ISSN 1866-8836
Клеточная терапия и трансплантация

BS-03. In vitro study of functional and immunophenotypic features of anti-CD19 CAR T cell therapy derived from T lymphocytes of healthy donors and patients with B cell malignancies

Ekaterina I. Fefelova, Yana V. Serdyuk, R. Salman, Tatyana A. Nenasheva, Nataliya O. Ivanova, Apollinariya V. Bogolyubova

National Medical Research Center for Hematology, Moscow, Russia

Contact: Ekaterina I. Fefelova, e-mail:

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


Cellular immunotherapy with T lymphocytes expressing chimeric antigen receptor (CAR) has demonstrated high efficacy in the treatment of B cell malignancies, in particular, acute B lymphoblastic leukemia (B-ALL) and diffuse large B cell lymphoma (DLBCL). CAR is a chimeric molecule consisting of the signaling domains of the T cell receptor complex fused to extracellular antigen-recognition domain, usually a single-chain fragment (scFv) of a monoclonal antibody. High- affinity CAR T cells can recognize and eliminate target antigen-carrying cells. CAR T cell therapy is available in both autologous and allogeneic forms. CAR T cells are frequently produced from the T cells of a healthy donor who has previously donated allogeneic hematopoietic stem cell for transplantation (allo-HSCT). It is known that original quality of T cells used for production has a considerable impact on the functionality of the acquired cellular product; thus, the parameters of the product obtained from T lymphocytes of healthy donors and patients may differ significantly. The aim of the present research was to study functional and immunophenotypic features of second generation anti-CD19 CAR T cell preparations derived from cells of healthy donors and patients with B cell malignancies.

Materials and methods

The study was based on samples obtained from 3 healthy donors, 3 patients with DLBCL and 5 patients with B-ALL. CAR T cells were obtained by isolation of CD3+ cells, their activation with Enceed reagent (Genscript) and subsequent transduction with a third-generation lentiviral vector carrying the CAR gene. The level of CAR T cell transduction was evaluated by flow cytometry by staining cells with rCD19-Fc protein conjugated with AF-647 dye. CAR T cell expansion was assessed within 7-14 days. To estimate functional activity, a cytotoxicity test was performed at 0- and 24-hours using cell lines carrying CD19 antigen on their surface (Raji, NALM6) as targets. Different ratios of CAR T cells to target cells (E:T) were used. Functional activity and immunophenotype were evaluated by flow cytometry.


This research showed that the transduction level of CAR T cell preparations derived from healthy donors and patients did not vary and ranged from 19 to 49% (median 40%). The expansion level of CAR T cell product was measured on day 8-10 from the beginning of the experiment. Expansion levels of healthy donors were 11- to 28-fold (median, 15), 15- to 18-fold (median, 15.5) in patients with DLBCL, and 5- to 8-fold (median, 7.5) in patients with B-ALL. Cytotoxicity test results showed that the number of target cells was reduced at all E:T ratios, with the most pronounced effect at ratio of a 5:1, regardless of the source of cells for CAR T cell production. The immunophenotype differed in all samples, 22-65% of CAR T cells were central memory cells.


This study concerned cytotoxic and immunophenotypic features of anti-CD19 CAR T cell product derived from T lymphocytes of healthy donors and patients. It was shown that there were no differences in the level of transduction and expansion. The immunophenotype was different for all samples. Thus, T lymphocytes from any of the above sources may be used for the production of anti-CD19 CAR T cells using the investigated lentiviral vector.


Immunotherapy, B-ALL, DLBCL, chimeric antigenic receptor, CAR T cells, anti-CD19.

Supplement 12-3

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

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