Polypeptides based on a scaffold
11505576 · 2022-11-22
Assignee
Inventors
- Caroline Ekblad (Saltsjö-Boo, SE)
- Elin Gunneriusson (Saltsjöbaden, SE)
- Sophia Hober (Stockholm, SE)
- Sarah Lindbo (Lidingö, SE)
Cpc classification
C07K14/00
CHEMISTRY; METALLURGY
International classification
C07K14/00
CHEMISTRY; METALLURGY
Abstract
The disclosure provides a population of polypeptide variants based on a common scaffold, each polypeptide in the population comprising the scaffold amino acid sequence X.sub.sc1AELDX.sub.sc2X.sub.sc3GVG AXXIKXIX.sub.sc4XA XXVEXVQXXK QXILAX. The disclosure also provides methods for selecting and identifying polypeptides from the population, as well as such polypeptides themselves.
Claims
1. A population of polypeptide variants based on a common scaffold, each polypeptide in the population comprising the scaffold amino acid sequence TABLE-US-00017 (SEQ ID NO: 165) X.sub.sc1AELDX.sub.sc2X.sub.sc3GVG AXXIKXIX.sub.sc4XA XXVEXVQXXK QX ILAX wherein, independently of one another, X.sub.sc1 is a scaffold amino acid residue selected from I and L; X.sub.sc2 is a scaffold amino acid residue selected from C and S; X.sub.sc3 is a scaffold amino acid residue selected from K and Y; X.sub.sc4 is a scaffold amino acid residue selected from E and Q; and each X individually is a binding amino acid residue corresponding to any amino acid residue.
2. A population according to claim 1, in which each polypeptide comprises the scaffold amino acid sequence TABLE-US-00018 (SEQ ID NO: 166) LAEAKEAAX.sub.sc1A ELDX.sub.sc2X.sub.sc3GVGAX XIKXIX.sub.sc4XAXX VE XVQXXKQX ILAXLP wherein X.sub.sc1, X.sub.sc2, X.sub.sc3, X.sub.sc4 and each individual X are as defined in claim 1.
3. A population according to claim 1, which comprises at least 1×10.sup.4 unique polypeptide molecules.
4. A population of polynucleotides, characterized in that each member thereof encodes a member of a population of polypeptides according to claim 1.
5. A combination of a polypeptide population according to claim 1 with a polynucleotide population that encodes said polypeptide population, 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.
6. A method for selecting a desired polypeptide having an affinity for a predetermined target from a population of polypeptides, comprising the steps: (a) contacting the population of polypeptides of claim 1 with a predetermined target under conditions that enable specific interaction between the target and at least one desired polypeptide having an affinity for the target; and (b) selecting, on the basis of said specific interaction, the at least one desired polypeptide from the remaining population of polypeptides.
7. A method according to claim 6, further comprising the steps of providing a population of polynucleotides that encodes said polypeptide population and expressing said population of polynucleotides to yield said population of polypeptides.
8. A method according to claim 7, 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.
9. 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 according to the method of claim 6; and isolating the thus separated polynucleotide encoding the desired polypeptide.
10. A method for identifying a desired polypeptide having an affinity for a predetermined target, comprising the steps: isolating a polynucleotide encoding said desired polypeptide according to the method of claim 9; and sequencing the polynucleotide to establish by deduction the amino acid sequence of said desired polypeptide.
11. 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 1 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.
12. A 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 claim 10; and producing said desired polypeptide.
13. A method for production of a desired polypeptide having an affinity for a predetermined target, comprising the steps: isolating a polynucleotide encoding said desired polypeptide according to the method of claim 9; and expressing the thus isolated polynucleotide to produce said desired polypeptide.
14. A Polypeptide comprising an amino acid sequence which is at least 97% identical to TABLE-US-00019 (SEQ ID NO: 165) X.sub.1AELDX.sub.6X.sub.7GVG AX.sub.12X.sub.13IKX.sub.16IX.sub.18X.sub.19A X.sub.21X.sub.22VEX.sub.25VQ X.sub.28X.sub.29K QX.sub.32ILAX.sub.3 wherein, independently of one another, X.sub.1 is selected from I and L; X.sub.6 is selected from C and S; X.sub.7 is selected from K and Y; X.sub.18 is selected from E and Q; and each of X.sub.12, X.sub.13, X.sub.16, X.sub.19, X.sub.21, X.sub.22, X.sub.25, X.sub.28, X.sub.29, X.sub.32 and X.sub.36 is any amino acid residue.
15. A Polypeptide according to claim 14, which comprises an amino acid sequence which is at least 97% identical to TABLE-US-00020 (SEQ ID NO: 166) LAEAKEAA X.sub.1AELDX.sub.6X.sub.7GVG AX.sub.12X.sub.13IKX.sub.16IX.sub.18X.sub.19A X.sub.21 X.sub.22VEX.sub.25VQX.sub.28X.sub.29KQX.sub.32ILAX.sub.36 LP wherein all amino acid residues denoted X are as defined in claim 14.
16. The Polypeptide according to claim 14 further comprising a second polypeptide moiety, such that the polypeptide is a fusion polypeptide comprising a first moiety which fulfils the sequence definition of claim 14, and a second moiety with a desired function.
17. A method for production of a desired polypeptide having an affinity for a predetermined target, comprising the steps: selecting and identifying a desired polypeptide according to the method of claim 11; and producing said desired polypeptide.
18. A method for production of a desired polypeptide having an affinity for a predetermined target, comprising the steps: (a) backtranslating a polypeptide identified using the selection and identification method according to the method of claim 11; and (b) expressing the thus isolated polynucleotide to produce said desired polypeptide.
Description
BRIEF DESCRIPTION OF THE FIGURES
(1)
(2)
(3)
(4)
EXAMPLES
(5) The following Examples disclose design and construction of a protein library based on a scaffold sequence inspired by the albumin binding domain PP013 (SEQ ID NO:159), in turn derived from ABD of G148-GA3 (SEQ ID NO:158). The Examples show the successful assembly of a high complexity library while retaining high stability and solubility. The successful use of the designed library for selection of new ligands for three different target molecules is also demonstrated.
(6) A critical part of the approach was the decision concerning which residues to randomize and which residues to keep fixed, as well as the decision concerning the identity of the fixed residues. In this regard, randomization at positions critical for intramolecular stabilization should be avoided, but, at the same time, the potential of the library to offer optimal coverage and evaluation of possible alternative sequences can be limited by a suboptimal choice of surface-exposed residues. In the process of identifying suitable positions to randomize, comprehensive knowledge of the scaffold protein was applied, including structural information for the domain G148-GA3 (Kraulis et al, supra; Johansson et al, supra) and information related to its albumin binding activity (Lejon et al, supra). In the design of new scaffolds, the surface area involved in the native interaction has often been the main focus for randomization. However, randomizing exactly the same residues as those providing binding affinity in the ancestor polypeptide may be suboptimal when aiming to design a broad library that can be used to find novel binders against a variety of targets with different sizes and structures. Furthermore, the native binding interface may not only be central for target binding, but may also be important for maintaining the framework and structural stability of the scaffold. Thus, focusing on regions that are not directly involved in the native interaction may be equally important, in order to identify the optimal region for randomization. In brief, the following procedure was applied: 1) Design of a first library with the aim to establish what positions could be varied to provide new binding abilities, and to incorporate flexibility and improvements in the scaffold positions. 2) Creation of this first library, denoted “Ylib001Naive.I”. 3) Selections against a first set of targets. 4) Assessment of selected ligands, primarily in terms of binding and stability. 5) Sequential mutational programs for additional improvements; including generation, production and assessment of mutated ligands. 6) Design of a second library based on the results from steps 3)-5), with decisions on what binding positions to vary in order to generate novel binding abilities, and on what scaffold residues should be kept fixed and to what amino acid residues. 7) Creation of this second library, denoted “Ylib002Naive.I”. 8) Selections against a second set of targets. 9) Assessment of selected ligands in terms of binding, stability, producibility and solubility, in order to verify the quality of the library and the selected variants. 10) Sequential mutational programs for fine-tuning of library. 11) Design of a scaffold according to the disclosure based on the entire preceding procedure.
(7) The scaffold sequence and populations or libraries described herein are referred to as “Y scaffold” and “Y populations” or “Y libraries”, respectively, and binding variants derived therefrom are denoted “Y variants”.
Example 1
Description of General Procedures
(8) Summary
(9) This Example describes general procedures for cloning, production and analysis. These general procedures were used throughout the Examples 2-8 unless otherwise specified in the respective Example.
(10) Materials and Methods
(11) Biotinylation of Target Protein:
(12) Target proteins were biotinylated using No-Weigh EZ-Link Sulfo-NHS-LC-Biotin (Thermo Scentific) at a 10× molar excess, according to the manufacturer's recommendations. The reactions were performed at room temperature (RT) for 30 min. Buffer exchange to phosphate buffered saline (PBS; 10 mM phosphate, 137 mM NaCl, 2.68 mM KCl, pH 7.4) was performed after biotinylation, using either dialysis cassettes (Pierce, Slide-a-lyzer (3500 MWCO)) or illustra NAP-5 desalting columns (GE Healthcare) according to the manufacturers' instructions.
(13) Cloning of Y Variants:
(14) Cloning was performed using methods known in the art. In brief, one of the following procedures was applied:
(15) 1) The DNA encoding the Y variant(s) of interest was amplified from the library vector pAY03686 using a standard PCR protocol and AmpliTaq Gold polymerase (Life Technologies). Fragments were restricted using enzymes SalI-HF and BamHI-HF (New England Biolabs) and purified using QIAquick PCR Purification Kit (QIAGEN) according to the supplier's recommendations. An expression vector (with T7 promoter) providing an N-terminal His.sub.6 tag was prepared and digested with the same restriction enzymes. The vector was run on a preparative 1% agarose (BioNordika AB) gel electrophoresis and purified using QIAGEN Gel Extraction Kit (QIAGEN) according to the supplier's recommendations. Gene fragments and vector were ligated using T4 DNA ligase (Thermo Scientific) in ligase buffer and electroporated into electrocompetent Escherichia coli (E. coli) TOP10 cells. The transformed cells were spread on TBAB plates (30 g/l tryptose blood agar base) supplemented with 50 μg/ml of kanamycin, followed by incubation at 37° C. overnight.
(16) 2) DNA encoding the Y variant(s) of interest was ordered as fragment genes from GeneArt (Life Technologies) or Twist Bioscience, and restricted using enzymes BamHI-HF and NgoMIV (New England Biolabs). An expression vector (with T7 promoter) providing an N-terminal His.sub.6 tag was prepared and digested with the same restriction enzymes. Ligation and transformation were performed as described above.
