The present description relates to anti-Thermus thermophilus SlyD FKBP domain antibodies and methods of using the same.

FIELD OF THE INVENTION

The present description relates to anti-Thermus thermophilusSlyD FKBP domain antibodies and methods of using the same.

BACKGROUND OF THE INVENTION

In life sciences and in related applied fields there is a need for non-antibody polypeptide molecules capable of performing specific protein-protein interactions. A main focus relies on identifying polypeptide domains that bind to a predetermined target. However, a major obstacle of linear polypeptides comprising about 5 to about 50 amino acids is their intrinsic flexibility. In solution such polypeptides are usually transitioning a large number of structural states that are almost equivalent from an energetic perspective. Nevertheless, such structural states are generally highly dependent on the environment of the polypeptides. As the structural state is an important factor for presenting a certain epitope, e.g. if such polypeptides are used for immunization of an animal for antibody production, it is an essential requirement that the structural state of the polypeptide is not affected by environmental changes, such that an unambiguous presentation of a certain structural state representing a defined epitope can be ensured.

To meet those demands, a protein scaffold can be used where the polypeptide of interest is grafted into a rigid structure. The scaffold forces the polypeptide insertion into an entropy-restricted, structural state, limiting its torsional degrees of freedom. These constructs can be used for applications, such as for the immunization of an experimental animal for producing antibodies against the polypeptide insertion. Furthermore, such a scaffold can be used for the purpose to map antibody epitopes. In another application, such a scaffold can be used as a chimeric calibrator polypeptide for diverse immunological assays. In another application such a scaffold can display constrained peptides with predefined target binding specificity, which allows the scaffold to be used in diverse affinity purification approaches, like affinity chromatography or pull-down assays. In another application, antibody CDR loops can be grafted into such a scaffold. In another application, subdomains of other proteins can be grafted into such a scaffold in order to circularly permutate the chimeric target polypeptide. Domains such as variable loops of antigen binding regions of antibodies have been extensively engineered to produce amino acid sequence segments having improved binding (e.g. affinity and/or specificity) to known targets (e.g. disclosed in Knappik, A. & Plückthun A. J. Mol. Biol. 296 (2000) 57-86; EP 1025218). Engineering of non-antibody frameworks has been reviewed e.g. by Hosse, R. J. et al. Protein Sci., 15 (2006) 14-27. Non-antibody or alternative protein scaffolds have considerable advantages over traditional antibodies due to their small size, high stability, and ability to be expressed in prokaryotic hosts. Novel methods of purification are readily applied; they are easily conjugated to drugs/toxins, penetrate efficiently into tissues and are readily formatted into mono- or multi-specific binders (Skerra, A, et al. J. Mol. Recognit. 13 (2000) 409-410; Binz, H. K. et al. Nature Biotechnol. 23 (2005) 1257-1268).

As known in the art, human FKBP12 can be used as a protein scaffold to improve its enzymatic activity. Knappe, T. A., et al. (J. Mol. Biol. 368 (2007) 1458-1468) reported that the Flap-region of human FKBP12 can be replaced by the IF domain of the structurally relatedE. colichaperone SlyD. This chimeric FKBP12-IF polypeptideThermus thermophilesSlyD-FKBP has a 200 times increased peptidyl-prolyl-cis/trans isomerase activity (PPI activity) compared to the isolated polypeptide. TheE. coliSlyD and human FKBP12 (wild type and mutants C23A and C23S) can be recombinantly produced inE. coliin high yield in soluble form (Standaert, R. F., et al., Nature 346 (1990) 671-674).

The amino acid sequence of the human FKBP12 polypeptide comprises a single tryptophan residue at position 60. Thus, human FKBP12 mutants can be analyzed for structural integrity simply by analyzing the tryptophan fluorescence (DeCenzo, M. T., et al., Protein Eng. 9 (1996) 173-180). A test for remaining catalytic activity of the human FKBP12 mutant can be performed by determining the remaining rotamase activity (Brecht, S., et al., Neuroscience 120 (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 human FKBP12 mutants by determining the 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 constrained by an aliphatic linkage between two C (alpha) sites: design, synthesis, and unexpected conformational properties of an i,(i+4)-linked peptide.

Suzuki, et al. (Suzuki, R., et al., J. Mol. Biol. 328 (2003) 1149-1160) report the three-dimensional solution structure of an archaic SlyD with a dual function of peptidyl-prolyl-cis-trans isomerase and chaperone-like activities. Expression vector, host, fused polypeptide, process for producing fused polypeptide and process for producing protein are reported in EP 1 516 928. Knappe, T. A., et al., reports that the insertion of a chaperone domain converts human FKBP12 into a powerful catalyst of protein folding (J. Mol. Biol. 368 (2007) 1458-1468). A chimeric polypeptide with superior chaperone and folding activities is reported in WO 2007/077008. In WO 03/000878 the use of SlyD chaperones as expression tool is reported. In EP 1 621 555 an immunogen, composition for immunological use, and method of producing antibody using the same are reported. Rebuzzini, G. (PhD work at the University of Milano-Bicocca (Italy) (2009)) reports a study of the hepatitis C virus NS3 helicase domain for application in a chemiluminescent immunoassay.

In WO 2007/077008 chimeric fusion proteins with superior chaperone and folding activities are reported. The conversion of human FKBP12 into a powerful catalyst of protein folding by insertion of a chaperone domain is reported by Knappe et al. (Knappe, T. A., et al., J. Mol. Biol. 368 (2007) 1458-1468).

WO 2012/150320 discloses a fusion polypeptide comprising one or more fragments of one or more peptidyl-prolyl cis/trans isomerase or FKBP domain family members and its use in methods for antibody screening/selection, for epitope mapping as well as its use as immunogen for the production of antibodies specifically binding an immunogenic peptide or secondary structure presented by the fusion polypeptide.

Among other SlyD chaperones from different species, especially suited is theThermus thermophilusSlyD FKBP domain (herein also referred to as TtSlyD-FKBP) due to its superior biophysical properties regarding thermodynamic stability and solubility (Low et al. (2010) J Mol Biol 398(3): 375-390). TheThermus thermophilusSlyD FKBP domain can be used as scaffold for the presentation of constrained peptides WO 2012/150320 which is useful for various applications, such as display methods including phage display, ribosome display, mRNA display and cell surface display. Such methods can be applied to select and optimize target-binding polypeptides from libraries with a large number of candidate amino acid sequences. Another application of theThermus thermophilusSlyD FKBP domain with a certain constrained peptide bound thereto is its use as immunogen for the production of antibodies in animals (WO 2012/150320). Further, theThermus thermophilusSlyD FKBP domain with a certain constrained peptide can be used as a ligand in protein-protein interaction experiments, whereas the constrained peptide represents one specific binding site of the corresponding entire protein binding partner.

