medicine, Beijing 100853, PR China *Authors for correspondence: Tel.: +86 21 8187 0801 Fax: +86 21 8187 0801 yjguo@ smmu.edu.cn liwei@ smmu.edu.cn ‡ Authors contributed equally. 1

part of

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Review Li, Wei, Li, Gao, Feng & Guo is secreted by NK cells and effector T cells in response to recognition of tumor antigen, which can promote NK cell activity and T helper type 1 (Th1) cell differentiation. TLRs belong to a group of transmembrane proteins that trigger maturation of DCs, stimulate proliferation of CD4 + and CD8 + T cells, and modulate the suppressive function of Treg cells [4] . Currently, ten distinct TLRs are expressed in humans. Three TLR agonists (Bacillus Calmette–Guérin [agonist of TLR2 and TLR4], monophosphoryl lipid A [MPLA; agonist of TLR4] and imiquimod [agonist of TLR7]) have been approved by the US FDA for the treatment of cancer [5] . Tumor-specific immune activation involves the recognition of tumor cells/antigens by immune cells. The cytotoxic T lymphocytes (CTLs) can destroy tumors by releasing perforin and granzymes when the T-cell receptors specifically recognize and bind with the peptide–MHC I complex on the tumor [2] . It is widely accepted that the activation of CTLs requires two signals: the first signal consists of the recognition of the antigenic peptide MHC molecules on antigen-presenting cells (APCs) through T-cell receptors. The second signal is called costimulation signal, which involves the interaction between costimulatory ligands on the T cells and their receptors on APCs (e.g., B7/CD28 or CD40/CD40). Therefore, the induction of tumorspecific immune responses requires potent interactions between T cells and professional APCs [5,6] . The most effective APC is the DC [7] . When exposed to tumor antigens (TAs), immature DCs in peripheral tissues may differentiate to mature DCs, which are characterized by the high expression of MHC and costimulatory molecules. Thus, targeting DCs is a promising strategy for antigen acquisition, presentation and robust antigen-specific T-cell response initiation [4,8,9] . Cellular resistance to immunotherapies Relapse is an issue in traditional immunotherapies. The dynamic interaction between the host immune system and tumor was defined as immunoediting by Schreiber in 2002 [10] . This concept is based on the hypothesis that when protecting the host from cancer, the immune system may also induce tumor survival in immunologically environments. Figure 1 shows the process of tumor initiation, growth, suppression and immunoediting. Immunoediting involves three phases: elimination, equilibrium,and escape [2,10] . The elimination phase refers to the complex process of innate and adaptive immunity, which suppresses tumor cells by the generation/activation of various immune cells including B and T lymphocytes and NK cells, and by the secretion of tumor-killing cytokine such as IFN-γ, IFN-α/β and perforin [11] . Although many of the orig-

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inal tumor cells are destroyed, some tumor cells with highly genetic instability may withstand the immunosurveillance. The new tumor variants carry more mutations, which lead to resistance to immune attack in the equilibrium phase [10,12] . The ‘edited’ tumor cells that survived the equilibrium phase may circumvent both innate and adaptive immunologic defenses. They enter the escape phase and become clinically detectable in the host [10,13] . Overcoming the aforementioned tumor escape strongly relies on TA identification [15] . TAs are generally divided into tumor-associated antigens (TAAs) and tumor-specific antigens (TSAs). TAAs are antigens expressed by tumor cells and/or normal cells. TSAs mainly refer to the mutated or altered proteins specifically expressed in tumors, which include tyrosinase, MART1 and gp100, prostate-specific antigen and idiotype antibodies. In addition, the immunosuppressive molecules play a pivotal role in tumor escape. In the tumor microenvironment, the ability of molecules such as granulocyte-macrophage colony-stimulating factor and IL-4 to promote DC differentiation and function is low. The ability of cytokines including IL-12 and IL-18 and interferons for inducing Th1-type responses is also low. However, molecules suppressing DC differentiation and function such as VEGF, IL-6, IL-10, TGF-β and macrophage colony-stimulating factor, arginase, indoleamine-2,3-deoxygenase, prostaglandin E2, cyclooxygenase-2 and nitric-oxide synthase-2 are abundant. Most of the suppressive molecules come from tumor cells, macrophages and stromal cells in the tumor microenvironment, which profoundly affects tumor-specific immunity. All of these factors including the loss of MHC molecules, the high levels of Treg cells or myeloid-derived suppressor cells, and the large amount of TGF-β in the tumor microenvironment cooperatively promote tumor survival [16] . Drug resistance in monoclonal antibody (mAb)-based immunotherapy occurs via complex mechanisms including the diminished expression of targeting TAs, activation of cell-survival pathways and inhibition of apoptotic pathways [17] . Moreover, the growth and survival of tumor cells can often be impaired by the inactivation of the oncogene addiction [18] . Clinical data have demonstrated that the k-ras mutations confer intrinsic resistance to EGF receptor mAbs in colorectal cancer [19] . As a result, how to overcome tumor escape and cellular resistance is a key issue in cancer immunotherapy. Strategies for enhanced immunotherapy Immunomodulating antibodies

