Polypeptide libraries with a predetermined scaffold
10556933 · 2020-02-11
Assignee
Inventors
- Lars Abrahmsén (Bromma, SE)
- Nina Herne (Stockholm, SE)
- Christofer Lendel (Farsta, SE)
- Joachim Feldwisch (Tyresö, SE)
Cpc classification
C40B40/10
CHEMISTRY; METALLURGY
C12N15/1044
CHEMISTRY; METALLURGY
C12N15/1041
CHEMISTRY; METALLURGY
C12N15/1075
CHEMISTRY; METALLURGY
G01N33/6845
PHYSICS
C07K1/047
CHEMISTRY; METALLURGY
International classification
C40B40/10
CHEMISTRY; METALLURGY
C12N15/10
CHEMISTRY; METALLURGY
Abstract
Populations of polypeptide variants based on a common scaffold, each polypeptide in the population comprising the scaffold amino acid sequence EXXXAXXEIX XLPNLTXXQX XAFIXKLXDD PSQSSELLSE AKKLNDSQ (SEQ ID NO: 1) or AKYAKEXXXAXX EIXXLPNLTX XQXXAFIXKL XDDPSQSSEL LSEAKKLNDS Q (SEQ ID NO: 2), wherein each X individually corresponds to an amino acid residue which is varied in the population are disclosed. Also populations of polynucleotides, wherein each member encodes a member of a polypeptide population are disclosed. Furthermore, combinations of such polypeptide populations and such polynucleotide populations are disclosed, wherein each member of polypeptide population is physically or spatially associated with the polynucleotide encoding that member via means for genotype-phenotype coupling.
Claims
1. A polypeptide having affinity for a predetermined target, comprising the first scaffold amino acid sequence TABLE-US-00015 (SEQ.ID.No.1) EXXXAXXEIXXLPNLTXXQXXAFIXKLXDDPSQSSELLSE AKKLNDSQ wherein each X corresponds to a randomizable amino acid residue in a second polypeptide based on an original scaffold amino acid sequence and wherein said second polypeptide has affinity for said predetermined target.
2. The polypeptide according to claim 1, wherein the first scaffold amino acid sequence comprises TABLE-US-00016 (SEQ.ID.No.2) AKYAKEXXXAXXEIXXLPNLTXXQXXAFIXKLXDDPSQSSELLSE AKKLNDSQ wherein each X corresponds to a randomizable amino acid residue in a second polypeptide based on an original scaffold amino acid sequence and wherein said second polypeptide has affinity for said predetermined target.
3. A polypeptide according to claim 1, wherein said first scaffold amino acid sequence is derived from SpA.
4. The polypeptide according to claim 1 comprising additional amino acid residues.
5. The polypeptide according to claim 4 comprising additional amino acid residues at the C-terminus of said polypeptide.
6. The polypeptide according to claim 4, wherein said additional amino acid residues are added for the purpose of binding, production, purification, stabilization, coupling or detection of the polypeptide.
7. The polypeptide according to claim 4, wherein said additional amino acid residues constitute one or more polypeptide domain(s).
8. The polypeptide according to claim 7, wherein said one or more polypeptide domain(s) has a function selected from the group of a binding function, an enzymatic function, a metal ion chelating function and a fluorescent function, or mixtures thereof.
9. The polypeptide according to claim 1, further comprising a label.
10. The polypeptide according to claim 1, further comprising a therapeutic agent.
11. The polypeptide according to claim 1, wherein said target is TNF-.
12. The polypeptide according to claim 1, wherein said target is insulin.
13. The polypeptide according to claim 1, wherein said target is taq-polymerase.
14. The fusion polypeptide comprising a polypeptide according to claim 1 as a moiety.
15. A method for production of the polypeptide of claim 1, comprising the steps of providing a second polypeptide having affinity for a predetermined target wherein said second polypeptide is based on an original scaffold derived from SpA, and mutating original scaffold amino acids to generate the polypeptide of claim 1 comprising the first scaffold amino acid sequence TABLE-US-00017 (SEQ.ID.No.1) EXXXAXXEIXXLPNLTXXQXXAFIXKLXDDPSQSSELLSE AKKLNDSQ wherein each X individually corresponds to an amino acid residue which is conserved from the second polypeptide.
16. The method of claim 15, wherein the first scaffold amino acid sequence comprises TABLE-US-00018 (SEQ.ID.No.2) AKYAKEXXXAXXEIXXLPNLTXXQXXAFIXKLXDDPSQSSELLSE AKKLNDSQ wherein each X individually corresponds to an amino acid residue which is conserved from the second polypeptide.
17. The method according to claim 15, wherein said polypeptide comprises the amino acid sequence ELGWAIGEIG TLPNLTHQQF RAFILKLWDD PSQSSELLSE AKKLNDSQ (SEQ ID NO: 44), and wherein said predetermined target is TNF-.
18. The method according to claim 15, wherein said polypeptide comprises the amino acid sequence EKYMAYGEIR LLPNLTHQQV MAFIDKLVDD PSQSSELLSE AKKLNDSQ (SEQ ID NO: 45), and wherein said predetermined target is insulin.
19. The method according to claim 15, wherein said polypeptide comprises the amino acid sequence EKGEAVVEIF RLPNLTGRQV KAFIAKLYDD PSQSSELLSE AKKLNDSQ (SEQ ID NO: 46), and wherein said predetermined target is taq-polymerase.
20. The method according to claim 15, wherein said original scaffold is derived from SpA domain B and wherein mutating original scaffold amino acids comprises a G29A mutation, corresponding to A in position 22 in SEQ ID NO:1 and A in position 27 in SEQ ID NO: 2.
Description
BRIEF DESCRIPTION OF THE FIGURES
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EXAMPLES
Example 1Construction of a Combinatorial Polypeptide Library
(8) A combinatorial library of polypeptides was constructed essentially as described in Grnwall C et al (2007) J Biotechnol 128:162-183, by PCR amplification of a 123-nucleotide template oligonucleotide with certain degenerate codons (5-GTA GAT GCC AAA TAC GCC AAA GAA NNN NNN NNN GCG NNN NNN GAG ATC NNN NNN TTA CCT AAC TTA ACC NNN NNN CAA NNN NNN GCC TTC ATC NNN AAA TTA NNN GAT GAC CCA AGC CAG AGC-3 SEQ ID No. 6) encoding helices 1 and 2 of the Staphylococcus aureus protein A-derived protein Z (Nilsson et al (1987), supra), with point mutations N3A, F5Y, N6A, N23T and S33K. The PCR amplification was performed using the primers AFFI-1364 and AFFI-1365 with an Xho I site and a Sac I site, respectively, underlined in Table 1.
(9) The resulting gene fragment encoding the library was restricted with Xho I and Sac I. Subsequently, the library-encoding gene fragment was ligated into an Xho I- and Sac I-restricted phagemid vector adopted for phage display denoted pAY2016, essentially based on the phagemid vector pAffi1 (Grnwall C et al (2007), supra), in frame with amino acid residues 41-58 of protein Z, encoding helix 3 with point mutations A425, N43E, A465 and A54S. Helix 3 was constructed by annealing of the two complementary oligonucleotides AFFI-1333 and AFFI-1334 (Table 1).
(10) The resulting library vector was electroporated into Escherichia coli strain RR1M15 (Ruther U (1982) Nucl Acids Res 10:5765-5772), yielding a library of 2.410.sup.10 members.
(11) Preparation of phage stocks from the library was performed using standard procedures involving M13K07 helper phage (New England Biolabs, Beverly, Mass., USA), routinely yielding phage titers of approximately 10.sup.11 cfu per ml cultivation.
(12) TABLE-US-00003 TABLE1 Listofoligonucleotides Name Sequence5-3 AFFI-1333 AGCTCTGAATTACTGAGCGAAGCTAAAAAGCTAAATGAT AGCCAGGCGCCGAAAGTAGACTAC(SEQIDNo.7) AFFI-1334 GTAGTCTACTTTCGGCGCCTGGCTATCATTTAGCTTTTT AGCTTCGCTCAGTAATTCAGAGCT(SEQIDNo.8) AFFI-1364 AAATAAATCTCGAGGTAGATGCCAAATACGCCAAAG (SEQIDNo.9) AFFI-1365 TAAATAATGAGCTCTGGCTTGGGTCATC (SEQIDNo.10)
Example 2Phage Display Selection and Characterization of Human HER2 Binding Polypeptide Variants
(13) Summary
(14) Biotinylated HER2 protein is used as target in phage display selections using the library constructed in Example 1. Selections are carried out using a variety of conditions in order to maximize the likelihood of obtaining molecules having a high affinity for HER2. After elution of selected phages, the corresponding expressed proteins are tested for affinity to HER2 in an ELISA setup. Positive clones are identified and sequenced, and the predicted amino acid sequences of the corresponding polypeptides and their HER2 binding motifs are deduced, which yields a large number of sequences of HER2 binding molecules.
(15) Biotinylation of HER2
(16) Lyophilized human HER2 protein (R&D Systems, #1129-ER) is dissolved in PBS (2.68 mM KCl, 1.47 mM KH.sub.2PO.sub.4, 137 mM NaCl, 8.1 mM Na.sub.2HPO.sub.4, pH 7.4) to a final concentration of 10 mg/ml. EZ-link Sulfo-NHS-LC-Biotin (Pierce, #21335) is dissolved in water to a final concentration of 1 mg/ml and a 5 and 30 fold molar excess is added to 500 g HER2 in a total volume of 0.5 ml. The mixtures are incubated at room temperature (RT) for 30 min. Unbound biotin is removed by dialyzing against PBS using a dialysis cassette (Slide-A-Lyser, 10 kDa; Pierce).
