Mutants of lymphotoxin-α with augmented cytotoxic activity via TNFR1 for use in cancer therapy.

The cytokine lymphotoxin-α (LTα) is a promising candidate for use in cancer therapy. However, the instability of LTαin vivo and the insufficient levels of tumor necrosis factor receptor 1 (TNFR1)-mediated bioactivity of LTα limit its therapeutic potential. Here, we created LTα mutants with increased TNFR1-mediated bioactivity by using a phage display technique. We constructed a phage library displaying lysine-deficient structural variants of LTα with randomized amino acid residues. After affinity panning, we screened three clones of lysine-deficient LTα mutant, and identified a LTα mutant with TNFR1-mediated bioactivity that was 32 times that of the wild-type LTα (wtLTα). When compared with wtLTα, the selected clone showed augmented affinity to TNFR1 due to slow dissociation rather than rapid association. In contrast, the mutant showed only 4 times the TNFR2-mediated activity of wtLTα. In addition, the LTα mutant strongly and rapidly activated caspases that induce TNFR1-mediated cell death, whereas the mutant and wtLTα activated nuclear factor-kappa B to a similar extent. Our data suggest that the kinetics of LTα binding to TNFR1 play an important role in signal transduction patterns, and a TNFR1-selective LTα mutant with augmented bioactivity would be a superior candidate for cancer therapy.

Suppression of nanosilica particle-induced inflammation by surface modification of the particles.

It has gradually become evident that nanomaterials, which are widely used in cosmetics, foods, and medicinal products, could induce substantial inflammation. However, the roles played by the physical characteristics of nanomaterials in inflammatory responses have not been elucidated. Here, we examined how particle size and surface modification influenced the inflammatory effects of nanosilica particles, and we investigated the mechanisms by which the particles induced inflammation. We compared the inflammatory effects of silica particles with diameters of 30-1,000 nm in vitro and in vivo. In macrophages in vitro, 30- and 70-nm nanosilica particles (nSP30 and nSP70) induced higher production of tumor necrosis factor-α (TNFα) than did larger particles. In addition, intraperitoneal injection of nSP30 and nSP70 induced stronger inflammatory responses involving cytokine production than did larger particles in mice. nSP70-induced TNFα production in macrophage depended on the production of reactive oxygen species and the activation of mitogen-activated protein kinases (MAPKs). Furthermore, nSP70-induced inflammatory responses were dramatically suppressed by surface modification of the particles with carboxyl groups in vitro and in vivo; the mechanism of the suppression involved reduction in MAPK activation. These results provide basic information that will be useful for the development of safe nanomaterials.

Structure-activity relationship of T-cell receptors based on alanine scanning.

T-cell receptors (TCR) recognize complexes between human leukocyte antigens (HLA) and peptides derived from intracellular proteins. Their therapeutic use for antigen targeting, however, has been hindered by the very low binding affinity of TCRs, typically in the 1- to 100-µM range. Therefore, to construct mutant TCRs with high binding affinity, we need to understand the relationship between the structure and activity of these molecules. Here, we attempted to identify the amino acids of the TCR that are important for binding to the peptide/HLA complex. We used a TCR that recognizes complexes between HLA-A(∗)0201 and the peptide from tyrosinase, antigen overexpressed in melanoma. We changed 16 amino acids in the third complementarity-determining region within the TCR to alanine and examined the effect on binding affinity. Five alanine substitutions decreased the binding affinity to below 10% compared with that of wild-type TCR. In contrast, one alanine substitution caused a faster on-rate and slower off-rate, and increased the binding affinity to three times that of the wild-type TCR. Our results provide fundamental information for constructing mutant TCRs with high binding affinity.

Lysine-deficient lymphotoxin-α mutant for site-specific PEGylation.

The cytokine lymphotoxin-α (LTα) is a promising anticancer agent; however, its instability currently limits its therapeutic potential. Modification of proteins with polyethylene glycol (PEGylation) can improve their in vivo stability, but PEGylation occurs randomly at lysine residues and the N-terminus. Therefore, PEGylated proteins are generally heterogeneous and have lower bioactivity than their non-PEGylated counterparts. Previously, we created phage libraries expressing mutant LTαs in which the lysine residues of wild-type LTα (wtLTα) were substituted for other amino acids. Here, we attempted to create a lysine-deficient mutant LTα with about the same bioactivity as wtLTα by using these libraries and site-specific PEGylation of the N-terminus. We isolated a lysine-deficient mutant LTα, LT-K0, with almost identical bioactivity to that of wtLTα against mouse LM cells. The bioactivity of wtLTα decreased to 10% following random PEGylation, whereas that of LT-K0 decreased to 50% following site-specific PEGylation; PEGylated LT-K0 retained five times the bioactivity of randomly PEGylated wtLTα. These results suggest that site-specific PEGylated LT-K0 may be useful in cancer therapy.

