Searching for Vicugna pacos VHHs against cancer stem cell markers CD44 and ERBB2 with the help of ribosome display
Today one of the most promising ways of cancer treatment is immunotherapy. In vitro experiments showed that co-cultivation of cancer cell line RKO with lymphocytes, activated by dendritic cells, which were maturated in presence of the RKO cancer cell protein lysates, lead to significant 98% RKO cells growth inhibition. However, in vivo experiments require methods with better specificity, such as chimeric antigen receptors (CARs) carrying selective antibody structures. CARs endue effector immune cells with significantly elevated specificity towards their molecular targets. Probably one of the most convenient ways of designing such CARs is based on Camelidae single-chain antibodies. Not only are they shorter than usual antibodies – about 500 bp – which ensures a wider range of applications, but also sometimes such antibodies demonstrate higher selectivity to their molecular targets. Ribosome display is a simple and straightforward method for rapid evolution and selection of protein ligands, and in particular, for selection of antibodies. With this technology selecting from libraries with high complexity (about 10^15) becomes possible. This in turn allows analyzing naive libraries, the diversity of which is higher, as well as reduces time and efforts. Specific heavy-chain variable domain (VHH) of Vicugna pacos antibody against CD47 – one of the well-known cancer stem cells (CSCs) markers – was previously found with phage display technology. The upstream region for cell-free coupled transcription-translation and downstream sequence containing either Flag-tag alone, or in conjunction with tRFP-tag, followed by the constant region of immunoglobulin kappa light chain (Ck) with removed stop codon were added to the aforementioned anti-CD47 VHH sequence with OE-PCR. Ribosome display selection cycle based on this construct turned out to be fully functional, as mRNA-ribosome-VHH complexes required for ribosome display were produced. Afterwards, VHH libraries were prepared: naive library from the lymph node of neonate alpaca, and immune library from ERBB2-immunised animals. Several selection cycles were carried out with ribosome display technology using constructs, which were obtained on the bases of such naive and immune libraries in order to find selective antibodies against cancer stem cell markers CD44 and ERBB2 correspondingly.