The role of TNC in atherosclerosis and drug development opportunities

Tenascin C (TNC), a rich glycoprotein of the extracellular matrix, exhibits a pro-atherosclerosis or anti-atherosclerosis effect depending on its location. TNC, especially its C domain/isoform (TNC-C), is strongly overexpressed in atherosclerotic plaque active areas but virtually undetectable in most normal adult tissues, suggesting that TNC is a promising delivery vector target for atherosclerosis-targeted drugs. Many delivery vectors were investigated by recognizing TNC-C, including G11, G11-iRGD, TN11, PL1, and PL3. F16 and FNLM were also investigated by recognizing TNC-A1 and TNC, respectively. Notably, iRGD was undergoing clinical trials. PL1 not only recognizes TNC-C but also the extra domain-B (EDB) of fibronectin (FN), which is also a promising delivery vector for atherosclerosis-targeted drugs, and several conjugate agents are undergoing clinical trials. The F16-conjugate agent F16IL2 is undergoing clinical trials. Therefore, G11-iRGD, PL1, and F16 have great development value. Furthermore, ATN-RNA and IMA950 were investigated in clinical trials as therapeutic drugs and vaccines by targeting TNC, respectively. Therefore, targeting TNC could greatly improve the success rate of atherosclerosis-targeted drugs and/or specific drug development. This review discussed the role of TNC in atherosclerosis, atherosclerosis-targeted drug delivery vectors, and agent development to provide knowledge for drug development targeting TNC.


Introduction
Coronary heart disease (CHD) is the leading cause of death worldwide and is primarily caused by atherosclerosis.Atherosclerotic plaque growth blocks blood flow, leading to ischemia of the surrounding tissue by promoting narrowing of the vessel lumen.Plaque ruptures lead to life-threatening myocardial infarction (MI), coronary artery disease (CAD), cerebral infarction (ischemic stroke), and acute coronary syndrome (ACS) by promoting platelet aggregation, fluid coagulation, and thrombosis.Atherosclerosis is characterized by the accumulation of lipids in the artery walls, accompanied by infiltration of immune cells and chronic inflammation.The early stage of atherosclerosis is the formation of "fatty streaks" that consist of cholesterol-laden foam cells.As the disease progresses, necrotic cells accumulate in the plaque to form necrotic nuclei that infiltrate numerous inflammatory cells and release proinflammatory cytokines and chemokines.Therefore, lipid-lowering, anti-inflammatory, or immunomodulatory therapy is necessary to delay the progression of atherosclerosis [1][2][3].However, atherosclerosis mainly occurs in the heart aorta.Many drugs have many side effects.These include Ivyspring International Publisher statin-induced liver and neuromuscular toxicity, cramps, myalgia, necrotizing myopathy, and rhabdomyolysis [4,5].Ezetimibe has a placebo-like side effect profile.PCSK9 inhibitors induce nasopharyngitis [6,7].Therefore, the development of drugs that target aortic plaques without affecting other tissues will greatly improve the efficacy and reduce the side effects of drugs.Conjugative drugs that conjugate the genes highly expressed in pathological tissue but rarely expressed in normal tissue have a specificity and potency that traditional drugs cannot achieve.Current conjugative drugs include antibody-drug conjugates (ADCs), antibody degraducer conjugates (ADeCs), aptamer-drug conjugates (ApDCs), antibody-oligonucleotide conjugates (AOCs), antibody fragment-drug conjugates (FDCs), immune-stimulating antibody conjugates (ISACs), radionuclide-drug conjugates (RDCs), small molecule-drug conjugates (SMDCs), and virus-like drug conjugates (VDCs).ADCs, RDCs, SMDCs, and ISACs are the most successful coupling drugs because many related drugs have been approved on the market or entered into clinical trials [8][9][10].Therefore, the development of these conjugative agents will greatly improve the success rate of drug development.
Tenascin C (TNC) is a member of the extracellular matrix (ECM) protein family and plays a key role in wound healing and tissue remodeling.TNC has multiple biological roles by binding to and interacting with multiple genes.Heat shock protein 33 (HSP33) is responsible for TNC accumulation in cells.The epidermal growth factor (EGF)-like repeat domains of TNC regulated cell adhesion and cell motility and were anti-adhesion regions of fibroblasts, neurons, and glial cells.This domain also regulates neuronal migration and axon pathfinding during development.TNC has many isomers with different functions and sizes due to the alternative splicing of fibronectin type III (FNIII)-like repeats (Fig. 1) [11][12][13][14][15][16][17][18][19][20][21][22].Many studies have shown that TNC plays a key role in atherosclerosis.TNC expression was increased in the plaque in apoE-/-mice [23,24].Serum TNC levels were increased in 307 patients with CHD [25][26][27] and 170 patients with ACS [28].The single nucleotide polymorphisms (SNPs) of TNC, such as rs3789875 and rs12347433, were related to atherosclerosis and CAD [29].TNC was rapidly upregulated after ischemic events, such as MI, suggesting that circulating TNC is a diagnostic or prognostic auxiliary biomarker in patients with cardiovascular disease [30].However, the friend and foe of TNC in atherosclerosis depended on its location in vivo.Interestingly, TNC was strongly overexpressed in atherosclerotic plaque active areas but was virtually undetectable in most normal adult tissues, especially the TNC C domain/isoform (TNC-C).G11, G11-iRGD, TN11, PL1, PL3, F16, and FNLM are promising delivery vectors for atherosclerosis-targeted drugs.In particular, G11-iRGD, PL1, and F16 have great development value because they contain or recognize components of clinical studies or their conjugate agents being investigated in clinical trials.In addition, to the best of our knowledge, ATN-RNA, a TNC-specific agent, was investigated in clinical trials as a therapeutic drug, while IMA950, a TNC agent, was investigated in clinical trials as a vaccine.Therefore, using TNC could greatly improve the success rate of atherosclerosis-targeted drugs and/or specific drug development.In this review, we focused on the potential of TNC in atherosclerosis, the delivery vector for atherosclerosis-targeted drugs, and TNC agents in the hope of providing knowledge for drug development by targeting TNC.

