Development of therapeutic antibodies for the treatment of diseases

Abstract

It has been more than three decades since the first monoclonal antibody was approved by the United States Food and Drug Administration (US FDA) in 1986, and during this time, antibody engineering has dramatically evolved. Current antibody drugs have increasingly fewer adverse effects due to their high specificity. As a result, therapeutic antibodies have become the predominant class of new drugs developed in recent years. Over the past five years, antibodies have become the best-selling drugs in the pharmaceutical market, and in 2018, eight of the top ten bestselling drugs worldwide were biologics. The global therapeutic monoclonal antibody market was valued at approximately US$115.2 billion in 2018 and is expected to generate revenue of $150 billion by the end of 2019 and $300 billion by 2025. Thus, the market for therapeutic antibody drugs has experienced explosive growth as new drugs have been approved for treating various human diseases, including many cancers, autoimmune, metabolic and infectious diseases. As of December 2019, 79 therapeutic mAbs have been approved by the US FDA, but there is still significant growth potential. This review summarizes the latest market trends and outlines the preeminent antibody engineering technologies used in the development of therapeutic antibody drugs, such as humanization of monoclonal antibodies, phage display, the human antibody mouse, single B cell antibody technology, and affinity maturation. Finally, future applications and perspectives are also discussed.

Keywords: Affinity maturation; Antibody market; Human antibody mouse; Humanized antibody; Phage display; Single B cell antibody technology; Therapeutic antibody.

Fig. 1

Timeline from 1975 showing the successful development of therapeutic antibodies and their applications. Many biotech companies that promised antibodies as anticancer “magic bullets” were launched from 1981 to 1986. The height of the line and numerical annotations represent the estimated market value of mAb therapeutics in each indicated year (shown as billions of US dollars). Antibodies colored in red represent the top 10 best-selling antibody drugs in 2018. Ab, antibody; ALCL, systematic anaplastic large-cell lymphoma; aTTP, acquired thrombotic thrombocytopenic purpura; BC, breast cancer; CD, cluster of differentiation; CGRP, calcitonin gene-related peptide; CGRPR, calcitonin gene-related peptide receptor; CRC, colorectal cancer; CTLA-4, cytotoxic T-lymphocyte-associated protein 4; EGFR, epidermal growth factor receptor; FGF, fibroblast growth factor; GC, gastric cancer; GD2, disialoganglioside G_D2; HER2, human epidermal growth factor receptor 2; IgE, immunoglobulin E; IL, interleukin; IL-17R, interleukin-17 receptor; mAb, monoclonal antibody; MCC, merkel-cell carcinoma; NSCLC, non-small cell lung cancer; PD-1, programmed cell death protein 1; PD-L1, programmed death-ligand 1; TNFα, tumor necrosis factor α; RA, rheumatoid arthritis; RANKL, receptor activator of nuclear factor kappa-B ligand; VEGF-A, vascular endothelial growth factor A; VEGFR2, vascular endothelial growth factor receptor 2; vWF, von Willebrand factor; XLH, X-linked hypophosphatemia

Fig. 2

Schematic overview of antibody humanization from murine antibodies (green domains) to fully human antibodies (orange domains) and associated suffixes. a The murine monoclonal antibody. b The chimeric monoclonal antibody: variable regions are of murine origin, and the rest of the chains are of human origin. c Humanized monoclonal antibody: only includes the hypervariable segments of murine origin. d Human monoclonal. C_H: domains of the constant region of the heavy chain; C_L: constant domain of the light chain; Fab and Fc: fragments resulting from proteolysis; V_H: variable domain of the heavy chain; V_L: variable domain of the light chain

Fig. 3

Approaches for the development of therapeutic antibodies. a The traditional mouse hybridoma technique starts by immunization of mice with desired antigens to trigger an immune response. Harvested splenocytes are fused with myeloma cells to produce hybridoma cells that persistently secrete antibodies. After the screening, selected leads are used to generate chimeric or humanized antibodies. b Phage display. A human phage-displayed human antibody library is used to select antigens of interest. After 3–5 rounds of biopanning, immuno-positive phage clones are screened by ELISA; then DNA sequences are analyzed to construct and express human IgGs. c Transgenic mouse. Similar to the mouse hybridoma technique or single B cell methods. d The single B cell technique. From infected or vaccinated donors, PBMCs are prepared for isolation of suitable B cells by flow cytometry. Following the RT-PCR, V_H and V_L information of each B cell informs the generation of human mAbs

Fig. 4

Construction and affinity selection with phage-display antibody library. a Outline of the procedure for constructing a phage-displayed antibody (Fab or scFv) library. b Structure of IgG molecule. Fab consists of the light chain and the first two domains of the heavy chain. scFv is composed of the variable heavy (V_H) and variable light (V_L) domains joined by a short flexible polypeptide linker. c Biopanning with a phage-displayed library. Initial pools of antibodies on the surface of phages are applied to antigens immobilized on a solid surface, e.g., ELISA plates or magnetic beads. Non-specific phages are removed by stringent washing. Antigen-bound phages are eluted and re-infected into E. coli to produce a subset of phages for the next cycle of panning. After several rounds, the antigen-binding clones are sufficiently enriched and individual clones can be selected for further analysis

Fig. 5

Schematic overview showing the development of antibody-based therapeutics for the treatment of cancer. Therapeutic antibodies can be roughly separated into two broad categories. The first category involves the direct use of the naked antibody for disease therapy. Antibodies in this category are used for cancer treatment and elicit cell death by different mechanisms, including ADCC/CDC, direct targeting of cancer cells to induce apoptosis, targeting the tumor microenvironment, or targeting immune checkpoints. For antibodies in the second category, additional engineering is performed to enhance their therapeutic efficacy. Some general approaches for the use of these antibodies include immunocytokine, antibody-drug conjugate (ADC), antibody-radionuclide conjugate (ARC), bispecific antibody, immunoliposome, and CAR-T