Based on protein interaction, Bioassays mainly include two types: ligand-binding assay (LBA)-based method and MOA (Mode of Action)-based method with cell experiment.
LBA-based methods are used for drug affinity test, blood drug concentration test, biomarker concentration detection, anti-drug antibody (ADA) / in vivo neutralizing antibody (NAb) testing and etc.
MOA-based methods are for determining cell membrane surface antigen and drug affinity, drug cell activity, and in vivo NAb determination, etc. It is implemented throughout the entire drug development process.
Compared to the direct interaction between the drug and the target, the interaction between the drug and the cell is more complicated.
After the large molecular drug binds to the receptor on the cell membrane surface, it will cause the activity change of the receptor cell’s intracellular region, resulting in the change in concentration or the conformational activity of specific signal molecules. This causes cascade changes in the cell cytoplasm and ultimately transmits the signal to the nucleus, regulates the transcription and translation of the protein, and finally exerts its biological function.
Intracellular physiological changes can be summarized as:
- Changes in concentration of second messengers such as cAMP, Ca2+ flow, kinases phosphorylation/dephosphorylation and other activation transcription factors;
- Transcription factors enter the nucleus and the promoter region of the genome to activate the promoter;
- The expression of downstream gene mRNA is transcribed after the promoter is activated;
- The mRNA is translated from the nucleus into active proteins, which regulate cell growth and development, in the endoplasmic reticulum and Golgi apparatus.
In the actual drug development process, biofunctional experiments are designed to measure the physiological changes of cells caused by drug doses. These physiological changes are displayed through experimental methods in the form of establishing dose-response curves.
Since these physiological changes are multi-level and multi-dimensional, cell function experiments are also diversified. The intracellular signals are not proportionally amplified, so it will generate inconsistent dose curves and EC50 during the detection of different physiological changes.
If the target molecule that reflects physiological changes and the signal change are nonlinearly- related, then the dose-response curve may potentially distorted (such as TR-FRET experiment and competitive ELISA).
In this case, it is necessary to introduce the target molecule as standard, and then convert the signal size to the concentration of the target molecule before performing the drug-target molecule dose curve fit, so that it can reflect the actual physiological changes caused by the drug.
The construction of engineered stable cell lines for recombinant protein expression has been relatively mature. The basic process includes the following procedures:
- Determination of the minimum lethal concentration of eukaryotic resistance of mother cells
- Vector construction
- Recombinant protein gene transfection into mother cells
- The transfected pool undergoes pressurized screening for eukaryotic resistance
- Monoclonal selection, amplification, suspension and acclimatization
- Determination of protein expression, protein quality characterization and analysis
- Optimum clone expansion library
Functional stable cell lines and engineered stable cell lines
The construction of functional stable cell lines have similar process. The different consideration between functional stable cell lines and engineered stable cell lines are as follows:
- The choices of mother cell lines for functional stable transgenic cell lines are various, which need to be selected according to the purpose of specific experiments and the genetic background of the mother cells. For example, it is better to use Jurkat T cell lymphoid line to study the function of PD1 drugs.
- The difficulty of transfection between mother cells varies. The functional stable transfected cell line can use lentivirus, plasmid as the target gene carrier, and use methods such as liposome, lentivirus infection and electrotransfection for transfection/infection.
- The design of functional cell line vectors will also be relatively complicated.For instance, reporter gene stable transfection cell lines generally require the construction of multiple vectors such as target receptor vectors and reporter gene system vectors. Some target receptors exist in the form of heterodimers on the cell membrane, and scaffold proteins are needed to help them stably express on the cell membrane or exert their biological activity. It is necessary to transfer multiple target receptor genes such as CD16, FGFR1/β -Kloth etc.
- The quality characteristics of functional cell lines and engineered cell lines are different.
For monoclonal, functional cell lines need to be evaluated in a comprehensive manner for application purposes. These evaluations include:
- The measurement of the abundance of the cell membrane cells;
- The signal-to-noise ratio of the intracellular reporter gene under activated/inactive conditions;
- The stability of the parallel well such as CV, Z factor;
- Cell doubling time, cell serum tolerance for NAb determination, preliminary evaluation of cut point, etc.
- Engineered stable transgenic cell lines has mature quality indicators and guidance methods, while the quality characterization of functional stable transgenic cell lines does not often have mature methods and standards. Based on different biological detection principles, customized characterization plan is often required.The selection of single clones expressing target receptor genes and reporter genes often requires complex and specialized characterization schemes to suit the purpose of functional evaluation.