(17) Phage Display Selection
(18) In total, five rounds of selection are carried out, using increasingly stringent conditions, such as decreasing HER2 concentration and increasing numbers of washes. Three initial rounds are performed, chiefly with a view to establish a suitable selection protocol. Selection is then carried out for two more cycles using the combinations of selection buffer, target concentration and solid support that are listed in Table 2.
(19) TABLE-US-00004 TABLE 2 Selection conditions for HER2 selection Sample Selection buffer Streptavidin name supplement Target conc. (nM) beads (g) Cycle 4 A Gelatin 20 100 B Gelatin 10 100 C BSA 5 100 D BSA 2.5 100 Cycle 5 A Gelatin 10 50 B Gelatin 5 50 C BSA 1 50 D BSA 0.5 50
(20) All tubes and beads (DYNABEADS M-280 Streptavidin, #112.06; Dynal) used in the selection procedure are pre-blocked in TPBSB (5%) (0.05% Tween20, 5% bovine serum albumin (BSA), 0.02% Na azide in PBS) or gelatin (0.5%) for at least 30 min at RT.
(21) Selection solutions (1 ml) contained biotinylated human HER2, phages, Na azide (0.02%), Tween 20 (0.05%) and either BSA (3%) or gelatin (0.1%) according to Table 2, and are prepared in PBS. The phages are incubated with biotinylated human HER2 target at 4 C. during three days for Cycle 4 and during one day for Cycle 5, followed by 1 h incubation under agitation at RT. The selection samples are transferred to blocked streptavidin beads for 15 min under agitation at RT. The beads are washed 10 times with 1 ml of selection buffer TPBSB (3%) (0.05% Tween20, 3% bovine serum albumin (BSA), 0.02% Na azide in PBS) or GT 0.1 (0.1% gelatin, 0.1% Tween 20 and 0.02% Na azide in PBS)), followed by 10 washes with PBS where the second last wash is performed for 5 min. Phages are either eluted with 1000 l 50 mM glycine-HCl, pH 2.2, for 10 min at RT, followed by immediate neutralization with 900 l PBS supplemented with 100 l 1 M Tris-HCl, pH 8.0, or eluted with 1000 l trypsin (2 mg/ml) for 30 min at RT followed by addition of 1000 l aprotinin (0.4 mg/ml). The eluted phages ( of the volume) are used to infect 50 ml log phase E. coli RR1M15 cells (Rther, 1982, supra) after each cycle of selection. After 30 min incubation with gentle agitation and 30 min with vigorous agitation at 37 C., the cells are centrifuged and the pellet is dissolved in a smaller volume and spread on TYE plates (15 g/l agar, 10 g/l tryptone water (Merck), 5 g/l yeast extract, 3 g/l NaCl supplemented with 2% glucose and 100 g/ml ampicillin) and finally incubated over night at 37 C.
(22) Phage Stock Preparation
(23) Cells from plates are re-suspended in TSB medium (30 g/l tryptic soy broth) and the cell concentration is determined by measuring the optical density at 600 nm assuming that OD.sub.600=1 corresponds to 510.sup.8 cells/ml. Cells are inoculated (approximately 100 times excess of cells compared to eluted phages) in 100 ml TSB+YE medium supplemented with 2% glucose and 100 g/ml ampicillin and grown at 37 C. to approximately OD.sub.600=0.5-0.7. Thereafter, 10 ml are transferred to a new flask and infected by 10 times molar excess of M13K07 helper phage (New England Biolabs, # NO315S) and incubated for 30 min with low agitation. Cells are pelleted at 2000 g for 10 min and resuspended in 100 ml TSB+YE medium supplemented with 100 M isopropyl -D-1-thiogalactopyranoside (IPTG), 50 g/ml kanamycin and 100 g/ml ampicillin and grown over night at 100 rpm and 25 C. A portion of the resuspended cells is stored at 80 C. as a glycerol stock.
(24) The overnight culture is centrifuged at 2500 g for 10 min and phages in the supernatant are precipitated by adding of the volume of precipitation buffer (20% PEG/2.5 M NaCl) and incubated on ice for 1 hour. Precipitated phages are pelleted by centrifugation at 10000 g at 4 C. for 30 min, resuspended in 20 ml PBS and thereafter the precipitation procedure is repeated. The phages are finally resuspended in 1 ml PBS and filtered through a 0.45 m filter.
(25) Selection, wash and elution solutions are titrated after each round of selection. Phage solutions are diluted in sterile water in a microtiter plate and 100 l log phase E. coli RR1M15 cells are added to each phage dilution. After 20 min incubation at RT, 5 l from each titration are transferred to a TYE plate and incubated over night at 37 C. The resulting colonies are counted and the titers (cfu/ml) calculated.
(26) ELISA Analysis of HER2 Binding
(27) Clones from the final selection cycles are expressed and screened for HER2 binding activity using an ELISA setup as described in Example 4 below (but using HER2 as target protein), or as described below. Randomly picked colonies are expressed in 96 deep-well plates by inoculating each colony into 1 ml TSB+YE medium supplemented with 100 g/ml ampicillin and 1 mM IPTG and grown for 18-24 hours at 37 C. After incubation, replicate plates are made by transferring a small fraction of each culture to 96-well plates with 15% glycerol for storage at 20 C.
(28) Remaining cells are pelleted by centrifugation at 3000 g for 10 min, re-suspended in 400 l PBS-T 0.05 (PBS supplemented with 0.05% Tween 20) and frozen at 80 C. Frozen samples are thawed in a water bath and cells are pelleted at 3700 g for at least 20 min. Supernatants containing expressed molecules are collected and used in ELISA.
(29) Half area microtiter wells (Costar, #3690) are coated over night at 4 C. with 50 l of HSA at a concentration of 6 g/ml in ELISA coating buffer (Sigma, #3041). The wells are blocked with 100 l blocking buffer (2% non fat dry milk in PBS) for 2 h at RT. After removal of the blocking buffer, 50 l of the prepared proteins are added to the wells, and plates are incubated for 1.5 h at RT. Supernatants are discarded, and biotinylated HER2 at a concentration of 0.5-10 g/ml in PBS-T 0.05 is added to the wells and incubated for 1.5 h. Bound complexes are detected with horse radish peroxidase conjugated streptavidin (HRP, Dako, # P0397) diluted 1:5000 in PBS-T 0.05, incubated for 1 h at RT. 50 l IMMUNOPURE TMB substrate (Pierce, #34021) are added to the wells and the plates are treated according to the manufacturer's recommendations. Absorbance of the wells is read at 450 nm in a Tecan Ultra 384 ELISA reader (Tecan) and evaluated using Magellan v. 5.0 software (Tecan). Prior to addition of each new reagent, four washes are done with PBS-T 0.05.
(30) Based on the result of this experiment, clones are picked for sequencing as described next.
(31) Sequencing of ELISA Positive Clones
(32) PCR fragments from selected colonies are amplified using appropriate oligonucleotides. Sequencing of amplified fragments is performed using a BIGDYE Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems) according to the manufacturer's recommendations and with the appropriate biotinylated oligonucleotide. The sequencing reactions are purified by binding to DYNABEADS REGEN streptavidin-coated paramagnetic beads using a Magnatrix 8000 instrument (Magnetic Biosolutions), and finally analyzed on ABI PRISM 3100 Genetic Analyser (Applied Biosystems).
(33) Sub-Cloning into Plasmid pAY1448
(34) DNA encoding selected and HER2-specific molecules is sub-cloned into the expression vector pAY1448 to create His.sub.6-tagged monomeric molecules expressed as MGSSHHHHHHLQ-[Z #####]-VD (His.sub.6-Z #####) (SEQ ID No. 11), wherein Z ##### denotes an identified member of the starting population of variant molecules. Plasmids containing inserts are purified from 2 ml overnight cultures of E. coli RR1M15 cells in TSB supplemented with 100 g/ml ampicillin using Qiagen Mini Kit (Qiagen) according to manufacturer's recommendations.
(35) DNA of selected molecules is sub-cloned into the expression vector pAY1448 by Accl-Notl PCR sticky end cloning using the appropriate PCR primer pairs.
(36) The expression vector pAY1448 is digested in two steps at 37 C. for 4 h using Accl and Notl in NEB4 and NEB3 buffer (New England Biolabs), respectively, and dephosphorylated with calf intestinal alkaline phosphatase (CIAP; Fermentas) for 1 h at 37 C. The cleaved plasmid and fragments are purified by QIAquick PCR purification kit (Qiagen) according to the manufacturer's recommendations.
(37) The PCR products are hybridized and ligated into Accl-Notl digested and dephosphorylated pAY1448 for 1 h at RT using T4 DNA ligase (5 units/l; Fermentas). Aliquots of the ligations are electroporated into E. coli BL21(DE3) cells. The cells are plated on tryptose blood agar base (TBAB) plates supplemented with 50 g/ml kanamycin and incubated over night at 37 C. Positive clones are first verified for inserts with PCR screening and then analyzed for correct sequences as described above.
