Binding polypeptides having a mutated scaffold
RE049495 · 2023-04-18
Assignee
Inventors
Cpc classification
G01N33/6845
PHYSICS
C07K1/047
CHEMISTRY; METALLURGY
C12Q1/6874
CHEMISTRY; METALLURGY
International classification
C07K14/00
CHEMISTRY; METALLURGY
C12Q1/6874
CHEMISTRY; METALLURGY
Abstract
The present disclosure relates to a class of engineered polypeptides and provides a polypeptide comprising the sequence EX.sub.2X.sub.3X.sub.4AX.sub.6X.sub.7EIX.sub.10 X.sub.11LPNLX.sub.16X.sub.17X.sub.18QX.sub.20 X.sub.21AFIX.sub.25X.sub.26LX.sub.28X.sub.29X.sub.30 PX.sub.32QSX.sub.35X.sub.36LLX.sub.39E AKKLX.sub.45X.sub.46X.sub.47Q (SEQ ID NO: 55). The present disclosure also relates to populations of polypeptide variants based on a common scaffold, each polypeptide in the population comprising the amino acid sequence EX.sub.2X.sub.3X.sub.4AX.sub.6X.sub.7EIX.sub.10 X.sub.11LPNLX.sub.16X.sub.17X.sub.18QX.sub.20 X.sub.21AFIX.sub.25X.sub.26LX.sub.28X.sub.29X.sub.30 PX.sub.32QSX.sub.35X.sub.36LLX.sub.39E AKKLX.sub.45X.sub.46X.sub.47Q (SEQ ID NO: 55), and methods for selecting a desired polypeptide having an affinity for a predetermined target from said population.
Claims
1. A polypeptide comprising an amino acid sequence selected from: i) TABLE-US-00022 .[.(SEQ ID NO: 55).]. .[.EX.sub.2X.sub.3X.sub.4AX.sub.6X.sub.7EIX.sub.10 X.sub.11LPNLX.sub.16X.sub.17X.sub.18QX.sub.20.]. .[.X.sub.21AFIX.sub.25X.sub.26LX.sub.28X.sub.29X.sub.30 PX.sub.32QSX.sub.35X.sub.36LLX.sub.39E.]. .[.AKKLX.sub.45X.sub.46X.sub.47Q,.]. .Iadd.(SEQ ID NO: 55).Iaddend. .Iadd.EX.sub.2X.sub.3X.sub.4AX.sub.6X.sub.7EIX.sub.10X.sub.11LPNLX.sub.16X.sub.17X.sub.18QX.sub.20.Iaddend. .Iadd.X.sub.21AFIX.sub.25X.sub.26LX.sub.28X.sub.29X.sub.30PX.sub.32QSX.sub.35X.sub.36LLX.sub.39E.Iaddend. .Iadd.AKKLX.sub.45X.sub.46X.sub.47Q,.Iaddend. wherein each of X.sub.2, X.sub.3, X.sub.4, X.sub.6, X.sub.7, X.sub.10, X.sub.11, X.sub.17, X.sub.18, X.sub.20, X.sub.21, X.sub.25 and X.sub.28 independently corresponds to any amino acid residue; and wherein, independently of each other, X.sub.16 is selected from N and T; X.sub.26 is selected from K and S; X.sub.29X.sub.30PX.sub.32 is selected from DDPS .Iadd.(SEQ ID NO: 74) .Iaddend.and RQPE .Iadd.(SEQ ID NO: 75).Iaddend.; X.sub.35 is selected from A and S; X.sub.36 is selected from E and N; X.sub.39 is selected from A, C and S; X.sub.45 is selected from E.[., N.]. and S; X.sub.46 is selected from D, E and S.[., provided that X.sub.46 is not D when X.sub.45 is N.].; X.sub.47 is selected from A and S; and ii) an amino acid sequence which has at least 91% identity to the sequence defined in i), provided that X.sub.46 is not D .Iadd.or E .Iaddend.when X.sub.45 is N.Iadd., provided that for i) and ii) X.sub.10 is not D when X.sub.20 is W for a polypeptide capable of binding human complement component 5.Iaddend..
2. The polypeptide according to claim 1, wherein X.sub.45 is S.
3. The polypeptide according to claim 1, wherein X.sub.45X.sub.46 is selected from ES and SE.
4. The polypeptide according to claim 3, wherein X.sub.45X.sub.46 is SE.
5. The polypeptide according to claim 1, which comprises an amino acid sequence selected from: TABLE-US-00023 .[.(SEQ ID NO: 56).]. .[.YAK EX.sub.2X.sub.3X.sub.4AX.sub.6X.sub.7EIX.sub.10 X.sub.11LPNLX.sub.16X.sub.17X.sub.18QX.sub.20.]. .[.X.sub.21AFIX.sub.25X.sub.26LX.sub.25X.sub.29X.sub.30 PX.sub.32QSX.sub.35X.sub.36LLX.sub.39E.]. .[.AKKLX.sub.45X.sub.46X.sub.47Q AP;.]. .[.and.]. .[.(SEQ ID NO: 57).]. .[.FNK EX.sub.2X.sub.3X.sub.4AX.sub.6X.sub.7EIX.sub.10 X.sub.11LPNLX.sub.16X.sub.17X.sub.18QX.sub.20.]. .[.X.sub.21AFIX.sub.25X.sub.26LX.sub.25X.sub.29X.sub.30 PX.sub.32QSX.sub.35X.sub.36LLX.sub.39E.]. .[.AKKLX.sub.45X.sub.46X.sub.47Q AP,.]. .Iadd.(SEQ ID NO: 56).Iaddend. .Iadd.YAK EX.sub.2X.sub.3X.sub.4AX.sub.6X.sub.7EIX.sub.10X.sub.11LPNLX.sub.16X.sub.17X.sub.18QX.sub.20.Iaddend. .Iadd.X.sub.21AFIX.sub.25X.sub.26LX.sub.25X.sub.29X.sub.30PX.sub.32QSX.sub.35X.sub.36LLX.sub.39E.Iaddend. .Iadd.AKKLX.sub.45X.sub.46X.sub.47Q AP;.Iaddend. .Iadd.and.Iaddend. .Iadd.(SEQ ID NO: 57).Iaddend. .Iadd.FNK EX.sub.2X.sub.3X.sub.4AX.sub.6X.sub.7EIX.sub.10X.sub.11LPNLX.sub.16X.sub.17X.sub.18QX.sub.20.Iaddend. .Iadd.X.sub.21AFIX.sub.25X.sub.26LX.sub.25X.sub.29X.sub.30PX.sub.32QSX.sub.35X.sub.36LLX.sub.39E.Iaddend. .Iadd.AKKLX.sub.45X.sub.46X.sub.47Q AP,.Iaddend. wherein each X.sub.y is as defined in claim 1.
