293F Cells Research Articles

Anti-HIV Double Variable Domain Immunoglobulins Binding Both gp41 and gp120 for Targeted Delivery of Immunoconjugates

BackgroundAnti-HIV immunoconjugates targeted to the HIV envelope protein may be used to eradicate the latent reservoir of HIV infection using activate-and-purge protocols. Previous studies have identified the two target epitopes most effective for the delivery of cytotoxic immunoconjugates the CD4-binding site of gp120, and the hairpin loop of gp41. Here we construct and test tetravalent double variable domain immunoglobulin molecules (DVD-Igs) that bind to both epitopes.MethodsSynthetic genes that encode DVD-Igs utilizing V-domains derived from human anti-gp120 and anti-gp41 Abs were designed and expressed in 293F cells. A series of constructs tested different inter-V-linker domains and orientations of the two V domains. Antibodies were tested for binding to recombinant Ag and native Env expressed on infected cells, for neutralization of infectious HIV, and for their ability to deliver cytotoxic immunoconjugates to infected cells.FindingsThe outer V-domain was the major determinant of binding and functional activity of the DVD-Ig. Function of the inner V-domain and bifunctional binding required at least 15 AA in the inter-V-domain linker. A molecular model showing the spatial orientation of the two epitopes is consistent with this observation. Linkers that incorporated helical domains (A[EAAAK]nA) resulted in more effective DVD-Igs than those based solely on flexible domains ([GGGGS]n). In general, the DVD-Igs outperformed the less effective parental antibody and equaled the activity of the more effective. The ability of the DVD-Igs to deliver cytotoxic immunoconjugates in the absence of soluble CD4 was improved over that of either parent.ConclusionsDVD-Igs can be designed that bind to both gp120 and gp41 on the HIV envelope. DVD-Igs are effective in delivering cytotoxic immunoconjugates. The optimal design of these DVD-Igs, in which both domains are fully functional, has not yet been achieved.

Hyperglycosylated resurfaced stabilized GP120 core as an immunogen elicits antibodies targeted at the CD4-binding site

Methods In this study, we used structural information of HIV gp120 core, together with the computer-assisted protein design tool to strategically add various glycosylation sites on the protein surface and create a panel of resurfaced stabilized core (RSC3)-based derivatives. The modified proteins were expressed in 293F or GnTi(-/-) cells and purified by Nickel-affinity and gel-filtration chromatography. To verify the glycosylation sites and antigenic analysis, the purified proteins were subject to mass spectrometry and SDS PAGE analysis using a panel of welldefined monoclonal antibodies. The proteins with desirable antigenicity and well-tolerated glycan additions were tested for their immunogenicity in mice and NHPs.

Molecular cloning of chicken interleukin-5 receptor α-chain and analysis of its binding specificity

Interaction between interleukin (IL)-5 and its receptor (IL-5R) is important for the regulation of immunity against worm infections, allergic reactions and B cell response in mammals. In this study, we identified a full-length cDNA encoding chicken IL-5R α-chain (chIL-5Rα). The deduced amino acid sequence showed 41–43% identity to mammalian homologues. It has four well-conserved cysteines and a WSXWS motif in the extracellular region, and a PPXP motif in the cytoplasmic region. Quantitative RT-PCR analysis revealed that chIL-5Rα mRNA expression was markedly high in bone marrow and relatively high in spleen and lung. Recombinant proteins of soluble chIL-5Rα and cytokines (artificially produced chIL-5 (achIL-5) and another IL-5-like molecule KK34) were expressed by 293F cells to examine the cytokine-receptor interactions. Interaction assay using a Biacore biosensor showed that chIL-5Rα has the capability to bind with monomeric achIL-5, but not with KK34. In conclusion, chicken has an IL-5Rα homologue but KK34 does not complement the IL-5/IL-5R system.

