HK1195337B

HK1195337B - Amino acid sequence presenting fusion polypeptide and its use

Info

Publication number: HK1195337B
Application number: HK14108864.0A
Authority: HK
Inventors: Herbert Andres; David CASAGOLDA VALLRIBERA; Hartmut Duefel; Michael Gerg; Christian Scholz; Michael Schraeml
Original assignee: F. Hoffmann-La Roche Ag
Priority date: 2011-05-05
Filing date: 2012-05-04
Publication date: 2017-10-13

Description

Amino acid sequences presenting fusion polypeptides and uses thereof

Herein is reported a fusion polypeptide comprising one or more fragments of one or more peptidyl prolyl cis/trans isomerases or FKBP family members, and its use in methods for antibody screening/selection, for epitope mapping, and its use as an immunogen in the generation of antibodies specifically binding to immunogenic peptides or secondary structures presented by the fusion polypeptide.

Background

In recent years, the production of therapeutic antibodies has steadily increased, and it is likely that therapeutic antibodies will become the largest group of therapies available for treating various diseases in the near future. The impact of therapeutic antibodies derives from their specificity, such as specific target recognition and binding functions.

Antibodies can be obtained from experimental animals immunized with an immunogen. The immunogen is in most cases a polypeptide or a fragment of a polypeptide. To provide the immunogen in sufficient quantity and purity, recombinantly produced immunogens can be utilized.

In general, prokaryotic and eukaryotic cells can be used for recombinant production of polypeptides. The recombinant polypeptide may be obtained in soluble form or as a precipitate (inclusion body). Prior to chromatographic purification, the insoluble polypeptide contained in the inclusion bodies must be solubilized.

Typically, the immunogen is a synthetic or peptidic or recombinantly produced or fusion or chimeric or support conjugated polypeptide. For immunization, the immunogen may be administered alone or in combination with an adjuvant (e.g., Freund's adjuvant).

Knappe, T.A. et al (J.mol.biol.368(2007)1458-1468) reported that the Flap region of FKBP12 could be replaced by the IF domain of the structurally related E.coli (E.coli) chaperone SlyD. The chimeric FKBP12-SlyD fusion polypeptide has a 200-fold increased peptidyl-prolyl cis/trans isomerase activity compared to the isolated polypeptide.

Coli SlyD and FKBP12 (wild type and mutants C23A and C23S) can be produced recombinantly in soluble form at high yields in E.coli (Standaert, R.F., et al, Nature346(1990) 671-674).

FKBP and E.coli SlyD derived from thermophilic organisms can be used as chaperones in the recombinant expression of fusion polypeptides in E.coli (Ideno, A. et al, appl.Microbiol.Biotechnol.64(2004) 99-105). Coli SlyD and FKBP12 polypeptides are reversibly folded polypeptides (Scholz, C. et al, J.biol.chem.271(1996) 12703-12707).

The amino acid sequence of FKBP12 polypeptide comprises a single tryptophan residue at position 60. Therefore, the structural integrity of the FKBP12 mutant can be analyzed simply by analyzing tryptophan fluorescence (Decenzo, M.T., et al, Protein Eng.9(1996) 173-180). Testing of FKBP12 mutants for retained catalytic activity can be performed by measuring retained rotamase activity (Brecht, S. et al, Neuroscience120(2003) 1037-1048; Schories, B. et al, J.Pept. Sci.13(2007) 475-480; Timerman, A.P. et al, J.biol. chem.270(1995) 2451-2459). It is also possible to determine the structural integrity of FKBP12 mutants by determining FK506 or rapamycin binding (Decenzo, M.T., et al, Protein Eng.9(1996) 173-180).

McNamara, A. et al (J.org.chem.66(2001)4585-4594) report peptides that are restricted by aliphatic linkage between two C (. alpha.) positions: design, synthesis and unexpected conformational properties of i, (i +4) -linker peptides.

Suzuki et al (Suzuki, R. et al, J.mol.biol.328(2003) 1149) -1160) report the three-dimensional solution structure of bifunctional archaea (archaic) FKBP with peptidyl-prolyl cis/trans isozyme and chaperone-like activity. Expression vectors, hosts, fusion polypeptides, methods for producing fusion polypeptides and methods for producing proteins are reported in EP 1516928. Knappe, t.a. et al report that insertion of chaperone domain converts FKBP12 into a potent protein folding catalyst (j.mol.biol.368(2007) 1458-. Chimeric fusion polypeptides with excellent chaperone and folding activities are reported in WO 2007/077008. In WO 03/000878, the use of FKBP chaperones as expression tools is reported. In EP1621555, immunogens, compositions for immunological use and methods for producing antibodies therewith are reported. Rebuzzini, G. (Ph. study (2009) at the university of Milano-Bicocca (Italy)) reported the study of the helicase domain of hepatitis C virus NS3 for application to chemiluminescent immunoassays.

In WO 2007/077008, a chimeric fusion protein having excellent chaperone activity and folding activity is reported. Knappe et al (Knappe, T.A. et al, J.mol.biol.368(2007)1458-1468) report the conversion of FKBP12 to a potent protein folding catalyst by insertion of a chaperone domain.

Summary of The Invention

A fusion polypeptide as reported herein is a fusion polypeptide comprising: i) one or more moieties derived from one (i.e., the same) or different polypeptide having PPI enzyme activity or belonging to the FKBP family; and ii) an immunogenic polypeptide interposed therebetween.

The fusion polypeptides reported herein may be used to immunize animals to generate antibodies that specifically bind to immunogenic polypeptides inserted into one or more portions derived from one or more polypeptides having PPI enzymatic activity or belonging to the FKBP family.

One aspect as reported herein is a fusion polypeptide of formula I

NH₂-S₂-X₁-S₁-COOH (formula I)

Wherein

X₁Comprises a random amino acid sequence, or comprises an amino acid sequence derived from a first polypeptide;

S₂and S₁Is a non-overlapping amino acid sequence derived from a second polypeptide; and is

-represents a peptide bond;

wherein the second polypeptide is a polypeptide having peptidyl-prolyl cis/trans isomerase activity (PPI enzyme activity), or is derived from a family of FKBP domains.

It has been found that antibodies that specifically bind to internal (so-called hidden or buried) epitopes of a (naturally occurring) amino acid sequence can be obtained with the fusion polypeptides reported herein. Internal epitopes are inaccessible in classical immune protocols because these epitopes are accessible only upon activation of an antigenic polypeptide (e.g. a receptor) and concomitant conformational changes, for example. Furthermore, antibodies can be obtained that specifically bind immunogenic polypeptides derived from structures that would otherwise be difficult to provide in sufficient quantity or quality.

The fusion polypeptides reported herein are chimeric, recombinant polypeptides which can be used for peptide, secondary and tertiary structure display, e.g. in methods for antibody screening/selection or for epitope mapping, and as immunogens for generating antibodies specifically binding to presented antigenic amino acid sequences or secondary structures. The polypeptides reported herein can be recombinantly produced, thermodynamically stable, monomeric, and soluble in aqueous solution.

One aspect as reported herein is a fusion polypeptide of formula II

NH₂-S₄-X₂-S₃-S₂-X₁-S₁-S₀-COOH (formula II)

Wherein

S₂and S₁Is a non-overlapping amino acid sequence derived from a second polypeptide;

S₃and S₀Absent, or derived from a third polypeptide;

S₄lacking, or derived from, an amino acid sequence of a fourth polypeptide;

X₂absent, or a peptide linker sequence; and is

-represents a peptide bond;

wherein said second polypeptide and said third and fourth polypeptides are different from each other and are polypeptides having peptidyl-prolyl cis/trans isomerase activity (PPI enzyme activity) or are polypeptides derived from the FKBP domain family.

In one embodiment of all aspects reported herein, the second polypeptide having peptidyl-prolyl cis/trans isomerase activity or derived from the FKBP domain family is SlyD.

In one embodiment of all aspects reported herein, the second polypeptide is a polypeptide from a thermophilic organism.

In one embodiment, the thermophilic organism is a thermophilic bacterium. In one embodiment, the thermophilic bacterium is from the family Thermus (Thermaceae). In one embodiment, the thermophilic organism is a thermophilic thermus thermophilus (Thermusthermophilus).

In one embodiment, the thermophilic organism is a thermophilic Archaea (Archaea). In one embodiment, the thermophilic archaebacteria are hyperthermophilic archaebacteria. In one embodiment, the thermophilic organism is from the class Thermococcus (Thermococi). In one embodiment, the thermophilic organism is a Thermococcus gamma.

In one embodiment, the thermophilic organism has an optimum growth temperature of at least 60 ℃.

In one embodiment of all aspects reported herein, the immunogenic sequence is comprised in X₁In the amino acid sequence. In one embodiment, the X is₁The amino acid sequence comprises an immunogenic sequence and one or more portions derived from other polypeptides having peptidyl-prolyl cis/trans isomerase activity (PPI enzyme activity) or one or more portions derived from other polypeptides from the FKBP folding domain family, which other polypeptides are thereby different from the second polypeptide.

Insert X₁In place of the intervening flap domain (IF domain) of the second polypeptide. Thus, if X₁Identical to the IF domain, i.e.having the amino acid sequence of the IF domain, the fusion polypeptide S₂-X₁-S₁Identical to the corresponding portion of the naturally occurring second polypeptide.

In one embodiment of all aspects reported herein, S is derived from a second polypeptide₂And S₁The amino acid sequences are linked (directly) to each other via the IF domain in the wild-type (naturally occurring) second polypeptide.

In this contextIn one embodiment of all aspects reported, X is inserted₁In place of the intervening flap domain (IF domain) of the second polypeptide.

One aspect as reported herein is the exclusion of X₁A polypeptide having at least 70% amino acid sequence identity to a polypeptide of formula I, as determined, or excluding X₁、X₂And a polypeptide having at least 70% amino acid sequence identity to the polypeptide of formula II as determined by the sequence of the deletion. In one embodiment, the polypeptides have at least 80% amino acid sequence identity. In one embodiment, the polypeptides have at least 90% amino acid sequence identity. In one embodiment, the polypeptides have at least 95% amino acid sequence identity. In one embodiment, the polypeptides have at least 98% amino acid sequence identity.

In one embodiment of all aspects reported herein, X is₁Comprising an amino acid sequence corresponding to a cryptic epitope.

In one embodiment of all aspects reported herein, X is₁Has an amino acid sequence length of from 4 to about 500 amino acid residues. In one embodiment, X₁Has an amino acid sequence length of from 5 to about 100 amino acid residues. In one embodiment, X₁Has an amino acid sequence length of about 7 to about 60 amino acid residues.

In one embodiment of all aspects reported herein, X is₁Comprises a post-translational modification. In one embodiment, X₁Comprises a post-translational modification, or two or three or four or five or six or seven or eight or nine or ten amino acid residues.

In one embodiment, the fusion polypeptide is according to the formula

NH₂-S₃-S₂-X₁-S₁-S₀-COOH

Wherein

X₁Comprising a random amino acid sequence, or comprising an amino acid sequence derived from a first polypeptide,

S₂and S₁Is a non-overlapping amino acid sequence derived from a second polypeptide,

S₃and S₀Is deleted, or is derived from a non-overlapping amino acid sequence of a third polypeptide, and

-represents a peptide bond,

wherein the second polypeptide and the third polypeptide are different from each other and are polypeptides having peptidyl-prolyl cis/trans isomerase activity (PPI enzyme activity) or are polypeptides derived from the FKBP domain family.

In one embodiment of all aspects reported herein, the second and the third and the fourth polypeptide are from different species.

In one embodiment of all aspects reported herein, the second polypeptide is a human polypeptide, or a plant polypeptide, or a bacterial polypeptide, or an archaeal polypeptide.

In one embodiment of all aspects reported herein, the third polypeptide is a human polypeptide, or a bacterial polypeptide, or an archaeal polypeptide.

In one embodiment of all aspects reported herein, the fourth polypeptide is a bacterial polypeptide, or an archaeal polypeptide.

In one embodiment of all aspects reported herein, the fourth polypeptide is a bacterial polypeptide. In one embodiment, the bacterial polypeptide is a polypeptide from a thermophilic bacterium. In one embodiment, the thermophilic organism is from the family Thermus. In one embodiment, the thermophilic organism is a thermophilic thermus.

In one embodiment of all aspects reported herein, the fourth polypeptide is an archaeal polypeptide. In one embodiment, the archaeal polypeptide is a polypeptide from a hyperthermophilic archaeal. In one embodiment, the thermophilic organism is from the class Thermococcus. In one embodiment, the archaebacteria is a Thermococcus gamma.

In one embodiment of all aspects reported herein, the thermophilic organism has an optimal growth temperature of at least 60 ℃.

In one embodiment of all aspects reported herein, the first polypeptide is a human polypeptide.

In one embodiment of all aspects reported herein, X is₁Is a random amino acid sequence or is derived from a first polypeptide to which a dipeptide GS is added at the N-terminus and a tripeptide GSS is added at the C-terminus.

In one embodiment of all aspects reported herein, X is₁Is a polypeptide of formula III

X_aX_bX_cX_d-X₀-X_eX_fX_gX_h(formula III)

Wherein X₀Is a random amino acid sequence or an amino acid sequence of a first polypeptide; and is

Wherein X_aTo X_hEach of which represents a (naturally occurring) amino acid residue, and X_a-hEither of which may be present or absent independently.

In one embodiment of all aspects reported herein, X is₁Is a polypeptide selected from formula IV to formula XIII (see below)

GS-X₀-GSS (formula IV);

AGS-X₀-GSS (formula V);

CG-X₀-GC (formula VI);

C-X₀-GC (formula VII);

G-X₀-G (formula VIII);

S-X₀-GSS (formula IX);

GG-X₀-GG (formula X);

G-X₀-TGG (formula XI);

GGGS-X₀-GGGS (formula XII);

GGNP-X₀-GPT (formula XIII);

wherein X₀Is a random amino acid sequence, or is derived from an amino acid sequence of a first polypeptide.

In one embodiment, X₀Flanked at their N-and C-termini by a single (single) cysteine residue.

In one embodiment, X₁Comprising a cysteine residue within the N-terminal amino acid residue and a cysteine residue within the C-terminal amino acid residue. In one embodiment, the N-terminal or C-terminal amino acid residue is eight terminal residues. In one embodiment, X₁It contains a cysteine residue at its N-terminus and a cysteine residue at its C-terminus.

In one embodiment, X₁Is a circularly constrained (circular constrained) polypeptide.

In one embodiment, X₁Is a cyclic polypeptide.

In one embodiment, X₁Has a α carbon atom distance of 4.3 angstroms to 6.5 angstroms in one embodiment, X₁Has an α carbon distance of 4.5 angstroms in one embodiment, X₁Has an average α carbon atom distance of 5.6 angstroms.

In one embodiment of all aspects reported herein, X is₁Or X₀Having from 4 to about 500 amino acid residuesThe length of the base.

In one embodiment of all aspects reported herein, X is₂Is a linker amino acid sequence of from about 10 to about 30 amino acid residues.

In one embodiment of all aspects reported herein, the second polypeptide is Arabidopsis thaliana (Arabidopsis thaliana) FKBP13(SEQ ID NO: 01), or a polypeptide having at least 70%, or at least 80%, or at least 90%, or at least 95%, or at least 98% amino acid sequence identity, S₄、X₂、S₃And S₀And (4) lack. In one embodiment, S₂Has the amino acid sequence of SEQ ID NO: 02(DRGAGC), S₁Has the amino acid sequence of SEQ ID NO: 03(CLIPPASV), S₄、X₂、S₃And S₀And (4) lack. In one embodiment, S₂Has the amino acid sequence of SEQ ID NO: 04, S₁Has an amino acid sequence of SEQ ID NO: 05, S₄、X₂、S₃And S₀Deficiency, X₁Is of formula IV (GS-X)₀-GSS).

In one embodiment of all aspects reported herein, the second polypeptide is human FKBP12(SEQ ID NO: 06), or a polypeptide having at least 70%, or at least 80%, or at least 90%, or at least 95%, or at least 98% amino acid sequence identity, S₄、X₂、S₃And S₀And (4) lack. In one embodiment, S₂Has the amino acid sequence of SEQ ID NO: 07, S₁Has the amino acid sequence of SEQ ID NO: 08(LVFDVELLKLE), S₄、X₂、S₃And S₀Deficiency, X₁Is of the formula III (GS-X)₀GSS) or formula VI ((P) CG-X)₀-GC).

In one embodiment of all aspects reported herein, the second polypeptide is Thermus thermophilus SlyD (SEQ ID NO: 09), or a polypeptide having at least 70%, or at least 80%, or at least 90%, or at least 95%, or at least 98% amino acid sequence identity, S₄、X₂、S₃And S₀And (4) lack. In one embodiment, S₂Has the amino acid sequence of SEQ ID NO: 10, S₁Has the amino acid sequence of SEQ ID NO: 11, S₄、X₂、S₃And S₀Deficiency, X₁Is of the formula V (AGS-X)₀GSS) or formula VI ((P) CG-X)₀-GC).

In one embodiment of all aspects reported herein, the second polypeptide is Arabidopsis FKBP13(SEQ ID NO: 01), or a polypeptide having at least 70%, or at least 80%, or at least 90%, or at least 95%, or at least 98% amino acid sequence identity, and the third polypeptide is human FKBP12(SEQ ID NO: 06), or a polypeptide having at least 70%, or at least 80%, or at least 90%, or at least 95%, or at least 98% amino acid sequence identity, S₄And X₂And (4) lack. In one embodiment, S₃Has the sequence shown in SEQ ID NO: 07, S₂Has the amino acid sequence of SEQ ID NO: 02(DRGAGC), S₁Has the amino acid sequence of SEQ ID NO: 03(CLIPPASV), S₀Has the amino acid sequence of SEQ ID NO: 08(LVFDVELLKLE), S₄And X₂Deficiency, X₁Is of formula IV (GS-X)₀GSS) or formula VI ((P) CG-X)₀-GC).

In one embodiment of all aspects reported herein, the second polypeptide is Arabidopsis thaliana FKBP13(SEQ ID NO: 01), or a polypeptide having at least 70%, or at least 80%, or at least 90%, or at least 95%, or at least 98% amino acid sequence identity, and the third polypeptide is Thermus thermophilus SlyD (SEQ ID NO: 09), or a polypeptide having at least 70%, or at least 80%, or at least 90%, or at least 95%, or at least 98% amino acid sequence identity, S₄And X₂And (4) lack. In one embodiment, S₃Has the sequence shown in SEQ ID NO: 10, S₂Has the amino acid sequence of SEQ ID NO: 02(DRGAGC), S₁Has the amino acid sequence of SEQ ID NO: 03(CLIPPASV), S₀Has the amino acid sequence of SEQ ID NO: 11(LVFDVELLKLE), S₄And X₂Deficiency, X₁Is of the formula IV (GS-X₀GSS) or formula VI ((P) CG-X)₀-GC).

In one embodiment of all aspects reported herein, the second polypeptide is Arabidopsis thaliana FKBP13(SEQ ID NO: 01), or a polypeptide having at least 70%, or at least 80%, or at least 90%, or at least 95%, or at least 98% amino acid sequence identity, the third polypeptide is human FKBP12(SEQ ID NO: 06), or a polypeptide having at least 70%, or at least 80%, or at least 90%, or at least 95%, or at least 98% amino acid sequence identity, and the fourth polypeptide is Escherichia coli SlyD (SEQ ID NO: 12), or a polypeptide having at least 70%, or at least 80%, or at least 90%, or at least 95%, or at least 98% amino acid sequence identity. In one embodiment, S₄Has the amino acid sequence of SEQ ID NO: 12, X₂Has the amino acid sequence of SEQ ID NO: 13, S₃Has the sequence shown in SEQ ID NO: 07, S₂Has the amino acid sequence of SEQ ID NO: 02(DRGAGC), S₁Has the amino acid sequence of SEQ ID NO: 03(CLIPPASV), S₀Has the amino acid sequence of SEQ ID NO: 08(LVFDVELLKLE), X₁Is of formula IV (GS-X)₀GSS) or formula VI ((P) CG-X)₀-GC).

In one embodiment of all aspects reported herein, the second polypeptide is Arabidopsis thaliana FKBP13(SEQ ID NO: 01), or a polypeptide having at least 70%, or at least 80%, or at least 90%, or at least 95%, or at least 98% amino acid sequence identity, the third polypeptide is Thermus thermophilus SlyD (SEQ ID NO: 09), or a polypeptide having at least 70%, or at least 80%, or at least 90%, or at least 95%, or at least 98% amino acid sequence identity, and the fourth polypeptide is Escherichia coli SlyD (SEQ ID NO: 12), or a polypeptide having at least 70%, or at least 80%, or at least 90%, or at least 95%, or at least 98% amino acid sequence identity. In one embodiment, S₄Has the amino acid sequence of SEQ ID NO: 12, X₂Has the amino acid sequence of SEQ ID NO: 13, S₃Has the sequence shown in SEQ ID NO: 10, S₂Has an amino acid sequence of SEQ ID NO: 02(DRGAGC), S₁Has the amino acid sequence of SEQ ID NO: 03(CLIPPASV), S₀Has an amino acid sequence of SEQ ID NO: 11(LVFDVELLKLE), X₁Is of formula IV (GS-X)₀GSS) or formula VI ((P) CG-X)₀-GC).

In one embodiment of all aspects reported herein, the second polypeptide is Thermus thermophilus SlyD (SEQ ID NO: 09), or a polypeptide having at least 70%, or at least 80%, or at least 90%, or at least 95%, or at least 98% amino acid sequence identity, and the fourth polypeptide is E.coli SlyD (SEQ ID NO: 12), or a polypeptide having at least 70%, or at least 80%, or at least 90%, or at least 95%, or at least 98% amino acid sequence identity, S₃And S₀And (4) lack. In one embodiment, S₄Has the amino acid sequence of SEQ ID NO: 12, X₂Has the amino acid sequence of SEQ ID NO: 13, S₂Has the amino acid sequence of SEQ ID NO: 10, S₁Has the amino acid sequence of SEQ ID NO: 11, S₃And S₀Deficiency, X₁Is of the formula V (AGS-X)₀GSS) or formula VI ((P) CG-X)₀-GC).

What is reported hereinIn one embodiment of an aspect, the second polypeptide is Arabidopsis thaliana FKBP13(SEQ ID NO: 01), or a polypeptide having at least 70%, or at least 80%, or at least 90%, or at least 95%, or at least 98% amino acid sequence identity, and the fourth polypeptide is Escherichia coli SlyD (SEQ ID NO: 12), or a polypeptide having at least 70%, or at least 80%, or at least 90%, or at least 95%, or at least 98% amino acid sequence identity, S₃And S₀And (4) lack. In one embodiment, S₄Has the amino acid sequence of SEQ ID NO: 12, X₂Has the amino acid sequence of SEQ ID NO: 13, S₂Has the amino acid sequence of SEQ ID NO: 04, S₁Has the amino acid sequence of SEQ ID NO: 05, S₃And S₀Deficiency, X₁Is of formula IV (GS-X)₀GSS) or formula VI ((P) CG-X)₀-GC).

In one embodiment of all aspects reported herein, the second polypeptide is Thermococcus gamma-amino acids SlyD (SEQ ID NO: 106), or a polypeptide having at least 70%, or at least 80%, or at least 90%, or at least 95%, or at least 98% amino acid sequence identity, S₄、X₂、S₃And S₀And (4) lack. In one embodiment, S₂Has the amino acid sequence of SEQ ID NO: 107, S₁Has the amino acid sequence of SEQ ID NO: 108, S₄、X₂、S₃And S₀Deficiency, X₁Is a polypeptide selected from formula IV to formula XIII.

In one embodiment of all aspects reported herein, the second polypeptide is Arabidopsis thaliana FKBP13(SEQ ID NO: 01), or a polypeptide having at least 70%, or at least 80%, or at least 90%, or at least 95%, or at least 98% amino acid sequence identity, and the third polypeptide is Thermococcus gamma-amino SlyD (SEQ ID NO: 106), or a polypeptide having at least 70%, or at least 80%, or at least 90%, or at least 95%, or at least 98% amino acid sequence identity, S₄And X₂And (4) lack. In one embodiment, S₃Has the sequence shown in SEQ ID NO: 107, S₂Having an amino acid sequenceSEQ ID NO：02(DRGAGC)，S₁Has the amino acid sequence of SEQ ID NO: 03(CLIPPASV), S₀Has the amino acid sequence of SEQ ID NO: 108, S₄And X₂Deficiency, X₁Is a polypeptide selected from formula IV to formula XIII.

In one embodiment of all aspects reported herein, the second polypeptide is Arabidopsis FKBP13(SEQ ID NO: 01), or a polypeptide having at least 70%, or at least 80%, or at least 90%, or at least 95%, or at least 98% amino acid sequence identity, the third polypeptide is human FKBP12(SEQ ID NO: 06), or a polypeptide having at least 70%, or at least 80%, or at least 90%, or at least 95%, or at least 98% amino acid sequence identity, and the fourth polypeptide is Thermococcus gamma-amino SlyD (SEQ ID NO: 106), or a polypeptide having at least 70%, or at least 80%, or at least 90%, or at least 95%, or at least 98% amino acid sequence identity. In one embodiment, S₄Has the amino acid sequence of SEQ ID NO: 106, X₂Has the amino acid sequence of SEQ ID NO: 13, S₃Has the sequence shown in SEQ ID NO: 07, S₂Has the amino acid sequence of SEQ ID NO: 02(DRGAGC), S₁Has the amino acid sequence of SEQ ID NO: 03(CLIPPASV), S₀Has the amino acid sequence of SEQ ID NO: 08(LVFDVELLKLE), X₁Is a polypeptide selected from formula IV to formula XIII.