6. A fusion polypeptide comprising a polypeptide according to claim 1 as a moiety.
7. A polynucleotide encoding a polypeptide according to claim 1 or fusion polypeptide according to claim 6.
8. A population of polypeptide variants based on a common scaffold, each polypeptide in the population comprising an amino acid sequence selected from: i) TABLE-US-00024 .[.(SEQ ID NO: 55).]. .[.EX.sub.2X.sub.3X.sub.4AX.sub.6X.sub.7EIX.sub.10 X.sub.11LPNLX.sub.16X.sub.17X.sub.18QX.sub.20.]. .[.X.sub.21AFIX.sub.25X.sub.26LX.sub.28X.sub.29X.sub.30 PX.sub.32QSX.sub.35X.sub.36LLX.sub.39E.]. .[.AKKLX.sub.45X.sub.46X.sub.47Q,.]. .Iadd.(SEQ ID NO: 55).Iaddend. .Iadd.EX.sub.2X.sub.3X.sub.4AX.sub.6X.sub.7EIX.sub.10X.sub.11LPNLX.sub.16X.sub.17X.sub.18QX.sub.20.Iaddend. .Iadd.X.sub.21AFIX.sub.25X.sub.26LX.sub.28X.sub.29X.sub.30PX.sub.32QSX.sub.35X.sub.36LLX.sub.39E.Iaddend. .Iadd.AKKLX.sub.45X.sub.46X.sub.47Q,.Iaddend. wherein each of X.sub.2, X.sub.3, X.sub.4, X.sub.6, X.sub.7, X.sub.10, X.sub.11, X.sub.17, X.sub.18, X.sub.20, X.sub.21, X.sub.25 and X.sub.28 independently corresponds to any amino acid residue; and wherein, independently of each other, X.sub.16 is selected from N and T; X.sub.26 is selected from K and S; X.sub.29X.sub.30PX.sub.32 is selected from DDPS .Iadd.(SEQ ID NO: 74) .Iaddend.and RQPE .Iadd.(SEQ ID NO: 75).Iaddend.; X.sub.35 is selected from A and S; X.sub.36 is selected from E and N; X.sub.39 is selected from A, C and S; X.sub.45 is selected from E.[., N.]. and S; X.sub.46 is selected from D, E and S.[., provided that X.sub.46 is not D when X.sub.45 is N.].; X.sub.47 is selected from A and S; and ii) an amino acid sequence which has at least 91% identity to the sequence defined in i), provided that X.sub.46 is not D .Iadd.or E .Iaddend.when X.sub.45 is N.Iadd., provided that for i) and ii) X.sub.10 is not D when X.sub.20 is W for a polypeptide capable of binding human complement component 5.Iaddend..
9. The population according to claim 8, which comprises at least 1×10.sup.4 unique polypeptide molecules.
10. A population of polynucleotides, characterized in that each member thereof encodes a member of a population of polypeptides according to claim 8.
11. A composition comprising a polypeptide population according to claim 8 and a polynucleotide population according to claim 10, wherein each member of said population of polypeptides is physically or spatially associated with the polynucleotide encoding that member via means for genotype-phenotype coupling.
12. The composition according to claim 11, wherein said means for genotype-phenotype coupling comprises a phage display system.
13. A method for selecting a desired polypeptide having an affinity for a predetermined target from a population of polypeptides, comprising the steps: (a) providing a population of polypeptides according to claim 8; (b) bringing the population of polypeptides into contact with the predetermined target under conditions that enable specific interaction between the target and at least one desired polypeptide having an affinity for the target; and (c) selecting, on the basis of said specific interaction, the at least one desired polypeptide from the remaining population of polypeptides.
14. A method for isolating a polynucleotide encoding a desired polypeptide having an affinity for a predetermined target, comprising the steps: selecting said desired polypeptide and the polynucleotide encoding it from a population of polypeptides using the method according to claim 13; and isolating the thus separated polynucleotide encoding the desired polypeptide.
15. A method for identifying a desired polypeptide having an affinity for a predetermined target, comprising the steps: isolating a polynucleotide encoding said desired polypeptide using the method according to claim 14; and sequencing the polynucleotide to establish by deduction the amino acid sequence of said desired polypeptide.
16. A method for selecting and identifying a desired polypeptide having an affinity for a predetermined target from a population of polypeptides, comprising the steps: (a) synthesizing each member of a population of polypeptides according to claim 8 on a separate carrier or bead; (b) selecting or enriching the carriers or beads based on the interaction of the polypeptide with the predetermined target; and (c) identifying the polypeptide by protein characterization methodology.
.Iadd.17. The polypeptide according to claim 5, which comprises the amino acid sequence of SEQ ID NO: 56..Iaddend.
.Iadd.18. The polypeptide according to claim 5, which comprises the amino acid sequence of SEQ ID NO: 57..Iaddend.
Description
BRIEF DESCRIPTION OF THE FIGURES
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EXAMPLES
(10) The following Examples disclose novel Z variant polypeptides exhibiting improved stability. Herein, the properties of Z variant polypeptides based on previous generations of scaffolds were compared with Z variant polypeptides based on the scaffold disclosed herein.
Comparative Example 1
Stability Test of Known C5 Binding Z Variant
(11) A C5 binding Z variant designated PSI0242 (SEQ ID NO:1) was formulated in 25 mM NaP/125 mM NaCl pH 7.0 and subjected to an accelerated stability study for 2 weeks at 37° C. The stability was measured by the appearance of new variants after the stability testing by SDS-PAGE and Reversed Phase HPLC (RPC). In both analyses, the initial sample and the one subjected to the stability study were run in parallel. For the SDS-PAGE, 7.5 μg protein was loaded into each well. The RPC was run on an Agilent 1100 HPLC using a Mobile Phase A consisting of 0.1% trifluoroacetic acid (TFA) in water, and a Mobile Phase B consisting of 0.1% TFA/45% MeOH/45% isopropylamine (IPA)/10% water.
(12) The results show that new forms of the protein were formed during incubation, visualized as bands in SDS-PAGE (
(13) Positions 1-60 in SEQ ID NO:1 correspond to the polypeptide Z06175a, previously disclosed in WO2013/126006 as SEQ ID NO:753.
Example 2
Stability Test of Modified C5 Binding Polypeptides and Compounds
(14) Modified C5 binding polypeptides and compounds were synthesized and purified essentially as described in WO2013/126006.
(15) Briefly, DNA encoding C5 binding Z variants were E. coli codon optimized and synthesized by GeneArt, GmbH. The synthetic genes representing the new C5 binding Z variants were subcloned and expressed in E. coli.
(16) Intracellularly expressed Z variants were purified using conventional chromatography methods. Homogenization and clarification was performed by sonication followed by centrifugation and filtration. Anion exchange chromatography was used as capture step. Further purification was obtained by hydrophobic interaction chromatography. The purifications were executed at acidic conditions (pH 5.5). Polishing and buffer exchange was performed by size exclusion chromatography.
(17) The purified proteins were formulated in 25 mM NaP/125 mM NaCl pH 7.0 and subjected to an accelerated stability study for 2 weeks at 37° C. The stability was measured by the appearance of new variants after the stability testing by SDS-PAGE and Reversed Phase HPLC (RPC). In both analyses, the initial sample and the one subjected to the stability study were run in parallel. For the SDS-PAGE, 7.5 μg protein was loaded into each well. An example of a resulting gel is shown in
(18) The RPC was run on an Agilent 1100 HPLC using a Mobile Phase A consisting of 0.1% trifluoroacetic acid (TFA) in water, and a Mobile Phase B consisting of 0.1% TFA/45% MeOH/45% isopropylamine (IPA)/10% water. An example of a resulting chromatogram for SEQ ID NO:5 is shown in
(19) The results of the stability testing are summarized in Table 1.
(20) TABLE-US-00007 TABLE 1 Stability of Z variant polypeptides after 2 weeks of incubation at 37° C. Results from SDS-PAGE and HPLC are compared. SEQ SDS- ID PAGE RPC Main peak (% of RPC NO: Designation bands prepeaks total protein) postpeaks 1 PSI0242 2 2 57 1 4 PSI0332 2 1 57 1 5 PSI0334 1 1 73 0 6 PSI0335 2 2 57 1 7 PSI0336 2 2 57 1 8 PSI0337 2 2 57 1 9 PSI0339 2 2 57 1 10 PSI0340 2 2 67 1 11 PSI0369 2 1 90 1 12 PSI0377 1 0 77 0 13 PSI0378 1 0 89 0 14 PSI0379 1 0 88 0 15 PSI0381 1 0 87 0 16 PSI0383 1 0 91 0 22 PSI0400 1 0 91 0 23 PSI0410 1 1 72 1 24 PSI0403 1 1 77 1 25 PSI0404 1 1 88 0
(21) It can be concluded from Table 1 that certain modified C5 binding polypeptides or compounds have improved properties, such as increased stability, when compared with PSI0242. Such improved C5 binding polypeptides or compounds include PSI0334 (SEQ ID NO:5), PSI0340 (SEQ ID NO:10), PSI0369 (SEQ ID NO:11), PSI0377 (SEQ ID NO:12), PSI0378 (SEQ ID NO:13), PSI0379 (SEQ ID NO:14), PSI0381 (SEQ ID NO:15), PSI0383 (SEQ ID NO:16), PSI0400 (SEQ ID NO:22), PSI0410 (SEQ ID NO:23), PSI0403 (SEQ ID NO:24) and PSI0404 (SEQ ID NO:25). Six of the mentioned variants (SEQ ID NO:5, 12, 13, 14, 16 and 22) have in common that the amino acid residues in positions 52-53 have been substituted from ND (cf. PSI0242) to SE. In SEQ ID NO:15, the corresponding substitution is from ND to ES. In SEQ ID NO:24 only the amino acid residue in position 53 has been substituted from D to E, while in SEQ ID NO:25 the amino acid residue in position 52 has been substituted from N to S.
