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Automated production of CCR5-negative CD4⁺-T cells in a GMP-compatible, clinical scale for treatment of HIV-positive patients.

ABSTRACT: Ex-vivo gene editing in T lymphocytes paves the way for novel concepts of immunotherapy. One of those strategies is directed at the protection of CD4⁺-T helper cells from HIV infection in HIV-positive individuals. To this end, we have developed and optimised a CCR5-targeting TALE nuclease, CCR5-Uco-hetTALEN, mediating high-efficiency knockout of C-C motif chemokine receptor 5 (CCR5), the HIV co-receptor essential during initial infection. Clinical translation of the knockout approach requires up-scaling of the manufacturing process to clinically relevant cell numbers in accordance with good manufacturing practice (GMP). Here we present a GMP-compatible mRNA electroporation protocol for the automated production of CCR5-edited CD4⁺-T cells in the closed CliniMACS Prodigy system. The automated process reliably produced high amounts of CCR5-edited CD4⁺-T cells (>1.5 × 10⁹ cells with >60% CCR5 editing) within 12 days. Of note, about 40% of total large-scale produced cells showed a biallelic CCR5 editing, and between 25 and 42% of produced cells had a central memory T-cell phenotype. In conclusion, transfection of primary T cells with CCR5-Uco-hetTALEN mRNA is readily scalable for GMP-compatible production and hence suitable for application in HIV gene therapy.

SUBMITTER: Schwarze LI

PROVIDER: S-EPMC8455337 | biostudies-literature | 2021 Sep

REPOSITORIES: biostudies-literature

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Automated production of CCR5-negative CD4<sup>+</sup>-T cells in a GMP-compatible, clinical scale for treatment of HIV-positive patients.

Schwarze Lea Isabell LI Sonntag Tanja T Wild Stefan S Schmitz Sabrina S Uhde Almut A Fehse Boris B

Gene therapy 20210419 9

Ex-vivo gene editing in T lymphocytes paves the way for novel concepts of immunotherapy. One of those strategies is directed at the protection of CD4<sup>+</sup>-T helper cells from HIV infection in HIV-positive individuals. To this end, we have developed and optimised a CCR5-targeting TALE nuclease, CCR5-Uco-hetTALEN, mediating high-efficiency knockout of C-C motif chemokine receptor 5 (CCR5), the HIV co-receptor essential during initial infection. Clinical translation of the knockout approach ...[more]

PMID: 33867524

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Project description:BACKGROUND:Gallium-68 labeled synthetic somatostatin analogs for PET/CT imaging are the current gold standard for somatostatin receptor imaging in neuroendocrine tumor patients. Despite good imaging properties, their use in clinical practice is hampered by the low production levels of 68Ga eluted from a 68Ge/68Ga generator. In contrast, 18F-tracers can be produced in large quantities allowing centralized production and distribution to distant PET centers. [18F]AlF-NOTA-octreotide is a promising tracer that combines a straightforward Al18F-based production procedure with excellent in vivo pharmacokinetics and specific tumor uptake, demonstrated in SSTR2 positive tumor mice. However, advancing towards clinical studies with [18F]AlF-NOTA-octreotide requires the development of an efficient automated GMP production process and additional preclinical studies are necessary to further evaluate the in vivo properties of [18F]AlF-NOTA-octreotide. In this study, we present the automated GMP production of [18F]AlF-NOTA-octreotide on the Trasis AllinOne® radio-synthesizer platform and quality control of the drug product in accordance with GMP. Further, radiometabolite studies were performed and the pharmacokinetics and biodistribution of [18F]AlF-NOTA-octreotide were assessed in healthy rats using μPET/MR. RESULTS:The production process of [18F]AlF-NOTA-octreotide has been validated by three validation production runs and the tracer was obtained with a final batch activity of 10.8 ± 1.3 GBq at end of synthesis with a radiochemical yield of 26.1 ± 3.6% (dc), high radiochemical purity and stability (96.3 ± 0.2% up to 6 h post synthesis) and an apparent molar activity of 160.5 ± 75.3 GBq/μmol. The total synthesis time was 40 ± 3 min. Further, the quality control was successfully implemented using validated analytical procedures. Finally, [18F]AlF-NOTA-octreotide showed high in vivo stability and favorable pharmacokinetics with high and specific accumulation in SSTR2-expressing organs in rats. CONCLUSION:This robust and automated production process provides high batch activity of [18F]AlF-NOTA-octreotide allowing centralized production and shipment of the compound to remote PET centers. Further, the production process and quality control developed for [18F]AlF-NOTA-octreotide is easily implementable in a clinical setting and the tracer is a potential clinical alternative for somatostatin directed 68Ga labeled peptides obviating the need for a 68Ge/68Ga-generator. Finally, the favorable in vivo properties of [18F]AlF-NOTA-octreotide in rats, with high and specific accumulation in SSTR2 expressing organs, supports clinical translation.

Dataset Information

Automated production of CCR5-negative CD4⁺-T cells in a GMP-compatible, clinical scale for treatment of HIV-positive patients.

Publications

Automated production of CCR5-negative CD4<sup>+</sup>-T cells in a GMP-compatible, clinical scale for treatment of HIV-positive patients.

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Dataset Information

Automated production of CCR5-negative CD4+-T cells in a GMP-compatible, clinical scale for treatment of HIV-positive patients.

Publications

Automated production of CCR5-negative CD4<sup>+</sup>-T cells in a GMP-compatible, clinical scale for treatment of HIV-positive patients.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets

Automated production of CCR5-negative CD4⁺-T cells in a GMP-compatible, clinical scale for treatment of HIV-positive patients.