Applications of SNAP‐tag technology in skin cancer therapy

Abstract Background Cancer treatment in the 21st century has seen immense advances in optical imaging and immunotherapy. Significant progress has been made in the bioengineering and production of immunoconjugates to achieve the goal of specifically targeting tumors. Discussion In the 21st century, antibody drug conjugates (ADCs) have been the focus of immunotherapeutic strategies in cancer. ADCs combine the unique targeting of monoclonal antibodies (mAbs) with the cancer killing ability of cytotoxic drugs. However, due to random conjugation methods of drug to antibody, ADCs are associated with poor antigen specificity and low cytotoxicity, resulting in a drug to antibody ratio (DAR) >1. This means that the cytotoxic drugs in ADCs are conjugated randomly to antibodies, by cysteine or lysine residues. This generates heterogeneous ADC populations with 0 to 8 drugs per an antibody, each with distinct pharmacokinetic, efficacy, and toxicity properties. Additionally, heterogeneity is created not only by different antibody to ligand ratios but also by different sites of conjugation. Hence, much effort has been made to find and establish antibody conjugation strategies that enable us to better control stoichiometry and site‐specificity. This includes utilizing protein self‐labeling tags as fusion partners to the original protein. Site‐specific conjugation is a significant characteristic of these engineered proteins. SNAP‐tag is one such engineered self‐labeling protein tag shown to have promising potential in cancer treatment. The SNAP‐tag is fused to an antibody of choice and covalently reacts specifically in a 1:1 ratio with benzylguanine (BG) substrates, eg, fluorophores or photosensitizers, to target skin cancer. This makes SNAP‐tag a versatile technique in optical imaging and photoimmunotherapy of skin cancer. Conclusion SNAP‐tag technology has the potential to contribute greatly to a broad range of molecular oncological applications because it combines efficacious tumor targeting, minimized local and systemic toxicity, and noninvasive assessment of diagnostic/prognostic molecular biomarkers of cancer.


| Skin cancer
Skin cancer occurs as two main types, ie, non-melanoma and melanoma. Non-melanoma types include basal cell carcinoma (BCC) and squamous cell carcinoma (SCC), which are of keratinocyte origin, as well as Merkel cell carcinoma (MCC), sebaceous gland tumors, and malignant pilomatrixoma. 1,2 Melanoma types include superficial spreading melanoma (SSM), lentigo maligna melanoma (LMM), nodular melanoma (NM), acral lentiginous melanoma (ALM), mucosal melanoma, desmoplastic melanoma, and nevoid melanoma. 3 In addition, non-melanoma skin cancers (NMSC) can have more than one histological subtype, in which case they are referred to as mixed types. 4 There is variability in the behavior of different types of skin cancer, as well as histopathological variants, depending on growth patterns. 4 For example, BCC carcinomas grow slowly, with damage to surrounding tissue, but rarely spreads to vital structures, whereas SCC and melanomas are aggressive and are more likely to metastasize. 5,6 Solar and actinic keratitis, viral warts, and Bowen disease increase the risk of NMSC, while clinically atypical mole (CAM), giant congenital melanocytic nevi, and lentigo maligna have been shown to increase the risk of developing melanoma. 7 The risk of developing skin cancer is also higher in people with poor immune function (such as HIV/AIDS and solid organ transplant patients) [8][9][10] and people of fair skin color. [10][11][12][13][14] The observed increases in skin cancer rates are associated with several factors, including the fact that older populations are at higher risk of NMSC, and also increased occupational and recreational UV light exposure 15,16 (Figure 1). For instance, studies have shown that indoor tanning is associated with a significantly increased risk of BCC and SCC, with a higher risk with use in early life (<25 y). 17 Each year in the United States, over 5.4 million cases of NMSC are treated in more than 3.3 million people. 18 In 2017, it was estimated that 87 110 new cases of invasive melanoma were diagnosed in the United States and an estimated 9730 people were casualties of this aggressive type of skin cancer. 19 The annual cost of treating skin cancers in the United States is estimated at $8.1 billion: about $4.8 billion for NMSC and $3.3 billion for melanoma. 20