In one embodiment of all aspects reported herein, the second polypeptide is Arabidopsis thaliana FKBP13(SEQ ID NO: 01), or a polypeptide having at least 70%, or at least 80%, or at least 90%, or at least 95%, or at least 98% amino acid sequence identity, the third polypeptide is Thermococcus gamma-amino slyD (SEQ ID NO: 106), or a polypeptide having at least 70%, or at least 80%, or at least 90%, or at least 95%, or at least 98% amino acid sequence identity, and the fourth polypeptide is Escherichia coli SlyD (SEQ ID NO: 12), or a polypeptide having at least 70%, or at least 80%, or at least 90%, or at least 95%, or at least 98% amino acid sequence identity. In a particular embodiment, S₄Has the amino acid sequence of SEQ ID NO: 12, X₂Has the amino acid sequence of SEQ ID NO: 13, S₃Has the sequence shown in SEQ ID NO: 107, S₂Has the amino acid sequence of SEQ ID NO: 02(DRGAGC), S₁Has the amino acid sequence of SEQ ID NO: 03(CLIPPASV), S₀Has the amino acid sequence of SEQ ID NO: 108, X₁Is a polypeptide selected from formula IV to formula XIII.

In one embodiment of all aspects reported herein, the second polypeptide is Thermococcus gamma-amino acids SlyD (SEQ ID NO: 106), or a polypeptide having at least 70%, or at least 80%, or at least 90%, or at least 95%, or at least 98% amino acid sequence identity, and the fourth polypeptide is E.coli SlyD (SEQ ID NO: 12), or a polypeptide having at least 70%, or at least 80%, or at least 90%, or at least 95%, or at least 98% amino acid sequence identity, S₃And S₀And (4) lack. In one embodiment, S₄Has the amino acid sequence of SEQ ID NO: 12, X₂Has the amino acid sequence of SEQ ID NO: 13, S₂Has the amino acid sequence of SEQ ID NO: 107, S₁Has the amino acid sequence of SEQ ID NO: 108, S₃And S₀Deficiency, X₁Is a polypeptide selected from formula IV to formula XIII.

In one embodiment of all aspects reported herein, the second polypeptide is Arabidopsis thaliana FKBP13(SEQ ID NO: 01), or a polypeptide having at least 70%, or at least 80%, or at least 90%, or at least 95%, or at least 98% amino acid sequence identity, and the fourth polypeptide is Thermococcus gamma-amino SlyD (SEQ ID NO: 106), or a polypeptide having at least 70%, or at least 80%, or at least 90%, or at least 95%, or at least 98% amino acid sequence identity, S₃And S₀And (4) lack. In one embodiment, S₄Has the amino acid sequence of SEQ ID NO: 107, X₂Has the amino acid sequence of SEQ ID NO: 13, S₂Has the amino acid sequence of SEQ ID NO: 04, S₁Has the amino acid sequence of SEQ ID NO: 05, S₃And S₀Deficiency, X₁Is a polypeptide selected from formula IV to formula XIII.

The fusion polypeptides of the aspects reported herein have many applications, as they can be recombinantly produced in good yield, e.g., in e. For example, the fusion polypeptide may be used to present amino acid sequences for immunization, antibody production, antibody screening, antibody epitope mapping, or immunohistochemical screening.

In one embodiment of all aspects reported herein, X is₂Has the amino acid sequence GGGSGGGSGGGSGGGSGGGSGGG (SE Q ID NO: 14).

In one embodiment of all aspects reported herein, X is₁Having the amino acid sequence GGGSGGNPX₀GPTGGGS (SEQ ID NO: 32), wherein X₀Is an amino acid sequence of from 4 to 85 amino acid residues.

In one embodiment, a fusion polypeptide comprising the amino acid sequences of e.coli SlyD and human FKBP12 is used to present the polypeptide in an unstable conformation.

In one embodiment, the stable secondary structure is presented with a fusion polypeptide comprising amino acid sequences of human FKBP12 and Arabidopsis FKBP13, or Thermus thermophilus SlyD alone, or Thermus thermophilus SlyD and Arabidopsis FKBP13, or Thermococcus gamma-leaverans SlyD alone, or Thermococcus gamma-leaverans SlyD and Arabidopsis FKBP 13.

One aspect as reported herein is a fusion polypeptide as reported herein eliciting a targeting against X in an animal₁Or X₀The use in an immune response of (1).

One aspect as reported herein is a method for eliciting an immune response against a polypeptide in an animal comprising the step of administering to the animal at least once a fusion polypeptide as reported herein, X₀Thus an immunogenic amino acid sequence.

One aspect as reported herein is a method for obtaining a nucleic acid encoding an antibody specifically binding to a target antigen, comprising the steps of:

a) is applied to animalsAt least once with the fusion polypeptides reported herein, X₁Whereby the amino acid sequence of (a) comprises the amino acid sequence of the target antigen;

b) recovering from the animal three to ten days after the last administration of the polypeptide B cells that produce antibodies that specifically bind to the target antigen;

c) obtaining from the B cell a nucleic acid encoding an antibody that specifically binds to a target antigen.

One aspect as reported herein is a method for generating an antibody specifically binding to a target antigen, comprising the steps of:

a) administering to an animal at least once a fusion polypeptide as reported herein, X₁Whereby the amino acid sequence of (a) comprises the amino acid sequence of the target antigen;

c) optionally obtaining from the B cell a nucleic acid encoding an antibody that specifically binds the target antigen; and

d) culturing a cell comprising a nucleic acid encoding an antibody that specifically binds to the target antigen, and recovering the antibody from the cell or the culture medium, thereby producing an antibody that specifically binds to the target antigen.

a) recovery of a polypeptide producing a specific binding having X from an experimental animal following administration of a fusion polypeptide as reported herein₀B cells of an antibody to the target antigen of the amino acid sequence of (a); and

b) culturing comprises encoding specific binding X₀And recovering the antibody from the cell or the culture medium, thereby producing an antibody that specifically binds to the target antigen.

One reported hereinAspects are the use of the fusion polypeptides as reported herein for epitope mapping, X₁The amino acid sequence of (a) thus comprises the epitope.

One aspect as reported herein is a method for selecting an antibody specifically binding to a target antigen, comprising the steps of:

a) determination of binding affinity of various antibodies to target antigens, X of fusion polypeptides reported herein₁Whereby the amino acid sequence of (a) comprises the amino acid sequence of the target antigen;

b) selecting an antibody having an apparent complex stability above a predetermined threshold level.

One aspect as reported herein is a method for selecting an antibody suitable for immunohistochemical analysis of a target polypeptide comprising the steps of:

a) determining binding kinetics of the plurality of antibodies;

c) selecting an antibody having an apparent complex stability above a predetermined threshold level.

One aspect as reported herein is a method for localizing the binding site of an antibody to a target amino acid sequence comprising the steps of:

a) contacting a solid support having immobilized thereon a fusion polypeptide as reported herein with an antibody, X₁Whereby the amino acid sequence of (a) comprises the target amino acid sequence;

b) determining the kinetic properties of the antibody and the fusion polypeptides reported herein;

One aspect as reported herein is the use of a fusion polypeptide as reported herein for determining a structure-function relationship, X₁The amino acid sequence of (a) thus comprises the polypeptide whose structure-function relationship is to be determined.

Herein reported aOne aspect is the use of the fusion polypeptides as reported herein for presenting polypeptides in their correct secondary and/or tertiary structure, X₁Thereby comprising the polypeptide.

One aspect as reported herein is the use of a fusion polypeptide as reported herein in a screening method.

In one embodiment, the screening method is for identifying or selecting specific binding of X₁The method of screening for a molecule of (1). In one embodiment, the molecule is a small molecule or polypeptide. In one embodiment, the polypeptide is an antibody, or antibody fragment, or antibody fusion polypeptide.

One aspect as reported herein is the use of a fusion polypeptide as reported herein for ribosome display.

One aspect as reported herein is the use of a fusion polypeptide as reported herein in phage display.

One aspect as reported herein is the use of a fusion polypeptide as reported herein for cell surface display. In one embodiment, the cell is a prokaryotic cell. In one embodiment, the prokaryotic cell is a bacterial cell. In one embodiment, the cell is a eukaryotic cell. In one embodiment, the eukaryotic cell is a CHO cell, or a HEK cell, or a BHK cell, or an Sp2/0 cell, or an NS0 cell, or a yeast cell.

One aspect as reported herein is an antibody produced by the method as reported herein.

One aspect as reported herein is a pharmaceutical formulation comprising a fusion polypeptide as reported herein and a pharmaceutically acceptable carrier.

One aspect as reported herein is a diagnostic formulation comprising a fusion polypeptide as reported herein conjugated to a detectable label.

One aspect as reported herein is the use of a fusion polypeptide as reported herein for the preparation of a medicament.

One aspect as reported herein is the use of a fusion polypeptide as reported herein for the treatment of a disease.

One aspect as reported herein is a method of treating an individual comprising administering to the individual an effective amount of a fusion polypeptide as reported herein.

Detailed Description

Typically, the insertion is, for example, contained in X₁Replacing the inserted flap domain (IF domain) of the SlyD portion of the second polypeptide in the fusion polypeptide reported herein.

In one embodiment of all aspects reported herein, X is₀Selected from fragments of naturally occurring polypeptides, or random amino acid sequences. In one embodiment, the naturally-occurring polypeptide is a human polypeptide.

Comprising X₁The fusion polypeptides of amino acid sequence can be used on the one hand for immunizing animals to produce antibodies and on the other hand for screening antibody libraries obtained by randomization or after immunization. Specific binding agents can also be identified using any screening and display method, such as ribosome display, phage display, cell surface display, viral display, and fusion polypeptide-based display as reported herein.

Thermus thermophilus SlyD and Thermococcus gamma adurans SlyD are highly stable proteins with the ability to fold reversibly even when their Flap domain is replaced with an exogenous amino acid insertion X1. These molecules can be used for ribosome display essentially as described by Mattheakis, L.C. et al Proc.Natl.Acad.Sci USA91(1994) 9022-. The so-called ternary complex consists of (1) ribosomal subunits attached to (2) mRNA encoding (3) the genetic information of the ribosome-presented fusion polypeptide.

The ternary complex may be used in panning procedures against antibodies or antibody fragments that specifically recognize the X1 amino acid sequence.

The fusion polypeptides reported herein may be used for screening/selecting antibodies obtained by immunizing an animal with the fusion polypeptides reported herein, wherein the fusion polypeptide for immunizing an animal and the fusion polypeptide for screening the obtained antibodies have the same X₁The amino acid sequence, and the remaining amino acid sequences are different. This allows deselection of specific binding scaffolds without binding to the immunogenic peptide X₁The antibody of (1).

In one embodiment, the fusion polypeptide for immunization and the fusion polypeptide for screening have less than 20% sequence identity. In one embodiment, the sequence identity is less than 10%.

As an aspect herein is reported a fusion polypeptide comprising at its N-terminus a fragment of the Thermus thermophilus SlyD polypeptide (SEQ ID NO: 10), residues 2 to 64 of the Thermus thermophilus SlyD polypeptide (numbering beginning with M as residue 1 of SEQ ID NO: 09). Then, the insertion is contained in X₁The immunogenic sequence of (1). The C-terminus of the fusion polypeptide is formed by amino acid residues 123-149 (SEQ ID NO: 11) of Thermus thermophilus SlyD polypeptide and an optional purification tag having an amino acid sequence GSRKHHHHHHHH (SEQ ID NO: 16).

As an aspect herein is reported a fusion polypeptide comprising at its N-terminus an N-terminal fragment of a Thermococcus gamma-maleorans SlyD polypeptide (SEQ ID NO: 106), residues 2 to 85 of the Thermococcus gamma-maleorans SlyD polypeptide (numbering beginning with M as residue 1 of SEQ ID NO: 107). Then, the insertion is contained in X₁The immunogenic sequence of (1). The C-terminus of the fusion polypeptide is formed by amino acid residues 137 to 156(SEQ ID NO: 108) of the Thermococcus gamma-telorans SlyD polypeptide and a purification tag having an amino acid sequence GSRKHHHHHHHH (SEQ ID NO: 16).

As an aspect herein is reported a fusion polypeptide comprising at its N-terminus an N-terminal fragment of an e.coli SlyD polypeptide, i.e. residues 1 to 165 of the e.coli SlyD polypeptide (numbering starting with M as residue 1). Then, a linker was inserted which links the C-terminus of the e.coli SlyD fragment to the N-terminal fragment of the human FKBP12 polypeptide, i.e. residues 2 to 84 (numbered starting with M as residue 1) of the human FKBP12 polypeptide. An amino acid sequence of 5 to 500 amino acid residues may then be inserted. The C-terminus of the polypeptide is formed by amino acid residues 97 to 108 of the human FKBP12 polypeptide and an optional purification tag having the amino acid sequence GSRKHHHHHHHH (SEQ ID NO: 16).

One aspect as reported herein is a variant of a fusion polypeptide as reported herein having at least 70% amino acid sequence identity with respect to the parent polypeptide and having an increased melting point compared to the parent polypeptide. In one embodiment, the melting point is at least 55 ℃. In one embodiment, the melting point is at least 60 ℃. In one embodiment, the melting point is at least 65 ℃.

The term "derived from a polypeptide" refers to a fragment of the full-length amino acid sequence in which each polypeptide is present, whereby the fragment has at least 70% amino acid sequence identity to each sequence in the full-length polypeptide.

X₁And X₀The amino acid sequences can be freely selected as long as it is at least 5 amino acid residues in length, for example, the insertion sequence can be derived from a fragment which can comprise the leukocyte markers CD2, CD3, CD4, CD5, CD6, CD7, CD8, CD11a, B, c, CD13, CD14, CD18, CD19, CD20, CD22, CD23, CD27 and ligands thereof, CD27 and ligands B7.1, B7.2, B7.3, CD27 and ligands thereof, CD27 and ligands gp 27, CD27, CD27 and isoforms, CD27 (Campath antigen), CD27, CD27, CD27, CD27, CTLA-4, CTLA-1 and TCR, tissue antigen compatibility, MHC class I or II, Lewis antigen, VLeex, VLelex, VLSLeI and SLE 3, VLIW-1, VLICA-72, VLEA-1, VLEA-72, VLA-3, VLI-1-7, VLA-72, VLICA 3, CD27, CD27, CD27, CD27, VLI-1-7, VLI-7, CD-3, VLI-7, CD-3, CD-7, CD-3-III, CD27, CD27, and VLI-III, and L-3, as wellIL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-14 and IL-15, interleukin receptors selected from IL-1R, IL-2R, IL-3R, IL-4R, IL-5R, IL-6-R, IL-7R, IL-8R, IL-9R, IL-10R, IL-11R, IL-12R, IL-13R, IL-14R and IL-15R, chemokines selected from PF R, IL, RANE, MIP R, IL, MCP R, IL, NAP-2, Gro R, IL and IL-8, growth factors selected from TNF R, IL, TGF R, IL, VEGF/VPF, VEGFA, TSGFB, VEGF111, VEGF165, VEGF189, VEGF206, PTHrP, GF family, PDGF family, FGF, Fibrosin (GFF-1), human VEGF111, VEGF121, VEGF165, VEGF189, EGF glycoprotein family, GFR, VEGF family, VEGF receptor glycoprotein, VEGF receptor, VEGF, protein receptor, protein.

The term "peptide linker sequence" refers to a peptide linker of natural and/or synthetic origin. They consist of a linear chain of amino acids, of which 20 naturally occurring amino acids are monomeric building blocks. The chain has a length of 10 to 50 amino acids, in particular 10 to 30 amino acids. The linker may comprise a repeating amino acid sequence or a sequence of naturally occurring polypeptides, such as polypeptides having a hinge function. In one embodiment, the peptide linker amino acid sequence is a "synthetic linker amino acid sequence" designed to be rich in glycine, glutamine and/or serine residues. Examples of such residuesE.g., arranged in small repeating units of up to 5 amino acids, such as GGGGS, QQQG or SSSSSSSG (SEQ ID NO: 17, 18 or 19). These small repeat units can be repeated two to six times to form a multimeric unit. Up to six additional any naturally occurring amino acids may be added at the amino terminus and/or the carboxy terminus of the multimer unit. Other synthetic peptide linkers consist of a single amino acid repeated 10 to 20 times and may comprise up to six additional any naturally occurring amino acids at the amino and/or carboxy terminus, such as serine in linker GSSSSSSSSSSSSSSSG (SEQ ID NO: 20). Specific linker amino acid sequences are shown in the table below. In one embodiment, the linker amino acid sequence is selected from [ GQ₄]₃GNN(SEQ ID NO：21)、G₃[SG₄]₂SG(SEQ ID NO：22)、G₃[SG₄]₂SG₂(SEQ ID NO：23)、(G₃S)₅GGG (SEQ ID NO: 24). All peptide linkers can be encoded by nucleic acid molecules and thus can be expressed recombinantly.

Watch (A)

Linker amino acid sequence	SEQ ID NO：
		(GQ₄)₃	25
(GQ₄)₃G	26
		(GQ₄)₃GNN	27
(G₂S)₃	28
		(G₂S)₄	29
(G₂S)₅	30
		(G₃S)₃	31
(G₃S)₄	32
		(G₃S)₅	33
(G₃S)₅GGG	34
		(G₄S)₂	35
(G₄S)₂G	36
		(G₄S)₂GG	37
(G₄S)₂GGG	38
		(G₄S)₂GN	39
(G₄S)₃	40
		(G₄S)₃G	41
(G₄S)₃T	42
		(G₄S)₃GG	43
(G₄S)₃GGT	44
		(G₄S)₃GGN	45
(G₄S)₃GAS	46
		(G₄S)₄	47
(G₄S)₅	48
		(G₄S)₅G	49
(G₄S)₅GG	50
		(G₄S)₅GAS	51
G(S)₁₅G	52
		G(S)₁₅GAS	53

"percent (%) amino acid sequence identity" with respect to a reference polypeptide sequence is defined as the percentage of amino acid residues in a candidate sequence that are identical with the amino acid residues in the reference polypeptide sequence after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, without considering any conservative substitutions as part of the sequence identity. Alignment for the purpose of determining percent amino acid sequence identity can be achieved in a number of ways within the skill in the art, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN, or megalign (dnastar) software. One skilled in the art can determine appropriate parameters for aligning sequences, including any algorithms necessary to achieve maximum alignment over the full length of the sequences being compared. However, for purposes herein, the sequence comparison computer program ALIGN-2 was used to generate% amino acid sequence identity values. The ALIGN-2 sequence comparison computer program was written by Genentech, inc, and the source code was submitted with the user file to the us copyright office, Washington d.c., 20559, which was registered in the us copyright office as us copyright registration number TXU 510087. The ALIGN-2 program is publicly available from Genentech, Inc., South San Francisco, California, or may be compiled from source code. The ALIGN-2 program is compiled for use on a UNIX operating system and includes the numeral UNIXV4.0D. All sequence comparison parameters were set by the ALIGN-2 program and were not changed.

In the case of amino acid sequence comparisons using ALIGN-2, the% amino acid sequence identity of a given amino acid sequence a to (or compared to) a given amino acid sequence B (which may alternatively be stated as a given amino acid sequence a having or comprising some% amino acid sequence identity to (or compared to) a given amino acid sequence B) is calculated as follows:

100 times a fraction X/Y

Wherein X is the number of matched amino acid residues scored as identical by sequence alignment program ALIGN-2 in the alignment of a and B of that program, and wherein Y is the total number of amino acid residues in B. It will be understood that where the length of amino acid sequence A is not equal to the length of amino acid sequence B, the% amino acid sequence identity of A to B will not be equal to the% amino acid sequence identity of B to A. Unless specifically stated otherwise, all% amino acid sequence identity values used herein are obtained as described in the preceding paragraph using the ALIGN-2 computer program.

The term "animal" refers to an animal selected from the group consisting of mice, rats, rabbits, sheep, cats, dogs, hamsters, macaques and chimpanzees. In particular, the animal is a mouse or rabbit or hamster or rat. In one embodiment, the animal is a non-human animal.

In one embodiment, the term "recovering" includes: a) (ii) immortalizing B cells from an animal immunized with a target antigen, and (ii) screening the resulting immortalized cells for secretion of an antibody that specifically binds to the target antigen; or B) (i) co-culturing the monocultured B cells in the presence of feeder cells, and (ii) screening the culture supernatant for the presence of antibodies that specifically bind the target antigen.

The term "specifically binds to a target antigen" refers to an antibody that binds at least 10 times specifically^-8Dissociation constant (= K) of mol/l_Diss.) especially at least 10^-10mol/l of K_DissBinding to the target antigen. At the same time, through 10^-7mol/l or less, e.g. 10^-5mol/l of K_DissTo ensure the property of not specifically binding to the target antigen.

The term "pharmaceutical formulation" refers to a formulation that is in a form such as to permit the biological activity of the active ingredient contained therein to be effective, and which does not contain additional ingredients that have unacceptable toxicity to the individual to whom the formulation is to be administered.

"pharmaceutically acceptable carrier" refers to an ingredient of a pharmaceutical formulation other than the active ingredient, which is not toxic to the individual. Pharmaceutically acceptable carriers include, but are not limited to, buffers, excipients, stabilizers, or preservatives.

The term "escherichia coli SlyD" refers to a polypeptide having the following amino acid sequence:

MKVAKDLVVSLAYQVRTEDGVLVDESPVSAPLDYLHGHGSLISGLETALEGHEVGDKFDVAVGANDAYGQYDENLVQRVPKDVFMGVDELQVGMRFLAETDQGPVPVEITAVEDDHVVVDGNHMLAGQNLKFNVEVVAIREATEEELAHGHVHGAHDHHHDHDHD(SEQ ID NO：12).。

the term "refers to a polypeptide having the amino acid sequence given below" refers to a polypeptide of the given amino acid sequence and also includes variants thereof with respect to X₁Has the same properties as the polypeptide. In one embodiment, the term refers to polypeptides having at least 70% amino acid sequence identity. In one embodiment, the term refers to polypeptides having at least 80% amino acid sequence identity. In one embodiment, the term refers to polypeptides having at least 90% amino acid sequence identity. In one embodiment, the term refers to polypeptides having at least 95% amino acid sequence identity. In one embodiment, the term refers to polypeptides having at least 98% amino acid sequence identity.

If the polypeptide is produced in or derived from E.coli, the amino-terminal methionine residue is generally not efficiently cleaved by the protease, and thus the amino-terminal methionine residue is partially present in the produced polypeptide. To explain this, all sequences are given with the initial methionine residue.

The term "thermus thermophilus SlyD" refers to a polypeptide having the following amino acid sequence:

MKVGQDKVVTIRYTLQVEGEVLDQGELSYLHGHRNLIPGLEEALEGREEGEAFQAHVPAEKAYGPHDPEGVQVVPLSAFPEDAEVVPGAQFYAQDMEGNPMPLTVVAVEGEEVTVDFNHPLAGKDLDFQVEVVKVREATPEELLHGHAH(SEQID NO：09).。

the term "Thermococcus gammatolerans SlyD" refers to a polypeptide having the amino acid sequence:

MKVERGDFVLFNYVGRYENGEVFDTSYESVAREQGIFVEEREYSPIGVTVGAGEIIPGIEEALLGMELGEKKEVVVPPEKGYGMPREDLIVPVPIEQFTSAGLEPVEGMYVMTDAGIAKILKVEEKTVRLDFNHPLAGKTAIFEIEVVEIKKAGEA(SEQ ID NO：106).。

the term "human FKBP 12" refers to a polypeptide having the amino acid sequence:

MGVQVETISPGDGRTFPKRGQTCVVHYTGMLEDGKKFDSSRDRNKPFKFMLGKQEVIRGWEEGVAQMSVGQRAKLTISPDYAYGATGHPGIIPPHATLVFDVELLKLE(SEQ ID NO：06).。

the term "arabidopsis FKBP 13" refers to a polypeptide having the amino acid sequence:

ETTSCEFSVSPSGLAFCDKVVGYGPEAVKGQLIKAHYVGKLENGKVFDSSYNRGKPLTFRIGVGEVIKGWDQGILGSDGIPPMLTGGKRTLRIPPELAYGDRGAGCKGGSCLIPPASVLLFDIEYIGKA(SEQ ID NO：01).。

the term "FKBP 12 fusion polypeptide" refers to a polypeptide having the amino acid sequence:

MRSGVQVETISPGDGRTFPKRGQTAVVHYTGMLEDGKKFDSSRDRNKPFKFMLGKQEVIRGWEEGVAQMSVGQRAKLTISPDYAYG-X₁-TLVFDVELLKLE(SEQ ID NO：109)，

wherein X₁Is the linker, or peptide, or antigen, or amino acid sequence of secondary or tertiary structure to be presented by the FKBP12 fusion polypeptide.