Tumor vascular targeted delivery of polymer-conjugated adenovirus vector for cancer gene therapy.

Previously, we generated a cancer-specific gene therapy system using adenovirus vectors (Adv) conjugated to polyethylene glycol (Adv-PEG). Here, we developed a novel Adv that targets both tumor tissues and tumor vasculatures after systemic administration by conjugating CGKRK tumor vasculature homing peptide to the end of a 20-kDa PEG chain (Adv-PEG(CGKRK)). In a primary tumor model, systemic administration of Adv-PEG(CGKRK) resulted in ~500- and 100-fold higher transgene expression in tumor than that of unmodified Adv and Adv-PEG, respectively. In contrast, the transgene expression of Adv-PEG(CGKRK) in liver was about 400-fold lower than that of unmodified Adv, and was almost the same as that of Adv-PEG. We also demonstrated that transgene expression with Adv-PEG(CGKRK) was enhanced in tumor vessels. Systemic administration of Adv-PEG(CGKRK) expressing the herpes simplex virus thymidine kinase (HSVtk) gene (Adv-PEG(CGKRK)-HSVtk) showed superior antitumor effects against primary tumors and metastases with negligible side effects by both direct cytotoxic effects and inhibition of tumor angiogenesis. These results indicate that Adv-PEG(CGKRK) has potential as a prototype Adv with suitable efficacy and safety for systemic cancer gene therapy against both primary tumors and metastases.

Optimized PEGylated adenovirus vector reduces the anti-vector humoral immune response against adenovirus and induces a therapeutic effect against metastatic lung cancer.

Application of adenovirus vectors (Adv) in metastatic cancer treatment is limited. We previously demonstrated that covalent conjugation of polyethleneglycol (PEG) to Adv enhances therapeutic effects and decreases toxic side-effects after systemic administration, but the level of immune response to PEGylated Adv (PEG-Ad) was not examined. Here, we examined the effect of PEGylation of Adv on the production of anti-Adv antibodies and antitumor response. We constructed a set of PEG-Ad using 5-kDa PEG, with modification rates of 30%, 45% and 90%. After systemic administration of Advs to rats, we examined the level of anti-Adv immunoglobulin (Ig)G and IgM in serum. The levels of anti-Adv IgG and anti-Adv IgM in rats treated with unmodified Adv were higher than those in control group. Rats treated with PEG-Ad that had a 90% modification rate showed lower level of anti-Adv IgG and anti-Adv IgM than those treated with unmodified Adv, whereas rats treated with PEG-Ad that had a 30% or 45% modification rate showed a similar level of anti-Adv IgG and IgM to those treated with unmodified Adv. Systemic administration of PEG-Ad that had a 90% modification rate, and expressed tumor necrosis factor-alpha, significantly reduced the number of metastatic colonies in the lung compared to unmodified Adv, with negligible side effects. These results suggest that systemic administration of PEG-Ad with an appropriate PEG modification rate has the potential to reduce the production of antibodies against Adv and increase the therapeutic response against metastatic cancer.

Adenovirus vector covalently conjugated to polyethylene glycol with a cancer-specific promoter suppresses the tumor growth through systemic administration.

Cancer gene therapy with adenovirus vectors (Adv) is limited to local administration because systemic administration of Adv produces a weak therapeutic effect and severe side effects. Previously, we generated a dual cancer-specific Adv system by using Adv covalently conjugated to polyethylene glycol (PEG) for transductional targeting and the telomere reverse transcriptase (TERT) promoter as a cancer-specific promoter for transcriptional targeting (PEG-Ad-TERT). We demonstrated that systemic administration of PEG-Ad-TERT showed superior antitumor effects against lung metastatic cancer with negligible side effects. Here, we investigated the therapeutic efficacy of systemic administration of PEG-Ad-TERT for the treatment of primary tumors. We first evaluated the transgene expression of PEG-Ad-TERT containing the luciferase gene (PEG-Ad-TERT/Luc) in primary tumors. Systemic administration of PEG-Ad-TERT/Luc resulted high transgene expression, similar to that observed in tumors for the conventional cytomegalovirus (CMV) promoter-driven Adv containing the luciferase gene (Ad-CMV/Luc). By comparison, transgene expression was 2500-fold lower than that of Ad-CMV/Luc in liver. We then examined the therapeutic effect of systemic administration of PEG-Ad-TERT containing the herpes simplex virus thymidine kinase (HSVtk) gene (PEG-Ad-TERT/HSVtk) for the treatment of primary tumors. We showed that PEG-Ad-TERT/HSVtk produced a notable antitumor effect against primary tumors with negligible side effects. These results demonstrated that PEG-Ad-TERT can be regarded as a prototype Adv with suitable efficacy and safety for systemic cancer gene therapy against both metastatic and primary tumors.

