Novel biomanufacturing platform for large-scale and high-quality human T cells production

Abstract

The adoptive transfer of human T cells or genetically-engineered T cells with cancer-targeting receptors has shown tremendous promise for eradicating tumors in clinical trials. The objective of this study was to develop a novel T cell biomanufacturing platform using stirred-tank bioreactor for large-scale and high-quality cellular production. First, various factors, such as bioreactor parameters, media, supplements, stimulation, seed age, and donors, were investigated. A serum-free fed-batch bioproduction process was developed to achieve 1000-fold expansion within 8 days after first stimulation and another 500-fold expansion with second stimulation. Second, this biomanufacturing process was successfully scaled up in bioreactor with dilution factor of 10, and the robustness and reproducibility of the process was confirmed by the inclusion of different donors' T cells of various qualities. Finally, T cell quality was monitored using 12 surface markers and 3 intracellular cytokines as the critical quality assessment criteria in early, middle and late stages of cell production. In this study, a new biomanufacturing platform was created to produce reliable, reproducible, high-quality, and large-quantity (i.e. > 5 billion) human T cells in stirred-tank bioreactor. This platform is compatible with the production systems of monoclonal antibodies, vaccines, and other therapeutic cells, which provides not only the proof-of-concept but also the ready-to-use new approach of T cell expansion for clinical immune therapy.

Anti-CD47 Monoclonal Antibody–Drug Conjugate: A Targeted Therapy to Treat Triple-Negative Breast Cancers

“…The flow cytometry was conducted to analyze the anti-CD47 mAb’s binding rate to TNBC cells and quantify the immune cells using a BD LSRII flow cytometer (BD Biosciences, San Jose, CA, USA). In surface binding analysis, the produced CD47 mAb was labelled with an Alexa Fluor™ 647 labelling kit (Life Technologies, part of Fisher) and used to stain MDA-MB-468, MDA-MB-231, 4T1, and 184B5 cells with 5 µg of mAb-AF647 per million of cells at room temperature for 30 min [ 57 , 58 , 59 ]. The harvested lymph nodes were dissociated with a Tissue Dissociation/Single Cell Isolation Kit (101 Bio LLC, Sunnyvale, CA, USA) following the manufacturer’s procedure, and stained with Cy5 anti-CD69 antibody, APC anti-CD11c antibody, or PE/Cy7 anti-CD4 antibody (BioLegend, San Diego, CA, USA) for flow cytometry analysis.…”

“…Confocal microscopy imaging was collected to validate the targeting and internalization of anti-CD47 mAb in TNBC cells following our reported protocol [ 39 , 54 , 58 , 59 ]. Briefly, the MDA-MB-468 cells were stained with BacMam GFP Transduction Control, which expresses eGFP protein in the cytoplasm, NucBlue Live ReadyProbes to stain the nucleus, and AF647 labelled anti-CD47 mAb to target TNBC cells via surface receptors.…”

Induced Pluripotent Stem Cells Provide an Unlimited T-cell Source for CAR-T cell Development and A Potential Source for Off-the-shelf Products

“…The last format is the use of Cas9 protein and sgRNA complex, known as ribonucleoprotein, which showed to be beneficial compared to the other two systems [84]. CAR iPSC-derived T cells are expanded by different commercial procedures using supplemental factors and cytokine supplementation [85]. Finally, CAR iPSC-derived T cells are ready to be introduced into the recipient patient through IV injection or intratumoral administration (Figure 4).…”

Chimeric Antigen Receptor Based Therapy as a Potential Approach in Autoimmune Diseases: How Close Are We to the Treatment?

“…After expansion system selection, modified-T cells are expanded into media using supplemental factors and strict control over temperature, pH, agitation, dissolved oxygen (DO) levels, gas sparging, and cytokine supplementation (68). Expansion protocols for CAR-T cells rely typically on cytokines, such as IL-2, IL-7, IL-15, and IL-21.…”