Cetuximab increases LGR5 expression and augments LGR5-targeting antibody-drug conjugate efficacy in patient-derived colorectal cancer models

Abstract

Colorectal cancer (CRC) remains the second-leading cause of cancer-associated deaths, indicating an urgent need for improved therapeutic options. We previously generated antibody-drug conjugates (ADCs) targeting the cancer stem-like cell marker leucine-rich repeat-containing G protein-coupled receptor 5 (LGR5). However, tumor relapse due to LGR5 downregulation and suboptimal payload selection warranted strategies to improve ADC efficacy. Here we report cetuximab, an EGFR-targeting monoclonal antibody indicated for RAS ^WJ metastatic CRC, augments LGR5 expression independent of RAS/PIK3CA mutation status and promotes EGFR-LGR5 interactions. Furthermore, we describe the development of LGR5 ADCs incorporating a camptothecin-derived payload that is well-tolerated and significantly inhibits tumor growth. Importantly, cetuximab in combination with LGR5 ADCs results in enhanced tumor inhibition or regression versus single-agent treatment and extends survival in RAS ^MUT patient-derived xenografts. These findings support growing evidence that ADC combination therapies may be more effective than monotherapies and suggests a broader clinical use for cetuximab in treating RAS ^MUT CRC.

Keywords: Antibody-drug conjugate; EGFR; LGR5; cetuximab; colorectal cancer; combination treatment.

Figure 1.. EGFR inhibitors reversibly increase LGR5 expression in CRC cells independent of mutation status.

(A) Western blot of endogenous EGFR and LGR5 expression in a panel of CRC cell lines and patient-derived xenograft (PDX) models XST-GI-010 and CRC-001. (B) EGFR and LGR5 RNA-seq expression data from Cancer Cell Line Encyclopedia (CCLE). Values are read per kilobase of transcript per million (RPKM). Western blots and quantification of LGR5 protein fold-changes normalized to actin in (C) LIM1215, LoVo, and SW620 cells following 48 h treatment with a panel of EGFR- and HER2-targeting mAbs (3 μg/ml) and TKIs (1 μΜ), (D) 5 μg/ml CTX over indicated time-course, and (E) LIM1215, LoVo, and DLD-1 cells following 15 min CTX treatment and subsequent washout for indicated time-course. (F) Immunocytochemistry staining of total (permeabilized) and surface (non-permeabilized) LGR5 in LoVo cells treated for 15 min with vehicle or 5 μg/ml CTX. (G-H) Western blot and quantification of LGR5 protein fold-changes normalized to actin in (G) cycloheximide (CHX, 30 μg/ml) chase for 4 or 24 h performed in LoVo cells pre-treated for 15 min with vehicle or 5 μg/ml CTX (s, short exposure; I, long exposure) and (H) LoVo cells pre-treated for 15 min with 0.1 μM Bafilomycin A1 or 10 μM MG132 to inhibit lysosomal or proteasomal degradation, respectively, in the presence or absence of 5 μg/ml CTX.

Figure 2.. LGR5 levels are reduced simultaneously with loss of EGFR expression in CRC cells.

Western blot and quantification following (A) 72 h treatment with 100 nM EGFR siRNA or non-targeting control siRNA in LIM1215, LoVo, and DLD-1 cells, (B) treatment with 30 ng/ml EGF for 8 or 24 h in LoVo cells, (C) 48 h treatment with 1 ng/ml EGF in LIM1215 and DLD-1 cells, and (D) 48 h treatment of LoVo, DLD-1, DLD-1 shLGR5, and SW620 cells with increasing doses of MEK1/2 inhibitor trametinib. EGFR and LGR5 protein fold-changes were normalized to actin and p-ERK/2 protein fold-change was normalized to total ERK1/2 for western blot quantification.

Figure 3.. EGFR interaction with LGR5 is enhanced by CTX.

Co-immunoprecipitation (co-IP) experiments with EGFR antibody or IgG isotype control shows (A) recombinant LGR5 in 293T-LGR5 cells and (B) endogenous LGR5 in LoVo cells specifically interacts with endogenous EGFR. (C) Co-IP experiment with LGR5 mAb in LoVo cells shows EGFR pulls down with endogenous LGR5. (D) Co-IP shows enhanced LGR5 interaction with EGFR in LoVo cells after 15 min treatment with 5 μg/ml CTX. TL, total lysate. Proximity ligation assays and quantification performed in (E) LIM1215 and (F) LoVo cells treated with vehicle or 5 μg/ml CTX. Statistical analysis was performed using two-tailed unpaired t-test; *p<0.05; **p<0.01. Data presented as mean +/− SD.

