Generation of human high‐affinity antibodies specific for the fibroblast activation protein by guided selection

Schmidt, Alexej; Müller, Dafne; Mersmann, Michael; Wüest, Thomas; Gerlach, Elke; Garin‐Chesa, Pilar; Rettig, Wolfgang; Pfizenmaier, Klaus; Moosmayer, Dieter

doi:10.1046/j.1432-1327.2001.02046.x

Cited by 37 publications

(22 citation statements)

References 39 publications

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“…It shows the enzyme to have an active-site within a tube-like structure that courses through each monomeric half with apertures of 14 and 24 diameters at the ends of each tube. Although previously stated that FAP or its derivatives do not circulate in blood [6], we concluded that plasma APCE, which lacks the cytoplasmic tail and transmembrane sequence of FAP, may indeed result from FAP [7], possibly as a consequence of unidentified cell membrane "sheddase" activity as occurs with certain other membrane peptidases [8;9], or that it might be synthesized and secreted as a non-membrane-bound variant of FAP. To date Met-α 2 AP is the only definitive physiologic substrate identified for native APCE, and it is also cleaved by recombinant FAP [1;7].…”

Section: Introductionmentioning

confidence: 66%

“…Recent studies from our laboratory strongly suggest that the human plasma prolyl serine proteinase, termed antiplasmin cleaving enzyme (APCE), could be a derivative of cell membrane FAP or possibly, an alternately spliced gene product [1;7]. FAP is reported to not circulate [6], yet it is functionally indistinguishable from plasma-derived APCE despite the latter's lacking the intra-membrane and cytoplasmic aminoterminal domains of FAP [7]. Our ability to prepare relatively large amounts of homogeneously pure APCE from human plasma, and recombinant FAP from a yeast expression system [1;7], allowed us to examine a range of well-defined concentrations for either enzyme's proteolytic activity towards purified collagens I, III and IV.…”

Section: Discussionmentioning

confidence: 99%

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Effect of fibroblast activation protein and α2-antiplasmin cleaving enzyme on collagen Types I, III, and IV

Christiansen

Jackson

Lee

et al. 2007

Archives of Biochemistry and Biophysics

110

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The circulating enzyme, α 2 -antiplasmin cleaving enzyme (APCE), has very similar sequence homology and proteolytic specificity as fibroblast activation protein (FAP), a membrane-bound proteinase. FAP is expressed on activated fibroblasts associated with rapid tissue growth as in embryogenesis, wound healing, and epithelial-derived malignancies, but not in normal tissues. Its presence on stroma suggests that FAP functions to remodel extracellular matrix (ECM) during neoplastic growth. Precise biologic substrates have not been defined for FAP, although like APCE, it cleaves α 2 -antiplasmin to a derivative more easily crosslinked to fibrin. While FAP has been shown to cleave gelatin, evidence for cleavage of native collagen, the major ECM component, remains indistinct. We examined the potential proteolytic effects of FAP or APCE alone and in concert with selected matrix metalloproteinases (MMPs) on collagens I, III and IV. SDS-PAGE analyses demonstrated that neither FAP nor APCE cleaves collagen I. Following collagen I cleavage by MMP-1, however, FAP or APCE digested collagen I into smaller peptides. These peptides were analogous to, yet different from, those produced by MMP-9 following MMP-1 cleavage. Aminoterminal sequencing and mass spectrometry analyses of digestion mixtures identified several peptide fragments within the sequences of the two collagen chains. The proteolytic synergy of APCE in the cleavage of collagen I and III was not observed with collagen IV. We conclude that FAP works in synchrony with other proteinases to cleave partially degraded or denatured collagen I and III as ECM is excavated, and that derivative peptides might function to regulate malignant cell growth and motility.

