ABSTRACT
Background & Aim: The recent supply chain crisis highlights a need to establish alternative manufacturing (MFG) protocols ensuring continuity of existing and new cell therapy (CT) clinical trials. Our academic CT program, and likely others, experienced purchasing delays and restrictions caused by diversion of critical supplies to meet COVID-19- related research demands and/or reduced vendor capacity due to resource constraints, including attrition of skilled workforce. Mitigation strategies aimed at creating process redundancies overcome production challenges resulting from a scarcity of goods. Here, we validated an alternative ex vivo culture system to clinically MFG lentiviral vector (LV) modified CAR T cells due to limited availability of cell expansion culture bags for the Wave bioreactor, a critical unit of operation that we have used to successfully MFG thousands of gene-modified T cell products for 30+ clinical trials. Methods, Results & Conclusion: The disposable G-REX culture vessels were compatible and seamlessly integrated with our closed system platform. Mesothelin CAR T cells were manufactured in parallel via the G-REX or conventional Wave bioreactor using consented patient starting material. Critical quality attributes of the final T cell products, including viability, transduction efficiency, phenotype and function were assessed. Transduction efficiencies assessed by flow cytometry and/or molecular qPCR were lower in products generated in the G-REX compared to the wave using the same multiplicity of infection. However, at least 50-fold expansion was achieved, with cell viabilities greater than 90% and with comparable cellular phenotypes. The Meso CAR T cells generated by either process were capable of eliciting CAR-mediated cytotoxicity and effector cytokine production. Strikingly, 2-4 billion T cells were harvested from a starting seed number of just 50 million T cells in the 1L G-REX, which may be sufficient to meet most protocol- specified cell therapy doses, suggesting that a full apheresis collection may not be needed. Notably, this process required just 1/3 of the starting material, 1/5 of the media and decreased manual effort through culture duration compared to the Wave. Additionally, the reduced reliance on specialized capital equipment combined with a small footprint enables simultaneous MFG of several immunotherapy products. These advantages propose consideration in replacement of current expansion platform as well as validating an alternative process for MFG CAR T cells.
ABSTRACT
Background: Inflammatory bowel disease (IBD) is a chronic inflammatory disorder of the gastrointestinal tract characterized by immune dysregulation and decreased T cell receptor (TCR) repertoire diversity. Patients with immune-mediated disorders such as IBD have attenuated convalescent antibody responses after COVID-19 infection. We sought to understand the immune configuration associated with high versus low convalescent SARS-CoV- 2 antibodies in patients with IBD using single-cell immunophenotyping. Methods: We performed a study of 9 patients with IBD who were SARS-CoV-2 convalescent (recovered from COVID-19 and converted RNA positive to negative) and 9 matched SARS-CoV-2 naïve controls (no prior COVID-19, confirmed RNA negative). We measured plasma SARS-CoV- 2 antibody (N protein IgG, S1RBD IgG, S1RBD IgA) levels from patients with IBD two months after recovering from COVID-19 (RNA negative). We selected three patients with the highest SARS-CoV-2 antibodies and three matched (for age, sex, IBD subtype and disease activity, medications, COVID-19 severity) patients with the lowest antibodies and performed their peripheral blood mononuclear cell (PBMC) single-cell transcriptomics with paired TCR and BCR sequencing using 10X Genomics. Normalization, dimensionality reduction, and clustering were performed using Seurat. TCR and BCR immune repertoire analyses were performed using Immunarch. Results: SARS-CoV-2 convalescent patients with IBD had detectable but variable SARS-CoV-2 antibody levels (range 0-469 U/mL), whereas SARSCoV- 2 naïve IBD patients had no detectable antibodies. The mean SARS-CoV-2 antibody concentration among the three IBD patients with the highest and three patients with the lowest groups differed by more than 10-fold (206.0 vs 17.5 U/mL, P<0.001). PBMC singlecell immunophenotyping revealed decreased naïve CD4+ T cell and increased CD14+ monocyte and memory CD4+ T cell proportions in IBD patients in the low versus high SARSCoV- 2 antibody group. There were higher numbers of HLA-DQA1+ B cells and CD8 T cells and lower GPR183+ B cells and CD8 T cells in the high SARS-CoV-2 antibody group. There was a trend towards decreased TCR and BCR repertoire diversity in the low SARS-COV-2 antibody group. Finally, we identified immunoglobulin gene signatures (IGHV1-69D/IGLV3- 25, IGHV3-48, IGHV3-7/IGKV41/IGLV1-47, IGHV3-7/IGKV4-1, IGHV3-7/IGKV4-44) that were enriched only in the high SARS-CoV-2 antibody group. Conclusions: Single-cell immunophenotyping of PBMC from convalescent patients with IBD reveal differences in CD4+ T cell, CD14+ monocyte, and HLA-DQA1+ and GPR183+ B and CD8 T cell immunophenotypes, immune repertoire diversity, and immunoglobulin gene signatures in patients with high versus low SARS-CoV-2 antibody levels.(Figure Presented)Figure 1. SARS-COV-2 Antibodies in Convalescent Patients with IBD and Single-Cell Immunophenotypes. A) SARS-COV-2 antibody levels in COVID-19 convalescent versus SARS-CoV-2 naïve patients with IBD B) T-SNE plot of PBMC immunophenotypes in all convalescent patients with IBD C) Differences in proportion of single-cell PBMC immunophenotypes in high versus low SARS-COV-2 antibody patients D) Differences in HLA-DQA1 and GPR183 immunophenotypes in high versus low SARS-COV-2 antibody patients.