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Biological drug and drug delivery-mediated immunotherapy

The initiation and development of major inflammatory diseases, i.e., cancer, vascular inflammation, and some autoimmune diseases are closely linked to the immune system. Biologics-based immunotherapy is exerting a critical role against these diseases, whereas the usage of the immunomodulators is always limited by various factors such as susceptibility to digestion by enzymes in vivo, poor penetration across biological barriers, and rapid clearance by the reticuloendothelial system. Drug delivery strategies are potent to promote their delivery. Herein, we reviewed the potential targets for immunotherapy against the major inflammatory diseases, discussed the biologics and drug delivery systems involved in the immunotherapy, particularly highlighted the approved therapy tactics, and finally offer perspectives in this field.


Introduction
Inflammatory diseases including cardiovascular diseases, cancer, allergies, autoimmune, and neuropsychiatric diseases commonly feature dysregulation of immune response 1 . For instance, atherosclerosis (AS) starting with dysfunctional alternation in the endothelium demonstrates increased recruitments of immune cells encompassing lymphocytes, antigen-presenting cells (APCs) and monocytes/macrophages 2 . Immunotherapy refers to disease treatment through activating or inhibiting the immune system. Unlike traditional treatments, the immunotherapy exerts therapeutic efficacy through modifying the endogenous immune response, or reversing the immunosuppression conditions of diseases 3 , always characterized by changed infiltration of immune cells and modified expression of immune factors at the allergen 4 . Immunotherapy possesses significant advantages over traditional treatment regimes, such as higher efficacy against disease with fewer off-target effects and more durable response. Increasing evidence demonstrates immunotherapy is potent to treat malignant diseases, and cancer immunotherapy is becoming widely accepted in the clinic 5 . Furthermore, immunotherapy has promising potential to treat other inflammatory disorders, e.g., AS 6 , rheumatoid arthritis (RA) 7 , intestinal inflammation 8 , and pulmonary arterial hypertension (PAH) 9 .
To target the pathogenesis of diseases, innate or adaptive immune system 10,11 , numerous biological drugs were approved, including anti-tumor necrosis factor (anti-TNF) agents (etanercept, adalimumab, infliximab) 12 , immune checkpoint (ICP) blockers (ipilimumab, tremelimumab, pembrolizumab) 13 , and interleukin (IL)-family agonists (nivolumab) or antagonists (tocilizumab) 14,15 . The approved immunotherapy is present in Table 1 16e24 . However, the use of immunotherapies is always limited by several factors. For instance, repeated administration of the immunomodulators at high dose is always required and, as a result, may induce a series of autoimmune-mediated side effects 25 , such as flu-like reactions, and vascular leak syndrome 26 , and show a significant individual variation. Furthermore, most immunomodulators belong to biological drugs separated from or manufactured by biological systems and always are characterized by increased size, low stability, poor penetration into the diseased site and limited ability to cross cell membrane. Drug delivery using carriers including liposomes, hydrogels, living cells, microspheres, inorganic materials, polymeric micelles, drug crystals, and protein vehicles is robust to improve the treatment efficacy of biological drugs 27e32 , owing to the advantages including extended blooded circulation, improved accumulation, promoted penetration in diseased tissues, increased uptake, and high drug-loading ability, large surface areas, and easy decoration of physicochemical properties 1,27,33e36 . Especially, more than 65 nanoscale drug delivery systems (DDSs) were marked for commercial use. In this review, we summarize the immunotherapy implications in major inflammatory diseases, highlight the biopharmaceuticals and DDS utilized to improve the delivery of immunomodulators, and finally provide perspectives in this field.

Potential targets for cancer immunotherapy
Immunotherapy of cancer is attracting huge attention for its remarkable success in the clinic. Via targeting the immune system and overcoming the acquired drug resistance, the immunologically "cold" tumors lacking immune infiltrate can be converted into "hot" tumors having dense T cell infiltrate through efficient approaches 37 , exhibiting as mobilization of the immune cells and eliminating the cancer cells 10 . In general, the immune response could be prompted by modulating the production of immune factors and ICPs and motivating the immune cells (Fig. 1).

Cytokines and vaccines
Cytokines are potent to modulate the immune system. Three main types of cytokine are involved in cancer immunotherapy, including ILs such as IL-2, IL-12, IL-15, and IL-21, interferons (IFNs), and granulocyteemacrophage colony-stimulating factor (GM-CSF) 38 . The recombinant cytokine IFNa is the first approved for clinical use in 1986 39 , followed by recombinant IL-2 40 . However, a high dose of cytokines was required for effective treatment efficacy, and frequently leads to a series of unwanted effects, e.g., capillary-leak syndrome and cytokine-release syndrome 41 . The combined use of cytokines with checkpoint inhibitors or anticancer monoclonal antibodies (mAbs) might improve anti-tumor efficacy 42 .
Therapeutic cancer vaccines whose activities closely rely on cytotoxic T cells combat the disease via abolishing cancer cells or abnormal cells 43 . The cancer vaccines are divided into four classes: peptide vaccines, cell-based vaccines, viral vector vaccines, and nucleic acid vaccines 44 . APCs, especially dendritic cells (DCs), are essential to the vaccination because they are  43 . Three cancer vaccines, Gardasil, Cervarix, and Sipuleucel-T were commercially marketed. However, the mutation of antigens is always unique to individuals and, therefore, compromises the treatment efficacy of the commonly used vaccine. The personalized vaccine is a potential route to overcome the shortage.