(38) Expression and Purification of His.sub.6-Tagged Polypeptides
(39) Selected molecules, all sub-cloned into pAY1448 as described above, are expressed in E. coli BL21(DE3) as fusions to an N-terminal His.sub.6-tag and purified by IMAC. A colony of each molecule is used to inoculate 5 ml TSB medium supplemented with 50 g/ml kanamycin. The cultures are grown over night at 37 C. The following day, 50 l of each culture are inoculated separately to 100 ml TSB+YE medium supplemented with 50 g/ml kanamycin in a 1 liter flask. The cultures are grown at 100 rpm at 37 C. to an OD.sub.600 of 0.7-1, after which IPTG is added to a final concentration of 0.5 mM and cells are incubated at RT over night at 100 rpm. Cultures are harvested by centrifugation at 8000 g for 5 minutes and pellets are stored in a freezer until protein preparation.
(40) The His.sub.6-tagged proteins are IMAC purified under denatured conditions using 1.5 ml Ni-NTA Superflow columns (Qiagen). The buffer is exchanged to PBS using PD-10 columns (GE Healthcare).
(41) Protein concentration is determined using A.sub.280 and the BCA Protein Assay Reagent Kit (Pierce) as recommended by the manufacturer. The purity of the proteins is analyzed by SDS-PAGE stained with Coomassie Blue R.
(42) Biosensor Analysis of Selected Molecules' Affinity for Human HER2
(43) Biosensor analysis on a BIACORE 2000 instrument (GE Healthcare) is performed with human HER2 immobilized by amine coupling onto the carboxylated dextran layer on the surface of a CM-5 chip (research grade; GE Healthcare) according to the manufacturer's recommendations. Surface 1 on the chip is activated and deactivated and used as reference cell during injections. The selected molecules, expressed and purified as described above, are diluted in HBS-EP (GE Healthcare) to 25 nM and injected at a constant flow-rate of 25 l/min for 10 minutes, followed by dissociation in HBS-EP for 30 minutes. The surfaces are regenerated with two injections of 25 mM HCl.
Example 3Cloning, Production and Evaluation of Melting Temperature and In Vitro Antigenicity of Original and Inventive Scaffold Variants
(44) Summary
(45) This example describes the cloning, production and evaluation of original and inventive scaffold variants. The introduced scaffold mutations are believed to improve several properties of the polypeptide molecules, such as antigenicity, hydrophilicity and alkaline and structural stability. Thus, different molecules were evaluated for melting temperature and in vitro antigenicity and the results showed that inventive molecules had increased melting temperatures and displayed lower in vitro antigenicities (lower IgG binding) as compared to original molecules.
(46) Cloning of Polypeptides
(47) For original constructs, DNA sequences encoding molecules specific for Tumor Necrosis Factor-alpha (TNF-), HER2, insulin, Taq polymerase and Platelet Derived Growth Factor-Receptor beta (PDGF-R) (Table 3) were amplified by PCR in two individual reactions each (PCR1 and PCR2). Primer pairs AFFI-267/AFFI-1014 and AFFI-1015/AFFI-270 (Table 4), encoding parts of the Accl restriction site in the 5-ends, were applied in PCR1 and PCR2 respectively. To prepare the plasmid templates, bacteria harboring the plasmid DNA were grown overnight in TSB medium, supplemented with 50 g/ml of kanamycin. The cells were pelleted by centrifugation and plasmids were prepared using QIAprep Spin Miniprep Kit (Qiagen).
(48) For inventive constructs, PCR amplification (PCR1 and PCR2) of nucleotide sequences encoding modified, inventive molecules was performed using partially overlapping oligonucleotides as templates (AFFI-1320-AFFI-1323, AFFI-1326 and AFFI-1327) or using vector with original construct and oligonucleotides containing relevant mutations (AFFI-69, AFFI-70, AFFI-1151 and AFFI-1152) (Table 4). Primer pairs AFFI-1328/AFFI-1331 and AFFI-1329/AFFI-1330, encoding parts of the Accl restriction site in the 5-ends, were included in the PCR reactions.
(49) The PCR reactions were amplified using Pfu Turbo DNA polymerase (Stratagene, #600854-52) according to a standard PCR protocol and the PCR products were analyzed by 1% agarose gel electrophoresis.
(50) The PDGF-R binding Z variants were generated using oligonucleotides with varied codons and a PCR based mutagenesis technique. Obtained PCR fragments were ligated into a cleaved expression vector using In-Fusion technology (Clontech, #639607).
(51) To generate DNA fragments containing upstream and downstream Accl sticky-ends for all constructs, forward and reverse nucleotide strands of the PCR1 and PCR2 products were separated using magnetic streptavidin beads. After 30 min incubation at RT under continuous rotation, the beads were washed with wash buffer (50 mM Tris-HCl pH 7.5, 10 mM MgCl.sub.2, 10 mM DTT) and heated to 95 C. for 5 min. The supernatant containing the non-biotinylated fragments was collected and heated to 95 C., followed by stepwise cooling to 25 C. during 30 min to hybridize the DNA strands.
(52) The DNA fragments encoding binding molecules were subsequently ligated at RT, either for 2 h or overnight, into a CIP-treated (calf intestinal alkaline phosphate) and purified expression vector, previously digested with Accl restriction enzyme. The achieved constructs were MGSSHHHHHHLQ-[Z #####]-VD (SEQ ID No. 11), MGSSLQ-[Z #####]-VDC (for Z.sub.PDGF-R:2465) (SEQ ID No. 12) or M-[Z #####]-C (for Z.sub.PDGF-R:3358).
(53) The ligations were transformed into electrocompetent E. coli TOP10 cells and cultivated on plates as described in Example 2. Bacterial colonies harboring the newly constructed plasmids were PCR screened, and the insert DNA sequences were verified as described in Example 2.
(54) Verified plasmids were prepared as described earlier.
(55) Expression of Polypeptides
(56) E. coli BL21(DE3) cultures transformed with relevant plasmids were inoculated into 800 ml TSB-YE medium supplemented with 50 g/ml kanamycin and 0.3 ml/l anti-foam agent (Breox FMT 30) and grown at 37 C. to an OD600 of approximately 2. Protein expression was then induced by addition of 1 M IPTG to a final concentration of 0.5 mM. The cultivations were performed using the multifermenter system Greta (Belach). The cultures were harvested 5 h after induction by centrifugation at 15 900g for 20 min. Supernatants were discarded and the cell pellets collected and stored at 20 C. The protein expression level was determined using SDS-PAGE and ocular inspection of stained gels.
(57) Purification of Expressed Polypeptides
(58) Proteins with His.sub.6 tag were purified as follows: Pelleted bacterial cells harboring soluble His.sub.6 tagged polypeptides were suspended in His GRAVITRAP binding buffer (20 mM sodium phosphate, 0.5 M NaCl, 20 mM imidazole and 40 U/ml BENZONASE) and disrupted by ultrasonication. After clarification, the supernatants were loaded on His GRAVITRAP columns (GE Healthcare) previously equilibrated with His GRAVITRAP binding buffer. After washing the columns with 10 column volumes (CV) of His GRAVITRAP washing buffer (20 mM sodium phosphate, 0.5 M NaCl, 60 mM imidazole), the polypeptides were eluted with 3 CV His GRAVITRAP elution buffer (20 mM sodium phosphate, 0.5 M NaCl, 500 mM imidazole).
(59) Proteins without His.sub.6 tag were purified as follows: Pelleted bacterial cells harboring soluble Z.sub.PDGF-R:2465-Cys or Z.sub.PDGF-R:3358-Cys were suspended in 20 mM Tris-HCl, pH 7.1. To disrupt the cells and release the intracellular content, the cell suspensions were heated to 85 C. for 3 minutes. The lysates were clarified by centrifugation followed by filtration, and loaded on 25 ml Q Sepharose FF (GE Healthcare) packed in an XK26 column (GE Healthcare), previously equilibrated with 20 mM Tris-HCl, pH 7.1. After column wash with 5 CV 20 mM Tris-HCl, pH 7.1, bound proteins were eluted with a linear gradient of 0-0.5 M NaCl in 20 mM Tris-HCl, pH 7.1, during 10 CV. The flow rate was 5 ml/min and the 280 nm signal was monitored. Fractions containing Z.sub.PDGF-R: 2465-CYS or Z.sub.PDGF-R:3358-Cys were identified by SDS-PAGE analysis. Relevant fractions were pooled and pH was adjusted to 8.0 by addition of 1 M Tris-HCl, pH 8.0, to a final concentration of 50 mM. The C-terminal cysteine on the constructs was reduced by addition of DTT to 20 mM, followed by incubation at 40 C. for 3 minutes. After reduction, acetonitrile (ACN) was added to a final concentration of 5% and reduced Z.sub.PDGF-R:2465-Cys or Z.sub.PDGF-R: 3358-Cys was loaded on 1 ml Resource 15 RPC columns (GE Healthcare), previously equilibrated with RPC A Buffer (0.1% TFA, 5% ACN, 95% water). After column wash with 10 CV RPC A Buffer, bound proteins were eluted with a linear gradient of 0-40% RPC B Buffer (0.1% TFA, 80% ACN, 20% water). The flow rate was 1 ml/min and the 280 nm signal was monitored. Fractions containing pure Z.sub.PDGF-R:2465-Cys or Z.sub.PDGF-R:3358-Cys were identified by SDS-PAGE analysis and pooled.
