Intraductal administration of transferrin receptor-targeted immunotoxin clears ductal carcinoma in situ in mouse models of breast cancer-a preclinical study.

The human transferrin receptor (TFR) is overexpressed in most breast cancers, including preneoplastic ductal carcinoma in situ (DCIS). HB21(Fv)-PE40 is a single-chain immunotoxin (IT) engineered by fusing the variable region of a monoclonal antibody (HB21) against a TFR with a 40 kDa fragment of Pseudomonas exotoxin (PE). In humans, the administration of other TFR-targeted immunotoxins intrathecally led to inflammation and vascular leakage. We proposed that for treatment of DCIS, intraductal (i.duc) injection of HB21(Fv)-PE40 could avoid systemic toxicity while retaining its potent antitumor effects on visible and occult tumors in the entire ductal tree. Pharmacokinetic studies in mice showed that, in contrast to intravenous injection, IT was undetectable by enzyme-linked immunosorbent assay in blood following i.duc injection of up to 3.0 µg HB21(Fv)-PE40. We demonstrated the antitumor efficacy of HB21(Fv)-PE40 in two mammary-in-duct (MIND) models, MCF7 and SUM225, grown in NOD/SCID/gamma mice. Tumors were undetectable by In Vivo Imaging System (IVIS) imaging in intraductally treated mice within 1 wk of initiation of the regimen (IT once weekly/3 wk, 1.5 µg/teat). MCF7 tumor-bearing mice remained tumor free for up to 60 d of observation with i.duc IT, whereas the HB21 antibody alone or intraperitoneal IT treatment had minimal/no antitumor effects. These and similar findings in the SUM225 MIND model were substantiated by analysis of mammary gland whole mounts, histology, and immunohistochemistry for the proteins Ki67, CD31, CD71 (TFR), and Ku80. This study provides a strong preclinical foundation for conducting feasibility and safety trials in patients with stage 0 breast cancer.

Highly active CAR T cells that bind to a juxtamembrane region of mesothelin and are not blocked by shed mesothelin.

SignificanceMesothelin (MSLN) is a cell-surface protein that is a popular target for antibody-based therapies. We have identified shed MSLN as a major obstacle to successful antibody therapies and prepared a monoclonal antibody that inhibits shedding and makes very active CAR T cells whose activity is not blocked by shed MSLN and merits further preclinical development.

Immunotoxin SS1P is rapidly removed by proximal tubule cells of kidney, whose damage contributes to albumin loss in urine.

Recombinant immunotoxins (RITs) are chimeric proteins composed of an Fv and a protein toxin being developed for cancer treatment. The Fv brings the toxin to the cancer cell, but most of the RITs do not reach the tumor and are removed by other organs. To identify cells responsible for RIT removal, and the pathway by which RITs reach these cells, we studied SS1P, a 63-kDa RIT that targets mesothelin-expressing tumors and has a short serum half-life. The major organs that remove RIT were identified by live mouse imaging of RIT labeled with FNIR-Z-759. Cells responsible for SS1P removal were identified by immunohistochemistry and intravital two-photon microscopy of kidneys of rats. The primary organ of SS1P removal is kidney followed by liver. In the kidney, SS1P passes through the glomerulus, is taken up by proximal tubular cells, and transferred to lysosomes. In the liver, macrophages are involved in removal. The short half-life of SS1P is due to its very rapid filtration by the kidney followed by degradation in proximal tubular cells of the kidney. In mice treated with SS1P, proximal tubular cells are damaged and albumin in the urine is increased. SS1P uptake by kidney is reduced by coadministration of l-lysine. Our data suggests that l-lysine administration to humans might prevent SS1P-mediated kidney damage, reduce albumin loss in urine, and alleviate capillary leak syndrome.

Recombinant immunotoxins with albumin-binding domains have long half-lives and high antitumor activity.

