Comparison of a mouse and a novel human scFv-SNAP-auristatin F drug conjugate with potent activity against EGFR-overexpressing human solid tumor cells

Abstract

Antibody–drug conjugates (ADCs) can deliver toxins to specific targets such as tumor cells. They have shown promise in preclinical/clinical development but feature stoichiometrically undefined chemical linkages, and those based on full-size antibodies achieve only limited tumor penetration. SNAP-tag technology can overcome these challenges by conjugating benzylguanine-modified toxins to single-chain fragment variables (scFvs) with 1:1 stoichiometry while preserving antigen binding. Two (human and mouse) scFv-SNAP fusion proteins recognizing the epidermal growth factor receptor (EGFR) were expressed in HEK 293T cells. The purified fusion proteins were conjugated to auristatin F (AURIF). Binding activity was confirmed by flow cytometry/immunohistochemistry, and cytotoxic activity was confirmed by cell viability/apoptosis and cell cycle arrest assays, and a novel microtubule dynamics disassembly assay was performed. Both ADCs bound specifically to their target cells in vitro and ex vivo, indicating that the binding activity of the scFv-SNAP fusions was unaffected by conjugation to AURIF. Cytotoxic assays confirmed that the ADCs induced apoptosis and cell cycle arrest at nanomolar concentrations and microtubule disassembly. The SNAP-tag technology provides a platform for the development of novel ADCs with defined conjugation sites and stoichiometry. We achieved the stable and efficient linkage of AURIF to human or murine scFvs using the SNAP-tag technology, offering a strategy to improve the development of personalized medicines.

Advances in epidermal growth factor receptor specific immunotherapy: lessons to be learned from armed antibodies

“…Antibody-drug-conjugates (ADCs) emerged as a promising therapeutic modality prepared from naked antibodies by chemically or enzymatically conjugating a cytotoxic payload using specific linker chemistries. Most currently, cytotoxic molecules are too toxic for systemic application, thus ADCs provide a method to harness the specificity of a mAb for targeted delivery of such highly potent cytotoxic agents to tumor cells expressing a unique cognate antigen [ 89 , 91 , 97 , 104 , 107 , 108 ]. According to the generally accepted mechanism of action, binding of an ADC to such a tumor associated cell surface antigen, induces internalization of the ADC-antigen complex into the targeted cell by receptor-mediated endocytosis and subsequent trafficking of the ADC-loaded endosomes to the lysosomal compartment ( Figure 3 ).…”

Applications of SNAP‐tag technology in skin cancer therapy

“…After their release, their corresponding signals accumulate within the tumor and allow for optical detection. These are a few examples that prove the versatility of SNAP‐tag technology, which depending on the type of the BG modified substrate conjugated will either elicit signal accumulation for diagnosis or induce apoptosis to eliminate cancer cells …”

“…Singlet O 2 is converted to reactive oxygen species (ROS), which induces apoptosis/necrosis of tumor cells . This application is referred to as photoimmunotherapy; in Figure B, auristatin F (AURIF) (microtubule destabilizer) conjugated to the SNAP‐tag antibody fusion protein gets internalized and released into the cytosol where it induces apoptosis, referred to as ADC therapy. In Figure ; C & D, fluorophores and magnetofluorescent nanoparticles enter the cell by receptor‐mediated uptake and used for optical imaging .…”

Antibody-Based Immunotherapy: Alternative Approaches for the Treatment of Metastatic Melanoma

“…To address the limitations associated with conventional therapies and naked mAb-based immunotherapy, recombinant ADCs were developed in the form of scFv–SNAP-tag fusion proteins conjugated to MMAF endowed with the ability to specifically detect and kill melanoma and other cancer cells overexpressing EGFR using nano and picomolar concentrations [ 242 , 243 ]. The efficacy of these MMAF ADCs was confirmed on melanoma cells that overexpress multiple receptors such as melanotransferrin and the epidermal growth factor receptor 3 (HER3) [ 1 , 16 , 33 ].…”

“…Recently, HER2-conjugated MMAE/F was shown to have superior potency on gastric, pancreatic, and other cancers that overexpress HER2 when compared to trastuzumab [ 16 , 244 , 253 ]. Moreover, using scFv-SNAP-tag fusion proteins, MMAF was shown to specifically detect and kill breast and skin cancer cells expressing EGFR, while sparing antigen-negative cells [ 242 , 243 ]. The potency of these MMAF-based ADCs was confirmed on melanoma cells that overexpress the receptor tyrosine-protein kinase erbB-3, which is also known as HER3 (HER3 allows the escape of tumor cells from vemurafenib-targeted treatment of BRAF-positive melanoma cells) [ 1 , 16 , 33 ].…”