Title of Invention

"METHOD FOR GENERATING A MUTEIN OF A PROTEIN"

Abstract Method for generating a mutein of a protein selected from the group consisting of human neutrophil gelatinase-associated lipocalin (hNGAL), rat a2-microglobulin-related protein (A2m) and mouse 24p3/uterocalin (24p3), said mutein having detectable affinity to a given target, characterized in that the method comprises the step of subjecting the protein to mutagenesis at one or more of the sequence positions which correspond to the sequence positions 40 to 50,70 to 79,101 to 103, and 125 to 132 of hNGAL, resulting in one or more mutein(s) of the protein.
Full Text JFor the production of recombinant Tlpc, cells of E. coli JMS3 (Yanisch-Perron et al., Gene [33 (1985), 103-119) were transformed with the expression plasraid pTLpc3 harbouring the jcDNA of Tlpc (for the cDNA of Tlpc, see Holzfeind and Redl, Gene 139 (1994), 177-183) land used for protein production and purification according to example 3. The protein yield (was approximately 2.2 mg per 11 culture volume.
[Unoccupied binding sites on the surface of the Immuno-Stick were saturated by incubation Kvith 1.2 ml 2 % w/v BSA in PBST (PBS with 0.1 % v/v Tween 20) for 2 hours at RT. Afterwards the Immuno-Stick was incubated with a mixture of 250 ul of the phagemid solution and of 500 ul of blocking buffer (3 % w/v BSA in PBST) for 1 hour at RT.
por the removal of unbound phagemids, washing was performed eight times, each time with 950 ul PBST for 2 minutes. Adsorbed phagemids were finally eluted by 10 minute treatment of me Immuno-Stick with 950 f*l 0.1 M glycine/HCl pH 2.2, followed by immediate neutralisation of the pH of the elution fraction by mixing it with 150 ul 0.5 M Tris.
For the amplification, this phagemid solution (1.1 ml, containing between 106 and 108 Colony-forming Units, depending on the selection cycle) was shortly warmed to 37 °C, mixed with 3 ml of an exponentially growing culture of E. coli XLl-blue (OD550 - 0.5), and incubated for 30 minutes at 37 °C, 200 rpm. The cells infected with the phagemids were subsequently sedimented (2 minutes, 4420 g, 4 °C), resuspended in 600 u.1 of the culture medium, and plated out onto three agar plates with LB-medium containing 100 jag/ml ampicillin (LB/Amp; 140 mm diameter).
kfter incubation for 14 hours at 32 °C, the cells were scraped from the agar plates, each with addition of 10 ml 2xYT/Amp-medium, were transferred to a sterile Erlenmeyer-flask, and were shaken for 20 minutes at 37 °C, 200 rpm for complete suspension. 200 ml of 2xYT/Amp-medium prewarmed to 37 °C were inoculated to an OD550 - 0.08 with an appropriate volume of this suspension.
For the repeated production and affinity enrichment of phagemid particles the same procedure as described at the beginning of this example was used. In.these cases 50 ml 2xYT/Amp-medium was inoculated with 0.2 to 1 ml of the suspension of the cells grown on the agar plates and phagemids were produced during a period of seven instead of five hours at 30 °C. Four further selection cycles with the Tlpc were carried out in this way.

Example 3: Identification of human Tear Lipocalin-binding hNGAL muteir.s by use of the "colony screening"-method
For the analytical production of the hNGAL muteius as fusion proteins with the Strep-Tag® II as well as with the albumin-binding domain and their characterization by colony screening, the gene cassette between the two BstXI cleavage sites was subcloned from the vector phNGAL5 on pliNGAL7.
For this purpose the phasmid DNA was isolated from the mixture of the E. coli clones obtained by infection with the phagemids from Example 2 eluted as a result of the last selection cycle, using the Perfectprep Plasmid Midi Kit (Eppendorf). The DNA was cut with the restriction enzyme BstXI and the smaller of the two fragments (347 bp) was purified by preparative agarose-gel electrophoresis as described in Example 1. The DNA of the vector phNGAL7 was cut with BstXI and the larger of the two fragments (3971 bp) was isolated in the same way.
For the ligation, each 100 fmol of the two DNA-fragments were mixed with 1.5 Weiss Units T4 DNA ligase (Promega) in a total volume of 20 ul (30 mM Tris/HCl pH 7.S, 10 mM MgCl2, 10 mM DTT, 1 mM ATP), followed by incubation overnight at 16 °C. E. coli TG1-F" (E. coli K12 TGI, which had lost its episome through repeated culturing under non-selective conditions) was transformed with 2 ul of this ligation mixture according to the CaCh-method (Sambrook et al, supra).
A hydrophilic PVDF membrane (Millipore, type GVWP, pore size 0.22 urn), labelled at one position and cut to size, was laid onto an LB/Amp agar plate. 150 ul of the cell suspension from the transformation batch, which had been centrifuged (5000 g, 2 min, 4 °C) and resuspended in 500 u,i of the culture medium, were uniformly plated onto this membrane. The agar plate was incubated for 7.5 hours at 37 °C until the colonies had reached a size of approximately 0.5 mm.
In the meantime a hydrophobic membrane (Millipore, Immobilon P, pore size 0.45 urn), also cut to size, was moistened with PBS according to the instructions of the manufacturer. It was subsequently agitated for 4 hours at RT in a solution of 10 mg/ml human serum albumin (HSA, Sigma) in PBS. Remaining binding sites on the membrane were saturated by incubation with 3 % w/v BSA, 0.5 % v/v Tween 20 in PBS for 2 hours at RT. The membrane was washed twice for 10 minutes each with 20 ml PBS and agitated afterwards for 10 minutes in 10 ml LB/Amp medium, to which 200 ug/1 anhydrotetracycline were

added. It was subsequently marked at one position and was laid onto a culture plate with LB/Amp agar, which additionally contained 200 ug/1 anhydrotetracycline. The hydrophilic membrane on which the colonies were grown was laid onto the hydrophobic membrane in such a way that both of the marks superimposed. The culture plate was incubated with both membranes at 22 °C for 15 hours. During this phase the respective hNGAL muteins were secreted from the colonies and were immobilized via the albumin-binding domain on the HSA on the lower membrane.
After this, the upper membrane with the colonies was transferred to a fresh LB/Amp agar plate and stored at 4 °C. The hydrophobic membrane was removed, washed three times for 5 minutes each with 20 ml PBST, and subsequently incubated for 1 hour in 10 ml of a solution of a conjugate (10 ug/ml) from Tear lipocalin and biotin in PBST. For the production of the conjugate, a solution of 0.285 mg D-biotinoyl-e-amidocaproic acid-N-hydroxysuccinimide ester (Roche) in 9 ul DMSO was slowly added to 2.5 ml of 450 ug/ml Tlpc in 5 % w/v NaHCC>3 (pH 8.2). After stirring for 1 hour at RT, excess reactant was removed by means of a PD-10 gel filtration column (Pharmacia) using PBS as running buffer.
After incubation with the conjugate, the membrane was washed three times with PBST, followed by incubation for 1 hour with 10 ml avidin-alkaline-phosphatase conjugate (Sigma, dilution 1:40000 in PBST). The membrane was subsequently washed each twice with PBST and once with PBS for 5 minutes and agitated for 10 minutes in AP-buffer (0.1 M Tris/HCl pH 8.8, 0.1 M NaCl, 5 mM MgCb). For the chromogenic reaction, the membrane was incubated in 10 ml AP-buffer, to which 30 ul 5-bronio-4-chloro-3-indoIyl phosphate 4-toluidine salt (Roth, 50 fig/ml in dimethylformamide) and 5 ul nitro blue tetrazolium (Roth, 75 ug/ml in 70 % v/v dimethylformamide) were added, until distinct colour signals could be recognized at the positions of some of the colonies. In this way the rinding activity for the protein ligand, i.e. Tlpc, of the hNGAL muteins produced by these colonies was detected.
Twelve of the colonies giving rise to colour spots were cultured from the first membrane. Their plasmid DNA was isolated and the hNGAL gene cassette was subjected to sequence jmalysis by use of the Genetic Analyzer 310 system (Applied Biosystems) according to the Instructions of the manufacturer using the oligodeoxynucleotide SEQ ID NO: 11 as primer. The twelve sequenced clones exhibited only eight different sequences, which were named TlpcA, TlpcB, TlpcC, TlpcD, TlpcE, TlpcF, TIpcG, TlpcH. The clone TlpcA was found five times. The nucleotide sequences of the clones were translated into amino acid

sequences and those amino acids deviating from hNGAL are given in Table 1. The amino acid sequence and the nucleotide sequence of the mutein TlpcA are also given as SEQ ID NO:12 and SEQ ID NO:13. The sequencing revealed amber stop codons, which were suppressed in the employed E. coli strains, at different positions in ail of the selected variants.
Example 4: Production of the hNGAL muteins
For the preparative production of hNGAL and its muteins one selected colony as well as khe hNGAL originally encoded on phNGAL7, as a control, were produced in the E. coli (strain TGI-F-.
To this end, 100 ml of LB/Amp-medrum were inoculated with a single colony of the TG1-F~ transformant carrying the respective plasmid, and incubated overnight at 30 °C, 200 pm. 2 I of LB/Amp-medium in a 5 1-Erlenmeyer flask were then inoculated with each 40 nl of this preculture and were shaken at 22 °C, 200 rpm to an OD550 = 0.5. Induction was performed by adding 200 jig/1 anhydrotetracycline (200 u.1 of a 2 mg/ml stock solution in PMF) followed by shaking for 3 further hours at 22 °C, 200 rpm.
"the cells from one flask were centrifuged (15 minutes, 4420 g, 4 °C) and, after decanting tjhe supernatant, were resuspended in 20 ml of periplasmic release buffer (100 mM 'jis/HCl pH 8.0, 500 mM sucrose, 1 mM EDTA) with cooling for 30 minutes on ice. subsequently the spheroplasts were removed in two successive centrifugation steps (15 minutes, 4420 g, 4 °C and 15 minutes, 30000 g, 4 °G). The supernatant comprising the j eriplasmatic protein extract was dialyzed against CP-buffer (100 mM Tris/HCl pH 8.0, 150 mM NaCl, 1 raM EDTA), sterile-filtered, and served for the chromatographic I urification.
The purification took place by means of the Strep-Tag® Il-affinity tag (Schmidt et al., supra) which was situated between the hNGAL variant and the albumin binding domain. In tie present case the streptavidin mutein "I" was employed (German Patent 196 41 876.3; Yoss and Skerra, Protein Eng. 10 (1997), 975-982), which was coupled to an NHS-ajctivated sepharose (Pharmacia) yielding 5 mg/ml immobilized streptavidin, relative to the bed volume of the matrix.
A 4 ml bed volume chromatography column filled with this material was equilibrated with 2)0 ml CP-buffer at 4 °C at a flow rate of 40 ml/li. Chromatography was monitored by

measuring the absorption at 280 11m of the eluate in a flow-through photometer. After the application of the periplasmatic protein extract, the column was washed with CP-buffer until the base line was reached and the bound hNGAL mutein was subsequently eluted with 10 ml of a solution of 2.5 mM D-desthiobiotin (Sigma) in CP-buffer. The fractions containing the purified hNGAL mutein were checked via SDS-polyacrylamide gel electrophoresis (Fling und Gregerson, Anal. Biochem. 155 (1986), 83-88) and were pooled. The protein yields were between 30 u.g and 70 jig per 11 culture.
Table 1: Sequence characteristics of selected hNGAL muteins
Pos. hNGAL TlpcA TlpcB TlpcC TlpcD TlpcE TlpcP TlpcG TlpcH
40 Ala Cys Gly Leu Ser Val Gly Arg Ala
42 Leu Val Tyr Pro Val Leu Ser Ser Cys
44 Glu Gin Tyr lie Phe Gin* Phe He Pro
46 Lys Leu Gin* Gin* Gin* Cys Arg Phe Leu
47 Asp Leu Arg Ala Ser Trp Phe Gin* Phe

