More than three decades have passed since the U.S. Food and Drug Administration (FDA) approved the first monoclonal antibody in 1986. During this period, antibody engineering has undergone tremendous development. Due to their high specificity, current antibody drugs are associated with fewer adverse reactions. Consequently, therapeutic antibodies have become a major category among new drugs developed in recent years. Over the past five years, antibodies have emerged as the best-selling pharmaceuticals in the market, with the global therapeutic monoclonal antibody market value growing annually. Therefore, the market for therapeutic antibody drugs has experienced explosive growth, with new drugs being approved to treat various human diseases, including numerous cancers, autoimmune diseases, metabolic disorders, and infectious diseases. As of December 2019, 79 therapeutic monoclonal antibodies had received FDA approval in the United States, yet significant growth potential remains. Current outstanding antibody engineering technologies used in therapeutic antibody drug development include monoclonal antibody humanization, phage display, human antibody mice, single B-cell antibody technology, and affinity maturation technologies. These developments, combined with a deeper understanding of the immunomodulatory properties of antibodies, have paved the way for the development of next-generation, improved antibody-based drugs for treating human diseases.

Therapeutic Antibody Development Data and History

Globally, at least 570 therapeutic monoclonal antibodies from commercial companies are being investigated in clinical trials. Seventy-nine therapeutic monoclonal antibodies have received approval from the U.S. Food and Drug Administration (USFDA) and are currently on the market, with 30 of these monoclonal antibodies used for cancer treatment. The increasing importance of therapeutic monoclonal antibodies is evident, as they have become a major treatment modality for various diseases over the past 25 years. During this period, significant technological advancements have enabled faster and more efficient discovery and development of monoclonal antibody therapies.

The first therapeutic monoclonal antibody was muromonab-CD3 (Orthoclone OKT3), approved by the U.S. FDA in 1986. It is a mouse monoclonal antibody targeting CD3 expressed on T cells (Figure 1A) and was used as an immunosuppressant to treat acute transplant rejection. To overcome issues of immunogenicity potential and reduced efficacy, thereby extending the therapeutic use of antibodies, researchers developed techniques to convert rodent antibodies into structures more similar to human antibodies without losing their binding properties. The first chimeric antibody (abciximab) was approved by the U.S. FDA in 1994 for inhibiting platelet aggregation in cardiovascular diseases. This drug was developed by combining mouse variable region sequences with human constant region sequences (Figure 2B). The generation of humanized antibodies via complementarity determining region (CDR) grafting was a special advancement accelerating therapeutic monoclonal antibody approvals. In CDR grafting, non-human antibody CDR sequences are transplanted into human framework sequences to retain target specificity (Figure 2C). Antibody humanization enabled the clinical application of a new class of biologics directly targeting diseases requiring long-term treatment, such as cancer and autoimmune diseases.

Figure 1. Schematic Structures of Various Antibodies

Antibody Development Technologies

(1) Phage Display Technology

Phage display technology is currently the most widely used in vitro antibody selection technology. This technology utilizes recombinant DNA technology to fuse foreign peptides with the coat protein (pIII) of M13 phage, displaying the peptide on the phage surface. Subsequently, it was discovered that small molecular weight antibodies like scFv and VHH could also be expressed on phage, leading to the establishment of different M13 phage display antibody libraries. Antibody libraries are typically subjected to iterative screening to enrich for target-binding phages, followed by amplification of the bound phages in E. coli cells (Figure 2).

Figure 2. Flowchart of Phage Library Screening

(2) Transgenic Animals and Antibody Humanization Technology

Transgenic animals represent another technology for obtaining fully human monoclonal antibodies (Figure 3). These cell lines are genetically modified by inserting human immunoglobulin (Ig) genes into the genome, replacing the endogenous Ig genes. This enables these animals to synthesize fully human antibodies upon immunization. Depending on the immunization protocol, high-affinity human antibodies can be obtained by further selecting hybridoma clones generated from immunized transgenic mice. Transgenic animals provide a reliable platform for antibody drug development. Compared to other technologies for producing human antibodies, transgenic animals offer advantages such as no requirement for humanization, greater diversity, in vivo affinity maturation, and antibody optimization through clonal selection.

