Recombinant Antibody Technology Research Articles

Physicochemical Stimulus-Responsive Systems Targeted with Antibody Derivatives.

The application of monoclonal antibodies and antibody fragments with the advent of recombinant antibody technology has made notable progress in clinical trials to provide a regulated drug release and extra targeting to the special conditions in the function site. Modification of antibodies has facilitated using mAbs and antibody fragments in numerous models of therapeutic and detection utilizations, such as stimuli-responsive systems. Antibodies and antibody derivatives conjugated with diverse stimuli-responsive materials have been constructed for drug delivery in response to a wide range of endogenous (electric, magnetic, light, radiation, ultrasound) and exogenous (temperature, pH, redox potential, enzymes) stimuli. In this report, we highlighted the recent progress on antibody-conjugated stimuli-responsive and dual/multi-responsive systems that affect modern medicine by improving a multitude of diagnostic and treatment strategies.

RAbDesFlow: a novel workflow for computational recombinant antibody design for healthcare engineering.

Recombinant antibodies (rAbs) have emerged as a promising solution to tackle antigen specificity, enhancement of immunogenic potential and versatile functionalization to treat human diseases. The development of single chain variable fragments has helped accelerate treatment in cancers and viral infections, due to their favorable pharmacokinetics and human compatibility. However, designing rAbs is traditionally viewed as a genetic engineering problem, with phage display and cell free systems playing a major role in sequence selection for gene synthesis. The process of antibody engineering involves complex and time-consuming laboratory techniques, which demand substantial resources and expertise. The success rate of obtaining desired antibody candidates through experimental approaches can be modest, necessitating iterative cycles of selection and optimization. With ongoing advancements in technology, in silico design of diverse antibody libraries, screening and identification of potential candidates for in vitro validation can be accelerated. To meet this need, we have developed rAbDesFlow, a unified computational workflow for recombinant antibody engineering with open-source programs and tools for ease of implementation. The workflow encompasses five computational modules to perform antigen selection, antibody library generation, antigen and antibody structure modeling, antigen-antibody interaction modeling, structure analysis, and consensus ranking of potential antibody sequences for synthesis and experimental validation. The proposed workflow has been demonstrated through design of rAbs for the ovarian cancer antigen Mucin-16 (CA-125). This approach can serve as a blueprint for designing similar engineered molecules targeting other biomarkers, allowing for a simplified adaptation to different cancer types or disease-specific antigens.

The Potential of Single-Chain Variable Fragment Antibody: Role in Future Therapeutic and Diagnostic Biologics.

The advancement of genetic engineering has revolutionized the field of immunology by allowing the utilization of intrinsic antibody structures. One of the biologics that are being produced by recombinant antibody technology is single-chain fragments variable (scFv). Genes of variable regions, the heavy and light chains that are genetically linked into a single transcript by a short flexible linker peptide, are used to generate this fragment from cellular and synthetic libraries. The specificity and affinity of these molecules are comparable to those of parental antibodies. Fusion with marker proteins and other potent molecules improves their stability, circulation half-life, activity, and efficient purification. Besides, this review comprises construction protocols, therapeutics, and diagnostic applications of scFv, as well as related challenges. Nonetheless, there are still issues with efficacy, stability, safety, intracellular administration, and production costs that need to be addressed.

Using novel diagnostic antibodies to identify lymphocyte markers of rheumatic fever and heart disease.

Abstract Acute rheumatic fever (ARF) is an autoimmune disease that occurs in a subset of patients as a delayed complication of an improperly treated throat infection with a rheumatogenic strain of Streptococcus pyogenes. ARF / Rheumatic heart disease is a leading cause of preventable cardiovascular morbidity and mortality in children worldwide, affecting >33 million people. A method of detecting children susceptible to ARF (3–5% of the total population) would have major global health and economic benefits. One potential ARF diagnostic is the IgM antibody, D8/17, which binds B cells from ARF patients of diverse ethnic groups and geographical locations at a higher % versus matched controls. While this antibody has had mixed results in the past, no group has identified the antigen that D8/17 binds. To aid in this endeavor, we used recombinant antibody engineering to produce new IgM and IgG1 versions of D8/17 that give more consistent results in our in vitrodiagnostic tests. These new derivatives were then utilized in a multi-omics approach to characterize the differences between immortalized B cell lines of ARF patients and controls. Western blot and MS/MS proteomic analyses of lymphocyte lysates identified cytoskeletal proteins from ARF B cells that cross-react more readily with our recombinant D8/17. RNA sequencing and microarray analysis of B cells from ARF patients versus controls confirmed our proteomic results. Further, we discovered differences in the gene expression of other cell surface proteins, kinases and signaling pathways in ARF B cells that bind these antibodies. We hope our analyses will help identify markers or genetic factors related to ARF development and pathogenesis, or aid in designing diagnostic assays for ARF susceptibility. Supported by a grant from the NIH /NIAD (SC2 1SC2AI134947-01)

