A recombinant antibody is a synthetic monoclonal antibody that is generated by recombinant DNA technology; an in vitro alternative to the traditional hybridoma-based technology for creating monoclonal antibodies. Recombinant antibodies are generated outside the immune system using synthetic genes and therefore do not require animal immunization for their production. These synthetic antibodies can be used in all applications and have several advantages compared to traditional animal-derived antibodies due to their high specificity, sensitivity, and reproducibility. In recombinant DNA technology, antibody genes encoding for heavy and light chain immunoglobulin fragments are amplified from antibody-producing cells, or synthesized artificially, and cloned into phage vectors. These vectors are then introduced into expression hosts such as bacteria, yeast, or mammalian cells for the production of the functional antibody.
Recombinant antibodies can come in different formats, including: full-length immunoglobulins (Ig), monovalent antibody fragments such as single-chain fragment variable (scFv) and fragment antigen-binding (Fab), and multimeric formats such as diabodies (dimeric scFvs) or triabodies (trimeric scFvs).
Single-chain fragment variable (scFv) is an antibody fragment composed only of the antibody’s binding site. scFv is the smallest form of a recombinant antibody that is still able to bind the antigen and, despite the removal of the constant regions, retains the specificity of the original Ig. scFv is a fusion protein obtained by recombining the light chains (VL) and heavy chains (VH) of immunoglobulins through a short peptide linker. Importantly, scFv fragments maintain the specificity of the entire monoclonal antibody and are very easy to generate in a prokaryotic expression system because they are rarely glycosylated.
Other miniaturized forms of recombinant antibodies include fragment antigen-binding (Fab), which is the antigen-binding region on an antibody composed of two sets of variable and constant components, and multimeric formats like diabodies (dimeric scFvs) or triabodies (trimeric scFvs). Each of these formats can have different utilities and can be used in several fields of research as well as in therapeutic applications. For example, Fab can be used to stabilize proteins in structural biology applications and scFv fragments can be used in the screening on the CAR in Car-T therapy or in microscopic applications due to their small size.
Importantly, recombinant technologies permit the generation of different antibody forms (i.e. Ig, scFc, Fab, etc.) which enables researchers to use the most suitable form for their application and recombinant antibodies, like classical monoclonal antibodies, can be used in all research applications including immunohistochemistry, immunofluorescence, immunocytochemistry, western blotting, and flow cytometry as well as in diagnostic and therapeutic applications.
Recombinant antibodies are produced through various genetic engineering / recombinant approaches, known as display technologies, such as: phage display, yeast display, and mammalian cell display. The most commonly used technique is phage display technology, an in vitro technology that displays recombinant proteins, in this case, antibody fragments, on the surface of filamentous bacteriophages (viruses that infect bacteria) such as M13.
The first step is to generate an antibody library containing a large number of phages, each carrying a different antibody gene, before screening these antibodies for their interaction with the target of interest. As an overview, a library of antibody genes encoding for heavy (VH) and light chain (VL) immunoglobulin fragments, amplified from antibody-producing cells or synthesized artificially, are cloned into an appropriate expression vector and displayed on the surface of filamentous bacteriophages as antibodies.
VH and VL cDNA are cloned into the bacteriophage M13 and used to infect Escherichia coli. Subsequently, a large collection of combined heavy and light chain fragments (the antibodies) are expressed and displayed on the bacteriophages. Each phage only contains one VH and VL pairing and therefore, taken together, there is a whole set of antibodies or antibody fragments (the antibody library).
Next, the antigen of interest is immobilized on a solid surface and the antibody library is applied on to it. In these conditions, non-specific and unbound phages are removed during the washing steps while specific phages, that remain bound to the immobilized antigen, are retained; providing a collection of highly reactive antibodies. Selected phages can then be amplified again in E. coli and new selection cycles, such as biopanning, can be applied to facilitate further enrichment of specific antibodies.
Finally, target-specific clones are amplified and screened for target binding by ELISA before these clones are further validated by cell sorting, DNA sequencing, or immunoblotting. At the end of this process, the DNA sequence of highly specific and versatile antibodies, derived from selected phages, can be used for recombinant antibody production.
Due to the limitations of prokaryotic cell folding machinery, only small antibody fragments, such as Fab’s, scFv’s, and diabodies, can be used for antibody phage display. For example, prokaryotic cells such as E. coli, Bacillus magaterium, and Bacillus subtilis have been used for the production of scFv antibodies. Prokaryotic cells cannot be used to produce full length antibodies because IgG-like antibodies require a complex and human-like glycosylation and bacteria cannot produce any type of glycans.
Recombinant antibodies can be also generated using yeasts (Saccharomyces cerevisiae and Pichia Pastoris), insect cell lines (order Diptera and Lepidoptera), plants (Daucus carota, Nicotiana tabacum BY2, and Nicotiana benthamiana), and mammalian cells (human embryonic kidney-HEK293, baby hamster kidney-BHK-2, chinese hamster ovary cells-CHO, and mouse myeloma-NS0).
