Antibody Engineering: Humanized Monoclonal Antibodies (Part 5- mAbs)
Welcome to the 5th part of the 19-part series on monoclonal antibodies (mAbs).
Previous Parts: Part 1, Part 2, Part 3, and Part 4
The second type of engineered therapeutic antibody is humanized antibodies. In an attempt to reduce the immunogenicity of mouse monoclonal antibodies, scientists have engineered chimeric monoclonal antibodies through grafting the mouse antibody’s variable region onto the constant region of the human antibody.
To further improve humanization proportion and reduce the immunogenicity of the chimeric antibodies, humanized antibodies were developed . A humanized antibody can be developed through grafting the complementarity determining regions abbreviated as CDRs from the mouse antibody into the corresponding region of the human antibody.
Because CDRs are majorly responsible for recognizing and binding to the target antigen, the scientists thought why not to take the mouse antibody’s CDRs and graft them onto the corresponding variable region of the human antibody. Such generated antibodies are called humanized antibodies.
How are humanized antibodies engineered?
It is achieved through recombinant DNA methods using an appropriate vector and expression in mammalian cells. For this, first, the mouse is immunized with a specific antigen (therapeutic target) against which the antibodies are to be generated. After a few days of immunizing, the mouse is sacrificed. Next, its spleen is separated, from which plasma cells are isolated and are fused with the myeloma cells to generate hybridoma cells. The hybridoma cells that produce the antibodies against the desired therapeutic target are then isolated from the mixture of fused hybridoma cells.
From the selected hybridoma cell, mRNA is isolated and is reverse transcribed into cDNA. After this step, using specific primers, Polymerase chain reaction, abbreviated as PCR, amplifies the DNA sequence corresponding to the CDRs of the desired mouse antibody. Once the desired CDRs are amplified, these CDR sequences are inserted appropriately into a construct containing the DNA for a human antibody.
In the next step, the cloned target-specific antibody DNA sequence is expressed as a monoclonal Ab into the mammalian cells like Chinese hamster ovary cells, abbreviated as CHO cells. In this way, the humanized antibody is produced, containing the CDRs of mouse origin and antibody scaffold of human origin.
In some cases, apart from CDRs, certain other amino acids in the framework region of mouse antibodies are crucial to maintaining antibody binding activity. These amino acid residues cooperate with CDRs to directly interact with antigens. These essential framework amino acid residues can be identified by observing the structure of the antibody-antigen complex by X-ray crystallography, cryo-electron microscopy, and computer-aided protein homology modeling. Therefore, specific framework residues in humanized antibodies are replaced with the crucial amino acids present in the framework region of mouse antibodies, thereby improving the affinity and stability of the humanized monoclonal antibody.
Examples of humanized monoclonal antibodies
Humanized therapeutic antibodies have the potential to improve humanization proportion to 85%-90%. Humanized antibodies contain “zu” in their name. One such humanized antibody is Otelixizumab which is currently in clinical trials to treat rheumatoid arthritis and diabetes mellitus. Another humanized antibody in clinical trials is daclizumab to treat adults with relapsing forms of multiple sclerosis.
Currently, chimeric and humanized antibodies are the primary forms of human therapeutic antibodies playing an essential role in cancer therapy.
Immunogenicity of humanized monoclonal antibodies
Humanized antibodies harbor human sequence in constant regions and also exhibit nearly all human sequences in the variable region, except the CDRs which are mouse-derived. On the other hand, chimeric antibodies contain the complete variable regions from mouse antibodies and the constant regions are from human antibodies.
Since the humanized antibodies are more human-like, therefore as compared to chimeric monoclonal antibodies, the ADAs generated against the humanized monoclonal antibodies are reduced although the incidence of ADAs is not completely eliminated. Because the humanized antibodies still retain mouse CDRs which could be regarded as foreign antigens by host immune systems and can eventually lead to the production of ADAs against them.
Therefore to further reduce the immunogenicity of humanized antibodies, fully human monoclonal antibodies are developed, which will be discussed in part-6 of this series on antibody engineering.
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