Application of Rabbit Monoclonal Antibody

Monoclonal antibody technology originated from the in vitro murine hybridoma technology created by Kohler et al. After years of research, murine monoclonal antibody has evolved from simple biological laboratory research and clinical diagnosis to therapeutic antibodies drugs. However, the main drawback of murine mAb is that the mouse immune system does not recognize certain immunogens, especially murine immunogens, and the affinity of murine monoclonal antibodies is not as high as that of rabbit-derived antibodies. However, due to the lack of technical difficulties in transfecting rabbit-derived plasmacytoma and virus in vitro in rabbit B cells, until 1995, Spieker-Polet et al. The rabbit plasmacytoma cell line was successfully obtained in c-myc/v-abl transgenic rabbits, and a stable rabbit-rabbit hybridoma was obtained, which made a breakthrough in rabbit monoclonal antibody technology. In recent years, rabbit monoclonal antibody technology has been improved day by day, and there are thousands of commercial rabbit monoclonal antibodies. This article reviews the current development of rabbit monoclonal antibody technology, technical advantages and the application prospects of rabbit monoclonal antibody technology in basic research, clinical diagnosis and treatment.

Development 

Since the establishment of murine monoclonal antibody technology, many rabbit monoclonal antibodies have been tried by hybridoma technology, but rabbit-mouse hybridoma cells obtained by heterologous myeloma fusion are unstable due to the lack of rabbit myeloma cell line. Can not secrete antibodies and other issues for a long time.

Knight et al. overexpressed v-abl and c-myc two oncogenes c on a transgenic service rabbit and placed them downstream of the heavy and light chain enhancers, and finally obtained myeloma-like tumors from the transgenic rabbits. A myeloma cell line was isolated and named 240E-1. The hybridoma obtained by fusing this strain of 240E-1 cells with rabbit B lymphocytes secretes a rabbit monoclonal antibody. This is the first time that a rabbit monoclonal antibody has been obtained by means of a rabbit-rabbit hybridoma. However, like the early murine myeloma cell line, 240E-1 cells also have two major problems: first, instability, after several passages or subclones, the hybridoma gradually loses the ability to secrete antibodies; secondly, the cell line itself Endogenous rabbit IgG was expressed. In order to solve the stability problem, Zhu et al. [22] improved the medium method and used repeated subcloning techniques to obtain a new cell line, named 240E-W, by 11 rounds of subcloning and screening. Chromosome typing experiments have shown that its chromosome number is more stable, with 79 to 90 pairs (mean 84 pairs), 240E-W is more stable than 240E-1 44 to 70 pairs (average 60 pairs). DNA fingerprinting and gene expression profiling also showed great differences between the two cells. The fusion cell strain was prepared with 240E-W, and the hybridoma cells obtained were more efficient and more stable. In order to solve the problem of endogenous IgG, Huang et al. obtained a cell line without the endogenous IgG heavy chain gene from the hybridoma of 240E-W cells and named it 240E-W2. Hybridoma cells obtained by fusion with 240E-W2 did not express detectable endogenous IgG heavy chains and improved fusion efficiency and hybridoma positive rate by various large-scale fusion experiments.

Applications

Application in the development of biomarkers

Rabbit monoclonal antibodies are widely used in various fields of life science research [28-31], and have unique advantages especially in immunohistochemistry and post-translational modification of proteins. To date, more than 100 rabbit monoclonal antibodies have been developed for the clinical diagnosis and research of IHC for cancer. The most representative ones are Her2 and c-Kit IHC diagnostic kits. In the field of protein modification research, such as protein phosphorylation, plays a crucial role in the disease-related protein signaling pathway. The rabbit monoclonal antibody using phosphorylated protein can clearly show the degree of phosphorylation of the protein. More than 200 phosphorylated specific rabbit monoclonal antibodies have been available.

Application in the development of therapeutic antibody drugs

Most therapeutic antibodies used in clinical practice require the step of in vitro affinity maturation to increase the affinity of the antibody. This process often takes 8 to 12 months and the final affinity is in the nanomolar range (10-9 mol/L). Rabbit monoclonal antibodies can reach 10-11 to 10-13 mol/L without the need for in vitro affinity maturation. This high affinity not only reduces the clinical use of antibodies, but also reduces the side effects caused by the use of large amounts of antibodies. But this high affinity advantage needs to be further confirmed in animal and clinical trials. In addition, humanization of rabbit monoclonal antibodies is easier than humanization of murine monoclonal antibodies. Because rabbit monoclonal antibody is mainly composed of a heavy chain and light chain genes, it is found that rabbits and human antibodies have higher homology, about 60% to 76%, by analyzing a large number of rabbit monoclonal antibody sequences, and mice. The homology with human antibodies is slightly lower, ranging from 57% to 72%, indicating that the development of drugs with rabbit monoclonal antibodies is more promising.