(17) 3) DNA encoding the Y variant(s) of interest were ordered from GeneArt as fully cloned genes in a custom vector (expression vector (with T7 promoter) providing an N-terminal His.sub.6 tag). Transformation was performed as described above.
(18) Sequencing:
(19) Bacterial clones harboring plasmids of interest were picked for sequencing. PCR fragments were amplified from single colonies using a standard PCR program and a complementary pair of primers. Sequencing of amplified fragments was performed using a biotinylated oligonucleotide and a Big Dye® Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems), in accordance with the manufacturer's protocol. The sequencing reactions were purified by binding to magnetic streptavidin coated beads (Detach Streptavidin Beads, Nordiag) using a Magnatrix 8000 (Magnetic Biosolutions) instrument and analyzed on an ABI PRISM® 3130xl Genetic Analyzer (PE Applied Biosystems).
(20) Protein Expression:
(21) E. coli T7E2 cells were transformed with plasmids containing the gene fragment of each respective Y variant. The resulting recombinant strains were generally cultivated in media supplemented with 50 μg/ml kanamycin at 30-37° C. in 50 ml scale using the EnPresso protocol (Enpresso GmbH). In order to induce protein expression, isopropyl-β-D-1-thiogalactopyranoside (IPTG) was added to a final concentration of 0.5 mM at an optical density at 600 nm (OD.sub.600) of approximately 10. After induction, the cultures were incubated for 16 h. The cells were harvested by centrifugation. Alternatively, the culture was performed at 37° C. in 980 ml of TSB-YE medium (tryptic soy broth 30 g/l supplemented with yeast extract 5 g/l) supplemented with 50 μg/ml kanamycin, and protein expression induced with 0.2 mM IPTG at OD.sub.600=2, followed by incubation for 5 h before harvesting of the cells by centrifugation. The total yield as well as the fraction of soluble and insoluble product of the respective Y variant was estimated based on SDS-PAGE analysis.
(22) Purification of Y Variants with an N-Terminal His.sub.6-Tag:
(23) Cells were re-suspended in binding buffer (20 mM sodium phosphate, 0.5 M NaCl, 20 mM imidazole, pH 7.4) supplemented with Benzonase® (Merck). After cell disruption, cell debris was removed by centrifugation and each supernatant was applied on a 1 ml His GraviTrap IMAC column (GE Healthcare). Contaminants were removed by washing with wash buffer (20 mM sodium phosphate, 0.5 M NaCl, 60 mM imidazole, pH 7.4) and the Y variants were subsequently eluted with elution buffer (20 mM sodium phosphate, 0.5 M NaCl, 500 mM imidazole, pH 7.4). Buffer exchange to PBS was performed using PD-10 desalting columns (GE Healthcare) according to the manufacturer's instructions.
(24) Protein Analysis and Verification:
(25) Protein concentrations were determined by measuring the absorbance at 280 nm, using a NanoDrop® ND-1000 spectrophotometer (Saveen Werner AB) and the extinction coefficient of the respective protein. The purity was analyzed by SDS-PAGE stained with Coomassie Blue and the identity of each purified Y variant was confirmed using LC/MS analysis.
(26) Circular Dichroism (CD) Spectroscopy Analysis:
(27) The respective Y variant was diluted to 0.5 mg/ml in PBS. A CD spectrum at 250-195 nm was obtained at 20° C. In addition, a variable temperature measurement (VTM) was performed to determine the melting temperature (Tm). In the VTM, absorbance was measured at 221 nm while the temperature was raised from 20° C. to 90° C. with a temperature gradient of 5° C./min. A new CD spectrum was obtained at 20° C. after the heating procedure, in order to study the refolding ability of the Y variants. 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.
(28) Kinetic Analysis Using Surface Plasmon Resonance:
(29) Kinetic constants (k.sub.a and k.sub.d) and affinities (K.sub.D) were determined for His.sub.6-tagged Y variants using a Biacore T200 instrument (GE Healthcare). Target proteins C4, IL-6 and insulin, respectively, were immobilized in separate flow cells on the carboxylated dextran layer of different CM5 chip surfaces (GE Healthcare). Immobilization was performed using amine coupling chemistry according to the manufacturer's protocol and PBS pH 7.4 supplemented with 0.1 Tween20 (PBST 0.1%) as running buffer. The ligand immobilization levels on the surfaces were 3280-8830 RU for C4, 495-1184 RU for IL-6, and 270-282 RU for insulin. One flow cell surface on each chip was activated and deactivated for use as blank during analyte injections. In the kinetic experiment, PBST 0.1% was used as running buffer at a flow rate of 30 μl/min. The analytes, i.e. Y variants, were each diluted in PBST 0.1 buffer at concentrations of 1000, 500, 100, 50 and 10 nM and injected for 5 min, followed by dissociation in running buffer for 3 min. After dissociation, the surfaces were regenerated with one injection of 30 μl 10 mM HCl. Kinetic constants were calculated from the sensorgrams using the Langmuir 1:1 model of Biacore T200 Evaluation software 2.0 (GE Healthcare).
Example 2
Design and Construction of a Library Based on an ABD Variant Scaffold
(30) Summary
(31) This Example describes the design and construction of a first library to be used for a first selection described below in Example 3. The aim was to establish what positions to vary in order to achieve new binding capabilities, and to incorporate flexibility and improvements in the scaffold.
(32) Materials and Methods
(33) Library Design:
(34) A library was designed based on the albumin binding domain PP013 (SEQ ID NO:159) and on information concerning the structure of the G148-GA3 domain. Surface-exposed amino acid positions involved in the natural binding to albumin, positions in the near vicinity of the binding surface, as well as additional positions in helix one and in the loop between helix one and helix two, were targeted for variegation. The amino acid positions in PP013 selected for randomization were: N9, Y15, V17, S18, D19, F20, Y21, K22, R23, L24, K27, A28, K29, T30, G33, A36, L37, A40, A43, and A44. Each position was randomized allowing different compositions of amino acid residues (all excluding the amino acids C, M and P). Full randomization at each of these twenty positions was not possible, because the theoretical size of the library would then widely exceed the possible practical size. Therefore, positions thought not to be involved in binding (i.e. “scaffold positions”) were randomized more restrictively, whereas the degree of randomization in positions potentially involved in binding (i.e. “binding positions”) ranged from 11 to 17 allowed amino acid residues. Limitations applied depended on the nature of the position in the structure of the G148-GA3 domain (helix position versus loop position) and expected relevance to binding function. In some positions, homologous residues were excluded, e.g. allowing K but not R, or allowing L and V but not I. The selection of amino acids at the respective position and their theoretical distributions in the resulting library, denoted “Ylib001Naive.I”, are displayed in Table 1.
(35) Using split-pool synthesis, the following DNA oligo of 177 bp was generated, encoding a partially randomized amino acid sequence: 5′-AA ATA AAT GGA TCC AGC CTG GCT GAG GCG AAA GAA GCC GCG NNN GCC GAG CTG GAT AGC NNN GGT NNN NNN NNN NNN NNN NNN NNN NNN ATC GAG NNN NNN NNN NNN GTT GAG NNN GTT GAA NNN NNN AAA GAA NNN ATT CTG NNN NNN CTG CCG GCG AGC GGT AGC GTC GAO ATT ATT TA-3′ (SEQ ID NO:163; randomized codons are illustrated as NNN) flanked by restriction sites SalI and BamHI. The oligonucleotide was ordered from Atum (formerly DNA2.0). The resulting theoretical library size is 7.8×10.sup.16 variants.
(36) TABLE-US-00004 TABLE 1 The Ylib001Naive.I library. Percentages of the amino acids used in each of the 20 varied positions are indicated. Position with reference to SEQ ID NO: 165 1 7 9 10 11 12 13 14 15 16 Position in full length Y variant (with ref to SEQ ID NO: 159) Codon 9 15 17 18 19 20 21 22 23 24 A Ala 0 33.3 0 8.3 33.3 5.9 6.3 0 0 6.3 C Cys 0 0 0 0 0 0 0 0 0 0 D Asp 0 0 0 8.3 33.3 5.9 6.3 0 0 6.3 E Glu 0 0 0 8.3 33.3 5.9 6.3 0 33.3 6.3 F Phe 0 0 0 0 0 5.9 6.3 0 0 6.3 G Gly 0 0 0 8.3 0 5.9 0 0 0 0 H His 0 0 0 8.3 0 5.9 6.3 0 0 6.3 I Ile 33.3 0 50.0 0 0 5.9 6.3 50.0 0 6.3 K Lys 0 33.3 0 8.3 0 5.9 6.3 50.0 33.3 6.3 L Leu 33.3 0 0 0 0 5.9 6.3 0 0 6.3 M Met 0 0 0 0 0 0 0 0 0 0 N Asn 33.3 0 0 8.3 0 5.9 6.3 0 0 6.3 P Pro 0 0 0 0 0 0 0 0 0 0 Q Gln 0 0 0 8.3 0 5.9 6.3 0 0 6.3 R Arg 0 0 0 0 0 5.9 6.3 0 0 6.3 S Ser 0 0 0 8.3 0 5.9 6.3 0 33.3 6.3 T Thr 0 0 0 8.3 0 5.9 6.3 0 0 6.3 V Val 0 0 50.0 8.3 0 5.9 6.3 0 0 6.3 W Trp 0 0 0 0 0 5.9 6.3 0 0 6.3 Y Tyr 0 33.3 0 8.3 0 5.9 6.3 0 0 6.3 Position with reference to SEQ ID NO: 165 19 20 21 22 25 28 29 32 35 36 Position in full length Y variant (with ref to SEQ ID NO: 159) Codon 27 28 29 30 33 36 37 40 43 44 A Ala 5.9 50.0 8.3 8.3 7.1 9.1 6.3 6.3 50 8.3 C Cys 0 0 0 0 0 0 0 0 0 0 D Asp 5.9 0 0 8.3 0 0 6.3 6.3 0 8.3 E Glu 5.9 0 8.3 0 7.1 9.1 6.3 6.3 0 8.3 F Phe 5.9 0 8.3 8.3 7.1 9.1 6.3 6.3 0 0 G Gly 5.9 50.0 8.3 8.3 7.1 0 0 0 0 8.3 H His 5.9 0 8.3 8.3 7.1 9.1 6.3 6.3 0 8.3 I Ile 5.9 0 0 8.3 0 0 6.3 6.3 0 0 K Lys 5.9 0 8.3 0 7.1 9.1 6.3 6.3 50 8.3 L Leu 5.9 0 8.3 0 7.1 9.1 6.3 6.3 0 0 M Met 0 0 0 0 0 0 0 0 0 0 N Asn 5.9 0 0 0 7.1 0 6.3 6.3 0 8.3 P Pro 0 0 0 0 0 0 0 0 0 0 Q Gln 5.9 0 8.3 8.3 7.1 9.1 6.3 6.3 0 8.3 R Arg 5.9 0 0 8.3 0 0 6.3 6.3 0 0 S Ser 5.9 0 8.3 8.3 7.1 9.1 6.3 6.3 0 8.3 T Thr 5.9 0 0 0 7.1 0 6.3 6.3 0 8.3 V Val 5.9 0 8.3 8.3 7.1 9.1 6.3 6.3 0 8.3 W Trp 5.9 0 8.3 8.3 7.1 9.1 6.3 6.3 0 0 Y Tyr 5.9 0 8.3 8.3 7.1 9.1 6.3 6.3 0 8.3
(37) Library Construction:
(38) A phagemid vector denoted pAY03686 was constructed in a step-wise manner beginning with pUC119 (Vieira and Messing Meth. Enzymol. 1987, 153:3-11) and using standard molecular biology methods to introduce essential parts in the translation cassette. Thus, the resulting library vector pAY03686 encodes, under regulation of the E. coli lac promoter, the E. coli OmpA leader peptide in frame with the variable Y library, a 58 amino acid residue Taq polymerase binding domain (Z03639, SEQ ID NO:160) and residues 249-406 of M13 filamentous phage coat protein III (Lowman et al, Biochemistry, 1991, 30:10832-10838), the latter preceded by an amber stop codon.