Figure 4.. Generation and characterization of LGR5-targeting ADC 8E11-CPT2

(A) Schematic of microbial transglutaminase (mTG)-mediated conjugation of a branched linker to Q295 and N297Q of the Fc region of 8E11 mAb followed by strain promoted alkyne-azide cycloaddition of camptothecin 2 (CPT2) payload attached to a cleavable tripeptide valine-lysine-glycine (VKG) linker to generate 8E11-CPT2 ADC (DAR = 8). (B) LC-MS/MS analysis of 8E11 mAb, 8E11-azido spacer conjugate, and 8E11-CPT2 ADC under reducing conditions. LC, light chain; HC, heavy chain. (C) Coomassie staining of 8E11 mAb and 8E11-CPT2 ADC under reducing conditions shows increase in HC molecular weight, indicating linker-payload conjugation. (D) 8E11-CPT2 and R20-CPT2 cytotoxicity in LGR5-negative 293T parental and LGR5-overexpressing 293T-LGR5 cells. (E) Comparison of 8E11-CPT2 cytotoxicity in a panel of gastrointestinal cancer cell lines with different levels of LGR5 expression. Data presented as mean +/− SD.

Figure 5.. CTX enhances the efficacy of 8E11-CPT2 ADC in vitro.

Confocal images and quantification of rat 8F2 LGR5 mAb internalization and co-localization with lysosome marker LAMP1 after 45 min incubation at 37° C in (A) LIM1215 and (B) LoVo cells pre-treated with vehicle or 5 μg/ml CTX for 15min. Statistical analysis was performed using two-tailed unpaired t-test; *p<0.05; **p<0.01. Data presented as mean +/− SD. Loewe synergy score heat maps and associated cell survival plots for (C) LIM1215, (D) AGS, (E) LoVo, and (F) DLD-1 cells co-treated with increasing concentrations of CTX and 8E11-CPT2 for 5 days. Example experiments showing % cell survival values for CTX and 8E11-CPT2 combinations with marked additive effects for (G) LIM1215 and (H) AGS cells and synergistic effects for (I) LoVo and (J) DLD-1 cells. Statistical significance was performed using ANOVA. *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001.

Figure 6.. CTX treatment increases LGR5 expression in CRC models in vivo.

Western blot and quantification of LGR5 expression in (A) LoVo cell line-derived xenograft models treated with 2 or 20 mg/kg CTX or PBS vehicle (N=4/group) once every three days for a total of two doses (tumors were harvested on day 4), (B) DLD-1 cell line-derived xenograft models from a previously reported survival study treated weekly for 2 doses of 10 mg/kg CTX or PBS vehicle (N=5/group), and (C) KRAS^MUT XST-GI-010 PDOs treated for 72 h with vehicle or 5 μg/ml CTX. LGR5 protein fold-change was normalized to actin. Data presented as mean +/− SD. (D) LGR5 mRNA microarray expression data following acute (24 to 72 h) or chronic (6-week) CTX treatment (20 mg/kg twice per week) in RAS^WT mCRC PDX models (GSE108277; N=21). Statistical analyses performed using ANOVA. *p<0.05; **p<0.01.

Figure 7.. CTX enhances 8E11-CPT2 ADC antitumor efficacy and extends survival in patient-derived CRC models.

Safety assessment shows (A) bodyweights, (B) ALT, (C) AST, (D) CRE, and (E) WBC plasma levels of C57BL/6J mice (N=5/group) following 2-week treatment with single-dose 8E11-CPT2 as indicated. (F) Anti-tumor efficacy of CTX or 8E11-CPT2 ADC compared to combination therapy in XST-GI-010 PDX models up to day 24 when first vehicle-treated animal reached maximal tumor burden. All treatments were performed at 5 mg/kg by intraperitoneal injection weekly for a total of 3 doses (N=7/group). Statistical significance performed using ANOVA. ***p<0.001; ****p<0.0001. Data presented as mean +/− SD. (G) Percent tumor growth inhibition (TGI) at day 24 in XST-GI-010 PDX models. Data presented as mean +/− SD. (H) Kaplan–Meier survival plot and log-rank test for the XST-GI-010 study. (I) Anti-tumor efficacy of CTX, 8E11-CPT2, and R20-CPT2 monotherapies and combination therapies in an NRAS^MUTCRC-001 PDX model up to day 24 when first vehicle-treated animal reached maximal tumor burden. All treatments were performed at 5 mg/kg by intraperitoneal injection weekly for a total of 3 doses (N=5 for all groups except 8E11-CPT2 and 8E11-CPT2+CTX, N=6). Statistical significance performed using ANOVA. *p<0.05; **p<0.01. Data presented as mean +/− SD. (J) TGI at day 24 in CRC-001 PDX models. Data presented as mean +/− SD. (K) Kaplan–Meier survival plot and log-rank test for the CRC-001 study.