A strategy for antitumor vascular therapy by targeting the vascular endothelial growth factor: receptor complex.

Vascular endothelial growth factor (VEGF) is produced by cancer cells in response to hypoxia and is the primary stimulant of vascularization in solid tumors. Endothelial cells lining the blood vessels of these tumors have a high concentration of receptor-bound VEGF on their surface, providing a target for antibody- directed cancer therapy. To obtain a cloned antibody to this target when bound to its receptor on tumor endothelium, we used phage display technology to create a single-chain Fv (sFv) antibody library from mice immunized with the 165-amino acid isoform of human VEGF-A. We selected, purified, and characterized LL4, an anti-VEGF sFv that was shown to react with receptor-bound VEGF. LL4 bound selectively to blood vessel endothelium, as shown by immunohistochemistry on tissue sections of human tumors. Furthermore, using autoradiography and grain counting of histological sections, systemically administered LL4 was shown to localize selectively to the endothelial lining of tumor blood vessels in human colorectal carcinoma xenografts in vivo. This study demonstrates the feasibility of targeting tumor vasculature using recombinant antibodies to the VEGF:receptor complex.

Identification of the C3b binding site in a recombinant vWF-A domain of complement factor B by surface-enhanced laser desorption-ionisation affinity mass spectrometry and homology modelling: implications for the activity of factor B.

Factor B is a key component of the alternative pathway of the complement system. During complement activation, factor B complexed with activated C3 is cleaved into the Ba and Bb fragments by the protease factor D to form the C3 convertase from the complex between C3b and Bb. The Ba fragment contains three short consensus/complement repeat (SCR) domains, and the Bb fragment contains a von Willebrand factor type A (vWF-A) domain and a serine protease (SP) domain. Surface-enhanced laser desorption-ionization affinity mass spectrometry (SELDIAMS) was used to investigate the reaction of factor B with immobilised activated C3(NH3) in the presence of Mg(2+). A recombinant vWF-A domain (residues G229-Q448), the native Ba and Bb fragments and native factor B all demonstrated specific interactions with C3(NH3), while no interactions were detected using bovine serum albumin as a control. A mass analysis of the proteolysis of the vWF-A domain when this was bound to immobilised C3(NH3) identified two peptides (residues G229-K265 and T355-R381) that were involved with vWF-A binding to C3(NH3). A homology model for the vWF-A domain was constructed using the vWF-A crystal structure in complement receptor type 3. Comparisons with five different vWF-A crystal structures showed that large surface insertions were present close to the carboxyl and amino edges of the central beta-sheet of the factor B vWF-A structure. The peptides G229-K265 and T355-R381 corresponded to the two sides of the active site cleft at the carboxyl edge of the vWF-A structure. The vWF-A connections with the SCR and SP domains were close to the amino edge of this vWF-A beta-sheet, and shows that the vWF-A domain can be involved in both C3b binding and the regulation of factor B activity. These results show that (i) a major function of the vWF-A domain is to bind to activated C3 during the formation of the C3 convertase, which it does at its active site cleft; and that (ii) SELDIAMS provides an efficient means of identifying residues involved in protein-protein interactions.

Effect of glycosylation of a synthetic MUC1 mucin-core-related peptide on recognition by anti-mucin antibodies.

Human epithelial mucins are heterogeneously glycosylated proteins associated with breast and ovarian cancer. Several peptide-reactive anti-mucin MUC1 monoclonal antibodies are used in experimental and diagnostic assays but it is not known how glycosylation of the mucin influences antibody recognition. In this report we show that increasing glycosylation of a synthetic 25-amino acid fragment of the MUC1 core protein with N-acetylgalactosamine (GalNAc) elicits different responses in its recognition by two anti-MUC1 antibodies, C595 and HMFG1. We propose that increasing glycosylation of the synthetic mucin fragment produces an alteration in the structure of the epitope which enhances binding in C595, but not in HMFG1.

Clinical applications of phage-derived sFvs and sFv fusion proteins.

