Crystal structures and molecular dynamics simulations of a humanised antibody fragment at acidic to basic pH

Antibody-fragment (Fab) therapy development has the potential to be accelerated by computational modelling and simulations that predict their target binding, stability, formulation, manufacturability, and the impact of further protein engineering. Such approaches are currently predicated on starting with good crystal structures that closely represent those found under the solution conditions to be simulated. A33 Fab, is an undeveloped immunotherapeutic antibody candidate that was targeted to the human A33 antigen homogeneously expressed in 95% cases of primary and metastatic colorectal cancers. It is now used as a very well characterised testing ground for developing analytics, formulation and protein engineering strategies, and to gain a deeper understanding of mechanisms of destabilisation, representative of the wider therapeutic Fab platform. In this article, we report the structure of A33 Fab in two different crystal forms obtained at acidic and basic pH. The structures overlapped with RMSD of 1.33 Å overall, yet only 0.5 Å and 0.76 Å for the variable- and constant regions alone. While most of the differences were within experimental error, the switch linker between the variable and the constant regions showed some small differences between the two pHs. The two structures then enabled a direct evaluation of the impact of initial crystal structure selection on the outcomes of molecular dynamics simulations under different conditions, and their subsequent use for determining best fit solution structures using previously obtained small-angle x-ray scattering (SAXS) data. The differences in the two structures did not have a major impact on MD simulations regardless of the pH, other than a slight persistence of structure affecting the solvent accessibility of one of the predicted APR regions of A33 Fab. Interestingly, despite being obtained at pH 4 and pH 9, the two crystal structures were more similar to the SAXS solution structures obtained at pH 7, than to those at pH 4 or pH 9. Furthermore, the P65 crystal structure from pH 4 was also a better representation of the solution structures at any other pH, than was the P1 structure obtained at pH 9. Thus, while obtained at different pH, the two crystal structures may represent highly (P65) and lesser (P1) populated species that both exist at pH 7 in solution. These results now lay the foundation for confident MD simulations of A33 Fab that rationalise or predict behaviours in a range of conditions

Multi-functional Frequency Selective Absorber Enabling FR1 and FR2 5G OTA Tests in a Hybrid Reverberation Chamber

Due to the special advantages of the reverberation chamber (RC), it has been recognized as a standardized over-the-air (OTA) testing methodology by the cellular telecommunications industry association (CTIA). Currently, OTA tests of the fifth-generation (5G) wireless terminals are usually performed in different testing environments, e.g., in a reverberation chamber for fast isotropic tests in the sub-6 GHz frequency band, and in an anechoic chamber for directional tests in the millimeter-wave (mm-wave) frequency band, which are inconvenient and costly. In this work, two multi-functional frequency selective absorbers (FSAs) are designed to realize absorption in the 5G mm-wave band and reflection/transmission in the sub-6 GHz band. By loading the FSAs to the RC, an integrated testing environment for 5G terminals can be established. Namely, a quasi-anechoic environment in the mm-wave band and a reverberation environment in the sub-6 GHz band can be simultaneously achieved in the RC loaded with the proposed FSAs. Experimental and simulated results are presented to demonstrate the effectiveness of the proposed scheme

Artemisinin resistance-associated markers in Plasmodium falciparum parasites from the China-Myanmar border: predicted structural stability of K13 propeller variants detected in a low-prevalence area.

BACKGROUND: Malaria reduction and future elimination in China is made more difficult by the importation of cases from neighboring endemic countries, particularly Myanmar, Laos, and Vietnam, and increased travel to Africa by Chinese nationals. The increasing prevalence of artemisinin resistant parasites across Southeast Asia highlights the importance of monitoring the parasite importation into China. Artemisinin resistance in the Mekong region is associated with variants of genes encoding the K13 kelch domain protein (pf13k), found in specific genetic backgrounds, including certain alleles of genes encoding the chloroquine resistance transporter (pfcrt) and multidrug resistance transporter PgH1 (pfmdr1). METHODS: In this study we investigated the prevalence of drug resistance markers in 72 P. falciparum samples from uncomplicated malaria infections in Tengchong and Yingjiang, counties on the Yunnan-Myanmar border. Variants of pf13k, pfcrt and pfmdr1 are described. RESULTS: Almost all parasites harboured chloroquine-resistant alleles of pfcrt, whereas pfmdr1 was more diverse. Major mutations in the K13 propeller domain associated with artemisinin resistance in the Mekong region (C580Y, R539T and Y493H) were absent, but F446I and two previously undescribed mutations (V603E and V454I) were identified. Protein structural modelling was carried out in silico on each of these K13 variants, based on recently published crystal structures for the K13 propeller domain. Whereas F446I was predicted to elicit a moderate destabilisation of the propeller structure, the V603E substitution is likely to lead to relatively high protein instability. We plotted these stability estimates, and those for all previously described variants, against published values for in vivo parasitaemia half-life, and found that quadratic regression generates a useful predictive algorithm. CONCLUSION: This study provides a baseline of P. falciparum resistance-associated mutations prevalent at the China-Myanmar border. We also show that protein modelling can be used to generate testable predictions as to the impact of pfk13 mutations on in vivo (and potentially in vitro) artemisinin susceptibility

Structural and functional studies of the human mitotic kinesins MPP1 and KifC1, kinase DYRK2, and antibody A33 Fab and their potential as targets for development of cancer chemotherapy drugs

