Next-Generation Therapeutic Bio Agents
Superantibodies, the Next-Generation Therapeutic Agents for Diagnostics and Treatment of Toxic Infections
Tech Area / Field
- BIO-CGM/Cytology, Genetics and Molecular Biology/Biotechnology
- MED-DRG/Drug Discovery/Medicine
8 Project completed
Senior Project Manager
Melnikov V G
State Research Center for Applied Microbiology and Biotechnology, Russia, Moscow reg., Obolensk
- Institute of Bioorganic Chemistry, Russia, Moscow
Infectious diseases of bacterial origin especially those with epidemic propensity are the matter of continuing concern in the modern world. New antibiotics while serve as efficient drugs to counter the majority of known threats, at the same time, work as important force under which new dangerous strains may evolve. Moreover, antibiotics may be unsuccessful in management of bacteria that produce fulminantly acting toxins, because the main goal in this case is immediate toxin neutralization rather than plain killing of the pathogen. That is why new approaches are sought to monitor the infection spread and to counter efficiently possible disease outbreaks. Of special interest are systemic approaches that allow both rapid diagnostics and treatment of a particular disease and that can be applied to a plethora of pathogens without significant alterations of both R&D efforts and treatment protocols.
Passive immunotherapy has long been considered as efficient supplement to vaccination and antibiotic therapy. Antibodies are ideal medicines for rapid neutralization of toxic effects in infectious diseases that are exacerbated by pathological monocyte activation and harmful cytokine release, symptoms otherwise known as “toxic shock”. However, homologous, i.e. human monoclonal antibodies specific to a particular bacterial antigen or toxin are barely available, thus posing formidable limitation to this approach. Animal antisera and monoclonal antibodies while efficient under certain circumstances, yet cannot be considered as completely satisfactory therapeutics due to dangerous adverse effects. Moreover, immunodominant epitopes to which both human and animal antibodies usually form in many cases appear to be inefficient targets because they frequently are the integral part of the mechanism by which bacteria evade the host defense.
Recently emerged combinatorial approaches of artificial antibody design and evolution using semisynthetic antibodies and phage display techniques can provide efficient alternative to natural human antibodies. Antibodies that bind to several epitopes of the antigen can thus be rapidly evolved. Hence, artificial antibody evolution can provide building blocks to the desired systemic approach of diagnostics and therapy of infections, toxic ones in particular. Not only this implies simultaneous selection of several diagnostics and therapeutic antibodies directed against a single target, but also provides a framework for rapid generation of safe and efficient medicines suited to counter known and emerging bacterial infections.
Most important features that the human-like antibody therapeutics should bear are high affinity and specificity to the target antigen. Natural antibodies rarely reach the affinity higher than 1010 M-1. Affinities of artificial combinatorial antibodies derived from naпve repertoire are even lower and only antibodies containing one or several synthetic randomized complementarity determining regions (CDRs) were reported to possess binding constants in the upper picomolar range. That is why semisynthetic antibodies especially those with all six CDRs randomized can be considered as the most promising alternative to the artificial “nonimmune” antibody repertoires.
However, current technologies of preparation of antibody genes with completely randomized combining site are cumbersome. They require simultaneous assembly of several long synthetic oligonucleotides into the gene that is both cost-ineffective and yields antibody repertoires of low complexity due to extremely inefficient procedure of PCR-mediated shuffling of several oligonucleotides. Also, current vector systems further decrease the complexity of the libraries because cloning of the repertoire is mediated by frequently cutting hexanucleotide restriction endonucleases. Furthemore, most widespread technology yields single-chain antibodies with decreased binding capacity due to conformational hindrance introduced by linker and relatively low library complexity that arises from inherent limitations of PCR-mediated antibody chain shuffling technique.
