RESUMEN
INTRODUCTION: Phage display technology is a well-established versatile in vitro display technology that has been used for over 35 years to identify peptides and antibodies for use as reagents and therapeutics, as well as exploring the diversity of alternative scaffolds as another option to conventional therapeutic antibody discovery. Such successes have been responsible for spawning a range of biotechnology companies, as well as many complementary technologies devised to expedite the drug discovery process and resolve bottlenecks in the discovery workflow. AREAS COVERED: In this perspective, the authors summarize the application of phage display for drug discovery and provide examples of protein-based drugs that have either been approved or are being developed in the clinic. The amenability of phage display to generate functional protein molecules to challenging targets and recent developments of strategies and techniques designed to harness the power of sampling diverse repertoires are highlighted. EXPERT OPINION: Phage display is now routinely combined with cutting-edge technologies to deep-mine antibody-based repertoires, peptide, or alternative scaffold libraries generating a wealth of data that can be leveraged, e.g. via artificial intelligence, to enable the potential for clinical success in the discovery and development of protein-based therapeutics.
Asunto(s)
Técnicas de Visualización de Superficie Celular , Descubrimiento de Drogas , Biblioteca de Péptidos , Proteínas , Descubrimiento de Drogas/métodos , Humanos , Técnicas de Visualización de Superficie Celular/métodos , Animales , Desarrollo de Medicamentos/métodos , Anticuerpos , Péptidos/farmacología , Péptidos/química , Biotecnología/métodos , Inteligencia ArtificialRESUMEN
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) continues to evolve, escape coronavirus disease 2019 therapeutics and vaccines, and jeopardize public health. To combat SARS-CoV-2 antigenic escape, we developed a rapid, high-throughput pipeline to discover monospecific VHH antibodies and iteratively develop VHH-Fc-VHH bispecifics capable of neutralizing emerging SARS-CoV-2 variants. By panning VHH single-domain phage libraries against ancestral or beta spike proteins, we discovered high-affinity VHH antibodies with unique target epitopes. Combining two VHHs into a tetravalent bispecific construct conferred broad neutralization activity against multiple variants and was more resistant to antigenic escape than the monospecific antibody alone. Following the rise of the Omicron variant, a VHH in the original bispecific construct was replaced with another VHH discovered against the Omicron BA.1 receptor binding domain; the resulting bispecific exhibited neutralization against both BA.1 and BA.5 sublineage variants. A heavy chain-only tetravalent VHH-Fc-VHH bispecific platform derived from humanized synthetic libraries held a myriad of unique advantages: (i) synthetic preconstructed libraries minimized risk of liabilities and maximized discovery speed, (ii) VHH scaffolds allowed for a modular "plug-and-play" format that could be rapidly iterated upon as variants of concern arose, (iii) natural dimerization of single VHH-Fc-VHH polypeptides allowed for straightforward bispecific production and purification methods, and (iv) multivalent approaches enhanced avidity boosting effects and neutralization potency, and conferred more robust resistance to antigenic escape than monovalent approaches against specific variants. This iterative platform of rapid VHH discovery combined with modular bispecific design holds promise for long-term viral control efforts.
RESUMEN
New immune checkpoints are emerging in a bid to improve response rates to immunotherapeutic drugs. The adenosine A2A receptor (A2AR) has been proposed as a target for immunotherapeutic development due to its participation in immunosuppression of the tumor microenvironment. Blockade of A2AR could restore tumor immunity and, consequently, improve patient outcomes. Here, we describe the discovery of a potent, selective, and tumor-suppressing antibody antagonist of human A2AR (hA2AR) by phage display. We constructed and screened four single-chain variable fragment (scFv) libraries-two synthetic and two immunized-against hA2AR and antagonist-stabilized hA2AR. After biopanning and ELISA screening, scFv hits were reformatted to human IgG and triaged in a series of cellular binding and functional assays to identify a lead candidate. Lead candidate TB206-001 displayed nanomolar binding of hA2AR-overexpressing HEK293 cells; cross-reactivity with mouse and cynomolgus A2AR but not human A1, A2B, or A3 receptors; functional antagonism of hA2AR in hA2AR-overexpressing HEK293 cells and peripheral blood mononuclear cells (PBMCs); and tumor-suppressing activity in colon tumor-bearing HuCD34-NCG mice. Given its therapeutic properties, TB206-001 is a good candidate for incorporation into next-generation bispecific immunotherapeutics.