Resources Blog Antibody Basics: Part 4​ - Antibody formats: Single-Chain Variable Fragments (scFvs)

Antibody Basics: Part 4​ - Antibody formats: Single-Chain Variable Fragments (scFvs)

Biointron 2024-02-19 Read time: 10 mins

Welcome back to Antibody Basics by Biointron, part 4. In this episode, we’ll continue to cover antibody formats, specifically, Single-Chain Variable Fragments (scFvs).

What are single-chain variable fragments?

A form of engineered antibody fragment that have become increasingly important in therapeutic and diagnostic applications due to their unique structural and functional properties. scFvs consist of the variable regions of the heavy (VH) and light (VL) chains of immunoglobulins, connected by a short, flexible linker peptide. This configuration maintains the antigen-binding specificity of conventional antibodies while offering a smaller and versatile structure.

The emergence and evolution of scFvs:

  • 1988: Huston et al., designed an scFv with a single polypeptide connected with a 15 amino acid linker. It showed clear specificity against digoxin.

  • 1990s: Refinement of scFv technology, addressing issues such as stability and solubility. There was increasing interest in potential applications in diagnostics and therapeutics.

  • Early 2000s: Growing use of scFvs in diagnostic applications, including immunoassays and imaging, in addition to therapeutic purposes, especially in cancer targeting.

  • Mid 2000s: Introduction of bispecific scFvs, capable of binding two different antigens simultaneously.

  • 2010s - present: scFvs began entering clinical trials for therapeutic applications, with ongoing research to improve stability, bioavailability, and reduce potential immunogenicity.

Advantages over conventional formats

Traditional antibodies are Y-shaped molecules composed of two identical heavy chains and two identical light chains. Each arm of the Y structure contains a variable region (VH and VL) responsible for antigen recognition. The stem of the Y comprises constant regions that mediate immune responses.

  • Size: Being significantly smaller, scFvs can penetrate tissues more effectively, reaching targets that are inaccessible to full-sized antibodies.

  • Production: scFvs can be produced rapidly and inexpensively in various expression systems, including bacteria, yeast, and mammalian cells.

  • Flexibility and Adaptability: The genetic manipulation of scFvs is simpler, allowing for the creation of customized antibody fragments with specific properties, such as increased stability or altered specificity.

  • Reduced Immunogenicity: The immunogenicity generated by the Fc portion of the antibody is absent in the conventional scFv molecule.

Possible expression systems:

Bacterial: E. coli is the most commonly used host due to its simplicity, rapid growth, low-cost, and high yields up to 10–30% of total cellular protein. However, scFvs produced in bacteria may require proper folding and might lack glycosylation, which is essential for some applications. 

Yeast: Yeasts, like Pichia pastoris, offer a middle ground, providing post-translational modifications and higher yields than bacterial systems. 

Mammalian Cell Lines: Systems like Chinese Hamster Ovary (CHO) cells are used when complex post-translational modifications, such as glycosylation, are necessary for the scFv's function.

Plant: Galeffi et al. successfully produced recombinant scFvs to ErbB-2 with functional expression in stable transgenic tobacco plants.

Insect: Choo et al. expressed a functional recombinant cytolytic immunotoxin, scFv-mel-FLAG, in Spodoptera frugiperda ovarian cells.

Display libraries for scFvs:

Phage Display Libraries: Phage display involves displaying scFvs on the surface of bacteriophages, allowing for the rapid selection of scFvs with high affinity for specific antigens. Immune libraries: Constructed from variable domains of antibody genes of B cells derived from immunized animals e.g. mice, camel, sheep, and humans. Naïve libraries: Derived from nonimmunized donors of B cells constructed from a pool of V-genes of IgM mRNA. These libraries are not biased towards any antigen. Synthetic libraries: Derived from nonimmune sources and prepared synthetically by combining germ line gene sequences together with randomized complementary determining regions (CDRs) that are responsible for antigen binding.

In vitro Ribosomal Display Technology: Ribosome display technology (RDT) is a cell-free system that overcomes many limitations of cell-based methods by producing in vitro protein-mRNA complexes. The method is more efficient when screening large libraries as it does not compromise transformation efficiency, selecting high-affinity combining sites, and eukaryotic cell-free systems, which are capable of post-translational modifications. DNA library of scFv is transcribed and translated in vitro to create the complex for selection on immobilized antigen. The mRNAs specifically bound to the antigen are eluted, followed by reverse transcription, and further selection from the enriched regenerated DNA pool.

Diagnostic applications

ScFvs are diagnostic reagents in several different assay formats, including ELISA tests. They can bind to a variety of molecules, e.g. haptens, proteins, whole pathogens. They have improved stability through protein fusions, e.g. constant light chain domain, leucine zipper dimerization domain (ZIP), Fc fragments, alkaline phosphatase. Detection can be done using secondary antibodies recognizing specific tags fused to the C- or N-terminus of the scFv, e.g. c-myc, E-tag (Pharmacia). Improved protein folding via phage format in ELISA assays - the scFv remains attached to the coat protein of filamentous phage. With new phage display antibody libraries, one can generate a range of scFvs specifically directed against any antigen economically, quickly, and without the bother of immunizing animals and manipulating hybridomas.

Therapeutic applications: Cancer

ScFvs have revolutionized the landscape of targeted therapy, particularly in oncology. Their ability to bind specifically to tumor antigens has led to the development of highly effective, minimally invasive treatments. Examples include:

  • Antibody-Drug Conjugates (ADCs): These are scFvs linked to cytotoxic drugs. The scFv guides the drug directly to cancer cells, improving efficacy and reducing systemic toxicity.

  • Bispecific Antibodies: Comprising two different scFvs, these molecules can engage two different antigens simultaneously, such as a tumor antigen and a T-cell activator, enhancing the immune response against cancer cells.

Only 3 scFv-related therapeutics are approved worldwide, with 3 more in review

  • Blinatumomab (Blincyto): Approved by FDA in 2014. Bispecific tandem scFv targeting CD19 and CD3. Treats acute lymphoblastic leukemia.

  • Brolucizumab (Beovu): Approved by FDA in 2019. scFv targeting VEGF-A. Treats neovascular age-related macular degeneration.

  • Tebentafusp (Kimmtrak): Approved by FDA in 2022. Bispecific scFv-TCR fusion protein targeting gp100 and CD3. Treats metastatic uveal melanoma.

  • Currently in review: Ivonescimab: Bispecific antibody for lung cancer; Tarlatamab: Bispecific T-cell engager for small cell lung cancer; Zanidatamab: Bispecific antibody for biliary tract cancers.

Biointron focuses on antibody discovery, expression, and optimization.


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