Species Comparison and Pharmacological Characterization of Human, Monkey, Rat, and Mouse TRPA1 Channels

The transient receptor potential ankyrin-1 (TRPA1) channel has emerged as an attractive target for development of analgesic and anti-inflammatory drugs. However, drug discovery efforts targeting TRPA1 have been hampered by differences between human and rodent species. Many compounds have been identified to have antagonist activity at human TRPA1 (hTRPA1), but when tested at rat TRPA1 (rTRPA1) and mouse TRPA1 (mTRPA1), they show reduced potency as antagonists, no effect, or agonist activity. These compounds are excluded from further drug development because they cannot be tested in preclinical studies using conventional rat/mouse models. To broaden our understanding of species-specific differences, we cloned and functionally characterized rhesus monkey TRPA1 (rhTRPA1) and compared its pharmacological profile to hTRPA1, rTRPA1, and mTRPA1 channels. The functional activities of a diverse group of TRPA1 ligands (both reactive and nonreactive) were determined in a fluorescent Ca²⁺ influx assay, using transiently transfected human embryonic kidney 293-F cells. 4-Methyl-N-[2,2,2-trichloro-1-(4-nitro-phenylsulfanyl)-ethyl]-benzamide, menthol, and caffeine displayed species-specific differential pharmacology at TRPA1. The pharmacological profile of the rhTRPA1 channel was found to be similar to the hTRPA1 channel. In contrast, the rTRPA1 and mTRPA1 channels closely resembled each other but were pharmacologically distinct from either hTRPA1 or rhTRPA1 channels. Our findings reveal that TRPA1 function differs between primate and rodent species and suggest that rhesus monkey could serve as a surrogate species for humans in preclinical studies.

A Prospective, Multi-Center, Randomized Clinical Trial to Compare the Pharmacokinetic Properties of Human-Cl rhFVIII, a New Human-Cell Line Derived Recombinant Factor VIII, with Those of a Full-Length Recombinant Factor VIII Expressed in Baby Hamster Kidney Cells,

Abstract 3309▪▪This icon denotes a clinically relevant abstract Introduction:human-cl rhFVIII is the first recombinant factor VIII (FVIII) concentrate expressed in a human cell line (Human Embryonic Kidney (HEK) 293F cells). The manufacturing process includes two steps for virus inactivation and removal (solvent/detergent treatment and a nanofiltration). No animal- or human-derived materials are added to the culture medium or during the purification and final formulation. Objective:The primary objective of this study is to compare the pharmacokinetics (PKs) of human-cl rhFVIII, a B-domain deleted rFVIII product expressed in a human cell line, with a commercially available full-length rFVIII product (Kogenate FS), which is expressed in Baby Hamster Kidney (BHK) cells. Methods:This study is an open, multinational, prospective, multicenter clinical phase-II trial. The PK properties of human-cl rhFVIII and Kogenate FS were determined using a randomized two-period, cross-over design. After a 96 hours or more wash-out phase of any previously administered FVIII product, subjects received a single intravenous dose of 50 IU/kg body weight of one of the products. Blood samples were collected over a 48-h period and FVIII coagulant activity (FVIII:C) was measured by validated chromogenic substrate and one-stage clotting assay in a central laboratory, which also assigned drug potency with the same assays. Following completion of the PK segment of the study, subjects will be followed for at least 50 Exposure Days and at least 6 months of treatment with human-cl rhFVIII to evaluate the efficacy and safety of on-demand treatment. The planned interim analysis stipulates the comparison of the complete PK profile of human-cl rhFVIII and Kogenate FS with the bioequivalence of the AUC as the primary endpoint. The full efficacy and safety profile will be reported when the study is completed. Results:Twenty-two adolescent and adult PTPs with severe hemophilia A (FVIII:C <1%) were enrolled at 10 study centers in the United States and Europe. The key PK parameters (AUC, elimination half-life, in-vivo recovery, clearance) are comparable between human-cl rhFVIII and Kogenate FS both for the one-stage and chromogenic assays. The two products were judged to be bioequivalent as the 90% confidence interval of the ratio of the geometric mean values of the AUC fell within 80–125%. human-cl rhFVIII has been well tolerated and no safety or efficacy issues have been reported so far, in particular no inhibitors or non inhibitory antibodies were detected or reported. Conclusion:The PK data from this study indicate that human-cl rh FVIII is bioequivalent to a licensed rFVIII concentrate measured by the one stage and the chromogenic assays. In this study as well as in the other ongoing clinical studies, which examine the efficacy and safety of human-cl rhFVIII during prophylactic treatment in children and adults with severe hemophilia A, the product has been safe and well tolerated so far. Disclosures:Knaub:Octapharma AG: Employment. Valentino:Baxter Bioscience: Consultancy; GTC Biotherapeutics: Consultancy; Bayer Healthcare: Consultancy; NovoNordisk: Consultancy; Inspiration Bioscience: Consultancy; Biogen: Consultancy. Manco-Johnson:Octapharma AG: Consultancy.

