Inhibition of fibroblast activation protein and dipeptidylpeptidase 4 increases cartilage invasion by rheumatoid arthritis synovial fibroblasts.

OBJECTIVE: Since fibroblasts in the synovium of patients with rheumatoid arthritis (RA) express the serine proteases fibroblast activation protein (FAP) and dipeptidylpeptidase 4 (DPP-4)/CD26, we undertook the current study to determine the functional role of both enzymes in the invasion of RA synovial fibroblasts (RASFs) into articular cartilage. METHODS: Expression of FAP and DPP-4/CD26 by RASFs was analyzed using fluorescence-activated cell sorting and immunocytochemistry. Serine protease activity was measured by cleavage of fluorogenic substrates and inhibited upon treatment with L-glutamyl L-boroproline. The induction and expression of matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs) in RASFs were detected using real-time polymerase chain reaction. Densitometric measurements of MMPs using immunoblotting confirmed our findings on the messenger RNA level. Stromal cell-derived factor 1 (SDF-1 [CXCL12]), MMP-1, and MMP-3 protein levels were measured using enzyme-linked immunosorbent assay. The impact of FAP and DPP-4/CD26 inhibition on the invasiveness of RASFs was analyzed in the SCID mouse coimplantation model of RA using immunohistochemistry. RESULTS: Inhibition of serine protease activity of FAP and DPP-4/CD26 in vitro led to increased levels of SDF-1 in concert with MMP-1 and MMP-3, which are downstream effectors of SDF-1 signaling. Using the SCID mouse coimplantation model, inhibition of enzymatic activity in vivo significantly promoted invasion of xenotransplanted RASFs into cotransplanted human cartilage. Zones of cartilage resorption were infiltrated by FAP-expressing RASFs and marked by a significantly higher accumulation of MMP-1 and MMP-3, when compared with controls. CONCLUSION: Our results indicate a central role for the serine protease activity of FAP and DPP-4/CD26 in protecting articular cartilage against invasion by synovial fibroblasts in RA.

Targeted therapy of renal cell carcinoma: synergistic activity of cG250-TNF and IFNg.

Immunotherapeutic targeting of G250/Carbonic anhydrase IX (CA-IX) represents a promising strategy for treatment of renal cell carcinoma (RCC). The well characterized human-mouse chimeric G250 (cG250) antibody has been shown in human studies to specifically enrich in CA-IX positive tumors and was chosen as a carrier for site specific delivery of TNF in form of our IgG-TNF-fusion protein (cG250-TNF) to RCC xenografts. Genetically engineered TNF constructs were designed as CH2/CH3 truncated cG250-TNF fusion proteins and eucariotic expression was optimized under serum-free conditions. In-vitro characterization of cG250-TNF comprised biochemical analysis and bioactivity assays, alone and in combination with Interferon-gamma (IFNgamma). Biodistribution data on radiolabeled [(125)J] cG250-TNF and antitumor activity of cG250-TNF, alone and in combination with IFNgamma, were measured on RCC xenografts in BALB/c nu/nu mice. Combined administration of cG250-TNF and IFNgamma caused synergistic biological effects that represent key mechanisms displaying antitumor responses. Biodistribution studies demonstrated specific accumulation and retention of cG250-TNF at CA-IX-positive RCC resulting in growth inhibition of RCC and improved progression free survival and overall survival. Antitumor activity induced by targeted TNF-based constructs could be enhanced by coadministration of low doses of nontargeted IFNgamma without significant increase in side effects. Administration of cG250-TNF and IFNgamma resulted in significant synergistic tumoricidal activity. Considering the poor outcome of renal cancer patients with advanced disease, cG250-TNF-based immunotherapeutic approaches warrant clinical evaluation.

Sequential cancer immunotherapy: targeted activity of dimeric TNF and IL-8.

