Synthetic antibody

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Synthetic antibodies are affinity reagents generated entirely in vitro, thus completely eliminating animals from the production process. [1] Synthetic antibodies include recombinant antibodies, nucleic acid aptamers and non-immunoglobulin protein scaffolds. As a consequence of their in vitro manufacturing method the antigen recognition site of synthetic antibodies can be engineered to any desired target and may extend beyond the typical immune repertoire offered by natural antibodies. [2] Synthetic antibodies are being developed for use in research, diagnostic and therapeutic applications. Synthetic antibodies can be used in all applications where traditional monoclonal or polyclonal antibodies are used and offer many inherent advantages over animal-derived antibodies, including comparatively low production costs, reagent reproducibility and increased affinity, specificity and stability across a range of experimental conditions. [3]

Contents

Recombinant antibodies

Recombinant antibodies are monoclonal antibodies generated in vitro using synthetic genes. Recombinant antibody technology involves recovering the antibody genes from the source cells, amplifying and cloning the genes into an appropriate vector, introducing the vector into a host, and achieving expression of adequate amounts of functional antibody. Recombinant antibodies can be cloned from any species of antibody-producing animal, if the appropriate oligonucleotide primers or hybridization probes are available. [4] The ability to manipulate the antibody genes make it possible to generate new antibodies and antibody fragments, such as Fab fragments and scFv in vitro. This can be done at the level of the whole combining site by making new combinations of H and L chains. It can also be done by mutating individual CDRs. Display libraries, commonly expressed in phage or yeast, can be analysed to select for desirable characteristics arising from such changes in antibody sequence. [5] [6]

Non-immunoglobulin derived synthetic antibodies

These molecules typically differ in structure to that of an antibody and can be generated either from nucleic acids, as in the case of aptamers, or from non-immunoglobulin protein scaffolds / peptide aptamers, into which hypervariable loops are inserted to form the antigen binding site. Constraining the hypervariable binding loop at both ends within the protein scaffold improves the binding affinity and specificity of the synthetic antibody to levels comparable to or exceeding that of a natural antibody. [7] Common advantages of these molecules compared to use of the typical antibody structure include a smaller size, giving improved tissue penetration, rapid generation times of weeks compared to months for natural and recombinant antibodies and cheaper costs. [3]

Affimer proteins

Affimer proteins are small robust affinity reagents, with a molecular weight of 12–14kDa. They are engineered to bind to their target proteins with high affinity and specificity and as such are a member of the synthetic antibody family.

The Affimer protein scaffold is derived from the cysteine protease inhibitor family of cystatins. [8] [9] [10] [11] Within the protein scaffold there exist two variable peptide loops and a variable N-terminal sequence that provide a high affinity binding surface for the specific target protein. Affimer binders have been produced to a large number of targets including ubiquitin chains, immunoglobulins and C-reactive protein [12] for use in a number of molecular recognition applications. Affimer technology has been commercialised and developed by Avacta Life Sciences, who are developing Affimer binders as reagents for research, diagnostic and therapeutic applications.

Applications

Synthetic antibodies have shown their utility in a number of applications. Their use within the field of research lies predominantly in the life sciences as reagents for protein capture and as protein inhibitors. Within diagnostics they have been utilised in applications ranging from infection [12] and cancer screening [13] to mycotoxin detection in grain samples. [14] Synthetic antibodies are currently the fastest growing class of therapeutics. [15]

See also

Related Research Articles

<span class="mw-page-title-main">Antibody</span> Protein(s) forming a major part of an organisms immune system

An antibody (Ab), also known as an immunoglobulin (Ig), is a large, Y-shaped protein used by the immune system to identify and neutralize foreign objects such as pathogenic bacteria and viruses. The antibody recognizes a unique molecule of the pathogen, called an antigen. Each tip of the "Y" of an antibody contains a paratope that is specific for one particular epitope on an antigen, allowing these two structures to bind together with precision. Using this binding mechanism, an antibody can tag a microbe or an infected cell for attack by other parts of the immune system, or can neutralize it directly.

