ISSN 1866-8836
Клеточная терапия и трансплантация

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

Ivan N. Gaponenko, Artem A. Potanin, Vladislav V. Markelov, Dina A. Senichchina, Vladislav S. Sergeev, Alena I. Shakirova, Kirill V. Lepik, Alexander D. Kulagin

RM Gorbacheva Research Institute, Pavlov University, St. Petersburg, Russia

Contact: Ivan N.Gaponenko, e-mail:

doi 10.18620/ctt-1866-8836-2023-12-3-1-176


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).


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.


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.


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

Supplement 12-3

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doi 10.18620/ctt-1866-8836-2023-12-3-1-176

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