Recombinant immunotoxins (RITs) are chimeric proteins consisting of a Fv that binds to a cancer cell and a portion of a protein toxin. One of these, Moxetumomab pasudotox, was shown to be effective in treating patients with some leukemias, where the cells are readily accessible to the RIT. However, their short half-life limits their efficacy in solid tumors, because penetration into the tumors is slow. Albumin and agents bound to albumin have a long half-life in the circulation. To increase the time tumor cells are exposed to RITs, we have produced and evaluated variants that contain either an albumin-binding domain (ABD) from Streptococcus or single-domain antibodies from Llama. We have inserted these ABDs into RITs targeting mesothelin, between the Fv and the furin cleavage site. We find that these proteins can be produced in large amounts, are very cytotoxic to mesothelin-expressing cancer cell lines, and have a high affinity for human or mouse serum albumin. In mice, the RIT containing an ABD from Streptococcus has a longer half-life and higher antitumor activity than the other two. Its half-life in the circulation of mice ranges from 113 to 194 min compared with 13 min for an RIT with no ABD. Cell uptake studies show the RIT enters the target cell bound to serum albumin. We conclude that RITs with improved half-lives and antitumor activity should be evaluated for the treatment of cancer in humans.

A Bispecific Antibody That Targets the Membrane-Proximal Region of Mesothelin and Retains High Anticancer Activity in the Presence of Shed Mesothelin.

Mesothelin (MSLN) is a cell-surface protein that is expressed in many cancers, which makes it a popular target for Ab-based cancer therapy. However, MSLN is shed from cancer cells at high levels via proteases that cleave at its membrane-proximal C-terminal region. Shed MSLN accumulates in patients' fluids and tumors and can block Ab-based MSLN-targeting drugs from killing cancer cells. A previously established mAb, 15B6, binds MSLN at its protease-sensitive C-terminal region and does not bind shed MSLN. Moreover, 15B6 variable fragment (Fv)-derived chimeric antigen receptor T cells are not inhibited by shed MSLN and kill tumors in mice more effectively than mAb SS1 Fv-derived chimeric antigen receptor T cells, which bind an epitope retained in shed MSLN. In this study, we have established 15B6 Fv-derived MSLN × CD3 bispecific antibodies (BsAb) that target MSLN-expressing cancers. We identified our lead candidate BsAb 5 after screening multiple 15B6-derived BsAb formats in vitro for cytotoxic activity. BsAb 5 activates T cells to kill various cancer cell lines in a MSLN-specific manner. MSLN 296-591 His, a recombinant protein mimicking shed MSLN, does not inhibit 15B6-derived BsAb 5 but completely inhibits humanized SS1-derived BsAb 7. Furthermore, BsAb 5 inhibits and delays tumor growth and is not inhibited by MSLN 296-585 His in mice. Our findings indicate that by targeting the protease-sensitive region of MSLN, BsAb 5 has high MSLN-specific anticancer activity that is not inhibited by shed MSLN. BsAb 5 may be a promising immunotherapy candidate for MSLN-expressing cancers.

A bispecific antibody that targets the membrane-proximal region of mesothelin and retains high anticancer activity in the presence of shed mesothelin.

Mesothelin (MSLN) is a cell-surface protein that is expressed on many cancers, which makes it a popular target for antibody-based cancer therapy. However, MSLN is shed from cancer cells at high levels via proteases that cleave at its membrane-proximal C-terminal region. Shed MSLN accumulates in patient fluids and tumors and can block antibody-based MSLN-targeting drugs from killing cancer cells. A previously established monoclonal antibody (mAb), 15B6, binds MSLN at its protease-sensitive C-terminal region and does not bind shed MSLN. 15B6 variable fragment (Fv)-derived chimeric antigen receptor (CAR) T cells are not inhibited by shed MSLN and kill tumors in mice more effectively than mAb SS1 Fv-derived CAR T cells, which bind an epitope retained in shed MSLN. Here, we have established 15B6 Fv-derived MSLN x CD3 bispecific antibodies (BsAbs) that target MSLN-expressing cancers. We identified our lead candidate, BsAb 5, after screening multiple 15B6-derived BsAb formats in vitro for cytotoxic activity. BsAb 5 activates T cells to kill various cancer cell lines in a MSLN-specific manner. MSLN 296-591 His, a recombinant protein mimicking shed MSLN, does not inhibit 15B6-derived BsAb 5 but completely inhibits humanized SS1-derived BsAb 7. Furthermore, BsAb 5 inhibits and delays tumor growth and is not inhibited by MSLN 296-585 His in mice. Our findings indicate that by targeting the protease-sensitive region of MSLN, BsAb 5 has high MSLN-specific anticancer activity that is not inhibited by shed MSLN. BsAb 5 may be a promising immunotherapy candidate for MSLN-expressing cancers.

