Principles of the therapeutic use of monoclonal antibodies in oncology

Monoclonal antibodies have reached the stage of therapeutic agents, mostly in oncology, as illustrated by their wide use in lymphoma, breast cancer or colorectal cancer. The unravelling of their mechanisms of action and their interactions with their cellular receptors allows us to engineer new classes of therapeutic antibodies with increased efficacy. The identification of some of the tumour escape mechanisms may also help to define new approaches for patient selection and immunomonitoring. The present review addresses these various aspects.     

Coumarin hybrids as novel therapeutic agents

Naturally occurring coumarins, having wide spectrum of activities such as antioxidant, anti-inflammatory, anticancer, MAO-B inhibitory and antimicrobial, are frequently used by the researchers to develop novel synthetic and semisynthetic coumarin based therapeutic agents. Many of these agents are hybrid molecules, which are designed through concept of molecular hybridization and have shown multiple pharmacological activities. This multifunctional attribute of these hybrid compounds makes them potential drug candidates for the treatment of multifactorial diseases such as cancer, Alzheimer’s disease, metabolic syndromes, AIDS, malaria, and cardiovascular diseases. The present review compiles research reports on development of different coumarin hybrids, classify these on the basis of their therapeutic uses and propose structure–activity relationships. It is intended to help medicinal chemist in designing and synthesizing novel and potent hybrid compounds for the treatment of different disorders.     

Nanobodies as novel therapeutic agents in envenomation

Background

An effective therapy against envenoming should be a priority in view of the high number scorpion stings and snakebites. Serum therapy is still widely applied to treat the envenomation victims; however this approach suffers from several shortcomings. The employment of monoclonal antibodies might be an outcome as these molecules are at the core of a variety of applications from protein structure determination to cancer treatment. The progress of activities in the twilight zone between genetic and antibody engineering have led to the development of a unique class of antibody fragments. These molecules possess several benefits and lack many possible disadvantages over classical antibodies. Within recombinant antibody formats, nanobodies or single domain antigen binding fragments derived from heavy chain only antibodies in camelids occupy a privileged position.

Biologic protease inhibitors as novel therapeutic agents

Deregulated proteolytic activities frequently have causative or exacerbative functions in pathological conditions such as cancer and inflammatory disease. Many proteases therefore represent therapeutic targets, but the generation of successful small molecule drugs is often limited by the ability to achieve sufficient specificity of action. Consequently, several proteases have been deemed as unsuitable drug targets due to the inability to target them successfully. In an effort to circumvent these issues, much interest has recently focused on the development and application of biologic inhibitors. In this review, the latest research in the development of biologic protease inhibitors is examined. This includes a review of engineered kunitz and other inhibitory domains as well as the application of antibodies as therapeutically viable inhibitors.     

Anti-Ebola therapies based on monoclonal antibodies: current state and challenges ahead

The 2014 Ebola outbreak, the largest recorded, took us largely unprepared, with no available vaccine or specific treatment. In this context, the World Health Organization declared that the humanitarian use of experimental therapies against Ebola Virus (EBOV) is ethical. In particular, an experimental treatment consisting of a cocktail of three monoclonal antibodies (mAbs) produced in tobacco plants and specifically directed to the EBOV glycoprotein (GP) was tested in humans, apparently with good results. Several mAbs with high affinity to the GP have been described. This review discusses our current knowledge on this topic. Particular emphasis is devoted to those mAbs that have been assayed in animal models or humans as possible therapies against Ebola. Engineering aspects and challenges for the production of anti-Ebola mAbs are also briefly discussed; current platforms for the design and production of full-length mAbs are cumbersome and costly.     

Antibody mimetics: promising complementary agents to animal-sourced antibodies

Despite their wide use as therapeutic, diagnostic and detection agents, the limitations of polyclonal and monoclonal antibodies have inspired scientists to design the next generation biomedical agents, so-called antibody mimetics that offer many advantages over conventional antibodies. Antibody mimetics can be constructed by protein-directed evolution or fusion of complementarity-determining regions through intervening framework regions. Substantial progress in exploiting human, butterfly (Pieris brassicae) and bacterial systems to design and select mimetics using display technologies has been made in the past 10 years, and one of these mimetics [Kalbitor® (Dyax)] has made its way to market. Many challenges lie ahead to develop mimetics for various biomedical applications, especially those for which conventional antibodies are ineffective, and this review describes the current characteristics, construction and applications of antibody mimetics compared to animal-sourced antibodies. The possible limitations of mimetics and future perspectives are also discussed.     

