New Manufacturing Approach Enhances CAR-T Cell Functionality

A study published recently in Nature Communications has demonstrated that personalizing CAR-T cell stimulation during manufacturing can significantly enhance the consistency and potency of the resulting CAR-T cell products. By using artificial antigen-presenting cell mimicking scaffolds (APC-ms), the team from Harvard University and Dana-Farber Cancer Insitute was able to fine-tune the levels of T cell stimulation to match the phenotype of T cells obtained from leukemia patients, and significantly enhanced their ex vivo and in vivo tumor-clearing abilities.

“We show that CAR-T cell products made from T cells derived from cancer patients are generally less functional than CAR-T cells products derived from healthy individuals,” said senior author David Mooney. “Matching the CAR-T cell antigen-stimulation dose to the phenotype of patients’ T cells using a precisely controllable biomaterials approach that closely mimics natural antigen presentation can significantly improve their function. This approach could further personalize CAR-T cell therapy and remove an existing inadequacy from current T cell manufacturing.”

The team investigated the phenotypes of T cells that they isolated from samples obtained from patients with from acute lymphoblastic leukemia (ALL) and chronic lymphoblastic leukemia (CLL), as well as from healthy donors. Next, they utilized APC-ms to provide the T cells with different doses of anti-CD3/anti-CD28 antigen stimulation and thus created a CAR-T cell library. All CAR-T cell products contained in the library were then probed again for functional differences, including their ability to kill cancer cells in vitro. The researchers directly compared their approach with one that is commonly used in CAR-T cell manufacturing, which presents the same antigens on rigid magnetic beads (Dynabeads) to T cells.

A key finding was that cancer patients’ T cells were much more easily over-stimulated at antigen doses commonly used during CAR-T cell manufacturing than “healthy” T cells. This made them lose their functionality, or become more “exhausted” as immunologists say, and decreased their ability to proliferate. CAR-T cells not only need to be transformed into a functional state but also amplified by millions to be able to eliminate tumor cells and metastasis in the entire body.

“By exploring a precise, narrow range of stimulation doses made possible with APC-ms, we show that there is something like a personalized ‘sweet spot’ for patient-derived T cells that maximizes functionality and amplification, which is, on average, lower than the usual doses,” said first-author David Zhang.   “The APC-ms approach functions much more naturally than Dynabeads, because highly controllable levels of T-cell signals are embedded into a lipid bilayer, which allows the CAR-T cells to push and pull at them as just as T cells usually do across the ‘immunological synapse’ between them and antigen-presenting cells when T cell stimulation is at its best.”

While the team did not observe any significant differences between CAR-T cells created from ALL and CLL patient samples, overall their approach generated more cells with high cytotoxic potential toward tumor cells, a more balanced ratio between cytotoxic CD8⁺ T cells and CD4⁺ T cells that support their function, and more memory T cells that themselves are not cytotoxic but can be activated in later responses. In a mouse in vivo study, infused CAR-T cell products created with different levels of stimulation also exhibited significantly different abilities to control CD19-expressing Burkitt’s lymphoma, with cells again stimulated at lower than usual levels during manufacturing showing the strongest potential.