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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 β-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., (©2012 AACR.)

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.

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.

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.

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.

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.

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.

Bispecific antibodies directed against tumour associated antigens and the T cell receptor component CD3 for recruitment and tumour targeted activation of T cells represent a novel class of highly specific immunotherapeutics for cancer. We here describe the construction, eukaryotic expression and in vitro functional activity of a new T cell activating bispecific reagent, termed TTS for T cell targeting to the tumour stroma, comprised of a CD3 specific single chain antibody derivative (scFv) fused C-terminally to a 'fibroblast activation protein' (FAP) specific scFv that targets cytotoxic effector cells to FAP. FAP is highly expressed in the vascularised tumoural stroma of most lung, breast and colon carcinomas. It thus represents a selectively tumour associated, yet common marker of many solid tumours and is a potentially ideal candidate marker for efficient targeting of immune effector cells.

TNF-related apoptosis-inducing ligand (TRAIL) is a typical member of the tumor necrosis factor (TNF) ligand family that is expressed as a type II membrane protein (memTRAIL) and signals apoptosis via the death domain-containing receptors TRAIL-R1 and -2. Soluble recombinant derivatives of TRAIL (sTRAIL) are considered as novel tumors therapeutics because of their selective apoptosis inducing activity in a variety of human tumors but not in normal cells. Using antagonistic antigen-binding fragment (Fab) preparations of TRAIL-R1- and TRAIL-R2-specific antibodies, we demonstrate in this study that TRAIL-R1 becomes activated by both the soluble and the membrane-bound form of the ligand, whereas TRAIL-R2 becomes only activated by memTRAIL or soluble TRAIL secondarily cross-linked by antibodies. Furthermore, we show that the restricted signal capacity of sTRAIL can be readily converted into a fully signal competent memTRAIL-like molecule, i.e. a TRAIL-R2 stimulating ligand, by genetic fusion to an antibody derivative that allows antigen-dependent 'immobilization' of the fusion protein to cell surfaces. We conclude that antibody targeting-dependent activation can be used to design selective therapeutics derived of those ligands of the TNF family that are biologically inactive in their soluble form.

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 carcinomas. Importantly, compared with the CDR-grafted humanized 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., (Copyright 2001 Wiley-Liss, Inc.)

Four completely human antibody derivatives [single-chain-antibody fragments (scFvs)] with specificity for the general tumor stroma marker fibroblast activation protein (FAP) were isolated by guided selection. Highly diverse IgG, IgM and IgD isotypes comprising heavy-chain variable domain libraries were generated using cDNAs derived from diverse lymphoid organs of a multitude of donors. Three of the human scFvs were converted into bivalent minibodies and expressed in eukaryotic cells for further functional characterization. Binding-competition studies and analysis by fluorescence-activated cell sorting showed high-affinity binding (10--20 nM) for two clones and recognition of the same epitope as the murine guiding antibody. The minibodies were successfully used for immunohistology of a variety of human carcinoma biopsies, revealing specific staining of stromal fibroblasts. Therefore, they should be suitable for in vivo diagnostic and tumor-targeting studies and, because of their completely human origin, be superior to murine or humanized antibody derivatives.

Pseudomonas putida S-313 is able to desulphonate a broad range of aromatic sulphonates to provide sulphur for growth by monooxygenolytic cleavage to yield the corresponding phenol. After miniTn5 transposon mutagenesis of this strain, 11 mutants were isolated that were no longer able to utilize benzenesulphonate as a sulphur source. Three of these mutants were defective in the utilization of all aromatic sulphonates tested, but they grew normally with other sulphur sources. These strains contained independent insertions in the novel 4.2 kb asfRABC gene cluster, encoding a putative reductase (AsfA), a ferredoxin (AsfB), a putative periplasmic binding protein (AsfC), which was localized to the periplasm using alkaline phosphatase fusions, and a divergently oriented fourth gene, asfR, that encoded a LysR-type regulator protein. A further mutant was interrupted in the ssu locus, which includes the gene for a putative desulphonative monooxygenase. Transformation of Pseudomonas aeruginosa with the asfRAB genes was sufficient to allow arylsulphonate utilization by this species, which does not normally use these compounds, suggesting that the AsfAB proteins may constitute an arylsulphonate-specific electron transport system that interacts with a less specific oxygenase. Expression of the asfABC genes in P. putida was induced by benzenesulphonate or toluenesulphonate, and it was repressed in the presence of sulphate in the growth medium. AsfR was a negative regulator of asfABC expression, and toluenesulphonate induced expression of these genes indirectly by reducing the expression of the asfR gene.

When Pseudomonas aeruginosa is grown with organosulfur compounds as sulfur sources, it synthesizes a set of proteins whose synthesis is repressed in the presence of sulfate, cysteine, or thiocyanate (so-called sulfate starvation-induced proteins). The gene encoding one of these proteins, PA13, was isolated from a cosmid library of P. aeruginosa PAO1 and sequenced. It encoded a 381-amino-acid protein that was related to several reduced flavin mononucleotide (FMNH2)-dependent monooxygenases, and it was the second in an operon of three genes, which we have named msuEDC. The MsuD protein catalyzed the desulfonation of alkanesulfonates, requiring oxygen and FMNH2 for the reaction, and showed highest activity with methanesulfonate. MsuE was an NADH-dependent flavin mononucleotide (FMN) reductase, which provided reduced FMN for the MsuD enzyme. Expression of the msu operon was analyzed with a transcriptional msuD::xylE fusion and was found to be repressed in the presence of sulfate, sulfite, sulfide, or cysteine and derepressed during growth with methionine or alkanesulfonates. Growth with methanesulfonate required an intact cysB gene, and the msu operon is therefore part of the cys regulon, since sulfite utilization was found to be CysB independent in this species. Measurements of msuD::xylE expression in cysN and cysI genetic backgrounds showed that sulfate, sulfite, and sulfide or cysteine play independent roles in negatively regulating msu expression, and sulfonate utilization therefore appears to be tightly regulated.