Research and Development Status and Progress of Antibody Technology (Part I)

Antibody-based biological therapeutic drugs have been playing an increasingly important role in biopharmaceuticals due to their strong targeting, good specificity, and significant therapeutic effects. In recent years, the number of approved antibody drugs has increased significantly, and their market share has been rising steadily, making them the fastest-growing area in biopharmaceuticals. With the increasing demand for antibody drugs, the development of antibody technology is also advancing rapidly. From polyclonal antibody preparation to monoclonal antibody screening, a series of breakthrough achievements have been made, including mouse-derived antibodies, chimeric antibodies, humanized antibodies, and fully human antibodies. Antibody technology has become a hot topic in the field of biotechnology, especially in the field of biomedical drugs.

According to its developmental history, antibody technology can be divided into three stages: polyclonal antibody technology, hybridoma monoclonal antibody technology, and genetic engineering antibody technology. Polyclonal antibodies are mostly obtained through immunization of animals. When an antigen is injected into an animal, antibodies against that antigen will be produced. Since most antigen surfaces have complex components with multiple different antigenic epitopes, the antibodies produced in the immune serum are comprehensive, heterogeneous polyclonal antibodies targeting multiple antigen components or antigenic epitopes. Currently, the use of convalescent plasma therapy for the emergency treatment of acute infectious diseases is still widely concerned, such as in the prevalence of influenza, Ebola, severe acute respiratory syndrome (SARS), and coronavirus disease 2019 (COVID-19), where convalescent plasma therapy has been used for treatment or clinical research.

Hybridoma monoclonal antibody technology involves fusing B lymphocytes capable of producing antibodies with tumor cells capable of indefinite reproduction. The resulting hybrid cells can both produce antibodies and replicate indefinitely, resulting in monoclonal antibodies with high purity, uniform physicochemical properties, and strong specificity.

Genetic engineering antibody technology utilizes recombinant DNA and protein engineering techniques to process, modify, and reassemble antibody-encoding genes according to different needs. The expressed antibody molecules are then obtained by transducing appropriate recipient cells. This technology is used to reduce the murine component of antibodies, increase their humanization, and lower immunogenicity, making it the third-generation antibody technology. Common techniques currently include mouse-derived antibody humanization technology, as well as human antibody screening techniques such as antibody library display technology, transgenic mouse technology, and single-cell sequencing technology.

With the continuous development of modern biotechnology and considering the biological characteristics of antibody drugs, researchers are constantly optimizing and innovating traditional antibody technology to obtain therapeutic antibodies with lower immunogenicity, better human compatibility, and lower costs through simpler and faster methods.

1. Hybridoma Monoclonal Antibody Technology

Hybridoma monoclonal antibody technology involves fusing B lymphocytes from immunized animals with myeloma cells to form hybridoma cells that can survive indefinitely in vitro and secrete immunoglobulins. Through cloning, a hybridoma monoclonal cell line derived from a single hybridoma cell is obtained, which produces a large amount of monoclonal antibodies. In this process, the two rounds of hybridoma cell screening are crucial. The most traditional secondary hybridoma screening method is the limited dilution method. This method is simple to operate and does not require special equipment, but it has slow and inefficient manual operation, long time consumption, and easy to make mistakes, which cannot meet the requirements of high-throughput screening. Flow cytometry is also commonly used for hybridoma cell screening, which has the advantages of fast automation and high accuracy, but it requires fluorescent labeling of cells, and the detection results are easily affected by the cell cycle. The fully automated cell screening system (Cellcelector) is a widely used hybridoma screening technology at present. This technology has the advantages of high throughput, automation, high efficiency, programmability, quantification, archiving, and traceability, which can shorten the research and development cycle and reduce costs.

Hybridoma monoclonal antibody technology is convenient, simple, and cost-effective, but it can only be used for the screening of mouse-derived antibodies. In 1986, the US Food and Drug Administration (FDA) approved the first mouse-derived antibody drug, muromomab-CD3, which was screened using hybridoma technology. However, mouse-derived antibodies can be recognized by the human immune system, triggering a strong human anti-mouse antibody response that weakens the efficacy of the drug and causes severe adverse reactions.

