An evolved ribosome-inactivating protein targets and kills human melanoma cells in vitro and in vivo
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Few treatment options exist for patients with metastatic melanoma, resulting in poor prognosis. One standard treatment, dacarbazine (DTIC), shows low response rates ranging from 15 to 25 percent with an 8-month median survival time. The development of targeted therapeutics with novel mechanisms of action may improve patient outcome. Ribosome-inactivating proteins (RIPs) such as Shiga-like Toxin 1 (SLT-1) represent powerful scaffolds for developing selective anticancer agents. Here we report the discovery and properties of a single chain ribosome-inactivating protein (scRIP) derived from the cytotoxic A subunit of SLT-1 (SLT-1A), harboring the 7-amino acid peptide insertion IYSNKLM (termed SLT-1AIYSNKLM) allowing the toxin variant to selectively target and kill human melanoma cells.
SLT-1AIYSNKLM was able to kill 7 of 8 human melanoma cell lines. This scRIP binds to 518-A2 human melanoma cells with a dissociation constant of 18 nM, resulting in the blockage of protein synthesis and apoptosis in such cells. Biodistribution and imaging studies of radiolabeled SLT-1AIYSNKLM administered intravenously into SCID mice bearing a human melanoma xenograft indicate that SLT-1AIYSNKLM readily accumulates at the tumor site as opposed to non-target tissues. Furthermore, the co-administration of SLT-1AIYSNKLM with DTIC resulted in tumor regression and greatly increased survival in this mouse xenograft model in comparison to DTIC or SLT-1AIYSNKLM treatment alone (115 day median survival versus 46 and 47 days respectively; P values < 0.001). SLT-1AIYSNKLM is stable in serum and its intravenous administration resulted in modest immune responses following repeated injections in CD1 mice.
These results demonstrate that the evolution of a scRIP template can lead to the discovery of novel cancer cell-targeted compounds and in the case of SLT-1AIYSNKLM can specifically kill human melanoma cells in vitro and in vivo.
KeywordsMelanoma SCID Mouse Human Melanoma Cell Human Melanoma Cell Line Pseudomonas Exotoxin
Single Chain Ribosome-Inactivating Protein
Shiga-Like Toxin 1
Shiga-Like Toxin 1 A subunit
Shiga-Like Toxin 1 A subunit with an IYSNKLM insertion between residues 245 and 246
Poly(ADP) Ribose Polymerase
Maximum Intensity Projection
Ordered Subset Expectation Maximization
Pseudomonas exotoxin A
High Molecular Weight Melanoma Associated Antigen
Melanoma Chondroitin Sulfate Proteoglycan
CXC chemokine receptor
Carcinoembryonic-Antigen-Related Cell Adhesion Molecule 1
Identification of SLT-1AIYSNKLM: a human melanoma-specific scRIP toxin variant
SLT-1 is a bacterial type II ribosome-inactivating protein produced by enteropathogenic E. coli strains such as O157:H7. Previously, the full-length SLT-1 (70 kDa; AB5) was used by our group as a scaffold to construct a combinatorial library. Random peptide elements were embedded within its receptor-binding B domain to alter receptor-binding specificity, while the wild type cytotoxic A subunit was retained to screen the library for SLT-1 variants able to selectively kill cancer cells . However, the expression of protein variants from such a library was typically less than 1% when mutations were introduced in the receptor binding regions of the B subunit. We therefore describe here a novel combinatorial library designed using the smaller cytotoxic SLT-1 A subunit (31 kDa) as a scaffold (Additional File 2).
Catalytically active SLT-1AIYSNKLM triggers apoptosis in human melanoma cells
SLT-1AIYSNKLM binds to receptors on human melanoma 518-A2 cells
SLT-1AIYSNKLM rapidly localizes at tumor sites in vivo
To visualize the tumor-localization of SLT-1AIYSNKLM, a group of 518-A2 xenograft-bearing SCID mice received a single i.v. injection of either 125I-SLT-1AIYSNKLM (900 μCi) or wt 125I-SLT-1A (900 μCi; negative control). The distribution of radiolabeled scRIPs was recorded 45 min post-injection with a nanoSPECT/CT scanner. The resulting CT MIP (Maximum Intensity Projection) images were then overlaid with the SPECT image slice that transversed the central plane of the tumor (Figure 6C). These images highlight the rapid localization and specificity of 125I-SLT-1AIYSNKLM into 518-A2 cell xenografts as compared to the control, wt 125I-SLT-1A, which shows no tumor localization.