mAb and ICP suppressors
By targeting surface antigens differentially expressed on cancer cells, such as CD20, CD33, CD52, human epidermal growth factor-2 (HER2), vascular endothelial growth factor (VEGF), epidermal growth factor receptor (EGFR), the antibody exerts cancer immunotherapy via means including the antibodydependent cellular cytotoxicity and complement-mediated cytotoxicity 45 . mAbs represent the most frequently employed cancer immunotherapy in the clinic, and over 30 products were approved.
2.1.2.1. CTLA-4. T cells are activated via binding their surface CD28 with B7-1 (CD80) or B7-2 (CD86) on the APCs 50 . However, the CD28 homolog CTLA-4, possesses a significantly greater binding affinity toward B7 51 and, as a result, leads to blockade of T cell upregulation and activation. Anti-CTLA-4 acts through blocking the connection between B7 and CTLA-4. Human CTLA-4 antibodies, ipilimumab, was approved to treat advanced metastatic cancer; and while another CLTLA-4 blockade, tremelimumab, is under clinical trial. The long-lasting anti-tumor response always occurs after dosing, yet accompanying unwanted effects, such as enterocolitis, inflammatory hepatitis, and dermatitis; however, it was argued these toxicities could be discounted by using corticosteroids and whereas did not reduce the anti-tumor effects 52 .
2.1.2.2. PD-1 and PD-L1. PD-1 is categorized into the CD28 superfamily as well, whereas PD-L1 and PD-L2 are classified as the B7 family. The expression of the PD-1 was found predominantly on three immune cells in the periphery, activated CD8 þ and CD4 þ T cells and B cells 53 . The binding with the ligand, PD-L1 or PD-L2, allow PD-1 to recruit the sarcoma gene (Src) homology 2 domain-containing tyrosine phosphatase 2 (SHP-2) and inhibit the T-cell activities 54 , e.g., T-cell expansion and effector functions including release of IFN-g and cytotoxicity 55 . It should be noteworthy that PD-1 mainly reduce effector T-cell functions at the later-phase of immune reaction while CTLA-4 engages at the early stage 56 .
Blockades of CTLA-4 or PD-1-based signaling are effective to combat cancer, however, monotherapy may induce adverse effects occasionally probably due to the individual variations 25,58 . Combinatorial treatment, e.g., anti-CTLA-4 plus anti-PD-1 and PD-1 plus PD-L1 antibodies, provides the potential to eliminate or alleviate the side effects.

Adoptive T cell therapy (ACT)
ACT refers to the transfer of isolated T cells from the patient that are genetically engineered in vitro to the same patient 59 , including tumor-infiltrating lymphocytes (TILs), T cell receptor (TCR) T cells, and chimeric antigen receptor (CAR) T cells 60 (Fig. 1). The FDA has approved CAR therapy for adult patients with leukemia and lymphoma.
For TIL therapy, the TILs are extracted from the separated tumors, sorted with endogenous TCRs, purified, and ultimately undergo a rapid expansion protocol in vitro using with IL-2 and CD3 antibody 61 . For TCR therapy, TCR composed of an aand a b-chains is anchored on T cells through noncovalently binding with CD3 complex. T cells become cytotoxic T cells when the anchored TCR is recognized and binds with the MHC on APCs or tumor cells 60 . Rapoport et al. 62 developed NY-ESO-1/LAGE-1 TCR-engineered T cells to treat multiple myeloma (MM). The engineered T cells were infused into twenty patients with MM at a cell number of 2.4 Â 10 9 two days after dosing autologous stem cell 62 . The results indicated that the engineered T cells could proliferate, move to the marrow, and kill the cancer cells selectively, with clinical response of up to 80% and median survival of 19.1 months 62 .
To overcome the limitations of TCR therapy, e.g., the requirement of MHC expression, MHC identity, and costimulation, CAR therapy was developed via adding CAR genes on T cells comprised of an extracellular single-chain variable fragment (scFv), a transmembrane spacer, and intracellular signaling/ activation domain(s) 63 .
ACT therapy has demonstrated its success in the treatment of cancers and several products were approved for clinical use. However, increasing limitations of the therapy are revealed as well, i.e., on-target/off-tumor toxicity, cytokine-release syndrome, neurologic toxicities, off-targeting reactivity, complicated fabrication, extremely high cost, etc. 64e66 .

Drug delivery-mediated cancer immunotherapy
To promote the immune response toward cancer, repeated administration of immunomodulators at a high dose is always required because most immunostimulants are unstable in physiological conditions, have the poor tumor-targeting ability, and can't translocate the plasma membrane, etc. 27 . Such a dosing approach frequently induces side effects and compromises the patient's compliance. As a result, dosing the immunomodulators in a controllable and safe way is highly expected. Drug carriers, e.g., liposomes 67 and dendrimers 68 , are effective to improve the delivery due to their well-known advantages (Fig. 1). Numerous drug carriers were reported to improve the efficacy of immunotherapeutic agents such as T cell activators, ICP inhibitors, and cytokines 69 , via increasing their circulation time 70 and target-ability to immune cells 71 . Several reviews summarized the use of a drug delivery strategy to improve cancer immunotherapy 69,72e75 . The most commonly used carriers include polymer nanoparticles (NPs), inorganic NPs, and lipid-based NPs 72,76,77 . Recently, to lower the cost and facilitate the expansion of T lymphocytes for CAR therapy without complicated procedures, Smith et al. 78 developed plasmid DNA-loaded polymeric nanocarriers decorated with T-cell-targeting anti-CD3e f(ab')2 fragments to deliver leukemia-specific CAR genes into host T cells in situ. They found that the 155-nm NPs were able to rapidly and selectively program circulating T cells in vivo and demonstrated improved leukemia regression over the treatment with conventional CAR therapy 78 . This work represents a new use of the DDS aiming to reduce the cost of ACT and avoid complications of clinical-scale manufacturing. Furthermore, changing the basic properties of NPs, e.g., diameter, shape and surface charge, are potent to modulate the immunotherapy 73 . For instance, smaller NPs (<50 nm) have enhanced ability to elicit the immune activities over the large NPs (>100 nm) because the smaller ones tend to traffic to lymph nodes via DCs, whereas the larger ones are difficult to move once accumulating at the diseased site 72 . The NPs with a diameter of over 500 nm can target macrophages and are internalized via phagocytosis 74 .
Another significant advantage of using drug carriers is the efficiency to facilitate the combinational therapy. The durable immune response is only indicated in limited cancer types when an immunostimulant is used alone. Such that immunomodulator and other anti-tumor inhibitors are always combined for use in the clinic; however, their active targets are spatio-temporarily discrepant and, as a result, often leads to sub-optimized treatment efficacy. Drug carriers have a remarkable potential to deliver the agents to their respective active sites precisely by co-delivery approach or physicochemical triggering means. For example, by using nanoclews based on long-chain single-strand DNA as a carrier that could be enzymatically degraded in inflammation conditions, CpG oligodeoxynucleotides (CpG ODNs) and anti-PD-1 antibody were released in a sustained pattern 79 . The results demonstrated that the codelivery system synergistically induced long-lasting anti-tumor T lymphocyte responses in a melanoma model 79 .
The nanocarriers demonstrated their promising potential to promote the treatment efficacy of cancer immunotherapy. Nonetheless, few of them are translated, mainly owing to the poor reproducibility and scalability, unpredictable toxicity in vivo, etc 80 . Several techniques such as bubble blown assembly, capillary-force-assisted assembly, electric-field-assisted assembly, and LangmuireBlodgett assembly were developed to scale up the nanomaterials 81 . Yet, it is difficult to acquire a commonly used approach to fabricate the devices since they always have their unique features. Furthermore, many studies for cancer immunotherapy was performed on the mouse models while there are huge discrepancy between the animal and human immune systems, the efficacy appeared on mouse may have poor correlation with human patients 82 .

