Synthesis and Characterization of Cetuximab–Docetaxel and Panitumumab–Docetaxel Antibody–Drug Conjugates for EGFR-Overexpressing Cancer Therapy

Abstract

The safety and efficacy of anticancer antibody-drug conjugates (ADCs) depend on the selection of tumor-targeting monoclonal antibody (mAb), linker, and drug, as well as their specific chemical arrangement and linkage chemistry. In this study, we used a heterobifunctional cross-linker to conjugate docetaxel (DX) to cetuximab (CET) or panitumumab (PAN). The resulting ADCs were investigated for their in vitro EGFR-specific cytotoxicity and in vivo anticancer activity. Reaction conditions, such as reducing agent, time, temperature, and alkylation buffer, were optimized to yield potent and stable ADCs with consistent batch-to-batch drug-to-antibody ratios (DARs). ADCs were synthesized with DARs from 0.4 to 3.0, and all retained their EGFR affinity and specificity after modification. ADCs were sensitive to cell surface wildtype EGFR expression, demonstrating more cytotoxicity in EGFR-expressing A431 and MDA-MB-231 cell lines compared to U87MG cells. A431 tumor-bearing mice treated once weekly for four weeks with 100 mg/kg cetuximab-docetaxel ADC (C-SC-DX, DAR 2.5) showed durable anticancer responses and improved overall survival compared to the same treatment regimen with 1 mg/kg DX, 100 mg/kg CET, or a combination 1 mg/kg DX and 100 mg/kg CET. New treatment options are emerging for patients with both wild-type and mutated EGFR-overexpressing cancers, and these studies highlight the potential role of EGFR-targeted ADC therapies as a promising new treatment option.

Litmus-Body: A Molecularly Targeted Sensor for Cell-Surface pH Measurements

“…50 Thus, trafficking of cetuximab provides an ideal testbed for validating our Litmus-body, given the diverse range of pH values that are expected in cellular compartments along the endocytic pathway. 51 Besides its use in treating multiple cancer types including skin, colorectal, head, and neck, 52,53 cetuximab is under active investigation as an ADC for cancer therapies, 54 although the pH values it encounters on the cell surface and following endocytosis have not been reported to our knowledge.…”

“…Antibody trafficking into acidic intracellular compartments may be important for payload release in the design of antibody–drug conjugates (ADCs) used in cancer therapies . In our proof-of-principle studies, Litmus-body reported on the pH surrounding the internalized cetuximab, an EGFR specific antibody used in cancer treatment that is undergoing active research for ADC based therapies . This intracellular tracking was enabled by the single-wavelength coexcitable and dual-emission nature of the Litmus-body that permitted the accurate colocalization of cfSEP and mCyRFP1.…”

“…61 In our proof-of-principle studies, Litmus-body reported on the pH surrounding the internalized cetuximab, an EGFR specific antibody used in cancer treatment that is undergoing active research for ADC based therapies. 54 This intracellular tracking was enabled by the single-wavelength coexcitable and dualemission nature of the Litmus-body that permitted the accurate colocalization of cfSEP and mCyRFP1. Simultaneous excitation of fluorophores with a single-wavelength light can minimize the effect of sample movement, uneven sensor distribution and sample thickness variations.…”

Understanding and targeting resistance mechanisms in cancer

“…It is considered one of the essential directions for developing monoclonal antibodies (especially in the field of tumor‐targeted therapy) in the next decade. The tissue specificity and cytotoxicity of the new generation of ADCs have been improved compared with the previous generation products, allowing them to show fantastic activity in treating refractory cancers 405,406 . The currently available evidence shows that the potency of ADCs is based on complex and delicate interactions among antibodies, conjugates, various components of the cytotoxin, and the tumor and its microenvironment 407 .…”

Litmus-Body: a Molecularly Targeted Sensor for Cell-Surface pH Measurements

“…Antibody trafficking into acidic intracellular compartments may be important for payload release in the design of antibody-drug conjugates (ADCs) used in cancer therapies 48 . In our proof-of-principle studies, Litmus-body reported on the pH surrounding the internalized Cetuximab, an EGFR specific antibody used in cancer treatment that is undergoing active research for ADC based therapies 49 . This intracellular tracking was enabled by the single-wavelength co-excitable and dual-emission nature of the Litmus-body that permitted the accurate co-localisation of cfSEP and mCyRFP1.…”