A novel platform to produce human monoclonal antibodies: The next generation of therapeutic human monoclonal antibodies discovery

A new technology has been developed by immunologix that allows human antibodies to be quickly generated against virtually any antigen. Using a novel process, naïve human B cells are isolated from tonsil tissue and transformed with efficiency up to 85%, thus utilizing a large portion of the human VDJ/VJ repertoire. Through ex vivo stimulation, the B cells class switch and may undergo somatic hypermutation, thus producing a human “library” of different IgG antibodies that can then be screened against any antigen. Since diversity is generated ex vivo, sampling immunized or previously exposed individuals is not necessary. Cells producing the antibody of interest can be isolated through limiting dilution cloning and the human antibody from the cells can be tested for biological activity. No humanization is necessary because the antibodies are produced from human B cells. By eliminating immunization and humanization steps and screening a broadly diverse library, this platform should reduce both the cost and time involved in producing therapeutic monoclonal antibody candidates.Key words: human, antibody, monoclonal, novel platform, naïve, B cell, therapeutic     

Development of human neutralizing monoclonal antibodies for prevention and therapy of MERS-CoV infections

The recent Middle East respiratory syndrome coronavirus (MERS-CoV) outbreak poses a serious threat to public health. Here, we summarize recent advances in identifying human neutralizing monoclonal antibodies (mAbs) against MERS-CoV, describe their mechanisms of action, and analyze their potential for treatment of MERS-CoV infections.     

Preclinical pharmacokinetic characterization of an adipose tissue-targeting monoclonal antibody in obese and non-obese animals

Target receptor levels can influence pharmacokinetics (PK) or pharmacodynamics (PD) of monoclonal antibodies (mAbs), and can affect drug development of this class of molecules. We generated an effector-less humanized bispecific antibody that selectively activates fibroblast growth factor receptor (FGFR)1 and βKlotho receptor, a FGF21 receptor complex highly expressed in both white and brown adipocytes. The molecule shows cross-species binding with comparable equilibrium binding affinity (Kd) for human, cynomolgus monkey, and mouse FGFR1/βKlotho. To understand the PK/PD relationship in non-obese and obese animals, we evaluated the adipose tissue distribution of the antibody, serum exposures, and an associated PD marker (high-molecular-weight adiponectin), in both non-obese and obese mice and monkeys. Antibody uptake into fat tissue was found to be higher on a per gram basis in non-obese animals compared to obese animals. Since obesity has been reported to be associated with reduced expression of FGFR1 and βKlotho receptor in white adipose tissues in mice, our results suggest that the distribution in adipose tissues was influenced by target expression levels. Even so, the overall dose-normalized serum exposures were comparable between non-obese and obese mice and monkeys, suggesting that adipose tissue uptake plays a limited role in overall systemic PK determination. It remains to be determined if and how obesity and receptor expression in humans influence the PK and PD profile of this novel therapeutic candidate.     

Novel monoclonal antibody-based therapies: implications for the treatment and prevention of HCMV disease

Human cytomegalovirus (HCMV) is an important pathogen worldwide. Although HCMV infection is often asymptomatic in immunocompetent individuals, it can cause severe or even life-threatening symptoms in immunocompromised patients. Due to limitations of antiviral treatments, it is necessary to search for new therapeutic alternatives. Recent studies have highlighted the contribution of antibodies in protecting against HCMV disease, including neutralizing and non-neutralizing antibodies. Given the immunocompromised target population, monoclonal antibodies (mAbs) may represent an alternative to the clinical management of HCMV infection. In this context, we provide a synthesis of recent data revising the literature supporting and arguing about the role of the humoral immunity in controlling HCMV infection. Additionally, we review the state of the art in the development of therapies based on mAbs.     

Discovery of novel serine palmitoyltransferase inhibitors as cancer therapeutic agents

We pursued serine palmitoyltransferase (SPT) inhibitors as novel cancer therapeutic agents based on a correlation between SPT inhibition and growth suppression of cancer cells. High-throughput screening and medicinal chemistry efforts led to the identification of structurally diverse SPT inhibitors 4 and 5. Both compounds potently inhibited SPT enzyme and decreased intracellular ceramide content. In addition, they suppressed cell growth of human lung adenocarcinoma HCC4006 and acute promyelocytic leukemia PL-21, and displayed good pharmacokinetic profiles. Reduction of 3-ketodihydrosphingosine, the direct downstream product of SPT, was confirmed under in vivo settings after oral administration of compounds 4 and 5. Their anti-tumor efficacy was observed in a PL-21 xenograft mouse model. These results suggested that SPT inhibitors might have potential to be effective cancer therapeutics.     

