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An Overview of Monoclonal Antibody Drugs: Related Research and Applications (I)

Monoclonal refers to a cell population with identical genetic traits formed by a single cell through asexual reproduction. Separating a B lymphocyte from the body, the cell can produce a single antibody against a certain antigenic determinant, and then through pure breeding, it can produce only one antibody. This type of antibody produced by a single clone of B lymph hybridoma cells that can recognize a specific antigenic determinant on the antigen molecule is called a monoclonal antibody (mAb).

It has been more than 40 years since the discovery of monoclonal antibodies, during which it has gone through four stages of development: murine monoclonal antibodies, human-mouse chimeric monoclonal antibodies, humanized monoclonal antibodies and fully human monoclonal antibodies.

The source of the antibody of the murine monoclonal antibody is entirely from mouse. Mouse-derived monoclonal antibodies are prone to produce human anti-mouse antibody (HAMA) response, and are easily eliminated as heterologous proteins in the body, and their half-life is short, thereby reducing the therapeutic effect, which greatly restricted the application of mouse-derived monoclonal antibodies in clinical treatment.

Chimeric antibody is an antibody that combines the variable region sequence of a murine monoclonal antibody and the constant region of a human antibody to form a human-mouse hybrid antibody. Since most of the immune response of a heterologous antibody is directed at the constant region, use of the constant region of human antibodies reduces the immunogenicity of mouse-derived monoclonal antibodies as heterologous proteins to humans and reduces the occurrence of HAMA reactions.

Humanized monoclonal antibody refers to the re-expressed antibody after the murine monoclonal antibody is modified using transgene and DNA recombination technology. Most of its amino acid sequence is replaced by human sequence, which basically retains the affinity and specificity of the parent mouse monoclonal antibody, which greatly reduces the immune side reaction caused by its heterogeneity to the human body, and is beneficial to be applied to the human body.

The fully human monoclonal antibody transfers all the human antibody-encoding genes to the genetically engineered animal with the antibody gene deletion through transgene or transchromosome technology, so that the animal can fully express the human antibody, thereby achieving the goal of fully humanized antibody.

  1. The key technology of monoclonal antibody
  2. 1 Antibody preparation

The current methods for preparing humanized monoclonal antibodies mainly include antibody library technology and human antibody transgenic mouse technology. The antibody library technology also includes phage antibody library, ribosomal antibody library and yeast display technology.

  1. 1. 1 Phage antibody library technology

Phage antibody library is a new technology that uses phage to express foreign antibody genes. The specific operation is to randomly recombine antibody heavy chain variable region (VH) and light chain variable region (VL) genes and phage coat protein Ⅲ (PIII) or coat protein Ⅷ (PⅧ) gene. After infection with E. coli, it is re-amplified and expressed on the surface of phage as antibody fragment Fab or single-chain antibody variable region (scFv)-coat protein fusion protein. This kind of phage undergoes the process of “adsorption-elution-amplification” to screen out specific antibodies, also known as phage antibodies. At present, the technology of phage antibody library is very mature, which can screen out antibody gene sequences with high specificity and strong affinity to prepare fully human antibodies.

  1. 1. 2 Ribosomal antibody library technology

In 1994, the peptide library of ribosome display technology was established for the first time. The technology is carried out in vitro, and the preparation is simple and the storage capacity is large, and the required high-affinity antibodies can be screened from it. This technology gradually matured in the following two decades and became an important way to prepare fully human monoclonal antibodies. The preparation method is first to construct a DNA template, and then perform in vitro transcription and translation, perform affinity screening, screen protein-ribosome-mRNA-trimers, and finally perform reverse transcription to amplify mRNA.

  1. 1. 3 Yeast surface display technology

At present, the development of the display system of Saccharomyces cerevisiae is relatively complete. Its preparation method is to introduce the target protein into yeast cells after fusion with a specific gene, and then express and display it on the yeast cell wall. Because it is expressed in a eukaryotic system, this technology is efficient and convenient for screening. It can process complex eukaryotic proteins and has the advantages of small expression deviation.

  1. 1.4 Transgenic mice

Transgenic mice referred to the use of gene knockout technology, yeast chromosome technology and other genetic engineering techniques to artificially modify the genes of mice and transcribe human antibody genes into the mice. At present, the technology of preparing fully human antibodies from transgenic mice is the mainstream of monoclonal antibody preparation. Compared with antibody library platform technology, humanized mouse technology is developing fully human sources. It has more advantages and has become the most advantageous antibody drug research and development.

  1. 2 Antibody modification
  2. 2. 1 Glycosylation modification

Glycosylation is one of the important modifications of proteins. According to the modified site of glycosylation, glycosylation can be divided into N-position glycosylation and O-position glycosylation. The N-position glycosylation site is at Asn-297, where N-acetylglucosamine is linked to the amide nitrogen on the asparagine residue to modify the protein, starting from the endoplasmic reticulum and ending in the Golgi apparatus; O-position glycosylation is accomplished in the Golgi apparatus by connecting N-acetylgalactose in oligosaccharides to hydroxyl groups on serine or threonine residues. Glycosylation at the N position in immunoglobulins secreted by animal cells is the most common glycosylation modification, and it is also the most studied glycosylation modification. Taking IgG1 as an example, its important glycosylation modification site is located at the Fc end, and can be divided into complex type, hybrid type and high mannose type according to the difference in the fine structure (length, branching and monosaccharide arrangement) of the terminal. Studies have shown that N-glycosylation can enhance the stability of drugs, increase acceptance affinity, and reduce antibody-dependent cytotoxicity.

To be continued in Part II…

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