Cell type-dependent gene regulation by Staufen2 in conjunction with Upf1

BackgroundStaufen2 (Stau2), a double-stranded RNA-binding protein, is a component of neuronal RNA granules, which are dendritic mRNA transport machines. Although Stau2 is thought to be involved in the dendritic targeting of several mRNAs in neurons, the mechanism whereby Stau2 regulates these mRNAs is unknown. To elucidate the functions of Stau2, we screened for novel binding partners by affinity purification of GST-tagged Stau2 from 293F cells.ResultsThree RNA helicases, RNA helicase A, Upf1 and Mov10, were identified in Stau2-containing complexes. We focused our studies on Upf1, a key player in nonsense-mediated mRNA decay. Stau2 was found to bind directly to Upf1 in an RNA-independent manner in vitro. Tethering Stau2 to the 3'-untranslated region (UTR) of a reporter gene had little effect on its expression in HeLa cells. In contrast, when the same tethering assay was performed in 293F cells, we observed an increase in reporter protein levels. This upregulation of protein expression by Stau2 turned out to be dependent on Upf1. Moreover, we found that in 293F cells, Stau2 upregulates the reporter mRNA level in an Upf1-independent manner.ConclusionsThese results indicate that the recruitment of Stau2 alone or in combination with Upf1 differentially affects the fate of mRNAs. Moreover, the results suggest that Stau2-mediated fate determination could be executed in a cell type-specific manner.

Efficient Production and Characterization of Recombinant Human NELL1 Protein in Human Embryonic Kidney 293-F Cells

NELL1 is a secretory protein that induces osteogenic differentiation and bone formation by osteoblastic cells. Because of its potent osteoinductive activity, NELL1 may be useful for bone regeneration therapy. However, at present, we have little knowledge regarding NELL1 receptors and NELL1-mediated signaling pathways. We have previously produced NELL1 using an insect's cell expression system; however, the protein was relatively unstable and was degraded by proteases released from dead cells. In the present study, NELL1 protein was expressed in human embryonic kidney 293-F cells. Stable cell lines expressing NELL1 fused to a C-terminal hexahistidine-tag were obtained by G418 selection of transfected cells. Cells grown in serum-free medium showed high levels of NELL1 protein production (approximately 4 mg/l cell culture) for up to 6 months. NELL1 protein was purified from culture medium using a one-step nickel-chelate affinity chromatography protocol. Purified NELL1 protein immobilized onto culture dishes induced the expression of both early and late osteogenic markers on mouse mesenchymal C3H10T1/2 cells. When NELL1-expressing 293-F cells were grown on gelatin-coated glass cover slips, recombinant NELL1 was deposited in the extracellular matrix after detachment of cells. These results suggest that NELL1 acts as an extracellular matrix component. Recombinant NELL1 formed multimers and was glycosylated. An abundant source of functionally active NELL1 protein will be useful for more advanced studies, such as the development of novel techniques for bone regeneration.

Trimerization of murine TNF ligand family member LIGHT increases the cytotoxic activity against the FM3A mammary carcinoma cell line

LIGHT is a member of the tumor necrosis factor ligand superfamily, which plays important roles in inflammatory and immune responses. LIGHT forms a membrane-anchored homotrimeric complex on the cell surface and is often processed as a soluble protein. Recombinant soluble human LIGHT produced by mammalian cells or Escherichia coli is functional at nanomolar concentrations. However, there is little information about the biological activity of mouse LIGHT (mLIGHT) because of the difficulty in producing bioactive soluble mLIGHT. In this study, recombinant trimeric soluble mLIGHT, or Foldon-mLIGHT, was produced by fusing mLIGHT with the trimerization domain foldon from bacteriophage T4 fibritin. Foldon-mLIGHT was secreted from 293F cells as a 68-kDa trimeric protein. The recombinant protein potently inhibited the growth of the FM3A mouse mammary carcinoma cell line with an IC(50) of 77pM; however, the monomer or dimer forms of mLIGHT produced by E. coli or mammalian cell systems showed weak or no inhibitory activity. These data clearly indicated that trimerization of soluble mLIGHT is essential for its biological activity.

