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 that affect its functionality, recombinant antibodies produced in phage display maintain a high standard of specificity and low immunogenicity.
Structure and characterization
Formats There are several known formats of recombinant antibodies which are commonly produced. These are the Fab recombinant antibodies, scFv and diabodies. Each of the formats has a slightly different potential in applications and may be used in various fields of research as well as human and animal medicine. Another researched possibility is the development of anti-idiotypic antibodies. Anti-idiotypic antibodies bind to a paratope of another specific antibody. Therefore, it can be used for measuring presence of antibodies and drug loads in patients' sera. Based on their binding specificity 3 types of anti-idiotypic antibodies can be distinguished, which partially overlap with the previously mentioned formats: the classical ones, a group including Fab fragment antibodies, antibodies binding to idiotope outside of the drug binding site and antibodies, which only bind to the already assembled complex of drug bound to the target. The most commonly used are the scFv, Fab fragments and bispecific antibodies.
Single chain variable fragment (scFv) scFv is the smallest of the recombinant antibody formats, which is capable of antigen binding. They have a molecular weight of approximately 27kDa. They are formed by light and heavy chain of the variable region of an immunoglobulin. The two chains are linked by a flexible peptide linker. The flexible peptide linker usually consists of short sequence repetition. The sequence is made up of four glycines and a serine and it serves the purpose of stabilization of the fragment. The functionality may be enhanced by site-specific chemical modifications, adding a peptide-tag or by fusion with a gene to achieve production of bifunctional recombinant antibodies. It is important to establish the binding activity in order to ensure good functionality of the product. To determine the binding activity, ELISA assay is routinely performed.
Fab fragments Structurally Fab fragments consist of two sets of variable and constant components, which create two polypetide chains. Together they form a stable structure. As a member of the anti-idiotypic antibodies, Fab fragment recombinant antibodies bind directly to the paratope of the target antibody. That means that they compete with the drug for binding site and have an inhibitory function. Fab fragment antibodies can be used for detection of not bound drugs or free drugs in the serum. Fab antibodies have also been used to avoid the adverse effects caused by unspecific binding of the Fc portion of the antibody, which is missing in the Fab fragment. In case the IgG immunoglobulin was more suitable for the treatment or some other particular application, experiments have also been conducted, in which the recombinant Fab fragments were converted into recombinant IgG form. This possibility further broadens the pool of potential target structures.
Bispecific recombinant antibodies Along scFv and Fab fragments, diabodies or bispecific recombinant antibodies are the third major format. Bispecific antibodies combine two different antigen binding specificities within one molecule. The bispecific antibodies are used to crosslink the target molecules with two different cells and mediate direct cytotoxicity.
Production and development
Production of recombinant antibodies The production of recombinant antibodies follows principally similar workflow. It consists of determining the sequence of the desired product followed by refinement of the codon, then gene synthesis and construct generation. Once the construct is delivered to the laboratory, expression constructs are produced, then they are transferred to a cell culture in the process called transfection and once the cell culture produces the desired recombinant antibody, it is regularly collected, purified and analyzed or used for further experimentation. For recombinant antibody production the stable cell lines such as CHO and HEK293 are used. Optimizations of mammalian cell cultures have led to increase the yield of antibodies from HEK293 or CHO cell lines to over 12g/liter. In the beginning phases of the recombinant antibody production it was important to achieve the assembly of a functional Fv fragment in Escherichia coli. The correct fold is essential for functionality of the antibody. Second essential prerequisite for the modern day production of scFv was the successful assembly of recombinant antibodies from heavy and light chain of immunoglobulin. These two experiments allowed for further development and refinement of the recombinant antibodies until modern day form. Today's in vitro production process eliminates the need for laboratory animals. Using a synthetic or human Ab library, as opposed to immunization of animals and the subsequent generation of stable hybridoma cell lines, requires fewer resources and produces less waste, making the entire process more sustainable.
Hybridoma
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