Proatherogenic effect and mechanism of TNC
TNC not only stimulated pro-inflammatory cytokine expression, such as IL-6, IL-8, and TNFα but also anti-inflammatory cytokine expression, such as IL-4 and IL-13, suggesting that TNC has both pro-inflammatory and anti-inflammatory activity [37].However, knockout of TNC not only did not change the lipoprotein profile but also the adhesion, migration, and proliferation of SMCs [37], suggesting that the effect of TNC on atherosclerosis was independent of lipid metabolism and SMCs.

G11
G11 (mini antibody SIP format) was a human antibody that only recognized TNC-C.G11 was used to observe the angiogenesis of advanced atherosclerotic plaques in mice, as well as gliomas and lung tumors [30]. 125I (iodine)-G11 is a G11 with 125 I labeling. 125I-G11 (intravenous injection, I.V., 0.3 MBq, 8 μg) was taken up in fat and activated macrophages in aortic plaques in apoE-/-mice.The sensitivity of autoradiography to detect plaque fatty stained areas was 74% and 76% at 4 hours and 24 hours after intravenous administration of 125 I-G11.The signal-to-noise ratio (SNR) of vessel walls with plaque and without plaque was 43:1 and 104:1 at 4 hours and 24 hours, respectively.The SNR of activated macrophages in aortic plaques was 50:1 and 90:1 at 4 hours and 24 hours, respectively.The aorta-to-blood ratios of 125 I-G11 were 0.45 and 1.53.After 4-24 hours of administration, the clearance of 125 I-G11 was 86%, suggesting that the blood half-life of 125 I-G11 was less than 20 min [30,40].Therefore, G11 may facilitate molecular imaging of advanced atherosclerotic plaques and the delivery of related drugs.No G11 conjugate agents have been investigated in clinical trials.More studies are needed to confirm G11's development value.Of note, TNC-C was undetectable in normal adult tissue and exhibited a more restricted expression pattern than other TNC domains.TNC-C was also strongly abundant in macrophage-rich plaques but was not detected in normal adult tissues [30,40,41], suggesting that targeting TNC-C facilitated the development of diagnostic and targeting agents.Importantly, several other antibodies or antibodies can specifically recognize TNC-C, including G11-iRGD, TN11, PL1, and PL3 (Fig. 2).
G11-iRGD is a fusion peptide that contains the G11 antibody and iRGD peptide (sequence: CRGDKGPDC) [42].iRGD is also known as CEND-1 and LSTA1 and is a promising delivery vector.Interestingly, iRGD was investigated in clinical trials in cancer [43,44].Therefore, G11-iRGD may be a codelivery system than G11 and iRGD.TN11 is a human antibody fragment antibody (scFV) that only recognizes TNC-C [45].However, to the best of our knowledge, the role of G11-iRGD, iRGD, and TN11 in atherosclerosis has not been investigated.No G11-iRGD or TN11 conjugate agents were investigated in clinical trials.More studies are needed to confirm the development value of G11-iRGD and TN11.
