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.

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.

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.

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.

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.

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.

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.

Anti-Mesothelin Recombinant Immunotoxin Therapy for Colorectal Cancer.

BACKGROUND: Mesothelin (MSLN) is a cell surface glycoprotein expressed at a high level on many malignancies, including pancreatic adenocarcinoma, serous ovarian cancer, and epithelioid mesothelioma. MSLN-targeted recombinant immunotoxins (RITs) consist of an anti-MSLN Fv fused to the catalytic domain of Pseudomonas exotoxin A. Recent data has also shown that MSLN is expressed at clinically relevant levels on the surface of colorectal cancer (CRC). In this study, CRC cell lines were tested for MSLN expression and susceptibility to MSLN-targeted RITs. MATERIALS AND METHODS: CRC cell lines were tested for membranous MSLN expression via flow cytometry. Cell lines expressing MSLN were tested by WST-8 cell viability assay for sensitivity to various RITs and chemotherapeutic agents. CRC cell line SW-48 was tested in a mouse model for response to RIT as a single agent or in combination with actinomycin D and oxaliplatin. RESULTS: CRC cell lines were susceptible to anti-MSLN RITs at half maximal inhibitory concentration levels comparable with those previously described in pancreatic cancer cell lines. In a nude mouse model, MSLN-targeted RIT treatment of SW48 CRC tumors resulted in a significant decrease in tumor volume. Although combination therapy with standard of care chemotherapeutic oxaliplatin did not improve tumor regressions, combination therapy with actinomycin D resulted in > 90% tumor volume reduction with 50% complete regressions. CONCLUSIONS: These data support the development of anti-MSLN RITs as well as other MSLN-targeted therapies for CRC.

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.

Targeted inhibition of hantavirus replication and intracranial pathogenesis by a chimeric protein-delivered siRNA.

Hantavirus (HV) infection, which underlies hantavirus hemorrhagic fever with renal syndrome and hantavirus pulmonary syndrome, remains to be a severe clinical challenge. Here, we synthesized small interfering RNAs (siRNAs) that target the encoding sequences of HV strain 76-118, and validated their inhibitory role in virus replication in HV-infected monkey kidney Vero E6 cells. A chimeric protein, 3G1-Cκ-tP, consisting of a single-chain antibody fragment (3G1) against the HV surface envelop glycoprotein, the constant region of human immunoglobulin κ chain (Cκ), and truncated protamine (amino acids 8-29, tP), was further generated. The fusion protein showed high affinity to HV antigen on the infected cell membrane, and internalized through clathrin-mediated endocytosis; it bound to siRNAs via the basic nucleic acid-rich protamine fragment, leading to their specific delivery into HV-infected cells and efficient inhibition of virus replication. An encephalitis mouse model was established via intracranial HV administration. Intraperitoneal injection of siRNAs complexed with 3G1-Cκ-tP achieved specific distribution of siRNAs in HV-infected brain cells, significantly reduced HV antigen levels, and effective protection from HV infection-derived animal death. These results provide a compelling rationale for novel therapeutic protocols designed for HV infection and related disorders.

[miR-141-3p regulates the expression of androgen receptor by targeting its 3'UTR in prostate cancer LNCaP cells].

OBJECTIVE: To detect the target degradation of miR-141-3p on androgen receptor (AR) gene in LNCaP cells and demonstrate whether AR gene is a target of miR-141-3p. METHODS: After prostate cancer cell line LNCaP was transfected with miR-141-3p mimics, expression levels of AR mRNA and protein in the LNCaP cells were detected by reverse transcription PCR and Western blotting, respectively. The 3'untranslated regions (3'UTR) of AR mRNA containing the binding site of miR-141-3p was amplified by PCR and inserted into pmiR-report vector (a 3'downstream luciferase reporter gene). The product was termed pmiR-AR-3'UTR. Double luciferase reporter system was employed to verify the potential target effect of miR-141-3p on pmiR-AR-3'UTR. RESULTS: Transfection of miR-141-3p mimics decreased both mRNA and protein expression levels of AR in LNCaP cells. Compared with control group, miR-141-3p transfection significantly inhibited the activity of luciferase of pmiR-AR-3'UTR. CONCLUSION: AR is a direct target gene of miR-141-3p.

