Since the emergence of monoclonal antibodies, the most commonly used mouse derived monoclonal antibodies have played a significant role in clinical diagnosis and treatment. However, commonly used mouse monoclonal antibodies have strong immunogenicity and can cause human anti mouse antibody (HAMA) reactions in clinical applications, resulting in a shortened half-life of the antibody (less than 20 hours) and weakened therapeutic effects. By utilizing DNA recombination and protein engineering techniques, antibody genes can be recombined to preserve the effective binding site of mouse antibodies to antigens while minimizing the non binding site's murine origin. This type of antibody, which contains both mouse and human components expressed through recombinant genes, is called humanized antibody or genetically engineered antibody. Humanization of antibodies has become an important way to convert mouse derived monoclonal antibodies into safe and effective therapeutic drugs.
Humanized antibodies include chimeric antibodies, modified antibodies, surface remodeling antibodies, and fully humanized antibodies
1、 Chimeric antibody
Due to 90% of HAMA targeting the C region of antibodies, functional Ig V region genes were isolated from the genome of hybridoma cells secreting mouse monoclonal antibodies, then spliced with human C region genes in a certain way, cloned into expression vectors, and transferred into host cells to prepare antibodies called Chimeric Antibodies. The Fab region of this antibody is mouse derived, while the Fc region is human derived. Due to the fact that about 2/3 of the entire antibody molecule is human derived, HAMA reaction is effectively 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 connected the C region gene of human Ig with the V region gene of mouse monoclonal antibodies, successfully expressing human mouse chimeric antibodies. Since then, the research on genetically engineered antibodies has developed rapidly. Multiple genetically engineered antibodies with different specificities have been approved by the FDA for clinical use, demonstrating promising application prospects.
2、 Modified antibody
Modified antibodies, also known as CDR grafting antibodies, have variable region CDRs that recognize and bind to antigens, directly determining the specificity of the antibody. Transplanting the CDRs of mouse monoclonal antibodies into the variable region of human antibodies, replacing human antibody CDRs, enables human antibodies to acquire the antigen binding specificity of mouse monoclonal antibodies while reducing their heterogeneity. However, although antigens mainly come into contact with the CDRs of antibodies, the FR region also frequently participates, affecting the spatial configuration of CDRs. Therefore, when replaced with the human derived FR region, the V region where mouse derived CDRs and human derived FRs are embedded may alter the original CDR configuration of the monoclonal antibody, leading to a decrease or even a significant reduction in its ability to bind to antigens. Although molecular design of antibodies is currently possible by introducing certain key residues from the mouse FR region into the human FR region (if properly configured, their affinity can be comparable to that of the original mouse antibody), humanized antibodies often fail to achieve the affinity of the original mouse antibody.
3、 Surface reshaping antibody
Surface reshaping antibodies refer to the humanized modification of amino acid residues on the surface of heterologous antibodies. The principle of this method is to only replace regions that differ significantly from human antibody SAR, while maintaining antibody activity and reducing heterogeneity, and selecting amino acid substitutions similar to human antibody surface residues; In addition, the number of segments to be replaced should not be too large, and residues that affect the size, charge, hydrophobicity, or may form hydrogen bonds and affect the conformation of the antibody complementarity determining region (CDR) should be avoided as much as possible.
4、 Fully humanized antibody
Fully humanized antibodies refer to the transfer of all human encoded antibody genes to genetically engineered antibody gene deficient animals through genetic engineering or chromosome transfer technology, allowing animals to express human antibodies and achieve the goal of fully humanized antibodies.
FDA approved human monoclonal antibodies and the techniques used for their use
Among the various antibodies currently undergoing clinical research, chimeric antibodies and humanized antibodies account for over 70%. At present, humanized antibodies are mainly used for the treatment of tumors, autoimmune diseases, cardiovascular diseases, as well as anti transplant rejection and antiviral infections. From the perspective of research and development methods, we can obtain fully humanized antibodies from two common approaches. The first method is the mouse immune platform, which allows mice to express variable, diverse, and linker sequences (VDJ antibody sequences) of human antibodies and immunize them; The second method is "phage display", which involves screening in large-scale antibody libraries. Pujian Biotechnology, through a comprehensive protein expression and purification platform, stable cell line construction platform, and phage display technology platform, combined with an experienced team of experts (who have successfully developed and marketed therapeutic antibody drugs), can provide you with one-stop services, including:
(1) Antibody variable region/antibody full-length sequencing
Perform antibody variable region/full-length gene sequencing using mouse monoclonal antibody cell lines, 2-4 weeks.
(2) Chimeric antibody expression and antigen antibody binding analysis
Design chimeric antibodies by combining variable region sequences and human constant region sequences, and express the chimeric antibodies through the mammalian system; Perform antigen antibody binding analysis on chimeric antibodies, and provide samples for affinity and biological activity testing to customers for 8-10 weeks.
(3) Humanized antibody design
Humanized design of VH and VL regions based on CDR transplantation, response mutation, bioinformatics analysis and other methods, providing different packages for customers to choose from according to their needs, 2-3 weeks.
(4) Humanized antibody expression
Codon optimization, gene synthesis, subcloning, transfection, antibody expression and purification, 8-10 weeks.
(5) Humanized antibody property detection
Combining force testing: ELISA/FACS to determine EC50 value, or Biacore/Octet to determine Kd value; In vitro biological activity analysis of humanized antibodies and comparison with chimeric antibodies; Cross reactivity analysis between humanized antibodies and homologous species; Biophysical analysis of humanized antibodies: SEC-HPLC analysis, DS-PAGE analysis, DSC analysis.
The screening of humanized antibodies is based on the following evaluation criteria:
The expression level of mammalian protein expression system (HEK293F or CHO) for transient expression.
Compare the binding affinity of humanized antibodies with chimeric antibodies based on detection indicators EC50 (detected by ELISA or FACS) or Kd (detected by Biacore or Octet).
Based on in vitro bioactivity testing, compare the activity of humanized antibodies with that of chimeric antibodies.
Based on in vitro binding assays, detect the cross reactivity between humanized antibodies and related homologous species.
Based on the analysis of biophysical properties, the comparison between humanized antibodies and chimeric antibodies mainly includes the following points:
a) Identify the degree of polymer aggregation through SEC-HPLC.
b) Identification of reducing and non reducing SDS-PAGE.
c) Detect Tm values through DSC analysis.
I believe that with the development of molecular biology technology, especially the humanization of mouse antibodies, the rapid development of antibody library technology, transgenic technology, and the continuous in-depth research on disease mechanisms, humanized antibodies will be more widely used in clinical applications. Let's wait and see.