Example 3
Binding of Modified Compounds to Human C5
(22) Human serum albumin was immobilized to Amine Reactive 2.sup.nd generation (AR2G) Dip and Read Biosensors (Pall Life sciences (ForteBio) Cat #18-5092) by amine coupling. PSI0242 (SEQ ID NO:1; 1 μM) and modified C5 binding compounds (1 μM) in read buffer (HBS-EP Buffer [10 mM HEPES pH 7.4, 150 mM NaCl, 3 mM EDTA, 0.005% Surfactant P20], GE Healthcare, cat. no. BR100188) were loaded, each onto a separate sensor with HSA, for 120 seconds followed by a base line recording for 60 seconds in read buffer before being subjected to human C5 (Quidel, cat. no. A403) at concentrations ranging from 0.79 nM to 25 nM in read buffer with a regeneration cycle and a base line recording between each concentration. Regeneration conditions for the sensors were 10 mM Glycine, pH 2 (three pulses with 30 seconds and running buffer for 60 seconds). Each spectrogram was reference subtracted against that of an analogous construct containing an albumin binding domain (SEQ ID NO:2) but without the C5 binding capacity. The data were analyzed according to Langmuir 1:1 model using ForteBio Analysis 7.1 (Pall Life sciences (ForteBio) kinetics software).
(23) The relative K.sub.D of the interaction of PSI0242 (SEQ ID NO; 1) with C5 is shown in Table 2. The K.sub.D of PSI0242 (SEQ ID NO:1) varied from 1-3 nM in different runs.
(24) The results in Table 2 indicate that C5 binding compounds according to the present disclosure have a binding capacity to human C5 which is similar to that of the polypeptide PSI0242 (SEQ ID NO:1) disclosed in WO2013/126006.
(25) TABLE-US-00008 TABLE 2 K.sub.D value of the interaction of SEQ ID NO: 5, 13, 15 and 16 with C5 compared to K.sub.D value of C5 interaction with SEQ ID NO: 1 SEQ ID NO: Designation Rel. K.sub.D 1 PSI0242 1.0 5 PSI0334 1.1 13 PSI0378 1.3 15 PSI0381 23 16 PSI0383 2.1
Example 4
Stability of Chemically Synthesized C5 Binding Polypeptide
(26) A chemically synthesized PSI0400 (SEQ ID NO:22) was ordered from BACHEM AG. The stability of the polypeptide was tested according to the same methodology as in Example 2. The results of the stability testing are shown in Table 3.
(27) TABLE-US-00009 TABLE 3 Stability of the chemically produced C5 binding polypeptide PSI0400 (SEQ ID NO: 22) after 2 weeks of incubation SEQ Main peak ID SDS-PAGE RPC (% of RPC NO Designation bands prepeaks total protein) postpeaks 22 PSI0400 1 0 91 0
The stability of PSI0400 was comparable to the same polypeptide produced in E. coli in Example 2.
(28) The integrity of the fold of PSI0400 (SEQ ID NO:22) was compared to a recombinant C5 binding polypeptide (PSI0257, SEQ ID NO:26), produced in accordance with the methods of Example 2, using far UV circular dichroism (CD) spectra.
(29) The CD spectra were recorded by a J-720 CD spectropolarimeter (Jasco, Japan). The samples were diluted to 0.17 mg/ml protein concentration using Pi buffer (5 mM Na—K—PO.sub.4, pH 7.0). A CD spectrum of Pi buffer was firstly recorded, then spectra were recorded for each of the samples and lastly for the Pi buffer again. As the two buffer spectra coincide, the firstly recorded spectrum was used as the buffer spectrum. The buffer spectrum was smoothened using the Savitzky-Golay procedure with convolution width of 25. The other spectra were smoothened according to the same procedure with a convolution width of 15. The smoothened buffer spectrum was then subtracted from each of the other smoothened spectra. The CDNN program was used to estimate the secondary content of the proteins and the resulting estimations are presented in Table 4. The results showed that neither the two amino acid substitutions at position 52 and 53 nor the polypeptide production by chemical synthesis influence the secondary structure content of the chemically synthesized polypeptide. The integrity of the secondary structure content was compared to the recombinantly produced PSI0257 (SEQ ID NO:26).
(30) TABLE-US-00010 TABLE 4 Comparison of secondary structure content for two C5 binding polypeptides as determined by CD SEQ ID NO: 26 SEQ ID NO: 22 Helix 63% 69% Antiparallel 3% 2% Parallel 3% 3% Beta-Turn 13% 12% Rndm. Coil 13% 11%
Example 5
Binding of Modified Z Variants and Polypeptides to Human C5
(31) The binding affinity of the C5 binding compounds PSI0242 (SEQ ID NO:1), PSI0340 (SEQ ID NO:10), PSI0378 (SEQ ID NO:13), and PSI0410 (SEQ ID NO:23) and the C5 binding polypeptide PSI0400 (SEQ ID NO:22) for human C5 was analyzed using a Biacore T200 instrument (GE Healthcare). Human C5 (Quidel, cat. no. A403) was coupled to a CM5 sensor chip (900 RU) using amine coupling chemistry according to the manufacturer's protocol. The coupling was performed by injecting hC5 at a concentration of 7.5 μg/ml in 10 mM Na-acetate buffer pH 5 (GE Healthcare). The reference cell was treated with the same reagents but without injecting human C5. Binding of the C5 polypeptide and compounds to immobilized hC5 was studied with the single cycle kinetics method, in which five concentrations of sample, typically 25, 12.5, 6.25, 3.12 and 1.56 nM in HBS-EP buffer were injected one after the other at a flow rate of 30 μl/min at 25° C. in the same cycle without regeneration between injections. Data from the reference cell were subtracted to compensate for bulk refractive index changes. In most cases, an injection of HBS-EP was also included as control so that the sensorgrams were double blanked. The surfaces were regenerated in HBS-EP buffer. Kinetic constants were calculated from the sensorgrams using the Langmuir 1:1 analyte model of the Biacore T200 Evaluation Software version 1.0. The resulting K.sub.D values of the interactions are presented in Table 5.
(32) TABLE-US-00011 TABLE 5 K.sub.D value of the interaction of SEQ ID NO: 10, 13, 22 and 23 with C5 compared to K.sub.D value of C5 interaction with SEQ ID NO: 1 SEQ ID NO: Designation K.sub.D (nM) 1 PSI0242 1.3 10 PSI0340 2.5 13 PSI0378 2.1 22 PSI0400 0.53 23 PSI0410 1.3
(33) The present data show that the stability-enhancing amino acid substitutions do not have any significant negative effect on the ability of the molecules to bind to C5, and thus do not influence their biological activities.
Example 6
Inhibition of Hemolysis
(34) For studies of classical complement pathway function and inhibition thereof by the C5 binding compounds PSI0378 (SEQ ID NO:13) and PSI0410 (SEQ ID NO:23), and C5 binding polypeptide PSI0400 (SEQ ID NO:22), sheep erythrocytes were prepared from fresh sheep whole blood in Alsever's solution (Swedish National Veterinary Institute). The erythrocytes were thereafter treated with rabbit anti-sheep erythrocyte antiserum (Sigma) to become antibody sensitized sheep erythrocytes (EA). The whole process was conducted under aseptic conditions. All other reagents were from commercial sources.
(35) The in vitro assay was run in 96-well U-form microtiter plate by consecutive additions of a test protein, a complement serum and EA suspension. The final concentrations of all reagents, in a total reaction volume of 50 μl per well and at pH 7.3-7.4, were: 0.15 mM CaCl 2; 0.5 mM MgCl 2; 3 mM NaN 3; 138 mM NaCl; 0.1% gelatin; 1.8 mM sodium barbital; 3.1 mM barbituric acid; 5 million EA; complement protein C5 serum at suitable dilution, and C5 binding compound or polypeptide at desired concentrations.