This amino acid sequence and variants thereof are a single aspect as reported herein.

The term "SlyD-FKBP 12 fusion polypeptide" refers to a polypeptide having the amino acid sequence:

MKVAKDLVVSLAYQVRTEDGVLVDESPVSAPLDYLHGHGSLISGLETALEGHEVGDKFDVAVGANDAYGQYDENLVQRVPKDVFMGVDELQVGMRFLAETDQGPVPVEITAVEDDHVVVDGNHMLAGQNLKFNVEVVAIREATEEELAHGHVHGAHDHHHDHDHD-X₂-RSGVQVETISPGDGRTFPKRGQTAVVHYTGMLEDGKKFDSSRDRNKPFKFMLGKQEVIRGWEEGVAQMSVGQRAKLTISPDYAYG-X₁-TLVFDVELLKLE(SEQ ID NO：110)，

wherein X₁Is a linker, or a peptide, or an antigen, or an amino acid sequence of secondary or tertiary structure to be presented by a SlyD-FKBP12 fusion polypeptide; and is

Wherein X₂Is the amino acid sequence of the linker.

The term "FKBP 12/13 fusion polypeptide" refers to a polypeptide having the amino acid sequence:

MRSGVQVETISPGDGRTFPKRGQTAVVHYTGMLEDGKKFDSSRDRNKPFKFMLGKQEVIRGWEEGVAQMSVGQRAKLTISPDYAYGDRGAGCGS-X₁-GSSCLIPPASVLVFDVELLKLE(SEQ ID NO：54)，

wherein X₁Is the linker, or peptide, or antigen, or amino acid sequence of secondary or tertiary structure to be presented by the FKBP 12/13 fusion polypeptide.

The term "SlyD-FKBP 12/13 fusion polypeptide" refers to a polypeptide having the amino acid sequence:

MKVAKDLVVSLAYQVRTEDGVLVDESPVSAPLDYLHGHGSLISGLETALEGHEVGDKFDVAVGANDAYGQYDENLVQRVPKDVFMGVDELQVGMRFLAETDQGPVPVEITAVEDDHVVVDGNHMLAGQNLKFNVEVVAIREATEEELAHGHVHGAHDHHHDHDHD-X₂-GVQVETISPGDGRTFPKRGQTAVVHYTGMLEDGKKFDSSRDRNKPFKFMLGKQEVIRGWEEGVAQMSVGQRAKLTISPDYAYGDRGAGCGS-X₁-GSSCLIPPASVLVFDVELLKLE(SEQ ID NO：55)，

wherein X₁Is a linker, or a peptide, or an antigen, or an amino acid sequence of secondary or tertiary structure to be presented by a SlyD-FKBP 12/13 fusion polypeptide; and is

Wherein X₂Is the amino acid sequence of the linker.

The term "thermus thermophilus SlyD fusion polypeptide" refers to a polypeptide having the following amino acid sequence:

MRSKVGQDKVVTIRYTLQVEGEVLDQGELSYLHGHRNLIPGLEEALEGREEGEAFQAHVPAEKAY-X₁-GKDLDFQVEVVKVREATPEELLHGHAH(SEQ ID NO：56)，

wherein X₁Is a linker, or a peptide, or an antigen, or an amino acid sequence of secondary or tertiary structure to be presented by a thermus thermophilus SlyD fusion polypeptide.

The term "SlyD-thermus thermophilus SlyD fusion polypeptide" refers to a polypeptide having the following amino acid sequence:

MKVAKDLVVSLAYQVRTEDGVLVDESPVSAPLDYLHGHGSLISGLETALEGHEVGDKFDVAVGANDAYGQYDENLVQRVPKDVFMGVDELQVGMRFLAETDQGPVPVEITAVEDDHVVVDGNHMLAGQNLKFNVEVVAIREATEEELAHGHVHGAHDHHHDHDHD-X₂-KVGQDKVVTIRYTLQVEGEVLDQGELSYLHGHRNLIPGLEEALEGREEGEAFQAHVPAEKAY-X₁-GKDLDFQVEVVKVREATPEELLHGHAH(SEQ ID NO：57)，

wherein X₁Is a linker to be presented by a SlyD-Thermus thermophilus SlyD fusion polypeptide, or a peptide, or an antigen, or an amino acid sequence of a secondary or tertiary structure; and is

Wherein X₂Is the amino acid sequence of the linker.

The term "Thermococcus gamma-telarans SlyD fusion polypeptide" refers to a polypeptide having the amino acid sequence:

MKVERGDFVLFNYVGRYENGEVFDTSYESVAREQGIFVEEREYSPIGVTVGAGEIIPGIEEALLGMELGEKKEVVVPPEKGYGMP-X₁-AGKTAIFEIEVVEIKKAGEA(SEQ ID NO：111)，

wherein X₁Is a linker, or a peptide, or an antigen, or an amino acid sequence of secondary or tertiary structure to be presented by a Thermococcus gamma-lelarans SlyD fusion polypeptide.

The term "SlyD-Thermococcus gamma SlyD fusion polypeptide" refers to a polypeptide having the amino acid sequence:

MKVAKDLV VSLAYQVRTEDGVLVDESPVSAPLDYLHGHGSLISGLETALEGHEVGDKFDVAVGANDAYGQYDENLVQRVPKDVFMGVDELQVGMRFLAETDQGPVPVEITAVEDDHVVVDGNHMLAGQNLKFNVEVVAIREATEEELAHGHVHGAHDHHHDHDHD-X₂-KVERGDFVLFNYVGRYENGEVFDTSYESVAREQGIFVEEREYSPIGVTVGAGEIIPGIEEALLGMELGEKKEVVVPPEKGYGMP-X₁-AGKTAIFEIEVVEIKKAGEA(SEQ ID NO：112)，

wherein X₁Is a linker to be presented by a Thermococcus gamma-lelarans SlyD fusion polypeptide, or a peptide, or an antigen, or an amino acid sequence of secondary or tertiary structure; and is

Wherein X₂Is the amino acid sequence of the linker.

This amino acid sequence and variants thereof are a single aspect as reported herein. The term "thermus thermophilus SlyD-FKBP13 fusion polypeptide" refers to a polypeptide having the following amino acid sequence:

GDRGAGCGS-X₁-GSSCLIPPASVLDFQVEVVKVREATPEELLHGHAH(SEQ ID NO：58)，

wherein X₁Is a linker, or a peptide, or an antigen, or an amino acid sequence of secondary or tertiary structure to be presented by a thermus thermophilus SlyD-FKBP13 fusion polypeptide.

The term "SlyD-thermus thermophilus SlyD-FKBP13 fusion polypeptide" refers to a polypeptide having the following amino acid sequence:

MKVAKDLVVSLAYQVRTEDGVLVDESPVSAPLDYLHGHGSLISGLETALEGHEVGDKFDVAVGANDAYGQYDENLVQRVPKDVFMGVDELQVGMRFLAETDQGPVPVEITAVEDDHVVVDGNHMLAGQNLKFNVEVVAIREATEEELAHGHVHGAHDHHHDHDHD-X₂-KVGQDKVVTIRYTLQVEGEVLDQGELSYLHGHRNLIPGLEEALEGREEGEAFQAHVPAEKAYGDRGAGCGS-X₁-GSSCLIPPASVLDFQVEVVKVREATPEELLHGHAH(SEQ ID NO：59)，

wherein X₁Is a linker, or a peptide, or an antigen, or an amino acid sequence of secondary or tertiary structure to be presented by a SlyD-thermus thermophilus SlyD-FKBP13 fusion polypeptide; and is

Wherein X₂Is the amino acid sequence of the linker.

The term "arabidopsis FKBP13 fusion polypeptide" refers to a polypeptide having the amino acid sequence:

ETTSCEFSVSPSGLAFCDKVVGYGPEAVKGQLIKAHYVGKLENGKVFDSSYNRGKPLTFRIGVGEVIKGWDQGILGSDGIPPMLTGGKRTLRIPPELAYGDRGAGCGS-X₁-GSSCLIPPASVLLFDIEYIGKA(SEQ ID NO：60)，

wherein X₁Is the linker, or peptide, or antigen, or amino acid sequence of secondary or tertiary structure to be presented by the arabidopsis FKBP13 fusion polypeptide.

The term "SlyD-arabidopsis FKBP13 fusion polypeptide" refers to a polypeptide having the amino acid sequence:

MKVAKDLVVSLAYQVRTEDGVLVDESPVSAPLDYLHGHGSLISGLETALEGHEVGDKFDVAVGANDAYGQYDENLVQRVPKDVFMGVDELQVGMRFLAETDQGPVPVEITAVEDDHVVVDGNHMLAGQNLKFNVEVVAIREATEEELAHGHVHGAHDHHHDHDHD-X₂-ETTSCEFSVSPSGLAFCDKVVGYGPEAVKGQLIKAHYVGKLENGKVFDSSYNRGKPLTFRIGVGEVIKGWDQGILGSDGIPPMLTGGKRTLRIPPELAYGDRGAGCGS-X₁-GSSCLIPPASVLLFDIEYIGKA(SEQID NO：61)，

wherein X₁Is a linker, or a peptide, or an antigen, or an amino acid sequence of secondary or tertiary structure to be presented by a SlyD-arabidopsis FKBP13 fusion polypeptide; and is

Wherein X₂Is the amino acid sequence of the linker.

MKVAKDLVVSLAYQVRTEDGVLVDESPVSAPLDYLHGHGSLISGLETALEGHEVGDKFDVAVGANDAYGQYDENLVQRVPKDVFMGVDELQVGMRFLAETDQGPVPVEITAVEDDHVVVDGNHMLAGQNLKFNVEVVAIREATEEELAHGHVHGAHDHHHDHDHD-X₂-GVQVETISPGDGRTFPKRGQTAVVHYTGMLEDGKKFDSSRDRNKPFKFMLGKQEVIRGWEEGVAQMSVGQRAKLTISPDYAYGDRGAGC-X₁-CLIPPASVLVFDVELLKLEGGGSRPLLPPLPGG(SEQ ID NO：113)，

Wherein X₂Is the amino acid sequence of the linker.

In one embodiment of the above aspect, X₂Has an amino acid sequence GGGSGGGSGGGS (SEQ ID NO: 13).

For direct detection, the detectable label may be selected from any known detectable label group, such as dyes, luminescent label groups, such as chemiluminescent groups, e.g., acridinium esters or dioxanes (dioxanes), or fluorescent dyes, e.g., fluorescein, coumarin, rhodamine, oxazines, resorufin, cyanine, and derivatives thereof. Other examples of detectable labels are luminescent metal complexes, such as ruthenium or europium complexes, for example enzymes for ELISA or for CEDIA (clonase donor immunoassay), and radioisotopes. In one embodiment of the process of the present invention,metal chelates that can be detected by electrochemiluminescence are also signal-emitting groups that serve as detectable labels, with ruthenium chelates being particularly preferred. In one embodiment, the labeling group is ruthenium (bipyridyl)₃ ²⁺A chelate compound.

Indirect detection systems include, for example, labeling a detection reagent, such as a detection antibody, with a first partner of a binding pair. Examples of suitable binding pairs are haptens or antigens/antibodies, biotin or biotin analogues (such as aminobiotin, iminobiotin or desthiobiotin)/avidin or streptavidin, sugars/lectins, nucleic acids or nucleic acid analogues/complementary nucleic acids, and receptors/ligands, such as steroid hormone receptors/steroid hormones. In one embodiment, the first binding pair member is selected from the group consisting of a hapten, an antigen, and a hormone. In one embodiment, the hapten is selected from digoxin and biotin and analogs thereof. The second partner of such a binding pair (e.g., an antibody, streptavidin, etc.) is typically labeled to allow direct detection, e.g., by the labels mentioned above.

The fusion polypeptides reported herein are based on polypeptides from the FKBP domain protein family (i.e.proteins having PPI enzyme activity), such as human FKBP12(Handschumacher, R.E. et al, Science226(1984) 544-. The fusion polypeptides reported herein are useful for presenting polypeptides comprised in X₁A scaffold for the polypeptide in the amino acid sequence of (a).

X₁The amino acid sequence of (a) may replace the Flap domain (amino acid residues a85 to a96) and β -knob (amino acid residues S39 to P46) in the FKBP12 part and/or the IF domain (amino acid residues G69 to D120) in the SlyD part, it is then possible to dispense with the time-consuming recombinant preparation and purification of full-length protein immunogens.

Polypeptide X₁Or X₀The defined presentation of the inserted amino acid sequences of (a) and/or the structural motifs associated therewith, respectively, in the fusion polypeptides reported herein allows to sufficeSufficient quantity, mass and efficient and cost-effective production of the constituent X in the correct three-dimensional structure₁And X₀The immunogenic amino acid sequence of (1).

Any amino acid sequence can be inserted, such as a helix, helix-turn-helix motif, coiled-coil structure, helix bundle, turn-loop motif, beta-hairpin structure, beta-sheet, fold-helix motif, fold-turn-sheet motif, and the like. It is also possible to present a defined native tertiary structure, a single domain or subdomain of a multidomain polypeptide, a binding domain, an antibody fragment, an enzyme, etc.

For example in terms of solubility and/or reversible folding (renaturation/denaturation), respectively with X derived therefrom₁Or X₀The immunogenic polypeptide may be improved compared to the full-length polypeptide of the amino acid sequence of (a). Fusion polypeptides as reported herein provide X derived from a polypeptide₁Into which the amino acid sequence of (an antibody against the amino acid sequence is to be obtained) is inserted, and which stabilizes X, respectively₁And X₀Because of the reduced conformational entropy.

The N-terminal SlyD mediates chaperone functionality if present and maintains the complete fusion polypeptide as a monomeric, soluble and stable polypeptide. Furthermore, it increases the molecular weight of the fusion polypeptide, which is beneficial for its use in mass-sensitive assays (e.g., SPR measurements).

Independently of the presence of FKBP12 containing its flap region, one or more SlyD-derived polypeptide portions of the fusion polypeptide fold into the correct (native) three-dimensional conformation. The chimeric FKBP12 domain of the fusion polypeptide appears to be incorrectly folded. Unlike the wild-type FKBP12 polypeptide which shows an intrinsic Trp fluorescence emission peak at 320nm, fluorescence spectroscopic analysis of the SlyD-FKBP12 fusion polypeptide shows no peak at 320nm, but a typical extrinsic Trp emission shift at 350 nm. The 350nm peak is broadened. The single Trp moiety in the SlyD-FKBP12 fusion polypeptide was exposed to a solvent. This indicates that the FKBP domain within the SlyD-FKBP12 fusion polypeptide is partially or fully unfolded.

BIAcore binding assays of SlyD-FKBP12 fusion polypeptide derivatives to immobilized bi-FK506 also showed no binding activity, indicating a loss of structure-function of the FKBP12 derived part of the polypeptides reported herein.

Based on these findings, without being bound by theory, the current SlyD-FKBP12 insertion structure model is that the polypeptide consists of a fully folded SlyD portion and a structurally hindered FKBP12 fold, which FKBP12 fold provides at least its single core Trp residue to the solvent. The polypeptides are monomeric, soluble, reversibly folded, and exhibit sufficient thermal stability for their applications.

Thus, the fusion polypeptides reported herein are scaffolds suitable for mimicking structurally diverse peptide secondary structure motifs, as long as the inserted peptide secondary structure motif is not folded into a separate, autonomously folded structure.

Structurally diverse peptide secondary structural motifs are assumed to be present, for example, in paraffin-embedded, formalin-fixed tissues in immunohistochemical experiments ((Abe et al, anal. biochem.318(2003) 118-123)).

X₁Or X₀Can be respectively represented by the amino acid sequence of (A) and (B)₃The S linker sequence is flanked to ensure sufficient distance and flexibility from the amino acid sequence of the second polypeptide to avoid pairing X, respectively₁Or X₀The structural integrity of the amino acid sequence of (a).

The fusion polypeptide reported herein has a molecular weight of at least 15 kDa. This eliminates the need to conjugate the polypeptide to a carrier protein (such as KLH or particles) or a substance for immunization, and reduces the appearance of neo-epitopes (neo-epitopes) by KLH conjugation.

However, conjugation should be required, which is made possible by selective activation of the lysine residue in sequence motif GSRKHHHHHHHH (SEQ ID NO: 16), which can be activated by the adjacent histidine and arginine residues with LC-SPDP (6- (3- [ 2-pyridyldithio ] -propionamido) hexanoic acid succinimide).

The structure or partial/complete deformation or creation of new epitopes under immunohistochemical analysis conditions is not usually recognized during the preparation of protein immunogens. Especially during formalin treatment, the side chains of the amino acid residues Lys, Tyr, His and Cys are cross-linked. Furthermore, the tertiary and quaternary structure of polypeptides is distorted during tissue preparation using tissue fixation reagents, (heat) incubation, paraffin embedding and ethanol treatment tissue dehydration (see e.g. Fowler, c.b. et al, lab. invest.88(2008) 785-. It is unclear whether the antigen retrieval method can completely remove the formalin-induced cross-linking to restore the native conformation of the protein structure. Thus, new secondary structures can be formed, and new epitopes can be generated, or even retained after repair procedures.

These new and non-native structures are not present during immune activity. By using conventional antibody preparation techniques, no or even a limited number of IHC-compatible antibodies can be obtained.

Furthermore, free polypeptides in solution have large conformational entropies, leading to transient structures that make immune responses to secondary structural epitopes of defined enthalpies difficult (see, e.g., Scott, K.A. et al, Proc. Natl. Acad. Sci. USA104(2007) 2661-2666; Gamacho, C.J. et al, PLoS Compout. biol.4(2008) e 10002311-8). Free peptides used as immunogens can result in the production of antibodies only against the termini of the respective peptides.

In addition to use in immunizing animals and generating immune responses, the fusion polypeptides reported herein can be used to localize antibody epitopes, such as linear epitopes or conformational epitopes. Can be respectively used with X₁Or X₀Presents different structural motifs (= epitopes, antigenic (immunogenic) amino acid sequences). These different structural motifs can also be used to generate pairs X, respectively₁Or X₀The amino acid sequence of (a) is specific for an antibody suitable for Immunohistochemistry (IHC). For this purpose, X is selected in such a way₁Such that only a limited number of neo-epitopes can be expected to be formed during formalin treatment. In particular, lysine can be minimized by selecting an appropriate sequenceNumber of tyrosine, histidine and cysteine residues. It is also possible to replace a single residue, such as a cysteine residue, with a serine residue. As X₁Sequences, for example, small secondary structural motifs, which show a high probability of refolding into their conformational source structure, can be used. By mixing X₁Implantation of the FKBP domain, the ends of the inserted polypeptide are no longer free and accessible and the structural enthalpy increases.

One aspect as reported herein is a fusion polypeptide of formula I

NH₂-S₂-X₁-S₁-COOH (formula I)

Wherein

s2 and S1 are non-overlapping amino acid sequences derived from a second polypeptide; and is

-represents a peptide bond;

wherein the second polypeptide is a polypeptide having peptidyl-prolyl cis/trans isomerase activity (PPI enzyme activity), or is a member of the FKBP domain family.

In one embodiment, the polypeptide having peptidyl-prolyl cis/trans isomerase activity is SlyD.

In one embodiment, the second polypeptide is a polypeptide from a thermophilic organism.

In one embodiment, the thermophilic organism is a thermophilic bacterium. In one embodiment, the thermophilic bacterium is from the family Thermus. In one embodiment, the thermophilic organism is a thermophilic thermus.

In one embodiment, the thermophilic organism is a thermophilic archaea. In one embodiment, the thermophilic archaebacteria are hyperthermophilic archaebacteria. In one embodiment, the thermophilic organism is from the class Thermococcus. In one embodiment, the thermophilic organism is a Thermococcus gamma.

One aspect as reported herein is a fusion polypeptide of formula II

S₄-X₂-S₃-S₂-X₁-S₁-S₀(formula II)

Wherein

X₁Is a random amino acid sequence, or is derived from an amino acid sequence of a first polypeptide;

S₃and S₀Absent, or derived from a third polypeptide;

S₄lacking, or derived from, an amino acid sequence of a fourth polypeptide;

X₂absent, or a peptide linker sequence; and is

-represents a peptide bond;

wherein the second polypeptide and the third and fourth polypeptides are different from each other and are a polypeptide having peptidyl-prolyl cis/trans isomerase activity (PPI enzyme activity) or a member of the FKBP domain family.

In one embodiment, the fusion polypeptide as reported herein comprises an amino acid sequence tag.

In one embodiment, the amino acid sequence tag is selected from the group consisting of a polyhistidine tag, an Avi tag, a polyglutamic acid tag, a polyarginine tag, a Strep tag, a streptavidin binding peptide, an epitope tag, and combinations thereof.

In one embodiment, the amino acid sequence tag is an octahistidine tag.

The term "amino acid sequence tag" refers to a sequence of amino acid residues linked to each other by peptide bonds having specific binding properties. In one embodiment, the amino acid sequence tag is an affinity or purification tag. In one embodiment, the amino acid sequence tag is selected from an arginine tag, a histidine tag, a Flag tag, a 3xFlag tag, a Strep tag, a Nano tag, an SBP tag, a c-myc tag, an S tag, a calmodulin-binding peptide, a cellulose-binding domain, a chitin-binding domain, a GST tag, or an MBP tag. In another embodiment, the amino acid sequence tag is selected from the group consisting of SEQ ID NO: 16(GSRKHHHHHHHH), or SEQ ID NO: 62(RRRRR), or SEQ ID NO: 63(RRRRRR), or SEQ ID NO: 64 (hhhhhhhh), or SEQ ID NO: 65(KDHLIHNVHKEFHAHAHNK), or SEQ ID NO: 66(DYKDDDDK), or SEQ ID NO: 67(DYKDHDGDYKDHDIDYKDDDDK), or SEQ ID NO: 68(AWRHPQFGG), or SEQ ID NO: 69(WSHPQFEK), or SEQ ID NO: 70(MDVEAWLGAR), or SEQ ID NO: 71(MDVEAWLGARVPLVET), or seq id NO: 72(MDEKTTGWRGGHVVEGLAGELEQLRARLEHHPQGQREP), or SEQ ID NO: 73(EQKLISEEDL), or SEQ ID NO: 74(KETAAAKFERQHMDS), or SEQ ID NO: 75(KRRWKKNFIAVSAANRFKKISSSGAL), or SEQ ID NO: 76 (cellulose binding domain), or SEQ ID NO: 77 (cellulose binding domain), or SEQ ID NO: 78(TNPGVSAWQVNTAYTAGQLVTYNGKTYKCLQPHTSLAGWEPSNV PA LWQLQ), or SEQ ID NO: 79(GST tag), or SEQ ID NO: 80(MBP labeling). In one embodiment of all aspects previously reported, the amino acid sequence tag has a sequence selected from the group consisting of SEQ ID NO: 62 to SEQ ID NO: 80. In one embodiment, the amino acid sequence tag has the amino acid sequence of SEQ ID NO: 16, or SEQ ID NO: 64, or SEQ ID NO: 68.

If a polypeptide is produced in an E.coli strain, the protease usually does not cleave the methionine residue at the amino terminus efficiently, so that the methionine residue at the amino terminus is partially present in the produced polypeptide. Thus, all sequences presented herein list an N-terminal methionine residue, although this residue may be absent in the isolated polypeptide. However, an amino acid sequence comprising an N-terminal methionine would also encompass amino acid sequences in which this methionine is deleted.

If X is₁Or X₀The amino acid sequences have a non-helical structure, respectively, and can be in X₁Or X₀The N-and C-termini of the amino acid sequence of (1) were introduced with GGGS linkers (SEQ ID NO: 81).

If X is₁Or X₀The amino acid sequences have helical structures, respectively, and X can be inserted into the amino acid sequences₀Or X₁The N-terminal GGGSGGNP linker (SEQ ID NO: 82) and GPTGGGS linker (SEQ ID NO: 83) of the amino acid sequence of (A).

Antigens can be presented using SlyD/FKBP 12-antigen, Thermus thermophilus SlyD-antigen and Thermococcus gamma attolorans SlyD-antigen fusion polypeptides.

Thermus thermophilus SlyD (Loew, C. et al, J.mol.biol.398(2010)375-390) was derived from the archaea Thermus thermophilus. Thermococcus gamma-telerans SlyD originates from the archaea Thermococcus gamma-telerans. In contrast to human FKBP12, FKBP13, chimeric FKBP 12/13 and E.coli SlyD, both proteins show improved thermodynamic stability. When Thermus thermophilus SlyD or Thermococcus gamma-amino SlyD is included in the fusion polypeptides reported herein, the N-terminal E.coli SlyD-derived polypeptide (i.e.when the fourth polypeptide is E.coli SlyD) may be omitted.

Typically, the polypeptide comprising SEQ ID NO: 16 has the following amino acid sequence:

MGVQVETISPGDGRTFPKRGQTAWHYTGMLEDGKKFDSSRDRNKPFKFMLGKQEVIRGWEEGVAQMSVGQRAKLTISPDYAYGGGGS-X₁-GGGSTLVFDVELLKLEGGGSRKHHHHHHHH(SEQ ID NO：84)，

wherein X₁Is the linker, or peptide, or antigen, or amino acid sequence of secondary or tertiary structure to be presented by the FKBP 12-antigen fusion polypeptide.