It is known that the balance between costimulatory or coinhibitory molecules in the host immune system

future science group

Pharmaceutical nanotechnology for cancer immunotherapy

Chronic inflammation Bacterial infection Genetic mutation Radiation Virus

Tumor antigens Uric acid Heterogeneity ECM products

Elimination

Escape

CD8+ C CD4+ NKT NK

NKTCD4

+

NK Normal cells

Equilibrium

Review

Tumor formation

+ NK KCD4

NKTCD4

NKTCD8

+

Immunosurveillance

+ NK KCD4

NKTCD4

CD4+

NK

+ NKTCD8

NKTCD8

NK

+

Genetic instability

Tumor variants

Figure 1. Multiiple stages of tumorigenesis and immune escape. (A & B) Tumor formation process and (C–E) smart tumor escape. ECM: Extracellular level matrix. Adapted with permission from [14] under Creative Commons Licences.

can be utilized to regulate immune activation or tolerance. Such costimulatory or coinhibitory molecules enable the host immunity system to respond to invading pathogens. Consequently, both antagonist mAbs targeting the coinhibitory receptors and agonist mAbs targeting the costimulatory receptors are promising proteins for overcoming tumor escape. Antagonist mAbs can disrupt the inhibitory signals, while agonist mAbs can accentuate the stimulatory signals [20] . One successful example of immunomodulating antibodies is ipilimumab which is an antagonist mAb against CTLA4. Other antibodies such as the programmed cell death-1, T-cell immunoglobulin mucin-3 and lymphocyte activation gene-3 are also potential antagonist mAbs [21] . Agonist mAbs targeting costimulatory receptors have also been generated. These mAbs mainly target the costimulatory receptors of a group of TNF family members including CD40, CD134, CD137 and glucocorticoid-induced TNF receptor. Among these antibodies, agonist anti-CD40 mAbs have been extensively studied and have exhibited clinical activities in a range of tumor types. To date, more than 14 mAbs and their conjugates have been approved by the FDA/European Medilines Agency, as shown in Table 1. Antibodies in cancer treatment act by different mechanisms: blocking the signal pathway (required for malignant cell growth and function) by inhibiting the ligand–receptor interaction by receptor modulation and/or dimerization; interrupting the trophic interaction between malignant cells and their stroma; recognizing TAs and exerting antitumor effects by immune effector mechanisms including antibody-dependent cellular cytotoxicity and complement-dependent cytotoxicity, or by inducing direct apoptosis of malignant cells; and conjugation to form immunoconjugates that specifically deliver toxins to tumor cells [22] . Although mAbs offer new hope for


cancer therapy, drug resistance and high cost are still big challenges in clinics. New cancer vaccine formulation

