Bispecific immune cell engager enhances the anticancer activity of CD16+ NK cells and macrophages in vitro, and eliminates cancer metastasis in NK humanized NOG mice.

BACKGROUND: In a prior report, we detailed the isolation and engineering of a bispecific killer cell engager, referred to as BiKE:E5C1. The BiKE:E5C1 exhibits high affinity/specificity for the CD16a activating receptor on natural killer (NK) cells and human epidermal growth factor receptor 2 (HER2) on cancer cells. In vitro studies have demonstrated that BiKE:E5C1 can activate the NK cells and induce the killing of HER2+ ovarian and breast cancer cells, surpassing the performance of the best-in-class monoclonal antibody, Trazimera (trastuzumab). To advance this BiKE technology toward clinical application, the objective of this research was to demonstrate the ability of BiKE:E5C1 to activate CD16+ immune cells such as NK cells and macrophages to kill cancer cells, and eradicate metastatic HER2+ tumors in NK humanized NOG mice. METHODS: We assessed BiKE:E5C1's potential to activate CD16-expressing peripheral blood (PB)-NK cells, laNK92 cells, and THP-1-CD16A monocyte-macrophages through flowcytometry and antibody-dependent cell-mediated cytotoxicity/phagocytosis (ADCC) assays. Subsequently, laNK92 cells were selected as effector cells and genetically modified to express the nanoluciferase gene, enabling the monitoring of their viability in NK humanized NOG mice using quantitative bioluminescent imaging (qBLI). To evaluate the functionality of BiKE:E5C1 in vivo, we introduced firefly luciferase-expressing ovarian cancer cells via intraperitoneal injection into hIL-15 and hIL-2 NOG mice, creating a model of ovarian cancer metastasis. Once tumor establishment was confirmed, we treated the mice with laNK92 cells plus BiKE:E5C1 and the response to therapy was assessed using qBLI. RESULTS: Our data demonstrate that BiKE:E5C1 activates not only laNK92 cells but also PB-NK cells and macrophages, significantly enhancing their anticancer activities. ADCC assay demonstrated that IgG1 Fc region had no impact on BiKE:E5C1's anticancer activity. In vivo results reveal that both hIL-15 and hIL-2 NOG mouse models support the viability and proliferation of laNK92 cells. Furthermore, it was observed that BiKE:E5C1 activates laNK92 cells in mice, leading to eradication of cancer metastasis in both NK humanized hIL-15 and hIL-2 NOG mouse models. CONCLUSIONS: Collectively, our in vivo findings underscore BiKE:E5C1's potential as an immune cell engager capable of activating immune cells for cancer cell elimination, thereby expanding the arsenal of available BiKEs for cancer immunotherapy.

A high affinity and specificity anti-HER2 single-domain antibody (VHH) that targets trastuzumab's epitope with versatile biochemical, biological, and medical applications.

In the past decade, various single-domain antibodies from llamas, also known as VHH or nanobody, have been discovered with applications in tumor imaging and cancer therapy. However, the potential application of anti-HER2 VHHs as a diagnostic tool suitable for ELISA, flow cytometry, cell imaging, bispecific antibody engineering, and immunohistochemistry has not been fully elucidated. To investigate this potential, HER2 antigen was expressed in HEK293 F cells, purified, and used to immunize llama. Using phage display, anti-HER2 VHHs with high affinity and specificity were isolated, sequenced, and constructed with a Histag and c-Myc tag. The constructed anti-HER2 VHHs were then expressed in E. coli, purified, and evaluated for their use in ELISA, flow cytometry, cell imaging, and immunohistochemistry. The affinities of the anti-HER2 VHHs toward the HER2 antigen were determined using biolayer interferometry. Furthermore, the binding sites of the anti-HER2 VHHs were evaluated by epitope mapping and in silico modeling and docking. Here, we report the sequence of an anti-HER2 VHH with high affinity (sub-nanomolar), specificity, and selectivity. This VHH binds to the same epitope as trastuzumab and can be utilized to generate bispecific antibodies or used as a diagnostic tool to differentiate HER2+ from HER2- antigens on plates, cells, and tissues. This discovery has broad applications in biochemical, biological, and medical sciences.

Small extracellular vesicles (sEVs): discovery, functions, applications, detection methods and various engineered forms.

