Hybridoma method

The hybridoma technology of mice was established in 1975, and it is the most commonly used method for producing monoclonal antibodies in mice. This method involves fusing the B lymphocytes from immunized mice with myeloma cells derived from BALB/c mice to form immortal hybridoma cells. The hybridoma cells that produce specific antibodies are then selected through limited dilution screening.

Due to the lack of rabbit myeloma cell lines, there have been certain difficulties in rabbit hybridoma cell production. The method of transforming rabbit B cells with viruses to produce myeloma-like cell lines also presents significant challenges. The method of forming hybridomas through mouse-rabbit heterohybridomas was first reported in 1988 for producing rabbit monoclonal antibodies. However, these hybridomas exhibit instability, low efficiency, and inability to secrete antibodies for extended periods. In 1995, scientists established a method for producing rabbit monoclonal antibodies through rabbit-rabbit hybridomas. The first stable rabbit plasmacytoma cell line, 240E-1, was discovered in v-abl/c-myc double transgenic rabbits. However, compared to traditional mouse hybridomas, rabbit-rabbit hybridomas are also unstable and have relatively low fusion efficiency, leading to more severe decline in IgG secretion over time compared to mouse-rabbit hybridomas. The initial 240E-1 cell line was improved to 240E-W and its subsequent cell lines 240E-W2 and 240-W3, which have higher fusion efficiency and do not secrete endogenous rabbit heavy and light chains. This improvement is part of Abcam's proprietary RabMab platform.

Phage display

Phage display technology is a method for producing monoclonal antibodies that was invented in the early 1990s. The minor coat protein (pIII) of filamentous phage can be modified at its N-terminus, and the added peptide segment does not affect the infectivity of the phage. This system consists of two components, one being the phagemid encoding the pIII fusion protein. The phagemid is a plasmid with the replication origin of the filamentous phage. The phagemid itself does not contain genes encoding phage proteins. The other component is the helper phage, which helps in producing all the proteins required for the infectious phage particle from the phage genome. These helper phages contain modified packaging signals, leading to the preferential assembly of phage particles containing the phagemid.

In this technology, a V gene library is obtained from lymphocytes, and the combinations of VH and VL are cloned and expressed on the surface of the phage by fusion with the coat protein. Phage expressing specific monoclonal antibodies are selected and detected accordingly. Compared to the more restricted hybridoma technology, phage display technology has been successfully used for screening and isolating monoclonal antibodies from any species with known immunoglobulin genes. The technical route for selecting rabbit monoclonal antibodies using phage display technology was established in 2000. The random combination of antibody variable region genes during library construction leads to the loss of natural homologous heavy and light chain pairings selected during in vivo evolution and selection during the immune response. Due to this random pairing, antibodies from the initial antibody library usually need to undergo in vitro maturation to confer higher affinity and stability before progressing as therapeutic molecules.

In addition to phage display, in vitro antibody display technology also includes ribosome display and in vivo display platforms, such as bacterial, yeast, and mammalian cell surface display.

Generating monoclonal antibodies from individual rabbit B cells through single-cell cloning

During the production of monoclonal antibodies, both hybridoma and phage display technologies have some limitations. The cell fusion efficiency of hybridoma technology is low, while phage display technology can lead to the loss of natural homologous pairing of heavy and light chains. The method of producing rabbit monoclonal antibodies through single-cell cloning can circumvent these two drawbacks. The production of rabbit monoclonal antibodies based on single-cell cloning consists of the following steps: 1. Isolation of individual B cells from lymphoid tissue or peripheral blood; 2. RNA extraction, amplification of the corresponding B cell cDNA through reverse transcription; 3. Amplification of immunoglobulin encoding genes from cDNA through PCR; 4. Cloning the amplified genes into expression vectors and sequencing; 5. Expression of the sequenced plasmids in mammalian cells (such as HEK293 and CHO) or bacteria (such as Escherichia coli); 6. Evaluation of the corresponding antibodies through ELISA. The greatest advantage of this method is the preservation of natural pairing of antibody heavy and light chains, which is conducive to the production of monoclonal antibodies with high affinity, specificity, and stability.

Memory B cells and plasma/plasmablast cells are the main sources of antibodies. Various methods have been used for the screening of B cells. Some methods utilize fluorescence-activated cell sorting (FACS) and endoplasmic reticulum (ER)-specific fluorescent dyes to identify and separate antigen-specific plasma cells or plasmablast cells. Other methods use immunospot array assay for the detection of antigen-specific rabbit monoclonal antibodies. There are also methods for the isolation of rabbit plasma cells secreting antigen-specific antibodies through fluorescence and micromanipulation. Additionally, there are methods for the isolation of antigen-specific B cells from peripheral blood through lymphocyte panning. A two-color antigen staining method has recently been used to identify antigen-specific rabbit memory B cells.

A method called HybriFree has been successfully used for the production of monoclonal antibodies in mice, rabbits, and chickens. The workflow of HybriFree includes capturing antigen-specific B cells on a solid matrix with the antigen, amplifying single-cell cDNA containing VH and VL encoding genes, constructing VH-VL combination libraries in mammalian expression systems, and determining suitable VH-VL combinations. A system called "single-cell RT-PCR linked in vitro expression (SICREX)" has also been used for the production of rabbit monoclonal antibodies. Antigen-specific B cells are isolated and amplified using a linear immunoglobulin expression box for cell-free production. The linear expression box contains all the necessary elements for transcription and translation regulation, including the T7 promoter and T7 terminator, and the antigen-binding fragment or single-chain variable fragment can be expressed without cloning. The entire process of SICREX is carried out in vitro, which can shorten the production time of monoclonal antibodies to a few days.

Humanization of rabbit monoclonal antibodies

The function of the human immune system is to recognize and selectively remove potential pathogenic organisms and substances, including proteins from other species. Monoclonal antibodies derived from mice and rabbits are also recognized by the human immune system as foreign substances, and the corresponding immune response can result in the production of high-titer human antibodies against these exogenous antibodies. This phenomenon can lead to two consequences: firstly, the production of side effects equivalent to allergic reactions of varying severity, and secondly, the rapid elimination of exogenous monoclonal antibodies used, limiting their diagnostic or therapeutic effects. Therefore, non-human monoclonal antibodies are usually humanized by combining the non-human variable regions with human constant regions to form chimeric monoclonal antibodies, or by further transplanting all or part of the non-human variable regions CDR into human antibodies to form humanized monoclonal antibodies. The strategies for humanizing mouse monoclonal antibodies are mostly applicable to rabbit monoclonal antibodies. Transplanting all six CDRs and then iteratively fine-tuning the framework residues is the most common method for humanizing rabbit monoclonal antibodies. Studies have shown that patients can still produce humanized antibodies or even fully human antibodies that may elicit immune responses, which may be due to the homologous, unique (idiotype) and glycosylation of the antibodies. However, the incidence of these adverse events is significantly lower than that of non-humanized antibodies.