We developed the approach that is aimed to overcome the shortcomings of current methods of synthetic antibody repertoire preparation. First, oligonucleotide-mediated gene construction will be replaced by whole plasmid PCR-driven introduction of CDRs into the existing antibody framewok. This approach allows a step-by-step modification of CDRs in the antibody with the primers amplifying the whole plasmid and containing randomized 5'-termini. After amplification the plasmid can be ligated and subjected to the next round of PCR-mediated CDR insertion. The proposed technique offers extreme flexibility in modifying antibody chains, e.g. in replacement of only selected CDRs, choice of the initial antibody, introduction of CDRs of different length at any step of the gene construction etc. Within this method technical contrivances were elaborated that permit high yield of correctly mutated sequences. For example, phosphorothioated 5'-termini of PCR primers were employed to minimize shedding of several terminal bases frequently seen in “native” mutants. Second, a new vector system suited both for phage display and cloning of semisynthetic antibody genes assembled into Fab fragments is developed. The vectors feature two pairs of sites for rare-cutting octanucleotide restriction nucleases for independent cloning of heavy and light chain fragments, a leucine zipper assembly for increased Fab stability and a system of affinity tags for protein detection and purification. Vectors allow periplasmic localization of the products without any non-native amino acid residues remaining at the N-termini of the chains. Third, a library construction using a flow-through electroporation is developed that allows preparation of the libraries with complexity above 1012 inpidual clones. Taken together, these modifications of the existing combinatorial approaches would result in creation of antibodies with high specificity and increased binding constants. This project is aimed at construction of artificial human-like antibody Fab fragments that interfere with toxin binding to the cell and, in the case of anthrax, block formation of toxin complexes. It is expected that the combination of the above mentioned techniques would allow to raise antibodies with binding constants in the lower picomolar range, thus increasing the neutralization efficiency and improving pharmacological properties of the prototype therapeutic.
Special attention will be paid to construction of proteolytic abzyme capable to inactivate the anthrax protective antigen by cleavage in defined region. This part of investigation will utilize latest developments in the field of antibody-mediated cleavage of macromolecules, to which members of the Project Team made some direct contribution.
Construction of highly efficient antitoxin antibodies for diagnostics and therapy of infectious diseases basing on the described approach will be the primary goal of the proposed project. We suggest here to use the described system of artificial antibody construction for preparation of diagnostic and therapeutic antibodies against the antigens of Bacillus Anthracis, and Corynebacterium diphtheriae etiologic agents of anthrax and diphteria, respectively.
The reasons to chose these pathogens are the following:
Both pathogens feature highly dangerous toxins as major disease causative agents.
Toxins of B. anthracis and С. diphtheriae are well-studied thus providing the firm basis for construction of efficient multilayer antibody-based therapeutic system without any need to conduct additional fundamental research.
Toxic system of B. anthracis is especially hazardous because of its high virulence and mode of action that directs the primary attack against host macrophages, the first-line defense cells. The danger is doubled by toxic shock that inevitably accompanies massive macrophage lysis. Due to the toxic shock antibiotics frequently fail to counter the disease.
Anthrax has natural isolates with increased risk of the disease outbreaks in Russian Federation and neighbor countries.
Lack of vaccines that produce a lifetime immune response to both pathogens.
Scientists at the Institute of Applied Microbiology accumulated significant experience regarding work with the mentioned pathogens.
The project milestones will include production of target antigens, construction and screening of phage libraries displaying semisynthetic antibody Fab fragments, selection and analysis of clones with diagnostic and therapeutic potential using in vitro approaches and, finally, validation of therapeutic action of engineered antibodies in direct experiments in vivo that will be performed at specialized facility of the Institute of Applied Microbiology. A completely new approach based on generation of the toxin-cleaving catalytic antibodies is planned to be developed. The Project implies collaboration with US and French scientists that are working in the fields of toxic infectious diseases and catalytic antibodies.
It is expected that the proposed research would be the cornerstone for the above mentioned systemic approach that implies the design of antibody-based diagnostics and therapeutics by redirecting of already constructed antibody libraries against other known, emerging and artificially modified bacterial pathogens.
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