Single-chain Approach Research Articles

Backbone and methyl side-chain resonance assignments of the single chain Fab fragment of trastuzumab

Trastuzumab is a therapeutic monoclonal antibody developed to target human epidermal growth factor receptor 2 (HER2) present at higher levels in early cancers. Here we report the near complete resonance assignment of trastuzumab-scFab fragment backbone and the methyl groups of isoleucine, leucine and valine residues, as well as their stereo-assignments. The antibody fragment was produced using a single chain approach in Escherichia coli.

Inference of unexploded ordnance (UXO) by probabilistic inversion of magnetic data

SUMMARY Magnetic modelling of unexploded ordnance (UXO) is a well-documented method used to interpret magnetic anomalies occurring in UXO excavation surveys. By treating UXO as a ferrous spheroidal object, the induced dipole moment can be estimated by approximation of UXO characteristics such as shape, size and orientation. Inversion of magnetic data with respect to UXO requires one to solve the equation for the induced dipole moment, while also determining the location and orientation of the object. This is a highly nonlinear, non-unique problem, where many solutions often are present, which make it difficult for standard inversion methods, such as linearized approaches and maximum likelihood estimators, in assessing uncertainties and correlations in estimated model parameters that often result in an incomplete solution. In this study, we treat the problem concerning magnetic UXO inversion by a probabilistic approach using Markov chain Monte Carlo (McMC) sampling. To deal with the potential multimodality, a combination of two well-known McMC sampling methods is employed in a single-chain approach: the extended Metropolis algorithm is used for efficient local sampling, and the Gibbs sampler is used to help exploring the possible multimodal density of the posterior. By adding a Gibbs step we significantly increase the efficiency of the extended Metropolis, making it viable to use as a single-chain sampler for this problem. We refer to the algorithm as the Gibbs-within-Metropolis algorithm. We test and compare the proposed algorithm to a multichain McMC parallel tempering setup using the extended Metropolis algorithm. We then present synthetic cases and a real data case, where the algorithm is used. We demonstrate how the probabilistic approach allows full inference of the parameters describing a UXO while also including the possible presence of remanent magnetization.

Effects of fluctuations of cross-linking points on viscoelastic properties of associating polymer networks

We study the effects of fluctuations of cross-linking points, or junctions, on the dynamic mechanical and viscoelastic properties of polymer networks formed by multisticker associating polymers on the basis of a single-chain approach. Fluctuations of junctions are implemented by introducing virtual springs. We consider two possible cases for the treatment of virtual springs: the direct contribution from virtual springs is either neglected or included in the stress. We show that, if neglected, the fluctuation of junctions decreases (or softens) the dynamic modulus over a wide range of frequencies. This result agrees qualitatively with the result of several multichain models that predicts the decrease of the static or plateau modulus. We also show that the fluctuation accelerates the associative Rouse mode at low frequencies originating from the association/dissociation process of stickers. These results are apparently reasonable, but it is expected that there are some errors arising from thermodynamical inconsistency due to the neglect of virtual-spring contribution from the total stress against the virtual work principle and the second law of thermodynamics. On the other hand, if the direct contribution from the virtual springs is included in the stress, thermodynamics is satisfied but the plateau modulus does not change, contrary to the multichain prediction. The softening occurs only at the low-frequency regime. Thus, each method has both merits and demerits, and hence the treatment of junction fluctuations in the framework of single-chain approaches requires care and further investigation.