Wilms' tumour suppressor gene 1 (WT1) is involved in the carcinogenesis of Lung cancer through interaction with PI3K/Akt pathway.

ABSRACT: Although studies have shown the oncogene WT1 is overexpressed in lung cancer, there is no data showing the implication of WT1 in lung cancer biology. In the present study, we first demonstrated that isotype C of WT1 was conservely overexpressed in 20 lung cancer patient specimens. Knockdown of WT1 by small interference RNA (siRNA) transfection resulted in a significant inhibition of cell proliferation, induction of cell cycle arrest at G1 phase, and the expression change of BCL-2 family genes in WT1+ A549 cells. Furthermore, we found that DDP treatment could decrease the WT1 mRNA expression level by 5% and 15% at a dose of 1 µg/ml, by 25% and 40% at a dose of 2 µg/ml for 24 and 48 h, respectively. In the mean time, DDP treatment also reduced the PI3K/AKT pathway activity. Further analysis by using siRNA targeting the AKT-1 and the PI3K pathway inhibitor Ly294002 revealed that the AKT-1 siRNA reduced the WT1 expression effectively in A549 cells, and the same result was observed in Ly294002 treated cells, indicating that DDP treatment could down regulate WT1 expression through the PI3K/AKT pathway. Of particular interest, knockdown of WT1 also inhibited the AKT expression effectively, Chip assay further confirmed that WT1 is a transcription factor of AKT-1. We thus concluded that there is a positive feedback loop between WT1 and AKT-1. Taken together, DDP treatment downregulates the WT1 expression through the PI3K/AKT signaling pathway, and there is a feedback between WT1 and AKT-1; WT1 is involved in cellular proliferation in A549 cells, WT1 inhibition in combination with DDP will provide a new light for lung cancer therapy.

Recent research and development of inhibitors targeting sentrin-specific protease 1 for the treatment of cancers.

Small ubiquitin-like modifier (SUMO)/sentrin-specific protease 1 (SENP1), is a cysteine protease that promotes SUMO maturation and deSUMOylation of target proteins and regulates transcription factors or co-regulatory factors to mediate gene transcription. Many studies have shown that SENP1 is the driving factor for a multitude of cancers including prostate cancer, liver cancer, and breast cancer. Inhibition of SENP1 activity has been proved to inhibit the survival, proliferation, invasion, and migration of cancer cells, and increase their chemical and radiation sensitivity. Therefore, SENP1 is a promising anti-tumor target. At present, peptide inhibitors of SENP1 have entered clinical trials. Recently, many small molecule compounds and natural products were synthesized and identified as SENP1 inhibitors, and showed good tumor inhibitory activity in vitro and in vivo. This review summarizes the structure, physiological function, and role of SENP1 in tumorigenesis and development, focusing on the design and discovery of small molecule inhibitors of SENP1 from the perspective of medicinal chemistry, providing ideas for the development and research of small molecule inhibitors of SENP1 in the future.

Development of inhibitors targeting glycogen synthase kinase-3ß for human diseases: Strategies to improve selectivity.

Glycogen synthase kinase-3ß (GSK-3ß) is a conserved serine/threonine kinase that participates in the transmission of multiple signaling pathways and plays an important role in the occurrence and development of human diseases, such as metabolic diseases, neurological diseases and cancer, making it to be a potential and promising drug target. To date, copious GSK-3ß inhibitors have been synthesized, but only few have entered clinical trials. Most of them exerts poor selectivity, concomitant off-target effects and side effects. This review summarizes the structural characteristics, biological functions and relationship with diseases of GSK-3ß, as well as the selectivity profile and therapeutic potential of different categories of GSK-3ß inhibitors. Strategies for increasing selectivity and reducing adverse effects are proposed for the future design of GSK-3ß inhibitors.

Synergistic tumor growth-inhibitory effect of the prostate-specific antigen-activated fusion peptide BSD352 for prostate cancer therapy.

