Many acquired EGFR mutations block cetuximab and/or panitumumab binding and confer drug resistance
Current research have proven that a lot of EGFR ECD mutations could also be acquired in sufferers with metastatic colorectal most cancers (mCRC) after cetuximab and/or panitumumab therapy. These EGFR ECD mutations occurred in EGFR area III (amino acids 334–504) which is the binding epitope for cetuximab and panitumumab (Fig. 1a), mediating acquired resistance to cetuximab and/or panitumumab by way of attenuating or abolishing antibody binding20,21,22,23,24,25,26.
To higher examine the impact of those mutations on cetuximab and/or panitumumab, we evaluated the binding of cetuximab and panitumumab (1, 10, and 100 nmol/L) with NIH3T3 cells expressing wild-type EGFR or mutant EGFR (Supplementary Fig. 1) by movement cytometry. A number of launched single mutations practically abolished anti-EGFR antibody binding to EGFR—e.g., S492R, G465R, S464L or V441D mutation considerably diminished the cetuximab binding affinity, and G465R, G465E, S464L or S442R mutation abolished panitumumab binding to EGFR (Fig. 1b, prime). Among the many mutations in mCRC sufferers with a historical past of cetuximab or panitumumab therapy, S492R and G465R seemed to be the 2 most typical level mutations, current in ~20% of sufferers with scientific resistance20,21. A retrospective evaluation of plasma samples from a part III trial (ASPECCT) in numerous colorectal most cancers sufferers revealed that the EGFR S492R mutation developed in 16% of sufferers within the cetuximab arm (Fig. 1b, backside)20.
Excessive-resolution EGFR/cetuximab buildings present a big, flat binding interface primarily comprising CDRL3, CDRH3, and CDRH2 of cetuximab28. Each the S492R and G465R mutations are situated within the cetuximab binding web site (epitope) on EGFR. We carried out homology modeling to foretell the impact of the S492R and G465R mutations on cetuximab binding to EGFR (Fig. 1c). The crystal construction of the wild-type EGFR/Fab complicated for cetuximab (PDB code: 1YY9) revealed that the CDR of cetuximab fashioned a central cavity. The S492 and G465 residues of EGFR are accommodated into the above central cavity29. In line with our structural modeling outcomes, the mutated arginine (R492 and R465) with a big aspect chain (guanidino group), nevertheless, couldn’t match into the small central cavity within the cetuximab CDR, thus leading to steric clashes between cetuximab and EGFRS492R or EGFRG465R.
Utility of the SGAPAE strategy effectively recognized cetuximab variants to reverse the resistance conferred by EGFRS492R or EGFRG465R
We used the SGAPAE strategy to direct the evolution of cetuximab to reverse EGFRS492R– or EGFRG465R-mediated resistance (Fig. 2). Based mostly on the crystal construction of wild-type EGFR certain to cetuximab Fab (PDB code: 1YY9), the Rosetta platform30 decided essential interface residues which can be important for re-establishing the EGFRS492R/cetuximab or EGFRG465R/cetuximab interplay by way of an exhaustive scan of residues on the interface. On this approach, we constructed an epitope-focused cetuximab mutant library to enhance the effectivity of subsequent phage show.
To facilitate the development of the targeted phage-display library, we used the RosettaScripts protocol31,32 and the InterfaceAnalyzer33 module in Rosetta30, software program designed for protein–protein interplay (PPI) interface evaluation, to slender down the record of residues essential for restoring cetuximab binding with the EGFR mutants. Among the many a number of indicators (e.g., dG_separated, packstat), dG_separated, which represents the vitality distinction between protein interface separation and binding, was chosen to guage the impression of mutations on cetuximab affinity. EGFR S492R or G465R mutation considerably decreased the binding affinity of cetuximab to EGFR, ensuing from an elevated free vitality of binding. The expected ∆∆G values for 100 fashions of EGFRS492R/cetuximab and EGFRG465R/cetuximab complexes have been in keeping with the scientific knowledge (∆∆G > 0) (Supplementary Desk 1), which justified the applying of the RosettaScripts protocol and InterfaceAnalyzer technique for predicting adjustments in binding vitality accompanying EGFR mutations (Fig. 3a). We additional selected the InterfaceAnalyzer technique to carry out subsequent mutation scanning for the interface residues of cetuximab as a result of it’s simple to make use of and has excessive calculation effectivity.
