Chimeric Antigen Receptor (CAR-) T cell immunotherapy is revolutionising cancer treatment. However, the vast majority of CAR-T products in development are autologous therapies; inevitably they will struggle to reach mass adoption given the cost and complexity – two recent FDA-approved products have price tags of US$373,000 and US$475,000 per treatment. Other major issues include: time to manufacture the product, complexity of release specifications for each product, and starting cell variability as a result of compromised immune systems following chemotherapy prior to creating autologous CAR-T cells. Logically, a precisely defined, consistent, ‘off-the-shelf’ CAR-T product with broad histocompatibility is the future of this technology.
In vitro directed differentiation of T-cells from stem cells sources, such as induced pluripotent stem cells (iPSCs), provides a platform to generate a ‘limitless’ supply of CAR-T cells. Indeed, it has been shown that co-culture of iPSCs with genetically-engineered mouse support cells can be used to direct differentiation into hematopoietic lineage then into T-cells. However, the current manufacturing system is inherently inconsistent and not suitable for clinical use, given the support lines are of mouse origin. Human cell line analogues have been attempted with limited success. Small molecule patterning has been shown to create HSCs from iPSCs, although conversion of these HSCs to T-cells remains challenging.
Accordingly, we have established a molecularly defined, xeno-free, stroma-free, serum-free T cell differentiation culture system suitable for upscale manufacture. For the first time we demonstrate the generation of mature CD8αβ+ CD4- TCRαβ cytotoxic T-cells from cord blood HSCs without an animal stromal cell component. When activated via CD3/CD28 co-stimulation, the in vitro generated T-cells have strong dose-dependent cytotoxic function. Using this system we can create ~100 CD3+TCRαβ+CD8αβ+ cytotoxic T-cells per cord HSC, over 47 days of differentiation. With the prospect of post T-cell expansion, using our system 1 cord blood sample can yield up to 2.5 x 1011 T-cells. We have, however, observed donor cord variability, appearing to affect the end-state of T-cell maturity. This work potentially unlocks a pivotal step in generating unlimited CAR-T cells from iPSC, along with new avenues for allogenic CAR-T products from HLA matched cord blood tissue. Furthermore, this manufacture system serves as a stand-alone technique to enable immune reconstitution for a variety of diseases.