PL1 (sequence: PPRRGLIKLKTS), a peptide, recognizes not only TNC-c but also Extra Domain-B (EDB) of fibronectin (FN), which plays a key role in atherosclerosis development [46].However, FN is a double-edged sword in atherosclerosis.FN exhibited an anti-atherosclerotic effect by preventing plaque rupture and vascular occlusion by promoting the formation of thick fiber caps, while FN exhibited a proatherogenic effect by promoting lipoprotein retention and inflammatory cell infiltration by expanding the ECM [47][48][49].Interestingly, FN was also a promising delivery vector for atherosclerosistargeted drugs, such as TPTS/C/T.TPTS/C/T is a simvastatin nanoprodrug (TPTS) with ROSresponsive cleavage properties and a fibronectintargeted system.TPTS/C/T could codeliver the simvastatin prodrug and ticagrelor.TPTS/C/T exhibited stronger anti-inflammatory and antioxidant effects than free simvastatin by decreasing the M1-type polarization of macrophages, intracellular reactive oxygen species (ROS), and proinflammatory cytokines, such as IL-1β, MCP-1, and TNFα levels.In the apoE-/-mouse model of atherosclerosis, TPTS/C/T exhibited good synergistic therapy, anti-atherosclerosis effects, and biosafety by targeting the release of simvastatin and ticagrelor in plaques [50].In addition, L19 is a human recombinant antibody that specifically recognizes FN-EDB.L19 exhibited similar results with G11 in atherosclerotic plaques in apoE-/-mice [40].More importantly, many L19 antibody cytokine fusion proteins are currently being investigated in clinical trials, such as L19-IL2 (also named Darleukin) [51,52], L19-TNF (also named Fibromun) [52,53], and Nidlegy TM (combination of L19-IL2 and L19-TNF) (Table 1) [52,54].Several L19 antibody cytokine fusion proteins were also investigated in preclinical trials, such as L19-TNF-IL2 (also named Tripokin) [55], L19-IFNγ (also named L19-IFNγ KRG) [56], L19-IL12 [57], and L19-IL15 [58,59].Therefore, PL1 may be a codelivery system other than G11, FN, and L19 because it specifically recognizes TNC-C and FN-EDB.However, the role of PL1 in atherosclerosis has not been investigated.No PL1 conjugate agents have been investigated in clinical trials.More studies are needed to confirm the development value of PL1.
PL3 (sequence: AGRGRLVR), a peptide, recognizes not only the C domain of TNC but also neuropilin-1 (Nrp1) [60].Nrp1, a type I transmembrane protein, is also a double-edged sword in atherosclerosis.Nrp1 from T cells promoted atherosclerosis development by promoting the recruitment of CD4 T cells into the aorta [61].Nrp1 suppressed leukocyte rolling and atherosclerotic plaque size by reducing proinflammatory cytokine and adhesion molecule levels by interacting with VE-cadherin and transforming growth factor-β (TGF-β) receptor II (TGFBR2) [62].In addition, the role of PL3 in atherosclerosis has not been investigated.No PL3 conjugate agents have been investigated in clinical trials.More studies are needed to confirm the development value of PL3.

F16
F16 is a fully human monoclonal antibody that only recognizes TNC A1 domain//isoform (TNC-A1) [63].F16 was investigated in atherosclerosis in clinical trials [64].F16 could bind to macrophages, blood vessels, and proliferating cells in human atherosclerotic plaque active areas but not to normal arteries and resting plaque areas (28 atherosclerotic plaques and 11 normal arteries), suggesting that the F16 antibody has strong specificity and selectivity for active plaques and may be a strong candidate for plaque imaging radiopharmaceuticals [64].Interestingly, the F16 antibody cytokine fusion protein F16IL2 (also named Teleukin) was investigated in clinical trials (Table 1).Therefore, F16 is a promising delivery vector for atherosclerosis-targeted drugs.However, in our capacity, we did not find any information about the F16 conjugate agents in atherosclerosis.