49 Gin Ser Trp He Ser Cys Val Val Phe
50 Lys Met Ser Phe Ala Pro Gin Phe Leu
70 Leu Leu Leu Glu Gly Asp Ser Pro Gin*
72 Arg Met Arg Ala Asn Glu Gin* Ala Arg
73 Lys Asp Asp Tyr Lys Lys Gin Asn Pro
77 Asp Arg Pro Val Asn Asn Trp He Ser
79 Trp Tyr Met Lys Thr Val Arg Ala Arg
101 Pro Gly Leu Tyr Tyr Pro Phe Val Leu
102 Gly He Ser Leu Trp Val Pro Val Thr
103 Leu Val Leu Tyr Gin* Leu Ser Thr Met
12 5 Lys Ser Trp Leu Gly Glu Arg Ala Lys
127 Ser Met Ala Cys Arg Ala Lys Lys Ser
128 Gin Thr Asp Pro Met Ser Ala Thr Asp
130 Arg Gin* Gin Gly Glu Gin His Lys Asp
132 Tyr Ala Trp Lys Thr Leu Ser Leu He
55 He Val0
98 Lys Asn0
*These glutamine residues were encoded by amber stop codons. °These amino acid substitutions arose due to random mutations.

(Eppendorf), followed by a final incubation for 5 minutes at 60 °C. The desired amplification product was isolated in each case by preparative agarose gel electrophoresis from GTQ Agarose (Roth) using the Jetsorb DNA extraction kit (Genomed).
The subsequent amplification step was also earned out in a 100 j.tl mixture, wherein approximately 6 ng of the two DNA fragments were used as template in the presence of 50 pmol of each of the primers SEQ ID NO:5 and SEQ ID NO:6. Both of these primers carried a biotin group at their 5'-end, in contrast to Example I. The remaining components of the PCR mixture were added in the same amounts as in the previous amplification steps. PCR was performed with 20 temperature cycles of 1 minute at 94 °C, 1 minute at 60 °C, 1.5 minutes at 72 °C, followed by a subsequent incubation for 5 minutes at 60 °C. The PCR product was purified using the E.Z.N.A. Cycle-Pure Kit (PeqLab).
For the cloning of the DNA fragment which represented the library of the hNGAL muteins in nucleic acid form, the S'-biotinylated PCR product was cut with the restriction enzyme BstXI (Promega) according to the instructions of the manufacturer and the resulting fragment of 347 nucieotides in size was purified by preparative agarose gel electrophoresis as described above. Residual DNA-fragments which were not or incompletely digested were removed via their 5'-biotin tags by incubating their solution with streptavidin-coated paramagnetic beads (Dynal), thus obtaining the doubly cut DNA fragment suitable for the subsequent ligation reaction.
To this end, 100 ul of the commercially available suspension of the paramagnetic particles in a concentration of 10 mg/ml were washed three times with 100 p.1 TE-buffer. The paramagnetic particles were then drained and mixed with 11 to 22 pmol of the DNA-fragment in 100 ul TE-buffer for 15 min at room temperature. The paramagnetic particles were collected at the wall of the Eppendorf vessel with the aid of a magnet and the supernatant containing the purified DNA fragment was recovered for further use in the following ligation reaction.
The DNA of the vector phNGAL12 (Fig.6) was cut with BstXI as described above and the larger of the two resulting fragments (3971 bp) was isolated by preparative agarose gel electrophoresis. For the ligation, 6.85 ug (30 pmol) of the PCR fragment and 78.65 ug (30 pmol) of the vector fragment were incubated in the presence of S55 Weiss Units of T4 DNA ligase (Promega) in a total volume of 8550 ul (50 mM Tris/HCl pH 7.8, 10 mM MgCl2, 10 mM DTT, 1 mM ATP, 50 ug/ml BSA) for four days at 16 °C. The DNA was then precipitated by adding 110 ug tRNA from yeast (Boehringer Mannheim), 350 ul 5 M

ammonium acetate, and 1300 ul ethanol per 350 JE-L! of the ligation mixture. Incubation at -20 °C for two days was followed by centrifugation (30 minutes, 16000 g, 4 °C). Each precipitate was washed with 750 u.1 ethanol (70 % v/v, -20°C), centrifiiged (5 minutes, 16000 g, 4 °C), and dried under vacuum (2 minutes). The DNA was finally dissolved in a total volume of 427.5 u.1 water to a final concentration of 200 jig/ml.
The preparation of electrocompetent cells of the E. coli K12 strain XLl-blue (Bullock et al, supra) was carried out according to the methods described under Example 1.
The Micro Pulser system (BioRad) was used in conjunction with the cuvettes from the same vendor (electrode separation 2 mm) for the electroporation. All steps were carried out in the cold room at 4 °C. Each 10 ui of the DNA solution (2 p.g) from above was mixed I with 100 ul of the cell suspension, incubated for 1 minute on ice, and transferred to the pre-chilled cuvette. Then the electroporation was performed (5 ms, 12.5 kV/cm) and the suspension was immediately diluted in 2 ml of ice-cold SOC-medium followed by shaking for 60 minutes at 37 °C and 200 rpm. The culture was diluted in 1.5 1 2xYT-medium containing 100 ug/ml ampicillin (2YT/Amp) to an OD550' of 0.5 and cultivated at 37°C until the ODJSJ was raised to 0.7 as caused by the replicating cells. By employing in total 85.5 fig of the ligated DNA, 1.8-1010 transformants were obtained in this manner using altogether 43 electroporation runs. The transformants were further used according to Example 7.
Example 7: Phagemid presentation and selection of hNGAL muteins against an eight-itmino-acid peptide from the cell- binding domain of human Thrombospondinl "thrombospondin peptide")
The 1500 ml culture, containing the cells which were transformed with the phasmid vectors corresponding to phNGAL12, but coding for the library of the lipocalin muteins as 'usion proteins, were infected with VCS-M13 helper phage (Stratagene) at a multiplicity of infection of approximately 10. The culture was shaken for additional 30 minutes at 37 °C, 60 rpm. Then the incubator temperature was lowered to 26 °C and Kanamycin (70 ug/ml) was added. After 10 minutes, anhydrotetracycline was added at 25 jag/1 (1875 j.il of a 20 ug/m! stock solution in DMF) in order to induce gene expression. Incubation continued for Another 15 hours at 26 °C, 160 rpm.
The ceils were sedimented by centrifugation (60 minutes, 12500 g, 4 °C). The supernatant containing the phagemid particles was sterile-filtered (0.45 um), mixed with 1/4 volume

(375 ml) ice-cold 20 % w/v PEG 8000, 15 % w/v NaCl, and incubated for one hour at 4 °C. After centrifugation (30 minutes, 18500 g, 4 °C) the precipitated phagemid particles were dissolved in 60 ml of cold PBS. The solution was incubated on ice for 60 minutes and was distributed into two SS34 centrifugation rubes. After centrifugation of undissolved components (5 minutes, 18500 g, 4 °C) each supernatant was transferred to a new centrifugation tube.
The phagemid particles were reprecipitated by mixing with 1/4 volume 20 % w/v PEG 8000, 15 % w/v NaCl, followed by incubation for 60 minutes on ice. The phagemids were aliquoted (2 ml) and 1 mM EDTA and 50 mM benzamidine (Sigma) were added for long term storage at -20°C.
The biotinylated synthetic thrombospondin peptide derived from the cell binding domain of human Thrombospondin I (Tulasne et al., Blood 98 (2001), 3346-3352; H2N-Arg-Phe-Tyr-Val-Val-Met-Trp-Lys-Aca-Aca-Lys-Biotin-COOH, SEQ ID NO: 18, Aca: amino caproic acid) was used together with Streptavidin-coated paramagnetic particles (Dynal) as target for affinity enrichment from the library of phagemids representing the hNGAL muteins.
For this purpose a 2 ml aliquot of the precipitated phagemids from above was centrifuged (20 minutes, 18.5QG g, 4 °C), the supernatant was removed, and the sedimented phagemid particles were dissolved in 1 ml PBS. After incubation for 30 minutes on ice the solution was centrifuged (5 minutes, 1S500 g, 4 °C) to remove residual aggregates and the supernatant was directly used for the affinity enrichment.
In order to enrich peptide-binding phagemids, 40 ul of a 825 nM solution (33 pmol) of the biotinylated thrombospondin peptide in PBS (prepared by mixing 100 ul of a 10 uM solution of the synthetic peptide in DMF with 1112 ul PBS) was mixed with 260 ul of a solution of the freshly prepared phagemids (between 2«1012 and 5*1012 colony forming units, cfu) and incubated at RT for 1 h so that complex formation between the peptide and the muteins presented by the phagemids was able to occur. Then, 100 ul of a solution of 8 % w/v BSA, 0.4 % v/v Tween 20 in PBS was added.
Parallel thereto, 100 ul of the commercially available suspension of streptavidin-paramagnetic particles (Dynal) were washed three times with 100 ul PBS. Herein, the particles were kept suspended for 1 min by rotating the 1.5 ml Eppendorf vessel and then collected at the wall of the vessel with the aid of a magnet, and the supernatant was