Challenges and opportunities for the future of monoclonal antibody development: Improving safety assessment and reducing animal use

The market for biotherapeutic monoclonal antibodies (mAbs) is large and is growing rapidly. However, attrition poses a significant challenge for the development of mAbs, and for biopharmaceuticals in general, with large associated costs in resource and animal use. Termination of candidate mAbs may occur due to poor translation from preclinical models to human safety. It is critical that the industry addresses this problem to maintain productivity. Though attrition poses a significant challenge for pharmaceuticals in general, there are specific challenges related to the development of antibody-based products. Due to species specificity, non-human primates (NHP) are frequently the only pharmacologically relevant species for nonclinical safety and toxicology testing for the majority of antibody-based products, and therefore, as more mAbs are developed, increased NHP use is anticipated. The integration of new and emerging in vitro and in silico technologies, e.g., cell- and tissue-based approaches, systems pharmacology and modeling, have the potential to improve the human safety prediction and the therapeutic mAb development process, while reducing and refining animal use simultaneously. In 2014, to engage in open discussion about the challenges and opportunities for the future of mAb development, a workshop was held with over 60 regulators and experts in drug development, mechanistic toxicology and emerging technologies to discuss this issue. The workshop used industry case-studies to discuss the value of the in vivo studies and identify opportunities for in vitro technologies in human safety assessment. From these and continuing discussions it is clear that there are opportunities to improve safety assessment in mAb development using non-animal technologies, potentially reducing future attrition, and there is a shared desire to reduce animal use through minimised study design and reduced numbers of studies.     

Design of novel iron compounds as potential therapeutic agents against tuberculosis

In the search for new therapeutic tools against tuberculosis two novel iron complexes, [Fe(L-H)₃], with 3-aminoquinoxaline-2-carbonitrile N¹,N⁴-dioxide derivatives (L) as ligands, were synthesized, characterized by a combination of techniques, and in vitro evaluated. Results were compared with those previously reported for two analogous iron complexes of other ligands of the same family of quinoxaline derivatives. In addition, the complexes were studied by cyclic voltammetry and EPR spectroscopy. Cyclic voltammograms of the iron compounds showed several cathodic processes which were attributed to the reduction of the metal center (Fe(III)/Fe(II)) and the coordinated ligand. EPR signals were characteristic of magnetically isolated high-spin Fe(III) in a rhombic environment and arise from transitions between m_S = ± 1/2 (g_eff ~ 9) or m_S = ± 3/2 (g_eff ~ 4.3) states. Mössbauer experiments showed hyperfine parameters that are typical of high-spin Fe(III) ions in a not too distorted environment. The novel complexes showed in vitro growth inhibitory activity on Mycobacterium tuberculosis H₃₇Rv (ATCC 27294), together with very low unspecific cytotoxicity on eukaryotic cells (cultured murine cell line J774). Both complexes showed higher inhibitory effects on M. tuberculosis than the “second-line” therapeutic drugs.     

SPION-Smac mimetic nano-conjugates: Putative pro-apoptotic agents in oncology

Non-covalent (NP-1/3) and covalent (NP-A-1/3) pro-apoptotic SPION-Smac mimetic nano-conjugates antitumor agents are reported. The solution synthesis of key Smac mimetics, their support onto SPIONs through non-covalent adsorption (NP-1/3) or APTES-mediated covalent binding (NP-A-1/3), the analytical characterization of SPION-Smac mimetic conjugates, their target affinity in cell-free assays, and their cytotoxicity against tumor cells are thoroughly described.     

The structure and function of novel proteins of Bacillus anthracis and other spore-forming bacteria: development of novel prophylactic and therapeutic agents

The overall goal of this review is to summarize the current body of knowledge about the structure and function of major proteins of Bacillus anthracis and/or similar spore-forming organisms. B. anthracis is a key spore-forming biological threat agent, as well as human and animal Gram-positive bacterial pathogen. The structural information described here is limited to approximately the last 5 years. This information is then related to the role of the selected proteins in pathogenesis and in the possible development of novel vaccine and/or other antimicrobial agents against spore-forming organisms, including anthrax, a disease caused by B. anthracis. Among spore-forming bacteria, Bacillus and Clostridium species are the predominant spore-forming bacilli that cause serious diseases. The biochemical properties and mechanism of catalysis of the novel spore germination protease that degrades small, acid-soluble proteins protecting DNA against damage, a cofactor independent phosphoglycerate mutase, NAD+ synthetase, and the three know B. anthracis toxins, protective antigen, lethal factor, and edema factor are described. The studies described in this work review and unify selected information critical for the prevention of microbial diseases such as anthrax. A strategy for the structure-guided development of new prophylactic and therapeutic agents is discussed.     