Immunotherapy for autoimmune disease therapy
Autoimmune diseases encompassing RA, multiple sclerosis, inflammatory bowel diseases (IBD), mainly results from dysregulation of the T cell checkpoint pathways 83 . Especially, the helper T cells have profound effect on the progression of these diseases, since they often affect the function of other immune cells, e.g., regulatory T cells (Tregs), monocytes and macrophages 84 .

RA
RA is a chronic inflammation and frequently demonstrates damage of both articular cartilage and bone 85 . The exact pathological mechanism of RA is unclear, but it is well accepted that RA is closely linked to the breakdown of immune tolerance 86 . The immunotherapy by modulating the differentiation of lymphocytes and secretion of cytokines may combat RA (Fig. 2).
3.1.1. Implications for immunotherapy in RA 3.1.1.1. Regulation of lymphocytes. Four lymphocyte subpopulations, Tregs, T helper 17 (Th17) cells, and regulatory B lymphocytes (Bregs), affect the process of RA 87 . Furthermore, Lamas et al. 88 discovered that the activation extends of peripheral blood mononuclear cells (PBMCs) and the disease activity allowed for immunomodulatory effect of bone marrow-derived mesenchymal stem cells (MSCs) on T-cell activation. Accordingly, the immunotherapy should concentrate on the modulation of these lymphocytes.
A deficit of Tregs was demonstrated to promote the RA development and increasing proliferation of Tregs via anti-TNF treatment benefits to the suppression of RA 89 . Consequently, the activation of Tregs is the potential to ameliorate RA. These activators include IL-2 90 , T cell superagonists (CD28SAs), and nondepleting anti-CD4 mAbs 91 . Second, the subsets of B cells, i.e., Bregs 92 , memory B cells (CD24 hi CD27 þ phenotype) 93 , and B10 þ cells 94 , are potential targets to treat RA due to increased secretion of IL-10 92 , improved proliferating of Tregs 94 , or reduced expansion of Th1, Th17, TNFa þ T cells 95 . However, it was reported that, in patients with RA, the CD24 hi CD27 þ and the CD24 hi CD38 hi B cells may not enhance the Treg's proliferation or decrease Th1 and TNFa þ T cells although the abundance of the two sets is similar to that in the healthy 96 . In this situation, the adoptive transfer of Bregs has the potential to alleviate the symptoms of the disease 97 . Besides, the synovial macrophages advance the process of RA via the secreting IL-6 and TNF-a and the resultant damage of the joint 1 .
Overall, via reeducating or depleting the autoreactive cells, the process of RA can be inhibited via inducing immune tolerance to self-antigens 98 . The antigen-specific immunotherapy (ASIT) using peptides, antibodies, vaccines, etc. is extensively employed to target the autoreactive cells, i.e., T and B cells and DCs 98 . Recently, a pcDNA-CCOL2A1 DNA vaccine was developed to treat collagen-induced RA 99 . The administration via intramuscular injection at 300 mg/kg pcDNA-CCOL2A1 enabled decreased percentages of CD4 þ CD29 þ and transferred Th1 to Th2 and Tc1 to Tc2, along with the reduced level of Th1 cytokines and downregulation of proinflammatory modulators IL-10 and transforming growth factor b (TGF-b) derived from Th2 and Th3, respectively 99 .

Cytokines and chemokines.
Cytokines and chemokines have a robust ability to regulate intercellular interactions, cell activation, localization, and phenotype in the lymphoid environment 100 . The cytokines, in particular TNF and IL-6 101 , IL family, and GM-CSF 101 , promote the process of RA 102 .
TNF, a multifunctional cytokine, often exacerbates inflammation via increasing T-cell proliferation and differentiation at various stages 103 , i.e., single positive thymocytes and CD3/CD4/CD8 (triple-negative) T cells 104 , and activating the immune system via the control of secondary lymphoid organs structures 103 . Anti-TNF-a treatment is potent to treat RA via increasing Treg proportion and suppressing effector T cell (T eff ) 105 , affecting T peripheral helper (T ph ) cells that may prevent the differentiation of plasma-blasts 106 , decreasing activated B cells, and expanding regulatory B10 cells 107 .
IL-6, an activator of B and T cells, facilitates the differentiation of B cells into lg-producing plasmablasts, directs the expansion of antigen inexperienced CD4 þ T cells, as a consequence, promotes the transition of innate immunity to adaptive immunity 108 . IL-6 inhibitors, such as IL-6 mAbs and miRNA targeting IL-6 108 , toll-like receptor (TLR) 4 inhibitor 109 , demonstrated promising inhibition of RA 109 . In particular, the IL-6 mAbs exhibited outstanding efficacy against RA 108 .
So far, some mAbs capable of neutralizing TNF-a have been approved for the clinical use, including etanercept, infliximab, certolizumab pegol, golimumab, adalimumab, and other blockers such as mAbs IL-6 (tocilizumab) and IL-1R (anakinra) 110 . The turbulence of immune cells, and the immune response is closely linked to RA progression. Consequently, the regulation of immune cells or immune response is promising to alleviate RA. However, individual treatment should not be ignored, since the gene expression and sensitivities are differentiated among person to person.
3.1.1.3. Janus kinase (JAK) inhibitors. The JAK pathway also links to the development of diverse immune-dependent disorders, e.g., RA and IBD, by promoting the signal transduction of immunostimulators 111 . The JAK, mainly composed of JAK1, JAK2, JAK3, and TYK2, acts through the receptors of type I and II. Type-I receptor generally associates with ILs, hormones and colony-stimulating factors, whereas Type-II receptor binds with IL-10-family cytokines including IL-10, IL-19, IL-20, IL-22 and IL-26 112 and IFNs. Two inhibitors of JAK, tofacitinib and baricitinib, were marked in 2018 and 2012, respectively, to treat RA 113 .