These methods and experiments require as a tool an antibody which specifically binds to theThermus thermophilusSlyD FKBP domain (herein referred to as anti-TtSlyD-FKBP antibody). No such antibody is described in the state of the art. The problem to be solved by the present description is therefore the provision of an antibody which binds to the native TtSlyD-FKBP polypeptide.

SUMMARY OF THE INVENTION

The present description relates to anti-TtSlyD-FKBP antibodies and methods of using the same.

In one aspect the description relates to an isolated monoclonal rabbit antibody that binds to TtSlyD-FKBP, wherein the antibody specifically binds to the native conformation of TtSlyD-FKBP.

In one embodiment, the antibody exhibits a ka from 1×1031/Ms to 5×1071/Ms, a kd from 1×10−21/s to 1×10−61/s, a t1/2dfrom 1 min to 1500 min and a KD from 1×10−6M to 1×10−13M at a temperature of 25° C. or 37° C.

In a specific embodiment, the antibody comprises (a) HVR-H3 comprising the amino acid sequence of SEQ ID NO:06, (b) HVR-L3 comprising the amino acid sequence of SEQ ID NO:03, and (c) HVR-H2 comprising the amino acid sequence of SEQ ID NO:05, or the antibody comprises (d) HVR-H3 comprising the amino acid sequence of SEQ ID NO:12, (e) HVR-L3 comprising the amino acid sequence of SEQ ID NO:09, and (f) HVR-H2 comprising the amino acid sequence of SEQ ID NO:11.

In another specific embodiment, the antibody comprises (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO:04, (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO:05, and (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO:06, or the antibody comprises (d) HVR-H1 comprising the amino acid sequence of SEQ ID NO:10, (e) HVR-H2 comprising the amino acid sequence of SEQ ID NO:11, and (f) HVR-H3 comprising the amino acid sequence of SEQ ID NO:12.

In yet another specific embodiment, the antibody comprises (a) HVR-L1 comprising the amino acid sequence of SEQ ID NO:01; (b) HVR-L2 comprising the amino acid sequence of SEQ ID NO:02; and (c) HVR-L3 comprising the amino acid sequence of SEQ ID NO:03, or the antibody comprises (d) HVR-L1 comprising the amino acid sequence of SEQ ID NO:07; (e) HVR-L2 comprising the amino acid sequence of SEQ ID NO:08; and (f) HVR-L3 comprising the amino acid sequence of SEQ ID NO:09.

In yet another specific embodiment, the antibody comprises (a) a VH sequence having at least 95% sequence identity to the amino acid sequence of SEQ ID NO:14; (b) a VL sequence having at least 95% sequence identity to the amino acid sequence of SEQ ID NO:13; or (c) a VH sequence as in (a) and a VL sequence as in (b), or the antibody comprises (d) a VH sequence having at least 95% sequence identity to the amino acid sequence of SEQ ID NO:16; (e) a VL sequence having at least 95% sequence identity to the amino acid sequence of SEQ ID NO:15; or (f) a VH sequence as in (d) and a VL sequence as in (e).

In another embodiment, the antibody comprises a VH sequence of SEQ ID NO:14, or wherein the antibody comprises a VH sequence of SEQ ID NO:16. In yet another embodiment, the antibody comprises a VL sequence of SEQ ID NO:13, or wherein the antibody comprises a VL sequence of SEQ ID NO:15.

In another aspect, the description relates to an antibody comprising a VH sequence of SEQ ID NO:14 and a VL sequence of SEQ ID NO:13, or a VH sequence of SEQ ID NO:16 and a VL sequence of SEQ ID NO:15.

In another aspect, the description relates to an antibody that binds to the same epitope as the antibody described herein.

In another aspect, the description relates to isolated nucleic acid encoding the antibody described herein. In one embodiment, the isolated nucleic acid comprises the nucleic acid sequences of SEQ ID NO:17 and SEQ ID NO:18, or the isolated nucleic acid comprises the nucleic acid sequences of SEQ ID NO:19 and SEQ ID NO:20.

In another aspect, the description relates to a host cell comprising the nucleic acids as described in the previous paragraph.

In yet another aspect, the description relates to a method of producing an antibody comprising culturing the host cell of the previous paragraph so that the antibody as described in the present description is produced.

In yet another aspect, the description relates to the use of the antibody as described in the present description in a method, wherein the antibody is used to bind to TtSlyD-FKBP carrying a specific constrained polypeptide.

DETAILED DESCRIPTION OF THE INVENTION

The term “about” as used herein in conjunction with a numerical value modifies that value by extending the boundaries above and below the values. In general, the term “about” modifies a numerical value above and below the stated value by a variance of 5% higher or lower. For example a value of “about 100” means a range of “95 to 105”.

The terms “anti-TtSlyD-FKBP antibody” and “an antibody that binds to TtSlyD-FKBP” refers to an antibody that is capable of binding TtSlyD-FKBP (Thermus thermophilusSlyD FKBP domain) with sufficient affinity such that the antibody is useful as a diagnostic and/or therapeutic agent in targeting TtSlyD-FKBP. In one embodiment, the extent of binding of an anti-TtSlyD-FKBP antibody to an unrelated, non-TtSlyD-FKBP polypeptide is less than about 10% of the binding of the antibody to TtSlyD-FKBP as measured, e.g., by a radioimmunoassay (MA) or by SPR. In certain embodiments, an antibody that binds to TtSlyD-FKBP has a dissociation constant (KD) of ≤1 μM, ≤100 nM, ≤10 nM, ≤1 nM, ≤0.1 nM, ≤0.01 nM, or ≤0.001 nM (e.g. 10−8M or less, e.g. from 10−8M to 10−13M, e.g., from 10−9M to 10−13M).

The term “Thermus thermophilusSlyD” or “TtSlyD” refers to a polypeptide that comprises the amino acid sequence SEQ ID No:21.

The term “Thermococcus gammatoleransSlyD” refers to a polypeptide that comprises the amino acid sequence SEQ ID No:22.

