Role of paraoxonase 1 in organophosphate G-series nerve agent poisoning and future therapeutic strategies.

Chemical warfare nerve agents (CWNA) are neurotoxic chemicals unethically used as agents of mass destruction by terrorist outfits and during war. The available antidote against CWNA-mediated toxicity is not sufficiently effective and possesses several limitations. As a countermeasure, paraoxonase 1 (PON1), a catalytic bioscavenger, is being developed as a prophylactic treatment. However, the catalytic activity and substrate specificity of human PON1 are insufficient to be used as a potential antidote. Several laboratories have made different approaches to enhance the CWNA hydrolytic activity against various nerve agents. This review explores the holistic view of PON1 as a potential prophylactic agent against G-series CWNA poisoning, from its initial development to recent advancements and limitations. Apart from this, the review also provides an overview of all available PON1 variants that could be used as a potential prophylactic agent and discusses several possible ways to counteract immunogenicity.

Polybodies: Next-generation clinical antibodies.

Conventional antibodies [full-length and fragments: F(ab')2, fragment antigen-binding (Fab), single-chain variable fragment (scFv), variable heavy domain of heavy chain (VHH)] are monospecific first-generation antibodies that have dominated the biopharmaceuticals field. However, advanced protein engineering technology has led to the advent of the next-generation polybody, which is a significant improvement over the conventional antibody. Polybodies comprise polyspecific and polyvalent antibodies that enable a single antibody to target multiple specific antigens simultaneously. Polybodies are superior to first-generation antibodies (more efficacious, broad-spectrum, resistance resistant, customizable, etc.) and provide a cost-effective healthcare solution. This review thoroughly addresses developments in polybodies, highlighting their superiority over conventional antibodies and offering future perspectives to encourage the generation of innovative immunotherapies.

Broad-spectrum anti-cancer activity of fused human arginase variants.

The rapid increase in cancer cases worldwide necessitates the development of novel therapeutic approaches. Therapies targeting cancer's altered metabolism, especially those that deplete critical amino acids, have emerged as promising ones, some of which are already being used in clinical practice and many others are under development. This study reports the anti-cancer activity of two novel fused human arginase I (FHA) variants, FHA-3 and FHA-12, assessed using the NCI-60 human tumor cell line panel. Both variants have demonstrated a range of potencies in a single-dose assay (10 µM), but FHA-3 was found to be more potent with significant growth inhibition in most tested cell lines. To calculate 50% growth inhibition (GI50), FHA-3 was further evaluated in a five-dose assay, where notable anti-cancer activity was observed across the nine cancer types of the NCI-60 panel. Our results demonstrated the broad-spectrum anti-cancer activity of novel FHA variants, with FHA-3 being the most potent. Further studies elucidating its efficacy in animal models will help explore its therapeutic potential.

Warfare Nerve Agents and Paraoxonase-1 as a Potential Prophylactic Therapy against Intoxication.

Nerve agents are a class of lethal neurotoxic chemicals used in chemical warfare. In this review, we have discussed a brief history of chemical warfare, followed by an exploration of the historical context surrounding nerve agents. The article explores the classification of these agents, their contemporary uses, their toxicity mechanisms, and the disadvantages of the current treatment options for nerve agent poisoning. It then discusses the possible application of enzymes as prophylactics against nerve agent poisoning, outlining the benefits and drawbacks of paraoxonase- 1. Finally, the current studies on paraoxonase-1 are reviewed, highlighting that several challenges need to be addressed in the use of paraoxonase-1 in the actual field and that its potential as a prophylactic antidote against nerve agent poisoning needs to be evaluated. The literature used in this manuscript was searched using various electronic databases, such as PubMed, Google Scholar, Web of Science, Elsevier, Springer, ACS, Google Patent, and books using the keywords chemical warfare agent, butyrylcholinesterase, enzyme, nerve agent, prophylactic, and paraoxonase-1, with the time scale for the analysis of articles between 1960 to 2023. The study has suggested that concerted efforts by researchers and agencies must be made to develop effective countermeasures against NA poisoning and that paraoxonase-1 has suitable properties for the development of efficient prophylaxis against NA poisoning.

Molecular and functional insight into anti-EGFR nanobody: Theranostic implications for malignancies.