FNLM
FH (sequences, FHKHKSPALSPV) is a peptide that recognizes the large isoform of TNC.TNC is highly expressed in some inflammatory conditions, such as MI injury, but is sparsely expressed in normal adult myocardium.FNLM-miR consists of a hybrid membrane shell and MSNs-miR.The hybrid membrane shell is an artificial lipid membrane modified with FH peptide and fused with neutrophil membrane proteins (NMPs).MSNs-miR are mesoporous silica nanoparticles (MSNs) loaded with miR-1, miR-133a, miR-208, and miR-499 (miRCombo).FNLM-miR delivered more miRCombo into cardiac fibroblasts (CFs) in the injured heart to induce reprogramming into induced cardiomyocyte-like cells (iCMs).FNLM-miR reduced the expression of multiple cytokines, including CCR2, CXCR1, CXCR2, LFA-1, IL-1β, and IL-6.FNLM-miR also increased the expression of sarcomere-related genes, such as Myh6 and Tnnis, transcription factor-related genes, such as Gata4, Mef2c, and Tbx5, and ion channel-related genes, such as Kcnj2 and Scn5a.FNLM-miR (I.V.) induced reprogramming to improve cardiac function and alleviate fibrosis by delivering miRCombo into fibroblasts in a myocardial ischemia/reperfusion (MIR) injury mouse model.FNLM-miR has good safety, long blood circulation, and a fine fibroblast targeting profile in this model [65].These results suggest that FNLM-miR could deliver miRCombo into the cardiovascular system.FNLM is a promising delivery vector for atherosclerosis-targeted drugs.No FNLM conjugate agents have been investigated in clinical trials.More studies are needed to confirm the development value of FNLM.

ATN-RNA
ATN-RNA, an RNA interference (RNAi), is a double-stranded RNA (dsRNA) that targets TNC.ATN-RNA inhibited TNC synthesis in vivo [66,67].ATN-RNA (injection into the brain after resection) for the treatment of brain tumors (N=46) inhibited tumor growth and disease recurrence.ATN-RNA prolonged the survival period of patients and improved the quality of life [67][68][69].The median overall survival (OS) of patients treated with ATN-RNA was 106.6 weeks and was higher than that of the control group (48.2 weeks).The longest survival was 180.9 weeks (Table 2) [69].These results support the continued development of ATN-RNA.However, the role of ATN-RNA in atherosclerosis was not investigated.Of note, the development of ATN-RNA has obtained patent protection, such as US8946400 (B2), EP2121927 (B1), US2010076053 (A1), EP2121927 (A2), WO20080 16317 (A3), WO2008016317 (A2), and WO2008016317.However, since the last report of clinical results (2010), we have not found any clinical reports or clinical trials.
The median OS and disease control rates for all patients were 19 months (21 months after surgery) and 42%, respectively, including 17 months (19 months after surgery) and 31.2% of glioblastoma patients.The PFS for all patients and glioblastoma patients was 9 months and 9 months, respectively, while it was 10 months and 9.5 months after surgery, respectively.The mode of administration had no significant effect on OS and PFS [74].Bevacizumab (BEV), a monoclonal anti-VEGF-A IgG1 antibody, was approved for the treatment of multiple cancers, such as cervical cancer, colorectal cancer, glioblastoma, glioma, liver cancer, NSCLC, ovarian cancer, and renal cell carcinoma.However, IMA950/poly-ICLC did not improve the response rate, median OS, or median PFS of BEV for the treatment of relapsing high-grade glioma patients (vaccinated=16, nonvaccinated=40) [75].These novel results support the continued development of IMA950, except in combination with BEV.many clinical trials on IMA950 are ongoing (Table 2).However, the further development of IMA950 in glioblastoma was discontinued on 23 September 2020 [76].