(36) The C5 binding compounds and polypeptide were pre-incubated with the above described complement serum for 20 min on ice prior to starting the reaction by the addition of EA suspension. The hemolytic reaction was allowed to proceed at 37° C. under conditions of agitation for 45 min and was then optionally ended by addition of 100 μl ice-cold saline containing 0.02% Tween 20. The cells were centrifuged to the bottom of the vial and the upper portion, corresponding to 100 μl supernatant, was transferred to a transparent microplate having half-area and flat-bottom wells. The reaction results were analyzed as optical density using a microtiter plate reader at a wavelength of 415 nm.
(37) A control sample (PSI0242, SEQ ID NO:1) and vehicle were included in each plate to define values for uninhibited and fully inhibited reactions, respectively. These values were used to calculate the % inhibition of the complement hemolysis at any given sample concentration. The inhibitory potencies (IC 50-values) of tested C5 binding compounds and polypeptide were defined by applying the same assay in the presence of a controlled concentration of human C5 added to C5 depleted serum. For highly potent inhibitors (low nanomolar to sub-nanomolar), a final C5 concentration of the reaction mixture was controlled at 0.1 nM, which was optionally established by using C5 depleted or deficient sera. The results are presented below in Table 6.
(38) TABLE-US-00012 TABLE 6 The inhibitory capacity of C5-binding compounds and polypeptide SEQ ID NO: Designation Potency (%) IC 50 (nM) 1 PSI0242 100 0.47 13 PSI0378 83 0.58 22 PSI0400 — 4 23 PSI0410 107 0.49
(39) The results from the hemolysis assay show that the improved C5 binding compounds PSI0378 (SEQ ID NO:13) and PSI0410 (SEQ ID NO:23) do not significantly differ from the reference compound PSI0242 (SEQ ID NO:1) in terms of function. The C5 binding polypeptide PSI0400 (SEQ ID NO:22) is functional in the assay and since it does not comprise an albumin binding domain, the results cannot be directly compared to those of the reference compound.
Example 7
Binding to Human Albumin
(40) For assessment of the affinity of the C5 binding compounds for albumin, a human albumin ELISA was used, utilizing recombinant human albumin as coating (Novozymes) and commercially available antibodies from Affibody AB (primary) and DakoCytomation (detecting). A method standard prepared from PSI0242 (SEQ ID NO:1) and comprising a C5 binding polypeptide and an albumin binding domain of streptococcal protein G, was used for quantification of samples.
(41) A 96-well microplate was coated with recombinant human albumin. The plate was then washed with phosphate buffered saline containing 0.05% Tween 20 (PBST) and blocked for 1-2 hours with 1% casein in PBS. After a plate wash, the standard, method controls, control sample and test samples are added to the plate. After incubation for 2 hours, unbound material was removed by a wash. A goat anti-AFFIBODY IgG (Affibody AB, cat no. 20.1000.01.0005) was added to the wells and the plate was incubated for 1.5 hours to allow binding to the bound C5 binding compounds. After a wash, rabbit anti-goat IgG HRP (DakoCytomation) was allowed to bind to the goat antibodies for 1 h. After a final wash, the amount of bound HRP was detected by addition of TMB substrate (3,3′,5,5′-tetramethylbenzidine), which was converted to a blue product by the enzyme. Addition of 1 M hydrochloric acid after 30 minutes stopped the reaction and the color of the well contents changed from blue to yellow. The absorbance at 450 nm was measured photometrically, using the absorbance at 650 nm as a reference wavelength. The color intensity was proportional to the amount of PSI0242 (SEQ ID NO:1) and the sample concentrations were determined from the standard curve.
(42) The C5 binding compounds comprising a derivative of the albumin binding domain from streptococcal protein G (ABD) were shown to be capable of binding to human albumin. Data is presented in Table 7.
(43) TABLE-US-00013 TABLE 7 Summary of results from ELISA SEQ ID NO: Designation % of total protein content 1 PSI0242 103 13 PSI0378 85 23 PSI0410 150
(44) The interpretation of the assay is that both the investigated C5 binding polypeptides with improved stability maintain their ability to bind human serum albumin.
Example 8
Three Month Stability Test of C5 Binding Z Variants and Polypeptides
(45) The C5 binding variants and polypeptides that showed an improved stability compared to PSI0242 in the 2 week stability test at 37° C. (Example 2) were subjected to a longer 3 month stability test at 37° C. The setup of the stability test and the analysis by RPC was as described in Example 2. The evaluation of the stability was made by measuring the main peak of the chromatogram and calculating the corresponding percentage of the total protein content. The data from Example 2 is included in Table 8 below to make the interpretation easier.
(46) TABLE-US-00014 TABLE 8 Stability of C5 binding polypeptides and compounds after 3 months of incubation at 37° C. 2 weeks, 37° C. 3 months, 37° C. Main peak Main peak SEQ ID NO: Designation (% of total protein) (% of total protein) 5 PSI0334 73 16 13 PSI0378 89 59 14 PSI0379 88 58 15 PSI0381 87 46 16 PSI0383 91 59 23 PSI0410 72 16 24 PSI0403 77 35 25 PSI0404 88 46
(47) C5 binding compounds comprising the amino acid substitutions ND to SE in positions 52-53 (SEQ ID NO:13, 14, and 16) compared to PSI0242 showed a higher proportion of protein in the original form after 3 months at 37° C. than PSI0242 (SEQ ID NO:1), after 2 weeks under the same conditions (see Table 1). The other tested compounds also display an increased stability compared to the PSI0242.
Example 9
Generation, Stability Study and Binding Assessment of Scaffold-Modified Polypeptides with Specificity for Different Targets
(48) Generation of Scaffold-Modified Polypeptides with Specificity for Different Targets:
(49) Polypeptide variants comprising the new scaffold described herein are generated by taking Z variant polypeptides with specificity for different targets, and performing site-directed mutagenesis at selected positions within the scaffold. The new molecules may, alternatively, be made by chemical synthesis of the entire molecule or by using other molecular biology methods, known to a person skilled in the art, to graft a binding motif of a Z variant polypeptide onto the new scaffold.
(50) Comparative Stability Study of Scaffold-Modified Polypeptides with Specificity for Different Targets:
(51) For each new polypeptide created as described above, the stability is compared to the stability of the original polypeptide or another comparable polypeptide. The polypeptides are subjected to different conditions, such as formulation in [25 mM NaP, 125 mM NaCl, pH 7.0] and incubation at 37° C. for 2 weeks as described in Example 2 and/or for 3 months as described in Example 8. The stability is assessed by analyzing the appearance of new variants by performing SDS-PAGE and RPC analyses as described in Example 2.
(52) Polypeptides with the introduced modifications in scaffold positions are expected to show improved stability in similar to the results presented in Example 2 and Example 12.
(53) Binding Assessment of Scaffold-Modified Polypeptides:
(54) Polypeptides which have shown improved stability properties are further assessed in terms of preserved binding capacities to its target after introduction of alterations in the scaffold. Binding studies are performed on a biosensor instrument, or any other instrument known to the person skilled in the art and measuring the interaction between two or more molecules. For example, the target molecule, or a fragment thereof, is immobilized on a sensor chip of the instrument, and the sample containing the polypeptide to be tested is passed over the chip. Alternatively, the polypeptide to be tested is immobilized on a sensor chip of the instrument, and a sample containing the predetermined target, or a fragment thereof, is passed over the chip. The binding affinity may be tested in an experiment in which samples of the polypeptide are captured on antibody-coated ELISA plates and biotinylated predetermined target, or a fragment thereof, is added, followed by streptavidin conjugated HRP. TMB substrate is added and the absorbance at 450 nm is measured using a multi-well plate reader, such as Victor.sup.3 (Perkin Elmer). If a quantitative measure is desired, for example to determine the EC50 value (the half maximal effective concentration) for the interaction, ELISA may also be used. The response of the polypeptide against a dilution series of the predetermined target, or a fragment thereof, is measured using ELISA as described above. The results obtained by such experiments and EC50 values may be calculated from the results using for example GraphPad Prism 5 and non-linear regression. If the polypeptide contains an albumin binding domain, the effect on albumin binding will be assessed likewise, as described in Example 3 or as described in Example 7.