Cancer vaccines are generally applied to treat existing cancer (therapeutic vaccine) or prevent cancer progression (preventive vaccine) [12,23] . Usually, the components of cancer vaccines include TSAs and adjuvants. TSAs generally come from cancer cells and include proteins, carbohydrates, glycoproteins and gangliosides. According to the type of antigens, cancer vaccines can be divided into several types: peptide/protein vaccines, DNA vaccines, DC-based vaccines, tumorbased vaccines and virus-like particle (VLP) vaccines [24] . Adjuvants can enhance immune response and increase, accelerate and prolong the response of corresponding vaccines while remaining nontoxic to the host. Many different kinds of adjuvants have been developed over the years such as mineral salts (aluminum hydroxide and aluminum phosphate), oil emulsions (MF59) [25] particulate adjuvants (DOTAP) [26] , virusomes (assembled from viral membrane proteins) and cytokines (granulocyte-macrophage colony-stimulating factor, IL-12 and IL-2). DOTAP and/or IL-12 can induce a Th1 immune response, which contributes to the induction of cellular immune responses such as antigen-specific CTLs [26] . Alum, cholera toxin and labile toxin can induce a Th2 immune response, which is responsible for the humoral immune response and enhances antigen-specific antibody generation [27] . Some prophylactic VLP-based vaccines are currently commercially available such as the Engerix® (hepatitis B virus; GlaxoSmithKline Biologicals, Rixensart, Belgium) and Cervarix® (human papillomavirus; GlaxoSmithKline Biologicals, Rixensart, Belgium), Recombivax HB® (hepatitis B virus; Merck & Co Inc., NY, USA) and Gardasil® (human papillomavi-

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Table 1. Current monoclonal antibodies for cancer therapy approved by the US FDA. mAb

Target

Year approved Indications

Rituximab

CD20

1997

CD20-positive B-cell non-Hodgkin’s lymphoma and chronic lymphocytic leukemia

Trastuzumab

HER2

1998

HER2-overexpressing breast cancer, metastatic gastric or gastroesophageal junction adenocarcinoma

Gemtuzumab†

CD33

2000

Acute myeloid leukemia (coupled with calicheamicin)

Alemtuzumab

CD52

2001

Chronic lymphocytic leukemia

Ibritumomab tiuxetan

CD20

2002

Relapsed or refractory, low grade or transformed B-cell non-Hodgkin’s lymphoma

Tositumomab

CD20

2003

Follicular non-Hodgkin’s lymphoma

Bevacizumab

VEGF

2004

Certain metastatic cancers and certain lung cancers, renal cancers and glioblastoma multiforme of the brain

Cetuximab

EGFR

2004

Head and neck cancer, wild-type colon cancer

Panitumumab

EGFR

2006

EGFR-expressing metastatic colorectal cancer with disease progression despite prior treatment

Catumaxomab

CD3 EpCAM

2009

Malignant ascites

Ofatumumab

CD20

2009

Chronic lymphocytic leukemia that is refractory to fludarabine and alemtuzumab

Brentuximab vedotin

CD30

2011

Anaplastic large cell lymphoma and Hodgkin’s lymphoma

Denosumab

RANKL

2011

Osteoporosis, treatment-induced bone loss, bone metastases, rheumatoid arthritis, multiple myeloma and giant cell tumor of bone

Ipilimumab

CTLA 4

2011

Melanoma

‡

Withdrawn from the market in June 2010 when a clinical trial showed the drug increased patient death and added no benefit over conventional cancer therapies. ‡ Withdrawn from markets in the USA and Europe in 2012 to prepare for a higher-priced relaunch aimed at multiple sclerosis. EGFR: EGF receptor; mAb: Monoclonal antibody. †

rus; Merck & Co, Inc., NY, USA) [28] . They are able to elicit long-term memory without causing autoimmunity [11,29] . Side effects such as flu-like symptoms (e.g., fever, chills, dizziness, nausea and vomiting) and inflammation (e.g., pain, swelling, itchiness and rash) were also avoided. Cervarix, which contains aluminum hydroxide and AS04 (3-O-desacyl-4’-MPLA), was subsequently approved by the FDA in late 2009 [30,31] . It is important to note that not all viruses are suitable for VLP vaccine owing to in vivo instable assembly and technical limitations in formulation [32] . Moreover, serious symptoms such as asthma, autoimmune diseases and severe hypersensitivity have been reported [2] . Recently, molecular delivery systems were developed to avoid serious symptoms. Tumor microenvironment reversion

