The Solution Strategy For Cetuximab Resistance Mediated By EGFR Ectodomain Mutations In Colorectal Cancer

Colorectal cancer is a common malignant tumor that seriously threatens human health.Especially for metastatic colorectal cancer,the treatment options are limited and the five-year survival rate is less than 20%.The emergence of anti-EGFR monoclonal antibody therapy has brought hope to patients with metastatic colorectal cancer.Cetuximab is a monoclonal antibody drug targeting EGFR.Currently,cetuximab combined with chemotherapy is the first-line regimen for patients with left sided RAS/RAF wild-type metastatic colorectal cancer,and cetuximab combined with RAF kinase inhibitor conefenib is a second-line regimen for patients with BRAF V600E mutant metastatic colorectal cancer.These treatment regimens effectively prolong the survival of patients and improve their quality of life.However,the increasingly frequent problem of drug resistance severely limits the clinical application of cetuximab.In particular,the acquisition of point mutations located in the cetuximab epitope in the EGFR ectodomain completely destroys the binding ability of cetuximab resulting in the loss of the opportunity for cetuximab to be used in combination therapy.To our knowledge,there is a lack of relevant drug guidance and effective resolving strategies.Therefore,it is of significance to study the mechanism of cetuximab resistance mediated by point mutations located in the EGFR extracellular domain and explore strategies to solve drug resistance.Based on these,this paper carries out research in the following three aspects:（1）In the first part of this paper,we constructed a series of NIH/3T3 cell lines that stably expressed wild-type or mutant EGFR（i.e.,S492R,I491M,K489E,K467T,G465R,G465E,S464L,I462R,R451C,S442R,or V441D）.We comprehensively analyzed the effect of different point mutations on cetuximab,including its binding ability to EGFR mutants and its inhibitory ability on the ligand-induced phosphorylation of EGFR mutants.Moreover,we prepared wild-type and mutant EGFR proteins and quantified the affinities of cetuximab to them.Finally,we obtained the structures of EGFR mutants by computer homology modeling and explained the potential molecular mechanism of resistance to cetuximab.These results provide the basis for establishing clinical guidelines for anti-EGFR monoclonal antibody drugs related to the point mutations located in EGFR extracellular domain,and also lay the foundation for designing next generation drugs.（2）In the second part of this paper,we developed a structure-guided and phage-assisted evolution（SGAPAE）approach to evolve cetuximab to overcome resistance mediated by EGFR^S492R or EGFR^G465R,the two most common antibody-resistance point mutations in cetuximab treatment.We applied the SGAPAE approach to calculate the energy difference between the bound and unbound states of the interface using the Rosetta platform to identify potential evolutionary sites,and constructed a semi-rationally designed library of cetuximab variants with a restricted epitope.The focused library enabled us to efficiently discover three cetuximab variants with minimal mutations（Ctx-VY,Ctx-Y104D and Ctx-W52D）that reversed EGFR^S492R or EGFR^G465R-driven resistance to cetuximab in vitro and in vivo,while largely preserving the optimized physical and chemical properties of cetuximab and thus ensuring their druggability.（3）In the third part of this paper,in order to overcome the heterogeneous resistance and improve the tumor selectivity of anti-EGFR therapy,we designed a T-cell binder,Ctx-DD-TCB,which can preferentially bind to EGFR^G465R.Our study showed that T cells treated with Ctx-DD-TCB could selectively kill colorectal cancer cells expressing EGFR^G465R,as well as effectively control KRAS/BRAF mutant colorectal cancer cells.Encouragingly,in the subcutaneous tumor models,Ctx-DD-TCB exhibited superior antitumor efficacy against colorectal cancer cells expressing EGFR^G465R,compared to cells expressing EGFR^WT.