INTRODUCTION: Extracellular vesicles (EVs) are cell-created delivery systems of proteins, lipids, or nucleic acids, and means of extracellular communication. Though sEVs were initially considered to be the waste disposal mechanism, today they are at the forefront of research with different biological and pathological functions. Such EVs play a key role in the immunoregulation, CNS development, nervous system physiology, mammary gland development, induction of immunosuppression in pregnancy, the developmental signaling pathways, regeneration of different tissues, inflammation, angiogenesis, coagulation, apoptosis, stem cell differentiation, and extracellular matrix turnover. AREAS COVERED: SEVs contribute to the pathogenesis of different cancers and the progression of various neurodegenerative diseases, infections, as well as metabolic and cardiovascular diseases. Expert Opinion: There is no exact classification for EVs; however, according to size, density, morphological features, content, and biogenesis, they can be categorized into three major classes: microvesicles (ectosomes or microparticles), apoptotic bodies, and sEVs. SEVs, as an important class of EVs, have a crucial role in distinct biological functions. Moreover, shedding light on different structural and molecular aspects of sEV has led to their application in various therapeutic, diagnostic, and drug delivery fields. In this review, we have endeavored to elaborate on different aspects of EVs, especially sEVs.

Immunotherapeutic approaches to curtail COVID-19.

COVID-19, the disease induced by the recently emerged severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has imposed an unpredictable burden on the world. Drug repurposing has been employed to rapidly find a cure; but despite great efforts, no drug or vaccine is presently available for treating or prevention of COVID-19. Apart from antivirals, immunotherapeutic strategies are suggested considering the role of the immune response as the host defense against the virus, and the fact that SARS-CoV-2 suppresses interferon induction as an immune evasion strategy. Active immunization through vaccines, interferon administration, passive immunotherapy by convalescent plasma or synthesized monoclonal and polyclonal antibodies, as well as immunomodulatory drugs, are different immunotherapeutic approaches that will be mentioned in this review. The focus would be on passive immunotherapeutic interventions. Interferons might be helpful in some stages. Vaccine development has been followed with unprecedented speed. Some of these vaccines have been advanced to human clinical trials. Convalescent plasma therapy is already practiced in many countries to help save the lives of severely ill patients. Different antibodies that target various steps of SARS-CoV-2 pathogenesis or the associated immune responses are also proposed. For treating the cytokine storm induced at a late stage of the disease in some patients, immune modulation through JAK inhibitors, corticosteroids, and some other cognate classes are evaluated. Given the changing pattern of cytokine induction and immune responses throughout the COVID-19 disease course, different adapted approaches are needed to help patients. Gaining more knowledge about the detailed pathogenesis of SARS-CoV-2, its interplay with the immune system, and viral-mediated responses are crucial to identify efficient preventive and therapeutic approaches. A systemic approach seems essential in this regard.

In Silico Elucidation of Deleterious Non-synonymous SNPs in SHANK3, the Autism Spectrum Disorder Gene.

SHANK3, a member of SH3 and multiple ankyrin repeat domains (SHANK) proteins, plays a crucial role in synaptic development and functions. Mutations in SHANK3 have been linked to a number of neuropsychiatric and neurodevelopmental disorders, including autism spectrum disorder. In this study, the functional and structural impacts of non-synonymous single-nucleotide polymorphisms (SNPs) on SHANK3 were predicted. Various databases were used to extract 16,894 non-redundant SNPs, out of which 1179 were annotated as missense variants. Missense variants were categorized as deleterious or non-deleterious. Twenty-nine missense variants were unanimously recognized as deleterious and subjected to structural and stability analyses. Mutations, including L47P, G54W, G172D, G250C/D, and G627E, which posed drastic effects on the secondary structure of SHANK3, were modeled. Stability analyses introduced L47P, G54W, and G250D as the most destabilizing mutations, thus they were subjected to molecular dynamics simulation. Simulation revealed significant changes in intramolecular interactions and high fluctuations in residues of 1-350 that significantly affect the ANK functional domain. G250C/D and G635R consensus deleterious mutations were found in the first and second binding domains of SHANK3, and none were found in the post-translational modification sites. This study suggests L47P, G54W, and G250C/D deleterious mutations as priorities for future studies on SHANK3.

Effect of alumina (Al2O3) nanoparticles and macroparticles on Trigonella foenum-graceum L. in vitro cultures: assessment of growth parameters and oxidative stress-related responses.