| Current skin cancer treatment
The use of conventional cancer therapies for skin cancers has been fraught with poor specificity in targeting the cancer cells, partly due to variations in surface receptor expressed by tumor cells. 21 Surgical therapy may be used depending on the type and location of cancer, age of person, and whether the cancer is in the primary or recurring stages. 22 For example, a person (<50 y of age), diagnosed with BCC, can undergo a surgical excision known as Mohs surgery. Mohs micrographic surgery removes skin cancer one layer at a time, each time examining these layers under a microscope immediately after removal.
Although this procedure allows for minimal scarring by preserving healthy tissue, it is time consuming (3-4 h to remove a single lesion) and expensive. [23][24][25] Superficial removal of cancerous tissue can be done with cautery and curettage using a spoon-like instrument with a sharp edge. 26 Another surgical procedure that may be used for skin cancer is electrosurgery, which is a procedure that cuts/destroys/cauterizes tissue using a high-frequency electric current applied locally with a pencil-shaped metal instrument. 27 For low-risk disease or treatment of elderly patients, radiation therapy (external beam radiotherapy or brachytherapy), 28 topical chemotherapy (5-fluorouracil), 29 and cryotherapy (freezing the cancer off) 30 can provide adequate control of the disease. However, the topical application of 5-fluorouracil often fails due to the inadequate frequency and/or length of treatment, insufficient drug concentration, and a poor penetration of the cream into the epidermis, which contributes to tumor recurrence. 29 Hence, personalized/precision medicine has emerged because of its potential to improve the accuracy of tumor targeting and minimize toxicity to normal tissue. The beneficial role of novel-targeted therapies and the potential use of SNAP-tag fusion FIGURE 1 A sketch showing the squamous cells, melanocytes, and basal cells found in the epidermal layer of the skin. Ultraviolet (UV) light from the sun can damage the DNA in these skin cells and give rise to SCC, BCC, or melanoma proteins in cutaneous cancers is described hereafter. This review seeks to elaborate more on the applications of these diagnostic and therapeutic treatment modalities.