Several issues of concern
TNC, especially TNC-C, is a promising target and delivery vector for drug development and atherosclerosis-targeted drugs.However, several issues of concern need to be noted.(1) Anti-TNC-USPIO, ultrasmall superparamagnetic iron oxide (USPIO) nanoparticle-labeled mouse anti-TNC monoclonal antibody, is a promising molecular imaging tool for detecting and monitoring atherosclerotic plaques by magnetic resonance imaging (MRI) in preclinical trials [23,77].However, the role of anti-TNC-USPIO in clinical trials has not been investigated.(2) Many TNC domains/isoforms were investigated in atherosclerosis, such as EGF-like, A1, A2, FBG, C, and TnfnIII 1-5 domains.However, there are many domains/isoforms that have not been investigated.(3) Targeting TNC-C, such as G11, G11-iRGD, TN11, PL1, and PL3, is a promising delivery vector for atherosclerosis-targeted agents.However, no conjugate agents have been investigated in clinical trials.The role of G11-iRGD, TN11, PL1, and PL3 in atherosclerosis is also not unclear.(4) TTA1, a 13-kDa oligonucleotide (39-mer) aptamer, could recognize the TNC fibrinogen-like domain.TTA1 could target delivery to the ECM of atherosclerotic lesions and tumors [78].Therefore, TTA1 is a promising delivery vector for atherosclerosis-targeted agents.However, no TTA1 conjugate agents have been investigated.The role of TTA1 in atherosclerosis is also not unclear.(5) IL-2 not only plays a key role in cancer but also in atherosclerosis [79][80][81][82][83]. F16IL2 can deliver IL-2 to tumor tissue very well.F16 is a promising delivery vector for atherosclerosis-targeted agents.However, the role of F16IL2 in atherosclerosis has not been investigated.( 6) Knockout and knockdown of TNC in vivo may suppress atherosclerosis development, suggesting that ATN-RNA may suppress atherosclerosis development by reducing TNC expression in vivo.However, the role of ATN-RNA in atherosclerosis has not been investigated.(7) The low uptake of ATN-RNA in cells weakens its anticancer ability and requires delivery vectors to enhance its uptake [84].Magnetic nanoparticles coated with polyethyleneimine (PEI) could improve the delivery of ATN-RNA in human U-118 MG cell lines [84,85].However, the role of this nanomediated delivery of ATN-RNA in vivo is unclear.( 8) TNC monoclonal antibodies may improve the delivery of ATN-RNA.However, attention should be given to cross-reactions within the TNC gene.( 9) ATN-RNA also suppressed MDA-MB-231 breast cancer cell proliferation, migration, and adhesion in vitro [86].However, the role of ATN-RNA in suppressing breast cancer in vivo is unclear.(10) TNC is one of the antigens of the IMA950 vaccine.However, to the best of our knowledge, the role of TNC in IMA950 has not been investigated.(11) The investigation of the reasons for stopping the development of IMA950 is of great significance for restarting its development.Of note, GM-CSF and poly-ICLC were used as adjuvants for the IMA950 vaccine.However, these adjuvants are only ongoing in clinical research and are not found in marketed vaccines.Many adjuvants are approved for use in marketed vaccines, such as AS01 (TLR4 and NLRP3 agonist), AS03 (apoptosis-associated specklike protein (ASC) agonist), AS04 (TLR4 and NLRP3 agonist), CpG ODN 1018 (TLR9 agonist), and MF59 (apoptosis-associated speck-like protein (ASC) agonist) [87][88][89][90].These adjuvants may be more suitable for the development of the IMA950 vaccine.

Conclusions
The pro-atherosclerosis or anti-atherosclerosis effect of TNC depended on its location.TNC was overexpressed in blood and atherosclerotic plaque active areas from patients with CHD.However, its expression is virtually undetectable in most normal adult tissues, especially TNC-C.Targeting TNC-C with G11, G11-iRGD, TN11, PL1, and PL3 is a promising delivery vector for atherosclerosis-targeted drugs.In particular, G11-iRGD and PL1 have great development value because they contain or recognize components of clinical studies.F16 and FNLM are also targeted drug delivery vectors with great development value due to their conjugate agents being investigated in preclinical and clinical trials, especially F16 (clinical trials).Targeting TNC is a promising target for drug development due to the specific agents ATN-RNA and IMA950 being investigated in clinical trials.With the deepening of research and cooperation in scientific research, it is believed that more scientists will develop more therapeutic (conjugate and/or specific agents), vaccine, and diagnostic agents using TNC as a delivery vector or target.