(39) The library oligo was amplified using AmpliTaq Gold polymerase during 12 cycles of PCR, and pooled products were purified with QIAquick PCR Purification Kit according to the supplier's recommendations. The purified pool of randomized library fragments was digested with restriction enzymes SalI-HF and BamHI-HF and concentrated using QIAquick PCR Purification Kit. Subsequently, the product was run on a preparative 2.5% agarose (NuSieve® GTG® Agarose, Lonza) gel electrophoresis and purified using QIAGEN Gel Extraction Kit (QIAGEN) according to the supplier's recommendations.
(40) The phagemid vector pAY03686 was restricted with the same enzymes and purified using phenol/chloroform extraction and ethanol precipitation. The restricted fragments and the restricted vector were ligated in a molar ratio of 5:1 with T4 DNA ligase for 2 h at RT, followed by overnight incubation at 4° C. The ligated DNA was recovered by phenol/chloroform extraction and ethanol precipitation, followed by dissolution in 10 mM Tris-HCl, pH 8.5. Thus, the resulting library in vector pAY03686 encoded Y variants each fused to a Taq polymerase binding domain (Z03639).
(41) The ligation reactions (approximately 160 ng DNA/transformation) were electroporated into electrocompetent E. coli ER2738 cells (Lucigen). Immediately after electroporation, approximately 1 ml of recovery medium (supplied with the E. coli ER2738 cells) was added. The transformed cells were incubated at 37° C. for 60 min. Samples were taken for titration and for determination of the number of transformants. The cells were thereafter pooled and cultivated overnight at 37° C. in 3 I of TSB-YE medium supplemented with 10 μg/ml tetracycline and 100 μg/ml ampicillin. The cells were pelleted for 15 min at 4,000 g and re-suspended in a 40% glycerol solution. The cells were aliquoted and stored at −80° C. Clones from the library of Y variants were sequenced in order to verify the content and to evaluate the outcome of the constructed library vis-à-vis the library design. Sequencing was performed as described in Example 1 and the amino acid distribution was verified.
(42) Results
(43) Library Design:
(44) A library denoted Ylib001Naive.I was designed based on the G148-GA3 variant PP013, which has an albumin binding surface positioned from helix two to helix three of the triple alpha-helical protein. The amino acid positions involved in the albumin binding together with positions in the vicinity of the binding surface were used to create a combinatorial library for exploring the possibility to redefine the binding function. In addition, a set of surface-exposed residues in helix one and in the loop preceding helix two was variegated, resulting in a total of 20 amino acid positions that were targeted for randomization (Table 1). Taking all theoretically possible combinations into account, the theoretical size of the designed library was 7.8×10.sup.16 unique Y variants.
(45) Library Construction:
(46) A new phagemid vector, denoted pAY03686, was constructed for monovalent display using the M13 filamentous phage coat protein III. The library Ylib001Naive.I was constructed using pAY03686. The actual size of the library, determined by titration after transformation to E. coli ER2738 cells, was 1.0×10.sup.10 transformants. The library quality was tested by sequencing of 192 transformants and by comparing their actual sequences with the theoretical design. Sequence analysis of individual library members verified a distribution of codons that was in accordance with the theoretical design. A library of potential binders in a novel scaffold sequence was thus successfully constructed.
Example 3
Phage Display Selection and Screening from a First Library
(47) Summary
(48) In this Example, complement component 4 (C4), interleukin 6 (IL-6) and insulin were used as targets in phage display selections using a phage library of Y variants. Selected clones were DNA sequenced, produced in E. coli as soluble fractions and assayed against each respective target using ELISA and SPR. Based on sequence observations in these selected Y variants and the results described in this Example, it was concluded to subject positions 20, 21, 24, 27, 29, 30, 33, 36, 37, 40 and 44 to randomization as “binding positions” in a second library, further described in Examples 5 and 6.
(49) Materials and Methods
(50) Production of Library Phage Stock:
(51) Production of phage stock was performed as follows. A glycerol stock containing the phagemid library Ylib001Naive.I in E. coli cells ER2738 was inoculated in 19 l of cultivation medium (2.5 g/l (NH.sub.4).sub.2SO.sub.4; 5.0 g/l yeast extract; 30 g/l tryptone; 2 g/l K.sub.2HPO.sub.4; 3 g/l KH.sub.2PO.sub.4; 1.25 g/l Na.sub.3C.sub.6H.sub.5O.sub.7.2H.sub.2O; 0.1 ml/l Breox FMT30 antifoaming agent), supplemented with 25 μg/ml carbenicillin, 5 ml/l of 1.217 M MgSO4 and 19 ml of a trace element solution (129 mM FeC13; 37 mM ZnSO.sub.4; 10.6 mM CuSO.sub.4; 78 mM MnSO.sub.4; 94 mM CaCl.sub.2, dissolved in 1.2 M HCl). pH was maintained at 7 through the automatic addition of 25% NH.sub.4OH, air was supplemented (19 l/min), and the stirrer was set to keep the dissolved oxygen level above 30%. When the cells reached an OD.sub.600 of 0.50, the culture was infected using a 5× molar excess of M13K07 helper phage (New England Biolabs). The cells were incubated for 30 min before expression was induced by the addition of IPTG to a concentration of 100 μM. 1 h after the induction, the culture was supplemented with 25 μg/ml kanamycin, and a glucose-limited fed-batch cultivation was started where a 600 g/l glucose solution was fed to the reactor (30 g/h the first 20 h and then 90 g/h until the end of the cultivation). The culture was harvested 24 h after the addition of helper phages. The cells in the culture were removed by centrifugation (15,900 g, 50 min).
(52) The phage particles in the supernatant were precipitated twice in PEG/NaCl (20% polyethylene glycol/2.5 M NaCl sodium chloride) using standard procedures. Phage stocks were filtered using a 0.45 μm filter, dissolved in PBS and glycerol, and stored at −80° C. before use.
(53) Phage Display Selection of C4, IL-6 and Insulin Binding Y Variants:
(54) C4 (Complement Technology Inc, cat. no. A105), IL-6 (R&D Systems, cat. no. 206-IL-200/CF) and insulin (Roche, cat. no. 1376497) were biotinylated as described in Example 1. The phage stock described in this Example, displaying random variants of protein Y according to the Ylib001Naive.I definition on bacteriophage, was used to select C4, IL-6 and insulin binding polypeptides. Streptavidin coated paramagnetic beads (SA beads) (Dynabeads® M-280 Streptavidin; Life Technology) were used as solid support. Before each round, except for the first round, a negative selection was performed to remove unspecific binders against SA or the beads. The phage particles were incubated with the beads for 30 min at RT and the supernatant was used as input in selection rounds. All tubes and beads were blocked with PBSTB (PBS supplemented with 0.1% Tween20 and 3% BSA (bovine serum albumin) to avoid unspecific binding.
(55) The selection buffer consisted of PBSTB supplemented with 1.5 μM human serum albumin (Albucult, Novozymes), and selection was performed in four rounds. In round one to three, binders to each biotinylated target were selected separately as well as from a mix with equal amounts of target. In the first round, 100 nM of the respective target were used and incubated with phage particles for 2 h under rotation at RT. In rounds two and three, the target concentrations were reduced to 50 nM and 25 nM, respectively, and the incubation time was shortened to 90 min and 60 min, respectively. For round four, two separate target concentrations were used, 25 and 12.5 nM, and the phage particles were incubated with the target proteins for 60 min under rotation at RT. In round four, the output from the mixed track from round three was split and incubated with each target separately at 25 nM. To capture phage-target complexes, blocked SA beads were added and incubated for 15 min. The beads were washed with PBST 0.1% with increased stringency for each round (twice in round one, four times in round two, six times in round three and eight times in round four).
(56) Bead-captured phage particles were eluted with 500 μl 0.1 M glycine-HCl, pH 2.2 during 10 min followed by immediate neutralization with 50 μl Tris-HCl, pH 8.0 and 450 μl PBS. Selected phage particles were amplified as described below and new phage stocks were prepared between each cycle. Phage stock, i.e. phages entering the selection cycle, and eluted phage particles were titrated after each selection cycle.
(57) Amplification of Phage Particles Between Rounds:
(58) E. coli XL1-Blue cells (Agilent technologies), cultivated to log phase in TSB supplemented with tetracycline 10 μg/ml, were infected with eluted phage particles for 30 min at 37° C. after each cycle of selection. TSB medium was added after infection to double the cultivation volume and ampicillin was added to a final concentration of 100 μg/ml. The infected bacteria were incubated for 1 h before addition of helper phage at a 10× excess compared to number of eluted phage particles used. Superinfection was allowed to take place during 1.5 h before the bacteria were pelleted in a centrifuge. Bacteria were re-suspended in TSB+YE supplemented with 100 μg/ml ampicillin, 25 ug/ml kanamycin and 0.1 mM IPTG and grown over night at 30° C. The amount of bacteria used for infection was 100× excess compared to the number of eluted phage particles. The overnight cultures were made in 100 ml for round one and 50 ml for each of rounds two and three. The overnight cultures were pelleted by centrifugation, and phage particles in the supernatant were precipitated twice with PEG/NaCl buffer. Finally, the phages were re-suspended in selection buffer before entering the next selection round. In the final selection cycle, ER2738 bacteria were used for infection and bacteria were spread on TBAB plates supplemented with 200 μg/ml ampicillin in order to form single colonies to be used in ELISA screening.