The tumor microenvironment comprises immune cells, tumor cells, stromal cells and the scaffold extra-

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cellular matrix, which is main battle ground for tumor escape. The tumor microenvironment is characterized by deformed structure, which result in fibrosis and contraction of the interstitial matrix with a lack of lymph vessels [33] . These structural defects lead to poor or dysfunctional T-cell priming, which drives the escape of tumors from the immune system. Thus, restructuring the tumor into a real active lymphoid organ and imitating the peripheral TAA-specific priming within the tumor microenvironment could overcome the potential TAA ignorance (immune system does not recognize and repond to TAA) in the tumor-draining lymph nodes [33] . Notably, Treg cells inhibit TAA-specific priming in the tumor-draining lymph nodes and TAA-specific effector functions in the tumor microenvironment [34] . Since the transcription factor Foxp3 is indispensable for the development and function of Treg cells, Foxp3 knockdown may reverse the suppressive function of Treg cells. Alternative approaches such



as blocking or reducing the differentiation of Treg cells are being developed as new cancer immunotherapeutic strategies [35] . Several strategies have already been used in clinical trials as single agents or in combination with cancer vaccines. In nonspecific immune activation strategies, positive factors such as cytokines, interferons and ligands of TLRs are being used against the negative factors in the tumor microenvironment [36] . They can efficiently inhibit overexpressed immunosuppressive molecules in tumors including inflammatory molecules, cytokines, chemokines, tumor-infiltrating T cells, DCs and macrophages. The host immune system is thus activated for suppressing tumor escape [37] . The key issue for efficient escape suppression is to successfully transfer positive factors to the tumor. Nanoimmunotherapy Nanotechnology for promoting immunotherapy

With the development of materials science and nano technology, nanoparticulate-based delivery systems offer new hopes for improving the efficiency of aforementioned immunotherapies with the following potential advantages. First, the nanocarrier protects the bioactive molecules (e.g., antibody and antigen) from enzymatic degradation and immediate elimination from the circulation. Second, the nanocarriers deliver the bioactive molecules en masse with high efficiency. Third, nanocarriers can be easily decorated by ligands with specific cellular targeting. Fourth, nanocarriers can be facilely tailored to the appropriate size and to possess the appropriate surface properties with low reticuloendothelial system circulation, which prolongs the half-life and changes the biodistribution of the bioactive molecules [38–40] . Nanoimmunotherapy can thus be defined as the application and further development of nanotechnology for enhancing immunotherapy, which includes the development of nanocarriers for targeted and controlled delivery of antibodies to cancer cells (passive immunotherapy), and of antigens to DCs to evoke immune response to the disease (active immunotherapy) [38,39] . Active nanoimmunotherapy

Many types of particulate-based active nanoimmunotherapies have been developed to promote the efficiency of vaccine [41,42] . The antigen-loaded polymeric nanoparticles have shown significant potential in vaccines and adjuvants [43,44] , as summarized in Table 2. The protein-encapsulated biodegradable poly(gammaglutamic acid) nanoparticles were efficiently taken up by immature DCs, resulting in high levels of DC maturation [45,46] . HIV-1 gp120 encapsulated by nanoparticles strongly induced antigen-specific cel-


