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• W2564105863 abstract "The NK-92/5.28.z cell line is an ErbB2 (HER2) specific, CAR-expressing continuously growing derivative of the clinically applicable NK-92 line, and exhibits profound and highly specific cytotoxicity against ErbB2-expressing tumor targets which are resistant to parental NK-92 cells. This study aimed to establish good manufacturing practice procedures enabling the generation of therapeutic doses of ErbB2-CAR expressing NK-92 cells for upcoming phase I/II clinical trials in ErbB2-overexpressing malignancies. The concept of patient dose manufacturing presented here encompasses the generation of a GMP-compliant master cell bank, which serves as a well-defined source of NK-92/5.28.z cells for further expansion of individual patient doses.To reach sufficient numbers of genetically modified NK-92 cells, expansion protocols were developed by systematic testing of culture conditions including GMP-grade culture media (X-Vivo 10 with human holo-transferrin, X-Vivo 10 with recombinant transferrin, CellGro) and human serum substitutes (human serum albumin, platelet lysate, heat inactivated human plasma). Since growth and potency of NK-92/5.28.z cells are IL-2 dependent, identification of a suitable concentration of this cytokine was an important issue addressed in this work. As a result of performed analyses, X-Vivo 10 containing recombinant transferrin supplemented with 5 % of heat inactivated human plasma and 500 U/ml IL-2 was selected as the culture medium for clinical expansions. This combination supported reproducible cell growth with doubling time of 28.84 h ± 0.5 h ±, maximal -fold expansion in batch culture of 24.97 ± 0.65 (max. conc.: 12.49 x 105 ± 0.32 x 105 cells/ml ) stable CAR expression and potency of the cells, even in long-term culture (CAR expression: 99.77 % ± 0.03 %; specific cytotoxicity: 89.75 % ± 3.77 % after 12 months of culturing). Considering the sensitivity of NK-92 and its CAR-expressing derivative to cryopreservation procedures, a part of this thesis focused on selection of cryopreservation solutions providing satisfactory post-thaw recovery of frozen cells, where human serum albumin as a base medium supplemented with 7.5 % DMSO gave the best results (post-thaw recovery of NK-92/5.28.z cells: 68.84 % ± 5.07 %). Following established GMP-compliant culture and cryopreservation procedures, a master cell bank comprising 200 vials ofNK-92/5.28.z cells was generated. By means of concurrently developed quality control methods, 6 representative vials from the master cell bank were tested in terms of manufacturing related parameters (mean cryovial content, post-thaw recovery and cell growth, cell identity, specific cytotoxicity) confirming the high quality of the cells and thereby the validity of the performed process. Multiparameter analysis revealed that therapeutic doses of 5 x 109 NK-92/5.28.z cells can be expanded from a master cell bank within 5 days. Application of X-Vivo 10 rTF medium supplemented with100 U/ml of IL-2 as an excipient, supported 6-hour shelf life of irradiated (10 Gy)NK-92/5.28.z cells formulated as a final product at the density of 5 x 107/ml.This concentration translates to the final dosage form of 1 x 108 cells/dose in 2 ml,a volume which is suitable for intracranial injection in the upcoming clinical trial in glioblastoma. The systemic (intravenous) route of administration planned inan upcoming breast cancer trial will allow the infusion of even higher volumes of up to 100 ml, and thereby higher cell numbers up to 5 x 109 cells/dose. In addition, investigation of factors impacting the efficacy and safety of clinical application was another important part of this thesis. Analysis of soluble factors released by target stimulated NK-92/5.28.z cells showed selective and significant increase in the secretion of GZMB (2-fold), IFN-γ (4-fold), IL-8 (24-fold) and IL-10 (5-fold) NK-92/5.28.z upon stimulation with ErbB2(+) targets. This phenomenon was not observed in the case of parental NK-92 cells and primary NK cells, indicating resistance of ErbB2(+) target cell line to natural killing mechanism, which can be overcome by specific CAR-mediated activation. Abundant amounts of immunomodulatory cytokines secreted by tumor activated NK-92/5.28.z may stimulate the host immune system by interaction with dendritic cells, induction of CTL and tumor-specific antibody production by B cells.Of possible relevance for clinical tolerability, IL-6 which plays the leading role in cytokine release syndrome was not detected. Treatment with clinically relevant doses of corticosteroids (maximal dose of 200 µg/ml corresponds with the highest dose of prednisolone applied clinically) did not affect retargeted killing of ErbB2(+) targets by NK-92/5.28.z cells, while natural killing against K562 cells was attenuated ina dose-dependent manner, as expected. Colony forming assays with mobilized peripheral blood progenitor cells pretreated with NK-92 or NK-92/5.28.z showed no evidence of NK cell impact on the colony forming capacity of PBSCs. In this study, GMP-compliant procedure enabling production of therapeutic doses of NK-92/5.28.z was successfully established. The data identify genetically modified, CAR-expressing NK-92 cells as a powerful, clinically feasible candidate for efficient and safe adoptive immunotherapy of ErbB2-overexpressing malignancies like breast cancer or glioblastoma." @default.
• W2564105863 created "2017-01-06" @default.
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• W2564105863 date "2016-01-01" @default.
• W2564105863 modified "2023-09-27" @default.
• W2564105863 title "Establishment of a good manufacturing practice-compliant procedure for expansion of therapeutic doses of genetically modified, CAR expressing NK-92 cells for the treatment of ErbB2-positive malignancies" @default.
• W2564105863 hasPublicationYear "2016" @default.
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