Overexpression of Factor VIII Is Associated with Immune Responses to Factor VIII and Cellular Stress in Hemophilia A Mice

Abstract 22For many years it has been evident that it is challenging to express recombinant factor VIII (FVIII) in vitro and in vivo. In vitro studies suggest that high levels of FVIII expression can lead to cellular stress. We have demonstrated that adeno-associated viral (AAV) vector delivery results in long-term expression of therapeutic levels of FVIII in hemophilia A (HA) dogs up 10% of normal. In this model transgene expression is restricted to a portion of liver cells resulting in sustained FVIII levels. Notably, in dogs FVIII expression is sustained for periods longer than 7 years. Conceivably, the increased load per cell for cellular processing of FVIII may impact the cell’s ability to synthesize and secrete functional FVIII, activation of cellular stress and the potential for immune responses. We sought to determine whether cellular stress and immune responses to the transgene are increased by overexpression of FVIII in mice by AAV vectors. Using AAV to deliver FVIII as a B-domain deleted single chain (single chain approach) or as two separate chains (light and heavy chains) (two chain approach), we analyzed dose-dependent FVIII synthesis and its cellular impact. We hypothesized that high levels of FVIII expression after AAV-mediated delivery of FVIII may induce cellular stress. HA mice were administered AAV serotype 8 (AAV8) vector in 3 groups: (1) two-chain approach (2) single chain approach or (3) AAV empty capsid (control). Three total AAV doses were administered: 1×1010 vector genomes/mouse (low dose), 5×1010 vg/mouse (mid-dose) and 2.5×1011 vg/mouse (high dose). FVIII antigen levels (heavy and light chain) and anti-cFVIII IgG antibodies were detected by ELISA at a series of time points post vector administration until the terminal time points (1, 2, 4, 8, 12 and 24 wks). As we previously observed, after single chain delivery we detect equivalent amounts of heavy chain and light chain in the circulation by ELISA that correlates with activity. However, in the two-chain approach we observe that the light chain is >2-fold higher than the heavy chain in the circulation and that the activity correlates with the amount of heavy chain. We observe a dose-dependent increase in FVIII expression and dose-dependent anti-cFVIII IgG antibody titers. The kinetics of FVIII expression and antibody formation are different for the two-chain delivery compared to the single chain delivery. For the two-chain delivery, we observe AAV dose-dependent peak FVIII expression within 1 week (30, 100, or 400 ng/ml, respective of dose) followed by the onset of anti-FVIII IgG2 antibodies by 4 weeks (16, 27, 52 μg/ml, respective of dose). For single chain delivery, we observe FVIII expression within 1 week and low level antibody titers by 4 weeks that increase dramatically by 12 weeks (14, 21, 38 μg/ml, respective of dose). Thus, we observe a more gradual increase in antibody titers in the single chain compared to the two-chain delivery but at late time points the immunogenicity of both approaches is indistinguishable. RNA was isolated from the livers of the mice at the terminal time points for quantitative PCR analysis of two key components of the unfolded protein response signaling network, BiP and CHOP. We compared the two-chain and single chain AAV delivery of FVIII alongside animals injected with AAV empty capsid that did not express FVIII. A tunicamycin treated positive control had an increase in BiP (54-fold) and CHOP (74-fold) expression compared to untreated HA mice. There were mild elevations (2–3-fold) of BiP and/or CHOP in some animals observed at week 1 in all three treatment groups. Only at 12 weeks post vector administration were significant increases in BiP (>70 fold) and CHOP (>4 fold) observed in the single chain treated group at the high dose expressing >300% but not the two-chain approach or empty capsid. These studies suggest that supraphysiological expression (>100%) of BDD-cFVIII may increase the likelihood of an immune response to FVIII and cellular stress. Further studies may determine if there is a relationship between high FVIII expression, cellular stress and immune responses to FVIII. Thus, there is a threshold of FVIII expression levels that may prove unsafe. Moreover, these data are in agreement with the lack of evidence of cellular toxicity and immunogenicity in HA dogs expressing therapeutic levels of FVIII by AAV vectors. Disclosures:Lange:Bayer Healthcare: Research Funding. Altynova:Bayer Healthcare: Research Funding. Sabatino:Bayer Healthcare: Research Funding.

Long Term Dose-Dependent Correction of Hemophilia A Dogs Using AAV-8 and AAV-9-Mediated FVIII Gene Transfer.

