Epitope tags recognized by specific binding agents

Abstract

The present invention provides peptides that can be used as epitope tags and antibodies that specifically bind to these peptides, and the peptides can be fused with target polypeptides. The peptides and / or antibodies can be used to detect, immobilize, isolate, or purify molecules conjugated with such peptides and / or antibodies.

Description

[0001] Cross - Reference to Related Applications

[0002] This application claims the priority benefits of European Patent Application No. 18193663.4 filed on September 11, 2018 and European Patent Application No. 19160485.9 filed on March 4, 2019, the contents of which are incorporated herein by reference in their entirety. Field of the Invention

[0003] The present invention provides peptides that can be used as epitope tags and antibodies that specifically bind to these peptides, and the peptides can be fused with target polypeptides. The peptides and / or antibodies can be used to detect, immobilize, isolate, or purify molecules conjugated to the peptides and / or antibodies. Background Art

[0004] Epitope tags actually play important roles in all aspects of life sciences. For example, they are used in biotechnological applications to facilitate the expression and purification of recombinant proteins (Waugh, D. S. Making the most of affinity tags. Trends Biotechnol 23, 316 - 320 (2005)). In cell biology, epitope tags are commonly used to monitor the biogenesis or topology of a given protein of interest (POI) (Nooh, M. M. & Bhaouth, S. W. Visualization and quantification of GPCR trafficking in mammalian cells by confocal microscopy. Methods Cell Biol. 142, 67 - 78 (2017); Kocaoglu, O. & Carlson, E. E. Progess and prospects for small - molecule probes of bacterial imaging. Nat Chem Biol 12, 472 - 478 (2016)). Tags also assist in the immunoprecipitation of protein complexes to be studied by mass spectrometry techniques (Shi, Y. et al. A stragety for dissecting the architctures of native macromolecular assemblies. Nat Methods 12, 1135 - 1138 (2015); Smits, A. H. & Vermeulen, M. Characterizing Protein - Protein Interactions Using Mass Spectrometry: Challenges and Opportunities. Trends Biotechnol 34, 825 - 834 (2016)). Over the years, at least a dozen different tags have been developed, providing researchers with a variety of tools for the most rigorous protocols (Waugh, D. S. Making the most of affinity tags. Trends Biotechnol 23, 316 - 320 (2005); Brizzard, B. Epitope tagging. BioTechniques 44, 693 - 695 (2008)). However, a given tag may perform very well in a particular application and fail completely in others. As a result, most researchers rely on a variety of tags to cover the range of desired applications.

[0005] It seems that there has not been a truly universal tag so far. One explanation could be the fact that most tags were discovered as a by-product when screening conjugates (usually monoclonal antibodies) against naturally occurring proteins. For example, the c-myc-tag (Evan, G.I., Lewis, G.K., Ramsay, G. & Bishop, J.M. Isolation of monoclonal antibodies specific for human c-myc proto-oncogene product. Mol Cell Biol 5, 3610-3616 (1985)), the HA-tag (Field, J. et al. Purification of a RAS-responsive adenylyl cyclase complex from Saccharomyces cerevisiae by use of an epitope addition method. Mol Cell Biol 8, 2159-2165 (1988)) or the Spot-tag (Virant, D. et al. A peptide tag-specific nanobody enables high-quality labeling for dSTORM imaging. Nat Commun 1-14 (2018). doi:10.1038 / s41467-018-03191-2, Braun, M.B. et al. Peptides in headlock-a novel high-affinity and versattle peptide-binding nanobody for proteomics and microscopy. Sci Rep 6, 19211 (2016)) fall into this category. Typically, a tag is thus by default the smallest peptide that can be effectively recognized by the corresponding conjugate. As a result, the properties of these tags mainly randomly depend on the selected conjugate, and they usually cannot be re-tuned to fit specific experimental requirements or conditions. Alternatively, some tags have also been rationally designed for a specific application. For example, the His-tag is ideally suited for the initial crude purification of recombinantly expressed proteins on metal ion chelating resins, which is exactly the purpose for which this tag was developed (Hochuli, E., H. & Schacher, A. New metal chelate adsorbents selective for proteins and peptides containing neighbouring histidine residues. J. Chromatogr. 411, 177 - 184 (1987)). However, due to the lack of high - affinity conjugates, His - tags have not been widely used in microscopic applications in cell biology to date.

[0006] The object of the present invention is to provide improved epitope tags and their specific binding molecules. Summary of the Invention

[0008] The present invention relates to a fusion protein comprising: (a) a peptide comprising a sequence of X1 - X2 - X3 - X4 - X5 - X6 - X7 - X8 - X9 - R - L - X12 - X13 (SEQ ID NO: 01), wherein X1 is G or S or T or P, X2 is R or G or A or E or P, X3 is L or V, X4 is E or Q, X5 is E or Q, X6 is E or Q, X7 is L or I or V, X8 is R or A or Q or E, X9 is R or A or Q or E, X12 is S or T or D or E or P or A or no amino acid, and wherein X13 is E or K or P or S or A or D or no amino acid; and (b) a polypeptide.

[0009] The present invention also relates to an antibody that specifically binds to the peptide contained in the fusion protein of the present invention.

[0010] The present invention also relates to a fusion protein comprising the peptide to which the antibody of the present invention binds.

[0011] The present invention also relates to a complex comprising the fusion protein of the present invention and the antibody of the present invention.

[0012] The present invention also relates to a nucleic acid encoding the fusion protein of the present invention or the antibody of the present invention.

[0013] The present invention also relates to a vector comprising the nucleic acid of the present invention.

[0014] The present invention also relates to a host cell comprising the nucleic acid of the present invention or the vector of the present invention, or a host cell expressing the fusion protein of the present invention or the antibody of the present invention.

[0015] The present invention also relates to the use of the antibody of the present invention for detecting, immobilizing, separating or purifying the fusion protein of the present invention.

[0016] The present invention also relates to a method for detecting the fusion protein of the present invention, comprising contacting the fusion protein with the antibody of the present invention.

[0017] The present invention also relates to a method for separating the fusion protein described in the present invention, including contacting the fusion protein with the antibody described in the present invention. When the fusion protein described in the present invention contains an antibody portion, the present invention also relates to a method for separating the specific target of the antibody portion.

[0018] The present invention also relates to a kit, which contains a nucleic acid or nucleic acid expression construct encoding a peptide contained in the fusion protein described in the present invention and optionally the antibody described in the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 : ALFA ST and ALFA PE Interaction with NbALFA ST

[0020] A, Schematic diagram of the protein for ALFA binding assay. In this figure, the ALFA tag for the shGFP2 fusion can be ALFA ST (SEQ ID NO: 05 - 07), or it can be ALFA PE (SEQ ID NO: 33). B, 20 μl of ALFA Selector ST (SEQ ID NO: 133) presenting NbALFA ST resin was saturated with GFP variants (shGFP2, Frey and ST or ALFA PE fused at different positions (internal (left); N - terminus (middle) or C - terminus (right)) with ALFA Cell. June 28, 2018; 174(1):202 - 217.e9.doi:10.1016 / j.cell.2018.05.045). After washing 4 times with PBS, the beads were suspended in 10 - fold excess of PBS containing 200 μM ALFA ST peptide (acetyl - PSRLEEELRRRLTEP - amide, SEQ ID NO: 179) and gently mixed at room temperature. At the designated time points, the specific elution from the beads was quantified using the GFP fluorescence released into the supernatant. Shown are the average fluorescence values and the standard deviation at each time point for three independent experiments performed in parallel. Efficient peptide elution of the ALFA PE fusion was observed after 20 - 30 minutes at room temperature. In contrast, compared with AFFA STAll GFP variants in the fusion remained tightly bound to the resin. Notably, the elution kinetics were largely independent of the position of the corresponding ALFA-tag variant in the fusion protein. C, 10 μl of ALFA ST -shGFP2 (top row) or ALFA PE -shGFP2 (bottom row) saturated ALFA Selector ST resin was transferred to an 8-well PCR strip. After removing the remaining liquid, the beads were incubated with 100 μl of the indicated substances for 60 min at room temperature. Photos were taken after the beads had settled. Asterisks (*) denote conditions known to cause partial or complete loss of GFP fluorescence.

[0021] Figure 2 : Use ALFA Selector ST to pull down ALFA-tagged target proteins and protein complexes from complex lysates.

[0022] A, shGFP2 was fused to ALFA at the N-terminus ST (SEQ ID NO: 07; ALFA ST -sfGFP2, left) or ALFA PE (SEQID NO: 33; ALFA PE -GFP, right). Proteins were overexpressed in E. coli and purified by nickel affinity chromatography using their C-terminal His6-tag, followed by gel filtration on a Superdex 75 size exclusion column. B + C, To obtain defined input materials for one-step affinity purification using ALFA Selector ST , E. coli (B) or HeLa (C) mock extracts were mixed with 3 μM of the corresponding substrate. Mock lysates were used as specific controls. 1 mL of each lysate / substrate mixture was incubated with 25 μl of ALFA-Selector ST containing NbALFA ST (SEQ ID NO: 133) for 1 h at 4°C. After washing 4 times with 1 mL of PBS, the bound proteins were eluted with 25 μl of a PBS solution of 200 μM ALFA ST peptide (SEQ ID NO: 179) twice for 10 min at room temperature. Subsequently, the proteins remaining on the beads were eluted with SDS sample buffer. 0.5 μl (B) or 1.5 μl (C) of the input material and the unbound fractions were analyzed by SDS-PAGE (12%) and Coomassie staining. The indicated eluate fractions correspond to the material eluted from 1 μl of ALFA Selector ST resin. Notably, ALFA STTag or ALFA PE The protein with the tag is pulled down specifically by the ALFA Selector ST under natural conditions. Efficient and specific elution of the protein fused with the ALFA PE tag can be achieved by competition with free ALFA ST peptide under natural conditions. It is also worth noting that, compared with the corresponding substrate protein purified by conventional two-step chromatography, the protein purified from either of the two lysates using the ALFA Selector ST resin contains significantly fewer impurities.

[0023] D, left: Schematic diagram of the YfgM-PpiD complex. Right: Untagged (-) (SEQ ID NO: 198) or C-terminally ALFA PE tagged YfgM (+) (SEQ ID NO: 197) was expressed in the yfgGM deletion strain. Membrane protein complexes were solubilized from the total lysate using 1% DDM. The complex containing YfgM-ALFA PE was purified in one step using the ALFA Selector affinity resin containing the nanobody of SEQ ID NO: 133. The serum against the YfgM-PpiD complex recognized both PpiD and YfgM in the input part. The ALFA Selector ST specifically immunoprecipitated the native protein complex of YfgM-ALFA ST and its interacting partner PpiD. PE

[0024] Figure 3 : Nanobody-based detection of proteins with the ALFA ST tag in immunofluorescence applications

[0025] A: Schematic diagram of NbALFA ST binding to the ALFA PE tag (left) or ALFA ST tag (right). The ALFA tag sequences for tagging at different positions are given (N-terminal ALFA ST tag: SEQ ID NO: 05, middle ALFA ST tag: SEQ ID NO: 06, C-terminal ALFA ST tag: SEQ ID NO: 07)

[0026] B: NbALFA STsequence (NbALFA clone 1G5; SEQ ID NO: 133). Gray boxes indicate CDR 1-3 (AbM definition, SEQ ID NO: 115-117).

[0027] C: Tom70-EGFP-ALFA ST (upper row) or Tom70-EGFP-ALFA PE (lower row) transfected COS-7 cells were fixed with 4% paraformaldehyde. After permeabilization, NbALFA conjugated with AbberiorStar635P (FluoTag-X2 anti-ALFAAbberiorStar635P) ST was used for staining. First column: FluoTag-X2 anti-ALFA; Second column: target detection using intrinsic EGFP fluorescence; Third column: DAPI staining added to the overlay; Fourth column: fluorescently labeled NbALFA ST Schematic diagram of the target protein detected. All scale bars: 20 μM.

[0028] D: After fixation with 4% paraformaldehyde (PFA), 2% glutaraldehyde (GA), or 100% methanol (MEOH), vimentin with an ALFA ST tag at the N-terminus (upper row) or vimentin with an ALFA PE tag (lower row) was detected with FluoTag-X2 anti-ALFAAbberiorStar635P. Right column: fluorescently labeled NbALFA ST Schematic diagram of the ALFA-tagged vimentin detected.

[0029] E: Intracellular antibody-based detection of proteins with an ALFA ST tag. COS-7 cells were co-transfected with an NbALFA ST -mScarlet-I fusion and a target protein with an ALFA ST tag. The target protein was detected by EGFP fluorescence (for TOM70-EGFP-ALFA ST ) or by immunofluorescence using FluoTag anti-ALFA AbberiorStar635P (for ALFA ST -FLAG-vimentin). In parallel, NbALFA ST -mScarlet-I was detected by red mScarlet-I fluorescence. Note the good co-localization between the target protein (left column) and the mScarlet-I signal (middle column).

[0030] Figure 4 : Proteins with an ALFA tag can be detected by fluorescently labeled NbALFAST It is detected regardless of the position of ALFA in the fusion protein.

[0031] COS-7 cells were transfected with constructs encoding proteins fused to the ALFA tag, where the ALFA tag was fused at the N-terminus of the protein (ALFA ST -FLAG-vimentin or ALFA PE -FLAG-vimentin; A) or fused in a separate protein domain (EGFP-ALFA ST -myc-TM; B). Cells were fixed with 4% PFA and stained as indicated. For A and B, cells were permeabilized with 0.1% Triton X-100; for C, cells were stained under non-permeabilized conditions. TM: transmembrane domain. The schematic diagram shows the topology of the substrate and the NbALFA ST detected by (FluoTag-X2 anti-ALFA).

[0032] Figure 5 : GFP fused to N- or C-terminal ALFA ST tag shows normal intracellular localization

[0033] 3T3 cells were transiently transfected with EGFP fusions with N- or C-terminal ALFA ST tag. Untagged EGFP from pEGFP-N1 was used as a control. For each construct, the localization of each EGFP variant was analyzed based on 6-7 individual images. A total of 120-130 cells were imaged for each construct, and the localization of EGFP was analyzed. Generally, each EGFP construct showed a distribution across the cytosol and nucleus. Based on the observed nucleocytoplasmic localization of EGFP, the cells were divided into three groups ("slight nuclear localization", "uniform", and "other"). The standard deviation was derived from the values obtained from individual images. The difference in the localization between tagged and untagged EGFP variants was not statistically significant (Student's t-test).

[0034] Figure 6 : Western-blot and dot-blot detection of ALFA-tagged target proteins using fluorescently labeled NbALFA ST (FluoTag-X2 anti-ALFA)

[0035] A, with ALFA ST -FLAG-vimentin or ALFA PE-FLAG-Vimentin-transfected COS-7 cells were lysed in SDS buffer. Cells transfected with irrelevant plasmids were used as controls. Lysates corresponding to the same number of cells were analyzed by SDS-PAGE and Western-Blot. The vimentin fusion protein was visualized using NbALFA conjugated to IRDye800 (FluoTag-X2 anti-ALFA IRDye800). ST Tubulin was used as a loading control and detected by mouse anti-tubulin followed by FluoTag-X2 anti-mouse conjugated to IRDye680. The complete lanes are shown in Figure 7 A.

[0036] B, Schematic of recombinant Escherichia coli maltose-binding protein (MBP) with multiple epitope tags (FLAG, HA, myc, and ALFA ST ) for the experiments shown in C and D.

[0037] C, Dilution series of the protein shown in B were spotted onto nitrocellulose membranes. Commercially available monoclonal antibodies (anti-FLAG M2 - Sigma#F1804, anti-myc 9E10 - SynapticSystems#343 011, anti-HA F-7 - SantaCrus#sc-7392) were used in combination with secondary anti-mouse IgG IRDye800CW (Li-Cor#925-32210, diluted 1:1000) to detect the FLAG, myc, and HA-tags, respectively. The ALFA ST tag was detected using FluoTag-X2 anti-ALFA directly conjugated to IRDye800CW. The nanobody and all primary antibodies were used at a final concentration of 2.7 nM, which is well within the range recommended by the suppliers. The complete experiment including internal controls is shown in Figure 7 B.

[0038] D, Quantification of the signals obtained in C, shown as a double-logarithmic plot. The lines represent linear fits to the values obtained. Even without amplification by a secondary antibody, the signals obtained by NbALFA ST were 3- to >10-fold stronger than the signals obtained by commercially available reagents recognizing epitope tags. At the same time, the detection sensitivity using NbALFA ST was 10-fold higher and showed good linearity over ~3 orders of magnitude.

[0039] Figure 7 : Highly sensitive Western and dot blot detection of ALFA-tagged target proteins using fluorescently labeled NbALFA ST (FluoTag-X2 anti ALFA)

[0040] A, the same experiment as Figure 6 that shown in A. However, the complete lanes are shown here. It should be noted that in the absence of any vector encoding the protein with the ALFA ST tag, only very small bands (*) can be detected with fluorescently labeled NbALFA (FluoTag-X2 anti-ALFA).

[0041] B, the same experiment as Figure 6 that shown in C. In addition to Figure 6 the data shown in C, the detection of MBP by the combination of rabbit polyclonal serum against MBP (SynapticSystems) and anti-rabbit IgG IRDye680RD (Li-Cor #925-68071) is shown as an internal loading control. The overlay shows the MBP signal in red and the signal corresponding to the epitope tag in green.

[0042] Figure 8 : Interaction of the protein with the ALFA ST tag and the ALFA Selector ST and the ALFA Selector PE resin.

[0043] A; Schematic of the ALFA Selector resin conjugated with shGFP2-ALFA ST . In this schematic, the ALFA Selector resin can be ALFA Selector ST or ALFA Selector PE .

[0044] B and C; Peptide elution from NbALFA-conjugated affinity resin. Load shGFP2 with a C-terminal ALFA ST (SEQ ID NO: 133; ALFA Selector ST , left) or NbALFA PE mutant (SEQ ID NO: 134; ALFA Selector PE , right) conjugated agarose-based resin. To evaluate the dissociation rate (off-rate), suspend the resin in the presence of excess free ALFA ST tag. STIn PBS of the peptide and incubated at 25 °C. Control reactions were carried out without the peptide. At the indicated time points, the shGFP2 released from the resin was quantified. B shows the average fluorescence readings of three experiments and the standard deviation at each time point. The lines represent the fit to a single exponential. shGFP2-ALFA was observed after 15 - 20 minutes at room temperature ST from the ALFA Selector PE efficient peptide elution. In contrast, even after long incubation, the peptide elution from the ALFASelector ST was inefficient. In the absence of free ALFA ST peptide during the elution process, the target protein with the ALFA ST tag remained tightly bound to both resins. After 3 hours of elution, a photograph was taken under UV irradiation (C).

[0045] D; Tolerance to the stringent washing step. The ALFA Selector variants described in B were loaded with ALFA ST -shGFP2 or shGFP2-ALFA ST and incubated with shaking for 1 h at 25 °C with 10 volumes of the substances. Without further washing steps, a photograph was taken under UV irradiation after the beads had settled.

[0046] E; Tolerance to non-physiological pH. Similar to D. However, here after incubation for 30 minutes at the specified pH, the resin was washed to remove unbound substances. A photograph was taken after re-equilibration in PBS to restore GFP fluorescence.

[0047] Figure 9 : Using the ALFA Selector ST and the ALFA Selector PE to pull down the target protein and protein complexes with the ALFA ST tag from complex lysates.

[0048] A; The input protein used for the experiment (in B and C).

[0049] B and C; One-step affinity purification using ALFA Selector resin. E. coli (A) or HeLa (B) lysates mixed with 3 μM purified shGFP2 with the ALFA ST tag (A) were incubated with the ALFA Selector ST resin, the ALFA Selector PE resin or a similar resin without immobilized sdAb (control Selector). After washing with PBS, the resin was incubated with 200 μM ALFAST The peptides were incubated for 20 minutes. The proteins retained on the beads were eluted with SDS sample buffer. The indicated fractions were analyzed by SDS-PAGE and Coomassie staining. The indicated eluate fractions correspond to the material eluted from 1 μl of resin.

[0050] D; Using ALFA Selector PE Pull-down of the native Escherichia coli YfgM-PpiD inner membrane protein complex. Left: Schematic diagram of the YfgM-PpiD membrane protein complex. Right: The yfgM deletion strain was complemented with C-terminally tagged ALFA ST (left panel) or untagged YfgM (right panel; control reaction) expressed from a low-copy vector. The membrane protein complex was solubilized from the total lysate using DDM. The complex containing YfgM-ALFA ST was purified in one step using ALFA Selector PE affinity resin and eluted with 200 μM ALFA ST peptide under native conditions. Samples corresponding to 1 / 800 of the input and unbound material or 1 / 80 of the eluate fractions were resolved by SDS page and analyzed by Western-blot. Rabbit serum against the YfgM-PpiD complex (et al. YfgM is an ancillary subunit of the SecYEG translocon in Escherichia coli. J Biol Chem 289, 19089-19097 (2014)) recognized both PpiD and YfgM in the input fraction. ALFA Selector PE specifically immunoprecipitated the native protein complex containing ALFA ST tagged YfgM and its interacting partner PpiD. In the control reaction (without ALFA ST tag on YfgFGM), neither protein was present in the eluate.

[0051] Figure 10 : Peptide elution of ALFA ST tagged GFP from the ALFA Selector resin.

[0052] Twenty microliters of ALFA Selector ST (presenting NbALFA ST , SEQ ID NO: 133) or ALFA Selector PE (presenting NbALFA PE, SEQ ID NO: 134) shGFP2-ALFA on the load ST (A), bdSUMO-ALFA ST -shGFP2 (B) or ALFA ST -shGFP2 (C). After washing with PBS, the beads were suspended in 10-fold excess of PBS containing 200 μM free ALFA ST peptide and gently mixed at 25 °C. A control reaction without peptide was carried out. At the indicated time points, the specific elution from the beads was quantified using the GFP fluorescence released into the supernatant. Shown are the average fluorescence readings of three experiments and the standard deviation for each time point. The lines represent the fit to a single exponential. Only the half-time of elution of the peptide from the ALFA Selector PE is given. For all substrate proteins, elution of the peptide from the ALFA Selector ST was inefficient even after extended incubation. In the absence of the ALFA ST peptide, the target protein with the ALFA ST tag remained tightly bound to both resins. Figure 10 A repeat Figure 8 Data shown in B are repeated here to allow direct comparison. Left panel: Schematic diagram showing the experimental setup. The ALFA Selector resin can be ALFA Selector ST or ALFA Selector PE ; Middle panel: Experiment carried out with ALFA Selector ST ; Right panel: Experiment carried out with ALFA Selector PE .

[0053] Figure 11 : X-ray structure of NbALFA ST bound to the ALFA ST peptide

[0054] A - C; View of the NbALFA ST -ALFA ST peptide structure. A, View of the N-terminus of the ALFA ST peptide; B, Side view of the ALFA ST peptide; C, View of the C-terminus of the ALFA ST peptide. NbALFA ST is shown in light gray and the side chains are shown as lines. Residues contacting the ALFA ST peptide are shown as sticks. The ALFA ST peptide is depicted in dark gray and the side chains are shown as sticks. The ALFA STPeptide (SEQ ID NO: 179).

[0055] D;NbALFA ST The sequence of (SEQ ID NO: 133). Figure 3 B, boxes represent CDRs 1-3 (SEQ ID NOs: 115-117). Direct contact with ALFA ST The residues of the peptide are boxed. The residues in the filled boxes were mutated to reduce the affinity of ALFA ST Peptide affinity.

[0056] Figure 12 : Isolation of naive lymphocytes using ALFA-tagged nanobody recognizing CD62L.

[0057] Total human PBMCs were either untreated (before sorting) or sorted using ALFASelector loaded with ALFA-tagged anti-human CD62L nanobody. PE Resin separation (after sorting). A schematic diagram of the affinity purification strategy is shown in (a). Cells were stained with anti-CD62L antibodies and analyzed by flow cytometry (b). The same cells as in (b) were stained with antibodies for CD3, CD19 and CD62L and analyzed by flow cytometry (c). In all analyses, a forward scatter / side scatter gate was set on lymphocytes. DETAILED DESCRIPTION

[0058] In order to overcome some shortcomings in the prior art, the inventors of the present application have created small epitope tags that are recognized by high-affinity nanobodies. Such a system can allow the function of proteins to be analyzed in many aspects, including but not limited to analyzing their localization, analyzing their interaction partners by purification from lysates, or in vivo operations, including protein mislocalization or depletion induced using a minimal set of recombinant constructs and cell lines, without inadvertently interfering with the physiological function of the target protein.

[0059] Given the limitations of current epitope tags, the inventors of the present application decided to address this problem and identify the characteristics that an ideal epitope tag system should possess. A truly universal tag should be small to minimize potential side effects (Kocaoglu, O. and Carlson, E. E. Progress and Prospects for small-molecule probes of bacterial imaging. Nat Chem Biol 12, 472-478 (2016)). It should preferably be monomeric to minimize artificial oligomerization of the tagged protein. It should also be electrically neutral to avoid adding a net charge to the tagged protein; and it should be soluble (Esposito, D. and Chatterjee, D. K. Enhancement of soluble protein expression through the use of fusion tags. Curr. Opin. Biotechnol. 17, 353-358 (2006)). An ideal tag should not affect the native structure, topology, or localization of the protein to which it is attached (Stadler, C. et al. Immunofluorescence and fluorescent-protein tagging show high correlation for protein localization in mammalian cells. Nat Methods 10, 315-323 (2013); Hoffmann, C. et al. A FlAsH-based FRET approach to determine G protein-coupled receptor activation in living cells. Nat Methods 2, 171-176 (2005)). In addition, the tag should be well-expressed in both eukaryotic and prokaryotic hosts and should be resistant to proteolytic degradation. Ideally, it should be fixation-resistant, and its sequence should not be present in common model organisms to avoid unintended detection of endogenous host proteins.

[0060] Similar to an ideal epitope tag, its corresponding conjugate should also possess several properties to enable perfect tag detection. For example, the conjugate should be small to easily access crowded regions and provide optimal binding affinity for different applications. Current sophisticated applications (such as in vivo imaging) require specific and genetically accessible probes with high affinity for the tag, which should be able to fold autonomously in various host organisms. However, for biochemical applications, the preferred conjugate should preferably have intermediate affinity to allow competitive elution of immunoprecipitated materials under native conditions. When evaluating the commonly used epitope tags available to date, a person skilled in the art applying the prior art will ultimately need to sacrifice at least one of the mentioned features (see Table 1 below). To prepare an epitope tag with ultimate versatility that meets all the stated boundary conditions, the inventors of the present application have recognized that the most straightforward approach is to redesign it.

[0061] Table 1: Properties of Universal Epitope Tag Systems

[0062]

[0063]

[0064] n.a.: Not applicable

[0065] n.d.: No data available

[0066] mAb: Monoclonal antibody

[0067] sdAb: Single domain antibody

[0068] 1For optimal performance, multiple tags are often used in tandem (Hernan, R., Heuermann, K. and Brizzard, B. Multiple epitope tagging of expressed proteins for enhanced detection. BioTechniques 28, 789–793 (2000); Ross-Macdonald, P., Sheehan, A., Roeder, G. S. and Snyder, M. A multipurpose transposon system for analyzing protein production, localization, and function in Saccharomyces cerevisiae. Proc Natl Acad Sci USA 94, 190 - 195 (1997); Sharrock, R. A. and Clack, T. Heterodimerization of type II phytochromes in Arabidopsis. Proc Natl Acad Sci USA 101, 11500–11505 (2004); Graumann, J. et al. Applicability of tandem affinity purification MudPIT to pathway proteomics in yeast. Mol. Cell Proteomics 3, 226–237 (2004))

[0069] 2 The conjugate also recognizes biotinylated proteins

[0070] 3 The conjugate recognizes endogenous proteins with multiple accessible histidines.

[0071] 4 Depending on the chelator and polyhistidine tag used

[0072] 5 The conjugate also recognizes endogenous β-catenin

[0073] 6The conjugate needs to dimerize to be suitable for high-definition imaging applications (Virant, D. et al. A peptide tag-specific nanobody enables high-quality labeling for dSTORM imaging. Nat Commun 1–14 (2018). doi:10.1038 / s41467-018-03191-2)

[0074] 7 Fixed with amine-reactive fixatives and crosslinkers; deduced from the sequence

[0075] 8 Schmidt, T.G.M. et al. Development of the and its application for purification of recombinant proteins from cell culture supernatants. Protein Expr. Purif. 92, 54–61 (2013)

[0076] 9 Porath, J., Carlsson, J., Olsson, I. and Belfrage, G. Metal chelate affinity chromatography, a new approach to protein fractionation. Nature 258, 598–599 (1975); Hochuli, E., H. and Schacher, A. New metal chelate adsorbent selective for proteins and peptides containing neighbouring histidine residues. J. Chromatogr. 411, 177–184 (1987)

[0077] 10 Hopp, T.P. et al. A Short Polypeptide Marker Sequence Useful for Recombinant Protein Identification and Purification. Nat Biotechnol 6, 1204–1210 (1988)

[0078] 11 Wilson, I.A. et al. The structure of an antigenic determinant in a protein. Cell 37, 767 - 778 (1984)

[0079] 12 Evan, G.I., Lewis, G.K., Ramsay, G. and Bishop, J.M. Isolation of monoclonal antibodies specific for human c-myc proto-oncogene product. Mol Cell Biol 5, 3610–3616 (1985)

[0080] 13 Virant, D. et al. A peptide tag-specific nanobody enables high-quality labeling for dSTORM imaging. Nat Commun 1–14 (2018). doi:10.1038 / s41467-018-03191-2; Braun, M.B. et al. Peptides in headlock-a novel high-affinity and versatile peptide-binding nanobody for proteomics and microscopy. Sci Rep 6, 19211 (2016)

[0081] 14 Petukhov, M. et al. Design of stable alpha-helices using global sequence optimization. J.Pept.Sci. 15, 359–365 (2009)

[0082] 15 Hunter, M.R., Grimsey, N.L. and Glass, M. Sulfation of the FLAG epitope is affected by co-expression of G protein-coupled receptors in a mammalian cell model. Sci Rep 6, 27316 (2016)

[0083] 16 Schembri, L. et al. The HA tag is cleaved and loses immunoreactivity during apoptosis. Nat Methods 4, 107–108 (2007)

[0084] 17 https: / / www.iba - lifesciences.com / tl_files / ProteinProductionAssays / 5 - Immobilization / DynamicBiosensors - Application - Note - StrepTactinXT - switchSENSE.pdf

[0085] 18 N: N-terminus; M: between two folded domains; C: C-terminus.

[0086] With such a clear goal, the inventors of the present application designed the epitope tags described herein. The epitope tags of the present invention preferably consist of about 8-25 amino acids and are collectively referred to as the ALFA tag.

[0087] The inventors of the present application decided to adopt a completely new method to generate new epitope tags. The epitope tags according to the present invention are small monomeric epitope tags, preferably having a minimum size of ≤15 aa. This sequence is preferably uncharged and hydrophilic at physiological pH, and most preferably, it has no residues that are easily modified by amine-reactive fixatives and crosslinking agents. This size is in contrast to larger tags (such as the FLAG tag trimer, which is usually used in a 3x tandem form to increase affinity) or even larger fluorescent proteins. In addition, the epitope tags of the present invention have no counterparts in eukaryotic or prokaryotic sequence databases, which minimizes the risk of cross-binding with native structures. This feature is in contrast to, for example, those described in WO 2017 / 085086 A1 Form a contrast.

[0088] Another advantage of the epitope tags of the present invention is that, unlike, for example, the Myc tag, it is compatible with common amine-reactive fixatives (paraformaldehyde (PFA), glutaraldehyde (GA)). It is also compatible with methanol fixation. Another advantage of the epitope tags of the present invention is that it is not restricted in its location (N, C, or between proteins), which is different from the EPEA tag described in WO 2011 / 147890 A1.

[0089] Without wishing to be bound by theory, it is believed that the epitope tags according to the present invention form stable α-helical structures. The formation of a stable helix is considered to be superior to differently folded, unfolded or unstably folded structures because certain antibodies, such as single domain antibodies (sdAb) or nanobodies, are thought to prefer defined three-dimensional surfaces for binding. In the past, it has been difficult to generate single domain antibodies that bind to a native unfolded peptide, which has led to sbAbs having only weak to moderate binding affinities. Accordingly, the inventors of the present application have sought to provide epitope tags that are believed to form small α-helices that fold stably in solution, as such structures are the smallest entities that form stable secondary structures as monomers. Without wishing to be bound by theory, it is believed that the α-helical structure refolds efficiently and spontaneously even after exposure to harsh chemical treatments. In addition, it is believed that due to its helical structure, the tag is smaller than most unstructured linear epitope tags. Furthermore, the epitope tags of the present invention can be placed at the N- or C-terminus of a target protein, or even between two folded protein domains, without compromising the correct targeting and folding of the target protein.

[0090] However, finding the ideal conjugate has proven challenging. While conventional antibodies do meet most requirements, their large size makes them suboptimal for current super-resolution microscopy techniques (Fornasiero, E.F. and Opazo, F. Super-resolution imaging for cell biologists: Concepts, applications, current challenges and developments. Bioessays 37, 436–451 (2015); Mikhaylova, M. et al. Resolving bundled microtubules using anti-tubulin nanobodies. Nat Commun 6, 7933 (2015)), and they cannot be genetically encoded to target intracellular targets in living cells. Therefore, the inventors of the present application chose to develop camelid single-domain antibodies (sdAbs, also known as nanobodies) (Muyldermans, S. Nanobodies: Natural Single-Domain Antibodies. Annu Rev Biochem (2013). doi:10.1146 / annurev-biochem-063011-092449) that meet the set criteria. To this end, a new in-house selection method called "Celline" enabled the inventors to generate saAbs from alpacas with extremely high affinity in a very time-efficient manner. The antibodies of the present invention in combination with the epitope tags of the present invention have proven to be very suitable for imaging and intracellular detection of target proteins bearing the ALFA ST tag and allow for very efficient and clean immunoprecipitation.