Yeasts are able to perform glycosylation, however, the structure of these glycans is significantly different than those of human origin, as such, yeast systems are used for the production of antibody fragments that do not require post-translational modifications or IgG-like antibodies for research and analytical applications. Mammalian systems, such as CHO and HEK293 cell lines, that can perform human-like glycosylation, are commonly used to produce full-length recombinant antibodies. Plant and insect cell lines may also be used to produce recombinant antibodies and these systems have high potential for scalability and are low cost; for example, three different recombinant monoclonal antibodies have been developed against Ebola virus (ZMapp) using plant cells. Despite the fact that several biotherapeutics companies are producing recombinant antibodies in insect cells, little is known about antibody expression in this system. Insect systems cannot perform human-like glycosylation and, therefore, their use for production of full-length antibodies (Ig) is still very limited.
To summarize, therapeutic full-length Ig antibodies, which require complex post-translational modifications, are predominantly produced using stable mammalian expression systems whereas antibodies intended for diagnostics and research use, where it is possible to omit the complex glycosylation process, may be produced using bacterial and yeast expression systems.
Recombinant antibodies provide many important advantages compared to traditional antibodies, including:
The advantages of recombinant antibodies are confirmed by an increasing number of publications citing their use. Recombinant antibodies can be produced in different formats (i.e. Ig, scFc, Fab, etc.) enabling researchers to use the most suitable form for their application (i.e. flow cytometry, western blotting, immunoprecipitation, etc.). Importantly, antibody fragments such as scFv or Fab have complete binding specificity, since the antigen-binding surface is unchanged, and show better pharmacokinetics for tissue penetration due to their small size compared to full-length antibodies. Due to their good penetration, small multivalent scFv fragments are currently used as tumour-imaging reagents for solid tumours. In addition, recombinant antibodies are ideal for clinical use since they are derived from synthetic genes and, therefore, can be enginerred not to induce an immunogenic reaction.
Recombinant antibodies, like monoclonal antibodies, can be used for the treatment of several diseases including autoimmune disorders and cancer. For example, a recombinant monoclonal antibody called Bevacizumab (also known as Avastin) is currently used to treat breast, lung, and colorectal cancer.
In therapeutic applications, antibody fragments such as scFv can offer multiple advantages compared to full-length antibodies due to the fact that these antibody fragments are able to penetrate more rapidly into tumours and can be coupled with radionuclides and drugs. scFV recombinant antibodies can be also used as diagnostic reagents as they are able to bind to several different types of antigens including proteins, haptens, and pathogens and, as such, they can be used for antigen recognition and detection in immunoblotting or ELISA. Consequently, scFv recombinant antibodies are currently being investigated in multiple clinical trials as tools to treat and diagnose disease.
Recombinant antibodies have also been utilized to treat infectious diseases such as HIV-1, bacterial toxins, coronaviruses, and Ebola viruses. A very interesting recombinant antibody, known as Tocilizumab, is currently being used against the aberrant and excessive immune response induced by a high expression of cytokines, such as Interleukin 6 (IL-6), in SARS-CoV-2 infected patients. This recombinant humanized monoclonal antibody acts as an IL-6 receptor antagonist and can be used to interfere with IL-6 signaling.
The quality, consistency, and specificity of a research antibody is essential for the reliability and reproducibility of pre-clinical research. Recombinant antibodies have high specificity, high sensitivity, and excellent batch-to-batch reproducibility and their use in biomedical research decreases experimental variability and enables high reproducibility. Recombinant antibodies also display reduced cross-reactivity and provide a better signal-to-noise ratio, as such, recombinant antibodies are regarded as a better and more effective choice than their conventional hybridoma-derived counterparts.
Recombinant antibodies can be used for many research applications, including: western blot (WB), immunohistochemistry (IHC), immunofluorescence (IF), immunocytochemistry (ICC), enzyme-linked immunosorbent assay (ELISA), flow cytometry (FC), chromatin immunoprecipitation (ChIP), and many more applications. scFv antibodies are commonly used in immunohistochemistry and flow cytometry while Fab antibodies are commonly used in structural biology; where these fragment antibodies are used to stabilize protein complexes. For example, recombinant antibodies are commonly used for the crystallization of membrane proteins, such as GPRC, and soluble proteins, such as matriptase and B synthase. Fab antibodies are also used in electron cryomicroscopy (cryoEM) and in the field of proteomics.
Recombinant antibodies can also be used to eliminate background signal (also known as false-positive signal) in research applications. For example, unspecific binding of conventional antibodies to Fcγ receptors (FcRs) found on cells, such as macrophages, B cells, and dendritic cells, can produce unwanted false positive signals in flow cytometry experiments. Recombinant antibodies can have a mutated human IgG1 Fc region that prevents them binding to any FcRs expressed on cells, as such, recombinant antibodies can help researchers eliminate background signal in experiments.
Below we have listed some of our most popular recombinant antibodies for research use.