Plant-Produced Therapeutic Crizanlizumab Monoclonal Antibody Binds P-Selectin to Alleviate Vaso-occlusive Pain Crises in Sickle Cell Disease.

Sickle Cell Disease (SCD) is a severe genetic disorder causing vascular occlusion and pain by upregulating the adhesion molecule P-selectin on endothelial cells and platelets. It primarily affects infants and children, causing chronic pain, circulatory problems, organ damage, and complications. Thus, effective treatment and management are crucial to reduce SCD-related risks. Anti-P-selectin antibody Crizanlizumab (Crimab) has been used to treat SCD. In this study, the heavy and light chain (HC and LC) genes of anti-P-Selectin antibody Crimab were cloned into a plant expression binary vector. The HC gene was under control of the duplicated 35S promoter and nopaline synthase (NOS) terminator, whereas the LC gene was under control of the potato proteinase inhibitor II (PIN2) promoter and PIN2 terminator. Agrobacterium tumefaciens LBA4404 was used to transfer the genes into the tobacco (Nicotiana tabacum cv. Xanthi) plant. In plants the genomic PCR and western blot confirmed gene presence and expression of HC and LC Crimab proteins in the plant, respectively. Crimab was successfully purified from transgenic plant leaf using protein A affinity chromatography. In ELISA, plant-derived Crimab (CrimabP) had similar binding activity to P-selectin compared to mammalian-derived Crimab (CrimabM). In surface plasmon resonance, the KD (dissociation binding constant) and response unit values were lower and higher than CrimabP, respectively. Taken together, these results demonstrate that the transgenic plant can be applied to produce biofunctional therapeutic monoclonal antibody.

Fc engineered anti-virus therapeutic human IgG1 expressed in plants with altered binding to the neonatal Fc receptor.

Production of therapeutic monoclonal antibody (mAb) in transgenic plants has several advantages such as large-scale production and the absence of pathogenic animal contaminants. However, mAb with high mannose (HM) type glycans has shown a faster clearance compared to antibodies produced in animal cells. The neonatal Fc receptor (FcRn) regulates the persistence of immunoglobulin G (IgG) by the FcRn-mediated recycling pathway, which salvages IgG from lysosomal degradation within cells. In this study, Fc-engineering of antirabies virus therapeutic mAb SO57 with the endoplasmic reticulum (ER)-retention peptide signal (Lys-Asp-Glu-Leu; KDEL) (mAbpK SO57) in plant cell was conducted to enhance its binding activity to human neonatal Fc receptor (hFcRn), consequently improve its serum half-life. Enzyme-linked immunosorbent assay (ELISA) and Surface plasmon resonance assay showed altered binding affinity of the Fc region of three different mAbpK SO57 variants [M252Y/S254T/T256E (MST), M428L/N434S (MN), H433K/N434F (HN)] to hFcRn compared to wild type (WT) of mAbpK SO57. Molecular modeling data visualized the structural alterations in these mAbpK SO57. All of the mAbpK SO57 variants had HM type glycan structures similar to the WT mAbpK SO57. In addition, the neutralizing activity of the three variants against the rabies virus CVS-11 was effective as the WT mAbpK SO57. These results indicate that the binding affinity of mAbpK SO57 variants to hFcRn can be modified without alteration of N-glycan structure and neutralization activity. Taken together, this study suggests that Fc-engineering of antirabies virus mAb can be applied to enhance the efficacy of therapeutic mAbs in plant expression systems.

Single-cell transcriptome sequencing of plant leaf expressing anti-HER2 VHH-FcK cancer therapeutic protein.

The transgenic plant is a promising strategy for the production of highly valuable biotherapeutic proteins such as recombinant vaccines and antibodies. To achieve an efficient level of protein production, codon sequences and expression cassette elements need to be optimized. However, the systematical expression of recombinant proteins in plant biomass can generally be controlled for the production of therapeutic proteins after the generation of transgenic plants. Without understanding the transgene expression patterns in plant tissue, it is difficult to enhance further production levels. In this study, single-cell RNA-sequencing (scRNA-seq) analysis of transgenic tobacco (Nicotiana tabacum) leaf, expressing an immunotherapeutic llama antibody against breast cancer, anti-HER2 VHH-Fc, was conducted to obtain data on the expression pattern of tissue-specific cells. These high-quality scRNA-seq data enabled the identification of gene expression patterns by cell types, which can be applied to select the best cell types or tissues for the high production of these recombinant antibodies. These data provide a foundation to elucidate the mechanisms that regulate the biosynthesis of recombinant proteins in N. tabacum.

Spike Optimization to Improve Properties of Ferroelectric Tunnel Junction Synaptic Devices for Neuromorphic Computing System Applications.

The continuous advancement of Artificial Intelligence (AI) technology depends on the efficient processing of unstructured data, encompassing text, speech, and video. Traditional serial computing systems based on the von Neumann architecture, employed in information and communication technology development for decades, are not suitable for the concurrent processing of massive unstructured data tasks with relatively low-level operations. As a result, there arises a pressing need to develop novel parallel computing systems. Recently, there has been a burgeoning interest among developers in emulating the intricate operations of the human brain, which efficiently processes vast datasets with remarkable energy efficiency. This has led to the proposal of neuromorphic computing systems. Of these, Spiking Neural Networks (SNNs), designed to closely resemble the information processing mechanisms of biological neural networks, are subjects of intense research activity. Nevertheless, a comprehensive investigation into the relationship between spike shapes and Spike-Timing-Dependent Plasticity (STDP) to ensure efficient synaptic behavior remains insufficiently explored. In this study, we systematically explore various input spike types to optimize the resistive memory characteristics of Hafnium-based Ferroelectric Tunnel Junction (FTJ) devices. Among the various spike shapes investigated, the square-triangle (RT) spike exhibited good linearity and symmetry, and a wide range of weight values could be realized depending on the offset of the RT spike. These results indicate that the spike shape serves as a crucial indicator in the alteration of synaptic connections, representing the strength of the signals.