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lular immunity [47] . Poly(d,l-lactic-co-glycolic acid) (PLGA) nanoparticles loaded with ovalbumin (OVA) antigen and MPLA as adjuvants significantly induced potent CD4 + and CD8 + T-cell responses [48] . Plasmid DNA (pDNA)-based vaccine can elicit potent cellular immune responses with high immunogenicity [49] . Carrier systems such as liposomes and cationic polymers successfully condensed pDNA into a small particle, which allowed efficient and safe delivery of pDNA to the targeting cells [50–52] . The OVA-encoded pDNA complexed with cationic poly-L-lysine (PLL) and coated with polystyrene particles produced high levels of CD8 T cells and OVA-specific antibodies in C57BL/6 mice [53] . Cationic polymer-based nanocarriers have attracted a great deal of interest owing to their role as an agonist for TLR5, which stimulated TLR3 and TLR7 and reversed the tolerogenic phenotype of human and mouse ovarian tumor-associated DCs [54] . In addition, the micellar system was also used in active nanoimmunotherapy [55,56] . The block copolymer poly(ethylene glycol)-b-poly(propylene sulfide) (PEG-PPS) micellar system efficiently traveled to draining lymph nodes and interacted with APCs to induce potent immune responses [57] . The OVA-conjugated micelles generated more (2.4-fold) OVA-specific CD8 + T cells in the blood and higher (1.7-fold) IFN-γ levels from splenocytes upon restimulation than splenocytes immunized with free OVA and CpG. For enhancing the intracellular release of immune factors from DCs, pH-sensitive micelles were formed from polymethacrylic acid-b-polyethylene oxide, PLL and a fluorescent peptide (OVA-fluorescein isothiocyanate (FITC) peptide). The peptides were efficiently loaded onto MHC class II molecules by pH-sensitive micelles [58] . The intracellular delivery of mRNA was realized by cationic micelles containing diethylaminoethyl methacrylate segments to mediate mRNA condensation and a hydrophilic poly(ethylene glycol) methyl ether methacrylate segment to enhance stability and biocompatibility [59] . Specific targeting of immune cells is an efficient strategy for promoting vaccination [65,68] . Lectin receptors, which act as molecular targets for sugar molecules, are found on the surface of phagocytic cells of the immune system. Dutta et al. reported that mannosylated fifth generation poly(propyleneimine) (MPPI) dendrimers can significantly improve the cellular uptake and consequently anti-HIV activity of lamivudine compared to free drug [66] . The mannose receptor, which is highly expressed on the surface of immune cells, is a useful molecule target for improving vaccination [62] . It was found that the intracellular trafficking and the cross-presentation efficiency were significantly increased by endoplasmic reticulum-targeted PLGA


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Table 2. Summary of selected formulation for active nanoimmunotherapy. Carriers

Polymer

Properties

Factors

Indications

Nanoparticles

Polycations

pH responsive

Ag

To disrupt endosomes and mediate drug/cell binding

Ref.

Gamma-PGA

Biodegradable

Ag

Inducing strong cellular and humoral immune responses

[44,60]

Gamma-PGA

Biodegradable

OVA

Great potential as novel and efficient protein-based vaccines

[45]

Gamma-PGA

Biodegradable

OVA

Eliciting effector T cells without histopathological changes

[46]

Gamma-PGA

Biodegradable

OVA

Universal delivery system for protein-based vaccines

[47]

PLGA

Biodegradable

OVA

Inducing potent specific CD4 + and CD8 + T-cell responses

Mesoporous silica/PEI

pH sensitive

pDNA

Potential gene carrier to antigen-presenting cells

[51]

PLL-PSt

pH sensitive

pDNA

Highly effective for the delivery of DNA at small sizes

[53]

PEI

pH sensitive

siRNA

Reversing tumor-induced immunosuppression

[54]

PLA

Biodegradable

HBsAg

Continuous release of a high concentration of antigen

[41]

Liposomes

Lipids

–

Ag, siRNA

Generate a protective immune response

[50]

Lipids/ ethosomes

–

HBsAg

Ethosomes demonstrated higher efficiency

[52]

R8-Lip

–

Ag

Enhanced antigen presentation and CTL activity

[62]

MTLV

pH sensitive

Ag/ Eliciting CD8 + T-cell responses adjuvant

[63]

Micelles

Gamma-PGAL-PAE

Biodegradable

OVA

Eliciting potent immune responses

[64]

PEG-PAEM

pH sensitive

pDNA

Practically useful DNA vaccine delivery systems

[56]

PEG-PPS

Redox sensitive

OVA

Efficient antigen delivery vehicles

[57]

PEA-PEO/PLL

pH sensitive

Peptide

Efficient peptide vectors immunogically

[58]