The effect of surface modification of amorphous silica particles on NLRP3 inflammasome mediated IL-1beta production, ROS production and endosomal rupture.

Although amorphous silica particles (SPs) are widely used in cosmetics, foods and medicinal products, it has gradually become evident that SPs can induce substantial inflammation accompanied by interleukin-1beta (IL-1beta) production. Here, to develop safe forms of SPs, we examined the mechanisms of SP-induced inflammation and the relationship between particle characteristics and biological responses. We compared IL-1beta production levels in THP-1 human macrophage like cells in response to unmodified SP of various diameters (30- to 1000-nm) and demonstrated that unmodified microsized 1000-nm SP (mSP1000) induced higher levels of IL-1beta production than did smaller unmodified SPs. Furthermore, we found that unmodified mSP1000-induced IL-1beta production was depended on the sequence of reactive oxygen species (ROS) production, endosomal rupture, and subsequent activation of pro-inflammatory complex NLRP3 inflammasome. In addition, we compared IL-1beta production levels in THP-1 cells treated with mSP1000s modified with a functional group (-COOH, -NH(2), -SO(3)H, -CHO). Although unmodified and surface-modified mSP1000s were taken up with similar frequencies equally into the THP-1 cells, surface modification of mSP1000 dramatically suppressed IL-1beta production by reducing ROS production. Our results reveal a part of NLRP3 activation pathway and provide basic information that should help to create safe and effective forms of SPs.

Creation of a LIGHT mutant with the capacity to evade the decoy receptor for cancer therapy.

The cytokine LIGHT activates various anti-tumor functions through its two receptors, lymphotoxin beta receptor (LTbetaR) and herpes virus entry mediator (HVEM), and is expected to be a promising candidate for cancer therapy. However, LIGHT is also trapped by decoy receptor 3 (DcR3), which is highly expressed in various tumors. Here, we used phage display technique to create LIGHT mutants that specifically bind LTbetaR and HVEM, and is not trapped by DcR3 for optimized cancer therapy. We constructed phage library displaying structural variants of LIGHT with randomized amino acid residues. After the affinity panning, we created 6 clones of LIGHT mutants as candidates for DcR3-evading LIGHT. Analysis of binding affinities showed that all candidates had 10-fold lower affinities for DcR3 than wild-type LIGHT, while 5 of the 6 clones had almost the same affinity for LTbetaR and HVEM. Furthermore, analysis of detailed binding kinetics showed that lower affinity for DcR3 is dependent on their faster off-rate. Further, we showed that the LIGHT mutant had almost the same cytotoxicity via LTbetaR, and had 62-fold higher DcR3-evading capacity compared to the wild type. Our data provide valuable information for construction of more functional LIGHT mutants that might be powerful tools for cancer therapy.

Creation of a lysine-deficient LIGHT mutant with the capacity for site-specific PEGylation and low affinity for a decoy receptor.

The cytokine LIGHT is a promising candidate for cancer therapy. However, the therapeutic effect of LIGHT as a systemic anticancer agent is currently insufficient because of its instability and its binding to nonfunctional soluble decoy receptor 3 (DcR3), which is overexpressed in various tumors. Modification of proteins with polyethylene glycol (PEGylation) can improve their in vivo stability, but PEGylation may occur randomly at all lysine residues and the NH(2)-terminus; therefore, PEGylated proteins are generally heterogeneous and have decreased bioactivity. In this study, we attempted to create a lysine-deficient LIGHT mutant that could be PEGylated site-specifically and would have lower affinity for DcR3. We prepared phage libraries expressing LIGHT mutants in which all the lysine residues were replaced with other amino acids. A lysine-deficient LIGHT mutant [mLIGHT-Lys(-)] was isolated by panning against lymphotoxin beta receptor (LTbetaR). mLIGHT-Lys(-) could be site-specifically PEGylated at its NH(2)-terminus, yielding molecular uniformity and in vitro bioactivity equal to that of non-PEGylated, wild-type LIGHT. Furthermore, mLIGHT-Lys(-) was not trapped by the nonfunctional DcR3, despite binding to its functional receptors. These results suggest that mLIGHT-Lys(-) might be a useful candidate for cancer therapy.

