Benchmarking antibody clustering methods using sequence, structural, and machine learning similarity measures for antibody discovery applications.

Antibodies are proteins produced by our immune system that have been harnessed as biotherapeutics. The discovery of antibody-based therapeutics relies on analyzing large volumes of diverse sequences coming from phage display or animal immunizations. Identification of suitable therapeutic candidates is achieved by grouping the sequences by their similarity and subsequent selection of a diverse set of antibodies for further tests. Such groupings are typically created using sequence-similarity measures alone. Maximizing diversity in selected candidates is crucial to reducing the number of tests of molecules with near-identical properties. With the advances in structural modeling and machine learning, antibodies can now be grouped across other diversity dimensions, such as predicted paratopes or three-dimensional structures. Here we benchmarked antibody grouping methods using clonotype, sequence, paratope prediction, structure prediction, and embedding information. The results were benchmarked on two tasks: binder detection and epitope mapping. We demonstrate that on binder detection no method appears to outperform the others, while on epitope mapping, clonotype, paratope, and embedding clusterings are top performers. Most importantly, all the methods propose orthogonal groupings, offering more diverse pools of candidates when using multiple methods than any single method alone. To facilitate exploring the diversity of antibodies using different methods, we have created an online tool-CLAP-available at (clap.naturalantibody.com) that allows users to group, contrast, and visualize antibodies using the different grouping methods.

LAP: Liability Antibody Profiler by sequence & structural mapping of natural and therapeutic antibodies.

Antibody-based therapeutics must not undergo chemical modifications that would impair their efficacy or hinder their developability. A commonly used technique to de-risk lead biotherapeutic candidates annotates chemical liability motifs on their sequence. By analyzing sequences from all major sources of data (therapeutics, patents, GenBank, literature, and next-generation sequencing outputs), we find that almost all antibodies contain an average of 3-4 such liability motifs in their paratopes, irrespective of the source dataset. This is in line with the common wisdom that liability motif annotation is over-predictive. Therefore, we have compiled three computational flags to prioritize liability motifs for removal from lead drug candidates: 1. germline, to reflect naturally occurring motifs, 2. therapeutic, reflecting chemical liability motifs found in therapeutic antibodies, and 3. surface, indicative of structural accessibility for chemical modification. We show that these flags annotate approximately 60% of liability motifs as benign, that is, the flagged liabilities have a smaller probability of undergoing degradation as benchmarked on two experimental datasets covering deamidation, isomerization, and oxidation. We combined the liability detection and flags into a tool called Liability Antibody Profiler (LAP), publicly available at lap.naturalantibody.com. We anticipate that LAP will save time and effort in de-risking therapeutic molecules.

Expression of the Tyrosine Hydroxylase Gene from Rat Leads to Oxidative Stress in Potato Plants.

Catecholamines are biogenic aromatic amines common among both animals and plants. In animals, they are synthesized via tyrosine hydroxylation, while both hydroxylation or decarboxylation of tyrosine are possible in plants, depending on the species, though no tyrosine hydroxylase-a counterpart of the animal enzyme-has been identified yet. It is known that in potato plants, it is the decarboxylation of tyrosine that leads to catecholamine production. In this paper, we present the effects of the induction of an alternative route of catecholamine production by introducing the tyrosine hydroxylase gene from rat. We demonstrate that an animal system can be used by the plant. However, it does not function to synthesize catecholamines. Instead, it leads to elevated reactive oxygen species content and a constant stress condition in the plant, which responds with elevated antioxidant levels and improved resistance to infection.

V79 Fibroblasts Are Protected Against Reactive Oxygen Species by Flax Fabric.

Chinese hamster pulmonary fibroblasts (V79 cells) pre-treated with flax fabrics derived from non-modified or genetically engineered flax fibres and treated with H2O2 revealed a markedly lower level of intracellular reactive oxygen species (ROS) than control, non-pre-treated cells. The fabrics were prepared from fibres derived from two kinds of transgenic plants: W92 plants, which overproduce flavonoids, and M type plants, which produce hydroxybutyrate polymer in their vascular bundles and thus in fibres. Incubating the V79 cells with the flax fabrics prior to H2O2 treatment also reduced the amount of DNA damage, as established using the comet assay (also known as alkaline single-cell gel electrophoresis) and pulsed-field electrophoresis of intact cellular DNA. Selected gene expression analysis revealed the activator impact of fabrics on the apoptotic (BCL2 family, caspases) gene expression. This promoting activity was also detected for histone acetyltransferase (HAT; MYST2) gene expression. The flax fabric derived from both GM flax plants exhibited a protective effect against oxidative stress and ROS-mediated genotoxic damage, but the W92 fabric was the strongest. It is thus suggested that these fabrics might be useful as a basis for new biomedical products (e.g. wound dressings) that actively protect cells against inflammation and degeneration.

Methyl Salicylate Level Increase in Flax after Fusarium oxysporum Infection Is Associated with Phenylpropanoid Pathway Activation.

Flax (Linum usitatissimum) is a crop plant valued for its oil and fiber. Unfortunately, large losses in cultivation of this plant are caused by fungal infections, with Fusarium oxysporum being one of its most dangerous pathogens. Among the plant's defense strategies, changes in the expression of genes of the shikimate/phenylpropanoid/benzoate pathway and thus in phenolic contents occur. Among the benzoates, salicylic acid, and its methylated form methyl salicylate play an important role in regulating plants' response to stress conditions. Upon treatment of flax plants with the fungus we found that methyl salicylate content increased (4.8-fold of the control) and the expression profiles of the analyzed genes suggest that it is produced most likely from cinnamic acid, through the ß-oxidative route. At the same time activation of some genes involved in lignin and flavonoid biosynthesis was observed. We suggest that increased methyl salicylate biosynthesis during flax response to F. oxysporum infection may be associated with phenylpropanoid pathway activation.

Flax Fiber Hydrophobic Extract Inhibits Human Skin Cells Inflammation and Causes Remodeling of Extracellular Matrix and Wound Closure Activation.

Inflammation is the basis of many diseases, with chronic wounds amongst them, limiting cell proliferation and tissue regeneration. Our previous preclinical study of flax fiber applied as a wound dressing and analysis of its components impact on the fibroblast transcriptome suggested flax fiber hydrophobic extract use as an anti-inflammatory and wound healing preparation. The extract contains cannabidiol (CBD), phytosterols, and unsaturated fatty acids, showing great promise in wound healing. In in vitro proliferation and wound closure tests the extract activated cell migration and proliferation. The activity of matrix metalloproteinases in skin cells was increased, suggesting activation of extracellular components remodeling. The expression of cytokines was diminished by the extract in a cannabidiol-dependent manner, but ß-sitosterol can act synergistically with CBD in inflammation inhibition. Extracellular matrix related genes were also analyzed, considering their importance in further stages of wound healing. The extract activated skin cell matrix remodeling, but the changes were only partially cannabidiol- and ß-sitosterol-dependent. The possible role of fatty acids also present in the extract is suggested. The study shows the hydrophobic flax fiber components as wound healing activators, with anti-inflammatory cannabidiol acting in synergy with sterols, and migration and proliferation promoting agents, some of which still require experimental identification.