High-Density Cryopreservation of Off-the-Shelf CAR-NK Cells Facilitates On-demand Treatment Access

Multiplexed engineered, clonal master induced pluripotent stem cell (iPSC) lines are a renewable source for the routine, mass production of immune effector cells that address many shortcomings associated with current autologous and allogenic donor-derived cell-based immunotherapies, including off-the-shelf availability for broad patient access and multi-dose administration. To enable the feasibility of multi-dose administration and dose escalation of iPSC-derived NK (iNK) and T (iT) cells without extending the patient administration time and ensuring continuous drug product (DP) availability at all treatment sites, we evaluated the feasibility and stability of manufacturing high-cell density fill (HD-fill) doses of multiplexed-engineered iNK cells. To select the ideal cryopreservation formulation in HD-fill studies, we performed iterative formulation screening supported by orthogonal analytical characterizations looking at cell viability, proliferation capacity, and cytotoxicity function upon thaw and over time. The tested HD-fills were produced using two iNK cell products, anti-CD19 CAR (CAR19) iNK cells and anti-MICA/B CAR (CAR-MICA/B) iNK cells, directed to the treatment of either B cell malignancies or solid tumors, respectively. At the final formulation stage, cells were added to various formulations at various densities, cryopreserved in CryoMed™ Controlled-Rate Freezers and stored in vapor-phase liquid nitrogen at <-150°C. We examined standard dose concentration of 1.5E+07 viable cells/mL and HD-fill dose concentrations up to 1.1E+08 viable cells/mL in the top cryopreservation formulation using assessment parameters such as immediate post-thaw recovery and tolerance of hold-time (in-use stability), transgene expression, and potency. Results demonstrated comparable recovery and viability immediately post thaw (>99.0%) as well as after 90 minutes hold time at room temperature (>95.0%) between standard density and HD-fill densities. Transgene expression was also comparable across all HD-fill densities and no differences in drug product purity were observed (>90% CAR for CAR19 and CAR-MICA/B). In vitro cellular cytotoxicity assays were used to evaluate CAR-mediated target cell clearance and antibody-dependent cell cytotoxicity (ADCC). HD-fill of FT536 showed comparable CAR specific cytotoxicity against CaSki cervical carcinoma cells at E:T ratio of 5:1 and ADCC with cetuximab at E:T ratio of 3:1 in the single round and re-stimulation killing assays. HD-Fill of FT596 also showed similar cytolytic potency against Nalm6 leukemia cells compared to the standard dose. Furthermore, HD-fill storage stability was assessed in both liquid nitrogen (<-150°C) and -80°C freezers, with the latter temperature tested to facilitate DP storage at majority of hospitals. Preliminary data show viability, recovery, and potency are consistent for all control and test configurations, including short-term hold in -80°C freezers, demonstrating the suitability of our fill/finish and cryopreservation formulation platform to support HD-fill dose presentations. In summary, we demonstrate the successful generation of HD-fill CAR-iNK cells without interfering with drug product integrity, identity, and function. HD-fill for off-the-shelf immunotherapies will enable flexibility to clinicians and patients in the out-patient setting for the treatment of hematologic and solid tumors.