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In horses, Clostridium perfringens is associated with acute and fatal enterocolitis, which is caused by a beta toxin (CPB), and myonecrosis, which is caused by an alpha toxin (CPA). Although the most effective way to prevent these diseases is through vaccination, specific clostridial vaccines for horses against C. perfringens are not widely available. The aim of this study was to pioneer the immunization of horses with three different concentrations (100, 200 and 400 µg) of C. perfringens recombinant alpha (rCPA) and beta (rCPB) proteins, as well as to evaluate the humoral immune response over 360 days. Recombinant toxoids were developed and applied to 50 horses on days 0 and 30. Those vaccines attempted to stimulate the production of alpha antitoxin (anti-CPA) and beta antitoxin (anti-CPB), in addition to becoming innocuous, stable and sterile. There was a reduction in the level of neutralizing anti-CPA and anti-CPB antibodies following the 60th day; therefore, the concentrations of 200 and 400 µg capable of inducing a detectable humoral immune response were not determined until day 180. In practical terms, 200 µg is possibly the ideal concentration for use in the veterinary industry’s production of vaccines against the action of C. perfringens in equine species.

Today, the production and use of various samples of recombinant protein/polypeptide toxins is known and is actively developing. This review presents state-of-the-art in research and development of such toxins and their mechanisms of action and useful properties that have allowed them to be implemented into practice to treat various medical conditions (including oncology and chronic inflammation applications) and diseases, as well as to identify novel compounds and to detoxify them by diverse approaches (including enzyme antidotes). Special attention is given to the problems and possibilities of the toxicity control of the obtained recombinant proteins. The recombinant prions are discussed in the frame of their possible detoxification by enzymes. The review discusses the feasibility of obtaining recombinant variants of toxins in the form of protein molecules modified with fluorescent proteins, affine sequences and genetic mutations, allowing us to investigate the mechanisms of toxins’ bindings to their natural receptors.

Despite nearly four decades of effort, broad inhibition of oncogenic RAS using small-molecule approaches has proven to be a major challenge. Here we describe the development of a pan-RAS biologic inhibitor composed of the RAS-RAP1–specific endopeptidase fused to the protein delivery machinery of diphtheria toxin. We show that this engineered chimeric toxin irreversibly cleaves and inactivates intracellular RAS at low picomolar concentrations terminating downstream signaling in receptor-bearing cells. Furthermore, we demonstrate in vivo target engagement and reduction of tumor burden in three mouse xenograft models driven by either wild-type or mutant RAS. Intracellular delivery of a potent anti-RAS biologic through a receptor-mediated mechanism represents a promising approach to developing RAS therapeutics against a broad array of cancers.

Botulinum neurotoxins (BoNTs) show increasing therapeutic applications ranging from treatment of locally paralyzed muscles to cosmetic benefits. At first, in the 1970s, BoNT was used for the treatment of strabismus, however, nowadays, BoNT has multiple medical applications including the treatment of muscle hyperactivity such as strabismus, dystonia, movement disorders, hemifacial spasm, essential tremor, tics, cervical dystonia, cerebral palsy, as well as secretory disorders (hyperhidrosis, sialorrhea) and pain syndromes such as chronic migraine. This review summarizes current knowledge related to engineering of botulinum toxins, with particular emphasis on their potential therapeutic applications for pain management and for retargeting to non-neuronal tissues. Advances in molecular biology have resulted in generating modified BoNTs with the potential to act in a variety of disorders, however, in addition to the modifications of well characterized toxinotypes, the diversity of the wild type BoNT toxinotypes or subtypes, provides the basis for innovative BoNT-based therapeutics and research tools. This expanding BoNT superfamily forms the foundation for new toxins candidates in a wider range of therapeutic options.

Snakes, scorpions, and spiders are venomous animals that pose a threat to human health, and severe envenomings from the bites or stings of these animals must be treated with antivenom. Current antivenoms are based on plasma-derived immunoglobulins or immunoglobulin fragments from hyper-immunized animals. Although these medicines have been life-saving for more than 120 years, opportunities to improve envenoming therapy exist. In the later decades, new biotechnological tools have been applied with the aim of improving the efficacy, safety, and affordability of antivenoms. Within the avenues explored, novel immunization strategies using synthetic peptide epitopes, recombinant toxins (or toxoids), or DNA strings as immunogens have demonstrated potential for generating antivenoms with high therapeutic antibody titers and broad neutralizing capacity. Furthermore, these approaches circumvent the need for venom in the production process of antivenoms, thereby limiting some of the complications associated with animal captivity and venom collection. Finally, an important benefit of innovative immunization approaches is that they are often compatible with existing antivenom manufacturing setups. In this review, we compile all reported studies examining venom-independent innovative immunization strategies for antivenom development. In addition, a brief description of toxin families of medical relevance found in snake, scorpion, and spider venoms is presented, as well as how biochemical, bioinformatic, and omics tools could aid the development of next-generation antivenoms.

The production of therapeutic recombinant toxins requires careful host cell selection. Bacteria, yeast, and mammalian cells are common choices, but no universal solution exists. Achieving the delicate balance in toxin production is crucial due to potential self-intoxication. Recombinant toxins from various sources find applications in antimicrobials, biotechnology, cancer drugs, and vaccines. “Toxin-based therapy” targets diseased cells using three strategies. Targeted cancer therapy, like antibody–toxin conjugates, fusion toxins, or “suicide gene therapy”, can selectively eliminate cancer cells, leaving healthy cells unharmed. Notable toxins from various biological sources may be used as full-length toxins, as plant (saporin) or animal (melittin) toxins, or as isolated domains that are typical of bacterial toxins, including Pseudomonas Exotoxin A (PE) and diphtheria toxin (DT). This paper outlines toxin expression methods and system advantages and disadvantages, emphasizing host cell selection’s critical role.