A complete of 2014 and 1957 predictions have been performed for the EGFRS492R/cetuximab (Cluster 1:684, Cluster 2:665, Cluster 3:665) and EGFRG465R/cetuximab (Cluster 1:684, Cluster 2:627, Cluster 3:646) complexes, respectively (Fig. 3b). In line with the adjustments in binding vitality (∆∆G) within the three clusters (Supplementary Tables 2–7; Supplementary Information 1–6), the intersection of the three clusters, indicating essential residues for affinity restoration, was obtained. Mild chain residues (N32, W94 and T96) and heavy chain residues (V50, D58 and Y014) of cetuximab have been recognized as potential “replaceable” residues for directed evolution of cetuximab towards binding with EGFR S492R. The heavy chain residues V50 and W52 of cetuximab have been recognized as mutation web site candidates to reverse cetuximab resistance conferred by the acquired EGFR G465R mutation. Subsequently, we chosen the above potent residues within the receptor binding web site of cetuximab for additional development of the phage-display mutagenesis library (Fig. 3c).
We utilized a phage-display-based coselection technique34,35 to display screen for cetuximab scFv mutants that may bind EGFRG465R or EGFRS492R whereas sustaining excessive affinity for wild-type EGFR (Fig. 4a). After three rounds of panning, two cetuximab scFv variants focusing on EGFRS492R have been recognized from the S492R library and named Ctx-VY-scFv (V50Q, CDRH2; Y104V, CDRH3) and Ctx-Y104D-scFv (Y104D, CDRH3) (Fig. 4b). One other cetuximab variant focusing on EGFRG465R was chosen from the G465R library and named Ctx-W52D-scFv (W52D, CDRH3) (Fig. 4b). For additional characterization, Ctx-VY-scFv, Ctx-Y104D-scFv and Ctx-W52D-scFv have been reworked into human IgG1/κ format (Ctx-VY, Ctx-Y104D and Ctx-W52D) and have been then transiently expressed in HEK293F cells. The heavy and light-weight chains of every cetuximab variant appeared at roughly 55 and 30 kDa, respectively, beneath lowering situations, and the intact cetuximab variants appeared at approximately150 kDa (Supplementary Fig. 2a). The SDS–PAGE outcomes, along with the size-exclusion chromatography (SEC) outcomes and dynamic mild scattering (DLS) outcomes, indicated that the launched mutations didn’t have an effect on the soundness or polymerization state of the parental antibody (Supplementary Fig. 2a–c). On this foundation, we additional mixed the W52D mutation with the V50Q/Y104V or Y104D mutations to generate cetuximab variants Ctx-VWY (V50Q and W52D, CDRH2; Y104V, CDRH3) and Ctx-DD (W52D, CDRH2; Y104D, CDRH3) so as to cowl these most typical EGFR mutations (Fig. 4b and Supplementary Fig. 3a).
Cetuximab variants restored binding to EGFRG465R or EGFRS492R
The binding of Ctx-VY, Ctx-Y104D, Ctx-W52D, Ctx-VWY and Ctx-DD to wild-type EGFR or EGFR mutants was analyzed by movement cytometry and ELISA. Panitumumab, wild-type and mutant EGFR-ECD-Fc, and cetuximab variants have been efficiently produced in HEK293F cells and demonstrated to be intact and accurately assembled in response to the SDS–PAGE outcomes beneath lowering and nonreducing situations (Supplementary Fig. 3b, c). As proven in Fig. 4c, cetuximab, panitumumab, Ctx-VY and Ctx-W52D confirmed an analogous binding affinity with wild-type EGFR-expressing steady NIH3T3 (WT-EGFR-NIH3T3) cells and WT-EGFR-ECD-Fc. The introduction of Y104D barely weakened the binding capability of cetuximab with both wild-type EGFR-expressing steady NIH3T3 cells or WT-EGFR-ECD-Fc compared with that of the opposite developed mutants Ctx-VY and Ctx-W52D. Moreover, when two mutations, Y104D and W52D (Ctx-DD), or three mutations, V50Q, W52D and Y104V (Ctx-VWY), have been launched on the similar time, the binding affinity of the mutants, particularly Ctx-DD, for WT-EGFR-NIH3T3 and WT-EGFR-ECD-Fc was significantly diminished. As anticipated, cetuximab didn’t exhibit binding with EGFRS492R-expressing steady NIH3T3 cells (S492R-EGFR-NIH3T3), whereas Ctx-VY, Ctx-Y104D, Ctx-DD and Ctx-VWY exhibited efficiently restoration of a robust interplay with S492R-EGFR-NIH3T3 cells just like that of panitumumab, an anti-EGFR antibody with a binding web site totally different from that of cetuximab. Intriguingly, Y104D resulted in a better Emax with EGFRS492R-expressing steady NIH3T3 cells and S492R-EGFR-ECD-Fc than panitumumab, Ctx-VY, Ctx-DD and Ctx-VWY. Amongst all of the cetuximab variants, solely Ctx-W52D exhibited restoration of sturdy binding to EGFR-expressing steady cells and for EGFR-ECD-Fc with the G465R mutation, which abolished the binding of each cetuximab and panitumumab. As well as, we examined the binding affinity of Ctx-W52D within the EGFRG465E mannequin. Nevertheless, Ctx-W52D couldn’t reverse G465E-driven resistance to cetuximab (Supplementary Fig. 4a).