Drug delivery-mediated immunotherapy in RA
The used drugs against RA mainly consist of nonsteroidal antiinflammatory drugs (NSAIDs), corticosteroids, disease-modifying antirheumatic drugs (DMARDs), and biological DMARDs (bDMARDs), and are always dosed via oral delivery or injection. Various DDSs were adopted to improve the delivery of modulators to the immune cells, e.g., lipid-based NPs 114e116 , polymeric NPs 117,118 , hydrogels 119 , gold NPs 120 , pH-sensitive NPs 118 , and biological membrane-coated NPs 121e123 . Surface modification with ligands or peptide modification allows for improved targeting-ability to immune cells 124 , and cytokines and chemokines pathways, such as nuclear factor k-B (NF-kB), ERK signal pathway, IL related pathway, etc 91 . Nonetheless, the most frequently reported ligand is folate receptor b that is overexpressed on the activated macrophages 125 .
Liposomes or liposome-like NPs are the most widely used DDS for RA treatment due to their excellent encapsulation-ability and biocompatibility 126 . Recently, lipidoid-polymer hybrid NPs were designed to deliver siRNA against IL-1b to the activated macrophages to inhibit the pathogenesis of RA induced by collagen antibody 115 . Dosing via intravenous injection of the NPs enabled the efficient delivery of siRNA to macrophages inhibiting in the arthritic joints, downregulation of inflammation-induced modulators in the joints, and a significant reduction in the cartilage destruction, swelling of ankle and bone damage 115 . Furthermore, such DDS facilitates codelivery for combined therapy. For instance, hybrid-NPs consisted of calcium phosphate/liposomes were developed to deliver methotrexate and NF-kB-specific siRNA to the lipopolysaccharide (LPS)-activated macrophages at the diseased site 127 . Egg phosphatidylcholine liposomes were used to co-load an antigen, OVA or methylated BSA, and a waterinsoluble inhibitor of NF-kB, curcumin or quercetin, and targeted APCs 116 , demonstrating as suppressing the response of the cells to proinflammatory pathway and promoting the proliferation of Ag-specific Foxp3 þ Tregs 116 .
Biological membrane from living cells, e.g., red blood cells, platelet, neutrophils, and macrophages, is always rich in various biomarkers and receptors; and as a result, its coating is able to alter the biological properties of DDSs and elevate their celltargeting ability 121 . Motivated by the association of platelet with RA, platelet-like NPs were fabricated to deliver the antiinflammatory tacrolimus to the joints of a collagen-induced arthritis 122 . Through GVPI recognition and P-selectin, these NPs allowed for efficient drug accumulation in the joint and inhibited RA's development 122 . Interestingly, drug-free neutrophil membrane-coated poly lactic-co-glycolic acid (PLGA) based NPs were developed recently 123 . Via neutralizing the inflammationinduced TNF-a and IL-1b, these neutrophil-NPs exhibited synovial inflammation, robust chondroprotection against joint damage, and enhanced penetration into the cartilage matrix 123 . Furthermore, their anti-RA effectiveness in both human-transgenic arthritic model and collagen-induced model is comparable to that from the treatment with anti-TNF-a or anti-IL-1b 123 . These biomimetic-targeting DDSs, due to their natural targeting-ability to the inflamed sites, represent a promising approach against RA and may have the potential of clinical translation because of the simple preparation process. Nevertheless, their translation is still limited by the scalability of DDS.

Intestinal inflammation
3.2.1. Potential targets for IBD immunotherapy IBD is always characterized by long-lasting inflammation and divided into ulcerative colitis (UC) and Crohn's disease (CD). The IBD pathogenesis has not been illustrated fully, however, is often characterized by an imbalance between the mucosal immune system and the commensal ecosystem 128 . The regulatory immune cells including intestinal intraepithelial lymphocytes, T and B cells, macrophages, DCs and innate lymphoid cells could affect the progression of IBD 129 . DCs contribute the maintaining of immune environment homeostasis in the intestine via connecting humoral and cellular immune response. Especially, Tregs play a critical role in limiting the populations of Teffs and innate inflammatory signaling 130 . Antigen-specific T-helper cells and natural killer (NK) cells contribute to inflammation in IBD as well and their influx can be used as a potential treatment target 131 . In addition, agitations in intestinal epithelial cells, in particular Paneth cells, may initiate intestinal inflammation 131 . The therapy strategies toward IBD are classified into anti-inflammatory treatment with mesalazine and glucocorticoids, antibiotics therapy using ciprofloxacin and metronidazole, gene therapy, and immunotherapy with immunosuppressants and anti-TNF agents 8 . The immunotherapy is acquired through interfering with IL-12/23 axis, JAK and TGF-b/Smad 7 pathways, and modulating IL-6, IL-13, chemokines and chemokine receptors CC receptor 9eCC chemokine ligand 25 (CCR9eCCL25) 132 , and cell adhesion and leukocyte recruitment 133 . The IBD immunotherapy can also be achieved by using adoptive cell transfer, such as MSCs and engineered Tregs 8 . Previous reviews summarized the use of biological drugs for IBD immunotherapy 133,134 . Currently, about seven mAbs were approved for IBD immunotherapy, including ustekinumab, natalizumab, infliximab, vedolizumab, golimumab, certolizumab pegol and adalimumab. The implication of immunotherapy for IBD is illustrated in Fig. 3.

