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Developing a fully human monoclonal antibody (mAb) using (a) phage display technology and (b) transgenic mouse technology. (a) Step 1: A suitable source of starting material (for example, human blood) is subjected to polymerase chain reaction using appropriate primers, providing 'libraries' of heavy chain V domain (VH) and light chain V domain (VL) sequences. Step 2: Randomly combined VH and VL sequences, connected via a short linker, are incorporated into the genome of a bacteriophage such that they will be expressed at the phage surface. The combination marked with an asterisk encodes the desired specificity. Step 3: The phage library is used to infect a bacterial culture, and the resulting supernatant, containing single-chain Fv-expressing phage particles, is incubated with an appropriate source of target antigen (panning). This can be on a column, Petri dish, and so on. Phage with appropriate specificity adheres to the antigen source. Step 4: Adherent phage is eluted and enriched for the appropriate specificity by further rounds of panning. Step 5: After several rounds of panning, adherent phage is sequenced. A successful procedure should lead to the presence of just one or a few Fv specificities, which can be individually cloned and their specificity checked. At this stage, in vitro affinity maturation procedures can be performed if required (see 'Human antibodies' section for details). Ultimately, the desired specificity is recloned into an appropriate vector containing full-length mAb sequence for expression in a mammalian cell line. (b) Step 1: A transgenic mouse that produces human antibodies is created by targeted disruption of the endogenous murine immunoglobulin heavy- and light-chain genetic loci and their replacement by the equivalent human sequences. Step 2: The mouse, now containing human immunoglobulin genes, is immunised in a conventional manner using the target antigen. Step 3: Splenocytes from the immunised mouse are used to generate hybridomas via conventional fusion technology. Step 4: Resulting hybridomas are screened, leading to isolation and cloning of a hybridoma-secreting high-affinity mAb against the target antigen. Note: In theory, phage display rather than fusion technology can be applied from stage 3 onwards.
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There has been a therapeutic revolution in rheumatology over the past 15 years, characterised by a move away from oral immuno-suppressive drugs toward parenteral targeted biological therapies. The potency and relative safety of the newer agents has facilitated a more aggressive approach to treatment, with many more patients achieving disease remiss...
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... phage could be washed away, leaving bound phage, a proportion of which was specific for the target antigen. Bound phage then could be eluted and further enriched by infecting a second bacterial culture and repeat- ing the panning process a number of times (Figure 4a). Once an Fv of appropriate specificity and affinity was identified, it could be recloned into a vector containing appropriate C domains for further drug development. ...
Citations
... On the other hand, IgG 1 portion endows prolonged circulating half-life of abatacept and the ability for the purification by protein A chromatography. (Isaacs et al., 2009;Ramos-de-la-Peña et al., 2019). Abatacept selectively modulates a key co-stimulatory signal required for full activation of T lymphocytes expressing CD28. ...
... Generally, the heavy chains comprise four different domains, while the light chains consist of two. The first domain in each chain is the variable domain (V), namely VH/VL, and the rest of the heavy chain contains three constant (C) domains (CH1-CH3), while the light chains comprise one constant domain (CL) each [81]. A flexible peptide segment between CH1 and CH2 domains is called 'hinge'. ...
With the onset of the COVID-19 pandemic, the amount of data on genomic and proteomic sequences of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) stored in various databases has exponentially grown. A large volume of these data has led to the production of equally immense sets of immunological data, which require rigorous computational approaches to sort through and make sense of. Immunoinformatics has emerged in the recent decades as a field capable of offering this approach by bridging experimental and theoretical immunology with state-of-the-art computational tools. Here, we discuss how immunoinformatics can assist in the development of high-performance vaccines and drug discovery needed to curb the spread of SARS-CoV-2. Immunoinformatics can provide a set of computational tools to extract meaningful connections from the large sets of COVID-19 patient data, which can be implemented in the design of effective vaccines. With this in mind, we represent a pipeline to identify the role of immunoinformatics in COVID-19 treatment and vaccine development. In this process, a number of free databases of protein sequences, structures and mutations are introduced, along with docking web servers for assessing the interaction between antibodies and the SARS-CoV-2 spike protein segments as most commonly considered antigens in vaccine design.