(60) To enable lyophilization of the proteins, the buffer was exchanged to either 10 mM ammonium hydrogen carbonate buffer, pH 8.0, or 10 mM ammonium acetate buffer, pH 6.0, using disposable PD-10 desalting columns (GE Healthcare). The lyophilization buffer was chosen in regard to the isoelectric point of relevant proteins. Finally, the binding polypeptides His.sub.6-Z.sub.TNF-:185, His.sub.6-Z.sub.HER2:342, His.sub.6-Z.sub.Insulin:810, His.sub.6-Z.sub.Taq:1154, Z.sub.PDGF-R:2465-Cys, His.sub.6-Z.sub.HER2:2628, His.sub.6-Z.sub.Taq:3229, His.sub.6-Z.sub.TNF-:3230, His.sub.6-Z.sub.Insulin:3232 and Z.sub.PDGF-R:3358-Cys were lyophilized using a Christ Alpha 2-4 LSC instrument and stored at 4 C. until use (Table 5). The free C-terminal cysteine was blocked using N-ethylmalemide (NEM) according to the manufacturer's recommendations (Pierce).
(61) Analysis of Purified Polypeptides
(62) Determination of the concentration of polypeptide solutions was performed by measuring the absorbance at 280 nm using a NANODROP ND-1000 Spectrophotometer. The proteins were further analyzed with SDS-PAGE and LC-MS.
(63) For the SDS-PAGE analysis, approximately 10 g polypeptide was mixed with LDS Sample Buffer and DTT (45 mM final concentration), incubated at 70 C. for 15 min and loaded onto NUPAGE 4-12% Bis-Tris Gels. The gels were run with MES SDS Running Buffer in a Novex Mini-Cell employing the SEEBLUE Plus2 Prestained Standard as molecular weight marker and PAGEBLUE Protein Staining Solution for staining.
(64) To verify the identity of the polypeptides, LC/MS analyses were performed using an Agilent 1100 LC/MSD system, equipped with API-ESI and a single quadruple mass analyzer. After buffer exchange, protein samples were diluted in lyophilization buffer to a final concentration of 0.5 mg/ml and 10 l were loaded on a Zorbax 300SB-C8 Narrow-Bore column (2.1150 mm, 3.5 m) at a flow-rate of 0.5 ml/min. Proteins were eluted using a linear gradient of 10-70% solution B for 30 min at 0.5 ml/min. The separation was performed at 30 C. The ion signal and the absorbance at 280 and 220 nm were monitored. The molecular weights of the purified proteins were determined by analysis of the ion signal.
(65) Determination of Melting Temperature (Tm)
(66) Lyophilized polypeptides were dissolved in PBS to a final concentration of approximately 0.5 mg/ml and stored on ice. CD analysis was performed on a Jasco J-810 spectropolarimeter in a cell with an optical path-length of 1 mm. In variable temperature measurements, the absorbance was measured at 221 nm from 20 to 80 C., with a temperature slope of 5 C./min. Melting temperatures (Tm) for the tested polypeptides were calculated by determining the midpoint of the transition in the CD vs temperature plot.
(67) The polypeptide molecules modified in accordance with the invention had increased melting temperatures as compared to the original molecules (Table 6). In
(68) In Vitro Antigenicity ELISA (Analysis of IgG Binding in Serum)
(69) The general conditions for the ELISA were as follows: the ELISA assays were performed in half area, 96-well plates. Volumes used were 50 l per well for all incubations except for blocking where 100 l was used. Coating was done over night at 4 C. in coating buffer (15 mM Na.sub.2CO.sub.3 and 35 mM NaHCO.sub.3), and all other incubations were performed at room temperature. Dilution of primate serum and detection antibodies was made in PBS+0.5% casein. All washes were done using an automatic ELISA Scan Washer 300, where each well was washed four times with 175 l washing buffer (PBS-T; 0.05% Tween 20 in 1PBS) per wash.
(70) The wells of the ELISA plate were coated with 2 g/ml of the binding polypeptides His.sub.6-Z.sub.TNF-:185, His.sub.6-Z.sub.HER2:342, His.sub.6-Z.sub.Insulin:810, His.sub.6-Z.sub.Taq:1154, Z.sub.PDGF-R: 2465-Cys-NEM, His.sub.6-Z.sub.HER2:2628, His.sub.6-Z.sub.Taq:3229, His.sub.6-Z.sub.TNF-:3230, His.sub.6-Z.sub.Insulin:3232 and Z.sub.PDGF-R:3358-Cys-NEM. Z.sub.HER2:342 was used as standard. After coating, the wells were washed twice with tap water and blocked with PBS+0.5% casein. The plate was emptied and a 2-fold dilution series of a primate serum pool from cynomolgus monkey (MAccaca fascicularis; obtained from Swedish Institute for Infectious Disease Control) was added to the wells. The titration series started with a 1/100 dilution and ended at 1/102400. The dilution was done directly in the 96-well plate. After incubating one hour with the primate serum pool, the plate was washed and a goat anti-human Ig-HRP antibody was added in dilution 1/5000 for detection. After 50 minutes incubation with the detection antibody, the plate was washed and the substrate added. Equal volumes of the two components in the IMMUNOPURE TMB kit were mixed, and 50 l was added per well. Subsequently, the plate was incubated in the dark for 12 minutes, and the reaction was stopped by addition of 50 l stop solution (2 M H.sub.2SO.sub.4). The absorbance at 450 nm was recorded using an ELISA reader. As negative control, PBS+0.5% casein was used instead of the primate serum pool.
(71) To evaluate the results and to obtain an IVA value that represents the level of primate Ig-molecules binding to the polypeptide, the program GraphPad Prism 5 was used. Sample values, with background OD values subtracted, were added to a template based on a XY-non-linear regression (sigmoidal dose response) formula. A dilution value for OD 0.3 was obtained from the formula and the IVA values were calculated by setting standard dilution value to 100 and by relating all samples to 100. A value below 100 indicates a decreased ability of the tested polypeptide to bind to immunoglobulins as compared to the Z.sub.HER2:342 molecule used as a positive control.
(72) The inventive molecules showed less potential to bind immunoglobulins as compared with original molecules (Table 7). The results are shown as in vitro antigenicity (IVA) values, and a reduced in vitro antigenicity (IgG binding) is read as a decrease in the IVA value.
(73) TABLE-US-00005 TABLE3 Listofbindingpolypeptidesequences Name Aminoacidsequence Z.sub.TNF-:185 VDNKFNKELGWAIGEIGTLPNLNHQQFRAFILSLWDD PSQSANLLAEAKKLNDAQAPK(SEQIDNo.13) Z.sub.HER2:342 VDNKFNKEMRNAYWEIALLPNLNNQQKRAFIRSLYDD PSQSANLLAEAKKLNDAQAPK(SEQIDNo.14) Z.sub.Insulin:810 VDNKFNKEKYMAYGEIRLLPNLNHQQVMAFIDSLVD DPSQSANLLAEAKKLNDAQAPK (SEQIDNo.15) Z.sub.Taq:1154 VDNKFNKEKGEAVVEIFRLPNLNGRQVKAFIASLYDD PSQSANLLAEAKKLNDAQAPK(SEQIDNo.16) Z.sub.PDGF-R:2465 VDNKFNKELIEAAAEIDALPNLNRRQWNAFIKSLVD DPSQSANLLAEAKKLNDAQAPK (SEQIDNo.17)
(74) TABLE-US-00006 TABLE4 Listofoligonucleotides Name Sequence5-3 AFFI-069 GTGAGCGGATAACAATTCCCCTC(SEQIDNo.18) AFFI-070 CAGCAAAAAACCCCTCAAGACCC(SEQIDNo.19) AFFI-115 CAGCAAAAAACCCCTCAAGACCC(SEQIDNo.20) AFFI-267 AGATAACAAATTCAACAAAG(SEQIDNo.21) AFFI-270 CTACTTTCGGCGCCTGAGCATCATTTAG (SEQIDNo.22) AFFI-1014 ACTTTCGGCGCCTGAGCATCATTTAG (SEQIDNo.23) AFFI-1015 ATAACAAATTCAACAAAGAA(SEQIDNo.24) AFFI-1043 ACTTTCGGCGCCTGAGAATCATTTAGCTTTTTA (SEQIDNo.25) AFFI-1044 CTACTTTCGGCGCCTGAGAATCATTTAGCTTTTTA (SEQIDNo.26) AFFI-1143 AGATGCCAAATACGCCAAAGAAATGCGAA (SEQIDNo.27) AFFI-1144 ATGCCAAATACGCCAAAGAAATGCGAA (SEQIDNo.28) AFFI-1151 CCCAAGCCAAAGCTCTGAATTGCTATCAGAAGCTAAAAAGC (SEQIDNo.29) AFFI-1152 GCTTTTTAGCTTCTGATAGCAATTCAGAGCTTTGGCTTGGG (SEQIDNo.30) AFFI-1320 AGATGCCAAATACGCCAAAGAAAAGGGGGAGGCGGTGGTT GAGATCTTTAGGTTACCTAACTTAACCGGGAGGCAAGTGAA GGCCTTCATCGCGAAATTATA(SEQIDNo.31) AFFI-1323 CTACTTTCGGCGCCTGGCTATCATTTAGCTTTTTAGCTTCG CTCAGTAATTCAGAGCTCTGGCTTGGGTCATCCCATAATTT AAGGATGAAGGCCCGAAATT(SEQIDNo.32) AFFI-1326 AGATGCCAAATACGCCAAAGAAAAGTATATGGCGTATGGTG AGATCCGGTTGTTACCTAACTTAACCCATCAGCAAGTTATG GCCTTCATCGATAAATTAGT(SEQIDNo.33) AFFI-1327 CTACTTTCGGCGCCTGGCTATCATTTAGCTTTTTAGCTTCG CTCAGTAATTCAGAGCTCTGGCTTGGGTCATCCACTAATTT ATCGATGAAGGCCATAACTT(SEQIDNo.34) AFFI-1328 AGATGCCAAATACGCCAAAG(SEQIDNo.35) AFFI-1329 ATGCCAAATACGCCAAAGAA(SEQIDNo.36) AFFI-1330 CTACTTTCGGCGCCTGGCTATCATTTAG (SEQIDNo.37) AFFI-1331 ACTTTCGGCGCCTGGCTATCATTTAG (SEQIDNo.38)
