How much do you know about IgG, Fab, scFv, and VHH?-Special Topic on Phage Display Technology-Tekbiotech-Yeast and Phage Display CRO, Expert in Nano-body and Antibody Drug Development

How much do you know about IgG, Fab, scFv, and VHH?

Cancer remains one of the major causes of human mortality. To improve the survival and cure rates of cancer patients, scientists have proposed tumor-targeted therapy. Monoclonal antibodies (mAbs) represent the first generation of antibody drugs. However, after long-term clinical application and research, the limitations of mAbs have gradually been discovered. One major issue is that mouse monoclonal antibodies, when introduced into the human body, can cause severe allergic reactions and are rapidly eliminated. Another limitation is the large size (four polypeptide chains) and long half-life of mAbs. With technological advancements, smaller and modular antibody fragments have emerged.

IgG

As shown in the figure, IgG is composed of two identical light chains and two identical heavy chains. Both the light and heavy chains, as well as the heavy chains themselves, are connected via disulfide bonds^[1](Figure 1A). The heavy and light chains are further subdivided into constant regions and variable regions (VH and VL), with three CDRs within the variable region responsible for antigen binding.

Figure 1: Schematic diagram and functions of IgG.

Application: IgG possesses comprehensive functions, including both antigen targeting (via Fab) and effector functions (via Fc) capable of activating the immune system (e.g., ADCC, CDC). It is the mainstay of therapeutic antibody drugs and can be used to generate ADC drug molecules.

Fab

Fab fragments can be obtained through enzymatic reaction. Digestion of a complete antibody with papain results in its breakdown into two identical Fab fragments and one Fc fragment. Fab consists of one complete light chain, along with the variable region and CH1 domain of a heavy chain.

Figure 2: Schematic diagram of Fab

Application: These fragments can be synthesized in vitro ^[3] (Figure 3). Removal of the Fc region is primarily to ensure targeted antigen binding without activating the immune system, thereby reducing immunogenicity. When used as therapeutics, they can act and be cleared rapidly. Another major application is in diagnostics and imaging.

Figure 3: In vitro synthesis of Fab

scFv

scFv consists of VH + VL, connected by a linker peptide. The flexible peptide linker, rich in serine and glycine, ensures the structural stability of the scFv. The monovalent antigen-binding scFv possesses complete specificity for a single antigen, demonstrating promising prospects in cancer therapy.

Application: Composed of the variable regions of the light and heavy chains, scFv has a simple structure, is easy to assemble, and can be modularly designed, making it a perfect building block for constructing multi-functional antibodies. Its main applications are in CAR-T cell therapy, bispecific antibodies (typically composed of two scFvs), and immunotoxins (where scFv is fused to a toxin protein).

Figure 4: Schematic diagram of scFv and two connection formats

Nanobody (VHH)

Nanobodies are single variable domains isolated from naturally occurring heavy-chain antibodies found in camelids using phage display technology, representing the smallest antigen-binding fragments. Similar single-domain antibodies have also been discovered in sharks. Compared to conventional antibodies, nanobodies lack the CH1 domain and therefore cannot associate with light chains. Furthermore, the CDR3 region of nanobodies is longer than that of conventional antibodies, and the concave spatial structure of CDR3 facilitates antigen binding ^[2].

Application: VHHs possess superior penetration capability, capable of crossing the blood-brain barrier for trans-boundary therapy and molecular imaging. Due to their high stability, they can be used to generate intrabodies for neutralizing toxins.

TekBiotech, based on phage display and yeast display technologies, has established a comprehensive targeted antibody drug discovery platform. We provide high-quality monoclonal antibody development services for scientists worldwide, covering various formats including scFv, VHH, and Fab. Furthermore, we can develop antibodies with diverse functional and structural characteristics, including but not limited to neutralizing antibodies, conformation-specific antibodies, and cross-reactive antibodies. Additionally, TekBiotech offers supporting downstream services such as antibody expression validation, antibody humanization design and validation, antibody affinity maturation, and CAR-T candidate sequence design, meeting the diverse needs of clients for antibody drug development.

References

[1] Guzzeloni V, Veschini L, Pedica F, Ferrero E, Ferrarini M. 3D Models as a Tool to Assess the Anti-Tumor Efficacy of Therapeutic Antibodies: Advantages and Limitations. Antibodies (Basel). 2022 Jul 8;11(3):46.

[2] Zhao X, Ning Q, Mo Z, et al. A promising cancer diagnosis and treatment strategy: targeted cancer therapy and imaging based on antibody fragment. Artif Cells Nanomed Biotechnol. 2019;47(1):3621-3630.

[3] Kim JH, Sim DW, Park D, et al. Bacterial production and structure-functional validation of a recombinant antigen-binding fragment (Fab) of an anti-cancer therapeutic antibody targeting epidermal growth factor receptor. Appl Microbiol Biotechnol. 2016;100(24):10521-10529.

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