The term “Thermus thermophilusSlyD FKBP” or “TtSlyD-FKBP” refers to a polypeptide that comprise the amino acid sequence SEQ ID NO:23 (part 1) and SEQ ID NO:24 (part 2), wherein both sequences (parts) are linked by X1, (i.e. SEQ ID NO:23-X1-SEQ ID NO:24) and wherein X1is the amino acid sequence of a linker, or a peptide, or an antigen, or a secondary or tertiary structure to be presented by theThermus thermophilusSlyD fusion polypeptide.

An “antibody that binds to the same epitope” as a reference antibody refers to an antibody that blocks binding of the reference antibody to its antigen in a competition assay by 50% or more, and conversely, the reference antibody blocks binding of the antibody to its antigen in a competition assay by 50% or more. An exemplary competition assay is provided herein.

The term “chimeric” protein refers to a protein in which a portion of the protein is derived from a particular source or species, while another portion of the protein is derived from a different source or species. In case of the “chimeric TtSlyD-FKBP TtSlyD-FKBP polypeptide” the polypeptide consists of theThermus thermophilusSlyD FKBP domain and a polypeptide graft, which replaces the insert in Flap Domain of the wild typeThermus thermophilusSlyD chaperone.

The term “TtSlyD-FKBP” is used herein as known by the person skilled in the art synonymous for the terms “Thermus thermophilusSlyD FKBP domain”, “T.th.SlyD FKBP domain”, “chimeric TtSlyD-FKBP polypeptide”, and the like. The term“TtSlyD-FKBP” as used herein refers to the peptidyl-prolyl cis-trans isomerase SlyD as it derives from the extremophile archaebacteriaThermus thermophilus(Low et al. (2010) J Mol Biol 398(3): 375-390; Scholz, C., et al. (2006). Biochemistry 45(1): 20-33.) and as it is referred in UNIPROT (Q5SLE7).

TtSlyD-FKBP-A is a 14 kDa derivative of theThermus thermophilusSlyD FKBP domain, wherein the IF domain is replaced by an 8 amino acid insertion.

TtSlyD-FKBP-B is a 21 kDa derivative of theThermus thermophilusSlyD FKBP domain, wherein the IF domain is replaced by a 68 amino acid insertion.

Unless otherwise indicated, HVR residues and other residues in the variable domain (e.g., FR residues) are numbered herein according to Kabat et al., supra.

“Isolated nucleic acid encoding an anti-TtSlyD-FKBP antibody” refers to one or more nucleic acid molecules encoding antibody heavy and light chains (or fragments thereof), including such nucleic acid molecule(s) in a single vector or separate vectors, and such nucleic acid molecule(s) present at one or more locations in a host cell.

The term “variable region” or “variable domain” refers to the domain of an antibody heavy or light chain that is involved in binding the antibody to antigen. The variable domains of the heavy chain and light chain (VH and VL, respectively) of a native antibody generally have similar structures, with each domain comprising four conserved framework regions (FRs) and three hypervariable regions (HVRs). (See, e.g., Kindt, T. J. et al. Kuby Immunology, 6th ed., W.H. Freeman and Co., N.Y. (2007), page 91) A single VH or VL domain may be sufficient to confer antigen-binding specificity. Furthermore, antibodies that bind a particular antigen may be isolated using a VH or VL domain from an antibody that binds the antigen to screen a library of complementary VL or VH domains, respectively. See e.g. Portolano, S. et al., J. Immunol. 150 (1993) 880-887; Clackson, T. et al., Nature 352 (1991) 624-628).

In one aspect, an antibody is described that binds to TtSlyD-FKBP, wherein the antibody specifically binds to the native conformation of TtSlyD-FKBP without interfering with the ligand binding site at the insertion site of TtSlyD-FKBP.

Since the antibody binds conformational epitopes aside from the peptide insertion site, they principally can be used in screening approaches, where it is to select and differentiate correctly folded chimeric TtSlyD-FKBP polypeptides from misfolded TtSlyD-FKBP ones. Under the assumption, that misfolded TtSlyD-FKBP polypeptides are instable and prone to, the antibody of the description is useful, e.g., as a tool for detecting intact TtSlyD-FKBP or for detecting stable TtSlyD-FKBP carrying a specific peptide. Thus, in one embodiment, the description refers to the use of the antibody described herein in a method, wherein the antibody is used to bind to intact TtSlyD-FKBP carrying a specific constrained polypeptide. In a specific embodiment, the method is a quality control method for the detection of TtSlyD-FKBP carrying a specific constrained polypeptide.

In one embodiment, TtSlyD-FKBP carrying a specific constrained binding polypeptide can be used in immunological assays for the detection of the binding partner to TtSlyD-FKBP. In a specific embodiment, TtSlyD-FKBP carrying a specific constrained binding polypeptide can be used as ligand immobilized on affinity chromatography media known in the art. After a purification process known in the art the antibody can be used in immunological quality analyses known in the art to determine the absence or presence of TtSlyD-FKBP ligand impurities in the final product. Furthermore, chromatography media known in the art can be surface-functionalized by said antibody in order to generate a multi-purpose chromatography media by capturing TtSlyD-FKBP ligands with different target binding specificities. For that purpose regeneration conditions of the antibodies were optimized. TtSlyD-FKBP derivatives, which present a constrained binding peptide in order to interact, antagonize or agonize protein-protein interactions can also be cloned into fusion polypeptide constructs. The fusion polypeptides can be immunologically detected or purified or pinpointed to diverse surfaces by said antibodies.

In another specific embodiment the antibodies can be used as secondary reagents to detect the presence of TtSlyD-FKBP ligands e.g. on tissue, in IHC experiments, in in vivo imaging studies, in ELISA experiments and in general, in interaction experiments. As it is shown in the examples, the antibodies can be directly coated or indirectly captured on Biosensor surfaces, e.g. SPR, SAW or QCM sensors and TtSlyD-FKBP ligands can be site directed presented on the sensor surface, while the polypeptide insertion in the TtSlyD-FKBP ligand remains accessible for further interacting partners, respectively analytes in solution. It is known in the art, that evolutionary library generation strategies like error prone PCR or the usage of random mutated primers generate a large output of unwanted undefined library members. In another embodiment the antibodies can be used as display targets in TtSlyD-FKBP molecular display approaches in order to enrich in frame TtSlyD-FKBP binding derivatives with intact conformation and stability. This is i.e. of importance to deselect busted TtSlyD-FKBP polypeptides to overcome library quality issues.

In the following two antibodies capable of binding to TtSlyD-FKBP are described separately, RabMab 0612pS3A8 and RabMab 0712pS4D3.