| Targeted drug and molecular therapies in skin cancers
The field of cancer immunotherapy attempts to target and kill cancer cells by manipulating the body's immune system and has been immensely successful for the treatment of skin cancer. 31 To date, eight drugs have been approved by the US Food and Drug Agency (FDA) for the treatment of metastatic melanoma. These include the chemotherapeutic drug dacarbazine (DTIC) (FDA approved in 1975) and the immunotherapeutic agents such as vermurafenib, ipilimumab (FDA approved 2011), dabrafenib, and trametinib (FDA approved 2013), interleukin-2 (IL-2) (FDA approved 1998), [32][33][34][35] and nivolumab and pembrolizumab (FDA approved 2014) 36 (Table 1). DTIC is more commonly used as the current standard treatment for metastatic melanoma. [32][33][34] Until recently, single-agent chemotherapy using DTIC has produced the best therapeutic outcome, with 5% to 15% of patients responding to the therapy, although less than 2% survive 6 years post treatment. 33,37 Since the discovery of the BRAF V600E mutation in melanomas, three BRAF inhibitors-vemurafenib, trametinib, and dabrafenib-have been used to stop signals that cause cancer cells to grow and divide. However, these drugs are associated with serious side effects, as highlighted in Table 1. 38,39 As persistent inflammation has emerged as a cardinal hallmark of cancer, 40 targeting toll-like receptors (TLRs) is also hypothesized as a plausible potential molecular approach for skin cancer therapies. 41 Another successful treatment option in patients with melanoma is the use of monoclonal antibodies (mAbs), which are immune checkpoint inhibitors. For example, the monoclonal antibody ipilimumab is directed toward the cytotoxic T-lymphocyte antigen (CTLA)-4 and was the first (CTLA)-4 inhibitor to demonstrate an improved overall survival rate in melanoma patients. 42 Other mAbs such as nivolumab bind to the programmed-cell death (PD)-1 receptor and block interaction with PD-L1 and PD-L2 ligands. 43 This binding releases PD-1 pathway-mediated immune responses against tumor cells. 43 Recently, anti-programmed cell death-1 (Anti-PD-1) was approved for the treatment of patients with advanced melanoma. 37 Although PD1 blockers have comparatively better safety, the main concern with PD1 monotherapy is patient response rate (around 30%-40%). 44 The pegylated version of interferon α-2b (PEG-IFN) has been approved as an adjuvant for surgically resected "high-risk" melanoma patients. 45 However, these mAbs are associated with severe side effects, including dermatologic, gastrointestinal, hepatic, endocrine, and, less commonly, inflammatory events. 46 EGFR (epidermal growth factor receptor) is the first molecular target against which mAbs have been developed for cancer therapy. 47 Anti-EGFR mAbs are known to bind to the extracellular domain of EGFR in its inactive state, then compete for receptor binding by occluding the ligand-binding region, and block ligand-induced EGFR tyrosine kinase activation. 48,49 The anti-EGFR mAb called cetuximab is used in combination with radiotherapy and is considered a promising treatment modality for locally advanced inoperable NMSC. 50 However, side effects such as a persistent rash are still associated with cetuximab as well as other cutaneous toxicities such as painful fissures in palms and soles and paronychia. 51 mAbs targeting tumor-associated cell surface antigens overexpressed on tumor cells but also expressed on normal cells can thus also interact with normal cells. 52,53 In addition to the related off-target, nonspecific toxicities, the high proportion of nonhuman sequences eventually incorporated in mAbs is likely to be recognized as "foreign" and therefore induce a host immune response.
This can result in reduced efficacy of the mAb, due to increased clearance. 52,53 Table 1 summarizes the different types of treatments approved for skin cancer and their corresponding adverse side effects.
To provide an improved targeting approach, antibody drug conjugates (ADCs) have been designed, in which a cytotoxic payload is attached to an antibody via a chemical linker. 54 This is exemplified, for instance, by the novel ADC EV20-Sap that displayed promising antitumor activity in metastatic melanoma, obtained by chemically coupling the HER-3 targeting antibody EV20 to the plant toxin saporin. 55 This conjugate maintained the biological activity of the naked HER-3 antibody. It binds to melanoma cells with the same affinity as free EV20 and eliminated cancer cells, upon internalization with IC50 values in the range of 0.15nM to 20nM. This attests to its powerful specificity and target-dependent cytotoxic activity. Furthermore, in a murine melanoma model, EV20-Sap treatment led to a significant reduction of pulmonary metastasis. 55 Despite the therapeutic efficacy of ADCs, their major challenges were size and heterogeneity. 56,57 The large size of mAbs (150 kDa) might exhibit relatively limited tissue penetration and is prone to nonspecific binding owing to their Fc domain. 58 Furthermore, the cytotoxic agents in ADCs are typically conjugated randomly to the antibodies, using either the reduced sulfhydryl groups of cysteine residues or the amino groups of lysine side chains. This generates heterogeneous ADC populations with variable drug to antibody ratios (DAR) that results in reduced efficacy and unpredictable pharmacokinetic profiles. 56,57 While one might presume that high-affinity binding is ideal, several studies have shown that very high affinities might be suboptimal for therapeutic antibodies to penetrate deep into solid tumors. 59,60 This results in rapid and tight binding to the outer surface of a tumor and reduced numbers of antibodies diffusing to the core of the tumor.
This is because rate of diffusion is approximately inversely proportional to the cube root of molecular weight. 60,61 Thus, the intended effects of the mAb would not be universal to all tumor cells. 60,62 Many nanoparticle-based drug delivery systems have been approved by the FDA and are currently undergoing clinical trials for skin cancer therapy. 63 It has been shown that delivering the chemotherapeutic agent doxorubicin by gold nanoparticles was very effective against a melanoma cell line. 64 Lo Prete et al applied a cholesterol-rich nanoemulsion to deliver etoposide in a mouse model of melanoma. 65 The nanoemulsion delivery was associated with decreased side effects, increasing maximum tolerated dose fivefold and increased inhibition of tumor growth by concentrating etoposide at the tumor site (a fourfold higher concentration in tumor than with free etoposide). 65 Nonetheless, nanoparticles as efficient drug delivery systems are hindered by incomplete toxicological assessment, low drug-loading capacities, difficulty in scale-up production, and low stability. 66 Natural compounds have been suggested for use alone 67,68 or in combination with photodynamic therapy (PDT) 69 in the treatment of skin cancer. PDT is a treatment modality that uses an effector molecule called a photosensitizer (PS), followed by local illumination with visible light of specific wavelength(s). When a PS is exposed to a specific wavelength of light, it produces reactive oxygen species (ROS) that induce apoptosis of cancerous lesions. 70 To date, targeting cancer cells using PDT has relied on the passive accumulation of PS in tumor tissues, which might not lead to optimal dosage of PS, thus leading to the application of relatively high dosage of PS within the tumor. 71 As a result, PDT may damage healthy tissues, by causing prolonged skin photosensitivity. 72 Efforts to bypass this lack of specificity have focused on the identification of specific cancer biomarkers, drug conjugates, and resistant mechanisms contributing to cancer survival after therapeutic treatments.
The addition of SNAP-tag technology to skin cancer management potentially presents a more structurally reliable method for conjugation and delivery of photosensitizer or cytotoxic payload for targeted cancer chemotherapeutic purposes, as discussed below. inhibitor ipilimumab, to treat melanoma. 94 It was observed that after laser immunotherapy on one patient, cutaneous melanoma in the head and neck completely disappeared. 94 The patient was then adminis- Targeting CSPG4 was also shown to be clinically relevant by an increase in survival of melanoma patients who received CSPG4 mimics as a form of active specific immunotherapy. 102 Targeting CSPG4 also inhibited the growth and recurrence of melanoma in a human melanoma xenograft model. 103 Two photosensitizers (PS) that are currently used in NIR-PIT are IR700 and hypericin. 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. 90,[104][105][106][107] 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. 76,88 In vitro success of this therapeutic approach in killing melanoma cells was attributed to the scFv-425

