Human antibody derivatives against the fibroblast activation protein for tumor stroma targeting of carcinomas

Abstract

The fibroblast activation protein (FAP) is selectively expressed on activated fibroblasts of the tumor stroma on more than 90% of lung, breast and colon carcinomas. The high prevalence and abundance of FAP stroma make it a promising target for in vivo diagnosis and therapy of a variety of carcinomas. We describe the humanization of the murine FAP-specific MAb, F19, which has already been clinically used for in vivo diagnostic purposes. Using phage display technology and human V-repertoires, VL and VH regions of F19 were replaced by analogous human V-regions while retaining the original HCDR3 sequence in order to maintain F19 epitope specificity. The resulting human single-chain fragments of immunoglobulin variable regions (scFv 34, scFv 18) showed affinities of 6 nM on cell membrane-bound FAP. scFv 34 was expressed as a bivalent minibody (Mb 34). The antigen-binding characteristics of Mb 34 were comparable to the parental and a complementarity-determining region (CDR)-grafted version of F19. This was revealed by binding competition studies, FACS analyses and immunohistochemistry on various tumor samples including breast, colon and lung carcino-mas. Importantly, compared with the CDR-grafted human-ized scFv version of F19, the V-regions of the selected human scFv 34 showed sequence identity with the parental antibody (Ab) only over the short, 15-amino acid long HCDR3. Thus, a largely reduced xenoantigenic potential is expected. These human Ab derivatives are suitable to develop novel therapeutic concepts with broad applicability for a wide variety of histological carcinomas based on tumor stroma targeting. The growth of tumors (diameter 1-2 mm) is dependent on angiogenesis and blood supply, postulated already by Folkman in 1971 1 and experimentally proven in later years. 2,3 Moreover, a correlation between the degree of tumor vascularization and the formation of metastases has been observed. 4 Cells of the tumor-supplying tissue, in particular the endothelial cells of the tumor vasculature and stromal fibroblasts, are regarded as expedient targets. This is because they are non-transformed and thus considered as genetically stable and shared by practically all solid tumors. Markers selectively expressed on these cells are exploited for in vivo diagnostic and novel therapeutic approaches based on specific antibodies (tumor stroma targeting). The feasibility of this strategy is supported by studies with animal tumor models. 5,6 The FAP is considered as a suitable Ag for a stroma-targeting approach for several reasons: (1) in more than 90% of all epithelial tumors including lung, colorectal and breast carcinomas (primary tumors and metastases), the tumor stroma is FAP ; in post-natal non-tumor tissues, FAP is expressed during wound healing and on pancreatic cells; (2) stromal fibroblasts are non-transformed cells, making the rapid emergence of Ag loss variants and therapy resistance unlikely; (3) the FAP stromal compartment, commonly contributing approximately 50% to 90% of the tumor mass, presents an abundant target...

Antiplasmin-cleaving enzyme is a soluble form of fibroblast activation protein

“…The analyses identified 30 peptides from APCE and 21 from rFAP, equivalent to 48% and 30%, respectively, of the amino acid sequence predicted from FAP cDNA (Figure 3). APCE and rFAP were both purified using the F19 monoclonal antibody specific for FAP, 35 and both enzymes were recognized by the same antibody in Western blot analyses ( Figure 1B). Hence, data comparing amino acid sequences and antibody cross-reactivities support APCE being a soluble derivative of membrane-bound FAP.…”

Seprase: An overview of an important matrix serine protease

“…Several groups have developed antibodies directed against Seprase. Antibodies were developed using the Seprase specific mAbF19 as a model and humanising the antibody [101][102][103][104][105].…”

Androgen receptor signalling in prostate: Effects of stromal factors on normal and cancer stem cells

“…FAP is a cell surface glycoprotein with protease activity, whose expression is confined to tumour reactive stroma. FAP is expressed not only in CaP (Tuxhorn et al, 2002), but was also found to be expressed in more than 90% of lung, breast and colon carcinomas (Mersmann et al, 2001). FAP may also represent a pathway for a stromal targeted therapy by selective activation of a prodrug consisting of a FAP specific peptide substrate coupled to a potent cytotoxin to target FAP positive tumour stromal cells (Brennen et al, 2006).…”