| Autor: |
Eliseev IE; Laboratory of Renewable Energy Sources, Alferov University, St. Petersburg 194021, Russia.; Center for Personalized Medicine, FSBSI Institute of Experimental Medicine, St. Petersburg 197376, Russia.; Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, Moscow 117997, Russia., Ukrainskaya VM; Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, Moscow 117997, Russia., Yudenko AN; Research Center for Molecular Mechanisms of Aging and Age-Related Diseases, Moscow Institute of Physics and Technology, Dolgoprudny 141700, Russia., Mikushina AD; Laboratory of Renewable Energy Sources, Alferov University, St. Petersburg 194021, Russia., Shmakov SV; Laboratory of Renewable Energy Sources, Alferov University, St. Petersburg 194021, Russia., Afremova AI; CJSC Biocad, St. Petersburg 198515, Russia., Ekimova VM; CJSC Biocad, St. Petersburg 198515, Russia., Vronskaia AA; Laboratory of Renewable Energy Sources, Alferov University, St. Petersburg 194021, Russia., Knyazev NA; Saint-Petersburg Clinical Scientific and Practical Center for Specialized Types of Medical Care (Oncological), St. Petersburg 197758, Russia., Shamova OV; Center for Personalized Medicine, FSBSI Institute of Experimental Medicine, St. Petersburg 197376, Russia. |
| Abstrakt: |
The human ErbB3 receptor confers resistance to the pharmacological inhibition of EGFR and HER2 receptor tyrosine kinases in cancer, which makes it an important therapeutic target. Several anti-ErbB3 monoclonal antibodies that are currently being developed are all classical immunoglobulins. We took a different approach and discovered a group of novel heavy-chain antibodies targeting the extracellular domain of ErbB3 via a phage display of an antibody library from immunized llamas. We first produced three selected single-domain antibodies, named BCD090-P1, BCD090-M2, and BCD090-M456, in E. coli , as SUMO fusions that yielded up to 180 mg of recombinant protein per liter of culture. Then, we studied folding, aggregation, and disulfide bond formation, and showed their ultimate stability with half-denaturation of the strongest candidate, BCD090-P1, occurring in 8 M of urea. In surface plasmon resonance experiments, two most potent antibodies, BCD090-P1 and BCD090-M2, bound the extracellular domain of ErbB3 with 1.6 nM and 15 nM affinities for the monovalent interaction, respectively. The receptor binding was demonstrated by immunofluorescent confocal microscopy on four different ErbB3 + cancer cell lines. We observed that BCD090-P1 and BCD090-M2 bind noncompetitively to two distinct epitopes on the receptor. Both antibodies inhibited the ErbB3-driven proliferation of MCF-7 breast adenocarcinoma cells and HER2-overexpressing SK-BR-3 cells, with the EC 50 in the range of 0.1-25 μg/mL. BCD090-M2 directly blocks ligand binding, whereas BCD090-P1 does not compete with the ligand and presumably acts through a distinct allosteric mechanism. We anticipate that these llama antibodies can be used to engineer new biparatopic anti-ErbB3 or bispecific anti-ErbB2/3 antibodies. |