stripped off. In order to saturate unspecific binding sites, the paramagnetic particles were incubated with 100 |xl of 2 % w/v BSA in PBST at RT for 1 h.
After removing the supernatant, the mixture of the biotinylated thrombospondin peptide and the phagemids was added to the paramagnetic particles, and the particles were resuspended and incubated at RT for lOmin. Finally, free biotin-binding sites of streptavidin were saturated by adding 10 ul of a 4 inM D-desthiobiotin (Sigma) solution in PBS to the mixture and incubating said mixture at RT for 5 min. This step served for preventing the Strep-tag® II - as part of the fusion protein of the muteins and the phage coat protein pin fragment - from forming a complex with streptavidin.
Unbound phagemids were removed by washing the paramagnetic particles eight times with I ml of fresh PBST containing 1 mM D-desthiobiotin. Each time the particles were collected with the aid of the magnet and the supernatant was stripped off. Finally the bound phagemids were eluted by resuspending the particles in 950 ul of 0.1 M glycine/HCl pH 1.2 and incubation for 15 minutes. After collecting the particles with the magnet, the supernatant was recovered and immediately neutralized by addition of 150 ul of 0.5 M Tris.
For the purpose of amplification, this phagemid solution (1.1 ml, containing between 107 ^nd 1010 cfo, depending on the selection cycle) was shortly wanned to 37 °C, mixed with 3 ml of an exponentially growing culture of E. coli XLI-blue (OD550 = 0.5 at 37 °C), and incubated for 30 minutes at 37 °C, 200 ipm. The cells infected with the phagemids were subsequently sedimented (2 minutes, 4420 g, 4 °C), resuspended in 600 \x\ of the culture medium, and plated out onto three agar plates with LB-medium containing 100 ug/ml ajmpicillin (LB/Amp; 140 mm diameter).
After incubation for 14 hours at 32 °C, the cells were scraped from the agar plates, each with addition of 10 ml 2xYT/Amp-medium, were transferred to a sterile Erlenmeyer-flask, apd were shaken for 20 minutes at 37 °C, 200 rpm for complete resuspension.
For another cycle of production and affinity enrichment of the phagemid particles 50 ml of 2xYT/Amp medium prewarmed to 37°C were inoculated with 0.2 to 1 ml of said suspension so that the cell density was OD550 = 0.08. This culture was incubated at 37°C5 150 rpm to a cell density of OD550 = 0.5. Then the culture was infected with VCS-M13 helper phage (Stratagene) at a multiplicity of infection of approximately 10 and the culture was shaken for further 30 minutes at 37 °C, 160 rpm. Kanamycin (70 j.ig/ml) was

subsequently added, the incubator temperature was lowered to 26 °C and, after 10 minutes, anhydrotetracycline was added to 25 ng/1 to induce gene expression. Incubation continued for another 15 hours at 26 °C, 160 rpm.
The cells were sedimented by centrifugation (15 minutes, 12000 g, 4 °C). The supernatant containing the phagemid particles was sterile-filtered (0.45 urn), was mixed with 1/4 volume (12.5 ml) 20 % w/v PEG 8000, 15 % w/v NaCl, and was incubated for 1 h on ice. After centrifugation (20 minutes, 18000 g, 4 °C) the precipitated phagemid particles were dissolved in 2 ml of cold PBS. The solution was incubated on ice for 30 minutes and was distributed into two 1.5 ml reaction vessels. After centrifugation of undissolved components (5 minutes, 18500 g, 4 °C) each supernatant was transferred to anew reaction vessel.
The phagemid particles were reprecipitated by mixing with 1/4 volume 20 % w/v ?EG 8000, 15 % w/v NaCl, followed by incubation for 60 mimites on ice. After centrifugation (20 minutes, 18500 g, 4 °C) the supernatant was removed and the precipitated phagemid particles were dissolved in 1 ml PBS. After incubation for 30 minutes on ice the solution was centrifuged (5 minutes, 18500 g, 4 °C) and the supernatant was used directly for the affinity enrichment. Five further selection cycles with the thrombospondin peptide were carried out in this way.
Example 8: Identification of thrombospondin peptide-bindine hNGAL muteins by use of the "colony screening" method
For the analytical production of the hNGAL rauteins as fusion proteins with the Strep-Tag® II and the albumin-binding domain the gene cassette between the two BstXl cleavage sites was subcloned from the vector phNGAL12 on phNGALJ.
For this purpose the phasmid DNA was isolated from the mixture of the E. coli clones obtained by infection with the phagemids from Example 7, eluted as a result of the fourth and the fifth selection cycle, using the Plasmid Midi Kit (Qiagen). The DNA of both preparations was cut with the restriction enzyme BstXl and in each case the smaller of the two fragments (347 bp) was purified by preparative agarose gel electrophoresis as iescribed in Example 6. The DNA of the vector phNGAL7 was cut with BstXl and the arger of the two fragments (3971 bp) was isolated in the same way.

For the ligation, each 100 finol of the isolated small DNA-fragments was mixed with 100 fraol of the large DNA-fragment and with 1.5 Weiss Units T4 DNA ligase (Promega) in a total volume of 20 ul (30 mM Tris/HCl pH 7.8,10 mM MgCl2s 10 mM DTT, 1 mM ATP), followed by incubation overnight at 16 °C. E. coli TG1-F' (JE. coli K12 TGI, which had lost its episome through repeated culturing under non-selective conditions) was transformed with 2 uJ of each of this ligation mixtures according to the CaCl2-method (Sambrook et al., supra), obtaining 2.0 ml of a cell suspension. 100 JJ.1 of this suspension were plated out on an agar plate containing LB/Amp medium and incubated at 37°C for 14 h.
Two hydrophilic PVDF membranes (Millipore, type GVWP, pore size 0.22 um), labelled at one position and cut to size, were laid onto LB/Amp agar plates. 150 ul of the cell suspension from the transformation batches, which had been centrifuged (5000 g, 2 min, 4 I °C) and resuspended in 500 ul of the culture medium, were uniformly plated onto these membranes. The agar plates were incubated for 7.5 hours at 37 °C until the colonies had reached a size of approximately 0.5 mm.
In the meantime two hydrophobia membranes (Millipore, Lnmobilon P, pore size 0.45 um), also cut to size, were moistened with PBS according to the instructions of the manufacturer. They were then each agitated lor 4 hours at room temperature in 10 ml of a solution of 10 mg/ml human serum albumin (HSA, Sigma) in PBS. Remaining binding sites on the membranes were saturated by incubation with 20 ml 3 % w/v BSA, 0.5 % v/v Tween 20 in PBS for 2 hours at RT. The membranes were washed twice for 10 minutes each with 20 ml PBS and immersed afterwards for 10 minutes in 10 ml LB/Amp medium, to which 200 jig/1 anhydrotetracycline was added.
finally, they w«e marked at one position and were laid onto culture plates with LB/Amp igar, which additionally contained 200 ug/1 anhydrotetraeycline. The hydrophilic nembranes from above, on which the colonies were grown, were laid onto the iydrophobic membranes in such a way that both of the marks superimposed. The culture ])lates were incubated each with a stack of both membranes at 22 °C for 15 hours. During i his phase the respective hNGAL muteins were secreted from the colonies on the upper membrane and were immobilized via their albumin-binding domain on the HSA on the lower membranes.

After this, the upper membranes with the colonies were transferred to fresh LB/Anip agar plates and stored at 4 °C. The hydrophobic membranes were removed, washed three times for 5 minutes each with 20 ml PBST.
For analysis of the binding activity of the immobilized liNGAL muteins the membranes were then incubated for 1 hour in 10 ml of a 30 jig/ml solution of the biotinylated thrombospondin peptide in PBST (a 1 mM stock solution of the biotinylated peptide in DMF was therefore 1:100 diluted in PBST). The membranes were washed three times with PBST, followed by incubation for 1 hour with 10 ml avidin-alkaline phosphatase conjugate (Sigma, dilution 1:40,000 in PBST) for detection of bound peptides via their biotin groups. The membranes were washed twice witii PBST and once with PBS, each for 5 minutes, and agitated for 10 minutes in AP-buffer (0.1 M Tris/HCl pH 8.8, 0.1 M NaCl, 5 mM MgCfe). For the chromogenic reaction, the membranes were incubated in 10 ml AP-buffer, to which 30 fil 5-bromo-4-chioro-3-indolyl phosphate 4-toluidine salt (Roth, dissolved at 50 ug/mlindimethylformamide)and 5 ul nitro bluetetrazolium (Roth, 75 ug/ml in 70 % v/v dimethylformaniide) were added, until distinct colour signals could be recognized at the positions of some of the colonies.
19 of the colonies (9 isolated from panning cycle four and 10 from cycle five) giving rise to intense colour spots on the hydrophobic membranes were cultured from the corresponding hydrophilic membranes. Their plasmid DNA was isolated and the hNGAL gene cassette was subjected to sequence analysis by use of the Genetic Analyzer 310 system with Big Dye Terminator Cycle Sequencing Kit (Applied Biosystems) according to the instructions of the manufacturer and using the oligodeoxynucleotide SEQ ID NO:11 as primer.
The 19 sequenced clones exhibited only twelve different sequences, which were named RFY-A, RFY-B, RFY-C, RFY-D, RFY-E, RFY-F, RFY-G, RFY-H, RFY-I, RFY-J, RFY-K and RFY-L. The clone RFY-B was found twice and the clone RFY-C was found six times. The clone RFY-J exhibited a deletion of 3 nucleotides, resulting in the deletion of one ammo acid situated within peptide loop No. 4 at position 125. An amber stop codon, which is translated into the amino acid glutamine in a supE suppressor strain, was identified in clone RFY-L.
The nucleotide sequences of the clones were translated into amino acid sequences and those amino acids which deviate from the original hNGAL protein are given in Table 2a,b. The nucleotide sequences of the muteins RFY-B, RFY-C, and RFY-E are also given as

43
SEQ ED NO:23, SEQ ID NO:25 and SEQ ID NO:27 in the sequence listing. The amino acid sequences of these muteins are given as SEQ ID NO:245 SEQ ID NO:26 and SEQ ID NO:28 in the sequence listing.
The muteins RFY-B and RFY-C, which were found twice and six times respectively were chosen for detailed characterization of their binding activity. In addition the mutein RFY-E was chosen, because its amino acid composition in the randomized regions appeared to be largely unique.
Table 2a: Sequence characteristics of selected hNGAL muteins
POS. hNGAL RPY-A RFY-B RFY-C RPY-D RFY-E RFY-F RFY-G RFY-H


40 Ala Phe Gly Gly
42 Leu Glu Thr His
44 Glu Leu Tyr Leu
46 Lys Ser Arg Asn
47 Asp Arg Leu Val
49 Gin Phe Ser Pro
50 Lys Thr Val Asn
70 Leu Met Asp Asp
72 Arg Phe Trp Leu
73 Lys Ser Ala Met
77 Asp Leu Asn Pro
19 Trp Leu Lys Phe
L01 Pro Phe Leu Pro
.02 Gly Leu Gly Phe
.03 Leu Asp Leu Leu
.25 Lys Asn Pro Asn
}27 Ser Ser Gly Pro
1281 Gin Gin Gly Arg
Arg Arg lie Tyr
¥2 Tyr Tyr Trp Glu
tO6 Phe°

