Phage Display Derived Monoclonal Antibodies: From Bench to Bedside

Abstract

Monoclonal antibodies (mAbs) have become one of the most important classes of biopharmaceutical products, and they continue to dominate the universe of biopharmaceutical markets in terms of approval and sales. They are the most profitable single product class, where they represent six of the top ten selling drugs. At the beginning of the 1990s, an in vitro antibody selection technology known as antibody phage display was developed by John McCafferty and Sir. Gregory Winter that enabled the discovery of human antibodies for diverse applications, particularly antibody-based drugs. They created combinatorial antibody libraries on filamentous phage to be utilized for generating antigen specific antibodies in a matter of weeks. Since then, more than 70 phage-derived antibodies entered clinical studies and 14 of them have been approved. These antibodies are indicated for cancer, and non-cancer medical conditions, such as inflammatory, optical, infectious, or immunological diseases. This review will illustrate the utility of phage display as a powerful platform for therapeutic antibodies discovery and describe in detail all the approved mAbs derived from phage display.

Keywords: antibody libraries; biopanning; biopharmaceuticals; monoclonal antibodies; phage display.

Figure 1

Schematic representation of different antibody formats. (A) Single chain fragment variable (scFv) composed of variable regions of the light chain (V_L) linked to variable regions of the heavy chain (V_H) by a flexible glycine-serine linker (Gly₄Ser)₃. (B) Nanobody fragments. (C) Fragment of antigen binding (Fab) composed of V_L and a constant domain of the light chain (CL) linked to V_H and constant domain 1 of the heavy chain (CH1) by a disulphide bond between the CL and CH1 domains. (D) Diabody composed of V_L linked to variable heavy V_H by a pentameric (Gly₄Ser). (E) F(ab)2 fragment composed of 2 × Fab fragments joined by an Immunoglobulin G (IgG) hinge region. (F) scFv fusion with an Fc IgG. (G) IgG composed of constant fragment (Fc), which is able to bind and stimulate immune effector cells, and Fab, which comprises the variable domains that contain the antigen binding regions. (H) Bispecific IgG antibody.

Figure 2

Strategy used for construction of naïve scFv-phage display libraries. Total RNA is isolated from B-lymphocytes from non-immunized healthy donors. Then cDNA is synthesized from the isolated RNA using reverse transcriptase enzyme. Then the repertoire of the V_H and V_L genes is amplified from the cDNA using forward and reverse primers hybridizing to the variable domains. scFvs are constructed and cloned into phagemid vector and a naïve phage library of 10⁸-10¹⁰ is usually generated.

Figure 3

Schematic representation of phage biopanning. This is the basic method for sequential affinity screening of the phage display libraries for specific binding phage from a large excess of non-binding clones is often referred to as “panning or biopanning.” The phage antibody selection involves the immobilization of the ligand of interest on a solid support, followed by applying the phage display library (in the form of purified virions) to the immobilized ligand to allow binding of specific variants. To eliminate the adherent non-binders, multiple rounds of washing are usually performed, and remaining bound phages are eluted and re-amplified. At least three rounds of biopanning are usually required in order to amplify the binding variants and to exclude any non-specific binders.

Figure 4

Highest development phase achieved for antibodies isolated from various major company-owned libraries.

Figure 5

Indications for antibodies isolated from various major company-owned libraries. Clinical domains (cancer vs. non-cancer) of the approved or in clinical trials antibody-derived phage display.