Rapid generation of high-quality recombinant antibodies using an Expi293F expression system for a 17 β-estradiol immunoassay

The rapid generation of high-quality target antibodies is essential for research employing immunoassays. The use of recombinant antibody technology that relies on genetic engineering is one such means to produce high-quality antibodies. Obtaining the gene sequence information of immunoglobulin is a prerequisite for the preparation of genetically engineered antibodies. At present, many researchers have shared their amino acid sequence data for various high-performance antibodies and their related properties. In this study, we obtained the protein sequence of a variable region of a 17 β-estradiol (E2) antibody from the Protein Data Bank (PDB) and subsequently constructed heavy (H) and light (L) chain expression vectors through codon optimization. The transient expression, purification, and performance identification of the immunoglobulin G (IgG), antigen-binding fragment (Fab), and single-chain variable fragment (scFv) antibodies were carried out, respectively. The effects of the different expression vectors on the expression yield of the IgG antibody were further compared. Among them, the expression yield based on the pTT5 vector was the highest, reaching 27 mg/L. Based on the expressed IgG and Fab antibodies, an indirect competitive enzyme-linked immunosorbent assay (ic-ELISA) standard curve of E2 was constructed, and the half-maximal inhibitory concentrations (IC50) for these two antibodies were determined to be 0.129 ng/mL and 0.188 ng/mL, respectively. In addition, an immunochromatographic assay (ICA) based on the IgG antibody was constructed with an IC50 of 3.7 ng/mL. Therefore, in featuring the advantages of simplicity, high efficiency, rapid obtainment, and high titer yield, we propose the system for the rapid generation of high-quality recombinant antibodies by reusing the published antibody information and show that it has good implementation prospects in improving upon existing immunoassay techniques.

Therapeutic antibodies for precise cancer immunotherapy: current and future perspectives.

Antibodies, as one of the most important components of host adaptive immune system, play an important role in defense of infectious disease, immune surveillance, and autoimmune disease. Due to the development of recombinant antibody technology, antibody therapeutics become the largest and rapidly expanding drug to provide major health benefits to patients, especially for the treatment of cancer patients. Many antibody-based therapeutic strategies have been developed including monoclonal antibodies, antibody-drug conjugates, bispecific and trispecific antibodies and pro-antibodies with promising results from both clinical and pre-clinical trials. However, the response rate and side-effect still vary between patients with undefined mechanisms. Here, we summarized the current and future perspectives of antibody-based cancer immunotherapeutic strategies for designing next-generation drugs.

Rapid-format recombinant antibody-based methods for the diagnosis of Clostridioides difficile infection: Recent advances and perspectives.

Clostridioides difficile, the most common cause of nosocomial diarrhea, has been continuously reported as a worldwide problem in healthcare settings. Additionally, the emergence of hypervirulent strains of C. difficile has always been a critical concern and led to continuous efforts to develop more accurate diagnostic methods for detection of this recalcitrant pathogen. Currently, the diagnosis of C. difficile infection (CDI) is based on clinical manifestations and laboratory tests for detecting the bacterium and/or its toxins, which exhibit varied sensitivity and specificity. In this regard, development of rapid diagnostic techniques based on antibodies has demonstrated promising results in both research and clinical environments. Recently, application of recombinant antibody (rAb) technologies like phage display has provided a faster and more cost-effective approach for antibody production. The application of rAbs for developing ultrasensitive diagnostic tools ranging from immunoassays to immunosensors, has allowed the researchers to introduce new platforms with high sensitivity and specificity. Additionally, DNA encoding antibodies are directly accessible in these approaches, which enables the application of antibody engineering to increase their sensitivity and specificity. Here, we review the latest studies about the antibody-based ultrasensitive diagnostic platforms for detection of C. difficile bacteria, with an emphasis on rAb technologies.

Standardizing flow cytometric assays during cell manufacturing

Watch the video or read the poster to learn how to minimize variation in flow cytometric assays during cell manufacturing with: Dry recombinant antibody technology Fully automated data acquisition and analysis Robust protocols and technical supportAnnika Graband is Miltenyi Biotec’s Product Manager responsible for the cell and gene therapy reagents portfolio, including CAR detection reagents and StainExpress™ dry antibody cocktails. During the past year, she has been working together with R&D to advance the development of tools that enable highly reliable and standardized cell and gene therapy analytics. Annika received her PhD from the University of Cologne.