Antitumor activity of SA12, a novel peptide, on SKBr-3 breast cancer cells via the mitochondrial apoptosis pathway.

Breast cancer is considered to be the most common malignancy in women. Treatment of breast cancer has been focused on molecular targeted therapy, and anticancer peptides are considered to be some of the most promising candidate drugs. In the current study, we used mRNA-peptide display technology to screen antibreast cancer peptides and identified a novel peptide, SA12, which showed significant activity in the inhibition of proliferation and induction of apoptosis in SKBr-3 breast cancer cells. The mechanism by which SA12 peptide triggers apoptosis was further investigated using a pull-down assay, reverse transcription-polymerase chain reaction, and Western blotting analysis. The results demonstrated that this peptide could interact with tumor-associated proteins MECP2 and CDC20B, which further induced apoptosis of tumor cells via the mitochondrial pathway involving the Bcl-2 family and related caspases. We propose that the novel SA12 peptide has the potential to provide a new strategy for the development of targeted therapy in breast cancer.

Early growth response gene 1, a TRBP binding protein, is involved in miRNA activity of miR-125a-3p in human cells.

BACKGROUND: MicroRNAs (miRNAs) are key regulators of many cellular pathways. However, the picture for components or regulators involved in the process of miRNA biogenesis and function remains to be further elucidated. Early growth response gene 1 (Egr1) has long been considered as tumor suppressor and transcriptional factor involved in cell proliferation and regulation of apoptosis. RESULTS: Here we show that Egr1 is able to modulate guide strand loading of certain miRNAs or siRNAs in human HEK293 and A549 cells, which is related with thermodynamic parameters of miRNA or siRNA. Further, we found that Egr1 modulates the silencing activity of miR-125a-3p in vivo. Immunoprecipitation experiment demonstrated that Egr1 could bind miRNA biogenesis protein TAR RNA-binding protein2 (TRBP2), and knockdown TRBP by RNAi abolished the regulating effects of Egr1 on miR-125a-3p efficiency. Further experiments revealed that deleting sequence 97-227aa containing dsRBD B domain of TRBP eliminated the binding phenomenon between Egr1 and TRBP and impaired the effect of Egr1 on miR-125a-3p efficiency. CONCLUSIONS: Taken together, our study has demonstrated that Egr1 is able to regulate miRNA activity of miR-125a-3p in human cells through binding TRBP, which highlights an unexpected function of Egr1 in miRNA pathway.

Chimeric ubiquitin ligases inhibit non-small cell lung cancer via negative modulation of EGFR signaling.

Targeting epidermal growth factor receptor (EGFR) represents a promising therapeutic strategy for non-small cell lung cancers (NSCLC). The ubiquitin-proteasome system (UPS) is a major pathway that mediates protein degradation. To target the degradation of EGFR, we generated two artificial ubiquitin ligases, which are composed of an EGFR-binding domain, i.e., the SH2 domain from growth factor receptor binding protein 2 (Grb2), and an ubiquitin ligase catalytic domain, i.e., the RING domain from Cbl or the U-box domain from CHIP. When the chimeric ubiquitin ligases were introduced into lung cancer SPC-A1 cells, they effectively associated with EGFR, promoted its ubiquitination and degradation, and as a result, blocked the downstream PI3K-Akt signal pathway. Moreover, cell proliferation and invasion were inhibited, the sensitivity to docetaxel-induced apoptosis was enhanced and the tumorigenicity was suppressed. In conclusion, negative modulation of EGFR signaling by the chimeric ubiquitin ligases can inhibit malignancy of SPC-A1 cells and sensitize these cells to chemotherapy, thus it may be applied to targeted therapy for NSCLC.

Downregulation of microRNA-429 inhibits cell proliferation by targeting p27Kip1 in human prostate cancer cells.