(55) Polypeptides having the scaffold mutations described herein and, in addition, similar or improved binding capacities for its target, are considered to be better candidates for further development into e.g. biopharmaceutical products.
Example 10
Generation of Scaffold-Modified Polypeptides with Specificity for Four Different Targets
(56) Polypeptide variants comprising the new scaffold described herein were generated by taking Z variant polypeptides with specificity for different targets, and performing site-directed mutagenesis at selected positions within the scaffold. Amino acid substitutions at scaffold positions in the polypeptide variants Z02891 (SEQ ID NO:27), targeting the human epidermal growth factor receptor 2 (HER2); Z15805 (SEQ ID NO:30), targeting the platelet-derived growth factor receptor beta (PDGF-Rβ); Z10103 (SEQ ID NO:33), targeting the neonatal Fc receptor (FcRn); and Z09782 (SEQ ID NO:36), targeting the carbonic anhydrase IX (CAIX), are specified in Table 9.
(57) TABLE-US-00015 TABLE 9 Original and inventive polypeptides produced and analyzed in terms of stability and function in the Examples described below SEQ Amino acid Original vs ID NO Designation Target substitutions inventive 27 Z02891 HER2 — Original 28 Z17341 HER2 N52S, D53E Inventive 29 Z17342 HER2 D36R, D37Q, S39E, Inventive N52S, D53E 30 Z15805 PDGF-Rβ — Original 31 Z17343 PDGF-Rβ N52S, D53E Inventive 32 Z17344 PDGF-Rβ D36R, D37Q, S39E, Inventive N52S, D53E 33 Z10103 FcRn — Original 34 Z17347 FcRn N52S, D53E Inventive 35 Z17348 FcRn D36R, D37Q, S39E, Inventive N52S, D53E 36 Z09782 CAIX — Original 37 Z17351 CAIX N52S, D53E Inventive 38 Z17352 CAIX D36R, D37Q, S39E, Inventive N52S, D53E 39 Z17355 CAIX D53E Inventive 40 Z17357 CAIX D36R, D37Q, Inventive S39E, D53E 41 Z17359 CAIX N52S Inventive 42 Z17360 CAIX D36R, D37Q, Inventive S39E, N52S
(58) All variants were cloned with an N-terminal 6× Histidine-tag (His.sub.6) and obtained constructs encoded polypeptides in the format MGSSHHHHHHLQ-[Z#####] (SEQ ID NO: 73). Mutations were introduced in the plasmids of the inventive polypeptides using overlapping oligonucleotide primer pairs encoding the desired amino acid substitutions and by applying established molecular biology techniques. The correct plasmid sequences were verified by DNA sequencing.
(59) E coli (strain T7E2) cells (GeneBridge) were transformed with plasmids containing the gene fragments encoding the original and the inventive polypeptides. The cells were cultivated at 37° C. in TSB-YE medium supplemented with 50 μg/ml kanamycin and protein expression was subsequently induced by addition of IPTG. Pelleted cells were disrupted using a FASTPREP-24 homogenizer (Nordic Biolabs) and cell debris was removed by centrifugation. Each supernatant containing the Z variant as a His.sub.6-tagged protein was purified by immobilized metal ion affinity chromatography (IMAC) using His GRAVITRAP columns (GE Healthcare) according to the manufacturers instructions. Purified Z variants were buffer exchanged to phosphate-buffered saline (PBS; 1.47 mM KH.sub.2PO.sub.4, 8.1 mM Na.sub.2HPO.sub.4, 137 mM NaCl, 2.68 mM KCl, pH 7.4) using PD-10 desalting columns (GE Healthcare). The correct identity of each polypeptide was verified by SDS-PAGE and HPLC-MS.
Example 11
Circular Dichroism Spectroscopy Analysis of Scaffold-Modified Polypeptides
(60) Circular dichroism (CD) analysis was carried out to determine the melting temperatures (Tm) and assess potential changes in the secondary structure of the inventive polypeptides as a result of the amino acid substitutions.
(61) Purified His.sub.6-tagged Z variants were diluted to 0.5 mg/ml in PBS. For each diluted Z variant, a CD spectrum at 250-195 nm was recorded at 20° C. A variable temperature measurement (VTM) was performed to determine the Tm. In the VTM, the absorbance was measured at 221 nm while the temperature was raised from 20 to 90° C., with a temperature slope of 5° C./min. After the VTM, a second CD spectrum at 250-195 nm was recorded at 20° C. The CD measurements were performed on a Jasco J-810 spectropolarimeter (Jasco Scandinavia AB) using a cell with an optical path-length of 1 mm.
(62) The Tm of each respective polypeptide as determined from the midpoint of the transition in the CD signal vs. temperature plot is shown in Table 10. All mutated polypeptides showed preserved alphahelical structure and refolded reversibly or nearly reversibly even after heating to 90° C. A selected set of CD spectra are shown in
(63) TABLE-US-00016 TABLE 10 Melting temperatures for original and invenitve Z variants determined by CD SEQ ID NO Designation Target Tm (° C.) Original vs inventive 27 Z02891 HER2 70 Original 28 Z17341 HER2 66 Inventive 29 Z17342 HER2 62 Inventive 30 Z15805 PDGF-Rβ 48 Original 31 Z17343 PDGF-Rβ 46 Inventive 32 Z17344 PDGF-Rβ 42 Inventive 33 Z10103 FcRn 48 Original 34 Z17347 FcRn 50 Inventive 35 Z17348 FcRn 44 Inventive 36 Z09782 CAIX 43 Original 37 Z17351 CAIX 40 Inventive 38 Z17352 CAIX 45 Inventive 39 Z17355 CAIX 43 Inventive 40 Z17357 CAIX 47 Inventive 41 Z17359 CAIX 41 Inventive 42 Z17360 CAIX 46 Inventive
Example 12
Comparative Stability Study of Scaffold-Modified Polypeptides with Specificity for Four Different Targets
(64) For each new polypeptide created as described in Example 10, the stability was compared to the stability of the original polypeptide. The polypeptides, formulated in PBS pH 7.4, were diluted to 1 mg/ml and 200 μl aliquotes were incubated at 37° C. for 2 weeks. Samples collected prior to and after the stability test were analyzed by SDS-PAGE using 10% Bis-Tris NuPAGE gels (Invitrogen) and by loading 5 μg protein into each well. The resulting Coomassie blue stained gels are shown in
(65) All polypeptides with modifications introduced in scaffold positions as outlined in Table 9 showed improved stability compared to the respective original polypeptide. In samples of the original polypeptides a second band was visible on the gel just above the main band. A corresponding second band was not visible in the samples of the inventive polypeptides with the substitution D53E and/or N52S. This is in analogy with results presented in Examples 2 and 4. Thus, the stabilizing effect observed for the inventive scaffold mutations appears to be a general effect regardless of the target specificity of the Z variant or polypeptide comprising said Z variant. Polypeptides with the substitutions D53E and/or N52S, alone or combined with the substitutions D36R, D37Q and S39E, showed similar profiles on the SDS-PAGE gel. The substitution D53E alone or in combination with the substitutions D36R, D37Q and S39E appeared to reduce the amount of the species with an alternative confirmation observed as a second band on the SDS-PAGE gel, but could not completely prevent the formation of this species.
