Antibody Engineering: Fully Human Monoclonal Antibodies and Phage Display (Part 6- mAbs)
Welcome to the 6th part of the 19-part series on monoclonal antibodies (mAbs).
Previous Parts: Part 1, Part 2, Part 3, Part 4, and Part 5
The third type of engineered therapeutic antibody is fully human monoclonal antibodies. In an attempt to reduce the immunogenicity of mouse monoclonal antibodies, chimeric monoclonal antibodies were developed through grafting the mouse antibody’s variable region onto the human constant region (See part 4 for knowing about chimeric monoclonal antibodies in detail). And to further improve humanization proportion and to further reduce the immunogenicity of the chimeric antibodies, humanized antibodies were developed through grafting the complementarity determining regions abbreviated as CDRs from mouse antibody into the variable region of a human antibody (See part 5 for knowing about humanized antibodies in detail). But still, the immune system can generate antibodies against them because the humanized antibodies still retain mouse CDRs which could be regarded as foreign antigens by host immune systems that can eventually lead to the production of antibodies against them.
Therefore, fully human monoclonal antibodies were engineered that can further reduce the incidence of production of antibodies against them by the host’s immune system.
These antibodies can be produced by 2 methods: Either from transgenic mice or from human antibody libraries.
Transgenic mice
The transgenic mice are created by replacing the entire mouse antibody genes with human antibody genes.
For this, the human heavy and light chain construct is cloned, which contains genes encoding the variable regions and the constant regions of human antibodies. The genes encoding the immunoglobulin chains are present as multiple gene segments. The light chains are encoded by V, J, and C gene segments. V and J gene segments encode for the variable region and C gene segments encode for the constant region of the light chain of the antibody. On the other hand, heavy chains are encoded by V, D, J, and C gene segments. V, D and J gene segments encode for the variable region and C gene segments encode for the constant region of the heavy chain of the antibody.
For the creation of transgenic mice, the human heavy chain construct is cloned, which contains V, D, J, and C gene segments encoding the heavy chain of the human antibody. Also, the human light chain construct is cloned that contains V, J, and C gene segments.
Then these constructs are micro-injected into fertilized eggs of the mouse in which endogenous mouse Ig genes are disrupted. Microinjection allows the insertion of human heavy and light chain constructs into the mouse genome to produce the transgenic mouse that is unable to produce mouse antibodies but is capable of producing human antibodies upon immunization. Thus, upon immunizing the transgenic humanized mouse with a target antigen, it produces the human antibodies. No part of the antibodies produced is mouse-derived. The monoclonal antibodies are produced through hybridoma technology.
Plasma cells are isolated from the mouse and are fused with myeloma cells to create a mouse hybridoma that secretes human monoclonal antibodies. Then the hybridoma producing the desired human monoclonal antibodies is selected and cloned to make multiple identical daughter cells that produce the desired human monoclonal antibodies.
Phage display method
The second method by which fully human monoclonal antibodies can be created is the phage display method. Using recombinant DNA techniques, the gene encoding a foreign protein (protein of interest) is integrated into the genome of filamentous bacteriophage M13. It leads to the expression of foreign peptides in conjugation with coat protein pIII, as a fusion protein, causing the bacteriophage to display the foreign peptides on its surface. A bacteriophage is a type of virus that infects bacteria.
For the generation of humanized antibodies, the phage-display antibody library is generated. In this, mRNA from the B cells is isolated and is reverse transcribed into cDNA. After this step, Polymerase chain reaction, abbreviated as PCR, is done to amplify different VH and VL chain-region gene families using specific primers to amplify all the possible transcribed variable regions within the Ig repertoire. The amplified VH and VL domains are joined by a protease-resistant glycine-serine linker into a single DNA sequence. These antibody sequences are then introduced and cloned as a gene fusion with the bacteriophage pIII gene in phagemid vectors. Phagemid vector is a plasmid that carries an antibiotic resistance gene, bacterial, and phage origins of replication. Additionally, phagemid vectors can be packaged into the capsid of a bacteriophage because of the presence of a genetic sequence that signals for packaging.
These phagemid vectors are electroporated into the E. coli cells. The E. coli cells are then infected with bacteriophages. After infection, the bacteriophages generate a phage-display library in which the Fv fragments containing VH and VL region are displayed on the phage coat protein.
After this, affinity screening of the phage-display antibody library is done by a process called biopanning. In this, the phages expressing the Fv region of antibodies on the surface are added to the antigens immobilized on a solid surface, for example, on ELISA plates. The antigen can be a protein on tumor cells or other cells against which a therapeutic antibody is required to be generated. The phages that have Fv regions specific for the antigens bind to the coated antigen. Non-specific phages are removed by stringent washing. Antigen-bound phages are then eluted and are re-infected into E. coli to produce a subset of phages for the next cycle of panning.
