Occludin as a functional marker of vascular endothelial cells on tube-forming activity.

Cell therapy using endothelial progenitor cells (EPCs) is a promising strategy for the treatment of ischemic diseases. Two types of EPCs have been identified: early EPCs and late EPCs. Late EPCs are able to form tube structure by themselves, and have a high proliferative ability. The functional marker(s) of late EPCs, which relate to their therapeutic potential, have not been fully elucidated. Here we compared the gene expression profiles of several human cord blood derived late EPC lines which exhibit different tube formation activity, and we observed that the expression of occludin (OCLN) in these lines correlated with the tube formation ability, suggesting that OCLN is a candidate functional marker of late EPCs. When OCLN was knocked down by transfecting siRNA, the tube formation on Matrigel, the S phase + G2 /M phase in the cell cycle, and the spheroid-based sprouting of late EPCs were markedly reduced, suggesting the critical role of OCLN in tube formation, sprouting, and proliferation. These results indicated that OCLN plays a novel role in neovascularization and angiogenesis.

Cell-Surface MMP-9 Protein Is a Novel Functional Marker to Identify and Separate Proangiogenic Cells from Early Endothelial Progenitor Cells Derived from CD133(+) Cells.

To develop cell therapies for ischemic diseases, endothelial progenitor cells (EPCs) have been expected to play a pivotal role in vascular regeneration. It is desirable to use a molecular marker that is related to the function of the cells. Here, a quantitative polymerase chain reaction array revealed that early EPCs derived from CD133(+) cells exhibited significant expression of MMP-9. Some populations of early EPCs expressed MMP-9 on the cell surface and others did not. We also attempted to separate the proangiogenic fraction from early EPCs derived from CD133(+) cells using a functional cell surface marker, and we then analyzed the MMP-9(+) and MMP-9(-) cell fractions. The MMP-9(+) cells not only revealed higher invasion ability but also produced a high amount of IL-8. Moreover, the stimulative effect of MMP-9(+) cells on angiogenesis in vitro and in vivo was prohibited by anti-IL-8 antibody. These data indicate that MMP-9 is one of the useful cell surface markers for the separation of angiogenic cells. Our treatment of early EPCs with hyaluronidase caused not only a downregulation of cell-surface MMP-9 but also a decrease in invasion ability, indicating that membrane-bound MMP-9, which is one of the useful markers for early EPCs, plays an important role in angiogenesis. Stem Cells 2016;34:1251-1262.

Angiogenic Role of MMP-2/9 Expressed on the Cell Surface of Early Endothelial Progenitor Cells/Myeloid Angiogenic Cells.

Since the introduction of angiogenic cell therapy using early endothelial progenitor cells (EPCs), myeloid angiogenic cells (MACs) have been expected to be useful in treating ischemic diseases. In order to elucidate the angiogenic properties of MACs/EPCs, we clarified the characteristics of MACs as compared to M2 macrophages (Mϕs). Comparison of the gene expression profiles of MACs and late EPCs revealed that MACs expressed greater amounts of metalloproteinase (MMP)-9. It should be noted that the profile of MMP-2/9 expression on the cell surface of MACs was similar to that of M2 Mϕs, and that cell surface MMP-2/9 might be an active form based on molecular size. In addition, the invasion of MACs was prohibited not only by MMP-2/9 inhibitor, but also by the hyaluronidase treatment that caused the down-regulation of MMP-9 on the cell surface of MACs and inhibited their invasion activity. These results indicate that cell surface MMP-2/9 plays an important role in the high invasion ability of MACs. The conditioned medium of both MACs and M2 Mϕs stimulated tube formation of endothelial cells in vitro. MACs caused an increase in vessel formation in in vivo models through the production of IL-8. We propose that the role of MACs with cell surfaces expressing MMP-2/9 is rapidly invading ischemic tissue.

Regulatory Frameworks for Gene and Cell Therapies in Japan.