... Rituximab is a genetically engineered chimeric antibody and contains murine light-and heavy-chain variable region sequences and human constant region sequences. Ocrelizumab is humanized through 'CDR grafting', which involves transferring murine complementarity-determining regions (CDR) to the framework regions of a human antibody [44]. Ofatumumab is a fully human antibody. ...
Introduction: Multiple sclerosis (MS) is a chronic inflammatory disease of the central nervous system. The latest development of B-cell depletion by anti-CD20 monoclonal antibodies has been a large step forward in the treatment of this devastating disease.
Areas covered: In this manuscript, we review mechanisms of action, efficacy, safety, and tolerance of anti-CD20 therapies for MS, including rituximab, ocrelizumab, and ofatumumab.
Expert opinion: B-cell depletion efficiently suppresses acute inflammatory disease activity in relapsing-remitting MS (RRMS), and may slowdown progression in primary progressive MS (PPMS). The treatment is generally well tolerated, with manageable adverse events related to infusion reactions and infections. Ocrelizumab, a humanized anti-CD20 monoclonal antibody, is the first therapy to be approved for the treatment of both RRMS and PPMS.
... 12 Although the antibody's high affinity to a particular target leads to a very selective binding, the induction of immune response against humanized antibodies (HAHAs) after treatment by human monoclonal antibodies restricts their application in cancer therapeutics. 13,14 VHHs (variable fragments of heavy chain antibodies) are the smallest intact antigen binding fragments from heavy-chain-only antibodies. These single chain antibodies naturally occur in Camelidae. ...
To combine the targeted gene delivery and cell-specific transcriptional targeting, a delivery system was designed based on Generation 5 polyamidoamine dendrimer (PAMAM), polyethylene glycol, and anti-HER2 (human epidermal growth factor receptor 2) variable domains of camelid heavy chain antibodies (single domain antibody, VHHs). Anti-HER2 VHHs were covalently attached to the distal ends of polyethylene glycols in PAMAM-polyethylene glycol structure. The prepared immunotargeted nanobiopolymer was then used to condense gene construct, encoding a transcriptionally targeted truncated-Bid killer gene under the control of the breast cancer–specific MUC1 (transmembrane glycoprotein mucin 1) promoter. Transfection and transcription efficiencies of the engineered dendriplexes were quantified by truncated-Bid gene expression measurements, using real-time polymerase chain reaction. The engineered dendriplexes exhibited desirable physicochemical characteristics evaluated by dynamic light scattering and agarose gel retardation assay. Flow cytometry results showed a significantly higher uptake of VHH-modified dendriplexes than untargeted particles in HER2-positive cell lines. In vitro gene expression studies using PAMAM-polyethylene glycol-VHH in complex with a luciferase expressing plasmid resulted in 1.6- and 4.8-fold higher transfection efficiencies than intact PAMAM dendrimers in BT-474 and SK-BR-3 (both as HER2-positive cell lines), respectively. Transfection by PAMAM-polyethylene glycol-VHH/pMUC1-truncated-Bid also increased the level of truncated-Bid gene expression at 2.2- and 3.6-folds in BT-474 and SK-BR-3 cell lines, respectively, compared to the untargeted PAMAM treatment. A remarkable cell death was also observed in case of HER2-positive cells transfected by the targeted dendriplexes, indicating not only an effective targeted delivery but also a better transcriptional targeting efficiency of truncated-Bid killer gene, when expressed under the control of MUC1 promoter.
... Przełączanie klas i wzrost siły wiązania (affinity) wytwarzanych przeciwciał następuje również w miarę upływu czasu i kolejnych immunizacji. Stosując powszechnie przyjęte schematy immunizacji najczęściej uzyskuje się hybrydomy wytwarzające przeciwciała klasy IgG [29,38]. Klasa uzyskiwanych przeciwciał zależy również od natury antygenu; antygeny typu II (grasiczozależne) stymulują przede wszystkim powstawanie przeciwciał klasy IgG, a także IgA czy IgE, natomiast antygeny typu I (grasiczoniezależne) przeciwciał klasy IgM. ...