Polymorphonuclear neutrophils (PMNs) are potent effectors of inflammation and their attempts to respond to cancer are suggested by their systemic, regional and intratumoral activation. We previously reported on the recruitment of CD11b+ leukocytes due to tumor site-specific enrichment of TNF activity after intravenous administration of a dimeric TNF immunokine with specificity for fibroblast activation protein (FAP). However, TNF-induced chemo-attraction and extravasation of PMNs from blood into the tumor is a multistep process essentially mediated by interleukin 8. With the aim to amplify the TNF-induced and IL-8-mediated chemotactic response, we generated immunocytokines by N-terminal fusion of a human anti-FAP scFv fragment with human IL-8 (IL-8(72)) and its N-terminally truncated form IL-8(3-72). Due to the dramatic difference in chemotaxis induction in vitro, we favored the mature chemokine fused to the anti-FAP scFv for further investigation in vivo. BALB/c nu/nu mice were simultaneously xenografted with FAP-positive or -negative tumors and extended chemo-attraction of PMNs was only detectable in FAP-expressing tissue after intravenous administration of the anti-FAP scFv-IL-8(72) construct. As TNF-activated PMNs are likewise producers and primary targets for IL-8, we investigated the therapeutic efficacy of co-administration of both effectors: Sequential application of scFv-IL-8(72) and dimeric IgG1-TNF fusion proteins significantly enhanced anti-tumor activity when compared either to a single effector treatment regimen or sequential application of non-targeted cytokines, indicating that the tumor-restricted sequential application of IL-8(72) and TNF is a promising approach for cancer therapy.

The influence of IL-2 family cytokines on activation and function of naturally occurring regulatory T cells.

IL-2 is essential for CD4+CD25+forkhead box P3+ (FoxP3+) naturally occurring regulatory T cell (Treg) homeostasis and activation. Binding of IL-2 to its receptor leads to phosphorylation of STAT5, and binding of phosphorylated STAT5 to the foxp3 promoter increases foxp3 transcription, resulting in elevated levels of FoxP3 protein in Tregs. Transcriptional regulation by the elevated levels of FoxP3 is thought to be essential for the strong suppressor function seen in activated Tregs. IL-2 belongs to a family cytokines, which all depend on the common gamma-receptor chain (gammac). Given the well-documented effects of IL-2 on Treg function, the effect of other IL-2 family cytokines (IL-7, -15, and -21) on Tregs was examined. We observed that IL-7 and IL-15 induce STAT5 phosphorylation and up-regulation of FoxP3 in Tregs. STAT5 activation correlated with enhanced viability. However, only in the presence of IL-2 did Tregs acquire potent suppressor function. This finding is surprising, as IL-15 as well as IL-2 use the same IL-2R betac and gammac for signaling. In contrast, IL-21 activated STAT3 but did not activate STAT5 and had no effect on Treg viability, activation, or function. We therefore conclude that phosphorylation of STAT5, mediated through the IL-2Rgamma, promotes Treg survival in a resting and activated state. However, activation of STAT5 alone in conjunction with TCR signaling is not sufficient for the induction of potent suppressor function in Tregs, as IL-7 and IL-15 are not capable of inducing potent Treg suppressor function.

TNF-Selectokine: a novel prodrug generated for tumor targeting and site-specific activation of tumor necrosis factor.

We describe a TNF fusion protein designated TNF-Selectokine, which is a homo-trimeric molecule comprised of a single chain antibody (scFv) targeting module, a trimerization domain and TNF. TNF-Selectokine exerts high bioactivity towards the targeted and adjacent, antigen negative cells. Membrane targeting dependent immobilization of the TNF-Selectokine induced cell death in TNFR1 and TNFR2 dependent manner, thus cell bound TNF-Selectokine mimicks membrane TNF. To restrict TNF activity to the tumor, a prototype of a TNF-Selectokine prodrug was constructed by insertion of a TNFR1 fragment, separated from TNF by a protease-sensitive linker. The prodrug exerts minimal TNF activity, but can be activated in vitro several thousand-fold by proteolytic digest, showing the principal feasibility of this approach. Choice of cleavage site(s) recognized by protease(s) typically associated with a given carcinoma should allow high dose systemic application of the respective TNF prodrug that unveils its specific bioactivity only in targeted tissues.