Single chain Fv antibodies (sFvs) have been produced from filamentous bacteriophage libraries obtained from immunised mice. MFE-23, the most characterised of these sFvs, is reactive with carcinoembryonic antigen (CEA), a glycoprotein that is highly expressed in colorectal adenocarcinomas. MFE-23 has been expressed in bacteria and purified in our laboratory for two clinical trials; a gamma camera imaging trial using 123I-MFE-23 and a radioimmunoguided surgery trial using 125I-MFE-23, where tumour deposits are detected by a hand-held probe during surgery. Both these trials show MFE-23 is safe and effective in localising tumour deposits in patients with cancer. We are now developing fusion proteins which use MFE-23 to deliver a therapeutic moiety; MFE-23::CPG2 targets the enzyme carboxypeptidase G2 (CPG2) for use in the ADEPT (antibody directed enzyme prodrug therapy) system and MFE::TNF alpha aims to reduce sequestration and increase tumor concentrations of systemically administered TNF alpha.

Recombinant anti-carcinoembryonic antigen antibodies for targeting cancer.

Antibodies can be used to target cancer therapies to malignant tissue; the approach is attractive because conventional treatments such as chemo- and radiotherapy are dose limited due to toxicity in normal tissues. Effective targeting relies on appropriate pharmacokinetics of antibody-based therapeutics, ideally showing maximum uptake and retention in tumor and rapid clearance from normal tissue. We have studied the factors influencing these dynamics for antibodies against carcinoembryonic antigen (CEA). Protein engineering of anti-CEA antibodies, in vivo biodistribution models, and mathematical models have been employed to improve understanding of targeting parameters, define optimal characteristics for the antibody-based molecules employed, and develop new therapies for the clinic. Engineering antibodies to obtain the desired therapeutic characteristics is most readily achieved using recombinant antibody technology, and we have taken the approach of immunizing mice to provide high-affinity anti-CEA single-chain Fv antibodies (sFvs) from filamentous bacteriophage libraries. MFE-23, the most characterized of these sFvs, has been expressed in bacteria and purified in our laboratory for two clinical trials: a gamma camera imaging trial using 123I-MFE-23 and a radioimmunoguided surgery trial using 125I-MFE-23, where tumor deposits are detected by a hand-held probe during surgery. Both these trials showed that MFE-23 is safe and effective in localizing tumor deposits in patients with cancer. We are now developing fusion proteins that use the MFE-23 antibody to deliver a therapeutic moiety; MFE-23:: carboxypeptidase G2 (CPG2) targets the enzyme CPG2 for use in the antibody-directed enzyme prodrug therapy system and MFE::tumor necrosis factor alpha (TNFalpha) aims to reduce sequestration and increase tumor concentrations of systemically administered TNFalpha.

Purification of monoclonal antibodies by epitope and mimotope affinity chromatography.

Murine monoclonal antibodies raised against the carcinoma-associated MUC1 mucin have applications in the diagnosis and therapy of human cancer. Many of these antibodies define linear epitopes of three, four or five amino acids within an immunodominant region of the MUC1 protein core. Various synthetic peptides which incorporated this region were prepared and covalently linked to agarose beads for use as affinity matrices. An unrelated peptide was identified as a mimotope for one of the anti-MUC1 antibodies using phage display technologies and this was also evaluated as a potential ligand in an affinity matrix. Epitope affinity chromatographic purification of an anti-MUC1 antibody was performed using hybridoma tissue culture supernatants as sample. Following sample application and column washing, antibody was desorbed from the matrix by gradient elution with increasing concentrations of NaSCN. The procedure has proved efficient for the purification of anti-MUC1 antibodies and the concentration of NaSCN required for antibody desorption gives a measure of the relative binding affinity of the antibody for the peptide epitope matrix so that separation strategies may be optimised.

Design of ligands for the purification of anti-MUC1 antibodies by peptide epitope affinity chromatography.