The regulations for the human use of advanced therapy medical products such as gene and cell therapy products have evolved in accordance with advance of clinical experience, scientific knowledge, and social acceptance to these technologies. In Japan, two laws, the Pharmaceuticals and Medical Devices (PMD) Act and the Act on the Safety of Regenerative Medicine (ASRM), were enacted in November 2014. The PMD Act defines regenerative medical products for the first time and introduces a system for the conditional and time-limited marketing authorization of regenerative medical products. Under ASRM, the responsibilities of medical institutions to ensure the safety and provide transparency of such medical technologies are described. Amendments to accompanying guidelines for these two Acts are currently in preparation. It is expected that the new legislative frameworks will promote the timely development of new products and technologies, to bring safe and effective regenerative medicines to Japanese patients.

[Current situations and the future prospect of monoclonal antibody products].

Monoclonal antibody products and monoclonal antibody-based biopharmaceuticals have shown considerable effectiveness in the treatment for variety of diseases; cancer, auto-immune/auto-inflammation diseases and so on. Significant advance in monoclonal antibody products for cancer treatments was made with antibody-drug conjugates (ADC), and antibodies for blockade of immune checkpoints. Already 3 ADCs and 2 anti-immune-checkpoint antibodies products have been approved, and these monoclonal antibody-related product pipelines reach over 30. On the other hand, EU approved first monoclonal-antibody biosimilar, RemsimaTM (infliximab), suggesting that other monoclonal-antibody biosmilars will follow to the market. In this paper, several new issues about monoclonal antibody products will be discussed.

Angiogenic cell therapy for severe ischemic diseases.

Cell therapies for severe ischemic diseases such as limb ischemia, acute myocardial infarction, and cerebral ischemia have been developed through in vitro and in vivo animal and clinical studies. Active cells for angiogenic cell therapy are believed endothelial progenitor cells (EPCs). EPCs have been extensively investigated to clarify their origin and biology. Many sources of EPCs have been proposed, including mononuclear cells (MNCs) fraction containing CD34(+) or CD133(+) (AC133(+)), isolated CD34(+) and AC133(+) cells, and induction and differentiation of EPCs from hematopoietic stem cells (HSCs). However, in vivo mechanisms by which EPCs contribute to neovascularization should be clarified. Many in vitro, in vivo, and clinical studies have been performed using these cells; angiogenic cell therapy will become an important regimen for severe ischemic diseases.

Determination of galactosamine impurities in heparin sodium using fluorescent labeling and conventional high-performance liquid chromatography.

Heparin is a sulfated glycosaminoglycan (GAG), which contains N-acetylated or N-sulfated glucosamine (GlcN). Heparin, which is generally obtained from the healthy porcine intestines, is widely used as an anticoagulant during dialysis and treatments of thrombosis such as disseminated intravascular coagulation. Dermatan sulfate (DS) and chondroitin sulfate (CS), which are galactosamine (GalN)-containing GAGs, are major process-related impurities of heparin products. The varying DS and CS contents between heparin products can be responsible for the different anticoagulant activities of heparin. Therefore, a test to determine the concentrations of GalN-containing GAG is essential to ensure the quality and safety of heparin products. In this study, we developed a method for determination of relative content of GalN from GalN-containing GAG in heparin active pharmaceutical ingredients (APIs). The method validation and collaborative study with heparin manufacturers and suppliers showed that our method has enough specificity, sensitivity, linearity, repeatability, reproducibility, and recovery as the limiting test for GalN from GalN-containing GAGs. We believe that our method will be useful for ensuring quality, efficacy, and safety of pharmaceutical heparins. On July 30, 2010, the GalN limiting test based on our method was adopted in the heparin sodium monograph in the Japanese Pharmacopoeia.

Development of defective and persistent Sendai virus vector: a unique gene delivery/expression system ideal for cell reprogramming.