[0091] The present invention also provides high-affinity antibodies to the epitope tags of the present invention. Some of the antibodies are monovalent sdAb-based conjugates. Monovalent binding is in contrast to the epitope tags bound by conventional antibodies. While bound by the sdAb, to increase the affinity, the binding sdAb is used as a dimer in some applications. The use of monovalent antibodies has the advantage of preventing cluster formation. It is also believed that due to the α-helical structure of the epitope tag, the inventors of the present invention were able to generate single-domain antibodies that bind to the epitope tags of the present invention in the range of approximately 10 pM, an affinity that is approximately ~1000 times higher than that of comparable epitope tag / sdAb systems (such as those described in WO 2017 / 085086 A1 or WO 2011 / 147890 A1). d ​

[0092] For some of the epitope tags of the present invention, such as SEQ ID NO: 05 - 07, it is virtually impossible to effectively separate an antibody containing the SEQ ID NO: 133 sequence from the tag under natural conditions. In some cases, this may limit the application of SEQ ID NO: 05 - 07 to the purification of native proteins and their interaction partners. Resolving the crystal structure of the high - affinity complex of the antibody against SEQ ID NO: 133 and the peptide of SEQ ID NO: 179 allowed the inventors to map the interaction determinants in detail and design novel single - domain antibodies that allow for competitive elution of the target protein and interaction partners bearing the ALFA ST tag under natural conditions. Exemplary antibodies for this purpose contain the amino acid sequence of SEQ ID NO: 134.

[0093] The epitope tags and antibody systems provided herein are applicable to a very wide range of applications from biotechnology to cell biology. Thus, a single tag can simultaneously replace many traditional epitope tags.

[0094] Accordingly, the present invention relates to a fusion protein comprising (a) a peptide comprising the sequence of X1 - X2 - X3 - X4 - X5 - X6 - X7 - X8 - X9 - R - L - X12 - X13 (SEQ ID NO: 01), wherein X1 is G or S or T or P, X2 is R or G or A or E or P, X3 is L or V, X4 is E or Q, X5 is E or Q, X6 is E or Q, X7 is L or I or V, X8 is R or A or Q or E, X9 is R or A or Q or E, X12 is S or T or D or E or P or A or no amino acid, and wherein X13 is E or K or P or S or A or D or no amino acid; and (b) a polypeptide. In the fusion protein of the present invention, the peptide can be used as an epitope tag.

[0095] As used herein, the term "peptide" refers to a series of linearly linked amino acids, preferably linked to each other by peptide bonds between the α - amino and carboxyl groups of adjacent residues. As used herein, the term "amino acid" refers to natural and / or non - natural or synthetic amino acids, including glycine and D or L optical isomers, as well as amino acid analogs and peptidomimetics, preferably proteinogenic amino acids. A "proteinogenic amino acid" is an amino acid that can be biosynthetically incorporated into a protein during translation. Currently, there are 22 known genetically encoded (proteinogenic) amino acids, 20 in the standard genetic code and 2 additional ones that can be incorporated via special translation mechanisms. The "peptide" used herein preferably contains no more than about 50 amino acids.

[0096] As used herein, the term "polypeptide" generally refers to a peptide having at least about 30, at least about 40, or at least about 50 amino acids. The term "protein" as used herein encompasses one or more polypeptides.

[0097] As used herein, the term "fusion protein" refers to a polypeptide or protein comprising two or more subunits. At least one of said subunits is preferably a protein or polypeptide, and at least one of said subunits is preferably a peptide. In said fusion protein, these subunits may be linked by covalent or non-covalent bonds. Preferably, said fusion protein is a translational fusion between two or more subunits. Said translational fusion can be generated by genetically engineering the coding nucleotide sequence of one subunit in-frame with the coding nucleotide sequence of another subunit. The subunits may be flanked by linkers.

[0098] If one or more of said subunits is part of a protein (complex) consisting of more than one polypeptide chain, the term "fusion protein" may also refer to a protein comprising the fusion sequence of said protein (complex) and all other polypeptide chains.

[0099] As used herein, an "epitope tag" refers to a stretch of amino acids of a proteinaceous molecule that is capable of generating a specific antibody or has antibody-like function. Such an epitope tag can allow for the specific identification and / or tracking of a tagged polypeptide or protein that may be present in a living organism or cultured cell. Detection of the tagged molecule can be achieved using a variety of different techniques. Examples of these techniques include: immunohistochemistry, immunoprecipitation, flow cytometry, immunofluorescence microscopy, electron microscopy, ELISA, Western blot, and affinity chromatography. The epitope tag adds a known epitope (antibody binding site) to the test polypeptide for binding by a known and generally high-affinity antibody. Epitope tags can also be used to isolate and / or purify the tagged molecule, for example, by pull-down applications.

[0100] In the fusion proteins of the present invention, the peptide, i.e., the epitope tag, can be located at any position of the fusion protein. The peptide can be fused to the N-terminus or C-terminus of the polypeptide. Alternatively, the peptide can be fused to the polypeptide at a position between the N-terminus and C-terminus of the polypeptide. As an illustrative example, the peptide can be fused between two domains of the polypeptide.

[0101] The polypeptide contained in the fusion protein can have a stable fold independent of the presence or absence of the peptide. This means that the peptide preferably does not alter or interfere with the native structure of the polypeptide.

[0102] The peptide itself preferably has an α - helical structure. The crystal structure of an antibody with the SEQ ID NO: 133 sequence complexed with the ALFA peptide containing the acetyl - PSRLEEELRRRLTEP - amide sequence (SEQ ID NO: 179) has been resolved, showing that the epitope tag binds to the nanobody as a stably folded α - helix. It is believed that this structure explains the unusually tight binding. As used herein, "α - helical structure" refers to a secondary structure in the form of an α - helix. The α - helical secondary structure of the peptide is preferably independent of the fusion partner. This means that if the peptide is in the form of an isolated peptide and if the peptide is part of a fusion protein, the peptide preferably can form an α - helical secondary structure in a physiological buffer. ST The crystal structure of an antibody with the SEQ ID NO: 133 sequence complexed with the ALFA peptide containing the acetyl - PSRLEEELRRRLTEP - amide sequence (SEQ ID NO: 179) has been resolved, showing that the epitope tag binds to the nanobody as a stably folded α - helix. It is believed that this structure explains the unusually tight binding. As used herein, "α - helical structure" refers to a secondary structure in the form of an α - helix. The α - helical secondary structure of the peptide is preferably independent of the fusion partner. This means that if the peptide is in the form of an isolated peptide and if the peptide is part of a fusion protein, the peptide preferably can form an α - helical secondary structure in a physiological buffer.

[0103] The peptide can be specifically recognized by a camelid VHH domain containing the CDR sequences GVTISALNAMAMG (SEQ ID NO: 115), AVSERGNAM (SEQ ID NO: 116), and LEDRVDSFHDY (SEQ ID NO: 117). The peptide can be further specifically recognized by other antibodies described herein.

[0104] In the fusion protein of the present invention, the peptide can be fused to the polypeptide either directly or via a linker. As used herein, "linker" joins two or more subunits of the fusion protein described herein. The linkage can be covalent. A preferred covalent linkage is via a peptide bond, such as a peptide bond between amino acids. A preferred linker is a peptide linker. The linker preferably contains one or more amino acids, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more amino acids. Preferred peptide linkers include glycine - serine (GS) linkers, glycosylated GS linkers, and proline - alanine - serine polymers (PAS) linkers. The GS linker can be the (G4S)3 linker as described in SEQ ID NO: 159.

[0105] The polypeptide contained in the fusion protein of the present invention may comprise at least one protein domain. As used herein, "protein domain" refers to a part of a given protein sequence and (tertiary) structure that can function and / or exist independently of the rest of the protein chain. The protein domain preferably forms a stable three-dimensional structure and can generally be stably folded independently. A protein domain can further form a functional unit. The polypeptide contained in the fusion protein may comprise more than one protein domain, such as 2, 3, 4 or even more protein domains. The preferred position of the peptide can be outside the protein domain. This can be the N-terminus or C-terminus of at least one protein domain of the polypeptide, or between two protein domains of the polypeptide. The polypeptide contained in the fusion protein of the present invention can be a globular protein, a membrane protein, a fibrous protein or a naturally unfolded protein, or a subunit or domain of the globular protein, membrane protein, fibrous protein or naturally unfolded protein.

[0106] The peptide contained in the fusion protein of the present invention can have a length of about 8 to about 25 amino acids, preferably about 10 to about 18 amino acids, preferably about 12 to about 17 amino acids, preferably 12 to 15 amino acids. The polypeptide contained in the fusion protein of the present invention can have a length of at least 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 40, 50, 60, 70, 80, 90 or 100 amino acids. The polypeptide can be a polypeptide or protein that naturally exists in the cells expressing the fusion protein of the present invention.

[0107] The fusion protein of the present invention may comprise a peptide containing the sequence (SEQ ID NO: 02) of X1-X2-L-E-X5-E-X7-R-R-R-L-X12-X13, where X1 is G or S or P or T, X2 is R or G or P, X5 is E or Q, X7 is L or I, X12 is S or T or P or A or D or E, and where X13 is P or A or S or A or D or E or no amino acid. This sequence defines the core structure of the peptide and may also contain up to two additional amino acids at the N-terminus and up to two additional amino acids at the C-terminus. Such additional amino acids at the ends of the peptide core structure generally do not necessarily affect the secondary structure of the peptide or the specific binding of the peptide to a peptide-specific antibody, but can be used as a linker structure in the fusion protein. Therefore, the type and number of the additional amino acids can depend on the position of the peptide in the fusion protein and can vary depending on whether the peptide is located at the N-terminus or C-terminus of the polypeptide, or at a position between the N-terminus and C-terminus of the polypeptide. The dissociation T of the peptide containing the SEQ ID NO: 02 sequence from the single-domain antibody containing the SEQ ID NO: 133 sequence 1 / 2, for example, when measured using the assay substantially described in Example 1, it can be at least about 2 minutes. The binding of the peptide to the single-domain antibody comprising the sequence of SEQ ID NO: 133 can have a K of about 30 nM or less d .

[0108] The peptide contained in the fusion protein of the present invention can comprise the N-terminal amino acids Xa-Xb of X1, where Xa is D or S or G or M or P or no amino acid, and Xb is S or D or P or M or R or G or no amino acid.

[0109] The peptide contained in the fusion protein of the present invention can comprise the C-terminal amino acids Xy-Xz of X13, where Xy is G or S or P or D or A or E or K or non-amino acid, and Xz is S or P or no amino acid.

[0110] The fusion protein of the present invention can comprise a peptide containing the sequence (SEQ ID NO: 03) of X1-X2-L-E-X5-E-L-R-R-R-L-X12-X13 as a core structure, where X1 is S or T, X2 is R or G, X5 is E or Q, X12 is T or D or E, and where X13 is A or D or E or no amino acid. The T at which such a peptide dissociates from the single-domain antibody comprising the sequence of SEQ ID NO: 133 1 / 2 , for example, when measured using the assay substantially described in Example 1, it can be at least about 100 minutes. The binding of the peptide to the single-domain antibody comprising the sequence of SEQ ID NO: 133 can have a K of about 1 nM or less d . Such a peptide can comprise the sequence (SEQ ID NO: 04) of S-R-L-E-E-E-L-R-R-R-L-T-E or a variant thereof, where the variant has 1 to 5 mutations compared to (SEQ ID NO: 04), and the mutations are selected from: S1→T, R2→G, E5→Q, T12→D, and T12→E, E13→A, E13→D, and deletion of E13. The variant can have the following mutations compared to SEQ ID NO: 04: (a) S1→T and E13→A; (b) R2→G; (c) R2→G and E5→Q; (d) R2→G, E5→Q and E13→A; (e) R2→G, E5→Q and T12→D, and E13→A; (f) R2→G, E5→Q and T12→E, and E13→A; (g) T12→D and E13→A; (h) T12→E and E13→A; (i) and E13→A; (j) and E13→D; or (k) deletion of E13.

[0111] A peptide containing the sequence SEQ ID NO: 03 as the core structure may contain the N-terminal amino acids Xa-Xb of X1, where Xa is S or G or M or P or no amino acid, and Xb is R or G or S or P or M or no amino acid. Xa-Xb may be selected from P, M-P, G-R, P-G, P-S, S-P, G-P, S-P, M, and M-S, preferably P or M-P. Such a peptide may also contain the C-terminal amino acids Xy-Xz of X13, where Xy is P or D or A or no amino acid, and Xz is P or S or no amino acid. Xy-Xz may be selected from no amino acid, P, D-P, A, and A-S, preferably no amino acid or P. The peptide may contain a combination of Xa-Xb and Xy-Xz, and the combination is selected from: (a) M-P and P; (b) P and P; and (c) P and no amino acid.

[0112] The peptide contained in the fusion protein described in the present invention may have the core structure of SEQ ID NO: 03 and may contain a sequence selected from the following:

[0113] (a) MPSRLEEELRRRLTEP (SEQ ID NO: 05);

[0114] (b) PSRLEEELRRRLTEP (SEQ ID NO: 06);

[0115] (c) PSRLEEELRRRLTE (SEQ ID NO: 07);

[0116] (d) GRSRLEEELRRRLTA (SEQ ID NO: 08);

[0117] (e) PGSRLEEELRRRLTAP (SEQ ID NO: 09);

[0118] (f) PSTRLEEELRRRLTAP (SEQ ID NO: 10);

[0119] (g) SPSRLEEELRRRLTAP (SEQ ID NO: 11);

[0120] (h) SPSRLEEELRRRLDAP (SEQ ID NO: 12);

[0121] (i) SPSRLEEELRRRLEAP (SEQ ID NO: 13);

[0122] (j) SPSRLEEELRRRLTDP (SEQ ID NO: 14);

[0123] (k) SPSRLEEELRRRLTEP (SEQ ID NO: 15);

[0124] (l) SPSRLEEELRRRLTADP (SEQ ID NO: 16);

[0125] (m) SPSGLEEELRRRLTEP (SEQ ID NO: 17);

[0126] (n) GPSRLEEELRRRLT (SEQ ID NO: 18);

[0127] (o) GPSRLEEELRRRLTA (SEQ ID NO: 19);

[0128] (p) GPSRLEEELRRRLTAA (SEQ ID NO: 20);

[0129] (q) GPSRLEEELRRRLTAAS (SEQ ID NO: 21);

[0130] (r) SPSGLEQELRRRLTAP (SEQ ID NO: 22);

[0131] (s) SPSGLEQELRRRLDAP (SEQ ID NO: 23);

[0132] (t) SPSGLEQELRRRLEAP (SEQ ID NO: 24);

[0133] (u) SPSGLEQELRRRLTEP (SEQ ID NO: 25);

[0134] (v) GPSRLEEELRRRLTAP (SEQ ID NO: 26);

[0135] (w) GPSRLEEELRRRLTEP (SEQ ID NO: 27);

[0136] (x) GPSRLEEELRRRLTE (SEQ ID NO: 28);

[0137] (y) MSRLEEELRRRLTEP (SEQ ID NO: 29); and

[0138] (z) MSSRLEEELRRRLTEP (SEQ ID NO: 30).

[0139] The fusion protein described in the present invention may comprise a peptide containing the sequence (SEQ ID NO: 31) of X1-X2-L-E-X5-E-X7-R-R-R-L-X12-X13 as the core structure, where X1 is G or S or P, X2 is R or G, X5 is E or Q, X7 is L or I, X12 is S or T or P or A, and X13 is P or A or S or no amino acid. Such a peptide dissociates from the single-domain antibody comprising the SEQ ID NO: 133 sequence at a T 1 / 2 , for example, when measured using the assay substantially described in Example 1, can be from about 2.5 minutes to about 30 minutes. The binding of the peptide to the single-domain antibody comprising the SEQ ID NO: 133 sequence can have a K of about 3 - 40 nM or less d .

[0140] Such a peptide may comprise the sequence (SEQ ID NO: 32) of G-R-L-E-E-E-L-R-R-R-L-S or a variant thereof, where the variant has 1 to 6 mutations compared to (SEQ ID NO: 32), and the mutations are selected from: G1→S, G1→P, R2→G, E5→Q, L7→I, S12→T, S12→P, and S12→A, addition of P13, addition of A13, and addition of S13. The variant may have the following mutations compared to SEQ ID NO: 32: (a) G1→S, R2→G, E5→Q, and addition of P13; (b) R2→G, E5→Q, S12→T, and addition of A13; (c) G1→P, R2→G, E5→Q, S12→T, and addition of A13; (d) G1→S, R2→G, E5→Q, S12→T, and addition of P13; (e) G1→S, R2→G, S12→T, and addition of A13; (f) G1→S, R2→G, E5→Q, and S12→T; (g) G1→S, R2→G, E5→Q, S12→T, and addition of A13; (h) G1→S and S12→P, and addition of P13; (i) G1→S, R2→G, E5→Q, S12→T, and addition of P13; (J) E5→Q, L7→I, and addition of P13; (k) addition of P13; or (I) S12→A.

[0141] A peptide containing the sequence SEQ ID NO: 31 as the core structure may contain the N-terminal amino acids Xa-Xb of X1, where Xa is M or S or P or D or G or no amino acid, and Xb is S or D or P or no amino acid. Xa-Xb may be selected from M-S, S-D, P-D, P-S, D-S, S-P, and G-P, preferably M-S. Such a peptide may also contain the C-terminal amino acids Xy-Xz of X13, where Xy is G or P or A or E or K or S or no amino acid, and Xz is P or S or no amino acid. Xy-Xz may be selected from no amino acid, G, P, A, E-P, A-S, K, and S, preferably no amino acid. The peptide may contain a combination of Xa-Vb and Xy-Xz, and the combination is M-S and no amino acid.

[0142] The peptide contained in the fusion protein of the present invention may have the core structure of SEQ ID NO: 31 and may contain a sequence selected from the following:

[0143] (a) GRLEEELRRRLS (SEQ ID NO: 32);

[0144] (b) MSGRLEEELRRRLSP (SEQ ID NO: 33);

[0145] (c) SDSGLEQELRRRLSPG (SEQ ID NO: 34);

[0146] (d) PDGGLEQELRRRLTAP (SEQ ID NO: 35);

[0147] (e) PSGGLEQELRRRLTAP (SEQ ID NO: 36);

[0148] (f) DSPGLEQELRRRLTAP (SEQ ID NO: 37);

[0149] (g) PDSGLEQELRRRLTPA (SEQ ID NO: 38);

[0150] (h) SPSGLEEELRRRLTAEP (SEQ ID NO: 39);

[0151] (i) GPSGLEQELRRRLT (SEQ ID NO: 40);

[0152] (j) GPSGLEQELRRRLTAAS (SEQ ID NO: 41);

[0153] (k) SPSRLEEELRRRLPSK (SEQ ID NO: 42);

[0154] (l) SPSGLEQELRRRLTPS (SEQ ID NO: 43);

[0155] (m) SPGRLEQEIRRRLSPS (SEQ ID NO: 44);

[0156] (n) PSGRLEEELRRRLSPS (SEQ ID NO: 45);

[0157] (o) PSGRLEEELRRRLS (SEQ ID NO: 46);

[0158] (p) PSGRLEEELRRRLA (SEQ ID NO: 47); and

[0159] (q) PSGRLEEELRRRLSP (SEQ ID NO: 48).

[0160] The fusion protein according to the present invention may comprise a peptide containing the sequence of X1-X2-L-E-X5-E-L-R-R-R-L-X12-X13 (SEQ ID NO: 49) as a core structure, wherein X1 is S or G or P, X2 is R or G or P, X5 is E or Q, X12 is S or T or D or E, and X13 is P or A or D or no amino acid. The T1 / 2 of such a peptide dissociated from a single domain antibody comprising the SEQ ID NO: 133 sequence, for example when measured using an assay substantially as described in Example 1, may be about 20 minutes to about 100 minutes. The K of the peptide binding to a single domain antibody comprising the SEQ ID NO: 133 sequence d may be about 1-5 nM or less.

[0161] A peptide comprising the sequence SEQ ID NO: 49 as a core structure may comprise N-terminal amino acids Xa-Xb of X1, wherein Xa is P or D or S or G or no amino acid, and Xb is D or S or P or no amino acid. Such a peptide may also comprise C-terminal amino acids Xy-Xz of X13, wherein Xy is G or P or E or D or S or no amino acid, and Xz is P or no amino acid.

[0162] The peptide contained in the fusion protein according to the present invention may have the core structure of SEQ ID NO: 49 and may comprise a sequence selected from the following:

[0163] (a) PDSGLEQELRRRLSPG (SEQ ID NO: 50);

[0164] (b) PDSGLEQELRRRLTAP (SEQ ID NO: 51);

[0165] (c) PSSGLEQELRRRLTAP (SEQ ID NO: 52);

[0166] (d) DPSGLEQELRRRLTAP (SEQ ID NO: 53);

[0167] (e) DSGPLEQELRRRLTAP (SEQ ID NO: 54);

[0168] (f) SPSRLEEELRRRLTAEP (SEQ ID NO: 55);

[0169] (g) SPSGLEEELRRRLTAP (SEQ ID NO: 56);

[0170] (h) SPSGLEEELRRRLDAP (SEQ ID NO: 57);

[0171] (i) SPSGLEEELRRRLEAP (SEQ ID NO: 58);

[0172] (j) SPSGLEEELRRRLTDP (SEQ ID NO: 59);

[0173] (k) SPSGLEEELRRRLTADP (SEQ ID NO: 60);

[0174] (l) GPSGLEQELRRRLTA (SEQ ID NO: 169);

[0175] (m) SPSGLEQELRRRLTDP (SEQ ID NO: 170);

[0176] (n) SPSGLEQELRRRLTADP (SEQ ID NO: 171);

[0177] (o) SPSGLEQELRRRLTAEP (SEQ ID NO: 172);

[0178] (p) DSPGLEQELRRRLTAP (SEQ ID NO: 173);; and

[0179] (q) SPSGLEQELRRRLSPS (SEQ ID NO: 174).

[0180] The fusion protein of the present invention may comprise a peptide containing a sequence (SEQ ID NO: 61) of X1-X2-X3-X4-X5-X6-X7-X8-X9-R-L-X12-X13 as a core structure, where X1 is G or S, X2 is R or G or A or E, X3 is L or V, X4 is E or Q, X5 is E or Q, X6 is E or Q, X7 is L or I or V, X8 is R or A or Q or E, X9 is R or A or Q or E, X12 is S or T or L or no amino acid, and X13 is K or P or S or no amino acid. Such a peptide dissociates from a single-domain antibody comprising the SEQ ID NO: 133 sequence at T 1 / 2 , for example, when measured using an assay substantially as described in Example 1, can be from about 2 minutes to about 10 minutes. The K of the peptide binding to a single-domain antibody comprising the SEQ ID NO: 133 sequence d can be about 10 - 50 nM or less.

[0181] A peptide comprising a sequence SEQ ID NO: 61 as a core structure may comprise N-terminal amino acids Xa-Xb of X1, where Xa is D or S or G or M or no amino acid, and Xb is S or D or P or M or no amino acid. Such a peptide may also comprise C-terminal amino acids Xy-Xz of X13, where Xy is G or S or P or no amino acid, and Xz is S or no amino acid.

[0182] The peptide contained in the fusion protein of the present invention may have a core structure of SEQ ID NO: 61 and may comprise a sequence selected from the following:

[0183] (a) GRLEEELRRRLS (SEQ ID NO: 32);

[0184] (b) MSGRLEEELRRRLSP (SEQ ID NO: 33);

[0185] (c) DSGRLEEELRRRLSKG (SEQ ID NO: 62);

[0186] (d) DSGRLEEELRRRLSPG (SEQ ID NO: 63);

[0187] (e) SDSGLEEELRRRLSPG (SEQ ID NO: 64);

[0188] (f) SDSGVEEELRRRLSPG (SEQ ID NO: 65);

[0189] (g) SDSAVEEELRRRLSPG (SEQ ID NO: 66);

[0190] (h) SDSGLQEELRRRLSPG (SEQ ID NO: 67);

[0191] (i) SDSGLEEQLRRRLSPG (SEQ ID NO: 68);

[0192] (j) SDSGLEEEIRRRLSPG (SEQ ID NO: 69);

[0193] (k) SDSGLEEEVRRRLSPG (SEQ ID NO: 70);

[0194] (l) DSGELEEELRRRLSPG (SEQ ID NO: 71);

[0195] (m) DSGRLEQELRRRLSPG (SEQ ID NO: 72);

[0196] (n) DSGRLEEEIRRRLSPG (SEQ ID NO: 73);

[0197] (o) DSGRLEQEIRRRLSPG (SEQ ID NO: 74);

[0198] (p) DSGRLEQEIARRLSPG (SEQ ID NO: 75);

[0199] (q) DSGRLEQEIQRRLSPG (SEQ ID NO: 76);

[0200] (r) DSGRLEQEIERRLSPG (SEQ ID NO: 77);

[0201] (w) DSGRLEQEIRARLSPG (SEQ ID NO: 78);

[0202] (t) DSGRLEQEIRQRLSPG (SEQ ID NO: 79);

[0203] (u) DSGRLEQEIRERLSPG (SEQ ID NO: 80);

[0204] (v) GPSRLEEELRRRL (SEQ ID NO: 81);

[0205] (w) MSGLEQELRRRLTPS (SEQ ID NO: 82);

[0206] (x) MSGRLEEELRRRLSPS (SEQ ID NO: 83);

[0207] (y)SPSAVEEELRRRLSPS (SEQ ID NO: 84);

[0208] (z)GPSAVEEELRRRLS (SEQ ID NO: 85);

[0209] (aa)MPSGLEQELRRRLTPS (SEQ ID NO: 86);

[0210] (bb)MSSGLEQELRRRLTPS (SEQ ID NO: 87);

[0211] (cc)MPSGRLEEELRRRLSPS (SEQ ID NO: 88);

[0212] (dd)MSGRLEEELRRRLSP (SEQ ID NO: 89).

[0213] The fusion protein of the present invention can be complexed with a binding partner that specifically binds to the peptide contained in the fusion protein. Such a specific binding partner can be an antibody disclosed herein.

[0214] The fusion protein of the present invention can comprise an antibody portion. The antibody portion can be a single-domain antibody. Such an antibody portion can specifically bind to a target. Such a target can be a cell. For example, the specific target can comprise or be a structure on the cell surface, such as a cell surface receptor. A preferred target is CD62L.

[0215] The present invention further provides an antibody that specifically binds to the peptide contained in the fusion protein of the present invention. Such an antibody can be a monovalent antibody. In a preferred embodiment, the antibody of the present invention comprises or consists of a camelid VHH domain. In a preferred embodiment, the antibody of the present invention is a single-domain antibody, such as a camelid single-domain antibody.

[0216] The term "antibody" generally refers to a proteinaceous binding molecule having immunoglobulin-like function. Exemplary antibodies are, but are not limited to, immunoglobulins, as well as derivatives or functional fragments thereof that still retain binding specificity. Techniques for producing antibodies are well known in the art. The term "antibody" also includes immunoglobulins (Ig classes) of different classes (i.e., IgA, IgG, IgM, IgD, IgE, IgY, etc.) and subclasses (e.g., IgG1, IgG2, etc.), even if recombinantly produced in a foreign host using techniques known to those skilled in the art. Illustrative examples of antibodies are full-length immunoglobulins, F ab fragments, F(ab')2, F VFragment, single-chain F V fragment (scF V ), diabody or domain antibody (Holt LJ et al., Trends Biotechnol. 21(11), 2003, 484 - 490). A domain antibody can be a single domain antibody, a single variable domain antibody or an immunoglobulin single variable domain having only one variable domain (which can be VH or VL), which domain specifically binds to an antigen or epitope independently of other V regions or domains. A particularly preferred single domain antibody is the VHH domain of an antibody of only the heavy chain. Such an immunoglobulin single variable domain can not only comprise an isolated antibody single variable domain polypeptide, but also a larger polypeptide which comprises one or more monomers of the antibody single variable domain polypeptide sequence or is composed of said one or more monomers. It will be understood that a single domain antibody can comprise a VHH domain and a fusion partner, such as a protein or peptide tag. Accordingly, the definition of the term "antibody" also includes embodiments such as chimeric antibodies, single-chain antibodies and humanized antibodies. The term "antibody" can also include antibody fragments.

[0217] A single domain antibody is an antibody in which the complementarity determining regions are part of a single domain polypeptide. Examples include but are not limited to heavy chain antibodies, antibodies that are naturally devoid of light chains, single domain antibodies derived from conventional 4-chain antibodies, engineered antibodies and single domain scaffolds of those not derived from antibodies. A single domain antibody can be any single domain antibody existing in the art or any future single domain antibody. A single domain antibody can be derived from any species, including but not limited to mouse, human, camel, llama, goat, rabbit or bovine. According to the present invention, the single domain antibodies used herein are preferably derived from antibodies that are naturally occurring and are called heavy chain antibodies that are devoid of light chains. Such single domain antibodies are disclosed, for example, in WO 94 / 04678. For clarity, the variable domain derived from such a heavy chain antibody that is naturally devoid of light chains is referred to herein as VHH to distinguish it from the conventional VH of a four-chain immunoglobulin. Such VHH molecules can be derived from antibodies produced in Camelidae species, such as camel, dromedary, llama, alpaca, vicuña and guanaco. Other species outside of Camelidae can also produce heavy chain antibodies that are naturally devoid of light chains. As an illustrative example, it is known that sharks produce heavy chain antibodies that are naturally devoid of light chains (commonly named IgNAR), which also contain VHH domains. In addition, VHHs can be obtained from synthetic libraries. All such VHHs are within the scope of the present invention.

[0218] VHHs, according to the present invention, are preferably derived from the variable heavy domains of immunoglobulins that are naturally devoid of light chains, such as those derived from Camelidae as described in WO 94 / 04678 (hereinafter referred to as VHH domains or nanobodies). VHH molecules are approximately 10 times smaller than IgG molecules. They are single polypeptides and are very stable, resistant to extreme pH and temperature conditions. Moreover, they are highly resistant to the action of proteases, unlike conventional antibodies. In addition, the in vitro expression of VHHs or their expression in prokaryotic or eukaryotic organisms suitable for recombinant protein expression results in highly productive, correctly folded functional VHHs. It should be understood that the single-domain antibodies according to the present invention are preferably VHHs.

[0219] The antibodies according to the present invention may carry one or more domains having a sequence with at least about 60%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 92%, at least about 95%, at least about 96%, at least about 97%, at least about 98% or at least about 99% sequence identity to the corresponding naturally occurring domains of immunoglobulins that are naturally devoid of light chains. It should be noted in this regard that the term "about" as used herein refers to a deviation within 20% of a given value or range, for example within 10% or within 5% of the value.

[0220] The "percent sequence identity" with respect to amino acid sequences disclosed herein is defined as: the percentage of amino acid residues in a candidate sequence that are pairwise identical to the amino acid residues in a reference sequence (i.e., the antibody molecules disclosed herein) after aligning the sequences and introducing gaps (if necessary) to obtain the maximum percentage of sequence identity, and not considering any conservative substitutions as part of the sequence identity. The alignment for determining the percentage of amino acid sequence identity can be achieved in various ways within the skill in the art, for example, using publicly available computer software such as BLAST, ALIGN or Megalign (DNASTAR) software. Those skilled in the art can determine the appropriate parameters for measuring the alignment, including any algorithms required to achieve the maximum alignment over the full length of the sequences being compared. The same applies to the nucleotide sequences disclosed herein.

[0221] The term "variable" refers to the portions of the immunoglobulin domains that exhibit variability in their sequences and are involved in determining the specificity and binding affinity of a particular antibody (i.e., the "variable domains"). The variability is not evenly distributed throughout the variable domains of an antibody; it is concentrated in subdomains within each of the variable regions of the heavy and light chains. These subdomains are called "hypervariable regions", "HVRs", or "HVs", or "complementary determining regions" (CDRs). The more conserved (i.e., non-hypervariable) portions of the variable domains are called "framework" regions (FRs). The variable domains of the naturally occurring heavy and light chains each include four FR regions that predominantly adopt a β-sheet conformation and are connected by three hypervariable regions, thereby forming loops that connect the β-sheet structures and in some cases form part of the β-sheet structure. The hypervariable regions in each chain are tightly associated with one another by the FRs and together with the hypervariable regions from the other chain form the antigen-binding site (see Kabat et al., below). Typically, naturally occurring immunoglobulins include six CDRs (see below); three in VH (H1, H2, H3) and three in VL (L1, L2, L3). In naturally occurring immunoglobulins, H3 and L3 exhibit the greatest diversity among the six CDRs, and in particular H3 is thought to play a unique role in conferring good specificity to the immunoglobulin. However, immunoglobulins that are naturally light-chain-free include three CDRs in the VHH region. The constant domains do not directly participate in antigen binding but rather exhibit various effector functions such as antibody-dependent, cell-mediated cytotoxicity and complement activation.

[0222] Each VHH, VH, and VL has three CDRs and four FRs, which are arranged in the following order from the amino terminus (N-terminus) to the carboxyl terminus (C-terminus): FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of the heavy and / or light chains contain binding domains that interact with antigenic epitopes. The term "immunoglobulin" can refer to a protein of two heavy chains that may be light-chain-free, such as a light-chain-free antibody, or an antigen-binding portion thereof. An immunoglobulin can also include at least two heavy (H) chains and two light (L) chains linked by disulfide bonds, or an antigen-binding portion thereof.

[0223] When used herein, an immunoglobulin can be a dimeric glycosylated protein consisting of two heavy chains, such as IgG with only camel heavy chains (hcIgG) or shark IgNAR. An immunoglobulin as used herein can also be a tetrameric glycosylated protein consisting of two light chains (L) each of approximately 25 kDa and two heavy chains (H) each of approximately 50 kDa.