Animal venoms are complex mixtures containing peptides and proteins known as toxins, which are responsible for the deleterious effect of envenomations. Across the animal Kingdom, toxin diversity is enormous, and the ability to understand the biochemical mechanisms governing toxicity is not only relevant for the development of better envenomation therapies, but also for exploiting toxin bioactivities for therapeutic or biotechnological purposes. Most of toxinology research has relied on obtaining the toxins from crude venoms; however, some toxins are difficult to obtain because the venomous animal is endangered, does not thrive in captivity, produces only a small amount of venom, is difficult to milk, or only produces low amounts of the toxin of interest. Heterologous expression of toxins enables the production of sufficient amounts to unlock the biotechnological potential of these bioactive proteins. Moreover, heterologous expression ensures homogeneity, avoids cross-contamination with other venom components, and circumvents the use of crude venom. Heterologous expression is also not only restricted to natural toxins, but allows for the design of toxins with special properties or can take advantage of the increasing amount of transcriptomics and genomics data, enabling the expression of dormant toxin genes. The main challenge when producing toxins is obtaining properly folded proteins with a correct disulfide pattern that ensures the activity of the toxin of interest. This review presents the strategies that can be used to express toxins in bacteria, yeast, insect cells, or mammalian cells, as well as synthetic approaches that do not involve cells, such as cell-free biosynthesis and peptide synthesis. This is accompanied by an overview of the main advantages and drawbacks of these different systems for producing toxins, as well as a discussion of the biosafety considerations that need to be made when working with highly bioactive proteins.

Background/Aim: Pseudomonas exotoxin (PE) is one of the most widely used toxins in the construction of therapeutic fusion proteins in pre-clinical studies followed by phase trials. In principle, PE acts by blocking protein synthesis through catalyzing the inactivation of elongation factor-2 (EF-2). The interleukin-13 fused PE (IL13-PE) cytotoxin was previously designed to target GBM cells. In this study, the cytotoxic effects of IL13-PE were evaluated in 5 different types of cancers and the therapeutic effects were further analyzed in a lung cancer cell line, NCI-H460. Conceptually, in another lung cancer cell line (A549), IL13Rα2 was overexpressed by lentiviruses (A549-IL13Rα2) and evaluated for cytotoxic efficacy of IL13-PE. Materials and Methods: The expression profile of IL13Rα2 in different cancer cell lines was determined by RT-PCR. Secretable toxin fusion was expressed in the toxin resistant HEK-293T cell line (293T-TxR) by using a plasmid coding for IL13-PE and IRES-GFP (LV-IL13-PE-IRES/GFP). Next, the cells were shown to produce and secrete functional IL13-PE by dot blot analysis, followed by cell viability assays and cell death analysis. Results: Upon treatment with IL13-PE, a significant decrease in cell viability was selectively demonstrated in cancer cells with cognate receptor expression. IL13-PE treatment increased the apoptotic/necrotic cell populations in the NCI-H460 cell line. Conclusion: Our results demonstrate that IL13-PE can be a therapeutic target for tumors bearing mostly IL13Rα2 positive cell populations. Our findings also suggest a cell-based delivery option for the recombinant toxins in the treatment of different cancers which can provide a solution for the clinical use of toxin therapy.

Since its introduction as a treatment for strabismus, botulinum toxin (BoNT) has had a phenomenal journey and is now recommended as first-line treatment for focal dystonia, despite short-term clinical benefits and the risks of adverse effects. To cater for the high demand across various medical specialties, at least six US Food and Drug Administration (FDA)-approved formulations of BoNT are currently available for diverse labelled indications. The toxo-pharmacological properties of these formulations are not uniform and thus should not be used interchangeably. Synthetic BoNTs and BoNTs from non-clostridial sources are not far from clinical use. Moreover, the study of mutations in naturally occurring toxins has led to modulation in the toxo-pharmacokinetic properties of BoNTs, including the duration and potency. We present an overview of the toxo-pharmacology of conventional and novel BoNT preparations, including those awaiting imminent translation from the laboratory to the clinic.

Brown spider envenomation results in dermonecrosis, characterized by an intense inflammatory reaction. The principal toxins of brown spider venoms are phospholipase-D isoforms, which interact with different cellular membrane components, degrade phospholipids, and generate bioactive mediators leading to harmful effects. The Loxosceles intermedia phospholipase D, LiRecDT1, possesses a loop that modulates the accessibility to the active site and plays a crucial role in substrate. In vitro and in silico analyses were performed to determine aspects of this enzyme’s substrate preference. Sphingomyelin d18:1/6:0 was the preferred substrate of LiRecDT1 compared to other Sphingomyelins. Lysophosphatidylcholine 16:0/0:0 was preferred among other lysophosphatidylcholines, but much less than Sphingomyelin d18:1/6:0. In contrast, phosphatidylcholine d18:1/16:0 was not cleaved. Thus, the number of carbon atoms in the substrate plays a vital role in determining the optimal activity of this phospholipase-D. The presence of an amide group at C2 plays a key role in recognition and activity. In silico analyses indicated that a subsite containing the aromatic residues Y228 and W230 appears essential for choline recognition by cation-π interactions. These findings may help to explain why different cells, with different phospholipid fatty acid compositions exhibit distinct susceptibilities to brown spider venoms.