Photoimmunotheranostic agents for triple-negative breast cancer diagnosis and therapy that can be activated on demand

Abstract

Triple-negative breast cancer (TNBC) is a heterogeneous disease in which the tumors do not express estrogen receptor (ER), progesterone receptor (PgR) or human epidermal growth factor receptor 2 (HER2). Classical receptor-targeted therapies such as tamoxifen or trastuzumab are therefore unsuitable and combinations of surgery, chemotherapy and/or radiotherapy are required. Photoimmunotheranostics is a minimally invasive approach in which antibodies deliver nontoxic photosensitizers that emit light to facilitate diagnosis and produce cytotoxic reactive oxygen species to induce apoptosis and/or necrosis in cancer cells. We developed a panel of photoimmunotheranostic agents against three TNBC-associated cell surface antigens. Antibodies against epidermal growth factor receptor (EGFR), epithelial cell adhesion molecule (EpCAM) and chondroitin sulfate proteoglycan 4 (CSPG4) were conjugated to the highly potent near-infrared imaging agent/photosensitizer IRDye®700DX phthalocyanine using SNAP-tag technology achieving clear imaging in both breast cancer cell lines and human biopsies and highly potent phototherapeutic activity with IC50values of 62–165 nM against five different cell lines expressing different levels of EGFR, EpCAM and CSPG4. A combination of all three reagents increased the therapeutic activity against TNBC cells by up to 40%.

Chondroitin Sulfate Proteoglycan 4 and Its Potential As an Antibody Immunotherapy Target across Different Tumor Types

“…As with other antibody therapeutic applications, CSPG4 has been recognized as a promising candidate for theranostic applications, based on high expression by tumor cells and low expression by healthy cells. In an in vitro photo-immunotheranostic study, single-chain variable fragment (scFv) antibodies recognizing TNBC targets, including CSPG4, as well as epidermal growth factor receptor (EGFR) and epithelial cell adhesion molecule (EpCAM), were conjugated to a potent photosensitizing agent and were used to target TNBC cell lines and tumor biopsy samples ( 118 ). The conjugated scFvs demonstrated high quality imaging capacity, and triggered apoptosis of cancer cells via induction of reactive oxygen species.…”

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 …”

“…SNAP‐tag is genetically fused to the scFv and conjugated to the cytotoxic agent via an autocatalytically generated covalent bond through the cysteine residue of the enzymes catalytic site with the benzylguanine‐modified agent to generate a homogenous construct with a higher therapeutic index . This creates a single specific site for conjugation and allows an optimal 1:1 stoichiometry, which does not affect the activity of the recombinant ligand and overcomes the challenges of current ADC conjugation strategies that are highlighted above (Figure ; A3 & B3). (3) The Fc region is the domain that determines the effector function of the IgG, that is, how it engages with specific cell receptors or other defense proteins, eg, B lymphocytes, in order to elicit an immune response to cause cell death .…”

“…IR700 is a promising PS that, besides having no off‐target effects, possesses ideal properties such as high purity, photostability, and a strong absorption peak close to 700 nm allowing improved light penetration into tissues . Recently, the IR700 fluorophore was conjugated to a scFv fragment against three overexpressed cancer antigens, ie, the EGFR, EpCAM, and CSPG4, using SNAP‐tag technology . In vitro success of this therapeutic approach in killing melanoma cells was attributed to the scFv‐425 (EGFR) targeted effect, as well as the nontoxic effect of free IR700 even after irradiation.…”

“…The energy‐enriched photosensitizer releases the extra energy to its surroundings and returns to the ground state. 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.…”