The incidence of melanoma has been rapidly rising worldwide with no effective treatment in sight for patients with metastatic forms of this disease . Despite decades of evaluating new therapeutic modalities for advanced melanoma, patients are mainly being treated with agents that display low response rates, i.e. DTIC and IL-2 . In view of the urgent need for new therapeutic strategies, we designed and screened a combinatorial library based on the scaffold of a single chain ribosome-inactivating protein (scRIP), namely the cytotoxic A subunit of SLT-1, to identify SLT-1 A variants that target and kill human melanoma cells. In this study, we demonstrated that the screening of this combinatorial SLT-1A protein library led to the identification of a toxin variant termed SLT-1AIYSNKLM that selectively binds to human melanoma cells, is internalized by them and results in their apoptotic death.
Immunotoxins derived from the fusion of the cytotoxic domain of Pseudomonas exotoxin A (PE) to a tumor-targeted Fv fragment, have been shown to kill cells using a combination of cytotoxic and cytostatic mechanisms of action, namely the induction of apoptosis and cell death (cytotoxic mechanism) due to the inhibition of protein synthesis (cytostatic effect) [25, 26]. Wild type SLT-1 utilizes both mechanisms. Firstly, the SLT-1 A subunit undergoes retrograde transport to the endoplasmic reticulum, where it then retrotranslocates to the cytoplasm and halts protein synthesis via its catalytic activity by cleaving 28S rRNA . The blockage of protein synthesis subsequently triggers apoptosis through the activation of caspases 8, 9, and 3 leading to the display of morphological characteristics such as membrane blebbing, DNA fragmentation, chromatin condensation and cell shrinkage [14, 17]. As presented in Figure 3, only the catalytically active SLT-1AIYSNKLM kills cells, activates caspases-3 and cleaves PARP, as observed for ricin, another common RIP family member . Thus, one key advantage of SLT-1A as a combinatorial library template is that it yields small scRIP agents with short new ligand binding domains without altering the inherent intracellular localization and cytotoxic properties of the A subunit. This mechanism of action is distinct from most commonly prescribed anticancer agents in the clinic, suggesting that the cytotoxic A subunit of SLT-1 could complement the action of existing drugs in combination therapy.
The melanoma-specific scRIP SLT-1AIYSNKLM was directly derived using RESCRIPT (Additional File 1: Figure S1) from a cell-based screen and its therapeutic potential confirmed in vivo without the need to re-engineer parts of its structure. This discovery and optimization approach differs from preexisting immunotoxin design strategies. Specifically, the traditional two-step assembly of immunotoxins and related conjugates has been based on an a priori selection of a known tumor marker. However, the fusion or chemical coupling of a protein ligand (typically > 25 kDa) to a toxin domain often generates large protein constructs displaying altered functions in terms of cell targeting, cellular processing and cytotoxic functions [9, 22, 29]. Secondly, RESCRIPT, as a discovery tool, is distinct from phage display approaches which are typically used to identify peptide and protein ligands and are not presently compatible with cell killing assays. RESCRIPT thus limits the need for post-discovery, protein engineering steps required to ensure that functions such as cell targeting, cellular routing and toxicity are retained by the resulting conjugates.
Mechanistically, SLT-1AIYSNKLM behaves as a catalytically active RIP causing apoptosis in 518-A2 melanoma cells (Figure 3). SLT-1AIYSNKLM was shown to bind to a surface receptor on 518-A2 cells with a Kd of 18 nM. These cells express about 140,000 copies of the receptor. More importantly, the specific binding of this A subunit variant to receptors on melanoma cells was rapid, saturable and reversible (Figure 4). Preliminary biochemical studies aimed at determining the nature of the surface determinant recognized by SLT-1AIYSNKLM on 518-A2 melanoma cells, suggests that the receptor may not be a protein. Specifically, pre-treating 518-A2 cells extensively with trypsin yielded an identical SLT-1AIYSNKLM cell cytotoxicity profile to that of untreated 518-A2 cells. Furthermore, preliminary cell surface radioiodination followed by crosslinking/pull-down experiments with His-tagged SLT-1AIYSNKLM and wt SLT-1A did not reveal any unique radiolabeled membrane species that may act as putative SLT-1AIYSNKLM receptors on SDS-PAGE. Both approaches have limitations in terms of specificity and detection sensitivity. Biochemical and proteomic approaches are on-going to define this melanoma-specific receptor. Studies are also being conducted to determine whether the SLT-1AIYSNKLM receptor may be a cell-surface melanoma marker that has already been described in the literature.
Although several melanoma biomarkers have previously been reported, only a few are expressed on the cell-surface and therefore would qualify as possible SLT-1AIYSNKLM receptor candidates . These surface markers include HMW-MAA (high molecular weight melanoma associated antigen; melanoma chondroitin sulfate proteoglycan; MCSP), S100B (originally known as S100), CD44, CXCR4 (CXC chemokine receptor) and CEACAM1 (carcinoembryonic-antigen-related cell adhesion molecule 1) [31–37]. However, these known markers are present on normal tissues as well as other cancer types, highlighting challenges in designing melanoma-specific therapies through the traditional method of conjugating a ligand to a cell-killing component [30, 33, 36, 38, 39]. Nevertheless, antibodies raised against some of these biomarkers, such as anti-Id mAb MK2-23 for HMW-MAA, have shown promise in increasing patient survival. To date, there is still a lack of clinically successful melanoma-specific markers, and none for the detection of primary tumors for high risk patients [30, 40, 41].
The pharmacological properties of SLT-1AIYSNKLM were also investigated in a mouse model and suggest that the molecule is cleared with the expected half-life profile of antibody fragments with a mass of ~30 kDa. SLT-1AIYSNKLM is also stable in human serum, displays modest immunogenicity, and accumulates readily in tumor xenografts of 518-A2 cells implanted in SCID mice (Figure 6, Figure S4 and Figure 5 and 7). This scRIP also retains its cytotoxic activity both in vitro (Figure 2 and 3) and in vivo as shown by the level of tumor regression observed upon treating tumor-bearing mice (Figure 7). These remarkable properties suggest that SLT-1AIYSNKLM be pursued as a possible treatment modality for melanoma patients displaying metastatic forms of this disease.