[0224] When used in reference to proteins or peptides, the term "amino acid" or "amino acid residue" generally refers to an α-amino carboxylic acid having a definition recognized in the art, such as an amino acid selected from L-alanine (Ala or A), L-arginine (Arg or R), L-asparagine (Asn or N), L-aspartic acid (Asp or D), L-cysteine (Cys or C), L-glutamine (Gln or Q), L-glutamic acid (Glu or E), glycine (Gly or G), L-histidine (His or H), L-isoleucine (Ile or I), L-leucine (Leu or L), L-lysine (Lys or K), L-methionine (Met or M), L-phenylalanine (Phe or F), L-proline (Pro or P), L-serine (Ser or S), L-threonine (Thr or T), L-tryptophan (Trp or W), L-tyrosine (Tyr or Y), and L-valine (Val or V), although modified, synthetic, or rare amino acids, such as taurine, ornithine, selenocysteine, homocysteine, hydroxyproline, thioproline, iodotyrosine, 3-nitrotyrosine, ornithine, citrulline, canavanine, 5-hydroxytryptophan, carnosine, cycloleucine, 3,4-dihydroxyphenylalanine, N-acetylcysteine, prolinol, allylglycine, or acetamidomalonic acid, can be used as needed. Generally, amino acids can be classified as having nonpolar side chains (e.g., Ala, Cys, Ile, Leu, Met, Phe, Pro, Val), negatively charged side chains (e.g., Asp, Glu), positively charged side chains (e.g., Arg, His, Lys), or uncharged polar side chains (e.g., Asn, Cys, Gln, Gly, His, Met, Phe, Ser, Thr, Trp, and Tyr).

[0225] A target according to the present invention is a substance of any biological or chemical origin that can be directly or indirectly detected by an antibody of the present invention. The target can be, for example, a protein, peptide, nucleic acid, oligonucleic acid, sugar, polysaccharide, glycoprotein. Examples include, but are not limited to, therapeutic targets, diagnostic targets, receptors, receptor ligands, viral coat proteins, immune system proteins, hormones, enzymes, antigens, cell signaling proteins, or fragments thereof. The target can be a native protein or a fragment thereof, a homologous sequence thereof, a functional portion thereof, or a functional portion of a homologous sequence.

[0226] The term "epitope", also referred to as "antigenic determinant", refers to the antigenic portion of an antigen to which an antibody specifically binds to form a complex. Thus, the term "epitope" includes any molecular or protein determinant that can specifically bind to an immunoglobulin or T cell receptor. The binding site (antibody determinant) of the antibody molecules described herein can specifically bind to / interact with conformational or continuous epitopes that are unique to the target structure. Epitope determinants are usually composed of the chemically reactive surface groups of a molecule such as amino acids or sugar side chains, and usually have specific three-dimensional structural features as well as specific charge characteristics. Epitope determinants can include the chemically reactive surface groups of a molecule such as amino acids, sugar side chains, phosphoryl or sulfonyl groups, and in certain embodiments, can have specific three-dimensional structural features and / or specific charge characteristics. For polypeptide antigens, conformational or discontinuous epitopes are characterized by the presence of two or more discrete amino acid residues that are separated in the primary sequence but assemble into a continuous structure on the molecular surface when the polypeptide folds into the native protein / antigen (Sela, M., Science (1969) 166, 1365-1374; Laver, W.G., et al. Cell (1990) 61, 553-556). The two or more discrete amino acid residues that make up the epitope can be present in separate segments of one or more polypeptide chains. These residues come together on the molecular surface when the polypeptide chain folds into a three-dimensional structure to form the epitope. In contrast, continuous or linear epitopes are composed of two or more discrete amino acid residues that are present in a single linear segment of the polypeptide chain. As an illustrative example, the "context-dependent" CD3 epitope refers to the conformation of the epitope. This context-dependent epitope located on the ε-chain of CD3 can only form its correct conformation when it is embedded in the rest of the ε-chain and maintained in the correct position by heterodimerization of the ε-chain with the γ-chain or δ-chain of CD3. In contrast, a non-context-dependent CD3 epitope can be the polypeptide of the first 1-27 amino acid residues at the N-terminus of CD3ε or a functional fragment thereof. Generally, an epitope can be a naturally occurring linear or can be a discontinuous epitope. Thus, the term "conformational epitope" as used herein refers to a discontinuous epitope formed by the spatial relationship between the amino acids of an antigen, rather than an unbroken series of amino acids. The term "epitope" also includes the antigenic determinant of a hapten, which is considered to be a small molecule that can be used as an antigen by displaying one or more immunologically recognizable epitopes when bound to a larger substance such as a larger molecule e.g. a protein.

[0227] An antibody or antibody molecule / fragment is said to "specifically" bind to an antigen when it recognizes its target antigen in a complex mixture of proteins and / or macromolecules. Typically, an antibody is capable of specifically interacting with and / or binding to its target, but essentially does not bind to another epitope or antigen. Antibodies are said to "bind to the same epitope" if they cross-compete such that only one antibody can bind to the epitope at a given point in time, i.e., one antibody blocks the binding or regulatory action of another antibody.

[0228] Typically, a binding that is considered specific can also have a high affinity, e.g., when the binding affinity is higher than 10 -6 M (in K d ). In particular, the binding affinity can be about 10 -8 to 10 -11 M (K d ), or about 10 -9 to 10 -11 M or even higher. Thus, antibody molecules with an affinity in the picomolar range (K d from 9.9×10 -10 M to 10 -12 M) are also included in the present invention. If desired, non-specific binding of the binding site can be reduced by changing the binding conditions without substantially affecting specific binding.

[0229] An antibody according to the present invention can be an isolated antibody molecule. As used herein, the term "antibody according to the present invention" refers to an antibody molecule that has been identified, isolated, and / or recovered from the components of its natural environment. The contaminating components of its natural environment are substances that interfere with the application of the antibody and can include enzymes, hormones, and other proteinaceous or non-proteinaceous solutes. In some embodiments, the antibody molecule is purified to greater than 95% by weight, e.g., greater than 99% by weight, as determined by the Lowry method. In some embodiments, the antibody molecule is purified to the extent that at least 15 residues of the N-terminal or internal amino acid sequence can be obtained using a spinning cup sequencer. In some embodiments, the antibody is purified to be judged homogeneous by SDS-PAGE under reducing or non-reducing conditions using Coomassie blue or preferably silver staining. In some embodiments, the isolated antibody molecule can be present in a foreign host cell together with one or more components that are not present in the natural environment of the antibody. Isolated antibodies are typically prepared by at least one purification step.

[0230] Unless otherwise specified, the CDR sequences shown in this disclosure follow the definitions given by AbM as used in Oxford Molecular’s AbM antibody modelling software. See generally, for example, Protein Sequence and Structure Analysis of Antibody Variable Domains, as incorporated in Antibody Engineering Lab Manual (Eds. Duebel, S. and Kontermann, R., Springer-Verlag, Heidelberg). There are other standards for defining CDRs, such as the definition according to Maass 2007 (Journal of Immunological Methods 324 (2007) 13 - 25). Another standard is the definition according to Kabat CDRs, as described in Sequences of Proteins of immunological Interest, U.S. Department of Health and Human Services (1991) (Eds. Kabat et al.). Another standard for characterizing the antigen binding site refers to the hypervariable loops as described by Chothia (see, e.g., Chothia et al. (1992); J. Mol. Biol. 227:799 - 817; and Tomlinson et al. (1995) EMBO J. 14:4628 - 4638). It should be understood that the embodiments described with the AbM CDR definition may optionally be implemented using similar relationships described, such as the Maass, Kabat, or Chothia definitions.

[0231] The titer of an antibody is generally expressed as the number of antigen binding sites of one molecule of any given antibody or the number of antibody binding sites of any given antigen. Most antibody molecules, and those belonging to the IgG, IgA, IgD, and IgE immunoglobulin classes, have two antigen binding sites per molecule. Generally, a monovalent antibody contains a single antigen binding site. Examples of monovalent antibodies are single domain antibodies, VHH domains, single Fab fragments, Fv fragments, scFv fragments, or single VH or VL domains.

[0232] The terms “Fab,” “Fab region,” “Fab portion,” or “Fab fragment” are understood to define a moiety that contains V H , C H 1, V L and C LPolypeptides of immunoglobulin domains. Fab may refer to this region alone, or in the context of an antibody molecule according to the invention, as well as full-length immunoglobulins or immunoglobulin fragments. Typically, the Fab region contains the entire light chain of the antibody. The Fab region can be used to define "one arm" of the immunoglobulin molecule. It contains the epitope-binding portion of the Ig. The Fab region of a naturally occurring immunoglobulin can be obtained as a proteolytic fragment by partial papain digestion. The "F(ab')2 portion" is a proteolytic fragment of a partially pepsin-digested immunoglobulin. The "Fab' portion" is the product obtained by reducing the disulfide bonds of the F(ab')2 portion. As used herein, the terms "Fab", "Fab region", "Fab portion" or "Fab fragment" may further include the hinge region (see above) that defines the C-terminus of the antibody arm. This hinge region corresponds to the hinge region seen at the C-terminus of the C H 1 domain C-terminus, where the arms of the antibody molecule in the full-length immunoglobulin can be brought together to form a "Y". The term hinge region is used in the art because immunoglobulins have a certain elasticity in this region.

[0233] "Fv" or "Fv fragment" consists only of the V of the "single arm" of the immunoglobulin L and V H domains. Thus, "Fv" is the smallest antibody fragment that contains a complete antigen recognition and binding site. A "two-chain" Fv fragment consists of a dimer of the variable domains of one heavy chain and one light chain that are tightly and non-covalently associated. Single-chain Fv classes (scFv) include the V of the immunoglobulin H and V L domains, which are present in a single polypeptide chain in which they are covalently linked to each other by a flexible peptide linker. Typically, in an scFv fragment, the variable domains of the light and heavy chains associate in a dimer structure similar to that of the two-chain Fv class. In a single-chain Fv fragment, either the variable domain of the light chain can be positioned at the N-terminus of the single polypeptide chain, followed by the linker and the variable domain of the heavy chain positioned at the C-terminus of the polypeptide chain, or conversely, the variable domain of the heavy chain can be positioned at the N-terminus, the variable domain of the light chain at the C-terminus and the peptide linker in the middle. The peptide linker can be any flexible linker known in the art, for example, a linker consisting of glycine and serine residues. The connection between V H and V LDomain linkers between domains (see Reiter et al., Stabilization of the Fv fragments in recombinant immunotoxins by disulfide bonds engineered into conserved framework regions, Biochemistry 1994, 33, 6551-5459). These scFv fragments are also referred to as disulfide-stabilized scFv fragments (ds-scFv).

[0234] As used herein, the terms "Fc region" or "Fc fragment" are used to define the C-terminal region of an immunoglobulin heavy chain, including native sequence Fc regions and variant Fc regions. The Fc portion mediates effector functions of the antibody, such as activation of the complement system and immune effector cells with Fc-receptors (e.g., NK cells). In a human IgG molecule, the Fc region is generated by papain cleavage at the N-terminal Cys226. Although the boundaries of the Fc region of an immunoglobulin heavy chain may vary, the human IgG heavy chain Fc region is generally defined as extending from the amino acid residue at position Cys226 or from Pro230 to its carboxyl terminus. The C-terminal lysine of the Fc region (residue 447 according to the EU numbering system) may be removed, e.g., during the preparation or purification of the antibody molecule, or by recombinant engineering of the nucleic acid encoding the heavy chain of the antibody molecule. Thus, the composition of a full-length antibody can include a population of antibodies with all K447 residues removed, a population of antibodies without K447 residues removed, and a population of antibodies comprising a mixture of antibodies with or without K447 residues. Suitable native sequence Fc regions for the antibodies of the invention include IgG1, IgG2 (IgG2A, IgG2B), IgG3, and IgG4 of mammals (e.g., human or murine). The Fc region contains two or three constant domains, depending on the class of the antibody. In embodiments where the immunoglobulin is IgG, the Fc region has a C H 2 domain and a C H 3 domain.

[0235] The antibodies according to the invention can be produced using any known and well-established expression systems and recombinant cell culture techniques, e.g., by expression in a bacterial host (prokaryotic system) or a eukaryotic system such as yeast, fungi, insect cells, or mammalian cells. The antibody molecules of the invention can be produced in transgenic organisms such as goats or plants. Antibodies can also be produced by chemical synthesis.

[0236] The antibodies of the present invention may comprise a CDR1 sequence GVTISALNAMAMG (SEQ ID NO: 115) or a sequence having 1 or 2 mutations relative to said sequence, a CDR2 sequence AVSERGNAM (SEQ ID NO: 116) or a sequence having 1 or 2 mutations relative to said sequence, and a CDR3 sequence LEDRVDSFHDY (SEQ ID NO: 117) or a sequence having 1 or 2 mutations relative to said sequence. The antibodies of the present invention may comprise a CDR1 sequence GVTISALNAMAMG (SEQ ID NO: 118) or a sequence having 1 or 2 mutations relative to said sequence, a CDR2 sequence AVSSRGNAM (SEQ ID NO: 119) or a sequence having 1 or 2 mutations relative to said sequence, and a CDR3 sequence. The antibodies of the present invention may comprise a CDR1 sequence GVTVSALNAMAMG (SEQ ID NO: 121) or a sequence having 1 or 2 mutations relative to said sequence, a CDR2 sequence AVSERGNAM (SEQ ID NO: 122) or a sequence having 1 or 2 mutations relative to said sequence, and a CDR3 sequence LEDRVDSFHDY (SEQ ID NO: 123) or a sequence having 1 or 2 mutations relative to said sequence. It should be understood that such antibodies preferably comprise a camelid VHH domain (e.g., a single domain antibody) or an antibody naturally lacking a light chain or are composed of said camelid VHH domain or an antibody naturally lacking a light chain. It should also be understood that antibodies in which 1 or 2 mutations are introduced into one, two or all three CDR sequences are still capable of specifically binding to the peptides contained in the fusion proteins of the present invention, particularly peptides having the core structure of SEQ ID NO: 3, such as peptides of any one of SEQ ID NOs: 05 - 07. In such antibodies of the present invention, the E or S at amino acid position 4 of CDR2 may be mutated to G, A, L, I, S, T, V, C, M, D, N, E, Q, F, Y, H, W, K, R or P, preferably D, N or H. Such antibodies of the present invention may comprise an FR1 sequence EVQLX1ESGGGLVX2PGGSX3RLSCTAS, where X1 is Q, V, E or L, X2 is Q or P, X3 is L or M (SEQ ID NO: 124), or a sequence having at least 80%, 85%, 90% or 95% sequence identity to said sequence. Such antibodies of the present invention may comprise an FR1 sequence EVQLQESGGGLVQPGGSLRLSCTAS (SEQ ID NO: 125) or a sequence having at least 80%, 85%, 90% or 95% sequence identity to said sequence.Such an antibody of the invention may comprise the FR2 sequence WYRQX1PGEX2RVMVA, wherein X1 is A or R and X2 is R or E (SEQ ID NO: 126), or a sequence having at least 80%, 85%, 90% or 95% sequence identity with said sequence. Such an antibody of the invention may comprise the FR2 sequence WYRQAPGERRVMVA (SEQ ID NO: 127) or a sequence having at least 80%, 85%, 90% or 95% sequence identity with said sequence. Such an antibody of the invention may comprise the FR2 sequence WYRQAPGEERVMVA (SEQ ID NO: 128) or a sequence having at least 80%, 85%, 90% or 95% sequence identity with said sequence. Such an antibody of the invention may comprise the FR3 sequence YRESVQGRFTVTRDFTNKMVSLQMDNLX1PEDX2AVYYCHV, wherein X1 is K or Q and X2 is T or M (SEQ ID NO: 129), or a sequence having at least 80%, 85%, 90% or 95% sequence identity with said sequence. Such an antibody of the invention may comprise the FR3 sequence YRESVQGRFTVTRDFTNKMVSLQMDNLKPEDTAVYYCHV (SEQ ID NO: 130) or a sequence having at least 80%, 85%, 90% or 95% sequence identity with said sequence. Such an antibody of the invention may comprise the FR4 sequence WGQGX1QVTVSS, wherein X1 is T or I (SEQ ID NO: 131), or a sequence having at least 80%, 85%, 90% or 95% sequence identity with said sequence. Such an antibody of the invention may comprise the FR4 sequence WGQGTQVTVSS (SEQ ID NO: 132) or a sequence having at least 80%, 85%, 90% or 95% sequence identity with said sequence.

[0237] An antibody of the invention may comprise or consist of a VHH sequence selected from the following:

[0238] (a) EVQLQESGGGLVQPGGSLRLSCTASGVTISALNAMAMGWYRQAPGERRVMVAAVSERGNAMYRESVQGRFTVTRDFTNKMVSLQMDNLKPEDTAVYYCHVLEDRVDSFHDYWGQGTQVTVSS (SEQ ID NO: 133);

[0239] (b) EVQLQESGGGLVQPGGSLRLSCTASGVTISALNAMAMGWYRQAPGEERVMVAAVSSRGNAMYRESVQGRFTVTRDFTNKMVSLQMDNLKPEDTAVYYCHVLEDRVDSFHDYWGQGTQVTVSS (SEQ ID NO: 134);

[0240] (c) EVQLQESGGGLVQPGGSLRLSCTASGVTISALNAMAMGWYRQAPGEERVMVAAVSERGNAMYRESVQGRFTVTRDFTNKMVSLQMDNLKPEDTAVYYCHVLEDRVDSFHDYWGQGTQVTVSS (SEQ ID NO: 135);

[0241] (d) EVQLQESGGGLVQPGGSLRLSCTASGVTISALNAMAMGWYRQAPGERRVMVAAVSSRGNAMYRESVQGRFTVTRDFTNKMVSLQMDNLKPEDTAVYYCHVLEDRVDSFHDYWGQGTQVTVSS (SEQ ID NO: 136);

[0242] (e) EVQLVESGGGLVPPGGSMRLSCTASGVTVSALNAMAMGWYRQRPGERRVMVAAVSERGNAMYRESVQGRFTVTRDFTNKMVSLQMDNLQPEDMAVYYCHVLEDRVDSFHDYWGQGIQVTVSS (SEQ ID NO: 137);

[0243] (f) EVQLVESGGGLVPPGGSMRLSCTAPGVTVSALNAMAMGWYRQRPGERRVMVAAVSERGNAMYRESVQGRFTVTRDFTNKMVSLQMDNLQPEDMAVYYCHVLEDRVDSFHDYWGQGIQVTVSS (SEQ ID NO: 138);

[0244] (g) EVQLVESGGGVVQPGGSLRLSCTASGVTISALNAMAMGWYRQAPGERRVMVAAVSERGNAMYRESVQGRFTVTRDFTNKMVSLQMDNLKPEDTAVYYCHVLEDRVDSFHDYWGQGTQVTVSS (SEQ ID NO: 139);

[0245] (h) EVQLVESGGGLVQPGGSLRLSCTASGVTISALNAMAMGWYRQAPGERRVMVAAVSERGNAMYRESVQGRFTVTRDFTNKMVSLQMDNLKPEDTAVYYCHVLEDRVDSFHDYWGQGTQVTVSS (SEQ ID NO: 140);

[0246] (i) EVQLEESGGGLVQPGGSLRLSCTASGVTISALNAMAMGWYRQAPGERRVMVAAVSERGNAMYRESVQGRFTVTRDFTNKMVSLQMDNLKPEDTAVYYCHVLEDRVDSFHDYWGQGTQVTVSS (SEQ ID NO: 141);

[0247] (j) EVQLLESGGGLVQPGGSLRLSCTASGVTISALNAMAMGWYRQAPGERRVMVAAVSERGNAMYRESVQGRFTVTRDFTNKMVSLQMDNLKPEDTAVYYCHVLEDRVDSFHDYWGQGTQVTVSS (SEQ ID NO: 142);

[0248] (k) EVQLQESGGGLVQPGGSLRLSCTASGVTISALNAMAMGWYRQAPGERRVMVAAVSDRGNAMYRESVQGRFTVTRDFTNKMVSLQMDNLKPEDTAVYYCHVLEDRVDSFHDYWGQGTQVTVSS (SEQ ID NO: 175);

[0249] (l) EVQLQESGGGLVQPGGSLRLSCTASGVTISALNAMAMGWYRQAPGERRVMVAAVSNRGNAMYRESVQGRFTVTRDFTNKMVSLQMDNLKPEDTAVYYCHVLEDRVDSFHDYWGQGTQVTVSS (SEQ ID NO: 176); or

[0250] (m) EVQLQESGGGLVQPGGSLRLSCTASGVTISALNAMAMGWYRQAPGERRVMVAAVSHRGNAMYRESVQGRFTVTRDFTNKMVSLQMDNLKPEDTAVYYCHVLEDRVDSFHDYWGQGTQVTVSS (SEQ ID NO: 177);

[0251] or a sequence having at least 80%, 85%, 90% or 95% sequence identity to any of the aforementioned sequences.

[0252] The antibody of the present invention may comprise a CDR1 sequence GTMSAINALN (SEQ ID NO: 143) or a sequence having 1 or 2 mutations relative to said sequence, a CDR2 sequence AITDNGNAH (SEQ ID NO: 144) or a sequence having 1 or 2 mutations relative to said sequence, and a CDR3 sequence LEEEKLGVWVDY (SEQ ID NO: 145) or a sequence having 1 or 2 mutations relative to said sequence. The antibody of the present invention may comprise a CDR1 sequence GTMSAINALN (SEQ ID NO: 146) or a sequence having 1 or 2 mutations relative to said sequence, a CDR2 sequence AITDNGNAH (SEQ ID NO: 147) or a sequence having 1 or 2 mutations relative to said sequence, and a CDR3 sequence LEEKLGAWVDY (SEQ ID NO: 148) or a sequence having 1 or 2 mutations relative to said sequence. The antibody of the present invention may comprise a CDR1 sequence GTMSAINALN (SEQ ID NO: 149) or a sequence having 1 or 2 mutations relative to said sequence, a CDR2 sequence AITDNGNAH (SEQ ID NO: 150) or a sequence having 1 or 2 mutations relative to said sequence, and a CDR3 sequence LEKEKLGVWVDY (SEQ ID NO: 151) or a sequence having 1 or 2 mutations relative to said sequence. It should be understood that such antibodies preferably comprise a camelid VHH domain (e.g., a single domain antibody) or an antibody naturally lacking a light chain or consist of said camelid VHH domain or an antibody naturally lacking a light chain. It should also be understood that antibodies in which 1 or 2 mutations are introduced into one, two or all three CDR sequences are still capable of specifically binding to the peptides contained in the fusion proteins of the present invention, especially peptides having the core structure of SEQ ID NO: 3, such as peptides of any one of SEQ ID NO: 05 - 07. Such an antibody of the present invention may comprise an FR1 sequence EVQLX1ESGGGLVQPGGSLTLSCAAS, where X1 is V or L (SEQ ID NO: 152), or a sequence having at least 80%, 85%, 90% or 95% sequence identity to said sequence. Such an antibody of the present invention may comprise an FR2 sequence WYRQX1PGKERKMVA, where X1 is P or A (SEQ ID NO: 153), or a sequence having at least 80%, 85%, 90% or 95% sequence identity to said sequence. Such an antibody of the present invention may comprise an FR3 sequence YADSVKGRFTISRDNARNMVFLQMNSLX1PDDTAVYYCHY, where X1 is K or E (SEQ ID NO: 154), or a sequence having at least 80%, 85%, 90% or 95% sequence identity to said sequence.This antibody of the present invention may comprise the FR4 sequence WGQGTQVTVSS (SEQ ID NO: 155) or a sequence having at least 80%, 85%, 90% or 95% sequence identity with said sequence.

[0253] The antibody of the present invention may comprise or consist of a VHH sequence selected from the following:

[0254] (a) EVQLVESGGGLVQPGGSLTLSCAASGTMSAINALNWYRQPPGKERKMVAAITDNGNAHYADSVKGRFTISRDNARNMVFLQMNSLKPDDTAVYYCHYLEEEKLGVWVDYWGQGTQVTVSS (SEQ ID NO: 156);

[0255] (b) EVQLLESGGGLVQPGGSLTLSCAASGTMSAINALNWYRQAPGKERKMVAAITDNGNAHYADSVKGRFTISRDNARNMVFLQMNSLEPDDTAVYYCHYLEEKLGAWVDYWGQGTQVTVSS (SEQ ID NO: 157); or

[0256] (c) EVQLVESGGGLVQPGGSLTLSCAASGTMSAINALNWYRQPPGKERKMVAAITDNGNAHYADSVKGRFTISRDNARNMVFLQMNSLKPDDTAVYYCHYLEKEKLGVWVDYWGQGTQVTVSS (SEQ ID NO: 158);

[0257] or a sequence having at least 80%, 85%, 90% or 95% sequence identity with any of said sequences.

[0258] The antibodies of the present invention can be conjugated with a detectable label. Generally, such a "detectable label" can be any suitable chemical substance or enzyme that directly or indirectly produces a detectable compound or signal in a chemical, physical, optical, or enzymatic reaction. For example, a fluorescent or radioactive label can be conjugated to an antibody to produce fluorescence or X-rays as detectable signals. Alkaline phosphatase, horseradish peroxidase, and β-galactosidase are examples of enzyme labels (which are also optical labels) that catalyze the formation of a colorimetric reaction product. In a preferred embodiment, the detectable label refers to a detectable entity that can be used to detect a target in microscopy, immunohistochemistry, or flow cytometry. Preferably, the label does not adversely affect the properties of the antibody to which it is conjugated. Examples of labels are fluorescent labels, to name just a few, such as phycoerythrin, allophycocyanin (APC), Brilliant Violet 421, Alexa Fluor 488, coumarin, or rhodamine. There are many types of detectable labels, including fluorescent labels, chromophore labels, isotope labels, or metal labels, preferably fluorescent labels. The presence of the fusion protein of the present invention can be detected by contacting the fusion protein with the antibody of the present invention conjugated with a detectable label and detecting the signal of the detectable label. For a fluorescent label, this means detecting the emitted light by exciting the fluorescent label. Non-exhaustive examples of suitable fluorescent labels are "green light" emitters (Atto488, Alexa488, Cy2, etc.), "orange light" emitters (Atto542, Alexa555, Cy3, etc.), "red-far red light" emitters (Alexa633, Atto 647N, Cy5, etc.), infrared emitters (Atto700, LiCor IRDye700, LiCor IRDye 800, etc.), ultraviolet-absorbing fluorescent dyes (Atto390 or Alexa405). The fluorescent label can also be a fluorescent protein, such as GFP, eGFP, YFP, RFP, CFP, BFP, mCherry, or a near-infrared fluorescent protein. Non-exhaustive examples of suitable chromophore labels are alkaline phosphatase or peroxidase exposed to TMB (3,3',5,5'-tetramethylbenzidine), DAB (3,3',4,4'-diaminobenzidine), and 4CN (4-chloro-1-naphthol). ABTS (2,2'-azino-bis[3-ethylbenzothiazoline-6-sulfonate]), OPD (o-phenylenediamine), and BCIP / NBT (5-bromo-4-chloro-3-indolyl phosphate / nitroblue tetrazolium). Non-exhaustive examples of isotope labels are carbon 13, nitrogen 15, fluorine 19, aluminum 27, boron 11, iodine 127, or different lanthanide isotopes. Non-exhaustive examples of metal labels are gold, palladium, lead, platinum, silver, mercury, and osmium. The label can be a direct label, i.e., a directly detectable label.Optionally, the tag can be an indirect tag, i.e., a tag that is an affinity tag (or epitope tag) that can be specifically bound by another specific binding partner conjugated to another detectable tag (e.g., a fluorescent or chromophore tag). Examples of suitable epitope tags include, but are not limited to, the FLAG-tag (sequence: DYKDDDDK, SEQ ID NO: 160), the Strep-tag (sequence: WSHPQFEK, SEQ ID NO: 178), the Myc-tag (sequence: EQKLISEEDL, SEQ ID NO: 161), the HA-tag (sequence: YPYDVPDYA, SEQ ID NO: 162), the VSV-G-tag (sequence: YTDIEMNRLGK, SEQ ID NO: 163), the HSV-tag (sequence: QPELAPEDPED, SEQ ID NO: 164), the V5-tag (sequence: GKPIPNPLLGLDST, SEQ ID NO: 165), the SPOT-tag (sequence: PDRVRAVSHWSS, SEQ ID NO: 166), the BC2 tag (sequence: PDRKAAVSHWQQ, SEQ ID NO: 167), and the EPEA tag (sequence: EPEA, SEQ ID NO: 168). The antigen can also be a protein, e.g., glutathione-S-transferase (GST), maltose binding protein (MBP), chitin binding protein (CBP), or thioredoxin as an antigen. The detectable tag can also be a nucleic acid, e.g., an oligonucleotide having a recognition sequence. Such a recognition sequence can be a random sequence. The random sequence can be a barcode sequence that has been incorporated into a nucleic acid molecule and can be used to identify a target molecule that has been conjugated to the nucleic acid. "The antibody of the present invention can be conjugated to a detectable tag" can also mean that the antibody itself is a detectable tag. This may imply that the antibody is an affinity target that can be specifically recognized by another specific binding partner that specifically binds to the antibody. For example, such a specific binding partner can be an antibody that specifically recognizes the camel VHH domain. Such a specific binding partner can also be conjugated to a detectable tag (e.g., a fluorescent tag).

[0259] The antibodies of the present invention can be conjugated to a solid support. The term "solid support" or in the context of the present invention refers to any type of carrier material that can be used to immobilize an affinity ligand such as an antibody or a portion thereof, and it can refer to particulate (e.g., beads or granules, commonly used in extraction columns) or sheet-like (e.g., membranes or filters, glass or plastic sheets, microtiter plates, dipsticks, capillary filling devices, etc.) materials, which can be flat, pleated or hollow fibers or tubes. Non-exhaustive suitable and well-known matrices are: silica (porous amorphous silica), agarose or polyacrylamide carriers or macroporous polymers. Examples include dextran, collagen, polystyrene, polypropylene, polyvinyl chloride, polyacrylamide, methacrylate, cellulose, calcium alginate, controlled pore glass, aluminum, titanium and porous ceramics, synthetic polymers and copolymers, latex, silica, agarose, metals, glass and carbon. Alternatively, the solid surface can include part of a mass-related sensor, such as a surface plasmon resonance detector. Conveniently, an array is provided that comprises a plurality of individual affinity ligands, such as antibodies or antibody fragments, bound or immobilized to a solid surface, said affinity ligands being capable of specifically binding to the epitope tag of the present invention. The array can be used to capture tagged polypeptides contained in a solution, provided that the solution is brought into contact with the immobilized affinity ligand such as an antibody or antibody fragment. The solid support can also be magnetic beads or polymer beads or a chromatographic stationary phase.

[0260] The antibodies of the present invention can be complexed with the epitope to which it specifically binds. Such an epitope can be a peptide contained in the fusion protein of the present invention. Thus, the antibodies of the present invention can be complexed with the fusion protein of the present invention. Accordingly, the present invention encompasses fusion proteins that contain a peptide to which the antibodies of the present invention specifically bind.

[0261] The present invention also relates to a complex comprising (a) a fusion protein and (b) an antibody, wherein the fusion protein is a fusion protein of the present invention and / or wherein the antibody is an antibody of the present invention.

[0262] The present invention also relates to nucleic acid molecules comprising sequences encoding the inventive fusion proteins or the inventive antibodies described herein. The nucleic acid molecules can be DNA or RNA molecules. The nucleic acid molecules of the present invention can be part of a vector or any other kind of cloning or expression vector, such as a plasmid, phagemid, phage, baculovirus, cosmid or artificial chromosome. The nucleic acid molecule can permit the expression of the fusion protein or antibody. It can include sequence elements containing information about transcriptional and / or translational regulation, and such sequences can be "operably linked to" the nucleotide sequence encoding the protein. An operable linkage is a linkage in which the regulatory sequence element and the sequence to be expressed are linked in such a way as to enable gene expression. The precise nature of the regulatory regions required for gene expression can vary between species, but generally these regions include a promoter, which in prokaryotes contains both the promoter itself, i.e., the DNA element that directs the start of transcription, and a DNA element that will signal the start of translation when transcribed into RNA. Such promoter regions typically include 5' non-coding sequences involved in the initiation of transcription and translation, such as the -35 / -10 box and the Shine-Dalgarno element in prokaryotes or the TATA box, CAAT sequence and 5'-capping element in eukaryotes. These regions can also include enhancer or repressor elements as well as translation signals and leader sequences for targeting the native protein to specific compartments of the host cell.

[0263] Such a vector described herein can include, in addition to the regulatory sequences and the nucleic acid sequence encoding the peptide or protein described herein, replication and control sequences derived from a species compatible with the host cell for expression, and a selection marker that confers a selectable phenotype on the transformed or transfected cell. A large number of suitable cloning vectors are known in the art and are commercially available. Accordingly, the present invention also relates to vectors comprising the nucleic acid molecules of the present invention.

[0264] Cloning or expression of the nucleic acid molecules or vectors of the present invention can be carried out at least in part in vivo using host cells that have been transformed with the nucleic acid or vector or into which the nucleic acid molecule or vector has been transferred by other means including transduction or transfection. Transfer of DNA can be carried out using standard techniques. Accordingly, the present invention also relates to host cells comprising the nucleic acid molecules or vectors disclosed herein.

[0265] The peptides disclosed herein can be used in a number of applications. A commonality among these cellular applications is that the peptides are used as epitope tags. Non-limiting examples of applications in which the peptides function include, but are not limited to, detecting, immobilizing, separating or purifying the inventive fusion proteins. Similarly, the antibodies of the present invention can be used in all applications that include specific binding to an epitope tag. The antibodies of the present invention can be used, for example, to detect, immobilize, separate or purify the inventive fusion proteins.

[0266] The present invention also relates to a method for detecting the fusion protein of the present invention. The detection can be optical detection, isotope detection or electron microscopy detection. The method comprises contacting the fusion protein with an antibody of the present invention, preferably under conditions that permit the formation of a complex between the peptide contained in the fusion protein and the antibody. The antibody of the present invention preferably carries a detectable label as defined herein. When the detectable label is a fluorescent label, a chromophore label, an isotope label or a metal label attached to the antibody, it should be understood that the antibody preferably has a defined number of labels attached thereto. The method may comprise the step of detecting the detectable label. The method may comprise expressing the fusion protein of the present invention prior to contacting the fusion protein with the antibody.

