Botulinum toxin (BoNT) has been used for the treatment of a variety of neurologic, medical and cosmetic conditions. Two serotypes, type A (BoNT-A) and type B (BoNT-B), are currently in clinical use. While considered safe and effective, their use has been rarely complicated by the development of antibodies that reduce or negate their therapeutic effect. The presence of antibodies has been attributed to shorter dosing intervals (and booster injections), higher doses per injection cycle, and higher amounts of antigenic protein. Other factors contributing to the immunogenicity of BoNT include properties of each serotype, such as formulation, manufacturing, and storage of the toxin. Some newer formulations with purified core neurotoxin devoid of accessory proteins may have lower overall immunogenicity. Several assays are available for the detection of antibodies, including both structural assays such as ELISA and mouse-based bioassays, but there is no consistent correlation between these antibodies and clinical response. Prevention and treatment of antibody-associated non-responsiveness is challenging and primarily involves the use of less immunogenic formulations of BoNT, waiting for the spontaneous disappearance of the neutralizing antibody, and switching to an immunologically alternate type of BoNT.

https://www.mdpi.com/2072-6651/11/9/491/pdf

Immunogenicity Associated with Botulinum Toxin Treatment.

Botulinum toxin type A (BoNTA) products are widely used for therapeutic and aesthetic indications, but there is a need for longer-lasting treatments that maintain symptom relief between injections and reduce the frequency of re-treatment. DaxibotulinumtoxinA for Injection (DAXI) is a novel BoNTA product containing highly purified 150-kDa core neurotoxin and is the first to be formulated with a proprietary stabilizing excipient peptide (RTP004) instead of human serum albumin. The positively charged RTP004 has been shown to enhance binding of the neurotoxin to neuronal surfaces, which may enhance the likelihood of neurotoxin internalization. DAXI produces robust, extended efficacy across both aesthetic and therapeutic indications. In an extensive glabellar lines clinical program, DAXI showed a high degree of efficacy, a consistent median time to loss of none or mild glabellar line severity of 24 weeks, and median time until return to baseline of up to 28 weeks. In adults with cervical dystonia, DAXI at 125 U and 250 U significantly improved Toronto Western Spasmodic Torticollis Rating Scale (TWSTRS) total scores, with a median duration of efficacy of 24 and 20 weeks, respectively, which compares favorably with the 12-14 weeks' duration reported for approved BoNTA products. Overall, DAXI was well tolerated, and the consistent extended duration of effect suggests that DAXI has the potential to improve the management of both aesthetic and therapeutic conditions.

/pdf/overview-of-daxibotulinumtoxina-for-injection-a-novel-sea8x1f29a.pdf

Overview of DaxibotulinumtoxinA for Injection: A Novel Formulation of Botulinum Toxin Type A

Botulinum neurotoxins (BoNTs) have been used as therapeutic agents in the clinical treatment of a wide array of neuromuscular and autonomic neuronal transmission disorders. These toxins contain three functional domains that mediate highly specific neuronal cell binding, internalization and cytosolic delivery of proteolytic enzymes that cleave proteins integral to the exocytosis of neurotransmitters. The exceptional cellular specificity, potency and persistence within the neuron that make BoNTs such effective toxins, also make them attractive models for derivatives that have modified properties that could potentially expand their therapeutic repertoire. Advances in molecular biology techniques and rapid DNA synthesis have allowed a wide variety of novel BoNTs with alternative functions to be assessed as potential new classes of therapeutic drugs. This review examines how the BoNTs have been engineered in an effort to produce new classes of therapeutic molecules to address a wide array of disorders.

Engineering of Botulinum Neurotoxins for Biomedical Applications.

Botulinum neurotoxins (BoNT) are produced by several species of clostridium. There are seven immunologically unique BoNT serotypes (A–G). The Centers for Disease Control classifies BoNTs as ‘Category A’ select agents and are the most lethal protein toxins for humans. Recently, BoNT-like proteins have also been identified in several non-clostridia. BoNTs are di-chain proteins comprised of an N-terminal zinc metalloprotease Light Chain (LC) and a C-terminal Heavy Chain (HC) which includes the translocation and receptor binding domains. The two chains are held together by a disulfide bond. The LC cleaves Soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs). The cleavage of SNAREs inhibits the fusion of synaptic vesicles to the cell membrane and the subsequent release of acetylcholine, which results in flaccid paralysis. The LC controls the catalytic properties and the duration of BoNT action. This review discusses the mechanism for LC catalysis, LC translocation, and the basis for the duration of LC action. Understanding these properties of the LC may expand the applications of BoNT as human therapies.

https://www.mdpi.com/2072-6651/10/7/268/pdf

Light Chain Diversity among the Botulinum Neurotoxins

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.

Engineering Botulinum Neurotoxins for Enhanced Therapeutic Applications and Vaccine Development.

