Bispecific antibodies represent a significant advancement in therapeutic biotechnology, differing from conventional antibodies by their ability to engage two distinct targets simultaneously. This dual-targeting capability allows them to bridge different cells or molecules, enabling novel mechanisms of action in various medical applications, particularly in areas like cancer therapy and autoimmune disorders.
Understanding the Basic Building Blocks
Antibodies are proteins produced by the immune system to identify and neutralize foreign invaders. Their fundamental structure is a Y-shape, composed of two identical heavy chains and two identical light chains. The tips of the “Y” arms, known as the antigen-binding regions or Fab (Fragment antigen-binding) fragments, are responsible for recognizing and binding to specific targets. The stem of the “Y” is the constant region, or Fc (Fragment crystallizable) region, which mediates various immune functions.
To construct bispecific antibodies, researchers often utilize smaller, engineered components derived from these natural antibody structures. Single-chain variable fragments (scFvs) are one such building block, created by linking the variable heavy and light chains of an antibody with a short peptide linker. These scFvs retain the full antigen-binding capability of the parent antibody in a much smaller format. Similarly, Fab fragments, which consist of one light chain and the variable and first constant domains of a heavy chain, also serve as versatile modules due to their inherent binding specificity.
Early Approaches to Bispecific Antibody Production
The initial efforts to create bispecific antibodies involved methods that faced considerable challenges in product purity and yield. One early strategy was quadroma technology, also known as hybrid-hybridoma. This method involved fusing two different hybridoma cell lines, each producing a distinct monoclonal antibody, to generate a single quadroma cell. The quadroma cell then secreted a mixture of the two parental antibodies, as well as the desired bispecific antibody.
A significant limitation of quadroma technology stemmed from the random assortment and pairing of four different antibody chains. This random assembly led to the production of ten distinct antibody species, only one of which was the desired bispecific format. Consequently, purifying the specific bispecific antibody from this heterogeneous mixture proved complex and inefficient, resulting in low yields.
Another pioneering approach was chemical conjugation, where two different antibodies or antibody fragments were chemically linked together. This typically involved using cross-linking reagents to form covalent bonds. Chemical conjugation often led to uncontrolled reactions. The resulting products could be heterogeneous in molecular weight and composition, potentially leading to aggregation or a loss of binding activity.
Modern Design Strategies and Engineering
Current methods for producing bispecific antibodies predominantly rely on genetic engineering techniques, allowing for precise control over their assembly and improved purity. These strategies involve designing specific DNA sequences that encode the desired antibody components, which are then introduced into host cells, such as mammalian cell lines or yeast. The host cells then express and assemble the engineered proteins, leading to the production of the bispecific antibody.
One widely adopted strategy for ensuring correct heavy chain pairing is the Knob-into-Hole (KiH) technology. This approach involves introducing specific amino acid mutations into the Fc regions of two different antibody heavy chains. One heavy chain is engineered with a larger “knob” residue, while the other is modified with a smaller “hole” residue. This complementary knob-and-hole interaction promotes the preferential pairing of the two different heavy chains, significantly increasing the yield of the desired heterodimeric bispecific antibody.
Another effective design is the common light chain approach, which simplifies the assembly process by utilizing a single light chain that can pair with two different heavy chains. This design strategy mitigates the issue of mispairing light chains. By employing a common light chain, the number of potential mispaired byproducts is reduced, leading to a more homogeneous and easier-to-purify product.
Single-chain variable fragments (scFvs) are versatile building blocks in modern bispecific antibody design. These fragments, which link an antibody’s variable heavy and light chains, can be genetically fused together in various configurations to create diverse bispecific formats. For instance, tandem scFvs involve linking two scFvs in series to form a single, bivalent molecule. Diabodies are created when two scFvs are designed with short linkers that prevent intramolecular pairing, forcing them to dimerize and form a bivalent structure. Triabodies extend this concept, forming trivalent structures from three scFvs, enhancing binding avidity or allowing for engagement of three targets. Other innovative designs, such as CrossMab and DVD-Ig, demonstrate the modularity of genetic engineering, allowing for the creation of various bispecific formats with different protein domains and orientations.
Refining and Ensuring Quality
After bispecific antibodies are produced in host cells, they exist within a complex mixture alongside host cell proteins, cell culture media components, and potentially undesired antibody byproducts. Therefore, rigorous refining steps are essential to isolate the desired therapeutic product and ensure its quality. The primary method for achieving this purity is chromatography.
Chromatography techniques, such as Protein A chromatography and ion exchange chromatography, are critical for separating the bispecific antibody from impurities. Protein A chromatography selectively binds to the Fc region of antibodies, allowing for efficient capture and purification. Subsequent ion exchange chromatography further refines the product by separating molecules based on their charge, effectively removing remaining contaminants and undesired antibody forms.
Following purification, extensive characterization and quality control measures are implemented to confirm the bispecific antibody’s identity, structural integrity, and functional activity. Analytical techniques like mass spectrometry are used to verify the exact molecular weight and composition, ensuring correct assembly of the heavy and light chains. Size exclusion chromatography assesses the purity and aggregation state of the antibody, confirming it exists as a single, stable molecule. Binding assays are performed to confirm that the bispecific antibody retains high affinity for both of its intended targets, ensuring its therapeutic efficacy.