Increased stable integration efficiency in CHO cells through enhanced nuclear localization of Bxb1 serine integrase.

BACKGROUND: Mammalian display is an appealing technology for therapeutic antibody development. Despite the advantages of mammalian display, such as full-length IgG display with mammalian glycosylation and its inherent ability to select antibodies with good biophysical properties, the restricted library size and large culture volumes remain challenges. Bxb1 serine integrase is commonly used for the stable genomic integration of antibody genes into mammalian cells, but presently lacks the efficiency required for the display of large mammalian display libraries. To increase the Bxb1 integrase-mediated stable integration efficiency, our study investigates factors that potentially affect the nuclear localization of Bxb1 integrase. METHODS: In an attempt to enhance Bxb1 serine integrase-mediated integration efficiency, we fused various nuclear localization signals (NLS) to the N- and C-termini of the integrase. Concurrently, we co-expressed multiple proteins associated with nuclear transport to assess their impact on the stable integration efficiency of green fluorescent protein (GFP)-encoding DNA and an antibody display cassette into the genome of Chinese hamster ovary (CHO) cells containing a landing pad for Bxb1 integrase-mediated integration. RESULTS: The nucleoplasmin NLS from Xenopus laevis, when fused to the C-terminus of Bxb1 integrase, demonstrated the highest enhancement in stable integration efficiency among the tested NLS fusions, exhibiting over a 6-fold improvement compared to Bxb1 integrase lacking an NLS fusion. Subsequent additions of extra NLS fusions to the Bxb1 integrase revealed an additional 131% enhancement in stable integration efficiency with the inclusion of two copies of C-terminal nucleoplasmin NLS fusions. Further improvement was achieved by co-expressing the Ran GTPase-activating protein (RanGAP). Finally, to validate the applicability of these findings to more complex proteins, the DNA encoding the membrane-bound clinical antibody abrilumab was stably integrated into the genome of CHO cells using Bxb1 integrase with two copies of C-terminal nucleoplasmin NLS fusions and co-expression of RanGAP. This approach demonstrated over 14-fold increase in integration efficiency compared to Bxb1 integrase lacking an NLS fusion. CONCLUSIONS: This study demonstrates that optimizing the NLS sequence fusion for Bxb1 integrase significantly enhances the stable genomic integration efficiency. These findings provide a practical approach for constructing larger libraries in mammalian cells through the stable integration of genes into a genomic landing pad.

The delivery assessment for small targets on Halcyon radiotherapy system - Measured and calculated dose comparison.

BACKGROUND: With the ever-increasing requirements of accuracy and personalization of radiotherapy treatments, stereotactic radiotherapy (SRT) with volumetric modulated arc therapy (VMAT) on O-ring Halcyon radiotherapy system could potentially provide a fast, safe, and feasible treatment option. PURPOSE: The purpose of this study was to assess the delivery of Halcyon VMAT plans for small targets. METHODS: Well-defined VMAT-SRT plans were created on Halcyon radiotherapy system with the stacked and staggered dual-layer MLC design for the film measurement set-up and the target sizes and shapes designed to emulate the targets of the stereotactic treatments. The planar dose distributions were acquired with film measurements and compared to a current clinical reference dose calculation with AcurosXB (v18.0, Varian Medical Systems) and to Monte Carlo simulations. With the collapsed arc versions of the VMAT-SRT plans, the uncertainty in dose delivery due to the multileaf collimator (MLC) without the gantry rotation could be separated and analyzed. RESULTS: The target size was mainly limited by the resolution originated from the design of the MLC leaves. The results of the collapsed arc versions of the plans show good consistency among measured, calculated, and simulated dose distributions. With the full VMAT plans, the agreement between calculated and simulated dose distributions was consistent with the collapsed arc versions. The measured dose distribution agreed with the calculated and simulated dose distributions within the target regions, but considerable local differences were observed in the margins of the target. The largest differences located in the steep gradient regions presumably originating from the deviation of the isocenter. CONCLUSIONS: The potential of the Halcyon radiotherapy system for VMAT-SRT delivery was evaluated and the study revealed valuable insights on the machine characteristics with the delivery.

Monte Carlo modeling of Halcyon and Ethos radiotherapy beam using CAD geometry: validation and IAEA-compliant phase space.

