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Humanized antibody mainly refers to the re-expressed mouse monoclonal antibodies modified by gene cloning 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 antibodies, and reduces heterogeneity at the same time. Its application would be beneficial to the human body.
Humanized antibody means that the constant region of the antibody (CH and CL regions) or all of the antibody is encoded by human antibody genes. And humanized antibodies can greatly reduce the immune side effects caused by heterologous antibodies to the human body.
According to different principles of action, humanized antibodies can be divided into four categories: human-mouse chimeric antibody, modified antibodies, resurfaced antibodies and human antibodies.
What is a chimeric antibody? Human-mouse chimeric antibody, that is, the crossable region of the antibody is derived from small rat McAb, while the constant region is derived from human antibody. Such antibodies not only maintain the specificity and affinity of the original McAb, but also greatly reduce the immunogenicity in the human body.
Chimeric antibody inserts the light and heavy chain variable region genes of the heterologous monoclonal antibodies into an expression vector containing the constant region of a human antibody, transforming mammalian cells to express the chimeric antibody by DNA recombination technology. The V regions of the light and heavy chains are heterologous, while the C region is of human origin, so that nearly 2/3 of the entire antibody molecule is of human origin. The antibody produced in this way reduces the immunogenicity of the heterologous antibody while retaining the ability of the parent antibody to specifically bind to the antigen.
Modified antibody is also called CDR grafting antibody. The CDR of the variable region in the antibody is the region where the antibody recognizes and binds to the antigen, which directly determines the specificity of the antibody. The CDR of the mouse monoclonal antibodies is transplanted to the variable region of the human antibody to replace the CDR of the human antibody, so that the human antibody obtains the antigen binding specificity of the mouse monoclonal antibody while reducing its heterogeneity. However, although the antigen is mainly in contact with the CDR of the antibody, the FR region often affects the spatial configuration of the CDR.
Therefore, after replacing the human-derived FR region, the V region where the mouse-derived CDR and the human-derived FR are embedded may change the CDR configuration of the single antigen, and the ability to bind the antigen will decrease or sharply decrease. Although it has been possible to carry out molecular design of antibodies and introduce certain key residues of mouse FR regions into human FR regions, if properly configured, their affinity can be equivalent to that of the original mouse antibody, but humanized antibody often fails to reach the affinity of the original mouse monoclonal antibody.
Surface remodeling antibody refers to the humanized modification of amino acid residues on the surface of heterologous antibody. The principle of this method is to replace only the regions that are significantly different from the SAR of human antibodies.
On the basis of maintaining antibody activity and reducing heterogeneity, amino acid substitutions similar to the surface residues of human antibodies should be selected; in addition, the replaced segments should not be excessive. And try not to replace residues that affect the size, charge, hydrophobicity of the side chain, or may form hydrogen bonds to affect the conformation of the antibody complementarity determining region (CDR).
Human antibody refers to the transfer of human antibody genes through transgene or transchromosome technology to transfer all the human antibody-encoding genes to genetically engineered animals with antibody gene deletions, so that the animals can express human antibodies to achieve human antibodies purpose.
According to the different sources of monoclonal antibodies, they can be divided into mouse antibodies, chimeric antibody, humanized antibodies and human antibodies. The so-called human monoclonal antibody means that 100% of the gene source is from human. However, in fact, human monoclonal antibodies are also produced in mice.
By transferring human genes related to antibody production to mice, the structure of antibodies in mice can be the same as those of human antibodies. Therefore, human antibodies ≠ antibodies made directly from human cells, but the structure of the antibody is 100% encoded according to human genes. Humanized monoclonal antibodies are mostly human-derived and are fused with mouse-derived components. In recent years, scientists have deliberately kept the key beneficial gene structure of mouse resistance in the human frame through genetic engineering, which plays a special role, just like optimizing reprocessing.
For example, Ichizumab (humanized monoclonal antibody) retains 1.8% of the mouse-derived ingredients, which integrates advantageous genes, so it achieves high affinity and rapid onset of action.
Humanization of antibodies is an important part of experimental research on the production and preparation of recombinant antibodies (monoclonal antibodies). The antibody humanization is the process of developing from mouse antibody to human antibody.
More than a hundred years ago, the discovery of the principles of antibody-antigen specific binding and the passive immune characteristics of antibodies opened up a new way of disease diagnosis. The advent of monoclonal antibody technology in 1975 accelerated the widespread application of this method. In the initial stage, most of the monoclonal antibodies used clinically were mouse monoclonal antibodies. Due to the species specificity of humans and mice, there are various restrictions on the use of them.
Although the mouse antibody is specific to the target antigen and can specifically bind to the target antigen, it cannot activate the corresponding human effector system, such as antibody-dependent cell-mediated cytotoxicity (ADCC), complement-dependent cytotoxicity (CDC), etc., so that the antigen-antibody reaction cannot occur normally.
In addition, the mouse antibody enters the human body as a foreign protein, which will cause the human immune system to respond and produce specific antibodies taking the mouse antibody as the antigen, that is, the human anti-mouse antibody. Usually heterologous protein with a short half-life will be quickly eliminated in the human body. Due to various limitations in the clinical application of mouse antibodies, people use recombinant DNA technology to humanize mouse antibodies to humanize antibodies.
The humanization of mouse antibody is to make it have a very similar profile to the antibody molecule in the human body through genetic modification, thereby evading recognition by the human immune system and avoiding the induction of HAMA response. The humanization of antibodies should follow two basic principles, that is, to maintain or improve the affinity and specificity of the antibody: greatly reduce or substantially eliminate the immunogenicity of the antibody.
The process of antibody humanization has gone through three stages: human-mouse chimeric antibody, humanized antibody and human antibody. According to different principles of action, humanized antibodies can be divided into four types: human-mouse chimeric antibody, modified antibodies, resurfaced antibodies, and human antibodies.
| Mouse antibody | Humanized antibody | Human antibody | |
|---|---|---|---|
| Antibody Types | Recombinant antibody | Recombinant antibody | Recombinant antibody |
| Structure | Both variable and constant regions are derived from mouse antibodies | The variable region is from small (large) mouse McAb, and the constant region is from a human antibody | Both variable and constant regions are derived from human antibodies |
| Immunogenicity | Strong | Strong | Strong |
| Stimulate the human immune response | Easily cause human immune response and produce HAMA | Weak | Weak |
| Clinical application | Large restriction, small application range | Wide | Wide |
| Difficulty of preparation | Easy | Difficult | Difficult |
In recent years, the emergence of humanized antibody and human antibodies has brought new hopes for clinical applications. They show excellent effect in the treatment of tumors, autoimmune diseases and cardiovascular diseases, as well as anti-transplant rejection and anti-viral infections. They can reduce the adverse reactions of traditional tumor drug therapy, protect the function of transplanted organs, and be the induction of solid organ transplantation therapy.
The US FDA approved the first human-mouse chimeric Fab antibody (ReoPro) in 1994. This drug has been widely used in the treatment of various cardiovascular diseases, mainly used to prevent restenosis after coronary artery formation. Research in recent years shows that humanized antibodies are prospecting in anti-viral infection (such as HBV, HCV, HIV and other viral infectious diseases).
Human antibody belongs to humanized antibody. Humanized antibody means that the constant region of the antibody (CH and CL regions) or all of the antibody is encoded by human antibody genes. While human antibodies mean that all antibodies are encoded by human antibody genes, allowing animals to express human antibodies.
Want to learn more about antigens and antibodies? Read about FC stand information.
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