The human FKBP 12-derived polypeptide may be fused at the N-terminus to an e.coli SlyD-derived polypeptide.

Comprises the amino acid sequence of SEQ ID NO: 16 has the following amino acid sequence:

MKVAKDLVVSLAYQVRTEDGVLVDESPVSAPLDYLHGHGSLISGLETALEGHEVGDKFDVAVGANDAYGQYDENLVQRVPKDVFMGVDELQVGMRFLAETDQGPVPVEITAVEDDHVVVDGNHMLAGQNLKFNVEVVAIREATEEELAHGHVHGAHDHHHDHDHDGGGSGGGSGGGSGGGSGGGSGGGGVQVETISPGDGRTFPKRGQTAVVHYTGMLEDGKKFDSSRDRNKPFKFMLGKQEVIRGWEEGVAQMSVGQRAKLTISPDYAYGGGGS-X₁-GGGSTLVFDVELLKLEGGGSRKHHHHHHHH(SEQ ID NO：85)，

wherein X₁Is the linker, or peptide, or antigen, or amino acid sequence of secondary or tertiary structure to be presented by the SlyD/FKBP 12-antigen fusion polypeptide.

Comprises the amino acid sequence of SEQ ID NO: amino acid sequence of 16 the labeled SlyD/FKBP 12-control polypeptide (SDS and Western blot see fig. 1) has the following amino acid sequence:

MKVAKDLVVSLAYQVRTEDGVLVDESPVSAPLDYLHGHGSLISGLETALEGHEVGDKFDVAVGANDAYGQYDENLVQRVPKDVFMGVDELQVGMRFLAETDQGPVPVEITAVEDDHVVVDGNHMLAGQNLKFNVEVVAIREATEEELAHGHVHGAHDHHHDHDHDGGGSGGGSGGGSGGGSGGGSGGGGVQVETISPGDGRTFPKRGQTAVVHYTGMLEDGKKFDSSRDRNKPFKFMLGKQEVIRGWEEGVAQMSVGQRAKLTISPDYAYGGGGSGGNPGPTGGGSTLVFDVELLKLEGGGSRKHHHHHHHH(SEQ ID NO：86).。

MRGSKVGQDKVVTIRYTLQVEGEVLDQGELSYLHGHRNLIPGLEEALEGREEGEAFQAHVPAEKAYGPHG-X1-GAGKDLDFQVEVVKVREATPEELLHGHAHGGGSRKHHHHHHHH(SEQ ID NO：87)，

wherein X₁Is a linker, or a peptide, or an antigen, or an amino acid sequence of secondary or tertiary structure to be presented by a Thermus thermophilus-SlyD-antigen fusion polypeptide.

MKVERGDFVLFNYVGRYENGEVFDTSYESVAREQGIFVEEREYSPIGVTVGAGEIIPGIEEALLGMELGEKKEVVVPPEKGYGMP-X₁-AGKTAIFEIEVVEIKKAGEAGGGSRKHHHHHHHH(SEQ ID NO：114)，

wherein X₁Is a linker to be presented by a Thermococcus gamma-SlyD-antigen fusion polypeptide, or a peptide, or an antigen, or an amino acid sequence of secondary or tertiary structure.

Thermus thermophilus-SlyD-antigen fusion polypeptide and Thermococcus gamma-SlyD-antigen fusion polypeptide do not need an Escherichia coli SlyD chaperone structural domain at the N end. In these fusion polypeptides, the immunogenic sequence (antigen sequence) insertion can be stabilized by disulfide bonds in the neck of the antigen loop, which is an embodiment reported herein. In the thermus thermophilus-SlyD-antigen fusion polypeptide, two cysteine mutations can be set at amino acid positions H66C and a70C, and glycine residues can each be introduced to optimize the linkage between the scaffold and the insertion.

MRGSKVGQDKVVTIRYTLQVEGEVLDQGELSYLHGHRNLIPGLEEALEGREEGEAFQAHVPAEKAYGPCG-X₁-GCGKDLDFQVEVVKVREATPEELLHGHAHGGGSRKHHHHHHHH(SEQ ID NO：88)，

wherein X₁Is a linker to be presented by a thermus thermophilus-SlyD-antigen fusion polypeptide stabilized by disulfide bonds, or a peptide, or an antigen, or an amino acid sequence of secondary or tertiary structure.

MKVERGDFVLFNYVGRYENGEVFDTSYESVAREQGIFVEEREYSPIGVTVGAGEIIPGIEEALLGMELGEKKEVVVPPEKGYGMPCG-X₁-GCAGKTAIFEIEVVEIKKAGEAGGGSRKHHHHHHHH(SEQ ID NO：115)，

anti-ERCC 1 antibodies suitable for IHC applications

anti-ERCC 1 antibodies suitable for IHC staining targeting the helix-loop-helix region within the C-terminal domain of ERCC1(ERCC = excision repair cross-complementation; Tripsilanes, K. et al, Structure13(2005)1849-1858) were prepared using the SlyD/FKBP12-ERCC1 fusion polypeptide as a screening reagent.

The function of the ERCC1 polypeptide is primarily in the repair of nucleotide excision of damaged DNA (Aggarwal, C. et al, J.Natl.Compr.Canc.Netw.8(2010) 822-581; Rahn, J.J. et al, environmental. mol.Mutagen.51(2010) 567-581; Westerveld, A. et al, Nature310(1984) 425-429).

ERCC1 has diagnostic relevance because it is a predictive and prognostic marker closely linked to multiple disease indications (Gandara, D.R. et al, J.Thorac Oncol.5(2010) 1933-.

In general, it is possible to generate new epitopes by formalin-induced cross-linking events under IHC conditions (Lin, W. et al, J.Histochem.Cytochem.45(1997) 1157-7613; Webster, J.D. et al, J.Histochem.Cytochem.57(2009) 753-761). The actual structure can be partially or completely denatured or at least structurally modified by stringent conditions during tissue preparation and subsequent antigen retrieval (Rait, V.K., et al, Lab.Invest.84(2004) 300-306). The epitope region is likely to resemble a number of unstable primary or secondary structures that are not adequately represented by linear, synthetically produced peptides. Therefore, immunogens or suitable antibody screening reagents must be used that are capable of coping with all these tasks, while being stable and biochemically reliable.

As postulated in IHC applications, the SlyD/FKBP12-ERCC1 scaffold may mimic structurally diverse linearization, complete denaturation, partial refolding or complete secondary structural motifs. At the same time, the support ensures thermodynamic stability and reliable handling.

SlyD/FKBP12-ERCC1 fusion polypeptides were used to screen anti-ERCC 1 antibodies suitable for IHC applications from multiple antibodies obtained by a linear peptide immunization strategy.

The helix-loop-helix secondary structural motif was extracted from the ERCC1(PDB1Z00) structure (fig. 2). The C-terminal ERCC1 domain may be structurally characterized as being primarily a helical polypeptide. This makes it difficult to identify continuous linear epitopes commonly used in linear peptide immunization methods. To obtain antibodies that specifically recognize the desired sequence motif, animals were immunized with linear KHL-coupled peptides.

The extracted human ERCC1C274S sequence is amino acid sequence IAASREDLALSPGLGPQKARRLFD (SEQ ID NO: 89). This sequence represents a helix-turn-helix motif. The cysteine residue originally present at position 11 of the inserted sequence (underlined in SEQ ID NO: 89) has been changed to a serine residue to avoid oxidative aggregation.

Since the inserted sequence has a helical conformation, an additional amino acid sequence GGGSGGNP (SEQ ID NO: 82) is introduced at the N-terminus of the inserted amino acid sequence, and an amino acid sequence GPTGGGS (SEQ ID NO: 83) is introduced at the C-terminus of the inserted amino acid sequence. Thus, the terminal flanking motif GGGGSGGNP and GPTGGGS were obtained. Without being bound by theory, it is speculated that such motifs promote helical propensity through the proline helix of the capping sequence motif.

MGVQVETISPGDGRTFPKRGQTAVVHYTGMLEDGKKFDSSRDRNKPFKFMLGKQEVIRGWEEGVAQMSVGQRAKLTISPDYAYGGGGSGGNPIAASREDLALSPGLGPQKARRLFDGPTGGGSTLVFDVELLKLEGGGSRKHHHHHHHH(SEQID NO：90).。

MKVAKDLWSLAYVRTEDGVLVDESPVSApLDYLHGHGSLISGLETALEGHEVGDKFDVAVGANDAYGYDENLVRVPKDVFMGVDELVGMRFLAETDGPVPVEITAVEDDHVWDGNHMLAGNLKFNVEWAIREATEEELAHGHVHGAHDHHHDHDHDGGGSGGGSGGGSGGGSGGGSGGGGVVETISPGDGRTFPKRGTAWHYTGMLEDGKKFDSSRDRNKPFKFMLGKEVIRGWEEGVAQMSVGRAKLTISPDYAYGGGGSGGNpIAASREDLALSPGLGPQKARRLFDGPTGGGSTLVFDVELLKLEGGGSRKHHHHHHHH(SEQ ID NO：91).。

the SlyD/FKBP12-ERCC1 fusion polypeptide and the SlyD/FKBP12 control polypeptide were used in a screening method to identify cell clones producing anti-ERCC 1 antibodies.

The SlyD/FKBP12 control polypeptide has the amino acid sequence of SEQ ID NO: 86.

Typically, for purification of the fusion polypeptide, an affinity layer system step is used under denaturing conditions in the presence of a chaotropic agent. The fusion polypeptide is captured on an affinity matrix. The chaotropic buffer was transferred to non-denaturing buffer conditions by washing the column with physiological buffer solution. The E.coli SlyD portion of the SlyD/FKBP12-ERCC1 fusion polypeptide was then refolded. The refolded fusion polypeptide is recovered from the affinity chromatography column in physiological buffer.

The affinity-purified fusion polypeptide was dialyzed and filtered (SDS gel see FIG. 3). UV/Vis Spectroscopy quantitative SlyD/FKBP12-ERCC 1. Protein fluorescence measurements were used to test the conformational properties of SlyD/FKBP12-ERCC1 (FIG. 4). FKBP12 mutant C22A is particularly useful as a vector for polypeptide insertion because the structural integrity of the FKBP12 portion can be determined using a single FKBP12Trp portion (Scholz, C. et al, J.biol. chem.271(1996) 12703-. FKBP12C22A in its native structure showed a single fluorescence emission peak at 320nm (Zoldak, G. et al, J.mol.biol.386(2009) 1138-1152).

Without being bound by theory, the inherent Trp solvolochromic (solvetochromic) fluorescence emission at 350nm will be strongly quenched in the context of the folded FKBP12 protein, which increases with unfolding of FKBP 12. Temperature screening from 25 ℃ to 85 ℃ did not show any other fluorescence emission peaks, but showed temperature-dependent fluorescence quenching of the 350nm emission. No 320nm emission was detected indicating the structural integrity of FKBP12 (see fig. 4).

BIAcore binding assays (FIGS. 5 and 6) with the fusion polypeptide SlyD/FKBP12-ERCC1 as an analyte in solution on the sensor surface-presented ligand bi-FK506 showed no FK506 binding activity, indicating a loss of structure-function of the FKBP12 moiety in the fusion polypeptide. The control polypeptide FKBP12(C22A) showed FK506 binding activity.

The binding of the SlyD/FKBP12-ERCC1 fusion polypeptide to immobilized FK-506 provided evidence of a SlyD/FKBP12-ERCC1 structure that is different from the structure of the FKBP12(C22A) conformation. This is accompanied by a loss of binding activity of the chimeric FKBP12 domain.

Fluorescence measurements and FK506 binding assays provide evidence of loss of structure-function of the SlyD/FKBP12-ERCC1 fusion polypeptide. The N-terminal e.coli SlyD domain keeps the fusion polypeptide in its soluble and monomeric state. This was demonstrated by HPLC analysis of the SlyD/FKBP12-ERCC1 and SlyD/FKBP 12-ctrl fusion polypeptides (see FIGS. 7 and 8).

Mice were immunized intraperitoneally with KHL-coupled peptide encompassing amino acids 219-245 of human ERCC1 (excision repair cross-complementing protein). The production of hybridoma primary cultures was performed according to the method of Koehler and Milstein. Hybridomas were isolated and screened for antigen binding by the ELISA method. Primary hybridoma cell cultures showing positive color formation in ELISA for the peptides ERCC1[219-245] were transferred to kinetic screening (see FIGS. 9 to 12). To avoid selecting antibodies that are not suitable for IHC, screening is performed with the fusion polypeptides reported herein. The SlyD/FKBP12-ERCC1 fusion polypeptide and the SlyD/FKBP12 control polypeptide were used in a kinetic screening method to identify antibodies that bind to the ERCC1 amino acid sequence motif. Suitable primary cultures were further expanded to clonal cultures. The characteristics of the selected cell clones are shown in fig. 12. FIG. 13 illustrates BIAcore measurements of clones < ERCC1> -M-5.3.35. The SlyD/FKBP12-ERCC1 interaction is highly specific. No interaction was detected with the SlyD/FKBP12 control sample. No non-specific binding was detected. This interaction refers to the Langmuir kinetic model.

For Western blots, OVCAR-3 or HEK293 cell lysates were loaded onto lanes of SDS gels. FIG. 14 shows the Western blot results of clones < ERCC1> -M-5.1.35. An ERCC1 specific band at 37kDa was detected.

For immunohistochemical examination of ERCC1 in FFPE embedded human cancer tissue, tissue sections of SCLC cancer samples were prepared. Fig. 15 shows a positive IHC staining pattern. White arrows indicate specific staining sites for ERCC 1. Only with a peptide having seq id NO: 89, i.e. a similar screening and selection procedure was performed only with polypeptides that were not incorporated into the fusion polypeptides reported herein. From the 14 candidate cell clones identified in this peptide-based screen, 9 clones were selected by two screening methods. Although the capture level was the same on the BIAcore chip, 5 hits from the polypeptide-based screening method did not bind to the SlyD/FKBP12-ERCC1 fusion polypeptide.

It has been found that E.coli SlyD-FKBP 12-antigen fusion polypeptides and Thermus thermophilus SlyD-antigen fusion polypeptides (see, e.g., Kang, C.B. No., Neurosignals 16(2008)318-325) and Thermococcus gammatlerans SlyD-antigen fusion polypeptides can be used as combinatorial immunogens and screening tools to generate epitope-specific monoclonal antibodies that target antigens contained in the polypeptides.

Furthermore, it has been found that both E.coli SlyD-FKBP 12/13-antigen fusion polypeptides and Thermus thermophilus SlyD-antigen fusion polypeptides and Thermococcus gamma interferon SlyD-antigen fusion polypeptides can likewise be post-translationally modified and can then be used as a combined immunogen and screening tool to prepare post-translationally modified site-specific monoclonal antibodies targeting the post-translational modifications comprised in the polypeptide. In one embodiment, one of the fusion polypeptides reported herein is used for the production of an antibody and a second, different fusion polypeptide reported herein is used to select an antibody obtained with the first fusion polypeptide, wherein the two fusion polypeptides are different but comprise the same antigenic amino acid sequence, i.e. X₁Or X₀Identical in both fusion polypeptides.

The fusion polypeptides reported herein can be used to generate functional antibodies by epitope-targeting methods using structural modelling or scaffold techniques. The fusion polypeptides reported herein are particularly suitable, in particular, for the production of antibodies against antigens that are not easily accessible in conventional immunological activities (so-called cryptic epitopes).

anti-IGF-1 antibodies

Human IGF-1 and IGF-2 showed 67% amino acid sequence homology and high structural homology (see FIG. 16). In serum, IGF-2 is present 500-fold more than IGF-1 (Jones, j.i. and Clemmons, d.r., endocrin. rev.16(1995) 3-34).

Therefore, the generation of IGF-1 specific antibodies, i.e., antibodies that are not cross-reactive with IGF-2, is challenging. There was a small sequence deviation between IGF-1 and IGF-2 in the IGF-1 turn-loop motif at amino acid positions 74-90 of IGF-1 (UniProtKB entry P05019, IGF1_ human) numbered from the signal and propeptide. The corresponding amino acid sequence NKPTGYGSSSRRAPQTG (SEQ ID NO: 92) can be taken as the amino acid sequence X₀The insertion of the SlyD/FKBP-12 fusion polypeptide reported herein, or of the Thermus thermophilus SlyD fusion polypeptide, or of the Thermococcus gammatolerans SlyD fusion polypeptide.

The fusion polypeptide comprising amino acid sequence NKPTGYGSSSRRAPQTG (SEQ ID NO: 92) can be used to immunize an animal to obtain an antibody that specifically binds to this turn-ring sequence.

To improve presentation of the immunogenic polypeptide, the IGF-1 turn-ring sequence may be flanked by GGGS linkers (SEQ ID NO: 81) at the N-and C-termini of the amino acid sequence, or by HG dipeptide at the N-terminus of the IGF-1 amino acid sequence and GA dipeptide at the C-terminus of the IGF-1 amino acid sequence.

anti-IGF-1 antibodies that specifically bind IGF-1 amino acid sequence NKPTGYGSSSRRAPQTG (SEQ ID NO: 92) were prepared using SlyD/FKBP 12-IGF-1(74-90) fusion polypeptides as immunogens and as screening reagents.

Comprises the amino acid sequence of SEQ ID NO: 16 the amino acid sequence tagged FKBP12-IGF-1(74-90) fusion polypeptide may have the following amino acid sequence:

MGVQVETISPGDGRTFPKRGQTAVVHYTGMLEDGKKFDSSRDRNKPFKFMLGKQEVIRGWEEGVAQMSVGQRAKLTISPDYAYGGGGSNKPTGYGSSSRRAPQTGGGSTLVFDVELLKLEGGGSRKHHHHHHHH(SEQ ID NO：93).。

comprises the amino acid sequence of SEQ ID NO: 16 the amino acid sequence tagged SlyD/FKBP 12-IGF-1(74-90) fusion polypeptide may have the following amino acid sequence:

MKVAKDLVVSLAYQVRTEDGVLVDESPVSAPLDYLHGHGSLISGLETALEGHEVGDKFDVAVGANDAYGQYDENLVQRVPKDVFMGVDELQVGMRFLAETDQGPVPVEITAVEDDHVVVDGNHMLAGQNLKFNVEVVAIREATEEELAHGHVHGAHDHHHDHDHDGGGSGGGSGGGSGGGSGGGSGGGGVQVETISPGDGRTFPKRGQTAVVHYTGMLEDGKKFDSSRDRNKPFKFMLGKQEVIRGWEEGVAQMSVGQRAKLTISPDYAYGGGGSNKPTGYGSSSRRAPQTGGGGSTLVFDVELLKLEGGGSRKHHHHHHHH(SEQ ID NO：94).。

MRGSKVGQDKVVTIRYTLQVEGEVLDQGELSYLHGHRNLIPGLEEALEGREEGEAFQAHVPAEKAYGPHDPEGVQVVPLSAFPEDAEVVPGAQFYAQDMEGNPMPLTVVAVEGEEVTVDFNHPLAGKDLDFQVEVVKVREATPEELLHGHAHGGGSRKHHHHHHHH(SEQ ID NO：97).。

anti-IGF-1 antibodies targeting IGF-1 amino acid sequence NKPTGYGSSSRRAPQTG (SEQ ID NO: 92) were prepared using Thermus thermophilus-SlyD-IGF-1 (74-90) fusion polypeptides (SDS Page and Western blot, see FIG. 17) as immunogens and as screening reagents.

Comprises the amino acid sequence of SEQ ID NO: 16-C-terminal amino acid sequence tagged thermus thermophilus-SlyD-IGF-1 (74-90) fusion polypeptide (SDS and Western blot see fig. 3) can have the amino acid sequence:

MRGSKVGQDKVVTIRYTLQVEGEVLDQGELSYLHGHRNLIPGLEEALEGREEGEAFQAHVPAEKAYGPHGNKPTGYGSSSRRAPQTGGAGKDLDFQVEVVKVREATPEELLHGHAHGGGSRKHHHHHHHH(SEQ ID NO：95)，

or the amino acid sequence:

MKVGQDKVVTIRYTLQVEGEVLDQGELSYLHGHRNLIPGLEEALEGREEGEAFQAHVPAEKAYGPHGNKPTGYGSSSRRAPQTGGAGKDLDFQVEVVKVREATPEELLHGHAHPSGHHHHHH(SEQ ID NO：96).。

for screening and specificity testing, Thermus thermophilus SlyD-delta IF fusion polypeptides were generated. Thermus thermophilus SlyD-delta IF fusion polypeptides lack an IF domain, which is substituted with a short amino acid sequence motif.

Comprises the amino acid sequence of SEQ ID NO: 16C-terminal amino acid sequence tagged thermus thermophilus SlyD- Δ IF fusion polypeptide can have the amino acid sequence:

MRGSKVGQDKVVTIRYTLQVEGEVLDQGELSYLHGHRNLIPGLEEALEGREEGEAFQAHVPAEKAYGPHGAGSGSSGAGKDLDFQVEVVKVREATPEELLHGHAHGGGSRKHHHHHHHH(SEQ ID NO：116).。

for screening and specificity testing, Thermococcus gammatolerans SlyD fusion polypeptides with structurally homologous IGF-2(53-65) hairpin insertions were generated.

Comprises the amino acid sequence of SEQ ID NO: 16 can have an amino acid sequence of:

MKVERGDFVLFNYVGRYENGEVFDTSYESVAREQGIFVEEREYSPIGVTVGAGEIIPGIEEALLGMELGEKKEVVVPPEKGYGMP-G-SRVSRRSRG-G-AGKTAIFEIEVVEIKKAGEAGGGSRKHHHHHHHH(SEQ ID NO：117).。

all fusion polypeptides were produced in E.coli. All fusion polypeptides were purified and refolded using essentially the same procedures as described herein. Mice were immunized intraperitoneally with the SlyD/FKBP 12-IGF-1(74-90) fusion polypeptide and Thermus thermophilus-SlyD-IGF-1 (74-90) fusion polypeptide. Ten weeks after immunization, antibody titers were determined by ELISA (fig. 19 and 20). Mice immunized with the SlyD/FKBP 12-IGF-1(74-90) fusion polypeptide showed low titers for IGF-1, for the restricted IGF-1(74-90) peptide loop, for the SlyD/FKBP 12-IGF-1(74-90) fusion polypeptide, and for the SlyD/FKBP12 control polypeptide. Only one mouse provided sufficiently high IGF-1 titers (K1576M 1 in fig. 19) and was used to generate hybridomas.

It has been found that mice immunized with the Thermus thermophilus-SlyD-IGF-1 (74-90) fusion polypeptide show higher titers for the Thermus thermophilus-SlyD-IGF-1 (74-90) fusion polypeptide than for the SlyD/FKBP 12-IGF-1(74-90) fusion polypeptide (see FIGS. 19 and 20).

The production of hybridoma primary cultures was performed according to the method of Koehler and Milstein. Primary hybridomas were isolated by limiting dilution and screened for antigen binding by ELISA. The primary hybridoma cell cultures that showed positive color formation in ELISAs for the SlyD/FKBP 12-IGF-1(74-90) fusion polypeptide, thermus thermophilus-SlyD-IGF-1 (74-90) fusion polypeptide, and IGF-1, and lower signals for the thermus thermophilus-SlyD wild-type fusion polypeptide and the SlyD/FKBP12 control polypeptide were further evaluated using a kinetic screening method.

Only two primary cultures from the immune activities of SlyD/FKBP 12-IGF-1(74-90) fusion polypeptide were found to have positive ELISA signals for IGF-1. After development into clonal cultures, no kinetic binding signal was detected in Surface Plasmon Resonance (SPR) analysis. Several primary cultures were found which showed suitable ELISA binding signals for IGF-1 and thermus thermophilus-SlyD-IGF-1 (74-90) fusion polypeptides, but reduced signal intensity for thermus thermophilus-SlyD wild-type polypeptides (see fig. 21).

The primary cultures were analyzed by kinetic screening methods for native IGF-1, for native IGF-2, for Thermus thermophilus-SlyD-IGF-1 (74-90) fusion polypeptides, and for Thermus thermophilus-SlyD wild-type polypeptides (FIG. 22). Primary cultures producing IGF-1 specific antibodies were tested and expanded by limiting dilution to obtain clonal cultures.

Clone cultures were analyzed for specific binding to IGF-1 using ELISA (see FIG. 23). Exemplary BIAcore measurements of anti-IGF-1 antibodies obtained from thermus thermophilus-SlyD-IGF-1 (74-90) fusion polypeptides are shown in fig. 24. The antibody specifically binds Thermus thermophilus-SlyD-IGF-1 (74-90) fusion polypeptide and native IGF-1 with 10pM binding affinity. Does not bind native IGF-2 and wild-type Thermus thermophilus SlyD (see FIG. 25).

It has been found that for the production of IGF-1 specific antibodies, stabilizing the IGF-1 turn-loop motif to maintain its native folding by a rigid, enthalpy scaffold is of great importance. When presented on a metastable polypeptide scaffold (e.g., FKBP12), without being bound by this theory, it is speculated that sequence NKPTGYGSSSRRAPQTG (SEQ ID NO: 18) has too many rotational degrees of freedom. Finally, no native IGF-1 binding antibody was obtained.