Prostate-specific antigen (PSA), a serine protease, is a promising target for the development of prodrugs in prostate cancer treatment. In this study, we designed a novel fusion peptide, BSD352, containing three functional domains: a protein transduction domain from HIV transactivating regulatory protein (TAT) followed by the BH3 domain of the p53 upregulated modulator of apoptosis (TAT-BH3), an anti-vascular endothelial growth factor peptide (SP5.2), and an anti-basic fibroblast growth factor peptide (DG2). These different domains in BSD352 were linked together by a linker sequence corresponding to a PSA hydrolytic substrate peptide. The BSD352 fusion peptide could be selectively cleaved by PSA in PSA-producing LNCaP prostate cancer cells. Furthermore, the BSD352 fusion peptide was efficiently transduced into tumor cells both in vitro and in vivo, and the BH3 domain was found to induce tumor cell apoptosis by elevating the expression of Bax, cytochrome C release, and caspase-9 cleavage. Moreover, the SP5.2 and DG2 domains in the BSD352 fusion peptide also exhibited in-vitro endothelial cell growth inhibition and in-vivo antiangiogenic activities. Direct injection of BSD352 into an established LNCaP xenograft tumor in mice inhibited tumor growth, whereas a synergistic effect was observed with the combined use of wild-type BH3, SP5.2, and DG2 functional domains. These results suggest that BSD352 could be beneficial for the treatment of accessible prostate tumors and may provide a complementary strategy for prostate cancer therapy.

[The multi-spectra classification algorithm based on K-means clustering and spectral angle cosine].

The classification and de-aliasing methods with respect to multi-spectra and hyper-spectra have been widely studied in recent years. And both K-mean clustering algorithm and spectral similarity algorithm are familiar classification methods. The present paper improved the K-mean clustering algorithm by using spectral similarity match algorithm to perform a new spectral classification algorithm. Two spectra with the farthest distance first were chosen as reference spectra. The Euclidean distance method or spectral angle cosine method then were used to classify data cube on the basis of the two reference spectra, and delete the spectra which belongs to the two reference spectra. The rest data cube was used to perform new classification according to a third spectrum, which is the farthest distance or the biggest angle one corresponding to the two reference spectra. Multi-spectral data cube was applied in the experimental test. The results of K-mean clustering classification by ENVI, compared with simulation results of the improved K-mean algorithm and the spectral angle cosine method, demonstrated that the latter two classify two air bubbles explicitly and effectively, and the improved K-mean algorithm classifies backgrounds better, especially the Euclidean distance method can classify the backgrounds integrally.

[Hyperspectral image compression technology research based on EZW].

Along with the development of hyperspectral remote sensing technology, hyperspectral imaging technology has been applied in the aspect of aviation and spaceflight, which is different from multispectral imaging, and with the band width of nanoscale spectral imaging the target continuously, the image resolution is very high. However, with the increasing number of band, spectral data quantity will be more and more, and these data storage and transmission is the problem that the authors must face. Along with the development of wavelet compression technology, in field of image compression, many people adopted and improved EZW, the present paper used the method in hyperspectral spatial dimension compression, but does not involved the spectrum dimension compression. From hyperspectral image compression reconstruction results, whether from the peak signal-to-noise ratio (PSNR) and spectral curve or from the subjective comparison of source and reconstruction image, the effect is well. If the first compression of image from spectrum dimension is made, then compression on space dimension, the authors believe the effect will be better.

[Research on endmember extraction algorithm based on spectral classification].

Spectral unmixing is an important task for data processing of hyperspectral remote sensing, which is comprised of extracting the pure spectra (endmember) and calculating the abundance value of pure spectra. The most efficient endmember extracting algorithms (EEAs) is designed based on convexity geometry such as pure pixel index (PPI), N-finder algorithm (N-FINDR). Most EEAs choose pure spectra from all pixels of an image so that they have disadvantages like slow processing speed and poor precision. Partial algorithms need reducing the spectral dimension, which results in the difficulty in small target identification. This paper proposed an algorithm that classifies the hyperspetral image into some classes with homogeneous spectra and considers the mean spectra of a class as standard spectra for the class, then extracts pure spectrum from all standard spectra of classes. It reduces computation and the effect of system error, enhancing the speed and precision of endmember extraction. Using the least squares with constraints on spectral extraction and spectral unmixing, by controlling the band average value of the maximum spectral redundant allowance to control the number of endmembers, does not need to reduce the spectral dimension and predetermine the number of endmembers, so compared to N-finder algorithm, such algorithm is more rational.

p21WAF1/CIP1 gene transcriptional activation exerts cell growth inhibition and enhances chemosensitivity to cisplatin in lung carcinoma cell.