In one aspect, the description refers to an isolated monoclonal rabbit antibody that binds to TtSlyD-FKBP, wherein the antibody specifically binds to the native conformation of TtSlyD-FKBP. In one embodiment, the isolated monoclonal rabbit antibody binds to TtSlyD-FKBP, wherein the antibody specifically binds to the native conformation of theThermus thermophilusSlyD FKBP without interfering with the ligand binding site at the insertion site of FKBP domain.

In one embodiment, the isolated antibody that binds to TtSlyD-FKBP has one or more of the following properties (also each combination of each single property is contemplated herein): a) the antibody binds to a conformational epitope in TtSlyD-FKBP in native conformation; and/or b) the antibody exhibits a kafrom 1×1031/Ms to 5×1071/Ms, and/or c) the antibody exhibits a KD from 1×10−21/s to 1×10−61/s, and/or d) the antibody exhibits a t1/2dfrom 1 min to 1500 min, and/or e) the antibody exhibits a KD from 1×10−6M to 1×10−13M at a temperature of 25° C. or 37° C.

In a specific embodiment, the isolated antibody that binds to TtSlyD-FKBP has one or more of the following properties (also each combination of each single property is contemplated herein): a) the antibody binds to a conformational epitope in TtSlyD-FKBP in native conformation; and/or b) the antibody exhibits a kafrom 1×1051/Ms to 1×1061/Ms, and/or c) the antibody exhibits a kdfrom 1×10−41/s to 1×10−61/s, and/or d) the antibody exhibits a t1/2dfrom 800 min to 1200 min, and/or e) the antibody exhibits a KD from 1×10−10M to 1×10−12M at a temperature of 25° C. or 37° C.

In one aspect, an anti-TtSlyD-FKBP antibody is described comprising at least one, two, three, four, five, or six HVRs selected from (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO:04; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO:05; (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO:06; (d) HVR-L1 comprising the amino acid sequence of SEQ ID NO:01; (e) HVR-L2 comprising the amino acid sequence of SEQ ID NO:02; and (f) HVR-L3 comprising the amino acid sequence of SEQ ID NO:03.

In one aspect, an antibody is described comprising at least one, at least two, or all three VH HVR sequences selected from (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO:04; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO:05; and (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO:06. In one embodiment, the antibody comprises HVR-H3 comprising the amino acid sequence of SEQ ID NO:06. In another embodiment, the antibody comprises HVR-H3 comprising the amino acid sequence of SEQ ID NO:06 and HVR-L3 comprising the amino acid sequence of SEQ ID NO:03. In a further embodiment, the antibody comprises HVR-H3 comprising the amino acid sequence of SEQ ID NO:06, HVR-L3 comprising the amino acid sequence of SEQ ID NO:03, and HVR-H2 comprising the amino acid sequence of SEQ ID NO:05. In a further embodiment, the antibody comprises (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO:04; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO:05; and (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO:06.

In another aspect, an antibody is described comprising at least one, at least two, or all three VL HVR sequences selected from (a) HVR-L1 comprising the amino acid sequence of SEQ ID NO:01; (b) HVR-L2 comprising the amino acid sequence of SEQ ID NO:02; and (c) HVR-L3 comprising the amino acid sequence of SEQ ID NO:03. In one embodiment, the antibody comprises (a) HVR-L1 comprising the amino acid sequence of SEQ ID NO:01; (b) HVR-L2 comprising the amino acid sequence of SEQ ID NO:02; and (c) HVR-L3 comprising the amino acid sequence of SEQ ID NO:03.

In another aspect, an antibody according to the description comprises (a) a VH domain comprising at least one, at least two, or all three VH HVR sequences selected from (i) HVR-H1 comprising the amino acid sequence of SEQ ID NO:04, (ii) HVR-H2 comprising the amino acid sequence of SEQ ID NO:05, and (iii) HVR-H3 comprising an amino acid sequence selected from SEQ ID NO:06; and (b) a VL domain comprising at least one, at least two, or all three VL HVR sequences selected from (i) HVR-L1 comprising the amino acid sequence of SEQ ID NO:01, (ii) HVR-L2 comprising the amino acid sequence of SEQ ID NO:02, and (c) HVR-L3 comprising the amino acid sequence of SEQ ID NO:03.

In another aspect, an antibody is described comprising (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO:04; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO:05; (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO:06; (d) HVR-L1 comprising the amino acid sequence of SEQ ID NO:01; (e) HVR-L2 comprising the amino acid sequence of SEQ ID NO:02; and (f) HVR-L3 comprising an amino acid sequence selected from SEQ ID NO:03.

In another aspect, an anti-TtSlyD-FKBP antibody comprises a heavy chain variable domain (VH) sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO:14. In certain embodiments, a VH sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity contains substitutions (e.g., conservative substitutions), insertions, or deletions relative to the reference sequence, but an anti-TtSlyD-FKBP antibody comprising that sequence retains the ability to bind to TtSlyD-FKBP. In certain embodiments, a total of 1 to 10 amino acids have been substituted, inserted and/or deleted in SEQ ID NO:14. In certain embodiments, substitutions, insertions, or deletions occur in regions outside the HVRs (i.e., in the FRs). Optionally, the anti-TtSlyD-FKBP antibody comprises the VH sequence in SEQ ID NO:14, including post-translational modifications of that sequence. In a particular embodiment, the VH comprises one, two or three HVRs selected from: (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO:04, (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO:05, and (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO:06.

In another aspect, an anti-TtSlyD-FKBP antibody is provided, wherein the antibody comprises a light chain variable domain (VL) having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO:13. In certain embodiments, a VL sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity contains substitutions (e.g., conservative substitutions), insertions, or deletions relative to the reference sequence, but an anti-TtSlyD-FKBP antibody comprising that sequence retains the ability to bind to TtSlyD-FKBP. In certain embodiments, a total of 1 to 10 amino acids have been substituted, inserted and/or deleted in SEQ ID NO:13. In certain embodiments, the substitutions, insertions, or deletions occur in regions outside the HVRs (i.e., in the FRs). Optionally, the anti-TtSlyD-FKBP antibody comprises the VL sequence in SEQ ID NO:13, including post-translational modifications of that sequence. In a particular embodiment, the VL comprises one, two or three HVRs selected from (a) HVR-L1 comprising the amino acid sequence of SEQ ID NO:01; (b) HVR-L2 comprising the amino acid sequence of SEQ ID NO:02; and (c) HVR-L3 comprising the amino acid sequence of SEQ ID NO:03.