FKBP-12 in the SlyD/FKBP 12-IGF-1(74-90) fusion polypeptide was partially unfolded as measured by near-UV-CD spectroscopy. HPLC analysis shows that the SlyD/FKBP 12-IGF-1(74-90) fusion polypeptide is a monomer. DSC measurements show that the fusion polypeptide is capable of reversibly folding and unfolding. Without being bound by theory, the reversibly foldable N-terminal e.coli SlyD domain keeps the fusion polypeptide stable and monomeric in solution even when the C-terminal FKBP domain is partially or fully unfolded (see fig. 26).

As already found for the SlyD/FKBP12-ERCC1 fusion polypeptide, the SlyD/FKBP 12-IGF-1(74-90) fusion polypeptide can present structurally diverse linearized, fully denatured, partially refolded, or intact secondary structural motifs.

For the preparation of native IGF-1 binding antibodies, it has been found necessary to use scaffolds that present the insertion of the polypeptide in its native conformation. Thus, the presentation fusion polypeptide needs to be a stably folded polypeptide. It has been found that this can be achieved by using FKPB domains from extremophiles (i.e.thermophiles) such as Thermus thermophilus SlyD or Thermococcus gamma tellans SlyD.

To check whether the fusion polypeptides reported herein adopt a folded conformation, the CD spectrum in the near-UV region was determined. near-UV-CD determines the asymmetric environment of aromatic residues in polypeptides and is therefore a sensitive test for ordered tertiary structure. Native SlyD has typical CD characteristics in the near-UV region. Therefore, structural distortions or spatial conflicts caused by insertions in the IF domain should be visible in the near-UV CD spectrum. The superposition of the spectra of wild-type thermus thermophilus SlyD, the FKBP domain of wild-type thermus thermophilus SlyD lacking the IF domain (thermus thermophilus SlyD- Δ IF fusion polypeptide) and the thermus thermophilus SlyD-antigen fusion polypeptide, in which the insertion of 22 amino acids from the human extracellular receptor fragment is inserted, is shown in fig. 27. It has been found that substitution of the Thermus thermophilus IF domain does not result in a change in the overall structure of the remaining IF domains. It can be seen that the spectral characteristics are similar. Since unfolding will abolish any near-UV CD signal, this result provides evidence that native-like folding is maintained in the fusion polypeptide.

The thermus thermophilus SlyD-antigen fusion polypeptide is a fusion polypeptide comprising a 22 amino acid beta hairpin secondary structure insertion from the human growth factor receptor extracellular domain (ECD). The CD profile demonstrates that all polypeptides fold well into their native structure at 20 ℃.

FIG. 28 shows a temperature-dependent CD spectrum of Thermus thermophilus SlyD- Δ IF fusion polypeptides. After temperature-induced unfolding, the thermus thermophilus SlyD FKBP domain can refold upon cooling again. Thus, the fusion polypeptide can be affinity purified by on-column refolding, and furthermore, unlike the findings for the SlyD-FKBP12-IGF-1 fusion polypeptide, the thermus thermophilus SlyD-IGF-1(74-90) fusion polypeptide has structural stability to present the IGF-1 secondary structural motif in a stable conformation on the FKBP domain. The temperature-dependent near-UV CD spectrum of the pyrococcus gammatolerans SlyD-antigen fusion polypeptides showed even higher stability when compared to the thermus thermophilus-antigen fusion polypeptides (see fig. 29). Both scaffolds carry the same 22 amino acid beta hairpin secondary structure insertion from the human growth factor receptor ECD. Thermococcus gammatolerahs SlyD-antigen reversibly folds and unfolds. It has been found that under given physical conditions, complete unfolding of a Thermococcus gamma-antigen SlyD-fusion polypeptide is not achieved even at a temperature of 100 ℃.

It has been found that the stability of the archaeal FKBP domains enables the transplantation of immunogenic polypeptides by replacing their IF domains, thereby simultaneously maintaining the overall stability of the newly produced chimeric scaffold proteins.

Thermus thermophilus SlyD-IGF-1(74-90) fusion polypeptide was purified as a stable and monomeric polypeptide (see FIG. 18).

The monomeric fraction of the Thermococcus gammatolerans SlyD-antigen fusion polypeptide was re-chromatographed after repeated freeze-thaw cycles and temperature stress tests (see FIG. 30).

Using the nucleotide sequence of SEQ ID NO: 96 immunize mice. The obtained B cells were analyzed by ELISA. Thermus thermophilus-SlyD-IGF-1 (74-90) fusion polypeptide, Thermus thermophilus-SlyD wild-type polypeptide, IGF-1 and IGF-2 were used as controls.

Comprises the amino acid sequence of SEQ ID NO: 16C-terminal amino acid sequence-labeled thermus thermophilus-SlyD wild-type polypeptide (SDS and Western blot see fig. 31) has the following amino acid sequence:

all Clone Culture Supernatants (CCS) formed stable complexes with IGF-1 and Thermus thermophilus SlyD-IGF-1 fusion polypeptides at 37 ℃. No cross-reaction with Thermus thermophilus SlyD-wild-type polypeptide could be detected with either of the CCSs. Except for one clone, no cross-reactivity with IGF-2 was detected (see FIG. 32). As can be seen in FIG. 33, the first 8 clonal culture supernatants had a t/2-dis of 2 minutes, whereas the analyte IGF-1 bound faster and dissociated more slowly for the last 4 clonal culture supernatants. It stays in the complex for longer than 40 minutes. An exemplary sensorgram for one clone is shown in fig. 22. It can be seen that the antibody binds to IGF-1 and Thermus thermophilus-IGF-1 fusion polypeptide, while no binding to IGF-1 and Thermus thermophilus-wild type polypeptide is detected.

Thus, Thermus thermophilus SlyD-antigen fusion polypeptides and Thermococcus gammatolerans SlyD-antigen fusion polypeptides can be used as a combined immunogen and screening tool to prepare epitope-specific monoclonal antibodies that target the immunogen contained in the polypeptide.

FIG. 25 shows that the scaffold-derived monoclonal antibodies < IGF-1> M-11.11.17 and < IGF-1> M-10.7.9 have picomolar affinity for IGF-1. Monoclonal antibody < IGF-1> M-11.11.17 showed IGF-1 complex stability at t 1/2 dis =560 minutes. Cross-reactivity to IGF-2, wild-type Thermus thermophilus SlyD, wild-type Thermococcus gamma-malenans SlyD, Thermococcus thermophilus SlyD-. DELTA.IF and Thermococcus gamma-malenans SlyD-IGF-2(53-65) was not detected.

Monoclonal antibody M-2.28.44 was obtained by conventional immunization of mice with recombinant human IGF-1. While the antibody has 30pM binding affinity for IGF-1, the antibody is also cross-reactive with IGF-2 (500pM binding affinity). Using Thermus thermophilus SlyD-IGF-1(74-90) fusion polypeptide and Thermococcus gamma-interleans SlyD-IGF-2(53-65) fusion polypeptide as analytes, IGF-2 epitopes that could show cross-reactivity were not in the IGF-1/2 hairpin region.

This is confirmed by linear epitope mapping (see, e.g., Frank, R. and overhin, H., Methods in molecular Biology66(1996) 149-169). Both clones 11.11.17 and 11.9.15 recognized the epitope TGYGSSSR (SEQ ID NO: 124) for linear IGF-1 binding contribution. The linear binding portion of clone 10.7.9 bound epitope PTGYGSSSR (SEQ ID NO: 125). This epitope is located on top of the IGF-1 hairpin structure and is therefore not present in IGF-2.

In general, the fusion polypeptides reported herein can be used to generate functional antibodies by a targeted epitope approach using structural mimetics. The fusion polypeptides reported herein are particularly suitable, in particular, for the production of antibodies against antigens that are not accessible by conventional immunological activity using recombinant immunogens. It has been found that when presented well as an insertion in the fusion polypeptides reported herein, so-called cryptic epitopes (buried inside the native protein conformation) can be used as immunogens. In particular, neo-epitopes (which can only be targeted upon allosteric, ligand-induced conformational changes) can be used as immunogens by grafting these structures into the fusion polypeptides reported herein.

Chimeric FKBP 12/13 scaffold:

some of the fusion polypeptides reported herein are based on fusion polypeptides comprising a portion of human FKBP12 and a portion of arabidopsis FKBP 13. It has been found that a fusion polypeptide comprising at least part of human FKBP12 and at least part of arabidopsis thaliana FKBP13 can be used as an immunogen. In this fusion polypeptide, the human FKBP 12-derived portion is thermodynamically stable as a scaffold. FKBP13 contains a disulfide bond that stabilizes the IF domain. This sequence was implanted into a human FKBP 12-derived portion to stabilize the fusion polypeptide.

Comprises the amino acid sequence of SEQ ID NO: 16 has the amino acid sequence of FKBP 12/13 fusion polypeptide tagged with the C-terminal amino acid sequence of seq id no:

MRSGVQVETISPGDGRTFPKRGQTAVVHYTGMLEDGKKFDSSRDRNKPFKFMLGKQEVIRGWEEGVAQMSVGQRAKLTISPDYAYGDRGAGCGSGSSCLIPPASVLVFDVELLKLE GGGSRKHHHHHHHH(SEQ ID NO：118).。

the FKBP 12/13 fusion polypeptide was expressed as a soluble protein in E.coli (see FIG. 34). HPLC analysis (see FIG. 35) demonstrated that the FKBP 12/13 fusion polypeptide was a monomer. CD spectroscopy was performed as described above. CD spectra demonstrated that FKBP 12/13 fusion polypeptide folded at 20 ℃. Scaffolds based on Thermus thermophilus and Thermococcus gamma show higher temperature stability than FKBP 12/13 fusion polypeptides.

anti-PLGF antibodies:

in this case the inserted amino acid sequence has a turn-loop motif, which should result in an antibody suitable for use in IHC. The insert has the amino acid sequence DWSEYPSEVEHMFSPSS (SEQ ID NO: 98). The C-terminal cysteine residue in the immunogen has been changed to a serine residue.

MGVQVETISPGDGRTFPKRGQTAWHYTGMLEDGKKFDSSRDRNKPFKFMLGKQEVIRGWEEGVAQMSVGQRAKLTISPDYAYGGGGSDWSEYPSEVEHMFSPSSGGGSTLVFDVELLKLEGGGSRKHHHHHHHH(SEQ ID NO：99).。

the SlyD/FKBP 12-PLGF fusion polypeptide comprising a C-terminal amino acid sequence tag has the following amino acid sequence:

MKVAKDLVVSLAYQVRTEDGVLVDESPVSAPLDYLHGHGSLISGLETALEGHEVGDKFDVAVGANDAYGQYDENLVQRVPKDVFMGVDELQVGMRFLAETDQGPVPVEITAVEDDHVVVDGNHMLAGQNLKFNVEVVAIREATEEELAHGHVHGAHDHHHDHDHDGGGSGGGSGGGSGGGSGGGSGGGGVQVETISPGDGRTFPKRGQTAVVHYTGMLEDGKKFDSSRDRNKPFKFMLGKQEVIRGWEEGVAQMSVGQRAKLTISPDYAYGGGGSDVVSEYPSEVEHMFSPSSGGGSTLVFDVELLKLEGGGSRKHHHHHHHH(SEQ ID NO：100).。

mice were immunized with an immunogen comprising the sequence of PLGF (60-76). Hybridomas were subsequently generated and ELISA and kinetic screening was performed.

In the kinetic screening method, primary culture supernatants with binding activity for PLGF (60-76) were identified using a SlyD/FKBP 12-PLGF fusion polypeptide and biotinylated PLGF (60-76) peptide grafted on streptavidin alone. Both analytes produced 1: 1Langmuir kinetics, but the fusion polypeptide showed better dissociation fitted with a lower chi-square value than the SA probe grafted biotinylated PLGF peptide. Thus, fusion polypeptide-based screening methods take advantage of the monomeric state, and improved epitope accessibility of the immunogen in the fusion polypeptide when compared to the SA probe.

Antibodies prepared by this method (e.g., clone 53.4.1) were able to specifically detect PLGF in Western blots.

SlyD-FKBP 12/13-CSF 1R fusion polypeptide:

the term "SlyD-FKBP 12/13-CSF 1R fusion polypeptide" refers to a polypeptide having the amino acid sequence:

MKVAKDLVVSLAYQVRTEDGVLVDESPVSAPLDYLHGHGSLISGLETALEGHEVGDKFDVAVGANDAYGQYDENLVQRVPKDVFMGVDELQVGMRFLAETDQGPVPVEITAVEDDHVVVDGNHMLAGQNLKFNVEVVAIREATEEELAHGHVHGAHDHHHDHDHDGGGSGGGSGGGSGVQVETISPGDGRTFPKRGQTAVVHYTGMLEDGKKFDSSRDRNKPFKFMLGKQEVIRGWEEGVAQMSVGQRAKLTISPDYAYGDRGAGCGSGGVDYKNIHLEKKYVRRDSGFSSQGVDTYVEMRPVSTSSNDSFSEQDLDKEDGGSSCLIPPASVLVFDVELLKLEGGGSRPLLPPLPGGGSRKHHHHHHHH(SEQ ID NO：119).。

the polypeptide is expressed in E.coli as described herein and purified as described herein. Ni-NTA affinity purification was followed by size exclusion chromatography. The fusion protein was loaded on a HiLoad 26/60 Superdex 75pg column. The eluted fractions were analyzed on a non-denaturing SDS gel (see FIG. 37).

X for antibody production₁The amino acid sequence corresponds to the CSF1R intracellular kinase insertion domain. Src kinase, EGF1R and CSF1R may themselves phosphorylate tyrosine residues, particularly those in the loop structure of the kinase domain, flanked by threonine and valine (see Y residue at position 297 in SEQ ID NO: 119, also in italics and underlined).

After purification, the fusion protein can be phosphorylated using a suitable kinase. Thus, it is possible to provide X with posttranslational modifications₁Amino acid sequence, thereby making it possible to generate antibodies against the post-translationally modified polypeptides with the fusion polypeptides reported herein. The fusion polypeptides can be used for different applications, such as screening, specificity testing or as immunogens. In general, a peptide which is a substrate for enzymatic post-translational modification or chemical modification may be used as X₁The amino acid sequence is inserted into the scaffold. Protein fluorescence measurements were used to test the conformational properties of the SlyD/FKBP 12/13-CSF 1R fusion polypeptide. The 300nm to 425nm scan peaked at 305nm at 20 ℃ and 25 ℃ (see FIG. 38). The single tryptophan moiety in the FKBP12 domain exhibits the typical intrinsic tryptophan solvotochromic fluorescence emission, as it is buried in the hydrophobic core of the FKBP12 domain. Thus, it is assumed that the FKBP 12/13-CSF 1R polypeptide portion is folded. At 50 ℃, the emission peak shifts to 344nm, which demonstrates that the tryptophan moiety is now in an aqueous environment and that the FKBP domain is partially or fully unfolded. At 30 ℃ and 40 ℃, the intermediate state of the folded and unfolded protein can be determined.

It has been found that it is possible to stabilize the FKBP12 domain in a fusion polypeptide by engineering FKBP12 to an FKBP 12/13 fusion polypeptide, wherein the FLAP domain of FKBP12 is replaced by a disulfide-containing FKBP13 structure and a loop structure motif from the CSF1R receptor.

The fusion polypeptide already shows a significant unfolded protein fraction at 40 ℃ which is mainly referred to as the FKBP 12/13-CSF 1R domain, since e.coli SlyD does not contain tryptophan.

This finding emphasizes the need to further stabilize the chimeric FKBP12 to generate a non-metastable scaffold in order to use the chimeric FKBP domain as a scaffold to present a properly folded native secondary or tertiary structure as an immunogen. Furthermore, it was not possible to omit the e.coli SlyD domain in the fusion polypeptide, since otherwise the expression yield was significantly reduced (data not shown).

Positioning an epitope;

SlyD-FKBP fusion polypeptides may also carry complex amino acid insertion motifs, such as secondary structures containing disulfide bonds. Since the fusion polypeptide does not contain cysteine, on-column refolding under appropriate conditions facilitates correct formation of disulfide bonds within the insertion, which is additionally assisted by chaperone functionality of SlyD itself.

The fusion polypeptides SlyD-FKBP12-CD81 and SlyD-FKBP12-ctrl were used for epitope mapping purposes. Human CD81 is a receptor for hepatitis c virus envelope E2 glycoprotein. CD81 is a transmembrane protein belonging to the four transmembrane (tetraspanin) family. CD81 is a homodimeric protein 90 amino acids in length, showing a so-called mushroom-like structure (PDB1IV 5). Residues known to be involved in viral binding can be mapped to the so-called "head subdomain" of 35 amino acids in length, providing the basis for designing antiviral drugs and vaccines. Since the head subdomain sequence of the viral binding site is only 35 amino acids in length, it is difficult to locate the epitope of the antibody on the 10kDa CD81 protein using conventional cross-blocking experiments.

It is difficult to distinguish antibodies that bind directly on the mushroom-like head subdomain from antibodies that bind only nearby or elsewhere in the CD81LEL structure. All of these antibodies will show a competitive effect of the HCV E2 envelope protein, but will not specifically bind to the target domain. Thus, transplantation of the head domain structure into FKBP and contiguous epitope mapping is an advantageous approach. First, some biochemical problems of the CD81LEL protein are avoided, as the protein itself tends to oligomerize. Second, it is suitable for selecting antibody epitopes from a large number of antibodies that bind only the full-length CD81 protein.

SlyD-FKBP12-CD81 fusion polypeptide:

the term "SlyD-FKBP 12-CD81 fusion polypeptide" refers to a polypeptide having the amino acid sequence:

MKVAKDLVVSLAYQVRTEDGVLVDESPVSAPLDYLHGHGSLISGLETALEGHEVGDKFDVAVGANDAYGQYDENLVQRVPKDVFMGVDELQVGMRFLAETDQGPVPVEITAVEDDHVVVDGNHMLAGQNLKFNVEVVAIREATEEELAHGHVHGAHDHHHDHDHDGGGSGGGSGGGSGGGSGGGSGGGGVQVETISPGDGRTFPKRGQTAVVHYTGMLEDGKKFDSSRDRNKPFKFMLGKQEVIRGWEEGVAQMSVGQRAKLTISPDYAYGGGGSCCGSSTLTALTTSVLKNNLCPSGSNIISNLFKEDCGGGSTLVFDVELLKLEGGGSRKHHHHHHHH(SEQ ID NO：126).。

SlyD-FKBP12-ctrl fusion polypeptide (see FIG. 1):

the term "SlyD-FKBP 12-ctrl fusion polypeptide" refers to a polypeptide having the following amino acid sequence:

a BIAcore2000 instrument (GEHealthcare) with a BIAcore CM5 sensor mounted into the system was used at 25 ℃. Each protein ligand was immobilized into flow cells 2, 3 and 4 by EDC/NHS chemistry. Flow cell 1 was used as a reference. The following were immobilized on the sensor: the flow cell 2: SlyD-FKBP12 ctrl; flow cell 3: SlyD-FKBP12-CD 81; and a flow cell 4: CD81 LEL. 31 antibody analytes were injected. Sensorgrams were monitored as reference signals 2-1, 3-1 and 4-1 and evaluated by using BIAcore evaluation software 4.1. At the end of the analyte injection, a reporting point is set to quantify the maximum analyte binding signal. Data were normalized by setting the highest analyte binding signal to 100%. Normalized antibody binding responses showed that only 6 of the 30 anti-CD 81-LEL antibodies tested showed binding to an epitope on the CD81 head domain. Does not bind to the negative control polypeptide SlyD-FKBP12 ctrl. The positive control polypeptide CD81-LEL, which is simultaneously an immunogen, is bound by all antibodies. Slyd-FKBP12-CD81 only bound when the antibody epitope was located in the mushroom domain.

Confirmation of epitope mapping results by X-ray crystallization analysis

Fab fragments of antibodies K05 and K04 were co-crystallized with the CD81-LEL protein by known methods and analyzed by x-ray diffraction analysis (Seth Harris, Palo Alto). The resolution obtained isK04 recognizes the target epitope sequence directly, whereas K05 binds outside the target. Thus, x-ray analysis is directly linked to the scaffold-based epitope mapping method.

The following examples, figures and sequences are provided to aid the understanding of the present invention, the true scope of which is set forth in the appended claims. It is understood that changes may be made in the methods illustrated without departing from the spirit of the invention.

Brief Description of Drawings

FIG. 1 SDS PAGE (Coomassie staining) and Western blotting (incubation with anti-octahistidine-tagged antibody for 10 sec) of SlyD/FKBP 12-control polypeptides.

FIG. 2A: ERCC1(PDB1Z 00): a circled helix-corner helix motif (IAASREDLALSPGLGPQKARRLFD, C274S); b: FKBP12C 22A: a circled substitution sequence; the FKBP12 chimera was fused to E.coli SlyD at the C-terminus.

FIG. 3 SDS PAGE (Coomassie staining) and anti-histidine tag Western blot (10 sec exposure) of SlyD/FKBP12-ERCC1 polypeptide. M-Novex Sharp Standard; 1-2.5 μ g SlyD/FKBP12-ERCC1 fusion polypeptide; 2-5.0 μ g SlyD/FKBP12-ERCC1 fusion polypeptide; 3-10 μ g of SlyD/FKBP12-ERCC1 fusion polypeptide; m. Magic Mark.

FIG. 4 records the fluorescence emission intensity of the SlyD/FKBP12-ERCC1 fusion polypeptide at 25 deg.C, 35 deg.C, 45 deg.C, 55 deg.C, 85 deg.C driven by a wavelength sweep at 300nm-600nm at 600 nm/min.

FIG. 5 schematic representation of BIAcore assay for determining binding of SlyD/FKBP12-ERCC1 fusion polypeptide to FK-506.

FIG. 6300 nM SlyD/FKBP12-ERCC1 fusion polypeptide and 300nM wild-type FKBP12 as biotinylated ligand bi-FK506 presented to the sensor surface by the analyte in solution.

FIG. 7 analytical HPLC chromatogram of SlyD/FKBP 12-ctrl fusion polypeptide. After Ni-NTA purification, SlyD/FKBP 12-ctrl eluted as a monomeric peak.

FIG. 8 analytical HPLC chromatogram of SlyD/FKBP12-ERCC1 fusion polypeptide. After Ni-NTA purification, SlyD/FKBP12-ERCC1 eluted as a monomeric peak.

FIG. 9 schematic representation of BIAcore binding assay kinetic screening with SlyD/FKBP12-ERCC1 fusion polypeptide and 300nM SlyD/FKBP 12-ctrl as analytes in solution. CM5 sensor, capture RAMFCy: rabbit anti-mouse Fc γ capture antibody.

FIG. 10 is a plot of post Stability (Stability Late)/post Binding (Binding Late) showing the kinetic properties of anti-ERCC 1 antibodies as determined by kinetic screening using SlyD/FKBP12-ERCC1 as the analyte in solution. All clones had late binding values>40RU is positioned at 10^-51/s trend line, indicating extraordinary antigen complex stability. No binding to SlyD/FKBP 12-ctrl was detected.

FIG. 11 characterization of anti-ERCC 1 antibodies by kinetic screening. Post-binding/antibody capture level plots the binding titer channels are shown by trend lines. All 5.00x.35 sister clones (circled) mapped titer channels between mole ratio =0.5 and mole ratio =1 and were selected for further processing.

Fig. 12 contains a table of the kinetic properties of anti-ERCC 1 antibodies determined by kinetic screening. BL: post-binding, signal amplitude height in relative response units at the end of the SlyD/FKBP12-ERCC1 binding period. SL: post-stabilization, signal amplitude height in relative reaction units at the end of the dissociation period of SlyD/FKBP12-ERCC 1. Kd: langmuir-fitted dissociation rate constant (1/s) during dissociation. t 1/2 dis: the half-life of the antibody-SlyD/FKBP 12-ERCC1 complex, expressed in minutes, was calculated according to the formula t 1/2 dis = ln (2)/(60 kd).

FIG. 13 exemplary anti-ERCC 1 antibody single concentration kinetics of clone < ERCC1> M-5.3.35 with SlyD/FKBP12-ERCC1 as the analyte in solution.

FIG. 14 is a Western blot using clone < ERCC1> M-5.1.35. Mu.g of OVCAR-3 and HEK293 cell lysates were loaded per lane on NuPAGE SDS gels (Invitrogen). A specific ERCC1 band at 37kDa was detected.

FIG. 15 immunohistochemical detection of ERCC1 in FFPE-embedded human cancer tissue of SCLC cancer samples. White arrows indicate cells with elevated levels of EECC1 that appear in darker colors.

FIG. 16 overlap of IGF-1 (PDB: 1PMX) and IGF-2 (PDB: 1IGL) PyMOL1.4. Alignment of IGF-1 and IGF-2 (clustalW). Black boxes indicate the IGF-1(74-90) and IGF-2(53-65) hairpin sequences.

FIG. 17 SDS PAGE (Coomassie staining) and anti-histidine tag Western blotting (10 sec exposure) of Thermus thermophilus SlyD-IGF-1(74-90) fusion polypeptides. M-Novex Sharp standard; 1-2.5 μ g Thermus thermophilus SlyD-IGF-1(74-90) fusion polypeptide; 2-5.0 μ g Thermus thermophilus SlyD-IGF-1(74-90) fusion polypeptide; 3-10 μ g Thermus thermophilus SlyD-IGF-1(74-90) fusion polypeptide; m. Magic Mark.