Cross-talk between NR4A orphan nuclear receptors and β-catenin signaling pathway in osteoblasts

The canonical Wnt signaling pathway and its key mediator β-catenin are important regulators of osteoblast function. NR4A orphan nuclear receptors (Nurr1, NGFI-B, and Nor1) are expressed in osteoblasts and have been shown to regulate the expression of osteoblastic genes and osteoblastic differentiation. Recently, interplay between Nurr1 and the canonical Wnt signaling pathway was reported in 293F cells. We have studied the potential interplay between NR4A receptors and β-catenin in osteoblasts. NR4A receptors repressed β-catenin-mediated transactivation when cotransfected in U2-OS cells. In addition, Nurr1 inhibited β-catenin-mediated expression of Axin2 in MC3T3-E1 cells. The repression involved the DNA-binding domain of NR4A receptors. The repression of β-catenin did not result from reduced β-catenin expression or direct protein–protein interaction between β-catenin and NR4A receptors. β-Catenin was capable of inhibiting the transcriptional activity of NR4A receptors in U2-OS cells by a mechanism that involved the ligand-binding domain of NR4A receptors. As the canonical Wnt signaling pathway and β-catenin are crucial for the development and function of osteoblasts, the repressive effect of NR4A receptors on β-catenin is of potential biological and pathophysiological importance.

In-Cell NMR Spectroscopy in Protein Chemistry and Drug Discovery

"In-cell nuclear magnetic resonance (NMR)" is a unique method for characterization of conformation, interaction and dynamics of proteins inside living cells at atomic level. Since the method was proposed by Dötch and co-workers in 2001 [1], its application had been limited to bacterial cells and oocytes of Xenopus laevis [2]. Recently, we reported a method for efficient delivery of (15)N-labeled proteins into human HeLa cells using cell-penetrating peptides, and measured high-resolution two-dimensional (1)H-(15)N correlation spectra of proteins in the cells. The in-cell NMR spectroscopy in human cells is capable of analyzing structures, interactions, dynamics and stability of proteins inside cells. Of its possible applications, we propose that in-cell NMR spectroscopy can be utilized as an effective step in protein-targeted drug development process, by demonstrating that interaction of FKBP12 with immunosuppressants administered extracellularly was successfully observed in living cells. This observation suggests that drug delivery and capability of target proteins inside cells for interaction with drugs can be investigated by in-cell NMR spectroscopy. More recently, an alternative way for intracellular delivery of labeled proteins for in-cell NMR was reported on 293F cells by Shimada and co-workers. Here, we review recent technical developments of in-cell NMR spectroscopy, and discuss potential usefulness for protein chemistry and drug screening process.

Activin A Binds to Perlecan through Its Pro-region That Has Heparin/Heparan Sulfate Binding Activity

Activin A, a member of the transforming growth factor-β family, plays important roles in hormonal homeostasis and embryogenesis. In this study, we produced recombinant human activin A and examined its abilities to bind to extracellular matrix proteins. Recombinant activin A expressed in 293-F cells was purified as complexes of mature dimeric activin A with its pro-region. Among a panel of extracellular matrix proteins tested, recombinant activin A bound to perlecan and agrin, but not to laminins, nidogens, collagens I and IV, fibronectin, and nephronectin. The binding of recombinant activin A to perlecan was inhibited by heparin and high concentrations of NaCl and abolished by heparitinase treatment of perlecan, suggesting that activin A binds to the heparan sulfate chains of perlecan. In support of this possibility, recombinant activin A was capable of directly binding to heparin and heparan sulfate chains. Site-directed mutagenesis of recombinant activin A revealed that clusters of basic amino acid residues, Lys(259)-Lys(263) and Lys(270)-Lys(272), in the pro-region were required for binding to perlecan. Interestingly, deletion of the peptide segment Lys(259)-Gly(277) containing both basic amino acid clusters from the pro-region did not impair the activity of activin A to stimulate Smad-dependent gene expressions, although it completely ablated the perlecan-binding activity. The binding of activin A to basement membrane heparan sulfate proteoglycans through the basic residues in the pro-region was further confirmed by in situ activin A overlay assays using frozen tissue sections. Taken together, the present results indicate that activin A binds to heparan sulfate proteoglycans through its pro-region and thereby regulates its localization within tissues.

