An antibody-calmodulin fusion protein reveals a functional dependence between macromolecular isoelectric point and tumor targeting performance.
OBJECTIVE
Human monoclonal antibodies are promising agents for the development of improved anticancer therapeutics, because, unlike low-molecular-weight chemotherapeutic agents, they can selectively localize to solid tumors. In particular, the scFv(L19) antibody fragment, specific for the EDB domain of fibronectin, a marker of angiogenesis, has demonstrated an impressive tumor targeting performance in a variety of tumor-bearing animals and in patients with cancer. The purpose of this study was to develop a tumor pretargeting strategy, based on a novel anti-EDB fusion protein.


METHODS
We have fused the scFv(L19) to calmodulin, a small acidic protein for which specific binding peptides with a dissociation constant in the picomolar range are available. The resulting fusion protein has been expressed in mammalian cells and purified to homogeneity, before being characterized by quantitative biodistribution analysis in mice bearing the F9 murine teratocarcinoma.


RESULTS
Surprisingly, we have found that the fusion of scFv(L19) to calmodulin completely abrogated the tumor targeting ability of the antibody in vivo, although both scFv(L19) and calmodulin moieties within the fusion protein retained unaltered binding affinities toward their respective ligand. Furthermore, a systematic analysis of 13 derivatives of scFv(L19) recently produced in our laboratories showed that the 10 derivatives that retain the tumor targeting ability of the parental antibody have isoelectric points (pI) between 5.0 and 9.0, whereas scFv(L19)-calmodulin (pI = 4.49) and two other derivatives of scFv(L19) with pI >9.0 were unable to target tumors in vivo.


CONCLUSIONS
Because the EDB domain of fibronectin is a component of the modified extracellular matrix, predominantly located at the abluminal side of tumor blood vessels, our data suggest that extreme pI values of antibody-based pharmaceuticals may inhibit protein extravasation, perhaps by virtue of electrostatic interactions with endothelial cells and/or components of the extracellular matrix.
Melkko
Samu
S
Institute of Pharmaceutical Sciences, Swiss Federal Institute of Technology Zurich, Zurich, Switzerland.
Halin
Cornelia
C
Borsi
Laura
L
Zardi
Luciano
L
Neri
Dario
D
D000818:Animals; D000970:Antineoplastic Agents / Q000378:metabolism; D016466:CHO Cells; D002147:Calmodulin / Q000737:chemistry* / Q000378:metabolism; D003001:Cloning, Molecular; D006224:Cricetinae; D004797:Enzyme-Linked Immunosorbent Assay; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007457:Iodine Radioisotopes / Q000378:metabolism; D007525:Isoelectric Focusing; D051379:Mice; D008954:Models, Biological; D009389:Neovascularization, Pathologic; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000378:metabolism; D014018:Tissue Distribution; D014407:Tumor Cells, Cultured
D000970:Antineoplastic Agents; D002147:Calmodulin; D007457:Iodine Radioisotopes; D011993:Recombinant Fusion Proteins