PEGPDMAEMA

pH sensitive

mRNA

High transfection efficiencies in immune cells

[59]

Polymer conjugates

PPAA-PDSA

Re-oxd sensitive OVA

CD8-enhancing vaccine delivery system

[65]

Dendrimer

MPPI/PPI

–

3TC

Enhanced cellular uptake of 3TC

[66]

Nanogel

cCHP

–

BoHc/A

Adjuvant-free intranasal vaccines

[67]

[55]

[48,61]

3TC: Lamivudine; Ag: Antigen; BoHc/A: Neurotoxin; cCHP: Cholesteryl-group-bearing pullulan; CTL: Cytotoxic T lymphocyte; DMAEMA: Dimethylaminoethyl methacrylate; Gamma-PGA: Poly(gamma-glutamic acid); HBsAg: Hepatitis B surface antigen; L-PAE: L-phenylalanine ethyl ester; MPPI: Poly(propyleneimine) dendrimers; MTLV: Multilamellar vesicles; OVA: Ovalbumin; PAEM: Poly(aminoethyl methacrylate); pDNA: Plasmid DNA; PEA-PEO: Polymethacrylic acid-b-polyethylene oxide; PEI: Polyethylenimine; PLA: Polylactide; PLGA: Poly( d,l-lacticco-glycolic acid); PLL: Poly-l-lysine; PLL-PSt: PLL-coated polystyrene; PPAA-PDSA: Poly(propylacrylic acid-co-pyridyldisulfide acrylate); PPS: Poly(propylene sulfide); R8-Lip: Octaarginine-modified liposomes.

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nanoparticles [64] . Codelivery systems such as the lipopolysaccharide-modified PLGA nanoparticles loaded with OVA are preferentially internalized by DCs and elicit potent humoral and cellular immunity [60] . Furthermore, immunization with antigens and immunomodulators encapsulated in PLGA nanoparticles elicits potent cellular immune responses [69] . Coencapsulating TRP2 and a TLR ligand (7-acyl lipid A) into a PLGA nanoparticle activated TRP2-specific CD8 T cells, which secreted IFN-γ at lymph nodes and in the spleen of vaccinated mice [61] . By fine assembly, the interbilayer-crosslinked vesicles codelivered protein antigens (in the core) and lipid-based immunostimulatory molecules (in the interbilayer-crosslinked multilamellar), resulting in an extremely potent whole-protein vaccine that elicited endogenous T-cell and antibody responses that were comparable to those produced by the strongest vaccine vectors [63] . Delivery routes play an important role in vaccination. The nasal epithelium is characterized by relatively high permeability, low enzymatic activity and by the presence of an important number of immunocompetent cells. Besides improved protection and facilitated transport of the antigen, nanogel-based delivery systems could lead to a universal protein-based antigen delivery vehicle for adjuvant-free intranasal vaccination. [67] . For oral vaccination, the particulates used should protect the antigens from degradation. Nanoparticles made from N-trimethylated chitosanloaded with a model antigen OVA were prepared by ionic cationic interaction. Intraduodenal vaccination with OVA-loaded nanoparticles produced a significantly higher antibody response than immunization with OVA alone [70] . The size of the delivery system was a dominate factor of the vaccine. Immunization with nanoparticles (200–600 nm) was associated with higher levels of IFN-γ production, and upregulation of MHC class I molecules along with antibody isotypes that favor a Th1-type immune response. Immunization with microparticles (2–8 μm) promoted IL-4 secretion, upregulated MHC class II molecules and favored a Th2-type immune response [71] . The route of administration of vaccine-based delivery systems affected the immune response [72] . DCs were found to accumulate in the epidermis and dermis of human or animal skin. Thus, various methods of dermal or transcutaneous vaccination attract wide interest due to the enhanced humoral and cellular responses [73] . Passive nanoimmunotherapy

As mentioned above, mAbs and antibody fragments are powerful therapeutic agents for various debilitating diseases, as shown in Table 1. The antibodies used in traditional passive immunotherapy are generally pro-