Cyclic nucleotide phosphodiesterase (PDE) inhibitors: novel therapeutic agents for progressive renal disease

Cyclic nucleotides are recognized as critical mediators of many renal functions, including solute transport, regulation of vascular tone, proliferation of parenchymal cells, and inflammation. Although most studies have linked elevated cAMP levels to activation of protein kinase A, cAMP can also directly activate cyclic nucleotide gated ion channels and can signal through activation of GTP exchange factors. Cyclic AMP signaling is highly compartmentalized through plasma membrane localization of adenylyl cyclase and expression of scaffolding proteins that anchor protein kinase A to specific intracellular locations. Cyclic nucleotide levels are largely regulated through catabolic processes directed by phosphodiesterases (PDEs). The PDE superfamily is large and complex, with over 60 distinct isoforms that preferentially hydrolyze cAMP, cGMP, or both. PDEs contribute to compartmentalized cyclic nucleotide signaling. The unique cell- and tissue-specific distribution of PDEs has prompted the development of highly specific PDE inhibitors to treat a variety of inflammatory conditions. In experimental systems, PDE inhibitors have been employed to demonstrate functional compartmentalization of cyclic nucleotide signaling in the kidney. For example, mitogenesis in glomerular mesangial cells and normal tubular epithelial cells is negatively regulated by an intracellular pool of cAMP that is metabolized by PDE3, but not by other PDEs. In Madin-Darby canine kidney cells, an in vitro model of polycystic kidney disease, an intracellular pool of cAMP directed by PDE3 stimulates mitogenesis. In mesangial cells, an intracellular pool of cAMP directed by PDE4 inhibits reactive oxygen species and expression of the potent proin-flammatory cytokine monocyte chemoattractant protein 1. An intracellular pool of cGMP directed by PDE5 regulates solute transport. PDE5 inhibitors ameliorate renal injury in a chronic renal disease model. In this overview, we highlight recent studies to define relationships between PDE expression and renal function and to provide evidence that PDE inhibitors may be effective agents in treating chronic renal disease.     

Unraveling what makes a monoclonal antibody difficult-to-express: From intracellular accumulation to incomplete folding and degradation via ERAD

Although most therapeutic monoclonal antibodies (mAbs) can routinely be produced in the multigram per litre range, some mAb candidates turn out to be difficult-to-express (DTE). In addition, the class of more complex biological formats is permanently increasing and mammalian expression systems like Chinese hamster ovary (CHO) cell lines can show low performance. Hence, there is an urgent need to identify any rate limiting processing step during cellular synthesis. Therefore, we assessed the intracellular location of the DTE antibody mAb2 by fluorescence and electron microscopy (EM) and revealed an accumulation of the antibody, which led to an aberrant morphology of the endoplasmic reticulum (ER). Analysis of underlying cellular mechanisms revealed that neither aggregation nor antibody assembly, but folding represented the reason for hampered secretion. We identified that the disulfide bridge formation within the antibody light chain (LC) was impaired due to less recognition by protein disulfide isomerase (PDI). As a consequence, the DTE molecule was degraded intracellularly by the ubiquitin proteasome system via ER-associated degradation (ERAD). This study revealed that with the continuous emergence of DTE therapeutic protein candidates, special attention needs to be drawn to optimization processes to ensure manufacturability.     

Breast cancer-derived exosomes: Tumor progression and therapeutic agents

Tumor cells secrete extracellular vesicles (EVs) for intercellular communication. EVs by transporting different proteins, nucleic acids, and lipids contribute to affect target cell function and fate. ‎EVs which originate directly from multivesicular bodies so-called exosomes have dramatically fascinated the attention of researchers owing to their ‎pivotal roles in the tumorigenesis. Breast cancer, arising from milk-producing cells, is the most identified cancer among women and has become the leading cause of cancer-related death in women globally. Although different therapies are applied to eliminate breast tumor cells, however, the efficient therapy and survival rate of patients remain challenges. Growing evidence ‎shows exosomes from breast cancer cells contribute to proliferation, metastasis, angiogenesis, chemoresistance, and also radioresistance and, thus carcinogenesis. Additionally, these exosomes may serve as a cancer treatment tool because they are a good candidate for cancer diagnosis (as biomarker) and therapy (as drug-carrier). Despite recent development in the biology of tumor-derived exosomes, the detailed mechanism of tumorigenesis, and exosome-based cancer-therapy remain still indefinable. Here, we discuss the key function of breast cancer-derived exosomes in tumorgenesis and shed light on the possible clinical application of these exosomes in breast cancer treatment.