Hemophilia A (HA) is an X-linked bleeding disorder characterized by deficiency in clotting factor VIII (FVIII). Current treatment for hemophilia is protein replacement therapy while a gene-based therapy would provide continuous expression of even low levels of FVIII protein (>1% of normal) that is likely to improve the disease phenotype. It is challenging to utilize an AAV-mediated gene transfer approach for the FVIII cDNA (4.4kb) since the AAV vector can only efficiently accommodate a <5.3kb transgene cassette. The FVIII protein is composed of 2 chains -the heavy chain (HC) and the light chain (LC). FVIII undergoes proteolytic cleavage and processing of the 2 individual chains that form the active FVIII protein. In other studies in HA dogs (n=8), no dose-response and AAV serotype-dependent FVIII expression has been documented, which illustrates the difficulties in using a FVIII single-chain approach. We have utilized a 2-chain approach in which the 2.4kb LC cDNA is packaged in one AAV vector while the 2.5kb HC is packaged into a second AAV vector. Each construct contains a 695bp thyroxine-binding globulin gene promoter/enhancer fused to a 175bp intron along with a 263bp SV40 poly A signal. For this approach the LC and HC vectors packaged into either AAV8 or AAV9 were administered to HA dogs via the hepatic artery. Two male HA dogs received HC and LC in AAV8 and 2 male dogs received HC and LC in AAV9 at doses of 6x1012gc/vector/kg (low dose) or 1.25x1013gc/v/kg (high dose). At 150 days after vector infusion, the high dose group expressed FVIII at levels of 4.8% (AAV8) and 3% (AAV9) as detected by a functional assay (Coatest assay). FVIII remained stable for 797 days (AAV8) and >200 days (AAV9) (the longest time points to date) without any evidence of antibody formation to the transgene. In the low dose group at 150 days, FVIII levels were 1.5% (AAV8) and 0.5% (AAV9) cFVIII activity and were maintained in a follow up period of >150 days (AAV8) and >700 days (AAV9) without formation of antibodies to FVIII. Thus, no major differences between AAV8 and AAV9 vectors were observed. The transgene product is also functional based on shortening of whole blood clotting time (baseline values >50 min), in a dose-dependent manner, 10–15 min and 16–20 min for the high and low dose cohorts, respectively. Interestingly, high dose injection of AAV8 to 2 female HA dogs (1.25x1013 and 3x1013gc/v/kg) results in FVIII levels of 1–2%, which is consistent with data obtained in mice on the poor performance of AAV in mediating gene transfer to liver in female animals. Liver function tests and other blood chemistries were transiently elevated after the surgical procedure and were in normal limits within 4 days. Importantly, all dogs did not develop antibodies to FVIII. These findings suggest that FVIII chains efficiently assemble in vivo without increasing the protein immunogenicity. The 4 male dogs have remained asymptomatic with no spontaneous bleeds, whereas >20 bleeding episodes were expected for this group since untreated dogs require 5.5 plasma infusions/year. These data demonstrate for the first time, dose-dependent sustained expression of functional cFVIII in HA dogs by AAV8 and AAV9 vectors without formation of antibodies to cFVIII.

Production of Biological Active Single Chain Bovine LH and FSH

Luteinizing hormone as other glycoprotein hormones is characterized by a heterodimeric structure composed a common α-subunit noncovalently linked to a specific β-subunit. The correct conformation of the heterodimer is important for efficient secretion, hormonal-specific post-translational modifications, receptor binding and signal transduction. To determine whether α- and β- subunits can be synthesized as a single polypeptide chain (tethered-bLH and -bFSH) and also display biological activities, the tethered- bLH and -bFSH molecules were constructed and transfected into chinese hamster ovary (CHO-K1) cells. LH and FSH activities were assayed by using the human embryonic kidney (HEK) 293 cells expressing rat LH and FSH receptor genes. The tethered-bLH and - bFSH proteins were efficiently secreted and showed a similar activity to the dimeric bovine LH and FSH α/β wild type and native purified from bovine pituitary. The tethered-molecules can be permit development of potent new analogues that stimulate ovarian development. Taken together, a single-chain analog can also be constructed to include additional hormone-specific bioactive generating potentially efficacious compounds. These data indicate the potentiality of the single chain approach to further investigate structure- function relationships of LH and FSH. (Asian-Aust. J. Anim. Sci. 2003. Vol 16, No. 4 : 498-503)