Titanium dioxide induces different levels of IL-1beta production dependent on its particle characteristics through caspase-1 activation mediated by reactive oxygen species and cathepsin B.

Although titanium dioxide (TiO2) is widely used, its inhalation can induce inflammatory diseases accompanied by interleukin-1beta (IL-1beta) production. The particle characteristics of TiO2 are important factors in its biological effects. It is urgently necessary to investigate the relationship between the particle characteristics and biological responses for the development of safe forms of TiO2. Here, we systematically compared the production of IL-1beta in response to various forms of TiO2 by macrophage-like human THP-1 cells using various sizes (nano to micro), crystal structures (anatase or rutile), and shapes (spherical or spicular) of TiO2. The production of IL-1beta depended dramatically on the characteristics of the TiO2. Notably, smaller anatase and larger rutile particles provoked higher IL-1beta production. In addition, IL-1beta production depended on active cathepsin B and reactive oxygen species production independent of the characteristics of TiO2. Our results provide basic information for the creation of safe and effective novel forms of TiO2.

Creation of lysine-deficient mutant lymphotoxin-alpha with receptor selectivity by using a phage display system.

The cytokine lymphotoxin-alpha (LT alpha) activates various biological functions through its three receptor subtypes, tumor necrosis factor receptor 1 (TNFR1), TNFR2 and herpes virus entry mediator (HVEM), but the relative contribution of each receptor to each function is unclear. Therefore it is important to create mutant LT alpha with receptor selectivity for optimized cancer therapy and the analysis of receptor function. Here, we attempted to create a lysine-deficient mutant LT alpha with TNFR1-selective bioactivity using a phage display technique. We obtained the TNFR1-selective mutant LT alpha R1selLT, which contained the mutations K19N, K28Q, K39S, K84Q, K89V, and K119H. Compared with wild-type LT alpha (wtLT alpha), R1selLT showed several-fold higher bioactivity via TNFR1 but 40-fold lower bioactivity via TNFR2. Kinetic association-dissociation parameters of R1selLT with TNFR2 were higher than those of wtLT alpha, whereas these parameters of R1selLT with TNFR1 were lower than those of wtLT alpha, suggesting that destabilization of the R1selLT-TNFR2 complex causes the decreased bioactivity of R1selLT on TNFR2. We also showed that the K84Q mutation contributed to the enhanced activity via TNFR1, and K39S lowered activity via TNFR2. R1selLT likely will be useful in cancer therapy and in analysis of the LT alpha structure-function relationship.

Comparison of the anti-tumor activity of native, secreted, and membrane-bound LIGHT in mouse tumor models.

The TNF superfamily member LIGHT has potent anti-tumor activity through direct cytotoxicity and activation of the immune response, and is a promising candidate for cancer therapy. Natively, LIGHT exists as both a membrane-anchored form and a proteolytically processed, secreted form. However, the strength of the anti-tumor activity of each form of LIGHT has not been well defined. Here, to identify the optimal form of LIGHT for cancer gene therapy, we constructed fiber-mutant adenovirus vectors (AdRGD) encoding native full-length LIGHT (LIGHT/FL), stably membrane-anchored LIGHT (LIGHT/mem), and fully secreted LIGHT (LIGHT/sec). We then compared the anti-tumor effects of the different forms of LIGHT in mice by intratumoral injection of each AdRGD. We demonstrated that intratumoral injection of AdRGD-LIGHT/sec provided greater tumor suppression than AdRGD-LIGHT/FL, although this effect did not reach statistical significance. By comparison, AdRGD-LIGHT/mem had negligible anti-tumor activity. We also demonstrated that more CD4+ and CD8+ T cells accumulated inside tumors treated in vivo with AdRGD-LIGHT/sec than in tumors treated with AdRGD-LIGHT/FL or AdRGD-LIGHT/mem. These results suggest that the secreted form of LIGHT might be the optimal form for cancer gene therapy.

LIGHT protein suppresses tumor growth by augmentation of immune response.

