Evaluation of cell aggregation according to culture media after cell thawing.

NK-92 cells were frozen using seven types of cryoprotective agents. After thawing the frozen cells, (A) Cultured in Xuri T-cell media after 24 hours. (B) Cultured in X-vivo 10 media to observe cell aggregation after 24 hours. (TIF)</p

Assessment of cytokine production efficacy of NK-92 cells according to the culture medium.

(A) NK-92 cells cultured in three distinct media were subjected to flow cytometry analysis to evaluate the expression of effector molecules. To quantify the alterations in expression levels, we represented the MFI ratios for each individual molecule as determined through flow cytometry measurements. (TIF)</p

Assessment cell viability after cryopreservation for NK-92.

Cell viability was evaluated according to the freezing rate. Cell viability after thawing by type of cryoprotective agents (A) frozen at a rate of -0.5°C/min, (B) a rate of -1.0°C/min, and (C) a rate of -2.0°C/min. Cell viability was evaluated according to nucleation induction during cell freezing and viability results according to cell concentration. (D) Cell viability after thawing as a function of cell concentration, (E) Cell viability after thawing according to cell concentration induced nucleation. Cell viability was assessed after long-term storage of frozen cells. Cell viability and expansion efficacy of (F) 1 month, (G) 3 months, and (H) 6 months after storage of frozen cells in liquid nitrogen vapor phase. Evaluation of preservation efficacy in cryo-bags (I)Assessment cell viability after cryopreservation for NK-92. Cell viability was assessed after long-term storage of frozen cells. Cell viability and expansion efficacy of 1 month, 3 months, and 6 months after storage frozen cells. p value: ns > 0.05, ** < 0.01, *** < 0.001, **** < 0.0001.</p

Cells type used in the study.

Natural killer (NK) cells have recently shown renewed promise as therapeutic cells for use in treating hematologic cancer indications. Despite this promise, NK cell manufacturing workflows remain largely manual, open, and disconnected, and depend on feeders, as well as outdated unit operations or processes, often utilizing research-grade reagents. Successful scale-up of NK cells critically depends on the availability and performance of nutrient-rich expansion media and cryopreservation conditions that are conducive to high cell viability and recovery post-thaw. In this paper we used Cytiva hardware and media to expand the NK92 cell line in a model process that is suitable for GMP and clinical manufacturing of NK cells. We tested a range of cryopreservation factors including cooling rate, a range of DMSO-containing and DMSO-free cryoprotectants, ice nucleation, and cell density. Higher post-thaw recovery was seen in cryobags over cryovials cooled in identical conditions, and cooling rates of 1°C/min or 2°C/min optimal for cryopreservation in DMSO-containing and DMSO-free cryoprotectants respectively. Higher cell densities of 5x107 cells/ml gave higher post-thaw viability than those cryopreserved at either 1x106 or 5x106 cells/ml. This enabled us to automate, close and connect unit operations within the workflow while demonstrating superior expansion and cryopreservation of NK92 cells. Cellular outputs and performance were conducive to clinical dosing regimens, serving as a proof-of-concept for future clinical and commercial manufacturing.</div

Xuri W25 setpoints.

Natural killer (NK) cells have recently shown renewed promise as therapeutic cells for use in treating hematologic cancer indications. Despite this promise, NK cell manufacturing workflows remain largely manual, open, and disconnected, and depend on feeders, as well as outdated unit operations or processes, often utilizing research-grade reagents. Successful scale-up of NK cells critically depends on the availability and performance of nutrient-rich expansion media and cryopreservation conditions that are conducive to high cell viability and recovery post-thaw. In this paper we used Cytiva hardware and media to expand the NK92 cell line in a model process that is suitable for GMP and clinical manufacturing of NK cells. We tested a range of cryopreservation factors including cooling rate, a range of DMSO-containing and DMSO-free cryoprotectants, ice nucleation, and cell density. Higher post-thaw recovery was seen in cryobags over cryovials cooled in identical conditions, and cooling rates of 1°C/min or 2°C/min optimal for cryopreservation in DMSO-containing and DMSO-free cryoprotectants respectively. Higher cell densities of 5x107 cells/ml gave higher post-thaw viability than those cryopreserved at either 1x106 or 5x106 cells/ml. This enabled us to automate, close and connect unit operations within the workflow while demonstrating superior expansion and cryopreservation of NK92 cells. Cellular outputs and performance were conducive to clinical dosing regimens, serving as a proof-of-concept for future clinical and commercial manufacturing.</div

Expansion efficacy in the Xuri W25 cell expansion system.