SLT-1AIYSNKLM causes tumor regression and increased survival in vivo
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, 32, 33, 34, 35, 36, 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.
We have developed and screened a combinatorial single chain, ribosome-inactivating protein (scRIP) library with a view to use the intrinsic cytotoxicity of all members of the library (built-in search engine) as a tool to identify melanoma-specific scRIP variants in the context of cell viability assays. Library searches led to the discovery of SLT-1AIYSNKLM, a novel scRIP variant of the cytotoxic A subunit of Shiga-like Toxin 1 that is able to target and specifically kill human melanoma cells. SLT-1AIYSNKLM represents a candidate agent for treating patients with metastatic melanoma.
Materials and methods
Antibodies, Cell Lines and Bacterial Strains
The 518-A2 human melanoma cell line was kindly provided by Dr. Burkhard Jansen  and maintained in Dulbecco's Modified Eagle Medium containing 5% FBS, 100 U/mL penicillin and 0.1 mg/mL streptomycin. All other cell lines were purchased from ATCC and grown according to company guidelines.
Recombinant SLT-1A variants were generated using a modified version of the method previously described by our group . The initial library was designed to validate the variability of a heptapeptide insert as well as its effect on SLT-1 A subunit cytotoxicity (see Additional Files 2 and 3). The final heptapeptide library screened was genetically inserted into the A subunit of His-tagged SLT-1 between residues 245 and 246 by PCR. Specifically, the randomized insert was introduced by amplifying a fragment of the SLT-1 gene using primers A (CTG AAG CTT TAC GTT TTC GGC) and C (GAT GCC ATT CTG GCA ACT CGC GAT GCS NNS NNS NNS NNS NNS NNS NNS TGA TGA TGA CAA TTC AGT ATT AAT GC). This product was ligated to the remaining fragment, which had been amplified using primers B (GAG ACT GCA GAT TCC ATC TGT TGG) and D (GCA TCG CGA GTT GCC AGA ATG GCA), by PCR using primers A and B. After digestion with HindIII and PstI, the resulting library cassette was ligated into pECHE9A (pUC19-based plasmid that constitutively expresses SLT-1 constructs in bacteria), digested with NsiI to linearize wild-type material, transformed and expressed in JM101 E. coli (Promega).
Expression and Purification of SLT-1A Subunit Library Variants
Individual library colonies were picked from carbenicillin-selective LB-agar plates and grown overnight at 37°C in 1 mL of LB-broth in 96-well culture blocks. Bacterial pellets were lysed in a single freeze-thaw cycle with BugBuster Protein Extraction Reagent (Novagen) supplemented with 2.5 U/g pellet of Benzonase nuclease (EMD Biosciences). Cleared lysates were transferred to 96-well plates containing 2 μL of Ni-NTA magnetic agarose beads (Promega) and incubated for 30 minutes at room temperature. The beads were washed with Buffer A (50 mM phosphate buffer, pH 8, 300 mM NaCl, and 10 mM imidazole), and eluted with Buffer C (same as Buffer A but with 250 mM imidazole). The generation of a catalytically-inactive form of SLT-1AIYSNKLM is described in Additional File 2.