Drug delivery-mediated immunotherapy in IBD
The mAbs are effective to treat IBD; however, the response rate to initial treatment is only 50% and their use is always limited by systematic side-effects including immunogenicity, the induction of anti-drug antibodies, serum sickness, etc., induced by administration via intravenous injection 135 . DDS-mediated therapy may elevate treatment outcomes and reduce systemic toxicity. A previous review summarized various approaches and devices for targeting treatment of IBD using DDS, encompassing meanings of ligand-receptor-, charge-, degradation-, size-and microbiomemediation 136 . Another review discussed intestine targeting strategies, e.g., conventional DDS-mediated treatment, diseasemediated delivery of active agents by synthetic and biological DDS 137 . Given that oral administration is a well-accepted delivery route for both patients and physicians 138 , herein we mainly discuss targeted oral delivery of immunomodulators to the inflamed sites in the large intestine. The site-specific DDSs are often designed according to (i) the physiological changes in the gastrointestinal tract such as pH, microflora, transit time, pressure, and osmotic potential 139,140 or (ii) disease-induced alterations, i.e., increased permeability, changes in tight junctions and mucus composition and amount, reduced antimicrobial secretions and numbers of secretory cells and loss of the area of ulcerated epithelium 141e145 . The recently reported DDSs include hydrogel platform 146e149 , redox-and pH-sensitive NPs 150e155 , hyaluronic-based NPs 148 Recently, an oral inflammation-targeting hydrogel (IT-hydrogel) assembled from ascorbyl palmitate (AP) was developed to treat IBD 149 . IT-hydrogel microfibers encapsulating corticosteroid dexamethasone (Dex) could adhere to the inflamed mucosa from animal and human colon and exhibited increased drug release at the inflammation site due to the degradation by the enzymes secreted from active-macrophages and other immune cells. In clitic ulcerative mice administrated via a single enema with free Dex as control, dosing with the drug-loaded IT-hydrogel enabled significant reduction of colon weight, myeloperoxidase (MPO) activity, and expression of TNF in the distal colon, and lowered the systemic drug exposure 149 . Another reactive oxygen species (ROS) responsive assembles prepared from HA-bilirubin conjugate were fabricated to combat dextran sulfate sodium (DSS)induced acute colitis 156 . In vitro, the assembles dissociated rapidly after exposure to ROS, were well taken up by macrophages and granulocytes due to the hyaluronic acid (HA)-CD44 affinity, polarized pro-inflammatory M1 macrophages into the M2 phenotype. In vivo, in contrast with treatment with clinically used drugs, the treatment had remarkably boosted efficiency to combat DSS-induced acute colitis via decreasing the impairment of colon and MPO activity, recovering the body weight, and keeping the length of colon 156 . In addition, the treatment markedly reduced the infiltration of pro-inflammatory phenotypes, CD11b þ Ly6C þ Ly6G þ neutrophils and CD11b þ Ly6C þ Ly6G À monocytes, in the layer of lamina propria in DSS-induced model, and increased the accumulation of anti-inflammatory phenotypes including CD3 þ CD4 þ Foxp3 þ Tregs, MHCII þ CD11c þ CD11b À DCs and CD11b þ Ly6C À Ly6G-MHCII þ tissue-resident macrophages 156 . Overall, increasing oral DDSs against IBD is emerging, such as polymer-drug prodrug formulations 152,163 ,microspheric vehicles to suppress TNF-a 154,164 , thermoreversible mucoadhesive polymer-drug dispersion with prolonged retention at the inflamed site 165 , and biomimetic NPs, i.e., cell membrane-coated NPs and liposomes engineered with cell membrane proteins 166 . These oral inflammation-targeting DDSs represent a promising strategy to treat IBD, owing to their scalability, biocompatibility, and potent therapeutic efficacy. Numerous DDSs were designed to treat IBD via targeting macrophages 167,168 , whereas exosomes isolated from the TGF-b1 gene-modified DCs was demonstrated to inhibit the progression of IBD via eliciting immunosuppression 169 . The 50e100-nm exosomes can efficiently block the advance of DSStriggered IBD via inducing Tregs through the TGF-b1 pathway and reducing the Th17 in the inflammatory site, mesentery lymph nodes 169 .

AS
In AS, the immune cells encompassing monocytes, T cells, DCs, neutrophils, NK cells and innate lymphoid cells are recruited to these sites 170e173 , due to the elicited local inflammation by apolipoprotein-B-containing lipoproteins (ApoB LPs) that deposit in the artery wall and are liable to modification by oxidation, enzymes and aggregation 174 . The recruited monocytes constantly differentiate into macrophages, a major cell population in the atherosclerotic plaques, and finally become cholesterol-loaded macrophage foam cells, facilitating the plaque formation 175,176 . Whereas the factors, e.g., pro-inflammatory modulators, cholesterol crystals, oxidative stress, oxidized lipids, and dangerassociated molecular patterns (DAMPs) predominantly stemmed from the macrophage foam cells, construct a complicated microenvironment that maintains the local inflammation 177 . A previous review highlighted the role of macrophages in AS development 1 .
Here, we mainly focus on other immune cells involved in AS and related treatment approaches.

Implications for AS immunotherapy
According to the pathological mechanism of AS, the treatment with anti-hypertensive or cholesterol-lowering drugs is far from to cure AS. Several experimental results demonstrated the essential roles of immune activation in the AS development 178,179 . Always, the innate response to AS mediated by stimulating macrophages and endothelial cells (ECs) in the walls of the coronary arteries allows for adaptive immune reactions to the antigens presented to Teff by APCs, i.e., DCs 180 . As a result, targeting inflammation to modulate immune responses against plaque antigens may treat AS fundamentally. The potential targets and used DDSs are displayed in Fig. 4.  181 . In general, cytokine-based treatment drugs are mainly categorized into broad-based immunomodulatory agents, blockade of pro-inflammatory cytokines and activators to induce anti-inflammatory cytokines 182 . Clinical trials uncovered the administration of Canakinumab, a mAb targeting IL-1b, at 150-mg dose every 3 months reduced the inflammation and rate of cardiovascular events, though, did not lower the lipid-level 183 .
Another clinical test demonstrated that dosing Canakinumab with the same regimen allowed for decreased levels of IL-6 and inflammatory biomarker high-sensitivity C-reactive protein (hsCRP), an indicator that the mAb works via inhibiting the IL-1beIL-6 signaling of innate immunity 184 . Accordingly, proinflammatory cytokines can be effective targets for AS therapy and IL-1beIL-6eCRP signaling axis is a credible AS-associated inflammatory pathway 185 .