Monoclonal antibodies (mAbs) are biomolecules of great scientific and practical significance. In contrast to polyclonal antibodies from immune sera, they are homogeneous and monospecific, since they are produced by hybridoma cells representing a clone arising from a single cell. The successful technology was described for the first time in 1975; the inventors were later awarded the Nobel Prize. Currently, mAbs are broadly used as a research tool, in diagnostics and medicine in particular for the treatment of cancer or in transplantology. About 47 therapeutics based on monoclonal antibodies are now available in the US and Europe, and the number is still growing. Production of monoclonal antibodies is a multistage, time-consuming and costly process. Growing demand for these molecules creates space for research focused on improvements in hybridoma technology. Lower costs, human labor, and time are important goals of these attempts. In this article, a brief review of current methods and their advances is given.
... The safety and efficacy of tocilizumab has been assessed in many clinical trials in patients with RA, SLE, juvenile idiopathic arthritis, and Castleman's disease [8][9][10] . However, therapeutic mAbs are usually produced from nonhuman cell lines, and consequently, the exogenous biomacromolecules theoretically can invoke anti-globulin responses in some patients [11,12] . Therefore, it may be a reasonable strategy to develop small molecule drugs against IL-6R in the patients with anti-globulin responses to therapeutic mAbs. ...
Aim:Blockade of interleukin-6 (IL-6) or its receptor (IL-6R) is effective in preventing the progression of autoimmune diseases, such as systemic lupus erythematosus and rheumatoid arthritis. In the present study, we established a novel cell-based assay for identifying small molecule IL-6R antagonists.
Methods:HEK293A cells were transfected with recombinant plasmids pTaglite-SNAP-IL6R and pABhFc-IL6 to obtain membrane-bound IL-6R and recombinant human IL-6 coupled with human Fc fragment (rhIL-6), respectively. A novel screening assay based on the interaction between IL-6R and rhIL-6 was established, optimized and validated. The stability of the assay was also assessed by calculating the Z′-factor.
Results:RhIL-6 dose-dependently bound to IL-6R expressed at HEK293A cell surface. The IC50 value of the known antagonist ab47215 was 0.38±0.08 μg/mL, which was consistent with that obtained using the traditional method (0.36±0.14 μg/mL). The value of Z′-factor was 0.68, suggesting that the novel assay was stable for high throughput screening. A total of 474 compounds were screened using the novel screening assay, and 3 compounds exhibited antagonistic activities (IC50=8.73±0.28, 32.32±9.08, 57.83±4.24 μg/mL). Furthermore, the active compounds dose-dependently inhibited IL-6-induced proliferation of 7TD1 cells, and reduced IL-6-induced STAT3 phosphorylation in U937 cells.
Conclusion:
A novel cell-based screening assay for identifying small molecule IL-6R antagonists was established, which simplifies the procedures in traditional cellular ELISA screening and profiling and reduces the costs.
... This approach could have advantages over existing anti-IL-6 therapies; for example, monoclonal antibodies against the IL-6R, e.g. tocilizumab [40], are in regular clinical use; however, humanized antibodies are expensive to produce and store, have a limited shelf life and require regular injections for effective treatment. Current therapeutic small-molecule JAK/STAT3 inhibitors, such as tasocitinib [41], are cheap, easy to produce and can be taken orally, but effectively suppress SOCS3 activity [42] (Figure 3c). ...
The atherogenic cytokine interleukin-6 (IL6) induces pro-inflammatory gene expression in vascular endothelial cells (VECs) by activating the JAK/STAT3 signalling pathway, which is normally down-regulated by the STAT3-dependent induction of the E3 ubiquitin ligase component, SOCS3. Novel treatments based on the regulation of SOCS3 protein levels could therefore have value in the treatment of diseases with an inflammatory component, like atherosclerosis. To this end we carried out a screen of 1031 existing medicinal compounds to identify inducers of SOCS3 gene expression and identified the flavanoids, naringenin and flavone, as effective inducers of SOCS3 protein, mRNA and promoter activity. This was in contrast to the action of traditional JAK/STAT3 inhibitors, and the polyphenol resveratrol, which effectively suppress SOCS3 gene expression. Both naringenin and flavone also effectively suppressed IL6-stimulated phosphorylation of STAT3 (Tyr 705) which led to suppression of IL6-induction of the atherogenic, STAT3 target gene MCP1, suggesting that their ability to induce SOCS3 gene expression is STAT3 independent. Supporting this idea was the observation that the general kinase inhibitor, compound C, inhibits flavone- and cyclic AMP-dependent, but not JAK-dependent, SOCS3 induction in VECs. Indeed, the ability of flavanoids to induce SOCS3 expression requires activation of the ERK-dependent transcription factor, SP3, and not STAT3. We therefore describe novel molecular actions of flavanoids, which control SOCS3 gene induction and suppression of STAT3 signalling in VECs. These mechanisms could potentially be exploited to develop novel anti-atherogenic therapies.
