Commercial CAR T cells typically employ autologous T cells, which can be functionally unfit due to patient age, prior treatments, and tolerance within the tumor microenvironment. The autologous approach has practical drawbacks too, including manufacturing failures, time-consuming production, and high costs, prompting the need for more feasible and efficient protocols. Efforts are now focused on developing allogeneic CAR T cell therapies, though challenges like Graft-Versus-Host Disease (GVHD) and limited persistence remain. Our work has proven feasibility and safety of using Peripheral Blood (PB) healthy donor cells to generate CARCIK-CD19 cells1. Cord blood (CB) is a standard hematopoietic stem cell source and offers immunological advantages such as reduced risk of GVHD2 and enhanced graft versus leukemia particularly in patients with pre-transplant residual disease. CB T cells exhibit greater proliferation compared to adult PB T cells, especially when stimulated with cytokines like IL-7 and IL-152. Cytokine-induced killer (CIK) cells, which are CD3+CD56+-enriched T cells, are easily expandable in vitro from PBMCs and are associated with minimal GVHD. CIK cells, similar to natural killer cells, possess non-MHC-restricted cytolytic activity and have proven to be safe and effective against various solid and hematologic malignancies, representing an alternative effector T cell source for adoptive immunotherapy3. We demonstrated that functional CIK cells can be derived from both fresh and cryopreserved CB units4,5. The CB rapid availability and the low risk of GvHD represent appealing features for the generation of banked third-party “off the shelf” CB-derived CARCIK cells.
Fresh or thawed CB mononuclear cells were successfully modified with a third generation anti-CD19.CAR using a non-viral Sleeping Beauty (SB) transposon gene transfer platform optimized by our group, reaching up to 50% of CAR expression. Compared to the protocol for PB-derived CARCIK cells, we added a purification step to remove erythroblasts and used IL-7 and IL-15 instead of IL-2. These two variables significantly increased the cell yields of CARCIK cells obtained from frozen CB bags (from 5x108 to 1.6x109 total cells/CB bag subunit). Interestingly, we observed that CB-derived CARCIK cells are more metabolically fit. Using the NanoString CAR T Characterization Panel, we found differential gene expression profiles between PB and CB-derived CARCIK-CD19 cells. CB-derived cells exhibited a lower glycolysis score, validated by Seahorse analysis, and enhanced patterns of persistence, chemokine signaling, and T-cell migration. The in vitro functional profile of CB-derived CARCIK-CD19 was comparable to PB-derived ones. We then conducted three large-scale good manufacturing Practices (GMP)-grade validation runs using frozen CB bags. The total cell yields and CAR expression were 1.83 x 1010, 2 x 1010 and 2.5 x109 and 62.3%, 36.42% and 10.0% for the three respective runs. GMP-grade runs of both CB- and PB- derived CARCIKCD19 cells were functionally validated in vivo using a DAUDI xenograft NSG model, where both cell products prolonged the survival of treated mice and controlled disease progression. We are currently planning to incorporate these findings in our upcoming clinical studies with CARCIK cells.
In conclusion, we have successfully demonstrated the feasibility of deriving functional CARCIK-CD19 cells from the cord blood (CB) source. Metabolic and transcriptomic analyses revealed that CB-CARCIK cells exhibit a lower glycolytic score and a higher memory score compared to those derived from peripheral blood (PB), indicating advantageous CAR T cell characteristics. Furthermore, we established the scalability of a GMP-grade manufacturing process for deriving CARCIK cells from CB, enabling the production of readily available, banked, third-party CARCIK cells for treating hematological malignancies.
References:
1 Magnani CF, et al. J Clin Invest 2020; 130: 6021-6033.
2 Borrill R, et al. Front Pediatr 2023; 11: 1232281.
3 Schmeel LC, et al. J Cancer Res Clin Oncol 2015; 141: 839-49.
4 Introna M, et al. Bone Marrow Transplant 2006; 38. doi:10.1038/sj.bmt.1705503.
5 Introna M, et al. Biology of Blood and Marrow Transplantation 2010. doi:10.1016/j.bbmt.2010.05.015.
Biondi:CoImmune, Galapagos, Amgen, Novartis, BMS: Consultancy, Research Funding, Speakers Bureau. Rambaldi:Astellas: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Travel support, Speakers Bureau; Jazz: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Travel support, Speakers Bureau; Amgen: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Travel support, Speakers Bureau; Pfizer: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Travel support, Speakers Bureau; Janssen: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Travel support, Speakers Bureau; Incyte: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Travel support, Speakers Bureau; Kite-Gilead: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Travel support, Speakers Bureau; Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Travel support, Speakers Bureau; Roche: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Travel support, Speakers Bureau; Omeros: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Travel support, Speakers Bureau.
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