An evolved ribosome-inactivating protein targets and kills human melanoma cells in vitro and in vivo

Abstract

Background: 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-1A IYSNKLM) allowing the toxin variant to selectively target and kill human melanoma cells.

Results: SLT-1A IYSNKLM 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-1A IYSNKLM administered intravenously into SCID mice bearing a human melanoma xenograft indicate that SLT-1AI YSNKLM readily accumulates at the tumor site as opposed to non-target tissues. Furthermore, the co-administration of SLT-1A IYSNKLM with DTIC resulted in tumor regression and greatly increased survival in this mouse xenograft model in comparison to DTIC or SLT-1A IYSNKLM treatment alone (115 day median survival versus 46 and 47 days respectively; P values < 0.001). SLT-1A IYSNKLM is stable in serum and its intravenous administration resulted in modest immune responses following repeated injections in CD1 mice.

Conclusions: 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-1A IYSNKLM can specifically kill human melanoma cells in vitro and in vivo.

Figure 1

The scRIP SLT-1 A subunit served as a scaffold for the design and construction of an embedded 7-amino acid peptide combinatorial SLT-1 A subunit library. (A) Schematic diagram of the SLT-1 A subunit. Furin cleavage occurs within its protease-sensitive loop (C242 to C261) between residues 251 and 252 (FCS 251/252) of the A subunit [44]. A random 7-amino acid peptide library was inserted between residues 245 and 246, generating cytotoxic A subunit variants with a surface-displayed library insert. Catalytic residues are shown in red. (B) Modeled surface representations of the SLT-1 A subunit with and without a 7-residue long insertion. The models were derived from the crystal structure of Shiga Toxin [45]. The peptide insertion (blue) is solvent-exposed and positioned away from residues involved in catalysis (residues Y77, Y114, E167, R170, W203 colored in red) [20,46,47]. Models were rendered using PyMOL Software Version 1.0r1.

Figure 2

Screening of the SLT-1 A subunit library yielded a toxin variant, SLT-1A^IYSNKLM, displaying cytotoxicity towards human melanoma cell lines. (A) Dose-response curves illustrating the specific cytotoxicity of SLT-1A^IYSNKLM (black diamonds, black triangles, black squares, black circles) compared to wt SLT-1A (white diamonds, white triangles, white squares, white circles) for the 518-A2 human melanoma cell line (black squares, white circles). The carcinoma cell lines CAMA-1 (black diamonds, white diamonds; human breast), HepG2 (black triangles, white triangles; human liver), and PC-3 (black squares, white squares; human prostate) are insensitive to SLT-1A^IYSNKLM. (B) Dose-response curves illustrating the cytotoxicity profile of SLT-1A^IYSNKLM towards 8 different human melanoma cell lines: 518-A2 (black circles), A-375 (black inverted triangles), SK-Mel-28 (white circles), MeWo (black squares), A-2058 (white inverted triangles), MALME-3 M (white triangles), SK-Mel-2 (white squares), and C-32 (black triangles) as well as human melanocytes (white diamonds). Error bars represent s.e.m. for experiments performed in quadruplicate.

Figure 3

The cellular activities of SLT-1A^IYSNKLM are linked to its catalytic activity. (A) Dose-response curves indicating a comparable cytotoxicity for the purified, furin-cleaved A subunit of SLT-1A^IYSNKLM (A₁ and A₂ subunits remain linked by a disulfide bridge between C242 and C261; white squares) and the A₁ subunit alone of SLT-1A^IYSNKLM (white circles) towards 518-A2 cells. The catalytically-inactive forms (Y77S) of these same molecules (black squares, black circles) were not cytotoxic towards 518-A2 human melanoma cells at concentrations of up to 10 μM. (B) Western blot analysis demonstrating the cleavage of PARP following treatment of 518-A2 cells with SLT-1A^IYSNKLM. (C) Effect of exposing 518-A2 (black bars) and PC-3 cells (grey bars) to SLT-1A^IYSNKLM on the activation of caspase-3 as measured using a fluorescent peptide substrate for caspase-3. PC-3 cells are insensitive to SLT-1A^IYSNKLM. Error bars represent s.e.m. for experiments performed in quadruplicate.