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<span class="mw-page-title-main">Phage display</span> Biological technique to evolve proteins using bacteriophages

Phage display is a laboratory technique for the study of protein–protein, protein–peptide, and protein–DNA interactions that uses bacteriophages to connect proteins with the genetic information that encodes them. In this technique, a gene encoding a protein of interest is inserted into a phage coat protein gene, causing the phage to "display" the protein on its outside while containing the gene for the protein on its inside, resulting in a connection between genotype and phenotype. The proteins that the phages are displaying can then be screened against other proteins, peptides or DNA sequences, in order to detect interaction between the displayed protein and those of other molecules. In this way, large libraries of proteins can be screened and amplified in a process called in vitro selection, which is analogous to natural selection.

Affinity chromatography is a method of separating a biomolecule from a mixture, based on a highly specific macromolecular binding interaction between the biomolecule and another substance. The specific type of binding interaction depends on the biomolecule of interest; antigen and antibody, enzyme and substrate, receptor and ligand, or protein and nucleic acid binding interactions are frequently exploited for isolation of various biomolecules. Affinity chromatography is useful for its high selectivity and resolution of separation, compared to other chromatographic methods.

A protein microarray is a high-throughput method used to track the interactions and activities of proteins, and to determine their function, and determining function on a large scale. Its main advantage lies in the fact that large numbers of proteins can be tracked in parallel. The chip consists of a support surface such as a glass slide, nitrocellulose membrane, bead, or microtitre plate, to which an array of capture proteins is bound. Probe molecules, typically labeled with a fluorescent dye, are added to the array. Any reaction between the probe and the immobilised protein emits a fluorescent signal that is read by a laser scanner. Protein microarrays are rapid, automated, economical, and highly sensitive, consuming small quantities of samples and reagents. The concept and methodology of protein microarrays was first introduced and illustrated in antibody microarrays in 1983 in a scientific publication and a series of patents. The high-throughput technology behind the protein microarray was relatively easy to develop since it is based on the technology developed for DNA microarrays, which have become the most widely used microarrays.

<span class="mw-page-title-main">Aptamer</span> Oligonucleotide or peptide molecules that bind specific targets

Aptamers are short sequences of artificial DNA, RNA, XNA, or peptide that bind a specific target molecule, or family of target molecules. They exhibit a range of affinities, with variable levels of off-target binding and are sometimes classified as chemical antibodies. Aptamers and antibodies can be used in many of the same applications, but the nucleic acid-based structure of aptamers, which are mostly oligonucleotides, is very different from the amino acid-based structure of antibodies, which are proteins. This difference can make aptamers a better choice than antibodies for some purposes.

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Bacterial display is a protein engineering technique used for in vitro protein evolution. Libraries of polypeptides displayed on the surface of bacteria can be screened using flow cytometry or iterative selection procedures (biopanning). This protein engineering technique allows us to link the function of a protein with the gene that encodes it. Bacterial display can be used to find target proteins with desired properties and can be used to make affinity ligands which are cell-specific. This system can be used in many applications including the creation of novel vaccines, the identification of enzyme substrates and finding the affinity of a ligand for its target protein.

<span class="mw-page-title-main">Immunoproteomics</span> Study of large set of protein

Immunoproteomics is the study of large sets of proteins (proteomics) involved in the immune response.

<span class="mw-page-title-main">Systematic evolution of ligands by exponential enrichment</span> Technique for producing oligonucleotides that specifically bind to a target

Systematic evolution of ligands by exponential enrichment (SELEX), also referred to as in vitro selection or in vitro evolution, is a combinatorial chemistry technique in molecular biology for producing oligonucleotides of either single-stranded DNA or RNA that specifically bind to a target ligand or ligands. These single-stranded DNA or RNA are commonly referred to as aptamers. Although SELEX has emerged as the most commonly used name for the procedure, some researchers have referred to it as SAAB and CASTing SELEX was first introduced in 1990. In 2015, a special issue was published in the Journal of Molecular Evolution in the honor of quarter century of the discovery of SELEX.

Protein G is an immunoglobulin-binding protein expressed in group C and G Streptococcal bacteria much like Protein A but with differing binding specificities. It is a ~60-kDA cell surface protein that has found application in purifying antibodies through its binding to the Fab and Fc region. The native molecule also binds albumin, but because serum albumin is a major contaminant of antibody sources, the albumin binding site has been removed from recombinant forms of Protein G. This recombinant Protein G, either labeled with a fluorophore or a single-stranded DNA strand, was used as a replacement for secondary antibodies in immunofluorescence and super-resolution imaging.

