FAB PHAGE DISPLAY SCREENING SERVICE

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What are fragment antibodies (Fab) ?

Fab fragments, with a size of 50 kDa, represent the antigen binding fragment of a full-length antibody. They contain both the variable region of heavy chain (VH), a variable region of light chain (VL), a constant region of heavy chain 1 (CH1) and a constant region of light chain (CL). The fragments which contain disulfide bridge thiols are named Fab’ fragments, whereas those lacking the thiol functional group are called Fab fragments.

How are Fab antibodies generated?

There are three different methods to generate Fab fragments:

-Enzymatic cleavage of the native parent antibody:

Fab fragments can be generated by the enzymatic cleavage of the parent antibody (IgG) using papain, a cysteine protease. This enzymatic digestion leads to a separation of the 150 kDa antibody molecule into two 50 kDa fragments that bind the antigen (“fragment antigen-binding” Fab) and one that does not bind the antigen (“fragment crystallizable” Fc).

-Recombinant technology

Other Fab antibodies can be “synthesized” by recombinant technologies, leading to recombinant Fab antibodies, which sometimes might be different from the native Fab fragments. These methods encompass bacterial expression systems, using E. coli, usually used to get high antibody yields, or mammalian expression systems, such as CHO and HEK293 cells. Mammalian expression systems are the most widely used systems to keep the native properties of the Fab antibodies, for instance, the post-translational modifications that cannot occur in bacterial systems.

-Phage display screening

Nowadays, most recombinant antibody fragments are generated by phage display technology. This method is advantageous as it allows an easy and efficient selection of new Fab antibodies with higher binding affinities. It helps also selecting highly stable antibodies for a long-term storage, in a time and cost-efficient manner, when compared with other Fab antibody generating methods.

What are Fab applications in research and clinics?

Fab antibodies are used as powerful molecular tools in therapeutic and clinical applications as well as in research, thanks to their several advantages. First, their small size allows them a fast and efficient deep tissue penetration and clearance. Moreover, lacking the Fc-mediated effector functions reduces immunogenicity and immune reactions. In addition, the monovalent format of Fab and Fab’ antibodies lowers cross-linked immunocomplexes that can trigger anaphylaxis. To date, many Fab antibodies have received, or continue receiving FDA approval for a wide range of uses, mainly therapeutic, clinical, diagnostic and research applications. Here is a non-exhaustive list of some Fab applications reported to date:

-Fab in Protein crystallization

Fab proteins facilitate the determination of three-dimensional structures of hydrophobic proteins. They can create hydrophilic surfaces facilitating crystal contact formation essential for crystallization of hydrophobic proteins or transmembrane proteins having a hydrophobic micelle. They can also “Freeze” or “lock-in” some protein conformations, thus helping in their crystallization. Moreover, as they lack the Fc fragment and the hinge connecting the Fc and Fab fragments, interfering with crystallization, they act as “crystal chaperones” and facilitate the protein crystallization.

-Fab as antidote / antivenom

Fabs are used in emergency medicine as an antidote or antivenom. These Fab antidotes are generated from IgG enzymatic digestion. They are preferred to IgG antibodies mainly for their wide and fast distribution, low immunogenicity and their weak risks of inducing anaphylaxis following intravenous injections. Some of Fab antibodies which received the FDA approval and used in emergency medicine include: CroFab used as antivenom for rattlesnake bites and DigiFab which is an anti-Digoxin Fab antibody indicated in clinical toxicology, to treat patients who ingested fatal doses of cardiac glycoside digoxin.

-Fabs in therapeutics

Other Fabs are used in therapeutics to prevent or treat some diseases. These Fab antibodies include: ReoPro (abciximab) used as blood clotting inhibitor, which acts by binding to human integrin αIIbβ3 and thus, blocks the platelet aggregation; Ranibizumab (Lucentis) is used to treat Macular degeneration which targets vascular endothelial growth factor, involved in the blood vessel growth; Certolizumab pegol is a PEGylated Fab proposed to treat Crohn’s disease and rheumatoid arthritis by targeting the TNFα.

-Fabs in diagnostics

Fab antibodies have been modified to serve in diagnostic imaging of various cancers. Thanks to their shorter circulatory half-life properties, which makes them less toxic and fast cleared, these antibody fragments are preferred to radiolabeled IgGs. For instance, Arcitumomab is a Fab antibody that recognizes carcinoembryonic antigen overexpressed in 95% of colorectal cancers. This Fab fragment is radiolabeled with 99mTc and is used in diagnostic imaging to detect metastatic cancers.