Serum vitamin D levels in orthopaedic trauma patients living in the northwestern United States.

OBJECTIVE: To determine the incidence of vitamin D deficiency in orthopaedic trauma patients undergoing fracture surgery living in the Northwest United States. DESIGN: Retrospective observational cohort study. SETTING: Level 2 trauma center. PATIENTS: Two cohorts of patients undergoing fracture repair surgery during a 3-month period in winter and summer had serum vitamin D levels drawn at the time of surgery. One hundred three patients were reviewed in the winter cohort and 98 in the summer cohort. MAIN OUTCOME MEASURE: Serum 25(OH) vitamin D levels of patients undergoing fracture repair surgery. RESULTS: Normal levels of vitamin D were considered to be between 32 and 80 ng/mL. Most patients in both cohorts were vitamin D insufficient. The average level for the winter cohort was 26.4 ng/mL, which was significantly lower than the average level for the summer cohort, 29.8 ng/mL (P = 0.03). CONCLUSIONS: A high incidence of vitamin D insufficiency and deficiency likely exists across all age groups in orthopaedic trauma patients living in the Northwest United States and regions with similarly northern latitude. Further study is required to confirm improved fracture healing with normalization of serum vitamin D levels. LEVEL OF EVIDENCE: Prognostic Level IV. See Instructions for Authors for a complete description of levels of evidence.

Radioimmunotherapy of fibroblast activation protein positive tumors by rapidly internalizing antibodies.

PURPOSE: Fibroblast activation protein (FAP) is a serine protease that has emerged as a promising target for cancer therapy, either by direct abrogation of its proinvasive activity or by specific targeting of FAP-expressing cells with cytotoxic immunoconjugates. We aimed to select novel human-mouse cross-reactive antibodies and to test suitability for tumor therapy as radioimmunoconjugates in a preclinical model. EXPERIMENTAL DESIGN: Human Fab fragments that bind to human and murine FAP were selected from an antibody phage library. Two candidates (ESC11 and ESC14) were engineered into fully human IgG1 antibodies and further characterized. We investigated the intracellular trafficking of ESC11 and ESC14 in live cells by confocal microscopy and analyzed the biodistribution and therapeutic effects of anti-FAP antibodies labeled with the ß-emitting radionuclide (177)Lu in a melanoma xenograft nude mouse model. Results were compared with vF19, a humanized variant of an anti-FAP antibody that has been previously used in clinical trials. RESULTS: The two antibodies bound selectively to both human and mouse FAP, with affinities in the low nanomolar range. Binding to FAP-expressing melanoma cells resulted in rapid internalization of FAP-antibody complexes. (177)Lu-labeled ESC11 specifically accumulated in melanoma xenografts in vivo, with a higher tumor uptake than ESC14 and vF19. Radioimmunotherapy with 8 MBq (177)Lu-labeled anti-FAP antibodies delayed growth of established tumors, whereas (177)Lu-ESC11 extended mouse survival more pronounced than (177)Lu-ESC14 and (177)Lu-vF19. CONCLUSION: Our results show the potential of ESC11 and ESC14 as potent radioimmunoconjugates or antibody-drug conjugates for diagnostic and therapeutic use in patients with FAP-expressing tumors.

A randomized, prospective study comparing intertrochanteric hip fracture fixation with the dynamic hip screw and the dynamic helical hip system in a community practice.