In another aspect, an anti-TtSlyD-FKBP antibody is provided, wherein the antibody comprises a VH as in any of the embodiments provided above, and a VL as in any of the embodiments provided above. In one embodiment, the antibody comprises the VH and VL sequences in SEQ ID NO:14 and SEQ ID NO:13, respectively, including post-translational modifications of those sequences.

In a further aspect, an antibody is described that binds to the same epitope as an anti-TtSlyD-FKBP antibody provided herein. In a specific embodiment, an antibody is provided that binds to the same epitope as an anti-TtSlyD-FKBP antibody comprising a VH sequence of SEQ ID NO:14 and a VL sequence of SEQ ID NO:13. In another specific embodiment, an antibody is provided that binds to the same epitope as an anti-TtSlyD-FKBP antibody comprising at least one, two, three, four, five, or six HVRs selected from (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO:04; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO:05; (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO:06; (d) HVR-L1 comprising the amino acid sequence of SEQ ID NO:01; (e) HVR-L2 comprising the amino acid sequence of SEQ ID NO:02; and (f) HVR-L3 comprising the amino acid sequence of SEQ ID NO:03.

In a further aspect of the description, an anti-TtSlyD-FKBP antibody according to any of the above embodiments is a monoclonal antibody. In one embodiment, an anti-TtSlyD-FKBP antibody is a monoclonal rabbit antibody. In one embodiment, an anti-TtSlyD-FKBP antibody is an antibody fragment, e.g., a Fv, Fab, Fab′, scFv, diabody, or F(ab′)2fragment.

In one aspect, the description refers to an isolated monoclonal rabbit antibody that binds to TtSlyD-FKBP, wherein the antibody specifically binds to the native conformation of TtSlyD-FKBP. In one embodiment, the isolated monoclonal rabbit antibody binds to TtSlyD-FKBP, wherein the antibody specifically binds to the native conformation of TtSlyD-FKBP without interfering with the ligand binding site at the insertion site of TtSlyD-FKBP.

In one embodiment, the isolated antibody that binds to TtSlyD-FKBP has one or more of the following properties (also each combination of each single property is contemplated herein): a) the antibody binds to a conformational epitope in TtSlyD-FKBP in native conformation; and/or b) the antibody exhibits a ka from 1×1031/Ms to 5×1071/Ms, and/or c) the antibody exhibits a kd from 1×10−31/s to 1×10−61/s, and/or d) the antibody exhibits a t1/2dfrom 1 min to 1500 min, and/or e) the antibody exhibits a KD from 1×10−6M to 1×10−13M at a temperature of 25° C. or 37° C.

In a specific embodiment, the isolated antibody that binds to TtSlyD-FKBP has one or more of the following properties (also each combination of each single property is contemplated herein): a) the antibody binds to a conformational epitope in TtSlyD-FKBP in native conformation; and/or b) the antibody exhibits a ka from 1×1041/Ms to 1×1061/Ms, and/or c) the antibody exhibits a kd from 1×10−21/s to 1×10−61/s, and/or d) the antibody exhibits a t1/2dfrom 10 min to 1200 min, and/or e) the antibody exhibits a KD from 1×10−8M to 1×10−12M at a temperature of 25° C. or 37° C.

In one aspect, an anti-TtSlyD-FKBP antibody is described comprising at least one, two, three, four, five, or six HVRs selected from (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO:10; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO:11; (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO:12; (d) HVR-L1 comprising the amino acid sequence of SEQ ID NO:07; (e) HVR-L2 comprising the amino acid sequence of SEQ ID NO:08; and (f) HVR-L3 comprising the amino acid sequence of SEQ ID NO:09.

In one aspect, an antibody is described comprising at least one, at least two, or all three VH HVR sequences selected from (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO:10; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO:11; and (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO:12. In one embodiment, the antibody comprises HVR-H3 comprising the amino acid sequence of SEQ ID NO:12. In another embodiment, the antibody comprises HVR-H3 comprising the amino acid sequence of SEQ ID NO:12 and HVR-L3 comprising the amino acid sequence of SEQ ID NO:09. In a further embodiment, the antibody comprises HVR-H3 comprising the amino acid sequence of SEQ ID NO:12, HVR-L3 comprising the amino acid sequence of SEQ ID NO:09, and HVR-H2 comprising the amino acid sequence of SEQ ID NO:11. In a further embodiment, the antibody comprises (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO:10; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO:11; and (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO:12.

In another aspect, an antibody is described comprising at least one, at least two, or all three VL HVR sequences selected from (a) HVR-L1 comprising the amino acid sequence of SEQ ID NO:07; (b) HVR-L2 comprising the amino acid sequence of SEQ ID NO:08; and (c) HVR-L3 comprising the amino acid sequence of SEQ ID NO:09. In one embodiment, the antibody comprises (a) HVR-L1 comprising the amino acid sequence of SEQ ID NO:07; (b) HVR-L2 comprising the amino acid sequence of SEQ ID NO:08; and (c) HVR-L3 comprising the amino acid sequence of SEQ ID NO:09.

In another aspect, an antibody according to the description comprises (a) a VH domain comprising at least one, at least two, or all three VH HVR sequences selected from (i) HVR-H1 comprising the amino acid sequence of SEQ ID NO:10, (ii) HVR-H2 comprising the amino acid sequence of SEQ ID NO:11, and (iii) HVR-H3 comprising an amino acid sequence selected from SEQ ID NO:12; and (b) a VL domain comprising at least one, at least two, or all three VL HVR sequences selected from (i) HVR-L1 comprising the amino acid sequence of SEQ ID NO:07, (ii) HVR-L2 comprising the amino acid sequence of SEQ ID NO:08, and (c) HVR-L3 comprising the amino acid sequence of SEQ ID NO:09.

In another aspect, an antibody is described comprising (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO:10; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO:11; (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO:12; (d) HVR-L1 comprising the amino acid sequence of SEQ ID NO:07; (e) HVR-L2 comprising the amino acid sequence of SEQ ID NO:08; and (f) HVR-L3 comprising an amino acid sequence selected from SEQ ID NO:09.