VH-Y104 of cetuximab is a essential residue for the EGFR S492R mutation
The Y104 residue within the cetuximab VH area (VH-Y104) is alteredin each Ctx-VY and Ctx-Y104D. Therefore, we carried out web site saturation mutagenesis for VH-Y104 and Ctx-Y104X (X = A, C, D, E, F, G, H, I, Ok, L, M, N, P, Q, R, S, T, V, W, Y) to completely examine the impression of this residue on the binding affinity of cetuximab for EGFRS492R (Supplementary Fig. 3d). As proven in Supplementary Fig. 4b, Ctx-Y104D, Ctx-Y104C and Ctx-Y104N are the highest three high-affinity binders to EGFRS492R for each steady cells and S492R-EGFR-ECD-Fc, which means that the dimensions of the residue aspect chain will not be the foremost mechanism for restoring EGFRS492R binding. As well as, though Ctx-VY confirmed favorable binding with EGFRWT and EGFRS492R, the binding of Ctx-Y104V to EGFRS492R was weaker than that of the Ctx-Y104D/C/N mutants (Supplementary Fig. 4b). Subsequently, we additional explored the impression of the V50Q mutation (situated in CDRH3) on Ctx-VY binding affinity (Supplementary Fig. 4c). Cetuximab with both Y104V or V50Q confirmed a decreased affinity for EGFRS492R, indicating that each mutations are important for cetuximab to realize high-affinity binding with EGFRWT and EGFRS492R.
Cetuximab variants restored high-affinity binding to EGFRMut whereas exhibiting binding kinetics just like these of cetuximab within the interplay with wild-type EGFR
The binding kinetics of cetuximab and the cetuximab variants with WT-EGFR-ECD-Fc and Mut- EGFR-ECD-Fc have been quantitively decided by SPR evaluation (Fig. 4d and Supplementary Desk 8). Cetuximab, Ctx-VY, Ctx-Y104D and Ctx-W52D exhibited related high-affinity binding to WT-EGFR-ECD-Fc, with OkD values of 0.40 ± 0.28, 2.25 ± 1.57, 3.76 ± 2.42, and 0.79 ± 0.17 nM, respectively (Fig. 4e). The cetuximab variants had a comparatively low affiliation price (imply worth: Ctx-VY: kon = 5.77 × 104 Ms−1, koff = 1.29 × 10−4 s−1; Ctx-Y104D: kon = 8.20 × 104 Ms−1, koff = 3.07 × 10−4 s−1; Ctx-W52D: kon = 3.54 × 105 Ms−1, koff = 8.78 × 10−5 s−1) in comparison with that of cetuximab (imply worth: kon=1.68×105 Ms−1, koff = 7.29 × 10−5 s−1), which accounted for the unfinished restoration of binding affinity for EGFRWT. The interplay of cetuximab with EGFRS492R or EGFRG465R was not detectable by SPR; in distinction, Ctx-VY, Ctx-Y104D, and Ctx-W52D, exhibited main restoration of binding with EGFRS492R (OkD = 2.29 ± 1.20 nM; OkD = 2.00 ± 1.42 nM) and EGFRG465R (OkD = 4.56 ± 1.13 nM) with respect to the cetuximab/EGFRWT interplay (Fig. 4e). The deleterious impression of the S492R and G465R mutations on cetuximab binding affinity was additionally confirmed by SPR.