(75) TABLE-US-00007 TABLE 5 List of tested polypeptides Target Designation Variant TNF-alpha His.sub.6-Z.sub.TNF-:185 Original TNF-alpha His.sub.6-Z.sub.TNF-:3230 Inventive HER2 Z.sub.HER2:342 Original HER2 His.sub.6-Z.sub.HER2:342 Original HER2 His.sub.6-Z.sub.HER2:2628 Inventive Insulin His.sub.6-Z.sub.Insulin:810 Original Insulin His.sub.6-Z.sub.Insulin:3232 Inventive Taq polymerase His.sub.6-Z.sub.Taq:1154 Original Taq polymerase His.sub.6-Z.sub.Taq:3229 Inventive PDGF-R Z.sub.PDGF-R:2465-Cys Original PDGF-R Z.sub.PDGF-R:3558-Cys Inventive
(76) TABLE-US-00008 TABLE 6 Determined Tm values of tested polypeptides Target Designation Variant Tm ( C.) TNF-alpha His.sub.6-Z.sub.TNF-:185 Original 53 TNF-alpha His.sub.6-Z.sub.TNF-:3230 Inventive 60 HER2 His.sub.6-Z.sub.HER2:342 Original 63 HER2 His.sub.6-Z.sub.HER2:2628 Inventive 69 Insulin His.sub.6-Z.sub.Insulin:810 Original 42 Insulin His.sub.6-Z.sub.Insulin:3232 Inventive 48 Taq polymerase His.sub.6-Z.sub.Taq:1154 Original 46 Taq polymerase His.sub.6-Z.sub.Taq:3229 Inventive 50 PDGF-R Z.sub.PDGF-R:2465-Cys-NEM Original 42 PDGF-R Z.sub.PDGF-R:3558-Cys-NEM Inventive 42
(77) TABLE-US-00009 TABLE 7 IVA values of tested polypeptides Target Designation Variant IVA-value TNF-alpha His.sub.6-Z.sub.TNF-:185 Original 38 TNF-alpha His.sub.6-Z.sub.TNF-:3230 Inventive 21 HER2 His.sub.6-Z.sub.HER2:342 Original 99 HER2 His.sub.6-Z.sub.HER2:2628 Inventive 14 Insulin His.sub.6-Z.sub.Insulin:810 Original 43 Insulin His.sub.6-Z.sub.Insulin:3232 Inventive 16 Taq polymerase His.sub.6-Z.sub.Taq:1154 Original 26 Taq polymerase His.sub.6-Z.sub.Taq:3229 Inventive 18 PDGF-R Z.sub.PDGF-R:2465-Cys-NEM Original 35 PDGF-R Z.sub.PDGF-R:3558-Cys-NEM Inventive 3
Example 4Phage Display Selection and Characterization of Dynazyme Binding Polypeptide Variants
(78) Biotinylation of Dynazyme
(79) The target protein Dynazyme II DNA polymerase (Dynazyme) from species Thermus brockianus (Finnzymes, # F-501 L) was biotinylated using a 10 molar excess of NO-WEIGHT Sulfo-NHS-LC-biotin (Pierce, #21327) according to the manufacturer's protocol. Buffer was changed by dialysis using Slide-a-lyzer dialysis cassette (Pierce, 10K, 0.5-3 ml) to PBS prior to biotinylation and to TKMT (10 mM Tris-HCl, 50 mM KCl, 1.5 mM MgCl.sub.2, 0.1% Triton X-100, pH 8.8) after biotinylation to remove unbound biotin.
(80) Phage Display Selection
(81) Selection was performed against biotinylated Dynazyme using the inventive population of polypeptides (Example 1). Two approaches for selection were utilized; one with a high target concentration (track 1) and one with a low target concentration (track 2). Four selection cycles were performed. New phage stocks were prepared between each cycle. For selection overview and details, see
(82) Phage library stock was PEG/NaCl precipitated twice as described in Example 2 and dissolved in TKMT supplemented with 0.1% gelatin (TKMTg). Phages were pre-incubated with streptavidin-coated beads (SA beads, DYNABEADS M-280) for 30 minutes at RT. Beads used in the selection procedure and all tubes were pre-blocked in TKMTg.
(83) The target concentrations and the number of washes in each cycle are presented in
(84) ELISA Analysis of Dynazyme Binding Polypeptides
(85) Clones obtained after the last round of selection were randomly picked and used for periplasmic protein expression in a 96-well plate format as described in Example 2. Supernatants containing soluble polypeptide variants fused to ABD were assayed for target binding in an ELISA as follows. The putative binding polypeptides were expressed as AQLE-[Z #####]-VDYV-[ABD]-SQKA (SEQ ID NO. 39) (ABD=the albumin binding domain GA3 from Streptococcus sp. G148, Kraulis et al (1996) FEBS Lett. 378(2):190-194), wherein Z ##### denotes individual variants of the inventive polypeptide population.
(86) Half area microtiter wells (Costar, #3690) were coated with 50 l of 2-3 g/ml Dynazyme in ELISA coating buffer. The wells were blocked with 100 l TKMT complemented with 0.5% casein (Sigma) (TKMT-casein) for 1 h at RT. After removal of blocking solution, 50 l of supernatants were added to the wells and the plates were incubated for 1.5 h at RT. Captured polypeptide variants were detected by adding a primary and then a secondary antibody. The primary antibody, an affinity purified polyclonal rabbit Ig against Z variants, was diluted 1:5000 in TKMT-casein and incubated for 1.5 h at RT. The secondary antibody, a goat -rabbit-HRP Ig (DakoCytomation, # PO448), was diluted 1:5000 in TKMT-casein and incubated for 1 h at RT. The plates were washed four times with TKMT before incubation with the antibodies and the developing solution.
(87) Plates were developed as described in Example 2 and read at 450 nm in an ELISA spectrophotometer. All plates were prepared with relevant negative and positive controls as well as a blank where TKMT was used instead of periplasmic supernatant. In total, 1080 randomly picked clones were screened in ELISA for their binding to Dynazyme. Positive clones and some clones with low absorbance values were selected for sequencing.
(88) Sequencing of ELISA Positive Polypeptides
(89) Individual clones were subjected to sequencing according to Example 2. Eleven unique binding polypeptides regarded as positive in ELISA screening were found. Some of the clones occurred in several copies. In addition, several sequences from clones with lower ELISA values were identified.
(90) Sub-Cloning of Polypeptides into Plasmid pAY1448
(91) Fifteen unique polypeptides were subjected to subcloning as monomers into the expression vector pAY1448 providing an N-terminal His.sub.6-tag (as described in Example 2) using prepared plasmids as templates for the sticky-end PCR. The subcloning was performed as described in Example 3.
(92) Purification of Polypeptides
(93) The following text describes the purification of fifteen monomeric His.sub.6-tagged polypeptides, namely His.sub.6-Z04665, His.sub.6-Z04672, His.sub.6-Z04674, His.sub.6-Z04678, His.sub.6-Z04687, His.sub.6-Z04767, His.sub.6-Z04770, His.sub.6-Z04775, His.sub.6-Z04776, His.sub.6-Z04777, His.sub.6-Z04778, His.sub.6-Z04779, His.sub.6-Z04780, His.sub.6-Z04781 and His.sub.6-Z04899. Pelleted bacterial cells harboring the soluble His.sub.6-tagged molecules were suspended in His GRAVITRAP binding buffer (20 mM sodium phosphate, 0.5 M NaCl, 20 mM imidazole and 25 U/ml BENZONASE) and disrupted by ultrasonication. The sonicated cell suspensions were heated using hot water (95 C.) until the temperature of the suspensions stabilized at around 90 C. during five minutes. After clarification by centrifugation, the supernatants were loaded on His GRAVITRAP columns (GE Healthcare) previously equilibrated with His GRAVITRAP binding buffer. After washing the columns with 5 CV His GRAVITRAP binding buffer and 5 CV His GRAVITRAP washing buffer (20 mM sodium phosphate, 0.5 M NaCl, 60 mM imidazole), the polypeptides were eluted with 3 CV His GRAVITRAP elution buffer (20 mM sodium phosphate, 0.5 M NaCl, 500 mM imidazole).
(94) The polypeptide variants were further purified by reversed phase chromatography (RPC). Acetonitrile (ACN) was added to a final concentration of 2% in the eluted fractions from His GRAVITRAP. Samples were loaded on a RESOURCE RPC 3 ml column (GE Healthcare), previously equilibrated with RPC A buffer (0.1% trifluoroacetic acid (TFA), 2% ACN, 98% water). After column wash with 5 CV RPC A buffer, bound protein were eluted with a 20 CV linear gradient of 0-50% RPC B buffer (0.1% TFA, 80% ACN, 20% water). The flow rate was 5 ml/min and the 280 nm signal was monitored. Fractions containing pure polypeptides were identified by SDS-PAGE analysis and pooled.
(95) The buffer of the purified polypeptides was replaced to 50 mM Tris-HCl, pH 8.8, by size exclusion chromatography on disposable PD-10 Desalting Columns (GE Healthcare).
