While chimeric antigen receptor (CAR) T-cell therapies have significantly improved outcomes in hematological malignancies, attempts to translate this success to solid tumors have presented several challenges, such as inadequate efficacy and unacceptable toxicity. Researchers are continuously identifying new targets to overcome these challenges. However, in cell line development, translating their findings into viable, testable products poses difficulties. The team at the German Cancer Research Centre (DKFZ) is making significant progress in overcoming these challenges, with the help of Bruker’s Optofluidic technology.
Challenges in the Development of CAR-T Therapies
One area under investigation by DKFZ is CAR-T therapies. Dr. Patrick Schmidt, senior research associate and leader of the institution’s cellular therapies team, explained that while CAR-T products have improved outcomes in some haematological cancers, transferring their benefits to solid tumors has remained challenging.
“The effectiveness of these therapies is limited by the solid tumor environment,” he said, “and while combination therapies may address this, there is still the issue of safety.”
CAR-T cell therapy kills all cells expressing the target antigen, explained Dr. Schmidt, and, as such, the currently approved agents, such as those that target CD-19 and BCMA, often cause B-cell aplasia.
“In hematological cancer, that is an acceptable risk profile – you can live without B cells. But for solid tumors, it’s much more difficult. Even minimum expression of the target antigen on healthy cells would mean they are also killed during therapy,” said Dr. Schmidt.
So far, efforts to create CAR-T cells that target classical antigens, such as HER-2, which is over-expressed in a range of tumors including breast, gastric, and lung, for example, have failed because of this excess toxicity.
The Future of CAR-T Therapy
To overcome the toxicity issue and unlock the potential CAR-T cell therapy in solid tumors, researchers are seeking to find new targets.
“At the moment, there are no defined markers that are only expressed, or highly expressed compared to in healthy tissues, in solid tumors,” said Dr. Schmidt, adding that the “most promising” are cancer testis antigens (CTA).
This large family of antigens are expressed in human tumors of different histological origin, but not in normal tissues except for those of the testis and placenta. However, traditional approaches to agent development are not feasible. Because while existing CAR-Ts have been developed by converting commercially available antigens into T cells, no antigens for CTA are commercially available.
DKFZ’s solution is a CAR-T library, based on the organization’s work to identify groups of novel biomarkers of cancer development. “DKFZ’s basic research teams work to identify novel biomarkers and novel pathways of the metabolics of cancer development, and we are constantly asked whether or not we can make a CAR against the target X, Y, or Z. The library means we can now respond to these requests.”
It contains billions of frozen CAR cells, enabling Dr. Schmidt’s team to select one that binds to the requested target within just five days. After validation, the research team can then take the cell into animal studies and, if successful, roll out clinical trials.
Creating and exploiting the living library was a challenge that was made possible by Bruker’s Optofluidic platform. This novel technology enables the isolation and functional characterisation of thousands of single cells simultaneously on one platform.
Using Bruker’s Optofluidic Platform to Create an On-Chip CAR Library
Creating the library has been a multi-step process. “We had to start from zero,” said Dr. Schmidt. “There have been many attempts to build cellular or living libraries that use cells to carry diverse CARs, but they have never really been successful.”
One of the main reasons is the huge diversity of cells needed vastly exceeding the number of cells that could feasibly be grown in a reasonable timeframe using traditional lentiviral vectors. Dr. Schmidt said they overcame this by developing a novel plasma-derived vector. Nano-S/MARt (nS/MARt) combines prolonged CAR expression with minimal disruption of T cell activity. Importantly, it allows Dr. Schmidt’s team to transfer several copies of a single plasmid into each single cell. “With this new radical approach, we can just transfer a higher diversity into our cells,” he said.
Next, they used the Optofluidic workflow’s functional analysis to create a reporter line, derived from a single cell, that could act as an internal reporter, or on/off switch, during selection. “After establishing this clone, we were able to expand them up to millions, then use a large-scale electroplatation process to transfer our library into them,” said Scmidt. “We have stock frozen these cells so that everyone who is interested in using them can thaw them, then put them through the selection strategy – and the Optofluidic workflow is central to that process.”
First, the team uses classical fluorescence-activated cell sorting (FACS) to narrow the cells down from billions to hundreds of thousands. Next, they use Bruker’s Optofluidic platform for functional, single-cell analysis to spot the cells that response to the targeted reporter line. Cells that respond are considered “hits” and are then exported for sequencing and further analysis, to understand how and why the response was triggered.
“The unique selling point of this technology is that we can get all the functional information from the cell, and afterwards we can export it back to the culture without losing the ability to grow it again. That is very helpful in our workflow, where we go from billions of cells, down to a single cell for analysis, and back up to billions of cells in terms of generating the CAR-T product,” Dr. Schmidt explained. “So far, only the Optofluidic device is capable of performing this.”
Bruker’s Optofluidic functional assays can also inform the team of each cell’s important characteristics, such as whether it is fast to activate, or whether it has sustained proliferation capacity. Dr. Schmidt explained: “Sequencing will only give us a hint to these answers. With Optofluidic, we can keep the cells on a single chip and actually see the proliferation. You can’t get that from the book of the letters – you need to see the function of the cell, and that is what we can do with this device.”
Now the library has been established, Bruker’s Optofluidic platform will continue to be an essential part of the project. “We are now into the exploitation stage, and that can only be done with this device,” said Dr. Schmidt.
The Impact of Optofluidics on Understanding Single-Cell Function
Since the DKFZ team acquired the Optofluidic platform, and received training on the technology from Bruker, it has become “equipment of daily use”, and has even changed the researchers’ perceptions of their science.
“With Optofluidic, we were able to see for the first time that even single cells derived from the same parental cells behaved differently over time. This was new to us, and really changed our way of thinking about clonality,” said Dr. Schmidt.
As such, the institution is currently in the process of ranking projects to prioritise the order in which research teams will receive their target-specific CAR-Ts – and start the in vivo studies that could lead to the next groundbreaking cancer drug.
To learn more, watch our recent webinar with Dr. Patrick Schmidt on How to Speed Up Cell Therapy Development Processes.