Potential structural foundation of the binding mechanism of cetuximab variants to S492R- or G465R-mutated EGFR
To discover the potential structural foundation underlying the binding of the cetuximab variants to EGFRS492R and EGFRG465R, we simulated the construction of the cetuximab Fab variants certain to area III of EGFR with the S492R or G465R substitution through the protein construction homology-modelling server SWISS-MODEL (Fig. 5a). Due to the minimal mutations in cetuximab CDR, the general buildings of the EGFRS492R/cetuximab Fab variant complicated and EGFRG465R/cetuximab Fab variant complicated have been extraordinarily just like that of EGFRWT in complicated with cetuximab Fab. The restoration of the binding affinity of the cetuximab variants may very well be attributed to the numerous discount in steric hindrance through the V50Q/Y104V mutation (Ctx-VY) or Y104D mutation (Ctx-Y104D) and doable sturdy cost accumulation by way of the Asp-Arg (D-R) salt bridge (Ctx-Y104D versus EGFRS492R) (Fig. 5a). Relating to the prevalence of Ctx-W52D in recognizing EGFRG465R, the Asp-Arg (D-R) salt bridge-mediated reconnection and discount in steric hindrance most likely contributed concurrently to restoration of the binding capability, because the giant indolyl aspect chain of tryptophan (W) was downsized to the carboxyl group of aspartate (D). We additionally in contrast the paratopes of cetuximab and the cetuximab variants in floor mode (Fig. 5b). The scale of the central cavity surrounded by CDRL3, CDRH2 and CDRH3 is totally different between cetuximab and the cetuximab variants. The bigger central cavity of the cetuximab variants might accommodate the aspect chain of mutated R492 or R465, which can clarify the molecular mechanism underlying the restoration of cetuximab binding to EGFRS492R and EGFRG465R (Fig. 5b).
The developed cetuximab variants efficiently inhibited EGF-induced EGFRS492R and EGFRG465R activation
The first mechanism of motion of cetuximab is the blockade of ligand-induced EGFR activation and downstream signaling, together with signaling by way of the PI3K/Akt and MAPK/Erk pathways which have been linked to many processes essential to tumor development, together with metastasis and cell survival, proliferation, adhesion, differentiation, migration, transformation, and motility36. To guage the inhibitory actions of the cetuximab variants, we established steady NIH3T3 cells expressing full-length EGFRWT or EGFRMut as an in vitro cell mannequin. Strong phosphorylation of EGFR and activation of Akt and Erk within the downstream pathway have been induced in steady WT-EGFR-NIH3T3, S492R-EGFR-NIH3T3 and G465R-EGFR-NIH3T3 cells upon EGF stimulation, in response to the western blot outcomes (Fig. 5c). Pretreatment with cetuximab, panitumumab, Ctx-VY, Ctx-Y104D, or Ctx-W52D diminished the extent of phosphorylated EGFR (pEGFR) to basal ranges and decreased the degrees of phosphorylated Akt and Erk (pAkt and pErk) in WT-EGFR-NIH3T3 cells (Fig. 5c, left). Within the S492R-EGFR-NIH3T3 cells, panitumumab, Ctx-VY and Ctx-Y104D however not cetuximab abolished EGF-induced EGFR phosphorylation and downstream signaling (Fig. 5c, center), which was in keeping with the binding affinity outcomes (Fig. 4c). In G465R-EGFR-NIH3T3 cells, Ctx-W52D considerably diminished EGF-stimulated EGFRG465R activation, though cetuximab and panitumumab didn’t, in response to the western blot outcomes for downstream pathway indicators, similar to pEGFR, pAkt and pErk (Fig. 5c, proper). Owing to the unfinished restoration of the binding affinity of the cetuximab variants for EGFRWT, the Erk attenuation induced by the cetuximab variants was nonetheless restricted, notably in WT-EGFR-NIH3T3 cells.