Metabolic regulation of immune cells.
Besides the specific target substances, the altered metabolism of cells in AS may be used as therapeutic potential. The changed metabolism includes upregulated inflammatory activities, the elevated vulnerability of plaque, downregulated fatty acid oxidation (FAO), increased consumption of amino acids (AAs) and upregulated glycolysis in plaque 200 . Abnormal glycolysis always fosters the production of the inflammation-stimulated IL-1b and IL-6 201,202 . Accordingly, supplementary of FAO, during the activation of M2 macrophages 203 and T cells 204 , may stimulate anti-inflammation signals directly or make CD8 þ T cells exert indirect antiinflammatory activity 203e205 . Reduced metabolism of AAs may decrease foam cell formation and reduce plaque size 206,207 .
4.1.1.5. Vaccination against low density lipoprotein (LDL) particles. AS does not belong to an autoimmune disease, however, ApoB is always known as AS antigens. As a result, autoimmune responses against ApoB via vaccination can be a potential therapeutic implication for AS 171 . The vaccination therapy includes using mAbs against the cholesterol ester transfer protein (CETP) or proprotein convertase subtilisin kexin type 9 (PCSK9) and induction of antigen-specific Tregs. PCSK9 is able to damage the LDL receptor and raise plasma LDL cholesterol level 208 , whereas CETP strengthens the change of high density lipoprotein (HDL)-LDL 209 . Consequently, anti-PCSK9 allows for reductions of LDL cholesterol 210,211 , and vaccination with CETP could promote HDL cholesterol levels and decrease the plaque size 212 . In addition, Tregs can suppress the plaque development through limiting Teffs expansion, especially Th1 cells, and reduced production of inflammatory cytokine 213,214 . Accordingly, stimulation of the Tregs may inhibit AS progression by suppressing the activities of immune cells including T cells, NK cells, monocytes, B cells, and DCs and by inducing suppressive modulators such as IL-10, TGF-b and IL-35 215 .

Drug delivery-mediated immunotherapy in AS
The formation of plaque offers numerous opportunities for targeting therapy of AS by using DDS. The commonly reported DDSs are summarized in Fig. 4. The targeting approaches include enhanced accumulation of DDS in the plaque through enhanced permeability and retention effect (EPR)-like or biomimetic mechanism and promoted drug release from DDS in the plaque by microenvironment-responsive strategy. Beldman and coworkers 216 used a kind of HA-NPs to investigate the EPR effect in the plaque of AS progression. They found that the endothelial junction architecture normalized at the later period of AS compared with early AS and the accumulated HA-NPs was decreased. However, the HA-NPs can enter the plaque via endothelial junctions, distribute throughout the extracellular matrix (ECM) and eventually phagocytized by plaque-associated macrophages.
A recent study indicated the unusual and condensed cell morphology and junction irregularities in arterial endothelial layer of ApoE e/e mouse observed by transmission electron microscopy 216 . In the endothelial junctions of AS, the distance between vascular endothelial cadherin (VEC) units is up to 3 mm 216 . Whereas, in the normal vascular endothelial layer, the VEC units were firmly ranked and the space between the VEC units is only about 0.5 mm 216 . Nonetheless, advanced plaques have a small recovery of endothelial junction 216 . Whereas, in the normal vascular endothelial layer, the VEC units were firmly ranked and the space between the VEC units is only about 0.5 mm 216 . However, the stage of AS affects the accumulation and the trafficking pathway of NPs. In advanced plaque, the accumulation of NPs at the AS lesions was closely relied on the transcellular route and was reduced around 30% compared with that in early plaque 216 . Anyway, the findings rationalize the use of nanoscaled DDS for target treatment of AS. So far, DDSs have been widely utilized for AS immunotherapy, such as liposomes 217,218 , recombinant HDL (rHDL) NPs 219 , nanofiber 220 , membrane-coated NPs 221 , polymersomes 222 , and injectable filamentous hydrogel loaded micelles 81 .
Targeting macrophages are the most commonly used strategy in AS immunotherapy 1,167,219 . The ligand signaling of CD40eCD40L is a widely known enhancer of AS and other chronic inflammatory diseases; consequently, its inhibition allows for inhibition of AS 223 . Whereas tumor necrosis factor receptorassociated factor 6 (TRAF6) is potent to boost CD40's signaling cascade inside monocytes and macrophages 190 . As a result, disruption of CD40-TRAF6 interactions can reduce monocyte recruitment to plaques and inhibition the formation of plaque 190 . Recently, to suppress the interplay between CD40 and TRAF6 in macrophages and monocytes, a CD40-TRAF6 inhibitor 6877002 was loaded into 20-nm TRAF6i-rHDL NPs consisted of apolipoprotein AeI (ApoAeI), the 1,2-dimyristoyl-sn-glycero-3phosphatidylcholine (DMPC) and 1-myristoyl-2-hydroxy-snglycero-phosphocholine (MHPC) 224 . The results demonstrated that TRAF6i-HDL NPs could bind well to monocytes and macrophages in the lesion site. The one-week treatment decreased the content of plaque macrophage content and plaque inflammation through the reduction of monocyte accumulation instead of decrement of local macrophage proliferation 224 . The results demonstrated that TRAF6i-HDL NPs could bind well to monocytes and macrophages in the lesion site. The one-week treatment decreased the content of plaque macrophage content and plaque inflammation through the reduction of monocyte accumulation instead of decrement of local macrophage proliferation 219 . TRAF-STOPs enabled AS inhibition by limiting chemokine-induced accumulation of leukocyte to the plaques and suppressing release of cytokine from macrophages. Upon encapsulation in the rHDL NPs, their treatment efficacy was improved, displayed as reduced administration times and total dose over treatment with free TRAF-STOP 219 .
Targeting Tregs or DCs are promising for AS immunotherapy 215,225e227 . DCs have an extremely lower concentration compared with monocytes and macrophages within AS plaque, however, they have an unignored role in promoting the inflammation and AS advance 228e230 . Yi et al. 222 prepared three types NPs, 20 nm-micelles, 100-nm polymersomes, and 50 nm Â micron-length filomicelles using PEG-bl-PPS block copolymers. The results demonstrated that, among the NPs, the 100-nm polymersomes had the highest targeting ability to DCs in the arterial wall and lymphoid organs of animal model 222 . By surface decoration with a P-D2 peptide, the polymersomes could well target DCs and enhance the cytosolic delivery of anti-inflammatory agent 1,25-dihydroxy vitamin D3-(aVD) 231 . Lowdose intravenous administration for a week markedly inhibits the progression of plaque in a high-fat-diet-fed ApoE À/À mice 231 .
AS immunotherapy acquired via activating Tregs include adoptive transfer Tregs, AS relevant antigens such as x-LDL, HSP60, and ApoB100, pharmacological agents such as rapamycin, vitamin D3 and cholesterol-lowering drugs, and antibodies and cytokines such as IL-2 and anti-CD3 antibody 215 . To improve the delivery of the immunostimulators to Tregs, liposomes formulated with the anionic phospholipid 1,2-distearoyl-sn-glycero-3-phosphoglycerol 232 , pH-responsive NPs 233 and filomicelles 81 were developed to selectively deliver LDL-derived peptide antigen, miR-33, and vitamin D to Tregs.
AS immunotherapy displays promising treatment efficacy in preclinical studies, and some of them are undergoing clinical trials 234 . However, their translation is still hampered by various factors, e.g., unknown side effects and immunogenicity, poor scalability of DDS, high cost, and poor patient compliance because the injection is always required. AS immunotherapy obtained via oral administration benefits the translation, whereas it is challenged to prevent the degradation of the biopharmaceuticals or DDS in the gastrointestinal tract. Nonetheless, several oral formulations for AS immunotherapy have been investigated in preclinical studies, such as yeast-derived microcapsules (YCs) encapsulated an inhibitor of MCP-1/CCL2 bindarit 235 , recombinant Mycobacterium smegmatis, a live bacterial vector, that allowed to produce cloned Chlamydia pneumonia (AHC) antigen and induce regulatory immune response to self-proteins 236,237 , low-dose oral cyclophosphamide formulation 238 , algae-based vaccine 239 , and carrot-cell vaccine platform 240 .