The ectopic expression of transcription factors can reprogram differentiated tissue cells into induced pluripotent stem cells. However, this is a slow and inefficient process, depending on the simultaneous delivery of multiple genes encoding essential reprogramming factors and on their sustained expression in target cells. Moreover, once cell reprogramming is accomplished, these exogenous reprogramming factors should be replaced with their endogenous counterparts for establishing autoregulated pluripotency. Complete and designed removal of the exogenous genes from the reprogrammed cells would be an ideal option for satisfying this latter requisite as well as for minimizing the risk of malignant cell transformation. However, no single gene delivery/expression system has ever been equipped with these contradictory characteristics. Here we report the development of a novel replication-defective and persistent Sendai virus (SeVdp) vector based on a noncytopathic variant virus, which fulfills all of these requirements for cell reprogramming. The SeVdp vector could accommodate up to four exogenous genes, deliver them efficiently into various mammalian cells (including primary tissue cells and human hematopoietic stem cells) and express them stably in the cytoplasm at a prefixed balance. Furthermore, interfering with viral transcription/replication using siRNA could erase the genomic RNA of SeVdp vector from the target cells quickly and thoroughly. A SeVdp vector installed with Oct4/Sox2/Klf4/c-Myc could reprogram mouse primary fibroblasts quite efficiently; ∼1% of the cells were reprogrammed to Nanog-positive induced pluripotent stem cells without chromosomal gene integration. Thus, this SeVdp vector has potential as a tool for advanced cell reprogramming and for stem cell research.

Importance of neonatal FcR in regulating the serum half-life of therapeutic proteins containing the Fc domain of human IgG1: a comparative study of the affinity of monoclonal antibodies and Fc-fusion proteins to human neonatal FcR.

The neonatal FcR (FcRn) binds to the Fc domain of IgG at acidic pH in the endosome and protects IgG from degradation, thereby contributing to the long serum half-life of IgG. To date, more than 20 mAb products and 5 Fc-fusion protein products have received marketing authorization approval in the United States, the European Union, or Japan. Many of these therapeutic proteins have the Fc domain of human IgG1; however, the serum half-lives differ in each protein. To elucidate the role of FcRn in the pharmacokinetics of Fc domain-containing therapeutic proteins, we evaluated the affinity of the clinically used human, humanized, chimeric, or mouse mAbs and Fc-fusion proteins to recombinant human FcRn by surface plasmon resonance analysis. The affinities of these therapeutic proteins to FcRn were found to be closely correlated with the serum half-lives reported from clinical studies, suggesting the important role of FcRn in regulating their serum half-lives. The relatively short serum half-life of Fc-fusion proteins was thought to arise from the low affinity to FcRn. The existence of some mAbs having high affinity to FcRn and a short serum half-life, however, suggested the involvement of other critical factor(s) in determining the serum half-life of such Abs. We further investigated the reason for the relatively low affinity of Fc-fusion proteins to FcRn and suggested the possibility that the receptor domain of Fc-fusion protein influences the structural environment of the FcRn binding region but not of the FcgammaRI binding region of the Fc domain.

Microarray analysis of responsible genes in increased growth rate in the subline of HL60 (HL60RG) cells.

HL60RG, a subline of human promyelocytic leukemia HL60 cells, has a increased growth rate than their parental cells. To gain information of the mechanisms involved in the increased growth rate of HL60RG, we performed a multiplex fluorescence in situ hybridization (M-FISH), standard cytogenetics analysis (G-banding) and genome scan using 10K SNP mapping array on both cell types. Characteristic genomic alterations in HL60RG cells were identified including uniparental disomy (UPD) of chromosome 1, and hemizygous deletion in 10p and 11p. However, no such defects were observed in HL60 cells. Changes in gene expression in HL60RG cells were determined using expression arrays (Affymetrix GeneChip, HU133A). Candidate genes associated with the rapid growth of HL60RG cells were identified. Two tumor necrosis factor receptors, TNFRSF1B (type II tumor necrosis factor-α receptor) and TNFRSF8 (also known as a tumor marker CD30), which are adjacently located on chromosome 1 showed opposing changes in gene expression in HL60RG cells-over-expression of TNFRSF8 and repression of TNFRSF1B. Differences in the DNA methylation status in the transcriptional regulatory regions of both genes between HL60 and HL60RG was detected by a methylation-specific PCR assay. In conclusion, alterations in chromosome and gene expression in HL60RG may be associated with increased growth rate.