Induction of EGFR internalization and degradation, which ends up in diminished EGFR downstream signaling, is one other mechanism of cetuximab37. HEK293T cell strains stably expressing varied full-length EGFR-eGFP fusion proteins have been efficiently generated and named WT-EGFR-eGFP-HEK293T, S492R-EGFR-eGFP-HEK293T and G465R-EGFR-eGFP-HEK293T (Supplementary Fig. 5a, b). We used movement cytometry to measure the EGFR-eGFP sign in HEK293T cells after 48 h of antibody publicity (Fig. 5d). In WT-EGFR-eGFP-HEK293T cells, cetuximab, panitumumab, Ctx-VY, Ctx-Y104D and Ctx-W52D diminished the eGFP sign (EGFRWT-eGFP) in a concentration-dependent method, with EC50 values of 6.2, 6.3, 9.7, 8.3 and eight.1 nM, respectively (Fig. 5d, left). In S492R-EGFR-eGFP-HEK293T cells, cetuximab confirmed barely any efficacy in reducing the eGFP sign (EGFRS492R-eGFP), whereas panitumumab, Ctx-VY and Ctx-Y104D induced the degradation of S492R-mutated EGFR with EC50 values of 10.7, 4.0, and 11.8 nM, respectively (Fig. 5d, center). Ctx-W52D considerably diminished the eGFP sign (EGFRG465R-eGFP), with an EC50 of 15 nM in G465R-EGFR-eGFP-HEK293T cells; Nevertheless, cetuximab and panitumumab didn’t (Fig. 5d, proper).
Cetuximab variants with unimpaired effector actions potently inhibited tumor cell proliferation
Cell proliferation inhibition and immune effector exercise are essential mechanisms of motion for antitumor therapeutic antibodies (Fig. 6a). We additional evaluated the in vitro efficacy of cetuximab and the cetuximab variants in opposition to the wild-type EGFR cell line SW48 (WT-EGFR)38, SW48 cells stably expressing full-length EGFRG465R, i.e., G465R-EGFR-SW48 (WT/G465R-EGFR-Heterozygote), and COLO320DM cell strains stably expressing full-length wild-type or an EGFR mutant (WT-EGFR-COLO320DM, S492R-EGFR-COLO320DM and G465R-EGFR-COLO320DM) (Fig. 6b, c and Supplementary Fig. 6). COLO320DM is an EGFR-negative human colorectal most cancers cell line with wild-type KRAS/BRAF, which is a perfect cell mannequin for producing steady EGFR mutant cell strains. Each cetuximab and the cetuximab variants inhibited the proliferation of SW48 cells and WT-EGFR-COLO320DM cells and exhibited related in vitro efficacy and wild-type EGFR binding capability (Fig. 6b, left and Fig. 6c, prime). Contemplating the heterogeneous cell floor expression of EGFR and its variants in tumors, we generated the G465R-EGFR-SW48 cell line to guage the inhibitory impact of cetuximab and Ctx-W52D. Cetuximab confirmed decrease efficacy in inhibiting EGFR signaling within the G465-EGFR-SW48 mannequin, whereas Ctx-W52D maintained its excessive efficiency. This consequence indicated that exogenous introduction of full-length EGFRG465R within the SW48 cell mannequin impaired the inhibitory impact of cetuximab however not Ctx-W52D on EGFR downstream signaling (Fig. 6b, proper). Within the S492R-EGFR-COLO320DM cell mannequin, Ctx-VY and Ctx-Y104D exhibited related inhibition efficacies; nevertheless, cetuximab had nearly no inhibitory impact (Fig. 6c, center). In line with its EGFRG465R binding capability, solely Ctx-W52D inhibited cell proliferation in a dose-dependent method within the G465R-EGFR-COLO320DM cell mannequin compared with cetuximab and management IgG (Fig. 6c, backside).
Immune effector exercise additionally performs an essential position within the antitumor exercise of therapeutic antibodies. We additionally investigated the complement-dependent cytotoxicity (CDC) and antibody-dependent mobile cytotoxicity (ADCC) exercise of cetuximab variants in response to beforehand described strategies39,40. The outcomes confirmed that the cetuximab variants exhibited ADCC (Fig. 6d) and CDC (Fig. 6e) actions just like these of the parental antibody cetuximab in EGFRWT mannequin cells, and confirmed extra ADCC and CDC actions in EGFRMut-expressing mannequin cells, benefiting from their restored EGFRMut binding capability.
