The main purpose of preparing humanized antibodies (hAb) is to reduce the heterogeneity of antibodies to facilitate their clinical application. In order to further reduce the mouse sequence of hAb, reshaped antibodies (RAb) have been constructed on the basis of chimeric antibodies in recent years.

RAb refers to the use of genetic engineering technology to replace the complementarity-determining region (CDR) sequence in the variable (V) region of human antibodies with the CDR sequence of mouse monoclonal antibodies. The reconstructed hAb has both the specificity of mouse monoclonal antibodies and the affinity of antibodies.

一．Molecular Structural Basis of Reshaped Antibodies

The V region of the antibody molecule is composed of two parts: CDR and framework region (FR). The heavy chain V region (V_H) and the light chain V region (V_L) each have 3 CDRs and 4 FRs. The 6 CDRs on V_H and V_L are folded into a loop and protrude outward from one end of the V region to form an antigen binding site, which is mainly determined by the specificity and affinity of the antibody. FR sequences are basically conservative and have high homology between different antibodies.

二．Construction of Modified Antibodies

The difference in homology between humans and mice in the antibody V region makes it difficult for human FR to fully adapt to mouse CDR, which may lead to weakening or even disappearance of RAb affinity, which is the biggest problem encountered when constructing RAb. There are three methods for constructing modified antibodies:

2.1 Surface remodeling: A premise of the surface remodeling approach is that the immunogenicity of the mouse McAb variable region originates from its surface residues. According to the analysis results of existing antibody crystal structure data, the fidelity of the relative solvent accessibility distribution of human and mouse antibody variable region residues at the sequence pairing position is 98%. Replacing mouse-specific surface residues with human ones can simulate the surface profile of human antibodies, evade the recognition of the human immune system, and achieve the purpose of humanization.

2.2 CDR transplantation: In human FR, select residues that interact with CDR, are closely related to antibody affinity, or play a key role in the folding of FR spatial structure to compensate for complete CDR transplantation. Stereostructural data and homology analysis show that the residues that make up the antibody FR are not equally important in terms of antigen binding, stable CDR conformation, and FR folding. Based on this, these residues are divided into three categories: ① Low-risk residues, that is, residues that are exposed to the solvent and contribute little to antigen binding and antibody structure. Replacing residues at these positions can reduce immunogenicity without affecting the affinity of the antibody. ② High-risk residues, that is, residues that are directly involved in antigen binding, stable CDR conformation, or FR folding. In order to maintain the activity of humanized antibodies, replacements at these positions should be avoided as much as possible. ③ Moderate-risk residues. High-risk residues and some moderate-risk residues in FR are generally referred to as non-CDR region supplementary regulatory residues. They are scattered at different positions in the linear sequence, providing a suitable "platform" for each CDR loop. Their shape and side chain size synergistically determine the basic conformation of the CDR and affect its antigen binding specificity. Therefore, after the CDR is moved to the human FR, these positions in the human FR must be replaced with mouse non-CDR region supplementary regulatory residues to compensate for the complete CDR transplantation.

2.3 Positioning retention: Humanized McAb retains the amino acid residues involved in antigen binding in the mouse McAb variable region, including some key residues in CDR and FR. Although the rest of the mouse framework region can be moved from a human FR, there is no doubt that the humanized antibody sequence obtained in this way differs from the conserved sequence (consensusse-se-nces) of human antibodies in some positions. These atypical residues, which are derived from somatic mutations during the affinity maturation of individual antibodies, will induce immune responses when applied to patients. Therefore, the ideal approach is to use the human FR conserved sequence as a template for humanization. Since the conserved sequences of different subclasses of the light and heavy chain variable regions of human antibodies are not the same, a suitable method is needed to find the most suitable conserved sequence. The minimal positional template indicates which positions in the antibody variable region absolutely need to maintain the integrity of the antibody's antigen binding domain, and provides a method for determining key positions. When selecting the human framework region, first search for the sequence most similar to the mouse framework region from the existing human antibody conservative sequences; secondly, determine the key position residues of the mouse variable region based on the simplest position template; finally, retain all these positions and humanize the rest.

三．Application of Modified Antibodies

(1) Anti-tumor therapy;

(2) Anti-transplantation reaction, humanized antibodies, including anti-IL-2T, CD3 and other antibodies, have been widely used in the treatment of anti-transplantation reaction;

(3) Anti-virus, humanized anti-KSVgB and gD glycoprotein antibodies have strong affinity, neutralization of HSV and cell protection are significantly higher than their parental monoclonal antibodies;

(4) Anti-TNF, humanized anti-TNF-A antibody Mab780 has the ability to partially neutralize TNFL-A and can competitively antagonize its parental monoclonal antibody Binding of human or chimeric antibodies to TNF-A;

(5) Anti-rheumatoid arthritis (RA), humanized antibodies can be used for the clinical treatment of RA, but they have mild to moderate toxicity, so they can only temporarily improve RA symptoms, and the technology needs further improvement;

(6) Anti-allergic reaction, humanized antibody IgEMaEll antibody has shown this ability, and has great development potential for the treatment of allergic reactions;

(7) Treatment of leukemia, antibodies have shown in clinical trials for the treatment of leukemia that the antibodies have no obvious toxicity and antigenicity.

Antibody humanization is an important part of experimental research on the production and preparation of recombinant antibodies. Obtaining humanized antibodies with high specificity and affinity plays an important role in effective antibody treatment of many diseases. Tek Biotech has mature antibody humanization technology, which can humanize antibody sequences and ensure that the affinity of the modified humanized antibodies remains at the same order of magnitude. We provide guaranteed humanization services to facilitate your experiments.