Isolating, Engineering, & Expanding Cells for Adoptive T Cell Therapies

Adoptive T cell therapies are offering hope anew to patients with cancer and other severe forms of disease. After collecting naive T cells from a patient, researchers isolate the T cells, and engineer them to recognize specific antigens (for example, proteins expressed by cancer cells). Then the engineered T cell populations are expanded to generate the billions of cells required for a course of immunotherapy. Finally, the engineered T cells are introduced back into the patient, where they hopefully succeed in fighting off disease. But successful therapeutic outcomes depend upon optimized strategies for preparing these powerful, engineered T cells. Here’s a glimpse at tools and techniques for isolating, engineering, and expanding cells for adoptive T cell therapies, and new approaches on the horizon.

Isolation

Common cell isolation protocols (i.e., affinity-based, counter flow centrifugation, spinning filters) are still widely used, but newer methods are also emerging. “Optimizing T cell isolation is a critical step in the development of T cell therapies to ensure that the isolated T cells are of high purity, viability, and functionality,” says Klemens Wassermann, Managing Director and Co-founder of Cellectric Biosciences. Isolation tools can be label-based, using specific antibodies in methods like magnetic cell sorting (MACS), fluorescence-activated cell sorting (FACS), or crosslinking to red blood cells. In contrast, label-free methods isolate T cells according to their physical properties (e.g., size, density, electrical impedance), and may include density gradient centrifugation, microfluidics, dielectrophoresis, acoustic separation, and buoyancy-based technologies.

A unique cell isolation tool is Cellectric’s electromagnetic cell manipulation platform, which takes advantage of the electrical properties of cells. “Like the genome, the ‘electrome’—a cell’s electric fingerprint—is a basic cellular characteristic controlling cell identity and behavior,” says Wassermann. “Controlling and manipulating the electric fingerprint of cells would offer a new, label-free, and fully automatable approach to cell therapy—for isolation, engineering, and expansion.” Their platform allows the selective removal of unwanted cells from cell samples, which could improve production of modified T cells. “Overall, by combining precision, automation, and scalability, label-free electrophysical methods will have the potential to overcome some of the current limitations in T cell-based therapies, making these treatments more accessible and effective for a wider range of patients,” says Wassermann.

Researchers are also leveraging imaging technologies as cell isolation tools. For example, CYTENA’s UP.SIGHT platform offers automated single-cell dispensing and imaging for precise clone tracking and selection. “Advanced technologies that combine gentle dispensing and proof of clonality via well plate imaging, such as the UP.SIGHT, support T cell therapies by providing pure clonal cell lines, which are frequently required in single-cell analysis,” says Adrian Zambrano, Customer Success Manager at CYTENA. Platforms that incorporate automation can also optimize cell isolation for higher reproducibility, less contamination, greater yields, and regulatory compliance. “Utilization of imaging based-isolation combined with deeper cellular interrogatory techniques can support the development and bring costs down with high-throughput approaches,” he says.

Cell engineering

Cell engineering—delivering genes or other payloads for expression, gene editing, or other manipulations—is the next step required to change the isolated T cells into therapeutic agents. Methods to accomplish this include using viral and nonviral techniques. “While viral vectors are highly efficient, their insertion sites and copy numbers can vary, and they could also trigger undesirable immune and inflammatory responses,” says Shannon Eaker, Chief Technology Officer at Xcell Biosciences. “Nonviral vectors avoid those problems and can produce sustained gene expression, but they are not yet as efficient as their viral counterparts.”

Researchers continue to explore other nonviral methods for gene delivery to both cell lines and primary cells. “Electroporation is now widely used in place of viruses for ex vivo cell engineering due to compatibility with a wide variety of cell types and loading agents, including mRNA, plasmids, and RNPs,” says James Brady, Senior VP in Technical Applications and Customer Support at MaxCyte. “[Other] cell engineering technologies create transient perturbations to the cell membrane through mechanical pressure, microneedles, or chemical reagents.” Lipid nanoparticles are also being investigated for both in vivo and ex vivo gene delivery.

Gene-editing methods such as CRISPR-Cas9 are another valuable tool for creating T cells that express the receptors and other factors needed to constitute an effective therapeutic force upon reintroduction into the patient. “Gene-editing technologies are key for effective engineering of variant T cells,” says Zambrano. “A library of cells containing a broad set of receptors are important and, in some cases, a combination of them will make therapies more effective.”

Expansion

After engineering, T cell populations are typically expanded by growing them in bioreactors, not only to ensure optimal growth conditions, but also to comply with regulatory requirements. Increasingly, there are opportunities to monitor every stage of the workflow. “Several platforms now integrate sensors and other analytical capabilities to automatically monitor and adjust culture parameters to maximize cell viability, expansion rates, and other critical product attributes,” says Brady. “The ability to obtain real-time phenotypic and metabolic data could lead to many process improvements with significant benefits for both clinicians and patients.”

Zambrano notes that expansion in micro-bioreactors such as CYTENA’s C.BIRD results in enhanced activation levels of T cells. “The use of early cell mixing can accelerate the process development of the workflow, bringing therapies faster and more affordably to patients,” he adds. Indeed, a major obstacle to wider use of adoptive T cell therapies is cost, but advances may expand availability sooner.

Expanding T cells in the AVATAR Foundry system from Xcell Biosciences may help to prepare cells heading for battle against tumor cells. According to Eaker, T cells have a better chance of surviving in vivo if they are grown in conditions more similar to those they will encounter. “The microenvironment around a solid tumor is incredibly hostile, and requires therapeutic cells to metabolically adapt and function in a hypoxic, low-pH, high-pressure environment, which is why most cell therapies have failed for these disease indications,” he says. “But with the AVATAR Foundry system, cell therapies can be manufactured in and conditioned to the type of harsh environment they’ll encounter in vivo.” The Foundry system, for example, allows customized control of both oxygen concentration and atmospheric pressure, leading to the expansion of cells with greater fitness and potency, according to Eaker. This approach is currently under evaluation for effectiveness, but any method of preparing T cells capable of defeating solid tumors is surely welcome.