(59) Production of Soluble Y Variants:
(60) The Y variants were produced by inoculating single colonies from the selections into 1.2 ml TSB-YE medium supplemented with 100 μg/ml ampicillin and 1 mM IPTG in deep-well plates (Nunc). The plates were incubated with rotation for 24 h at 37° C. Cells were pelleted by centrifugation at 3300 g and re-suspended in 150 μl PBST 0.05 (PBS supplemented with 0.05% Tween20). The bacterial suspensions were heated to 82° C. during 20 min to lysate the cells. Soluble fractions of Y variants were isolated in 96 well plates by filtration using filter plates (EMD Millipore). The final supernatants contained the Y variants as fusions to Z03639, expressed as GSS-[Y#####]-ASGS-[Z03639]-YVPG (SEQ ID NO:173). Y##### refers to individual, 46 amino acid residue Y variants.
(61) Sequencing:
(62) In parallel with the ELISA screening, all clones were sequenced as described in Example 1.
(63) Sequence Analysis:
(64) Unique sequences from the selections were analyzed using an average-link hierarchical clustering method. This was done on the sequences from selections against each target separately, as well as with sequences from selection against different targets grouped together.
(65) ELISA Screening of Y Variants:
(66) The binding of Y variants to their respective target was analyzed in ELISA assays. Half-area 96-well ELISA plates (Greiner) were coated at 4° C. overnight with 2 μg/ml of an anti-Z03639 goat antibody (produced in-house) diluted in coating buffer (50 mM sodium carbonate, pH 9.6; Sigma). The antibody solution was poured off and the wells were washed in water and blocked with PBSC (PBS supplemented with 0.5% casein; Sigma) for 30 min at RT. The blocking solution was discarded, whereupon heated and filtered Y protein solutions, diluted 8× in PBST 0.05%, were added to the wells and incubated for 1.5 to 2.25 h at RT. As a negative control, ER2738 E. coli supernatants, cultivated, heat treated and filtered as described above, were added. The supernatants were poured off and the wells were washed 4 times with PBST 0.05%. Then, biotinylated target (C4 at a concentration of 50 nM, IL-6 at a concentration of 100 nM, or insulin at a concentration of 300 nM) in PBSC was added to each well. The plates were incubated for 1 h to 1.25 h at RT followed by washes as described above. Streptavidin conjugated HRP (Thermo Scientific) diluted 1:30,000 in PBSC, was added to the wells and the plates were incubated for 45 min. After washing as described above, 1-step Ultra TMB substrate (Thermo Scientific) was added to the wells and the plates were treated according to the manufacturer's recommendations. The absorbance at 450 nm was measured using an EnSpire multi-well plate reader (Perkin Elmer).
(67) Subcloning and Protein Production of a Subset of Y Variants with a His.sub.6-Tag:
(68) A subcloning strategy was applied to a subset of ELISA positive variants, for construction of monomeric Y variant molecules as described in Example 1. Proteins were expressed and purified using an N-terminal His.sub.6-tag according to methods described in Example 1. The Y variant gene fragments were subcloned into an expression vector, resulting in the encoded sequences MGSSHHHHHHGSS-[Y#####]-ASGSVD (SEQ ID NO:167).
(69) CD and SPR Analyses of Purified Y Variants:
(70) Produced Y variants were subjected to CD and SPR analyses according to the methods described in Example 1.
(71) Results
(72) Phage Display Selection of C4, IL-6 and Insulin Binding Y Variants:
(73) Phage display selection was performed using a newly designed library (Example 2) and the target proteins C4, IL-6 and insulin. In round one to three, the library was incubated with each target separately or with a mixture including all three targets. In the fourth round, the phage stock from the mixed-target track was split and incubated with each target separately. Individual clones were obtained after three and four cycles of phage display selection.
(74) Sequencing:
(75) Sequencing was performed for clones obtained after three and four cycles of selection. Each variant was given a unique identification number #####, and individual variants are referred to as Y#####. Examples of amino acid sequences of the 46 amino acid residues long Y variants are listed in
(76) Sequence Analysis:
(77) Clustering and consensus analysis was performed for the sequences of the clones obtained from the selection, in order to identify sequence similarities among all variants, as well as target-specific similarities. Consensus analyses performed for Y variants with verified binding in ELISA to its respective target are shown in
(78) Although positions 18, 20 and 21 in ABD are known to be important for its native binding to albumin, these positions did not show a strong consensus in the Y variants identified in the selection against the three targets used in this study. However, for the C4 and insulin binding Y variants, one third of the Y variants had the same residues in position 20 and 21, which indicates that these positions are involved in the interaction with their respective target, but not to the same extent as the residues in helix three. In the more restrictively randomized scaffold positions, V was preferred in position 17 and A was preferred in position 43, regardless of target. In position 28, a strong preference for A was observed in C4 and insulin binding Y variants, whereas G was tolerated in the IL-6 binding Y variants. In position 9, I was more frequently observed than N. In positions 15, 19, 22 and 23, no clear consensus was observed, which indicates that these positions are more tolerant to variation.
(79) ELISA Screening of Y Variants:
(80) The clones obtained after three or four cycles of selection were produced in 96-well plates and screened for b-C4, b-IL-6 or b-insulin binding activity in ELISA. Several unique Y variants were found to give a response of 0.15 AU or higher (corresponding to at least 2x the negative control) against b-C4 at a concentration of 50 nM or against b-IL-6 at a concentration of 100 nM, respectively. The average response of the negative controls was 0.059 AU and 0.067 AU for b-C4 and b-IL-6, respectively. The average response of the negative controls to b-insulin was 0.057 AU while the response of the selected Y variants spanned between 0.093 AU and 0.945 AU (corresponding to approximately 2× the negative control or more) at a concentration of 300 nM.
(81) CD and SPR Analyses of Purified Y Variants:
(82) Produced Y variants were subjected to CD and SPR analyses. The individual melting points and affinity values (K.sub.D) are shown in Table 2. All variants were able to refold after thermal denaturation, but the degree of α-helical content varied (
(83) TABLE-US-00005 TABLE 2 Melting points and affinity constants SEQ ID NO: Y variant Target Tm (° C.) K.sub.D (M) 1 Y00001 C4 61 n.a. 4 Y00004 C4 45 n.a. 5 Y00005 C4 60 n.a. 6 Y00006 C4 49 n.a. 50 Y00035 IL-6 60 3.6 × 10.sup.−7 61 Y00076 IL-6 47 4.3 × 10.sup.−7 90 Y00032 Insulin 47 5.0 × 10.sup.−8 106 Y00125 Insulin 35 1.1 × 10.sup.−7 n.a. not analyzed
Example 4
Mutational Studies of C4, IL-6 and Insulin Binding Y Variants
(84) Summary
(85) This Example describes a set of sequential mutational studies performed in order to optimize the scaffold properties in the light of positions decided to be randomized for binding according to the results described in Example 3.
(86) Materials and Methods
(87) Cloning of Mutated Y Variants:
(88) In a first mutational study, different mutations were introduced in sequence positions not varied for binding (i.e. “scaffold positions”) in the Y variants, in order to evaluate the impact of these mutations on the stability and binding ability. This was performed using the Y variants Y00001 (SEQ ID NO:1) and Y00032 (SEQ ID NO:4) binding C4 and insulin, respectively, as templates. Y00032 was regarded as a suitable model molecule because while it demonstrated a good ability to fold into an α-helical structure, the moderate Tm of 47° C. should nevertheless allow for improvements. Single or double mutations were introduced in the surface-exposed scaffold positions 13, 15, 17, 18, 19, 22, 23, 26, 28, 32, 35, 39 and 43. All variants were cloned with an N-terminal His.sub.6 tag, and the constructs encoded polypeptides in the format MGSSHHHHHHGSS-[Y#####] (SEQ ID NO:168).
(89) In a second mutational study, further mutations were introduced in the variants based on the results of the first mutational study, to evaluate the impact of these mutations on primarily stability, as well as to verify the results from the first study in different Y variants. Y variants Y00262 (SEQ ID NO:18; C4 binding) and Y00032 and Y00270 (SEQ ID NO:90 and SEQ ID NO:117, respectively; insulin binding), were used as templates. Single, double or triple mutations were introduced in the surface-exposed scaffold positions 17, 18, 19, 22, 23, 26, 35, 39 and 43. All variants were cloned with an N-terminal His.sub.6 tag, and constructs encoded polypeptides in the format MGSSHHHHHHGSS-[Y#####] (SEQ ID NO:168).
(90) In a third mutational study, a mutation in scaffold position 26 was introduced in Y variants Y00289 (SEQ ID NO:26) and Y000293 (SEQ ID NO:125) binding C4 and insulin, respectively. In addition, different N-terminal and C-terminal extensions were assessed with regard to what impact they had on the stability and binding ability as well as on the expression level. All variants were cloned with an N-terminal His.sub.6 tag and obtained constructs encoded polypeptides in one of the following formats MGSSHHHHHHGSS-[Y#####] (SEQ ID NO:168), MGSSHHHHHHGSS-[Y#####]-ASYGS (SEQ ID NO:169), MGSSHHHHHHGSS-[Y#####]-GYS (SEQ ID NO:170) or MGSSHHHHHHTIDEWL-[Y#####] (SEQ ID NO:171). Cloning was performed according to the methods described in Example 1.
(91) Production and Characterization of Mutated Y Variants:
(92) The Y variants were cloned, produced and characterized according to the general methods described in Example 1. Produced Y variants with point mutations and/or N-terminal or C-terminal extensions were subjected to CD and SPR analyses as described in Example 1.
(93) Results
(94) Cloning, Production and Characterization of Mutated Y Variants:
(95) Produced Y variants in mutation study 1, 2 and 3, respectively, were subjected to CD and SPR analyses to assess the effect of the point mutations and/or additional N-terminal or C-terminal amino acids on the stability and binding ability of the Y variants. The individual melting points and affinity values (K.sub.D) are shown in Tables 3-5.
(96) In mutation study 1, five mutants improved the stability with an increase in melting temperature between 1 to 5° C. The introduction of the non-charged residue Q in positions 35 and 39 in Y00032 increased the Tm as well as the α-helical content (
(97) In mutation study 2, promising mutations from study 1 were verified by mutations in the C4 binding Y variant Y00262. Q in positions 35 and 39 was shown to increase the Tm both as single mutations as well as in combination. The combination Q in position 35 and Q in position 39 was verified to increase the Tm also in insulin binding variants. Furthermore, A in position 19 was verified to have a positive impact on thermostability. All mutated variants were shown to have helical structure, and refolded reversibly after heating to 90° C. Results are summarized in Table 4.
