Microtubule-associated protein tau facilitates the targeted killing of proliferating cancer cells in vitro and in a xenograft mouse tumour model in vivo

Abstract

Background:Antibody drug conjugates (ADCs) and immunotoxins (ITs) are promising anticancer immunotherapeutics. Despite their encouraging performance in clinical trials, both ADCs and ITs often suffer from disadvantages such as stoichiometrically undefined chemical linkage of the cytotoxic payload (ADCs) and the potential immunogenicity of toxins derived from bacteria and plants (ITs).Methods:Human microtubule-associated protein tau (MAP) was cloned in-frame with human EGF, expressed in E. coli and purified by standard chromatographic methods. The in vitro activity was confirmed by flow cytometry, cell viability assays and tubulin polymerisation assay. The in vivo efficacy was demonstrated using noninvasive far-red in vivo imaging.Results:The EGF-MAP selectively induced apoptosis in EGFR-overexpressing proliferating cancer cells through stabilisation of microtubules. Nonproliferating cells were not affected, demonstrating superior selectivity of EGF-MAP for cancer cells. The EGF-MAP was well tolerated at high doses in mice compared with the ETA'-based control. The in vivo efficacy of EGF-MAP was demonstrated in a tumour xenograft mouse model.Conclusion:Our data indicate the general mechanism of action for a new class of human immunotherapeutic reagents suitable for the treatment of cancer. This approach combines the binding specificity of targeting ligands with the selective cytotoxicity of MAP towards proliferating cells.

Human microtubule‐associated protein tau mediates targeted killing of CD30+ lymphoma cells in vitro and inhibits tumour growth in vivo

“…The advantage of this new CFP, Ki4(scFv)-MAP, is that in addition to the specificity of the target-specific binding component Ki4(scFv), the genetically fused pro-apoptotic effector protein MAP selectively affects proliferating cells only, as reported previously (Hristodorov et al, 2013). This is especially interesting in the light of the fact that most tumour markers are also present on physiologically normal cells and superior selectivity for proliferating cells may thus reduce undesired off-target effects.…”

“…Recently, we have reported on the ability of the microtubule-binding protein tau (MAPT) to specifically kill proliferating cancer cells (Hristodorov et al, 2013). Here, we present a new CFP consisting of the CD30-specific Ki-4(scFv) genetically fused to human MAPT.…”

“…The open reading frame (ORF) for MAPT [Gene ID: 4137; modified as described previously (Hristodorov et al, 2013)] was modified with 5′-NotI and 3′-BlpI restriction sites by polymerase chain reaction (PCR) followed by ligation into a NotI/BlpI-linearized pMT vector already containing the Ki-4 (scFv) sequence. Successful cloning was confirmed by test digestion and sequencing.…”

“…Successful cloning was confirmed by test digestion and sequencing. Expression, purification and protein analysis was carried out as described previously (Hristodorov et al, 2013). L-glutamine, 50 lg/ml penicillin, and 100 lg/ml streptomycin at 37°C, 100% humidity and 5% CO 2 .…”

“…We previously reported the identification of a new cytolytic effector protein, called MAPT, which proved effective in specifically killing proliferating cancer cells, when fused to epidermal growth factor (Hristodorov et al, 2013). Here, the complete ORF of MAPT was cloned in frame with a 5′-ORF encoding the CD30-specific Ki4(scFv) into a bacterial expression vector.…”