In another aspect, an anti-TtSlyD-FKBP antibody comprises a heavy chain variable domain (VH) sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO:16. In certain embodiments, a VH sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity contains substitutions (e.g., conservative substitutions), insertions, or deletions relative to the reference sequence, but an anti-TtSlyD-FKBP antibody comprising that sequence retains the ability to bind to TtSlyD-FKBP. In certain embodiments, a total of 1 to 10 amino acids have been substituted, inserted and/or deleted in SEQ ID NO:16. In certain embodiments, substitutions, insertions, or deletions occur in regions outside the HVRs (i.e., in the FRs). Optionally, the anti-TtSlyD-FKBP antibody comprises the VH sequence in SEQ ID NO:16, including post-translational modifications of that sequence. In a particular embodiment, the VH comprises one, two or three HVRs selected from: (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO:10, (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO:11, and (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO:12.

In another aspect, an anti-TtSlyD-FKBP antibody is provided, wherein the antibody comprises a light chain variable domain (VL) having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO:15. In certain embodiments, a VL sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity contains substitutions (e.g., conservative substitutions), insertions, or deletions relative to the reference sequence, but an anti-TtSlyD-FKBP antibody comprising that sequence retains the ability to bind to TtSlyD-FKBP. In certain embodiments, a total of 1 to 10 amino acids have been substituted, inserted and/or deleted in SEQ ID NO:15. In certain embodiments, the substitutions, insertions, or deletions occur in regions outside the HVRs (i.e., in the FRs). Optionally, the anti-TtSlyD-FKBP antibody comprises the VL sequence in SEQ ID NO:15, including post-translational modifications of that sequence. In a particular embodiment, the VL comprises one, two or three HVRs selected from (a) HVR-L1 comprising the amino acid sequence of SEQ ID NO:07; (b) HVR-L2 comprising the amino acid sequence of SEQ ID NO:08; and (c) HVR-L3 comprising the amino acid sequence of SEQ ID NO:09.

In another aspect, an anti-TtSlyD-FKBP antibody is provided, wherein the antibody comprises a VH as in any of the embodiments provided above, and a VL as in any of the embodiments provided above. In one embodiment, the antibody comprises the VH and VL sequences in SEQ ID NO:16 and SEQ ID NO:15, respectively, including post-translational modifications of those sequences.

In a further aspect, an antibody is described that binds to the same epitope as an anti-TtSlyD-FKBP antibody provided herein. In a specific embodiment, an antibody is provided that binds to the same epitope as an anti-TtSlyD-FKBP antibody comprising a VH sequence of SEQ ID NO:16 and a VL sequence of SEQ ID NO:15. In another specific embodiment, an antibody is provided that binds to the same epitope as an anti-TtSlyD-FKBP antibody comprising at least one, two, three, four, five, or six HVRs selected from (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO:10; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO:11; (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO:12; (d) HVR-L1 comprising the amino acid sequence of SEQ ID NO:07; (e) HVR-L2 comprising the amino acid sequence of SEQ ID NO:08; and (f) HVR-L3 comprising the amino acid sequence of SEQ ID NO:09.

In a further aspect of the description, an anti-TtSlyD-FKBP antibody according to any of the above embodiments is a monoclonal antibody. In one embodiment, an anti-TtSlyD-FKBP antibody is a monoclonal rabbit antibody. In one embodiment, an anti-TtSlyD-FKBP antibody is an antibody fragment, e.g., a Fv, Fab, Fab′, scFv, diabody, or F(ab′)2fragment.

In a further aspect, an anti-TtSlyD-FKBP antibody according to any of the above embodiments may incorporate any of the features, singly or in combination, as described in Sections 1-4 below:

In certain embodiments, an antibody provided herein is an antibody fragment. Antibody fragments include, but are not limited to, Fab, Fab′, Fab′-SH, F(ab′)2, Fv, and scFv fragments, and other fragments described below. For a review of certain antibody fragments, see Hudson, P. J. et al., Nat. Med. 9 (2003) 129-134. For a review of scFv fragments, see, e.g., Plueckthun, A., In; The Pharmacology of Monoclonal Antibodies, Vol. 113, Rosenburg and Moore (eds.), Springer-Verlag, New York (1994), pp. 269-315; see also WO 93/16185; and U.S. Pat. Nos. 5,571,894 and 5,587,458. For discussion of Fab and F(ab′)2fragments comprising salvage receptor binding epitope residues and having increased in vivo half-life, see U.S. Pat. No. 5,869,046.

Single-domain antibodies are antibody fragments comprising all or a portion of the heavy chain variable domain or all or a portion of the light chain variable domain of an antibody.

In certain embodiments, antibody variants having one or more amino acid substitutions are provided. Sites of interest for substitutional mutagenesis include the HVRs and FRs. Exemplary changes are provided in Table 1 under the heading of “exemplary substitutions”, and as further described below in reference to amino acid side chain classes. Conservative substitutions are shown in Table 1 under the heading of “preferred substitutions”. Amino acid substitutions may be introduced into an antibody of interest and the products screened for a desired activity, e.g., retained/improved antigen binding, decreased immunogenicity, or improved ADCC or CDC.

In certain embodiments, substitutions, insertions, or deletions may occur within one or more HVRs so long as such alterations do not substantially reduce the ability of the antibody to bind antigen. For example, conservative alterations (e.g., conservative substitutions as provided herein) that do not substantially reduce binding affinity may be made in HVRs. Such alterations may be outside of HVR “hotspots” or SDRs. In certain embodiments of the variant VH and VL sequences provided above, each HVR either is unaltered, or contains no more than one, two or three amino acid substitutions.

b) Fc Region Variants

In certain embodiments, one or more amino acid modifications may be introduced into the Fc region of an antibody provided herein, thereby generating an Fc region variant. In another embodiment the FC portion can be exchanged by another species, like human, mouse, rabbit, hamster or any other species in order to facilitate a heterogeneous immunoassay. In some embodiments, alterations are made in the Fc region that result in altered (i.e., either improved or diminished) Clq binding and/or Complement Dependent Cytotoxicity (CDC), e.g., as described in U.S. Pat. No. 6,194,551, WO 99/51642, and Idusogie, E. E. et al., J. Immunol. 164 (2000) 4178-4184.

B. Recombinant Methods and Compositions

Anti-TtSlyD-FKBP antibodies provided herein may be identified, screened for, or characterized for their physical/chemical properties and/or biological activities by various assays known in the art.

1. Binding Assays and Other Assays

In one aspect, an antibody according to the description is tested for its antigen binding activity, e.g., by known methods such as ELISA, Western Blot, SPR etc.