Targeted therapy and imaging are the most popular techniques for the intervention and diagnosis of cancer. A potential therapeutic target for the treatment of cancer is the epidermal growth factor receptor (EGFR), primarily for glioblastoma, lung, and breast cancer. Over-production of ligand, transcriptional up-regulation due to autocrine/paracrine signalling, or point mutations at the genomic locus may contribute to the malfunction of EGFR in malignancies. This exploit makes use of EGFR, an established biomarker for cancer diagnostics and treatment. Despite considerable development in the last several decades in making EGFR inhibitors, they are still not free from limitations like toxicity and a short serum half-life. Nanobodies and antibodies share similar binding properties, but nanobodies have the additional advantage that they can bind to antigenic epitopes deep inside the target that conventional antibodies are unable to access. For targeted therapy, anti-EGFR nanobodies can be conjugated to various molecules such as drugs, peptides, toxins and photosensitizers. These nanobodies can be designed as novel immunoconjugates using the universal modular antibody-based platform technology (UniCAR). Furthermore, Anti-EGFR nanobodies can be expressed in neural stem cells and visualised by effective fluorescent and radioisotope labelling.

Treating liver cancer through arginine depletion.

Liver cancer, the sixth most common cancer globally and the second-leading cause of cancer-related deaths, presents a critical public health threat. Diagnosis often occurs in advanced stages of the disease, aligning incidence with fatality rates. Given that established treatments, such as stereotactic body radiation therapy and transarterial radioembolization, face accessibility and affordability challenges, the emerging focus on cancer cell metabolism, particularly arginine (Arg) depletion, offers a promising research avenue. Arg-depleting enzymes show efficacy against Arg-auxotrophic cancers, including hepatocellular carcinoma (HCC). Thus, in this review, we explore the limitations of current therapies and highlight the potential of Arg depletion, emphasizing various Arg-hydrolyzing enzymes in clinical development.

Polyvalency: an emerging trend in the development of clinical antibodies.

Medicine has benefited greatly from the development of monoclonal antibody (mAb) technology. First-generation mAbs have seen significant success in the treatment of major diseases, such as autoimmune, inflammation, cancer, infectious, and cardiovascular diseases. Developing next-generation antibodies with improved potency, safety, and non-natural characteristics is a booming field of mAb research. In this review, we discuss the significance of polyvalency and polyvalent antibodies, as well as important findings from preclinical studies and clinical trials involving polyvalent antibodies. We then review the role of tumor necrosis factor-alpha (TNF-α) in inflammatory diseases and the need for polyvalent anti-TNF-α antibodies.

Towards development of biobetter: L-asparaginase a case study.

BACKGROUND: L-asparaginase (ASNase) has played a key role in the management of acute lymphoblastic leukaemia (ALL). As an amidohydrolase, it catalyzes the hydrolysis of L-asparagine, a crucial step in the treatment of ALL. Various ASNase variants have evolved from diverse sources since it was first used in paediatric patients in the 1960s. This review describes the available ASNase and approaches being used to develop ASNase as a biobetter candidate. SCOPE OF REVIEW: The review discusses the Glycosylation and PEGylation techniques, which are frequently used to develop biobetter versions of the majority of the therapeutic proteins. Further, it explores current ASNase biobetters in therapeutic use and discusses the protein engineering and chemical modification approaches that were employed to reduce immunogenicity, extend protein half-life, and enhance protease stability of ASNase. Emerging strategies like immobilization and encapsulation are also highlighted as potential pathways for improving ASNase properties. MAJOR CONCLUSIONS: The purpose of the development of ASNase biobetter is to achieve a novel therapeutic candidate that could improve catalytic efficiency, in vivo stability with minimum glutaminase (GLNase) activity and toxicity. Modification of ASNase by immobilization and encapsulation or by fusion technologies like Albumin fusion, Fc fusion, ELP fusion, XTEN fusion, etc. can be exploited to develop a novel biobetter candidate suitable for therapeutic approaches. GENERAL SIGNIFICANCE: This review emphasizes the importance of biobetter development for therapeutic proteins like ASNase. Improved ASNase molecules have the potential to significantly advance the treatment of ALL and have broader implications in the pharmaceutical industry.