The tumor necrosis factor (TNF) superfamily member LIGHT has potent anti-tumor activities through activation of the immune response, and it is a promising candidate for use in cancer immunotherapy. However, there are no reports of the anti-tumor effects of LIGHT protein in vivo because of the lack of easy, efficient methods of manufacturing this protein. Here, we developed a method of manufacturing recombinant LIGHT protein using Escherichia coli through refolding of inclusion bodies; we then evaluated the anti-tumor activity of the protein. LIGHT protein expressed in E. coli showed the same biological activities and binding affinities to its receptors as did LIGHT expressed in mammalian cells. In addition, intratumoral injection of LIGHT significantly suppressed tumor growth, with augmentation of antigen-specific IFN-gamma-producing cells in the regional lymph nodes and spleen. These results indicate that LIGHT protein efficiently evokes the systemic tumor-specific immune response, and thus induces tumor suppression.

Tat conjugation of adenovirus vector broadens tropism and enhances transduction efficiency.

AIMS: Adenovirus vectors (Advs) have been very useful for basic research and clinical gene therapy because they propagate to high titers and efficiently transduce cells and tissues regardless of the mitotic status. However, poor transduction of cells that lack the coxsackievirus and adenovirus receptor (CAR), the primary receptor for Advs, has limited Adv application. In this study, we attempted to generate novel Tat-Advs (Advs conjugated with the HIV Tat-derived peptide, a protein-transduction domain (PTD)) to broaden Adv tropism and enhance transduction efficiency. MAIN METHODS: We constructed Tat-Advs by chemically conjugating Tat peptide to the surface-exposed lysine residues on Advs. We compared the gene transfer activity of Tat-Advs with that of unmodified Advs by measuring the luciferase expression in several types of cell lines. KEY FINDINGS: Tat-Advs showed gene expression 1 to 3 log orders higher than unmodified Advs in CAR-negative adherent cells and blood cells, which are refractory to conventional Advs. The inhibition of Tat-Adv-mediated gene expression by heparin and macropinocytosis inhibitor confirms that binding of Tat-Adv to cellular HSPGs and macropinocytosis are essential for efficient CAR-independent transduction. We also demonstrated that Adv modified with another PTD (R8) had the same high transduction efficiency as Tat-Adv. SIGNIFICANCE: These data suggest that Tat-Advs are important tools for transducing cells and will be useful as platform vectors for gene therapy.

TERT promoter-driven adenovirus vector for cancer gene therapy via systemic injection.

Adenovirus vectors (Adv) are used widely in cancer gene therapy research. However, the clinical application of Adv currently is limited to local, intratumoral administration; systemic administration leads to redundant transgene expression in the liver and subsequent hepatotoxicity. Here we replaced the conventional cytomegalovirus (CMV) promoter of Adv with a tumor-specific telomere reverse transcriptase (TERT) promoter, to restrict expression of the Adv-transduced transgene to tumor tissue alone. We evaluated the therapeutic and side effects after systemic administration of Adv expressing herpes simplex virus thymidine kinase (Ad-HSVtk) in mice bearing Meth-A tumors. Although systemically injected CMV promoter-driven Ad-HSVtk lacked therapeutic effect, mice injected with 2x10(11) viral particles containing TERT promoter-driven Ad-HSVtk showed inhibited tumor growth and prolonged survival with minimal side effects. Our results suggest that Adv in which transgene expression is driven by the TERT promoter are a promising prototype of tumor-targeting vectors for effective and safe cancer gene therapy.

Effective tumor targeted gene transfer using PEGylated adenovirus vector via systemic administration.

Conjugation of polyethylene glycol to protein or particles (PEGylation) prolongs their plasma half-lives and promotes their accumulation in tumors due to enhanced permeability and retention (EPR) effect. Although PEGylation of adenovirus vectors (Ads) is an attractive strategy to improve the in vivo kinetics of conventional Ads, the EPR effect of PEGylated Ad (PEG-Ad) had not previously been reported. In this study, we prepared PEG-Ads with PEG at various modification ratios, injected them intravenously into tumor-bearing mice, and determined the blood kinetics, viral distribution, and gene expression patterns, respectively. In addition, we conducted a cancer therapeutic study of PEG-Ad encoding tumor necrosis factor (TNF)-alpha. The plasma half-life of PEG-Ad was longer than that of unmodified-Ad, and accumulation of PEG-Ad in tumor tissue increased as the PEG modification ratio increased. In particular, PEG-Ad with about 90% modification ratio showed higher (35 times) gene expression in tumor and lower (6%) in liver, compared with values for unmodified Ad. Moreover, PEG-Ad encoding TNF-alpha demonstrated not only stronger tumor-suppressive activity but also fewer hepatotoxic side effects compared with unmodified-Ad. PEGylation of Ad achieved tumor targeting through the EPR effect, and these attributes suggest that systemic injection of PEG-Ad has great potential as an anti-tumor treatment.