Large-Scale Protein Purification
JM101 E. coli (Promega) transformants of SLT-1AIYSNKLM (or the wt SLT-1A control) were grown in LB-broth containing 100 μg/mL ampicillin with shaking (225 rpm) at 37°C overnight. Each gram of cell pellet was resuspended in 5 mL of BugBuster Reagent (Novagen), 2.5 mL Buffer A (50 mM phosphate buffer, pH 8, 300 mM NaCl, and 10 mM imidazole), and 2.5 U Benzonase (2.5 kU; Novagen), and an EDTA-free protease inhibitor tablet (Roche). After stirring at room temperature for 1 h followed by centrifugation at 8,000 × g for 30 min, the cleared lysate was then loaded onto Ni-NTA agarose (Sigma-Aldrich) pre-equilibrated with Buffer A. The noncovalently-associated B subunit was removed from the bound His-tagged A subunit by treating the column with 6 M guanidine-HCl in Buffer A. The retained A subunits were renatured on the nickel resin by washing with Buffer A containing decreasing concentrations of guanidine-HCl [4.8 M, 3.6 M, 2.4 M, and 1.2 M respectively]. After an additional wash with Buffer B (50 mM phosphate buffer, 300 mM NaCl, 20 mM imidazole, pH 8), the purified A subunit was eluted with Buffer C, concentrated and dialyzed against saline using an Amicon ultrafiltration unit (Millipore; 10 kDa MWCO). Proteolytic cleavage was achieved by incubating the purified A subunit with recombinant furin protease (New England Biolabs) in 100 mM MES buffer (pH 6.0), 5 mM CaCl2 and 1 mM β-mercaptoethanol. Typically, 1 mg of SLT-1 A subunit (or SLT-1AIYSNKLM) was cleaved with 10 U of furin for 48 h at 30°C. The bacterial strains producing either wt SLT-1 or AB5 SLT-1AIYSNKLM typically led to the recovery of 7.5 (+/- 2.5) mg of His-tagged purified toxins per liter of bacterial medium. On average, 2 mg of purified, furin-digested SLT-1AIYSNKLM and 1.5 mg of purified, furin-digested wt SLT-1A were obtained per liter of bacterial culture. Samples were characterized by SDS-PAGE, and stained with Coomassie Blue. The purity of the final products was assessed by densitometry to be > 98% pure (See Additional File 5; Figure S4).
Cell Viability and Apoptosis Measurements
Cells were exposed to escalating concentrations of purified SLT-1AIYSNKLM or purified scRIP library variants for 1 h at 37°C followed by a 48 h incubation period in fresh medium. The viability of adherent cells was subsequently determined using the sulforhodamine B assay . Apoptosis as measured by the activation of caspase 3 was determined after treating cells with 1 μM SLT-1AIYSNKLM for 6 h. Caspase 3 proteolytic activity was assessed via the cleavage of the peptide substrate Ac-DEVD-AFC (SensoLyte Homogeneous AFC caspase-3/7 assay kit; ANASPEC) and of PARP  by immunoblotting with a rabbit polyclonal antibody to human ARP (Cell Signaling Technology).
125I Radiolabeling and Radioligand Binding Assays
SLT-1AIYSNKLM and SLT-1A subunits were radioiodinated with carrier-free Na125I (37 MBq/10 uL; MP Bio) and Iodogen (Pierce). Radiolabeled A subunits were purified from unincorporated 125I by gel permeation (D-Salt Polyacrylamide, Pierce) and the specific activity determined and counted in a Wallac Wizard 3" 1480 instrument (PerkinElmer). The specific activity obtained was 2.1 × 1017 cpm/mol and 7.1 × 1016 cpm/mol for the SLT-1AIYSNKLM and wt SLT-1A subunits respectively. Data was plotted and analyzed using GraphPad Prism 5.0 (GraphPad Software, Inc). Details regarding radioligand binding assays are available in Additional File 2.
Pharmacokinetic, Biodistribution, Immunogenicity and Imaging Studies
Human melanoma 518-A2 cells (4 × 106 cells suspended in 0.2 mL culture medium) were implanted subcutaneously into five-week-old female SCID mice , resulting in tumor xenografts (volume of ~30 mm3) within 14 days. For pharmacokinetic analyses, tumor-bearing mice received a single i.v. injection of either 125I-SLT-1AIYSNKLM or wt 125I-SLT-1A (14 μg, 150 μl dose in USP grade saline; 100 μCi). Blood samples were collected via the saphenous vein and counted in a gamma counter to determine blood clearance rates (4 mice per group). For biodistribution studies, a separate group of tumor-bearing SCID mice was anesthetized using 5% isoflurane gas and blood samples collected via cardiac puncture. Mice were sacrificed and tissues collected, weighed and counted in a gamma counter (n = 4). Results were reported as percent injected dose per gram of wet tissue weight.
