In vivo Phage Display: A promising selection strategy for the improvement of antibody targeting and drug delivery properties

Abstract

The discovery of hybridoma technology, described by Kohler and Milstein in 1975, and the resulting ability to generate monoclonal antibodies (mAbs) initiated a new era in antibody research and clinical development. However, limitations of the hybridoma technology as a routine antibody generation method in conjunction with high immunogenicity responses have led to the development of alternative approaches for the streamlined identification of most effective antibodies. Within this context, display selection technologies such as phage display, ribosome display, yeast display, bacterial display, and mammalian cell surface display have been widely promoted over the past three decades as ideal alternatives to traditional hybridoma methods. The display of antibodies on phages is probably the most widespread and powerful of these methods and, since its invention in late 1980s, significant technological advancements in the design, construction, and selection of antibody libraries have been made, and several fully human antibodies generated by phage display are currently approved or in various clinical development stages. With evolving novel disease targets and the emerging of a new generation of therapeutic antibodies, such as bispecific antibodies, antibody drug conjugates (ADCs), and chimeric antigen receptor T (CAR-T) cell therapies, it is clear that phage display is expected to continue to play a central role in antibody development. Nevertheless, for non-standard and more demanding cases aiming to generate best-in-class therapeutic antibodies against challenging targets and unmet medical needs, in vivo phage display selections by which phage libraries are directly injected into animals or humans for isolating and identifying the phages bound to specific tissues offer an advantage over conventional in vitro phage display screening procedures. Thus, in the present review, we will first summarize a general overview of the antibody therapeutic market, the different types of antibody fragments, and novel engineered variants that have already been explored. Then, we will discuss the state-of-the-art of in vivo phage display methodologies as a promising emerging selection strategy for improvement antibody targeting and drug delivery properties.

Keywords: antibody discovery; antibody engineering; antibody selection; in vivo; phage display; therapeutic antibodies.

Figure 1

(A) Schematic representation of a conventional IgG antibody. Immunoglobulin G (IgG) have approximately 150 kDa and consist of two identical light chains (L) and two identical heavy chains (H) connected by disulfide bonds. Light chains are made up of a variable domain (VL) and a constant domain (CL). Heavy chains are made up of a variable domain (VH) and three constant domains (CH₁, CH₂ e CH₃). The IgG molecule can also be divided into two main fragments: the antigen-binding-domain (Fab) and the fragment crystallizable (Fc) domain. The Fab fragment consists of two constant domains (CH₁ and CL) and two variable domains (VH and VL). (B) Schematic representation of different antibody fragments. Fragment of antigen binding (Fab) composed of VL and a constant domain of the light chain (CL) linked to VH and a constant domain of the heavy chain (CH₁) by a disulfide bond between the CL and CH₁ domains. Single chain fragment variable (scFv) composed only of variable regions, one from the heavy chain (VH) and the other from the light chain (VL). The two variable regions are linked by a flexible glycine-serine linker (Gly₄Ser)₃. Camelid and shark immunoglobulin composed of only heavy chains. They present no light chain, and the displayed V domains bind their targets separately. Camelid heavy-chain antibodies composed a homodimer of one variable domain (VHH) and two C-like constant domains (CH). Shark new antigen receptor antibodies (IgNARs) composed of one variable domain (V-NAR) and five C-like constant domains (CH).

Figure 2

Schematic representation of the different approaches for producing monoclonal antibodies. Hybridoma technology allows the production of murine antibodies which can be modified to generate chimeric or humanized antibodies (shown at left); The procedure of obtaining human antibodies using genetically engineered mice with human immunoglobulin genes (shown at right); The central part of the figure shows the different antibody libraries (naïve, immune, and synthetic) which can be constructed from different sources (human, mouse, rabbit, camelid, or shark). The antibodies that are part of the libraries can be presented in different formats (IgG, VHH, IgNAR, Fab, scFv, or sdAb) and their screening is performed using different display technologies (phage, yeast, ribosome, or cell display).

Figure 3

Schematic representation of in vitro Phage Display Technology. (A) In phage display systems, antibody genes are linked to the amino terminus region of the phage minor coat protein pIII, as shown in the phagemid. When expressed, mature phages will incorporate the encoded fusion product, creating a link between antibody genotype and phenotype. (B) In vitro phage display, selection is composed of several steps: coating of the antigen or preparation of the cells; incubation of phage repertoire with antigen; washing to remove non-specific phages; and elution and reamplification of antigen-specific phages. The stringency of selection could be increased by the increase in the number of washing steps.

Figure 4

Schematic representation of in vivo Phage Display. In vivo phage display selects phage libraries using a living animal. In this methodology, the library is directly injected into animals and antibodies are allowed to bind directly to the specific organ or tissues. Non-binding phages are washed and, in the end, animals are euthanized, and the desired organs collected to recover the phages. This scheme contains a representation of an in vivo experiment performed on a xenograft mouse model where the phages are recovered from the tumor.