CDR grafting is a biotechnology technique used to make animal-derived antibodies more suitable for human medical use. This process involves genetically engineering an antibody to reduce the likelihood of an unwanted immune response when administered to a human patient, creating a “humanized” antibody that retains its ability to target specific foreign substances while minimizing foreign components that could trigger an adverse reaction.
The Antibody’s Key Components
Antibodies, also known as immunoglobulins, are Y-shaped proteins produced by the immune system to identify and neutralize foreign invaders like bacteria and viruses. Each antibody molecule consists of four polypeptide chains: two identical heavy chains and two identical light chains, connected by disulfide bonds. These chains are organized into distinct regions, broadly categorized as constant and variable regions.
The constant regions form the stem of the Y-shaped molecule and are largely the same across different antibodies of the same class, determining the antibody’s general function. In contrast, the variable regions, located at the tips of the “Y” arms, are highly diverse and responsible for recognizing and binding to specific targets, called antigens. Within these variable regions are seven amino acid segments: four Framework Regions (FRs) and three Complementarity-Determining Regions (CDRs). The FRs act as a structural scaffold, providing the overall shape and stability, while the CDRs are hypervariable loops directly involved in antigen binding.
The Grafting Process Explained
The core concept of CDR grafting involves taking the specific CDRs, the “binding parts,” from a non-human antibody (typically mouse) and inserting them into the Framework Regions (FRs) of a human antibody. This reduces the likelihood of an immune response, as complete non-human antibodies can trigger rapid clearance and reduced effectiveness. The resulting “humanized” antibody retains its target binding ability while being largely composed of human sequences.
The steps for CDR grafting begin with identifying the DNA sequences of the CDRs from the non-human antibody, often using techniques like reverse transcription polymerase chain reaction (RT-PCR). These non-human CDR sequences are then inserted into the corresponding FRs of a chosen human antibody framework using genetic engineering. Suitable human FRs are selected based on high sequence homology to the original non-human antibody’s FRs. Finally, the engineered DNA is introduced into host cells, such as Chinese Hamster Ovary (CHO) cells or HEK293 cells, to produce the new humanized antibody.
Impact on Therapeutic Antibodies
CDR grafting has significantly advanced the development of therapeutic antibodies, which are specialized proteins used to treat a wide array of diseases. These humanized antibodies are designed to target specific molecules involved in disease processes, offering a precise approach to treatment. For instance, in cancer therapy, humanized antibodies can target proteins on cancer cells, leading to their destruction or inhibiting their growth. Trastuzumab, used for certain breast cancers, is an example of a humanized antibody developed using this method.
In autoimmune disorders, where the immune system mistakenly attacks the body’s own tissues, humanized antibodies can block specific inflammatory pathways or deplete harmful immune cells. Adalimumab, used for conditions like rheumatoid arthritis and Crohn’s disease, is a humanized antibody that targets tumor necrosis factor-alpha (TNF-α), a protein involved in inflammation. Humanized antibodies are also employed against infectious diseases, where they can neutralize viruses or bacteria, or enhance the body’s immune response to clear pathogens.
Refining Grafted Antibodies
While CDR grafting is a powerful technique, simply transferring CDRs can sometimes lead to challenges, such as a reduction in the antibody’s ability to bind to its target (affinity) or a residual immune response in humans. This can occur because some framework residues, although not directly part of the CDRs, can still influence the precise three-dimensional structure of the binding site. To overcome these issues, scientists employ further engineering strategies to optimize the grafted antibodies.
One such strategy is “resurfacing,” also known as “veneering,” which involves identifying and modifying a limited number of surface-exposed framework residues to more closely resemble human sequences. This aims to reduce potential immunogenicity by altering only parts of the framework likely to be recognized as foreign, while preserving internal residues crucial for maintaining the CDRs’ correct conformation and binding function.
Another approach involves introducing “back mutations,” where a few specific human framework residues are changed back to their original non-human counterparts if those residues are important for maintaining the antibody’s binding affinity. These refinement processes, often guided by structural modeling and experimental testing, ensure the humanized antibody is highly effective and safe for therapeutic use.