(18). two of the three receptor binding sites in the cytokine. The different mode of binding to antigen and the engineering into bivalent constructs supports the design of highly potent VHH-based therapeutic entities. using the method described by Marmenout et al. (13). Purification was according to the procedures described by Curnis and Corti (14). Two llamas were immunized with TNF according to current animal welfare regulations, using the adjuvant Stimune (CEDI Diagnostics, Lelystad, The Netherlands). Two blood samples were collected from each animal as the source of B-cells. Total RNA was isolated according to the Pirozadil procedure described by Chomczynski and Sacchi (15). Random primed complementary DNA was prepared on total RNA, purified and subsequently used as template to amplify the Nanobody? repertoire. The procedure to amplify and clone the Nanobody? repertoire was based on a method described in Ref. (16). For the selection of Nanobodies? against TNF, a Nunc Maxisorp? 96-well plate was coated with neutravidin and blocked, and biotinylated TNF was added to the wells. Phages were prepared as described by Marks et al. (17) and allowed to bind to the wells for 2?h at room temperature. Phages were removed, and the wells were washed 20 times with phosphate-buffered saline (PBS)/0.1% tween; elution of bound phage was done with 10?M etanercept (Enbrel?) for 30?min at room temperature, or by denaturation with acid (0.2?M glycine pH 2.5) for 20?min at room temperature. Two rounds of selections were performed. The ability of the Nanobodies? to inhibit receptorCligand interaction was analysed in ELISA. A 96-well Maxisorp plate was coated overnight at 4C with 2?g/ml etanercept in PBS. Plates were blocked with 1% casein solution (in PBS) for 2?h at room temperature. Nanobody? samples were preincubated for 30?min at room temperature with biotinylated TNF (200?pM). The mixtures were added to the plates and incubated for 1?h at room temperature. Biotinylated TNF was detected using Extravidin alkaline phosphatase (Sigma; Pirozadil 1/2,000 diluted) and pNPP (Sigma; 2?mg/ml) as substrate. Formatting, Expression, and Purification of Nanobodies? For construction of bivalent anti-TNF Nanobodies?, two separate PCR reactions were used to amplify the N-terminal and the Pirozadil C-terminal Nanobody? subunits using oligo combinations containing sequences encoding a 9GS [(Gly)4Ser(Gly)3Ser], 12GS [(Gly)3(Ser)]3, and 30GS [(Gly)4(Ser)]6 linker to connect the different Nanobodies?. The N-terminal VHH PCR fragment was digested with SfiI and BamHI, and the C-terminal VHH PCR fragment was digested with BamHI Pirozadil and BstEII. Ligations and transformations were carried out as described earlier. For the generation of bispecific Nanobodies? consisting of two anti-TNF Nanobodies? combined with one anti-albumin Nanobody?, three PCR reactions were performed for the amplification of the N-terminal, the middle, and the C-terminal Nanobody? with oligonucleotide primers encoding the 9, 12, or 30??GlyCSer linker. The N-terminal VHH encoding PCR fragment was digested with SfiI and BamHI, the middle Nanobody? fragment was digested with BamHI and BspEI, and the C-terminal VHH PCR fragment was digested with BspEI and BstEII. Single clones were picked and grown in Luria Broth containing the appropriate antibiotics, and expression was induced with 1?mM isopropyl -d-1-thiogalactopyranoside. Periplasmic extraction and immobilized metal affinity chromatography purification of the VHH proteins were performed according to Ref. (18). The VHH proteins were further purified by cation exchange and/or gel filtration and dialyzed into PBS. Affinity Measurements Binding of Nanobodies? to TNF was characterized by surface plasmon resonance in a Biacore 3000 instrument (Biacore International AB, Uppsala, Sweden). In brief, TNF was covalently bound to a CM5 sensor LFA3 antibody chip surface amine coupling until an increase of Pirozadil 250 response units was reached. Remaining reactive groups were inactivated. Nanobody? binding was assessed, and B-cell display methods [unpublished], which do not suffer from polymerase chain reaction artifacts during library construction. This confirms the occurrence of circulating B cells derived from an ancestor B-cell clone as a consequence of the maturation process. The VHH encoding gene segments were recloned in an expression vector with or without the carboxy-terminal c-MYC and/or hexa-histidine tags. After expression and purification, VHH were tested in the bioassay for their neutralizing capacity. Murine L929 cells expressing the mouse receptor were used for testing the VHH in combination with the human cytokine. By sensitizing the cells with actinomycin D, picomolar amounts of.