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Depletion of high-content CD14⁺ cells from apheresis products is critical for successful transduction and expansion of CAR T cells during large-scale cGMP manufacturing.

ABSTRACT: With the US Food and Drug Administration (FDA) approval of four CD19- and one BCMA-targeted chimeric antigen receptor (CAR) therapy for B cell malignancies, CAR T cell therapy has finally reached the status of a medicinal product. The successful manufacturing of autologous CAR T cell products is a key requirement for this promising treatment modality. By analyzing the composition of 214 apheresis products from 210 subjects across eight disease indications, we found that high CD14⁺ cell content poses a challenge for manufacturing CAR T cells, especially in patients with non-Hodgkin's lymphoma and multiple myeloma caused by the non-specific phagocytosis of the magnetic beads used to activate CD3⁺ T cells. We demonstrated that monocyte depletion via rapid plastic surface adhesion significantly reduces the CD14⁺ monocyte content in the apheresis products and simultaneously boosts the CD3⁺ content. We established a 40% CD14⁺ threshold for the stratification of apheresis products across nine clinical trials and demonstrated the effectiveness of this procedure by comparing manufacturing runs in two phase 1 clinical trials. Our study suggests that CD14⁺ content should be monitored in apheresis products, and that the manufacturing of CAR T cells should incorporate a step that lessens the CD14⁺ cell content in apheresis products containing more than 40% to maximize the production success.

SUBMITTER: Wang X

PROVIDER: S-EPMC8411225 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:ObjectiveThe objectives of the present work were to optimize and validate the synthesis and stability of 14(R,S)-[18F]fluoro-6-thia-heptadecanoic acid ([18F]FTHA) and 16-[18F]fluoro-4-thia-palmitic acid ([18F]FTP) under cGMP conditions for clinical applications.MethodsBenzyl-14-(R,S)-tosyloxy-6-thiaheptadecanoate and methyl 16-bromo-4-thia-palmitate were used as precursors for the synthesis of [18F]FTHA and [18F]FTP, respectively. For comparison, a fatty acid analog lacking a thia-substitution, 16-[18F]fluoro-palmitic acid ([18F]FP), was synthesized from the precursor methyl 16-bromo-palmitate. A standard nucleophilic reaction using cryptand (Kryptofix/K222, 8.1?mg), potassium carbonate (K2CO3, 4.0?mg) and 18F-fluoride were employed for the 18F-labeling and potassium hydroxide (0.8?M) was used for the post-labeling ester hydrolysis. The final products were purified via reverse phase semi-preparative HPLC and concentrated via trap and release on a C-18 plus solid phase extraction cartridge. The radiochemical purities of the [18F]fluorothia fatty acids and [18F]FP were examined over a period of 4?h post-synthesis using an analytical HPLC. All the syntheses were optimized in an automated TRACERlab FX-N Pro synthesizer. Liquid chromatography mass spectrometry (LCMS) and high resolution mass spectrometry (HRMS) was employed to study the identity and nature of side products formed during radiosynthesis and as a consequence of post-synthesis radiation induced oxidation.ResultsRadiosyntheses of [18F]FTHA, [18F]FTP and [18F]FP were achieved in moderate (8-20% uncorrected) yields. However, it was observed that the HPLC-purified [18F]fluorothia fatty acids, [18F]FTHA and [18F]FTP at higher radioactivity concentrations (>1.11?GBq/mL, 30?mCi/mL) underwent formation of 18F-labeled side products over time but [18F]FP (lacking a sulfur heteroatom) remained stable up to 4?h post-synthesis. Various radiation protectors like ethanol and ascorbic acid were examined to minimize the formation of side products formed during [18F]FTHA and [18F]FTP synthesis but showed only limited to no effect. Analysis of the side products by LCMS showed formation of sulfoxides of both [18F]FTHA and [18F]FTP. The identity of the sulfoxide side product was further confirmed by synthesizing a non-radioactive reference standard of the sulfoxide analog of FTP and matching retention times on HPLC and molecular ion peaks on LC/HRMS. Radiation-induced oxidation of the sulfur heteroatom was mitigated by dilution of product with isotonic saline to reduce the radioactivity concentration to <0.518?GBq/mL (14?mCi/mL).ConclusionsSuccessful automated synthesis of [18F]fluorothia fatty acids were carried out in cGMP facility for their routine production and clinical applications. Instability of [18F]fluorothia fatty acids were observed at radioactivity concentrations exceeding 1.11?GBq/mL (30?mCi/mL) but mitigated through dilution of the product to <0.518?GBq/mL (14?mCi/mL). The identities of the side products formed were established as the sulfoxides of the respective thia fatty acids caused by radiation-induced oxidation of the sulfur heteroatom.

Project description:ObjectivesThis study sought to identify the ratio of M1/M2 cells in the infrapatellar fat pads (IFP) and subcutaneous fat tissues (SC) of osteoarthritis (OA) and rheumatoid arthritis (RA) patients. The clinical features of OA and RA patients treated with or without biological disease-modifying anti-rheumatic drugs (bDMARDs) were also assessed.MethodsIFP and SC were collected from patients with OA and RA who are undergoing total knee arthroplasty (TKA). CD14-positive cells were then isolated from these samples. Flow cytometry was used to determine the number of CD14++CD80+ cells and CD14++CD163+ cells. The expression levels of lipid transcription factors, such as sterol regulatory element-binding protein 1 (SREBP1) and liver X receptor alpha (LXRA), and inflammatory cytokines were also evaluated.ResultsTwenty OA patients and 22 RA patients were enrolled in this study. Ten of the RA patients (45.4%) received bDAMRDs before TKA. On average, a fivefold increase in the number of CD14-positive cells and lower expression levels of SREBP1C and LXRA were observed in OA IFP relative to OA SC; however, these results were not obtained from the RA samples. The median ratio of CD14++CD80+ cells/CD14++CD163+ cells of OA IFP was 0.87 (0.76-1.09, interquartile range), which is higher to that of OA SC with a lower ratio (p = 0.05835).ConclusionsThe quantity and quality of CD14-positive cells differed between IFP and SC in arthropathy patients. To our knowledge, this is the first study to characterize the ratio of M1/M2 cells in the IFP and SC of end-stage OA and RA patients. The increased ratio of CD14++CD80+ cells/CD14++CD163+ cells in the IFP from patients with OA and RA treated with bDMARDs indicated that inflammation was localized in the IFP. As adipose tissue-derived innate immune cells were revealed as one of the targets for regulating inflammation, further analysis of these cells in the IFP may reveal new therapeutic strategies for inflammatory joint diseases.

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Depletion of high-content CD14⁺ cells from apheresis products is critical for successful transduction and expansion of CAR T cells during large-scale cGMP manufacturing.

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Depletion of high-content CD14+ cells from apheresis products is critical for successful transduction and expansion of CAR T cells during large-scale cGMP manufacturing.

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Depletion of high-content CD14⁺ cells from apheresis products is critical for successful transduction and expansion of CAR T cells during large-scale cGMP manufacturing.