A Novel Approach to Anticancer Therapy: Molecular Modules Based on the Barnase:Barstar Pair for Targeted Delivery of HSP70 to Tumor Cells

Abstract

One important distinction between many tumor cell types and normal cells consists in the translocation of a number of intracellular proteins, in particular the 70 kDa heat shock protein (HSP70), to the surface of the plasma membrane. It has been demonstrated that such surface localization of HSP70 on tumor cells is recognized by cytotoxic effectors of the immune system, which increases their cytolytic activity. The mechanisms behind this interaction are not fully clear; however, the phenomenon of surface localization of HSP70 on cancer cells can be used to develop new approaches to antitumor immunotherapy. At the same time, it is known that the presence of HSP70 on a cell's surface is not a universal feature of cancer cells. Many types of tumor tissues do not express membrane-associated HSP70, which limits the clinical potential of these approaches. In this context, targeted delivery of exogenous HSP70 to the surface of cancer cells with the aim of attracting and activating the cytotoxic effectors of the immune system can be considered a promising means of antitumor immunotherapy. Molecular constructs containing recombinant mini-antibodies specific to tumor-associated antigens (in particular, antibodies specific to HER2/neu-antigen and other markers highly expressed on the surface of a wide range of cancer cells) can be used to target the delivery of HSP70 to tumor tissues. In order to assess the feasibility and effectiveness of this approach, recombinant constructs containing a mini-antibody specific to the HER2/ neu-antigen in the first module and HSP70 molecule or a fragment of this protein in the second module were developed in this study. Strong selective interaction between the modules was ensured by a cohesive unit formed by the barnase:barstar pair, a heterodimer characterized by an unusually high constant of association. During testing of the developed constructs in in vitro models the constructs exhibited targeted binding to tumor cells expressing the HER2/neu antigen and the agents had a significant stimulating effect on the cytotoxic activity of NK cells against the respective cancer cells.

Keywords: 70 kDa heat shock protein; HER2/neu antigen; NK cells; barnase:barstar; cancer immunotherapy; mini-antibody; targeted delivery.

Fig. 1

Scheme of the plasmid pET22_His_barstar_Hsp70 encoding the second module of the molecular construct carrying the Hsp70 effector protein. T7lac is the early promoter of bacteriophage T7 RNA polymerase; HSP70 is the gene encoding Hsp70; Amp is the ampicillin resistance gene

Fig. 2

Cytofluorimetric analysis of the binding of the 4D5 scFv-dibarnase:barstar-Hsp70 (A) and 4D5 scFv-dibarnase:barstar-Hsp70/16 (B) complexes to the surface of BT-474 tumor cells. The cells were incubated with the first and second modules, and the samples were stained with the first anti-HSP70 antibodies (BRM22) and second FITC-labeled antibodies. X axis – fluorescence intensity; Y axis – number of events. The histograms: 1 – autofluorescence control; 2 – 4D5 scFv-dibarnase control; 3 – barstar-Hsp70 (A) or barstar-Hsp70/16 (B) control; 4 – 4D5 scFv-dibarnase:barstar-Hsp70 (A) or 4D5 scFv-dibarnase:barstar-Hsp70/16 (B).

Fig. 3

Visualization of the interaction between the developed constructs and two types of tumor cells. Upper row: BT-474 cells were treated with 4D5 scFv-dibarnase:barstar-Hsp70 (A) or 4D5 scFv-dibarnase:barstar-Hsp70/16 (B). Bottom row: SKOV3 cells were treated with 4D5 scFv-dibarnase:barstar-Hsp70 (A) or 4D5 scFv-dibarnase:barstar-Hsp70/16 (B). The treated cell samples were stained with the first anti-Hsp70 antibodies (BRM22) and second Alexa Fluor 488-labeled antibodies. C – control cell samples stained only with the second antibodies. Cell nuclei were stained with DAPY. The images were recorded using an ECLIPSE TE2000-E laser confocal microscope (Nikon, Japan). Scale bars, 10 µm

Fig. 4

In vitro analysis of the effect of treating tumor target cells (BT-474) with the developed agents on the cytolytic activity of NK cells. I – control (no treatment); II – target cells treated with 4D5 scFv-dibarnase; III – target cells treated with barstar-Hsp70/16; IV – target cells treated with barstar-Hsp70; V – target cells treated with 4D5 scFv-dibarnase:barstar-Hsp70/16; and VI – target cells treated with 4D5 scFv-dibarnase:barstar-Hsp70