Engineered polyspecific antibodies: A new frontier in the field of immunotherapeutics.

The 21st-century beginning remarked with the huge success of monospecific MAbs, however, in the last couple of years, polyspecific MAbs (PsAbs) have been an interesting topic and show promise of being biobetter than monospecific MAbs. Polyspecificity, in which a single antibody serves multiple specific target binding, has been hypothesized to contribute to the development of a highly effective antibody repertoire for immune defence. This polyspecific MAb trend represents an explosion that is gripping the whole pharmaceutical industry. This review is concerned with the current development and quality enforcement of PsAbs. All provided literature on monospecific MAbs and polyspecific MAbs (PsAbs) were searched using various electronic databases such as PubMed, Google Scholar, Web of Science, Elsevier, Springer, ACS, Google Patent and books via the keywords Antibody engineering, Polyspecific antibody, Conventional antibody, non-conventional antibody, and Single domain antibody. In the literature, there are more than 100 different formats to construct PsAb by quadroma technology, chemical conjugation and genetic engineering. Till March 2023, nine PsAb have been approved around the world, and around 330 are in advanced developmental stages, showing the dominancy of PsAb in the growing health sector. Recent advancements in protein engineering techniques and the fusion of non-conventional antibodies have made it possible to create complex PsAbs that demonstrate higher stability and enhanced potency. This marks the most significant achievement for cancer immunotherapy, in which PsAbs have immense promise. It is worth mentioning that seven out of the nine PsAbs have been approved as anti-cancer therapy. As PsAbs continue to acquire prominence, they could pave the way for the development of novel immunotherapies for multiple diseases.

Recombinant human endostatin as a potential anti-angiogenic agent: therapeutic perspective and current status.

Angiogenesis is the physiological process that results in the formation of new blood vessels develop from pre-existing vasculature and plays a significant role in several physiological and pathological processes. Inhibiting angiogenesis, a crucial mechanism in the growth and metastasis of cancer, has been proposed as a potential anticancer therapy. Different studies showed the beneficial effects of angiogenesis inhibitors either in patients suffering from different cancers, alone or in combination with conventional therapies. Even though there are currently a number of efficient anti-angiogenic drugs, including monoclonal antibodies and kinase inhibitors, the associated toxicity profile and their affordability constraints are prompting researchers to search for a safe and affordable angiostatic agent for cancer treatment. Endostatin is one of the endogenous anti-angiogenic candidates that have been extensively pursued for the treatment of cancer, but even over three decades after its discovery, we have not made much advancement in employing it as an anticancer therapeutic despite of its remarkable anti-angiogenic effect with low toxicity profile. A recombinant human endostatin (rh-Es) variant for non-small cell lung cancer was approved by China in 2006 and has since been used effectively. Several other successful clinical trials related to endostatin for various malignancies are either ongoing or have already been completed with promising results. Thus, in this review, we have provided an overview of existing anti-angiogenic drugs developed for cancer therapy, with a summary of tumour angiogenesis in the context of Endostatin, and clinical status of rh-Es in cancer treatment. Furthermore, we briefly discuss the various strategies to improve endostatin features (poor pharmacokinetic properties) for developing rh-Es as a safe and effective agent for cancer treatment.

Development and characterization of fused human arginase I for cancer therapy.

Recombinant human arginase I (rhArg I) have emerged as a potential candidate for the treatment of varied pathophysiological conditions ranging from arginine-auxotrophic cancer, inflammatory conditions and microbial infection. However, rhArg I have a low circulatory half-life, leading to poor pharmacokinetic and pharmacodynamic properties, which necessitating the rapid development of modifications to circumvent these limitations. To address this, polyethylene glycol (PEG)ylated-rhArg I variants are being developed by pharmaceutical companies. However, because of the limitations associated with the clinical use of PEGylated proteins, there is a dire need in the art to develop rhArg I variant(s) which is safe (devoid of limitations of PEGylated counterpart) and possess increased circulatory half-life. In this study, we described the generation and characterization of a fused human arginase I variant (FHA-3) having improved circulatory half-life. FHA-3 protein was engineered by fusing rhArg I with a half-life extension partner (domain of human serum albumin) via a peptide linker and was produced using P. pastoris expression system. This purified biopharmaceutical (FHA-3) exhibits (i) increased arginine-hydrolyzing activity in buffer, (ii) cofactor - independency, (iii) increased circulatory half-life (t1/2) and (iv) potent anti-cancer activity against human cancer cell lines under in vitro and in vivo conditions.