Figure 4

Binding of ¹²⁵I-SLT-1A^IYSNKLM to 518-A2 cells. (A) Specific binding (white squares), total binding (black squares), and non-specific binding (white triangles) of ¹²⁵I-SLT-1A^IYSNKLM to 518-A2 cells at 4°C. (B) Displacement curves in which 518-A2 cells were treated with 45 nM of ¹²⁵I-SLT-1A^IYSNKLM in the presence of increasing concentrations of either unlabeled SLT-1A^IYSNKLM (black circles) or a synthetic 16-residue peptide containing the inserted 7-amino acid peptide ligand (HHHIYSNKLMASRVAR) (black squares). Data points represent the s.e.m. of experiments performed in triplicate.

Figure 5

SLT-1A^IYSNKLM generates modest IgG immune responses in CD1 mice. (A) Injection schedule of SLT-1A^IYSNKLM with and without adjuvant. Treatments consisted of i.v. injections of SLT-1A^IYSNKLM prepared in saline into CD1 mice as a series of 5 consecutive (daily) tail vein injections or a single s.c. injection of SLT-1A^IYSNKLM in Complete Freund's adjuvant (CFA) followed by two s.c. boosts of the antigen in Incomplete Freund's adjuvant (IFA) at days 21 and 35. (B) Histogram illustrating the average IgG immune responses (1:1000 titer dilution) from 3 CD1 mice injected with SLT-1A^IYSNKLM in the presence (black bars) or absence of adjuvant (white bars) as measured by ELISA at 405 nm. (C) Representative IgG immune responses (average ELISA signals at 405 nm versus antisera titers; day 42) engendered for groups of three CD1 mice after injection with SLT-1A^IYSNKLM in the presence (white circles) or absence of adjuvant (black circles). Data represent the s.e.m. of experiments performed in triplicate.

Figure 6

In vivo blood clearance rate and tumor-targeting of ¹²⁵I-SLT-1A^IYSNKLM (A) Blood clearance of ¹²⁵I-SLT-1A^IYSNKLM shown as percentage of injected dose per mL blood collected at various time points over a 72 h period post-i.v. injection. The clearance rate (t_1/2) of ¹²⁵I-SLT-1A^IYSNKLM was shown to be ~11 min. (B) Biodistribution per collected gram of wet tissue of ¹²⁵I-SLT-1A^IYSNKLM after 1 h (black bars), 6 h (light grey bars), 12 h (dark grey bars) and 24 h (white bars) post-i.v. administration. (C) Tumor localization of ¹²⁵I-SLT-1A^IYSNKLM versus wt ¹²⁵I-SLT-1A (negative control) as shown by nanoSPECT and CT imaging. The composite images show the tissue uptake of the radiolabeled proteins (as pink colored areas) as well as the location of tumor xenografts (white circles) and that of kidneys (grey arrows).

Figure 7

In vivo results following treatment regimens in SCID mice harboring established 518-A2 tumor xenografts. The regimens included 10-day, daily injections of a saline control (i.v) (white circles) or SLT-1A^IYSNKLM (i.v. 0.5 mg/kg) (black circles), or a 5-day course of DTIC (i.p. 8 mg/kg) (white squares), or the combination of both SLT-1A^IYSNKLM (i.v. 0.5 mg/kg) with DTIC (i.p. 8 mg/kg) regimens (black squares), (n = 7). (A) Mice showed no change in body weight related to treatment regimens over time. (B) Measured tumor volumes of mice demonstrate a significant synergistic effect of combining SLT-1A^IYSNKLM and DTIC treatments when compared to either treatment alone or to the saline control (P < 0.0001). (C) Kaplan-Meier plot comparing animal survival as a function of treatment regimens (ten-day regimen; black bar).