... mAbs bind to their targets with high specificity, and therefore have excellent potential as therapeutic agents. Biotechnological advances have allowed the production of large quantities of engineered mAbs for therapeutic use [14]. Recent research in RA has identified important mediators of synovitis. ...
Rheumatoid arthritis (RA) is a polyarticular inflammatory, angiogenic disease. Synovial angiogenesis contributes to inflammation in RA. In this study we have developed an arthritic model in rats using a novel angiogenic protein (NAP), isolated from human synovial fluid of RA patients. We produced anti-NAP monoclonal antibodies (mAbs) and investigated the therapeutic efficacy of the same in adjuvant-induced or NAP-induced arthritis as a model of human RA. The treatment of arthritic rats with anti-NAP mAbs resulted in effective amelioration of paw oedema, radiological arthritic characteristics, serum levels of vascular endothelial growth factor (VEGF) and NAP, compared to that of untreated arthritic animals. Further, profiling of angiogenic markers such as synovial microvessel density, angiogenesis, CD31, VEGF and fms-like tyrosine kinase (Flt1) by immunohistochemistry both in arthritic and anti-NAP mAb-treated animals revealed the efficacy of mAb as an anti-angiogenic functional antibody. Therefore, NAP may be an attractive target to design anti-angiogenic and anti-arthritic therapies to control the pathogenesis of arthritis.
... In addition, there are small molecules that specifically inhibit intracellular, cell-cell and cell-matrix interactions, fundamental to the inflammatory and immune process. 2,3 The majority of biologics bind to soluble cytokines or impede their binding to specific receptors. 4,5 A significant target in JIA and other rheumatic conditions is the proinflammatory cytokine tumor necrosis factor alpha (TNF-α). ...
Treatment of children and adolescents with juvenile idiopathic arthritis and other pediatric rheumatic diseases has evolved. Where once there was only a limited arsenal of medications, with significant side effects and inadequate efficacy, today, with an increased understanding of the pathogenesis of these diseases, there is a wider variety of more targeted and effective treatments. TNF-α is a cytokine involved in a number of inflammatory pathways in pediatric rheumatic diseases. The emergence of biologic modifiers that target TNF-α has been pivotal in providing the ability to deliver early and aggressive treatment. Adalimumab, a recombinant monoclonal antibody to TNF-α, is an important therapeutic option, which affords children and adolescents with chronic illnesses an improved quality of life.
... Infliximab is a chimeric human/murine monoclonal antibody which possesses a human IgG1 C region and murine V regions effective in binding TNFa [Haines, 2007]. Owing to the murine fragment, infliximab retains immunogenicity and can cause anaphylaxis, lack of efficacy, and infusion reactions [Isaacs, 2009]. Infliximab is given by intravenous infusion; with frequency ranging from every 2-8 weeks, at a dose of 3-6 mg/kg [Ruperto et al. 2010[Ruperto et al. , 2007. ...
THE GOALS OF TREATMENT FOR JUVENILE IDIOPATHIC ARTHRITIS (JIA) INCLUDE: suppression of inflammation, achievement of remission, relief of pain, maintenance of function and doing so with minimal toxicity. Important discoveries over the past 10-15 years have led to more targeted treatments for children with JIA. The International League of Associations for Rheumatology (ILAR) classification system for childhood arthritides, better assessment tools for clinical response, improved definitions of remission, new imaging techniques and evidence in gene expression profiling have all contributed to the development of more targeted treatments. Nonsteroidal anti-inflammatory agents still have a role in mild disease and intra-articular steroid injections continue to be used most commonly in patients with oligoarticular JIA. Disease-modifying agents such as methotrexate have demonstrated efficacy and safety; however, in many patients, the disease remains active despite this treatment. These children now receive more targeted treatment including the tumor necrosis factor alpha (TNFα) inhibitors, interleukin-1 blockade, interleukin-6 blockade, selective costimulation modulators and selective B-cell blockade. The biologic targeted therapies have changed the strategy in which we treat our children with JIA; however, there remains much to be learned about the long-term effects and safety of these medicines.