In an exemplary competition assay, immobilized TtSlyD-FKBP is incubated in a solution comprising a first labeled antibody that binds to TtSlyD-FKBP and a second unlabeled antibody that is being tested for its ability to compete with the first antibody for binding to TtSlyD-FKBP. The second antibody may be present in a hybridoma supernatant. As a control, immobilized TtSlyD-FKBP is incubated in a solution comprising the first labeled antibody but not the second unlabeled antibody. After incubation under conditions permissive for binding of the first antibody to TtSlyD-FKBP, excess unbound antibody is removed, and the amount of label associated with immobilized TtSlyD-FKBP is measured. If the amount of label associated with immobilized TtSlyD-FKBP is substantially reduced in the test sample relative to the control sample, then that indicates that the second antibody is competing with the first antibody for binding to TtSlyD-FKBP. See Harlow, E. and Lane, D., Antibodies: A Laboratory Manual, Chapter 14, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. (1988).

D. Methods and Compositions for Diagnostics and Detection

In certain embodiments, any of the anti-TtSlyD-FKBP antibodies provided herein is useful for detecting the presence of TtSlyD-FKBP in a biological sample. The term “detecting” as used herein encompasses quantitative or qualitative detection. In certain embodiments, a biological sample comprises a cell or tissue.

In one embodiment, an anti-TtSlyD-FKBP antibody for use in a method of diagnosis or detection is provided. In a further aspect, a method of detecting the presence of TtSlyD-FKBP in a biological sample is provided. In certain embodiments, the method comprises contacting the biological sample with an anti-TtSlyD-FKBP antibody as described herein under conditions permissive for binding of the anti-TtSlyD-FKBP antibody to anti-TtSlyD-FKBP, and detecting whether a complex is formed between the anti-TtSlyD-FKBP antibody and TtSlyD-FKBP. Such method may be an in vitro or in vivo method.

Description of the Amino Acid Sequences

The following examples 1 to 5 are provided to aid the understanding of the present invention, the true scope of which is set forth in the appended claims.

Anti-TtSlyD-FKBP Antibody Development with Rabbit B-Cell PCR

For the generation of antibodies against TtSlyD-FKBP, 16-week old ZiKa rabbits were immunized with native TtSlyD-FKBP. All rabbits were subjected to repeated immunizations. In the first months the animals were immunized weekly. From the second month onward the animals were immunized once per month. For each immunization 100 μg TtSlyD-FKBP dissolved in 1 ml 140 mM NaCl was emulsified in 1 ml CFA. The development of titers was evaluated on days 45 and 105 after the first immunization. When titers against the immunogen were detected antibodies were developed by B-cell cloning as described in Ligthwood et al. 2006, Journal of Immunological Methods 316, 133-143. Recombinant rabbit IgG was expressed by transient transfection of HEK293 cells. For the determination of the serum titers against TtSlyD-FKBP a small amount of serum of each rabbit was collected on day 45 and day 105 after start of the immunization campaign. For the ELISA the immunogen was immobilized on the plate surface. TtSlyD-FKBP was immobilized at a concentration of 1 μg/ml. The recombinant proteinThermococcus gammaduransSlyD (Uniprot C5A738) was used as a negative control. The sera from each rabbit were diluted in PBS with 1% BSA and the dilutions were added to the plates. The sera were tested at dilutions 1:300, 1:900, 1:2.700, 1:8.100, 1:24.300, 1:72900, 1:218.700 and 1:656.100. Bound antibody was detected with a HRP-labeled F(ab′)2goat anti-rabbit Fcγ (Dianova) and ABTS (Roche) as a substrate.

Biacore Binding Affinity

A Biacore B3000 instrument (GE Healthcare) was mounted with a CM5 research grade sensor and was normalized in HBS-ET buffer (10 mM HEPES pH 7.4, 150 mM NaCl, 3 mM EDTA, 0.005% w/v Tween 20) according to the manufacturer's instructions. The system operated at 25° C. 10000 RU GAR-Fcγ (relative units of Fcγ-fragment binding, polyclonal goat anti-Rabbit IgG/Jackson Laboratories) were immobilized according to the manufacturer's instructions using EDC/NHS chemistry on all 4 flow cells. The sensor is finally deactivated using 1 M ethanolamine. 10 nM of the respective antibody 0712pS4D5 or 0612pS3A8 in system buffer were injected for 2 min at 10 μl/min. TtSlyD-FKBP derivatives were injected at 100 μl/min for 2 min association and 5 min dissociation time in a concentration series of 0 nM, 4 nM, 11 nM, two times 33 nM, 100 nM, 300 nM. The GAR-Fcγ capture system was regenerated by 10 mM glycine pH 1.5 at 20 μl/min for 30 sec, followed by two consecutive injections of 10 mM glycine pH 1.7 at 20 μl/min for 30 sec. Affinity was determined using the Biacore evaluation software. The results of the kinetic analyses are depicted inFIG. 1. The analytes in solution were two engineered TtSlyD-FKBP, TtSlyD-FKBP-A and TtSlyD-FKBP-B. The Molar Ratio (MR) indicates a functional binding. 0612pS3A8 binds in a 1:1 ratio and 0712pS4D5 bind two scaffolds in a 1:2 mode. Therefore different binding sterics of the two antibodies can be assumed. The complex stability was too high and was therefore out of the instruments specifications. To calculate an apparent affinity the dissociation rate was set to the instrument limits of 1e-05 1/s. Therefore, the affinity is in the low picomolar range.FIG. 2depicts Biacore sensorgrams showing the 0612pS3A8 interactions with two different TtSlyD-FKBP derivatives.FIG. 3depicts Biacore sensorgrams showing the 0712pS4D5 interactions with two different TtSlyD-FKBP derivatives. Concentration-dependent sensorgram overlay plots of 0612pS3A8 are inFIG. 2, and for 0712pS4D5 inFIG. 3.

Biacore Binding Assay

FIG. 4shows the instrumental setup of a Biacore SPR binding assay. For such assay, a Biacore B3000 instrument (GE Healthcare) was used to kinetically assess TtSlyD-FKBP derivatives for binding specificity for Taq DNA polymerase A CM5 series sensor was mounted into the system and was normalized in HBS-ET buffer (10 mM HEPES pH 7.4, 150 mM NaCl, 3 mM EDTA, 0.005% w/v Tween 20) according to the manufacturer's instructions. The samples were diluted in the instrument's buffer. The system operated at 25° C. 10000 RU GAR-Fcγ (relative units of Fcγ-fragment binding, polyclonal goat anti-Rabbit IgG/Jackson Laboratories) were immobilized according to the manufacturer's instructions using EDC/NHS chemistry on all 4 flow cells. The sensor was finally deactivated using 1 M ethanolamine.

