The advantages of flow cytometry in drug development
One of the key reasons why flow cytometry has flourished within scientific research is that in addition to bulk profiling, it also reveals the existence of rare cell types with good sensitivity. The technique can also directly feed into other functional workstreams, allowing multiple datasets to be generated from a single sample. For example, a fluorescence-activated cell sorting (FACS) instrument can be used to not only capture data like a conventional flow cytometer, but also sort specific subpopulations of cells determined by predefined marker combinations, to a very high purity. These cells remain viable and functional and therefore can be further studied in downstream assays to understand the functional potential of rare subsets discovered, without the confounding effect of other cell types present.
Recent translational research
At Adaptimmune, our translational research is highly dependent on the information we gather from flow cytometry and FACS. We routinely deploy flow cytometry for the longitudinal study of peripheral blood mononuclear cell (PBMC) samples from patients on our clinical trials to understand how the phenotype of our SPEAR T-cell product develops or evolves over time. These samples are often challenging to work with and limited in size, so having a multi-parameter flow cytometer that provides high-quality reproducible data is essential to determine the biological differences between samples. These insights allow us to better understand the mechanism of action of our therapy and guide future investigations.
Additionally, we deploy FACS to gain an in‑depth understanding of our product at the point of infusion, to profile exactly what each patient is receiving. To achieve this, we identify and sort subpopulations of cells in our product into functional assays or for single-cell RNA sequencing (scRNA-Seq), to give us both phenotypic and transcriptional information. This can then be correlated with on-study clinical data to see how the properties of the infused cells affect patient response. Moreover, scRNA-Seq analysis allows for the unbiased identification of transcripts or novel markers that may be of clinical interest. Critically, we can discover these with no prior knowledge required. The findings from such experiments can then feed future investigations of these newly identified subsets, where flow cytometry and FACS are once again deployed.
Finally, we also use flow cytometry to aid our investigations into the mechanisms by which our therapy works and to understand how changes in our manufacturing process impact the T cells that it produces. We collaborate closely with other departments across the company to generate and discuss these data.
Ultimately, the data derived from this technology is compiled with data from other analytical platforms and patient clinical responses to provide a full translational understanding of our SPEAR T cells. Armed with this understanding, we seek to iteratively design better cell therapy products and deliver the best possible products to cancer patients.
The benefits of correlation
Flow cytometry continues to be a powerful and flexible tool for understanding our cells”
Being able to correlate flow cytometry data with other variables has been instrumental to increasing our understanding of the biology of the SPEAR T-cell products. These data and correlations enable critical decisions regarding the direction of therapeutic development.
One example of this relates to a recent change in Adaptimmune’s T-cell manufacturing process, where a new additive was tested to increase both yield and cell persistence post-infusion. Using flow cytometry, we were able to observe a change in the memory phenotype of cells produced in the presence of this additive, which indicated that these cells were less differentiated. Furthermore, scRNA‑Seq analysis on cells sorted by FACS indicated favourable transcriptional changes compared to cells manufactured without additive, which correlated with functional differences in how the cells responded to tumour antigens.1,2 Together, these data supported the adoption of this process change. Now, we are again using flow cytometry to track the memory phenotypes of T cells produced in this way in our on-study patient PBMC samples, to see how these populations maintain themselves or evolve over time.
Limitations associated with flow cytometry
Flow cytometry, particularly with panels of 15 or so markers, generates huge amounts of data when all possible marker combinations are considered. Therefore, an appropriate dataflow infrastructure must be established to process, analyse and visualise these data. This year, the Adaptimmune teams have focused on the development of computational methods to automate as much of the data processing and downstream correlations with clinical trial data as possible, to enable us to identify the key signals present. However, these automations still require a skilled human analyst to check the data is unaffected by any spectral overlaps or other technical issues.
Conclusion
Whenever designing new panels of markers for investigating immune cells, we always find ourselves limited by the number of different proteins that we can simultaneously detect. This in turn limits our understanding of the biology, which is key to the development of new therapeutics. The field of flow cytometry is ever evolving, with continuous improvements to the quality of lasers, fluorochromes, instrument function and detectors.
These advancements will significantly increase the quantity of information gained from each individual cell, on par with current mass cytometry approaches, thereby alleviating this limitation. Furthermore, data generated by Adaptimmune and others in the immuno-oncology field will lead to the identification of new markers that may uncover previously uncharacterised cell types, the functional analysis of which will be aided by the ability to isolate these populations by FACS. Therefore, we believe that flow cytometry will remain a key tool to help to continue to push the boundaries of therapeutic development in immuno-oncology.
References
- Mardilovich K, Wang L, Kenneil R, Betts G, Bath N, et al. Inhibition of AKT Signaling During Expansion of TCR-Engineered T-Cells from Patient Leukocyte Material Generates SPEAR T-Cells with Enhanced Functional Potential In Vitro. Poster presented at Society for Immunotherapy of Cancer (SITC), virtual congress, November 9−14, 2020.
- Schmidt EM, Mardilovich K, Bath N, Betts G, Spinner W, et al. Enhancement of TCR-engineered T-cells Targeting MAGE-A4 Antigen by Co-expression of CD8α and Inhibition of AKT Signaling during ex vivo T-cell Expansion. Poster presented at Society for Immunotherapy of Cancer (SITC), November 10−14, 2021, e-poster: 373.