Review

duced in vitro. The high production costs and functional limitations such as inadequate pharmacokinetics and tissue accessibility are the current principal disadvantages for the use of antibodies in a clinical setting. However, passive nanoimmunotherapy based on nanoparticulates can improve the mAb’s in vivo stability and intratumor accumulation, while lowering degradation and side effects [74] . Additionally, the application of new pharmaceutical nanotechnology for controlled, sustained and targeted delivery of antibodies to cancer cells represents a new area for immunotherapy [75] . Many delivery systems were thus developed for passive nanoimmunotherapy. The representative samples of passive nanoimmunotherapy are summarized in Table 3. Belaunzaran et al. designed a long-term antibodyfragment delivery system via an allogenous immunoisolated implant, which consisted of polymerencapsulated myoblasts. This system was engineered to chronically release single-chain variable fragment antibodies targeted against the N-terminus of the amyloid-β peptide [76] . Following a 6-month course of intracerebral therapy in a APP23 transgenic mouse model, the symptoms of Alzheimer’s disease were significantly suppressed with a significant reduction in the aggregation of the amyloid-β peptide. This research indicates that the sustained local release of antibodies using immunoisolated polymer implants has high therapeutic potential . Hollow mesoporous silica capsules (HMSCs) are also potential carriers for protein (antibody) delivery. Cell membrane-permeable HMSCs were prepared by Lim et al. for the delivery of mAbs. The surface hole size was about 20–50 nm while the particle size was around 100–300 nm. The HMSCs effectively delivered the antibodies into the HeLa cells [77] .The animal experiments also demonstrated the effectiveness of the local delivery of antibodies in cancer therapy, where local delivery of CTLA-4 antibody entrapped in functionalized mesoporous silica (FMS) with super high density (0.4–0.8 mg of antibody/mg of FMS) by noncovalent interactions, showed a significantly increased antitumor effect compared to systemic injection of free antibody [78] . Functional polymers have also been designed to control antibody delivery for promoting the intracellular release of immuno factors [79–85] . A successful example was the charge conversional polyion complex micelles, which can delivery antibodies to living cells via the pH-sensitive PEG-poly{N-[N’-2-(aminoethyl)-2-aminothyl]aspartamide} micelles [80] . A well-defined vesicle self-assembled from the pH-sensitive block copolymer poly[2-(methacryloyloxy)ethyl phosphorylcholine]-b[2-(diisopropylamino)ethyl methacrylate] successfully targeted delivery of the antibodies to their respective


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The principles for traditional immunotherapy are based on in vivo immunobalance regulated by negative (tumor) and positive (host) factors. Such linear immunobalance is difficult to maintain. In the nanoimmunotherapy, nanocarriers can produce sustained, controlled and targeted delivery of drugs for biological therapy and/or evoking immune responses [86] . In this scenario, a triangle relation appears among host, tumor and smart nano immune system when we introduce the nanocarriers with immunomodulatory features into the traditional lineal immunorelation (Figure 2) . The first is the interaction between the host and tumor involves reciprocal immunosurveilence and tumor immune-escape. The second is the relation between the host and nanocarriers where promotes the designment of smart nanoparticutes based on host conditions. The third relation between the tumor and smart nano immune system helps us to optimize the nanocarriers based on the tumor microenvironment by chemical design. By fine nanocarrier engineering and pharmaceutical nanotechnology, we can thus tip the triangle immunobalance toward increased host immunosurveillance and minimized

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tumor immune-escape, finally resulting in possible tumor cure [87] . Tumors possess significantly distinct properties from normal tissue with unique tumor microenviroments, as shown in Figure 3. It was reported that the microvasculature of solid tumors is abnormal with large gaps between the endothelial cells (200–700 nm or even up to 1.2 μm, depending on the tumor type) [89] . The heterogenetic structure and uneven distribution of the tumor blood vessels slows down the energy exchange within tumors. Both inhomogeneous blood vessels and a slow rate of mass exchange can result in unique tumor characteristics: the abnormal tumor blood vessels may display a gap of 200–700 nm, and the tumor may have a relatively high temperature (>37°C) and a relative low pH (pH