A deimmunised form of the ribotoxin, α-sarcin, lacking CD4+ T cell epitopes and its use as an immunotoxin warhead

Fungal ribotoxins that block protein synthesis can be useful warheads in the context of a targeted immunotoxin. α-Sarcin is a small (17 kDa) fungal ribonuclease produced by Aspergillus giganteus that functions by catalytically cleaving a single phosphodiester bond in the sarcin–ricin loop of the large ribosomal subunit, thus making the ribosome unrecognisable to elongation factors and leading to inhibition of protein synthesis. Peptide mapping using an ex vivo human T cell assay determined that α-sarcin contained two T cell epitopes; one in the N-terminal 20 amino acids and the other in the C-terminal 20 amino acids. Various mutations were tested individually within each epitope and then in combination to isolate deimmunised α-sarcin variants that had the desired properties of silencing T cell epitopes and retention of the ability to inhibit protein synthesis (equivalent to wild-type, WT α-sarcin). A deimmunised variant (D9T/Q142T) demonstrated a complete lack of T cell activation in in vitro whole protein human T cell assays using peripheral blood mononuclear cells from donors with diverse HLA allotypes. Generation of an immunotoxin by fusion of the D9T/Q142T variant to a single-chain Fv targeting Her2 demonstrated potent cell killing equivalent to a fusion protein comprising the WT α-sarcin. These results represent the first fungal ribotoxin to be deimmunised with the potential to construct a new generation of deimmunised immunotoxin therapeutics

In Vitro Quantification of Melanoma Tumor Cell Invasion

In order to quantify the invasiveness of melanoma tumor cells in vitro, a modification of the amniotic basement membrane (BM) model, described by Liotta et al. (Cancer Letters, 11, 141, 1980), was used in combination with radiolabeled tumor cells. B16-F10 metastatic murine melanoma cells and a derived clone (B16-F10L) were prelabeled with 0·1 μCi/ml of [14C]thymidine for 20-24 h in serum-free medium at 37°C. Following incubation, fetal bovine serum was added to a concentration of 5 per cent, and the cells were allowed to grow to confluency for the next 24-28 h. The labeled cells were seeded onto amniotic membranes situated in Membrane Invasion Culture System (MICS) chambers at a density of 2·5×104 per well. At various times points, radioactivity of tumor cells that completely traversed the membrane was determined using an under-the-membrane sampling method. The average percent invasion demonstrated by the B16-F10 line was 2·75 per cent, and 3·65 per cent exhibited by the B16-F10L cell line after 48-53 h in vitro. Since it was apparent that some variability in thickness existed among membrane samples, a morphological analysis was performed on five sectors of a three-inch-diameter sample from four different placentae. Differences and similarities in BM thickness within the same sector were noted by this technique and could possibly contribute to some variability observed in tumor cell invasion in this model. Another parameter examined was the proliferation of tumor cells in the upper and lower wells of the MICS chambers. By 48 h, approximately 32·1 per cent of the B16-F10 cell line as well as the clone had replicated in the upper wells associated with the BMs compared with a 32·9 per cent replication in the lower wells, which reaffirmed the viability of the tumor cells under experimental conditions and insured similarly replicating populations of cells. In order to quantify the invasiveness of radiolabeled tumor cells accurately through a biological membranous barrier, the proper concentration of cells must be used, tumor cell heterogeneity should be taken into consideration, the technique of sampling radiolabeled invasive cells should be critically analysed, and thickness of the membranous barrier should all be considered as possible important factors in the quantitative analyses

PET/CT imaging of mice bearing PC-3-tumors.

<p>Animals were imaged at 4 hours, 24 hours and 48 hours after injection of the ⁶⁴Cu-MeCOSar radiotracer for shWTCH2 and IgG. Shown are the maximum-intensity projections. The color scale for the PET image data shows radiotracer uptake with white corresponding to the highest activity and blue to the lowest activity. T: tumor; K: kidney; L: liver.</p

SUV of radiolabeled constructs in the tissues of mice bearing PC-3 tumors at 4 h, 24 h and 48 h after tracer injection.

<p>Results are presented as mean (+/-SD).</p