Culturing adequate CAR-T cells from less peripheral blood to treat B-cell malignancies

Abstract

Objective: Chimeric antigen receptor-modified T (CAR-T) cells have shown impressive results against relapsed/refractory B cell malignancies. However, the traditional manufacture of CAR-T cells requires leukapheresis to isolate large amounts of peripheral blood T cells, thus making some patients ineligible for the procedure. Methods: We developed a simple method for CAR-T cell preparation requiring small volumes of peripheral blood. First, CD3 + T cells isolated from 50 mL peripheral blood from patients (B-cell malignancies) were stimulated with immobilized anti-CD3/RetroNectin in 6-well plates and then transduced with CAR-expressing lentiviral vector. After 4 d, the T cells were transferred to culture bags for large-scale CAR-T cell expansion. In vitro and animal experiments were performed to evaluate the activity of the manufactured CAR-T cells. Finally, 29 patients with B-cell acute lymphoblastic leukemia (B-ALL) and 9 patients with B-cell lymphoma were treated with the CAR-T cells. Results: The CAR-T cells were expanded to 1-3 × 10 8 cells in 8-10 d and successfully killed B cell-derived malignant tumor cells in vitro and in vivo. For patients with B-ALL, the complete remission rate was 93% 1 month after CAR-T cell infusion; after 12 months, the overall survival (OS) and leukemia-free survival rates were 69% and 31%, respectively. For patients with lymphoma, the objective response rate (including complete and partial remission) was 78% 2 months after CAR-T cell infusion, and after 12 months, the OS and progression-free survival rates were 71% and 43%, respectively. Cytokine-release syndrome (CRS) occurred in 65.51% and 55.56% of patients with B-ALL and B-cell lymphoma, respectively; severe CRS developed in 20.69% of patients with B-ALL and in no patients with lymphoma. Conclusions: Our novel method can generate sufficient numbers of CAR-T cells for clinical use from 50-100 mL peripheral blood, thus providing an alternative means of CAR-T cell generation for patients ineligible for leukapheresis.

Catching Them Early: Framework Parameters and Progress for Prenatal and Childhood Application of Advanced Therapies

“…For both ex vivo and in vivo applications, various viral and non-viral vectors have been developed as delivery vehicles, each one with their own advantages and disadvantages [289,290]. Improvements can be achieved by ongoing technical innovations, such as in the delivery of cells or genetic material to the tissues and cells of interest for in vivo application [291][292][293][294][295][296][297] or for ex vivo application in the isolation of suitable cells [16,[298][299][300] or in more effective delivery into cells, and possibly nuclei, at improved efficiency and low toxicity [87,[301][302][303][304][305][306]. Viral vectors (γ-retroviruses, lentiviruses, adenoviruses and adeno-associated viruses) were among the first exploited delivery platforms and are still highly prevalent due to their inherently high efficiency of gene transduction to eukaryotic cells (extensively reviewed in [106]).…”

Update on diffuse large B-cell lymphoma: highlights from the 2022 ASCO Annual Meeting

“…Currently, CAR-T-cell therapy has significantly improved the prognosis of patients with R/R B-cell lymphoma, thus providing a new treatment strategy for patients unable to receive hematopoietic stem cell transplantation and whose disease progresses after multiple lines of therapy. Han et al 28 have developed a novel method to generate sufficient CAR-T cells from limited peripheral blood to treat B-cell malignancies, thereby providing an alternative to the traditional CAR-T cell generation method. However, limited sample sizes in clinical studies and severe toxicity remain barriers to developing effective CAR-T-cell therapies.…”

Facing challenges with hope: universal immune cells for hematologic malignancies

“…Chimeric antigen receptor (CAR)-T cells have a major role in hematologic malignancies. With the design of different CAR structures, applications of CAR-T therapies have been expanded from B-cell to other hematologic malignancies 3 ; however, cytokine release syndrome (CRS) in patients undergoing autologous CAR-T-cell therapy and GVHD to allogenic CAR-T cells limit for further application 4 , 5 .…”