(98) TABLE-US-00006 TABLE 3 Melting points and affinity constants of Y variants in mutation study 1 SEQ Y Y Tm K.sub.D ID NO: variant parental Mutation/s Target (° C.) (M) 1 Y00001 — — C4 57 n.a. 18 Y00262 Y00001 S23K C4 61 n.a. 90 Y00032 — — Insulin 49 6.3 × 10.sup.−8 110 Y00263 Y00032 D13K Insulin 43 1.3 × 10.sup.−7 111 Y00264 Y00032 K15Y Insulin 48 6.4 × 10.sup.−8 112 Y00265 Y00032 V17I Insulin 47 1.2 × 10.sup.−7 113 Y00266 Y00032 G18S Insulin 50 5.6 × 10.sup.−8 114 Y00267 Y00032 A19E Insulin 48 5.9 × 10.sup.−8 115 Y00268 Y00032 K22I Insulin 49 8.4 × 10.sup.−8 116 Y00269 Y00032 S23K Insulin 50 3.9 × 10.sup.−7 117 Y00270 Y00032 K22I + S23K Insulin 52 7.7 × 10.sup.−8 118 Y00271 Y00032 E26Q Insulin 49 4.7 × 10.sup.−8 119 Y00272 Y00032 A28G Insulin 34 1.1 × 10.sup.−7 120 Y00273 Y00032 E32Q Insulin 46 7.7 × 10.sup.−8 121 Y00274 Y00032 E35Q Insulin 53 5.6 × 10.sup.−8 122 Y00275 Y00032 E39Q Insulin 54 5.9 × 10.sup.−8 123 Y00276 Y00032 A43K Insulin 50 7.6 × 10.sup.−8 n.a. not analyzed
(99) TABLE-US-00007 TABLE 4 Melting points and affinity constants of Y variants in mutation study 2 SEQ Y Y Tm K.sub.D ID NO: variant parental Mutation/s Target (° C.) (M) 18 Y00262 — C4 61 n.a. 19 Y00282 Y00262 I17V C4 61 n.a. 20 Y00283 Y00262 N18S C4 61 n.a. 21 Y00284 Y00262 E19A C4 64 n.a. 22 Y00285 Y00262 I22K C4 57 n.a. 23 Y00286 Y00262 E26Q C4 62 n.a. 24 Y00287 Y00262 E35Q C4 62 n.a. 25 Y00288 Y00262 E39Q C4 66 n.a. 26 Y00289 Y00262 E35Q + E39Q C4 65 n.a. 27 Y00290 Y00262 E35Q + A43K C4 69 n.a. 28 Y00291 Y00262 E35Q + C4 66 n.a. E39Q + A43K 90 Y00032 — Insulin 49 6.3 × 10.sup.−8 124 Y00292 Y00032 S23K + Insulin 57 4.5 × 10.sup.−7 E35Q + E39Q 117 Y00270 — Insulin 52 7.7 × 10.sup.−8 125 Y00293 Y00270 E35Q + E39Q Insulin 56 4.9 × 10.sup.−7 126 Y00294 Y00270 E35Q + A43K Insulin 54 4.9 × 10.sup.−7 127 Y00295 Y00270 E35Q + Insulin 52 5.8 × 10.sup.−7 E39Q + A43K n.a. not analyzed
(100) In mutation study 3, different N-terminal and C-terminal extensions were shown to have a slightly positive effect on the thermostability, and Y variants with C-terminal extensions showed increased expression levels. Results are summarized in Table 5.
(101) TABLE-US-00008 TABLE 5 Melting points and affinity constants of Y variants in mutation study 3 N/C Expression SEQ Y Y terminal Tm K.sub.D (mg/g ID NO: variant parental Mutation tag Target (° C.) (M) pellet) 128 Y00296 Y00293 E26Q His.sub.6-GGS-Y##### Insulin 56 3.7 × 10.sup.−8 8.9 129 Y00296a Y00293 E26Q His.sub.6-GGS- Insulin 58 2.2 × 10.sup.−8 17 Y#####-ASYGS 130 Y00296b Y00293 E26Q His.sub.6-GGS- Insulin 56 3.1 × 10.sup.−8 9.2 Y#####-GYS 131 Y00296C Y00293 E26Q His.sub.6-GGS- Insulin 62 5.1 × 10.sup.−8 4.6 TIDEWL-Y##### 29 Y00297 Y00289 E26Q His.sub.6-GGS-Y##### C4 64 n.a. 23 30 Y00297a Y00289 E26Q His.sub.6-GGS- C4 65 n.a. 27 Y#####-ASYGS 31 Y00297b Y00289 E26Q His.sub.6-GGS- C4 69 n.a. 33 Y#####-GYS n.a. not analyzed
Example 5
Design and Construction of a Second Library
(102) Summary
(103) In this Example, a new library with a modified scaffold was designed and created. The outcome of the selections described in Example 3 from the library Ylib001Naive.I described in Example 2, together with the mutational studies performed in Example 4, were used as basis for the design of the new library. The library contained approximately 3.1×10.sup.10 individual clones.
(104) Materials and Methods
(105) Library Design:
(106) A second library was designed based on the results described in Example 3 and 4. In the library, 11 amino acid positions of the Y variant molecules were randomized (positions 20, 21, 24, 27, 29, 30, 33, 36, 37, 40 and 44 with reference to e.g. SEQ ID NO:159). Four oligonucleotides, two forward and two reverse complementary, both pairs having complementary 3′ ends, were generated using TRIM technology. These oligos were ordered from Ella Biotech GmbH (Martinsried, Germany).
(107) The DNA generated by the four separate oligonucleotides was a 117 bp long oligo, encoding an amino acid sequence partially randomized from helix two to helix three of ABD, with the sequence: 5′-GAT AGC AAA GGT GTT GGT GCA 001 001 ATT AAA 001 ATT CAG 002 GCA 002 002 GTT GAG 003 GTT CAA 001 001 AAA CAG 004 ATT CTG GCG 001 CTG CCG GCG AGC GGT AGC GTC-3′ (SEQ ID NO:164) where randomized codons are illustrated as 001 to 004. The different randomization strategies correspond to; 001) 18 possible amino acids, all except C and P, evenly distributed (5.6% each); 002) 19 possible amino acids, all except C, evenly distributed (5.3% each); 003) 19 possible amino acids, all except C, 50% of amino acid G and the rest evenly distributed (2.6% each); 004) 19 amino acids, all except C, 50% of amino acid A and the rest evenly distributed (2.6% each). A large number of errors are usually generated in longer oligos due to technical challenges during TRIM oligonucleotide synthesis. An overlap strategy for the oligos was therefore used, in which randomized positions 003 and 004 contained 50% of the amino acid G and A, respectively. In this way, the library could be assembled using two separate oligo pairs with a low number of errors and including all desired variable positions.
(108) The oligos were PCR amplified to introduce flanking restriction sites SacI and SalI. The resulting theoretical library size was 7.6×10.sup.13 variants.
(109) Library Construction:
(110) The phagemid vector pAY03686 was modified to contain the first part encoding amino acid residues 1 to 11 of helix one, followed by a SacI endonuclease cleavage site. The modified vector was denoted pAY04260.
(111) The library was constructed and verified essentially as described in Example 2, with the exception of using restriction endonucleases SacI-HF and SalI-HF (New England Biolabs) to cleave the fragment and the corresponding pAY04260 vector. The ligation reactions (approximately 200 ng DNA/transformation) were electroporated into electrocompetent E. coli XL1-Blue cells (Lucigen).
(112) Results
(113) Library Design:
(114) A second library was designed based on the findings described in Example 3 and 4. The amino acids used in the scaffold positions of the sequence and the distribution of variable amino acid residues in the binding positions were defined. A total of 11 amino acid positions were targeted for randomization, namely those corresponding to positions 20, 21, 24, 27, 29, 30, 33, 36, 37 40 and 44 in SEQ ID NO:159. The theoretical size of the designed library was 7.6×10.sup.13 different, unique Y variants.
(115) Library Construction:
(116) A new phagemid vector, denoted pAY04260, was constructed for monovalent display using the M13 filamentous phage coat protein III. The newly constructed vector contained DNA encoding the first 11 amino acids of the Y variants and was used for construction of the library. The library, or population, was denoted “Ylib002Naive.I”. The actual size of the library, determined by titration after transformation to E. coli XL1-Blue cells, was 3.1×10.sup.10 transformants. The library quality was tested by sequencing of 192 transformants and comparing their actual sequences with the theoretical design. Sequence analysis of individual library members verified a distribution of codons in accordance with the theoretical design. A library of potential binders in a novel scaffold was thus successfully constructed.
Example 6
Phage Display Selection and Screening from a Second Library
(117) Summary
(118) In this Example, C4, IL-6 and insulin were used as targets in phage display selections using the second phage library of Y variants. Selected clones were DNA sequenced, produced in E. coli as soluble protein fractions and assayed against each respective target using ELISA and SPR.
(119) Materials and Methods
(120) Production of Phage Stock:
(121) Production of phage stock was performed as follows. A glycerol stock containing the phagemid library Ylib002Naive.I in E. coli cells XL1 Blue was inoculated in 20 I of fermentor cultivation medium (30 g/l tryptic soy broth; 5 g/l yeast extract; 10 g/l glucose; 100 μg/ml carbenicillin; 10 μg/ml tetracycline hydrochloride). The culture was incubated at 37° C., air was supplemented (10 l/min), and the stirrer was set to keep the dissolved oxygen level above 30%. When the OD.sub.600 had reached 0.5, 16 liter of the culture was discarded. The remaining 4 liter culture was infected using a 10× molar excess of M13K07 helper phage. 16 liter of a new cultivation medium was added (3.05 g/l (NH.sub.4).sub.2SO.sub.4; 6.1 g/l yeast extract; 3.66 g/l K.sub.2HPO.sub.4; 5.48 g/l KH.sub.2PO.sub.4; 2.29 g/l Na.sub.3C.sub.6H.sub.5O.sub.7.2H.sub.2O), supplemented with 100 μg/ml carbenicillin, 3.2 ml/l of 1.217 M MgSO4, 0.9 ml/l of 25% NH.sub.4OH, and 1 μl/ml of a trace element solution (194 mM FeCl3; 55 mM ZnSO.sub.4; 10.6 mM CuSO.sub.4; 62 mM MnSO.sub.4; 47 mM CaCl.sub.2), dissolved in 1.2 M HCl), 0.2 mM thiamine, and 0.65 μl/ml of a vitamin solution (2.1 mM pantothenic acid; 3.6 mM choline chloride; 1.1 mM folic acid; 5.5 mM myo-inositol; 4.1 mM niacinamide; 0.13 mM riboflavin; 1.5 mM thiamine). After 60 min incubation, kanamycin was added to a concentration of 50 μg/ml and expression was induced by the addition of IPTG to a concentration of 100 μM. The cultivation temperature was lowered to 30° C. and 0.15 ml/l antifoam agent (Breox FMT 30) was added. pH was maintained at 7 through the automatic addition of % NH.sub.4OH, and a glucose-limited fed-batch cultivation was started where a 600 g/l glucose solution was fed to the reactor (15 g/h the first 20 h and then 75 g/h until the end of the cultivation). The culture was harvested 22 h after the addition of helper phage particles. The cells in the culture were removed by centrifugation (15,900 g, 50 min). The phage particles in the supernatant were precipitated twice in PEG/NaCl using standard procedures. Phage stocks were filtered using a 0.45 μm filter, dissolved in PBS and glycerol, and stored at −80° C. before use.