PAH
PAH is characterized by an average pulmonary arterial pressure of >25 mmHg while a capillary wedge pressure of 15 mmHg. PAH is an uncommon and serious disease demonstrated as pulmonary vascular remodeling, endothelial abnormality, vasoconstriction and in situ inflammation and thrombosis 241 . Most immune cells, i.e., T cells, DCs, NK cells, macrophages, B cells, mast cells, and eosinophils, are involved in the progression of PAH 1,242 . Accordingly, these immunity pathways can be potential targets for treat PAH. Implications of immunotherapy in PAH are illustrated in Fig. 5 243,244 . A previous review summarized the function of Tregs in PAH 245 . Tregs inhibit the development of PAH through producing cytokines and chemokines such as IL-10, bone morphogenetic protein type II receptor (BMPR-II) and CXCL12eCXCR4, relating with other immune cells to suppress the immune activity and, as a result, repair injured pulmonary artery endothelial cells (PAECs), control proliferation and apoptosis of pulmonary arterial smooth muscle cells (PASMCs), limit proliferation and activation of fibroblast, and stay immune homeostasis 245 . Consequently, Tregs is a potential treatment target against PAH. The Tregs-targeted treatment includes adoptive Treg therapy acquired by exogenous Treg transplantation and expansion of intrinsic Tregs induced by stimulators including Liver kinase B1 246 , IL-2 247,248 , vitamin D 249 , and CD28 superagonist 250 . Of the stimulators, IL-2 is most frequently applied and is demonstrated roust ability to promote the proliferation of Tregs. Until now, approximately fifty one clinical tests using Treg therapy have been recorded in Clinical-Trials.gov 251 . A phase I trial demonstrated that the infused Tregs in patients with T1D could last one year 252 , indicating safety and tolerance. Recently, adoptive Treg cell therapy was used on a patient with systemic lupus erythematosus (SLE) 253 . The results displayed that the treatment could increase activated Treg cells in inflamed skin and promote a shift from Th1 toward Th17 reactions 253 . Another clinical trial using rituximab to delete B cells for PAH immunotherapy is ongoing 254 .
Myeloid-derived suppressor cells (MDSCs) were reported to be involved in the development of PAH and several inflammatory diseases 255 . PD-L1 is overexpressed on MDSC from PAH patients 255 , and PD-1/PD-L1 interactions exacerbate the inflammation in PAH in animal model 256 . A report displayed that therapy using anti-PD-1 or PD-L1 might inhibit MDSC and alleviate the progression of PAH 256 .