The binding activity of the TtSlyD-FKBP clones 3 F12, 5F8 and 3 C1 versus Taq DNA polymerase was kinetically tested. The monoclonal rabbit anti-TtSlyD-FKBP antibody (0612pS3A8), was captured in all flow cells by a 3 min injection at 30 μl/min. Each TtSlyD-FKBP variant was captured on the flow cells 2, 3 and 4. Flow cell 1 served as a reference. The TtSlyD-FKBP clones were specifically captured on the sensor by a 3 min injection at 10 μl/min. The flow rate was set to 80 μl/min. Recombinant 93 a Taq DNA polymerase (Roche) was injected for 3 min at different concentration steps diluted in the sample buffer at 0 nM, 4 nM, 11 nM, two times 33 nM, 100 nM and 300 nM. 1 μM Streptavidin (Ser. No. 11/897,000, Roche) was injected as a non-interaction specificity control. The dissociation was monitored for 5 min.FIG. 6shows Biacore sensorgram overlay plots of three TtSlyD-FKBP derivatives interacting with Taq DNA polymerase at different concentrations.FIG. 7shows the kinetic data determined according to a Langmuir model.

Acidic regeneration of the sensor surface was achieved using a single injection of 10 mM glycine pH 1.5 at 20 μl/min for 30 sec, followed by two consecutive injections of 10 mM glycine pH 1.7 at 20 μl/min for 30 sec. Regeneration was complete.

As can be seen inFIG. 5, the overlay plot sensorgram shows six exemplary consecutive cycles of the SPR binding assay as depicted inFIG. 4. Capture denotes the injection of the TtSlyD-FKBP variant 3C1 and shows the reproducibility of the scaffold capturing step. Analyte denotes the injection of Taq DNA polymerase.

Each cycle injects an increasing Taq DNA polymerase concentration as described. Regeneration denotes the acidic regeneration of the sensor surface. Since Taq DNA polymerase was displayed as chemically biotinylated target protein on streptavidin coated paramagnetic particles, it was necessary to investigate potential streptavidin cross-reactive binding. No streptavidin binding to the variants could be detected at 1 μM streptavidin as analyte in solution.

Biacore Binding Study with Covalently Immobilized 0712pS4D5 and 0612pS3A8

A Biacore B3000 instrument (GE Healthcare) was mounted with a CM5 sensor and was normalized in HBS-ET buffer (10 mM HEPES pH 7.4, 150 mM NaCl, 3 mM EDTA, 0.005% w/v Tween 20) according to the manufacturer's instructions.

Both antibodies, 0712pS4D5 and 0612pS3A8 were incubated at 30 μg/ml in 10 mM NaAc pH 4.0, respectively pH 4.5, pH 5.0, pH 5.5 and were preconcentrated on the CM-5 sensor, followed by NHS-ECD immobilization according to the manufacturer's instructions.

10 mM NaAc pH 4.5 and 30 μg/ml antibody concentration was found the optimal condition for the immobilization of 10.000 RU of each antibody on the CM-5 sensor.

150 nM of the TtSlyD-FKBP derivative 5CRe1 was injected into the system for 5 min at 100 μl/min. The dissociation was monitored for another 5 min. The system was regenerated with 10 mM glycine buffer pH 1.7 at 20 μl/min for 1 min. Using this condition the immobilized antibodies are keeping their binding activity over 20 cycles of binding and regeneration. Therefore it is assumed, that the respective regeneration condition is optimal to keep up the antibodies binding activity.FIG. 8shows regeneration scouting of 0712pS4D5 and 0612pS3A8.

Kinetic Screening with Covalently Surface-Attached Antibodies

A Biacore 4000 instrument is mounted with a Biacore CM 5 sensor series S and was preconditioned like recommended by the manufacturer. The instrument buffer was HBS-ET (10 mM HEPES pH 7.4, 150 mM NaCl, 3 mM EDTA, 0.005% w/v Tween 20). The rabbit monoclonal antibodies, 0712pS4D5 and 0612pS3A8 were covalently immobilized on the sensor surface by using the standard amine coupling procedure as recommended by the manufacturer. The antibodies were each incubated at 30 μg/ml in 10 mM NaAc pH 4.5 and were preconcentrated on the CM-5 sensor for 10 min, followed by NHS-ECD immobilization according to the manufacturer's instructions. In average 8500 RU antibody were immobilized on the sensor spots 1, 2, 4 and 5 on the flow cells 1, 2, 3, and 4. Spot 3 serves as a reference.

Subsequently different TtSlyD-FKBP derivatives were injected at 10 μl/min for 3 min and were stably bound by the surface immobilized antibodies 0712pS4D5 on the flow cells 1 and 2 or 0612pS3A8 on the flow cells 3 and 4.FIG. 9shows a sensorgram overlay plot of 4 exemplary injections with 0712pS4D5 as capture antibody numbered with (1) and 0612pS3A8 as capturing antibody numbered with (2). Both antibodies bind different TtSlyD-FKBP based scaffold binders with high complex stability and stable baseline formation. This is of upmost importance to enable a kinetic measurement without any baseline drift. The surface presented scaffold binders are subsequently contacted with an analyte in solution. The analyte is injected for 5 min and dissociates for another 5 min at 30 The surface is fully regenerated by two injections of 10 mM glycine buffer pH 1.7 and the sensor is reusable.

To summarize, the rabbit monoclonal antibodies 0712pS4D5 and 0612pS3A8 can be used in a Biacore Kinetic Screening setup with a GAR-Fcγ capture system, which displays the rabbit antibodies via their FC portion. The antibodies bind to TtSlyD-FKBP derivatives with a 1:2 stoichiometry and with high complex stability, respectively picomolar affinity. The rabbit antibodies bind to an epitope of the TtSlyD-FKBP derivatives, which functionally displays the scaffolds, so that they can form a complex (sandwich) with further binding partners, like e.g. Taq DNA polymerase.

Another option is to directly immobilize the antibodies on the biosensor surface using NHS-EDC chemistry, whereby the optimal regeneration condition is a 10 mM glycine buffer pH 1.7. Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, the descriptions and examples should not be construed as limiting the scope of the invention. The disclosures of all patent and scientific literature cited herein are expressly incorporated in their entirety by reference.