Human arginase I: a potential broad-spectrum anti-cancer agent.

With high rates of morbidity and mortality, cancer continues to pose a serious threat to public health on a global scale. Considering the discrepancies in metabolism between cancer and normal cells, metabolism-based anti-cancer biopharmaceuticals are gaining importance. Normal cells can synthesize arginine, but they can also take up extracellular arginine, making it a semi-essential amino acid. Arginine auxotrophy occurs when a cancer cell has abnormalities in the enzymes involved in arginine metabolism and relies primarily on extracellular arginine to support its biological functions. Taking advantage of arginine auxotrophy in cancer cells, arginine deprivation, which can be induced by introducing recombinant human arginase I (rhArg I), is being developed as a broad-spectrum anti-cancer therapy. This has led to the development of various rhArg I variants, which have shown remarkable anti-cancer activity. This article discusses the importance of arginine auxotrophy in cancer and different arginine-hydrolyzing enzymes that are in various stages of clinical development and reviews the need for a novel rhArg I that mitigates the limitations of the existing therapies. Further, we have also analyzed the necessity as well as the significance of using rhArg I to treat various arginine-auxotrophic cancers while considering the importance of their genetic profiles, particularly urea cycle enzymes.

Polyspecificity - An emerging trend in the development of clinical antibodies.

The essence of the growth and development of therapeutic conventional monoclonal antibodies (MAbs) for the treatment of various disorders is the aptitude of MAbs to precisely bind a target antigen and neutralise or promote its activity. However, the conventional antibodies are monoclonal i.e., both paratopes bind to the same epitope. But most of the pathophysiological conditions are multifaceted, hence targeting/blocking/inhibition of more than one epitope/antigen is more promising than one epitope/antigen. Polyspecific antibodies (PsAbs) have the potential to concurrently bind to more than one target and are the next-generation antibodies that augment efficacy in both clinical and non-clinical contexts. Thus, the trend of engineering and developing various formats of PsAbs is emerging. In this review, we have briefly discussed the importance of antibody polyspecificity and PsAbs approved for clinical use. Subsequently, we have discussed the role of TNF-α and IL-23 in inflammatory diseases and stressed the need for developing anti-TNF-α and anti-IL-23 bispecific antibodies.

Fused human paraoxonase 1 as a prophylactic agent against organophosphate poisoning.

Organophosphates (OPs) are highly neurotoxic compounds and certain OP-compounds are also exploited as a weapon of mass destruction and chemical warfare in terrorist attacks. Available prophylactic and post-exposure treatments are less effective and also have serious side-effects. Thus, there is a dire need to develop effective and safe prophylactic agent(s) against OP-poisoning. Human Paraoxonase 1 (hPON1) can hydrolyze a wide range of OP molecules and can be developed as an effective and safe prophylactic agent. Thus, there is a dire need in the art to develop variant(s) of rhPON1 that not only possess 'good' OP-hydrolyzing activity but also have improved pharmacokinetic properties. In this report, we describe the characterization of the fused hPON1 (FHP) variant that not only exhibit enhanced in vivo pharmacokinetic properties but also delay / prevent the symptoms of OP-poisoning and prevents OP-induced mortality in rats.

Immunomodulatory potential of apolipoproteins and their mimetic peptides in asthma: Current perspective.

