Recombinant Antibodies

Recombinant Antibody Production

Phage Display

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. Phage display is 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 a library containing millions of phages, each carrying a different antibody gene before screening these antibodies for their interaction with the target of interest. Heavy (VH) and light chain (VL) cDNA are cloned into the bacteriophage M13 and used to infect E. coli. Subsequently, a large collection of combined heavy and light chain fragments (the antibodies) are expressed and displayed on the bacteriophages, constituting the library.

Next, the antigen of interest is immobilized on a solid surface and the antibody library is applied to it, resulting in some antibodies binding to the antigen. Non-specifically bound and unbound phages are removed during the washing steps, while specifically bound phages remain bound to the immobilized antigen and are retained. These phages are then eluted, usually by a change in pH, providing a collection of highly reactive antibodies. The process can be repeated by amplifying selected phages in E. coli, binding, washing (usually in harsher conditions) and eluting to identify antibodies with the highest affinity for the target.

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 antibodies, derived from selected phages, can be used for recombinant antibody production.

Eukaryotic Display Technologies

Due to the limitations of prokaryotic cell folding machinery, only small antibody fragments, such as Fabs, scFvs, 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.

Yeasts, such as Saccharomyces cerevisiae and Pichia pastoris are able to perform glycosylation, but the structure of these glycans is significantly different than those of human origin. Yeast systems are therefore 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 that can perform human-like glycosylation, such as HEK293, BHK-2, CHO or NS0 cell lines, are commonly used to produce full-length recombinant antibodies. Plant (Daucus carota, Nicotiana tabacum BY-2, and Nicotiana benthamiana) and insect (order Diptera and Lepidoptera) 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, 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.

Advantages of Recombinant Antibodies

Recombinant antibodies provide many important advantages compared to traditional antibodies, including:

• No animal immunization required during production
• Can be generated against non-immunogenic antigens
• Highly pure product (~98%) that does not contain animal pathogens or animal serum components (e.g. IgGs, BSA, etc.)
• Known DNA and protein sequence, so have excellent batch-to-batch reproducibility and lot-to-lot consistency
• Genetically stable and do not suffer from problems such as genetic drift, antibody expression variation, and antibody sequence mutation that are observed in traditional hybridoma production and may cause non-specific binding
• Easier and quicker to produce than classical monoclonal antibodies. To produce a functional antibody, hybridoma technology requires ~4 months whereas recombinant antibody technology requires ~8 weeks
• High sensitivity, high specificity, and low immunogenicity
• Wide range of applications
• Can be conjugated with other molecules such as biotin, enzymes (HRP, AP, etc.), and fluorochromes (FITC, PE, APC, etc.) for research purposes, and toxins and drugs for therapeutic use
• Different formats are available such as minimized antibodies (scFv, Fab, etc.), which are not available with traditional antibody technologies. As such, it is possible to maintain the complete antigen-binding site, known as paratope, while reducing the size of the antibody
• Smaller in size resulting in improved tissue penetration, better blood clearance, and lower retention times in non-target tissues
• Can be subjected to isotype conversion, which means that a recombinant antibody fragment can be converted into several species, isotypes, and subtypes by changing the constant domain
• Can be genetically modified to obtain a greater affinity to a specific antigen or to reduce their capacity to induce an immunogenic reaction in therapeutic use

Types of Recombinant Antibodies

While recombinant antibodies can be used in all research applications that classical monoclonal antibodies can, the flexibility of recombinant technology permits the generation of different recombinant antibody formats, which have distinct advantages in some applications. These different formats include:

• Full-length immunoglobulins (Ig)
• Monovalent antibody fragments such as fragment antigen-binding (Fab) and single-chain fragment variable (scFv)
• Multimeric formats such as diabodies (dimeric scFvs) or triabodies (trimeric scFvs)

Fab Fragments

Fab fragments lack the Fc region of the antibody due to enzymatic cleavage at the hinge region. Fabs can be either monovalent (F(ab)) or divalent (F(ab’)₂) depending on how they are generated, with F(ab) fragments being generated after papain cleavage and F(ab’)₂ fragments retaining some of the Fc hinge region after pepsin cleavage. Fab fragments have substantially reduced non-specific binding that is typically a result of Fc region binding to Fc receptors on cells. They are also able to penetrate tissue more easily due to their small size, and can be used to stabilize proteins in structural biology applications.

Single-chain fragment variable (scFv) recombinant antibodies

An 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. In addition to standard antibody applications, scFv fragments can be used as domains of chimeric antigen receptors in CAR-T therapy, and have enhanced tissue penetration due to their small size meaning they can be used as tumor-imaging reagents for solid tumors.

Multimeric formats

Diabodies, triabodies and tetrabodies are multimeric units of scFvs. They offer significant opportunities for multifunctional design by linking scFvs with different targets together, for example to form bispecific antibodies. Due to their much smaller size than IgGs, they tend to be in circulation for less time before being cleared from the body, making them suitable for imaging applications.

Recombinant Antibodies for Cancer Diagnosis and Infectious Diseases

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 tumors 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 so can be used for antigen recognition and detection in immunoblotting or ELISA.

Recombinant antibodies have also been used 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.

Recombinant Antibodies for Pre-clinical Research

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 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 IHC and FC while Fab antibodies are commonly used in structural biology to stabilize protein complexes. For example, recombinant antibodies are commonly used for the crystallization of membrane proteins, such as GPCRs, and soluble proteins, such as matriptase and B synthase. Fab antibodies are also used in cryo-electron microscopy and in the field of proteomics.

Non-specific 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 experiments. Recombinant antibodies can have a mutated human IgG1 Fc region that prevents them binding to any FcRs expressed on cells, significantly reducing background signal.

Recommended Products

We offer a wide variety of mouse, rabbit, human, humanized and chimeric monoclonal recombinant antibodies. Below we have listed some of our most popular recombinant antibodies for research use.

References

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