[0267] As used herein, the term "detection" includes both direct detection of a target (i.e., where the target is detected by a signal derived from the target) and indirect detection of a target (i.e., where the target is detected by a signal not directly derived from the target, e.g., by a signal derived from another molecule attached to the target). The term "detection" as used herein also includes both qualitative detection and quantitative detection. The term "detection" may refer to determining the presence, subcellular localization or amount of a given molecule or structure such as the fusion protein of the present invention. The fusion protein to be qualitatively, localized and / or quantified may be detected in its intracellular location within a host cell, e.g., in the nucleus, in the cell membrane or another cellular compartment. The fusion protein to be qualitatively and / or quantified may also be detected in a solution containing the tagged polypeptide or protein, e.g., a cell lysate obtained from a host cell or a tissue containing the host cell.

[0268] As used herein, the term "optical" preferably refers to visible light, but is not limited thereto in general. The term may also refer to infrared, ultraviolet and other regions of the electromagnetic spectrum.

[0269] As used herein, the term "isotope detection" relates to the detection of a molecule in which one or more atoms have been replaced by another isotope that typically has a detectable variation (i.e., "labeled with another label"). Isotope labels can be detected by a variety of means, such as by their mass (e.g., by mass spectrometry, matrix-assisted laser desorption / ionization (MALDI), desorption electrospray ionization (DESI), laser ablation inductively coupled plasma mass spectrometry (LA-ICP-MS) or secondary ion mass spectrometry (SIMS)), vibrational mode (e.g., by infrared spectroscopy), gyromagnetic ratio (e.g., by nuclear magnetic resonance) or radioactive decay (e.g., ionization chamber or gel autoradiography).

[0270] Electron microscopy involves detection methods using electron microscopes. Types of electron microscopes include transmission electron microscopes (TEM), scanning electron microscopes (SEM), reflection electron microscopes (REM), scanning transmission electron microscopes (STEM), and correlative light and electron microscopes (CLEM).

[0271] In the first step of the detection method, an antibody that specifically binds to a peptide contained in the fusion protein of the present invention can be contacted with a sample containing the fusion protein. The sample can be a host cell, tissue, a solution of a cell lysate containing the host cell, or any other sample containing the fusion protein, such as a supernatant obtained by centrifuging a liquid containing the host cell, wherein the host cell is capable of secreting a target polypeptide into the liquid or other sample (such as a body fluid).

[0272] This contacting step is preferably carried out under conditions that allow specific interaction between the antibody and the peptide to which it specifically binds. These conditions are well known to those skilled in the art. The washing step is typically carried out after the contacting step of the antibody with its antigen, and those skilled in the art know how and when to apply the washing step. When contacted with the sample, the antibody will specifically interact with the fusion protein. This interaction can be detected, monitored, and quantified by measuring or observing a reporter signal obtained from a detectable label. For example, if the detectable label is a fluorescent label, fluorescence can be measured and observed upon excitation.

[0273] If the detectable label is an affinity tag (or epitope tag), it can be specifically bound by another specific binding partner conjugated to another detectable label (such as a fluorescent or chromophore label). In this case, detection of the detectable label attached to the antibody of the present invention can be carried out by contacting the antibody with a specific binding partner that specifically binds to the detectable label, the detectable label being conjugated to the antibody of the present invention. The specific binding partner can be labeled with another detectable label, the other detectable label preferably being capable of being distinguished from the first detectable label, such as a fluorescent or chromophore label. However, the specific binding partner can be a structure that can be recognized by another specific (labeled) binder. For example, the specific binding partner that binds to the detectable label conjugated to the antibody of the present invention can be a (primary) antibody, which can be specifically recognized by a (secondary) antibody that carries a detectable label, the detectable label preferably being different from the first detectable label, such as a fluorescent label. Thus, a method for detecting the fusion protein of the present invention can include the step of contacting the fusion protein and the antibody with a specific binding partner of the detectable label contained in the antibody. In the presence of a second detectable label, the method can include the step of detecting the first and / or second detectable label.

[0274] According to the method in which the detectable label conjugated to the antibody of the present invention is an affinity tag, the method for detecting the fusion protein may include, in the first step, contacting the antibody of the present invention with a sample containing or suspected of containing the fusion protein of the present invention. In the second step, a (second) specific binding partner may be contacted with the sample containing the fusion protein bound to the antibody of the present invention. In the case where the (second) specific binding partner is not conjugated to the detectable label or the detectable label is an affinity tag, the method may further include the step of contacting another specific binding partner (such as another antibody that specifically binds to the (second) specific binding partner or its detectable label). The other specific binding partner may contain a detectable label, such as a fluorescent label that can be used to detect the fusion protein. The presence, amount, and / or localization of the tagged polypeptide or protein may be detected or determined by measuring or observing the reporter signal obtained from the detectable label contained in the (second) specific binding partner or other specific binding partner.

[0275] One advantage of the two-step detection method using two types of binding partners is that the tag-specific interaction is separated from the actual detection step. This keeps the antibody of the present invention intact as it does not need to contain an additional detectable moiety. In some cases, this may enhance its specificity or affinity compared to an antibody containing an additional detectable label, as the detectable label may in some cases affect the interaction between the peptide contained in the fusion protein of the present invention and the antibody of the present invention. Therefore, in some cases, the reliability and efficiency of the detection method can be improved. Moreover, if only the presence and amount of the fusion protein are to be determined, the antibody of the present invention is used only as a capture antibody and not as a capture and detection antibody, which separates the capture and detection steps. Thus, after the first step using the antibody of the present invention, a separation or enrichment step may be performed to obtain the captured target fusion protein. Then, the detection step may be performed on the separated and / or enriched fusion protein, thereby improving the reliability of the quantification obtained and making the detection step easier to operate.

[0276] Suitable physiological or biomolecular detection methods for the qualitative detection of epitope tag / antibody interactions include any suitable methods known in the art. These methods include, but are not limited to, methods for qualitative or quantitative determination, such as enzyme-linked immunosorbent assay (ELISA), ELISPOT assay, Western Blot, or immunoassay. These methods include, for example, optical, radioactive, or chromatographic methods, preferably when using any of the above-mentioned labels, markers, or linkers, more preferably fluorescence detection methods, radioactive detection methods, Coomassie blue staining, silver staining, or other protein staining methods, electron microscopy methods, methods for staining tissue sections by immunohistochemistry or by direct or indirect immunofluorescence, etc. These methods can be applied with antibodies, or can involve the use of other tools that specifically bind to a part of a fusion protein, antibody, or complex (such as the use of a second binding partner).

[0277] In some embodiments, the subcellular localization of the tagged target polypeptide or protein can also be determined. For example, different subcellular structures (such as the intermediate filament network or the core part of the replication machinery) can be visualized and monitored.

[0278] The detection of the fusion protein can also be performed using the antibodies of the present invention, i.e., intracellular antibodies. As used herein, "intracellular antibody" refers to an antibody that is located inside the cell to bind to intracellular proteins. Due to the lack of a reliable mechanism for bringing antibodies from the extracellular environment into living cells, it is usually necessary to express the antibody inside the target cell. After expression, the antibody can remain in the cytoplasm, or it can have a nuclear localization signal, or it can undergo co-translational translocation across the membrane into the lumen of the endoplasmic reticulum, provided that it is retained in that compartment by the KDEL sequence. The detectable label conjugated to the intracellular antibody can be a proteinaceous label, which can be expressed as a fusion protein with the intracellular antibody. Ideally, such a label can be optically detectable, for example, by fluorescence. Thus, the detectable label can be a fluorescent protein.

[0279] The present invention also relates to a method for isolating the fusion protein of the present invention. Such a method includes contacting the fusion protein with the antibody of the present invention, preferably under conditions that allow the formation of a complex between the antibody and the peptide contained in the fusion protein. Thereby, the binding of the fusion protein to the antibody can be achieved. This contacting step, also referred to as the capture step, can be carried out by contacting a sample (such as a solution) containing the fusion protein with the antibody.

[0280] The sample contacted with the tag-specific antibody can be any type of sample containing the fusion protein of the present invention and can be processed to isolate the polypeptide. Preferably, the sample is a solution, such as a host cell lysate or body fluid containing the fusion protein of the present invention, or a supernatant, such as a supernatant obtainable by centrifuging a liquid containing host cells that contain or are capable of expressing the fusion protein of the present invention, wherein the host cells are capable of secreting or otherwise transporting the fusion protein of the present invention into the liquid.

[0281] The antibody used for separation and / or purification in the method of the present invention can be used in solution or after immobilization. To immobilize the antibody, the antibody can be bound to a sample carrier, solid support or matrix. This immobilization step can be carried out before or after the antibody binds to the peptide contained in the fusion protein. Methods for immobilizing antibodies and their parts are well known to those skilled in the art and any method allowing immobilization without impairing the binding properties can be employed.

[0282] If the antibody of the present invention is not immobilized to a solid support, the method can further include a step of separating the complex, for example, by using a specific binding partner of the complex such as a second antibody that is specific for, for example, the complex or the antibody or a detectable label (such as an affinity tag) conjugated to the antibody. The second binding partner can be in solution or can be immobilized or immobilizable to a solid support.

[0283] In an optional further step after the capture step, the solid support containing the immobilized antibody bound to the fusion protein is washed to remove unbound and non-specifically bound components.

[0284] Optionally, in a further step, the fusion protein can be eluted to obtain the isolated fusion protein. Elution of the fusion protein bound to the immobilized antibody can be achieved by methods known in the art. For example, the fusion protein can be eluted by competing elution with the epitope peptide described herein in isolated form. Then, the isolated epitope peptide will compete with the fusion protein for binding to the immobilized tag-specific antibody. If the isolated peptide is added in an excess concentration, the binding reaction equilibrium will shift towards the binding of the immobilized antibody to the isolated epitope tag. This results in the release of the fusion protein. The epitope peptide used for elution can be the same peptide as the epitope peptide contained in the fusion protein. The epitope peptide used for elution can also be a different peptide from the epitope peptide contained in the fusion protein. If the epitope peptide used for elution is a different epitope peptide, preferably the epitope peptide used for elution has a higher binding affinity for the antibody compared to the epitope peptide contained in the fusion protein. Optionally, other steps for further purifying the released polypeptide can be added, such as method steps well known to those skilled in the art.

[0285] The fusion protein can also be kept immobilized to a solid support, such as (magnetic) beads, and further processed in downstream applications such as mass spectrometry without an elution step.

[0286] The fusion protein can comprise a linker having a cleavage site which can be cleaved with a suitable method, such as a protease, to remove the peptide. Thereby, the polypeptide of the fusion protein can be released from the immobilized antibody and the polypeptide can be obtained in its native form. For this embodiment, the nucleic acid sequence encoding the fusion protein should comprise not only the sequence encoding the epitope tag but also the sequence encoding a linker having a cleavable site (such as a cleavage site recognized by a protease). The enzymatic release step can replace or follow the elution step.

[0287] When the fusion protein of the invention comprises an antibody moiety, the invention also contemplates a method for isolating the target of the antibody moiety of the invention. In principle, the method can be carried out as described above for isolating the fusion protein. The method can comprise an additional step of contacting the fusion protein with the specific target of the antibody moiety comprised in the fusion protein. This contacting step can be carried out before or after contacting the fusion protein with an antibody that binds to the peptide tag comprised in the fusion protein, preferably the latter. In a preferred method, first the antibody specific for the peptide tag is immobilized on a solid support, then the fusion protein is immobilized by binding to the antibody specific for the peptide tag, and subsequently the target of the antibody moiety of the fusion protein is bound to the fusion protein. Elution can be carried out as described above. The specific target can be a cell. For example, a cell surface receptor on a cell, such as CD62L. The antibody moiety of the fusion protein can be specific for a structure on the cell such as CD62L. The antibody moiety of the fusion protein can be a single domain antibody.

[0288] The invention also contemplates that the detection and isolation of the fusion protein of the invention can be combined. Thus, the invention contemplates a method for detecting and isolating the fusion protein of the invention, comprising the detection method of the invention and the isolation and / or purification method of the invention.

[0289] A combination of the two methods can thus be carried out by using one antibody conjugated to a detectable label for detection and another antibody conjugated to a solid support for isolation of the same fusion protein. The two antibodies can be any antibody of the present invention. The advantage of such a combination can be that only one tagged fusion protein needs to be produced and detection and isolation are carried out with the same transgenic construct / cell. Sometimes, it may be desirable for the two antibodies to have the same sequence or at least one identical antigen-binding site. For example, the two antibodies may comprise the CDR 1-3 sequences shown in SEQ ID NO: 115-117, or may comprise the sequence shown in SEQ ID NO: 133 or a sequence having at least 80%, 85%, 90% or 95% sequence identity with SEQ ID NO: 133. Sometimes, it may be desirable for the two antibodies to have different sequences or different antigen-binding sites. The use of different antibodies may have the following advantages: A strong binder, such as an antibody having a high affinity (e.g., from about 1 pM to about 1 nM) for the peptide contained in the fusion protein, can be used for detection, while a mild binder, i.e., an antibody having a medium affinity (e.g., from about 1 nM to about 500 nM), can be used for isolation of the fusion protein. For example, one of the antibodies may comprise the CDR 1-3 sequences shown in SEQ ID NO: 115-117 or may comprise the sequence shown in SEQ ID NO: 133 or a sequence having at least 80%, 85%, 90% or 95% sequence identity with SEQ ID NO: 133, while the other antibody may comprise the CDR 1-3 sequences shown in SEQ ID NO: 118-120 or may comprise the sequence shown in SEQ ID NO: 134 or a sequence having at least 80%, 85%, 90% or 95% sequence identity with SEQ ID NO: 134. The peptide contained in the fusion protein can be a peptide having a core sequence of SEQ ID NO: 3 or 4 as described herein.

[0290] The combination of the two methods can also be carried out by detecting and purifying using the same antibody and two different peptides. The advantage of this is that only one antibody needs to be prepared, and depending on the application, the antibody can be conjugated to a detectable label or a solid support. When using two different peptides, it is preferred that the peptides have different binding affinities for the antibody. The peptide for detection can have a high binding affinity, for example, of about 1 pM to 1 nM, while the peptide for separation can have a moderate binding affinity of about 1 nM to 500 nM. Using peptides with different binding affinities may have the following advantages: If a peptide with moderate affinity is included in the fusion protein for separation, a peptide with high affinity can be used to elute the fusion protein. Thus, one peptide can be a peptide having the core sequence of SEQ ID NO: 3 or 4 as described herein, while the other peptide can be a peptide having the core sequence of SEQ ID NO: 31 or 32 as described herein. The antibody can be any antibody of the present invention. For example, the antibody can comprise the CDR 1-3 sequences shown in SEQ ID NO: 115-117, or can comprise the sequence shown in SEQ ID NO: 133 or a sequence having at least 80%, 85%, 90% or 95% sequence identity with SEQ ID NO: 133.

[0291] The combination of the two methods can also be carried out by using two peptides and two antibodies. The target fusion protein can comprise a peptide having a high affinity for a given antibody. For example, the peptide can be a peptide having the core sequence of SEQ ID NO: 3 or 4 as described herein. The antibody for detection can comprise the CDR 1-3 sequences shown in SEQ ID NO: 115-117, or can comprise the sequence shown in SEQ ID NO: 133 or a sequence having at least 80%, 85%, 90% or 95% sequence identity with SEQ ID NO: 133. The antibody for separation / purification can comprise the CDR 1-3 sequences shown in SEQ ID NO: 118-120, or can comprise the sequence shown in SEQ ID NO: 134 or a sequence having at least 80%, 85%, 90% or 95% sequence identity with SEQ ID NO: 134. The peptide for elution can be a peptide having a higher affinity for the antibody used for elution than the peptide included in the fusion protein, for example, another peptide as described herein.

[0292] The present invention also relates to a system comprising one peptide tag and two antibodies or two peptide tags and one antibody or two peptide tags and two antibodies, wherein the peptide tags and antibodies are as described herein.

[0293] The present invention also relates to a kit. The kit may comprise the components required for carrying out the method of the present invention. A kit for detecting or purifying a fusion protein may comprise a nucleic acid or a nucleic acid expression construct encoding a peptide / epitope tag as defined herein, said peptide / epitope tag being present in said fusion protein. The nucleic acid may comprise sites for promoting the genetic fusion of a polypeptide with said peptide / epitope tag, such as cleavage or recombination sites. The nucleic acid sequence encoding said peptide / epitope tag may be operably linked to a sequence element comprising information regarding transcriptional and / or translational regulation.

[0294] The kit may also comprise an antibody of the present invention, optionally conjugated to a detectable label as described herein, preferably an optically detectable label or an affinity tag. Optionally or additionally, the kit may comprise a detectable moiety that can be conjugated to the antibody of the present invention.

[0295] The kit may also include buffers and reagents required for the separation / purification and / or detection methods of the present invention.

[0296] The kit may also include buffers and reagents required for introducing the nucleic acid or nucleic acid expression construct contained in the kit into a host cell.

[0297] The kit may also include at least one (second) specific binding partner as described herein or another specific binding partner that specifically binds to the (second) specific binding partner as described herein.

[0298] The kit may also include a solid support comprising an antibody of the present invention immobilized or attached thereto. The kit may also include the isolated peptides described herein, which are suitable for competitively eluting the fusion protein bound to the antibody of the present invention, or other means for eluting the fusion protein, such as proteases.

[0299] Unless otherwise stated, the term "at least" before a series of elements should be understood to refer to each element in the series. Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the present invention.

[0300] The term "and / or" as used anywhere herein includes the meanings of "and", "or", and "any other combination of all or any of the elements connected by said term".

[0301] The term "about" or "approximately" as used herein means within 20% of a given value or range, preferably within 10%, more preferably within 5%. However, it also includes the exact value, e.g., about 20 includes 20.

[0302] Unless the context otherwise requires, throughout the specification and the appended claims, the word "comprise" and variations such as "comprises" and "comprising" will be understood to mean including the stated integer or step or group of integers or steps but not excluding any other integer or step or group of integers or steps. As used herein, the term "comprising" may be replaced by the term "containing" or "including" or sometimes when used herein by the term "having".

[0303] As used herein, "consisting of" does not include any element, step or ingredient not specified in the claim. As used herein, "consisting essentially of" does not exclude materials or steps that do not materially affect the basic and novel characteristics of the claim.

[0304] In each case herein, any one of the terms "comprising", "consisting essentially of" and "consisting of" may be replaced by any one of the other two terms.

[0305] It should be understood that the present invention is not limited to the specific methods, schemes, materials, reagents and substances described herein and will vary. The terms used herein are for the purpose of describing particular embodiments only and are not intended to limit the scope of the present invention, which is defined only by the claims.

[0306] All publications cited throughout the text of this specification (including all patents, patent applications, scientific publications, manufacturer's specifications, guidelines, etc.) are hereby incorporated by reference in their entirety. Nothing herein shall be construed as an admission that the present invention is not entitled to antedate such disclosure by virtue of prior invention. To the extent that the material incorporated by reference contradicts or is inconsistent with this specification, this specification shall supersede any such material.

[0307] Examples

[0308] Example 1: System

[0309] To search for new epitope tag / conjugate systems, we designed peptides of SEQ ID NOs: 05 - 07 that form stable α - helices in solution and are collectively referred to as "ALFA" STThe "tag". These peptides have no known counterparts in any eukaryotic model system, they are nearly neutral at physiological pH, and contain no primary amines that could be targets of common fixatives. The core ALFA tag sequence ( Figure 3 A, the amino acids in blue) consists of an artificial peptide that is reported to form a stable alpha-helix in solution (Petukhov, M. et al. Design of stable alpha-helices using global sequence optimization. J. Pept. Sci. 15, 359–365 (2009)). It was selected based on the properties that the sequence i) has no known counterparts in any eukaryotic model system, ii) it is neutral at physiological pH, and iii) contains no primary amines that could be targets of common amine-reactive fixatives and crosslinkers.

[0310] Anti-ALFA ST high-affinity nanobodies were generated in alpacas and selected by "Celline", a novel nanobody selection method that uses antigen-specific enrichment of B cells. To prepare a selective affinity resin, a preferred nanobody (NbALFA ST , clone 1G5, SEQ ID NO: 133) was site-specifically conjugated to an agarose-based resin with low non-specific protein binding properties via a flexible linker. The resulting ALFA Selctor ST resin efficiently pulled down the ALFA ST -shGFP2 fusion protein from an E. coli extract. However, surprisingly, all methods of competing elution of the bound protein with the ALFA ST peptide (acetyl-PSRLEEELRRRLTEP-amide shown in SEQ ID NO: 179) under native conditions failed. Even after competing with 10 column volumes of 200 μM peptide for 90 minutes, >95% of the ALFA ST -shGFP2 protein remained bound to the resin ( Figure 1 B, middle panel, grey line). Therefore, we hypothesized that the dissociation rate of the ALFA ST fusion protein binding to the resin was too low to allow effective competition by the peptide. Therefore, we performed systematic rational mutagenesis to identify an ALFA ST mutant that binds to NbALFA ST strongly enough to allow efficient pull-down of the target protein, but on the other hand can be eluted from the nanobody using the ALFA ST peptide. The peptides analyzed are shown in Table 2. ST ​

[0311] Table 2

[0312]

[0313]

[0314]

[0315]

[0316]

[0317] n.d.: Not determined

[0318] Position: N: N-terminus; M: between two folded domains; C: C-terminus

[0319] Rough estimate, based on an estimated on-rate of 2×10 5 (1 / (M×sec)), and typically observed for sdAb, t < 2 min 1 / 2 corresponding to K > ~30 nM d , t of 2 - 30 min 1 / 2 corresponding to K of ~2 - 30 nM d , t of 20 - 100 min 1 / 2 corresponding to K of ~0.6 - 3 nM d , t > 100 min 1 / 2 corresponding to K of ~1 nM or lower d .

[0320] We have found an ALFA mutant (ALFA PE , where PE stands for Peptide Elutable) (SEQ ID NO: 33) that meets these criteria. The protein fused to ALFA PE binds effectively to the ALFA Selector ST . This binding can even withstand stringent washing steps (e.g., up to 3M NaCl, 1M MgSO4, 4M urea, 1% TX-100 or even 100 mM DTT, Figure 1 C). However, effective elution under native conditions can be achieved within 15 - 20 min at room temperature by competing with 200 μM of the ALFA ST peptide (SEQ ID NO: 179) ( Figure 1 B). Interestingly, two ALFA tag variants (ALFA ST and ALFA PE) can be used at either end of the target protein or even between two protein domains, with only a slight effect on binding to NbALFA ST ( Figure 1 B).

[0321] Example 2: Application of NbALFA ST for Pull - down of ALFA - tagged Target Proteins

[0322] To address the specificity of the ALFA Selector ST , we performed pull - down experiments on complex lysates under physiological conditions. For this purpose, 3 μM of N - terminal ALFA ST - or ALFA PE - tagged shGFP2 (Frey, S. et al. Surface Properties Determining Passage Rates of Proteins through Nuclear Pores. Cell 174, 202–217.e9 (2018)) was incorporated into Escherichia coli or HeLa lysates prepared in PBS. The shGFP2 was expressed in E. coli and purified by nickel - chelating chromatography followed by size - exclusion chromatography. The purified input proteins are shown as Figure 2 A. From all lysates, both fusion proteins bound effectively to the ALFA Selector ST ( Figure 2 B and Figure 2 C). As expected from our previous experiments( Figure 1 ), GFP tagged with ALFA PE was efficiently eluted within 20 minutes using 200 μM of ALFA ST peptide under native conditions, while the ALFA ST - tagged target proteins required more stringent (denaturing) conditions for efficient elution. Surprisingly, the pull - down from both lysates was highly specific. Even after elution with SDS buffer, the number and intensity of detectable impurities from the lysate proteins were low. When the ALFA PE - GFP fusion protein was eluted with ALFA ST peptide, essentially all detectable bands in the eluate fractions obtained from both lysates were the same. Thus, these bands can be attributed to artifacts generated by the target protein itself (e.g., mature bands or DTT - resistant dimers). Interestingly, especially after peptide elution, the eluate fractions contained significantly fewer contaminating proteins than the substrate used as input material (which had been purified by two consecutive chromatography steps; Figure 2 A).

[0323] Example 3: Co-immunoprecipitation using target proteins with an ALFA PE tag and the ALFA Selector ST

[0324] To understand whether the ALFA system can also be used for more refined co-immunoprecipitation experiments, we attempted to pull down the binary Escherichia coli YfgM-PpiD inner membrane protein complex under native conditions ( H. et al. YfgM is an ancillary subunit of the SecYEG translocon in Escherichia coli. J Biol Chem 289, 19089–19097 (2014)). For this purpose, wild-type YfgM or YfgM-ALFA PE was expressed in the YfgMΔ strain. To ensure near-physiological expression levels, both YfgM variants were expressed from low-copy plasmids under the control of the endogenous promoter. When using total lysates containing YfgM-ALFA PE prepared in the presence of the mild non-ionic detergent DDM as input, the ALFA Selector ST was able to pull down the YfgM-PpiD complex in a specific and detergent-resistant manner. This indicates that the ALFA PE tag is compatible with the formation of this labile membrane complex. Importantly, the native and unmodified membrane protein complex could be recovered from the ALFA Selector ST resin within 20 minutes under physiological conditions using 200 μM ALFA ST peptide. YfgM and its interaction partner PpiD were specifically ligated to the ALFA Selector PE via the ALFA ST tag present on the C-terminus of (periplasmic) YfgM, as the complex could not be purified from control lysates expressing untagged YfgM. Thus, the ALFA PE tag together with the ALFA Selector ST resin can not only be used to purify proteins from various sources but is also suitable for the native pull-down of challenging (membrane) protein complexes.

[0325] Example 4: Detection of proteins with an ALFA ST tag by direct immunofluorescence

[0326] We first tested fluorescently labeled NbALFA STWhether it can be used for immunodetection of proteins with an ALFA tag in PFA-fixed samples. In fact, using NbALFA conjugated with two fluorophores (Fluo Tag-X2 anti-ALFA AbberiorStar635P) ST (SEQ ID NO: 133), a specific staining pattern can be obtained, independent of the localization of the ALFA ST tag or the ALFA PE tag on the target protein in mammalian cells. More specifically, we successfully tested that the ALFA tag was placed at the C-terminus (Tom70-EGFP-ALFA ST , Figure 3 C; Tom70-EGFP-ALFA PE , Figure 3 C), N-terminus (ALFA ST -FLAG-vimentin, Figure 3 D and Figure 4 A; ALFA PE -FLAG-vimentin, Figure 4 A) or between the folded domain and the transmembrane domain (EGFP-ALFA ST -TM, Figure 4 B) of the target protein.

[0327] Example 5: Proteins with an ALFA ST tag show normal folding, targeting, and multimeric states

[0328] Importantly, all tested target proteins showed their characteristic localization (Tom70-EGFP-ALFA: outer mitochondrial membrane; ALFA-vimentin: characteristic filamentous structures; EGFP-ALFA ST -TM: plasma membrane), indicating that the ALFF tag does not interfere with the general folding or correct targeting of the tagged protein. ALFA ST -vimentin and ALFA PE -vimentin correctly enter the characteristic intermediate filament structures( Figure 3 D and Figure 4 A) also suggests that the ALFA-tag does not interfere with correct filamentous assembly.

[0329] To more sensitively address whether the ALFA ST tag affects the intracellular localization of the target fusion protein, we statistically analyzed the localization of cytoplasmic EGFP carrying an N- or C-terminal ALFA ST tag transfected into mammalian cells( Figure 5 ). In this assay, with an ALFA at either end STThe nucleocytoplasmic distribution of tagged EGFP was indistinguishable from untagged EGFP. Importantly, we did not observe any signs of atypical association of the ALFA ST fusion proteins to cellular compartments such as membranes or organelles. In addition, gel filtration of the recombinant EGFP variants tagged with ALFA ST confirmed their monomeric state, indicating that the ALFA ST tag does not induce multimerization. Thus, we conclude that the ALFA ST tag generally does not impair the behavior of the target protein. However, we note that (as for any other tag), the specific effects on a given target protein must be analyzed on a protein-by-protein basis.

[0330] Example 6: ALFA tags are compatible with common fixation conditions

[0331] Immunofluorescence (IF) applications generally require optimization of fixation conditions. This can be complex, especially if proteins requiring different fixation conditions are to be localized in the same sample. In addition, existing epitope tags often contain lysines that render them potentially sensitive to modification by amine-reactive fixatives (Table 1). In contrast, the ALFA tag does not contain lysine. For these reasons, the ALFA ST tag and the ALFA PE tag can be detected after standard fixation with 4% paraformaldehyde (PFA) or precipitation fixation with 100% methanol, and are even resistant to 2% glutaraldehyde fixation ( Figure 3 D). Thus, both ALFA tags are compatible with most standard fixation methods and can even prove useful in EM applications where glutaraldehyde is preferred because of its ability to preserve structure at the nanoscale.

[0332] Example 7: In vivo detection of proteins tagged with ALFA ST tag

[0333] We next wanted to know whether NbALFA STCan it also be used as an intracellular antibody? Such nanobodies that are expressed in situ in the cytoplasm (or other compartments) of target cells are commonly used to localize or manipulate target proteins in living cells (Caussinus, E., Kanca, O. & Affolter, M. Fluorescent fusion protein knockout mediated by anti-GFP nanobody. Nat Struct Mol Biol 19, 117–121 (2012); Kirchhofer, A. et al. Modulation of protein properties in living cells using nanobodies. Nat Struct Mol Biol 17, 133–138 (2010)). Such applications depend on the stability and functionality of a given nanobody in the reducing environment of the cytoplasm of eukaryotic host cells. To test our nanobodies under such conditions, we used target proteins tagged with ALFA ST tag (ALFA ST -vimentin or Tom70-EGFP-ALFA ST ) and co-expressed with NbALFA ST fused to mScarlet-I (Bindels, D. S. et al. mScarlet: a bright monomeric red fluorescent protein for cellular imaging. Nat Methods 11, 121–122 (2016)). Indeed, in cells co-transfected with the two constructs, the mScarlet-I signal co-localized tightly with the corresponding target protein tagged with ALFA ST tag ([[]] Figure 3 E).

[0334] Example 8: Western Blot

[0335] To test whether the ALFA tag can be detected by Western blot with fluorescently labeled NbALFA ST , we analyzed vimentin tagged with ALFA ST tag ([[]] Figure 6 A and Figure 7 A) or vimentin tagged with ALFA PE tag ([[]] Figure 6A) Lysates of transfected COS-7 cells. Lysates of cells transfected with a non-related plasmid were used as a control. After polyacrylamide gel electrophoresis (SDS-PAGE) and Western-blotting, NbALFA labeled with IRDye800CW (FluoTag-X2 anti-ALFA IRDye800CW) ST specifically detected vimentin with an ALFA tag. Only a few minor non-specific bands were detected in the control lysate lacking the protein with an ALFA tag ( Figure 7 A).

[0336] For a direct comparison of the performance of NbALFA ST and commonly used monoclonal tools that recognize epitope tags, we prepared maltose-binding protein (MBP) fused to multiple epitope tags (HA, myc, FLAG, and ALFA ST , Figure 6 B). Detection of each epitope tag separately with the same concentration of the primary antibody (or nanobody) showed significant differences in signal intensity and sensitivity ( Figure 6 C, Figure 7 B). The detection signal obtained for the ALFA ST tag using fluorescently labeled NbALF ST was generally 3 - 10 times stronger than the signals obtained for all other epitope tags. This result is particularly remarkable because the detection of monoclonal antibodies (anti-FLAG M2, anti-HAF-7, anti-myc 9E10) involves signal amplification due to the use of polyclonal secondary antibodies, while the detection of the ALFA ST tag relies entirely on directly labeled NbALF ST . Without further optimizing the detection conditions, NbALF ST produced a significantly linear signal of at least three orders of magnitude ( Figure 6 D), and was able to detect as little as 100 pg of the target protein amount. Thus, the detection limit was ~10-fold lower than that observed for all other epitope tags.

[0337] Example 9: Capturing target proteins with an ALFA tag using ALFA Selector resin ST with an ALFA tag

[0338] Next, we specifically immobilized NbALFA ST onto an agarose-based resin with ultra-low background via a hydrophilic and flexible linker site. With an ALFA STGFP variant of the tag (shGFP2; Frey, S. et al. Surface Properties Determining Passage Rates of Proteins through Nuclear Pores. Cell 174, 202–217.e9 (2018); Figure 8 A, B) Analyze the binding to the resin conjugated with nanobody. As expected, shGFP2-ALFA ST binds effectively and tightly to the resulting resin. However, the binding is too strong so that even when using a significantly excessive amount of free ALFA ST peptide, the peptide cannot be competitively eluted from the resin ( Figure 8 A, black solid line). Even after competing with 10 column volumes of 200 μM peptide for 60 minutes, >95% of shGFP2-ALFA ST protein still remains on the resin. Therefore, we named the resin loaded with NbALFA ST "ALFA Selector ST " (ST stands for Super-Tight). Based on the structure of the NbALFA ST -ALFA ST complex, we followed a rational mutagenesis method to identify a weaker NbALFA ST mutant that allows efficient peptide elution while stably binding to the ALFA ST tagged protein in the absence of free ALFA ST peptide. We found a NbALFA mutant, NbALFA PE (PE stands for Peptide Elution) that meets these criteria: the agarose resin loaded with immobilized NbALFA PE (ALFA Selector PE ) binds tightly to shGFP2-ALFA ST . Even after washing for >1 hour, the target protein still stably binds to the resin. However, under natural conditions, at room temperature, upon competition with free ALFA ST peptide, it is effectively released within ~15 - 20 minutes (t 1 / 2 ~3 minutes) ( Figure 8 B and Figure 10 A, black solid line). Similar elution kinetics were observed when the ALFA ST tag was placed between two folded domains ( Figure 10 B), while shGFP2 with an ALFA ST tag at the N-terminus was released from the ALFA Seclector PEelutes slightly faster (t 1 / 2 ~50 s; Figure 10 C). Notably, in the absence of competing peptides, the spontaneous elution of all target proteins from both the ALFA Seclector ST and the ALFA Seclector PE is not significant ([[]] Figure 8 B and Figure 10 , dashed grey lines).

