Antibody Engineering: Chimeric Monoclonal Antibodies (Part 1)
Antibody engineering can be defined as modifying the monoclonal antibodies (mAbs) for research or clinical purposes. Monoclonal antibodies as the name indicates, are the antibodies that are derived from the clones of a single activated B cell that recognizes a particular epitope on an antigen.
In other words, monoclonal antibodies are identical antibodies with the same antigen specificity.
This property of monoclonal antibodies has revolutionized the fields of diagnosis and immunotherapy for the treatment of a variety of diseases, such as cancer, genetic diseases, HIV, autoimmune diseases, etc. Also making them more effective than conventional drugs. Because the conventional drugs not only attack the foreign pathogen or diseased cells but they also attack the body’s own cells that cause harsh side effects.
On the other hand, therapeutic monoclonal antibodies target only the foreign antigen or the specific protein markers on the target cells. For instance, in the case of cancer treatment, monoclonal antibodies are developed so that they are able to bind to the proteins present specifically on the tumor cells. After binding, the antibodies tag the target cancerous cells for destruction by the immune system.
Since the therapeutic monoclonal antibodies bind only to the intended target cells, therefore, unlike conventional drugs there are very few unexpected side effects while using them.
A challenging issue remains: most of the monoclonal antibodies are generated in mice through the Hybridoma Technology developed by Kohler and Milstein in 1975.
There are many clinical applications in which the mouse monoclonal antibodies are useful like in diagnostic and research purposes. However, when mouse monoclonal antibodies are introduced into the patients, the mouse antibodies are recognized as foreign by the patients’ bodies, generating an antibody response or anti-antibodies against the foreign mouse antibodies. Thus clearing the mouse monoclonal antibodies from the bloodstream.
In addition to this, circulating complexes of mouse and human antibodies can also cause allergic reactions. In some cases, these complexes can accumulate in organs such as the kidneys and can cause serious and even life-threatening reactions.
One way to avoid these undesirable reactions is to use human monoclonal antibodies for clinical applications. However, the mass preparation of human monoclonal antibodies has been hampered by numerous technical problems.
In response to the difficulty of producing human monoclonal antibodies and the complications resulting from the use of mouse monoclonal antibodies in humans, there is now a major effort undergoing by scientists to engineer monoclonal antibodies with recombinant DNA technology. With the knowledge of antibody structure, scientists and researchers have humanized the mouse antibodies to reduce their toxicity and side effects in patients.
Structure of Antibodies: Before discussing how antibody engineering can generate different types of therapeutic antibodies, let’s quickly discuss the structure of an antibody. Any antibody molecule consists of two identical light (L) chain polypeptides designated as L chains, and two identical heavy (H) chain polypeptides designated as H chains. And each light chain and heavy chain contains 2 distinct regions: Variable regions [V] and constant regions [C]. V regions are designated as VL in light chains and VH in heavy chains. It is the variable region in the light and heavy chain which together form the antigen-binding site. So there are 2 antigen-binding sites in an antibody molecule.
In other words, the variable region is responsible for giving the antibody its specificity to bind to a particular antigen. Additionally, all of the differences in specificity displayed by different antibodies to bind to different antigens can be traced to differences in the amino acid sequences of V regions. Both the heavy and light polypeptide chains of an antibody molecule contain several homologous units of 110 amino acid residues, and each unit is termed a domain. In the case of the light chain of an antibody, there is one variable domain (VL) and one constant domain (CL) and in the case of the heavy chain, there is one variable domain VH, and either 3 or 4 constant domains designated as CH1, CH2, CH3, and CH4.
Within the variable domain of each heavy and light chain, there are 3 complementarity determining regions, abbreviated as CDRs. Specifically, CDRs contribute to the antigen-binding site of an antibody. Since the variable domains of one heavy and one light chain together form 1 antigen-binding site and each variable domain contains 3 CDRs, therefore a single antigen-binding site is constituted by 6 CDRs, and similarly, 2 antigen-binding sites of an antibody molecule are constituted by a total of 12 CDRs.