The work described in this thesis focuses on the early-phase development of anti-cancer treatments based on small therapeutic molecules and therapeutic antibodies, which target key factors, including Kinesin Family Member C1 (KifC1), M-Phase Phosphoprotein 1 (MPP1), Dual Specificity Tyrosine Phosphorylation Regulated Kinase 2 (DYRK2), and human glycoprotein A33 antigen, during the formation and development of cancer. The first part of the thesis covers investigation of two human mitotic kinesins, KifC1 (also called human spleen, embryo and testes protein, HSET; kinesin-14 family) and MPP1 (kineisn-6 family), which are pivotal in cell division, with an emphasis on early-phase fragment-based drug discovery. Several small-molecule hits were identified and characterised using various assays. Attempts for acquiring the protein crystals of these kinesins are also described. The second part of the thesis describes the crystallisation and structure determination of a well-developed immunotherapy molecule, human A33 Fab, which targets a key factor during colorectal cancer development. Protein crystals of the human A33 Fab were obtained, and the structure of A33 Fab was determined. A comprehensive analysis of A33 Fab structure was conducted, which formed the basis of research conducted by our collaboration partners. The last part of the thesis covers a mechanistic investigation of selective inhibitors of human DYRK2, a cancer-related kinase. DYRK2 plays a pivotal role in the development of triple-negative breast cancer (TNBC). The crystal structures of DYRK2 in complex with three distinct inhibitors were determined. Ligand-protein interactions were analysed to explain the affinity and selectivity of the inhibitors, which may provide guidance for further drug development

Discrete Spectrum cover Signal Waveform Design and Generation Method

Radio Frequency-screen is one of the earliest radar active antijamming measures. It achieves antijamming by transmitting cover pulses of different frequencies before the radar pulse signal to induce enemy jammers. As the demand for antijamming measures has become increasingly urgent in recent years, Radio Frequency-screen technology has been further developed. The most representative is the use of discontinuous spectrum signals as a cover signal. However, energy utilization for sending the cover signal can be improved further. To address this problem, this paper proposes a discrete spectrum cover signal based on the discontinuous spectrum cover signal and establishes the waveform design function under the joint constraint of constant modulus and spectral amplitude. The cover signal with discrete spectrum and energy aggregation is generated using the Alternating Direction Method of Multipliers (ADMM) and spectrum shaping algorithm solution. The simulation results show that the discrete spectrum cover signal has a higher spectral amplitude of approximately 5～12 dB than the discontinuous-spectrum cover signal for the same energy and bandwidth. Moreover, the discrete spectrum cover signal can cover a larger spectral range with the same energy and close spectral amplitude, realizing a better antijamming cover effect

Precise capture of fish movement trajectories in complex environments via ultrasonic signal tag tracking

Ultrasonic tag tracking technology has been widely used in fish behavior research, but most existing studies obtain fish movement trajectories through use of supporting software or post-processing services provided by the manufacturers of the relevant equipment. Articles concerning fish positioning principles and optimization and processing methods suited to complex water environments, are rarely published. This paper proposes a set of solutions permitting precise capture of fish movement trajectories. The proposed methods can effectively mitigate the impact of difficult conditions such as partial loss of time synchronization signals and data containing gross error-bearing observations on the process of locating tagged fish. Test results show that (1) Even if time synchronization signals for a continuous 2 h period are lost, the accuracy of time synchronization between the hydrophones remains consistent with the requirements of meter-level positioning. (2) The proposed method can effectively detect the existence of gross errors exceeding 10 m in the observation data; the success rate of the method in identifying unique gross error-bearing observations was 99.7%. (3) Based on existing hydrophones with millisecond-level observation precision, three-dimensional (3D) positioning of fish-borne signal tags to m precision is possible

Crystal structures and molecular dynamics simulations of a humanised antibody fragment at acidic to basic pH

Abstract Antibody-fragment (Fab) therapy development has the potential to be accelerated by computational modelling and simulations that predict their target binding, stability, formulation, manufacturability, and the impact of further protein engineering. Such approaches are currently predicated on starting with good crystal structures that closely represent those found under the solution conditions to be simulated. A33 Fab, is an undeveloped immunotherapeutic antibody candidate that was targeted to the human A33 antigen homogeneously expressed in 95% cases of primary and metastatic colorectal cancers. It is now used as a very well characterised testing ground for developing analytics, formulation and protein engineering strategies, and to gain a deeper understanding of mechanisms of destabilisation, representative of the wider therapeutic Fab platform. In this article, we report the structure of A33 Fab in two different crystal forms obtained at acidic and basic pH. The structures overlapped with RMSD of 1.33 Å overall, yet only 0.5 Å and 0.76 Å for the variable- and constant regions alone. While most of the differences were within experimental error, the switch linker between the variable and the constant regions showed some small differences between the two pHs. The two structures then enabled a direct evaluation of the impact of initial crystal structure selection on the outcomes of molecular dynamics simulations under different conditions, and their subsequent use for determining best fit solution structures using previously obtained small-angle x-ray scattering (SAXS) data. The differences in the two structures did not have a major impact on MD simulations regardless of the pH, other than a slight persistence of structure affecting the solvent accessibility of one of the predicted APR regions of A33 Fab. Interestingly, despite being obtained at pH 4 and pH 9, the two crystal structures were more similar to the SAXS solution structures obtained at pH 7, than to those at pH 4 or pH 9. Furthermore, the P65 crystal structure from pH 4 was also a better representation of the solution structures at any other pH, than was the P1 structure obtained at pH 9. Thus, while obtained at different pH, the two crystal structures may represent highly (P65) and lesser (P1) populated species that both exist at pH 7 in solution. These results now lay the foundation for confident MD simulations of A33 Fab that rationalise or predict behaviours in a range of conditions