[0339] Example 10: Interaction of ALFA-tagged proteins with ALFA Seclector is compatible with stringent washing.

[0340] We decided to further analyze the biochemical properties of the two ALFA Selector resins. For this purpose, the two resins were loaded with ALFA ST -shGFP2 or shGFP2-ALFA ST , and subjected to a stringent washing step ([[]] Figure 8 D). For all resin and substrate combinations, this interaction is even resistant to stringent washing steps including up to 3 M NaCl, 1 M MgSO4, 2 M guanidine hydrochloride, or 1% non-denaturing detergents (such as TX-100, DDM, or sodium deoxycholate). No dissociation was observed even after incubation with 100 mM DTT at room temperature. Slight differences between different resin / substrate combinations were observed under denaturing conditions: partial release of ALFA ST -shGFP2 and to a lesser extent shGFP2-ALFA ST from the ALFA Seclector PE was observed upon washing with 4 M or 6 M urea, while both target proteins remained tightly bound to the ALFA Seclector ST under the same conditions. Surprising differences were observed after incubation with 0.1% SDS, as the ALFA PE attached to the ALFA Seclector ST -shGFP2 partially lost its fluorescence while remaining bound to the resin. This effect was not observed for any other combination of resin and substrate.

[0341] Example 11: pH tolerance

[0342] In a similar assay, the loaded ALFA Selector resins were washed with buffers adjusted to different pHs ([[]] Figure 8E). The interaction is pH-resistant from 7.5 to 9.5 and only slightly affected at pH 4.5. However, when washed with 100 mM glycine at pH 2.2, even after neutralization, both ALFA Selector resins remained completely non-fluorescent. In contrast, the eluted material regained its fluorescence successfully at neutral pH (not shown), indicating that acidic elution can be performed with pH 2.2 glycine even from tightly bound ALFA Selector. ST Perform acidic elution.

[0343] Example 12: Affinity Estimation

[0344] shGFP2-ALFA ST For NbALFA ST and NbALFA PE The affinity for was determined in solution by microscale thermophoresis (MST). This technique demonstrated that the dissociation constant of NbALFA PE was ~15 nM, while the K ST value of NbALFA ST binding to shGFP2-ALFA d was ~10 pM, which is the detection limit of the device. Assuming a binding rate of ~2×10 5 / M×sec (a rate common in nanobody-target interactions), these values were in good agreement with the dissociation kinetics observed during peptide elution ( Figure 8 B).

[0345] Example 13: Pull-down of ALFA-tagged target proteins from complex lysates ST Labeled target protein

[0346] To address the specificity of our ALFA Selector resin, we performed pull-down experiments on complex lysates under physiological conditions ([[]] Figure 9 A-C). For this purpose, 3 μM of recombinant purified ALFA ST -shGFP2 was incorporated into E. coli or HeLa lysates prepared in PBS ([[]] Figure 9 A). The fusion protein specifically bound to both ALFA Selector resins but not to the control resin without conjugated nanobody ("Selector control"). As expected based on our previous experiments, ALFA ST -shGFP2 was efficiently eluted from ALFA Selector ST using 200 μM of ALFA PE peptide under native conditions. In contrast, elution from ALFA Selector STSuccessful elution. Surprisingly, the pull-down from both E. coli and HeLa lysates was highly specific ( Figure 9 B and C). After eluting peptides from the ALFA Selector PE , essentially all visible bands could be attributed to the input protein, and even in the SDS eluate, the amount and intensity of detectable contaminants from the lysate proteins were low. In fact, the ALFA PE -shGFP2 obtained after one step of peptide elution from the ALFA Selector ST contained significantly fewer contaminants compared to the protein used for spiking the input lysate ( Figure 9 A). This observation was particularly striking since the input protein was purified by two consecutive chromatography steps.

[0347] Example 14: Co-immunoprecipitation using the ALFA Selector PE resin

[0348] To understand whether the ALFA system could also be applied to more refined co-immunoprecipitation experiments, we attempted to pull down the binary E. coli inner membrane protein complex YfgM-PpiD under native conditions ( H. et al. YfgM is an ancillary subunit of the SecYEG translocon in Escherichia coli. J Biol Chem 289, 19089–19097 (2014)) ( Figure 9 D). For this purpose, wild-type YfgM or YfgM-ALFA ST was expressed in the yfgMΔ strain. To ensure near-physiological expression levels, both YfgM variants were expressed from low-copy plasmids under the control of the endogenous promoter. When using the total lysate containing YfgM-ALFA ST prepared in the presence of the mild non-ionic detergent DDM as input, the ALFA Selector PE was able to pull down the YfgM-PpiD complex in a specific and detergent-resistant manner. This indicates that the ALFA ST tag is compatible with the formation of this unstable membrane complex. Importantly, the native and unmodified membrane protein complex could be recovered from the ALFA Selector ST resin within 20 minutes under physiological conditions using 200 μM ALFA PE peptide. YfgM and its interaction partner PpiD interact with the ALFA Selector ST via the AlfA tag present at the C-terminus of (periplasmic) YfgMPE Specific ligation, as the complex could not be purified from control lysates expressing untagged YfgM. Thus, the ALFA ST tag together with the ALFA Selector PE resin can be used not only for purifying proteins from various sources but also for native pull-down of challenging (membrane) protein complexes.

[0349] Example 15: Isolation of viable lymphocytes

[0350] ALFA Selector PE One envisioned application of the ALFA Selector is cell-specific enrichment under physiological conditions. This is particularly relevant for, e.g., generating chimeric antigen receptor-modified T (CAR-T) cells, the precursors of which are typically obtained from blood (Tokarew, N. et al. Teaching an old dog new tricks: next-generation CAR T cells. Br. J. Cancer (2018). doi:10.1038 / s41416-018-0325-1). To examine whether the ALFA system can be used to enrich viable blood cells, human peripheral blood mononuclear cells (PBMCs) were passed through an ALFA Selector PE column pre-loaded with an ALFA-tagged nanobody recognizing CD62L, a surface marker typically present on immature T cells ( L. Development, trafficking, and function of memory T-cell subsets. Immunological Reviews (2006). doi:10.1111 / j.0105-2896.2006.00393.x)( Figure 12 a). After washing, the bound cells were eluted with an ALFA ST peptide, stained with antibodies recognizing CD62L, the pan-T cell marker CD3, and the pan-B cell marker CD19, and analyzed by FACS ( Figure 12 ). Total PBMCs were used as a control. Using this strategy, the enrichment of CD62L+ lymphocytes changed from 71.8% to 97.7% ( Figure 12 B). Additionally, we confirmed that the majority of the cells eluted with the ALFA peptide were CD3-positive T cells, while B cells represented a minor population of the isolated cells ( Figure 12 C).

[0351] Discussion

[0352] We report the development and initial characteristics of the ALFA system. The system consists of the ALFA ST tag (a new highly versatile epitope tag), its mutant variant (the ALFA PE tag), and a set of related single-domain antibodies (nanobodies) that recognize the ALFA ST tag with either extremely high or moderate affinity, respectively. Importantly, the rational approach chosen allowed us to endow the ALFA system with features crucial for its general applicability. When selecting the ALFA tag sequence, the preferred tag is small, lysine-free, carries no net charge but is hydrophilic, and is not present in the proteomes of relevant model organisms, yet the tag will adopt a stable fold in solution. As a result, the ALFA tag is preferably highly specific by design, insensitive to amine-reactive fixatives, generally well tolerated by the tagged target protein, and can be easily refolded after denaturation.

[0353] As conjugates, we prefer nanobodies because, compared to conventional antibodies, they are small, monovalent, and durable probes that can be easily modified genetically and recombinantly produced in various expression systems. Thus, nanobodies can be site-specifically immobilized or fluorescent labels quantitatively introduced (Pleiner, T. et al. Nanobodies: site-specific labeling for super-resolution imaging, rapid epitope-mapping and native protein complex isolation. Elife 4, (2015)). NbALFA ST is our preferred high-affinity nanobody that recognizes the ALFA ST tag and can thus be conveniently used, for example, in direct immunofluorescence. Due to the small size of nanobodies (diameter ∼3 - 4 nm) and the defined number and position of attached dyes, fluorescently labeled NbAlFA ST is an ideal tool for high-resolution or quantitative imaging. We could demonstrate that NbALFA ST indeed interacts with various ALFA ST tagged target proteins expressed in mammalian cells. Importantly, NbALFA STIt can even fold in the cytoplasm of eukaryotic cells and can thus be used for in vivo detection or manipulation of target proteins carrying the ALFA tag (Rothbauer, U. et al. Targeting and tracing antigens in live cells with fluorescent nanobodies. Nat Methods 3, 887–889 (2006); Kirchhofer, A. et al. Modulation of protein properties in living cells using nanobodies. Nat Struct Mol Biol 17, 133–138 (2010); R. et al. Intracellular Delivery of Nanobodies for Imaging of Target Proteins in Live Cells. Pharm. Res. (2016). doi:10.1007 / s11095-016-2052-8). This finding is consistent with our biochemical evidence, which shows that NbALFA ST is at least tolerant to 100 mM DTT at room temperature and suggests that the conserved internal disulfide bonds common to all nanobodies are largely unnecessary for reliable interaction with the ALFA ST tag. For intracellular antibody applications, we found that the expression of NbALFA ST fused to a fluorescent reporter molecule under the control of the CMV or PGK promoter and the target protein both gave good results with low background. For optimal results or for detecting low-abundance target proteins carrying the ALFA ST tag, a more precise titration of the relative expression levels may be required.

[0354] Generally, most nanobodies recognize three-dimensional epitopes on the surface of their target proteins and thus do not recognize denatured proteins (e.g., in Western blotting). We were able to demonstrate that NbALFA ST is an exception to this rule as it can also be used for highly sensitive detection of target proteins in Western blotting applications. This fact suggests that the ALFA ST and ALFA PE tags can refold efficiently after transfer to the membrane and removal of SDS. A direct comparison shows that—despite its monovalent binding mode—in terms of absolute signal intensity and limit of detection, NbALFA STSignificantly outperforms existing monoclonal anti-epitope tagging tools. We envision similar advantages in other applications requiring high sensitivity such as ELISA or microarray analysis. Due to the tolerance to amine-reactive fixatives, we believe it may also be possible to apply the ALFA system to immunoelectron microscopy applications in the future.

[0355] NbALFA ST has a very high affinity for the ALFA ST tag. While this is ideal for high-profile imaging applications and high-sensitivity detection, it hinders elution under physiological conditions within a reasonable time frame and thus limits its use in biochemical applications. Therefore, we aimed to reduce the affinity of the nanobody for its substrate without affecting its specificity. We investigated this through two different approaches: 1) We screened a large number of ALFA peptides to reduce the binding strength. This approach identified an ALFA PE peptide that binds NbALFA ST effectively but can be eluted efficiently by competing with free ALFA ST peptide. 2) Based on the crystal structure of NbALFA ST complexed with the ALFA ST peptide, we introduced specific mutations in NbALFA ST that successfully increased the dissociation rate to a level that allows efficient elution of the peptide under physiological conditions. When immobilized on agarose resin with low background binding, the mutant nanobody (NbALFA PE ) was shown to be ideally suited for native purification of proteins and protein complexes from various lysates under physiological conditions. Subsequently, in special applications that require stringent washing with up to 6 M urea or up to 0.1% SDS, or when removing very low-abundance proteins from dilute lysates, the ALFA Selector ST of the wild-type high-affinity nanobody (NbALFA ST ) can have an advantage. However, elution from the ALFA Selector ST requires drastic denaturation or acidic elution (e.g., 1% SDS or glycine pH 2.2), which is generally incompatible with the native conformation of the target protein.

[0356] NbALFA ST The structure of the complex with the ALFA ST peptide indicates that NbALFA ST recognizes the α-helical conformation of the ALFA ST peptide. To minimize the potential influence of adjacent secondary structures on the ALFA ST tag conformation, we minimized the core ALFA STThe sequence (SRLEEELRRRLTE, SEQ ID NO: 04) is placed between two prolines and serves as an "insulator". Using this method, NbALFA ST interacts with ALFA ST tags largely independently of the tag's position in the protein, i.e., NbALFA ST recognizes ALFA PE tags located at the N- and C-termini of the target protein or even within two protein domains, and ST so does NbALFA

[0357] In summary, we herein provide a novel epitope tag system with particularly broad applicability. Using the ALFA system, a single transgenic cell line or organism carrying a target protein with an ALFA ST tag is sufficient for a large number of different applications, including (super-resolution) imaging, in vivo manipulation of proteins, in vitro detection by Western blotting, or even native pull-down applications aimed at detecting specific interaction partners. ALFA Selector PE can even be used to selectively enrich CD62L-positive lymphocytes from PBMC preparations ( Figure 12 ). We believe that this technology can be easily transferred to well-established recombinant Fab and scFv fragments currently used for cell separation methods and similar purposes (Mohr, F. et al. Minimally manipulated murine regulatory T cells purified by reversible Fab Multimers are potent suppressors for adoptive T-cell therapy. Eur. J. Immunol. (2017). doi:10.1002 / eji.201747137), or to novel nanobodies that recognize surface markers and can be easily conjugated with an ALFA tag. Thus, our new technology can contribute to recent advances in biomedical research and therapy, including CAR-T technology (Tokarew, N. et al. Teaching an old dog new tricks: next-generation CAR T cells. Br. J. Cancer (2018). doi:10.1038 / s41416-018-0325-1). We are highly confident that, due to its broad range of applications, the ALFA system is an important contribution that will significantly stimulate the scientific community.

[0358] Materials and Methods

[0359] Transfection of 3T3 and COS-7 cells

[0360] For immunofluorescence experiments, 3T3 or COS-7 cells were transiently transfected with the appropriate plasmids listed in Table 3 using a Polyjet transfection kit (SignaGen) according to the manufacturer's recommendations. Briefly, for each experiment, cells were seeded on 12-well plates. The volume was adjusted according to the well size. 1 μg of each plasmid was premixed with 38 μl of serum-free medium and subsequently supplemented with Polyjet transfection reagent diluted in 38 μl of serum-free medium. The suspension was incubated at room temperature for 15 minutes and then added dropwise to the cells. The cells were incubated at 37 °C and 5% CO2 for 24 hours. For co-expression experiments, plasmid DNAs were premixed at a 1:1 ratio and further processed as described above.

[0361] Fixation and staining of COS-7 cells

[0362] Transiently transfected cells were fixed at room temperature for 30 minutes in 4% paraformaldehyde (PFA) (w / v) or 2% glutaraldehyde (GA) (v / v) 24 hours after transfection. Alternatively, they were fixed in ice-cold methanol at -20 °C for 15 minutes. At room temperature, cells were blocked and permeabilized in PBS containing 10% normal goat serum (v / v) and 0.1% Triton-X 10 (v / v) for 15 minutes. Fluorescently labeled NbALFA ST (Fluo Tag-X2 anti-ALFA AbberiorStar635P, NanoTag Biotechnologies N1502-Ab635P-L) was diluted 1:500 in PBS containing 3% normal goat serum and 0.1% Triton-X 100 (v / v). The cells were incubated in this staining solution at room temperature for 1 hour, followed by 3 washes with PBS for 5 min. To stain the nuclei, DAPI (0.4 μg / ml) was added in one of the PBS wash steps. Coverslips were mounted on slides with MOWIOL solution, dried at 37 °C, and imaged using an epifluorescence microscope (Axio, Zeiss) equipped with a 20× lens. Constructs expressed on the cell surface were co-transfected with anti-FLAG M2 (primary antibody, Sigma, F1804) and Fluo Tag-X2 anti-mouse IgG Atto488 (secondary nanobody, NanoTag, N1202-At488-L) diluted 1:1000 and 1:5000, respectively, in PBS containing 3% normal goat serum and 0.1% Triton-X 100 (v / v).

[0363] Effect of the ALFA tag on EGFP localization

[0364] Transiently transfected 3T3 cells were imaged using an epifluorescence microscope (Axio, Zeiss) equipped with a 40×1.3 oil immersion lens. For cells transfected with pCMV ALFA ST -EGFP, pCMV EGFP-ALFA ST or pEGFP-N1, 107 - 133 cells were imaged on a total of 6 - 7 individual images. For each individual image, cells were grouped and counted according to the localization of EGFP (“slight nuclear localization”, “evenly distributed”, “other”). The portions of cells in each group were statistically analyzed using Student's t-test.

[0365] Western blot with COS-7 lysates

[0366] Transfected cells from a confluent 10 cm Petri dish were washed with PBS and lysed in 2 ml of SDS sample buffer. The lysates were resolved by SDS-PAGE and transferred to nitrocellulose membranes. After blocking with 5% milk powder in TBS-T, the membranes were incubated with mouse anti-tubulin (SYSY#302 211; 1:1000 dilution), followed by incubation with FluoTag-X2 anti-mouse IgG IRDye680 (NanoTag Biotechnologies#N1202; 1:1000 dilution) and FluoTag-X2 anti-ALFA IRDye800 (NanoTag Biotechnologies#1502; 1:1000 dilution). The membranes were scanned using Odyssey CLx (Li-COR).

[0367] Sensitivity assay

[0368] Serial dilutions of MBP fused to the FLAG, HA, myc, and ALFA ST tags were prepared in PBS at pH 7.4 containing 0.1 μg / mL BSA. 1 μl of each dilution was spotted onto nitrocellulose membranes.

[0369] The membranes were blocked and washed with TBS-T containing 5% milk powder. Existing monoclonal antibodies (anti-FLAG M2-Signa#F1804, anti-myc 9E10-SynapticSystems#343 011, anti-HA F-7-SantaCrus#sc-7392) were used in combination with secondary goat anti-mouse IgG IRDye800CW (Li-COR#925-32210, 1:500 dilution) to detect the FLAG, myc, and HA tags, respectively. ALFA STThe tags were detected with FluoTag-X2 anti-ALFA (NanoTag Biotechnologies #N1502) directly conjugated to IRDye800CW. The final concentration of all primary antibodies and nanobodies used was 2.7 nM. MBP was detected with a rabbit polyclonal serum (SynapticSystem) recognizing MBP, and anti-rabbit IgG IRDye680RD (Li-COR #925-68071) was used as an internal loading control. The membranes were scanned with an Odyssey CLx (Li-COR). Quantification was performed using ImageStudioLight (Li-COR).

[0370] Dissociation rate measurement

[0371] 20 μl ALFA Selector ST or ALFA Selector PE (NanoTag Biotechnologies) was saturated with the respective recombinant target protein. After washing 4 times with PBS, the beads were suspended in PBS containing a 10-fold excess of 200 μM free ALFA ST peptide and mixed at 25 °C. Control reactions were performed without peptide. At the designated time points, the specific elution from the beads was quantified by the GFP fluorescence released into the supernatant (Q-Bit 3.0; Thermo-Fischer Scientific). Three independent experiments were performed in parallel. The mean, standard deviation, and exponential fit were calculated using Graphpad Prism 5.0. Photos were taken under UV illumination using a Nikon D700 (Nikon) equipped with a 105 mm macro lens.

[0372] Tolerance to stringent washing and pH

[0373] According to the experiment, 10 - 15 μl of ALFA Selector saturated with the indicated ALFA-tagged shGFP2 fusion protein ST or ALFA Selector PE was washed with PBS and incubated with 100 μl of the indicated substance for 60 minutes at room temperature. After the beads settled, photos were taken under UV illumination. To determine the pH tolerance, the same beads were incubated with 150 mM NaCl buffered to different pHs (100 mM glycine - HCl, pH 2.2; 100 mM sodium acetate pH 4.5; 100 mM Tris - HCl pH 7.5; 100 mM carbonate pH 9.5) at room temperature for 30 minutes. The resin was washed twice with the same buffer. Photos were taken after equilibration with PBS several times.

[0374] One-step affinity purification using ALFA Selector resin.

[0375] To obtain defined input material for pull-down experiments from E. coli or HeLa lysates, the corresponding mock lysates were mixed with 3 μM of the indicated purified ALFA-tagged shGFP2 variants. 1 mL of each lysate / substrate mixture was incubated with 25 μL of ALFA Selector ST or ALFA Selector PE at 4 °C for 1 h. Depending on the experimental setup, a similar resin without immobilized sdAb (Selector control) or a mock lysate without the target protein was used as a specificity control. After washing three times with 600 μL of PBS, the resin was transferred to a MiniSpin column (NanoTag Biotechnologies). The excess buffer was removed by centrifugation (3000×g, 30 sek), and then the resin was incubated twice for 10 min each with 50 μL of 200 μM ALFA ST peptide in PBS at room temperature. The proteins retained on the beads were then eluted with SDS sample buffer. 0.5 μL (E. coli) or 5 μL (HeLa) of input and the unbound fractions were separated by SDS-PAGE (12%) and Coomassie staining. The indicated eluate fractions correspond to the material eluted from 1 μL of each resin.

[0376] Pull-down of YfgM using ALFA Selector PE The yfgM deletion strain was complemented with C-terminally ALFA

[0377] tagged or untagged YfgM expressed from a pSC-based low-copy vector under the control of the endogenous promoter. On ice, membrane protein complexes were solubilized from total lysates prepared with buffer LS (50 mM Tris pH 7.5, 300 mM NaCl, 5 mM MgCl2) using 1% DDM in 1 h (Maddalo, G. et al. Systematic analysis of native membrane protein complexes in Escherichia coli. J Proteome Res 10, 1848–1859 (2011)). The two lysates were incubated with 20 μL of ALFA Selector ST resin on a rotator at 4 °C for 1 h. After washing in PBS + 0.3% DDM, followed by incubation with 50 μL of 200 μM ALFA PE resin STThe peptide was incubated twice successively with PBS, and the bound protein was eluted under native conditions. Samples corresponding to 1 / 800 of the input and unbound material or 1 / 80 of the eluate fractions were separated by SDS-PAGE. Analysis was performed by Western blotting using polyclonal rabbit anti-serum against the YfgM-PpiD complex ( H. et al. YfgM is an ancillary subunit of the SecYEG translocon in Escherichia coli. J Biol Chem 289, 19089–19097 (2014)) followed by HRP-conjugated goat anti-rabbit IgG (Dianova). Blots were developed using the Western Lightning Plus-ECL kit (Perkin Elmer) and imaged using a LAS 4000 mini luminescent imager (Fuji Film).

[0378] Using ALFA Selector ST Pull down YfgM

[0379] The yfgM deletion strain was complemented with C-terminally ALFA PE tagged or untagged YfgM expressed from a pSC-based low-copy vector under the control of the endogenous promoter. On ice, membrane protein complexes were solubilized from total lysates prepared with buffer LS (50 mM Tris pH 7.5, 300 mM NaCl, 5 mM MgCl2) using 1% DDM for 1 h (Maddalo, G. et al. Systematic analysis of native membrane protein complexes in Escherichia coli. J Proteome Res 10, 1848–1859 (2011)). The two lysates were incubated with 20 μl of ALFA ST Selector resin on a rotator at 4 °C for 1 h. After washing in PBS + 0.3% DDM, the bound protein was eluted under native conditions following incubation twice successively with 50 μl of PBS containing 200 μM ALFA ST peptide. Samples corresponding to 1 / 800 of the input and unbound material or 1 / 80 of the eluate fractions were separated by SDS-PAGE. Analysis was performed by Western blotting using polyclonal rabbit anti-serum against the YfgM-PpiD complex ( H. et al. YfgM is an ancillary subunit of the SecYEG translocon in Escherichia coli. J Biol Chem 289, 19089–19097 (2014)) was followed by HRP-conjugated goat anti-rabbit IgG (Dianova) and analyzed by Western blotting. The blot was developed using the Western Lightning Plus-ECL kit (Perkin Elmer) and imaged using a LAS 4000 mini luminescent imager (Fuji Film).

[0380] Table 3: Plasmids

[0381]

[0382]

[0383] (a) H. et al. YfgM is an ancillary subunit of the SecYEG translocon in Escherichia coli. J Biol Chem 289, 19089–19097 (2014)

[0384] Escherichia coli strains

[0385] Escherichia coli MC4100ΔyfgMΔppiD( H. et al. YfgM is an ancillary subunit of the SecYEG translocon in Escherichia coli. J Biol Chem 289, 19089–19097 (2014))

[0386] Table 4 Antibodies

[0387]

[0388]

[0389] (a) H. et al. YfgM is an ancillary subunit of the SecYEG translocon in Escherichia coli. J Biol Chem 289, 19089–19097 (2014)

[0390] Table 5 Fusion proteins

[0391]

[0392]

[0393]

[0394]

[0395]

[0396]

[0397]

[0398]

[0399] Protein expression and purification

[0400] All recombinant proteins were expressed from an expression vector with a ColE1 origin conferring kanamycin resistance under the control of the Tac-promoter.

[0401] MBP fusion proteins carrying multiple epitope tags, ALFA ST -shGFP2, ALFA PE -shGFP2 and TwinStrepTag-bdNEDD8-ALFA min were expressed as N-terminal His 14 -bdSUMO fusions. For protein expression, Escherichia coli was cultured in Terrific broth (TB) supplemented with 0.3 mM IPTG at 23 °C for 14–16 h. After harvesting, Escherichia coli cells were lysed with LS buffer (50 mM Tris / HCl pH 7.5, 300 mM NaCl) supplemented with 15 mM imidazole / HCl pH 7.5 and 10 mM DTT and purified by binding to Ni(II)-chelating beads. After thorough washing, the proteins were eluted by on-column cleavage with bdSENP1 as described previously (Frey, S. and D. A new set of highly efficient, tag-cleaving proteases for purifying recombinant proteins. J Chromatogr A 1337, 95–105 (2014); Frey, S. and D. Purification of protein complexes of defined subunit stoichiometry using a set of orthogonal, tag-cleaving proteases. J Chromatogr A 1337, 106–115 (2014)).

[0402] ALFA ST -shGFP2-His6, ALFA PE -shGFP2-His6, His 14 -bdSUMO-ALFA ST -shsfGFP and His 14 -bdSUMO-ALFA PE -shsfGFP was expressed and purified in a similar manner; however, elution was performed using 250 mM imidazole in buffer LS.

[0403] For affinity assays and binding studies from complex lysates, the substrate protein was additionally purified by size-exclusion chromatography on a Superdex 200 10 / 30 column (GE Healthcare).

[0404] Selection of specific sdAb clones by B cell “Celline” affinity purification

[0405] 1 mL of T-Catch resin (IBA Lifesciences) was washed with B cell isolation buffer (PBS pH 7.4, 1% BSA, 1 mM EDTA) and saturated with a quantity of TwinStrepTag-bdNEDD8-ALFA min fusion protein and incubated with rolling at room temperature for 30 min. The resin was cleared from excess bait protein by thorough washing with B cell isolation buffer. A 100 mL blood sample was taken from an alpaca immunized with the ALFA peptide fusion and immediately incubated with 5000 IU / ml heparin (Sigma) to prevent clotting. PBMC were isolated from fresh blood (within 4 h of sampling) using Ficoll-Paque PLUS (GE Healthcare). To remove residual serum, PBMC were washed three times successively with B cell isolation buffer. PBMC were passed over the loaded T-Catch resin three times before washing the resin with 10 column volumes of B cell isolation buffer. By incubating at room temperature with 2 μM NEDP1 (Frey, S. and D. A new set of highly efficient, tag-cleaving proteases for purifying recombinant proteins. J Chromatogr A 1337, 95–105 (2014); Frey, S. and D. Purification of protein complexes of defined subunit stoichiometry using a set of orthogonal, tag-cleaving proteases. J Chromatogr A 1337, 106–115 (2014)) 30 minutes, elute the bound B cells from the resin. A sdAb-specific cDNA library was amplified from the eluted B cells by multi-step nested RT-PCR and cloned into a bacterial expression vector. Expression of 96 monoclonal ALFA-reactive sdAbs was tested by ELISA.

[0406] Preparation of human PBMCs

[0407] Human peripheral blood mononuclear cells (PBMCs) were obtained from fresh blood using standard density gradient centrifugation. Briefly, 60 mL of fresh blood was diluted with 40 mL of phosphate-buffered saline (PBS) supplemented with 1 mM EDTA and placed in a 50 mL LEUCOSEP tube (Greiner Bio-One) on top of CELLPURE Roti-Sep 1077 (Carl Roth) and centrifuged at 800 × g for 20 minutes at room temperature. Subsequently, the layer containing PBMCs was collected and washed 5 times in cold PBS + EDTA to remove platelets.

[0408] Isolation of CD62L-positive lymphocytes

[0409] Approximately 2 × 10 7 PBMCs were passed by gravity flow through an ALFASelector PE resin loaded with an ALFA-tagged anti-human CD62L nanobody and then washed thoroughly with PBS supplemented with 1 mM EDTA 1 and 1% (w / v) bovine serum albumin. Subsequently, the bound cells were eluted in the same buffer containing 200 μM ALFA peptide.

[0410] The invention described illustratively herein can be practiced appropriately in the absence of one or more elements, one or more limitations not specifically disclosed herein. Additionally, the terminology and phraseology used herein have been used as terms of description and not of limitation, and in using such terminology and phraseology, it is not intended to exclude any equivalents of the features shown and described or portions thereof, but it should be recognized that various modifications are within the scope of the claimed invention. Accordingly, it should be understood that although the invention has been specifically disclosed by way of exemplary embodiments and optional features, those skilled in the art can make improvements and variations to the invention as practiced herein, and such improvements and variations are considered to be within the scope of the invention.

[0411] The invention is described herein in general and generic terms. Each of the more specific types and subgeneric groups that fall within the general disclosure also forms part of the invention. This includes the general description of the invention, with the proviso or negative limitation that removes any subject matter from the genus, whether or not the deleted material is specifically recited herein.