AW551984: a novel regulator of cardiomyogenesis in pluripotent embryonic cells.

An understanding of the mechanism that regulates the cardiac differentiation of pluripotent stem cells is necessary for the effective generation and expansion of cardiomyocytes as cell therapy products. In the present study, we have identified genes that modulate the cardiac differentiation of pluripotent embryonic cells. We isolated P19CL6 cell sublines that possess distinct properties in cardiomyogenesis and extracted 24 CMR (cardiomyogenesis-related candidate) genes correlated with cardiomyogenesis using a transcriptome analysis. Knockdown of the CMR genes by RNAi (RNA interference) revealed that 18 genes influence spontaneous contraction or transcript levels of cardiac marker genes in EC (embryonal carcinoma) cells. We also performed knockdown of the CMR genes in mouse ES (embryonic stem) cells and induced in vitro cardiac differentiation. Three CMR genes, AW551984, 2810405K02Rik (RIKEN cDNA 2810405K02 gene) and Cd302 (CD302 antigen), modulated the cardiac differentiation of both EC cells and ES cells. Depletion of AW551984 attenuated the expression of the early cardiac transcription factor Nkx2.5 (NK2 transcription factor related locus 5) without affecting transcript levels of pluripotency and early mesoderm marker genes during ES cell differentiation. Activation of Wnt/ß-catenin signalling enhanced the expression of both AW551984 and Nkx2.5 in ES cells during embryoid body formation. Our findings indicate that AW551984 is a novel regulator of cardiomyogenesis from pluripotent embryonic cells, which links Wnt/ß-catenin signalling to Nkx2.5 expression.

Proteomic analysis of two types of exosomes in human whole saliva.

Saliva contains a large number of proteins that participate in the protection of oral tissue. Exosomes are small vesicles (30-100 nm in diameter) with an endosome-derived limiting membrane that are secreted by a diverse range of cell types. We have recently demonstrated that exosomes are present in human whole saliva. In this study, we found that whole saliva contained at least two types of exosomes (exosome I and exosome II) that are different in size and protein composition. Proteomic analysis revealed that both types of exosomes contained Alix, Tsg101 and Hsp70, all exosomal markers, immunoglobulin A and polymeric immunoglobulin receptor, whereas they had different protein compositions. Most of dipeptidyl peptidase IV known as CD26 in whole saliva, was present on the exosome II and metabolically active in cleaving chemokines (CXCL11 and CXCL12). Human whole saliva exosomes might participate in the catabolism of bioactive peptides and play a regulatory role in local immune defense in the oral cavity.

Experience of reviewing the follow-on biologics including Somatropin and erythropoietin in Japan.

To share the experience of reviewing clinical data required for the licensing of follow-on biologic products (biosimilar products and similar biotherapeutical products as EU and WHO terminology, respectively) in Japan, the data packages of two follow-on biologics, "Somatropin BS s.c. [Sandoz] (Omnitrope®)" and "Epoetin alfa BS [JCR]", which have been recently approved in Japan according to the "Guidelines for the Quality, Safety and Efficacy Assurance of Follow-on Biologics" published on March 4th 2009, are described. The clinical data package and indication of Somatropin BS/Omnitrope(®) were different in each country. In case of Epoetin alfa BS [JCR], non-clinical and clinical data-package was different from those of erythropoietin biosimilar products approved in EU. Submission of post-marketing surveillance plans for both products was required. Even though there seem to be differences in data requirements by each national regulatory authority, the accumulation of experience will provide the rationale and consensus on how to design the clinical trials for follow-on biologics.

