Medicilon Logo
|
search icon search icon language icon contact icon menu icon
Medicilon Logo
|
search icon close search icon language icon contact icon menu icon
logo icon CN
Contact Us
Close Button
Message
Back To Top
Online Message×
Click switch
Close Button
Medicilon's News information
News information

Humanization of antibody reduces the immunogenicity of antibody drugs

2021-02-18
|
Page View:

The application of antibodies has a history of hundreds of years. As a disease prevention, diagnosis and treatment agent, antibody drugs have undergone the development of murine antibodies, chimeric antibodies, humanized antibodies and fully humanized antibodies. Because the antibody drug is a kind of foreign protein, it is easy to activate the body’s own immune response and induce the production of resistant antibodies, which can seriously affect the safety and effectiveness of antibody drugs. Therefore, immunogenicity tests are carried out to detect antibodies during drug development. The immunogenicity of the drug is important. Studies have found that increasing the degree of humanization of antibodies can reduce their immunogenicity.

immunogenicity

Antibodies are glycoprotein molecules produced by mature B lymphocytes that can specifically bind to the antigen after the immune system of higher vertebrates is stimulated by an external antigen. Antibodies are important effector molecules in the body’s immune system. They have multiple biological functions such as binding antigens, binding complements, neutralizing toxins, mediating cytotoxicity, and promoting phagocytosis. They play an important role in anti-infection, anti-tumor, immune regulation and surveillance. effect.

Development of antibody drugs

The early preparation of antibodies is to immunize animals with a certain natural antigen through various ways. Mature B lymphocyte clones are stimulated by the antigen to produce antibodies and secrete them into serum and body fluids. Later, there was a highly uniform antibody produced by a single B-cell clone that only targeted a specific epitope, that is, a monoclonal antibody. Monoclonal antibody drugs currently on the market can be divided into four categories: murine antibodies, chimeric antibodies, humanized antibodies and fully humanized antibodies.

The birth of murine hybridoma technology based on cell fusion has enabled people to obtain large amounts of murine antibodies, which have been widely used in clinical practice. However, the emergence of human anti-mouse antibody (HAMA) reaction limits the effectiveness of early murine monoclonal antibodies In order to overcome the shortcomings of mouse-derived monoclonal antibodies in clinical applications, people have carried out preliminary humanization transformations using recombinant DNA technology. The development of humanized antibody technology has sent the research of antibody drugs into the track of rapid development. Although the immunogenicity of humanized antibodies is greatly reduced, in order to ensure drug safety, immunogenicity tests are still needed to monitor human anti-human antibody responses. The strength of immunogenicity is one of the decisive factors for the development of biotechnology drugs. Therefore, when evaluating drug safety, immunogenicity tests should be carried out to investigate its immunogenicity. Medicilon, which ranks high in China’s GLP organization, provides immunogenicity test services, mainly using mice, rats, guinea pigs, rabbits, dogs, and non-human primates for immunogenic toxicity tests.

Human-mouse chimeric antibodies are the earliest humanized antibodies that use DNA recombination technology to insert the murine monoclonal antibody’s light chain and heavy chain variable region genes into an expression vector containing the constant region of a human antibody. However, the chimeric antibody only partially eliminates the heterogeneity of the mouse monoclonal antibody, and the mouse-derived sequence of the variable region can still induce the human body to produce a HAMA response; then CDR transplantation is proposed, that is, the CDR of the mouse monoclonal antibody is transplanted to the human monoclonal antibody. Anti-skeletal area.

The humanization of murine antibodies is mainly through molecular cloning and other technologies, replacing some heterologous amino acid sequences with human sequences. While maintaining antibody affinity and biological activity, this modification greatly reduces immunogenicity and increases Safety in clinical use. With the development of molecular biology technology, antibody library technology, genetic engineering mouse technology, etc., fully human monoclonal antibodies have attracted more and more attention. Humanized antibodies and fully humanized antibodies require transgenic mice or phages, and the development cost is relatively high. However, compared to murine antibodies and chimeric antibodies, humanized antibodies and fully humanized antibodies are less likely to cause immunogenic reactions in the human body and have fewer side effects.

Humanization of mouse antibodies can reduce the immunogenicity of PD1

Researchers conducted a humanization study of PD1 mouse antibody and identified its activity [1]. First, the researchers obtained hybridoma cells expressing PD1 mouse monoclonal antibody, and successfully purified PD1 mouse monoclonal antibody. The researchers identified the subtype of this antibody. The light and heavy chains of the antibody are Kappa and IgG chains, respectively. Secondly, they extracted RNA reverse transcription of hybridoma cells, amplified the variable region genes of PD1 mouse monoclonal antibody, and then molecularly cloned the variable region of the antibody with human Kappa chain and IgG chain. Splicing and constructing on eukaryotic expression vector.

By flow cytometry analysis, the affinity of this murine chimeric antibody was compared with that of the murine monoclonal antibody, and it was found that the affinity would decrease with the change of the constant region. Subsequently, the researchers humanized four framework regions (FR) in the variable region of the chimeric antibody, and obtained two humanized light chains VK1-39 and VK2-28, and four humanized heavy chains. Chain VH3-23, VH3-23-M, VH4-34 and VH4-34-M. A total of 8 groups of PD1 humanized chimeric antibodies were expressed through cross combination. Several groups of antibodies containing VK2-28 heavy chain have very little expression or have very low affinity.

After comprehensive consideration, the researchers finally locked the VK1-39&VH3-23-M group and the VK1-39&VH4-34-M group, and expressed and purified a large number of these two groups of antibodies. After purification, the antibody was reduced with a reducing agent, which further verified the correct spatial structure of the antibody. Together with the previous PD1 murine chimeric antibody, the affinity of the three antibodies was further verified, and the various effects that may be caused to the antibody during the purification process were excluded. In the end, it was found that the affinity of VK1-39&VH3-23-M was the smallest difference from mouse chimera, and the final purified amount of humanized chimeric antibodies of this group was better than that of VK1-39&VH4-34-M.

Finally, the researchers tested the biological activity of the VK1-39&VH3-23-M group and mouse chimeric antibody during the mixed lymphatic reaction, and found that whether it is an allogeneic reaction or a homologous reaction, VK1-39&VH3-23 under high-concentration dose conditions The humanized antibodies of group M are superior to murine chimeric antibodies. In summary, by humanizing the constant region, we can successfully obtain PD1 mouse chimeric antibody, and further transform it, replace the human-derived sequence in the FR region in the variable region, and successfully verify that they have good The affinity and biological activity.

Chimerization technology greatly reduces the immunogenicity of therapeutic antibodies, and humanized antibodies further reduce the immunogenicity of antibody drugs. However, even if the chimeric antibody is highly or even fully humanized, it has a certain degree of immunogenicity. Among them, the content of T cell epitopes is the main factor causing the immunogenicity of therapeutic antibodies. The tolerable and negligible ratios of humanized and chimeric antibodies are almost equal, which means that it is necessary for humanized antibodies to further reduce immunogenicity. However, whether fully human antibodies will be less immunogenic than humanized antibodies requires more clinical trials to verify.

[1] Humanization of PD1 mouse antibody and its activity identification [J].

Related Articles:

Immunogenicity Testing Method of Biotech Drugs

Medicilon Immunogenicity Test Service

Immunogenicity Testing of Medicilon

Return
Relevant newsRelevant news