The impact of 100 µg ml-1 alumina (Al2O3) nanoparticles (NPs) on Trigonella foenum (fenugreek) in vitro cultures was studied within 3 weeks (on days 1, 7, 14, and 21) and compared with the control and bulk (macrometer-sized particles) alumina-treated groups. Transmission electron microscopy (TEM) and dynamic light scattering were used for the characterization of NPs. The results of TEM analysis represented a nearly spherical shape for the NPs with agglomeration. The zeta potential of alumina NPs was - 25.4 ± 2.5 mV and the averaged diameter was 20 ± 5 nm. Atomic absorption and inductively coupled plasma-optical emission spectroscopy provided evidence for the release and uptake of aluminum. Treatment of cultures with NPs led to an increase in the formation of lateral roots. Treatment of fenugreek with bulk alumina caused a significant decrease in the number of leaves on day 21 (p < 0.05) and the root length on days 14 and 21 compared with the control group (p < 0.05). Alumina NP has led to a significant increase in the malondialdehyde content on days 7, 14, and 21 (p < 0.001). The glutathione content was decreased significantly in NP and bulk-treated groups on days 1 and 7 (p < 0.05). FRAP assay results showed that NPs and bulk alumina led to a decrease in the antioxidant power on days 7, 14, and 21 (p < 0.001). The increased activity of catalase (p < 0.001) and ascorbate peroxidase (p < 0.001) was observed in the bulk-treated group. Lignin content had a significant increase in response to NPs on days 14 and 21 (p < 0.001). Conclusively, alumina nano/macro particles affected agronomical and physiological properties of T. foenum; however, smaller sized particles do not necessarily imply greater toxicity, while uptake of the aluminum ions should be considered seriously.

Selected application of peptide molecules as pharmaceutical agents and in cosmeceuticals.

Introduction: Peptide molecules are being vastly investigated as an emerging class of therapeutic molecules in recent years. Currently, 60 peptides have been approved by the US Food and Drug Administration (FDA), and more would enter the market in near future. Peptides have already opened their ways into cosmeceutical and food industries as well.Areas covered: Antibodies, vaccines, and antimicrobial agents are the major classes of therapeutic peptides. Additionally, peptides may be employed in drug development to support cell penetration or targeting. The interest in antimicrobial peptides is surging due to the increasing risk of antibiotic-resistant pathogens. Peptide vaccines with their significant advantages compared with traditional vaccines, are expected to find their place in coming years, especially for cancer, microbial and allergen-specific immunotherapy. The usage of peptides in cosmeceuticals is also growing rapidly.Expert opinion: Peptide synthesis has become accessible, and advances in peptide engineering, sequencing technologies, and structural bioinformatics have resulted in the rational designing of novel peptides. All these advancements would lead to the more prominent roles of peptides in the mentioned areas. In this review, we discuss applications of peptides in different fields including pharmaceuticals, cosmeceuticals, besides the critical factors in designing efficient peptide molecules.

A comprehensive in silico characterization of bacterial signal peptides for the excretory production of Anabaena variabilis phenylalanine ammonia lyase in Escherichia coli.

Anabaena variabilis double mutant (C503S/C565S) phenylalanine ammonia-lyase (PAL) is an appealing enzyme in the enzyme-replacement therapy of phenylketonuria. Yet abundant production of this enzyme has been of concern for industrial production. In this study, we have characterized 1175 bacterial signal peptides (SPs) and identified the most efficient ones for the excretory production of mutant AvPAL. Analysis by SignalP 4.1 revealed that more than 61% of SPs had a D-score greater than 0.7, denoting to be highly efficient. The optimum length of a bacterial SP was 25-30. The preferable net positive charge and the second residue of N-region were + 2 and Lys/Arg, respectively. Highly efficient SPs possessed 3-5 Leus in their H-region and A/L/VXA-FF cleavage site. Highly efficient SPs were from Escherichia coli, corroborating the necessity of an agreement between SPs and the host. Physiochemical characterization of mutant AvPAL conjugates via ProtParam and PROSOII, revealed that ~ 99.5% of proteins would not be entraped in inclusion bodies. Secretory pathways were identified by EffectiveDB and more than 98% of SPs were cleavable. Chimeras were modeled using the I-TASSER program, being evaluated by the Ramachandran plots. The mRNA secondary structure of mutant AvPAL upon linkage to SPs was assessed using the mfold program. It was shown that the linkage of a SP does not affect mutant AvPAL's stability at the protein or mRNA level. AllergenFP tool demonstrated that chimeras were not allergen. SPs, including FMF4_ECOLX, E2BB_ECOLX, and LPTA_ECOLI exhibited the highest propensity for secretion and appropriate features in all analyses.

A comprehensive review of signal peptides: Structure, roles, and applications.