(96) Twelve of the fifteen polypeptide variants were successfully purified: His.sub.6-Z04665, His.sub.6-Z04672, His.sub.6-Z04674, His.sub.6-Z04687, His.sub.6-Z04770, His.sub.6-Z04775, His.sub.6-Z04776, His.sub.6-Z04777, His.sub.6-Z04778, His.sub.6-Z04780, His.sub.6-Z04781 and His.sub.6-Z04899.
(97) Analysis of Purified Polypeptides
(98) Determination of the concentration of polypeptide solutions was performed by measuring the absorbance at 280 nm using a NANODROP ND-1000 Spectrophotometer. The proteins were further analyzed with SDS-PAGE and LC-MS.
(99) For the SDS-PAGE analysis, polypeptide solution was mixed with LDS Sample Buffer (Invitrogen) and incubated at 74 C. for 10 min. 10 g of each polypeptide variant were loaded on NUPAGE 4-12% Bis-Tris Gels (Invitrogen). The gels were run with MES SDS Running Buffer (Invitrogen) in an XCELL SURELOCK Mini-Cell (Invitrogen) employing NOVEX Sharp Prestained Protein Standard (Invitrogen) as molecular weight marker and PhastGel Blue R (GE Healthcare) protein staining solution for staining.
(100) To verify the identity of the polypeptide variants, LC/MS-analyses were performed using an Agilent 1100 LC/MSD system equipped with API-ESI and single quadruple mass analyzer. The protein samples were diluted in 50 mM Tris-HCl, pH 8.8, to a final concentration of 0.5 mg/ml and 10 l were loaded on a Zorbax 300SB-C18 column (4.6150, 3.5 m) (Agilent) at a flow-rate of 1 ml/min. Solution A contained 0.1% TFA in water and solution B contained 0.1% TFA in ACN. Proteins were eluted using a 22 minutes linear gradient of 15% to 65% solution B at 1 ml/min. The separation was performed at 30 C. The ion signal and the absorbance at 280 and 220 nm were monitored. The molecular weights of the purified proteins were verified by analysis of the ion signal.
(101) Purity of the polypeptide variants was determined to be greater than 95% according to the SDS-PAGE and LC/MS-analyses.
(102) Determination of Melting Temperature (Tm)
(103) Purified polypeptide variants were diluted in 50 mM Tris-HCl, pH 8.8, to a final concentration of 0.5 mg/ml. Circular dichroism (CD) analysis was performed on a Jasco J-810 spectropolarimeter in a cell with an optical path-length of 1 mm. In the variable temperature measurements, the absorbance was measured at 221 nm from 20 to 90 C., with a temperature slope of 5 C./min. Polypeptide melting temperatures (Tm) were calculated by determining the midpoint of the transition in the CD vs. temperature plot. For results, see Table 8.
(104) TABLE-US-00010 TABLE 8 Tm of Dynazyme binding polypeptide variants Designation Tm ( C.) His.sub.6-Z04665 38 His.sub.6-Z04672 33 His.sub.6-Z04674 35 His.sub.6-Z04687 60 His.sub.6-Z04770 44 His.sub.6-Z04775 45 His.sub.6-Z04776 55 His.sub.6-Z04777 58 His.sub.6-Z04778 43 His.sub.6-Z04780 65 His.sub.6-Z04781 66 His.sub.6-Z04899 39
Analysis of Heat Stability
(105) The ability to refold to the original alpha helical structure after being subjected to heat was a requested property of the above described polypeptide variants. To investigate structural reversibility, two CD spectra per sample were obtained at 20 C. Between the two measurements, the samples were heated to 96 C. The samples were kept at 96 C. for two minutes, and then cooled to 20 C. Similar CD spectra before and after heating would prove a sample to be structurally reversible. Three of twelve analyzed polypeptide variants were negatively affected by the heat treatment, whereas nine polypeptide variants were shown to regain their alpha helical structure completely. A typical overlay of two CD spectra before and after heating is shown in
(106) BIACORE Binding Analysis
(107) The interactions between 12 His.sub.6-tagged monomeric Z variants selected according to the invention and Dynazyme were analyzed in a BIACORE instrument (GE Healthcare). The target protein was immobilized in a flow cell on the carboxylated dextran layer of a CM5 chip surface (GE Healthcare). The immobilization was performed using amine coupling chemistry according to the manufacturer's protocol and using acetate pH 5.5. One flow cell surface on the chip was activated and deactivated for use as blank during analyte injections. The analytes, i.e. polypeptide variants diluted in HBS-EP running buffer (GE Healthcare) to a final concentration of 10 M, were injected in random order in duplicates at a flow-rate of 10 l/minute for 5 minutes. After 10 minutes of dissociation, the surfaces were regenerated with one injection of 0.05% SDS. The results were analyzed in BiaEvaluation software (GE Healthcare). Curves of the blank surface were subtracted from the curves from the ligand surfaces. The analysis showed an interaction for 5 of the polypeptide variants to the immobilized Dynazyme, as outlined in
Example 5Comparative Study of Chemical Synthesis of a Polypeptide of a Population According to the Invention
(108) Summary
(109) In the experiments making up this example, solid phase peptide synthesis (SPPS) of polypeptides of the populations according to the invention is described, and compared to synthesis of a polypeptide based on the original scaffold. The mutations introduced at four positions, i.e. [N23T], [A42S], [A46S] and [A54S], allowed for using an alternative synthesis strategy with pseudoproline precursors with the simplified abbreviation Fmoc-Xxx-Yyy-OH. Using pseudoprolines in three or four of the positions described above, it is possible to synthesize full length molecules with the sequences:
(110) TABLE-US-00011 SEQA: (SEQIDNo.3) maESEKYAKEMRNAYWEIALLPNLTNQQKRAFIRKLYDDPSQ SSELLSEAKKLNDSQAPK
(wherein ma designates mercaptoacetyl coupled to the N-terminus of the polypeptide); and
(111) TABLE-US-00012 SEQB: (SEQIDNo.4) AEAKYAKEMWIAWEEIRNLPNLNGWQMTAFIAKLLDDPSQ SSELLSEAKKLNDSQAPKC;
whereas standard synthesis failed to produce the peptide with SEQ A.
(112) Standard synthesis of SEQ C: AENKFNKEMW IAWEEIRNLP NLTGWQMTAF IASLLDDPSQ SANLLAEAKK LNDAQAPK (SEQ ID No. 5), which is similar to SEQ B but contains the original scaffold amino acids, resulted in a very impure preparation and in a low peptide yield.
(113) The introduction of novel serine or threonine residues also enables the use of isoacyl dipeptides, which is an alternative to pseudoprolines for increasing the synthetic efficiency by reducing aggregation during peptide synthesis (Sohma et al, Tetrahedron Lett. 47:3013, 2006). Several such building blocks are available from Novabiochem of Merck Biosciences AG.
(114) Rationale
(115) Peptide synthesis of the HER2 binding molecule Z.sub.HER2:342 (disclosed in WO 2005/003156 as Z.sub.HER2:107, and sometimes also called Z00342), as well as coupling of DOTA to the N-terminus for this molecule is possible and described in the literature (Orlova A et al (2006) Cancer Research 67:2178-2186). However, a huge variation in peptide yield after synthesis was observed. The difficulties to reproducibly synthesize the peptide can be related both to the length of the peptide as well as the primary amino acid sequence. In addition, long peptides with the reactive groups of the amino acid side chains still protected may generate unfavorable secondary structures, e.g. beta sheets, which can disturb solid phase peptide synthesis (Quibell M and Johnson T in Fmoc Solid Phase Peptide Synthesis-A Practical Approach, W. C. Chan, P. D. White Eds, Oxford University Press 2000:115-135). One way to prevent secondary structure formation during peptide synthesis is the use of pseudoprolines. Pseudoprolines, with the simplified abbreviation Fmoc-Xxx-Yyy-OH, can be used if the amino acid Yyy is serine, threonine or cysteine. These pseudoprolines display a closed proline-like structure with the side chain linked to the backbone, and can be converted into the normal amino acid structure by acid treatment (Haack T and Mutter M (1992) Tetrahedron Lett 33:1589-1592). Pseudoprolines are commercially available for 14 amino acids in position Xxx (all naturally occurring amino acids except Arg, Cys, His, Met, Pro, Thr) together with serine or threonine in position Yyy.
(116) The parent molecule Z.sub.HER2:342 has no threonine and cysteine in the primary sequence. Serine is only found in positions 33, 39 and 41. A pseudoproline precursor is only available for serine 41 (Q.sup.40-S.sup.41) For the two other serines, the amino acid in position Xxx prevents the use of pseudoproline, since there are no precursors available (R.sup.32-S.sup.33 and P.sup.38-S.sup.39).
(117) The mutations introduced in the polypeptides comprised in the population according to the invention are aimed to, but not restricted to, facilitate peptide synthesis. Especially the mutations in position 23, 42, 46 and 54, i.e. [N23T], [A42S], [A46S] and [A54S] may have the capacity to solve two of the identified problems in SPPS: they allow the use of pseudoprolines and the critical region around amino acid positions 21 to 26 is changed in position 23 by replacing asparagine with threonine.
(118) Synthesis Strategy 1
(119) The amino acid sequence SEQ A was assembled on an Fmoc-Lys(Boc)-Wang polystyrene resin in a fully automated peptide synthesizer. This resin is highly suitable for the formation of peptides with the Fmoc-strategy. 57 amino acids (with appropriate side-chain protection) were coupled onto the resin. In the last step, coupling of S-trityl-protected mercaptoacetic acid was performed manually.
