6. Narra K, Mullins SR, Lee HO, Strzemkowski-Brun B, Magalong K,Christiansen VJ, et al. Phase II trial of single agent Val-boroPro(talabostat) inhibiting fibroblast activation protein in patients withmetastatic colorectal cancer. Cancer Biol Ther 2007;6:1691–9.

7. Kelly T. Fibroblast activation protein-� and dipeptidyl peptidase IV(CD26): cell-surface proteases that activate cell signaling and arepotential targets for cancer therapy. Drug Resist Updat 2005;8:51–8.

8. Adams S, Miller GT, Jesson MI, Watanabe T, Jones B, Wallner BP.PT-100, a small molecule dipeptidyl peptidase inhibitor, has potentantitumor effects and augments antibody mediated cytotoxicity viaa novel immune mechanism. Cancer Res 2004;64:5471–80.

DOI 10.1002/art.27675

Reply

To the Editor:We thank Mr. Jakobs and Dr. Fritsche for raising

several important issues, and we appreciate their valuablecomments. In our study, we investigated the functional role ofthe serine protease activity of FAP and DPP-4/CD26 in theinvasion of RASFs into articular cartilage. FAP and DPP-4/CD26, together with more recently identified members of thedipeptidyl peptidase gene family, DPP-8 and DPP-9, are likelyto be multifunctional. They exhibit both enzymatic and extra-enzymatic (protein–protein interaction) modes of action. DPP-4/CD26 and FAP are closely related cell surface enzymes withDPP-4–like enzyme activity, and pharmacologic inhibition ofDPP-4–like protease activity has placed value on the structure

and function of DPP-4 gene family members (1). PT-630 is aL-glutamyl L-boroproline dipeptide that does not permeatecell membranes and appears to be restricted to inhibitingextracellular DPPs (FAP and DPP-4/CD26). It competitivelyinhibits FAP and DPP-4 with Ki in the nanomolar range and,in contrast to glycin-boroproline, as mentioned by Jakobs andFritsche, application of PT-630 by oral gavage for inhibition ofFAP and DPP-4/CD26 enzyme activity is feasible and welldocumented (2,3) (US patent application publication no. 2007/0072830A; FAP with a 50% inhibition concentration of 23 nMand DPP-4 with a 50% inhibition concentration of 3 nM).Enzyme activity remains inhibited at 80% for 48 hours after asingle dose of PT-630, and dosing twice daily ensures thechemical knockout of DPP-4–like activity mediated by FAPand DPP-4/CD26 in vivo. Similar to the findings of othergroups (2), we did not observe any changes in FAP orDPP-4/CD26 expression at the protein or gene level uponadministration of PT-630. We clearly demonstrated that theinhibition of DPP-4–like activity in vitro led to increased levelsof SDF-1 in concert with its downstream effectors MMP-1 andMMP-3. In experiments using the SCID mouse coimplantationmodel of RA, inhibition of DPP-4–like activity in vivo pro-moted the invasion of RASFs into human cartilage. Zones ofcartilage resorption were infiltrated by FAP-expressing RASFsthat were marked by significantly increased accumulation ofMMP-1 and MMP-3. We therefore concluded that there is acentral role for the serine protease activity (DPP-4–like activ-ity) of FAP and DPP-4/CD26 in protecting articular cartilageagainst invasion by RASFs.

Figure 1. Hypothetical model of activated fibroblasts expressing fibroblast activation protein(FAP) and matrix metalloproteinases (MMPs) on invadopodia and their mutual activation cascadein the setting of rheumatoid arthritis (RA) and giant cell bone tumor (GCT). TNF� � tumornecrosis factor �; IL-1 � interleukin-1; VEGF � vascular endothelial growth factor. Reproduced,with permission, from ref. 2.

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Jakobs and Fritsche’s characterization of our conclu-sion, however, is not complete, and unfortunately not precise.It is difficult to compare the inhibitory effects of PT-100, whichinhibits both intra- and extracellular DPP activities in anonselective manner, with those of PT-630. Indeed, we inves-tigated the effects of PT-100 on RASF behavior in vitro and invivo. When treating patients with PT-100, we did not observesignificant changes in chemokine or MMP profiles. Mostimportantly, using the SCID mouse coimplantation model ofRA, we found no significant difference between treatment andcontrol groups in articular cartilage invasion by RASFs (datanot shown).

In addition, the effects of treatment with PT-100, as citedby Jakobs and Fritsche, were observed in a sarcoma model. It iswell documented that, in regard to tumor entities, the biologicrole of FAP is still not clear and its expression sometimes evenappears to be paradoxical. A recent report on FAP-null miceshowed that a lack of FAP expression even resulted in reducedgrowth of syngeneic tumors (2). FAP expression causes eitherpromotion or suppression of tumor growth (4). In the instances oftumor promotion, FAP expression drives tumor growth by in-creased angiogenesis; in the case of suppression, FAP restores cellcontact inhibition, even when it is catalytically inactive. Notably, aphase II trial of single-agent PT-100 in patients with metastaticcolorectal cancer demonstrated only minimal clinical activity (5)and further clinical development of this drug is questionable.Concerning the extra-enzymatic activities of FAP and DPP-4, wealso discussed in detail that protein–protein interactions mediateeffects that contribute to cell proliferation and invasion, indepen-dent of DPP-4–like activity.

