Questions answered by Shitalben Patel:

Question: For the blood-based CETSA formats, how robust are the readouts across different donors or blood conditions?

Answer: It depends on the assay development and optimization, the target, the antibody selection and sensitivity of the assay. Based on what we have tried, the variability is less than 10% between the donors.

Question: Which conditions need to be optimized in MSD^® and AlphaLISA^® workflows for whole blood CETSA assay for a new target?

Answer: For a new target, I would focus on the lysis conditions, sample volume requirements, and the specificity of the antibody toward the folded protein. When using a commercial kit such as AlphaLISA, only minimal optimization is typically required. Please refer to our paper for further details on optimization:

Quantitative target engagement of RIPK1 in human whole blood via the cellular thermal shift assay for potential pre-clinical and clinical applications – PubMed

Question: What is the optimal concentration of compound you observe for blood CESTA assay?

Answer: It depends on the target and therapeutic dose. You need to select concentrations that cover the expected therapeutic dose to generate a clear dose–response curve and determine the EC₅₀.

Question: Why is the MSD platform preferred in the clinic?

Answer: Meso Scale Discovery’s platform is widely used in clinical trials, so the clinical community is already familiar with the technique. This means it will be easy to transfer assay protocols or implement an assay since the instrument is already available, and the staff is trained.

Question: Could you please provide more details regarding optimum freezing of whole blood CETSA?
Answer: Samples should be frozen after performing CETSA, and stored at -80°C. If you plan to analyze many samples together in one batch, freezing them like this allows you to store them for more than just one or two days before analysis. It can take several days, weeks, or even months before you have enough samples, similar to what happens in a clinical setting.

Question: What makes the whole blood CETSA format more practical or valuable compared to existing approaches, and how do scientists in your team help drive this innovation?
Answer: Western Blot combined with CETSA (WB-CETSA) is commonly used to assess drug binding. Although a great starting point, WB-CETSA requires large samples volumes since PBMC isolation is required, and it’s a semi-quantitative method. This makes it challenging to implement for use in clinical studies. The whole blood CETSA format is more practical since you do not need to extract PBMC’s, and MSD and AlphaLISA technologies deliver quantitative data, allowing use of the information to drive decision making.

The scientists in my team help drive this innovation by contributing with their individual specialty and expertise. For example, we work with the team that screen the donors and provide us with blood samples. We also collaborate with the preclinical team, the clinical staff and with our colleagues in precision medicine, because that is not our expertise. For the study, we collaborated with Pelago Bioscience. We did the MSD work and they did the AlphaLISA work.

Question: How much does the cellular context change the result? Rephrasing this, do lysing cells before or after the heating step influence the results?

Answer: The timing of cell lysis depends on your target. We performed in-cell CETSA followed by cell lysis. However, CETSA can also be performed directly in cell lysates.

Questions answered by Jami R. Erickson

Question: I’m curious about how you measure endogenous proteins. Do you fix and permeabilize the cells for this assay? After labeling with antibodies, do you generate single-cell suspensions through trypsinization, or is this just a high-content imaging assay?

Answer: For endogenous intracellular proteins, we do need to fix and permeabilize the cells. Typically, we prepare single-cell suspensions before antibody labeling, and this is done prior to compound treatment. To achieve this, we usually trypsinize the cells before compound treatment.

If you are concerned that trypsinization may damage extracellular proteins or epitopes, we have also successfully used a non-enzymatic cell dissociation buffer, which helps preserve surface proteins. All steps are carried out using single-cell suspensions from the very beginning of the experiment.

Question: For Flow Cytometry-based CETSA, what does the live/dead, fix/perm/stain process look like?

Answer: After treating the cells with compound and heat, we immediately do a live/dead stain for 15 to 20 minutes followed by washing. If it is an intracellular protein, we will fix and then permeabilize the cells. If it’s an intercellular protein, but not necessarily an inter-nuclear protein, we use BD Biosciences’ Fixation/Permeabilization Kit. After cell fixation and permeabilization, we use the BD Perm/Wash buffer when staining the cells. The cells are washed before analysis in the flow cytometer.

Question: What antibody affinity is required to conduct the FLOW CETSA assay?
Answer: I haven’t tested the antibody affinity, so I don’t have any quantitative values. We focus on staining and use as many controls as possible to confirm that the antibody binding is specific.

Question: Can you use CETSA to identify phosphoproteins for target engagement?

Answer: We have not yet used CETSA for phosphoproteins, but it’s something we are hoping to explore. Our goal is to determine whether we can use downstream pharmacodynamic markers, such as phosphoproteins, to confirm target engagement and inhibition or activation by our small molecules. If a suitable antibody can be identified, it should be feasible.

Question: Have you tried analyzing the whole blood CETSA samples with flow cytometry?

Answer: I haven’t tried that yet, but it’s something interesting to try.