(120) Step 1: Solid Phase Peptide Synthesis
(121) The Fmoc-Lys(Boc)-Wang polystyrene resin was transferred into an SPPS reactor with a stirrer. Synthesis was then started with Fmoc deprotection of the resin, followed by a coupling procedure with Fmoc-Pro-OH according to the general description given below. This step was again followed by an Fmoc deprotection and subsequent coupling of the amino acid derivatives according to the sequence. After final washings of the resin with isopropylether (IPE), the peptide resin was dried in a desiccator under reduced pressure.
(122) Both standard Fmoc peptide synthesis and synthesis using pseudoprolines in four positions were performed. For standard peptide synthesis, only Fmoc-amino acids were used. For the alternative peptide synthesis, apart from Fmoc-amino acids the following pseudoprolines were used: Fmoc-Leu-Thr-OH in position 22-23, Fmoc-Ser-Ser-OH in position 41-42, Fmoc-Leu-Ser-OH in position 45-46 and Fmoc-Asp-Ser-OH in position 53-54.
(123) Fmoc Deprotecting Procedure
(124) The resin was also treated with 20% piperidine in N-methyl-2-pyrrolidone (NMP) in order to achieve the cleavage of the N--Fmoc protecting group. The washing of the resin was then performed with NMP.
(125) Coupling Procedure
(126) Automated coupling of the amino acid derivates Pro57 to Glu1. Up to 3 eq of the Fmoc-AA derivative were dissolved in NMP. For the coupling, 2-(1H-benzotriazole-1-yl)-1,1,3,3-tetramethyluronium tetrafluoroborate (TBTU) in dimethylformamide (DMF) and sym.-collidine (2,4,6-trimethylpyridine) in NMP were added. The resulting solution was mixed at room temperature before it was poured onto the resin. NMP was used as solvent. After a coupling time of at least 15 minutes at 60 C., the resin was washed with NMP.
(127) After each coupling procedure, a repetition of the coupling with 2-(1H-7-azabenzotriazol-1-yl)-1,1,3,3-tetramethyluronium tetrafluoroborate (TATU) in DMF as coupling reagent and with dichloroethane as solvent takes place automatically, followed by acetic anhydride capping.
(128) Step 2: Coupling of Mercaptoacetic Acid
(129) Acylations were performed with 5 molar equivalents amino acid, 2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorphosphate (HBTU) and 1-hydroxybenzotriazole (HOBt) and 10 equivalents N-ethyldiisopropylamine (DIEA, from Lancaster Synthesis, Morecambe, England). S-trityl-mercaptoacetic acid was from AnaSpec Inc (San Jose, Calif., USA).
(130) Step 3: Cleavage from the Resin Including Cleavage of the Remaining Protection Groups
(131) The peptide resin was treated with trifluoroacetic acid (TFA) in the presence of purified water, ethanedithiol (EDT), and triisopropylsilane (TIS). After approx. 2 hours of cleavage time at room temperature, the reaction mixture was cooled to approx. 0 C., and ammonium iodide and dimethyl sulfide are added to reduce oxidized methionine. After an additional 60 min cleavage time at approx. 0 C., the formed iodine was reduced with ascorbic acid. After filtering off the product, it was precipitated in IPE in the cold, filtered off again, washed with IPE, and dried under reduced pressure.
(132) Purity Analysis by HPLC
(133) The purity of the 58 amino acid long peptides and some intermediates was determined by reversed phase HPLC using a Vydac 218 TP54 (5 m, 2504.6 mm) column and 0.1% TFA, 1% acetonitrile in H.sub.2O and 0.1% TFA in acetonitrile as solvent A and B respectively. The column oven temperature was set to 35 C. The column was eluted either with a gradient of 15 to 45% solvent B in 30 minutes or with a gradient from 20 to 50% B in 30 minutes. UV detection was at 220 nm. The purity was calculated by area normalization.
(134) Results
(135) The yield and purity of the molecule with the sequence SEQ A, synthesized with or without the use of pseudoprolines, were analyzed by analytical reversed phase chromatography. In order to follow the progress of the synthesis, a small portion of synthesis resin was taken after several coupling steps and analyzed for the presence, purity and yield of the desired peptide intermediate.
(136) Synthesis Strategy 2
(137) Two molecules were assembled using the Fmoc-strategy on a fully automated peptide synthesizer with an integrated microwave oven.
(138) The 59 amino acid residues of SEQ B, based on the inventive scaffold sequence, were assembled (with appropriate side chain protection) on an Fmoc-Cys(Trt)-Wang LL polystyrene resin.
(139) The 58 amino acid residues of SEQ C, based on the original AFFIBODY molecule scaffold, were assembled (with appropriate side chain protection) on an Fmoc-Lys(Boc)-Wang LL polystyrene resin.
(140) The Wang resin LL is highly suitable for the formation of peptides with the Fmoc strategy.
(141) Step 1: Solid Phase Peptide Synthesis
(142) The polystyrene resin was automatically transferred into an SPPS reaction vessel by the synthesizer (Liberty, CEM Corporation, NC USA). Synthesis was then started with Fmoc deprotection of the resin, followed by a coupling procedure with the next Fmoc-protected amino acid (Fmoc-AA) according to the general description given below. This step was again followed by an Fmoc deprotection and subsequent coupling of the amino acid derivatives according to the sequence. After final washings of the resin with dichloromethane (DCM), the peptide resin was dried under reduced pressure. The entire peptide SEQ C was made by standard Fmoc peptide synthesis, whereas pseudoprolines were used at positions in SEQ B where this was enabled by the improvements done to the scaffold. The following pseudoprolines were used: Fmoc-Ser-Ser-OH at position 41-42, Fmoc-Leu-Ser-OH at position 45-46 and Fmoc-Asp-Ser-OH at position 53-54.
(143) Fmoc Deprotecting Procedure
(144) The resin was treated with 5% piperazine in NMP, with microwave irradiation, in order to achieve the cleavage of the N--Fmoc protecting group. The washing of the resin was then performed with NMP.
(145) Coupling Procedure
(146) Automated coupling of the amino acid derivatives Cys59 to Ala1 (for SEQ B) and Lys58 to Ala1 (for SEQ C). Up to 5 equivalents of the Fmoc-AA were dissolved in NMP. For the coupling, O-(benzotriazole-N,N,N,N-tetramethyluronium hexafluorophosphate (HBTU) and N-hydroxybenzotriazole (HOBt) in dimethylformamide (DMF), N,N-diisopropylethylamine (DIPEA) in NMP were added to the resin at a molar ratio of 1:1:1:2 (AA/HBTU/HOBt/DIPEA). The mixture was agitated by bubbling nitrogen gas through the bottom of the reaction vessel. After a coupling time of at least 5 minutes at 75-80 C. with added energy using microwave irradiation, the resin was washed with NMP.
(147) After each coupling procedure, an automatic acetic anhydride capping was performed.
(148) Step 2: Cleavage from the Resin Including Cleavage of the Remaining Protection Groups
(149) The peptide resin was treated with trifluoroacetic acid (TFA) in the presence of purified water, ethanedithiol (EDT), and triisopropylsilane (TIS). After approx. 2 hours of cleavage at room temperature, the cleavage mixture was filtered and the resin rinsed with neat 95% TFA/water. The filtrate was slowly added to cooled methyl tert-butyl ether (MTBE). The precipitate was centrifuged and the MTBE removed. The solid was resuspended in ether and the operation repeated a total of three times. After the last removal of ether, the solid was resuspended in 0.1% TFA/water, the remaining ether was left to evaporate, and the solution was frozen before lyophilisation.
(150) Purity and Mass Analysis by HPLC-MS
(151) The purity of the peptides was determined by high performance liquid chromatography and on line mass spectrometry (HPLC-MS) using an Agilent 1100 HPLC/MSD equipped with electro spray ionization (ESI) and a single quadropol. The HPLC was run using a Zorbax 300SB C18 (3.5 m, 1504.6 mm) column and 0.1% TFA/water and 0.1% TFA/acetonitrile (ACN) as solvent A and B respectively. The column oven temperature was set to 30 C. The column was eluted with a gradient of 15 to 55% solvent B in 40 minutes. UV detection was at 220 nm. The purity was calculated by area normalization. The software used for the mass analysis and evaluation was ChemStation Rev. B.02.01. (Agilent).
(152) Results
(153) The yield and purity of the molecules SEQ B and SEQ C was analyzed by analytical reversed phase chromatography. The full length peptides were obtained in both syntheses, however with a much larger yield for SEQ B.
(154) Standard Fmoc-synthesis of SEQ C, however, resulted in a large number of small peptide peaks (
Example 6Immunogenicity of Original and Inventive Polypeptide Variants
(155) Summary
(156) In this example, the immunogenicity of one original and one inventive polypeptide variant was compared in vivo. Dimeric molecules were administered to rats, and the specific antibody responses were determined in an Anti-Drug Antibody (ADA) assay. The molecule with the introduced scaffold mutations according to the invention displays a lower and delayed antibody response compared to the original Z variant.