Single-chain Vascular Endothelial Growth Factor Variant with Antagonist Activity

Vascular endothelial growth factor is a specific endothelial cell mitogen that is essential for the formation of the vascular system but in the adult individual is involved in several pathological conditions, including cancer. It is a homodimeric protein that activates its receptor by binding two receptor molecules and inducing dimerization. By mixing two vascular endothelial growth factor monomers, each with different substitutions, heterodimers with only one active receptor binding site have previously been prepared. These heterodimers bind the receptor molecule but are unable to induce dimerization and activation. However, preparation of heterodimers is cumbersome, involving separate expression of different monomers, refolding the mixture, and separating heterodimers from homodimers. Here we show that a fully functional ligand can efficiently be expressed as a single protein chain containing two monomers. Single-chain vascular endothelial growth factor is functionally equivalent to the wild-type protein. By monomer-specific mutagenesis, one receptor binding site was altered. This variant competitively and specifically antagonizes the mitogenic effect of the wild-type protein on endothelial cells. The results obtained with the single-chain antagonist show the feasibility of the single-chain approach in directing alterations to single specific regions in natural homodimeric proteins that would be impossible to target in other ways.

Adsorption as a mechanism for nucleating activity: A thermodynamic explanation

Although polymeric crystallization processes heterogeneously induced by foreign surfaces have been the subject of an overwhelming amount of experimental investigations over the last 30 years, this topic suffers from the absence of theoretical or predictive tools describing the polymer–substrate interactions at work in surface-induced nucleation phenomena. In this work we consider the case when polymer chains are attracted to a flat surface (adsorption). Using a single-chain approach, we propose a model for deriving the thermodynamic potential barrier to nucleation within an adsorbed chain. It is shown that, because of the reduction of the configurational entropy of the entire chain prior to undercooling, forming a nucleus of a certain volume within the adsorbed chain costs less energy. In the case of weakly adsorbing surface, using the scaling treatment of de Gennes, we clearly relate the lowering of the thermodynamic potential for nucleation to the strength of the attractive interactions with the surface. This work points out adsorption as a possible mechanism for the nucleating activity of a surface. © 1997 John Wiley & Sons, Inc. J Polym Sci B: Polym Phys 35: 1333–1338, 1997

Adsorption as a mechanism for nucleating activity: A thermodynamic explanation

Although polymeric crystallization processes heterogeneously induced by foreign surfaces have been the subject of an overwhelming amount of experimental investigations over the last 30 years, this topic suffers from the absence of theoretical or predictive tools describing the polymer–substrate interactions at work in surface-induced nucleation phenomena. In this work we consider the case when polymer chains are attracted to a flat surface (adsorption). Using a single-chain approach, we propose a model for deriving the thermodynamic potential barrier to nucleation within an adsorbed chain. It is shown that, because of the reduction of the configurational entropy of the entire chain prior to undercooling, forming a nucleus of a certain volume within the adsorbed chain costs less energy. In the case of weakly adsorbing surface, using the scaling treatment of de Gennes, we clearly relate the lowering of the thermodynamic potential for nucleation to the strength of the attractive interactions with the surface. This work points out adsorption as a possible mechanism for the nucleating activity of a surface. © 1997 John Wiley & Sons, Inc. J Polym Sci B: Polym Phys 35: 1333–1338, 1997