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Section: Introductionmentioning

confidence: 66%

Section: Discussionmentioning

confidence: 99%

Effect of fibroblast activation protein and α2-antiplasmin cleaving enzyme on collagen Types I, III, and IV

Christiansen

Jackson

Lee

et al. 2007

Archives of Biochemistry and Biophysics

110

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“…Therefore, we next investigated the potential of targeted immobilization of CD27L, CD40L, 41BBL, and GITRL. To this end, soluble variants of the various ligands were fused to the scFv Ab fragment sc40 that specifically recognizes the tumor stroma Ag fibroblast activation protein (FAP) (23,24) (Fig. 6, A and B).…”

Section: Trimeric Scfv Fusion Proteins Gain High Activity After Cell mentioning

confidence: 99%

Trimer Stabilization, Oligomerization, and Antibody-Mediated Cell Surface Immobilization Improve the Activity of Soluble Trimers of CD27L, CD40L, 41BBL, and Glucocorticoid-Induced TNF Receptor Ligand

Wyzgol

Müller

Fick

et al. 2009

The Journal of Immunology

Self Cite

126

130

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For many ligands of the TNF family, trimer stability and oligomerization status are crucial determinants of receptor activation. However, for the immunostimulatory ligands CD27L, CD40L, 41BBL, and glucocorticoid-induced TNF receptor ligand (GITRL) detailed information regarding these requirements is lacking. Here, we comprehensively evaluated the effect of trimer stability and oligomerization on receptor activation by these ligands. Treatment with soluble Flag-tagged CD27L, 41BBL, and GITRL minimally activated receptor signaling, while Flag-CD40L was highly active. Oligomerization with anti-Flag Abs further enhanced the specific activity of Flag-CD40L 10-fold and of Flag-41BBL more than 200-fold, but it failed to activate Flag-CD27L and Flag-GITRL. We next investigated the relevance of trimer stability by introducing the tenascin-C (TNC) trimerization domain, yielding stabilized Flag-TNC-ligand trimers. Oligomerization with anti-Flag Ab potently activated signaling by Flag-TNC-CD27L and Flag-TNC-GITRL and, albeit to a lesser extent, Flag-TNC-CD40L and Flag-TNC-41BBL. Forced hexamerization, by introducing an Ig Fc domain, revealed that hexameric derivatives of Flag-TNC-41BBL, Flag-CD40L, and Flag-TNC-GITRL all activate receptor signaling with high efficiency, whereas hexameric Flag-CD27L variant left inactive. Finally, we attempted to selectively activate receptor signaling on targeted cells, by using Ab fragment (single-chain fragment variable region, scFv)-ligand fusion proteins, an approach previously applied to other TNF ligands. Target cell surface Ag-selective activation was achieved for scFv-41BBL, scFv-CD40L, and scFv-GITRL, although the latter two displayed already significant activity toward Ag-negative cells. In conclusion, our data establish that trimeric CD40L is active, 41BBL requires hexamerization, GITRL requires trimer stabilization, and CD27L requires trimer stabilization and oligomerization. Furthermore, surface immobilization might be exploited to gain locally enhanced ligand activity.

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“…Binding-competition studies ELISA binding-competition studies were performed to see whether the binding epitope of A6 was similar to that of TRAIL (15). Briefly, polystyrene plates were coated with 2 μg/ml purified DR5 in 0.05 M bicarbonate buffer (pH 9.6) at 37°C for 1 h and blocked with 10% FBS/PBS for 1 h at 37°C.…”

Section: Cell Lines and Reagentsmentioning

confidence: 99%

The Characteristic of an Anti-Human DR5 Antibody A6

Wang¹,

Zhao

Chen

et al. 2008

Cell Mol Immunol

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The efficacy of many cancer treatments is due to their ability to induce apoptosis. DR5 can activate apoptosis pathway after binding with its natural ligand, tumour necrosis factor-related apoptosis-inducing ligand (TRAIL/ Apo2L). Both TRAIL and agonistic anti-DR5 monoclonal antibody are currently being explored for cancer therapy. The mechanisms of cytotoxicity of our previously prepared monoclonal antibody A6 against DR5 were investigated here. A6 could cause viability loss of Jurkat cells in both time-and dose-dependent manner which could be attributed to the activation of apoptosis pathway.

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Generation of human high‐affinity antibodies specific for the fibroblast activation protein by guided selection

Cited by 37 publications

References 39 publications

Effect of fibroblast activation protein and α2-antiplasmin cleaving enzyme on collagen Types I, III, and IV

Effect of fibroblast activation protein and α2-antiplasmin cleaving enzyme on collagen Types I, III, and IV

Trimer Stabilization, Oligomerization, and Antibody-Mediated Cell Surface Immobilization Improve the Activity of Soluble Trimers of CD27L, CD40L, 41BBL, and Glucocorticoid-Induced TNF Receptor Ligand

The Characteristic of an Anti-Human DR5 Antibody A6

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