Engineering of human coagulation factor x variants activated by prostate-specific antigen.

In this study, we present a novel approach for the induction of tumor vessel thrombosis using genetically modified coagulation factor X. Human factor X was engineered in its activation peptide in a way that it can be specifically activated by prostate-specific antigen (PSA), a tumor-specific proteinase secreted into the bloodstream by prostate cancer cells. For this purpose we inserted different sequences of known PSA cleavage sites from the natural substrate of PSA, semenogelin I, into the activation peptide of factor X. One FX variant (FX-V4) was further optimized by site-directed mutagenesis of the P2 position and the P5 position (FX-V4-P2YP5R). After preincubation with PSA, FX-V4-P2YP5R was able to efficiently induce coagulation in vitro. These FX variants should be useful for site-specific induction of blood coagulation in the tumor vasculature.

Novel RGD lipopeptides for the targeting of liposomes to integrin-expressing endothelial and melanoma cells.

RGD peptides targeting alphav-integrins are promising ligands for the generation of vascular targeting agents. We isolated from phage display RGD motif libraries novel high-affinity cyclic RGD peptides by selection on either endothelial or melanoma cells. Although the starting sequences contained only two cysteine residues flanking the RGD motif, several of the isolated peptides possessed four cysteine residues. A high-affinity peptide (RGD10) constrained by only one disulfide bond was used to generate novel lipopeptides composed of a lipid anchor, a short flexible spacer and the peptide ligand conjugated to the spacer end. Incorporation of RGD10 lipopeptides into liposomes resulted in specific and efficient binding of the liposomes to integrin-expressing cells. In vivo experiments applying doxorubicin-loaded RGD10 liposomes in a C26 colon carcinoma mouse model demonstrated improved efficacy compared with free doxorubicin and untargeted liposomes.

Recombinant bispecific antibodies for the targeting of adenoviruses to CEA-expressing tumour cells: a comparative analysis of bacterially expressed single-chain diabody and tandem scFv.

We have generated two distinct recombinant bispecific antibody molecules for the retargeting of adenoviral vectors to CEA-expressing tumour cells. These antibody molecules were produced by combining the antigen-binding sites of a neutralising anti-fibre knob scFv (S11) and an anti-CEA antibody either in a single-chain diabody format (scDb CEA-S11) or a tandem scFv format (taFv CEA-S11). In order to facilitate expression of taFv CEA-S11 in bacteria we selected from a phage display library taFv molecules with an optimised linker that connects the two scFv fragments. ScDb CEA-S11 and taFv CEA-S11 were expressed and purified in soluble form from the bacterial periplasm with yields of approximately 100 micro g per litre of bacterial culture. In vitro, both bispecific molecules mediated selective and enhanced transduction of CEA-expressing tumour cells by recombinant adenoviruses. These assays did not reveal any differences in efficiency of adenoviral transduction by the two antibody formats. However, compared with taFv CEA-S11, scDb CEA-S11 exhibited a 2- to 3-fold increased stability in human plasma at 37 degrees C. In summary, we could demonstrate that both formats are suitable for adenovirus targeting to tumour cells with similar biological activity in vitro.

Targeting of immunoliposomes to endothelial cells using a single-chain Fv fragment directed against human endoglin (CD105).

We generated immunoliposomes targeting proliferating endothelial cells by chemically coupling a single-chain Fv fragment (scFv A5) directed against human endoglin to the liposomal surface. For this purpose, we introduced an additional cysteine residue at the C-terminus of the scFv fragment. This scFv' fragment was expressed in soluble form in bacteria and allowed for a site-directed coupling to sulfhydryl-reactive lipids incorporated into the lipid bilayer. The immunoliposomes (ILA5) showed rapid and strong binding to human endoglin-expressing endothelial cells (HUVEC, HDMEC), while no binding was observed with various endoglin-negative cell lines and blood lymphocytes. In vitro, ILA5 were stable for several hours in serum- or plasma-containing medium. Incubation of endothelial cells with ILA5 at 37 degrees C led to increased binding and internalisation of the liposomes as evidenced by a perinuclear accumulation. In vitro, doxorubicin-loaded ILA5 showed an increased cytotoxicity towards endothelial cells compared to untargeted liposomes and free doxorubicin. Since the vasculature of tumours is easily accessible to drug carrier systems, the described endothelial cell-specific immunoliposomes may be useful for the development of efficacious and safe vascular targeting agents in cancer therapy.

Isolation of endothelial cell-specific human antibodies from a novel fully synthetic scFv library.

We have isolated single-chain Fv fragments directed against human endothelial cells from a novel fully synthetic human scFv library (scFv 479). This library was constructed using the variable germline segments DP47 and DPkappa9 as scaffolds. Complementarity determining regions 3 (CDR) of the variable heavy and light chain were introduced with a length of 9 amino acid residues. In total, 16 amino acid positions of all six CDRs exposed in the antigen-binding site were randomized and the library was produced from synthetic oligonucleotides encoding the entire scFv fragment. From this library endothelial-specific scFv fragments were either selected using the recombinant extracellular domain of human endoglin (CD105) or by cell selections with human dermal microvascular endothelial cells (HDMEC). These scFv fragments might be useful for the generation of vascular or tumor targeting agents in cancer therapy.

Isolation from phage display libraries of lysine-deficient human epidermal growth factor variants for directional conjugation as targeting ligands.

Ligand-targeted anticancer therapeutics represent an opportunity for the selective and efficient delivery of drugs to tumours. The chemical coupling of ligands to drugs or drug carrier systems is, however, often hampered by the presence of multiple reactive groups within the ligand, for example, epsilon-NH(2) groups in lysine side chains. In this paper, we describe the isolation by phage display of human epidermal growth factor (EGF) variants without lysine and a reduced number of arginine residues. The selection on A431 carcinoma cells also revealed that R41 is indispensable for EGF binding activity as all EGF variants contained an arginine residue at this position. One EGF variant (EGFm1) with K28Q, R45S, K48S and R53S mutations was expressed in bacteria and showed an identical binding activity as wild-type EGF. EGFm1 could be labelled with fluorescein isothiocyanate demonstrating the accessibility of the N-terminal amino group for coupling reagents. Furthermore, coupling of EGFm1 to PEGylated liposomes resulted in target cell-specific binding and internalization of the liposomes. These human EGF variants should be advantageous for the generation of anticancer therapeutics targeting the EGF receptor, which is overexpressed by a wide variety of different tumours.