Optimization of polyethylenimine-mediated transient transfection using response surface methodology design

Response surface methodology was undertaken to optimize the polyethylenimine-mediated transient transfection of suspension cultured HEK 293-F cells. A total of 15 combinations were designed according to Box-Behnken design to identify the effects of DNA concentration, polyethylenimine concentration and incubation time on transient transfection efficiency. The highest integral optic density of green fluorescent protein presenting r-protein yield was accessed using a DNA concentration of 1.75 µg/mL, a polyethylenimine concentration of 10.5 µg/mL, and an incubation time of 11.8 min. Analysis of variance demonstrated that the experimental values fit well with a quadratic model. The RSM-optimized transient transfection resulted in greater production of human tissue prokallikrein (TproK) than non-RSM-optimized conditions: protein yield was 32.0 mg/L and the maximum viable cell density reached 3.57 x 10 6 cells/mL in a 5 L stirred-tank bioreactor culture.

Cell adhesion-dependent membrane trafficking of a binding partner for the ebolavirus glycoprotein is a determinant of viral entry

Ebolavirus is a hemorrhagic fever virus associated with high mortality. Although much has been learned about the viral lifecycle and pathogenesis, many questions remain about virus entry. We recently showed that binding of the receptor binding region (RBR) of the ebolavirus glycoprotein (GP) and infection by GP pseudovirions increase on cell adhesion independently of mRNA or protein synthesis. One model to explain these observations is that, on cell adhesion, an RBR binding partner translocates from an intracellular vesicle to the cell surface. Here, we provide evidence for this model by showing that suspension 293F cells contain an RBR binding site within a membrane-bound compartment associated with the trans-Golgi network and microtubule-organizing center. Consistently, trafficking of the RBR binding partner to the cell surface depends on microtubules, and the RBR binding partner is internalized when adherent cells are placed in suspension. Based on these observations, we reexamined the claim that lymphocytes, which are critical for ebolavirus pathogenesis, are refractory to infection because they lack an RBR binding partner. We found that both cultured and primary human lymphocytes (in suspension) contain an intracellular pool of an RBR binding partner. Moreover, we identified two adherent primate lymphocytic cell lines that bind RBR at their surface and strikingly, support GP-mediated entry and infection. In summary, our results reveal a mode of determining viral entry by a membrane-trafficking event that translocates an RBR binding partner to the cell surface, and they suggest that this process may be operative in cells important for ebolavirus pathogenesis (e.g., lymphocytes and macrophages).

Expression-System-Dependent Modulation of HIV-1 Envelope Glycoprotein Antigenicity and Immunogenicity

Recombinant expression systems differ in the type of glycosylation they impart on expressed antigens such as the human immunodeficiency virus type 1 (HIV-1) envelope glycoproteins, potentially affecting their biological properties. We performed head-to-head antigenic, immunogenic and molecular profiling of two distantly related Env surface (gp120) antigens produced in different systems: (a) mammalian (293 FreeStyle™ cells; 293F) cells in the presence of kifunensine, which impart only high-mannose glycans; (b) insect cells (Spodoptera frugiperda, Sf9), which confer mainly paucimannosidic glycans; (c) Sf9 cells recombinant for mammalian glycosylation enzymes (Sf9 Mimic™), which impart high-mannose, hybrid and complex glycans without sialic acid; and (d) 293F cells, which impart high-mannose, hybrid and complex glycans with sialic acid. Molecular models revealed a significant difference in gp120 glycan coverage between the Sf9-derived and wild-type mammalian-cell-derived material that is predicted to affect ligand binding sites proximal to glycans. Modeling of solvent-exposed surface electrostatic potentials showed that sialic acid imparts a significant negative surface charge that may influence gp120 antigenicity and immunogenicity. Gp120 expressed in systems that do not incorporate sialic acid displayed increased ligand binding to the CD4 binding and CD4-induced sites compared to those expressed in the system that do, and imparted other more subtle differences in antigenicity in a gp120 subtype-specific manner. Non-sialic-acid-containing gp120 was significantly more immunogenic than the sialylated version when administered in two different adjuvants, and induced higher titers of antibodies competing for CD4 binding site ligand–gp120 interaction. These findings suggest that non-sialic-acid-imparting systems yield gp120 immunogens with modified antigenic and immunogenic properties, considerations that should be considered when selecting expression systems for glycosylated antigens to be used for structure–function studies and for vaccine use.