Signal peptides (SP) are short peptides located in the N-terminal of proteins, carrying information for protein secretion. They are ubiquitous to all prokaryotes and eukaryotes. SPs have been of special interest in several scientific and industrial fields, including recombinant protein production, disease diagnosis, immunization, and laboratory techniques. Recently, the role of SPs in recombinant protein production has gained too much attention. Herein, several studies have been reviewed to elucidate the precise structure and function of SPs, particularly the optimized ones for recombinant protein production. However, some features of SPs still have remained obscure. In this review, some approaches concerning elucidation and optimization of SPs are discussed, and pragmatic conclusions and suggestions for future studies are also proposed. Moreover, a summary of secretory pathways, evolutionary changes, functions, applications, and different types of SPs is mentioned. At last, current limitations and prospects are discussed.

Identification and functional prediction of stress responsive AP2/ERF transcription factors in Brassica napus by genome-wide analysis.

Using homology and domain authentication, 321 putative AP2/ERF transcription factors were identified in Brassica napus, called BnAP2/ERF TFs. BnAP2/ERF TFs were classified into five major subfamilies, including DREB, ERF, AP2, RAV, and BnSoloist. This classification is based on phylogenetic analysis, motif identification, gene structure analysis, and physiochemical characterization. These TFs were annotated based on phylogenetic relationship with Brassica rapa. BnAP2/ERF TFs were located on 19 chromosomes of B. napus. Orthologs and paralogs were identified using synteny-based methods Ks calculation within B. napus genome and between B. napus with other species such as B. rapa, Brassica oleracea, and Arabidopsis thaliana indicated that BnAP2/ERF TFs were formed through duplication events occurred before B. napus formation. Kn/Ks values were between 0 and 1, suggesting the purifying selection among BnAP2/ERF TFs. Gene ontology annotation, cis-regulatory elements and functional interaction networks suggested that BnAP2/ERF TFs participate in response to stressors, including drought, high salinity, heat and cold as well as developmental processes particularly organ specification and embryogenesis. The identified cis-regulatory elements in the upstream of BnAP2/ERF TFs were responsive to abscisic acid. Analysis of the expression data derived from Illumina Hiseq 2000 RNA sequencing revealed that BnAP2/ERF genes were highly expressed in the roots comparing to flower buds, leaves, and stems. Also, the ERF subfamily was over-expressed under salt and fungal treatments. BnERF039 and BnERF245 are candidates for salt-tolerant B. napus. BnERF253-256 and BnERF260-277 are potential cytokinin response factors. BnERF227, BnERF228, BnERF234, BnERF134, BnERF132, BnERF176, and BnERF235 were suggested for resistance against Leptosphaeria maculan and Leptosphaeria biglobosa.

Genome-wide identification, functional prediction, and evolutionary analysis of the R2R3-MYB superfamily in Brassica napus.

R2R3-MYB transcription factors (TFs) have been shown to play important roles in plants, including in development and in various stress conditions. Phylogenetic analysis showed the presence of 249 R2R3-MYB TFs in Brassica napus, called BnaR2R3-MYB TFs, clustered into 38 clades. BnaR2R3-MYB TFs were distributed on 19 chromosomes of B. napus. Sixteen gene clusters were identified. BnaR2R3-MYB TFs were characterized by motif prediction, gene structure analysis, and gene ontology. Evolutionary analysis revealed that BnaR2R3-MYB TFs are mainly formed as a result of whole-genome duplication. Orthologs and paralogs of BnaR2R3-MYB TFs were identified in B. napus, B. rapa, B. oleracea, and Arabidopsis thaliana using synteny-based methods. Purifying selection was pervasive within R2R3-MYB TFs. Kn/Ks values lower than 0.3 indicated that BnaR2R3-MYB TFs are being functionally converged. The role of gene conversion in the formation of BnaR2R3-MYB TFs was significant. Cis-regulatory elements in the upstream regions of BnaR2R3-MYB genes, miRNA targeting BnaR2R3MYB TFs, and post translational modifications were identified. Digital expression data revealed that BnaR2R3-MYB genes were highly expressed in the roots and under high salinity treatment after 24 h. BnaMYB21, BnaMYB141, and BnaMYB148 have been suggested for improving salt-tolerant B. napus. BnaR2R3-MYB genes were mostly up regulated on the 14th day post inoculation with Leptosphaeria biglobosa and L. maculan. BnaMYB150 is a candidate for increased tolerance to Leptospheria in B. napus.