Cetuximab variants can inhibit the expansion of mutant EGFR-positive and WT-EGFR-positive tumors in vivo
We used colorectal most cancers (CRC) cell line-derived xenograft (CDX) fashions to guage the in vivo antitumor exercise of Ctx-VY, Ctx-Y104D and Ctx-W52D. Within the SW48 (WT-EGFR) xenograft mannequin (Fig. 7a), tumor progress was considerably suppressed within the Ctx-VY, Ctx-Y104D, Ctx-W52D and cetuximab teams in comparison with the PBS group, and the in vivo antitumor actions of the cetuximab variants have been in keeping with their binding affinities for EGFR. Specifically, the in vivo efficiency of Ctx-W52D and cetuximab was extremely related in response to the imply tumor quantity and tumor weight knowledge (Fig. 7b, c). No physique weight reduction was noticed in mice throughout cetuximab and cetuximab variants therapy (Fig. 7d). As well as, we couldn’t detect any apparent tissue harm within the essential organs, similar to coronary heart, liver, spleen, lung, and kidney, in mice, in response to histological examination of tissue sections through H&E staining (Supplementary Fig. 7a). Additional serological research revealed that serum ALT, AST, BUN and Cr didn’t present any vital distinction among the many automobile and therapy teams (Supplementary Fig. 7b), excluding the opportunity of extreme liver or kidney toxicity. Within the G465R-EGFR-SW48 (WT/G465R-EGFR-Heterozygote) xenograft mannequin (Fig. 7e), cetuximab couldn’t bind with EGFRG465R or have an effect on EGFRG465R signaling, due to this fact the exogenous introduction of EGFR G465R in SW48 lowered the efficiency of cetuximab on tumor progress inhibition within the G465R-EGFR-SW48 (WT/G465R-EGFR-Heterozygote) mannequin, compared to the SW48 (WT-EGFR) mannequin. Alternatively, the Ctx-W52D, able to binding with EGFRWT and EGFRG465R, confirmed related in vivo efficacy in two xenograft fashions (Fig. 7f, g). No physique weight reduction was noticed in mice throughout cetuximab and Ctx-W52D therapy (Fig. 7h). As well as, cetuximab and its variants confirmed related in vivo antitumor exercise within the WT-EGFR-COLO320DM xenograft mannequin (Fig. 7i), with respect to the SW48 (WT-EGFR) mannequin (Fig. 7j–l). The in vivo efficacy of Ctx-VY and Ctx-Y104D was maintained within the S492R-EGFR-COLO320DM xenograft mannequin; nevertheless, there was no vital distinction between the cetuximab group and the PBS group in tumor progress suppression (Fig. 7m–o). No physique weight reduction was noticed in mice throughout Ctx-VY and Ctx-Y104D therapy (Fig. 7p). The in vivo efficacy of Ctx-W52D was additionally maintained within the G465R-EGFR-COLO320DM xenograft mannequin; nevertheless, there was no vital distinction between the cetuximab group and PBS group in tumor progress suppression (Fig. 7q–s). No physique weight reduction was noticed in mice throughout Ctx-W52D therapy (Fig. 7t). Additional H&E staining outcomes demonstrated that the tumor tissue morphology was severely disrupted within the cetuximab variant teams in comparison with the cetuximab group and PBS group (Supplementary Fig. 8a). Immunostaining for EGFR confirmed that intratumoral EGFRWT expression was downregulated after the therapy of cetuximab and its variants within the EGFRWT mannequin. Nevertheless, within the EGFRMut fashions, immunostaining for EGFR confirmed that intratumoral EGFRMut expression was downregulated within the cetuximab variant teams, whereas no distinction was noticed between the cetuximab group and automobile group (Supplementary Fig. 8a). Immunohistochemical staining for Ki-67, an indicator of tumor development, revealed markedly fewer intratumoral Ki-67-positive cells in cetuximab variant teams, indicating that the cetuximab variants successfully inhibited EGFRWTand EGFRMut tumor cell proliferation within the xenograft mannequin (Supplementary Fig. 8a–d). The TUNEL assay revealed that there have been apoptotic tumor cells in cetuximab variant teams, additional confirming the in vivo efficacy of the cetuximab variants in opposition to EGFRWT and EGFRMut tumor cells (Supplementary Fig. 8a, e–g).