ISSN 1866-8836
Клеточная терапия и трансплантация

GC-02. Ofatumumab-based CD20-specific CARs: in vitro and in vivo activity

Tatyana N. Belovezhets, Andrey A. Gorchakov, Sergey V. Kulemzin

Institute of Molecular and Cellular Biology SB RAS, Novosibirsk, Russia

Dr. Tatyana Belovezhets, phone: +7 (903) 049-44-71, e-mail:

doi 10.18620/ctt-1866-8836-2021-10-3-1-148


Impressive clinical activity of CD19-specific CAR T-cell therapy has bolstered active research in the field of adoptive cellular immunotherapy of cancer. To date, FDA has approved 5 CAR T-cell products. However, it becomes clear that further improvements are needed to ensure CAR T-cell homing and long-term persistence, broader range of targetable epitopes, reduced costs, and switching to an allogeneic format. In the present work, we compared in vitro and in vivo activities of three different CD20-specific CARs vs a reference FMC63-based CD19-specific CAR (Kymriah), the first CAR approved for clinical practice.

Materials and methods

We produced lentiviral constructs encoding two CARs based on the mAbs 1F5 and Leu16, as well as the CAR based on the sequence of a fully human mAb ofatumumab (2F2). These constructs differ only in the antigen-recognition domain. Based on these constructs and T cells obtained from peripheral blood of healthy donors, the CAR T-cells were produced and their performance was compared using several assays.


First, we used FACS to measure surface expression of CARs on T-cells. Then, we observed that all CAR T-cells displayed similar level of cytotoxicity in a 4-hour co-incubation test against Nalm6 target cells expressing CD20 (Fig. 1). The proliferation rate of CAR T-cells with antigen-recognition regions from 1F5 and FMC63 was approximately 1.5 times higher than that of other CAR T-cells. In the replating assay, CAR T-cells based on 1F5 and Leu16 likewise showed more promising results (Fig. 2). All the CAR T-cells obtained controlled xenografted tumors in NOD. Cg-PrkdcscidIl2rgtm1Wjl/SzJ mice significantly better than irrelevant CAR T-cells (specific to PSMA) (Fig. 3).


Figure 1. Level of momentary cytotoxicity of CAR-T cells against Nalm6-CD20+ after 4 hours of incubation


Figure 2. Level of cytotoxicity of CAR-T cells against Nalm6-CD20+ in replating test


Figure 3. CAR-T cells effectively control the progression of B-ALL in a model experiment in vivo


CAR T-cells obtained display pronounced activity both in vitro and in vivo warranting their analysis in the clinic as well as the design and pre-clinical analysis of bi-specific CARs.

This study was supported by the RFBR grant №19-415-543015 р_мол_а.


Т-cells, chimeric antigen receptor, CAR T-cell therapy, ofatumumab.

Volume 10, Number 3

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doi 10.18620/ctt-1866-8836-2021-10-3-1-148

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