Isolation and characterization of full-length genes encoding the anti-human CD45 antibody from the hybridoma cell line 16E8-F2

Though the hybridoma technology has been widely applied in the production of monoclonal antibodies, it has existed some disadvantages including low yield and genetic instability. Therefore, an alternative approach should be taken into account. Recently, recombinant monoclonal antibody technology has emerged as the best choice to cure hybridoma related drawbacks. However, recombinant antibodies require known genes for their generation. The purpose of this study is to collect the full-length genes encoding the anti-human CD45 antibody derived from the hybridoma cell line 16E8-F2. In this research, we designed specific primer pairs to amplify the light and heavy chain genes of the antibody through the PCR method. Afterward, the genes were separately cloned into a cloning vector called pJET1.2/blunt. The generated recombinant pJET1.2 vectors will serve as the main material source to manufacture the recombinant monoclonal antibody recognizing human CD45 protein tomorrow.

Access to ultra-long IgG CDRH3 bovine antibody sequences using short read sequencing technology

The advances in high-throughput DNA sequencing and recombinant antibody technologies has presented new methods for characterizing antibody repertoires and significantly increased our understanding on the functional role of antibodies in immunity and their use in diagnostics, vaccine antigen design and as biological therapeutics. A subset of Bos taurus antibodies possesses unique ultra-long third complementary-determining region of the heavy chain (CDRH3) and are of special interest because they are thought to have unique functional abilities of broadly neutralizing properties – a functional role that has not been fully explored in vaccine development. Next generation sequencing technologies that are widely used to profile immunoglobulin (Ig) repertoires are based on short-read methods such as the Illumina technology. Although this technology has worked well in sequencing Ig V-D-J regions of most jawed vertebrates, it has faced serious technical challenges with sequencing regions in bovine Ig bearing ultra-long CDRH3 sequences, which are longer than 120 bp. To overcome this limitation, we have developed a sequencing strategy based on nested PCR products that allows sequence assembly of full-length bovine Ig heavy-chain (IgH) V-D-J regions. We have used this strategy to sequence IgH V-D-J regions of two Bos indicus breeds, Ankole and Boran. We confirm the presence of ultra-long CDRH3 sequences in IgG transcripts in both African cattle breeds, and provide preliminary evidence for differences and preferences in germline VH, DH and JH allele gene usage as well as differences in the length of the VH region in the two bovine breeds. Our method provides tools that should allow more robust analyses of ultra-long CDRH3 sequences aiding antibody and epitope discovery in different cattle breeds and their role in mediating immunity.

Recombinant Antibody Engineering Enables Reversible Binding for Continuous Protein Biosensing.

Engineering antibodies to improve target specificity, reduce detection limits, or introduce novel functionality is an important research area for biosensor development. While various affinity biosensors have been developed to generate an output signal upon varying analyte concentrations, reversible and continuous protein monitoring in complex biological samples remains challenging. Herein, we explore the concept of directed evolution to modulate dissociation kinetics of a high affinity anti-epidermal growth factor receptor (EGFR) single-chain variable antibody fragment (scFv) to enable continuous protein sensing in a label-free binding assay. A mutant scFv library was generated from the wild type (WT) fragment via targeted permutation of four residues in the antibody-antigen-binding interface. A single round of phage display biopanning complemented with high-throughput screening methods then permitted isolation of a specific binder with fast reaction kinetics. We were able to obtain ∼30 times faster dissociation rates when compared to the WT without appreciably affecting overall affinity and specificity by targeting a single paratope that is known to contribute to the binding interaction. Suitability of a resulting mutant fragment to sense varying antigen concentrations in continuous mode was demonstrated in a modified label-free binding assay, achieving low nanomolar detection limits (KD = 8.39 nM). We also confirmed these results using an independent detection mechanism developed previously by our group, incorporating a polarity-dependent fluorescent dye into the scFv and reading out EGFR binding based on fluorescence wavelength shifts. In future, this generic approach could be employed to generate improved or novel binders for proteins of interest, ready for deployment in a broad range of assay platforms.