Figure 3. Principle Flowchart of Human Antibody Preparation Using Transgenic Animals

(3) Chimeric Antibody Technology

To overcome the limitations of fully human heavy chain antibodies, it is necessary to retain the original mouse constant regions. This involves linking human VH, D, and JH genes with rat constant region loci. Bacterial Artificial Chromosome (BAC) and Yeast Artificial Chromosome (YAC) technologies are used for subcloning and ligating large fragments of human IgL and IgH. The small locus plasmid is then microinjected into fertilized oocytes. Simultaneously, zinc finger nucleases are used to silence the endogenous rat Ig loci. The resulting rat strain (OmniRat) with chimeric human elements exhibits antibody production, antigen affinity, and somatic mutation similar to wild-type rats.

(4) Single B-Cell Antibody Technology

In the human immune system, antibody responses are robust, highly specific, neutralizing, and self-tolerant. Producing therapeutic human antibodies using traditional hybridoma technology or transgenic mice requires lengthy immunization and screening procedures, and clinical use of mouse antibodies can trigger significant immunogenic reactions. To circumvent these obstacles, researchers developed a technology using Epstein-Barr Virus (EBV) to immortalize human B cells. In urgent situations like infectious disease outbreaks, the main advantage of single B-cell antibody technology is that it requires only a small number of cells, enabling efficient and rapid isolation of potential monoclonal antibodies. Furthermore, individual B-cell cloning preserves the biologically mediated pairing of heavy and light chains, avoiding the random pairing of monoclonal antibodies found in phage display antibody libraries. These randomly paired monoclonal antibodies can occasionally lose binding affinity or become self-reactive when transferred from scFv format to complete IgG format. After single B-cell sorting, each Ig heavy chain and its corresponding light chain are directly cloned. These genes are then cloned and expressed in mammalian cell lines for immediate production of recombinant monoclonal antibodies (Figure 4). Following testing for monoclonal antibody reactivity, the characteristics of each generated monoclonal antibody are determined.

Figure 4. Principle Flowchart of Human Antibody Preparation Using Single B-Cell Technology

Future Perspectives

Therapeutic antibodies can be broadly classified into two main categories (Figure 5). The first category involves the direct use of naked antibodies for disease treatment. These antibodies are used in cancer therapy and induce cell death through different mechanisms, including ADCC/CDC, directly targeting cancer cells to induce apoptosis, targeting the tumor microenvironment, or targeting immune checkpoints. For the second category of antibodies, additional engineering is required to enhance their therapeutic efficacy. Some general methods employing these antibodies include immunocytokines, antibody-drug conjugates (ADCs), antibody-radionuclide conjugates (ARCs), bispecific antibodies, immunoliposomes, and CAR-T cells.

Figure 5. Mechanisms of Action for Various Therapeutic Antibodies

This article summarizes five technology platforms related to the production of therapeutic antibodies, including chimeric antibodies, humanization, phage display, transgenic mice, and single B-cell antibody technologies. Phage display, transgenic mice, and single B-cell antibody technologies have proven to be reliable methods for generating human antibodies. A high-quality (antibody diversity) phage antibody library is critical for the successful identification of therapeutic monoclonal antibodies. Furthermore, optimal screening of phage display libraries depends on target antigen quality, antigen immobilization, and stringent control of binding and washing conditions. Additionally, the characteristics of antibodies discovered through biological screening, including conformational specificity, epitope specificity, internalization, neutralization, and interspecies cross-reactivity, can be tailored via screening protocol design.

Tek Biotech (Tianjin) Co., Ltd. has established a comprehensive targeted antibody drug discovery platform based on phage display technology and yeast display technology. We provide high-quality drug monoclonal antibody development services for various antibody formats, including scFv, VHH, and Fab, to scientists globally. We are also capable of developing various functional antibodies with specific structural characteristics (including but not limited to neutralizing antibodies, conformation-specific antibodies, cross-reactive antibodies, etc.). Concurrently, Tek Biotech offers supporting downstream services such as antibody expression and validation, antibody humanization design and validation, antibody affinity maturation, and CAR-T candidate sequence design, meeting the diverse needs of clients for therapeutic antibody development.

References

[1] Lu, RM., Hwang, YC., Liu, IJ. et al. Development of therapeutic antibodies for the treatment of diseases. J Biomed Sci 27, 1 (2020).