| Targeted drug conjugation and SNAP-tag technology
(EGFR) targeted effect, as well as the nontoxic effect of free IR700 even after irradiation. Escherichia coli. 110,111 Hypericin-based PDT treatment was shown to be effectively cytotoxic to metastatic melanoma through the localization of HYP in melanosomes. [112][113][114] Another study showed that hypericin can inhibit the growth of SCC tumors in culture and can reduce tumor size in mice in the complete absence of light. 115 Recently, an attempt to circumvent chemoresistance was made by Biteghe and Davids, who by combining DTIC with hypericin were able to overcome this resistance due to the genotoxic effect by DTIC and the oxidative stress induced by HYP-PDT. 116 Optical imaging methods have also seen widespread application in skin cancer diagnostics as they are noninvasive, with fast response times, and are potentially sensitive to biochemical and structural changes presented in skin cancer development. 117,118

| Optical imaging methods in cancer diagnosis
For skin cancer diagnostics, the primary optical imaging techniques used are widefield imaging, optical spectroscopy, and microscopy imaging. 119 Widefield imaging allows the examination of large areas and has the potential of improving detection of hidden lesions, margin delimitation, and also guide biopsy site determination. 120 A major advantage of the widefield microscope is the low cost, simplicity, and flexibility of the system. 121 In contrast, some disadvantages of widefield microscopy include low image resolution, potential for shading artifacts due to uneven illumination, and the alignment of different cameras to ensure pixel registration when using multiple indicators. 122 Microscopy imaging has the main advantage of the evaluation of the tissue characteristics at cellular level, but only a small fraction of the lesion volume is interrogated. 123 Optical spectroscopy presents more detailed information on tissue composition than widefield microscopy, as the light intensity for each collected emission wavelength is correlated to specific biomolecules. 124 Raman spectroscopy has gained considerable interest in disease diagnosis, particularly cancer, because of its ability to provide molecular specific information about tissues. Each Raman spectral peak can be associated with specific vibrations in molecular bonds. 125,126  The EGFR-specific scFv fusion protein 425-SNAP was labeled with the NIR dye BG-747, and its accumulation, specificity, and kinetics were monitored using NIR fluorescence imaging in a subcutaneous pancreatic carcinoma xenograft model. 74 The 425 (scFv) SNAP fusion protein accumulated rapidly and specifically at the tumor site. Its small size allowed efficient renal clearance and a high tumor to background ratio (TBR). 74 The SNAP-tag can also be combined with other protein tags, such as HaloTag, 134 or other reporter gene systems that use fluorescent substrates, such as β-galactosidase/DDAOG system, 135 to create multiplexed imaging systems. A second version of AGT-based tag named CLIP-tag reacts specifically with benzylcytosine (BC) derivatives. 136 Because SNAP-tag and CLIP-tag only react with their specific substrates, they could be used simultaneously for dual-color fluorescence imaging. 137 For example, the nonspecific blood flow tracer indocyanine green (ICG) was successfully used to visualize regional lymphatic flow from cancer lesions and identified sentinel lymph nodes in humans. 137 Simultaneous but separate visualization of different lymphatic drainages was made possible by fluorescent agents with multiple colors. 137 The clinical use of NIR fluorescence imaging for sentinel lymphatic mapping was first reported by Kitai et al in 18 breast cancer patients. 138 They injected 25 mg of ICG near the areola of breast cancer patients and successfully visualized the draining lymphatics in all patients and localized the sentinel lymph nodes in 17 of 18 patients. 138 Following this study, additional clinical studies have confirmed the utility of NIR sentinel mapping in melanoma. 139 Tumor detection with NIR fluorescence during a surgical procedure has been performed in several tumor types, with application in melanoma using ICG. 140 The subsequent conjugation of ICG to SNAP-tag thus creates new possibilities for image guided surgery in melanoma patients in the future.
As shown in Figure 4A, the scFv targets the fusion protein to the surface receptor on the tumor, cell and the conjugated photosensitizer (IR 700) is activated by a specific wavelength of light (500-700 nm). 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. 88 This application is referred to as photoimmunotherapy; in Figure 4B, auristatin F (AURIF) (microtubule destabilizer) conjugated to the SNAP-tag antibody fusion protein gets internalized and released into the cytosol where it induces apoptosis, 141 referred to as ADC therapy. In

| CONCLUSION
In the era of precision medicine, SNAP-tag technology is a potentially promising molecular targeting approach for early diagnosis and treatment of skin cancer, which has a high burden globally. In this review, we have identified and discussed the prospects for the use of SNAP-tag for targeted therapy of skin cancers, as well as some of its potential advantages over currently available conventional skin cancer treatment options.
Not least, the use of SNAP-tag technology in combination with other recently emerging 'omics-based technologies can potentially offer a treasure trove of targeted diagnostic, prognostic, and therapeutic options for the management of skin cancers in a systems-oriented manner.