Objective.A Monte Carlo (MC) model of a Halcyon and Ethos (Varian Medical Systems, a Siemens Healthineers Company) radiotherapy beam was validated and field-independent phase space (PHSP) files were recorded above the dual-layer multileaf collimators (MLC).Approach.The treatment head geometry was modeled according to engineering drawings and the dual-layer MLC was imported from CAD (computer-aided design) files. The information for the incident electron beam was achieved from an iterative electromagnetic solver. The validation of the model was performed by comparing the dose delivered by the square MLC fields as well as complex field measurements.Main results.An electron phase space was generated from linac simulations and achieved improved MC results. The output factors for square fields were within 1% and the largest differences of 5% were found in the build-up region of PDDs and the penumbra region of profiles. With the more complicated MLC-shaped field (Fishbone), the largest differences of up to 8% were found in the MLC leaf tip region due to the uncertainty of the MLC positioning and the mechanical leaf gap value. The impact of the collimator rotation on the PHSP solution has been assessed with both small and large fields, confirming negligible effects on in-field and out-of-field dose distributions.Significance.A computational model of the Halcyon and Ethos radiotherapy beam with a high accuracy implementation of the MLC was shown to be able to reproduce the radiation beam characteristics with square fields and more complex MLC-shaped fields. The field-independent PHSP files that were produced can be used as an accurate treatment head model above the MLC, and reduce the time to simulate particle transport through treatment head components.

Testing of an enhanced leaf model for improved dose calculation in a commercial treatment planning system.

BACKGROUND: With the ever-increasing complexity of dynamic radiotherapy treatments, dose calculation algorithms are challenged to accurately calculate the dose resulting from small, on- and off-axis multileaf collimator (MLC) aperture movements. Although the currently available Eclipse (Varian Medical Systems, Palo Alto) dose calculation algorithms still use a simplified, binary MLC model, a more advanced and detailed modeling of the MLC could be beneficial for the dose calculation precision of high-end treatments. PURPOSE: To improve the modeling of the MLC in the dose calculation algorithms of the Eclipse treatment planning system, an enhanced MLC attenuation model was constructed through ray tracing through the actual leaf designs for the most commonly used Varian MLC types. The enhanced leaf model (ELM) thus includes the rounded leaf tip shape, the drive screw cutout, and the leaf body thickness. The purpose of this work is to test out this new model and explore possible improvements compared to the previous model. METHODS: Dose calculations were performed in a research Eclipse environment equipped with the original and enhanced MLC model. Measurements were performed on TrueBeam and on Halcyon dual MLC treatment units. Dedicated static and dynamic MLC test plans were designed to challenge the dose calculation and highlight differences between both models while keeping the experimental setup simple in order to minimize measurement uncertainties. Measurements were performed with single ion chambers, 2D ion chamber arrays and film. RESULTS: The improved MLC model considerably improves the accuracy of the dose calculation for the test fields used in this study. For the TrueBeam MLC, improvements are most prominent for off-axis dose delivery through narrow (static or dynamic) MLC gaps. For 3 mm narrow sweeping gap deliveries at 12 cm off-axis, the advanced model agrees within 2% with the measurement, in contrast to the 12% deviation observed with the original MLC model. For the Halcyon MLC, improvements are especially prominent when the leaves of both MLC stacks are aligned, regardless of their position in the field. Sweeping gap measurements improve from a 7%-10% deviation with the original model to within 2% with the new model. CONCLUSIONS: Although test fields designed in this study emphasize the flaws in the original MLC dose calculation model, the enhanced MLC model resolves all of the observed discrepancies, showing excellent on- and off-axis agreements with all of the performed measurements.

Synonymous Codons and Hydrophobicity Optimization of Post-translational Signal Peptide PelB Increase Phage Display Efficiency of DARPins.

DsbA leader peptide targets proteins for cotranslational translocation by signal recognition particle (SRP) pathway and has been the standard signal sequence for filamentous phage display of fast-folding Designed Ankyrin Repeat Proteins (DARPins). In contrast, translocation of DARPins via the post-translational pathway, for example, with the commonly used PelB leader, has been reported to be highly inefficient. In this study, two PelB signal sequence libraries were screened covering different regions of the leader peptide for identifying mutants with improved display of DARPins on phage. A PelB variant with the most favorable combination of synonymous mutations in the n-region and hydrophobic substitutions in the h-region increased the display efficiency of a DARPin library 44- and 12-fold compared to PelBWT and DsbA, respectively. Based on thioredoxin-1 (TrxA) export studies the triple valine mutant PelB DN5 V3 leader was capable of more efficient cotranslational translocation than PelBWT, but the overall display efficiency improvement over DsbA suggests that besides increased cotranslational translocation other factors contribute to the observed enhancement in DARPin display efficiency.

Improvement of Fab expression by screening combinatorial synonymous signal sequence libraries.

