Species-crossreactive scFv against the tumor stroma marker "fibroblast activation protein" selected by phage display from an immunized FAP-/- knock-out mouse.
BACKGROUND
Fibroblast activation protein (FAP) is a type II membrane protein expressed on tumor stroma fibroblasts in more than 90% of all carcinomas. FAP serves as a diagnostic marker and is potential therapeutic target for treatment of a wide variety of FAP+ carcinomas. Murine tumor stroma models and FAP-specific antibodies are required to investigate the functional role of FAP in tumor biology and its usefulness for drug targeting. We here describe the development of antibodies with crossreactivity for human (hFAP) and murine FAP (mFAP), which share 89% amino acid identity.


METHODS
An FAP-/- mouse was sequentially immunized with recombinant murine and human FAP-CD8 fusion proteins. Immunoglobulin cDNA derived from hyperimmune spleen cells was used for the construction of a combinatorial single chain Fv (scFv) library. Phage display selection of FAP-specific scFv was performed on immobilized hFAP followed by selection on cells expressing murine FAP.


RESULTS
High-affinity, species-crossreactive, FAP-specific scFv were isolated upon sequential phage display selection. A bivalent derivative (minibody M036) constructed thereof was applied for immunohistochemical analyses and allowed detection of FAP expression on stroma cells of different human carcinomas as well as on murine host stroma in a tumor xenograft model.


CONCLUSIONS
MB M036, derived from phage display selected species crossreactive scFv, is suitable for tumor stroma targeting and will be a valuable tool in the analyses of the functional role of FAP in tumor biology as well as in the evaluation of the suitability of FAP for drug targeting.
Brocks
B
B
Institut für Zellbiologie und Immunologie, Universität Stuttgart, Germany.
Garin-Chesa
P
P
Behrle
E
E
Park
J E
JE
Rettig
W J
WJ
Pfizenmaier
K
K
Moosmayer
D
D
D000595:Amino Acid Sequence; D000818:Animals; D000951:Antigens, Neoplasm; D001483:Base Sequence; D014408:Biomarkers, Tumor / Q000235:genetics / Q000276:immunology; D003429:Cross Reactions; D017931:DNA Primers / Q000235:genetics; D010450:Endopeptidases; D018093:Gelatinases; D006133:Growth Substances / Q000235:genetics* / Q000276:immunology*; D006801:Humans; D007114:Immunization; D007135:Immunoglobulin Variable Region / Q000235:genetics* / Q000302:isolation & purification*; D008565:Membrane Proteins; D051379:Mice; D018345:Mice, Knockout; D008819:Mice, Nude; D008969:Molecular Sequence Data; D009369:Neoplasms / Q000235:genetics / Q000276:immunology; D009374:Neoplasms, Experimental / Q000235:genetics / Q000276:immunology; D019151:Peptide Library; D016133:Polymerase Chain Reaction; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000276:immunology; D012697:Serine Endopeptidases / Q000235:genetics* / Q000276:immunology*; D013045:Species Specificity; D014183:Transplantation, Heterologous; D000099167:Fibroblast Activation Protein Alpha
D000951:Antigens, Neoplasm; D014408:Biomarkers, Tumor; D017931:DNA Primers; D010450:Endopeptidases; D018093:Gelatinases; D006133:Growth Substances; D007135:Immunoglobulin Variable Region; D008565:Membrane Proteins; D019151:Peptide Library; D011993:Recombinant Fusion Proteins; D012697:Serine Endopeptidases; D000099167:Fibroblast Activation Protein Alpha