David vs. Goliath: The Structure, Function, and Clinical Prospects of Antibody Fragments

Abstract

Since the licensing of the first monoclonal antibody therapy in 1986, monoclonal antibodies have become the largest class of biopharmaceuticals with over 80 antibodies currently approved for a variety of disease indications. The development of smaller, antigen binding antibody fragments, derived from conventional antibodies or produced recombinantly, has been growing at a fast pace. Antibody fragments can be used on their own or linked to other molecules to generate numerous possibilities for bispecific, multi-specific, multimeric, or multifunctional molecules, and to achieve a variety of biological effects. They offer several advantages over full-length monoclonal antibodies, particularly a lower cost of goods, and because of their small size they can penetrate tissues, access challenging epitopes, and have potentially reduced immunogenicity. In this review, we will discuss the structure, production, and mechanism of action of EMA/FDA-approved fragments and of those in clinical and pre-clinical development. We will also discuss current topics of interest surrounding the potential use of antibody fragments for intracellular targeting and blood-brain barrier (BBB) penetration.

Keywords: ADC; BiTE®; ImmTAC®; Nanobody®; TandAb; V-NAR; antibody fragments; diabodies; domain antibodies; fab; scFv.

Figure 1

The single chain fragment variable format. The C-terminus of the light chain (VL) is linked the N-terminus of the heavy chain (VH) by a flexible glycine- and serine- rich linker.

Figure 2

The tandem scFv platform. (A) A monospecific bivalent tandem scFv composed of two identical scFvs joined by a helical linker. (B) A bispecific bivalent scFv composed of two different scFvs joined by a helical linker.

Figure 3

The structures of diabody, DART^®, and TandAb fragments. (A) A bispecific diabody composed of two different chains, each containing a VL and VH from different antibodies, in a head-to-tail arrangement. (B) A bispecific dual affinity re-targeting (DART^®) protein containing two distinct polypeptide chains held together by non-covalent interactions and a disulphide bond. (C) A TandAb composed of two diabodies linked in a linear arrangement to produce a tetravalent bispecific molecule.

Figure 4

scFv fusion bispecific formats with an Fc domain. (A) IgG-scFv. Canonical IgG with a scFv fused to the C-terminus of the CH3 domain to produce a tetravalent bispecific molecule. (B) Fab-scFv-Fc IgG with one IgG Fab arm exchanged for a scFv.

Figure 5

The structure of Fab and F(ab’)₂ fragments. (A) Fab fragment composed of an LC (containing VL and CL) linked to an Fd (containing VH and CH1) by a disulphide bond between the CL and CH1 domains. (B) F(ab’)₂ fragment composed of two Fab fragments joined by an IgG hinge region.

Figure 6

Camelid heavy-chain IgG and Nanobody^® fragments. (A) The structure of heavy-chain IgG, composed of two heavy chains, each containing a VHH domain, a CH2 domain, and a CH3 domain. (B) Mono-, bi-, and tri-valent Nb formats with each VHH having a different antigen specificity. (C) A Nanobody^® drug conjugate.

Figure 7

Shark heavy chain antibody (Ig-NAR), a dimer of heavy chains containing five constant domains and the antigen binding variable nucleotide antigen receptor (V-NAR).

Figure 8

The uses of domain antibodies (dAbs). (A) IgG-dAb (also called a mAb-dAb). IgG with a dAb fused to the C terminus of each heavy chain to produce a bispecific tetravalent molecule. (B) Tandem dAb with an anti-HSA domain. The dAb against the target of interest is linked to an anti-HSA dAb to improve half-life. (C) AlbuDab^®. A peptide linked to an anti-HSA dAb to improve the peptide’s half-life.

Figure 9

Simplified purification workflow for different fragment formats. A series of stages that can be used to purify common antibody fragments based on the presence of certain domains. The workflow typically includes affinity chromatography (where applicable) followed by multi-modal polishing stages, which may include size exclusion chromatography (SEC), cation (CIEX) or anion (AIEX) exchange, and hydrophobic interaction chromatography (HIC). Antibody fragments can also be purified using cation exchange chromatography (CIEX) or immobilised metal affinity chromatography (IMAC) as the initial capture step.

Figure 10

The structure and mechanism of action of MT103, an $\alpha$-CD3/$\alpha$-CD19 bispecific T-cell engager (BiTE^®). The antigen binding site of each parental antibody is isolated and converted into an scFv format. The two scFvs are then joined by a flexible peptide linker to produce a bispecific moiety. The anti-CD19 scFv binds to tumour cells whilst the anti-CD3 scFv will bind passing T-cells, re-directing them to attack the tumour cell.

Figure 11

The structure and mechanism of action of ImmTAC^®s. (A) The structure of an ImmTAC^®, comprising an $\alpha$-CD3 scFv linked to a disulphide-stabilised, affinity-enhanced soluble T-cell receptor. (B) The T-cell receptor binds its target with picomolar affinity causing the ImmTAC^® to cluster on the target cell. The anti-CD3 scFv then recruits passing T-cells by binding CD3 with nanomolar affinity. The clustering of CD3 on the T-cell leads to activation and re-direction of the T-cell to produce an immune response against the target cell.

Figure 11

The structure and mechanism of action of ImmTAC^®s. (A) The structure of an ImmTAC^®, comprising an $\alpha$-CD3 scFv linked to a disulphide-stabilised, affinity-enhanced soluble T-cell receptor. (B) The T-cell receptor binds its target with picomolar affinity causing the ImmTAC^® to cluster on the target cell. The anti-CD3 scFv then recruits passing T-cells by binding CD3 with nanomolar affinity. The clustering of CD3 on the T-cell leads to activation and re-direction of the T-cell to produce an immune response against the target cell.