FIG. 18 analytical HPLC chromatogram of Thermus thermophilus SlyD-IGF-1(74-90) fusion polypeptide.

FIG. 19 serum titers determined by ELISA 12 weeks after immunization of NMRI mice with SlyD/FKBP 12-IGF-1 (74-90). And mE: milliabsorbance, IGF-1: native human IGF-1 (Peprotech).

FIG. 20 serum titers determined by ELISA 12 weeks after immunization of Balb/C and NMRI mice. And mE: milliabsorbance, IGF-1: native human IGF-1 (Peprotech).

FIG. 21 ELISA screening of primary cultures with binding signals for IGF-1, Thermus thermophilus SlyD-IGF-1(74-90) fusion polypeptides and Thermus thermophilus SlyD wild-type polypeptides. And mE: milliabsorbance, IGF-1: native human IGF-1 (Peprotech).

FIG. 22 exemplary BIAcore kinetic screening of primary cultures < IGF-1> M-11.0.15 for IGF-1, IGF-2, Thermus thermophilus SlyD-IGF-1(74-90) fusion polypeptides and Thermus thermophilus SlyD wild-type polypeptides.

FIG. 23 ELISA screening of culture supernatants from clones for IGF-1, Thermus thermophilus SlyD-IGF-1(74-90) fusion polypeptides and Thermus thermophilus SlyD wild-type polypeptides. An increased binding uptake signal was detectable for IGF-1 and Thermus thermophilus SlyD-IGF-1(74-90) fusion polypeptides.

FIG. 24 BIAcore measurements of < IGF-1> M-11.11.17-IgG prepared from scaffolds on I GF-1, IGF-2, Thermus thermophilus SlyD-IGF-1(74-90) fusion polypeptides, Thermus thermophilus SlyD wild-type polypeptides, Thermococcus gamma-serum SlyD wild-type polypeptides, Thermococcus gamma-delta IF fusion polypeptides, Thermococcus gamma-serum SlyD-IGF-2(53-65) fusion polypeptides.

FIG. 25 contains a table of the binding kinetics of anti-IGF-1 antibodies prepared with fusion polypeptides mAb monoclonal antibody, RU relative reaction units of monoclonal antibody captured on the sensor, antigen in solution, kDa molecular weight of antigen injected as analyte in solution, ka association rate constant, KD dissociation rate constant, t 1/2 dis, antibody-antigen complex half-life calculated as formula t 1/2 dis = ln (2)/60 ＊ KDCounting; r_MAX: binding signal at the end of the dissociation period for 90nM analyte injection; MR: a molar ratio; chi²: the measurement fails; n.d.: it is not detected.

FIG. 26 DSC measurement, overlay of two experiments melting SlyD/FKBP 12-IGF-1(74-90) fusion polypeptide in a temperature gradient from 10 ℃ to 95 ℃. The SlyD/FKBP 12-IGF-1(74-90) fusion polypeptide folds reversibly.

FIG. 27 near UV CD spectra of Thermus thermophilus SlyD wild-type polypeptide, Thermus thermophilus SlyD- Δ IF fusion polypeptide (FKBP), and Thermus thermophilus SlyD-antigen fusion polypeptide. At 20 ℃, all polypeptides fold into their native structure.

FIG. 28 temperature-dependent CD spectra of Thermus thermophilus SlyD- Δ IF fusion polypeptides. Repeated heating and cooling showed that the FKBP domain of thermus thermophilus SlyD reversibly folds. Thermus thermophilus SlyD-delta IF fusion polypeptide is stabilized to 65 ℃ and unfolded at 85 ℃.

FIG. 29 temperature dependent CD spectra of Thermococcus gamma-lelarans SlyD-antigen fusion polypeptides. The lower signal plateau was not reached at 100 ℃ indicating that the fusion polypeptide had not yet completely unfolded. The fusion polypeptide is stable and folded up to 80 ℃.

FIG. 30 repeated freeze-thaw cycles and temperature stress tests followed by re-chromatography of fractions containing monomeric Thermococcus gamma-antigen SlyD-antigen fusion polypeptides. 0.75 ml/min 100. mu.l 50mM K₂HPO₄／KH₂PO₄And a 280nm SUX200 pattern of the Ni-NTA-eluted fraction of 300. mu.g of Thermococcus gamma-interferon SLyD-antigen fusion polypeptide in pH7.0, 100mM KCl, 0.5mM EDTA.

FIG. 31 SDS PAGE (Coomassie staining) and Western blotting of Thermus thermophilus SlyD wild-type polypeptides (incubation with anti-octahistidine-tagged antibody for 10 sec).

FIG. 32 quantification of this kinetic screening method for anti-IGF-1 antibodies. Empty cells indicate that the values are not detectable/determinable.

FIG. 3312 kinetics of IGF-1 binding of the supernatant of the clone cultures.

FIG. 34 SDS PAGE (Coomassie staining) of FKBP 12/13 fusion polypeptides expressed in E.coli.

FIG. 35 FKBP 12/13 fusion polypeptide: HPLC SEC elution Profile of Ni-NTA purified material. FKBP 12/13 fusion polypeptides are mostly monomeric.

FIG. 36 analytical HPLC chromatogram of SlyD/FKBP 12-IGF-1(74-90) fusion polypeptide.

FIG. 37 non-denaturing Coomassie staining8-16％Tris-Glycine Mini Gel(Invitrogen)。BM：BenchMark^TMPre-stabilized Protein Ladder (Invitrogen). F4 to F10: size exclusion chromatography of the eluted fractions. Fraction 8 and 9 showed a single distinct protein band at 37 kDa.

FIG. 38 fluorescence emission of SlyD-FKBP 12/13-CSF 1R at different temperatures.

FIG. 39 SDS PAGE (Coomassie staining) and anti-histidine Western blotting (10 sec exposure) of Thermus thermophilus SlyD- Δ IF fusion polypeptides. Black arrows indicate protein bands. M-Novex Sharp Standard; 1-2.5 μ g Thermus thermophilus SlyD- Δ IF fusion polypeptide; 2-5.0 μ g Thermus thermophilus SlyD- Δ IF fusion polypeptide; 3-10 μ g Thermus thermophilus SlyD- Δ IF fusion polypeptide; m. Magic Mark.

FIG. 40 SDS page (left) and Western blot (right) of Ni-NTA chromatographically purified SlyD-FKBP12-CD 81. M: novex Sharp standard, 1: SlyD/FKBP 12-CD 81; 2.5. mu.g MW: 36kD, 2: SlyD/FKBP 12-CD 81; 5.0. mu.g, 3: SlyD/FKBP 12-CD 81; 10 μ g M ANGSTROM: magic Mark.

Examples

Example 1

Expression and purification

The polypeptides were produced in E.coli (pQE80L vector/E.coli BL21Codonplus-RP cell line) according to known methods.

For purification of the crude polypeptide, an affinity chromatography step is used in the presence of a chaotropic agent under non-denaturing conditions or under denaturing conditions. For fusion polypeptides comprising SlyD moieties, purification in the presence of a chaotropic agent is particularly suitable, since the total amount of fusion polypeptide can be isolated from e. In addition, the entire fusion polypeptide is obtained in a random coil conformation. The fusion polypeptide still bound to the affinity chromatography material is transferred to non-denaturing conditions by washing the column with physiological salt solution. Due to the spontaneous folding of the SlyD and FKBP12 parts of the fusion polypeptide, the inserted amino acid sequence is also converted into its native conformation. The refolded fusion polypeptide is recovered from the affinity chromatography column using physiological buffer containing an imidazole gradient.

Example 2

Chemical derivatization

The C-terminal lysine residue was activated by LC-SPDP (6- (3- [ 2-pyridyldithio ] - (propionamido) hexanoic acid succinimide) (Pierce, cat # 68181-17-9) under acidic conditions (pH 6).

Arginine and lysine are bases that can take up a proton of the alkylammonium group of lysine. The free amino group can be derived from any hydroxy succinimide activated carbonic acid.

Example 3

Formalin treatment

The derived fusion polypeptide may be treated with formalin solution. The immobilized derivatized fusion polypeptide may then be purified by size exclusion chromatography to obtain a composition having a defined oligomeric state (monomer, oligomer, multimer).

Example 4

BIAcore characterization of antibody-producing clone culture supernatants

A BIAcore T100 instrument (GE Healthcare) was used with the BIAcore CM5 sensor installed into the system. 100 μ l/min of 0.1% SDS, 50mM NaOH, 10mM HCl and 100mM H₃PO₄To pre-condition the sensor for 1 minute.

The system buffer was HBS-ET (supplemented with 150mM NaCl, 1mM EDTA, 0.05% (w/v)20 mM HEPES (pH 7.4)). The sample buffer is the system buffer.

The BIAcore T100 system is driven under control software V1.1.1. Polyclonal rabbit IgG antibody < IgGFC γ M > R (Jackson ImmunoResearch Laboratories Inc.) was immobilized at 30 μ g/ml in 10mM sodium acetate buffer (pH4.5) at 6500RU on flowcells 1, 2, 3 and 4, respectively, by EDC/NHS chemistry as specified by the manufacturer. Finally, the sensor surface was blocked with 1M ethanolamine solution. The entire experiment was carried out at 25 ℃.

The clone culture supernatants containing 35nM to 190nM of each antibody were captured on < IgGFC γ M > R surface for 1 min at a flow rate of 5 μ l/min. As analytes in solution, recombinant antigens, biotinylated disulfide-bridged recombinant antigens, SlyD/FKBP 12-antigen, Thermus thermophilus SlyD-antigen, SlyD/FKBP 12-control and/or Thermus thermophilus SlyD-wt fusion polypeptides were used. Each analyte was injected at different concentration gradients of 90nM, 30nM, 10nM, 3.3nM, 1.1nM and 0 nM. The binding period was monitored at a flow rate of 100. mu.l/min for 3.5 minutes. Dissociation was monitored at a flow rate of 100. mu.l/min for 15 min. The system was regenerated with 10mM glycine buffer (pH 1.7). Kinetics were evaluated using BIAcore evaluation software.

Example 5

IHC sample preparation

The matrix-immobilized LC-SPDP fusion polypeptide is treated with an organic solvent, heated, and treated with an acidic buffer. The substrate-bound polypeptide is then recovered under reducing conditions. In order to obtain a substance with a defined composition, size exclusion chromatography may be performed. The substance thus obtained has a defined oligomeric state (monomers, oligomers and polymers) and can be used as an immunogen for immunizing experimental animals, but it can also be used as a test antigen for selecting and screening antibodies.

Example 6

Immunization

The pre-formulated immunogenic fusion polypeptides are administered intraperitoneally to experimental animals (e.g., mice, rats, rabbits, sheep, or hamsters) at various doses. Prior to B cell collection, booster immunizations were performed. B cell hybridomas are obtained according to the method of Koehler and Millstein (Kohler, G., and Millstein, C., Nature256(1975) 495-497). The obtained hybridomas are deposited as single clones or cells in wells of a multi-well plate. Primary hybridoma cultures that test positive for the binding of the secreted antibody to the antibody are further screened using kinetic screening methods.

Example 7

anti-IGF-1 antibodies

Cells obtained from four immunized NMRI mice were analyzed by ELISA. Nunc Maxisorb F-well plates were coated with SlyD/FKBP 12-IGF-1, SlyD/FKBP 12-IGF-1(74-90), Thermus thermophilus SlyD-IGF-1, SlyD/FKBP 12-control or Thermus thermophilus SlyD-wt by adding a solution containing 0.41. mu.g of polypeptide/ml. Isolated antigen IGF-1 was immobilized in the wells of StreptaWell High Bind SA multiwell plates by adding solutions containing either 90 ng/ml biotinylated IGF-1 or 500ng/ml biotinylated IGF-1-peptide loops.

The free binding sites were then blocked by adding 1% RPLA in PBS at room temperature. The wells were washed three times with a solution containing 0.9% (w/v) sodium chloride and 0.05% (w/v) Tween. With PBS 1: 50 dilutions of mouse serum were used as samples. According to the following steps: 4 gradient with optional further dilution, until 1: 819,200 final dilution. The incubation time was 1 hour at room temperature. The wells were washed three times with a solution containing 0.9% (w/v) sodium chloride and 0.05% (w/v) Tween. Polyclonal antibodies conjugated to the constant domains of an anti-target antibody of peroxidase were used as detection antibodies (PAK)<M-Fcγ>S-F(ab′)₂-POD). The detection antibody was added at a concentration of 80 ng/ml in PBS containing 1% (w/v) RSA. The incubation time was 1 hour at room temperature. The wells were washed three times with a solution containing 0.9% (w/v) sodium chloride and 0.05% (w/v) Tween. Wells were then incubated with ABTS solution for 15 minutes at room temperature. The intensity of the color development was measured with a spectrophotometer. Exemplary results are provided in the table below.

Watch (A)

-: no binding was detected in ELISA

Example 8

Kinetic screening of hybridoma culture supernatants

To select an antibody suitable for IHC, the target complex was set to a half-life of 10 minutes at 37 ℃.

Kinetic screening was performed on BIAcore a100 under the control of software version V1.1. The BIAcore CM5 chip was loaded into the machine and subjected to hydrodynamic studies according to the manufacturer's instructions, and then the chip was processed. HBS-EP buffer (10mM HEPES (pH7.4), 150mM NaCl, 1mM EDTA, 0.05% (w/v) P20) was used as a running buffer. Polyclonal anti-IgG Fc capture antibody compositions at a concentration of 30. mu.g/ml in 10mM sodium acetate buffer (pH4.5) were pre-concentrated to spots 1, 2, 4 and 5 in flow cells 1, 2, 3 and 4. Antibodies were covalently immobilized at 10,000RU by NHS/EDC. The sensor was then deactivated by saturation with a 1M ethanolamine solution. Spots 1 and 2 were used for the assay, and spots 2 and 4 were used as reference. Prior to addition to the sensor chip, hybridoma culture supernatant 1: 5 diluted in HBS-EP buffer. The diluted solution was added at a flow rate of 30. mu.l/min for 1 min. The antigen was then immediately injected at a flow rate of 30. mu.l/min for 2 minutes. The signal was then recorded for an additional 5 minutes. The sensor was regenerated by injecting 10mM glycine-HCl solution (pH1.7) at a flow rate of 30. mu.l/min for 2 minutes. The signal recorded shortly before the end of the injection of antigen is denoted as late Binding (BL). The recorded signal shortly before the end of the recording of the dissociation is denoted as post-Stability (SL). This was used to calculate the apparent complex stability according to the following formula:

(1-[BL(RU)-SL(RU)／BL(RU)]

hybridoma cells selected in the kinetic screen were deposited as single cells by FACS (facsaria (becton dickinson), software v4.1.2). The monoclonals were cultured in RPMI-1640 medium in 24-well plates or in 100ml roller bottles.

Example 9

Immunohistochemical analysis

IHC analysis was performed manually or automatically on a Ventana Benchmark XT or Discovery XT8R machine. Antibodies are tested on suitable positive or genetic, formalin-fixed or cryopreserved tissues or cells.

Alternatively, the cell is transfected with a nucleic acid encoding the target polypeptide. Transfected cells were lysed and tested for their suitability as positive or negative controls by Western blotting.

Example 10

SlyD/FKB 12-antigen scaffold-assisted production of anti-ERCC 1 antibody

Immunization

8-12 week old SJL mice were immunized intraperitoneally with 100 μ g KHL-conjugated ERCC 1-derived peptides encompassing amino acids 219-245 of human ERCC1 (excision repair cross-complement). ERCC1 derivatives were produced synthetically by peptide synthesis.

Mice were immunized 3 times (primary and 6 and 10 weeks after primary boost). The first immunization was performed with Freund's complete adjuvant, and the second and third immunization were performed with Freund's incomplete adjuvant. Three days prior to hybridoma fusion a final boost was performed intravenously with 100 μ g of KLH-conjugated peptide antigen. The generation of hybridoma primary cultures was performed according to Kohler and Milstein (Kohler, G and Milstein, C., Nature256(1975) 495-497). Hybridomas were isolated into 96-well MTPs by limiting dilution and screened for antigen binding using ELISA. The ELISA was driven by Tecan Sunrise running under firmware V3.1519/03/01, XREAD PLUS version V4.20. Primary hybridoma cell cultures showing positive color formation in ELISA by binding to biotinylated ERCC 1-derived peptides encompassing amino acids 219-245 were transferred to the kinetic screening method described herein.

To avoid selecting antibodies that are not suitable for IHC, but only bind to the linear peptide, further screening efforts were performed using a scaffold-based approach. The scaffold approach further deselects antibodies that bind immunogenic peptides at their termini.

Production of SlyD/FKBP12-ERCC1

Synthetic genes encoding SlyD/FKBP12-ERCC1 and SlyD/FKBP 12-ctrl were purchased from SloningBiotechnology GmbH (Germany) and cloned into pQE80L expression vector. The polypeptide was produced in E.coli BL21Codonplus-RP as a codon-optimized gene construct for E.coli (see FIGS. 3 and 8).

To purify the crude fusion polypeptide, an affinity chromatography step is used under denaturing conditions in the presence of a chaotropic agent. For fusion polypeptides containing SlyD moieties, purification in the presence of chaotropic agents is used in particular, since the total amount of fusion polypeptide can be isolated from e. In addition, the entire fusion polypeptide is obtained in a random coil conformation. The fusion polypeptide still bound to the affinity chromatography material is transferred to non-denaturing conditions by washing the column with physiological salt solution. The inserted amino acid sequence can also be converted to its native conformation due to spontaneous folding of the SlyD and FKBP12 portions of the fusion polypeptide. The refolded fusion polypeptide is recovered from the affinity chromatography column using physiological buffer containing an imidazole gradient. SDS gels and Western blots of SlyD/FKBP12-ERCC1 fusion polypeptides are shown in FIG. 3. < His6> -Western blot showing the C-terminal integrity of the fusion polypeptide. No other polypeptide bands were detected.

Fluorescence measurement

The affinity-purified fusion polypeptide was dialyzed against 75mM HEPES buffer (pH7.5, 150mM NaCl, 6.5% (w/v) sucrose, 10mM cysteine) and filtered. UV/Vis Spectroscopy at 7.4 mg/ml for an extinction coefficient calculated for the 35380.301Da polypeptide quantified SlyD/FKBP12-ERCC1 (FIG. 4). In the wavelength screening from 220nm to 340nm, a 280nm absorption peak derived from a single FKBP12Trp was obtained. No absorption at 340nm was detected.

Protein fluorescence measurements were used to test the conformational properties of SlyD/FKBP12-ERCC 1. FKBP12C22A is particularly useful as a vector for polypeptide insertion, since a single FKBP12Trp moiety can be used to diagnose the structural integrity of the FKBP12 moiety (Scholz, C. et al, J.biol. chem.271(1996) 12703-. FKBP12C22A in its native structure showed a single fluorescence emission peak at 320nm (Zoldak, G. et al, J.mol.biol.386(2009) 1138-1152).

Mu.l of HBS-E buffer (pH7.4) containing 2.5 mg/ml SlyD/FKBP12-ERCC1 were analyzed at different temperatures. Cary Eclipse instrument under scanning software version 1.1(132) was used at excitation and emission bandwidths of 5 nm. Wavelength scans of 300nm-600nm were driven at 600 nm/min. Excitation of intrinsic tryptophan fluorescence was set at 294 nm. A broad peak at 350nm was obtained (FIG. 4). By theory, the inherent Trp solvolochromic (solvatochromic) fluorescence emission at 350nm will be strongly quenched in the context of the folded FKBPl2 protein, which increases with unfolding of FKBPl 2. Temperature screening from 25 ℃ to 85 ℃ did not show any other fluorescence emission peaks, but showed temperature-dependent fluorescence quenching of the 350nm emission. No 320nm emission was detected indicating the structural integrity of FKBPl 2.

Thus, the single Trp residue in the SlyD/FKBPl 2-ERCCl fusion polypeptide had been exposed to solvent at 25 ℃, indicating that the chimeric FKBPl2 in the SlyD-FKBPl2 background was partially or fully unfolded.

Therefore, scaffolds are ideal platforms for modeling and presenting structurally diverse labile conformations, as they are typically present in paraffin-embedded, formalin-fixed tissues during immunohistochemical experiments (Abe, m. et al, anal. biochem.318(2003) 118-.

FK506BIAcore binding assay

The BIAcore3000 instrument under the control of the sensing SA chip assembly software version V4.1 was used according to the manufacturer's instructions. HBS-EP buffer (10mM HEPES, pH7.4, 150mM NaCl, 1mM EDTA, 0.05% (w/v) P20 (10% aqueous solution of nonionic surfactant polysorbate 20(Tween 20)) was used as a running buffer. The bi-linker-FK 506 conjugate of 1213RU was captured on flow cell 4 (Roche Diagnostics Mannheim, Germany).

300nM of purified SlyD/FKBPl 2-ERCCl fusion polypeptide and 300 nMSlD/FKBP 12 control polypeptide were injected into the system at 30. mu.l/min for 3 min binding time and 3 min dissociation time.

The sensor was regenerated by injecting 10mM glycine-HCl solution (pH1.7) at a flow rate of 30. mu.l/min for 2 minutes.

BIAcore binding assays with 300nM fusion polypeptide SlyD/FKBP12-ERCC1 as analyte in solution on the sensor surface presented ligand bi-FK506 (FIG. 5) showed no binding activity (FIG. 6), indicating loss of structural function of the FKBP12 moiety in the chimeric fusion polypeptide. The control polypeptide FKBP12(C22A) showed binding activity.

The inability of the SlyD/FKBP12-ERCC1 fusion polypeptide to bind FK-506 provides additional evidence of the structure of SlyD/FKBP12-ERCC1 that is different from the structure of the FKBP12(C22A) conformation. This is accompanied by a loss of binding activity of the chimeric FKBP12 domain.

Analytical HPLC chromatography

The fusion polypeptide was analyzed for its oligomeric state by analytical HPLC chromatography.

Chromeleon Dionex HPLC equipment equipped with a TSK3000SWXL column equilibrated in HBS-E buffer (pH7.4) was used at 25 ℃ as recommended by the manufacturer. The buffer flow rate was 0.7 ml/min. Mu.l of a solution containing SlyD/FKBP12-ERCC1 (7.4 mg/ml) was injected into the system (see FIG. 8). In another workflow, a solution containing the SlyD/FKBP12 control (9.5 mg/ml) was injected into the system (see fig. 7). In another workflow, a solution (3 mg/ml) containing Thermus thermophilus SlyD-IGF-1(74-90) was injected into the system (see FIG. 18). In another workflow, a solution (5.4 mg/ml) containing SlyD/FKBP 12-IGF-1(74-90) was injected into the system (see FIG. 36). The UV/VIS detector was set at 280 nm. The data were evaluated using Dionex software version 6.80SP2Build2284 according to the manufacturer's instructions. The system was calibrated with the molecular standard, Oriental Yeast, catalog number 46804000.

FIG. 8 shows the column elution profile of Ni-NTA affinity purified SlyD/FKBP12-ERCC 1. It can be seen that the area integral of 91.5% of the entire elution profile is localized in peak No. 5 (1310.319mAU) eluting at 12.37 minutes retention time. The figure shows a monomeric SlyD/FKBP12-ERCC1 fusion polypeptide. The monomeric fusion polypeptide is already obtained only after the initial Ni-NTA purification step.

Screening using kinetics

The SlyD/FKBP12-ERCC1 fusion polypeptide was used for SPR binding analysis. It is helpful to determine the antibody binding kinetics according to the Langmuir model with monomers in solution and monovalent analytes. Furthermore, improving the mass sensitivity of the measurement with analytes having an increased (e.g. high) molecular weight is helpful for SPR measurements. At the same time, epitope accessibility must be provided.

A schematic of the BIAcore screening assay is shown in figure 9.

Kinetic screening was performed on a BIAcore a100 instrument under the control of software version V1.1. Biacore cm5 chips were loaded into the machine and the hydrodynamic studies were performed as per the manufacturer's instructions. The chip is then processed. HBS-EP buffer (10mM HEPES pH7.4, 150mM NaCl, 1mM EDTA, 0.05% (w/v) P20) was used as the running buffer. Polyclonal compositions of anti-IgG Fc capture antibody at a concentration of 30. mu.g/ml in 10mM sodium acetate buffer (pH4.5) were pre-concentrated to spots 1, 2, 4 and 5 in flow cells 1, 2, 3 and 4. Antibodies were covalently immobilized at 10,000RU by NHS/EDC chemistry. The sensor was then deactivated with a 1M ethanolamine solution. Spots 1 and 2 were used for the assay, and spots 2 and 4 were used as reference. Prior to addition to the sensor chip, hybridoma supernatant 1: 5 diluted in HBS-EP buffer. The diluted solution was applied at a flow rate of 30. mu.l/min for 1 min. The formulated antigen (e.g., FKBP12 fusion polypeptide) was then injected immediately at a flow rate of 30. mu.l/min for 2 minutes. The signal was then recorded for an additional 5 minutes. The sensor was regenerated by injecting 10mM glycine-HCl solution (pH1.7) at a flow rate of 30. mu.l/min for 2 minutes. The signal recorded shortly before the end of the injection of antigen is denoted as late Binding (BL). The recorded signal shortly before the end of the recording of the dissociation is denoted as post-Stability (SL). Both data points are plotted against each other. The selected antibodies had a post-binding value equal to the post-stability value. These antibodies are located in the figure in the region near the trend line indicating BL = SL.