BACKGROUND: Antibody fragments can be expressed in Escherichia coli, where they are commonly directed to the periplasm via Sec pathway to enable disulphide bridge formations and correct folding. In order to transport antibody fragments to the periplasmic space via Sec pathway, they are equipped with N-terminal signal sequence. Periplasmic expression has many benefits but it's also subjected to many hurdles like inefficient translocation across the inner membrane and insufficient capacity of the translocation system. One solution to overcome these hurdles is a modulation of codon usage of signal sequence which has proved to be an efficient way of tuning the translocation process. Modulation of codon usage of signal sequences has been successfully employed also in improving the expression levels of antibody fragments, but unfortunately the effect of codon usage on the expression has not been thoroughly analyzed. RESULTS: In the present study we established three synonymous PelB signal sequence libraries by modulating codon usage of light chain and heavy chain PelB signal sequences of a Fab fragment. Each region (n-region, hydrophobic region and c-region) of the PelB signal sequence in the both chains of the Fab fragment in a bicistronic expression vector was mutated separately. We then screened for clones with improved expression profile. The best source for improved clones was the n-region library but in general, improved clones were obtained from all of the three libraries. After screening, we analyzed the effects of codon usage and mRNA secondary structures of chosen clones on the expression levels of the Fab fragment. When it comes to codon usage based factors, it was discovered that especially codon usage of fifth leucine position of the light chain PelB affects the expression levels of Fab fragment. In addition, we observed that mRNA secondary structures in the translation initiation regions of the light and heavy chain have an effect on expression levels as well. CONCLUSIONS: In conclusion, the established synonymous signal sequence libraries are good sources for discovering Fab fragments with improved expression profile and obtaining new codon usage related information.

Modification of the 4 MeV electron beam from a linear accelerator for irradiation of small superficial skin tumors.

Uniform dose distribution with steep lateral gradient within depth range of 0-0.5 cm is crucial to be able to treat small skin lesions. The standard nominal 4 MeV electron beam from Elekta Versa HD linear accelerator was modified with degrading filter to remove the lateral scatter from treatment head and minimize the penumbra. The energy degrading method was verified based on dosimetric properties and output factors (OFs) with comparison of four types of measurement methods. The properties of degraded 4 MeV electron beam and developed electron applicators seem optimal for treating small targets near the skin surface.

Abstract ID: 142 Monte Carlo modeling of Varian TrueBeam with Geant4-based VirtuaLinac and comparison to experiments.

Monte Carlo simulations can provide powerful insight into the physical phenomena and geometrical interactions of linear accelerator beams. This insight can be used to understand the phenomena that govern the beam characteristic and, for instance, to guide the development of treatment planning systems. In this study, we use the VirtuaLinac, a cloud-based application to model the treatment head of the Varian TrueBeam linear accelerator. VirtuaLinac implements the treatment head geometry into the Monte Carlo code Geant4, which is then utilized to provide the physics and numerical engine for the simulations. We consider both open fields and fields limited by multi-leaf collimators and compute the dose deposited in a water phantom. We then compare the simulation results with experimental measurements. The simulated data are also used to extract some of the characteristics of the multi-leaf collimators and to evaluate their impact on the beam properties and the dose distribution.

Effect of DNA sequence of Fab fragment on yield characteristics and cell growth of E. coli.

Codon usage is one of the factors influencing recombinant protein expression. We were interested in the codon usage of an antibody Fab fragment gene exhibiting extreme toxicity in the E. coli host. The toxic synthetic human Fab gene contained domains optimized by the "one amino acid-one codon" method. We redesigned five segments of the Fab gene with a "codon harmonization" method described by Angov et al. and studied the effects of these changes on cell viability, Fab yield and display on filamentous phage using different vectors and bacterial strains. The harmonization considerably reduced toxicity, increased Fab expression from negligible levels to 10 mg/l, and restored the display on phage. Testing the impact of the individual redesigned segments revealed that the most significant effects were conferred by changes in the constant domain of the light chain. For some of the Fab gene variants, we also observed striking differences in protein yields when cloned from a chloramphenicol resistant vector into an identical vector, except with ampicillin resistance. In conclusion, our results show that the expression of a heterodimeric secretory protein can be improved by harmonizing selected DNA segments by synonymous codons and reveal additional complexity involved in heterologous protein expression.

Sensitive luminometric method for protein quantification in bacterial cell lysate based on particle adsorption and dissociation of chelated europium.

A sensitive and rapid assay for the quantification of proteins, based on sample protein adsorption to Eu(3+)-chelate-labeled nanoparticles, was developed. The lanthanide ion of the surface-conjugated Eu(3+) chelate is dissociated at a low pH, decreasing the luminescence signal. The increased concentration of the sample protein prevents dissociation of the chelate, leading to a high luminescence signal due to the nanoparticle-bound protein. The assay sensitivity for the quantification of proteins was 130 pg for bovine serum albumin (BSA), which is an improvement of nearly 100-fold from the most sensitive commercial methods. The average coefficient of variation for the assay of BSA was 8%. The protein-to-protein variability was sufficiently low; the signal values varied within a 28% coefficient of variation for nine different proteins. The developed method is relatively insensitive to the presence of contaminants, such as nonionic detergents commonly found in biological samples. The existing methods tested for the total protein quantification failed to measure protein concentration in the presence of bacterial cell lysate. The developed method quantified protein also in samples containing insoluble cell components reducing the need for additional centrifugal assay steps and making the concept highly attractive for routine laboratory work.