[0412] Other embodiments are within the scope of the appended claims. Additionally, when features or aspects of the invention are described in terms of a Markush group, those skilled in the art will recognize that the invention is also thereby described in terms of any individual member or subgroup of members of the Markush group. Sequence Listing <110> Nanotech Biotech Co., Ltd. <120> Epitope Tags Recognized by Specific Binding Agents <130> LC21310006P <150> EP18193663.4 <151> 2018-09-11 <150> EP19160485.9 <151> 2019-03-04 <160> 199 <170> PatentIn version 3.5 <210> 1 <211> 13 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Peptide <220> <221> misc_feature <222> (1)..(9) <223> Xaa can be any naturally occurring amino acid <220> <221> misc_feature <222> (12)..(13) <223> Xaa can be any naturally occurring amino acid <400> 1 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Arg Leu Xaa Xaa 1 5 10 <210> 2 <211> 13 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptide <220> <221> MISC_FEATURE <222> (1)..(1) <223> Xaa is G or S or P or T <220> <221> MISC_FEATURE <222> (2)..(2) <223> Xaa is R or G or P <220> <221> MISC_FEATURE <222> (5)..(5) <223> Xaa is E or Q <220> <221> misc_feature <222> (7)..(7) <223> Xaa is L or I <220> <221> MISC_FEATURE <222> (12)..(12) <223> Xaa is S or T or P or A or D or E <220> <221> MISC_FEATURE <222> (13)..(13) <223> Xaa is P or A or S or A or D or E or no amino acid <400> 2 Xaa Xaa Leu Glu Xaa Glu Xaa Arg Arg Arg Leu Xaa Xaa 1 5 10 <210> 3 <211> 13 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptide <220> <221> MISC_FEATURE <222> (1)..(1) <223> Xaa is S or T <220> <221> MISC_FEATURE <222> (2)..(2) <223> Xaa is R or G <220> <221> MISC_FEATURE <222> (5)..(5) <223> Xaa is E or Q <220> <221> MISC_FEATURE <222> (12)..(12) <223> Xaa is T or D or E <220> <221> MISC_FEATURE <222> (13)..(13) <223> Xaa is A or D or E or no amino acid <400> 3 Xaa Xaa Leu Glu Xaa Glu Leu Arg Arg Arg Leu Xaa Xaa 1 5 10 <210> 4 <211> 13 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptide <400> 4 Ser Arg Leu Glu Glu Glu Leu Arg Arg Arg Leu Thr Glu 1 5 10 <210> 5 <211> 16 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Peptide <400> 5 Met Pro Ser Arg Leu Glu Glu Glu Leu Arg Arg Arg Leu Thr Glu Pro 1 5 10 15 <210> 6 <211> 15 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Peptide <400> 6 Pro Ser Arg Leu Glu Glu Glu Leu Arg Arg Arg Leu Thr Glu Pro 1 5 10 15 <210> 7 <211> 14 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Peptide <400> 7 Pro Ser Arg Leu Glu Glu Glu Leu Arg Arg Arg Leu Thr Glu 1 5 10 <210> 8 <211> 15 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Peptide <400> 8 Gly Arg Ser Arg Leu Glu Glu Glu Leu Arg Arg Arg Leu Thr Ala 1 5 10 15 <210> 9 <211> 16 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Peptide <400> 9 Pro Gly Ser Arg Leu Glu Glu Glu Leu Arg Arg Arg Leu Thr Ala Pro 1 5 10 15 <210> 10 <211> 16 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Peptide <400> 10 Pro Ser Thr Arg Leu Glu Glu Glu Leu Arg Arg Arg Leu Thr Ala Pro 1 5 10 15 <210> 11 <211> 16 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Peptide <400> 11 Ser Pro Ser Arg Leu Glu Glu Glu Leu Arg Arg Arg Leu Thr Ala Pro 1 5 10 15 <210> 12 <211> 16 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Peptide <220> <221> MISC_FEATURE <222> (1)..(1) <223> Xaa is G or S or T or P <220> <221> MISC_FEATURE <222> (2)..(2) <223> Xaa is R or G or A or E or P <220> <221> MISC_FEATURE <222> (3)..(3) <223> Xaa is L or V <220> <221> MISC_FEATURE <222> (4)..(4) <223> Xaa is E or Q <220> <221> MISC_FEATURE <222> (5)..(5) <223> Xaa is E or Q <220> <221> MISC_FEATURE <222> (6)..(6) <223> Xaa is E or Q <220> <221> MISC_FEATURE <222> (7)..(7) <223> Xaa is L or I or V <220> <221> MISC_FEATURE <222> (8)..(8) <223> Xaa is R or A or Q or E <220> <221> MISC_FEATURE <222> (9)..(9) <223> Xaa is R or A or Q or E <220> <221> MISC_FEATURE <222> (12)..(12) <223> Xaa is S or T or D or E or P or A or no amino acid <220> <221> MISC_FEATURE <222> (13)..(13) <223> Xaa is E or K or P or S or A or D or no amino acid <400> 12 Ser Pro Ser Arg Leu Glu Glu Glu Leu Arg Arg Arg Leu Asp Ala Pro 1 5 10 15 <210> 13 <211> 16 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptide <400> 13 Ser Pro Ser Arg Leu Glu Glu Glu Leu Arg Arg Arg Leu Glu Ala Pro 1 5 10 15 <210> 14 <211> 16 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptide <400> 14 Ser Pro Ser Arg Leu Glu Glu Glu Leu Arg Arg Arg Leu Thr Asp Pro 1 5 10 15 <210> 15 <211> 16 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptide <400> 15 Ser Pro Ser Arg Leu Glu Glu Glu Leu Arg Arg Arg Leu Thr Glu Pro 1 5 10 15 <210> 16 <211> 17 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptide <400> 16 Ser Pro Ser Arg Leu Glu Glu Glu Leu Arg Arg Arg Leu Thr Ala Asp 1 5 10 15 Pro <210> 17 <211> 16 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptide <400> 17 Ser Pro Ser Gly Leu Glu Glu Glu Leu Arg Arg Arg Leu Thr Glu Pro 1 5 10 15 <210> 18 <211> 14 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptide <400> 18 Gly Pro Ser Arg Leu Glu Glu Glu Leu Arg Arg Arg Leu Thr 1 5 10 <210> 19 <211> 15 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptide <400> 19 Gly Pro Ser Arg Leu Glu Glu Glu Leu Arg Arg Arg Leu Thr Ala 1 5 10 15 <210> 20 <211> 16 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptide <400> 20 Gly Pro Ser Arg Leu Glu Glu Glu Leu Arg Arg Arg Leu Thr Ala Ala 1 5 10 15 <210> 21 <211> 17 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptide <400> 21 Gly Pro Ser Arg Leu Glu Glu Glu Leu Arg Arg Arg Leu Thr Ala Ala 1 5 10 15 Ser <210> 22 <211> 16 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptide <400> 22 Ser Pro Ser Gly Leu Glu Gln Glu Leu Arg Arg Arg Leu Thr Ala Pro 1 5 10 15 <210> 23 <211> 16 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptide <400> 23 Ser Pro Ser Gly Leu Glu Gln Glu Leu Arg Arg Arg Leu Asp Ala Pro 1 5 10 15 <210> 24 <211> 16 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptide <400> 24 Ser Pro Ser Gly Leu Glu Gln Glu Leu Arg Arg Arg Leu Glu Ala Pro 1 5 10 15 <210> 25 <211> 16 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptide <400> 25 Ser Pro Ser Gly Leu Glu Gln Glu Leu Arg Arg Arg Leu Thr Glu Pro 1 5 10 15 <210> 26 <211> 16 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptide <400> 26 Gly Pro Ser Arg Leu Glu Glu Glu Leu Arg Arg Arg Leu Thr Ala Pro 1 5 10 15 <210> 27 <211> 16 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptide <400> 27 Gly Pro Ser Arg Leu Glu Glu Glu Leu Arg Arg Arg Leu Thr Glu Pro 1 5 10 15 <210> 28 <211> 15 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptide <400> 28 Gly Pro Ser Arg Leu Glu Glu Glu Leu Arg Arg Arg Leu Thr Glu 1 5 10 15 <210> 29 <211> 15 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptide <400> 29 Met Ser Arg Leu Glu Glu Glu Leu Arg Arg Arg Leu Thr Glu Pro 1 5 10 15 <210> 30 <211> 16 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptide <400> 30 Met Ser Ser Arg Leu Glu Glu Glu Leu Arg Arg Arg Leu Thr Glu Pro 1 5 10 15 <210> 31 <211> 13 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptide <220> <221> MISC_FEATURE <222> (1)..(1) <223> Xaa is G or S or P <220> <221> MISC_FEATURE <222> (2)..(2) <223> Xaa is R or G <220> <221> MISC_FEATURE <222> (5)..(5) <223> Xaa is E or Q <220> <221> MISC_FEATURE <222> (7)..(7) <223> Xaa is L or I <220> <221> MISC_FEATURE <222> (12)..(12) <223> Xaa is S or T or P or A <220> <221> MISC_FEATURE <222> (13)..(13) <223> Xaa is P or A or S or no amino acid <400> 31 Xaa Xaa Leu Glu Xaa Glu Xaa Arg Arg Arg Leu Xaa Xaa 1 5 10 <210> 32 <211> 12 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptide <400> 32 Gly Arg Leu Glu Glu Glu Leu Arg Arg Arg Leu Ser 1 5 10 <210> 33 <211> 15 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptide <400> 33 Met Ser Gly Arg Leu Glu Glu Glu Leu Arg Arg Arg Leu Ser Pro 1 5 10 15 <210> 34 <211> 16 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptide <400> 34 Ser Asp Ser Gly Leu Glu Gln Glu Leu Arg Arg Arg Leu Ser Pro Gly 1 5 10 15 <210> 35 <211> 16 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptide <400> 35 Pro Asp Gly Gly Leu Glu Gln Glu Leu Arg Arg Arg Leu Thr Ala Pro 1 5 10 15 <210> 36 <211> 16 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptide <400> 36 Pro Ser Gly Gly Leu Glu Gln Glu Leu Arg Arg Arg Leu Thr Ala Pro 1 5 10 15 <210> 37 <211> 16 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptide <400> 37 Asp Ser Pro Gly Leu Glu Gln Glu Leu Arg Arg Arg Leu Thr Ala Pro 1 5 10 15 <210> 38 <211> 16 <212> PRT <213> Synthetic sequence <220> <223> Synthetic peptide <400> 38 Pro Asp Ser Gly Leu Glu Gln Glu Leu Arg Arg Arg Leu Thr Pro Ala 1 5 10 15 <210> 39 <211> 17 <212> PRT <213> Synthetic sequence <220> <223> Synthetic peptide <400> 39 Ser Pro Ser Gly Leu Glu Glu Glu Leu Arg Arg Arg Leu Thr Ala Glu 1 5 10 15 Pro <210> 40 <211> 14 <212> PRT <213> Synthetic sequence <220> <223> Synthetic peptide <400> 40 Gly Pro Ser Gly Leu Glu Gln Glu Leu Arg Arg Arg Leu Thr 1 5 10 <210> 41 <211> 17 <212> PRT <213> Synthetic sequence <220> <223> Synthetic peptide <400> 41 Gly Pro Ser Gly Leu Glu Gln Glu Leu Arg Arg Arg Leu Thr Ala Ala 1 5 10 15 Ser <210> 42 <211> 16 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptide <400> 42 Ser Pro Ser Arg Leu Glu Glu Glu Leu Arg Arg Arg Leu Pro Ser Lys 1 5 10 15 <210> 43 <211> 16 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptide <400> 43 Ser Pro Ser Gly Leu Glu Gln Glu Leu Arg Arg Arg Leu Thr Pro Ser 1 5 10 15 <210> 44 <211> 16 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptide <400> 44 Ser Pro Gly Arg Leu Glu Gln Glu Ile Arg Arg Arg Leu Ser Pro Ser 1 5 10 15 <210> 45 <211> 16 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptide <400> 45 Pro Ser Gly Arg Leu Glu Glu Glu Leu Arg Arg Arg Leu Ser Pro Ser 1 5 10 15 <210> 46 <211> 14 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptide <400> 46 Pro Ser Gly Arg Leu Glu Glu Glu Leu Arg Arg Arg Leu Ser 1 5 10 <210> 47 <211> 14 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptide <400> 47 Pro Ser Gly Arg Leu Glu Glu Glu Leu Arg Arg Arg Leu Ala 1 5 10 <210> 48 <211> 15 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptide <400> 48 Pro Ser Gly Arg Leu Glu Glu Glu Leu Arg Arg Arg Leu Ser Pro 1 5 10 15 <210> 49 <211> 13 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptide <220> <221> MISC_FEATURE <222> (1)..(1) <223> Xaa is S or G or P <220> <221> MISC_FEATURE <222> (2)..(2) <223> Xaa is R or G or P <220> <221> MISC_FEATURE <222> (5)..(5) <223> Xaa is E or Q <220> <221> MISC_FEATURE <222> (12)..(12) <223> Xaa is S or T or D or E <220> <221> MISC_FEATURE <222> (13)..(13) <223> Xaa is P or A or D or no amino acid <400> 49 Xaa Xaa Leu Glu Xaa Glu Leu Arg Arg Arg Leu Xaa Xaa 1 5 10 <210> 50 <211> 16 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptide <400> 50 Pro Asp Ser Gly Leu Glu Gln Glu Leu Arg Arg Arg Leu Ser Pro Gly 1 5 10 15 <210> 51 <211> 16 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptide <400> 51 Pro Asp Ser Gly Leu Glu Gln Glu Leu Arg Arg Arg Leu Thr Ala Pro 1 5 10 15 <210> 52 <211> 16 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptide <400> 52 Pro Ser Ser Gly Leu Glu Gln Glu Leu Arg Arg Arg Leu Thr Ala Pro 1 5 10 15 <210> 53 <211> 16 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptide <400> 53 Asp Pro Ser Gly Leu Glu Gln Glu Leu Arg Arg Arg Leu Thr Ala Pro 1 5 10 15 <210> 54 <211> 16 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptide <400> 54 Asp Ser Gly Pro Leu Glu Gln Glu Leu Arg Arg Arg Leu Thr Ala Pro 1 5 10 15 <210> 55 <211> 17 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptide <400> 55 Ser Pro Ser Arg Leu Glu Glu Glu Leu Arg Arg Arg Leu Thr Ala Glu 1 5 10 15 Pro <210> 56 <211> 16 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptide <400> 56 Ser Pro Ser Gly Leu Glu Glu Glu Leu Arg Arg Arg Leu Thr Ala Pro 1 5 10 15 <210> 57 <211> 16 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptide <400> 57 Ser Pro Ser Gly Leu Glu Glu Glu Leu Arg Arg Arg Leu Asp Ala Pro 1 5 10 15 <210> 58 <211> 16 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptide <400> 58 Ser Pro Ser Gly Leu Glu Glu Glu Leu Arg Arg Arg Leu Glu Ala Pro 1 5 10 15 <210> 59 <211> 16 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptide <400> 59 Ser Pro Ser Gly Leu Glu Glu Glu Leu Arg Arg Arg Leu Thr Asp Pro 1 5 10 15 <210> 60 <211> 17 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Peptide <400> 60 Ser Pro Ser Gly Leu Glu Glu Glu Leu Arg Arg Arg Leu Thr Ala Asp 1 5 10 15 Pro <210> 61 <211> 13 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Peptide <220> <221> MISC_FEATURE <222> (1)..(1) <223> Xaa is G or S <220> <221> MISC_FEATURE <222> (2)..(2) <223> Xaa is R or G or A or E <220> <221> MISC_FEATURE <222> (3)..(3) <223> Xaa is L or V <220> <221> MISC_FEATURE <222> (4)..(4) <223> Xaa is E or Q <220> <221> MISC_FEATURE <222> (5)..(5) <223> Xaa is E or Q <220> <221> MISC_FEATURE <222> (6)..(6) <223> Xaa is E or Q <220> <221> MISC_FEATURE <222> (7)..(7) <223> Xaa is L or I or V <220> <221> MISC_FEATURE <222> (8)..(8) <223> Xaa is R or A or Q or E <220> <221> MISC_FEATURE <222> (9)..(9) <223> Xaa is R or A or Q or E <220> <221> MISC_FEATURE <222> (12)..(12) <223> Xaa is S or T or L or no amino acid <220> <221> MISC_FEATURE <222> (13)..(13) <223> Xaa is K or P or S or no amino acid <400> 61 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Arg Leu Xaa Xaa 1 5 10 <210> 62 <211> 16 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptide <400> 62 Asp Ser Gly Arg Leu Glu Glu Glu Leu Arg Arg Arg Leu Ser Lys Gly 1 5 10 15 <210> 63 <211> 16 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptide <400> 63 Asp Ser Gly Arg Leu Glu Glu Glu Leu Arg Arg Arg Leu Ser Pro Gly 1 5 10 15 <210> 64 <211> 16 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptide <400> 64 Ser Asp Ser Gly Leu Glu Glu Glu Leu Arg Arg Arg Leu Ser Pro Gly 1 5 10 15 <210> 65 <211> 16 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptide <400> 65 Ser Asp Ser Gly Val Glu Glu Glu Leu Arg Arg Arg Leu Ser Pro Gly 1 5 10 15 <210> 66 <211> 16 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptide <400> 66 Ser Asp Ser Ala Val Glu Glu Glu Leu Arg Arg Arg Leu Ser Pro Gly 1 5 10 15 <210> 67 <211> 16 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptide <400> 67 Ser Asp Ser Gly Leu Gln Glu Glu Leu Arg Arg Arg Leu Ser Pro Gly 1 5 10 15 <210> 68 <211> 16 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptide <400> 68 Ser Asp Ser Gly Leu Glu Glu Gln Leu Arg Arg Arg Leu Ser Pro Gly 1 5 10 15 <210> 69 <211> 16 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptide <400> 69 Ser Asp Ser Gly Leu Glu Glu Glu Ile Arg Arg Arg Leu Ser Pro Gly 1 5 10 15 <210> 70 <211> 16 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptide <400> 70 Ser Asp Ser Gly Leu Glu Glu Glu Val Arg Arg Arg Leu Ser Pro Gly 1 5 10 15 <210> 71 <211> 16 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Peptide <400> 71 Asp Ser Gly Glu Leu Glu Glu Glu Leu Arg Arg Arg Leu Ser Pro Gly 1 5 10 15 <210> 72 <211> 16 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Peptide <400> 72 Asp Ser Gly Arg Leu Glu Gln Glu Leu Arg Arg Arg Leu Ser Pro Gly 1 5 10 15 <210> 73 <211> 18 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Peptide <400> 73 Gln Gln Asp Ser Gly Arg Leu Glu Glu Glu Ile Arg Arg Arg Leu Ser 1 5 10 15 Pro Gly <210> 74 <211> 16 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Peptide <400> 74 Asp Ser Gly Arg Leu Glu Gln Glu Ile Arg Arg Arg Leu Ser Pro Gly 1 5 10 15 <210> 75 <211> 16 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Peptide <400> 75 Asp Ser Gly Arg Leu Glu Gln Glu Ile Ala Arg Arg Leu Ser Pro Gly 1 5 10 15 <210> 76 <211> 16 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Peptide <400> 76 Asp Ser Gly Arg Leu Glu Gln Glu Ile Gln Arg Arg Leu Ser Pro Gly 1 5 10 15 <210> 77 <211> 16 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Peptide <400> 77 Asp Ser Gly Arg Leu Glu Gln Glu Ile Glu Arg Arg Leu Ser Pro Gly 1 5 10 15 <210> 78 <211> 16 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Peptide <400> 78 Asp Ser Gly Arg Leu Glu Gln Glu Ile Arg Ala Arg Leu Ser Pro Gly 1 5 10 15 <210> 79 <211> 16 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptide <400> 79 Asp Ser Gly Arg Leu Glu Gln Glu Ile Arg Gln Arg Leu Ser Pro Gly 1 5 10 15 <210> 80 <211> 16 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptide <400> 80 Asp Ser Gly Arg Leu Glu Gln Glu Ile Arg Glu Arg Leu Ser Pro Gly 1 5 10 15 <210> 81 <211> 13 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptide <400> 81 Gly Pro Ser Arg Leu Glu Glu Glu Leu Arg Arg Arg Leu 1 5 10 <210> 82 <211> 15 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptide <400> 82 Met Ser Gly Leu Glu Gln Glu Leu Arg Arg Arg Leu Thr Pro Ser 1 5 10 15 <210> 83 <211> 16 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptide <400> 83 Met Ser Gly Arg Leu Glu Glu Glu Leu Arg Arg Arg Leu Ser Pro Ser 1 5 10 15 <210> 84 <211> 16 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptide <400> 84 Ser Pro Ser Ala Val Glu Glu Glu Leu Arg Arg Arg Leu Ser Pro Ser 1 5 10 15 <210> 85 <211> 14 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptide <400> 85 Gly Pro Ser Ala Val Glu Glu Glu Leu Arg Arg Arg Leu Ser 1 5 10 <210> 86 <211> 16 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptide <400> 86 Met Pro Ser Gly Leu Glu Gln Glu Leu Arg Arg Arg Leu Thr Pro Ser 1 5 10 15 <210> 87 <211> 16 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptide <400> 87 Met Ser Ser Gly Leu Glu Gln Glu Leu Arg Arg Arg Leu Thr Pro Ser 1 5 10 15 <210> 88 <211> 17 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptide <400> 88 Met Pro Ser Gly Arg Leu Glu Glu Glu Leu Arg Arg Arg Leu Ser Pro 1 5 10 15 Ser <210> 89 <211> 15 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptide <400> 89 Met Ser Gly Arg Leu Glu Glu Glu Leu Arg Arg Arg Leu Ser Pro 1 5 10 15 <210> 90 <211> 16 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptide <400> 90 Asp Ser Met Arg Leu Glu Glu Glu Leu Arg Arg Arg Leu Ser Lys Gly 1 5 10 15 <210> 91 <211> 16 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptide <400> 91 Asp Ser Met Arg Leu Glu Glu Glu Leu Arg Arg Arg Leu Ser Pro Gly 1 5 10 15 <210> 92 <211> 15 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptide <400> 92 Ser Asp Ser Gly Glu Glu Glu Leu Arg Arg Arg Leu Ser Pro Gly 1 5 10 15 <210> 93 <211> 14 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptide <400> 93 Ser Asp Ser Gly Glu Glu Leu Arg Arg Arg Leu Ser Pro Gly 1 5 10 <210> 94 <211> 15 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptide <400> 94 Asp Ser Gly Arg Leu Glu Glu Glu Leu Arg Arg Arg Ser Pro Gly 1 5 10 15 <210> 95 <211> 14 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptide <400> 95 Asp Ser Gly Arg Leu Glu Glu Glu Leu Arg Arg Ser Pro Gly 1 5 10 <210> 96 <211> 13 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptide <400> 96 Asp Ser Gly Arg Leu Glu Glu Glu Leu Arg Ser Pro Gly 1 5 10 <210> 97 <211> 16 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptide <400> 97 Ser Asp Ser Gly Leu Glu Glu Glu Ala Arg Arg Arg Leu Ser Pro Gly 1 5 10 15 <210> 98 <211> 16 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptide <400> 98 Ser Asp Ser Gly Ala Glu Glu Glu Leu Arg Arg Arg Leu Ser Pro Gly 1 5 10 15 <210> 99 <211> 16 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptide <400> 99 Asp Ser Gly Ala Leu Glu Glu Glu Leu Arg Arg Arg Leu Ser Pro Gly 1 5 10 15 <210> 100 <211> 16 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Peptide <400> 100 Asp Ser Gly Ala Leu Glu Gln Glu Ile Arg Arg Arg Leu Ser Pro Gly 1 5 10 15 <210> 101 <211> 16 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Peptide <400> 101 Asp Ser Gly Arg Leu Glu Gln Glu Ile Arg Arg Ala Leu Ser Pro Gly 1 5 10 15 <210> 102 <211> 16 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Peptide <400> 102 Asp Ser Gly Arg Leu Glu Gln Glu Ile Arg Arg Gln Leu Ser Pro Gly 1 5 10 15 <210> 103 <211> 16 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Peptide <400> 103 Asp Ser Gly Arg Leu Glu Gln Glu Ile Arg Arg Glu Leu Ser Pro Gly 1 5 10 15 <210> 104 <211> 16 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptide <400> 104 Pro Asp Ser Gly Leu Glu Gln Glu Leu Arg Arg Arg Leu Pro Thr Ala 1 5 10 15 <210> 105 <211> 16 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptide <400> 105 Asp Pro Ser Gly Leu Glu Gln Glu Leu Arg Arg Arg Leu Pro Thr Ala 1 5 10 15 <210> 106 <211> 16 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptide <400> 106 Asp Ser Pro Gly Leu Glu Gln Glu Leu Arg Arg Arg Leu Pro Thr Ala 1 5 10 15 <210> 107 <211> 16 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptide <400> 107 Asp Ser Gly Pro Leu Glu Gln Glu Leu Arg Arg Arg Leu Pro Thr Ala 1 5 10 15 <210> 108 <211> 13 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptide <400> 108 Gly Pro Ser Gly Leu Glu Gln Glu Leu Arg Arg Arg Leu 1 5 10 <210> 109 <211> 15 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptide <400> 109 Met Ser Ala Val Glu Glu Glu Leu Arg Arg Arg Leu Ser Pro Ser 1 5 10 15 <210> 110 <211> 15 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptide <400> 110 Met Ser Gly Leu Glu Gln Glu Leu Arg Arg Arg Leu Thr Pro Ser 1 5 10 15 <210> 111 <211> 15 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptide <400> 111 Met Ser Ala Val Glu Glu Glu Leu Arg Arg Arg Leu Ser Pro Ser 1 5 10 15 <210> 112 <211> 16 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptide <400> 112 Met Pro Ser Ala Val Glu Glu Glu Leu Arg Arg Arg Leu Ser Pro Ser 1 5 10 15 <210> 113 <211> 16 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptide <400> 113 Met Ser Ser Ala Val Glu Glu Glu Leu Arg Arg Arg Leu Ser Pro Ser 1 5 10 15 <210> 114 <211> 14 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptide <400> 114 Pro Ser Gly Arg Leu Glu Glu Glu Leu Arg Arg Arg Leu Pro 1 5 10 <210> 115 <211> 13 <212> PRT <213> Artificial sequence <220> <223> Antibody CDR <400> 115 Gly Val Thr Ile Ser Ala Leu Asn Ala Met Ala Met Gly 1 5 10 <210> 116 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Antibody CDR <400> 116 Ala Val Ser Glu Arg Gly Asn Ala Met 1 5 <210> 117 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> Antibody CDR <400> 117 Leu Glu Asp Arg Val Asp Ser Phe His Asp Tyr 1 5 10 <210> 118 <211> 13 <212> PRT <213> Artificial Sequence <220> <223> Antibody CDR <400> 118 Gly Val Thr Ile Ser Ala Leu Asn Ala Met Ala Met Gly 1 5 10 <210> 119 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Antibody CDR <400> 119 Ala Val Ser Ser Arg Gly Asn Ala Met 1 5 <210> 120 <211> 11 <212> PRT <213> Artificial sequence <220> <223> Antibody CDR <400> 120 Leu Glu Asp Arg Val Asp Ser Phe His Asp Tyr 1 5 10 <210> 121 <211> 13 <212> PRT <213> Artificial sequence <220> <223> Antibody CDR <400> 121 Gly Val Thr Val Ser Ala Leu Asn Ala Met Ala Met Gly 1 5 10 <210> 122 <211> 9 <212> PRT <213> Artificial sequence <220> <223> Antibody CDR <400> 122 Ala Val Ser Glu Arg Gly Asn Ala Met 1 5 <210> 123 <211> 11 <212> PRT <213> Artificial sequence <220> <223> Antibody CDR <400> 123 Leu Glu Asp Arg Val Asp Ser Phe His Asp Tyr 1 5 10 <210> 124 <211> 25 <212> PRT <213> Artificial sequence <220> <223> Antibody FR <220> <221> MISC_FEATURE <222> (5)..(5) <223> Xaa is Q, V, E or L <220> <221> MISC_FEATURE <222> (13)..(13) <223> Xaa is Q or P <220> <221> MISC_FEATURE <222> (18)..(18) <223> Xaa is L or M <400> 124 Glu Val Gln Leu Xaa Glu Ser Gly Gly Gly Leu Val Xaa Pro Gly Gly 1 5 10 15 Ser Xaa Arg Leu Ser Cys Thr Ala Ser 20 25 <210> 125 <211> 25 <212> PRT <213> Artificial sequence <220> <223> Antibody FR <400> 125 Glu Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Thr Ala Ser 20 25 <210> 126 <211> 14 <212> PRT <213> Artificial sequence <220> <223> Antibody FR <220> <221> MISC_FEATURE <222> (5)..(5) <223> Xaa is A or R <220> <221> MISC_FEATURE <222> (9)..(9) <223> Xaa is R or E <400> 126 Trp Tyr Arg Gln Xaa Pro Gly Glu Xaa Arg Val Met Val Ala 1 5 10 <210> 127 <211> 14 <212> PRT <213> Artificial sequence <220> <223> Antibody FR <400> 127 Trp Tyr Arg Gln Ala Pro Gly Glu Arg Arg Val Met Val Ala 1 5 10 <210> 128 <211> 14 <212> PRT <213> Artificial sequence <220> <223> Antibody FR <400> 128 Trp Tyr Arg Gln Ala Pro Gly Glu Glu Arg Val Met Val Ala 1 5 10 <210> 129 <211> 39 <212> PRT <213> Artificial sequence <220> <223> Antibody FR <220> <221> MISC_FEATURE <222> (28)..