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Antibodies made from non-human species whose protein sequences have been modified to increase their similarity to antibody variants produced naturally in humans.Browse Boster Featured Antibodies
Immunoglobulins are made up of four identical peptide chains: two identical heavy chains (H) and two identical light chains (L). Each chain comprises a variable domain (V) and a constant domain (C), which are responsible for interacting with the antigen or with other critical immune system components, respectively. Each V domain (VL and VH) has three non-contiguous complementarity-determining regions (CDRs), loops or hypervariable regions, which interact directly with an antigen.
The CDRs and adjacent sections of a molecule provide the most critical information about its antigen-specificity. This information has resulted in the creation of one of the most widely used procedures for humanizing antibodies – CDR-grafting – which incorporates these regions into the structure of a human antibody. As a result, the likelihood of the molecule being identified as foreign by the patient's immune system is decreased.
The majority of antibody humanization techniques begin by creating a chimeric IgG molecule that contains the V region of the xenogeneic molecule and the C region of a typical human IgG molecule. Following the expression of a stable chimeric molecule, humanization efforts are concentrated on the V region via a variety of approaches.
In 2020, the FDA or EMA approved 82 therapeutic antibodies for clinical use in the United States or the European Union, respectively. 39 of these are immunoglobulins that have been humanized in mice or rabbits. This data shows the fruits of a decade of antibody humanization research. Additionally, it demonstrates that humanized antibodies continue to compete on an equal footing with completely human antibodies in terms of clinical approval rates.
Various methods have been developed for humanization of antibodies up to now; each has its advantages and disadvantages.
Complementary determining regions (CDR) grafting, in which non-human antibodies' CDRs are grafted onto human frameworks, is a typical method for humanizing non-human antibodies. Human frameworks with the highest homology to the framework regions of non-human antibodies are typically chosen as CDR graft acceptors. The main issue with this process of humanizing antibodies is that they lose their affinity for their specific targets. In some circumstances, grafting CDR loops from murine antibodies onto human frameworks has little effect on antibody affinity, whereas in many others, it drastically diminishes affinity.
Vernier zone residues, which are found in murine framework areas, have been shown to alter the conformation of CDR loops and antibody affinity. These residues are found close to the CDRs in the -sheet framework regions. As a result, these residues are retained in humanized antibodies after the selection of desired human frameworks.
Because of the importance of vernier zone residues, technologies like X-ray diffraction and isothermal titration calorimetry should be used to assess and optimize the management of conformational entropy change during humanization of murine antibodies.
A new technique for humanizing antibodies based on CDR region homology. It was expected that murine and human antibody frameworks with similar CDRs might support each other's CDR structure with excellent affinity retention. Human frameworks are not chosen based on homology of framework areas, and key murine residues (vernier zone residues) are not restored in humanized antibodies. The creation of motifs that may be detected as foreign is decreased with this strategy. The antibodies generated by this technology have been discovered to have higher levels of affinity than those generated by the framework-homology-based humanization method.
SDR grafting is used to humanize antibodies by transferring only some of the CDR residues to a human scaVold. As a result, SDR grafting produces a humanized antibody with a significantly lower amount of non-human residues than its CDR-grafted equivalent. Both the SDR and CDR grafting techniques necessitate the selection of the most appropriate human frameworks to serve as templates, as well as the identification of the framework residues that are crucial to the antigen-binding site's structural preservation. Human templates contribute a major portion of the humanized antibody's framework sequences.
If any of the crucial residues of the target antibody are found to be divergent from their corresponding residues in the human template when the sequences of the frameworks of the target and template antibodies are compared, the crucial residues of the target antibody are incorporated in the frameworks of the templates for the Wnal design of the humanized antibody.
Human germline genes could be used to humanize murine antibodies as an alternate supply of framework regions. Germline genes have less intraclonal somatic hypermutation than framework areas derived from IgG. As a result, humanized antibodies with germline frameworks are projected to have lower immunogenicity than humanized antibodies with IgG frameworks. They created three humanized formats of this antibody, including single-chain antibody, Fab fragments, and chimeric Fab fragments incorporating human constant regions, while preserving murine vernier zone residues to preserve affinity.
The single-chain and Fab fragment variants of this antibody had a two-fold lower affinity for A peptide than recombinant chimeric Fab fragments and a five- to six-fold lower affinity than papain-cleaved parental Fab fragments, according to the researchers. Despite the fact that humanization of murine WO-2 reduced affinity, this was not a bad thing because the humanized derivates still had high affinity (at nanomolar range)
When compared to chimeric Fab fragments, humanized Fab fragments demonstrated a slight reduction in avidity. The biological activity of this humanized format was comparable to that of chimeric and hybrid Fabs, and it was able to display immunosuppressive function.
Another option for humanizing non-human antibodies is the antibody resurfacing method, which was first published by Paldan in 1991. This strategy entails replacing potentially allergenic surface framework residues with the most common human residues in their stead. The foundation for this strategy is that surface residues are the sole cause of human anti-mouse antibody (HAMA) reaction to the variable area. Antibodies that have been humanized in this way usually retain their stability and affinity.