Quality, safety and efficacy of follow-on biologics in Japan.

Recently, WHO, EU, Japan and Canada have published guidelines on biosimilar/follow-on biologics. While there seems to be no significant difference in the general concept in these guidelines, the data to be submitted for product approval are partially different. Differences have been noted in the requirements for comparability studies on stability, prerequisites for reference product, or for the need of comparability exercise for determination of process-related impurities. In Japan, there have been many discussions about the amount and extent of data for approval of follow-on biologics. We try to clarify the scientific background and rational for regulatory pathway of biosimilar/follow-on biologics in Japan in comparison with the guidelines available from WHO, EU and Canada. In this article, we address and discuss the scientific background underlying these differences to facilitate the harmonization of follow-on biologic principles in the guidelines in future.

A comparative study of monosaccharide composition analysis as a carbohydrate test for biopharmaceuticals.

The various monosaccharide composition analysis methods were evaluated as monosaccharide test for glycoprotein-based pharmaceuticals. Neutral and amino sugars were released by hydrolysis with 4-7N trifluoroacetic acid. The monosaccharides were N-acetylated if necessary, and analyzed by high-performance liquid chromatography (HPLC) with fluorometric or UV detection after derivatization with 2-aminopyridine, ethyl 4-aminobenzoate, 2-aminobenzoic acid or 1-phenyl-3-methyl-5-pyrazolone, or high pH anion exchange chromatography with pulsed amperometric detection (HPAEC-PAD). Sialic acids were released by mild acid hydrolysis or sialidase digestion, and analyzed by HPLC with fluorometric detection after derivatization with 1,2-diamino-4,5-methylenedioxybenzene, or HPAEC-PAD. These methods were verified for resolution, linearity, repeatability, and accuracy using a monosaccharide standard solution, a mixture of epoetin alfa and beta, and alteplase as models. It was confirmed that those methods were useful for ensuring the consistency of glycosylation. It is considered essential that the analytical conditions including desalting, selection of internal standards, release of monosaccharides, and gradient time course should be determined carefully to eliminate interference of sample matrix. Various HPLC-based monosaccharide analysis methods were evaluated as a carbohydrate test for glycoprotein pharmaceuticals by an inter-laboratory study.

Polyethyleneimine-modified resins effectively remove porcine circovirus and cellular prion protein.

Polyethyleneimine (PEI) possesses various molecular weights (MWs), structures, and virus capture capacities. However, whether PEI can capture porcine circovirus (PCV) and animal cell-derived prion protein (PrPC) that may contaminate source materials is unclear. Therefore, we conducted a feasibility study to assess the effectiveness of PEI in removing PCV and PrPC as a model of pathogenic prions. The removal performance of PCV was evaluated by quantitative PCR using PEIs with various MWs, structures, and ion exchange capacities in Tris (pH 7.5) and acetate (pH 5.5) buffers under neutral (pH 7.5) to acidic (pH 5.5) conditions. Removal performances of PrPC were also evaluated by western blotting using PEIs with various MWs and structures. Tris buffer did not affect the ability of PEI-modified resins to remove PCV, whereas acetate buffer affected removal performances, except those of PEI-10K-Br and PEI-70K-Br, which showed high ion-exchange capacities. PrPC was captured by PEIs with high MWs, especially PEI-70K-Br, which was the most effective. The results of this feasibility study suggested that PEI-modified resin could remove PCV and PrPC. PEI-70K-Br with an ion-exchange capacity of at least 0.3 meq/mL appears suitable as a PEI molecule for pathogen capture or removal of PCV or PrPC from biological materials.

Regulatory challenges with biosimilars: an update from 20 countries.

