Screening The Novel Nanobodies Targeting EGFR Domain ? And A Comparative Study Of Their Recombinant Expression Systems

Purpose:Through analysis of the relationship between the epitopes of anti-EGFR monoclonal antibodies/nanobodies and the internalization and downregulationof EGFR,EGFR domain III is determined as a key epitope.Compared with mono nanobodies,bispecific nanobodies are more effective in promoting EGFR internalization and conjugating with imaging probes and small molecular cytotoxic drugs.Hence,bispecific nanobodies are more valueble to be investigated as tumor binding moities.To investigate bispecific nanobodies with non-overlapping epitopes,the epitopes were analysed.The pair of the antibody Cetuximab and H11 can downregulate EGFR most effectively.Meanwhile,nanobodies can overcome the resistance caused by antibody-based therapy.Hence,the study aimed to screen the nanobodies which can block Cetuximab and H11 simultaneously.Method:The screening of novel nanobides is contained four main steps,which are the recombinant expression of the antigen,the construction of phage immune libraries,the prediction of epitope by molecular docking and the recombinant expression of nanobodies.The first part of the study is the production of EGFR domain III by two strategies.The affinity of H11 and the recombinant EGFR domain III will be measured to determine if the recombinant EGFR domain III can be used as the immunogen.The second part of the study is the construction and screening of the phage display libraries.By the immunization of a Bactrian camel and the construction of the immunization libraries,the nanobodies' fragments are transformed to the phage display vectors.Two phage display libraries were constructed successfully.After phage bio-panning,affinity ranking and epitope binning,the novel nanobody targeted to a specific epitope on EGFR domain III will be screened.Through the homology modeling and molecular docking of AS32611 and EGFR domain III,the epitope and paratope of AS32611 will be determined.The third part of the study is the influence of the recombinant systems on nanobodies.E.coli and Pichia pastoris are used to express two anti-EGFR nanobodies.The comparative study and evaluation of nanobodies from two different expression systems will be via LC-MS,affinity measurement and fluorescence imaging.The fourth part of the study is the recombinant expression of the screened novel nanobody.By being conjugated to fluorescent molecules and small molecular drugs,the novel nanobody will be assessed for its tumor-targeting activity.Results:A novel nanobody targeting EGFR domain III was obtained.In the first part of the study,EGFR domain III was obtained with high affinity for H11 via both two strategies.The better strategy was chosen for final production.2mg EGFR domain III was obtained as the immunogen.In the second part of the study,the camel had a strong humoral immune response after seven immunogen boosts and the serum with enough titer was obtained.Two phage display libraries were constructed with the qualified size and diversity.After bio-panning and affinity ranking,two screened nanobodies can block H11 and Cetuximab from the two constructed libraries.The sequences of two nanobides were compared and only the fifth amino acid showed difference.Thus,they are considered as the same nanobody AS32611.The homology model of AS32611 was built.The possible epitopes of AS32611 on EGFR domain III are described as follow: RTK,G,Q,DGD,TSGQK(405-407,410,411,434-436,459-463).The paratope region of AS32611 was mainly on the surface of CDR2.In the third part of the study,Pichia pastoris had 6 to 13.6-fold higher yield than E.coli.No glycosylation sites were identified during expression in Pichia pastoris.In the fourth part of the study,the novel nanobody AS32611 was obtained through expression in Pichia pastoris.A fluorescent molecule and two microtubule inhibitor(MMAE and DM1)were conjugated to AS32611.The experiments of fluorescent imaging in vitro proved the tumor-targeting activity of AS32611.Conclusion:A novel nanobody AS32611 was obtained by immunogen production and several rounds of screening with a distinct epitope on EGFR domain III.The eptiope was partly overlapped with nanobody 9G8 and matuzumab,indicating that it blocked EGFR downstream signal by preventing dimerization of the receptors.Experiments in vitro proved that AS32611 was able to enter into cells and carry small molecules as a tumor-targeting cargo.