The novel EpCAM-targeting monoclonal antibody 3-17I linked to saporin is highly cytotoxic after photochemical internalization in breast, pancreas and colon cancer cell lines.

The epithelial cell adhesion molecule (EpCAM) is expressed by a wide range of human carcinomas, making it an attractive diagnostic and therapeutic target in oncology. Its recent identification on cancer stem cells has raised further interest in its use for tumor targeting and therapy. Here, we present the characterization and therapeutic potential of 3-17I, a novel human EpCAM-targeting monoclonal antibody. Strong reaction of 3-17I was observed in all lung, colon, and breast human tumor biopsies evaluated. By flow cytometry and confocal fluorescence microscopy, we demonstrate that 3-17I specifically targets EpCAM-positive cell lines. We also show evidence for mAb-sequestration in endo-/lysosomes, suggesting internalization of 3-17I by receptor-mediated endocytosis. The ribosomal-inactivating toxin saporin was linked to 3-17I, creating the per se non-toxic immunotoxin 3-17I-saporin, a promising candidate for the drug delivery technology photochemical internalization (PCI). PCI is based on a light-controlled destruction of endolysosomal membranes and subsequent cytosolic release of the sequestered payload upon light exposure. EpCAM-positive human cancer cell lines MCF7 (breast), BxPC-3 (pancreas), WiDr (colon), and the EpCAM-negative COLO320DM (colon), were treated with 3-17I-saporin in combination with the clinically relevant photosensitizer TPCS2a (Amphinex), followed by exposure to light. No cytotoxicity was observed after treatment with 3-17I-saporin without light exposure. However, cell viability, proliferation and colony-forming capacity was strongly reduced in a light-dependent manner after PCI of 3-17I. Our results show that 3-17I is an excellent candidate for diagnosis of EpCAM-positive tumors and for development of clinically relevant antibody-drug conjugates, using PCI for the treatment of localized tumors.

Identification of scFv antibody fragments that specifically recognise the heroin metabolite 6-monoacetylmorphine but not morphine.

Use of phage display of recombinant antibodies and large repertoire naïve antibody libraries for identifying antibodies of high specificity has been extensively reported. Nevertheless, there have been few reported antibodies to haptens that have originated from naïve antibody libraries with potential use in diagnostics. We have used chain shuffling of lead single-chain fragment variable (scFv) antibodies, isolated from a naïve antibody library, to screen for antibodies that specifically recognise the major metabolite of heroin, 6-monoacetylmorphine (6MAM). The antibodies were identified by screening high-density colonies of Escherichia coli expressing soluble scFv antibody fragments without prior expression on bacteriophage (phage display). The antibodies recognise 6MAM with affinities of 1-3x10(-7) M with no crossreactivity to morphine. These antibodies can potentially be used for developing a rapid immunoassay in drug-testing programs. To our knowledge, this is the first report of an antibody that distinguishes 6MAM from its de-acetylated form, morphine.

Effect of domain order on the activity of bacterially produced bispecific single-chain Fv antibodies.

Bispecific single-chain Fv antibodies comprise four covalently linked immunoglobulin variable (VH and VL) domains of two different specificities. Depending on the order of the VH and VL domains and on the length of peptides separating them, the single-chain molecule either forms two single-chain Fv (scFv) modules from the adjacent domains of the same specificity, a so-called scFv-scFv tandem [(scFv)(2)], or folds head-to-tail with the formation of a diabody-like structure, a so-called bispecific single-chain diabody (scBsDb). We generated a number of four-domain constructs composed of the same VH and VL domains specific either for human CD19 or CD3, but arranged in different orders. When expressed in bacteria, all (scFv)(2) variants appeared to be only half-functional, binding to CD19 and demonstrating no CD3-binding activity. Only the diabody-like scBsDb could bind both antigens. Comparison of the scBsDb with a structurally similar non-covalent dimer (diabody) demonstrated a stabilizing effect of the linker in the middle of the scBsDb molecule. We demonstrated that the mechanism of inactivation of CD19xCD3 diabody under physiological conditions is initiated by a dissociation of the weaker (anti-CD3) VH/VL interface followed by domain swapping with the formation of non-active homodimers. The instability of one homodimer makes the process of diabody dissociation/reassociation irreversible, thus gradually decreasing the fraction of active molecules. The structural parameters influencing the formation of functional bispecific single-chain antibodies are indicated and ways of making relatively stable bispecific molecules are proposed.