Development of an optimized closed and semi-automatic protocol for Good Manufacturing Practice manufacturing of tumor-infiltrating lymphocytes in a hospital environment.

BACKGROUND AIMS: Several studies report on Good Manufacturing Process (GMP)-compliant manufacturing protocols for the ex vivo expansion of tumor-infiltrating lymphocytes (TILs) for the treatment of patients with refractory melanoma and other solid malignancies. Further opportunities for improvements in terms of ergonomy and operating time have been identified. METHODS: To enable GMP-compliant TILs production for adoptive cell therapy needs, a simple automated and reproducible protocol for TILs manufacturing with the use of a closed system was developed and implemented at the authors' institution. RESULTS: This protocol enabled significant operating time reduction during TILs expansion while allowing the generation of high-quality TILs products. CONCLUSIONS: A simplified and efficient method of TILs expansion will enable the broadening of individualized tumor therapy and will increase patients' access to state-of-the-art TILs adoptive cell therapy treatment.

Optimal priming of poxvirus vector (NYVAC)-based HIV vaccine regimens for T cell responses requires three DNA injections. Results of the randomized multicentre EV03/ANRS VAC20 Phase I/II Trial.

DNA vectors have been widely used as a priming of poxvirus vaccine in prime/boost regimens. Whether the number of DNA impacts qualitatively or quantitatively the immune response is not fully explored. With the aim to reinforce T-cell responses by optimizing the prime-boost regimen, the multicentric EV03/ANRS VAC20 phase I/II trial, randomized 147 HIV-negative volunteers to either 3xDNA plus 1xNYVAC (weeks 0, 4, 8 plus 24; n = 74) or to 2xDNA plus 2xNYVAC (weeks 0, 4 plus 20, 24; n = 73) groups. T-cell responses (IFN-γ ELISPOT) to at least one peptide pool were higher in the 3xDNA than the 2xDNA groups (91% and 80% of vaccinees) (P = 0.049). In the 3xDNA arm, 26 (37%) recipients developed a broader T-cell response (Env plus at least to one of the Gag, Pol, Nef pools) than in the 2xDNA (15; 22%) arms (primary endpoint; P = 0.047) with a higher magnitude against Env (at week 26) (P<0.001). In both groups, vaccine regimens induced HIV-specific polyfunctional CD4 and CD8 T cells and the production of Th1, Th2 and Th17/IL-21 cytokines. Antibody responses were also elicited in up to 81% of vaccines. A higher percentage of IgG responders was noted in the 2xDNA arm compared to the 3xDNA arm, while the 3xDNA group tended to elicit a higher magnitude of IgG3 response against specific Env antigens. We show here that the modulation of the prime strategy, without modifying the route or the dose of administration, or the combination of vectors, may influence the quality of the responses.

Quantification of the next-generation oral anti-tumor drugs dabrafenib, trametinib, vemurafenib, cobimetinib, pazopanib, regorafenib and two metabolites in human plasma by liquid chromatography-tandem mass spectrometry.

A sensitive and selective method of high performance liquid chromatography (HPLC) coupled to tandem mass spectrometry (MS/MS) has been developed for the simultaneous quantification of six anticancer protein kinase inhibitors (PKIs), dabrafenib, trametinib, vemurafenib, cobimetinib, pazopanib, regorafenib, and two active metabolites (regorafenib-M2 and regorafenib-M5) in human plasma. Plasma protein precipitation with methanol enables the sample extraction of 100 µL aliquot of plasma. Analytes are detected by electrospray triple-stage quadrupole mass spectrometry and quantified using the calibration curves with stable isotope-labeled internal standards. The method was validated based on FDA recommendations, including assessment of extraction yield (74-104%), matrix effects, analytical recovery (94-104%) with low variability (<15%). The method is sensitive (lower limits of quantification within 1 to 200 ng/mL), accurate (intra- and inter-assay bias: -0.3% to +12.7%, and -3.2% to +6.3%, respectively) and precise (intra- and inter-assay CVs within 0.7-7.3% and 2.5-8.0%, respectively) over the clinically relevant concentration range (upper limits of quantification 500 to 100,000 ng/mL). This method is applied in our laboratory for both clinical research programs and routine therapeutic drug monitoring service of PKIs.