Construction of a bispecific single chain antibody for recruitment of cytotoxic T cells to the tumour stroma associated antigen fibroblast activation protein.

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.

Differential activation of TRAIL-R1 and -2 by soluble and membrane TRAIL allows selective surface antigen-directed activation of TRAIL-R2 by a soluble TRAIL derivative.

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.

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

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.

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

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.

Genetic organization of sulphur-controlled aryl desulphonation in Pseudomonas putida S-313.

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.

A novel reduced flavin mononucleotide-dependent methanesulfonate sulfonatase encoded by the sulfur-regulated msu operon of Pseudomonas aeruginosa.

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.

Unilateral external fixation until healing with the dynamic axial fixator for severe open tibial fractures.

One hundred one cases of open tibia fractures were treated until healing with a unilateral external fixation device that permits fracture site compression with weight bearing. There were 38 type II and 63 type III (24 IIIA, 33 IIIB, six IIIC) open fractures. A standard protocol was followed including irrigation and debridement and, when necessary, flap coverage (19 cases) and bone grafting (31 cases). Fixators were applied at the first debridement and removed when the fracture was healed. All patients were permitted early partial weight bearing and progressed to full weight bearing with fixator dynamization. Ninety-six cases healed in the fixator (12-50 weeks; average, 24.6). Three of the five failures were associated with screw complications. Five patients required screw changes and 29 required oral antibiotic therapy for screw complications. Ninety-five percent of healed cases had angulation of less than 10 degrees (in any plane). There were only six fracture site infections during the course of treatment. Dynamic axial fixation may be applied at the first debridement and be used until healing in severe open tibia fractures. Change of the fixator to another treatment method is not required.