Thr Gly Leu
Pro Thr Phe
Leu Val Ala
Val Thr Leu
Leu Leu Phe
Thr Ser His
Glu Arg Arg
lie Pro Thr
Met Met Ser
Asp Gly Ser
Leu Lys Ser
Pro Lys Lys
Asp Met Pro
Ala Ser Phe
Leu Asn Ser
Pro Gin Pro
Lys His lie
Asn Lys Ser
Trp Pro Pro
Lys Ser Pro

Ser Met
Gly Val
Pro Leu
Ser He
Leu Pro
Arg Asn
Leu Val
His Pro
Lys Pro
Asn Ser
Pro Gly
Lys Glu
Cys Arg
Val Gly
Ala Asp
Glu Arg
Leu Leu
Asn Lys
Pro Gin
Leu He

Table 2b: Sequence characteristics of selected hNGAL muteins Pos. hNGAL RFY-I RPY-J RFY-K RPY-L

40 Ala Cys Arg Gin Ser
42 Leu Gly Leu Pro Pro
44 Glu Leu Cys Lys . Ser
46 Lys Ser Pro His Leu
47 Asp Phe Phe Leu Ala
49 Gin Asn Val Ser Gly
50 Lys Gly Arg Leu Gin*
70 Leu Lys Lys Pro Thr
72 Arg His Glu Lys Asn
73 Lys Gly Arg Met Ala
77 Asp Arg Ala Pro Ser
79 Trp Thr His He Arg
101 Pro Gly Lys Ala Asn
102 Gly Ser Phe Trp Phe
103 Leu Cys Ser Glu Ser
125 Lys Met AAA Thr Lys
127 Ser Leu Pro Gly Lys
128 Gin . Asn Ser Thr Lys
130 Arg Asn He Pro Asp
132 Tyr Ser Glu Thr Pro
106 Tyr Ser°
126 Val Pro0
* This glutamine residue was encoded by an amber stop codon. 0 These amino acid substitutions arose due to accidental
mutations outside the randomized positions. A This amino acid deletion arose due to an accidental
mutation outside the randomized positions.
Example 9: Production of the hNGAL muteins
jFor preparative production of the hNGAL muteins RFY-B, RFY-C, and RFY-E obtained from Example 8 the mutagenized coding region between the two BstXl cleavage sites was subcloned from the phNGAL7 vector on the expression plasmid phNGALlS (Fig.7). The

plasmid thus obtained encoded a fusion protein of the mutein with the OmpA signal sequence, at the amino terminus and the Strep-Tag® II affinity tag at the carboxy terminus.
For subcloning, the plasrnid DNA coding for the relevant rautein was cut with the restriction enzyme BstXX> and the smaller of the two fragments (347 bp) was purified by preparative agarose gel electrophoresis as described in Example 6, In the same manner, phNGALlS vector DNA was cut with BstXL, and the larger of the two fragments (3398 bp) was isolated.
1.5 Weiss units of T4 DNA ligase (Promega) were added to 50 finol of each of the two DNA fragments in a total volume of 20 ul (30 mM Tris/HCl pH 7.8, 10 mM MgCl2, 10 mM DTT, 1 mM ATP) and for ligation the mixture was incubated at 16°C for 16 h. 5 jil of the ligation mixture were then used to transform £. coli JMS3 (Yanisch-Perron et al., Gene 33 (1985), 103-119) according to the CaCl2 method, obtaining 2.0 ml of a cell suspension. 100 ul of this suspension were plated out on an agar plate containing LB/Amp medium and incubated at 37°C for 14 h.
A single colony of the E. coli-JM.%3 transformants was used for inoculating 100 ml of pLB/Amp-medium, followed by incubation overnight at 30°C, 200 rpm. 2 1 of LB/Amp-
i
nedium in a 5 1-Erlenmeyer flask were then inoculated with 40 ml of this preculture and /ere shaken at 22 °C» 200 rpm to an OD550 = 0.5. Induction was performed by adding 200 ig/1 anhydrotetr&cyciirie (200 uj of a 2 mg/ml stock solution in DMF) followed by shaking or 3 further hours at 22 °C, 200 rpm.
' The cells from one flask were centrifuged (15 minutes, 4420 g, 4 °C) and, after removing the supernatant, were resuspended is 20 ml of pre-chilled periplasmic release buffer (100 1 tiM Tris/HCl pH 8.0,500 mM sucrose, 1 raM EDTA) and incubated for 30 minutes on ice. "he spheroplasts were then removed in two successive centrifugation steps (15 minutes, 1420 g, 4 °C and 15 minutes, 30000 g, 4 °C). The supernatant comprising theperiplasrnatic j rotein extract was dialyzed against CP-buffer (100 mM Tris/HCl pH 8.0, 150 mM NaCl, 1 mM EDTA), sterile-filtered, and used for the chromatographic purification of the hfNGAL mutein.
The purification method was based on the C-terminal Strep-Tag® II-affinity tag (Schmidt e' al., supra). In the present case the streptavidin mutein "1" was employed (German Patent 136 41 876.3; Voss and Skerra, supra), which was coupled to an NHS-activated sepharose

(Pharmacia) yielding 5 mg/ml immobilized streptavidin per 1 ml of the bed volume of the matrix.
A 4 ml bed volume cliromatography column filled with this material was equilibrated with 20 ml CP-buffer at 4 °C at a flow rate of 40 ml/h. Chromatagraphy was monitored by measuring the absorption at 280 nm of the eluate in a flow-through photometer. After the application of the periplasmatic protein extract, the column was washed with CP-buffer until the base line was reached and the bound hNGAL mutein was subsequently eluted with 10 mi of a solution of 2.5 oM D-desthiobiotin (Sigma) in CP-buffer. The fractions containing the purified hNGAL mutein were checked via SDS-polyacryiamide gel electrophoresis (Fling and Gregerson, supra) and were pooled. To obtain suitable protein concentrations and buffer conditions for further applications the mutein pools were concentrated to a final volume of ca 500 |ii using centricon tubes YM 10 (MW-cutoff 1000, Millipore) for ultrafiltration and subsequently dialysed against HBS-buffer (150 mM NaCl, 10 mM HBPBS pH 7.4, 3 mM EDTA). The protein yields for RFY-B, RFY-C, and RFY-E were 70 ug, 60 ug and 200 ug respectively per 1 1 of culture. In this manner both the original hNGAL and its muteins RFY-B, RFY-C, and RFY-E were prepared.
Example 10: Measurement of the affinity of the hNGAL muteins for the thrombospondin peptide in ELISA
For the detection of binding in an ELISA (Enzyme-linked Immunosorbent Assay) the wells of a microtiter plate (Maxisorb, Nunc) were filled each with 50 ul of a 50 ug/ml solution of Avidin (Fluka) in PBS and were incubated over night at room temperature (RT). After washing three times with PBST, 50 ul of a 1 uM solution of the biotinylated thrombospondin peptide SEQ ED NO: 18 in PBST were filled into the wells and the peptide was immobilized via complex formation between biotin and the preadsovbed avidin. After incubation for one hour the solution was removed and the wells of the microtiter plate were washed three times with PBST. In order to saturate unspecific binding sites, the wells were filled with 100 ul of 4 % w/v BSA in PBST and incubated for one hour at RT, followed by three times washing with PBST.
Then a dilution series of the muteins from Example 9 was prepared, starting from 100 nM concentration, and followed by 1 hour incubation at RT. As a control for unspecific binding to avidin, a similar dilution series of hNGAL and its muteins was applied to wells where the thrombospondin peptide had been omitted. After washing three times with PBST, Anti-sti-epH-antibody-HRP-conjugate (IBA), diluted 1:8000 with PBST, was

applied to the wells. Incubation was performed for 1 hour at RT and followed by washing three times with PBST and twice with PBS.
Detection of bound IiNGAL muteins was accomplished using 3,3',5,5'-tetraniethylbenzidine and H2O2 as substrates for horseradish-peroxidase. For this purpose, 100 ui of a ready-to-use HRP-substrate solution (Biochem Immunosystems) was filled into the weils and the colour development was stopped after a few minutes by adding 100 ul of 0.3 M sulphuric acid. Product formation was measured via the endpoint absorption at 450 pm in a SpectraMax 250 photometer (Molecular Devices).
iThe curve was fitted by non-linear least squares regression with the help of the computer >rograra Kaleidagraph (Synergy software) according to the equation P • LHCPMLJtypMJ+fPJO- Thereby [P]t is the total concentration of immobilized
1 hrombospondin peptide (in A450 units), [LJt is the concentration of the applied mutein or
I ENGAL, respectively, [P • L] is the concentration of the formed complex (in A450 units),
^nd Ko is the apparent dissociation constant.
' "he resulting binding curves are depicted in Fig.8. The values obtained for the apparent association constants of the complexes between the IiNGAL muteins and the thrombospondin peptide are summarized in the following table:
t&fGAL variant Kn f nMI
hNGAL:
J FY-B: 4.3 ± 0.2
BFY-C: 4.6 ±0.6
R|FY-E: 8.2 ± 0.8
fro detectable binding activity
Ekample 11: Measurement of the affinity of the hNGAL muteins for the thrombospondin p The binding affinity of the hNGAL muteins to the thrombospondin peptide SEQ ID NO: 11 was determined by surface plasmon resonance (SPR) using the BIAcore X system (EIACORE). First 35 ul of the biotinylated thrombospondin peptide at a concentration of 1 W/ml (prepared by mixing 1 ul of a 200 ug/ml peptide solution in DMF with 199 ul HBS bjffer containing 150 raM NaCl, 10 mM HEPES pH 7.4, 3 raM EDTA) was immobilized

to the surface of one flow channel of a streptavidin-coated sensor chip SA (BIACORE) according to the instructions of the manufacturer, resulting in an amount of ca. 400 response units (RU). Binding curves of the liNGAL muteins were then measured hy applying 35 JJ.1 of each purified muteins from Example 9 in HBS buffer at concentrations between 500 nM and 25 nM using HBS-EP (HBS containing 0.005 % Surfactant P20) as a running buffer and a continuous fiowrate of 5 fil/min.
Steady state resonance values were measured at the end of the injection phase for the channel with the immobilized thrombospondin peptide for each protein concentration applied. Only negligible buffer effects were detected in the second channel of the sensor chip, which served as a control. These values were directly plotted against the concentration of the hNGAL mutein.
The curve was fitted by non-linear least squares regression with the help of the computer program Kaleidagraph (Synergy software) according to the equation [P • LH[P3t[L]t)/(KD+(P]t). Thereby [PJ, is the total concentration of immobilized thrombospondin peptide (in RU), [L]t is the concentration of the applied mutein or hNGAL, respectively, [P • L] is the concentration of the formed complex (in RU), and KD is the dissociation constant under equilibration conditions.
The resulting binding curves are depicted in Fig.9. The values obtained from SPR measurements for the dissociation constants of the complexes between the hNGAL muteins and the thrombospondin peptide are summarized in the following table:
hNGAL variant Kn fnM]
hNGAL:
RFY-B: 105.4 ±6.5
RFY-C: 106.1 ± 15.7
|RFY-E: 107.2 ±11.4
no detectable binding activity [Example 12: Selection of hNGAL muteins against human interleukin-8 (IL-8)
The recombinant human cytokine interleukin-8 (Baggiolmi and Clark-Lewis, FEBS Lett.
397, (1992), 97-101; H2K-
AVLPRSAKBLRCQCKTYSKPFHPKFIKELRVIESGPHCANTEnVKXSDGRELCLDP