Effect of Recombinant Human Lecithin Cholesterol Acyltransferase Infusion on Lipoprotein Metabolism in Mice

Lecithin cholesterol acyl transferase (LCAT) deficiency is associated with low high-density lipoprotein (HDL) and the presence of an abnormal lipoprotein called lipoprotein X (Lp-X) that contributes to end-stage renal disease. We examined the possibility of using LCAT an as enzyme replacement therapy agent by testing the infusion of human recombinant (r)LCAT into several mouse models of LCAT deficiency. Infusion of plasma from human LCAT transgenic mice into LCAT-knockout (KO) mice rapidly increased HDL-cholesterol (C) and lowered cholesterol in fractions containing very-low-density lipoprotein (VLDL) and Lp-X. rLCAT was produced in a stably transfected human embryonic kidney 293f cell line and purified to homogeneity, with a specific activity of 1850 nmol/mg/h. Infusion of rLCAT intravenously, subcutaneously, or intramuscularly into human apoA-I transgenic mice showed a nearly identical effect in increasing HDL-C approximately 2-fold. When rLCAT was intravenously injected into LCAT-KO mice, it showed a similar effect as plasma from human LCAT transgenic mice in correcting the abnormal lipoprotein profile, but it had a considerably shorter half-life of approximately 1.23 ± 0.63 versus 8.29 ± 1.82 h for the plasma infusion. rLCAT intravenously injected in LCAT-KO mice crossed with human apolipoprotein (apo)A-I transgenic mice had a half-life of 7.39 ± 2.1 h and increased HDL-C more than 8-fold. rLCAT treatment of LCAT-KO mice was found to increase cholesterol efflux to HDL isolated from mice when added to cells transfected with either ATP-binding cassette (ABC) transporter A1 or ABCG1. In summary, rLCAT treatment rapidly restored the normal lipoprotein phenotype in LCAT-KO mice and increased cholesterol efflux, suggesting the possibility of using rLCAT as an enzyme replacement therapy agent for LCAT deficiency.

Impaired production of BMP-15 and GDF-9 mature proteins derived from proproteins WITH mutations in the proregion

Mutations in the bone morphogenetic protein-15 (BMP-15) and growth and differentiation factor-9 (GDF-9) genes have been identified in women with primary ovarian insufficiency (POI) and mothers of dizygotic twins. Here, we show that biological activities of the conditioned media from human embryonic kidney 293F cells transfected with two representative BMP-15 and GDF-9 mutants identified in the affected women have significantly reduced biological activities compared with the corresponding wild-type. Moreover, this difference is due to decreased production of the mature proteins, attributed most likely to impaired posttranslational processing of the proprotein. As genetic studies of the BMP-15 and/or GDF-9 genes in ewes established that a reduction of these proteins is associated with an increased ovulation rate, it is conceivable that women affected with these mutations may have an increased probability of bearing dizygotic twins during active reproductive ages before diagnosis with POI at later ages due to an earlier exhaustion of ovarian reserve.

Abstract 1387: Development of novel antiangiogenic biologics: multifunctional VEGF traps

