AW-01. Simultaneous T-lymphocyte activation and B-lymphocyte elimination by bispecific monoclonal antibodies in the CAR-T production process

Summary

Chimeric antigen receptor T-cell (CAR-T) therapy is a promising option for the treatment of B-cell malignancies. One of the important steps in the production of a CAR-T product is T-cell activation and expansion. The main protocol options includes magnetic selection of CD4+/CD8+ lymphocytes followed by the addition of CD3/CD28 activator particles or activation of unselected peripheral blood mononuclear cells (PBMCs) using anti-CD3 monoclonal antibodies. The latter method is more economical, but the application of the products obtained in this way is limited to indications characterized by the presence of circulating tumor B cells. We propose to use monoclonal bispecific antibodies to simultaneously activate T cells and eliminate the B cell population during CAR-T production.

Materials and methods

PBMCs from 4 healthy donors were isolated by centrifugation in a ficoll density gradient. Cells were seeded onto a 24-well plate at a concentration of 1*10⁶ cells/mL, and activated by a monoclonal bispecific anti-CD3/CD20 antibody at 4 different concentrations (0.5;1, 5 and 10 pmol/mL) with 150 IU/mL IL-2 for 6 days. As a negative control, cells were cultured in a medium without the addition of an activating agent for 6 days; in the comparison group, activation was performed with OKT-3 at a concentration of 100 ng/mL. The cumulative population doubling level (CPDL) was used to evaluate the kinetics of PBMC proliferation. After isolation and on day 3 after activation, CD3+ and CD19+ cells were counted by flow cytometry. In 2 donors, the expression of CD4, CD45RO, and CCR7 on CD3 positive cells was evaluated to assess the effect of bispecific antibody on T cell phenotype. Based on these markers, 4 groups were identified among CD4 and CD8 cells: memory cells (TCM); naive/stem cell memory T lymphocytes; effector lymphocytes (EFFL) and effector memory cells (TEM).

Results

T-cell activation with expansion by day 6 was observed in all experiments. CPDL was significantly higher in all experimental groups compared to the control group (p<0.05). CDPL was statistically significantly different for the groups with the addition of bispecific anti-CD3/CD20 at concentrations of 0.5 pmol/mL (1.39±0.64), 1 pmol/mL (1.25±0.64), and 10 pmol/mL (2.2±0.19) from the group with the addition of OKT-3 (p<0.05). Initially, the CD19+ cell content among PBMCs was 5.97±1.05%. On the third day of culturing, elimination of B-lymphocytes was observed in all donors, where 3.28±1.2% and 5.93±1.2% were detected in controls with and without OKT-3, respectively. When evaluating the effect of the proposed activation protocol on the immunophenotype of T cells, a decrease in the number of EFFL by >2 times, TEM by >1.5 times was revealed. At the same time, the number of TCM increased by 8 times and more.

Conclusions

Activation of T cells in PBMC by the addition of monoclonal bispecific anti-CD3/CD20 antibody at all presented concentrations is not inferior to the standard activation and expansion protocol with OKT-3, and potentially allows the use of PBMC for CAR-T cell production without magnetic selection in patients with circulating CD20+ B cells. Together with the elimination of B-lymphocytes in the T-lymphocyte population, the number of effector cells decreases, while the number of memory cells increases, which is a favorable subpopulation profile of T cells for subsequent CAR-T production.

Keywords

CAR-T, chimeric antigen receptor, bispecific antibodies, B-cell tumors.