(28) <223> Xaa is K or Q <220> <221> MISC_FEATURE <222> (32)..(32) <223> Xaa is T or M <400> 129 Tyr Arg Glu Ser Val Gln Gly Arg Phe Thr Val Thr Arg Asp Phe Thr 1 5 10 15 Asn Lys Met Val Ser Leu Gln Met Asp Asn Leu Xaa Pro Glu Asp Xaa 20 25 30 Ala Val Tyr Tyr Cys His Val 35 <210> 130 <211> 39 <212> PRT <213> Artificial Sequence <220> <223> Antibody FR <400> 130 Tyr Arg Glu Ser Val Gln Gly Arg Phe Thr Val Thr Arg Asp Phe Thr 1 5 10 15 Asn Lys Met Val Ser Leu Gln Met Asp Asn Leu Lys Pro Glu Asp Thr 20 25 30 Ala Val Tyr Tyr Cys His Val 35 <210> 131 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> Antibody FR <220> <221> MISC_FEATURE <222> (5)..(5) <223> Xaa is T or I <400> 131 Trp Gly Gln Gly Xaa Gln Val Thr Val Ser Ser 1 5 10 <210> 132 <211> 11 <212> PRT <213> Artificial sequence <220> <223> Antibody FR <400> 132 Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser 1 5 10 <210> 133 <211> 122 <212> PRT <213> Artificial sequence <220> <223> Camelid VHH domain <400> 133 Glu Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Thr Ala Ser Gly Val Thr Ile Ser Ala Leu 20 25 30 Asn Ala Met Ala Met Gly Trp Tyr Arg Gln Ala Pro Gly Glu Arg Arg 35 40 45 Val Met Val Ala Ala Val Ser Glu Arg Gly Asn Ala Met Tyr Arg Glu 50 55 60 Ser Val Gln Gly Arg Phe Thr Val Thr Arg Asp Phe Thr Asn Lys Met 65 70 75 80 Val Ser Leu Gln Met Asp Asn Leu Lys Pro Glu Asp Thr Ala Val Tyr 85 90 95 Tyr Cys His Val Leu Glu Asp Arg Val Asp Ser Phe His Asp Tyr Trp 100 105 110 Gly Gln Gly Thr Gln Val Thr Val Ser Ser 115 120 <210> 134 <211> 122 <212> PRT <213> Artificial Sequence <220> <223> Camel VHH Domain <400> 134 Glu Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Thr Ala Ser Gly Val Thr Ile Ser Ala Leu 20 25 30 Asn Ala Met Ala Met Gly Trp Tyr Arg Gln Ala Pro Gly Glu Glu Arg 35 40 45 Val Met Val Ala Ala Val Ser Ser Arg Gly Asn Ala Met Tyr Arg Glu 50 55 60 Ser Val Gln Gly Arg Phe Thr Val Thr Arg Asp Phe Thr Asn Lys Met 65 70 75 80 Val Ser Leu Gln Met Asp Asn Leu Lys Pro Glu Asp Thr Ala Val Tyr 85 90 95 Tyr Cys His Val Leu Glu Asp Arg Val Asp Ser Phe His Asp Tyr Trp 100 105 110 Gly Gln Gly Thr Gln Val Thr Val Ser Ser 115 120 <210> 135 <211> 122 <212> PRT <213> Artificial Sequence <220> <223> Camel VHH Domain <400> 135 Glu Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Thr Ala Ser Gly Val Thr Ile Ser Ala Leu 20 25 30 Asn Ala Met Ala Met Gly Trp Tyr Arg Gln Ala Pro Gly Glu Glu Arg 35 40 45 Val Met Val Ala Ala Val Ser Glu Arg Gly Asn Ala Met Tyr Arg Glu 50 55 60 Ser Val Gln Gly Arg Phe Thr Val Thr Arg Asp Phe Thr Asn Lys Met 65 70 75 80 Val Ser Leu Gln Met Asp Asn Leu Lys Pro Glu Asp Thr Ala Val Tyr 85 90 95 Tyr Cys His Val Leu Glu Asp Arg Val Asp Ser Phe His Asp Tyr Trp 100 105 110 Gly Gln Gly Thr Gln Val Thr Val Ser Ser 115 120 <210> 136 <211> 122 <212> PRT <213> Artificial Sequence <220> <223> Camel VHH Domain <400> 136 Glu Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Thr Ala Ser Gly Val Thr Ile Ser Ala Leu 20 25 30 Asn Ala Met Ala Met Gly Trp Tyr Arg Gln Ala Pro Gly Glu Arg Arg 35 40 45 Val Met Val Ala Ala Val Ser Ser Arg Gly Asn Ala Met Tyr Arg Glu 50 55 60 Ser Val Gln Gly Arg Phe Thr Val Thr Arg Asp Phe Thr Asn Lys Met 65 70 75 80 Val Ser Leu Gln Met Asp Asn Leu Lys Pro Glu Asp Thr Ala Val Tyr 85 90 95 Tyr Cys His Val Leu Glu Asp Arg Val Asp Ser Phe His Asp Tyr Trp 100 105 110 Gly Gln Gly Thr Gln Val Thr Val Ser Ser 115 120 <210> 137 <211> 122 <212> PRT <213> Artificial sequence <220> <223> Camel VHH domain <400> 137 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Pro Pro Gly Gly 1 5 10 15 Ser Met Arg Leu Ser Cys Thr Ala Ser Gly Val Thr Val Ser Ala Leu 20 25 30 Asn Ala Met Ala Met Gly Trp Tyr Arg Gln Arg Pro Gly Glu Arg Arg 35 40 45 Val Met Val Ala Ala Val Ser Glu Arg Gly Asn Ala Met Tyr Arg Glu 50 55 60 Ser Val Gln Gly Arg Phe Thr Val Thr Arg Asp Phe Thr Asn Lys Met 65 70 75 80 Val Ser Leu Gln Met Asp Asn Leu Gln Pro Glu Asp Met Ala Val Tyr 85 90 95 Tyr Cys His Val Leu Glu Asp Arg Val Asp Ser Phe His Asp Tyr Trp 100 105 110 Gly Gln Gly Ile Gln Val Thr Val Ser Ser 115 120 <210> 138 <211> 122 <212> PRT <213> Artificial sequence <220> <223> Camelid VHH domain <400> 138 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Pro Pro Gly Gly 1 5 10 15 Ser Met Arg Leu Ser Cys Thr Ala Pro Gly Val Thr Val Ser Ala Leu 20 25 30 Asn Ala Met Ala Met Gly Trp Tyr Arg Gln Arg Pro Gly Glu Arg Arg 35 40 45 Val Met Val Ala Ala Val Ser Glu Arg Gly Asn Ala Met Tyr Arg Glu 50 55 60 Ser Val Gln Gly Arg Phe Thr Val Thr Arg Asp Phe Thr Asn Lys Met 65 70 75 80 Val Ser Leu Gln Met Asp Asn Leu Gln Pro Glu Asp Met Ala Val Tyr 85 90 95 Tyr Cys His Val Leu Glu Asp Arg Val Asp Ser Phe His Asp Tyr Trp 100 105 110 Gly Gln Gly Ile Gln Val Thr Val Ser Ser 115 120 <210> 139 <211> 122 <212> PRT <213> Artificial sequence <220> <223> Camelid VHH domain <400> 139 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Thr Ala Ser Gly Val Thr Ile Ser Ala Leu 20 25 30 Asn Ala Met Ala Met Gly Trp Tyr Arg Gln Ala Pro Gly Glu Arg Arg 35 40 45 Val Met Val Ala Ala Val Ser Glu Arg Gly Asn Ala Met Tyr Arg Glu 50 55 60 Ser Val Gln Gly Arg Phe Thr Val Thr Arg Asp Phe Thr Asn Lys Met 65 70 75 80 Val Ser Leu Gln Met Asp Asn Leu Lys Pro Glu Asp Thr Ala Val Tyr 85 90 95 Tyr Cys His Val Leu Glu Asp Arg Val Asp Ser Phe His Asp Tyr Trp 100 105 110 Gly Gln Gly Thr Gln Val Thr Val Ser Ser 115 120 <210> 140 <211> 122 <212> PRT <213> Artificial Sequence <220> <223> Camel VHH Domain <400> 140 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Thr Ala Ser Gly Val Thr Ile Ser Ala Leu 20 25 30 Asn Ala Met Ala Met Gly Trp Tyr Arg Gln Ala Pro Gly Glu Arg Arg 35 40 45 Val Met Val Ala Ala Val Ser Glu Arg Gly Asn Ala Met Tyr Arg Glu 50 55 60 Ser Val Gln Gly Arg Phe Thr Val Thr Arg Asp Phe Thr Asn Lys Met 65 70 75 80 Val Ser Leu Gln Met Asp Asn Leu Lys Pro Glu Asp Thr Ala Val Tyr 85 90 95 Tyr Cys His Val Leu Glu Asp Arg Val Asp Ser Phe His Asp Tyr Trp 100 105 110 Gly Gln Gly Thr Gln Val Thr Val Ser Ser 115 120 <210> 141 <211> 122 <212> PRT <213> Artificial sequence <220> <223> Camel VHH domain <400> 141 Glu Val Gln Leu Glu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Thr Ala Ser Gly Val Thr Ile Ser Ala Leu 20 25 30 Asn Ala Met Ala Met Gly Trp Tyr Arg Gln Ala Pro Gly Glu Arg Arg 35 40 45 Val Met Val Ala Ala Val Ser Glu Arg Gly Asn Ala Met Tyr Arg Glu 50 55 60 Ser Val Gln Gly Arg Phe Thr Val Thr Arg Asp Phe Thr Asn Lys Met 65 70 75 80 Val Ser Leu Gln Met Asp Asn Leu Lys Pro Glu Asp Thr Ala Val Tyr 85 90 95 Tyr Cys His Val Leu Glu Asp Arg Val Asp Ser Phe His Asp Tyr Trp 100 105 110 Gly Gln Gly Thr Gln Val Thr Val Ser Ser 115 120 <210> 142 <211> 122 <212> PRT <213> Artificial Sequence <220> <223> Camel VHH Domain <400> 142 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Thr Ala Ser Gly Val Thr Ile Ser Ala Leu 20 25 30 Asn Ala Met Ala Met Gly Trp Tyr Arg Gln Ala Pro Gly Glu Arg Arg 35 40 45 Val Met Val Ala Ala Val Ser Glu Arg Gly Asn Ala Met Tyr Arg Glu 50 55 60 Ser Val Gln Gly Arg Phe Thr Val Thr Arg Asp Phe Thr Asn Lys Met 65 70 75 80 Val Ser Leu Gln Met Asp Asn Leu Lys Pro Glu Asp Thr Ala Val Tyr 85 90 95 Tyr Cys His Val Leu Glu Asp Arg Val Asp Ser Phe His Asp Tyr Trp 100 105 110 Gly Gln Gly Thr Gln Val Thr Val Ser Ser 115 120 <210> 143 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> Antibody CDR <400> 143 Gly Thr Met Ser Ala Ile Asn Ala Leu Asn 1 5 10 <210> 144 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Antibody CDR <400> 144 Ala Ile Thr Asp Asn Gly Asn Ala His 1 5 <210> 145 <211> 12 <212> PRT <213> Artificial sequence <220> <223> Antibody CDR <400> 145 Leu Glu Glu Glu Lys Leu Gly Val Trp Val Asp Tyr 1 5 10 <210> 146 <211> 10 <212> PRT <213> Artificial sequence <220> <223> Antibody CDR <400> 146 Gly Thr Met Ser Ala Ile Asn Ala Leu Asn 1 5 10 <210> 147 <211> 9 <212> PRT <213> Artificial sequence <220> <223> Antibody CDR <400> 147 Ala Ile Thr Asp Asn Gly Asn Ala His 1 5 <210> 148 <211> 11 <212> PRT <213> Artificial sequence <220> <223> Antibody CDR <400> 148 Leu Glu Glu Lys Leu Gly Ala Trp Val Asp Tyr 1 5 10 <210> 149 <211> 10 <212> PRT <213> Artificial sequence <220> <223> Antibody CDR <400> 149 Gly Thr Met Ser Ala Ile Asn Ala Leu Asn 1 5 10 <210> 150 <211> 9 <212> PRT <213> Synthetic Sequence <220> <223> Antibody CDR <400> 150 Ala Ile Thr Asp Asn Gly Asn Ala His 1 5 <210> 151 <211> 12 <212> PRT <213> Synthetic Sequence <220> <223> Antibody CDR <400> 151 Leu Glu Lys Glu Lys Leu Gly Val Trp Val Asp Tyr 1 5 10 <210> 152 <211> 25 <212> PRT <213> Synthetic Sequence <220> <223> Antibody FR <220> <221> MISC_FEATURE <222> (5)..(5) <223> Xaa is V or L <400> 152 Glu Val Gln Leu Xaa Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Thr Leu Ser Cys Ala Ala Ser 20 25 <210> 153 <211> 14 <212> PRT <213> Artificial sequence <220> <223> Antibody FR <220> <221> MISC_FEATURE <222> (5)..(5) <223> Xaa is P or A <400> 153 Trp Tyr Arg Gln Xaa Pro Gly Lys Glu Arg Lys Met Val Ala 1 5 10 <210> 154 <211> 39 <212> PRT <213> Artificial sequence <220> <223> Antibody FR <220> <221> MISC_FEATURE <222> (28)..(28) <223> Xaa is K or E <400> 154 Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala 1 5 10 15 Arg Asn Met Val Phe Leu Gln Met Asn Ser Leu Xaa Pro Asp Asp Thr 20 25 30 Ala Val Tyr Tyr Cys His Tyr 35 <210> 155 <211> 11 <212> PRT <213> Artificial sequence <220> <223> Antibody FR <400> 155 Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser 1 5 10 <210> 156 <211> 120 <212> PRT <213> Artificial sequence <220> <223> Camel VHH domain <400> 156 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Thr Leu Ser Cys Ala Ala Ser Gly Thr Met Ser Ala Ile Asn 20 25 30 Ala Leu Asn Trp Tyr Arg Gln Pro Pro Gly Lys Glu Arg Lys Met Val 35 40 45 Ala Ala Ile Thr Asp Asn Gly Asn Ala His Tyr Ala Asp Ser Val Lys 50 55 60 Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Arg Asn Met Val Phe Leu 65 70 75 80 Gln Met Asn Ser Leu Lys Pro Asp Asp Thr Ala Val Tyr Tyr Cys His 85 90 95 Tyr Leu Glu Glu Glu Lys Leu Gly Val Trp Val Asp Tyr Trp Gly Gln 100 105 110 Gly Thr Gln Val Thr Val Ser Ser 115 120 <210> 157 <211> 119 <212> PRT <213> Artificial sequence <220> <223> Camelid VHH domain <400> 157 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Thr Leu Ser Cys Ala Ala Ser Gly Thr Met Ser Ala Ile Asn 20 25 30 Ala Leu Asn Trp Tyr Arg Gln Ala Pro Gly Lys Glu Arg Lys Met Val 35 40 45 Ala Ala Ile Thr Asp Asn Gly Asn Ala His Tyr Ala Asp Ser Val Lys 50 55 60 Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Arg Asn Met Val Phe Leu 65 70 75 80 Gln Met Asn Ser Leu Glu Pro Asp Asp Thr Ala Val Tyr Tyr Cys His 85 90 95 Tyr Leu Glu Glu Lys Leu Gly Ala Trp Val Asp Tyr Trp Gly Gln Gly 100 105 110 Thr Gln Val Thr Val Ser Ser 115 <210> 158 <211> 120 <212> PRT <213> Artificial sequence <220> <223> Camelid VHH domain <400> 158 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Thr Leu Ser Cys Ala Ala Ser Gly Thr Met Ser Ala Ile Asn 20 25 30 Ala Leu Asn Trp Tyr Arg Gln Pro Pro Gly Lys Glu Arg Lys Met Val 35 40 45 Ala Ala Ile Thr Asp Asn Gly Asn Ala His Tyr Ala Asp Ser Val Lys 50 55 60 Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Arg Asn Met Val Phe Leu 65 70 75 80 Gln Met Asn Ser Leu Lys Pro Asp Asp Thr Ala Val Tyr Tyr Cys His 85 90 95 Tyr Leu Glu Lys Glu Lys Leu Gly Val Trp Val Asp Tyr Trp Gly Gln 100 105 110 Gly Thr Gln Val Thr Val Ser Ser 115 120 <210> 159 <211> 15 <212> PRT <213> Artificial Sequence <220> <223> Linker <400> 159 Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser 1 5 10 15 <210> 160 <211> 8 <212> PRT <213> Artificial sequence <220> <223> FLAG-tag <400> 160 Asp Tyr Lys Asp Asp Asp Asp Lys 1 5 <210> 161 <211> 10 <212> PRT <213> Artificial sequence <220> <223> Myc-tag <400> 161 Glu Gln Lys Leu Ile Ser Glu Glu Asp Leu 1 5 10 <210> 162 <211> 9 <212> PRT <213> Artificial sequence <220> <223> HA-tag <400> 162 Tyr Pro Tyr Asp Val Pro Asp Tyr Ala 1 5 <210> 163 <211> 11 <212> PRT <213> Artificial sequence <220> <223> VSV-G-tag <400> 163 Tyr Thr Asp Ile Glu Met Asn Arg Leu Gly Lys 1 5 10 <210> 164 <211> 11 <212> PRT <213> Artificial sequence <220> <223> HSV-tag <400> 164 Gln Pro Glu Leu Ala Pro Glu Asp Pro Glu Asp 1 5 10 <210> 165 <211> 14 <212> PRT <213> Artificial Sequence <220> <223> V5-Tag <400> 165 Gly Lys Pro Ile Pro Asn Pro Leu Leu Gly Leu Asp Ser Thr 1 5 10 <210> 166 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> SPOT-Tag <400> 166 Pro Asp Arg Val Arg Ala Val Ser His Trp Ser Ser 1 5 10 <210> 167 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> BC2 Tag <400> 167 Pro Asp Arg Lys Ala Ala Val Ser His Trp Gln Gln 1 5 10 <210> 168 <211> 4 <212> PRT <213> Artificial Sequence <220> <223> EPEA Tag <400> 168 Glu Pro Glu Ala 1 <210> 169 <211> 15 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptide <400> 169 Gly Pro Ser Gly Leu Glu Gln Glu Leu Arg Arg Arg Leu Thr Ala 1 5 10 15 <210> 170 <211> 16 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptide <400> 170 Ser Pro Ser Gly Leu Glu Gln Glu Leu Arg Arg Arg Leu Thr Asp Pro 1 5 10 15 <210> 171 <211> 17 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptide <400> 171 Ser Pro Ser Gly Leu Glu Gln Glu Leu Arg Arg Arg Leu Thr Ala Asp 1 5 10 15 Pro <210> 172 <211> 17 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptide <400> 172 Ser Pro Ser Gly Leu Glu Gln Glu Leu Arg Arg Arg Leu Thr Ala Glu 1 5 10 15 Pro <210> 173 <211> 16 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Peptide <400> 173 Asp Ser Pro Gly Leu Glu Gln Glu Leu Arg Arg Arg Leu Thr Ala Pro 1 5 10 15 <210> 174 <211> 16 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Peptide <400> 174 Ser Pro Ser Gly Leu Glu Gln Glu Leu Arg Arg Arg Leu Ser Pro Ser 1 5 10 15 <210> 175 <211> 122 <212> PRT <213> Artificial Sequence <220> <223> Camel VHH Domain <400> 175 Glu Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Thr Ala Ser Gly Val Thr Ile Ser Ala Leu 20 25 30 Asn Ala Met Ala Met Gly Trp Tyr Arg Gln Ala Pro Gly Glu Arg Arg 35 40 45 Val Met Val Ala Ala Val Ser Asp Arg Gly Asn Ala Met Tyr Arg Glu 50 55 60 Ser Val Gln Gly Arg Phe Thr Val Thr Arg Asp Phe Thr Asn Lys Met 65 70 75 80 Val Ser Leu Gln Met Asp Asn Leu Lys Pro Glu Asp Thr Ala Val Tyr 85 90 95 Tyr Cys His Val Leu Glu Asp Arg Val Asp Ser Phe His Asp Tyr Trp 100 105 110 Gly Gln Gly Thr Gln Val Thr Val Ser Ser 115 120 <210> 176 <211> 122 <212> PRT <213> Artificial Sequence <220> <223> Camel VHH Domain <400> 176 Glu Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Thr Ala Ser Gly Val Thr Ile Ser Ala Leu 20 25 30 Asn Ala Met Ala Met Gly Trp Tyr Arg Gln Ala Pro Gly Glu Arg Arg 35 40 45 Val Met Val Ala Ala Val Ser Asn Arg Gly Asn Ala Met Tyr Arg Glu 50 55 60 Ser Val Gln Gly Arg Phe Thr Val Thr Arg Asp Phe Thr Asn Lys Met 65 70 75 80 Val Ser Leu Gln Met Asp Asn Leu Lys Pro Glu Asp Thr Ala Val Tyr 85 90 95 Tyr Cys His Val Leu Glu Asp Arg Val Asp Ser Phe His Asp Tyr Trp 100 105 110 Gly Gln Gly Thr Gln Val Thr Val Ser Ser 115 120 <210> 177 <211> 122 <212> PRT <213> Artificial Sequence <220> <223> Camel VHH Domain <400> 177 Glu Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Thr Ala Ser Gly Val Thr Ile Ser Ala Leu 20 25 30 Asn Ala Met Ala Met Gly Trp Tyr Arg Gln Ala Pro Gly Glu Arg Arg 35 40 45 Val Met Val Ala Ala Val Ser His Arg Gly Asn Ala Met Tyr Arg Glu 50 55 60 Ser Val Gln Gly Arg Phe Thr Val Thr Arg Asp Phe Thr Asn Lys Met 65 70 75 80 Val Ser Leu Gln Met Asp Asn Leu Lys Pro Glu Asp Thr Ala Val Tyr 85 90 95 Tyr Cys His Val Leu Glu Asp Arg Val Asp Ser Phe His Asp Tyr Trp 100 105 110 Gly Gln Gly Thr Gln Val Thr Val Ser Ser 115 120 <210> 178 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Strep - tag <400> 178 Trp Ser His Pro Gln Phe Glu Lys 1 5 <210> 179 <211> 15 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Peptide <220> <221> MOD_RES <222> (1)..(1) <223> Acetylation <220> <221> MOD_RES <222> (15)..(15) <223> Amidation <400> 179 Pro Ser Arg Leu Glu Glu Glu Leu Arg Arg Arg Leu Thr Glu Pro 1 5 10 15 <210> 180 <211> 490 <212> PRT <213> Artificial Sequence <220> <223> Fusion Protein <400> 180 Met Pro Ser Arg Leu Glu Glu Glu Leu Arg Arg Arg Leu Thr Glu Pro 1 5 10 15 Asp Tyr Lys Asp Asp Asp Asp Lys Gly Ser Thr Arg Ser Val Ser Ser 20 25 30 Ser Ser Tyr Arg Arg Met Phe Gly Gly Ser Gly Thr Ser Ser Arg Pro 35 40 45 Ser Ser Asn Arg Ser Tyr Val Thr Thr Ser Thr Arg Thr Tyr Ser Leu 50 55 60 Gly Ser Ala Leu Arg Pro Ser Thr Ser Arg Ser Leu Tyr Ser Ser Ser 65 70 75 80 Pro Gly Gly Ala Tyr Val Thr Arg Ser Ser Ala Val Arg Leu Arg Ser 85 90 95 Ser Val Pro Gly Val Arg Leu Leu Gln Asp Ser Val Asp Phe Ser Leu 100 105 110 Ala Asp Ala Ile Asn Thr Glu Phe Lys Asn Thr Arg Thr Asn Glu Lys 115 120 125 Val Glu Leu Gln Glu Leu Asn Asp Arg Phe Ala Asn Tyr Ile Asp Lys 130 135 140 Val Arg Phe Leu Glu Gln Gln Asn Lys Ile Leu Leu Ala Glu Leu Glu 145 150 155 160 Gln Leu Lys Gly Gln Gly Lys Ser Arg Leu Gly Asp Leu Tyr Glu Glu 165 170 175 Glu Met Arg Glu Leu Arg Arg Gln Val Asp Gln Leu Thr Asn Asp Lys 180 185 190 Ala Arg Val Glu Val Glu Arg Asp Asn Leu Ala Glu Asp Ile Met Arg 195 200 205 Leu Arg Glu Lys Leu Gln Glu Glu Met Leu Gln Arg Glu Glu Ala Glu 210 215 220 Ser Thr Leu Gln Ser Phe Arg Gln Asp Val Asp Asn Ala Ser Leu Ala 225 230 235 240 Arg Leu Asp Leu Glu Arg Lys Val Glu Ser Leu Gln Glu Glu Ile Ala 245 250 255 Phe Leu Lys Lys Leu His Asp Glu Glu Ile Gln Glu Leu Gln Ala Gln 260 265 270 Ile Gln Glu Gln His Val Gln Ile Asp Val Asp Val Ser Lys Pro Asp 275 280 285 Leu Thr Ala Ala Leu Arg Asp Val Arg Gln Gln Tyr Glu Ser Val Ala 290 295 300 Ala Lys Asn Leu Gln Glu Ala Glu Glu Trp Tyr Lys Ser Lys Phe Ala 305 310 315 320 Asp Leu Ser Glu Ala Ala Asn Arg Asn Asn Asp Ala Leu Arg Gln Ala 325 330 335 Lys Gln Glu Ser Asn Glu Tyr Arg Arg Gln Val Gln Ser Leu Thr Cys 340 345 350 Glu Val Asp Ala Leu Lys Gly Thr Asn Glu Ser Leu Glu Arg Gln Met 355 360 365 Arg Glu Met Glu Glu Asn Phe Ala Leu Glu Ala Ala Asn Tyr Gln Asp 370 375 380 Thr Ile Gly Arg Leu Gln Asp Glu Ile Gln Asn Met Lys Glu Glu Met 385 390 395 400 Ala Arg His Leu Arg Glu Tyr Gln Asp Leu Leu Asn Val Lys Met Ala 405 410 415 Leu Asp Ile Glu Ile Ala Thr Tyr Arg Lys Leu Leu Glu Gly Glu Glu 420 425 430 Ser Arg Ile Ser Leu Pro Leu Pro Thr Phe Ser Ser Leu Asn Leu Arg 435 440 445 Glu Thr Asn Leu Glu Ser Leu Pro Leu Val Asp Thr His Ser Lys Arg 450 455 460 Thr Leu Leu Ile Lys Thr Val Glu Thr Arg Asp Gly Gln Val Ile Asn 465 470 475 480 Glu Thr Ser Gln His His Asp Asp Leu Glu 485 490 <210> 181 <211> 489 <212> PRT <213> Artificial Sequence <220> <223> Fusion Protein <400> 181 Met Ser Gly Arg Leu Glu Glu Glu Leu Arg Arg Arg Leu Ser Pro Asp 1 5 10 15 Tyr Lys Asp Asp Asp Asp Lys Gly Ser Thr Arg Ser Val Ser Ser Ser 20 25 30 Ser Tyr Arg Arg Met Phe Gly Gly Ser Gly Thr Ser Ser Arg Pro Ser 35 40 45 Ser Asn Arg Ser Tyr Val Thr Thr Ser Thr Arg Thr Tyr Ser Leu Gly 50 55 60 Ser Ala Leu Arg Pro Ser Thr Ser Arg Ser Leu Tyr Ser Ser Ser Pro 65 70 75 80 Gly Gly Ala Tyr Val Thr Arg Ser Ser Ala Val Arg Leu Arg Ser Ser 85 90 95 Val Pro Gly Val Arg Leu Leu Gln Asp Ser Val Asp Phe Ser Leu Ala 100 105 110 Asp Ala Ile Asn Thr Glu Phe Lys Asn Thr Arg Thr Asn Glu Lys Val 115 120 125 Glu Leu Gln Glu Leu Asn Asp Arg Phe Ala Asn Tyr Ile Asp Lys Val 130 135 140 Arg Phe Leu Glu Gln Gln Asn Lys Ile Leu Leu Ala Glu Leu Glu Gln 145 150 155 160 Leu Lys Gly Gln Gly Lys Ser Arg Leu Gly Asp Leu Tyr Glu Glu Glu 165 170 175 Met Arg Glu Leu Arg Arg Gln Val Asp Gln Leu Thr Asn Asp Lys Ala 180 185 190 Arg Val Glu Val Glu Arg Asp Asn Leu Ala Glu Asp Ile Met Arg Leu 195 200 205 Arg Glu Lys Leu Gln Glu Glu Met Leu Gln Arg Glu Glu Ala Glu Ser 210 215 220 Thr Leu Gln Ser Phe Arg Gln Asp Val Asp Asn Ala Ser Leu Ala Arg 225 230 235 240 Leu Asp Leu Glu Arg Lys Val Glu Ser Leu Gln Glu Glu Ile Ala Phe 245 250 255 Leu Lys Lys Leu His Asp Glu Glu Ile Gln Glu Leu Gln Ala Gln Ile 260 265 270 Gln Glu Gln His Val Gln Ile Asp Val Asp Val Ser Lys Pro Asp Leu 275 280 285 Thr Ala Ala Leu Arg Asp Val Arg Gln Gln Tyr Glu Ser Val Ala Ala 290 295 300 Lys Asn Leu Gln Glu Ala Glu Glu Trp Tyr Lys Ser Lys Phe Ala Asp 305 310 315 320 Leu Ser Glu Ala Ala Asn Arg Asn Asn Asp Ala Leu Arg Gln Ala Lys 325 330 335 Gln Glu Ser Asn Glu Tyr Arg Arg Gln Val Gln Ser Leu Thr Cys Glu 340 345 350 Val Asp Ala Leu Lys Gly Thr Asn Glu Ser Leu Glu Arg Gln Met Arg 355 360 365 Glu Met Glu Glu Asn Phe Ala Leu Glu Ala Ala Asn Tyr Gln Asp Thr 370 375 380 Ile Gly Arg Leu Gln Asp Glu Ile Gln Asn Met Lys Glu Glu Met Ala 385 390 395 400 Arg His Leu Arg Glu Tyr Gln Asp Leu Leu Asn Val Lys Met Ala Leu 405 410 415 Asp Ile Glu Ile Ala Thr Tyr Arg Lys Leu Leu Glu Gly Glu Glu Ser 420 425 430 Arg Ile Ser Leu Pro Leu Pro Thr Phe Ser Ser Leu Asn Leu Arg Glu 435 440 445 Thr Asn Leu Glu Ser Leu Pro Leu Val Asp Thr His Ser Lys Arg Thr 450 455 460 Leu Leu Ile Lys Thr Val Glu Thr Arg Asp Gly Gln Val Ile Asn Glu 465 470 475 480 Thr Ser Gln His His Asp Asp Leu Glu 485 <210> 182 <211> 291 <212> PRT <213> Artificial Sequence <220> <223> Fusion Protein <400> 182 Met Lys Ser Phe Ile Thr Arg Asn Lys Thr Ala Ile Leu Ala Thr Val 1 5 10 15 Ala Ala Thr Gly Thr Ala Ile Gly Ala Tyr Tyr Tyr Tyr Gly Asn Ser 20 25 30 Pro Val Ala Thr Met Val Ser Lys Gly Glu Glu Leu Phe Thr Gly Val 35 40 45 Val Pro Ile Leu Val Glu Leu Asp Gly Asp Val Asn Gly His Lys Phe 50 55 60 Ser Val Ser Gly Glu Gly Glu Gly Asp Ala Thr Tyr Gly Lys Leu Thr 65 70 75 80 Leu Lys Phe Ile Cys Thr Thr Gly Lys Leu Pro Val Pro Trp Pro Thr 85 90 95 Leu Val Thr Thr Leu Thr Tyr Gly Val Gln Cys Phe Ser Arg Tyr Pro 100 105 110 Asp His Met Lys Gln His Asp Phe Phe Lys Ser Ala Met Pro Glu Gly 115 120 125 Tyr Val Gln Glu Arg Thr Ile Phe Phe Lys Asp Asp Gly Asn Tyr Lys 130 135 140 Thr Arg Ala Glu Val Lys Phe Glu Gly Asp Thr Leu Val Asn Arg Ile 145 150 155 160 Glu Leu Lys Gly Ile Asp Phe Lys Glu Asp Gly Asn Ile Leu Gly His 165 170 175 Lys Leu Glu Tyr Asn Tyr Asn Ser His Asn Val Tyr Ile Met Ala Asp 180 185 190 Lys Gln Lys Asn Gly Ile Lys Val Asn Phe Lys Ile Arg His Asn Ile 195 200 205 Glu Asp Gly Ser Val Gln Leu Ala Asp His Tyr Gln Gln Asn Thr Pro 210 215 220 Ile Gly Asp Gly Pro Val Leu Leu Pro Asp Asn His Tyr Leu Ser Thr 225 230 235 240 Gln Ser Lys Leu Ser Lys Asp Pro Asn Glu Lys Arg Asp His Met Val 245 250 255 Leu Leu Glu Phe Val Thr Ala Ala Gly Ile Thr Leu Gly Met Asp Glu 260 265 270 Leu Tyr Lys Gly Ser Pro Ser Arg Leu Glu Glu Glu Leu Arg Arg Arg 275 280 285 Leu Thr Glu 290 <210> 183 <211> 291 <212> PRT <213> Artificial Sequence <220> <223> Fusion Protein <400> 183 Met Lys Ser Phe Ile Thr Arg Asn Lys Thr Ala Ile Leu Ala Thr Val 1 5 10 15 Ala Ala Thr Gly Thr Ala Ile Gly Ala Tyr Tyr Tyr Tyr Gly Asn Ser 20 25 30 Pro Val Ala Thr Met Val Ser Lys Gly Glu Glu Leu Phe Thr Gly Val 35 40 45 Val Pro Ile Leu Val Glu Leu Asp Gly Asp Val Asn Gly His Lys Phe 50 55 60 Ser Val Ser Gly Glu Gly Glu Gly Asp Ala Thr Tyr Gly Lys Leu Thr 65 70 75 80 Leu Lys Phe Ile Cys Thr Thr Gly Lys Leu Pro Val Pro Trp Pro Thr 85 90 95 Leu Val Thr Thr Leu Thr Tyr Gly Val Gln Cys Phe Ser Arg Tyr Pro 100 105 110 Asp His Met Lys Gln His Asp Phe Phe Lys Ser Ala Met Pro Glu Gly 115 120 125 Tyr Val Gln Glu Arg Thr Ile Phe Phe Lys Asp Asp Gly Asn Tyr Lys 130 135 140 Thr Arg Ala Glu Val Lys Phe Glu Gly Asp Thr Leu Val Asn Arg Ile 145 150 155 160 Glu Leu Lys Gly Ile Asp Phe Lys Glu Asp Gly Asn Ile Leu Gly His 165 170 175 Lys Leu Glu Tyr Asn Tyr Asn Ser His Asn Val Tyr Ile Met Ala Asp 180 185 190 Lys Gln Lys Asn Gly Ile Lys Val Asn Phe Lys Ile Arg His Asn Ile 195 200 205 Glu Asp Gly Ser Val Gln Leu Ala Asp His Tyr Gln Gln Asn Thr Pro 210 215 220 Ile Gly Asp Gly Pro Val Leu Leu Pro Asp Asn His Tyr Leu Ser Thr 225 230 235 240 Gln Ser Lys Leu Ser Lys Asp Pro Asn Glu Lys Arg Asp His Met Val 245 250 255 Leu Leu Glu Phe Val Thr Ala Ala Gly Ile Thr Leu Gly Met Asp Glu 260 265 270 Leu Tyr Lys Gly Ser Pro Ser Arg Leu Glu Glu Glu Leu Arg Arg Arg 275 280 285 Leu Thr Glu 290 <210> 184 <211> 350 <212> PRT <213> Artificial Sequence <220> <223> Fusion Protein <400> 184 Met Glu Thr Asp Thr Leu Leu Leu Trp Val Leu Leu Leu Trp Val Pro 1 5 10 15 Gly Ser Thr Gly Asp Tyr Pro Tyr Asp Val Pro Asp Tyr Ala Ser Asn 20 25 30 Gly Thr Ser Lys Gly Glu Glu Leu Phe Thr Gly Val Val Pro Ile Leu 35 40 45 Val Glu Leu Asp Gly