Development of blocking streptabody specific to CD47 receptor on cancer cells
Development of high affinity molecules possessing the ability to block or modify the functional activity of receptors on the surface of cancer cells might have great application in perspective personalized therapy of tumor diseases. It is known that most types of cancer relapse is due to resistance to chemotherapy and high proliferative activity of so-called cancer stem cell (CSC) population. This resistance is ensured by overexpression of a number of receptors on the surface of cancer cells (CD47, CD44, CD133, etc.). CD47 protein is expressed at high level on the surface of many cancer cells. CD47 interacts with SIRPa receptor which is present on macrophages resulting in phagocytosis inhibition. Thus cancer cells can escape their recognition by the host immune system. CD47-SIRPa interaction is considered as a promising target for cancer therapy. Howewer, antibodies with properties suitable for clinical application are still under development. VHH antibodies and their various multivalent forms might be promising therapeutic agents which may be represented by a new high affinity anti-CD47 VHH-antibody which has a better blocking action, smaller size and minimal toxicity. VHH-antibodies have several advantages over single-chain antibodies derived from conventional molecules of IgG such as simplicity of genetic manipulation, high expression in different systems, good solubility, high stability in a wide range of conditions and better tissue penetration due to smaller size (15-30 kDa). Antibody aCD47cl19VHH was obtained in our lab by phage display technology and leads to apoptosis of lymphoma cell line U937. Now our aim is to increase the functional afﬁnity (avidity) of VHH fragments by tetramerization on streptavidin, following their site-speciﬁc biotinylation by the enzyme BirA. Expression vector have been constructed that enable addition of the 15 amino acid biotin acceptor domain (BAD) on aCD47cl19VHH. Different domains were cloned at the C-terminus of VHH in the following order: a semi-rigid hinge region (of 16 residues), the BAD, and a histidine tail. Monomeric form of aCD47cl19VHH-BAD were ﬁrst synthesized in Escherichia coli carrying the plasmid encoding the BirA enzyme, and then puriﬁed from the cytoplasmic extracts by Ni–NTA afﬁnity chromatography. aCD47cl19VHH-BAD has KD about 3 nM which is lower than for B6H12.2 monoclonal antibody. It will expected that streptabody form might have higher specificity and ﬂexibility allowing a better orientation of its multiple arms and the binding to several CD47 molecules expressed on the surface of cancer cells and CSC.