Crystallization is an ancient unit operation that remains vital for the chemical process industry. Traditional single-effect evaporation consumes a great deal of energy, and various alternatives to this method have been proposed. In this work, the total cost of producing a fixed quantity of different solid chemicals by crystallization from water is determined for several different technologies: evaporative crystallization, membrane distillation with porous hydrophobic membranes, reverse osmosis membrane-assisted crystallization, and eutectic freeze crystallization (EFC). Among the solute properties, the solubility has the greatest effect on the cost of the process since it determines the amount of water that must be removed per unit product produced. If waste heat is available at a unit price lower than that of low-pressure steam produced using coal or natural gas, then the assumed price of waste heat also has a significant effect on the economics. The results indicate that, if feasible, reverse osmosis has the lowest total annual cost. On the other hand, for high osmotic pressure cases, EFC has the lowest cost when cheap heating energy (waste heat) is not available. On the contrary, a conventional single or multiple effect evaporative crystallization has the lowest cost among the remaining options when heating energy is cheap or solubility is high. Moreover, eutectic temperature plays important role when determining the best technology when solubility is between 0.2 and 0.6 kg/kg solution.

Engineering cellular therapeutics by programming T cells has great potential in immunology. The primary mechanism employed by T cells for the specific transfer of proteins at the immunological synapse is via the lysosomal perforin pathway that facilitates the transfer of cytotoxic granzymes leading to apoptosis in target cells. Facilitating the delivery of non-cytotoxic proteins through perforin oligomers will dramatically expand the range of protein cargos that T cells can traffic to the target cells. Here, we have identified the intralysosomal protein, NPC2, as a chaperone that can facilitate the delivery of T-cell derived reporter proteins through perforin pores at the immunological synapse. Structural and biophysical considerations suggested that NPC2 could traverse through perforin pores and in vitro experiments confirmed the transport of purified NPC2 through perforin pores on cell membranes. To characterize the ability of NPC2 to facilitate the transfer of payloads in T cells, we constructed NPC2-mCherry fusion proteins in T cells. Using confocal microscopy and flow cytometry, we confirmed the colocalization of the NPC2 fused protein with lytic granules and the transfer of the fluorescent protein payload from T cells to target cells in co-culture experiments. The NPC2 fusion enabled the localization of mCherry to secretory lysosomes in mouse TCR CD8+ T cells and human CD4+ and CD8+ chimeric antigen receptor (CAR) T cells. These results illustrate that by using NPC2 as a molecular chaperone, the NPC2-perforin pathway can be exploited as a programmable molecular delivery system for cell-based therapies.

Background Chimeric antigen receptor (CAR) T cell therapies show remarkable progress in treating liquid tumors, with a complete remission rate of over 57%. 1 Translating the success of CAR T cells to solid tumors will need an understanding of the key mechanisms responsible for the cytotoxicity of CAR T cells. The primary factors contributing to tumor resistance against CAR T therapies are widely contested 2 , therefore, we seek to explore the impact of different CAR T cell killing mechanisms of tumors. Methods We examine CAR T cell killing of a leukemic cell line, NALM6, and an ovarian cancer cell line, SkOV3-CD19, in the presence of Granzyme B inhibitors and a Fas ligand inhibitor. We develop a fluorescent membrane reporter that translocates to the nucleus upon specific proteolytic cleaving by Granzyme A and B. Results . Overexpressing native Granzyme B inhibitor, ProteaseInhibitor-9 (PI-9), in NALM6 and SkOV3-CD19 does not affect killing frequencies in CAR(19-41BBz and 19-28z) T cell cytotoxicity assays. . Treating 19-41BBz with a small molecule inhibitor of Granzyme B does not impact killing frequencies in cytotoxicity assays against NALM6 and SkOV3-CD19. . Overexpressing PI-9 in NALM6 and SkOV3-CD19 does not affect 19-41BBz CAR T killing frequencies or killing kinetics in single cell time-lapse assays. . Inhibition of Fas ligand on 19-41BBz CAR T cells does not impact killing frequencies against NALM6 and SkOV3-CD19.Conclusions Our findings suggest that suppressing Granzyme B activity with small molecules or native proteins does not impair killing frequencies of 19-41BBz CAR T cells en masse or at the single cell level. We hypothesize that Granzyme A facilitates CAR T killing in the absence of Granzyme B, implying redundancy in granzyme expression. This study provides a comprehensive understanding of the main mechanisms associated with CAR T cell-mediated killing.