KENWVQRVVEKFLKRAENS-COOH; SEQ ID NO:29) was conjugated with biotin groups and used as a target for affinity enrichment from the library of phagemids representing the hNGAL muteins in the presence of streptavidin-coated paramagnetic particles (Dynal).
The conjugate was prepared by mixing 5.6 nmol (12.5 fig) of sulfosuccinimidyl-2-(biotinamido)ethyl-l,3-dithiopropionate (NHS-SS-Biotin, Pierce) dissolved in 3.4 p.1 H2O with 1.4 nmol (12.5 /.ig) human recombinant IL-8 (Promocell) dissolved iji 50 ul H7O, 36.6 |.tl H2O and with 10 pi lOx concentrated PBS/NaOH pH 8.5. The mixture was incubated under stirring at room temperature (RT) for 1 h. Excess reagent was then removed from the IL-8 conjugate by means of a PD-10 gel filtration column (Pharmacia) according to the manufacturer's instructions using PBS/NaOH pH 8.5 as running buffer. Biotinylated IL-8 eluted from the PD-10 column was adjusted to a final concentration of I f.iM with the same {buffer resulting in a total volume of 1.24 ml. Labelled IL-8 was stored in aiiquots at -SO °C jand thawed directly prior to use.
?or the isolation of phagemids displaying a mutein with affinity for IL-8, one aliquot of the irecipitated phagemids, obtained as in Example 7, which were kept at -20 °C for long term storage, was thawed and the phagemids were pelleted (20 minutes, 18500 g, 4 °C). After j emoval of the supernatant, the sedimented phagemid particles were dissolved in 270 ui ]'BS, incubated for 30 minutes on ice and finally centrifuged (5 minutes, 18500 g, 4 °C) to Remove residual aggregates.
M order to enrich IL-8-binding phagemids, 30 y.1 of a 1 uM solution (30 pmol) of liotinylated interleukin-S was mixed with 270 jal of the phagemids in PBS (ca, 1013 cm) and incubated at RT for 1 h so that complex formation between the cytokine and the nuteins presented by the phagemids was allowed to occur. Then, 100 u. 1 of a solution of 8 % w/v BSA, 0.4 % v/v Tween 20 in PBS was added.
Fjarallel thereto, 100 u.1 of the commercially available suspension of streptavidin-paramagnetic particles (Dynal) was washed three times with 1 ml PBS. Herein, the piarticles were kept suspended for 1 min by rotating the 1.5 ml Eppendorf vessel and then collected at the wall of the vessel with the aid of a magnet, and the supernatant was pipetted off In order to saturate unspecific binding sites, the paramagnetic particles were iijciibated with 1 ml of 2 % (w/v) BSA in PBST at RT for 1 h.

After removing the supernatant as above, the mixture of the biotinylated IL-8 and the phagemids was added to the paramagnetic particles, and the particles were resuspended and incubated at RT for lOmin. Finally, free biotin-binding sites of streptavidin were saturated by adding 10j.il of a 4mM D-desthiobiotin (Sigma) solution in PBS to the mixture and incubating said mixture at RT for 5 min. This step served for preventing the Strep-tag® II - as part of the fusion protein of the muteins and the phage coat protein pill fragment - from forming a complex with streptavidin.
Unbound phagemids were removed by washing the paramagnetic particles eight times for 1 min with 1 ml of fresh PBST containing 1 mM D-desthiobiotin. Each time the particles were collected with the aid of the magnet and the supernatant was pipetted off. Finally, the bound phagemids were erated under reducing conditions by resuspending the particles in 1 ml PBST containing 1 mM desthiobiotin and 100 mM DTT. The solution was incubated at 37 °C for 1 h to reduce the disuifide bond contained in the linker molecule between interleukin-S and biotin, thus releasing phagemids specifically bound to EL-8 from the beads.
For the purpose of amplification, the eluted phagemid solution (1.0 ml, containing between 107 and 10s cfu, depending on the selection cycle) was shortly brought to 37 °C, mixed with 3 ml of an exponentially growing culture of E. colt XLl-blue (OD55o = 0.5 at 37 °C), and was shaken at 200 rpm for 30 minutes at 37 °C. The infected cells were sedimented (2 minutes, 4420 g, 4 °C), resuspended in 600 p.1 of culture medium, and plated out onto three agar plates with LB-medium containing 100 ug/ml ampicillin (LB/Amp; 140 mm diameter).
After incubation for 14 hours at 32 °C, the lawn of colonies was scraped from the agar plates, each with addition of 10 ml 2xYT/Amp-medium. The suspension was transferred to a sterile Erlenmeyer-flask and shaken for 20 minutes at 37 °C, 200 rpm.
For another cycle of production and affinity enrichment of the phagemid particles, 50 ml of 2xYT/Amp medium prewarmed to 37 °C was inoculated with 0.2 to 1 ml of said [Suspension so that the cell density was OD550 = 0.0S. This culture was incubated at 37 °C, 160 rpm until a cell density of OD550 = 0.5 was reached. Then, the culture was infected with VCS-M13 helper phage (Stratagene) at a multiplicity of infection of approximately 10 and the culture was shaken for further 30 minutes at 37 °C, 160 rpm. Kanamycin (70 Lg/ml) was subsequently added, the incubator temperature was lowered to 26 °C and, after

3 0 minutes, anhydrotetracycline was added to 25 ug/1 to induce gene expression. Incubation continued for another 15 hours at 26 °C, 160 rpm.
The cells were sedimented by centrifugation (15 minutes, 12000 g, 4 °C). The supernatant containing the pliagemid particles was sterile-filtered (0.45 jim), was mixed with 1/4 volume (12.5 nil) 20 % w/v PEG 8000, 15 % w/v NaCl, and was incubated for 1 h on ice. Alter centrifugation (20 minutes, 18000 g, 4 °C) the precipitated phagemid particles were dissolved in 2 ml of cold PBS. The solution was incubated on ice for 30 minutes and was distributed into two 1.5 ml reaction vessels. After centrifugation of undissolved components (5 minutes, 1S500 g, 4 °C) each supernatant was transferred to a new reaction vessel.
The phagemid particles were reprecipitated by mixing with 1/4 volume 20 % w/v PEG 8000, 15 % w/v NaCl, followed by incubation for 60 minutes on ice. After centrifugation (20 minutes, 1S500 g, 4 °C) the supernatant was removed and the precipitated phagemid particles were dissolved in 270 ul PBS. After incubation for 30 minutes on ice the solution was centrifuged (5 minutes, 18500 g, 4 °C) and the supernatant was used directly for the affinity enrichment Four further selection cycles with EL-8 were carried out in this way.
Example 13: Identification of hNGAL muteins binding interleukin-8 bv use of the "colony screening" method
For the analytical production of the liNGAL muteins as fusion proteins with the Strep-Tag® II and the albumin-binding domain as described in Example 3, the gene cassette between the two BstXl cleavage sites was subcloned from the vector phNGAL12 on phNGAL7.
For this purpose the phasmid DNA was isolated from the mixture of the E. coli clones obtained by infection with the phagemids from Example 12, eluted after the fifth selection cycle, using the Plasmid Midi Kit (Qiagen). The DNA was cut with the restriction enzyme BstXl and the smaller one of the two fragments (347 bp) was purified by preparative lagarose gel electrophoresis as described in Example 6. The DNA of the vector pIiNGAL?
Iwas cut with BstXl and the larger one of the two fragments (3971 bp) was isolated in the
I
(same way.
for the ligation, 100 fmol of the isolated small DNA-fragment was mixed with 100 ftnol of the large DNA-fragment and incubated with 1.5 Weiss Units of T4 DNA ligase (Promega)

in a total volume of 20 ul (30 mM Tris/HCl pH 7.8, 10 mM MgCl2, 10 mM DTT, 1 mM ATP), followed by incubation overnight at 16 °C. E. coli TG1-F" (E. coli K12 TGI, which had lost its episome through repeated culturing under non-selective conditions) was transformed with 4 ul of this ligation mixture according to the CaCI2-method (Sambrook et a!., supra), obtaining 2.0 ml of a cell suspension. The cell suspension was centrifiiged (5000 g, 2 min, 4 °C),,resuspended in 100 ul of the culture medium, plated on an agar plate containing LB/Amp medium and incubated at 37 °C for 14 h to determine the transformation efficiency.
A hydrophilic PVDF membrane (Millipore, type GVWP, pore size 0.22 um), labelled at one position and cut to size, was laid onto an LB/Amp agar plate. The cell suspension from a fresh transformation batch, which had been transformed with 5-10 nl of the ligation mixture described above, was centrifiiged (5000 g, 2 min, 4 °C), resuspended in 100 ul of the culture medium, and uniformly plated onto this membrane in order to obtain 400 to 500 colonies. The agar plate was incubated for 7.5 hours at 37 °C until the colonies had reached a size of approximately 0.5 mm.
In the meantime, a hydrophobic membrane (Millipore, Immobilon P, pore size 0.45 u.m), also cut to size, was moistened with PBS according to the instructions of the manufacturer. boating with HSA was achieved by agitation for 4 hours at RT in 10 ml of a solution of 10 mg/ml HSA (Sigma) in PBS. Remaining binding sites on the membrane were saturated by incubation with 20 ml 3 % (w/v) BSA, 0.1 % (v/v) Tween-20 in PBS for 2 hours at RT. (Hie membrane was washed twice for 10 minutes with 20 ml PBS and immersed afterwards Ifor 10 minutes in 10 ml LB/Amp medium, to which 200 ug/1 anhydrotetracycline was Ldded.
Finally, it was marked at one position and laid onto a culture plate with LB/Amp agar, which additionally contained 200 ug/l anhydrotetracycline. The hydrophilic membrane from above, on which the colonies were grown, was laid onto the hydrophobic membrane in such a way that both marks superimposed. The culture plate was incubated with the »tack of both membranes at 22 °C for 15 hours. During this phase the respective hNGAL iiuteins were secreted from the colonies on the upper membrane and were immobilized via heir albumin-binding domain via the HSA at the lower membrane.
After this, the upper membrane with the colonies was transferred to a fresh LB/Amp agar plate and stored at 4 °C. The hydrophobic membrane was removed and washed three times for 5 minutes each with 20 ml PBST.