4.2.1.2.
Cytokine-and chemokine-based and vaccination therapy. The cytokines, e.g., IL-6, IL-8, IL-10, IL-13, IL-18, IL-1b and TNFa, are intimately associated with development of PAH 257e259 . Inhibition of the proinflammatory cytokines is potential to treat PAH. Clinical test was performed to study the effictiveness against PAH using a IL-6 receptor antagonist, tocilizumab 260 . The results revealed that the treatment with tocilizumab was safe and improved pulmonary hemodynamic parameters 260 . Dosing a TNF-a antagonist, recombinant human TNF-a receptor II-IgG Fc fusion protein (rhTNFRFc), alleviated PAH via lowering mean pulmonary artery pressure (mPAP) and inhibiting pulmonary vascular remodeling 261 . Furthermore, disruption of the IL-6/Th17/IL-21 pathway is promising to selectively treat PAH 262 .
Endothelin-1 (ET-1) receptor type A (ETAR) can activate the endothelin system and facilitate the initiation and development of PAH 266 . A vaccine against ETAR was designed by conjugating an ETR-002 peptide with a Qb bacteriophage virus-like particle 267 . The vaccination approach has potent efficacy to combat PAH in the monocrotaline (MCT)-induced-and Sugen/hypoxia-induced models by suppressing the pulmonary arterial remodeling and the RV hypertrophy through inhibition of Ca 2þ -dependent signal transduction events 267 . In addition, disruption of thea1D-adrenergic receptor (a1D-AR) might be a vaccination strategy against hypertension by using ADRQb-004 vaccine 268 274 . Loss of endothelial BMPR-II facilitates the initiation and development of PAH, enabling BMPR-II to be a therapeutic target 275 . Tacrolimus, an immunosuppressor, is able to activate BMPR-II and is allowed to repair the endothelial function in PAH patient cells and inhibit the remodeling of the pulmonary artery in animal model 276 . A clinical test demonstrated that administration of tacrolimus at a low dose to three patients with advanced PAH for twelve months, which the trough concentration was 1.5e5 ng/mL, upregulated BMPR-II in PBMCs and, as a result, ameliorated PAH through elevating heart function, prolonging 6-min walk distance, and inducing N-terminal pro-brain natriuretic peptide 277 . Another clinical trial displayed that this administration regimen was safe and could promote the expression of BMPR-II in subsets of PAH patients 278 .
To improve the pulmonary delivery of tacrolimus, nanocomposite microparticles (nCmPs) were prepared by formulating 200-nm drug-loaded polymeric NPs into microparticles through spray drying. After administration via inhalation, the nCmP could deposit in the lung regions, penetrate through the mucus barrier, and control drug release over time 279 . In addition, other immunomodulators, e.g., rapamycin, everolimus, anti-TNFa, TGF-b antagonist, rituximab, and tocilizumab 280 , were used to combat PAH as well.

Conclusions and outlook
We summarized the potential immune targets in several major inflammatory diseases, reviewed the biological drugs and DDSs used for immunotherapy. Immunotherapy is updating the concept of disease treatment and has acquired rapid development in the past five years, evident by that several products such as mAbs and adoptive cell transfer were approved for clinical use. In particular, immunotherapy is being developed as a most effective strategy against cancer. For RA and IBD immunotherapy, the progression is being promoted smoothly, along with several mAbs against TNF-a and IL-6 and two JAK inhibitors, baricitinib and tofacitinib, being marked, whereas there are seven mAbs approved for IBD immunotherapy. For immunotherapy of vascular diseases such as AS and PAH, the clinical test demonstrated promising potential, e.g., the treatment efficacy against AS with a mAb targeting IL-1b can persist three months 183 , bringing significant connivance to patients who have to take lipid-lowering drugs daily. So far, there is no report regarding clinical trials to ameliorate PAH. Always, the patients with the advanced PAH possess poor response to the frequently applied vasodilator agents probably due to the loss of elasticity in the remodeling pulmonary arteries. Such that the utilization of immunotherapy may reverse PAH; however, the rationalization is required from the physician. Overall, immunotherapy against serious vascular diseases don't move forward smoothly compared with cancer immunotherapy, mainly owing to the factors: (1) in pathogenesis lacking sufficiently understanding toward the immune pathways involved in these diseases; (2) absence of legitimacy from the clinic; (3) the potential immune-related adverse events 281 ; (4) remarkably high cost compared with the conventionally used treatment regimens; (5) patients' compliance because dosing via injection is always required in most immunotherapy.
In general, the strategies for immunotherapy are predominantly categorized into several types, including mAbs against cytokines or chemokines, inhibitory ICPs, JAK inhibitors, adoptive cell transfer, metabolic regulation of immune cells and vaccination. mAb-immunotherapy is the most widely applied approach and has gained huge success, evident by over seventy-four formulations have entered the market. Second, ACT, especially T cell-based transplantation, is attracting increasing attention and advances rapidly. The milestone event of this technique is the approval use of CAR therapy to treat for relapsed/refractory acute lymphoblastic leukemia (ALL) 282 . After that, other ACTs are constantly emerging, such as TIL-, TCR-, NK Cell-, Treg-, and MDSCtherapies. Although several problems regarding ACT, such as safety, efficacy, and persistence, are needed to be addressed, the ACT will be concentrated continuously and an increasing number of commercial products will be approved, mainly due to its advantages such as simple composition and controllable scalability. Particularly, Tregs are demonstrating potent efficacy to combat serious inflammation and over 50 of Treg techniques were registered for clinical trial 251 . This technique deserves much more attention and we believe increasing products will be marked for clinical usage.
Most of the immunomodulators belong to biological drugs and their application is always limited by their large size, poor stability, humble penetration ability across physiological barriers, rapid clearance by the reticuloendothelial system, etc. To improve immunotherapy, repeatedly dosings at high doses of the biological drugs via intravenous injection are always required, leading to safety concerns and significantly reducing patient's compliance. The drug delivery approach through engineering biomaterials is robust to enhance delivery of the biologics to the targeted site. Tremendous DDSs demonstrated their amazing immunotherapy efficacy toward various inflammatory diseases in pre-clinical studies. Nonetheless, extremely limited DDS-mediated immunotherapy is approved frequently due to DDS's potential toxicity to the body, unknown in vivo fate 283 , modest scale-up ability, and unfriendly dosing route. In this case, the selection of DDS is critical to the translation. As a result, DDSs with excellent safety and promising industrial perspectives may be an optional choice for the DDS immunotherapy. These DDSs include liposomes or liposome-like NPs, degradable polymeric carriers such as PLGA-NPs or microspheres, albumin-based NPs, cell carriers like red blood cells, etc. In addition, the dosing routes are of the essence to the translation, and well-accepted delivery pathways should be first choice, encompassing oral, buccal, transdermal, nasal, inhalation and subcutaneous routes 284 .

Author contributions
Wei He conceived the work. Qingqing Xiao, Xiaotong Li, Yi Li, Zhengfeng Wu, Chenjie Xu, Zhongjian Chen, and Wei He cowrote the paper. Xiaotong Li prepared the figures. All of the authors discussed the results and commented on the manuscript. All of the authors have read and approved the final manuscript.

Conflicts of interest
The authors have no conflicts of interest to declare.