Antibody Engineering: Humanized Monoclonal Antibodies (Part 2)
This is the second part of the three-part series on Antibody Engineering. You can read the first part here to understand the basics of antibody engineering and chimeric monoclonal antibodies.
The second type of engineered therapeutic antibodies 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 (See part 1 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 onto the variable region of a human antibody.
But before discussing how the humanized antibodies are engineered, we will first understand why CDRs are important and why they are chosen for grafting onto human antibodies to generate humanized antibodies?
What are CDRs?
We have already discussed in part 1 that it is the variable regions in the light (VL) and heavy chains (VH) which together form the antigen-binding site. There are 2 antigen-binding sites in an antibody molecule. Both the heavy and light polypeptide chains contain several homologous units of 110 amino acid residues, and each unit is termed a domain.
The overall shape of the variable domains of each heavy and light chain can be defined as the sandwich of two 𝜷 pleated sheets, each sheet containing antiparallel 𝜷 strands of amino acid residues connected by loops of varied lengths.
The interesting thing to know is that the maximum variation in the variable domain of heavy and light chains is present in the amino acid sequences of loops that join the β strands. These loop regions are called hypervariable regions. The light and heavy variable domains fold in a manner that brings the hypervariable regions of both chains together to create the Ag binding site also called a paratope.
The surface of the antigen-binding site formed by the hypervariable regions is complementary to the structure of epitopes to generate antigen specificity. The epitope is the specific part of an antigen to which the antibody binds. The antigen specificity of an antibody determines its ability to distinguish the subtle differences among antigens. Thus allowing the antibody to bind to the specific antigen only. Since the hypervariable regions are complementary to the structure of epitopes, therefore they are termed as complementarity determining regions abbreviated as CDRs. Within the variable domain of each heavy and light chain, there are 3 CDRs.
Each variable domain contributes to three complementarity-determining regions, CDR-L1, CDR-L2, and CDR-L3 in the variable region of the light chain
and CDR-H1, CDR-H2, and CDR-H3 in the variable region of the heavy chain.
CDRs from both VH and VL domains contribute to the antigen-binding site and it is the combination of the heavy and the light chain, and not either alone, that determines the final antigen specificity, thus a total of 6 CDRs constitute 1 antigen-binding site.
Similarly, the second antigen-binding site is formed by pairing of variable domains of the other light chain and heavy chain. Thus the second antigen-binding site will also be formed by another 6 CDRs. Therefore, a single antibody molecule contains a total of 12 CDRs. The CDRs account for the diversity of antigens that can be recognized by a repertoire of antibodies.
To summarize, CDRs are the specific regions within the variable region in each of the light and heavy chains that are responsible for generating the antigen-binding site of the 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? The humanized antibodies can be generated through recombinant DNA methods using an appropriate vector and expression in mammalian cells. For this, first, the mouse is immunized with a specific antigen against which the antibodies are to be generated. The antigen that is injected in the mouse is called a 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 population of hybridoma cells obtained is heterogeneous, i.e. they produce antibodies with different epitope specificities. But to select the hybridoma that secretes antibodies against the desired therapeutic target, these hybridomas are required to be isolated and grown individually. This is done by a method known as limiting dilution, which dilutes the concentrations of the heterogeneous population such that on an average, each well contains one cell. In practice, some wells may contain no cells, some may contain a single cell, and others may contain multiple cells.
In the next step, each hybridoma cell is screened for the secretion of antibodies with the desired specificity. This screening is done by the ELISA technique and selects only those hybridomas that produce antibodies against the desired therapeutic target.
For this, the hybridoma culture supernatant containing monoclonal Abs is added to the desired therapeutic target coated on microtiter wells, and monoclonal Abs are then allowed to interact with the therapeutic target. After this, the monoclonal Ab bound to the therapeutic target is detected by adding a secondary antibody labeled with an enzyme, which binds with primary monoclonal Ab. Then the chromogenic substrate is added. And upon addition of this substrate if a colored product is obtained it indicates a positive hybridoma.
The positive hybridoma producing antibodies against the desired therapeutic target is selected and from it, the DNA sequence corresponding to the CDRs of the desired mouse antibody is then amplified. 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 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 developed which contains 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 crucial framework amino acid residues can be identified by observing the structure of antibody-antigen complex by X-ray crystallography, cryo-electron microscopy, and computer-aided protein homology modeling. Therefore, certain 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.
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 for the treatment of rheumatoid arthritis and diabetes mellitus. Another humanized antibody in clinical trials is Daclizumab for the treatment of adults with relapsing forms of multiple sclerosis.
Currently, chimeric antibodies and humanized antibodies are the main forms of human therapeutic antibodies, that are playing an important role in cancer therapy.
Immunogenicity of humanized 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.
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-3 of this series on antibody engineering.
