Barnase-barstar Specific Interaction Regulates Car-T Cells Cytotoxic Activity toward Malignancy

Abstract

The development of CAR-T specific therapy made a revolution in modern oncology. Despite the pronounced therapeutic effects, this novel approach displayed several crucial limitations caused by the complications in pharmacokinetics and pharmacodynamics controls. The presence of the several severe medical complications of CAR-T therapy initiated a set of attempts aimed to regulate their activity in vivo. We propose to apply the barnase-barstar system to control the cytotoxic antitumor activity of CAR-T cells. To menage the regulation targeting effect of the system we propose to use barstar-modified CAR-T cells together with barnase-based molecules. Barnase was fused with designed ankyrin repeat proteins (DARPins) specific to tumor antigens HER2 (human epidermal growth factor receptor 2) The application of the system demonstrates the pronounced regulatory effects of CAR-T targeting.

Keywords: Barnase-barstar interaction; CAR-T cells; DARPins; solid tumors.

Fig. 1.

The scheme of regulating the specific targeting of a T-cell modified with a universal BsCAR by the protein darpin-barnase (Da-Bn) targeting the HER2 of the tumor cell. The HER2 receptor has 4 extracellular subdomains: membrane-proximal-IV specific Da G3-Bn, membrane-distal I Da 9.29-Bn. Barnase interacts with high affinity with its inhibitor-barstar, which acts as a recognition domain in the CAR construct. GGGSGGGSGGGS and CH2-CH3 IgG4 are used as a hinge, CD28 TM/cyt = transmembrane and cytoplasmic domains from CD28, 4-1BB = cytoplasmic activation domain from CD137, CD3ζ = cytoplasmic activation domain from CD3ζ.

Fig. 2.

BsCAR T cells targeted by DARPin 9.29.-Bn suppress HER2-positive tumors in vivo. (a) Scheme of the experiment, where Balb/c Nu mice (females, 20–24 g) were intraperitoneally injected with 8 million SKOV-ip-Kat cells that form solid tumor nodes in the peritoneum. The presence of the red fluorescent protein TurboFP635 in the cytoplasm of these cells makes it possible to visualize the growth and spread of tumor nodes in mice using in vivo imaging technology using the IVIS SPectrum CT system (Perkin Elmer, USA). On day 2, 8 million, on day 4, 14 million BsCAR-T cells were intravenously injected into two groups of mice: the control group – BsCAR-T and the experimental group – BsCAR-T + darpin 9.29 – barnase. The therapy was performed by intraperitoneal administration of Da 9.29-Ba according to the scheme in the figure. Visualization of tumors was performed on the 5th and 10th days after the introduction of SKOV-ip-Kat cells. (b) IVIS images of animals treated with BsCAR-T cells alone or in combination with 9.29-barnase. The fluorescence of tumors of individual mice in each group is shown. (c) Tumor burden quantified as the mean fluorescence over 5 and 10 days. Data were analyzed using the Mann-Whitney test and presented as mean ± range. Statistical significance: *p < 0.05.