(122) Phage Display Selection of C4, IL-6 and Insulin Binding Y Variants from Ylib002Naive.I:
(123) C4 (Lee Biosolutions Inc, cat. no. 194-41), IL-6 (R&D Systems, cat. no. 206-IL-200/CF) and insulin (Roche, cat. no. 1376497) were biotinylated as described in Example 1. The phage stock described in this Example, displaying random variants of the library sequence on bacteriophage, was used to select C4, IL-6 and insulin binding polypeptides. Selection was performed essentially as described in Example 3 with the following exceptions. Selections were performed with targets separately only (no mix). Four rounds were used for insulin while five rounds were used for each of C4 and IL-6. Target concentrations and washing steps were performed according to Table 6.
(124) Selected and eluted phage particles were amplified as described below and new phage stocks were prepared between each cycle. Phage stock, i.e. phage particles entering the selection cycle, and eluted phage particles were titrated after each selection cycle.
(125) TABLE-US-00009 TABLE 6 Overview of the selections against C4, IL-6 and insulin using the Ylib002Naive.I library Phage stock from library Target Number of Selection or selection Target conc. Number overnight Cycle track track protein (nM) of washes washes 1 1 Ylib002Naive.I b-C4 100 2 1 2 Ylib002Naive.I b-IL-6 100 2 1 3 Ylib002Naive.I b-insulin 100 2 2 1-1 1 b-C4 50 4 2 2-1 2 b-IL-6 50 4 2 3-1 3 b-insulin 50 4 3 1-1-1 1-1 b-C4 10 8 3 2-1-1 2-1 b-IL-6 10 8 3 3-1-1 3-1 b-insulin 10 8 4 1-1-1-1 1-1-1 b-C4 1 12 4 1-1-1-2 1-1-1 b-C4 1 11 1 4 1-1-1-3 1-1-1 b-C4 5 12 4 1-1-1-4 1-1-1 b-C4 5 11 1 4 1-1-1-5 1-1-1 b-C4 2.5 12 4 1-1-1-6 1-1-1 b-C4 2.5 11 1 4 2-1-1-1 2-1-1 b-IL-6 1 12 4 2-1-1-2 2-1-1 b-IL-6 1 11 1 4 2-1-1-3 2-1-1 b-IL-6 5 12 4 2-1-1-4 2-1-1 b-IL-6 5 11 1 4 2-1-1-5 2-1-1 b-IL-6 2.5 12 4 2-1-1-6 2-1-1 b-IL-6 2.5 11 1 4 1-1-1-1 1-1-1 b-insulin 5 12 4 1-1-1-2 1-1-1 b-insulin 1 12 4 1-1-1-3 1-1-1 b-insulin 1 11 1 5 1-1-1-3-1 1-1-1-3 b-C4 1 12 5 1-1-1-3-2 1-1-1-3 b-C4 1 11 1 5 2-1-1-3-1 2-1-1-3 b-IL-6 1 12 5 2-1-1-3-2 2-1-1-3 b-IL-6 1 11 1
(126) Amplification of Phage Particles Between Rounds:
(127) Amplification of phage particles between rounds was performed as described in Example 3 with the exception that carbenicillin was used at a concentration of 100 μg/ml instead of ampicillin during cultivations. The amount of bacteria used for infection was approximately 100-200× excess compared to the number of eluted phage particles. In selection cycle four (all targets) and selection cycle five (C4 and IL-6), ER2738 bacteria (C4) or XL1-Blue (IL-6 and insulin) were used for infection and bacteria were spread on TBAB plates supplemented with 200 μg/ml ampicillin in order to form single colonies to be used in ELISA screening.
(128) Production of Soluble Y Variants Supernatants and Sequencing:
(129) The Y variants were produced as soluble proteins as described in Example 3. In parallel with the ELISA screening, all clones were sequenced as described in Example 1
(130) Screening of Y Variants Using ELISA and SPR:
(131) The binding of Y variants to IL-6 and insulin, respectively, was analyzed in ELISA assays as described in Example 3 and using 300 nM IL-6 or insulin.
(132) Produced Y variants from the C4 and insulin selections were screened for target binding using a Biacore 8K instrument (GE Healthcare). Anti-Z03639 goat antibody was immobilized by amine coupling onto the carboxylated dextran layer on surfaces of CM-5 chips to levels of 14500-17500 RU. Prepared supernatants were diluted 10× in HBS-EP+ and injected at a flow rate of 10 μl/min for 5 min, followed by injection of a single concentration of target proteins (50 nM of C4 and 300 nM of insulin) for 5 min. The dissociation of targets was monitored for 7 min and the surfaces were thereafter regenerated with two injections of 30 μl glycine-HCl pH 2.5. Before performing the kinetic analyses, the signal from target injected over a reference surface containing goat anti-Z but no Y sample was subtracted from the sensorgrams of Y####-Z03639 binding to target. Target-binding analyses were performed using the Biacore 8K Evaluation Software. Binding clones showing the slowest off-rate were chosen for further analysis.
(133) Subcloning and Protein Production of a Subset of Y Variants with a His.sub.6-Tag:
(134) A subcloning strategy was applied on a subset of ELISA and/or SPR positive variants for construction of monomeric Y variant molecules according to the methods described in Example 1. Proteins were expressed and purified using an N-terminal His.sub.6-tag according to the methods described in Example 1. The Y variant gene fragments were subcloned into an expression vector, resulting in the encoded sequences MGSSHHHHHHGSS-[Y#####]-A (SEQ ID NO:172).
(135) CD and SPR Analyses of Cloned and Purified Y Variants:
(136) Produced Y variants were subjected to CD and SPR analyses according to the methods described in Example 1. In addition, CD spectra of selected IL-6 binding variants were also recorded at 60, 70, 80 and 90° C.
(137) Results
(138) Phage Display Selection of C4, IL-6 and Insulin Binding Y Variants from Ylib002Naive.I:
(139) Phage display selection was performed with a newly designed library (Example 5) against the target proteins C4, IL-6 and insulin. Individual clones were obtained after four and five cycles of phage display selection.
(140) Sequencing:
(141) Sequencing was performed for clones obtained after four and five cycles of selection. Each variant was given a unique identification number #####, and individual variants are referred to as Y#####. The amino acid sequences of the Y variants are listed in
(142) Screening of Y Variants Using ELISA and SPR:
(143) The clones obtained after four or five cycles of selection were produced in 96-well plates as soluble proteins. Y variants were screened for b-IL-6 or b-insulin binding activity in ELISA. Several unique Y variants were found to give a response corresponding to approximately 2× the negative control or more against b-IL-6 or b-insulin at a concentration of 300 nM, respectively. The average response of the negative controls was 0.083 AU and 0.054 AU for b-IL-6 and insulin, respectively.
(144) A selection of C4 and insulin binding Y variants was submitted to a kinetic screening using Biacore 8K as described in Example 1. A single concentration of C4 (50 nM) or insulin (300 nM) was injected over each Y#####-Z03639 captured from soluble extracts on a sensor chip surface containing an anti-Z03639 antibody. Y variants having a positive response in ELISA or showing the slowest off rate curves in SPR analysis were chosen for subcloning.
(145) CD and SPR Analyses of Purified Y Variants:
(146) Produced Y variants were subjected to CD and SPR analyses. Calculated kinetic parameters, affinities and Tm values as well as the estimated percentage of protein expressed as soluble product are presented in Table 7. Examples of melting curves as well as CD spectra recorded before and after the VTM are illustrated in
(147) TABLE-US-00010 TABLE 7 Expression data, calculated kinetic parameters, K.sub.D and Tm values Estimated Expression soluble SEQ Y (mg/g fraction k.sub.a k.sub.d K.sub.D Tm ID NO variant Target pellet) (%) (1/Ms) (1/s) (M) (° C.) 32 Y00792 C4 30 100 2.7 × 10.sup.4 1.7 × 10.sup.−3 6.2 × 10.sup.−8 58 34 Y02303 C4 3.3 100 9.6 × 10.sup.4 2.1 × 10.sup.−1 2.2 × 10.sup.−6 43 35 Y02309 C4 24 100 3.6 × 10.sup.4 2.5 × 10.sup.−3 7.0 × 10.sup.−8 56 36 Y02310 C4 15 100 n.a. n.a. n.a. 56 37 Y02330 C4 17 100 9.6 × 10.sup.4 4.6 × 10.sup.−3 4.8 × 10.sup.−8 49 38 Y02337 C4 6.1 100 4.2 × 10.sup.5 8.1 × 10.sup.−1 1.9 × 10.sup.−6 31 39 Y02358 C4 28 100 9.0 × 10.sup.4 1.4 × 10.sup.−3 1.6 × 10.sup.−8 51 72 Y02374 IL-6 21 100 1.4 × 10.sup.4 1.7 × 10.sup.−3 1.2 × 10.sup.−7 n.d.* 74 Y02415 IL-6 19 100 9.7 × 10.sup.3 1.5 × 10.sup.−3 1.6 × 10.sup.−7 n.d.* 75 Y02444 IL-6 25 100 9.0 × 10.sup.4 5.5 × 10.sup.−3 6.1 × 10.sup.−8 n.d.* 76 Y02447 IL-6 22 100 1.1 × 10.sup.4 1.7 × 10.sup.−3 1.6 × 10.sup.−7 n.d.* 78 Y02465 IL-6 12 100 7.5 × 10.sup.3 2.1 × 10.sup.−3 2.7 × 10.sup.−7 n.d.* 79 Y02495 IL-6 18 100 7.1 × 10.sup.3 1.7 × 10.sup.−3 2.4 × 10.sup.−7 n.d.* 132 Y00299 Insulin 34 75 3.1 × 10.sup.5 2.0 × 10.sup.−3 6.5 × 10.sup.−9 56 133 Y00301 Insulin 24 83 2.9 × 10.sup.7 5.6 × 10.sup.−2 .sup. 9.4 × 10.sup.−10 62 134 Y00304 Insulin 20 33 8.5 × 10.sup.4 1.1 × 10.sup.−3 2.1 × 10.sup.−8 41 135 Y00310 Insulin 31 86 1.7 × 10.sup.5 6.3 × 10.sup.−3 3.7 × 10.sup.−8 54 136 Y00330 Insulin 6.8 67 3.0 × 10.sup.4 3.7 × 10.sup.−3 1.2 × 10.sup.−7 56 138 Y00345 Insulin 18 80 2.9 × 10.sup.5 1.7 × 10.sup.−2 6.1 × 10.sup.−8 53 141 Y00356a Insulin 15.0 60 3.4 × 10.sup.7 6.1 × 10.sup.−3 .sup. 1.7 × 10.sup.−10 56 142 Y00358 Insulin 22 97 2.2 × 10.sup.5 2.3 × 10.sup.−3 1.0 × 10.sup.−8 61 144 Y00361 Insulin 6.3 100 1.2 × 10.sup.4 3.6 × 10.sup.−3 3.1 × 10.sup.−7 52 n.a. not analyzed n.d. not determinable *Tm not determinable due to small differences in CD amplitude upon temperature increase as partial structure is observed also at 90° C.
