Targeted Delivery of HSP70 to Tumor Cells via Supramolecular Complex Based on HER2-Specific DARPin9_29 and the Barnase:Barstar Pair.

(1) Background: We have previously shown that the use of an artificial supramolecular two-component system based on chimeric recombinant proteins 4D5scFv-barnase and barstar-heat shock protein 70 KDa (HSP70) allows targeted delivery of HSP70 to the surface of tumor cells bearing HER2/neu antigen. In this work, we studied the possibility to using DARPin9_29-barnase as the first targeting module recognizing HER2/neu-antigen in the HSP70 delivery system. (2) Methods: The effect of the developed systems for HSP70 delivery to human carcinomas SK-BR-3 and BT474 cells hyperexpressing HER2/neu on the activation of cytotoxic effectors of the immune cells was studied in vitro. (3) Results: The results obtained by confocal microscopy and cytofluorimetric analysis confirmed the binding of HSP70 or its fragment HSP70-16 on the surface of the treated cells. In response to the delivery of HSP70 to tumor cells, we observed an increase in the cytolytic activity of different cytotoxic effector immune cells from human peripheral blood. (4) Conclusions: Targeted modification of the tumor cell surface with molecular structures recognized by cytotoxic effectors of the immune system is among new promising approaches to antitumor immunotherapy.

A minimum set of stable blocks for rational design of polypeptide chains.

The aim of this work was to find a minimal set of structurally stable pentapeptides, which allows forming a polypeptide chain of a required 3D structure. To search for factors that ensure structural stability of the pentapeptide, we generated peptide sequences with no more than three functional groups, based on the alanine pentapeptide AAAAA. We analyzed 44,860 structures of peptides by the molecular dynamics method and found that 1,225 pentapeptides over 80% of the simulation time were in a stable conformation. Clustering of these conformations revealed 54 topological types of conformationally stable pentapeptides. These conformations relate to different combined elements of the protein secondary structure. So, we obtained a minimal set of amino acid structures of conformationally stable pentapeptides, creating a complete set of different topologies that ensure the formation of pre-folded conformation of protein structures.

Determination of T and B Cell Epitopes of Aspergillus fumigatus Ribotoxin and Heat Shock Protein.

Ribotoxin Asp f1 and heat shock protein Asp hsp1 from Aspergillus fumigatus represent the major components of A. fumigatus allergic complex. Eight computer predicted peptides corresponding to probable T epitopes of Asp f1 (4 peptides) and Asp hsp1 (4 peptides) have been synthesized according to the primary sequence of the proteins. The peptides Asp f1 15-27, 87-99 and Asp hsp 84-96, able to bind high affinity sera from mice BALB/c immunized with crude A. fumigatus extract, were considered to represent B cell epitopes. To screen T cell epitopes among the peptides splenocytes from mice immunized with crude A. fumigatus extract were stimulated in vitro with A. fumigatus or peptides, and their Ab production was analyzed. Spontaneous A. fumigatus-specific Ab production was found in unstimulated control. In cultures stimulated with 20 &mgr;g/ml of A. fumigatus preparation Ab production was blocked by 92%. The peptide Asp f1 87-99 decreased synthesis of A. fumigatus-specific Abs by 80%. The peptide Asp f1 15-27 and those overlapping hsp1 18-31 and 22-36 induced 34-37% decrease in Ab production. Other A. fumigatus-derived or irrelevant peptides did not affect specific Ab production. Thus, these peptides, containing T cell epitope, may be considered as potential candidates for peptide-based immunotherapy of allergic aspergillosis.