The fine specificity of epitope recognition of the anti-MUC1 mucin monoclonal antibody, C595 has been studied using solid-phase replacement net (RNET) analysis. Two peptides (RAAP and RPPP) showed increased reactivity with C595 antibody compared with the native epitope (RPAP). These were synthesized as integral motifs within MUC1 immunodominant peptides and analyzed by fluorescence quenching (FQ) and circular dichroism (CD). They were also tested as ligands for the purification of C595 antibody using epitope affinity chromatography. Affinity matrices were compared with respect to capacity, affinity, and quality of the purified product. In FQ tests the native epitope peptide (APDTRPAPG) and the alanine substituted peptide had similar association constants when reacting with C595 antibody, whereas the proline substituted peptide (APDTRPPPG) had a higher association constant. This order of affinity for C595 was confirmed in chromatography experiments in which antibody was eluted from the former two peptide matrices at approximately the same point on the NaSCN elution gradient, whereas antibody was desorbed from APDTRPPPG at a higher NaSCN concentration. Circular dichroism analysis showed that the thermodynamically preferred conformation of these peptides in aqueous solution is the P-II extended helix, the conformation preferred for an extended bound form of the peptide held by interactions with the peptide amides. The stronger binding peptide (APDTRPPPG) has the higher population of the P-II helix in solution. In conclusion, RNET analysis is useful in the rational design of peptide ligands so that the performance of affinity matrices may be regulated.

Production and characterization of a recombinant anti-MUC1 scFv reactive with human carcinomas.

Recombinant single-chain fragments (scFv) of the murine anti-MUC1 monoclonal antibody C595 have been produced using the original hybridoma cells as a source of variable heavy (V(H))- and variable light (V(L))-chain-encoding antibody genes. The use of the polymerase chain reaction (PCR), bacteriophage (phage) display technology and gene expression systems in E. coli has led to the production of soluble C595 scFv. The scFv has been purified from the bacterial supernatant by peptide epitope affinity chromatography, leading to the recovery of immunoreactive C595 scFv, which was similar in activity to the C595 parent antibody. Analysis by DNA sequencing, SDS-PAGE and Western blotting has demonstrated the integrity of the scFv, while ELISA, FACScan analysis, fluorescence quenching, quantitative immunoreactivity experiments and immunohistochemistry confirm that the activity of the scFv compares favourably with that of the parent antibody. The retention of binding activity to MUC1 antigen on human bladder and breast carcinoma tissue specimens illustrates the potential application of this novel product as an immunodiagnostic and immunotherapeutic reagent.

Structure/activity studies of the anti-MUC1 monoclonal antibody C595 and synthetic MUC1 mucin-core-related peptides and glycopeptides.

MUC1 mucin is a large complex glycoprotein expressed on normal epithelial cells in humans and overexpressed and under or aberrantly glycosylated on many malignant cancer cells which consequently allows recognition of the protein core by antibodies. In order to understand how glycosylation may modulate or regulate antibody binding of mucin protein core epitopes, we have analyzed the antibody C595 (epitope RPAP) for its structure, stability, and its binding to a series of synthetic peptides and glycopeptides by a number of spectroscopic methods. Thermal and pH denaturation studies followed by changes in the CD spectrum of the antibody indicate critical involvement of specific residues to the stability of the antibody. Fluorescence binding studies indicate that alpha-N-acetylgalactosamine (GalNAc) glycosylation of a MUC1 mucin synthetic peptide TAPPAHGVT9SAPDTRPAPGS20T21APPA at threonine residues 9 and 21 and serine residue 20 enhanced the binding of antibody. The structural effects of GalNAc glycosylation on the conformation of the MUC1 peptide were studied. CD of the peptides and glycopeptides in a cryogenic mixture cooled to approximately -97 degrees C revealed that a left-handed polyproline II helix (PPII) is adopted by the peptides in solution, which appears to be further stabilized by addition of the GalNAc residues. Consistent with the PPII helical structure, which has no intra-amide hydrogen bonds, high-field NMR spectroscopy of the glycopeptide revealed no sequential dNN, medium-range, or long-range nuclear Overhauser effect (NOE) connectivities. These studies indicate that stabilization of the PPII helix by GalNAc glycosylation present the epitope of C595 antibody with a favorable conformation for binding. Furthermore, they illustrate that glycosylation of the MUC1 tumor marker protein with a simple O-linked saccharide expressed in many cancers, can enhance the binding of the clinically relevant C595 antibody.