Immunogenicity studies were performed with CD1 mice (3 mice per time point) given a series of 5 consecutive (250 μg/kg; 100 μL daily) i.v. injections of SLT-1AIYSNKLM prepared in saline to mimic the therapeutic treatment procedure. As a positive control, a single dose (250 μg/kg; 100 μL) of this A chain emulsified (1:1) in Complete Freund's adjuvant (CFA) was injected subcutaneously into CD1 mice (control group). The control animals also received s.c. injections of the same antigen emulsified (1:1) in Incomplete Freund's adjuvant (IFA; 250 μg/kg; 100 μL dose) at day 21 and 35. To assess IgG immune responses raised against SLT-1AIYSNKLM, blood samples were collected at various time intervals post-injection (day 1, 10, 15, 21, 35, 42 for SLT-1AIYSNKLM; day 21, 35, 42 for the antigen with adjuvant). IgG responses were titrated for each mouse by serially diluting serum samples and measuring IgG levels by ELISA in 96-well plates pre-coated with 100 ng of SLT-1AIYSNKLM. Serum-treated wells (in triplicate) were subsequently exposed to an HRP-conjugated goat-anti-mouse IgG (1:1000) and ELISA signals recorded with a plate reader at 405 nm using the substrate ABTS (2,2'-Azinobis [3-ethylbenzothiazoline-6-sulfonic acid]-diammonium salt).
Imaging experiments were carried out at the UHN STTARR facility using a dual-modality NanoSPECT/CT (Bioscan) and are further described under Additional File 2 (2 mice). All animal protocols were reviewed and approved by the Animal Care Committee at the University Health Network.
Tumor Regression Studies
The effects of SLT-1AIYSNKLM and DTIC treatment on the survival of 518-A2 tumor-bearing SCID mice  were assessed when tumor xenografts reached a volume of ~30 mm3. At this stage, animals received daily i.v. injections of either saline, SLT-1AIYSNKLM (0.5 mg/kg; Days 8 to 12 and 15 to 19) or an i.p. dose of DTIC (8 mg/kg; Days 8 to 12) or the combination treatment of both SLT-1AIYSNKLM (i.v. dose: 0.5 mg/kg; Days 8 to 12 and 15 to 19) and DTIC (i.p. dose: 8 mg/kg; Days 8 to 12) (7 mice per treatment regimen). Mice were weighed and the dimension of their tumors measured with calipers. Tumor volumes were calculated by assuming a prolate spheroid shape (tumour volume = (large diameter × [short diameter]2)/2). Animal survival was monitored over a period of 230 days. Mice were euthanized by exposure to CO2 when tumor diameters reached 15 mm, or when ulcerations or other signs of distress such as poor grooming were observed in accordance with the regulatory parameters of the Animal Care Committee at the University Health Network.
Statistical analyses of tumor growth in mice were performed using one-way analysis of variance (ANOVA). Kaplan-Meier curves were used to summarize the distribution of mouse survival times. We used the Mantel-Cox test to compare the survival curves among treatment groups. All statistical tests were performed using GraphPad Prism 5.0 (GraphPad Software, Inc). P values less than 0.05 were considered statistically significant.
This study was supported by the Canadian Cancer Society Research Institute. We also would like to thank Dr. Gregory M.K. Poon for assisting with the radiolabeling, cell-binding experimental design and data analysis, Eric Huang for the optimization of protein production and purification, and for helping with pharmacokinetic, biodistribution, imaging, and mouse survival studies, Erin Green, and Qing Hong Zhang, for the production and purification of proteins and assistance with mouse survival and immunogenicity work, Amaalia E. Broad and Andrew J. McCluskey for their technical expertise and help with mouse work, Doug Vines for assistance and advice with respect to mouse imaging, and Dr. Nenad Cicmil for help with protein modeling software. We also thank Dr. Raymond Reilly and members of his laboratory who engaged in helpful discussions with respect to the design and data analysis relating to our radiolabeling studies.
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