Biochemical Characterization Of The Engineered Penetrative Nanobody Targeting Her2 Positive Breast Cancer Cells

Breast cancer is one of the most common cancerous diseases diagnosed in women,which pose a serious threat to the women’s health.As an important part of cancer treatment,antibody-based therapeutics against cancer are currently enjoying unprecedented recognition of their potential.However,as the average molecular weight reach around 150 k Da,therapeutics antibody lack the capacity to enter into the cells so that the clinical benefits of therapeutic antibodies have been blocking out of the cell membrane.To improve the cell permeability of therapeutic antibodies for intracellular targeting through optimized structural design,downsizing the antibody molecules and ligand modification would open vast opportunities for both monoclonal anybody drug discovery and cancer therapy.Unfortunately,it still remained as one of the major challenges in the antibody engineering practice thus far.Her2 and Kras are two oncoproteins driving the tumorigenesis and progression of breast cancer.HER2 is a cell membrane receptor mediating the growth signaling of the breast cancer cells.HER2 Targeting-monoclonal antibodies such as Herceptin,Lapatinib,Pertuzumab,etc.has contributed immensely in breast cancer therapy.Kras mutation has also been detected as a foundmental driving factor in a variety of breast tumors.Currently,despite comprehensive efforts,an effective anti-Kras drug has yet to be developed for clinic trial.Trim-away strategy,which aims to the degradation of targeted proteins mediated by therapeutic antibody,shied lights on the mutated Kras targeted cancer therapy.In this study,we proposes an antibody engineering strategy for the breast cancer targeting therapy.We construct therapeutic bispecific antibodies(Bs Ab)coupled with cell penetrating peptides(CPPs).Structurally,this fusion protein consists of the three functional modules: nanobody peptides against Her2(named NB2),cell penetrating peptides(CPPs)and nanobody peptides against mutated Kras protein.The engineered bispecific antibody is designed to recognize the Her2 positive cancer cells through the binding of Her2 extracellular region with NB2 and target intracellular mutated Kras protein for degradation via trim-away pathway.The core part of this fusion protein is the appropriate cell penetrating peptides which were introduced for the intracellular delivery of the engineered bispecific antibodies(Bs Ab).As the schematic working flow,the selective cancer cell recognition and targeted intervention of breast tumor cells are realized through the synergistic action of these three functional parts.Based on the above designing,we constructed multiple recombinant NB2 nanobody proteins fusion with different type of the cell penetrating peptides(NB2-c PPs).We purified recombinant Kras mutant protein for generating nanobody and four NB2-c PPs,including GST-NB2-BP100、GST-NB2-PEVC、GST-NB2-Penetrating、GST-NB2-R9TAT2 from E.coli.respectively.We thereafter performed biochemical and cellular characterization of the NB2-CPPs.We first demonstrated that four recombinant NB2-CPPs could recognize extracellular Her2 region cells by flow cytometry analysis.Secondly,through immunofluorescence staining and confocal imaging analysis,we confirmed the cell-penetrating peptide(CPPs)enable the recombinant NB2-CPPs to internalize into cells.Further,we evaluated the internalization efficiency of these four recombinant NB2-c PPs in HBL100(low Her2 expression)and SKBR3(high Her2 expression)cell lines respectively.Different internalization efficiency of the each NB2-CPPs within HBL100 cells or SKBR3 cells were revealed by one way anova analysis together with multiple comparison.Comparative evaluation were conducted on the internalization efficiency between the HBL100 and SKBR3 cells for each individual NB2-CPP.The two-way ANOVA statistic analysis revealed that,among all NB2-CPPs,only GST-NB2-PEVC showed significantly increased internalization efficiency in SKBR3 cells compared with that in HBL100 cells.Concerning the significant difference abundance of Her2 between HBL100 and SKBR3 cells,the data suggested that the internalization efficiency of GST-NB2-PEVC might correlated positively with the endogenous Her2 level of the host cells.Moreover,this result also indicated the PEVC could be suitable for the construction of our proposed therapeutic bispecific antibody.Taken together,our study identified the important NB2-PEVC fusion peptides which contribute the major part of our proposed therapeutic bispecific antibody.