Co-transient expression of PSA-Fc and PAP-Fc fusion protein in plant as prostate cancer vaccine candidates and immune responses in mice.

KEY MESSAGE: PAP-FcK and PSA-FcK prostate cancer antigenic proteins transiently co-expressed in plant induce their specific humoral immune responses in mice. Prostate-specific antigen (PSA) and prostatic acid phosphatase (PAP) have been considered as immunotherapeutic antigens for prostate cancer. The use of a single antigenic agent is unlikely to be effective in eliciting immunotherapeutic responses due to the heterogeneous and multifocal nature of prostate cancer. Thus, multiple antigens have been combined to enhance their anti-cancer effects. In the current study, PSA and PAP were fused to the crystallizable region (Fc region) of immunoglobulin G1 and tagged with KDEL, the endoplasmic reticulum (ER) retention signal motif, to generate PSA-FcK and PAP-FcK, respectively, and were transiently co-expressed in Nicotiana benthamiana. Western blot analysis confirmed the co-expression of PSA-FcK and PAP-FcK (PSA-FcK + PAP-FcK) with a 1:3 ratios in the co-infiltrated plants. PSA-FcK, PAP-FcK, and PSA-FcK + PAP-FcK proteins were successfully purified from N. benthamiana by protein A affinity chromatography. ELISA showed that anti-PAP and anti-PSA antibodies successfully detected PAP-FcK and PSA-FcK, respectively, and both detected PSA-FcK + PAP-FcK. Surface plasmon resonance (SPR) analysis confirmed the binding affinity of the plant-derived Fc fusion proteins to FcγRI/CD64. Furthermore, we also confirmed that mice injected with PSA-FcK + PAP-FcK produced both PSA- and PAP-specific IgGs, demonstrating their immunogenicity. This study suggested that the transient plant expression system can be applied to produce the dual-antigen Fc fusion protein (PSA-FcK + PAP-FcK) for prostate cancer immunotherapy.

Improvement of Resistance Change Memory Characteristics in Ferroelectric and Antiferroelectric (like) Parallel Structures.

Recently, considerable attention has been paid to the development of advanced technologies such as artificial intelligence (AI) and big data, and high-density, high-speed storage devices are being extensively studied to realize the technology. Ferroelectrics are promising non-volatile memory materials because of their ability to maintain polarization, even when an external electric field is removed. Recently, it has been reported that HfO2 thin films compatible with complementary metal-oxide-semiconductor (CMOS) processes exhibit ferroelectricity even at a thickness of less than 10 nm. Among the ferroelectric-based memories, ferroelectric tunnel junctions are attracting attention as ideal devices for improving integration and miniaturization due to the advantages of a simple metal-ferroelectric-metal two-terminal structure and low ultra-low power driving through tunneling. The FTJs are driven by adjusting the tunneling electrical resistance through partial polarization switching. Theoretically and experimentally, a large memory window in a broad coercive field and/or read voltage is required to induce sophisticated partial-polarization switching. Notably, antiferroelectrics (like) have different switching properties than ferroelectrics, which are generally applied to ferroelectric tunnel junctions. The memory features of ferroelectric tunnel junctions are expected to be improved through a broad coercive field when the switching characteristics of the ferroelectric and antiferroelectric (like) are utilized concurrently. In this study, the implementation of multiresistance states was improved by driving the ferroelectric and antiferroelectric (like) devices in parallel. Additionally, by modulating the area ratio of ferroelectric and antiferroelectric (like), the memory window size was increased, and controllability was enhanced by increasing the switchable voltage region. In conclusion, we suggest that ferroelectric and antiferroelectric (like) parallel structures may overcome the limitations of the multiresistance state implementation of existing ferroelectrics.

Synaptic Characteristic of Hafnia-Based Ferroelectric Tunnel Junction Device for Neuromorphic Computing Application.

Owing to the 4th Industrial Revolution, the amount of unstructured data, such as voice and video data, is rapidly increasing. Brain-inspired neuromorphic computing is a new computing method that can efficiently and parallelly process rapidly increasing data. Among artificial neural networks that mimic the structure of the brain, the spiking neural network (SNN) is a network that imitates the information-processing method of biological neural networks. Recently, memristors have attracted attention as synaptic devices for neuromorphic computing systems. Among them, the ferroelectric doped-HfO2-based ferroelectric tunnel junction (FTJ) is considered as a strong candidate for synaptic devices due to its advantages, such as complementary metal-oxide-semiconductor device/process compatibility, a simple two-terminal structure, and low power consumption. However, research on the spiking operations of FTJ devices for SNN applications is lacking. In this study, the implementation of long-term depression and potentiation as the spike timing-dependent plasticity (STDP) rule in the FTJ device was successful. Based on the measured data, a CrossSim simulator was used to simulate the classification of handwriting images. With a high accuracy of 95.79% for the Mixed National Institute of Standards and Technology (MNIST) dataset, the simulation results demonstrate that our device is capable of differentiating between handwritten images. This suggests that our FTJ device can be used as a synaptic device for implementing an SNN.