Antibody Engineering: Chimeric Monoclonal Antibodies (Part 4- mAbs)
Welcome to the 4th part of the 19-part series on monoclonal antibodies (mAbs).
Previous Parts: Part 1, Part 2, and Part 3
As already discussed in Part 1 of Antibody basics, an antibody molecule consists of two identical light (L) chain polypeptides and two identical heavy (H) chain polypeptides. And each light chain and heavy chain contains two distinct regions: Variable regions (V) and constant regions (C). It is the variable region in the light and heavy chains of an antibody molecule that together forms an antigen-binding site.
Because the target recognition only happens in the antibody’s variable domains, the scientists thought, why not take the mouse antibody’s variable region and graft them onto the human antibody’s constant region. Such generated antibodies are called chimeric antibodies. In other words, the chimeric antibodies contain variable regions from one species and the constant regions from the other species.
Production of chimeric monoclonal antibodies
Chimeric antibodies are generated using recombinant DNA technology. The immunoglobulin (Ig) variable regions of a selected mouse hybridoma are joined to the human Ig constant regions. For this, first, the mouse is immunized with a specific antigen against which the antibodies are to be generated. The antigen that is introduced in the mouse is called a human therapeutic target.
After a few days of immunizing, the mouse is sacrificed, and its spleen is isolated. Then, plasma cells are separated from the spleen and are fused with the myeloma cells to construct hybridoma cells. Hybridoma technology and its procedure have already been discussed in detail in Part 2. 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, using specific primers, Polymerase chain reaction, abbreviated as PCR, is done to amplify the DNA sequences, including promoter, leader, and VH and VL sequences encoding variable regions of the mouse antibody. In addition, the PCR primers create restriction enzyme sites in the amplified sequences to conveniently insert variable regions into the vectors.
On the other hand, a gene construct is made that contains DNA sequences including promoter and CH and CL sequences encoding constant regions of human antibodies. Then, the mouse/human chimeric genes are constructed by inserting human and mouse genes into a circular piece of DNA called a plasmid. The plasmid is then introduced into mammalian cells via a process called transfection.
After transfection, the mammalian cells produce the antibodies encoded by the recombinant gene construct. Finally, the antibodies are purified from the culture supernatant. The antibodies encoded by mouse/human chimeric gene construct are called chimeric antibodies or mouse-human chimera.
The antigenic specificity of this chimeric antibody is determined by the variable region derived from the mouse. But the constant region of the chimeric antibody is encoded by human genes, because of which the chimeric antibodies have fewer antigenic determinants and therefore are far less immunogenic than the mouse monoclonal antibodies when administered in humans.
Additionally, since the chimeric antibodies possess the mouse’s variable regions, they have the appropriate binding sites to recognize and bind to the specific target antigen. And the constant regions are encoded by the human DNA because of which they retain the biological effector functions of a human antibody and are more likely to trigger complement system activation and Fc receptor binding.
Examples of chimeric monoclonal antibodies
Mouse-human chimeric antibodies have been developed to treat patients suffering from various diseases, including cancer. The antibodies are designed in such a way that the mouse variable region recognizes the tumor antigens. In contrast, the human constant region activates the biological effector functions like activating natural killer cells to kill the tumor cells.
Due to their prolonged circulating half-life and relative ease of production, generally used therapeutic monoclonal antibodies are IgGs.
The IgG chimeric therapeutic antibody, “rituximab,” was the first monoclonal antibody approved by the US Food and Drug Administration (FDA) in 1997 to treat non-Hodgkin’s lymphoma. This medication has since been used to treat several CD20-positive B-cell malignancies and rheumatoid arthritis. The naming convention of Chimeric antibodies includes using the “xi” stem in their name. A few examples of chimeric antibodies that are approved for human therapy include abciximab, basiliximab, cetuximab, infliximab, rituximab, etc.
Immunogenicity of chimeric monoclonal antibodies
Immunogenicity is the ability of engineered monoclonal antibodies to provoke the immune system to generate antibodies against them. Since these therapeutic antibodies, also known as antibody drugs are glycoproteins, they have specific regions on them called antigenic determinants. These antigenic determinants can induce the immune system to produce antibodies against the antibody drugs or therapeutic antibodies. The antibodies generated against the antibody drugs are referred to as anti-drug- antibodies, abbreviated as ADAs.
Anti-drug antibodies can lead to the negation of all antibody drug-related effects, thus completely inhibiting the therapeutic aspect of the drug. Importantly, anti-drug antibodies may further cause adverse effects ranging from skin rashes to systemic inflammatory responses in the patients, which can impact both the safety and efficacy of the antibody drugs in clinical use. Therefore, it is extremely important for researchers to humanize antibodies as much as possible to lower the generation of anti-drug-antibodies in the patients.
The chimeric monoclonal antibodies contain variable regions from mouse antibodies and the constant regions from human antibodies. Undoubtedly, the chimeric monoclonal antibodies generate less ADAs than mouse monoclonal antibodies when injected into the patient’s body. But still, the possibility of the generation of ADAs is not eliminated. When the chimeric monoclonal antibody is injected into the patient, the variable region of the chimeric monoclonal antibody can be recognized as foreign by the patient’s body because of its mouse origin, thus the patient may generate ADAs against the injected chimeric monoclonal antibodies. Therefore to reduce the immunogenicity, humanized and fully human monoclonal antibodies are developed, which will be discussed in Part-5 of this series on monoclonal antibodies.
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