Figure 10 shows data for selected anti-ERCC 1 antibodies. It can be seen that the SlyD/FKBP12-ERCC1 interaction is highly specific. No interaction was detected with the SlyD/FKBP12 control sample. No non-specific binding is visible overall.

Figure 11 shows titer analysis of antibodies. The amount of antigen expressed in response units (post-binding, RU) of antibody presented at the saturation surface (capture level, RU) is shown. The titer (molar ratio) of the surface-presented antibodies is indicated by the trend lines and arrows in fig. 11. All sister clones (clone id5.00x.35) mapped to the titer channel MR0.5-MR1.0, while all other clones mapped to a channel below MR0.5 indicating less functionality. No functional binding to the SlyD/FKBP12 control was detected.

Figure 12 shows the quantification of this kinetic screening method. All 6 sister clones (5.001.35 to 5.006.35) showed appropriate values for post-binding and post-stability. The binding rate constant kd (1/s) shows high antigen complex stability, meeting the requirements of an antibody suitable for IHC. Calculated t for all 6 sister clones_1／2dissThe antigen complex stability half-life was 204 minutes.

FIG. 13 shows exemplarily the kinetic screening characteristics of clone 5.003.35 for the analytes SlyD/FKBP12-ERCC1 and SlyD/FKBP12 control. Since SlyD/FKBP12-ERCC1 is a stable, soluble and monomeric analyte, it perfectly fits 1: 1Langmuir dissociation model (black line on red dissociation raw data). No non-specific binding was detected. No interaction was detected with the SlyD/FKBP12 control.

Western blot

FIG. 14 shows a Western blot experiment using clone 5.001.35. Western blot can be used as an indication of suitability of the antibody IHC for subsequent use.

For Western blotting, 5. mu.g OVCAR-3 and 5. mu.g HEK-293 cell lysates were loaded in gel lanes on 4-12% NuPAGE SDS gels (Invitrogen). Neither cell line was pretreated, e.g., by irradiation or cisplatin.

Western blots were performed according to standard protocols using NuPAGE buffer and reagents (Invitrogen). Antibody 5.001.35 was used at a concentration of 50 ng/ml. Primary antibody incubations were performed at Room Temperature (RT) for 30 min. The membranes were developed using LumiImager together with LumiLight reagent according to the manufacturer's instructions (Roche Applied Science, Mannheim, Germany). Endogenous basal ERCC1 levels were specifically detected as a single 37kDa band in Western blots.

IHC experiment

Fig. 15 shows IHC detection of ERCC1 in FFPE human cancer tissue. For immunohistochemical detection, SCLC cancer is prepared2 μm sections of the samples. All staining procedures were performed on a Ventana Benchmark XT automated IHC staining instrument with Ventana buffer and reagents according to the manufacturer's standard instructions. Primary antibody (cloning)<ERCC1>M-5.1.35) was used at a concentration of 5. mu.g/ml. Primary antibody was incubated on the sections for 32 minutes at 37 ℃. Ventana iView as recommended by the manufacturer^TMThe detection kit detects the primary antibody. White arrows indicate cells with increased levels of ERCC1 that appear in darker colors.

Abstract

Unlike the small molecular weight ERCC1 peptide (2kDa), the scaffold used herein is a high molecular weight analyte (36kDa) that amplifies the signal in SPR-based kinetic screening methods.

Peptide-based screening reagents have the risk of selecting antibodies that recognize the peptide termini, which is completely circumvented by using the scaffold approach reported herein, where the peptides are embedded in an N-and C-terminal polypeptide background. Despite the provision of multiple metastable peptide insertions, the scaffold fusion polypeptide is generally stable, soluble and monomeric. 1: 1Langmuir kinetics.

The use of fusion polypeptides in this background (set up) is well suited to mimic the FFPE IHC environment and is therefore a very suitable screening reagent for the preparation of antibodies suitable for IHC.

Without being bound by theory, the fusion polypeptide comprises a folded SlyD-derived portion and an unfolded or partially unfolded human FKBP 12-derived portion, the FKBP 12-derived portion providing at least a single core Trp residue for solvent contact, as shown for SlyD/FKBP12-ERCC 1. SlyD folds reversibly and shows sufficient thermal stability for technical applications.

The SlyD/FKBP-12 scaffold is a platform suitable for modeling a variety of peptide secondary structure motifs such as those found in paraffin-embedded, formalin-fixed tissues in immunohistochemical experiments (see Abe et al (2003) supra).

Fusion polypeptides are particularly suitable as immunogens compared to full-length polypeptides from which the inserted (immunogenic) amino acid sequence is derived, e.g. in terms of solubility, reversible folding (renaturation/denaturation) and lack of disulfide bonds to be formed correctly. The fusion polypeptides reported herein provide a scaffold for insertion of immunogenic amino acid sequences. It stabilizes the structure of the inserted immunogenic amino acid sequence (by reducing conformational entropy, without being bound by theory). Without being bound by theory, it is hypothesized that the N-terminal SlyD fusion polypeptide keeps the entire chimeric fusion polypeptide in a soluble and thermodynamically stable, but partially unfolded form.

Rebuzzini, G. (Ph. study (2009) at the university of Milano-Bicocca (Italy)) reported the study of the helicase domain of hepatitis C virus NS3 for application to chemiluminescent immunoassays. In his study, Rebuzzini reported that chimeric FKBP12 used as an immunogen for presenting NS3 helicase domains with the insertion sequence of Knappe, t.a. et al (j.mol.biol.368(2007) 1458-. This is consistent with our finding that the chimeric FKBP12 in the SlyD-FKBP 12-antigen fusion polypeptide is partially or fully unfolded. Unlike the findings of Rebuzzini, the SlyD/FKBP 12-antigen fusion polypeptide is found herein to be monomeric and stable.

Example 11

Generation of IGF-1(74-90) -specific antibodies

Antigen-specific antibodies were obtained by immunizing mice with the chimeric Thermus thermophilus-SlyD-antigen fusion polypeptide. Multiple epitopes on the surface of the scaffold can be targeted, from which antibodies binding to the target antigen are identified by differential screening of wild-type thermus thermophilus-SlyD as a negative control or of the native recombinant antigen (IGF-1) as a positive control. In the following, the examples demonstrate the properties of archaeal SlyD derivatives compared to potentially metastable human FKBP 12. Thermus thermophilus-SlyD allows the presentation of enthalpy, rigidity and stable structure and is therefore suitable for the preparation of monoclonal antibodies against native protein structures.

Production of Thermus thermophilus SlyD fusion polypeptide

Guanidine hydrochloride (GdmCl) (grade a) was purchased from NIGU (Waldkraiburg, germany).EDTA-free protease inhibitor tablets, imidazole and EDTA were from Roche Diagnostics GmbH (Mannheim, Germany) and all other chemicals were of analytical grade from Merck (Darmstadt, Germany). Ultrafiltration membranes (YM10, YM30) were purchased from Amicon (Danvers, MA, USA), micro-permeable membranes (VS/0.025 μm) and ultrafiltration units (Biomax ultra filtration unit) from Millipore (Bedford, MA, USA). The nitrocellulose and cellulose acetate membranes (1.2 μm, 0.45 μm and 0.2 μm pore size) used for filtering the crude lysates were from Sartorius (Goettingen, germany).

Cloning of the expression cassette

The sequence of SlyD polypeptide from thermus thermophilus was retrieved from the SwissProt database (accession number Q72H 58). The sequence of the SlyD polypeptide from Thermococcus gammatolerans was retrieved from the Prosite database (accession No. C5A 738). Synthetic genes encoding Thermus thermophilus SlyD, Thermus thermophilus SlyD-IGF-1(74-90) and Thermus thermophilus SlyD- Δ IF were purchased from Sloning Biotechnology GmbH (Germany) and cloned into pQE80L expression vector. Codon usage was optimized for expression in E.coli host cells. Synthetic genes encoding Thermococcus gamma SlyD, Thermococcus gamma SlyD-IGF-2(53-65), Thermus thermophilus SlyD-antigen and Thermococcus gamma SlyD-antigen were purchased from Geneart (Germany) and cloned into pET24 expression vector (Novagen, Madison, Wisconsin, USA). Codon usage is preferred for expression in E.coli host cells.

In addition, a GS linker (GGGS, SEQ ID NO: 81) and a His tag were fused to the carboxyl terminus to allow affinity purification of the fusion polypeptide by immobilized metal ion exchange chromatography.

To generate monoclonal antibodies that specifically bind IGF-1 fragments 74-90 (amino acid sequence NKPTGYGSSSRRAPQTG, SEQ IDNO: 92), the amino acid sequences of each peptide were fused into the chaperone SlyD derived from Thermus thermophilus by deleting amino acids 68-120 of the original protein. Due to the angular adaptation of IGF-1 insertion, Asp at position 70 and Leu at position 88 were substituted with Gly (D70G and L88G). Thus, the fusion polypeptide has the amino acid sequence:

MRGSKVGQDKVVTIRYTLQVEGEVLDQGELSYLHGHRNLIPGLEEALEGREEGEAFQAHVPAEKAYGPHGNKPTGYGS S SRRAPQTGGAGKDLDFQVEVVKVREATPEELLHGHAHGGGSRKHHHHHHHH(SEQ ID NO：101).。

as a control, a native wild-type SlyD from thermus thermophilus was used:

MKVGQDKVVTIRYTLQVEGEVLDQGELSYLHGHRNLIPGLEEALEGREEGEAFQAHVPAEKAYGPHDPEGVQVVPLSAFPEDAEVVPGAQFYAQDMEGNPMPLTVVAVEGEEVTVDFNHPLAGKDLDFQVEVVKVREATPEELLHGHAHGGGSRKHHHHHH(SEQ ID NO：104).。

for screening and specificity testing, Thermus thermophilus SlyD-delta IF fusion polypeptides were generated. Thermus thermophilus SlyD- Δ IF lacks an IF domain (which is substituted with the amino acid sequence motif AGSGSS) and comprises the amino acid sequence of SEQ ID NO: 16C-terminal amino acid sequence tag:

MRGSKVGQDKVVTIRYTLQVEGEVLDQGELSYLHGHRNLIPGLEEALEGREEGEAFQAHVPAEKAYGPHGAGSGSSGAGKDLDFQVEVVKVREATPEELLHGHAHGGGSRKHHHHHHHH(SEQ ID NO：120).。

as a control, a nucleic acid comprising SEQ ID NO: 16, natural SlyD from thermococcus gammatolerans labelled with a C-terminal amino acid sequence:

MKVERGDFVLFNYVGRYENGEVFDTSYESVAREQGIFVEEREYSPIGVTVGAGEIIPGIEEALLGMELGEKKEVVVPPEKGYGMPREDLIVPVPIEQFTSAGLEPVEGMYVMTDAGIAKILKVEEKTVRLDFNHPLAGKTAIFEIEVVEIKKAGEAGGGSRKHHHHHH(SEQ ID NO：121).。

as a control for cross-reactivity, a structurally homologous hairpin sequence from human IGF-2(53-65) was inserted into a loop of Thermococcus gammatolerans SlyD fused at the C-terminus to a GS linker and a hexahistidine tag:

MKVERGDFVLFNYVGRYENGEVFDTSYESVAREQGIFVEEREYSPIGVTVGAGEIIPGIEEALLGMELGEKKEVVVPPEKGYGMP-G-SRVSRRSRG-G-AGKTAIFEIEVVEIKKAGEAGGGSRKHHHHHH(SEQ ID NO：122).。

expression, purification and refolding of fusion polypeptides

Coli BL21(DE3) cells containing the embodied expression plasmids were cultured at 37 ℃ in LB medium containing each antibiotic for selective growth (kanamycin 30 μ g/ml, or ampicillin (100 μ g/ml)), to OD600 of 1.5, cytoplasmic overexpression was induced by addition of 1mM isopropyl- β -D-thiogalactoside (IPTG), 3 hours after induction, cells were harvested by centrifugation (5,000g, 20 minutes), frozen and stored at-20 ℃ for cell lysis, the frozen pellet was resuspended in cooled 50mM sodium phosphate buffer (ph8.0) supplemented with 7M GdmCl and 5mM imidazole, then the suspension was stirred on ice for 2-10 hours until cells were completely lysed, centrifugation (25,000g, 1 hour) and filtration (nitrocellulose membrane, 8.0 μ M, 1.2 μ M, 0.2 μ M) after which the suspension was stirred on ice for 2-10 hours, the cells were completely lysed, centrifugation (25,000g, 1 hour) and filtration (nitrocellulose membrane, 8.0 μ M, 1.2 μ M, 0.2 μ M) was washed on a column with a buffer containing the lysate, a reducing buffer, and reducing the protein in a buffer containing the cell-folding buffer, reducing the cell, reducing the protein, reducing the protein, reducing protein, the protein, the proteinEDTA free, Roche). A total of 15 to 20 column volumes of refolding buffer were applied in the overnight procedure. Then, TCEP was removed by washing with 10 column volumes of 50mM sodium phosphate buffer (pH8.0) containing 100mM NaCl and 10mM imidazoleBoth without EDTA inhibitor mixture. In the final wash step, imidazole concentration was increased to 30nM (10 column volumes) to remove recalcitrant contaminants. The native polypeptide was eluted by applying 250mM imidazole in the same buffer. The purity of the protein-containing fractions was assessed by N-tris (hydroxymethyl) methylglycine-SDS-PAGE (Schaegger, H. and von Jagow, G., anal. biochem.166(1987) 368-. Subsequently, the protein was subjected to size exclusion chromatography (Superdex HiLoad, Amersham Pharmacia) using potassium phosphate as a buffer system (50mM potassium phosphate buffer (pH7.0), 100mM KCl, 0.5mM EDTA). Finally, the protein containing fractions were pooled and concentrated in Amicon cell (YM10) to a concentration of-5 mg/ml.

UV spectral measurement

Protein concentration measurements were performed with a UVIKON XL dual beam spectrophotometer. The molar extinction coefficient of the SlyD variant was calculated according to Pace (Pace, C.N. et al, Protein Sci.4(1995)2411-2423) (280).

CD spectral measurement

To examine whether the chimeric fusion protein of the present invention adopts a folded conformation, the CD spectrum in the near UV region was measured. CD spectra were recorded and evaluated using an A JASCO J-720 instrument and JASCO software as recommended by the manufacturer. A quartz cup with an optical path of 0.2cm was used. The instrument was set to 1 ℃ resolution, 1nm bandwidth, 5mdeg sensitivity and accumulation mode 1. The sample buffer was 50mM potassium phosphate pH7.5, 100mM NaCl, 1mM EDTA. The protein analyte concentrations for each assay were 36. mu.M Thermus thermophilus SlyD wild type, 23. mu.M Thermus thermophilus SlyD-. DELTA.IF, 16. mu.M Thermus thermophilus SlyD-antigen, 19. mu.M Thermococcus gamma. lamans SlyD wild type and 16. mu.M Thermococcus gamma. lamans SlyD-antigen. CD signals between 250nm and 330nm with 0.5nm resolution and 20nm scans per minute were recorded at 20 ℃. In the subsequent experimental embodiment, the CD signal was determined at a constant wavelength of 277nm in temperature gradients (20 ℃ C. -100 ℃ C.) and (100 ℃ C. -20 ℃ C.) for Thermococcus gammatolerans SlyD derivatives, respectively (20 ℃ C. -85 ℃ C.) and (85 ℃ C. -20 ℃ C.) for Thermus thermophilus SlyD derivatives. The temperature gradient was driven at 1 ℃ per minute.

FIG. 27 shows the superposition of three CD spectra of the fusion polypeptides Thermus thermophilus SlyD wild-type, Thermus thermophilus SlyD- Δ IF and Thermus thermophilus SlyD-antigen. CD characteristics show that at 20 ℃ all fusion polypeptides fold into their native structure even when the IF domain is deleted or replaced by amino acid transplantation.

FIG. 28 shows the temperature-dependent near-UV CD spectrum of the fusion polypeptide Thermus thermophilus SlyD-antigen in a temperature gradient from 20 ℃ to 85 ℃. Thermus thermophilus SlyD-antigen is reversibly unfolded and refolded.

Under the given physical conditions, complete unfolding of the Thermococcus gammatolerans SlyD-antigen was not achieved even at 100 ℃ (see FIG. 29). The extraordinary stability of the archaeal FKBP domains enables the transplantation of polypeptides by replacing their IF domains, wherein at the same time the overall stability of the newly generated chimeric scaffold proteins is maintained.

Thermus thermophilus SlyD-IGF-1(74-90) is used for immunizing mice and preparing anti-IGF-1 monoclonal antibody

8-12 week old Balb/c and NMRI mice were immunized intraperitoneally with 100. mu.g of Thermus thermophilus SlyD-IGF-1 (74-90). Mice were immunized three times at time points of 6 weeks and 10 weeks after the primary immunization. The first immunization can be performed with Freund's complete adjuvant, and the second and third immunization with Freund's incomplete adjuvant. After 12 weeks the mouse sera were tested for titer against native recombinant IGF-1 and Thermus thermophilus SlyD-IGF-1(74-90) by the ELISA method described below. After 12 weeks, serum titers were analyzed by ELISA. The ELISA was driven by TecanSunrise running under firmware V3.1519/03/01, XREAD PLUS version V4.20. Nunc Maxisorb F multi-well plates were coated with Thermus thermophilus SlyD-IGF-1(74-90) by adding a solution containing 0.5. mu.g of polypeptide/ml. Isolated antigen IGF-1 was immobilized in the wells of a StreptaWell High Bind SA multiwell plate by adding a solution containing 90 ng/ml biotinylated IGF-1. The free binding sites were then blocked by adding 1% RPLA in PBS for 1 hour at room temperature. With a medium containing 0.9% (w/v) sodium chloride and 0.05% (w/v)v) Wells were washed three times with a solution of Tween. With PBS 1: 50 dilutions of mouse serum were used as samples. According to the following steps: 4 gradient with optional further dilution, until 1: 819,200 final dilution. The incubation time was 1 hour at room temperature. The wells were washed three times with a solution containing 0.9% (w/v) sodium chloride and 0.05% (w/v) Tween. Polyclonal antibodies conjugated to the constant domains of an anti-target antibody of peroxidase were used as detection antibodies (PAK)<M-Fcγ>S-F(ab′)₂-POD). The detection antibody was applied at a concentration of 80 ng/ml in PBS containing 1% (w/v) RSA. The incubation time was 1 hour at room temperature. The wells were washed three times with a solution containing 0.9% (w/v) sodium chloride and 0.05% (w/v) Tween. Wells were then incubated with ABTS solution for 15 minutes at room temperature. The intensity of the color development was measured with a spectrophotometer. Figure 20 shows the obtained mouse serum titers.

Three days prior to splenocytes preparation and fusion with myeloma cell lines, a final boost was performed by intravenous injection of 100 μ g of Thermus thermophilus SlyD-IGF-1(74-90) fusion polypeptide. The generation of hybridoma primary cultures can be carried out according to the method of Koehler and Milstein (Koehler, G. and Milstein, C., Nature.256(1975) 495-497).

ELISA screening

The reactivity of the primary culture supernatants towards the respective blanks of the immunogen Thermus thermophilus SlyD-IGF-1(74-90), biotinylated native IGF-1 and wild type Thermus thermophilus SlyD was tested by ELISA. Elisa is driven by Tecan Sunrise, firmware V3.1519/03/01, XREAD PLUS version V4.20. Nunc MaxisorbF multi-well ELISA plates were coated with 5. mu.g/ml SlyD-fusion polypeptide. StreptaWell High Bind SA multi-well plates were coated with 125 ng/ml recombinant biotinylated IGF-1 antigen. The free binding sites were then blocked with 1% RPLA in PBS for 1 hour at room temperature. The wells were washed three times with a solution containing 0.9% (w/v) sodium chloride and 0.05% (w/v) Tween. Undiluted hybridoma supernatant in RPMI1640 medium was used as a sample. The incubation time was 1 hour at room temperature. The wells were washed three times with a solution containing 0.9% (w/v) sodium chloride and 0.05% (w/v) Tween. Polyclonal antibodies conjugated to the constant domains of an anti-target antibody of peroxidase were used as detection antibodies (PAK)<M-Fcγ>S-F(ab′)₂-POD). The detection antibody was added at a concentration of 80 ng/ml in PBS containing 1% (w/v) RSA. The incubation time was 1 hour at room temperature. The wells were washed three times with a solution containing 0.9% (w/v) sodium chloride and 0.05% (w/v) Tween. Wells were then incubated with ABTS solution for 15 minutes at room temperature. The intensity of the color development was measured in a 405nm spectrophotometer. The reference wavelength is 492 nm. Primary hybridoma supernatants which show rapid and intense color formation by binding to recombinant IGF-1, Thermus thermophilus SlyD-IGF-1(74-90) in ELISA and less binding to Thermus thermophilus SlyD were transferred to the kinetic screening method described below.

SPR-based kinetic screening

Thermus thermophilus SlyD-IGF-1(74-90), native recombinant IGF-1, native recombinant IGF-2, wild type Thermus thermophilus SlyD and Thermus thermophilus-SlyD-IGF-1 (74-90) were used for SPR-based kinetic screening analysis. For SPR analysis, it is generally accepted to measure antibody binding kinetics according to the Langmuir model with monomers in solution and monovalent analytes. Furthermore, for SPR measurements, it is very advantageous to use analytes with higher molecular weights to improve the sensitivity of the measurement, since SPR is a mass sensitive analysis.

Kinetic screening was performed on a BIAcore a100 instrument under the control of software version V1.1. The biacore cm5 chip was loaded into the instrument and processed for hydrodynamic studies according to the manufacturer's instructions. HBS-EP buffer (10mM HEPES (pH7.4), 150mM NaCl, 1mM EDTA, 0.05% (w/v) P20) was used as a running buffer. Polyclonal rabbit anti-mouse IgG Fc capture antibody at 30. mu.g/ml in 10mM sodium acetate buffer (pH4.5) was immobilized at 10,000RU to spots 1, 2, 4 and 5 in flow cells 1, 2, 3 and 4 (FIG. 23). Antibodies were covalently immobilized by NHS/EDC chemistry. The sensor was then deactivated with a 1M ethanolamine solution. Spots 1 and 5 were used for the assay and spots 2 and 4 were used as reference. Prior to application to the sensor chip, hybridoma supernatant 1: 2 diluted in HBS-EP buffer. The diluted solution was added at a flow rate of 30. mu.l/min for 1 min. The analyte (e.g., Thermus thermophilus SlyD-IGF-1(74-90) fusion polypeptide) was then injected immediately at a flow rate of 30. mu.l/min for 2 minutes. The signal was then recorded for 5 minutes dissociation time. The sensor was regenerated by injecting 10mM glycine-HCl solution (pH1.7) at a flow rate of 30. mu.l/min for 2 minutes. Kinetic screening performance was characterized by two reporting points, recorded signal shortly before the end of analyte injection, denoted as late Binding (BL), recorded signal shortly before the end of dissociation time, late Stability (SL).

In addition, the dissociation rate constant kd (1/s) is calculated according to the Langmuir model, and the half-life of the antibody/antigen complex expressed in minutes can be calculated according to the formula ln (2)/(60. ANG. kd).

Antibodies were obtained by immunization with the antigen Thermus thermophilus SlyD-IGF-1(74-90), and screening with Thermus thermophilus SlyD-IGF-1(74-90), Thermus thermophilus SlyD wild-type, native IGF-1 and native IGF-2. Scaffold-based screening methods allow for the specific production of antibodies that bind to a predetermined IGF-1 hairpin epitope.

The primary culture supernatant was further developed into a clonal culture supernatant by limiting dilution by known methods. Clonal culture supernatants were tested in functional assays for affinity and specificity.

BIAcore characterization of antibody-producing clone culture supernatants

A BIAcore T200 instrument (GE Healthcare) was used with the BIAcore CM5 sensor installed into the system. 100 μ l/min of 0.1% SDS, 50mM NaOH, 10mM HCl and 100mM H₃PO₄To pre-condition the sensor for 1 minute.

The system buffer was PBS-DT (10mM Na)₂HPO₄、0.1mM KH₂PO₄、2.7mM KCl、137mM NaCl、0.05％20. 5% DMSO). The sample buffer is the system buffer.

The BIAcore T200 system is driven under control software V1.1.1. Polyclonal rabbit IgG antibody < IgGFC γ M > R (Jackson ImmunoResearch Laboratories Inc.) was immobilized at 30 μ g/ml in 10mM sodium acetate buffer (pH4.5) at 6500RU on flowcells 1, 2, 3 and 4, respectively, by EDC/NHS chemistry as specified by the manufacturer. Finally, the sensor surface was blocked with 1M ethanolamine solution. The entire experiment was carried out at 25 ℃.