A general strategy to control antibody specificity against targets showing molecular and biological similarity: Salmonella case study

The control of antibody specificity plays pivotal roles in key technological fields such as diagnostics and therapeutics. During the development of immunoassays (IAs) for the biosensing of pathogens in food matrices, we have found a way to rationalize and control the specificity of polyclonal antibodies (sera) for a complex analytical target (the Salmonella genus), in terms of number of analytes (Salmonella species) and potential cross-reactivity with similar analytes (other bacteria strains). Indeed, the biosensing of Salmonella required the development of sera and serum mixtures displaying homogeneous specificity for a large set of strains showing broad biochemical variety (54 Salmonella serovars tested in this study), which partially overlaps with the molecular features of other class of bacteria (like specific serogroups of E. coli). To achieve a trade-off between specificity harmonisation and maximization, we have developed a strategy based on the conversion of the specificity profiles of individual sera in to numerical descriptors, which allow predicting the capacity of serum mixtures to detect multiple bacteria strains. This approach does not imply laborious purification steps and results advantageous for process scaling-up, and may help in the customization of the specificity profiles of antibodies needed for diagnostic and therapeutic applications such as multi-analyte detection and recombinant antibody engineering, respectively.

Engineered Protein Scaffolds as Next-Generation Therapeutics.

The concept of engineering robust protein scaffolds for novel binding functions emerged 20 years ago, one decade after the advent of recombinant antibody technology. Early examples were the Affibody, Monobody (Adnectin), and Anticalin proteins, which were derived from fragments of streptococcal protein A, from the tenth type III domain of human fibronectin, and from natural lipocalin proteins, respectively. Since then, this concept has expanded considerably, including many other protein templates. In fact, engineered protein scaffolds with useful binding specificities, mostly directed against targets of biomedical relevance, constitute an area of active research today, which has yielded versatile reagents as laboratory tools. However, despite strong interest from basic science, only a handful of those protein scaffolds have undergone biopharmaceutical development up to the clinical stage. This includes the abovementioned pioneering examples as well as designed ankyrin repeat proteins (DARPins). Here we review the current state and clinical validation of these next-generation therapeutics.

Bio-assay of the non-amidated progastrin-derived peptide (G17-Gly) using the tailor-made recombinant antibody fragment and phage display method: a biomedical analysis.

In this research, four novel and sensitive immunosensors for electrochemical determination of G17-Gly were designed based on signal amplification and tailor-made recombinant antibody technology. Anti-G17-Gly antibody fragments (i.e. scFv and VL specific to the N- and C-terminal of G17-Gly) were immobilized onto a polymeric nanocomposite comprising poly cetyl trimethyl ammonium bromide (P(CTAB)) as the conductive matrix, chitosan (CS) as a biocompatible agent and gold nanoparticles (AuNPs) as the signal amplification element. The high surface area provided by AuNPs and the small size of scFv/VL establish the basis for immobilizing a high amount of the anti-G17-Gly on the surface of the electrode for detecting G17-Gly in human plasma samples. Under optimal conditions, the designed immunosensors provide an excellent analytical capability for detecting and determining G17-Gly in human plasma samples with a linear range from 0.5 mM to 0.05 pM and a LLOQ of 0.05 pM. The sensitivity order of the immunosensors was Ag/2-mercaptoethanol/phage displaying scFv/P(CTAB-CS)-AuNP/GE, Ag/2-mercaptoethanol/phage displaying VL/P(CTAB-CS)-AuNP/GE, Ag/BSA/scFv/P(CTAB-CS)-AuNP/GE, and Ag/BSA/VL/P(CTAB-CS)-AuNP/GE. The aforementioned characteristics demonstrate that the proposed immune-devices can be used in biological and clinical diagnosis as reliable tools for identifying different oncobiomarkers.

The potential of recombinant antibody technology in venom therapeutics

The potential of recombinant antibody technology in venom therapeutics

Novel Microfluidic Analytical Sensing Platform for the Simultaneous Detection of Three Algal Toxins in Water.

Globally, the need for “on-site” algal-toxin monitoring has become increasingly urgent due to the amplified demand for fresh-water and for safe, “toxin-free” shellfish and fish stocks. Herein, we describe the first reported, Lab-On-A-Disc (LOAD) based-platform developed to detect microcystin levels in situ, with initial detectability of saxitoxin and domoic acid also reported. Using recombinant antibody technology, the LOAD platform combines immunofluorescence with centrifugally driven microfluidic liquid handling to achieve a next-generation disposable device capable of multianalyte sampling. A low-complexity “LED-photodiode” based optical sensing system was tailor-made for the platform, which allows the fluorescence signal of the toxin-specific reaction to be quantified. This system can rapidly and accurately detect the presence of microcystin-LR, domoic acid, and saxitoxin in 30 min, with a minimum of less than 5 min end-user interaction for maximum reproducibility. This method provides a robust “point of need” diagnostic alternative to the current laborious and costly methods used for qualitative toxin monitoring.