BACKGROUND: Non-small-cell lung carcinomas (NSCLCs) exhibit poor prognosis and are usually resistant to conventional chemotherapy. Absence of p21WAF1/CIP1, a cyclin-dependent kinase (cdk) inhibitor, has been linked to drug resistance in many in vitro cellular models. RNA activation (RNAa) is a transcriptional activation phenomena guided by double-strand RNA (dsRNA) targeting promoter region of target gene. METHODS: In this study, we explored the effect of up-regulation of p21 gene expression on drug-resistance in A549 non-small-cell lung carcinoma cells by transfecting the dsRNA targeting the promoter region of p21 into A549 cells. RESULTS: Enhanced p21 expression was observed in A549 cells after transfection of dsRNA, which was correlated with a significant growth inhibition and enhancement of chemosensitivity to cisplatin in A549 cells in vitro. Moreover, in vivo experiment showed that saRNA targeting the promoter region of p21 could significantly inhibit A549 xenograft tumor growth. CONCLUSIONS: These results indicate that p21 plays a role in lung cancer drug-resistance process. In addition, this study also provides evidence for the usage of saRNA as a therapeutic option for up-regulating lower-expression genes in lung cancer.

Production and characterization of a recombinant single-chain antibody against Hantaan virus envelop glycoprotein.

Hantaan virus (HTNV) is the type of Hantavirus causing hemorrhagic fever with renal syndrome, for which no specific therapeutics are available so far. Cell type-specific internalizing antibodies can be used to deliver therapeutics intracellularly to target cell and thus, have potential application in anti-HTNV infection. To achieve intracellular delivery of therapeutics, it is necessary to obtain antibodies that demonstrate sufficient cell type-specific binding, internalizing, and desired cellular trafficking. Here, we describe the prokaryotic expression, affinity purification, and functional testing of a single-chain Fv antibody fragment (scFv) against HTNV envelop glycoprotein (GP), an HTNV-specific antigen normally located on the membranes of HTNV-infected cells. This HTNV GP-targeting antibody, scFv3G1, was produced in the cytoplasm of Escherichia coli cells as a soluble protein and was purified by immobilized metal affinity chromatography. The purified scFv possessed a high specific antigen-binding activity to HTNV GP and HTNV-infected Vero E6 cells and could be internalized into HTNV-infected cells probably through the clathrin-dependent endocytosis pathways similar to that observed with transferrin. Our results showed that the E. coli-produced scFv had potential applications in targeted and intracellular delivery of therapeutics against HTNV infections.

[Research on spectral classification algorithm based on spatial feature].

With the wide use of imaging spectroscopy, applying data cubes to classification and identification of materials has been developed to be an important research content. The classification algorithms play a vital role in accuracy and precision of object identification. The most common classification algorithms mainly make use of the information gained from spectral dimension and classify the materials based on spectral match. The material reflectance spectra collected by imaging spectroscopy is determined not only by the sorts, but also by the geometry structure and roughness of material surface, and so on. Then classification and identification algorithms only using the reflection spectra have errors to some extent. This paper puts forward an algorithm based on the common classification algorithms that controls the classification process by using the spatial feature of image to promote the correctness of classification. This algorithm was applied to identify the true leaves from the fake ones. The result shows preferable spatial continuity. To a great extent, the algorithm overcomes "ma pixel" domino effect, and is proved valid.

Site-Specific PEGylation of Anti-Mesothelin Recombinant Immunotoxins Increases Half-life and Antitumor Activity.