In the present article, we focused on the role of FAPand DPP-4 protease activity and did not examine the rolesof FAP complex formation with other surface molecules inactivating cell signaling. We therefore stated in our sum-mary that our data lay the groundwork for therapeuticstrategies targeting the non-catalytic functions of DPP-4/CD26 or FAP for the development of novel treatmentroutes.

Drs. Bauer and Renner have a patent pending for the expression offibroblast activation protein by rheumatoid myofibroblast-like synoviocytes.

Stefan Bauer, MDNational Center for Tumor DiseasesHeidelberg, GermanyCaroline Ospelt, MD, PhDChristoph Renner, MDUniversity HospitalZurich, Switzerland

1. Wolf B, Clifford Q, Tran T, Wiesmann C, Sutherlin D. On theedge of validation: cancer protease fibroblast activation protein.Med Chem 2008;8:719–27.

2. Santos AM, Jung J, Aziz N, Kissil JL, Pure E. Targeting fibroblastactivation protein inhibits tumor stromagenesis and growth inmice. J Clin Invest 2009;119:3613–25.

3. Narra K, Lee HO, Lerro A, Valvardi J, Azeez O, Jesson M, et al.Inhibitors of the stromal protease fibroblast activation proteinattenuate tumor growth in vivo [abstract]. Proc Am Assoc CancerRes 2006;47:4382.

4. Kelly T. Fibroblast activation protein and dipeptidyl peptidase IV(CD26): cell surface proteases that activate cell signalling and arepotential targets for cancer therapy. Drug Resist Updat 2005;8:51–8.

5. Narra K, Mullins SR, Lee HO, Strzemkowski-Brun B, MagalongK, Christiansen VJ, et al. Phase II trial of single agent Val-boroPro(Talabostat) inhibiting fibroblast activation protein in patientswith metastatic colorectal cancer. Cancer Biol Ther 2007;6:1691–9.

DOI 10.1002/art.27669

Are antibodies to citrullinated antigens enriched insynovial fluids of patients with rheumatoid arthritis?:comment on the article by Snir et al

To the Editor:In the article by Snir et al, the authors conclude that the

antibodies to several different citrullinated antigens are enrichedin rheumatoid arthritis (RA) synovial fluids (SFs) as comparedwith serum samples, after adjustment for the concentrations ofIgG in the SFs (1). The authors then postulate that theseantibodies are in part produced by plasma cells in the synovium.

The authors state that the concentration of IgG wasmeasured by radial immunodiffusion assay or by rate nephe-lometry, but they do not specify which assay was used withserum samples or with SF samples. RA SFs contain significantamounts of immune complexes, consisting of IgG (2). Thepresence of these immune complexes decreases the amount ofIgG detected by radial immunodiffusion, thus giving a falselylow concentration of IgG (3). This result, in turn, provides ahigher concentration of specific antibodies in SFs when ad-justed to the IgG concentration. Assays for IgG concentrationby rate nephelometry are also likely to underestimate the trueconcentration of this protein, due to the presence of immunecomplexes. Furthermore, Snir et al do not state whether blankswere used for each SF specimen in the antibody assays. Lack ofcorrection for a blank will result in a falsely high value, more soin SF samples than in serum samples. The authors do notdescribe the method for detecting tetanus toxoid antibodies,which tended to have low arbitrary units in the immunoassaywhen adjusted to IgG concentration, as shown in Figure 1 oftheir article. Thus, it is hard to compare tetanus toxoidantibodies with other detected antibodies.

Since the adjusted concentration of specific antibodiesin SFs is subject to errors because of the difficulties in assayingthe IgG concentration in SFs, it is possible to use the directdetection of specific antibodies in plasma cells in the synovium.The synthesis of rheumatoid factors (RFs) by plasma cells inthe synovium was first described by Mellors et al, usingimmunofluorescence microscopy (4). In later studies, theproportion of IgG-producing plasma cells synthesizing IgG-RFs varied from 20% to 60% (5,6). In comparison, �3% ofIgG-producing plasma cells in the synovium synthesized anti-bodies to 5 different partially degraded IgG molecules (6).

The use of immunofluorescence microscopy would settlethe question of which antibodies to citrullinated molecules areproduced by plasma cells in the synovium of RA patients. Thisapproach may answer in part the question asked by Dr. HenryKunkel 35 years ago, after a presentation by Natvig, “Dr. Natvig,do you have information about the other 50% of plasma cells?What kind of antibody are they making?” (5).

Mart Mannik, MDUniversity of WashingtonSeattle, WA

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