To address this issue, scientists utilized genetic engineering methods to humanize mouse-derived antibodies, which to some extent reduced the human anti-mouse antibody response. It was until 1994 that the FDA approved the second antibody drug, abciximab, a human-mouse chimeric antibody, for market use. Since then, antibodies screened through hybridoma technology typically require further humanization before they can be used clinically as antibody drugs.

2. Antibody Library Display Technology

Antibody library display technology has rapidly developed over the past 30 years and is primarily used to screen for human-derived antibodies. This technology utilizes polymerase chain reaction (PCR) to amplify the heavy and light chain populations of antibodies from B cells and inserts them into appropriate vectors to express random combinatorial antibody libraries. Depending on the source of the antibody genes, the current antibody libraries mainly include natural antibody libraries, semi-synthetic antibody libraries, fully synthetic antibody libraries, and mixed antibody libraries. Additionally, with the development of computer-aided analysis and virtual design, epitope-specific targeted antibody library technology platforms have also emerged. Based on the different vectors used in the antibody library, the main technologies currently available are phage display technology, yeast surface display technology, ribosome display technology, and mRNA display technology.

The foundation of phage display technology lies in the process of phages infecting bacteria and utilizing them to express their own outer shell proteins. In 2002, the first fully human-derived antibody drug, adalimumab, approved by the US FDA, was screened using a phage display library. Additionally, nanobodies, which possess advantages such as simple structure, small relative molecular mass, strong stability, ease of recombinant modification, and strong tissue penetration, are often screened using a combination of immune llama and phage display library techniques. For instance, the nanobody drug caplacizumab, approved by the US FDA in September 2018, was screened using this method.

Yeast cell surface display technology is a eukaryotic protein display technique. As a eukaryotic expression system, yeast cell surface display technology allows for post-translational modification of proteins, and the eukaryotic secretory system has a "quality" control mechanism that prevents misfolded proteins from being transported out of the endoplasmic reticulum. Additionally, flow cytometry can be used for quantitative and real-time screening of constructed yeast cell libraries, making the process efficient and convenient. In 2020, the FDA approved the antibody drug eptinezumab, which was expressed in Pichia pastoris yeast cells, for market use, making it the first antibody drug expressed in yeast. Currently, this technology is also being used in the development of nanobodies.

Ribosome display technology and mRNA display technology, as emerging cloning display techniques, are completely conducted in vitro. By constructing a DNA library, transcription and translation are performed in vitro to ultimately form "mRNA-ribosome-protein" (ribosome display technology) or "cDNA-mRNA-protein" (mRNA display technology) tripartite complexes, connecting the genotype (mRNA) and phenotype (protein) of the target protein. Finally, mRNA or cDNA is isolated for reverse transcription PCR (RT-PCR) amplification. Compared to phage or yeast cell display technologies, these techniques have advantages such as simple library construction, large library capacity, strong molecular diversity, convenient screening methods, and the ability to improve target protein affinity through the introduction of mutation and recombination techniques. Additionally, the cDNA/mRNA hybrid double strand formed by reverse transcription in mRNA display technology avoids the interference of RNA secondary and tertiary structures during in vitro screening. Currently, an increasing number of researchers are applying these technologies in the field of new drug development.

Related Reading: "Research Status and Progress of Antibody Technology (Part 2)"

References

[1] Wu Ruijun, Sang Xiaodong, Li Zhifei, Ao Yi, Fan Ling. Research Status and Prospects of Antibody Technology [J]. Chinese Journal of Pharmacology and Toxicology, 2021, 35(05): 374-381.

[2] Feng Jiannan, Qiao Chunxia. Progress in the Development of Human Therapeutic Antibodies [J]. Journal of Nanjing Medical University (Natural Science Edition), 2020, 40(11): 1571-1574.

About the Author

Xiaomichong, a researcher in pharmaceutical quality, has been dedicated to pharmaceutical quality research and verification of drug analysis methods for a long time. Currently, she works in a large domestic pharmaceutical research and development company, engaged in drug inspection analysis and verification of analytical methods.