We have purified and characterized recombinant botulinum neurotoxin serotype FA (BoNT/FA). This protein has also been named as a new serotype (serotype H), but the classification has been controversial. A lack of well-characterized, highly pure material has been a roadblock to study. Here we report purification and characterization of enzymatically active, and of inactive nontoxic, recombinant forms of BoNT/FA as tractable alternatives to purifying this neurotoxin from native Clostridium botulinum. BoNT/FA cleaves the same intracellular target proteins as BoNT/F1 and other F serotype BoNTs; the intracellular targets are vesicle associated membrane proteins (VAMP) 1, 2 and 3. BoNT/FA cleaves the same site in VAMP-2 as BoNT/F5, which is different from the cleavage site of other F serotype BoNTs. BoNT/FA has slower enzyme kinetics than BoNT/F1 in a cell-free protease assay and is less potent at inhibiting ex vivo nerve-stimulated skeletal muscle contraction. In contrast, BoNT/FA is more potent at inhibiting neurotransmitter release from cultured neurons.

Purification and Characterization of Recombinant Botulinum Neurotoxin Serotype FA, Also Known as Serotype H.

Botulinum neurotoxins (BoNTs) are notorious toxins and powerful agents and can be lethal, causing botulism, but they are also widely used as therapeutics, particularly to treat neuromuscular disorders. As of today, the commercial BoNT treatments available are from native A or B serotypes. Serotype F has shown efficacy in a clinical trial but has scarcely been used, most likely due to its medium duration of effect. Previously, the uniqueness of the light chain of the F7 subtype was identified and reported, showing an extended interaction with its substrates, VAMPs 1, 2 and 3, and a superior catalytic activity compared to other BoNT/F subtypes. In order to more extensively study the properties of this neurotoxin, we engineered a modified F7 chimera, mrBoNT/F7-1, in which all the regions of the neurotoxin were identical to BoNT/F7 except the activation loop, which was the activation loop from BoNT/F1. Use of the activation loop from BoNT/F1 allowed easier post-translational proteolytic activation of the recombinant protein without otherwise affecting its properties. mrBoNT/F7-1 was expressed, purified and then tested in a suite of in vitro and in vivo assays. mrBoNT/F7-1 was active and showed enhanced potency in comparison to both native and recombinant BoNT/F1. Additionally, the safety profile remained comparable to BoNT/F1 despite the increased potency. This new modified recombinant toxin F7 could be further exploited to develop unique therapeutics to address unmet medical needs.

New Modified Recombinant Botulinum Neurotoxin Type F with Enhanced Potency

Recombinant expression and characterization of a botulinum neurotoxin serotype X chimera

Purification and characterization of recombinant botulinum neurotoxin serotype FA, also known as serotype H

Botulinum neurotoxins (BoNTs) are a family of bacterial toxins with seven major serotypes (BoNT/A-G). The ability of these toxins to target and bind to motor nerve terminals is a key factor determining their potency and efficacy. Among these toxins, BoNT/B is one of the two types approved for medical and cosmetic uses. Besides binding to well-established receptors, an extended loop in the C-terminal receptor-binding domain (HC) of BoNT/B (HC/B) has been proposed to also contribute to toxin binding to neurons by interacting with lipid membranes (termed lipid-binding loop [LBL]). Analogous loops exist in the HCs of BoNT/C, D, G, and a chimeric toxin DC. However, it has been challenging to detect and characterize binding of LBLs to lipid membranes. Here, using the nanodisc system and biolayer interferometry assays, we find that HC/DC, C, and G, but not HC/B and HC/D, are capable of binding to receptor-free lipids directly, with HC/DC having the highest level of binding. Mutagenesis studies demonstrate the critical role of consecutive aromatic residues at the tip of the LBL for binding of HC/DC to lipid membranes. Taking advantage of this insight, we then create a "gain-of-function" mutant HC/B by replacing two nonaromatic residues at the tip of its LBL with tryptophan. Cocrystallization studies confirm that these two tryptophan residues do not alter the structure of HC/B or the interactions with its receptors. Such a mutated HC/B gains the ability to bind receptor-free lipid membranes and shows enhanced binding to cultured neurons. Finally, full-length BoNT/B containing two tryptophan mutations in its LBL, together with two additional mutations (E1191M/S1199Y) that increase binding to human receptors, is produced and evaluated in mice in vivo using Digit Abduction Score assays. This mutant toxin shows enhanced efficacy in paralyzing local muscles at the injection site and lower systemic diffusion, thus extending both safety range and duration of paralysis compared with the control BoNT/B. These findings establish a mechanistic understanding of LBL-lipid interactions and create a modified BoNT/B with improved therapeutic efficacy.

/pdf/characterization-of-a-membrane-binding-loop-leads-to-18xn1lwbog.pdf

Characterization of a membrane binding loop leads to engineering botulinum neurotoxin B with improved therapeutic efficacy

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