(M1230-06-40) Cetuximab-Based Proteolysis Targeting Chimera for Effectual Downregulation of NSCLC with Varied EGFR Mutations

Purpose: Lung tumors are one of the prominent causes of cancer-related deaths worldwide in both women and men as treatment options remain confined in the advanced NSCLC stages. The most clinically advanced EGFR inhibition strategies for NSCLC include small-molecule-inhibitors and monoclonal antibody–mediated blockade of the extracellular ligand-binding domain. Antibody drug conjugates (ADC) are one-of-a-kind in their capability to directly kill tumor cells and simultaneously employ the host immune system in order to develop long-lasting effects. Characteristic targets of ADC include receptors exclusively overexpressed on the desired tumor cell surface. Compared to inhibitors that reveal unprecedented challenges, monoclonal antibodies (mAb) showcase numerous benefits. With the unveiling advancements in PROTACs, we explored a combinatorial approach by developing antibody-based PROTAC (ABTAC) which may lead to effective degradation of tumor-proliferative receptors and can serve as an innovative, never explored approach. We hypothesize that synthesizing an ABTAC using an EGFR targeting antibody and E3 ligase recruiter can aid in degrading overexpressed EGFR. In this context, the main objectives of our research were (i) to synthesize an ABTAC using EGFR targeting mAb and E3 ligase recruiter by click chemistry approach. (ii) to characterize the synthesized ABTAC and its corresponding mAb using various analytical techniques. (iii) to compare the developed ABTAC with corresponding mAb using various 2D and 3D cell-based assays.

Methods: Initially, cetuximab (CTX) was isolated from marketed formulation Erbitux. Cetuximab based PROTAC (CPRO) was prepared using Cu-free strain-promoted azide-alkyne cycloaddition (CuAAC) click chemistry by utilizing cysteine and lysine-based conjugation. In-process validation of developed ADC was ascertained using optimized gradient HPLC method and BCA assay. The drug-to-antibody ratio of CPRO was determined using Bruker UltrafleXtreme MALDI-TOF/TOF at 1mg/mL concentration. Post conjugation, changes in the structure of mAb were determined using Jasco J-1500 Circular Dichroism Spectrometer at 0.5 mg/mL concentration. The affinity binding evaluation of native CTX and CPRO to human EGFR were studied using openSPR. The molecular mass and stability of the conjugate was analyzed using SDS-PAGE. As CPRO was intended to be administered via parenteral route, it was assessed for %hemolysis using mice blood. In vitro cytotoxicity of CTX and CPRO was evaluated in NSCLC cell lines i.e., EGFR-TKIs sensitive (HCC4006), wild type/KRAS mutated (A549) and EGFR-TKIs resistant (NCI-H1650) using MTT assay. Downregulation of EGFR was confirmed using EGFR activation assay and ELISA. Comparative anti-metastatic efficacy was assessed by 2D culture assays like migration and colony formation. Apoptosis potential was computed using flow cytometry. Biocompatibility of CPRO on healthy cells was proven using HEK-293 cells. Multicellular 3D spheroid assay was performed to mimic the in vivo tumor conditions. Growth kinetics of the spheroids and apoptosis potential after treatment exposure was ascertained using EVOS microscope. %Protein aggregation in samples was determined using PROTEOSTAT® protein aggregation assay kit.

Results: Successful conjugation of CPRO could be achieved using lysine based click chemistry which was confirmed using HPLC and BCA. Analytical characterization using MALDI-QTOF suggested successful conjugation of five E3-ligase recruiter molecules on surface of one mAb. Circular dichroism of CTX displayed predominant β-sheet. In the case of CPRO, there was a shift in negative valley and reduction in the mean residual ellipticity which can correlate to successful conjugation of pomalidomide groups. Surface plasmon resonance (SPR) confirmed no significant impacts on the receptor binding ability of CTX post-conjugation. For the conjugate, molecular band was found to shift between 150-250 kDa indicating successful conjugation. CTX and CPRO even at 0.5 mg/mL concentration showed < 1% hemolysis. Compared to mAb, nearly 10-30 folds reduction in IC50 values i.e., in microgram/mL was observed in case of CPRO in all cell lines. Moreover, pomalidomide-azide alone did not exhibit an anticancer activity even at 50 μM concentration. Cell viability was found to be > 80% for CPRO even at 1 mg/mL concentration. High percentage of apoptotic population was observed in H1650 cells treated with CPRO compared to CTX treated cells. EGFR activation assay confirmed high percentage of EGFR non-expressing cells. For ELISA, nearly 3 folds higher EGFR downregulation was observed post CPRO exposure. CPRO showed higher repressing potential in spheroid size (****p < 0.0001) over time compared to control in multicellular 3D spheroid assay. Red fluorescence correlating to apoptotic/dead cell population was exceedingly prominent in CPRO treated spheroids compared to CTX and control. Minimal aggregate formation was observed in CPRO even after 4 months.

Conclusion: ABTACs as protein degrader modalities like CPRO come with advantages of targeting overexpressed receptors by using human derived scaffolds like mAbs thereby making them less viable to immune responses and widening their therapeutic window in clinical settings. Our research successfully illustrated the exalted effect of converting a mAb to degrader by portraying significant cytotoxicity and enhanced 2D and 3D apoptosis. Using the ADC approach, we could successfully conjugate 5 E3 ligase recruiting molecules on a single antibody structure thereby giving a new paradigm to the field of PROTAC technology.

Figure 1. (A) Synthetic scheme of cetuximab-based PROTAC (CPRO) synthesis. (B) Protein recovery during synthesis of CTX, CTX-DBCO and CPRO post ultracentrifugation. (C) MADLI -TOF/TOF intact mass spectrum of CTX, CTX-DBCO and CPRO. (D) Far-UV CD spectra of CTX and final conjugate, CPRO post lysine-based conjugation. (E) EGFR expression profile demonstrating inactivated, activated, and non-expressing EGFR cells post 4h treatment incubation in H1650 cells. (F) Assessment of EGFR downregulation using EGFR ELISA. Each data represents n=3.

Figure 2. (A) Surface plasmon resonance of CTX and CPRO. (B) SDS-PAGE analysis of CTX and CPRO on 4-12% polyacrylamide gel. (C) % Protein aggregation estimation post 4- and 8-months stability. (D) In vitro hemolysis study of CTX and CPRO compared to negative control, phosphate buffer and positive control, sodium lauryl sulfate. (E) Schematic representation comparing the advantages of CPRO over its native mAb, CTX. (F) In vitro biocompatibility study of CTX and CPRO in HEK-293 cells. Each data represents n=3.

Figure 3. In vitro cytotoxicity of CTX, CTX-DBCO and CPRO post 72 h incubation in NSCLC cell lines with varied EGFR mutations (A) HCC827, (B) H1650, (C) A549. (D) Representative images of spheroids exposed to CTX and CPRO individually. (E) Flow cytometric analysis of live, apoptotic and dead cells in different subtypes of NSCLC cells post-treatment of native CTX and CPRO. (F) Evaluation of spheroid growth area post treatment exposure compared to control. Fluorescence images portraying apoptotic profile of spheroids post treatment. Composite images of DAPI (blue), calcein-AM (green) and EthD-1 (red).