By contrast, within the same antibody molecule, the regions beyond the variable regions of both heavy and light chains are called constant regions. There is a single constant region present in each light chain which is designated as CL. On the other hand, multiple constant regions are present on each heavy chain and are designated as CH.
The constant regions are so-called because the amino acid sequence in these regions shows less variation among the different antibodies. It is the constant region of the heavy chain of the antibodies that forms the basis of the classification of antibodies IgG, IgM, IgA, IgE, and IgD. Each class of antibody is distinguished by the unique amino acids present in the constant region of the heavy chain that confer structural and functional properties to the antibodies. For instance, secretory IgA is present in body secretions and prevents attachment of pathogens to mucosal membranes. IgG antibody crosses the placenta and provides protection to the fetus. IgM antibody is most efficient in activating the complement system. IgE antibody plays a very crucial role in allergic reactions and in defense against parasitic worms. And IgD antibody signals B cells to get activated. The specific functions of each class of an antibody can be attributed to the unique amino acids present in the constant region of the respective heavy chains.
Additionally, there are 2 Fab regions and 1 Fc region in a basic Y-shaped antibody structure. The 2 Fab regions are the antigen-binding fragments that contain the variable regions of both light and heavy chains and the constant region of light and the first constant region of heavy chains.
On the other hand, the Fc region is composed of only constant regions of heavy chains and form the stem of the basic antibody molecule.
Fc region ensures that each antibody generates an appropriate immune response for a given antigen by binding to various cell receptors such as Fc receptors present on macrophages, natural killer cells, etc, and other immune molecules, such as complement proteins. By doing so, it mediates different antibody effector functions including opsonization, cell lysis, complement activation, antibody-dependent cellular cytotoxicity (ADCC), and degranulation of mast cells, basophils, and eosinophils.
Antibody engineering
With the knowledge of antibody structure and regulation, three types of therapeutic antibodies have been engineered. These are chimeric monoclonal antibodies, humanized monoclonal antibodies, and fully human monoclonal antibodies.
First are chimeric monoclonal antibodies
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 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.
Chimeric antibodies can be generated by genetic engineering, by joining the immunoglobulin (Ig) variable regions of a selected mouse hybridoma to human Ig constant regions. For this, first, the mouse is immunized with a specific antigen against which the monoclonal antibodies are to be generated. The antigen that is introduced in the mouse is called human therapeutic target. For instance, if the therapeutic antibodies are to be generated against tumor cells, then the human therapeutic target will be the protein markers found specifically on tumor cells.
After a few days of immunizing, the mouse is sacrificed and its spleen is isolated. From the spleen, the antibody-producing plasma cells are isolated and are then fused with the cancerous myeloma cells to construct hybridoma cells that secrete monoclonal antibodies. The antibody-producing plasma cells have a definite life span but the myeloma cells are cancerous plasma cells, which are immortal and can divide indefinitely. Thus the hybridoma cells produced
- Possess the capability to produce antibodies, a property of plasma cells
- And they also become immortal, a property of myeloma 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, the DNA sequences including promoter, leader, and VH and VL sequences encoding variable regions of the mouse antibody are amplified. 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, e.g. CHO 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 the mouse/human chimeric gene constructs 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 this antibody is encoded by human genes, because of which the chimeric antibodies have fewer antigenic determinants and therefore are far less immunogenic when administered in humans.
These chimeric antibodies have an advantage because they possess the mouse’s variable regions that have the appropriate binding sites to recognize and bind to the specific target antigen. And the constant regions are encoded by human DNA because of which they retain the biological effector functions of a human antibody and are more likely to trigger human complement activation, Fc receptor binding, etc.
For instance, mouse-human chimeric antibodies can be used to treat patients suffering from cancer. The antibodies are designed in such a way that the mouse variable regions recognize the tumor antigens while the human constant region activates the biological effector functions like activating natural killer cells to kill the tumor cells.
In 1997, the first IgG chimeric therapeutic antibody rituximab was approved for the treatment of non-Hodgkin's lymphoma and rheumatoid arthritis. The naming convention of Chimeric antibodies includes using xi stem in their name. 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-2 of this series on antibody engineering.
