br Materials and methods br Results br Discussion Anti EGFR
Materials and methods
Discussion Anti-EGFR therapy for epithelial carcinomas has important clinical significance, but widespread intrinsic or acquired resistance seriously affects efficacy. Mutation and heterodimerization of EGFR are the two main causes of resistance to anti-EGFR therapies [14,22], so targeting the highly conserved dimer interface of EGFR may solve the problem of hereditary and acquired resistance. Our previous work has shown that peptide vaccines and a monoclonal antibody (EGFR dimer mAb 5G9) prepared with the EGFR237−267 antigen peptide from the β-ring of the dimer interface of EGFR can effectively inhibit the growth of EGFR-overexpressing tumour cells [15,16]; however, BAY 41-8543 mg with a size smaller than that of monoclonal antibodies may be more attractive due to their good tissue penetration and low cost. Nanobodies have advantages over traditional monoclonal antibodies in terms of drug cost and tissue penetration and over other small antibodies, such as single chain antibodies and fab fragment antibodies, in terms of solubility and stability [23,24]. However, whether small antibodies, such as nanobodies, can effectively block the dimerization of EGFR needs to be explored. Therefore, nanobodies targeting the dimer interface of EGFR were screened from a synthesized humanized nanobody phage display library with the EGFR237−267 antigen peptide. Four nanobodies were screened, among which EGFR dimer Nb77 possessed the longest CDR3. A longer CDR3 is characteristic of nanobodies and highly correlated with specificity and affinity . Therefore, EGFR dimer Nb77 was selected for further analysis. The results from the specific binding analysis demonstrated that EGFR dimer Nb77 could bind to the EGFRs on the surface of A431 cells in a specific and EGF-dependent manner. When there is no ligand binding, EGFR is in an inactive state. The β-ring (dimer arm) located in extracellular domain II is bound by extracellular domain IV, and only ligand binding can cause its exposure [25,26]. Therefore, EGFR dimer Nb77 targeting of the dimerization arm must be EGF dependent. Exposure of the dimer interface, on the other hand, would immediately cause homo- or heterodimerization or oligomerization; therefore, the exposure of dimer interface after stimulation by a ligand is a transient [27,28], which means that the binding of antibodies targeting the dimer interface to EGFRs on the surface of cells is random and will not reach a saturation state as observed for other antibodies targeting the molecular surface of EGFR. This “unsaturation” may lead to an “accumulation effect” during the binding of such antibodies to EGFR on the surface of cells, causing the number of antibodies bound to vary greatly among different cells in the same cell cluster at the same point, which may also be the main reason for the widening of the flow cytometry signal peak in the high-dose antibody group mentioned above. Our previous work demonstrated that EGFR dimer mAb 5G9 can effectively inhibit the growth of a variety of EGFR-overexpressing tumour cells, and its inhibition rate in vitro is slightly higher than that of cetuximab . EGFR dimer Nb77 could also inhibit the growth of EGFR-overexpressing A431 tumour cells as effectively as EGFR dimer mAb 5G9, but whether the inhibitory effect of such antibodies on tumour cells in vivo has a cumulative effect similar to the binding reaction described above needs to be further studied. Furthermore, the value of such a small antibody for therapeutic purposes needs to be further evaluated. These studies should investigate the ability of such antibodies to inhibit the heterodimerization and phosphorylation of EGFR, to regulate the number of EGFR molecules on the surface, to block signalling, and to inhibit the growth of tumour cells with different EGFR phenotypes in vitro and in vivo. The Escherichia coli expression system has excellent industrial performance and good application prospects in the production of recombinant protein, but the over-expression products of a foreign protein often form inactive inclusion body proteins, which is not conducive to the efficient production of nanobodies . To obtain active expression products, the optimized gene of EGFR dimer Nb77 under the control of promoter T7 was cloned into the host cells E. coli Shuffle T7-B, which are more favourable for soluble expression; EGFR dimer Nb77 was expressed as an insoluble inclusion body protein at 37 °C but as a highly soluble protein at 30 °C. Even though the target gene was optimized according to the codons of E. coli and the strong T7 promoter was used, the expression level of the nanobody in E. coli Shuffle T7-B was far lower than that in E. coli 21B (DE3); however, the expression product in the latter cells formed an inclusion body under any temperature conditions (result not shown). The expression level was also much lower than that of other nanobodies reported in the literature . Obviously, this is an inherent contradiction between the solubility and efficiency of the E. coli expression system. Therefore, for the purpose of drug production, it is necessary to use a yeast expression system with high secretion-expression capacity, such as Pichia pastoris, to establish the production process.