Despite the diversity of antibody engineering technologies, researchers need to be aware of the limitations of the methods they choose to use and be informed about techniques that can help them circumvent unnecessary efforts.
In general, there are two methodologies to achieve these protein engineering goals: empirical methods and rational methods. Empirical methods are based on generating large libraries and rely on screening to select the desired variants by employing hybridoma generation and phage display which usually take longer time and higher cost. Rational methods are based on the existing knowledge of antibody sequences and structures which will typically lead to generating a smaller set of variants.
Therefore, choosing rational methods over empirical methods can significantly narrow down the antibody candidate pool to minimize investment of redundant time and cost.
Antibody de novo sequencing is a good example of rational methods that uses mass spectrometry to determine the amino acid sequence of an antibody. By directly analyzing the antibody sample, this method has made direct antibody sequencing possible without the need for hybridoma cell lines or prior knowledge of the DNA sequence. The central idea of protein de novo sequencing is to determine the mass of an amino acid residue in a protein by measuring the mass difference between two fragment ions from tandem mass spectra. In such a way, the entire protein sequence can be inferred by assigning each residue along the protein backbone.
The general steps of antibody protein de novo sequencing are as follows (Figure 3):
- The antibody protein sample is digested into short peptides of overlapping sequences with multiple proteases.
- Fragmented peptides are separated using chromatographic methods.
- The mass spectra for each peptide are generated using high mass accuracy tandem mass spectrometry (MS/MS).
- Peptide de novo sequencing is conducted on the mass spectra to identify the sequence of each peptide.
- Assembly of the full antibody protein sequence is generated by data analysis with de novo sequencing software.
Figure 3. Workflow of the REmAb® de novo sequencing platform.
The de novo protein sequencing platform, REmAb®, offered by Rapid Novor has proved its value in antibody engineering through partnerships with a myriad of projects. Listed below are some of the important applications that have been identified that help researchers more rationally develop antibody therapeutics.
(1) Antibody humanization
Typically, biopharmaceutical developers rely on hybridoma generation or phage display to develop human mAbs against human antigens of interest. However, homologous mAbs originating from other species may already be characterized and commercially available. Sequences of both commercially available or in-house generated anti-animal antibodies can be obtained with 100% accuracy and coverage using REmAb® de novo protein sequencing. With knowledge of the primary structure, different versions of the antibody may be generated to suit the research need; whether that be to engineer a chimeric version, or to tweak areas to make it a better therapeutic candidate. This application of de novo protein sequencing bypasses the generation of hybridoma cell line and phage display, which avoids the labour-intensive necessities of the empirical methods.
(2) Antibody fragments engineering
When developing therapeutic antibody fragments, or alternate variants such as bispecific antibodies or diabodies, the selection and screening process that usually relies on hybridoma and phage display can be tedious and inefficient due to the large number of choices for fragment combinations. De novo protein sequencing can be applied here to generate a series of proof-of-concept antibody fragment candidates to determine optimal target combinations. Well known and functionally validated antibodies against a variety of antigens may be de novo sequenced, which facilitates the generation of a panel of recombinant antibody fragments that engages two or more targets at once. Then In vivo and in vitro testing in primary animal and human assays can be done to determine the optimal combination of targets that are synergistic in a bispecific or multispecific format. Using well-characterized high quality antibody reagents can help safeguard the success of the early stage of a therapeutic campaign, and therefore derisking and expediting the following development (14).
(3) CAR-T Construction
Chimeric antigen receptor T cell (CAR-T) engineering is becoming quite widespread thanks to the success and hope this approach brings when no other cancer treatments have worked (15). CAR-T structures are typically composed of an ectodomain, existing in the extracellular space, a transmembrane domain, and an endodomain. Within the ectodomain, is an antigen recognition domain, typically a scFv (13). It is here where substantial research is done to determine the best target for this domain. The ideal targets should have high tumor coverage and high specificity to ensure both effectiveness and safety. However, selection of ideal targets is extremely hard to achieve in reality due to the toxic side effects toward non-tumor cells that can cause organ damage or even death (16). Thus, it is important to comprehensively assess the antibody-antigen recognition as well as the off-tumor effects. Implementing de novo protein sequencing into a CAR-T development workflow allows researchers to easily modify and create many CAR-T models by swapping the antigen recognition domain for variable region sequences found in mAbs of interest. This allows for the selection of the best scFv candidates, as well as simple engineering of these regions and detailed optimization of CAR-T models to achieve both enhanced target recognition and reduced non-tumor toxicity.