For analysis of the binding activity of the immobilized liNGAL muteins, the membrane was incubated for 1 hour in 3.5 ml of a solution of 100 nM digoxigenated IL-8 in PBS.
The conjugate was prepared by mixing 14 nmo! (9.2 ug) of digoxigenin-3-O-methylcarbonyl-E-aminocaproic acid N-liydroxysuccinimide ester (DIG-NHS, Roche) dissolved in 2.3 ul DMSO with 1.4 iimol (12.5 fig) human recombinant IL-S (Promocell) dissolved in 50 ul fyO, 37.7 ul H2O and with 10 pi lOx concentrated PBS/NaOH pH 8.5. The mixture was incubated under stirring at RT for 1 h. Excess reagent was removed from the IL-8 conjugate by means of a PD-10 gel filtration column (Pharmacia) according to the manufacturer's instructions using PBS/NaOH pH 8.5 as running buffer. Digoxigenated IL-8 eluted from the PD-10 column was adjusted to a final concentration of 1 pM with the same buffer resulting in a total volume of 1.56 ml. Labelled IL-8 was stored in aliquots at -80 °C and thawed directly prior to use.
The membrane was washed three times with PBST, followed by incubation for 1 hour with 10 ml anti-digoxigenin Fab-Alkaline-Phosphatase conjugate diluted 1:1000 in PBST for detection of bound IL-8 via its digoxigenin groups. The membrane was washed twice with PBST and once with PBS, each for 5 minutes, and agitated for 10 minutes in AP-buffer. For the chromogenic reaction, the membrane was incubated in 10 ml AP-buffer, to which J30 ul 5-bromo-4-chloro-3-indolyl phosphate 4-toluidine salt (Roth, dissolved at 50 ug/rnl in dunethylformamide) and 5 ul nitro blue tetrazolium (Roth, 75 ug/ml in 70 % v/v kimethylformamide) were added, until distinct colour signals could be recognized at the positions of some of the colonies.
/rom 200 colonies which appeared on the membrane 9 gave rise to an intense colour spot on the hydrophobic membrane and were cultured from the corresponding hydrophilic membrane. Their plasmid DNA was isolated and the hNGAL gene cassette was subjected to sequence analysis by use of the Genetic Analyzer 310 system with Big Dye Terminator (cycle Sequencing Kit (Applied Biosystems) according to the instructions of the nWufacturer and using the oligodeoxynucleotide SEQ ID NO:5 as primer.
4.11 9 clones exhibited the identical sequence, indicating that a single mutein was preferentially enriched during the selection procedure. This mutein was named variant N4.
Tine nucleotide sequences of the clone N4 was translated into its amino acid sequence and tliose amino acid residues which deviate from the original hNGAL protein are given in

Table 3. The nucleotide sequence and the full amino acid sequence of the mutein N4 is also given as SEQ ID NO:30 and SEQ ID NO: 34.

Table. 3: Sequence characteristics of hNGAL mutein N4
Pos. hNGAL N4
40 Ala Asp
42 Leu Tyr
44 Glu Ala
46 Lys Ser
47 Asp Leu
49 Gin Gly
50 Lys Leu
70 Leu Val
72 Arg Tyr
73 Lys Asp
77 Asp Val
79 Trp Ser
101 Pro Glu
102 Gly Ala
103 Leu Val
125 Lys Asp
127 Ser -Arg
128 Gin Pro
130 Arg Ser
132 Tyr Glu
Example 14: Production of the hNGAL mutein N4
For preparative production of the hNGAL mutein N4 obtained from Example 13, the BstXl cassette was isolated from the variant in phNGAL7 and subcloned onto the expression plasmid phNGAL15 (Fig.7). The resulting plasmid encodes a fusion protein of the mutein N4 with the OmpA signal sequence at the amino terminus and the Strep-Tag® II affinity tag at the carboxy terminus.
For subcloning, the phNGAL7 plasmid DNA coding for the relevant mutein was cut with the restriction enzyme BstXl, and the smaller one of the two fragments (347 bp) was purified by preparative agarose gel electrophoresis as described in Example 6. In the same

25

manner, phNGAL15 vector DNA was cut with BstXI, and the larger one of the two fragments (339S bp) was isolated.
1.5 Weiss units of T4 DNA ligase (Promega) were added to 50 fmol of each of the two DNA fragments in a total volume of 20 ^xl (30 mM Tris/HCl pH 7.8, 10 mM MgCl2, lOmM DTT, 1 mM ATP) and for ligation the mixture was incubated at 16 °C for 16 h. 5 j.tl of the ligation mixture was then used-to transform E. coli JM83 (Yanisch-Perron et al, supra) according to the CaCk method, obtaining 2.0 ml of a cell suspension. 100 ul of this suspension were plated on agar containing LB/Amp medium and incubated at 37 °C for 14 h.
A single colony of the E. coli JM83 transformants was used to inoculate 100 ml of LB/Amp-medium, followed by incubation overnight at 30 °C and 200 rpni. 2 1 of LB/Amp-medium in a 51-Erienmeyer flask were then inoculated with 40 ml of this preculture and were shaken at 22 °C and 200 rpm until an OD550 = 0.5 was reached. Expression of the mutein N4 was induced by adding 200 j.ig/1 anhydrotetracycline (200 ui of a 2 mg/ml stock solution in DMF) and incubation was continued for another 3 hours.
The cells from one flask were centrifuged (15 minutes, 5571 g, 4 °C) and, after removing
the supernatant, were resuspended in 20 ml of pre-chilled periplasmic release buffer (100
ImM Tris/HCl pH 8.0, 500 mM sucrose, 1 mM EDTA) and incubated for 30 minutes on ice.
The spheroplasts were then removed in two successive centrifugation steps (15 minutes,
|298S g, 4 °C and 15 minutes, 25000 g, 4 °C). The supernatant comprising the periplasmatic
protein extract was dialyzed against CP-buffer (100 mM Tris/HCl pH 8.0, 150 mM NaCl,
1 mM EDTA), sterile-filtered, and used for the chromatographic purification of the
IhNGAL mutein.
The purification method was based on the C-terminal Strep-Tag® II affinity tag (Schmidt et al., supra) employing Strep-Tactin® Superflow-material (DBA). A 4 ml bed volume ^hromatography column filled with of this material was equilibrated with 20 ml CP-buffer it 4 °C at a flow rate of 2 ml/min. Chromatography was monitored by measuring the absorption at 2S0 nm in a flow-tlirough photometer. After application of the periplasmatic jbrotein extract (flow rate 0.S ml/min) the column was washed with CP-buffer at a flow rate
kDa, Millipore) and analysed oa a 15 % SDS-poIyacrylamide gel (Fling and Gregsrson, supra). Finally, the material was sterilized by filtration (0.22 urn) and stored at 4 °C.
Example 15: Measurement of the affinity of the hNGAL mutein N4 for IL-8 in an ELISA assay
For the detection of binding in an ELISA experiment two rows (12 wells each) of a 96 microtiter plate (Maxisorb, Nunc) were coated with 50 yd of a 50 ug/ml solution of Avidin (Fluka) in PBS overnight at 4 °C. After washing three times with PBST, one row was treated with 50 ul/well of a 1 uM solution of biotinylated IL-8 in PBST, whereas the second row was incubated with PBST as a control. After 1 hour at RT, all wells were washed three times with PBST and unspecific binding sites were saturated with 100 (.il of 4 % w/v skimmed milk powder in PBST for I hour at RT.
To determine the Ko value of the mutein N4 for IL-8, a dilution series of the N4 protein from Example 14 was prepared in PBST, starting from a concentration of 1 uM. As a control for unspecific binding to avidin, a similar dilution series of the mutein N4 was applied to wells where the IL-8 had been omitted. Complex formation was allowed for 1 hour at RT, followed by three washes with PBST and incubation at RT for 1 hour with anti-strepII-antibody-HRP-conjugate (DBA) diluted 1:8000 in PBST.
Detection of bound mutein N4 was accomplished using 3,3',5,5'-tetramethylbenzidine and H2O2 as substrates for horseradish-peroxidase. For this purpose, all wells were filled with ] 00 pi of a ready-to-use HRP-substrate solution (Biochem Immunosystems). The colour development was stopped after a few minutes by adding 100 ul of 0.3 M sulphuric acid. Product formation was measured via endpoint absorption at 450 nra in a SpectraMax 250 photometer (Molecular Devices).
The curve was fitted by non-linear least squares regression with the help of the computer program Kaleidagraph (Synergy software) according to Example 10. The resulting binding curves are depicted in Fig.10. The value obtained for the apparent dissociation constant of [the complex between the hNGAL mutein N4 and IL-8 is 300 ± 34 nM.
[Example 16: Selection of hNGAL muteins against biotinvlated TNFct
|Recombinant human Tumor Necrosis Factor a (TNFa, SEQ ID NO:31, SEQ ID NO: 35) jwas conjugated with biotin groups and used as a target for affinity enrichment from the

library of phagemids representing the hNGAL muteins obtained as described in Example 7 in the presence of Streptavidin-coated paramagnetic particles (Dynal).
For the production of recorabinant TNFQ, cells of E. coli BL21 [DE3J were transformed with the expression plasraid pTNFD encoding the cytokine TNFQ (Wang et al, Science 228 (1985), 149-154) with an N-terminal Arg-Gly-Ser-His(6)-Gly(3)-tag. Expression of TNFD was performed as described by Schmidt and Skerra (Schmidt and Skeira., J Chromatogr. 676 (1994), 103-119) except that the production of the recombinant cytokine was induced at 30 °C for 4 hours. Under these conditions, TNFa accumulates as soluble protein in the cytoplasm and can be released by subsequently freezing and thawing the cell pellet of 1 1 culture medium in 30 ml lysis buffer (50 mM NaH2PO4/NaOH pH 8.0, 300 mM NaCl, 10 mM imidazole). TNFa was purified from that solution in its trimeric form (Lohrengel et al, Cytokine 12 (1999), 573-577) by subsequently employing Immobilized Metal Affinity Chromatography (JMAC; Porath et al, Nature 258, (1975) 59S-599) and size exclusion chromatography. The protein yield was approximately 1.5 mg per 1 1 culture volume.
The conjugate was prepared by reacting 40 nniol (24.3 jo.g) of NHS-SS-Biotin (Pierce) dissolved in 25 ul H2O with 10 nmol (187.7 ug) human recombinant TNFD dissolved in 235 ul PBS/NaOH pH 8.5 in a total volume of 1 ml PBS/NaOH pH 8.5. The mixture was incubated with stirring at room temperature (RT) for 1 h. Excess reagent was then removed from the TNFD conjugate by means of a PD-10 gel filtration column (Pharmacia) according to the manufacturer's instructions with PBS as running buffer.
For the isolation of phagemids displaying a mutein with affinity for TNFa, one aliquot of the precipitated phagemids obtained as in Example 7, which were kept at -20 °C for long term storage, was thawed and the phagemids were pelleted (20 minutes, 18500 g, 4 °C). After removal of the supernatant, the sedimented phagemid particles were dissolved in 270 j.tl PBS, incubated for 30 minutes on ice and finally eentrifuged (5 minutes, 18500 g, 4 °C) to remove residual aggregates.
30 \xl of a 1 pM solution (30 pmol) of biotinylated TNFa (prepared by mixing 200 \xl of a ;2,5 uM solution of the biotinylated cytokine in PBS with 300 y.1 PBS) was mixed with 270 ).il of the phagemids in PBS (ca. 10u cfu) and incubated at RT for 1 h so that complex formation between the cytokine and the muteins presented by the phagemids was allowed to occur. Then, 100 ul of a solution of 8 % w/v BSA, 0.4 % v/v Tween 20 in PBS was [added.