<span class="mw-page-title-main">Isotype (immunology)</span>

In immunology, antibodies are classified into several types called isotypes or classes. The variable (V) regions near the tip of the antibody can differ from molecule to molecule in countless ways, allowing it to specifically target an antigen . In contrast, the constant (C) regions only occur in a few variants, which define the antibody's class. Antibodies of different classes activate distinct effector mechanisms in response to an antigen . They appear at different stages of an immune response, differ in structural features, and in their location around the body.

<span class="mw-page-title-main">Immunolabeling</span> Procedure for detection and localization of an antigen

Immunolabeling is a biochemical process that enables the detection and localization of an antigen to a particular site within a cell, tissue, or organ. Antigens are organic molecules, usually proteins, capable of binding to an antibody. These antigens can be visualized using a combination of antigen-specific antibody as well as a means of detection, called a tag, that is covalently linked to the antibody. If the immunolabeling process is meant to reveal information about a cell or its substructures, the process is called immunocytochemistry. Immunolabeling of larger structures is called immunohistochemistry.

<span class="mw-page-title-main">Meir Wilchek</span> Israeli biochemist (born 1935)

Meir Wilchek is an Israeli biochemist. He is a professor at the Weizmann Institute of Science.

Affibody molecules are small, robust proteins engineered to bind to a large number of target proteins or peptides with high affinity, imitating monoclonal antibodies, and are therefore a member of the family of antibody mimetics. Affibody molecules are used in biochemical research and are being developed as potential new biopharmaceutical drugs. These molecules can be used for molecular recognition in diagnostic and therapeutic applications.

The Streptavidin-Binding Peptide (SBP)-Tag is a 38-amino acid sequence that may be engineered into recombinant proteins. Recombinant proteins containing the SBP-Tag bind to streptavidin and this property may be utilized in specific purification, detection or immobilization strategies.

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<span class="mw-page-title-main">Affitin</span>

Affitins are artificial proteins with the ability to selectively bind antigens. They are structurally derived from the DNA binding protein Sac7d, found in Sulfolobus acidocaldarius, a microorganism belonging to the archaeal domain. By randomizing the amino acids on the binding surface of Sac7d and subjecting the resulting protein library to rounds of ribosome display, the affinity can be directed towards various targets, such as peptides, proteins, viruses, and bacteria.

<span class="mw-page-title-main">Affimer</span> Type of protein

Affimer molecules are small proteins that bind to target proteins with affinity in the nanomolar range. These engineered non-antibody binding proteins are designed to mimic the molecular recognition characteristics of monoclonal antibodies in different applications. These affinity reagents have been optimized to increase their stability, make them tolerant to a range of temperatures and pH, reduce their size, and to increase their expression in E.coli and mammalian cells.

Recombinant antibodies are antibody fragments produced by using recombinant antibody coding genes. They mostly consist of a heavy and light chain of the variable region of immunoglobulin. Recombinant antibodies have many advantages in both medical and research applications, which make them a popular subject of exploration and new production against specific targets. The most commonly used form is the single chain variable fragment (scFv), which has shown the most promising traits exploitable in human medicine and research. In contrast to monoclonal antibodies produced by hybridoma technology, which may lose the capacity to produce the desired antibody over time or the antibody may undergo unwanted changes, which affect its functionality, recombinant antibodies produced in phage display maintain high standard of specificity and low immunogenicity.