Example 13
Binding Assessment of Scaffold-Modified Polypeptides
(66) A set of polypeptides showing improved stability properties in Example 12 were further assessed in terms of preserved binding capacities to their targets after introduction of alterations in the scaffold, as well as after having been subjected to the stability test, i.e. incubated at 37° C. for 2 weeks. Comparative kinetic constants (k.sub.on and k.sub.off) and affinities (K.sub.D) were determined using a Biacore 2000 instrument. The target proteins human HER2-Fc (R&D Systems, cat. no. 1129-ER-050), human PDGF-Rβ (R&D Systems, cat. no. 385-PR-100/CF), human FcRn (Biorbyt, cat. no. orb 84388) and human CAIX (R&D Systems, cat. no. 2188-CA), respectively, were immobilized on the carboxylated dextran layer surface of CM5 chips (GE Healthcare). The immobilization was performed using amine coupling chemistry according to the manufacturer's protocol and using HBS-EP as running buffer. One flow cell surface on the chip was activated and deactivated for use as blank during analyte injections. The immobilization level of target protein on the respective surface was approximately 850 RU for HER2, 2200 RU for PDGF-Rβ, 750 for FcRn and 580 RU for CAIX.
(67) HBS-EP (HER2, PDGF-Rβ, CAIX) or a pH 6.0 Na.sub.2HPO.sub.4/citric acid buffer (126 mM Na.sub.2HPO.sub.4, 37 mM citric acid) (FcRn) was used as running buffer and the flow rate was 30 μl/min in the binding experiments performed at 25° C. as further described below.
(68) The Z variants Z02891 (SEQ ID NO:27), Z17341 (SEQ ID NO:28), and Z17342 (SEQ ID NO:29) targeting HER2 were diluted in running buffer to final concentrations of 3.33, 10, 30 and 90 nM and injected for 5 minutes, followed by 30 minutes of dissociation in running buffer. Regeneration by four pulses alternating between 10 mM HCl and 10 mM NaOH followed by 5 min equilibration in running buffer was applied after each analyte injection.
(69) The Z variants Z15805 (SEQ ID NO:30), Z17343 (SEQ ID NO:31), and Z17344 (SEQ ID NO:32) targeting PDGF-R13 were diluted in running buffer to final concentrations of 6.67, 20, 60 and 180 nM and injected for 5 minutes, followed by 20 minutes of dissociation in running buffer. Regeneration by three pulses of 10 mM NaOH followed by 5 min equilibration in running buffer was applied after each analyte injection.
(70) The Z variants Z10103 (SEQ ID NO:33), Z17347 (SEQ ID NO:34), and Z17348 (SEQ ID NO:35) targeting FcRn were diluted in running buffer to final concentrations of 3.33, 10 and 30 nM and injected for 3 minutes, followed by 15 minutes of dissociation in running buffer. Regeneration by three pulses of HBS-EP followed by 10 min equilibration in running buffer was applied after each analyte injection.
(71) The Z variants Z09782 (SEQ ID NO:36), Z17351 (SEQ ID NO:37), Z17355 (SEQ ID NO:39), and Z17359 (SEQ ID NO:41) targeting CAIX were diluted in running buffer to final concentrations of 30, 90 and 270 nM and injected for 5 minutes, followed by 15 minutes of dissociation in running buffer. Regeneration by three pulses of 10 mM glycin-HCl pH 3.0 followed by 5 min equilibration in running buffer was applied after each analyte injection.
(72) Kinetic constants were calculated from the sensorgrams using the Langmuir 1:1 model (HER2, FcRn, CAIX) or the 1:1 binding with mass transfer model (PDGF-Rβ) of the BiaEvaluation software 4.1 (GE Healthcare). Curves of the blank surface were subtracted from the curves of the ligand surfaces and the data from the buffer cycles were subtracted from the data of the test-sample cycles to correct for any drift in signal.
(73) The comparative kinetic constants for Z variants binding to its target molecule are shown in Table 11 and sensorgrams for a subset of the analyzed interactions are shown in
(74) TABLE-US-00017 TABLE 11 Comparative kinetic analysis of original and inventive polypeptides SEQ Original vs K.sub.DInv/ K.sub.D(2w)/ ID NO: Test sample Inventive k.sub.a (Ms.sup.−1) k.sub.d (s.sup.−1) K.sub.D (M)* K.sub.DOrig** K.sub.D(0)*** HER2 binding Z variants 27 Z02891 (0) Original 1.33 × 10.sup.6 7.10 × 10.sup.−5 5.4 × 10.sup.−11 1.0 27 Z02891 (2w) Original 1.15 × 10.sup.6 7.19 × 10.sup.−5 6.2 × 10.sup.−11 1.0 1.17 28 Z17341 (0) Inventive 1.88 × 10.sup.6 8.35 × 10.sup.−5 4.5 × 10.sup.−11 0.83 28 Z17341 (2w) Inventive 2.06 × 10.sup.6 8.91 × 10.sup.−5 4.3 × 10.sup.−11 0.69 0.97 29 Z17342 (0) Inventive 8.94 × 10.sup.5 1.57 × 10.sup.−3 1.8 × 10.sup.−9 33 29 Z17342 (2w) Inventive 6.49 × 10.sup.5 1.50 × 10.sup.−3 2.3 × 10.sup.−9 37 1.31 PDGF-Rβ binding Z variants 30 Z15805 (0) Original 7.15 × 10.sup.6 1.39 × 10.sup.−3 1.9 × 10.sup.−10 1.0 30 Z15805 (2w) Original 5.81 × 10.sup.6 1.66 × 10.sup.−3 2.9 × 10.sup.−10 1.0 1.47 31 Z17343 (0) Inventive 4.80 × 10.sup.6 1.77 × 10.sup.−3 3.7 × 10.sup.−10 1.90 31 Z17343 (2w) Inventive 6.45 × 10.sup.6 1.71 × 10.sup.−3 2.3 × 10.sup.−10 0.93 0.72 32 Z17344 (0) Inventive 5.15 × 10.sup.7 6.16 × 10.sup.−2 1.2 × 10.sup.−9 6.19 32 Z17344 (2w) Inventive 5.62 × 10.sup.7 6.23 × 10.sup.−2 1.1 × 10.sup.−9 3.88 0.93 FcRn binding Z variants 33 Z10103 (0) Original 1.60 × 10.sup.6 4.56 × 10.sup.−3 2.9 × 10.sup.−9 1.0 33 Z10103 (2w) Original 3.15 × 10.sup.6 5.75 × 10.sup.−3 1.8 × 10.sup.−9 1.0 0.64 34 Z17347 (0) Inventive 1.18 × 10.sup.6 7.99 × 10.sup.−3 6.7 × 10.sup.−9 2.36 34 Z17347 (2w) Inventive 2.27 × 10.sup.6 8.79 × 10.sup.−3 3.9 × 10.sup.−9 2.13 0.57 35 Z17348 (0) Inventive 1.82 × 10.sup.6 1.00 × 10.sup.−2 5.5 × 10.sup.−9 1.93 35 Z17348 (2w) Inventive 1.28 × 10.sup.6 8.09 × 10.sup.−3 6.3 × 10.sup.−9 3.46 1.14 CAIX binding Z variants 36 Z09782 (0) Original 2.08 × 10.sup.5 1.46 × 10.sup.−3 7.0 × 10.sup.−9 1.0 36 Z09782 (2w) Original 1.40 × 10.sup.5 1.38 × 10.sup.−3 9.9 × 10.sup.−9 1.0 1.41 37 Z17351 (0) Inventive 1.51 × 10.sup.5 2.63 × 10.sup.−3 1.8 × 10.sup.−8 2.49 37 Z17351 (2w) Inventive 1.91 × 10.sup.5 2.86 × 10.sup.−3 1.5 × 10.sup.−8 1.52 0.86 39 Z17355 (0) Inventive 1.57 × 10.sup.5 1.23 × 10.sup.−3 7.9 × 10.sup.−9 1.12 39 Z17355 (2w) Inventive 1.16 × 10.sup.5 1.23 × 10.sup.−3 1.1 × 10.sup.−8 1.07 1.35 41 Z17359 (0) Inventive 1.68 × 10.sup.5 2.15 × 10.sup.−3 1.3 × 10.sup.−8 1.82 41 Z17359 (2w) Inventive 1.78 × 10.sup.5 2.33 × 10.sup.−3 1.3 × 10.sup.−8 1.32 1.02 *The K.sub.D values should not be regarded as absolute, as these were determined for comparative purposes and only included a limited number of sample concentrations. **Relative K.sub.D comparing the K.sub.D of respective inventive polypeptide with the K.sub.D of its original polypeptide (set to 1.0) either prior to (0) or after the stability test (2w) described in Example 12. ***Relative K.sub.D comparing the K.sub.D from (2w) with K.sub.D from (0) for each polypeptide pair identical in sequence.
