Recombinant antibodies (rAbs) are produced by cloning antibody genes
into expression vectors, which are subsequently introduced into expression hosts to provide animal-free antibody production. By employing genetic engineering methods, it is possible to express light and heavy chains of immunoglobulins as individual proteins, and create a whole set of antibody fragments with improved affinity, stability, and the desired spatial orientation of antigen binding sites or modify potential immunogenicity. The ability to manipulate the genes that encode antibodies (Ab), and thereby manipulate the structures of antibodies, has opened a new era in the study and application of antibodies.
Advantages of Using Recombinant Antibodies
The possibility of incorporating a desired amino acid is an important advantage offered by rAbs. To decrease immunogenicity, domains or sites inducing immune response are replaced by corresponding sequences of human Abs. Thus, the Ab modifications have significantly extended their applicability in clinical practice. One of the most promising directions in the development of rAb preparations is the design of Abs of higher affinity than natural Abs.
Compared with conventional Ab, rAb offers advantages such as smaller size, monovalency, ease of engineering and manufacturing, improved tissue penetration, no animal immunization and broader biodistribution. There is also a lack of potentially deleterious Fc effector function with rAbs. A small antibody fragment unlike full Ab does not require human-like post-translational modification. Therefore, it can potentially be expressed in prokaryotic expression system.
In addition to reducing the dependency of animal use in research, rAbs offer exceptional batch-to-batch reproducibility, and easier and more rapid production. They also obviate the problems of cell-line drift and mutations that are associated with classical hybridoma production and storage that can often necessitate cell rescue or re-cloning. Recombinant antibody production also bypasses many of the difficulties of antibody production associated with traditional methods. The demand for sequence-defined rAbs has advanced a new field of Ab modeling and understanding protein–protein interactions. Recombinant antibodies are also being designed to improve distribution and half-life of administered Abs. Not only does Ab engineering contribute to the design of potential therapeutics, but important basic science information has been gained using rAbs. For example, advances in understanding the role of specific effector functions in tumor cell destruction have been made possible using mutant and engineered recombinant antibodies.
Types of Recombinant Antibody Fragments
Preparation of several different types of recombinant antibody fragments have been described.
The following types of recombinant antibodies are now recognized:
- Fv, variable fragment – The Fv fragment is the smallest antibody fragment that retains antigen binding capacity. It consists of variable fragments of light (VL) and heavy (VH) chains, each of which is stabilized by an intramolecular disulfide bond. However, contacts between VH and VL involve only ionic interactions and therefore the structure of this fragment is very unstable. Structural stability of Fv fragments can be achieved by insertion of interchain disulfide bond.
- scFv, single chain Fv in which light and heavy chain fragments are connected by a polypeptide linker – The linker technology was a key step of success of constructing scfv antibody library. Scfvs have been developed as possible drug candidates in their own right, as well as components or domains of drug candidates. The scFv fragments are the “simplest” target for cloning and expression. However, they frequently exhibit lower stability and, sometimes, lower affinity; during therapeutic use, they are characterized by shorter half- lifetime in blood circulation compared with larger fragments.
- (scFv)2, fragment that consists of two scFv molecules connected by a disulfide bond – The (scFv)2 fragments obtained by disulfide bond linking between C-terminal cysteine residues demonstrate better in vivo transport into tissues compared with the scFv-fragments during their use as therapeutic agents.
- dsFv, variable fragment stabilized by additional intramolecular disulfide bond – The bacterial and yeast expression systems are used for expression of small nonglycosylated antibody fragments dsFv.
- Fab- and (Fab)2, fragments – These are identical in their structure with fragments formed during proteolysis of full-length IgG anti- bodies by papain and pepsin, respectively.
- VH, heavy chain variable domain – Single-chain fragment variable (scfv) molecules combine the coding sequence of the variable heavy (VH) and sequence of the variable light chain (VL) domains of an antibody in a single-gene encoded format.
- There are antibody derivatives known as “diabodies”, “triabodies”, and “tetrabodies”; their molecules consist of two, three, or four identical or different fragments of antibodies of the same or different specificity linked together.
- A Single domain antibody (sdAb) was discovered in both camelids and nurse sharks that consist of a lone VH domain, lacking a paired VL, attached to a constant region. The primary advantages of domain antibodies as compared with scFvs are generally better folding and stability characteristics.
Methods for Recombinant Antibody Production
Gram negative Bacteria such as E. coli
are one of the most important production systems for recombinant proteins reaching volumetric yields in the gram per liter scale for extracellular production. For production of functional Ab fragments, the key to success was the secretion of both V chains into the periplasmic space of E. coli
where the oxidizing environment allows the correct formation of disulfide bonds and the assembly to a functional Fv fragment. Gram-positive bacteria directly secrete proteins into the medium due to the lack of an outer membrane which could facilitate production of antibody fragments. The Gram-positive bacteria Bacillus brevis
, Bacillus subtilis
and Bacillus megaterium
have been successfully used for the production of different antibody fragments.
Eukaryotic Hosts for Recombinant Antibody Production
Eukaryotic cells have been developed with an advanced folding, posttranslational, and secretion apparatus which enhances the secretory production of Abs. Yeasts combine the properties of eukaryotic cells’ short generation time and ease of genetic manipulation with the robustness and simple medium requirements of unicellular microbial hosts. Pichia pastoris
represents the major yeast strain used for recombinant antibody production.
Today, 60–70% of all recombinant protein pharmaceuticals and 95% of the currently approved therapeutic antibodies are still produced in mammalian cell lines despite relatively high production costs. Advances in protein folding, secretion and post-translational modifications capable of producing Abs indistinguishable from those in the human body have been achieved. Chinese hamster ovary (CHO) cells are the most common cells used in the commercial production of biopharmaceuticals. The human embryonic kidney (HEK 293) cell lines have been widely used for transient protein expression. There are several applications where the glycosylation pattern does not play a critical role, such as for in vitro diagnostics or in research. Therefore, bacteria, yeasts, filamentous fungi, and insect cells can be employed in order to lower the production costs of these products. In principle, transgenic plants and animals offer promise for scaling up for production. Antibody phage display is now a widespread method for the development of antibody fragments such as scFv or Fab.
Antibody drugs have gained importance over the past three decades as a key therapeutic modality for the treatment of some human diseases and gaining. Antibody drug discovery and development has evolved dramatically over this time, including expanded knowledge of target biology. Enzo’s catalog
includes widely cited and thoroughly validated native and rAbs for research. Our decades of experience in the design and manufacturing of active enzymes and drug discovery kits offers tools to screen inhibitors of specific enzymes and identification of a potential therapeutic targets. Check out our Successful Research Tips including our Top 10 Tips for Choosing an Antibody
, How to select a secondary antibody?
and What is Hybridoma Technology?
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for further assistance.