(157) Cloning and Production of Polypeptides
(158) Two Taq-polymerase specific binding polypeptides fused to the albumin binding domain ABD035 (Jonsson et al (2008) Protein Eng Des Sel 8:515-27) were used in the study:
(159) 1. (Z01154).sub.2-ABD035: original scaffold
(160) 2. (Z03229).sub.2-ABD035: inventive scaffold
(161) PCR amplified and hybridized fragments of Z01154 and Z03229 with Accl-overhangs were cloned as dimers in the Accl digested pET (Novagen) derived expression vectors pAY492 and pAY1450 respectively. The resulting vectors were digested with Accl-Notl and ligated with ABD035 fragments that had been PCR amplified with Accl and Notl overhangs, generating the constructs pAY1827 (encoding MGSSLQ-[Z01154]-[Z01154]-VD-[ABD035] (SEQ ID No. 40)) and pAY2292 (encoding MGSSLQ-[Z03229]-[Z03229]-VD-[ABD035] (SEQ ID No. 41)). The plasmids were transformed into competent E. coli BL21(DE3) cells and proteins were produced by fermentation, essentially as described in Example 3. Pelleted cells were suspended in [25 mM Tris-HCl, 200 mM NaCl, 1 mM EDTA, 25 U/ml BENZONASE (Merck, #1.01654.0001), pH 8.0] and disrupted by sonication on ice. The clarified supernatants were loaded onto a column packed with CNBr-activated Sepharose (GE Healthcare, #17-0981-03) coupled in-house with human serum albumin. The column was pre-equilibrated in 1TST [25 mM Tris-HCl, 200 mM NaCl, 1 mM EDTA, 0.05% Tween 20, pH 8.0]. After sample application, washing was performed with 1TST followed by 5 mM NH.sub.4Ac pH 5.5 until no reduction of the Abs.sub.280 signal was observed. Bound proteins were eluted with 0.5 M HAc, pH 2.8. The eluted samples were supplemented with acetonitrile to a final concentration of 2% and further purified by reverse phase chromatography on a Resource RPC column (GE Healthcare, #17-1182-01). [2% acetonitrile, 0.1% TFA in water] was used as running buffer and samples were eluted using a linear gradient of 0-50% of [80% acetonitrile, 0.1% TFA in water] over 25 column volumes. Buffer exchange to [5 mM sodium phosphate, 150 mM NaCl, pH 7.2] was performed using a HiPrep 26/10 Desalting column (GH Healthcare, #17-5087-01). Sample purity was verified by SDS-PAGE and LC/MS analysis as described in Example 3. Endotoxin traces were removed on an AffinityPak Detoxi-Gel endotoxin removing gel (Thermo, #20344) according to the manufacturer's instructions. No endotoxins were detected in gel-clot LAL tests performed by APL (Apoteket Produktion & Laboratorier AB, Sweden). The samples were free of soluble aggregates as verified by size-exclusion chromatography carried out on a Superdex 75 10/300 column (GE Healthcare, #17-5174-01) using 1PBS as running buffer, a flow rate of 0.5 ml/min and a sample volume of 100 l with a concentration of 1 mg/ml.
Administration and Sampling Schemes
(162) The animal study was performed at Agrisera AB (Vnns, Sweden) with permission from the local animal ethics committee. Female Sprague Dawley rats divided into three groups were injected subcutaneously with (Z01154).sub.2-ABD035, (Z03229).sub.2-ABD035 or a buffer control as outlined in Table 9. Injections were given at days 0, 4, 7, 14, 21 and 28. 250 l blood samples were collected from each animal on day 1 (pre-serum) and on days 6, 13, 20 and 35. All animals were sacrificed on day 35. Collected blood samples were left to coagulate over night at 4 C. and obtained sera were stored at 20 C. until analysis.
(163) TABLE-US-00013 TABLE 9 Sample administration scheme mg/ No. of Means of animal/ ml/animal/ Group animals Molecule administration injection injection 1 8 (Z01154).sub.2-5 s.c. 0.125 0.1 ABD03 2 8 (Z03229).sub.2- s.c. 0.125 0.1 ABD035 3 4 Buffer s.c. 0.1 control: 5 mM sodium phosphate, 150 mM NaCl, pH 7.2
Anti-Drug Antibody (ADA) Assay
(164) To analyze the presence of anti-(Z01154).sub.2-ABD035 and anti-(Z03229).sub.2-ABD035 antibodies, three types of ELISA analyses were performed. All samples were initially screened for the presence of reacting antibodies followed by a confirmatory assay to verify specificity. Serum samples with specific antibodies against Z variants were subsequently titrated to quantify the titer of anti-(Z01154).sub.2-ABD035 and anti-(Z03229).sub.2-ABD035 antibodies.
(165) For screening of serum samples, ELISA plates (96-well, half-area plates, Costar, #3690) were coated over night with (Z01154).sub.2-ABD035 or (Z03229).sub.2-ABD035 diluted in coating buffer (Sigma, # C3041) to a final concentration of 2 g/ml. 50 l of the coating solution was added per well and plates were incubated over night at 4 C. The plates were washed twice manually with deionized water and subsequently blocked for 2 hours with 100 l/well of PBS-Casein (PBS with 0.5% Casein (Sigma, #8654)). The blocking solution was removed and serum samples (50 l/well) diluted 1:50 in blocking buffer were added. After 1.5 hour of incubation at RT, plates were washed in an automated ELISA washer (Scanwasher 300, Scatron) with PBST (PBS with 0.05% Tween 20 (Acros Organics, #233362500)). To detect rat antibodies against Z variants, 50 l per well of HRP-conjugated anti-rat IgG (Southern Biotech, #3050-05), diluted 1:6000 in PBS-Casein were added. After 1 hour of incubation, the plates were washed as described above and 50 l/well of substrate solution (IMMUNOPURE TMB, Pierce, #34021) were added. The plates were incubated at RT in the dark, and color development was stopped after 15 minutes with 50 l/well of 2 M H.sub.2SO.sub.4 (VWR, #14374-1). Plates were read at 450 nm in an ELISA reader (Victor.sup.3, Perkin Elmer).
(166) The ELISA method described above was also used for the confirmatory and titration ELISA assays, but with some alterations. For the confirmatory assay, serum samples were diluted 1:50 in PBS-Casein or in PBS-Casein including 1 g/ml of respective polypeptide variant. Serum samples with a reduction of the OD signal of 45% or more were considered to contain specific antibodies against Z variants. For the titration assay, serum samples were diluted 1:50 in PBS-Casein and then in series of 2-fold or 5-fold dilutions until they crossed the plate-specific cut point to allow a titer value to be calculated for the sample.
(167) During assay development the following key parameters were determined: Minimum dilution: 1:50 Non specific background (NSB): OD.sub.450 of a pool of normal rat sera (Sprague Dawley rats, Scanbur) used as dilution matrix and included on each plate throughout the analysis Assay cut point: mean OD.sub.450 of normal rat sera from 30 individuals plus 1.645 times standard deviations of the mean. A value of 0.11 was obtained for both (Z01154).sub.2-ABD035 and (Z03229).sub.2-ABD035. Normalisation factor. Assay cut point divided by the mean OD.sub.450 of the NSB: 1.87 and 1.86 for (Z01154).sub.2-ABD035 and (Z03229).sub.2-ABD035, respectively
During sample analysis, the plate specific cut point was then determined as: Mean OD.sub.450 of plate specific NSB, multiplied by the normalisation factor.
(168) Rat serum (hyperimmunised Sprague Dawley rats, Agrisera) confirmed to contain antibodies against the two polypeptide variants were used for preparing positive control (PC) samples included on each plate throughout the analysis: HighPC: positive control serum diluted 1:4 in matrix before minimum dilution in PBS-Casein. This PC has OD values high above the assay/plate cut point. LowPC: Positive control serum diluted 1:300 in matrix before minimum dilution in PBS-Casein. This PC has OD values that fall just above the assay/plate cut point.
(169) The LowPC and HighPC values were used to prepare the Titer quality controls LoQC1-5 and HiQC1-5). The LowPC and HighPC were diluted 1:50 in PBS-Casein to obtain LoQC1 and HiQC1 respectively. These were then further diluted in PBS-Casein to obtain LoQC2 (1:100), LoQC3 (1:200), LoQC4 (1:400) and LoQC5 (1:800), and HiQC2 (1:250), HiQC3 (1:1250), HiQC4 (1:6250) and HiQC5 (1:31250), respectively.
(170) The titer values were calculated using GraphPad Prism 5 (GraphPad Software Inc). Briefly, OD.sub.450 values were plotted against log dilution and the titer of the sample was defined as the log dilution at the plate specific cut point.
(171) Results
(172) The in vivo comparison between original ((Z01154).sub.2-ABD035) and inventive ((Z03229).sub.2-ABD035) molecules showed that the inventive molecule was less immunogenic. The response varied considerably between individuals and increased over time. The titer could be determined in three individuals that received the original molecule compared to two individuals that received the inventive molecule. The actual titer was also lower in the group that received the inventive molecule (Table 10). The reason for seeing few animals develop an antibody response may be due to the fused ABD molecule, which previously has been shown to reduce immunogenicity of a fused polypeptide (see e.g. WO 2005/097202).
(173) TABLE-US-00014 TABLE 10 Immune responses in rats given an original or an inventive polypeptide variant Group 1 Group 2 Group 3 (Z01154).sub.2-ABD035 (Z03229).sub.2-ABD035 Buffer control n = 8 n = 8 n = 4 Specific Specific Specific response response response Time (no. of Titer (no. of Titer (no. of Titer (days) animals) Mean SD animals) Mean SD animals) Mean SD 1 0 0 0 6 0 0 0 13 1 2.9 0 0 20 2 3.4 (1.0) 1 2.1 0 27 3 2.7 (1.0) 1 2.4 0 35 2 3.4 (1.3) 2 2.8 (0.1) 0