The World Health Organization (WHO) issued guidelines for the regulatory evaluation of biosimilars in 2009 and has provided considerable effort toward helping member states implement the evaluation principles in the guidelines into their regulatory practices. Despite this effort, a recent WHO survey (conducted in 2019-2020) has revealed four main remaining challenges: unavailable/insufficient reference products in the country; lack of resources; problems with the quality of some biosimilars (and even more with noninnovator products); and difficulties with the practice of interchangeability and naming of biosimilars. The following have been identified as opportunities/solutions for regulatory authorities to deal with the existing challenges: (1) exchange of information on products with other regulatory authorities and accepting foreign licensed and sourced reference products, hence avoiding conducting unnecessary (duplicate) bridging studies; (2) use of a "reliance" concept and/or joint review for the assessment and approval of biosimilars; (3) review and reassessment of the products already approved before the establishment of a regulatory framework for biosimilar approval; and (4) setting appropriate regulatory oversight for good pharmacovigilance, which is essential for the identification of problems with products and establishing the safety and efficacy of interchangeability of biosimilars.

Heparin identification test and purity test for OSCS in heparin sodium and heparin calcium by weak anion-exchange high-performance liquid chromatography.

Heparin sodium and heparin calcium, which are widely used as anti-coagulants, are known to potentially contain the natural impurity dermatan sulfate (DS). Recently serious adverse events occurred in patients receiving heparin sodium in the US, and a contaminant oversulfated chondroitin sulfate (OSCS) was found to be a cause of the events. To ensure the quality and safety of pharmaceutical heparins, there is need of a physicochemical identification test that can discriminate heparin from the heparin-related substances as well as a sensitive purity test for OSCS. Recently, HPLC with a strong-anion exchange column was proposed as the methods for identifying heparin and determination of OSCS in heparin sodium. Although this method is convenient and easy to perform, the only column suitable for this purpose is the Dionex IonPac AS11-HC column. In this study, we developed alternative identification test and test for OSCS in both heparin sodium and heparin calcium using a weak anion-exchange column. The identification test allowed for separation of heparin from the impurity DS and contaminant OSCS in a shorter time. The purity test provided enough sensitivity, specificity, linearity, recovery and repeatability for OSCS. We believe that our methods will be useful for quality control of pharmaceutical heparins.

Complete NMR analysis of oxytocin in phosphate buffer.

Complete NMR analysis of oxytocin (OXT) in phosphate buffer was elucidated by one-dimensional (1D)- and two-dimensional (2D)-NMR techniques, which involve the assignment of peptide amide NH protons and carbamoyl NH(2) protons. The (1)H-(15)N correlation of seven amide NH protons and three carbamoyl NH(2) protons were also shown by HSQC NMR of OXT without (15)N enrichment.

[Viral safety of biologicals: evaluation of hepatitis C virus (HCV) nucleic acid amplification test (NAT) assay and development of concentration method of HCV for sensitive detection by NAT].

The most important issue for the safety of biological products and blood products derived from human sources is how to prevent transmission of infectious agents. The hepatitis C virus (HCV) is a major public health problem due to its high prevalence. HCV is mainly transmitted by exposure to blood and highly infectious during the early window period with extremely low viral loads. Therefore it is important to develop more sensitive detection methods for HCV. In the case of blood products, both serological test and nucleic acid amplification test (NAT) are required to detect HCV. Since NAT is highly sensitive, establishment of a new standard is required for validation of NAT assay. NAT guideline and establishment of the standard for HCV RNA and HCV genotype panel is introduced in this review. On the other hand, to enhance the sensitivity of virus detection by NAT, a novel viral concentration method using polyethyleneimine (PEI)-conjugated magnetic beads (PEI beads) was developed. PEI beads concentration method is applicable to a wide range of viruses including HCV. Studies using the national standard for HCV RNA, HCV genotype panel and seroconversion panel, suggest that virus concentration method using PEI-beads is useful for improvement of the sensitivity of HCV detection by NAT and applicable to donor screening for HCV.