Recombinant immunotoxins (RIT) are chimeric proteins containing an Fv that binds to tumor cells, fused to a fragment of Pseudomonas exotoxin (PE) that kills the cell. Their efficacy is limited by their short half-life in the circulation. Chemical modification with polyethylene glycol (PEG) is a well-established method to extend the half-lives of biologics. Our goal was to engineer RITs with an increase in half-life and high cytotoxic activity. We introduced single cysteines at different locations in five anti-mesothelin RITs and employed site-specific PEGylation to conjugate them to 20-kDa PEG. Because our previous PEGylation method using ß-mercaptoethanol reduction gave poor yields of PEG-modified protein, we employed a new method using tris(2-carboxyethyl)phosphine to reduce the protein and could PEGylate RITs at approximately 90% efficiency. The new proteins retained 19% to 65% of cytotoxic activity. Although all proteins are modified with the same PEG, the radius of hydration varies from 5.2 to 7.1, showing PEG location has a large effect on protein shape. The RIT with the smallest radius of hydration has the highest cytotoxic activity. The PEGylated RITs have a 10- to 30-fold increase in half-life that is related to the increase in hydrodynamic size. Biodistribution experiments indicate that the long half-life is due to delayed uptake by the kidney. Antitumor experiments show that several PEG-RITs are much more active than unmodified RIT, and the PEG location greatly affects antitumor activity. We conclude that PEGylation is a useful approach to improve the half-life and antitumor activity of RITs.

Casein nanoparticles as oral delivery carriers of mequindox for the improved bioavailability.

Mequindox (Meq) is a promising broad-spectrum antibacterial agent, but the clinical application of Meq has been hampered by its low oral bioavailability. Casein (Cas) can bind to a variety of poorly water-soluble drugs to improve their water solubility through a micellar solubilization mechanism. Here, a low-cost and convenient method was introduced to prepare mequindox-loaded casein nanoparticles (Meq-Cas). Meq-Cas was characterized by several methods including differential scanning calorimetry (DSC), X-ray diffraction (XRD), and fourier transform infrared (FTIR) to illuminate the mutual effect between the drug and carriers. Meq-Cas presented nearly spherical nanoparticles with smooth surfaces and its mean particle size was lower than untreated Cas. Meq-Cas showed a nearly complete release of Meq, which displayed a biphasic drug release pattern in both phosphate-buffered solution (PBS) and simulated gastric fluid (SGF). The relative oral bioavailability of Meq-Cas was found to be about 1.20 times higher than that of the animals treated with Meq suspension (control). These results suggest that Cas is a good candidate to load in Meq for pharmaceutical purposes.

The proapoptotic activity of C-terminal domain of apoptosis-inducing factor (AIF) is separated from its N-terminal.

Apoptosis-inducing factor (AIF) is a mitochondrial flavoprotein that mediates both NADH-oxidizing and caspase-independent apoptosis. Further, the proapoptotic activity of AIF is located in the C-terminus of AIF, although the precise minimum sequence responsible for apoptosis induction remains to be investigated. In the present study, we generated two truncated AIFs, AIFDelta1-480-FLAG, which is a FLAG-tagged C-terminal peptide comprising amino acids from 481 to 613, and AIF360-480 containing amino acids from 360 to 480 of AIF. We used confocal microscopy to demonstrate that both the truncated proteins are expressed and located in the cytoplasm of transfected cells. AIFDelta1-480 but not AIF360-480 induces apoptosis in transfected cells. We also found that the expression of AIFDelta1-480 could initiate the release of cytochrome c from the mitochondria. The suppression of caspase-9 via siRNA blocked the proapoptotic activity of AIFDelta1-480. Therefore, AIFDelta1-480 is sufficient for inducing caspase-9-dependent apoptotic signaling, probably by promoting the release of cytochrome c. At last, we generated a chimeric immuno-AIFDelta1-480 protein, which comprised an HER2 antibody, a Pseudomonas exotoxin A translocation domain and AIFDelta1-480. Human Jurkat cells transfected with the immuno-AIFDeltal-480 gene could express and secrete the chimeric protein, which selectively recognize and kill HER2-overexpressing tumor cells. Our study demonstrates the feasibility of the immuno-AIFDeltal-480 gene as a novel approach to treating HER2-overexpressing cancers.