Example 7
Mutational Studies of C4, IL-6 and Insulin Binding Y Variants from the Second Library
(148) Summary
(149) This Example describes a set of two additional sequential mutational studies performed in order to further optimize the properties of the population sequence, primarily in terms of high production yields and high solubility, while maintaining a high thermostability and binding ability over a broad range of Y variants.
(150) Materials and Methods
(151) Cloning of Mutated Y Variants:
(152) In a fourth mutational study, different mutations were introduced in Y variants, to evaluate the impact of these mutations on protein expression. This was performed using the insulin binding Y variant Y00356 (SEQ ID NO:140) as template. The mutations were introduced in the scaffold positions 9 (X.sub.sc1), 15 (X.sub.sc3), and 26 (X.sub.sc4), and more precisely the mutations I9L, K15Y and Q26E. All variants were cloned with an N-terminal His.sub.6 tag and the genetic constructs obtained encoded polypeptides in the format MGSSHHHHHHGSS-[Y#####] (SEQ ID NO:168). Cloning was done according to the methods described in Example 1.
(153) In a fifth mutational study, the mutations I9L, K15Y and Q26E were further evaluated with respect to their potential impact on protein expression and stability using additional Y variants. The mutations K15Y and Q26E were also assessed in combination. Retaining a tyrosine at position 15 would guarantee the presence of an aromatic residue, which would be convenient in the analysis of selected binding polypeptides that happen to lack aromatic residues in the target binding positions. The study was performed using the C4 binding Y variants Y00792 (SEQ ID NO:32), Y02309 (SEQ ID NO:35) and Y02330 (SEQ ID NO:37), the IL-6 binding Y variants Y02374 (SEQ ID NO:72), Y02415 (SEQ ID NO:74) and Y002444 (SEQ ID NO:75) and the insulin binding Y variants Y00301 (SEQ ID NO:133), Y00310 (SEQ ID NO:135) and Y00358 (SEQ ID NO:142) as templates. All variants were cloned with an N-terminal His.sub.6 tag and the genetic constructs obtained encoded polypeptides in the format MGSSHHHHHHGSS-[Y#####]-A (SEQ ID NO:172). Cloning was done according to the methods described in Example 1.
(154) Production and Characterization of Mutated Y Variants:
(155) The Y variants were cloned, produced and characterized according to the general methods described in Example 1. Produced Y variants with point mutations were subjected to CD and SPR analyses as described in Example 1. In addition, CD spectra of the IL-6 binding variants were also recorded at 60, 70, 80 and 90° C.
(156) Results
(157) Production and Characterization of Mutated Y Variants:
(158) Produced Y variants from mutation studies 4 and 5 were subjected to SPR and/or CD analyses to assess the effect of the different point mutations on stability and binding ability of the Y variants. Furthermore, protein expression levels and fraction of soluble product were monitored. The expression levels, solubility, melting points, kinetic parameters and affinity values (K.sub.D) for the respective Y variant analyzed are summarized in Table 8 and 9, for the fourth and fifth mutational study, respectively.
(159) In the fourth mutational study, the mutations K15Y and Q26E were each shown to almost double the expression levels, whereas the mutation I9L increased solubility during expression.
(160) TABLE-US-00011 TABLE 8 Expression data and melting points of Y variants in mutation study 4 Estimated Expression soluble SEQ Y Y (mg/g fraction Tm ID NO: variant parental Mutation Target pellet) (%) (° C.) 140 Y00356 — Insulin 22 43 57 146 Y02674 Y00356 K15Y Insulin 42 40 55 147 Y02676 Y00356 Q26E Insulin 42 55 56 148 Y02683 Y00356 I9L Insulin 20 92 n.a. n.a. not analyzed
(161) In the fifth mutational study, single and double mutated Y variants were successfully expressed as soluble proteins, but the expression levels varied. All variants refolded reversibly after heating to 90° C. The IL-6 binding variants showed partial structure also at 90° C. I in position 9 was shown to be preferred over L. The preference of K versus Y in position 15 and Q versus E in position 26 varied depending on the Y variant.
(162) TABLE-US-00012 TABLE 9 Expression data, calculated kinetic parameters, K.sub.D and Tm values of Y variants in mutation study 5 Est Expression soluble SEQ Y Y (mg/g fraction k.sub.a k.sub.d K.sub.D Tm ID NO: variant parental Mutation(s) Target pellet) (%) (1/Ms) (1/s) (M) (° C.) 40 Y02685 Y00792 I9L C4 18 100 n.a. n.a. n.a. 53 41 Y02686 Y00792 K15Y C4 22 100 n.a. n.a. n.a. 59 42 Y02687 Y00792 Q26E C4 41 100 n.a. n.a. n.a. 57 43 Y02688 Y02330 I9L C4 5.8 100 n.a. n.a. n.a. 50 44 Y02689 Y02330 K15Y C4 13 100 n.a. n.a. n.a. 53 45 Y02690 Y02330 Q26E C4 13 100 n.a. n.a. n.a. 53 46 Y02691 Y00792 K15Y C4 25 100 n.a. n.a. n.a. 58 Q26E 47 Y02692 Y02309 K15Y C4 27 100 n.a. n.a. n.a. 57 Q26E 48 Y02693 Y02330 K15Y C4 19 100 n.a. n.a. n.a. 52 Q26E 81 Y02697 Y02444 I9L IL-6 23 100 5.0 × 10.sup.4 9.0 × 10.sup.−3 1.5 × 10.sup.−7 n.d.* 82 Y02698 Y02444 K15Y IL-6 12 100 2.8 × 10.sup.4 3.3 × 10.sup.−3 1.2 × 10.sup.−7 n.d.* 83 Y02699 Y02444 Q26E IL-6 26 100 1.9 × 10.sup.4 2.9 × 10.sup.−3 2.0 × 10.sup.−7 n.d.* 84 Y02700 Y02415 I9L IL-6 22 100 4.6 × 10.sup.4 2.5 × 10.sup.−3 6.2 × 10.sup.−8 n.d.* 85 Y02701 Y02415 K15Y IL-6 23 100 2.1 × 10.sup.4 3.6 × 10.sup.−3 2.1 × 10.sup.−7 n.d.* 86 Y02702 Y02415 Q26E IL-6 14 100 1.6 × 10.sup.4 7.0 × 10.sup.−3 4.4 × 10.sup.−7 n.d.* 87 Y02703 Y02444 K15Y IL-6 21 100 2.1 × 10.sup.4 2.5 × 10.sup.−3 1.2 × 10.sup.−7 n.d.* Q26E 149 Y02709 Y00358 I9L Insulin 25 100 2.7 × 10.sup.4 1.0 × 10.sup.−2 3.8 × 10.sup.−7 54 150 Y02710 Y00358 K15Y Insulin 33 100 1.9 × 10.sup.5 2.7 × 10.sup.−3 1.4 × 10.sup.−8 61 151 Y02711 Y00358 Q26E Insulin 34 100 1.5 × 10.sup.3 8.0 × 10.sup.−3 5.4 × 10.sup.−8 62 153 Y02713 Y00310 K15Y Insulin 30 100 1.1 × 10.sup.5 1.4 × 10.sup.−2 1.9 × 10.sup.−7 51 154 Y02714 Y00310 Q26E Insulin 14 100 2.9 × 10.sup.5 1.6 × 10.sup.−1 5.4 × 10.sup.−7 53 155 Y02715 Y00358 K15Y Insulin 27 100 6.7 × 10.sup.3 1.9 × 10.sup.−3 2.8 × 10.sup.−7 61 Q26E 156 Y02716 Y00301 K15Y Insulin 6.8 100 2.7 × 10.sup.7 2.1 × 10.sup.−1 7.6 × 10.sup.−9 58 Q26E 157 Y02717 Y00310 K15Y Insulin 3.6 100 3.3 × 10.sup.4 2.0 × 10.sup.−2 6.1 × 10.sup.−7 52 Q26E n.a. not analyzed n.d. not determinable *Tm not determinable due to small differences in CD amplitude upon temperature increase as partial structure is observed also at 90° C.
Example 8
Generation and Analysis of Y Variants Fused to an Albumin Binding Domain
(163) Summary
(164) This Example describes the cloning and production of Y variants in fusion with an albumin binding domain, advantageous for extending the in vivo half-life of Y variants.
(165) Materials and Methods
(166) Cloning of Y Variants in Fusion with PP013:
(167) Cloning was performed using methods known in the art. In brief, DNA encoding Y variants and the ABD variant PP013 were ordered as fragment genes from Twist Bioscience and restricted using the enzymes NdeI and NotI-HF (New England Biolabs). An expression vector (with T7 promoter) was prepared and digested with the same restriction enzymes. Ligation, transformation and sequencing were performed as described in Example 1. The constructs encoded by the expression vector were GSS-[Y#####]-G4S-PP013 or GSS-PP013-G4S-[Y#####]. One example of each construct is listed in the sequence listing as SEQ ID NO:161 and SEQ ID NO:162, respectively.