Surface plasmon resonance (SPR) based binding studies of refolded single chain antibody fragments

Recent advances in Recombinant antibody technology / Antibody Engineering has given impetus to the genetic manipulation of antibody fragments that has paved the way for better understanding of the structure and functions of immunoglobulins and also has escalated their use in immunotherapy. Bacterial expression system such as Escherichia coli has complemented this technique through the expression of recombinant antibodies. Present communication has attempted to optimize the expression and refolding protocol of single chain fragment variable (ScFv) and single chain antigen binding fragment (ScFab) using E.coli expression system. Efficiency of refolding protocol was validated by structural analysis by CD, native folding by fluorescence and functional analysis by its binding with full length HIV-1 gp120 via SPR. Results show the predominant β–sheet (CD) as secondary structural content and native folding via red shift (tryptophan fluorescence). The single chain fragments have shown good binding with HIV-1 gp120 thus validating the expression and refolding strategy and also reinstating E.coli as model expression system for recombinant antibody engineering. SPR based binding analysis coupled with E.coli based expression and purification will have implication for HIV therapeutics and will set a benchmark for future studies of similar kind.

Production of recombinant human antibodies suitable for medical applications. (review article)

Antibodies are fundamental proteins that have ability to recognise and attack variety of antigens via specific binding sites. Antibodies are considered essential diagnostic and therapeutic tools in the management of many disorders especially in malignancy. This review will discuss how recombinant human antibodies are produced. Recombinant antibody is introduced to perform various applications in scientific Field, where the hybridoma monoclonal antibodies are used for diagnosis bacterial and viral infections by using nanoparticles. The recombinant antibody technology is a valuable diagnostic and therapeutic tool, even though some challenges in their production and applications.

Selection, characterization, and thermal stabilization of llama single domain antibodies towards Ebola virus glycoprotein

BackgroundA key advantage of recombinant antibody technology is the ability to optimize and tailor reagents. Single domain antibodies (sdAbs), the recombinantly produced variable domains derived from camelid and shark heavy chain antibodies, provide advantages of stability and solubility and can be further engineered to enhance their properties. In this study, we generated sdAbs specific for Ebola virus envelope glycoprotein (GP) and increased their stability to expand their utility for use in austere locals. Ebola virus is extremely virulent and causes fatal hemorrhagic fever in ~ 50 percent of the cases. The viral GP binds to host cell receptors to facilitate viral entry and thus plays a critical role in pathogenicity.ResultsAn immune phage display library containing more than 107 unique clones was developed from a llama immunized with a combination of killed Ebola virus and recombinantly produced GP. We panned the library to obtain GP binding sdAbs and isolated sdAbs from 5 distinct sequence families. Three GP binders with dissociation constants ranging from ~ 2 to 20 nM, and melting temperatures from ~ 57 to 72 °C were selected for protein engineering in order to increase their stability through a combination of consensus sequence mutagenesis and the addition of a non-canonical disulfide bond. These changes served to increase the melting temperatures of the sdAbs by 15–17 °C. In addition, fusion of a short positively charged tail to the C-terminus which provided ideal sites for the chemical modification of these sdAbs resulted in improved limits of detection of GP and Ebola virus like particles while serving as tracer antibodies.ConclusionsSdAbs specific for Ebola GP were selected and their stability and functionality were improved utilizing protein engineering. Thermal stability of antibody reagents may be of particular importance when operating in austere locations that lack reliable refrigeration. Future efforts can evaluate the potential of these isolated sdAbs as candidates for diagnostic or therapeutic applications for Ebola.

Snakebite envenoming.

Snakebite envenoming is a neglected tropical disease that kills >100,000 people and maims >400,000 people every year. Impoverished populations living in the rural tropics are particularly vulnerable; snakebite envenoming perpetuates the cycle of poverty. Snake venoms are complex mixtures of proteins that exert a wide range of toxic actions. The high variability in snake venom composition is responsible for the various clinical manifestations in envenomings, ranging from local tissue damage to potentially life-threatening systemic effects. Intravenous administration of antivenom is the only specific treatment to counteract envenoming. Analgesics, ventilator support, fluid therapy, haemodialysis and antibiotic therapy are also used. Novel therapeutic alternatives based on recombinant antibody technologies and new toxin inhibitors are being explored. Confronting snakebite envenoming at a global level demands the implementation of an integrated intervention strategy involving the WHO, the research community, antivenom manufacturers, regulatory agencies, national and regional health authorities, professional health organizations, international funding agencies, advocacy groups and civil society institutions.