Parallel thereto, 100 ul of the commercially available suspension of streptavidin-paramagnetic particles (Dynal) was washed three times with 1 ml PBS. Herein, the particles were kept suspended for 1 rain by rotating the 1.5 ml Eppsndorf vessel and then collected at the wall of the vessel with the aid of a magnet, and the supernatant was pipetted off. Jn order to saturate unspecific binding sites, the paramagnetic particles were incubated with 1 ml of 2 % (w/v) BSA in PBST at RT for 1 h.
After removing the supernatant as above, the mixture of the biotinylated TNFD and the phagemids was added to the paramagnetic particles, and the particles were resuspended and incubated at RT for lOmin. Finally, free biotin-binding sites of streptavidin were saturated by adding 10 ul of a 4 mM D-desthiobiotin (Sigma) solution in PBS to the mixture and incubating said mixture at RT for 5 min. This step served for preventing the Strep-tag® II - as part of the fusion protein of the muteins and the phage coat protein pm fragment - from forming a complex with streptavidin.
Unbound phagemids were removed by washing the paramagnetic particles eight times for 1 min with 1 ml of fresh PBST containing 1 mM D-desthiobiotin. Each time the particles were collected with the aid of the magnet and the supernatant was pipetted off. Finally, the bound phagemids were eluted under reducing conditions by resuspending the particles in 1 ml PBST containing 1 mM desthiobiotin and 100 mM DTT. The solution was incubated at 37 °C for 1 h to reduce the disulfide bond contained in the linker molecule between TNFD and biotin, thus releasing phagemids specifically bound to TNFO from the beads.
For the purpose of amplification, the eluted phagemid solution (1.0 ml, containing between 107 and 109 cfu, depending on the selection cycle) was shortly brought to 37 °C, mixed with 3 ml of an exponentially growing culture of E. coli XLl-blue (OD55o = 0.5 at 37 °C), and was shaken at 200 rpm for 30 minutes at 37 °C. The infected cells were sedimented (2 minutes, 4420 g, 4 °C), resuspended in 600 ul of culture medium, and plated out onto three agar plates with LB-medium containing 100 ug/ml ampicillin (LB/Amp; 140 mm diameter).
After incubation for 14 hours at 32 °C, the lawn of colonies was scraped from the agar plates, each with addition of 10 ml 2xYT/Amp-medium. The suspension was transferred to a sterile Erlenmeyer-flask and was shaken for 20 minutes at 37 °C, 200 rpm.

For another cycle of production and affinity enrichment of the phagemid particles 50 ml of 2xYT/Amp medium prewarmed to 37 °C was inoculated with 0.2 to 1 ml of said suspension so that the cell density was ODsso^ 0.08. This culture was incubated at 37 °C, 160 rpm until a cell density of OD550 — 0.5 was reached. Then the culture was infected with VCS-M13 helper phage (Stratagene) at a multiplicity of infection of approximately 10 and the culture was shaken for further 30 minutes at 37 "C, 160 rpm. Kanamycin (70 ng/ml) was subsequently added, the incubator temperature was lowered to 26 °C and, after 10 minutes, anhydrotetracycline was added to 25 j.ig/1 to induce gene expression. Incubation continued for another 15 hours at 26 °C, 160 rpm.
The ceils were sedimented by centrifugation (15 minutes, 12000 g, 4 °C). The supernatant containing the phagemid particles was sterile-filtered (0.45 urn), was mixed with 1/4 volume (12.5 ml) 20 % w/v PEG 8000, 15 % w/v NaCl, and was incubated for 1 h on ice. After centrifugation (20 minutes, 18000 g, 4 °C) the precipitated phagemid particles were dissolved in 2 ml of cold PBS. The solution was incubated on ice for 30 minutes and was distributed into two 1.5 ml reaction vessels. After centrifugation of undissolved components (5 minutes, 18500 g, 4 °C) each supernatant was transferred to a new reaction vessel.
The phagemid particles were reprecipitated by mixing with 1/4 volume 20 % w/v PEG 8000, 15 % w/v NaCl, followed by incubation for 60 minutes on ice. After centrifugation (20 minutes, 18500 g, 4 °C) the supernatant was removed and the precipitated phagemid particles were dissolved in 270 ul PBS. After incubation for 30 minutes on ice the solution was centrifuged (5 minutes, 18500 g, 4 °C) and the supernatant was used directly for the affinity enrichment. Four further selection cycles with TNFD peptide were carried out in this way.
Example 17: Identification of hNGAL nmteins binding TNFq by use of the "colony screening" method
For the analytical production of the hNGAL muteins as fusion proteins with the Strep-Tag® II and the albumin-binding domain as described in Example 3, the gene cassette between the two BstXI cleavage sites was subcloned from the vector phNGAL12 on phNGAL7.
For this purpose the phasmid DNA was isolated from the mixture of the E, coli clones obtained by infection with the phagemids from Example 16, eluted after the fifth selection

cycle, using the Plasmid Midi Kit (Qiagen). The DNA was cut with the restriction enzyme BstXl and the smaller one of the two fragments (347 bp) was purified by preparative agarose gel electrophoresis as described in Example 6. The DNA of the vector phNGAL7 was cut with BstXl and the larger one of the two fragments (3971 bp) was isolated in the same way.
For the ligation, 100 fmol of the isolated small DNA-fragment was mixed with 100 fmol of the large DNA-fragment and incubated with 1.5 Weiss Units of T4 DNA ligase (Promega) in a total volume of 20 JJ.1 (30 mM Tris/HCl pH 7.8, 10 mM MgCl2, 10 niM DTT, 1 mM ATP), followed by incubation overnight at 16 °C. E. coli TG1-F" (E. coli K12 TGI, which had lost its episome through repeated culturing under non-selective conditions) was transformed with 4 ul of this ligation mixture according to the CaCb-method (Sambrook et al., supra), obtaining 2.0 ml of a cell suspension. The cell suspension was centriraged (5000 g, 2 mm, 4 °C), resuspended in 100 p.1 of the culture medium, plated on an agar plate containing LB/Amp medium and incubated at 37 °C for 14 h to determine the transformation efficiency.
A hydrophilic PVDF membrane (Millipore, type GVWP, pore size 0.22 pm), labelled at one position and cut to size, was laid onto an LB/Amp agar plate. The cell suspension from a fresh transformation batch, which had been transformed with 3-6 \xl of the above described ligation mixture, was centrifuged (5000 g, 2 min, 4 °C), resuspended in 100 ul of the culture medium, and uniformly plated onto this membrane in order to obtain 400 to 500 colonies. The agar plate was incubated for 7.5 hours at 37 °C until the colonies had reached a size of approximately 0.5 mm.
In the meantime a hydrophobic membrane (Millipore, Immobilon P, pore size 0.45 \xm), also cut to size, was moistened with PBS according to the instructions of the manufacturer. Coating with HSA was achieved by agitation for 4 hours at RT in 10 ml of a solution of 10 mg/ml HSA (Sigma) in PBS. Remaining binding sites on the membrane were saturated by incubation with 20 ml 4 % (w/v) skimmed milk powder, 0.1 % (v/v) Tween-20 in PBS for 2 hours at RT. The membrane was washed twice for 10 minutes with 20 ml PBS and immersed afterwards for 10 minutes in 10 ml LB/Amp medium, to which 200 j.ig/1 anhydrotetracycliue was added.
Finally, it was marked at one position and laid onto a culture plate with LB/Amp agar, which additionally contained 200 |.ig/l anliydrotetracycline. The hydrophilic membrane from above, on which the colonies were grown, was laid onto the hydrophobic membrane

in such a way that both of the marks superimposed. The culture plate was incubated with the stack of both membranes at 22 °C for 15 hours. During this phase the respective hNGAL muteins were secreted from the colonies on the uppei membrane and were immobilized via their albumin-binding domain via the PISA on the lower membrane
After this, the upper membrane with the colonies was transferred to a fresh LB/Ainp agar plate and stored at 4 °C. The hydrophobia membrane was removed and washed three times for 5 minutes each with 20 ml PBST.
For analysis of the binding activity of the immobilized hNGAL muteins the membrane was then incubated for 1 hour in 3.5 ml of a solution of 100 nM digoxigenated TNFct in PBST containing 0.5 % w/v skimmed milk powder.
The conjugate was prepared by reacting 40 nmol (27.5 jag) of DIG-NHS (Roche) dissolved in 27 /il DMSO with 10 nmol (187.7 ug) TNFa dissolved in 235 ul PBS/NaOH pH 8.5 in a total volume of 1 ml PBS/NaOH pH 8.5. The mixture was incubated with stirring at room temperature (RT) for I h. Excess reagent was then removed from the TNFa conjugate by means of a PD-10 gel filtration column (Pharmacia) according to the manufacturer's instructions with PBS as running buffer.
The membrane was washed three times with PBST, followed by incubation for 1 hour with 10 ml anti-digoxigenin Fab-Alkaline-Phosphatase conjugate diluted 1:1000 in PBST for detection of bound TNFa via its digoxigenin groups. The membrane was washed twice with PBST and once with PBS, each for 5 minutes, and agitated for 10 minutes in AP-buffer. For the chromogenic reaction, the membrane was incubated in 10 ml AP-buffer, to which 30 ul 5-bromo-4-chloro-3-indolyl phosphate 4-toluidine salt (Roth, dissolved at 50 ug/ml in dimethylformamide) and 5 ul nitro blue tetrazolium (Roth, 75 ug/ml in 70 % v/v dimethylformamide) were added, until distinct colour signals could be recognized at the positions of some of the colonies.
From 1800 colonies which appeared on the membranes of multiple colony screening experiments 14 giving rise to intense colour spots on the hydrophobic membrane were cultured from the corresponding hydrophilic membrane. Their plasmid DNA was isolated and the hNGAL gene cassette was subjected to sequence analysis by use of the Genetic Analyzer 310 system with Big Dye Terminator Cycle Sequencing Kit (Applied Biosystems) according to the instructions of the manufacturer and using the oligodeoxynucleotide SEQ ID NO:5 as primer.