Itemized List of Embodiments
(75) 1. Polypeptide comprising an amino acid sequence selected from: i)
(76) TABLE-US-00018 (SEQ ID NO: 55) EX.sub.2X.sub.3X.sub.4AX.sub.6X.sub.7EIX.sub.10 X.sub.11LPNLX.sub.16X.sub.17X.sub.18QX.sub.20 X.sub.21AFIX.sub.25X.sub.26LX.sub.28X.sub.29X.sub.30 PX.sub.32QSX.sub.35X.sub.36LLX.sub.39E AKKLX.sub.45X.sub.46X.sub.47Q, wherein each of X.sub.2, X.sub.3, X.sub.4, X.sub.6, X.sub.7, X.sub.10, X.sub.11, X.sub.17, X.sub.18, X.sub.20, X.sub.21, X.sub.25 and X.sub.28 independently corresponds to any amino acid residue; and wherein, independently of each other, X.sub.16 is selected from N and T; X.sub.26 is selected from K and S; X.sub.29X.sub.30PX.sub.32 is selected from DDPS and RQPE; X.sub.35 is selected from A and S; X.sub.36 is selected from E and N; X.sub.39 is selected from A, C and S; X.sub.45 is selected from E, N and S; X.sub.46 is selected from D, E and S, provided that X.sub.46 is not D when X.sub.45 is N; X.sub.47 is selected from A and S; and ii) an amino acid sequence which has at least 91% identity to the sequence defined in i), provided that X.sub.46 is not D when X.sub.45 is N.
(77) 2. Polypeptide according to item 1, wherein X.sub.16 is T.
(78) 3. Polypeptide according to item 1 or 2, wherein X.sub.26 is K.
(79) 4. Polypeptide according to any preceding item, wherein X.sub.29X.sub.30PX.sub.32 is DDPS.
(80) 5. Polypeptide according to item 1-3, wherein X.sub.29X.sub.30PX.sub.32 is RQPE.
(81) 6. Polypeptide according to any preceding item, wherein X.sub.35 is S.
(82) 7. Polypeptide according to any preceding item, wherein X.sub.36 is E.
(83) 8. Polypeptide according to any preceding item, wherein X.sub.39 is S.
(84) 9. Polypeptide according to any preceding item, wherein X.sub.45 is selected from E and S.
(85) 10. Polypeptide according to item 9, wherein X.sub.45 is E.
(86) 11. Polypeptide according to item 9, wherein X.sub.45 is S.
(87) 12. Polypeptide according to any preceding item, wherein X.sub.46 is selected from E and S.
(88) 13. Polypeptide according to item 12, wherein X.sub.46 is E.
(89) 14. Polypeptide according to item 12, wherein X.sub.46 is S.
(90) 15. Polypeptide according to item 12, wherein X.sub.46 is D.
(91) 16. Polypeptide according to any preceding item, provided that X.sub.46 is not D or E when X.sub.45 is N.
(92) 17. Polypeptide according to any preceding item, wherein X.sub.45X.sub.46 is selected from EE, ES, SE and SS.
(93) 18. Polypeptide according to item 17, wherein X.sub.45X.sub.46 is selected from ES and SE.
(94) 19. Polypeptide according to item 18, wherein X.sub.45X.sub.46 is ES.
(95) 20. Polypeptide according to item 18, wherein X.sub.45X.sub.46 is SE.
(96) 21. Polypeptide according to item 18, wherein X.sub.45X.sub.46 is SD.
(97) 22. Polypeptide according to any preceding item, wherein X.sub.47 is S.
(98) 23. Polypeptide according to any one of items 1-22, comprising additional amino acid residues.
(99) 24. Polypeptide according to item 23, comprising additional amino acid residues at the C-terminus of said polypeptide.
(100) 25. Polypeptide according to item 24, wherein the additional amino acid residues at the C-terminus of said polypeptide comprise AP.
(101) 26. Polypeptide according to item 23, comprising additional amino acid residues at the N-terminus of said polypeptide.
(102) 27. Polypeptide according to item 26, wherein the additional amino acid residues at the N-terminus of said polypeptide comprise AEAKYAK.
(103) 28. Polypeptide according to any one of items 23-27, wherein said additional amino acid residues are added for the purpose of binding, production, purification, stabilization, coupling or detection of the polypeptide.
(104) 29. Polypeptide according to any one of items 23-28, wherein said additional amino acid residues constitute one or more polypeptide domain(s).
(105) 30. Polypeptide according to item 29, 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.
(106) 31. Polypeptide according to any one of items 1-28, which comprises an amino acid sequence selected from:
(107) TABLE-US-00019 (SEQ ID NO: 56) YAK EX.sub.2X.sub.3X.sub.4AX.sub.6X.sub.7EIX.sub.10 X.sub.11LPNLX.sub.16X.sub.17X.sub.18QX.sub.20 X.sub.21AFIX.sub.25X.sub.26LX.sub.28X.sub.29X.sub.30 PX.sub.32QSX.sub.35X.sub.36LLX.sub.39E AKKLX.sub.45X.sub.46X.sub.47Q AP; and (SEQ ID NO: 57) FNK EX.sub.2X.sub.3X.sub.4AX.sub.6X.sub.7EIX.sub.10 X.sub.11LPNLX.sub.16X.sub.17X.sub.18QX.sub.20 X.sub.21AFIX.sub.25X.sub.26LX.sub.28X.sub.29X.sub.30 PX.sub.32QSX.sub.35X.sub.36LLX.sub.39E AKKLX.sub.45X.sub.46X.sub.47Q AP,
wherein each X.sub.y is as defined in any one of items 1-22.
(108) 32. Polypeptide according to item 31, which comprises an amino acid sequence selected from:
(109) TABLE-US-00020 (SEQ ID NO: 58) ADNNFNK EX.sub.2X.sub.3X.sub.4AX.sub.6X.sub.7EIX.sub.10 X.sub.11LPNLX.sub.16X.sub.17X.sub.18QX.sub.20 X.sub.21AFIX.sub.25X.sub.26LX.sub.28X.sub.29X.sub.30 PX.sub.32QSX.sub.35X.sub.36LLX.sub.39E AKKLX.sub.45X.sub.46X.sub.47Q APK; (SEQ ID NO: 59) ADNKFNK EX.sub.2X.sub.3X.sub.4AX.sub.6X.sub.7EIX.sub.10 X.sub.11LPNLX.sub.16X.sub.17X.sub.18QX.sub.20 X.sub.21AFIX.sub.25X.sub.26LX.sub.28X.sub.29X.sub.30 PX.sub.32QSX.sub.35X.sub.36LLX.sub.39E AKKLX.sub.45X.sub.46X.sub.47Q APK; (SEQ ID NO: 60) VDNKFNK EX.sub.2X.sub.3X.sub.4AX.sub.6X.sub.7EIX.sub.10 X.sub.11LPNLX.sub.16X.sub.17X.sub.18QX.sub.20 X.sub.21AFIX.sub.25X.sub.26LX.sub.28X.sub.29X.sub.30 PX.sub.32QSX.sub.35X.sub.36LLX.sub.39E AKKLX.sub.45X.sub.46X.sub.47Q APK; (SEQ ID NO: 61) VDAKYAK EX.sub.2X.sub.3X.sub.4AX.sub.6X.sub.7EIX.sub.10 X.sub.11LPNLX.sub.16X.sub.17X.sub.18QX.sub.20 X.sub.21AFIX.sub.25X.sub.26LX.sub.28X.sub.29X.sub.30 PX.sub.32QSX.sub.35X.sub.36LLX.sub.39E AKKLX.sub.45X.sub.46X.sub.47Q APK; and (SEQ ID NO: 62) AEAKYAK EX.sub.2X.sub.3X.sub.4AX.sub.6X.sub.7EIX.sub.10 X.sub.11LPNLX.sub.16X.sub.17X.sub.18QX.sub.20 X.sub.21AFIX.sub.25X.sub.26LX.sub.25X.sub.29X.sub.30 PX.sub.32QSX.sub.35X.sub.36LLX.sub.39E AKKLX.sub.45X.sub.46X.sub.47Q APK;
wherein each X.sub.y is as defined in any one of items 1-22.