OBJECTIVE: To evaluate the clinical performance of the Dynamic Helical Hip System (DHHS) spiral blade relative to the Dynamic Hip Screw (DHS) lag screw. DESIGN: Randomized prospective study. SETTING: One level-2 trauma center and one level-3 trauma center. PATIENTS: Fifty-one consecutive patients were recruited into the trial. Inclusion criteria included patients over 50 years of age with AO/OTA 31A1 or 31A2 fracture. INTERVENTION: Surgeries were performed by one of 15 participating community orthopaedic surgeons. The patients were randomized to either a DHHS or DHS implant. Follow-up occurred at two weeks and six weeks and then at six-week intervals until healing occurred. MAIN OUTCOME MEASURES: Primary outcome variables included sliding of die implant on the final AP radiographs, failure by cut-out and implant failure. RESULTS: There were 24 patients in the DHS group and 27 in the DHHS group. There was no difference in age, gender, ASA score, fracture classification or in the quality of reduction measured on the immediate postoperative radiographs (p=0.28) between the two groups. The tip apex distance was 18.7 mm in the DHHS group and 18.5 mm in the DHS group (p=0.40). The DHHS group had average blade sliding of 7.4 mm while the DHS group had an average lag-screw sliding of 7.7 (p=0.45). The DHHS group had two failures by central protrusion of the blade through the femoral head without significant varus collapse or superior migration. One was revised to a DHS and healed, the other was revised to a proximal femoral locking plate, which also failed and eventually required revision to a total hip arthroplasty. Investigation of the implants post failure showed evidence of binding of the blade shaft in the barrel as a mechanism of failure in both cases. No DHS implants cut out in this series, although one patient was revised to a total hip arthroplasty for symptomatic segmental osteonecrosis. CONCLUSION: Both implants performed well in a majority of cases. The higher incidence of failure in the DHHS group is concerning, despite the low numbers. The mechanism of failure of the DHHS implant left adequate bone stock for attempts at revision fixation.

Tumor-specific crosslinking of GITR as costimulation for immunotherapy.

Activation of murine glucocorticoid-induced tumor necrosis factor-related receptor (mGITR) by its natural ligand (GITRL) or antiGITR agonist mAb enhances T-cell responses, inhibits regulatory T-cell (Treg)-mediated suppression and induces tumor immunity in a variety of murine tumor models. However, systemic administration of these costimulatory agents can lead to global T-cell activation and autoimmunity. To specifically manipulate the T-cell compartment in the tumor microenvironment we propose to target the tumor infiltrating T cells with a bispecific mGITRL fusion protein. For that purpose, mGITRL is linked to a single-chain antibody targeting fibroblast activation protein (FAP) as FAP expression is restricted to cancer-associated fibroblasts (CAFs) found in the stroma of epithelial cancers. AntiFAP-mGITRL fusion protein forms dimers and binds to murine GITR with 1.2 µM affinity and to murine FAP with 4.5 nM. The construct is able to costimulate CD8+ and CD4+ effector T cells resulting in increased proliferation, IFN-γ and IL-2 production. This costimulatory effect is enhanced when the fusion protein is bound to a FAP-positive cell line mimicking FAP CAFs. In suppression assays, membrane-bound antiFAP-mGITRL is 100-fold more effective in overcoming Treg-mediated suppression than unbound fusion protein. These studies suggest that targeted tumor therapy with antiFAP-mGITRL fusion protein could induce tumor rejection while minimizing autoimmune side effects.

Recombinant ovine atadenovirus induces a strong and sustained T cell response against the hepatitis C virus NS3 antigen in mice.

Ovine atadenovirus (OAdV) is a novel gene transfer vector with excellent in vivo gene transfer characteristics. In the present study, we have investigated the ability of an OAdV vector to mediate a T cell response to an antigen of the hepatitis C virus (HCV) in mice. Specifically, an expression cassette coding for non-structural protein 3 (NS3) of hepatitis C virus was inserted into the OAdV genome and the resulting recombinant virus (OAdV-ns3) was shown to propagate stably and to express the ns3 gene at a high level in vitro. A single injection of this non-replicating vector into BALB/c mice resulted in a strong induction of NS3-specific, IFN-gamma secreting T-lymphocytes as measured by direct ex vivo ELISpot assay. The number of IFN-gamma secreting lymphocytes remained nearly unaltered for a period of at least 10 weeks. The immune response was shown to depend on virus dose but a single intramuscular injection of less than 10(8) infectious particles of OAdV-ns3 was sufficient to induce a significant NS3-specific T cell response. Moreover, this response was not affected by prior immunisation of animals with human adenovirus type 5 (HAdV-5). The results of our study provide proof for the concept that OAdV vectors may be valuable tools for vaccination and immunotherapy even in the face of natural immunity to human adenoviruses.