After several rounds, the specific antigen-binding phages are sufficiently enriched. In other words, the antibody Fv fragments displayed on the phage coat protein that exhibits a strong affinity for the antigen are selected. These Fv fragments are then isolated from the phage coat with the help of enzymes that cleave at the protease site engineered between the antibody and pIII protein of the phage. The isolated Fv fragments are then converted to intact IgG antibodies without impairing their antigen-binding activity. These intact IgG antibodies are called fully humanized antibodies.
This method can also be used to generate a phage-display library of complementarity determining regions abbreviated as CDRs. The phages express the CDRs on their surface, after which the expressed CDRs are screened to identify those CDRs that exhibit the strongest affinity for the desired antigen. Once the best CDRs are identified, they are grafted onto a human antibody scaffold to form fully human monoclonal antibodies. These are the several techniques by which fully human monoclonal antibodies can be generated.
The fully human antibodies contain -u- in their name. For instance, the first fully human therapeutic antibody developed was adalimumab. Also, this was the first phage-display derived fully human monoclonal antibody. Adalimumab binds and suppresses Tumor necrosis factor-alpha, abbreviated as TNFα, and is approved to treat inflammatory diseases, like rheumatoid arthritis, Crohn’s disease, and psoriasis. Other fully human monoclonal antibodies derived from phage display method are belimumab, necitumumab, avelumab, etc.
Isolation of monoclonal antibodies directly from humans
The biopanning approach can also be used to isolate and identify specific antibodies against novel or gene-mutated pathogens in an outbreak of emerging infectious diseases. The antigens on pathogens usually induce an immune response in patients; thus, the patients naturally produce high-affinity antibodies against the pathogens. Therefore, isolating mAbs directly from patients who were infected and cleared infections could be essential tools to neutralize the infectious agent. To obtain these antibodies, mRNA from the peripheral blood mononuclear cells (PBMCs) of pathogen-recovered people is collected. Then VH and VL chain-region gene families are amplified using specific primers to amplify all the possible transcribed variable regions within the immunoglobulin repertoire.
After this, these cloned fragments are cloned into phagemid vectors. Phagemid vectors are then electroporated into the E. coli cells. The E. coli cells are then infected with bacteriophages. After infection, the bacteriophages generate a phage-displayed library in which the Fv fragments containing VH and VL regions are displayed on the phage coat protein. After this, the phages expressing the Fv region of antibodies are added to the antigens immobilized on a solid surface. By the process of biopanning, the library can allow for the rapid identification of high-affinity antibodies, i.e., the Fv regions that strongly bind to the antigen.
These Fv fragments are then isolated from the phage coat and are used to develop intact antibodies or therapeutic drugs against the pathogens that infected the patients. Even these antibodies can be used as diagnostic reagents to diagnose if the person is infected with that pathogen.
Let’s take the example of isolating antibodies against pandemic influenza A virus H7N9, which broke out in 2013. For this, the researchers generated the phage-displayed antibody library from the human peripheral blood mononuclear cells of recovered patients that were infected with a novel influenza A virus H7N9. Using this library, antibodies targeting purified H7N9 virions were isolated. Two human antibodies were found to exhibit high neutralizing activity against live H7N9 virus due to their interactions with the receptor-binding site of viral hemagglutinin antigens. Thus, the isolation of the human antibodies can be used to develop human antibody-based drugs for the prevention and early treatment of influenza A or related viral pathogens.
Immunogenicity of humanized monoclonal antibodies
Since the chimeric antibodies retain the complete variable region and humanized antibodies still mouse CDRs of mouse antibody, therefore, they could be regarded as foreign antigens by host immune systems and can eventually lead to the production of ADAs against them. While in the case of fully human monoclonal antibodies, there are fewer chances of the production of ADAs when compared to the humanized antibodies or chimeric antibodies because in the case of fully human monoclonal antibodies both CDRs and antibody scaffold are derived from human immunoglobulin gene repertoires. However, immune responses to several fully human monoclonal antibodies have been reported when administered in patients. For instance, Adalimumab, a human IgG1 monoclonal antibody has been reported to generate significant immune responses through eliciting anti-idiotypic antibodies in some patients. The antibodies generated against the idiotypic determinants of the antibody, are called anti-idiotypic antibodies or simply written as anti-ID Ab. Idiotypic determinants of the antibody are discussed in detail in part 13 of Antibody basics.
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