The clone culture supernatants containing approximately 40nM of each antibody were captured on the < IgGFC γ M > R surface at a flow rate of 5 μ l/min for 2 min. As analytes in the solution, recombinant native IGF-1, recombinant native IGF-2, Thermus thermophilus SlyD-IGF-1(74-90), recombinant wild type Thermus thermophilus SlyD, recombinant Thermus thermophilus SlyD-delta IF, recombinant wild type Thermococcus gamma-Alternans SlyD, recombinant Thermococcus gamma-Alternans SlyD-IGF-2(53-65) fusion polypeptides were used. Thermus thermophilus SlyD- Δ IF is only the FKBP domain of Thermus thermophilus SlyD lacking the IF domain. Unlike IGF-2 hairpin insertions, Thermococcus gamma-interferon SlyD-IGF-2(53-65) was used to reverse screen and study specificity for the IGF-1 hairpin. Each analyte was injected at different concentration gradients of 90nM, 30nM, 10nM, 3.3nM, 1.1nM and 0 nM. The binding period was monitored at a flow rate of 100. mu.l/min for 3 minutes. Dissociation was monitored at a flow rate of 100. mu.l/min for 10 min. The system was regenerated with 10mM glycine buffer (pH 1.7). Kinetics were evaluated using BIAcore evaluation software.

The terms mAb monoclonal antibody, RU relative reaction units of monoclonal antibody captured on the sensor, antigen in solution, kDa molecular weight of antigen injected as analyte in solution, ka association rate constant, KD dissociation rate constant, t 1/2 dis, antibody-antigen complex half-life calculated according to the formula t 1/2 dis = ln (2)/60 ＊ KD, KD dissociation constant, R_MAX: binding signal at the end of the binding period for 90nM analyte injection; MR: a molar ratio; chi²: the measurement fails; n.d.: it is not detected.

FIG. 25 shows that scaffold-derived monoclonal antibodies M-11.11.17 and M-10.7.9 with picomolar affinity for IGF-1 were prepared. No cross-reactivity could be detected for IGF-2, or for wild-type Thermus thermophilus SlyD, or for wild-type Thermococcus gammatolerans SlyD, or for Thermococcus thermophilus SlyD-delta IF fusion polypeptide, or for Thermococcus gammatolerans SlyD-IGF-2(53-65) fusion polypeptide.

M-2.28.44 is a monoclonal antibody obtained by conventional immunization of mice with recombinant human IGF-1. Despite the fact that this antibody showed an affinity for IGF-1 of 30pM, a cross-reactivity of 500pM for IGF-2 could be detected. Using Thermus thermophilus SlyD-IGF-1(74-90) and Thermococcus gamma-interferon SLyD-IGF-2(53-65) as analytes, it was seen that the cross-reactive IGF-2 epitope was not an IGF hairpin region.

Example 12

Production of anti-PLGF antibodies

Mice were immunized with an immunogen comprising the sequence of PLGF (60-76). Hybridomas were subsequently generated and ELISA and kinetic screening performed (see examples 10 and 11 for detailed general methods).

In the kinetic screening method, primary culture supernatants with binding activity for PLGF (60-76) were identified using SlyD/FKBP 12-PLGF and the biotinylated peptide PLGF (60-76) -bi grafted alone on streptavidin. Both analytes produced 1: 1Langmuir kinetics, but the scaffold showed better dissociation fitted with a lower chi-square value than the SA-probe grafted dipeptide. Without being bound by theory, the scaffold-based screening method exploits the monomeric state, and the improved epitope accessibility of the scaffold when compared to carefully prepared SA-probes.

Example 13

anti-IGF-1 antibodies generated with SlyD-FKBP12-IGF-1(74-90)

The SlyD/FKBP 12-IGF-1(74-90) (see FIGS. 2 and 8) and SlyD/FKBP 12-ctrl (see FIG. 7) fusion polypeptides were produced in E.coli (pQE80L vector/E.coli BL21Codonplus-RP cell line) according to known methods. 8-12 week old Balb/c and NMRI mice were immunized intraperitoneally with 100. mu.g SlyD/FKBP 12-IGF-1(74-90) repeatedly.

Serum titers were analyzed by ELISA after 10 weeks. Nunc Maxisorb F-well plates were coated with SlyD/FKBP 12-IGF-1(74-90) by adding a solution containing 0.41. mu.g of polypeptide/ml. Isolated antigen IGF-1 was immobilized in the wells of a StreptaWell High Bind SA multiwell plate by adding a solution containing 90 ng/ml biotinylated IGF-1. The free binding sites were then blocked by adding 1% RPLA in PBS for 1 hour at room temperature. The wells were washed three times with a solution containing 0.9% (w/v) sodium chloride and 0.05% (w/v) Tween. With PBS 1: 50 dilutions of mouse serum were used as samples. According to the following steps: 4 gradient with optional further dilution, until 1: 819,200 final dilution. The incubation time was 1 hour at room temperature. The wells were washed three times with a solution containing 0.9% (w/v) sodium chloride and 0.05% (w/v) Tween. Polyclonal antibodies conjugated to the constant domains of an anti-target antibody of peroxidase were used as detection antibodies (PAK)<M-Fcγ>S-F(ab′)₂-POD). The detection antibody was added at a concentration of 80 ng/ml in PBS containing 1% (w/v) RSA. The incubation time was 1 hour at room temperature. The wells were washed three times with a solution containing 0.9% (w/v) sodium chloride and 0.05% (w/v) Tween. Wells were then incubated with ABTS solution for 15 minutes at room temperature. The intensity of the color development was measured with a spectrophotometer. Figure 19 shows the obtained mouse serum titers. Lower titers were obtained with SlyD/FKBP 12-IGF-1(74-90) compared to immunization with Thermus thermophilus SlyD-IGF-1(74-90) (FIG. 20). Additional antibody preparations were performed as described below in example 12. Finally, antibodies with binding to IGF-1 were not selected in the described BIAcore kinetic screening methods.

Example 14

Antibodies generated with FKBP 12/13 fusion polypeptides

The fusion polypeptide reported herein and used in this example comprises a portion derived from human FKBP12 and a portion derived from arabidopsis FKBP 13. The fusion polypeptide consisting of at least one amino acid sequence from human FKBP12 and at least one amino acid sequence from arabidopsis FKBP13 can serve as a scaffold to thermodynamically stabilize human FKBP12 and to avoid fusion of FKBP12 to the N-terminus of escherichia coli SlyD. In nature, FKBP13 contains disulfide bonds. This FKBP13 sequence was grafted into FKBP12 to stabilize the chimeric polypeptide for use in other sequence grafting methods.

Comprises the amino acid sequence of SEQ ID NO: 16 has the sequence:

MRSGVQVETISPGDGRTFPKRGQTAVVHYTGMLEDGKKFDSSRDRNKPFKFMLGKQEVIRGWEEGVAQMSVGQRAKLTISPDYAYGDRGAGCGSGSSCLIPPASVLVFDVELLKLEGGGSRKHHHHHHHH(SEQ ID NO：123).。

the FKBP 12/13 fusion polypeptide was expressed in the E.coli as a soluble and monomeric protein. CD spectroscopy was performed as described in example 12. CD spectra showed that FKBP 12/13 fusion polypeptide folded at 20 ℃.

Example 15

Production of SlyD-FKBP 12/13-CSF 1R fusion polypeptide

The polypeptide was expressed in E.coli as described above and purified as described above. After Ni-NTA affinity purification, size exclusion chromatography was performed. 50mg of protein was loaded in HiLoad 26/60 Superdex 75pg (GE Healthcare). The eluted fractions are loaded onto a non-denaturing gel and separated according to known methods.

For 50mM KH containing 100mM KCl and 0.5mM EDTA₂PO₄The buffer pH7.0 dialysis affinity purification of fusion polypeptide, and filtration through 0.22 u m filter. Extinction coefficient e =20525L · mol calculated with respect to 39744.9Da polypeptide^-1·cm^-1 quantitative SlyD/FKBP 12/13-CSF 1R by UV/Vis Spectroscopy at 1.19 mg/ml.

Protein fluorescence measurements were used to test the conformational properties of SlyD/FKBP 12/13-CSF 1R. FKBP12C22A as a vector for polypeptide insertion can be used as a reference, since a single FKBP12Trp moiety can be used to diagnose the structural integrity of the FKBP12 moiety (Scholz, C. et al, J.biol. chem.271(1996) 12703-. FKBP12C22A in its native structure showed a single fluorescence emission peak at 320nm (Zoldak, G. et al, J.mol.biol.386(2009) 1138-1152).

250 μ l KH containing 1.19 mg/ml SlyD-FKBP 12/13-CSF 1R were analyzed at different temperatures₂PO₄The buffer pH 7.0. By KH₂PO₄The buffer pH7.0 was used as reference. Cary Eclipse instrument under scanning software version 1.1(132) was used at excitation and emission bandwidths of 5 nm. The wavelength sweep was driven at 120 nm/min from 300nm to 425 nm. The excitation wavelength of the intrinsic tryptophan fluorescence was set at 280 nm.

Example 16

Stent-based reverse screening method for selecting antigen-binding antibodies

6-week-old NMRI mice were immunized intraperitoneally with 100. mu.g of a recombinant chimeric fusion polypeptide containing the element Thermococcus gamma adurans SlyD-antigen (TgSlyD-antigen). After 10 weeks, mice were boosted twice with 25 μ g TgSlyD-antigen. Hybridoma cells are produced according to known methods. Primary hybridomas were isolated by limiting dilution and screened for antigen binding by ELISA.

50 ng/ml TgSlyD-antigen fusion polypeptide, 50 ng/ml TgSlyD. DELTA.IF and 1. mu.g/ml of the isolated antigen were each coated overnight at 4 ℃ in 30X384 well (Nunc) plates. 1 piece of carbonate-bicarbonate tablet (Sigma, C3041-100CAP99) was dissolved in 100ml of double distilled H₂O(ddH₂O) to prepare coating buffer fresh. Mu.l of washing buffer (1L dH containing 150mM NaCl, 10ml Tween20(Sigma), 40ml Bromidox L (Roche))₂O). Mu.l of washing buffer (1L dH containing 150mM NaCl, 10ml Tween20(Sigma), 40ml Bromidox L (Roche)) were washed with a BioTek plate washer₂O) washing hole threeNext, the process is carried out. Using 30. mu.l of blocking buffer (1L ddH containing 10g BSA, 10 XPBS pellet (Gibco))₂O) the wells were blocked for 1 hour at room temperature and then washed 3 times with 100. mu.l of wash buffer. Mu.l of 1: hybridoma supernatants at 1000 dilutions were transferred into wells and incubated for 1 hour at room temperature. Antigen positive sera were used as positive controls. The wells were washed 3 times with 100. mu.l of wash buffer. Peroxidase conjugated F (ab')₂Fragment goat anti-mouse IgG antibody (Dianova) 1: 30000 was diluted in blocking buffer and 30. mu.l was transferred to each well. Incubate for 1 hour at room temperature, then wash 3 times with 100. mu.l wash buffer. 30 μ l of ready-to-use ABTS substrate was incubated in each well for 30 minutes at room temperature. The absorbance signal was monitored at 405 nm/492 nm with a Powerwave XS Reader (BioTek) as a reference signal. 15 hybridoma cultures were selected that showed a positive ELISA signal for the TgSlyD-antigen comprising the fusion polypeptide and the isolated antigen, but no signal for the TgSlyD Δ IF comprising the fusion polypeptide, and further cultured.

Primary hybridoma supernatants were isolated and screened for antigen binding by a second ELISA counter-screen performed in the same manner as described above. In the second screen, additional screening reagents were used to refine the specificity of the antibody-containing culture supernatant.

The cells were coated with 50 ng/ml TgSlyD-antigen fusion polypeptide, 50 ng/ml TgSlyD.DELTA.IF, 500ng ttSlyD-antigen fusion polypeptide and 1. mu.g/ml of the isolated antigen in 384 well (Nunc) plates for 1 hour at Room Temperature (RT). The ELISA was performed as described above. T.th.slyd and t.g.slyd show little sequence homology due to their different species origins. Only 36% of the amino acids are identical and only 48% sequence similarity exists, as calculated by blossom 62. But lack an insertion of TgSlyD Δ IF. Thus, the polypeptide is very well suited for use in ELISA reverse screening.

Thus, by immunizing with a scaffold instead of a polypeptide, specific epitopes in the native antigen domain can be pre-targeted.

Example 17

Epitope mapping

SlyD-FKBP fusion polypeptides may also carry complex amino acid insertion motifs, such as secondary structures containing disulfide bonds. Since the fusion polypeptide does not contain cysteine, on-column refolding under appropriate conditions facilitates correct formation of disulfide bonds within the insertion, which is additionally assisted by chaperone functionality of SlyD itself. The SlyD-FKBP fusion polypeptide was expressed in e.coli and refolded on the column as described above. SlyD-FKBP12ctrl was dialyzed at a concentration of 9.5 mg/ml against 75mM HEPES buffer (pH7.5) containing 150mM NaCl, 6.5% (w/v) sucrose, and 10mM cysteine. A1 mg/ml portion of the SlyD-FKBP12-CD81 fusion polypeptide was dialyzed against 50mM potassium phosphate buffer pH7.0 containing 100mM NaCl, 1mM EDTA, to avoid disulfide shuffling in the CD81 insertion, CD81 insertion comprising 4 cysteines forming two disulfide bonds with structural functional relevance. The fusion polypeptides SlyD-FKBP12-CD81 and SlyD-FKBP12ctrl were used for epitope mapping purposes.

Human CD81 is a receptor for hepatitis c virus envelope E2 glycoprotein. CD81 is a transmembrane protein belonging to the four transmembrane family. CD81 is a homodimeric protein 90 amino acids in length, showing a so-called mushroom-like structure (PDB1IV 5). Residues known to be involved in viral binding can be mapped to the so-called "head subdomain" of 35 amino acids in length, providing the basis for designing antiviral drugs and vaccines. Since the sequence of the head subdomain of the viral binding site is only 35 amino acids in length, it is difficult to locate the epitope of the antibody on the 10kDa CD81 protein using conventional cross-blocking experiments. It is difficult to distinguish antibodies that specifically bind directly on the relevant mushroom-like head domain from antibodies that bind nearby or elsewhere in the CD81LEL structure. All antibodies will show a competitive effect of the HCV E2 envelope protein independently of their binding site, but will not specifically bind to the target structure, i.e. the head domain. Thus, transplantation of related head domain structures into FKBPs and contiguous epitope mapping are useful methods for a variety of reasons. First, some biochemical problems of the CD81LEL protein are avoided, as the protein itself tends to oligomerize. Second, it is well suited for identifying antibody epitopes from a large number of antibodies that all bind to the full-length CD81 protein.

SDS page (left) and Western blot (right) of Ni-NTA chromatographically purified SlyD-FKBP12-CD81 are shown in FIG. 40.

A BIAcore2000 instrument (GEHealthcare) with a BIAcore CM5 sensor mounted into the system was used at 25 ℃. 100 μ l/min of 0.1% SDS, 50mM NaOH, 10mM HCl and 100mM H₃PO₄The sensor was preconditioned for 1 minute. HBS-EP buffer (10mM HEPES (pH7.4), 150mM NaCl, 1mM EDTA, 0.05% (w/v) P20) was used as a running buffer. The sample buffer is the system buffer. Each protein ligand was immobilized into flow cells 2, 3 and 4 at 30. mu.g/ml in 10mM sodium acetate buffer (pH4.0) by EDC/NHS chemistry. Flow cell 1 was used as a reference. The sensor was deactivated with a 1M ethanolamine pH8.0 solution. The following masses, expressed in Reaction Units (RU), were fixed on the sensor: the flow cell 2: 1800RU SlyD-FKBP12ctrl (32.8kDa), flow cell 3: SlyD-FKBP12-CD81(35.8kDa), flow cell 4: 900RU CD81LEL (10 kDa). 31 antibody analytes, each 50nM, were injected at 30. mu.l/min, 3 min on-phase, 3 min off-phase. 30 antibodies were derived from the immunological activity of the 10kda cd81LEL protein. The sensor surface was regenerated at 20. mu.l/min using 3 consecutive 30-second injections of 100mM HCl. Sensorgrams were monitored as reference signals 2-1 (flow cell 2 minus flow cell 1), 3-1, and 4-1 and evaluated by using BIAcore evaluation software 4.1. At the end of the analyte injection, a reporting point is set to quantify the maximum analyte binding signal. Data were normalized by setting the highest analyte binding signal to 100%.

In the following table, normalized antibody binding reactions are shown. Of the 30 < CD81-LEL > M antibodies tested, only 6 (bold) showed the exact epitope on the CD81 head domain. Does not bind to the negative control polypeptide SlyD-FKBP12 ctrl. The positive control polypeptide (which is simultaneously an immunogen) is recognized by all antibodies. Slyd-FKBP12-CD81 only bound when the antibody epitope was precisely located in the mushroom domain.

Watch (A)

Confirmation of epitope mapping results by X-ray crystallization analysis

Fab fragments of antibodies K05 and K04 were co-crystallized with the CD81-LEL protein and analyzed by x-ray diffraction analysis (Seth Harris, Palo Alto). The resolution obtained isAntibody K05 binds in the mushroom domain, while antibody K04 binds an epitope outside of the mushroom sequence.

Claims

1. A fusion polypeptide of formula II

NH₂-S₂-X₁-S₁-COOH formula II

Wherein

X₁Is a polypeptide selected from formula IV to formula XIII

GS-X₀-GSS of the formula IV,

AGS-X₀-a GSS of formula V,

CG-X₀-GC of the formula VI,

C-X₀-GC of the formula VII,

G-X₀-G is of the formula VIII,

S-X₀-a GSS of formula IX,

GG-X₀-GG of the formula X,

G-X₀-TGG of the formula XI,

GGGS-X₀-GGGS formula XII,

GGNP-X₀-GPT of formula XIII,

wherein X₀Is a random amino acid sequence, or is derived from an amino acid sequence of a first polypeptide; and

S₂and S₁Or a non-overlapping amino acid sequence derived from a second polypeptide;

-represents a peptide bond,

wherein the second polypeptide is selected from human FKBP12, Arabidopsis FKBP13, Thermus thermophilus SlyD, Escherichia coli SlyD, Thermococcus gammatolerans SlyD,

wherein is inserted with X₁To replace the inserted flap domain of the second polypeptide: an IF domain, which is a domain of an enzyme,

wherein the immunogenic sequence is contained in X₁In the amino acid sequence.

2. The fusion polypeptide of claim 1, wherein the second polypeptide is Thermus thermophilus SlyD.

3. The fusion polypeptide of claim 1, wherein said S derived from a second polypeptide₂And S₁The amino acid sequences are directly linked to each other via the IF domain in the naturally occurring wild-type second polypeptide.

4. The fusion polypeptide of claim 1, wherein X₁Has an amino acid sequence length of from 4 to 500 amino acid residues.

5. The fusion polypeptide of claim 1, wherein X₁Has an amino acid sequence length of from 5 to 100 amino acid residues.

6. The fusion polypeptide of claim 1, wherein X₁Has an amino acid sequence length of 7 to 60 amino acid residues.

7. The fusion polypeptide of claim 1, wherein said first polypeptide is a human polypeptide.

8. The fusion polypeptide of claim 1, wherein X₁Is a polypeptide of formula III

X_aX_bX_cX_d-X₀-X_eX_fX_gX_hFormula III

Wherein X₀Is a random amino acid sequence, or is an amino acid sequence of a first polypeptide; and

wherein X_aTo X_hEach of which represents a natural amino acid residue, and X_a-hEach of which may be present or absent independently.

9. The fusion polypeptide of claim 8, wherein X₀Flanked at their N-and C-termini by a single cysteine residue.

10. The fusion polypeptide of claim 8, wherein X₁Comprising a cysteine residue within the N-terminal amino acid residue and a cysteine residue within the C-terminal amino acid residue.

11. The fusion polypeptide of claim 8, wherein the N-terminal or C-terminal amino acid residue is eight terminal residues.

12. The fusion polypeptide of claim 8, wherein X₁It contains a cysteine residue at its N-terminus and a cysteine residue at its C-terminus.

13. The fusion polypeptide of claim 8, wherein X₁Or X₀Has a molecular weight of from 4 to500 amino acid residues in length.

14. The fusion polypeptide of claim 8, wherein X₁Having the amino acid sequence GGGSGGNPX₀GPTGGGS, SEQ ID NO:32, wherein X₀Is an amino acid sequence of from 4 to 85 amino acid residues.

15. Non-therapeutic use of a fusion polypeptide according to any one of claims 1 to 14 for eliciting responses against X in an animal₁Or X₀The immune response of (1).

16. A non-therapeutic method for eliciting an immune response against a polypeptide in an animal comprising administering to the animal the fusion polypeptide of any one of claims 1 to 14 at least once, X₀Thus, an immune amino acid sequence.

17. A method for obtaining a nucleic acid encoding an antibody that specifically binds a target antigen, comprising the steps of:

a) administering to an animal at least once a fusion polypeptide according to any one of claims 1 to 14, X₁Whereby the amino acid sequence of (a) comprises the amino acid sequence of the target antigen;

b) recovering B cells from the animal three to ten days after the last administration of the polypeptide, the B cells producing antibodies that specifically bind the target antigen; and

c) obtaining from the B cell a nucleic acid encoding an antibody that specifically binds to the target antigen.

18. A method for producing an antibody that specifically binds a target antigen, comprising the steps of:

b) recovering B cells from the animal three to ten days after the last administration of the polypeptide, the B cells producing antibodies that specifically bind the target antigen;

c) optionally obtaining nucleic acid encoding an antibody that specifically binds to the target antigen from the B cell; and

d) culturing a cell comprising a nucleic acid encoding an antibody that specifically binds to the target antigen, and recovering the antibody from the cell or culture medium, thereby producing an antibody that specifically binds to the target antigen.

19. A method for producing an antibody that specifically binds a target antigen, comprising the steps of:

a) recovering B cells from the experimental animal after administration of the fusion polypeptide of any one of claims 1 to 14, said B cells producing antibodies that specifically bind to a target antigen having X₀The amino acid sequence of (a); and

b) culturing comprises encoding specific binding X₀And recovering the antibody from the cells or the culture medium, thereby producing an antibody that specifically binds to the target antigen.

20. Use of a fusion polypeptide according to any one of claims 1 to 14 for epitope mapping, X₁Whereby the amino acid sequence of (a) comprises said epitope.

21. A method for selecting an antibody that specifically binds a target antigen, comprising the steps of:

a) determining the binding affinity of a plurality of antibodies to a target antigen, wherein the amino acid sequence of the target antigen is comprised in a fusion polypeptide according to any one of claims 1 to 14 and is present at X of the fusion polypeptide₁In the amino acid sequence;

22. A method for selecting an antibody suitable for use in an immunohistochemical analysis of a target polypeptide comprising the steps of:

a) determining binding kinetics of the plurality of antibodies;

b) selecting an antibody having an apparent complex stability above a predetermined threshold level,

wherein the target polypeptide is comprised in a fusion polypeptide according to any one of claims 1 to 14 and is present at X of the fusion polypeptide₁In the amino acid sequence.

23. A method for locating the binding site of an antibody to a target amino acid sequence comprising the steps of:

a) contacting a solid support, to which a fusion polypeptide according to any one of claims 1 to 14 is immobilized, with an antibody, X₁Whereby the amino acid sequence of (a) comprises the target amino acid sequence;

b) determining the kinetic properties of the antibody and the fusion polypeptide of any one of claims 1 to 14;

24. Use of a fusion polypeptide according to any one of claims 1 to 14, X, for determining a structure-function relationship₁The amino acid sequence of (a) thus comprises the polypeptide whose structure-function relationship is to be determined.

25. Non-therapeutic use of a fusion polypeptide according to any one of claims 1 to 14 for presenting the polypeptide in its correct secondary and/or tertiary structure, X₁Thereby comprising the polypeptide.

26. Use of a fusion polypeptide according to any one of claims 1 to 14 in a screening method for identifyingOr selecting specific binding of X₁The method of screening for a molecule of (1).

27. The method of claim 26, wherein the molecule is a small molecule or polypeptide.

28. The method of claim 27, wherein the polypeptide is an antibody, or antibody fragment, or antibody fusion polypeptide.

29. Use of a fusion polypeptide according to any one of claims 1 to 14 in ribosome display.

30. Use of a fusion polypeptide according to any one of claims 1 to 14 in phage display.

31. Use of the fusion polypeptide of any one of claims 1 to 14 for cell surface display.

32. The use of claim 31, wherein the cell is a prokaryotic cell.

33. The use of claim 32, wherein the prokaryotic cell is a bacterial cell.

34. The use of claim 33, wherein the cell is a eukaryotic cell.

35. The use of claim 34, wherein the eukaryotic cell is a CHO cell, or a HEK cell, or a BHK cell, or an Sp2/0 cell, or an NS0 cell, or a yeast cell.

36. A pharmaceutical formulation comprising the fusion polypeptide of any one of claims 1 to 14 and a pharmaceutically acceptable carrier.

37. A diagnostic formulation comprising the fusion polypeptide of any one of claims 1 to 14 conjugated to a detectable label.

38. Use of a fusion polypeptide according to any one of claims 1 to 14 for the manufacture of a medicament.

39. Use of a fusion polypeptide according to any one of claims 1 to 14 in the manufacture of a medicament for the treatment of a disease.

40. Use of the fusion polypeptide of any one of claims 1 to 14 in the manufacture of a medicament for treating a subject.