Cells were cultured using a Xuri W25 cell expansion system. The bioreactor was used to control parameters like rock rate, angle, temperature- and gas mix in an automated manner. (A) Expansion efficiency according to cell culture bag capacity. (B) Evaluation of reproducibility of expansion efficiency using NK-92 and TCR-NK-92 cells in the Xuri W25 cell expansion system. The expression level of cell (C) activation receptors, (D) effector molecules, and (E) inhibitory receptors was confirmed after culture up to 12 days. To estimate the change in receptor expression, MFI ratios for each receptor were depicted as measured by flow cytometry. Error bars represent standard error of the mean [25].</p

Assessment of expansion efficiency of NK-92 cells according to the culture medium.

For NK-92 cells, (A) cell concentration, (B) expansion, (C) viability, and (D) proliferation rate were evaluated every two days. NK cells from cultures of two media were analyzed by flow cytometry for expression of various cell-surface receptors. The expression level of cell (E) activator and (F) effector factors was confirmed after culture up to 12 days. To estimate the change in receptor expression, mean fluorescence intensity (MFI) ratios for each receptor were depicted as measured by flow cytometry. Error bars represent standard error of the mean [25]. p value:* < 0.05, ** < 0.01, **** < 0.0001.</p

Assessment of cell stability after thawing.

Cell stability using three types of cell culture media. (A) Cell expansion efficacy of the NK-92 cell line. (B) Cell morphology as a function of days in culture post-thaw. (C) The efficacy of NK-92 in target cell killing. (D) The expression level of effector molecules in co-culture. Error bars represent standard error of the mean [25]. p value: *** < 0.001, **** < 0.0001.</p

Assessment of culture efficiency of genetically engineered NK-92 cells.

Genetically engineered NK-92 cells were cultured in Xuri T cell expansion medium to assess (A) cell concentration in culture, (B) expansion efficiency, (C) viability, and (D) proliferation factors. Error bars represent standard error of the mean [25].</p

DataSheet1_YH29407 with anti-PD-1 ameliorates anti-tumor effects via increased T cell functionality and antigen presenting machinery in the tumor microenvironment.docx

Among cancer cells, indoleamine 2, 3-dioxygenase1 (IDO1) activity has been implicated in improving the proliferation and growth of cancer cells and suppressing immune cell activity. IDO1 is also responsible for the catabolism of tryptophan to kynurenine. Depletion of tryptophan and an increase in kynurenine exert important immunosuppressive functions by activating regulatory T cells and suppressing CD8+ T and natural killer (NK) cells. In this study, we compared the anti-tumor effects of YH29407, the best-in-class IDO1 inhibitor with improved pharmacodynamics and pharmacokinetics, with first and second-generation IDO1 inhibitors (epacadostat and BMS-986205, respectively). YH29407 treatment alone and anti-PD-1 (aPD-1) combination treatment induced significant tumor suppression compared with competing drugs. In particular, combination treatment showed the best anti-tumor effects, with most tumors reduced and complete responses. Our observations suggest that improved anti-tumor effects were caused by an increase in T cell infiltration and activity after YH29407 treatment. Notably, an immune depletion assay confirmed that YH29407 is closely related to CD8+ T cells. RNA-seq results showed that treatment with YH29407 increased the expression of genes involved in T cell function and antigen presentation in tumors expressing ZAP70, LCK, NFATC2, B2M, and MYD88 genes. Our results suggest that an IDO1 inhibitor, YH29407, has enhanced PK/PD compared to previous IDO1 inhibitors by causing a change in the population of CD8+ T cells including infiltrating T cells into the tumor. Ultimately, YH29407 overcame the limitations of the competing drugs and displayed potential as an immunotherapy strategy in combination with aPD-1.</p