Antibodies, also known as immunoglobulins, are essential components of the immune system responsible for recognizing and neutralizing foreign invaders, such as bacteria, viruses, and other pathogens. Traditionally, full-length antibodies have been the primary focus in immunotherapy and diagnostic applications. However, recent advancements have shed light on a smaller yet mighty alternative: antibody fragments. These fragments are compact, versatile, and hold immense potential in various biomedicine fields. This article explores the structure, types, production, and diverse applications of antibody fragments.

What are Antibody Fragments?

Antibody fragments are compact and versatile molecules derived from full-length antibodies, integral components of the immune system. These fragments result from the separation of the antigen-binding domains from the constant regions of the parent antibodies. Antibody fragments maintain the crucial antigen-binding specificity and affinity, while their smaller size grants them unique advantages in various biomedicine fields.

Types of Antibody Fragments

Based on the basic Y-shaped antibody structure, various antibody fragment types can be generated, including several engineered forms. Each type of antibody fragment offers distinct advantages, and their diverse properties have paved the way for innovative approaches in diagnostics, therapeutics, and other biomedical applications. Here are some representative types of antibody fragments:

Fig 1. Structures of the selected antibody fragments.

• Fab (Fragment antigen-binding)

Fab fragments are composed of a single antigen-binding region derived from the variable heavy (VH) and variable light (VL) chains of the antibody. These fragments retain the ability to specifically bind to antigens, enabling them to target and recognize foreign invaders or disease-related biomarkers.

• F(ab')2

F(ab')2 fragments consist of two linked Fab regions and are generated by enzymatic digestion of full-length antibodies with pepsin. Each F(ab')2 fragment contains two antigen-binding sites, making them valuable in crosslinking antigens and facilitating certain diagnostic and therapeutic applications.

• Fc (Fragment crystallizable)

Fc fragments lack the antigen-binding region but retain the constant region of the antibody. This portion is responsible for mediating various immune effector functions, such as antibody-dependent cell-mediated cytotoxicity (ADCC) and complement activation. Fc fragments play a crucial role in enhancing the immune response and promoting the removal of pathogens.

• Single-Chain Variable Fragment (scFv)

scFv is a recombinant antibody fragment that combines the variable heavy and light chains (VH and VL) into a single polypeptide chain. The VH and VL regions are connected by a short peptide linker, maintaining their antigen-binding affinity. scFv can be produced in microbial systems and has applications in diagnostics, targeted therapy, and imaging due to its small size and ease of manipulation.

• Single-Domain Antibodies (sdAbs)

Single-domain antibodies are derived from naturally occurring heavy-chain-only antibodies found in camelids and some other animals. These compact antibody fragments consist of a single variable domain (VHH) that can recognize and bind to specific antigens. Because of their small size and unique stability, sdAbs have gained popularity in various biomedical applications, especially in therapeutics and diagnostics.

• Bispecific Antibody Fragments

Bispecific antibody fragments are engineered to simultaneously target two different antigens. These fragments combine the antigen-binding specificities of two different antibodies or antibody domains into a single molecule. Bispecific fragments are designed to facilitate novel therapeutic strategies, such as redirecting immune cells to tumor cells or enhancing the targeting of multiple pathogens simultaneously.

Production of Antibody Fragments

Production of antibody fragments involves various techniques and methodologies to obtain these compact yet potent molecules. These methods are carefully designed to preserve the specificity and affinity of the parent antibodies while ensuring their functional integrity.

• Proteolytic Digestion

Proteolytic digestion is a classical method used to generate antibody fragments, specifically Fab and F(ab')2 fragments. This process involves the controlled enzymatic cleavage of full-length antibodies using proteolytic enzymes like papain or pepsin.

Fab Fragment: Papain, a proteolytic enzyme derived from the papaya plant, is used to cleave antibodies into two identical antigen-binding fragments known as Fab fragments. These Fab fragments retain the variable regions (VH and VL) responsible for antigen recognition.

F(ab')2 Fragment: On the other hand, pepsin, an enzyme found in the stomach, can cleave antibodies below the hinge region, generating F(ab')2 fragments. Each F(ab')2 fragment contains two antigen-binding regions connected by disulfide bonds.

• Recombinant Expression

Advancements in genetic engineering have revolutionized the production of antibody fragments through recombinant expression systems. Recombinant technology allows scientists to express specific antibody fragments in various host systems, providing numerous advantages:

Bacterial Expression: Antibody fragments, especially single-chain variable fragments (scFv), can be efficiently produced in bacteria, such as Escherichia coli. Bacterial expression systems are cost-effective and highly scalable, making them ideal for large-scale production.

Yeast Expression: Yeast, such as Saccharomyces cerevisiae, is another popular host for antibody fragment production. Yeast systems offer post-translational modifications, enabling proper folding and glycosylation of the antibody fragments.

Mammalian Cell Expression: Mammalian cell lines, including Chinese hamster ovary (CHO) cells, are widely used for the production of more complex antibody fragments. Mammalian cells ensure proper folding, assembly, and glycosylation, which are crucial for maintaining antibody fragment functionality.

• Phage Display Technology:

Phage display is an innovative and powerful technique that allows the selection of antibody fragments with high specificity and affinity for a particular target. This technique involves displaying a diverse library of antibody fragments on the surface of bacteriophages (viruses that infect bacteria) and subsequently isolating phages that bind to the desired antigen. A standard phage display includes three main steps:

Library Construction: Antibody fragment libraries are generated by combining various VH and VL sequences to create diverse repertoires of antibody fragments.

Biopanning: The selected antibody fragments are subjected to rounds of biopanning, where they are incubated with the target antigen and non-specifically bound phages are removed. The remaining phages, harboring antigen-specific antibody fragments, are then eluted and amplified for further selection rounds.

Antibody Characterization: The isolated antibody fragments from the phage display library are characterized for specificity, affinity, and binding kinetics. Promising candidates can be further engineered for improved functionality and tailored for various applications.

All listed services and products are For Research Use Only. Do Not use in any diagnostic or therapeutic applications.