Analyzing CAR-T Surface Proteins With Immunoprecipitation Coupled With Mass Spectrometry
By Nicolle Serrano SantoDomingo, Novartis Biomedical Research Center
Immunoprecipitation coupled with mass spectrometry (IP-MS) is a valuable technique that offers exciting possibilities for understanding cellular biology and protein interactions. This protocol enables the enrichment and quantification of target molecules, providing deeper insight into their biological structure and function. IP-MS can be applied to detect molecules generated by AAV or LVV systems in engineered cells such as CAR T cells.
By using IP-MS, we can isolate the CAR and analyze its protein interaction landscape, uncovering novel insights into how CAR T cells function and how their activity might be optimized. For example, when applied to primary T cells, examining the cellular surface proteins that co-elute with the CAR can reveal how these proteins influence signal modulation, enhance activation, and improve target specificity. This information is critical for designing robust T cell responses against tumors.
This approach can be applied to compare CAR constructs, evaluate the impact of different manufacturing processes, or monitor changes in protein interactions over time or under different stimulation conditions. It also holds potential for identifying off-target interactions that could inform safety assessments and guide future CAR design.
Why Correctly Identifying Surface Proteins Matters
CAR-T therapy is a transformative approach that modifies a patient's T cells to target and eliminate cancer cells using a synthetic receptor. The process begins with the transduction of T cells using a lentivirus encoding the chimeric antigen receptor (CAR) in its RNA. This viral RNA is reverse transcribed into DNA, which is then integrated into the host genome. However, splicing errors can occur during reverse transcription and transcription, potentially affecting CAR expression.
Our team used next-generation sequencing of proviral DNA to confirm the CAR sequence and detected splicing events in the CAR single-chain variable fragment region. These events can lead to a heterogeneous lentiviral vector (LVV) product, which may reduce therapeutic efficacy. Therefore, developing a method that accurately detects splicing events in CAR T cells, and can also help identify which cellular surface proteins co-elute with the CAR, is critical for ensuring the safety and effectiveness of these therapies.
Cell surface proteins represent a small and hydrophobic subset of total cellular proteins, making them challenging to analyze. Establishing a robust protocol to identify which surface proteins co-elute with CAR T cells is essential. This will help determine whether these proteins contribute to CAR T cell activation, signal transduction, or other mechanisms that enhance therapeutic efficacy.
The Goal: To Enrich And Characterize Surface Proteins
IP-MS allows for the enrichment of a specific molecule or protein of interest through immunoprecipitation, significantly enhancing sequence coverage. The enriched sample is then analyzed by mass spectrometry, providing detailed information about the protein’s sequence, presence, and post-translational modifications (PTMs), including glycosylation patterns.
IP-MS was particularly helpful in detecting whether a splicing variant was present in our CAR T cells. Typically, we use multiple enzymes in mass spectrometry to improve sequence coverage of our proteins or molecules of interest. In this case, a specific chymotryptic peptide forms when a splicing issue occurs in the CAR single-chain variable fragment (scFv). This peptide serves as a marker to confirm the presence of the splice variant.
However, this chymotryptic peptide is highly hydrophobic and unusually long, which makes it difficult to detect. Its absence in the data set does not necessarily indicate that it is not present — it may simply be below the detection threshold of the instrument. After applying the IP-MS protocol to our CAR T cells of interest, along with a splicing-positive control, we confirmed the presence of the full-length CAR but were unable to detect the splicing variant.
To further investigate, we generated a synthetic version of the peptide through GenScript and ran a standard curve to determine its limit of detection. Despite this, we were still unable to confirm its presence, suggesting that the peptide’s abundance may be below the instrument’s detection limit.
We were interested in analyzing cell surface proteins to not only confirm the presence of our CAR T cell on the cell surface, which is its intended location, but also to identify additional surface markers or signaling molecules that may enhance CAR T cell functionality. To develop and optimize this method, we used HEK293 cells and focused on enriching cell surface proteins.
Recognizing that no method is perfect, the goal of our IP-MS protocol was not to eliminate intracellular labeling entirely but rather to enrich and characterize cell surface proteins. To achieve this, we compared biotinylated proteins labeled prior to cell lysis with those labeled post-lysis to assess cell surface protein enrichment. Following enrichment, we performed LC-MS/MS analysis.
We focused on identifying cell surface proteins by leveraging various cell surface protein databases and filtering for unique proteins to reduce peptide redundancy assigned to each protein. This approach resulted in a threefold enrichment of observed cell surface proteins when comparing our CAR T cells of interest to lysed control cells.
By applying this IP-MS protocol to primary CAR T cells, we aim to quantify and analyze proteins that co-elute with the CAR, particularly those involved in signaling pathways. This can provide valuable insights into molecular interactions that influence CAR performance. Such information may support the design of next-generation CARs with optimized signaling architecture, improved activation profiles, and enhanced target specificity. A comprehensive understanding of the proteomic landscape in transduced cells is essential for guiding future CAR optimization strategies.
As the cell and gene therapy industry continues to evolve, the adoption of advanced analytical techniques like IP-MS will be essential for ensuring product consistency, safety, and efficacy that lead to the delivery of next-generation therapeutics.
Obstacles Include High Labor Costs, Insufficient Surface Protein Databases
Immunoprecipitation coupled with mass spectrometry remains a labor-intensive technique, involving multiple steps and requiring considerable analysis time. The hydrophobic nature of cell surface proteins poses additional challenges for extraction, often resulting in incomplete representation in the final data set. Moreover, protocol optimization is necessary to reduce off-target labeling of intracellular proteins.
A further limitation is the lack of comprehensive and curated databases for cell surface proteins, particularly within the context of CAR-T therapies. Consequently, development of or access to reliable resources that accurately annotate cell surface proteins relevant to the CAR-T landscape continues to be a significant hurdle.
Conclusion
Immunoprecipitation coupled with mass spectrometry is emerging as a critical analytical tool in the development and optimization of CAR T cell therapies. By enabling precise characterization of surface proteins and their interactions, IP-MS provides deeper insights into CAR expression, cell-to-cell communication, and the signaling cascades associated with CAR function. It also helps improve therapeutic efficacy by revealing how proteins that co-elute with the CAR influence its activity and stability, guiding the design of next-generation CAR constructs with enhanced performance.
Despite technical challenges and the limited availability of curated cell surface protein databases, continued exploration and refinement of IP-MS protocols for CAR-T analysis hold great promise. IP-MS plays a pivotal role in bridging molecular insights with clinical outcomes, ultimately contributing to the advancement of precision immunotherapy.
About The Author:
Nicolle Serrano SantoDomingo is a senior scientist at Novartis Pharmaceuticals, working within the analytical group at the Biomedical Research Center. She focuses on applying mass spectrometry to characterize diverse drug modalities, with the goal of advancing next-generation therapeutics. She began her scientific career at Vanderbilt University, concentrating on proteomics research. Later. She joined the Center for the Development of Therapeutics at the Broad Institute of MIT and Harvard, where she focused on small molecule drug discovery and the development of biochemical and biophysical assays. Currently, she is completing a master’s degree in biotechnology at Johns Hopkins University. Contact her on LinkedIn.