The most commonly used murine monoclonal antibodies (mAbs) have played a crucial role in clinical diagnosis and treatment, which, however, have strong immunogenicity and can cause human anti-mouse antibody (HAMA) reactions in clinical applications, resulting in shorter antibody half-life (less than 20h) and weakened therapeutic effects. The use of DNA recombination and protein engineering technology to recombine antibody genes can minimize the murine nature of non-binding sites while retaining the effective binding site of the murine antibody to the antigen. This recombinant gene-expressed antibody with both murine and human components is called humanized antibody, or genetically engineered antibody. Humanization of antibodies is an important approach to transforming murine mAbs into safe and effective therapeutic drugs.
Humanized antibodies include chimeric antibodies, modified antibodies, resurfaced antibodies, and fully humanized antibodies.
Chimeric antibodies (https://www.creativebiolabs.net/chimeric-antibodies_29.htm)
The functional Ig variable (V) region gene is isolated from the hybridoma cell genome secreting mouse mAb, and then spliced with the human Ig constant (C) region gene, cloned into the expression vector, and transferred into the host cell. The antibody prepared thereby is chimeric antibody, of which the Fab region is murine-origin, and the Fc region is human-origin. Since about two-thirds of the entire antibody molecule is of human origin, the HAMA reaction is better avoided, the half-life of the antibody in the body is prolonged, and the pharmacokinetics of the antibody is improved.
In 1984, American scientists Morrison et al. first bridged the C-region gene of human Ig with the V-region gene of mouse mAb. Since the successful expression of human-mouse chimeric antibody, the research on genetic engineering antibody has developed rapidly. A variety of genetically engineered antibodies with different specificities have been approved by the FDA for clinical use, revealing satisfactory application prospects.
Modified antibodies
Modified antibody is also called complementarity-determining region (CDR) grafting antibody. The CDR of the variable region of 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 murine mAb is transplanted into 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 murine mAb while reducing its heterogeneity. However, although the antigen mainly connects the CDR, the framework region (FR) often plays a part and affects the spatial configuration of the CDR. Therefore, after replacing the human-derived FR region, the V region in which the mouse-derived CDR and the human-derived FR are embedded may change the CDR configuration of the single antigen, resulting in a (sharp) decrease in the ability to bind to the antigen. Although the antibody can be modified molecularly to introduce some key residues of the mouse FR region into the human FR region. If configured properly, its affinity can be equivalent to that of the original mouse antibody). However, humanized antibodies often do not achieve the affinity of the original murine mAbs.
Resurfaced antibody
Antibody surface remodeling refers to the humanization modification of amino acid residues on the surface of a heterologous antibody. The principle is to replace only regions that are significantly different from the SAR of human antibodies, and use amino acid substitutions that are similar to human antibody surface residues on the basis of maintaining antibody activity and reducing heterogeneity. In addition, the replaced segments should not be excessive. Residues that affect the size, charge, and hydrophobicity of the side chain, or may form hydrogen bonds to affect the conformation of the antibody CDR should not be replaced as much as possible.
Fully humanized antibody
Fully humanized antibody refers to the transfer of all human antibody-encoding genes to genetically engineered antibody gene-deficient animals through transgene or transchromosome technology, so that the animals can express human antibodies and achieve the goal of full humanization.
Among the various antibodies currently under clinical research, chimeric antibodies and humanized antibodies account for more than 70%, which are mainly used in the treatment of tumors, autoimmune diseases, and cardiovascular diseases, as well as anti-transplant rejection and anti-viral infections. From the perspective of R&D methods, fully humanized antibodies can be obtained from two common methods. One is the mouse immunization platform, and the other is the phage display.
Those humanized antibodies can be obtained from specialized antibody product supplier (https://www.creativebiolabs.net/), which can be applied in various experiments to support the research on new drugs or therapeutics.
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