From these 14 muteins, 13 exhibited the same sequence, which was denominated TNF-V1, whereas the fourteenth sequence was different and was designated TNF-V2.
The nucleotide sequences of the clones TNF-V1 and TNF-V2 were translated into their amino acid sequences and those amino acid residues which deviate from the original hNGAL protein are given in Table 4. The nucleotide sequences of the muteins TNF-V1 and TNF-V2 are also given as SEQ ID NO:32 and SEQ ID NO:33, respectively. The fall amino acid sequences of these two muteins are given as SEQ ID NO: 36 and SEQ ED NO: 37, respectively.
Table 4: Sequence characteristics of hNGAL muteins TNF-V1 and TNF-V2
POS. hNGAL TNF-V1 TNF-V2
40 Ala Phe Gly
42 Leu Phe Phe
44 Glu Leu Val
46 Lys Leu Phe
47 Asp Glu Asn

49 Gin Phe He
50 Lys Phe Ser
70 Leu Thr Asp

72 Arg Leu Ala
73 Lys Glu Asn
77 Asp Phe Ala

79 Trp Glu Arg
80 He AAA* He

101 Pro Lys Gly
102 Gly His Asn
103 Leu Gly Val
125 Lys Asp Gly
127 Ser Ser Asp
128 Gin Gin Ser
130 Arg Arg Asn
132 Tyr Asn Arg
* Mutein TNF-V1 lacks the isoleucine residue ac position SC due to a deletion of nucleotides 238 to 240.

Example 18: Confirmation.of the binding activity of selected hNGAL muteius for TNFa. by use of the "colony spot assay"
The colony spot assay was conducted in a similar manner as the colony screening assay outlined in Example 17 except that single colonies of E. coli harbouring the corresponding expression plasmids were spotted from a master plate onto the hydrophilic membrane -marked with a grid - instead of plating a suspension of transformed cells. Each respective clone was spotted with a sterile tooth pick either four or five times onto a hydrophilic membrane (Millipore, type GVWP, pore size 0.22 fxm) that was placed on a culture plate with LB/Amp agar. Cells were grown at 37 °C for 5 hours.
In the meantime a hydrophobic membrane (Millipore, Immobilon P, pore size 0.45 um) was moistened with PBS according to the instructions of the manufacturer. Coating with HSA was achieved by agitation for 4 hours at RT in 10 ml of a solution of 10 mg/ml HSA (Sigma) in PBS. Remaining binding sites on the membrane were saturated by incubation with 20 ml 3 % (w/v) BSA, 0.1 % (v/v) Tween-20 in PBS for 2 hours at RT. The membrane was washed twice for 10 minutes with 20 ml PBS and immersed afterwards for 10 minutes in. 10 ml LB/Amp medium, to which 200 jig/t anliydrotetracycline was added.
Finally, it was marked at one position and laid onto a culture plate with LB/Amp agar, which additionally contained 200 jj.g/1 anhydrotetraeycline. The hydrophilic membrane from above, on which the colonies were grown, was laid onto the hydrophobic membrane in such a way that both of the marks superimposed. The culture plate was incubated with the stack of both membranes at 22 °C for 15 hours. During this phase the respective hNGAL muteius were secreted from the colonies on the upper membrane and were immobilized via their albumin-binding domain on the HSA on the lower membrane.
After this, the upper membrane with the colonies was transferred to a fresh LB/Amp agar plate and stored at 4 °C. The hydrophobic membrane was removed and washed three times for 5 minutes each with 20 ml PBST.
To confirm the binding activity of the spotted muteins towards TNFa the membrane was then incubated for 1 hour in 3.5 ml of a solution of 100 nM digoxigenated TNFa in PBST containing 0.5 % w/v skimmed milk powder (a 1.5 yM stock solution of the digoxigenated TNFa in PBS was therefore 1:15 diluted in PBST containing skimmed milk powder).
The membrane was washed three times with PBST, followed by incubation for 1 hour with 10 ml anti-digoxigenin Fab-Alkaline-Phosphatase conjugate diluted 1:1000 in PBST for detection of bound TNFa via its digoxigenin groups. The membrane was washed twice with PBST and once with PBS, each for 5 minutes, and agitated for 10 minutes in AP-buffer. For the chromogeiiic reaction, the membranes were incubated in 10 ml AP-buffer, to which 30 ul 5-bromo-4-chloro-3-iudolyI phosphate 4-toluidine salt (Roth, dissolved at 50 ug/ml in dimethylformamide) and 5 ul nitro blue tetrazolium (Roth, 75 ug/ml in 70 % v/v dimethylfomiamide) were added. Distinct colour signals could be recognized at those positions where the muteins TNF-V1 and TNF-V2 were spotted, whereas no binding signal could be observed for hNGAL wildtype (encoded by phNGAL7), bilin-binding protein (encoded by the vector pBBP22; Schlehuber et al. J. Mol. Biol. 297 (2000), 1105-1120), two unrelated muteins from the library of hNGAL muteins described in Example 6 (both subekmed on phNGALJ), and for the clone expressing no protein (harbouring pBluescript (Stratagene) as a plasmid), confirming fee binding activity of TNF-V1 and TNF-V2 to TNFa (Fig.12).








We claim:
1. Method for generating a mutein of a protein selected from the group consisting of human
neutrophil gelatinase-associated lipocalin (hNGAL), rat α2-microglobulin-related protein
(A2m) and mouse 24p3/uterocalin (24p3), said mutein having detectable affinity to a
given target,
characterized in that the method comprises the step (a) of:
subjecting the protein to mutagenesis at one or more of the sequence positions which correspond to the sequence positions40 to 50, 70 to 79, 101 to 103, and 125 to 132 of hNGAL, resulting in one or more mutein(s) of the protein.
2. The method as claimed in claim 1, wherein the mutagenesis in step (a) results in a plurality of muteins of the protein.
3. Method as claimed in any one of claims 1 to 2, wherein the protein is subjected to mutagenesis at one or more of the sequence positions which correspond to the sequence positions 40, 42, 44, 46, 47, 49, 50, 70, 72, 73, 77, 79, 101, 102, 103, 125, 127,128,130, and 132 of hNGAL
4. Method as claimed in any one of claims 1 to 3, wherein a nucleic acid coding for the one or more mutein(s) of the protein, which nucleic acid results from mutagenesis, is operably fused at the 3' end with a gene coding for the coat protein pIll of a filamentous bacteriophage of the M13-family or for a fragment of this coat protein, in order to select at least one mutein for the binding of the given target.
5. Mutein derived from human neutrophil gelatinase-associated lipocalin (hNGAL), rat α2-microglobulin-related protein (A2m) or mouse 24p3/uterocalin (24p3), characterized in that the mutein comprises a mutation at one or more of the sequence positions corresponding to sequence positions40 to 50, 70 to 79, 101 to 103, and 125 to 132 of hNGAL, and wherein the mutein has detectable binding affinity for a given target.

6. Method of generating a mutein of a protein selected from the group consisting of human neutrophil, substantially as herein described with reference to the foregoing description and the accompanying tables and drawings.

Documents:

9208-DELNP-2004-Correspondence-Others-(01-06-2010).pdf

9208-DELNP-2004-Form-3-(01-06-2010).pdf

928-DELNP-2004-1-Correspondence-Others-(07-03-2011).pdf

928-DELNP-2004-1-Drawings-(07-03-2011).pdf

928-DELNP-2004-Abstract-(27-09-2010).pdf

928-delnp-2004-abstract.pdf

928-delnp-2004-assignment.pdf

928-DELNP-2004-Claims-(11-05-2011).pdf

928-DELNP-2004-Claims-(27-09-2010).pdf

928-delnp-2004-claims.pdf

928-DELNP-2004-Correspondence-Others-(07-03-2011).pdf

928-DELNP-2004-Correspondence-Others-(10-09-2009).pdf

928-DELNP-2004-Correspondence-Others-(11-05-2011).pdf

928-DELNP-2004-Correspondence-Others-(27-09-2010).pdf

928-DELNP-2004-Correspondence-Others-(30-09-2010).pdf

928-delnp-2004-correspondence-others.pdf

928-delnp-2004-description (complete).pdf

928-delnp-2004-drawings.pdf

928-DELNP-2004-Form-1-(27-09-2010).pdf

928-delnp-2004-form-1.pdf

928-delnp-2004-form-13-(10-09-2009).pdf

928-DELNP-2004-Form-2-(27-09-2010).pdf

928-DELNP-2004-Form-3-(27-09-2010).pdf

928-delnp-2004-form-3.pdf

928-delnp-2004-form-5.pdf

928-DELNP-2004-GPA-(11-05-2011).pdf

928-delnp-2004-pct-101.pdf

928-delnp-2004-pct-105.pdf

928-delnp-2004-pct-210.pdf

928-delnp-2004-pct-301.pdf

928-delnp-2004-pct-304.pdf

928-delnp-2004-pct-308.pdf

928-delnp-2004-pct-332.pdf

928-delnp-2004-pct-401.pdf

928-delnp-2004-pct-408.pdf

928-delnp-2004-pct-409.pdf

928-delnp-2004-pct-416.pdf

928-DELNP-2004-Petition 137-(07-03-2011).pdf

abstract.jpg


Patent Number 248614
Indian Patent Application Number 928/DELNP/2004
PG Journal Number 30/2011
Publication Date 29-Jul-2011
Grant Date 28-Jul-2011
Date of Filing 08-Apr-2004
Name of Patentee PIERIS AG
Applicant Address LISE-MEITNER-STRASSE 30,, 85354 FREISING-WEIHENSTEPHEN GERMANY
Inventors:
# Inventor's Name Inventor's Address
1 SKERRA, ARNE MAX-LEHNER-STRASSE 18, 85354 FREISING GERMANY
2 NA NA
3 SCHLEHUBER, STEFFEN MURSTR. 27, 85356 FREISING GERMANY
PCT International Classification Number C12N 15/12
PCT International Application Number PCT/EP02/10490
PCT International Filing date 2002-09-18
PCT Conventions:
# PCT Application Number Date of Convention Priority Country
1 PCT/EP01/11213 2001-09-27 EUROPEAN UNION
2 PCT/EP02/04223 2002-04-16 EUROPEAN UNION