Many rodent monoclonal antibodies with potential clinical applications have now been generated. However, because mouse antibodies are highly immunogenic in humans. Although the invention of chimeric antibodies derived from mouse-human chimeric antibodies has helped to reduce the immunogenicity of mouse monoclonal antibodies in humans, the chimeric antibodies still induce HAMA responses because the mouse-derived variable regions are sufficient to trigger immune responses in humans. The CDRs of the heavy chain V structural domain and the light chain V structural domain together form the antigen-binding site, while the framework region constitutes the scaffold for the antigen-binding site. The concept of CDR grafting for the production of less immunogenic antibodies stems from the assumption that the CDR of a mouse monoclonal antibody (donor antibody) can replace the CDR of a human antibody (recipient antibody).Although CDR grafting has been successful in some cases, it has been found that the majority of CDR grafted antibodies do not retain the antigen-binding affinity of the parental mouse antibody. This is because certain framework residues interact closely with CDR residues in the V structural domain. Some researchers have found that transfer of mouse CDR residues alone into the human framework may alter the structure of the CDR, resulting in a loss of antigen-binding affinity. To identify these residues, Queen and colleagues used a computer-generated three-dimensional model of the V structural domain. Humanized antibodies were generated by transferring CDR residues along with key framework amino acids from a mouse antibody into a human framework. Since the introduction of computer-guided humanization, a large number of humanized antibodies have been successfully generated.

The past three decades have witnessed the evolution of several humanization methods, including CDR transplantation, specificity determining residue (SDR) transplantation, skinning, framework (FR) shuffling, FR libraries, and guided selection. Traditional humanization methods involve grafting complementary decision regions (CDRs) into suitable human templates. For example, researchers grafted the CDR of an EGFR mAb into a human template and did not find detectable signals until some frameshift residues of the humanized mAb were mutated back into the frameshift residues of the mouse; this process is known as reverse mutation. In addition, some humanized mAb still exhibit immunogenicity. Therefore, a more favorable method for selecting human mAb templates is needed. The germline humanization method involves grafting the CDR of a mouse monoclonal antibody into the sequence of a human Ab germline gene with the highest similarity. Because of the low rate of intraclonal somatic hypermutation of human germline genes, the grafted mAb may have low immunogenicity.Tan et al. used a germline humanization method to align the V region of a mouse anti-human CD28 mAb to a human germline gene sequence.Pelat et al. used a human germline sequence to humanize 35PA83 and regained affinity by an additional reverse mutation. These findings suggest the importance of reverse mutations in maintaining mAb binding affinity. However, selection of a human mAb template and subsequent reverse mutation requires extensive testing, which can be both time-consuming and expensive. It is also possible to use phage display-derived human antibody libraries, in which antibody libraries in humans are displayed on phages in vitro.

Humanization of mouse monoclonal antibodies (mAb) is essential to reduce their immunogenicity in humans. However, humanized mAb usually lose their binding affinity. Deletion of residues leads to incompatibility between human FR and non-human CDR. Reverse mutation of these critical residues is often used to restore binding affinity.CDR transplantation relies on computer modeling to identify the classical structure of the determining residues and to design reverse mutant variants. If the computational predictions are imprecise or if a lack of expertise leads to inadequate selection of key amino acids for reverse mutation. Unlike rational approaches that rely on antibody structure or sequence information, FR reorganization is an empirical approach that relies on constructing and screening large and diverse combinatorial libraries by phage display. The library contains six CDRs from mouse antibodies fused to a diverse set of human germline FRs containing almost all heavy- and light-chain human germline genes suitable for antibody humanization. The large amount of diversity aids in the selection of optimal combinations of human FRs that maintain the dominant conformation of the non-human CDRs, resulting in consistently high affinity. Some researchers have developed a computer-simulated V(D)J recombination platform, in which we used the V(D)J human germline gene sequences to design five humanized drug candidates against Tumor Necrosis Factor using different human germline templates.

CDR region is the key site of antibody-antigen binding, which directly determines the specificity of the antibody. In the process of CDR transplantation, if not handled properly, it may lead to changes in the structure or function of the CDR region. Although CDR transplantation technology can effectively mitigate the immunogenicity problem of non-human antibodies, however, if there is a mismatch between the CDR region and the framework region of the human antibody, an immune response may still be triggered. Prior to CDR transplantation, key amino acid residues in the CDR region are precisely identified by biological and chemical analyses to ensure that these residues are preserved during the transplantation process. After CDR transplantation, the human framework region is adapted to maintain the structure and function of the CDR. Molecular simulations were performed using computational tools and algorithms to predict the impact of changes in amino acid residues changes. High-throughput screening technology is used for initial screening of humanized antibodies to rapidly screen the required antibodies. By continuously optimizing CDR transplantation techniques, developing new bioinformatics tools and experimental methods.

In the process of antibody humanization, the first task is to ensure that its specificity is not weakened, which requires the implementation of precise modification of the variable region of the antibody so as to ensure that its ability to bind antigen is not affected. \n Reducing the immunogenicity of an antibody to reduce immune rejection during clinical application is another key goal of antibody humanization. To this end, the constant region of the antibody needs to be modified to further cut down its immunogenicity. At the same time, the process of antibody humanization also needs to ensure the stability of the antibody to maintain its long-term efficacy in vivo. This involves optimizing the structure of the antibody with the aim of improving its overall stability. In addition, improving the production efficiency of antibodies to reduce production costs is also an integral part of antibody humanization. This requires optimization of the antibody's gene sequence, which in turn enhances its expression efficiency in cells. Teck Bio uses CDR transplantation technology, which enables the insertion of a human antibody framework region (FR) into the CDR region of a non-human antibody, ensuring that antibody specificity is maintained. The revertant mutation process involves fine-tuning of key amino acids in the FR region, which is designed to enhance the binding efficacy and stability of the antibody to the antigen. By mosaicing or remodeling the surface residues of the mouse CDR and FR, the profile of the mouse CDR is close to that of the human antibody CDR and the form is consistent with the human FR, thus effectively weakening the immunogenicity.