MicroRNAs (miRNAs) are closely associated with cell proliferation, invasion and metastasis in various types of cancer, including prostate cancer. In this study, the role of miR-429 in the regulation of cell proliferation was investigated in prostate cancer cells. miR-429 expression levels were measured in the IF11 and IA8 prostate cancer cell lines and normal prostate epithelial tissues by quantitative polymerase chain reaction. miR-429 mimics or an miR-429 inhibitor were then transfected into the human prostate cancer cell lines. MTT and fluorescence-activated cell sorting were used to detect the effect of miR-429 on cell proliferation. A luciferase reporter system was employed to verify the potential target of miR-429. The results revealed that miR-429 was significantly upregulated in the human prostate cancer cell lines, compared with the normal prostate epithelial tissue. Downregulation of miR-429 expression in IF11 and IA8 cells inhibited cell proliferation and arrested the cells in the G1 phase of the cell cycle. The luciferase assay demonstrated that p27Kip1 was a direct target of miR-429. Furthermore, overexpression of p27Kip1 was observed to partially rescue the proliferation­promoting effect of miR-429 on IA8 cells. In conclusion, to the best of our knowledge this study was the first to show that miR-429 is involved in the oncogenesis of prostate cancer and thus may be a novel prognostic biomarker in prostate cancer.

Tumor suppressor miR-34a targets PD-L1 and functions as a potential immunotherapeutic target in acute myeloid leukemia.

miRNA (miR) 34a has been shown to modulate critical gene transcripts involved in tumorigenesis, but its role in tumor-mediated immunosuppression is largely unknown. PD-L1 plays an important role in immune responses, however, presently its transcriptional regulatory mechanisms are not well understood. In the present study, we analyzed the expression of PD-L1 and miR-34a in 44 acute myeloid leukemia (AML) samples, and observed an inverse correlation between PD-L1 and miR-34a expression. Overexpression of miR-34a in HL-60 and Kasumi-1 cells blocked PD-L1 expression, and reduced PD-L1 surface expression. Using luciferase reporter assay and mutagenesis, we identified miR-34a as a putative binder of the PD-L1-3'UTR. Surface expression of PD-L1 induced by chemotherapeutic agents could also be reversed by miR-34a; furthermore, PD-L1 specific T cell apoptosis was reduced as well following miR-34a transfection. We also found that there is a positive feedback between PD-L1 expression and AKT activation. Our data suggest that miR-34a can regulate PD-L1 expression by targeting PD-L1 mRNA, and our present findings shed new light on the complex regulation of PD-L1 in human tumors, and on miR-34a in cancer immuno-based therapy.

Rlim, an E3 ubiquitin ligase, influences the stability of Stathmin protein in human osteosarcoma cells.

Stathmin is an oncoprotein and is expressed at high levels in a wide variety of human malignancies, which plays important roles in maintenance of malignant phenotypes. The regulation of Stathmin gene overexpression has been wildly explored, but the exact mechanism still needs to be elucidated. It is believed that regulation of an oncogene protein abundance through post-translational modifications is essential for maintenance of malignant phenotypes. Here we identified the Rlim, a Ring H2 zinc finger protein with intrinsic ubiquitin ligase activity, as a Stathmin-interacting protein that could increase Stathmin turnover through binding with this targeted protein and then induce its degradation by proteasome in a ubiquitin-dependent manner. Inhibition of endogenous Rlim expression by siRNA could increase the level of Stathmin protein, which further led to cell proliferation and cell cycle changes in human osteosarcoma cell lines. On the other hand, forced overexpression of Rlim could decrease the level of Stathmin protein. These results demonstrate that Rlim is involved in the negative regulation of Stathmin protein level through physical interaction and ubiquitin-mediated proteolysis. Hence, Rlim is a novel regulator of Stathmin protein in a ubiquitin-dependent manner, and represents a new pathway for malignant phenotype turnover by modulating the level of Stathmin protein in human osteosarcomas.