Abstract Current anti-VEGF biologics have been successfully used as therapeutic agents for cancer and age-related macular degeneration (AMD). Since these strategies target VEGF systemically, side effects, long-term toxicities, such as proteinuria and thromboembolic events, and need for frequent eye injections in AMD treatment, prevail. Therefore, the aim of this study was to generate novel anti-VEGF biologics that inhibit VEGF activity specifically at the desired target site. Two classes of antibody-based recombinant proteins were engineered that simultaneously bind VEGF and either: 1) the extracellular matrix (ECM) or 2) target-site specific antigens. The first subgroup of proteins, “shV-H traps”, is comprised of a Fc domain shortened form of VEGF-trap linked to a sequence of hydrophobic amino acids, with various affinities for heparin sulfate proteoglycans of the ECM, designed to have a short systemic half-life. The second subgroup of molecules, “V-lassos”, is composed of a C-terminus positioned form of VEGF-trap linked to single-chain variable domain antibodies specific for either HER-2 or fibronectin extra domain B (EDB), expressed on tumour cell surfaces or in the vascular bed of solid tumours, respectively. Recombinant proteins were expressed in transgenic cancer cell lines in a doxycycline inducible manner and were shown to inhibit VEGF activity and also retain the native function of their constituent domains. Specifically, the shV-H traps adhered to the ECM and the HER-2 V-lasso inhibited the proliferation of HER-2 positive cancer cell lines. Xenograft studies were performed using the aforementioned transgenic cancer cell lines in order to determine the efficacy of shV-H traps and Vlasso. shV-H traps were able to inhibit or delay tumour growth of A-673, Pc-3 and HT-29 xenografts. In contrast to soluble VEGF-trap, sh-VH traps were retained at the tumour site and were undetectable in the circulation. Moreover, sh-VH traps did not cause any of the side-effects observed with soluble VEGF-trap, such as delay of wound healing and regression of trachea blood vessels. Both Vlassos were able to inhibit or delay the tumour growth of SKOV-3 and A-673 xenografts. Furthermore, the transgenic approach for inducible expression indicated that HER-2 Vlasso is more effective compared to anti-HER-2 Ab and VEGF-trap used alone or in combination. Preliminary results suggest that high recombinant protein levels can be generated via large-scale production in 293F cells, indicating that the new multifunctional VEGF-traps can be produced in large amounts for clinical studies. These novel classes of anti-angiogenic molecules could potentially be advantageous in a clinical setting. Furthermore, they can be useful in order to further study and understand the mechanisms involved in VEGF inhibition. Finally, similar dual function proteins can be designed for inhibition of other molecules with disease relevance. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1387.

Immobilization of 293 cells using porous support particles for adenovirus vector production

Adenovirus vector production by anchorage-independent 293 cells immobilized using porous biomass support particles (BSPs) was investigated in static and shake-flask cultures for efficient large-scale production of adenovirus vectors for gene therapy applications. The density of cells immobilized within BSPs was evaluated by measuring their WST-8 (2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium, monosodium salt) reduction activity. In shake-flask culture, 293-F cells, which were adapted to serum-free suspension culture, were not successfully retained within reticulated polyvinyl formal (PVF) resin BSPs (2×2×2mm cubes) with matrices of relatively small pores (pore diameter 60μm). When the BSPs were coated with a cationic polymer polyethyleneimine, a high cell density of more than 10(7) cells cm(-3)-BSP was achieved in both static and shake-flask cultures with regular replacement of the culture medium. After infection with an adenovirus vector carrying the enhanced green fluorescent protein gene (Ad EGFP), the specific Ad EGFP productivity of the immobilized cells was comparable to the maximal productivity of non-immobilized 293-F cells by maintaining favorable conditions in the culture environment.

Observation of NMR Signals from Proteins Introduced into Living Mammalian Cells by Reversible Membrane Permeabilization Using a Pore-Forming Toxin, Streptolysin O

We have developed a new in-cell NMR method that is applicable to any type of cell and does not require target protein modification or specialized equipment. The stable-isotope-labeled target protein, thymosin beta4 (Tbeta4), was delivered to 293F cells, which were permeabilized by a pore-forming toxin, streptolysin O, and resealed by Ca(2+) after Tbeta4 uptake. As a result, we successfully observed (1)H-(15)N HSQC signals originating from the Tbeta4, including those from the N-terminal acetylation, which had occurred inside the cell as a post-translational modification.

The heterogeneous nature of polyethylenimine-DNA complex formation affects transient gene expression

Polyethylenimine has been used widely in transient gene expression with mammalian cells. To further understand its mediation of gene transfer, the transfection of HEK 293-F cells with dynamically prepared PEI/DNA complexes was studied with the help of fluorescent labeling. The efficiency of complex endocytosis/phagocytosis was found to correlate with the average sizes of complexes applied and complexes greater than 1 μm in diameter were likely excluded by the cells. Coupled with complex growth in size, the degree of association between PEI and DNA increased with the time of complex formation in the presence of competing ions. The blocking of transcription by complex formation necessitated complex dissociation in the nuclear environment for transcription to happen. Intracellularly, the fates of PEI complexed DNA therefore may be mostly determined by the degree of association. Results also suggested that the uptake of PEI/DNA complexes and subsequent protein expression were independent of the cell cycle stages of HEK 293-F cells.