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.
Fibroblast activation protein (FAP) is a type II membrane protein expressed on tumor stroma fibroblasts in more than 90% of all carcinomas. FAP serves as a diagnostic marker and is potential ...therapeutic target for treatment of a wide variety of FAP+ carcinomas. Murine tumor stroma models and FAP-specific antibodies are required to investigate the functional role of FAP in tumor biology and its usefulness for drug targeting. We here describe the development of antibodies with crossreactivity for human (hFAP) and murine FAP (mFAP), which share 89% amino acid identity.
An FAP-/- mouse was sequentially immunized with recombinant murine and human FAP-CD8 fusion proteins. Immunoglobulin cDNA derived from hyperimmune spleen cells was used for the construction of a combinatorial single chain Fv (scFv) library. Phage display selection of FAP-specific scFv was performed on immobilized hFAP followed by selection on cells expressing murine FAP.
High-affinity, species-crossreactive, FAP-specific scFv were isolated upon sequential phage display selection. A bivalent derivative (minibody M036) constructed thereof was applied for immunohistochemical analyses and allowed detection of FAP expression on stroma cells of different human carcinomas as well as on murine host stroma in a tumor xenograft model.
MB M036, derived from phage display selected species crossreactive scFv, is suitable for tumor stroma targeting and will be a valuable tool in the analyses of the functional role of FAP in tumor biology as well as in the evaluation of the suitability of FAP for drug targeting.
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.
Overexpression of the tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) receptors, TRAIL-R1 and TRAIL-R2, induces apoptosis and activation of NF-κB in cultured cells. In this ...study, we have demonstrated differential signaling capacities by both receptors using either epitope-tagged soluble TRAIL (sTRAIL) or sTRAIL that was cross-linked with a monoclonal antibody. Interestingly, sTRAIL was sufficient for induction of apoptosis only in cell lines that were killed by agonistic TRAIL-R1- and TRAIL-R2-specific IgG preparations. Moreover, in these cell lines interleukin-6 secretion and NF-κB activation were induced by cross-linked or non-cross-linked anti-TRAIL, as well as by both receptor-specific IgGs. However, cross-linking of sTRAIL was required for induction of apoptosis in cell lines that only responded to the agonistic anti-TRAIL-R2-IgG. Interestingly, activation of c-Jun N-terminal kinase (JNK) was only observed in response to either cross-linked sTRAIL or anti-TRAIL-R2-IgG even in cell lines where both receptors were capable of signaling apoptosis and NF-κB activation. Taken together, our data suggest that TRAIL-R1 responds to either cross-linked or non-cross-linked sTRAIL which signals NF-κB activation and apoptosis, whereas TRAIL-R2 signals NF-κB activation, apoptosis, and JNK activation only in response to cross-linked TRAIL.
Background The natural course of nonoperatively treated rotator cuff tears is not fully understood. We explored the long-term development of tear anatomy and assessed functional outcomes. Methods ...Eighty-nine small to medium-sized full-thickness tears of the rotator cuff, all primarily treated by physiotherapy, were identified retrospectively. Twenty-three tears needed surgical treatment later on, and 17 patients were unable to meet for follow-up. The remaining 49 still unrepaired tears were re-examined after 8.8 (8.2-11.0) years with sonography. Re-examination by magnetic resonance imaging was possible for 37 patients. Shoulder function was assessed with shoulder scores. Primary outcome measures were progression of tear size, muscle atrophy, and fatty degeneration and the Constant score (CS). Results Mean tear size increased by 8.3 mm in the anterior-posterior plane ( P = .001) and by 4.5 mm in the medial-lateral plane ( P = .001). Increase of tear size was −5 to +9.9 mm in 33 patients, 10 to 19.9 mm in 8 patients, and ≥20 mm in 8 patients. The CS was 81 points for tear increases <20 mm and 58.5 points for increases ≥20 mm ( P = .008). Muscle atrophy and fatty degeneration progressed in 18 and 15 of the 37 patients, respectively. In tears with no progression of atrophy, the CS was 82 points compared with 75.5 points in tears with progression ( P = .04). Conclusions Anatomic tear deterioration was found in the majority of patients, but it was often moderate. Large tear size increases and progression of muscle atrophy were correlated to a poorer functional outcome.
Molecular interactions between near-IR fluorescent probes and specific antibodies may be exploited to generate novel smart probes for diagnostic imaging. Using a new phage display technology, we ...developed such antibody Fab fragments with subnanomolar binding affinity for tetrasulfocyanine, a near-IR
in vivo imaging agent. Unexpectedly, some Fabs induced redshifts of the dye absorption peak of up to 44 nm. This is the largest shift reported for a biological system so far. Crystal structure determination and absorption spectroscopy in the crystal in combination with microcalorimetry and small-angle X-ray scattering in solution revealed that the redshift is triggered by formation of a Fab dimer, with tetrasulfocyanine being buried in a fully closed protein cavity within the dimer interface. The derived principle of shifting the absorption peak of a symmetric dye
via packaging within a Fab dimer interface may be transferred to other diagnostic fluorophores, opening the way towards smart imaging probes that change their wavelength upon interaction with an antibody.
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.
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