(168) Production of Y Variants in Fusion with PP013:
(169) Expression of the fusion proteins was performed essentially as described in Example 1. Cell pellets containing the expressed protein are re-suspended in TST-buffer (25 mM Tris-HCl, 1 mM EDTA, 200 mM NaCl, 0.05% Tween20, pH 8.0) and the cells subjected to lysis. Clarified supernatants are applied to agarose immobilized with an anti-PP013 ligand (as described in WO2014/064237). After washing with TST-buffer and 5 mM NH.sub.4Ac pH 5.5 buffer, the PP013 fused Y variants are eluted with 0.1 M HAc. Further purification may be performed using RPC-HPLC. The correct identity of the respective purified protein is confirmed using SDS-PAGE and LC/MS analysis.
(170) Binding Analysis:
(171) Verification of binding to the target protein of the Y variant moiety of the fusion protein, as well as binding to albumin by the albumin binding moiety, is carried out by performing Biacore analyses essentially as described in Example 1.
(172) Pharmacokinetic Analysis:
(173) The serum half-lives of PP013-fused Y variants are investigated in mice. The respective fusion protein are administered intravenously (i.v.) to NMRI mice (Charles River) at a dose of ˜100 nmol/kg body weight. Sera from groups of three mice are obtained at 0.08, 6, 18, 78, 120, 168 and 240 hours after administration. The concentration of respective fusion protein is determined by ELISA.
(174) Results
(175) The results of the binding analyses are expected to show binding both to the target of the Y variant and to albumin. Furthermore, the fusion to PP013 and other albumin binding domain variants is expected to result in extended in vivo half-life.
Itemized Listing of Embodiments
(176) 1. A population of polypeptide variants based on a common scaffold, each polypeptide in the population comprising the scaffold amino acid sequence
(177) TABLE-US-00013 (SEQ ID NO: 165) X.sub.sc1AELDX.sub.sc2X.sub.sc3GVG AXXIKXIX.sub.sc4XA XXVEXVQXXK QXI LAX
wherein, independently of one another, X.sub.sc1 is a scaffold amino acid residue selected from I and L; X.sub.sc2 is a scaffold amino acid residue selected from C and S; X.sub.sc3 is a scaffold amino acid residue selected from K and Y; X.sub.sc4 is a scaffold amino acid residue selected from E and Q; and each X individually is a binding amino acid residue corresponding to any amino acid residue.
(178) 2. A population according to item 1, in which each polypeptide comprises the scaffold amino acid sequence
(179) TABLE-US-00014 (SEQ ID NO: 166) LAEAKEAAX.sub.sc1A ELDX.sub.sc2X.sub.sc3GVGAX XIKXIX.sub.sc4XAXX VEX VQXXKQX ILAXLP
wherein X.sub.sc1, X.sub.sc2, X.sub.sc3, X.sub.sc4 and each individual X are as defined in item 1.
(180) 3. A population according to any preceding item, in which X.sub.sc1 is I.
(181) 4. A population according to any preceding item, in which X.sub.sc1 is L.
(182) 5. A population according to any preceding item, in which X.sub.sc2 is S.
(183) 6. A population according to any preceding item, in which X.sub.sc2 is C.
(184) 7. A population according to any preceding item, in which X.sub.sc3 is K.
(185) 8. A population according to any preceding item, in which X.sub.sc3 is Y.
(186) 9. A population according to any preceding item, in which X.sub.sc4 is Q.
(187) 10. A population according to any preceding item, in which X.sub.sc4 is E.
(188) 11. A population according to any preceding item, which comprises at least 1×10.sup.4 unique polypeptide molecules.
(189) 12. A population according to item 11, which comprises at least 1×10.sup.6 unique polypeptide molecules.
(190) 13. A population according to item 12, which comprises at least 1×10.sup.8 unique polypeptide molecules.
(191) 14. A population according to item 13, which comprises at least 1×10.sup.10 unique polypeptide molecules.
(192) 15. A population according to item 14, which comprises at least 1×10.sup.12 unique polypeptide molecules.
(193) 16. A population according to item 15, which comprises at least 1×10.sup.14 unique polypeptide molecules.
(194) 17. A population according to item 16, which comprises at least 1×10.sup.15 unique polypeptide molecules.
(195) 18. A population of polynucleotides, characterized in that each member thereof encodes a member of a population of polypeptides according to any one of items 1-17.
(196) 19. A combination of a polypeptide population according to any one of items 1-17 with a polynucleotide population according to item 18, 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.
(197) 20. A combination according to item 19, wherein said means for genotype-phenotype coupling comprises a phage display system.
(198) 21. A combination according to item 19, wherein said means for genotype-phenotype coupling comprises a cell surface selection display system.
(199) 22. A combination according to item 21, wherein said cell surface display system comprises prokaryotic cells.
(200) 23. A combination according to item 22, wherein said prokaryotic cells are Gram.sup.+ cells.
(201) 24. A combination according to item 21, wherein said cell surface display system comprises eukaryotic cells.
(202) 25. A combination according to item 24, wherein said eukaryotic cells are yeast cells.
(203) 26. A combination according to item 19, wherein said means for genotype-phenotype coupling comprises a cell-free display system.
(204) 27. A combination according to item 26, wherein said cell free display system comprises a ribosome display system.
(205) 28. A combination according to item 26, wherein said cell free display system comprises an in vitro compartmentalization display system.
(206) 29. A combination according to item 26, wherein said cell free display system comprises a system for cis display.
(207) 30. A combination according to item 26, wherein cell free display system comprises a microbead display system.
(208) 31. A combination according to item 19, wherein said means for genotype-phenotype coupling comprises a non-display system.
(209) 32. A combination according to item 31, wherein said non-display system is protein-fragment complementation assay.
(210) 33. A method for selecting a desired polypeptide having an affinity for a predetermined target from a population of polypeptides, comprising the steps:
(211) (a) providing a population of polypeptides according to any one of items 1-17;
(212) (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
(213) (c) selecting, on the basis of said specific interaction, the at least one desired polypeptide from the remaining population of polypeptides.
(214) 34. A method according to item 33, wherein step (a) comprises the preparatory steps of providing a population of polynucleotides according to item 18 and expressing said population of polynucleotides to yield said population of polypeptides.
(215) 35. A method according to item 34, 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.
(216) 36. A method according to item 35, wherein said means for genotype-phenotype coupling is as defined in any one of items 20-32.
(217) 37. 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 item 35; and isolating the thus separated polynucleotide encoding the desired polypeptide.
(218) 38. 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 item 37; and sequencing the polynucleotide to establish by deduction the amino acid sequence of said desired polypeptide.
(219) 39. A method for selecting and identifying a desired polypeptide having an affinity for a predetermined target from a population of polypeptides, comprising the steps:
(220) (a) synthesizing each member of a population of polypeptides according to any one of items 1-17 on a separate carrier or bead;
(221) (b) selecting or enriching the carriers or beads based on the interaction of the polypeptide with the predetermined target; and
(222) (c) identifying the polypeptide by protein characterization methodology.
(223) 40. A method according to item 39, wherein the protein characterization methodology used in step (c) is mass spectrometric analysis.
(224) 41. A 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 38 or selecting and identifying a desired polypeptide using the method according to item 39 or 40; and producing said desired polypeptide.
(225) 42. A method according to item 41, wherein said production is carried out using chemical synthesis of the desired polypeptide de novo.
(226) 43. A method according to item 41, wherein said production is carried out using recombinant expression of a polynucleotide encoding the desired polypeptide.
(227) 44. A method for production of a desired polypeptide having an affinity for a predetermined target, comprising the steps:
(228) (a1) isolating a polynucleotide encoding said desired polypeptide using the method according to item 37; or
(229) (a2) backtranslating a polypeptide identified using the selection and identification method according to item 39 or 40; and
(230) (b), following either (a1) or (a2), expressing the thus isolated polynucleotide to produce said desired polypeptide.
(231) 45. Polypeptide comprising an amino acid sequence which is at least 97% identical to
(232) TABLE-US-00015 (SEQ ID NO: 165) X.sub.1AELDX.sub.6X.sub.7GVG AX.sub.12X.sub.13IKX.sub.16IX.sub.18X.sub.19A X.sub.21X.sub.22VEX.sub.25VQX.sub.28 X.sub.29K QX.sub.32ILAX.sub.36
wherein, independently of one another, X.sub.1 is selected from I and L; X.sub.6 is selected from C and S; X.sub.7 is selected from K and Y; X.sub.18 is selected from E and Q; and each of X.sub.12, X.sub.13, X.sub.16, X.sub.19, X.sub.21, X.sub.22, X.sub.25, X.sub.28, X.sub.29, X.sub.32 and X.sub.36 is any amino acid residue.
(233) 46. Polypeptide according to item 45, which comprises an amino acid sequence which is at least 97% identical to
(234) TABLE-US-00016 (SEQ ID NO: 166) LAEAKEAA X.sub.1AELDX.sub.6X.sub.7GVG AX.sub.12X.sub.13IKX.sub.16IX.sub.18X.sub.19AX.sub.21X.sub.22 VEX.sub.26VQX.sub.28X.sub.29K QX.sub.32ILAX.sub.36 LP
(235) wherein all amino acid residues denoted X are as defined in item 45.
(236) 47. Polypeptide according to any one of items 45-46, in which X.sub.1 is I.
(237) 48. Polypeptide according to any one of items 45-46, in which X.sub.1 is L.
(238) 49. Polypeptide according to any one of items 45-48, in which X.sub.6 is S.
(239) 50. Polypeptide according to any one of items 45-48, in which X.sub.6 is C.
(240) 51. Polypeptide according to any one of items 45-50, in which X.sub.7 is K.
(241) 52. Polypeptide according to any one of items 45-50, in which X.sub.7 is Y.
(242) 53. Polypeptide according to any one of items 45-52, in which X.sub.18 is E.
(243) 54. Polypeptide according to any one of items 45-52, in which X.sub.18 is Q.
(244) 55. Polypeptide according to any one of items 45-54, in which the amino acid residue in position 11 is A.
(245) 56. Polypeptide according to any one of items 45-55 further comprising a second polypeptide moiety, such that the polypeptide is a fusion polypeptide comprising a first moiety which fulfils the sequence definition of any one of items 45-55, and a second moiety with a desired function.
(246) 57. Polypeptide according to item 56, in which said second moiety is a polypeptide domain with binding affinity for albumin.
(247) 58. Polypeptide according to item 57, in which said polypeptide domain with binding affinity for albumin is a naturally occurring albumin binding domain from streptococcal Protein G, or an engineered variant thereof with retained or improved albumin binding affinity.
(248) 59. Polynucleotide encoding a polypeptide according to any one of items 45-58.
(249) 60. Method of producing a polypeptide according to any one of items 45-58, comprising the step of expressing a polynucleotide according to item 59.