Asthma prevailed as a common inflammatory disease affecting mainly the lower respiratory tract, with notable inflammation in the upper airways leading to significant morbidity and mortality. An extensive search for a new therapeutic target is continuously being carried out. Still, the majority have failed in the trials, and eventually, the drugs, including ß2-adrenergic agonists, muscarinic antagonists, and certain corticosteroids, remain the backbone for asthma control. Numerous endogenous factors aid in maintaining the normal homeostasis of the lungs and prevents disease progression. One among them is the apolipoproteins which are different sets of lipoprotein moieties that not only aid in the transport and metabolism of lipids but also impart immunomodulatory roles in various pathologies. Modern research joins the links between the immunomodulatory nature of apolipoproteins in chronic respiratory diseases like asthma and COPD, which can assist in ameliorating the disease progression. Recent studies have elucidated the protective roles of apoA-I and apoE in asthma. This has enabled the utilization of certain apolipoprotein-mimetic peptides to treat these severe pulmonary diseases in the long run. In this review, we have described the prominent and probable mechanistic roles of apolipoproteins like apoA-I, apoB, apoE, apoJ, and apoM in the pathogenesis and treatment of asthma along with the development of apoA-I and apoE-mimetics as a cardinal treatment strategy for eosinophilic as well as corticosteroid resistant neutrophilic asthma.

Apolipoprotein-mimetic Peptides: Current and Future Prospectives.

Apolipoprotein-mimetic peptides, mimicking the biological properties of apolipoproteins, have shown beneficial properties against various diseases (central and peripheral diseases) and have emerged as potential candidates for their treatments. Progress has been made from first-generation to second-generation apolipoprotein-mimetic peptides. Understanding these peptides from the first generation to the second generation is discussed in this review. First, we discussed the structural and therapeutic potentials of first-generation apolipoprotein-mimetic peptides. Further, we discussed the development of second-generation apolipoprotein-mimetic peptides, like dual-domain and bihelical peptides the emergence of second-generation apolipoprotein-mimetic peptides as potential candidates in different preclinical and clinical studies has also been emphasized.

Human arginase 1, a Jack of all trades?

Arginine, a conditionally essential amino acid, plays a crucial role in several metabolic and signalling pathways. Arginine metabolism in the body can be significantly increased under stress or during certain pathological conditions. Depletion of circulating arginine by administering arginine-hydrolysing enzyme has been shown to mitigate varied pathophysiological conditions ranging from cancer, inflammatory conditions, and microbial infection. This review provides an overview of such intriguing expanse of potential applications of recombinant human arginase 1 for different pathological conditions and its status of development.

Therapeutic potential of ApoE-mimetic peptides in CNS disorders: Current perspective.

The prevalence and burden of CNS disorders are increasing significantly due to the increase in life span and population. The contemporary need in CNS drug discovery is to develop the therapy that can halt the disease progression (disease-modifying therapy). While developing such CNS therapies, the major bottleneck is the blood-brain barrier (BBB) impermeability of drugs that influences the development of effective therapies to treat various CNS disorders. Since the influential innovation of insulin to treat diabetic patients in the 1920s, a lot of attention has been given for producing therapeutic proteins and peptides as remedies for several diseases, including neurological disorders. Recently, researchers have explored therapeutic potential of apolipoprotein E (ApoE)-mimetic peptides in the same context. ApoE is the major apolipoprotein produced in the brain by the astrocytes and plays a significant role in the formation of synapses, myelination, and neuronal proliferation. ApoE can be a potential candidate for treating CNS disorders. However, the large size of the ApoE leads to the BBB impermeability that restricts its use in native form. This problem can be overcome by developing small ApoE-mimetic peptides with good BBB permeability and similar biological function as native ApoE. Various ApoE-mimetic peptides have been developed and investigated in different CNS disorders. This review provide insights into the latest development of ApoE and its mimetic peptides in CNS disorders, along with their beneficial outcomes.

Optimization of medium composition to increase the expression of recombinant human interferon-ß using the Plackett-Burman and central composite design in E. coli SE1.

Recombinant human interferon-ß (rhIFN-ß) is therapeutically important and new commercially viable approaches are needed for its increased production. In this study, a codon-optimized gene encoding for rhIFN-ß(C17S) protein was designed and expressed in E. coli SE1. As a first step of medium optimization, growth of E. coli as a function of different media components was studied. Subsequently, to optimize the media composition, a response surface methodology (RSM) was used. Our results show that optimized medium (15.0 g/L tryptone, 12.3 g/L meat extract, 1.0 g/L MgSO4 and 0.5 g/L thiamine along with minimal medium) obtained in this study provide better growth of recombinant cells and the expression level of recombinant protein was ~ 1.7-fold more than Luria-Bertani medium. The optimized medium may be utilized for the large-scale production of rhIFN-ß.