(110) 33. Polypeptide according to any one of items 1-32 having an affinity for a predetermined target, wherein said target is optionally selected from the group consisting of ABD, HER2, TNFα, EGFR, IGF1R, IgG, PDGFRβ, HER3, C5, FcRn, CAIX, amyloid β, CD4, IL8, IL6 and insulin.
(111) 34. Fusion polypeptide comprising a polypeptide according to any one of items 1-33 as a moiety.
(112) 35. Polypeptide or fusion polypeptide according to any one of items 1-34, further comprising a label.
(113) 36. Polypeptide or fusion polypeptide according to any one of items 1-35, further comprising a therapeutic agent.
(114) 37. Use of a polypeptide or fusion polypeptide according to any one of items 1-36 as a detection reagent, capture reagent or separation reagent.
(115) 38. Polypeptide or fusion polypeptide according to any one of items 1-36 for use in therapy.
(116) 39. Polypeptide or fusion polypeptide according to any one of items 1-36 for use as a diagnostic agent.
(117) 40. Polynucleotide encoding a polypeptide or fusion polypeptide according to any one of items 1-34.
(118) 41. Population of polypeptide variants based on a common scaffold, each polypeptide in the population comprising an amino acid sequence selected from: i)
(119) TABLE-US-00021 (SEQ ID NO: 55) EX.sub.2X.sub.3X.sub.4AX.sub.6X.sub.7EIX.sub.10 X.sub.11LPNLX.sub.16X.sub.17X.sub.18QX.sub.20 X.sub.21AFIX.sub.25X.sub.26LX.sub.28X.sub.29X.sub.30 PX.sub.32QSX.sub.35X.sub.36LLX.sub.39E AKKLX.sub.45X.sub.46X.sub.47Q, wherein each of X.sub.2, X.sub.3, X.sub.4, X.sub.6, X.sub.7, X.sub.10, X.sub.11, X.sub.17, X.sub.18, X.sub.20, X.sub.21, X.sub.25 and X.sub.28 independently corresponds to any amino acid residue; and wherein, independently of each other, X.sub.16 is selected from N and T; X.sub.26 is selected from K and S; X.sub.29X.sub.30PX.sub.32 is selected from DDPS and RQPE; X.sub.35 is selected from A and S; X.sub.36 is selected from E and N; X.sub.39 is selected from A, C and S; X.sub.45 is selected from E, N and S; X.sub.46 is selected from D, E and S, provided that X.sub.46 is not D when X.sub.45 is N; X.sub.47 is selected from A and S; and ii) an amino acid sequence which has at least 91% identity to the sequence defined in i), provided that X.sub.46 is not D when X.sub.45 is N.
(120) 42. Population according to item 41, which comprises at least 1×10.sup.4 unique polypeptide molecules.
(121) 43. Population according to item 42, which comprises at least 1×10.sup.6 unique polypeptide molecules.
(122) 44. Population according to item 43, which comprises at least 1×10.sup.8 unique polypeptide molecules.
(123) 45. Population according to item 44, which comprises at least 1×10.sup.10 unique polypeptide molecules.
(124) 46. Population according to item 45, which comprises at least 1×10.sup.12 unique polypeptide molecules.
(125) 47. Population according to item 46, which comprises at least 1×10.sup.14 unique polypeptide molecules.
(126) 48. Population of polynucleotides, characterized in that each member thereof encodes a member of a population of polypeptides according to any one of items 41-47.
(127) 49. Combination of a polypeptide population according to any one of items 41-47 with a polynucleotide population according to item 48, wherein each member of said population of polypeptides is physically or spatially associated with the polynucleotide encoding that member via means for genotype-phenotype coupling.
(128) 50. Combination according to item 49, wherein said means for genotype-phenotype coupling comprises a phage display system.
(129) 51. Combination according to item 49, wherein said means for genotype-phenotype coupling comprises a cell surface selection display system.
(130) 52. Combination according to item 51, wherein said cell surface display system comprises prokaryotic cells.
(131) 53. Combination according to item 52, wherein said prokaryotic cells are Gram-positive cells.
(132) 54. Combination according to item 51, wherein said cell surface display system comprises eukaryotic cells.
(133) 55. Combination according to item 54, wherein said eukaryotic cells are yeast cells.
(134) 56. Combination according to item 49, wherein said means for genotype-phenotype coupling comprises a cell free display system.
(135) 57. Combination according to item 56, wherein said cell free display system comprises a ribosome display system.
(136) 58. Combination according to item 56, wherein said cell free display system comprises an in vitro compartmentalization display system.
(137) 59. Combination according to item 56, wherein said cell free display system comprises a system for cis display.
(138) 60. Combination according to item 56, wherein cell free display system comprises a microbead display system.
(139) 61. Combination according to item 49, wherein said means for genotype-phenotype coupling comprises a non-display system.
(140) 62. Combination according to item 61, wherein said non-display system is protein-fragment complementation assay.
(141) 63. Method for selecting a desired polypeptide having an affinity for a predetermined target from a population of polypeptides, comprising the steps:
(142) (a) providing a population of polypeptides according to any one of items 41-47;
(143) (b) bringing the population of polypeptides into contact with the predetermined target under conditions that enable specific interaction between the target and at least one desired polypeptide having an affinity for the target; and
(144) (c) selecting, on the basis of said specific interaction, the at least one desired polypeptide from the remaining population of polypeptides.
(145) 64. Method according to item 63, wherein step (a) comprises the preparatory steps of providing a population of polynucleotides according to item 48 and expressing said population of polynucleotides to yield said population of polypeptides.
(146) 65. Method according to item 64, wherein each member of said population of polypeptides is physically or spatially associated with the polynucleotide encoding that member via means for genotype-phenotype coupling.
(147) 66. Method according to item 65, wherein said means for genotype-phenotype coupling is as defined in any one of items 50-62.
(148) 67. Method for isolating a polynucleotide encoding a desired polypeptide having an affinity for a predetermined target, comprising the steps: selecting said desired polypeptide and the polynucleotide encoding it from a population of polypeptides using the method according to item 63; and isolating the thus separated polynucleotide encoding the desired polypeptide.
(149) 68. Method for identifying a desired polypeptide having an affinity for a predetermined target, comprising the steps: isolating a polynucleotide encoding said desired polypeptide using the method according to item 67; and sequencing the polynucleotide to establish by deduction the amino acid sequence of said desired polypeptide.
(150) 69. Method for selecting and identifying a desired polypeptide having an affinity for a predetermined target from a population of polypeptides, comprising the steps:
(151) (a) synthesizing each member of a population of polypeptides according to any one of items 41-47 on a separate carrier or bead;
(152) (b) selecting or enriching the carriers or beads based on the interaction of the polypeptide with the predetermined target; and
(153) (c) identifying the polypeptide by protein characterization methodology.
(154) 70. Method according to item 69, wherein the protein characterization methodology used in step (c) is mass spectrometric analysis.
(155) 71. Method for production of a desired polypeptide having an affinity for a predetermined target, comprising the steps: isolating and identifying a desired polypeptide using the method according to item 68 or selecting and identifying a desired polypeptide using the method according to any one of items 69 and 70; and producing said desired polypeptide.
(156) 72. Method according to item 71, wherein said production is carried out using chemical synthesis of the desired polypeptide de novo.
(157) 73. Method according to item 71, wherein said production is carried out using recombinant expression of a polynucleotide encoding the desired polypeptide.
(158) 74. Method for production of a desired polypeptide having an affinity for a predetermined target, comprising the steps:
(159) (a1) isolating a polynucleotide encoding said desired polypeptide using the method according to item 68; or
(160) (a2) backtranslating a polypeptide identified using the selection and identification method according to any one of items 69 and 70; and
(161) (b), following either (a1) or (a2), expressing the thus isolated polynucleotide to produce said desired polypeptide.