References

  1. ↑ Echko, M.M. and Dozier, S.K. (2010). "Recombinant antibody technology for the production of antibodies without the use of animals". AltTox.{{cite journal}}: CS1 maint: multiple names: authors list (link)
  2. ↑ Bradbury, A.R.M., Sidhu, S., Dübel, S. and McCafferty, J (2011). "Beyond natural antibodies: The power of in vitro display technologies". Nat. Biotechnol. 29 (3): 245–254. doi:10.1038/nbt.1791. PMC   3057417 . PMID   21390033.{{cite journal}}: CS1 maint: multiple names: authors list (link)
  3. 1 2 Gebauer, M. and Skerra (June 2009). "Engineered protein scaffolds as next-generation antibody therapeutics". Curr Opin Chem Biol. 13 (3): 245–55. doi:10.1016/j.cbpa.2009.04.627. PMID   19501012.
  4. ↑ Karu, A.E., Bell, C.W. and Chin, T.E. (2009). "Recombinant Antibody Technology". Curr Opin Chem Biol. 13 (3): 245–255.{{cite journal}}: CS1 maint: multiple names: authors list (link)
  5. ↑ Miersch, S. and Sidhu, S.S. (2012). "Synthetic antibodies: Concepts, potential and practical considerations". Methods. 57 (4): 486–498. doi:10.1016/j.ymeth.2012.06.012. PMID   22750306.{{cite journal}}: CS1 maint: multiple names: authors list (link)
  6. ↑ Hammers, C.M. and Stanley, J.R. (2014). "Antibody phage display: Technique and applications". J. Invest. Dermatol. 134 (2): 1–5. doi:10.1038/jid.2013.521. PMC   3951127 . PMID   24424458.{{cite journal}}: CS1 maint: multiple names: authors list (link)
  7. ↑ Ladner, R.C. (1995). "Constrained peptides as binding entities". Trends Biotechnol. 13 (10): 426–430. doi:10.1016/s0167-7799(00)88997-0. PMID   7546567.
  8. ↑ Woodman, R. Yeh, J.T.H., Laurenson, S. and KoFerrigno. (2005). "P. Design and validation of a neutral protein scaffold for the presentation of peptide aptamers". J Mol Biol. 352 (5): 1118–1133. doi:10.1016/j.jmb.2005.08.001. PMID   16139842.{{cite journal}}: CS1 maint: multiple names: authors list (link)
  9. ↑ Hoffmann, T. Stadler, L.K.J., Busby, M., Song, Q., Buxton A.T., Wagner, S.D., Davis, J.J. and Ko Ferrigno, P (2010). "Structure-function studies of and engineered scaffold protein derived from Stefin A. I: Development of the SQM variant". Protein Engineering, Design and Selection. 23 (5): 403–413. doi:10.1093/protein/gzq012. PMC   2851446 . PMID   20179045.{{cite journal}}: CS1 maint: multiple names: authors list (link)
  10. ↑ Hoffmann, Toni; Stadler, Lukas Kurt Josef; Busby, Michael; Song, Qifeng; Buxton, Anthony T.; Wagner, Simon D.; Davis, Jason J.; Ferrigno, Paul Ko (2010-05-01). "Structure-function studies of an engineered scaffold protein derived from stefin A. I: Development of the SQM variant". Protein Engineering Design and Selection. 23 (5): 403–413. doi:10.1093/protein/gzq012. ISSN   1741-0126. PMC   2851446 . PMID   20179045.
  11. ↑ Tiede, Christian; Tang, Anna A. S.; Deacon, Sarah E.; Mandal, Upasana; Nettleship, Joanne E.; Owen, Robin L.; George, Suja E.; Harrison, David J.; Owens, Raymond J. (2014-05-01). "Adhiron: a stable and versatile peptide display scaffold for molecular recognition applications". Protein Engineering Design and Selection. 27 (5): 145–155. doi:10.1093/protein/gzu007. ISSN   1741-0126. PMC   4000234 . PMID   24668773.
  12. 1 2 Johnson, Anthony; Song, Qifeng; Ferrigno, Paul Ko; Bueno, Paulo R.; Davis, Jason J. (2012-07-26). "Sensitive Affimer and Antibody Based Impedimetric Label-Free Assays for C-Reactive Protein". Analytical Chemistry. 84 (15): 6553–6560. doi:10.1021/ac300835b. PMID   22789061.
  13. ↑ Affibody ltd (2015). "Recombinant antibody technology for the production of antibodies without the use of animals". Online.
  14. ↑ NeoVentures Biotechnology Inc (2015). "NeoVentures Biotechnology Inc". Online.
  15. ↑ Frenzel, A., Hust, M. and Schirrmann, T. (2013). "Expression of recombinant antibodies". Front. Immunol. 4 (217): 217. doi: 10.3389/fimmu.2013.00217 . PMC   3725456 . PMID   23908655.{{cite journal}}: CS1 maint: multiple names: authors list (link)
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