Asp Val Asn Gly His Lys Phe Ser Val Ser Gly 50 55 60 Glu Gly Glu Gly Asp Ala Thr Tyr Gly Lys Leu Thr Leu Lys Phe Ile 65 70 75 80 Cys Thr Thr Gly Lys Leu Pro Val Pro Trp Pro Thr Leu Val Thr Thr 85 90 95 Leu Thr Tyr Gly Val Gln Cys Phe Ser Arg Tyr Pro Asp His Met Lys 100 105 110 Gln His Asp Phe Phe Lys Ser Ala Met Pro Glu Gly Tyr Val Gln Glu 115 120 125 Arg Thr Ile Phe Phe Lys Asp Asp Gly Asn Tyr Lys Thr Arg Ala Glu 130 135 140 Val Lys Phe Glu Gly Asp Thr Leu Val Asn Arg Ile Glu Leu Lys Gly 145 150 155 160 Ile Asp Phe Lys Glu Asp Gly Asn Ile Leu Gly His Lys Leu Glu Tyr 165 170 175 Asn Tyr Asn Ser His Asn Val Tyr Ile Met Ala Asp Lys Gln Lys Asn 180 185 190 Gly Ile Lys Val Asn Phe Lys Ile Arg His Asn Ile Glu Asp Gly Ser 195 200 205 Val Gln Leu Ala Asp His Tyr Gln Gln Asn Thr Pro Ile Gly Asp Gly 210 215 220 Pro Val Leu Leu Pro Asp Asn His Tyr Leu Ser Thr Gln Ser Ala Leu 225 230 235 240 Ser Lys Asp Pro Asn Glu Lys Arg Asp His Met Val Leu Lys Glu Phe 245 250 255 Val Thr Ala Ala Gly Ile Thr Leu Gly Met Asp Glu Leu Tyr Lys Gly 260 265 270 Ser Pro Ser Arg Leu Glu Glu Glu Leu Arg Arg Arg Leu Thr Glu Pro 275 280 285 Gly Asp Glu Gln Lys Leu Ile Ser Glu Glu Asp Leu Asn Ala Val Gly 290 295 300 Gln Asp Thr Gln Glu Val Ile Val Val Pro His Ser Leu Pro Phe Lys 305 310 315 320 Val Val Val Ile Ser Ala Ile Leu Ala Leu Val Val Leu Thr Ile Ile 325 330 335 Ser Leu Ile Ile Leu Ile Met Leu Trp Gln Lys Lys Pro Arg 340 345 350 <210> 185 <211> 382 <212> PRT <213> Artificial Sequence <220> <223> Fusion Protein <400> 185 Met Gly Ser Gly Asp Ala Ser Asp Ser Glu Val Gln Leu Gln Glu Ser 1 5 10 15 Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Thr 20 25 30 Ala Ser Gly Val Thr Ile Ser Ala Leu Asn Ala Met Ala Met Gly Trp 35 40 45 Tyr Arg Gln Ala Pro Gly Glu Arg Arg Val Met Val Ala Ala Val Ser 50 55 60 Glu Arg Gly Asn Ala Met Tyr Arg Glu Ser Val Gln Gly Arg Phe Thr 65 70 75 80 Val Thr Arg Asp Phe Thr Asn Lys Met Val Ser Leu Gln Met Asp Asn 85 90 95 Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys His Val Leu Glu Asp 100 105 110 Arg Val Asp Ser Phe His Asp Tyr Trp Gly Gln Gly Thr Gln Val Thr 115 120 125 Val Ser Ser Glu Pro Lys Thr Pro Lys Pro Gln Thr Ser Gly Ser Thr 130 135 140 Gly Glu Asn Val Ala Thr Met Val Ser Lys Gly Glu Ala Val Ile Lys 145 150 155 160 Glu Phe Met Arg Phe Lys Val His Met Glu Gly Ser Met Asn Gly His 165 170 175 Glu Phe Glu Ile Glu Gly Glu Gly Glu Gly Arg Pro Tyr Glu Gly Thr 180 185 190 Gln Thr Ala Lys Leu Lys Val Thr Lys Gly Gly Pro Leu Pro Phe Ser 195 200 205 Trp Asp Ile Leu Ser Pro Gln Phe Met Tyr Gly Ser Arg Ala Phe Ile 210 215 220 Lys His Pro Ala Asp Ile Pro Asp Tyr Tyr Lys Gln Ser Phe Pro Glu 225 230 235 240 Gly Phe Lys Trp Glu Arg Val Met Asn Phe Glu Asp Gly Gly Ala Val 245 250 255 Thr Val Thr Gln Asp Thr Ser Leu Glu Asp Gly Thr Leu Ile Tyr Lys 260 265 270 Val Lys Leu Arg Gly Thr Asn Phe Pro Pro Asp Gly Pro Val Met Gln 275 280 285 Lys Lys Thr Met Gly Trp Glu Ala Ser Thr Glu Arg Leu Tyr Pro Glu 290 295 300 Asp Gly Val Leu Lys Gly Asp Ile Lys Met Ala Leu Arg Leu Lys Asp 305 310 315 320 Gly Gly Arg Tyr Leu Ala Asp Phe Lys Thr Thr Tyr Lys Ala Lys Lys 325 330 335 Pro Val Gln Met Pro Gly Ala Tyr Asn Val Asp Arg Lys Leu Asp Ile 340 345 350 Thr Ser His Asn Glu Asp Tyr Thr Val Val Glu Gln Tyr Glu Arg Ser 355 360 365 Glu Gly Arg His Ser Thr Gly Gly Met Asp Glu Leu Tyr Lys 370 375 380 <210> 186 <211> 239 <212> PRT <213> Artificial Sequence <220> <223> EGFP <400> 186 Met Val Ser Lys Gly Glu Glu Leu Phe Thr Gly Val Val Pro Ile Leu 1 5 10 15 Val Glu Leu Asp Gly Asp Val Asn Gly His Lys Phe Ser Val Ser Gly 20 25 30 Glu Gly Glu Gly Asp Ala Thr Tyr Gly Lys Leu Thr Leu Lys Phe Ile 35 40 45 Cys Thr Thr Gly Lys Leu Pro Val Pro Trp Pro Thr Leu Val Thr Thr 50 55 60 Leu Thr Tyr Gly Val Gln Cys Phe Ser Arg Tyr Pro Asp His Met Lys 65 70 75 80 Gln His Asp Phe Phe Lys Ser Ala Met Pro Glu Gly Tyr Val Gln Glu 85 90 95 Arg Thr Ile Phe Phe Lys Asp Asp Gly Asn Tyr Lys Thr Arg Ala Glu 100 105 110 Val Lys Phe Glu Gly Asp Thr Leu Val Asn Arg Ile Glu Leu Lys Gly 115 120 125 Ile Asp Phe Lys Glu Asp Gly Asn Ile Leu Gly His Lys Leu Glu Tyr 130 135 140 Asn Tyr Asn Ser His Asn Val Tyr Ile Met Ala Asp Lys Gln Lys Asn 145 150 155 160 Gly Ile Lys Val Asn Phe Lys Ile Arg His Asn Ile Glu Asp Gly Ser 165 170 175 Val Gln Leu Ala Asp His Tyr Gln Gln Asn Thr Pro Ile Gly Asp Gly 180 185 190 Pro Val Leu Leu Pro Asp Asn His Tyr Leu Ser Thr Gln Ser Ala Leu 195 200 205 Ser Lys Asp Pro Asn Glu Lys Arg Asp His Met Val Leu Leu Glu Phe 210 215 220 Val Thr Ala Ala Gly Ile Thr Leu Gly Met Asp Glu Leu Tyr Lys 225 230 235 <210> 187 <211> 255 <212> PRT <213> Artificial Sequence <220> <223> Fusion Protein <400> 187 Met Val Ser Lys Gly Glu Glu Leu Phe Thr Gly Val Val Pro Ile Leu 1 5 10 15 Val Glu Leu Asp Gly Asp Val Asn Gly His Lys Phe Ser Val Ser Gly 20 25 30 Glu Gly Glu Gly Asp Ala Thr Tyr Gly Lys Leu Thr Leu Lys Phe Ile 35 40 45 Cys Thr Thr Gly Lys Leu Pro Val Pro Trp Pro Thr Leu Val Thr Thr 50 55 60 Leu Thr Tyr Gly Val Gln Cys Phe Ser Arg Tyr Pro Asp His Met Lys 65 70 75 80 Gln His Asp Phe Phe Lys Ser Ala Met Pro Glu Gly Tyr Val Gln Glu 85 90 95 Arg Thr Ile Phe Phe Lys Asp Asp Gly Asn Tyr Lys Thr Arg Ala Glu 100 105 110 Val Lys Phe Glu Gly Asp Thr Leu Val Asn Arg Ile Glu Leu Lys Gly 115 120 125 Ile Asp Phe Lys Glu Asp Gly Asn Ile Leu Gly His Lys Leu Glu Tyr 130 135 140 Asn Tyr Asn Ser His Asn Val Tyr Ile Met Ala Asp Lys Gln Lys Asn 145 150 155 160 Gly Ile Lys Val Asn Phe Lys Ile Arg His Asn Ile Glu Asp Gly Ser 165 170 175 Val Gln Leu Ala Asp His Tyr Gln Gln Asn Thr Pro Ile Gly Asp Gly 180 185 190 Pro Val Leu Leu Pro Asp Asn His Tyr Leu Ser Thr Gln Ser Ala Leu 195 200 205 Ser Lys Asp Pro Asn Glu Lys Arg Asp His Met Val Leu Leu Glu Phe 210 215 220 Val Thr Ala Ala Gly Ile Thr Leu Gly Met Asp Glu Leu Tyr Lys Gly 225 230 235 240 Ser Pro Ser Arg Leu Glu Glu Glu Leu Arg Arg Arg Leu Thr Glu 245 250 255 <210> 188 <211> 255 <212> PRT <213> Artificial Sequence <220> <223> Fusion Protein <400> 188 Met Pro Ser Arg Leu Glu Glu Glu Leu Arg Arg Arg Leu Thr Glu Pro 1 5 10 15 Met Val Ser Lys Gly Glu Glu Leu Phe Thr Gly Val Val Pro Ile Leu 20 25 30 Val Glu Leu Asp Gly Asp Val Asn Gly His Lys Phe Ser Val Ser Gly 35 40 45 Glu Gly Glu Gly Asp Ala Thr Tyr Gly Lys Leu Thr Leu Lys Phe Ile 50 55 60 Cys Thr Thr Gly Lys Leu Pro Val Pro Trp Pro Thr Leu Val Thr Thr 65 70 75 80 Leu Thr Tyr Gly Val Gln Cys Phe Ser Arg Tyr Pro Asp His Met Lys 85 90 95 Gln His Asp Phe Phe Lys Ser Ala Met Pro Glu Gly Tyr Val Gln Glu 100 105 110 Arg Thr Ile Phe Phe Lys Asp Asp Gly Asn Tyr Lys Thr Arg Ala Glu 115 120 125 Val Lys Phe Glu Gly Asp Thr Leu Val Asn Arg Ile Glu Leu Lys Gly 130 135 140 Ile Asp Phe Lys Glu Asp Gly Asn Ile Leu Gly His Lys Leu Glu Tyr 145 150 155 160 Asn Tyr Asn Ser His Asn Val Tyr Ile Met Ala Asp Lys Gln Lys Asn 165 170 175 Gly Ile Lys Val Asn Phe Lys Ile Arg His Asn Ile Glu Asp Gly Ser 180 185 190 Val Gln Leu Ala Asp His Tyr Gln Gln Asn Thr Pro Ile Gly Asp Gly 195 200 205 Pro Val Leu Leu Pro Asp Asn His Tyr Leu Ser Thr Gln Ser Ala Leu 210 215 220 Ser Lys Asp Pro Asn Glu Lys Arg Asp His Met Val Leu Leu Glu Phe 225 230 235 240 Val Thr Ala Ala Gly Ile Thr Leu Gly Met Asp Glu Leu Tyr Lys 245 250 255 <210> 189 <211> 558 <212> PRT <213> Artificial Sequence <220> <223> Fusion Protein <400> 189 Met Ser Lys His His His His Ser Asn His His Arg His Asn His His 1 5 10 15 His His Ser Gly Asn His His His Ser Gly Ser Ala Ala Gly Gly Glu 20 25 30 Glu Asp Lys Lys Pro Ala Gly Gly Glu Gly Gly Gly Ala His Ile Asn 35 40 45 Leu Lys Val Lys Gly Gln Asp Gly Asn Glu Val Phe Phe Arg Ile Lys 50 55 60 Arg Ser Thr Gln Leu Lys Lys Leu Met Asn Ala Tyr Cys Asp Arg Gln 65 70 75 80 Ser Val Asp Met Thr Ala Ile Ala Phe Leu Phe Asp Gly Arg Arg Leu 85 90 95 Arg Ala Glu Gln Thr Pro Asp Glu Leu Glu Met Glu Asp Gly Asp Glu 100 105 110 Ile Asp Ala Met Leu His Gln Thr Gly Gly Ala Ser Asp Tyr Lys Asp 115 120 125 Asp Asp Asp Lys Gly Ser Thr Gly Asp Tyr Pro Tyr Asp Val Pro Asp 130 135 140 Tyr Ala Ser Asn Gly Thr Lys Thr Glu Glu Gly Lys Leu Val Ile Trp 145 150 155 160 Ile Asn Gly Asp Lys Gly Tyr Asn Gly Leu Ala Glu Val Gly Lys Lys 165 170 175 Phe Glu Lys Asp Thr Gly Ile Lys Val Thr Val Glu His Pro Asp Lys 180 185 190 Leu Glu Glu Lys Phe Pro Gln Val Ala Ala Thr Gly Asp Gly Pro Asp 195 200 205 Ile Ile Phe Trp Ala His Asp Arg Phe Gly Gly Tyr Ala Gln Ser Gly 210 215 220 Leu Leu Ala Glu Ile Thr Pro Asp Lys Ala Phe Gln Asp Lys Leu Tyr 225 230 235 240 Pro Phe Thr Trp Asp Ala Val Arg Tyr Asn Gly Lys Leu Ile Ala Tyr 245 250 255 Pro Ile Ala Val Glu Ala Leu Ser Leu Ile Tyr Asn Lys Asp Leu Leu 260 265 270 Pro Asn Pro Pro Lys Thr Trp Glu Glu Ile Pro Ala Leu Asp Lys Glu 275 280 285 Leu Lys Ala Lys Gly Lys Ser Ala Leu Met Phe Asn Leu Gln Glu Pro 290 295 300 Tyr Phe Thr Trp Pro Leu Ile Ala Ala Asp Gly Gly Tyr Ala Phe Lys 305 310 315 320 Tyr Glu Asn Gly Lys Tyr Asp Ile Lys Asp Val Gly Val Asp Asn Ala 325 330 335 Gly Ala Lys Ala Gly Leu Thr Phe Leu Val Asp Leu Ile Lys Asn Lys 340 345 350 His Met Asn Ala Asp Thr Asp Tyr Ser Ile Ala Glu Ala Ala Phe Asn 355 360 365 Lys Gly Glu Thr Ala Met Thr Ile Asn Gly Pro Trp Ala Trp Ser Asn 370 375 380 Ile Asp Thr Ser Lys Val Asn Tyr Gly Val Thr Val Leu Pro Thr Phe 385 390 395 400 Lys Gly Gln Pro Ser Lys Pro Phe Val Gly Val Leu Ser Ala Gly Ile 405 410 415 Asn Ala Ala Ser Pro Asn Lys Glu Leu Ala Lys Glu Phe Leu Glu Asn 420 425 430 Tyr Leu Leu Thr Asp Glu Gly Leu Glu Ala Val Asn Lys Asp Lys Pro 435 440 445 Leu Gly Ala Val Ala Leu Lys Ser Tyr Glu Glu Glu Leu Ala Lys Asp 450 455 460 Pro Arg Ile Ala Ala Thr Met Glu Asn Ala Gln Lys Gly Glu Ile Met 465 470 475 480 Pro Asn Ile Pro Gln Met Ser Ala Phe Trp Tyr Ala Val Arg Thr Ala 485 490 495 Val Ile Asn Ala Ala Ser Gly Arg Gln Thr Val Asp Glu Ala Leu Lys 500 505 510 Asp Ala Gln Thr Asn Gly Ser Val Ser Ala Gly Asp Glu Gln Lys Leu 515 520 525 Ile Ser Glu Glu Asp Leu Asn Ala Val Gly Gln Asp Thr Ala Ser Thr 530 535 540 Pro Ser Arg Leu Glu Glu Glu Leu Arg Arg Arg Leu Thr Glu 545 550 555 <210> 190 <211> 259 <212> PRT <213> Artificial Sequence <220> <223> Fusion Protein <400> 190 Met Pro Ser Arg Leu Glu Glu Glu Leu Arg Arg Arg Leu Thr Glu Pro 1 5 10 15 Ser Lys Gly Glu Glu Leu Phe Thr Gly Thr Val Pro Ile Lys Val Glu 20 25 30 Leu Asp Gly Asp Val Asn Gly His Lys Phe Ser Val Arg Gly Glu Gly 35 40 45 Glu Gly Asp Ala Thr Glu Gly Lys Leu Thr Leu Lys Phe Ile Cys Thr 50 55 60 Thr Gly Lys Leu Pro Val Pro Trp Pro Thr Leu Val Thr Thr Leu Thr 65 70 75 80 Tyr Gly Val Gln Cys Phe Ser Arg Tyr Pro Asp His Met Lys Arg His 85 90 95 Asp Phe Phe Lys Ser Ala Met Pro Glu Gly Tyr Val Gln Glu Arg Thr 100 105 110 Ile Glu Phe Lys Asp Asp Gly Thr Tyr Lys Thr Arg Ala Glu Val Lys 115 120 125 Phe Glu Gly Asp Thr Leu Val Asn Arg Ile Glu Leu Lys Gly Asn Asp 130 135 140 Phe Lys Glu Asp Gly Asn Ile Leu Gly His Lys Leu Glu Tyr Asn His 145 150 155 160 Asn Ser His Asn Val Arg Ile Glu Ala Asp Lys Gln Lys Asn Gly Ile 165 170 175 Lys Ala Asn Phe Lys Ile Arg His Asn Val Glu Asp Gly Ser Gln Gln 180 185 190 Glu Ala Asp His Lys Gln Gln Asn Thr Pro Ile Gly Asp Gly Pro Val 195 200 205 Arg Leu Pro Asp Asn His Tyr Leu Ser Thr Gln Thr Thr Leu Ser Lys 210 215 220 Asp Pro Asn Glu Lys Arg Asp His Met Val Leu Lys Glu Phe Val Thr 225 230 235 240 Ala Ala Gly Ile Thr Lys Gly Glu Asp Glu Arg Asp Lys His His His 245 250 255 His His His <210> 191 <211> 258 <212> PRT <213> Artificial Sequence <220> <223> Fusion Protein <400> 191 Met Ser Gly Arg Leu Glu Glu Glu Leu Arg Arg Arg Leu Ser Pro Ser 1 5 10 15 Lys Gly Glu Glu Leu Phe Thr Gly Thr Val Pro Ile Lys Val Glu Leu 20 25 30 Asp Gly Asp Val Asn Gly His Lys Phe Ser Val Arg Gly Glu Gly Glu 35 40 45 Gly Asp Ala Thr Glu Gly Lys Leu Thr Leu Lys Phe Ile Cys Thr Thr 50 55 60 Gly Lys Leu Pro Val Pro Trp Pro Thr Leu Val Thr Thr Leu Thr Tyr 65 70 75 80 Gly Val Gln Cys Phe Ser Arg Tyr Pro Asp His Met Lys Arg His Asp 85 90 95 Phe Phe Lys Ser Ala Met Pro Glu Gly Tyr Val Gln Glu Arg Thr Ile 100 105 110 Glu Phe Lys Asp Asp Gly Thr Tyr Lys Thr Arg Ala Glu Val Lys Phe 115 120 125 Glu Gly Asp Thr Leu Val Asn Arg Ile Glu Leu Lys Gly Asn Asp Phe 130 135 140 Lys Glu Asp Gly Asn Ile Leu Gly His Lys Leu Glu Tyr Asn His Asn 145 150 155 160 Ser His Asn Val Arg Ile Glu Ala Asp Lys Gln Lys Asn Gly Ile Lys 165 170 175 Ala Asn Phe Lys Ile Arg His Asn Val Glu Asp Gly Ser Gln Gln Glu 180 185 190 Ala Asp His Lys Gln Gln Asn Thr Pro Ile Gly Asp Gly Pro Val Arg 195 200 205 Leu Pro Asp Asn His Tyr Leu Ser Thr Gln Thr Thr Leu Ser Lys Asp 210 215 220 Pro Asn Glu Lys Arg Asp His Met Val Leu Lys Glu Phe Val Thr Ala 225 230 235 240 Ala Gly Ile Thr Lys Gly Glu Asp Glu Arg Asp Lys His His His His 245 250 255 His His <210> 192 <211> 381 <212> PRT <213> Artificial Sequence <220> <223> Fusion Protein <400> 192 Met Ser Lys His His His His Ser Asn His His Arg His Asn His His 1 5 10 15 His His Ser Gly Asn His His His Ser Gly Ser Ala Ala Gly Gly Glu 20 25 30 Glu Asp Lys Lys Pro Ala Gly Gly Glu Gly Gly Gly Ala His Ile Asn 35 40 45 Leu Lys Val Lys Gly Gln Asp Gly Asn Glu Val Phe Phe Arg Ile Lys 50 55 60 Arg Ser Thr Gln Leu Lys Lys Leu Met Asn Ala Tyr Cys Asp Arg Gln 65 70 75 80 Ser Val Asp Met Thr Ala Ile Ala Phe Leu Phe Asp Gly Arg Arg Leu 85 90 95 Arg Ala Glu Gln Thr Pro Asp Glu Leu Glu Met Glu Asp Gly Asp Glu 100 105 110 Ile Asp Ala Met Leu His Gln Thr Gly Gly Gly Ser Lys Gly Glu Glu 115 120 125 Leu Phe Thr Gly Thr Val Pro Ile Lys Val Glu Leu Asp Gly Asp Val 130 135 140 Asn Gly His Lys Phe Ser Val Arg Gly Glu Gly Glu Gly Asp Ala Thr 145 150 155 160 Glu Gly Lys Leu Thr Leu Lys Phe Ile Cys Thr Thr Gly Lys Leu Pro 165 170 175 Val Pro Trp Pro Thr Leu Val Thr Thr Leu Thr Tyr Gly Val Gln Cys 180 185 190 Phe Ser Arg Tyr Pro Asp His Met Lys Arg His Asp Phe Phe Lys Ser 195 200 205 Ala Met Pro Glu Gly Tyr Val Gln Glu Arg Thr Ile Glu Phe Lys Asp 210 215 220 Asp Gly Thr Tyr Lys Thr Arg Ala Glu Val Lys Phe Glu Gly Asp Thr 225 230 235 240 Leu Val Asn Arg Ile Glu Leu Lys Gly Asn Asp Phe Lys Glu Asp Gly 245 250 255 Asn Ile Leu Gly His Lys Leu Glu Tyr Asn His Asn Ser His Asn Val 260 265 270 Arg Ile Glu Ala Asp Lys Gln Lys Asn Gly Ile Lys Ala Asn Phe Lys 275 280 285 Ile Arg His Asn Val Glu Asp Gly Ser Gln Gln Glu Ala Asp His Lys 290 295 300 Gln Gln Asn Thr Pro Ile Gly Asp Gly Pro Val Arg Leu Pro Asp Asn 305 310 315 320 His Tyr Leu Ser Thr Gln Thr Thr Leu Ser Lys Asp Pro Asn Glu Lys 325 330 335 Arg Asp His Met Val Leu Lys Glu Phe Val Thr Ala Ala Gly Ile Thr 340 345 350 Lys Gly Glu Asp Glu Arg Asp Lys Gly Ser Gly Asn Ser Asp Gly Pro 355 360 365 Ser Arg Leu Glu Glu Glu Leu Arg Arg Arg Leu Thr Glu 370 375 380 <210> 193 <211> 382 <212> PRT <213> Artificial sequence <220> <223> Fusion protein <400> 193 Met Ser Lys His His His His Ser Asn His His Arg His Asn His His 1 5 10 15 His His Ser Gly Asn His His His Ser Gly Ser Ala Ala Gly Gly Glu 20 25 30 Glu Asp Lys Lys Pro Ala Gly Gly Glu Gly Gly Gly Ala His Ile Asn 35 40 45 Leu Lys Val Lys Gly Gln Asp Gly Asn Glu Val Phe Phe Arg Ile Lys 50 55 60 Arg Ser Thr Gln Leu Lys Lys Leu Met Asn Ala Tyr Cys Asp Arg Gln 65 70 75 80 Ser Val Asp Met Thr Ala Ile Ala Phe Leu Phe Asp Gly Arg Arg Leu 85 90 95 Arg Ala Glu Gln Thr Pro Asp Glu Leu Glu Met Glu Asp Gly Asp Glu 100 105 110 Ile Asp Ala Met Leu His Gln Thr Gly Gly Gly Ser Lys Gly Glu Glu 115 120 125 Leu Phe Thr Gly Thr Val Pro Ile Lys Val Glu Leu Asp Gly Asp Val 130 135 140 Asn Gly His Lys Phe Ser Val Arg Gly Glu Gly Glu Gly Asp Ala Thr 145 150 155 160 Glu Gly Lys Leu Thr Leu Lys Phe Ile Cys Thr Thr Gly Lys Leu Pro 165 170 175 Val Pro Trp Pro Thr Leu Val Thr Thr Leu Thr Tyr Gly Val Gln Cys 180 185 190 Phe Ser Arg Tyr Pro Asp His Met Lys Arg His Asp Phe Phe Lys Ser 195 200 205 Ala Met Pro Glu Gly Tyr Val Gln Glu Arg Thr Ile Glu Phe Lys Asp 210 215 220 Asp Gly Thr Tyr Lys Thr Arg Ala Glu Val Lys Phe Glu Gly Asp Thr 225 230 235 240 Leu Val Asn Arg Ile Glu Leu Lys Gly Asn Asp Phe Lys Glu Asp Gly 245 250 255 Asn Ile Leu Gly His Lys Leu Glu Tyr Asn His Asn Ser His Asn Val 260 265 270 Arg Ile Glu Ala Asp Lys Gln Lys Asn Gly Ile Lys Ala Asn Phe Lys 275 280 285 Ile Arg His Asn Val Glu Asp Gly Ser Gln Gln Glu Ala Asp His Lys 290 295 300 Gln Gln Asn Thr Pro Ile Gly Asp Gly Pro Val Arg Leu Pro Asp Asn 305 310 315 320 His Tyr Leu Ser Thr Gln Thr Thr Leu Ser Lys Asp Pro Asn Glu Lys 325 330 335 Arg Asp His Met Val Leu Lys Glu Phe Val Thr Ala Ala Gly Ile Thr 340 345 350 Lys Gly Glu Asp Glu Arg Asp Lys Gly Ser Gly Asn Ser Asp Gly Met 355 360 365 Ser Gly Arg Leu Glu Glu Glu Leu Arg Arg Arg Leu Ser Pro 370 375 380 <210> 194 <211> 382 <212> PRT <213> Artificial sequence <220> <223> Fusion protein <400> 194 Met Ser Lys His His His His Ser Asn His His Arg His Asn His His 1 5 10 15 His His Ser Gly Asn His His His Ser Gly Ser Ala Ala Gly Gly Glu 20 25 30 Glu Asp Lys Lys Pro Ala Gly Gly Glu Gly Gly Gly Ala His Ile Asn 35 40 45 Leu Lys Val Lys Gly Gln Asp Gly Asn Glu Val Phe Phe Arg Ile Lys 50 55 60 Arg Ser Thr Gln Leu Lys Lys Leu Met Asn Ala Tyr Cys Asp Arg Gln 65 70 75 80 Ser Val Asp Met Thr Ala Ile Ala Phe Leu Phe Asp Gly Arg Arg Leu 85 90 95 Arg Ala Glu Gln Thr Pro Asp Glu Leu Glu Met Glu Asp Gly Asp Glu 100 105 110 Ile Asp Ala Met Leu His Gln Thr Gly Gly Ser Gly Asp Ala Ser Asp 115 120 125 Ser Pro Ser Arg Leu Glu Glu Glu Leu Arg Arg Arg Leu Thr Glu Pro 130 135 140 Ser Lys Gly Glu Glu Leu Phe Thr Gly Thr Val Pro Ile Lys Val Glu 145 150 155 160 Leu Asp Gly Asp Val Asn Gly His Lys Phe Ser Val Arg Gly Glu Gly 165 170 175 Glu Gly Asp Ala Thr Glu Gly Lys Leu Thr Leu Lys Phe Ile Cys Thr 180 185 190 Thr Gly Lys Leu Pro Val Pro Trp Pro Thr Leu Val Thr Thr Leu Thr 195 200 205 Tyr Gly Val Gln Cys Phe Ser Arg Tyr Pro Asp His Met Lys Arg His 210 215 220 Asp Phe Phe Lys Ser Ala Met Pro Glu Gly Tyr Val Gln Glu Arg Thr 225 230 235 240 Ile Glu Phe Lys Asp Asp Gly Thr Tyr Lys Thr Arg Ala Glu Val Lys 245 250 255 Phe Glu Gly Asp Thr Leu Val Asn Arg Ile Glu Leu Lys Gly Asn Asp 260 265 270 Phe Lys Glu Asp Gly Asn Ile Leu Gly His Lys Leu Glu Tyr Asn His 275 280 285 Asn Ser His Asn Val Arg Ile Glu Ala Asp Lys Gln Lys Asn Gly Ile 290 295 300 Lys Ala Asn Phe Lys Ile Arg His Asn Val Glu Asp Gly Ser Gln Gln 305 310 315 320 Glu Ala Asp His Lys Gln Gln Asn Thr Pro Ile Gly Asp Gly Pro Val 325 330 335 Arg Leu Pro Asp Asn His Tyr Leu Ser Thr Gln Thr Thr Leu Ser Lys 340 345 350 Asp Pro Asn Glu Lys Arg Asp His Met Val Leu Lys Glu Phe Val Thr 355 360 365 Ala Ala Gly Ile Thr Lys Gly Glu Asp Glu Arg Asp Lys Ala 370 375 380 <210> 195 <211> 382 <212> PRT <213> Artificial Sequence <220> <223> Fusion Protein <400> 195 Met Ser Lys His His His His Ser Asn His His Arg His Asn His His 1 5 10 15 His His Ser Gly Asn His His His Ser Gly Ser Ala Ala Gly Gly Glu 20 25 30 Glu Asp Lys Lys Pro Ala Gly Gly Glu Gly Gly Gly Ala His Ile Asn 35 40 45 Leu Lys Val Lys Gly Gln Asp Gly Asn Glu Val Phe Phe Arg Ile Lys 50 55 60 Arg Ser Thr Gln Leu Lys Lys Leu Met Asn Ala Tyr Cys Asp Arg Gln 65 70 75 80 Ser Val Asp Met Thr Ala Ile Ala Phe Leu Phe Asp Gly Arg Arg Leu 85 90 95 Arg Ala Glu Gln Thr Pro Asp Glu Leu Glu Met Glu Asp Gly Asp Glu 100 105 110 Ile Asp Ala Met Leu His Gln Thr Gly Gly Ser Gly Asp Ala Ser Asp 115 120 125 Ser Met Ser Gly Arg Leu Glu Glu Glu Leu Arg Arg Arg Leu Ser Pro 130 135 140 Ser Lys Gly Glu Glu Leu Phe Thr Gly Thr Val Pro Ile Lys Val Glu 145 150 155 160 Leu Asp Gly Asp Val Asn Gly His Lys Phe Ser Val Arg Gly Glu Gly 165 170 175 Glu Gly Asp Ala Thr Glu Gly Lys Leu Thr Leu Lys Phe Ile Cys Thr 180 185 190 Thr Gly Lys Leu Pro Val Pro Trp Pro Thr Leu Val Thr Thr Leu Thr 195 200 205 Tyr Gly Val Gln Cys Phe Ser Arg Tyr Pro Asp His Met Lys Arg His 210 215 220 Asp Phe Phe Lys Ser Ala Met Pro Glu Gly Tyr Val Gln Glu Arg Thr 225 230 235 240 Ile Glu Phe Lys Asp Asp Gly Thr Tyr Lys Thr Arg Ala Glu Val Lys 245 250 255 Phe Glu Gly Asp Thr Leu Val Asn Arg Ile Glu Leu Lys Gly Asn Asp 260 265 270 Phe Lys Glu Asp Gly Asn Ile Leu Gly His Lys Leu Glu Tyr Asn His 275 280 285 Asn Ser His Asn Val Arg Ile Glu Ala Asp Lys Gln Lys Asn Gly Ile 290 295 300 Lys Ala Asn Phe Lys Ile Arg His Asn Val Glu Asp Gly Ser Gln Gln 305 310 315 320 Glu Ala Asp His Lys Gln Gln Asn Thr Pro Ile Gly Asp Gly Pro Val 325 330 335 Arg Leu Pro Asp Asn His Tyr Leu Ser Thr Gln Thr Thr Leu Ser Lys 340 345 350 Asp Pro Asn Glu Lys Arg Asp His Met Val Leu Lys Glu Phe Val Thr 355 360 365 Ala Ala Gly Ile Thr Lys Gly Glu Asp Glu Arg Asp Lys Ala 370 375 380 <210> 196 <211> 222 <212> PRT <213> Artificial Sequence <220> <223> Fusion Protein <400> 196 Met Glu Ile Tyr Glu Asn Glu Asn Asp Gln Val Glu Ala Val Lys Arg 1 5 10 15 Phe Phe Ala Glu Asn Gly Lys Ala Leu Ala Val Gly Val Ile Leu Gly 20 25 30 Val Gly Ala Leu Ile Gly Trp Arg Tyr Trp Asn Ser His Gln Val Asp 35 40 45 Ser Ala Arg Ser Ala Ser Leu Ala Tyr Gln Asn Ala Val Thr Ala Val 50 55 60 Ser Glu Gly Lys Pro Asp Ser Ile Pro Ala Ala Glu Lys Phe Ala Ala 65 70 75 80 Glu Asn Lys Asn Thr Tyr Gly Ala Leu Ala Ser Leu Glu Leu Ala Gln 85 90 95 Gln Phe Val Asp Lys Asn Glu Leu Glu Lys Ala Ala Ala Gln Leu Gln 100 105 110 Gln Gly Leu Ala Asp Thr Ser Asp Glu Asn Leu Lys Ala Val Ile Asn 115 120 125 Leu Arg Leu Ala Arg Val Gln Val Gln Leu Lys Gln Ala Asp Ala Ala 130 135 140 Leu Lys Thr Leu Asp Thr Ile Lys Gly Glu Gly Trp Ala Ala Ile Val 145 150 155 160 Ala Asp Leu Arg Gly Glu Ala Leu Leu Ser Lys Gly Asp Lys Gln Gly 165 170 175 Ala Arg Ser Ala Trp Glu Ala Gly Val Lys Ser Asp Val Thr Pro Ala 180 185 190 Leu Ser Glu Met Met Gln Met Lys Ile Asn Asn Leu Ser Ile Gly Ser 195 200 205 Pro Ser Arg Leu Glu Glu Glu Leu Arg Arg Arg Leu Thr Glu 210 215 220 <210> 197 <211> 222 <212> PRT <213> Artificial Sequence <220> <223> Fusion protein <400> 197 Met Glu Ile Tyr Glu Asn Glu Asn Asp Gln Val Glu Ala Val Lys Arg 1 5 10 15 Phe Phe Ala Glu Asn Gly Lys Ala Leu Ala Val Gly Val Ile Leu Gly 20 25 30 Val Gly Ala Leu Ile Gly Trp Arg Tyr Trp Asn Ser His Gln Val Asp 35 40 45 Ser Ala Arg Ser Ala Ser Leu Ala Tyr Gln Asn Ala Val Thr Ala Val 50 55 60 Ser Glu Gly Lys Pro Asp Ser Ile Pro Ala Ala Glu Lys Phe Ala Ala 65 70 75 80 Glu Asn Lys Asn Thr Tyr Gly Ala Leu Ala Ser Leu Glu Leu Ala Gln 85 90 95 Gln Phe Val Asp Lys Asn Glu Leu Glu Lys Ala Ala Ala Gln Leu Gln 100 105 110 Gln Gly Leu Ala Asp Thr Ser Asp Glu Asn Leu Lys Ala Val Ile Asn 115 120 125 Leu Arg Leu Ala Arg Val Gln Val Gln Leu Lys Gln Ala Asp Ala Ala 130 135 140 Leu Lys Thr Leu Asp Thr Ile Lys Gly Glu Gly Trp Ala Ala Ile Val 145 150 155 160 Ala Asp Leu Arg Gly Glu Ala Leu Leu Ser Lys Gly Asp Lys Gln Gly 165 170 175 Ala Arg Ser Ala Trp Glu Ala Gly Val Lys Ser Asp Val Thr Pro Ala 180 185 190 Leu Ser Glu Met Met Gln Met Lys Ile Asn Asn Leu Ser Ile Gly Ser 195 200 205 Met Ser Gly Arg Leu Glu Glu Glu Leu Arg Arg Arg Leu Ser 210 215 220 <210> 198 <211> 206 <212> PRT <213> Artificial sequence <220> <223> YfgM <400> 198 Met Glu Ile Tyr Glu Asn Glu Asn Asp Gln Val Glu Ala Val Lys Arg 1 5 10 15 Phe Phe Ala Glu Asn Gly Lys Ala Leu Ala Val Gly Val Ile Leu Gly 20 25 30 Val Gly Ala Leu Ile Gly Trp Arg Tyr Trp Asn Ser His Gln Val Asp 35 40 45 Ser Ala Arg Ser Ala Ser Leu Ala Tyr Gln Asn Ala Val Thr Ala Val 50 55 60 Ser Glu Gly Lys Pro Asp Ser Ile Pro Ala Ala Glu Lys Phe Ala Ala 65 70 75 80 Glu Asn Lys Asn Thr Tyr Gly Ala Leu Ala Ser Leu Glu Leu Ala Gln 85 90 95 Gln Phe Val Asp Lys Asn Glu Leu Glu Lys Ala Ala Ala Gln Leu Gln 100 105 110 Gln Gly Leu Ala Asp Thr Ser Asp Glu Asn Leu Lys Ala Val Ile Asn 115 120 125 Leu Arg Leu Ala Arg Val Gln Val Gln Leu Lys Gln Ala Asp Ala Ala 130 135 140 Leu Lys Thr Leu Asp Thr Ile Lys Gly Glu Gly Trp Ala Ala Ile Val 145 150 155 160 Ala Asp Leu Arg Gly Glu Ala Leu Leu Ser Lys Gly Asp Lys Gln Gly 165 170 175 Ala Arg Ser Ala Trp Glu Ala Gly Val Lys Ser Asp Val Thr Pro Ala 180 185 190 Leu Ser Glu Met Met Gln Met Lys Ile Asn Asn Leu Ser Ile 195 200 205 <210> 199 <211> 260 <212> PRT <213> Artificial Sequence <220> <223> Fusion protein <400> 199 Met Ser Lys His His His His Ser Asn His His Arg His Asn His His 1 5 10 15 His His Ser Gly Asn His His His Ser Gly Ser Ala Ala Gly Gly Glu 20 25 30 Glu Asp Lys Lys Pro Ala Gly Gly Glu Gly Gly Gly Ala His Ile Asn 35 40 45 Leu Lys Val Lys Gly Gln Asp Gly Asn Glu Val Phe Phe Arg Ile Lys 50 55 60 Arg Ser Thr Gln Leu Lys Lys Leu Met Asn Ala Tyr Cys Asp Arg Gln 65 70 75 80 Ser Val Asp Met Thr Ala Ile Ala Phe Leu Phe Asp Gly Arg Arg Leu 85 90 95 Arg Ala Glu Gln Thr Pro Asp Glu Leu Glu Met Glu Asp Gly Asp Glu 100 105 110 Ile Asp Ala Met Leu His Gln Thr Gly Gly Ala Cys Ala Trp Ser His 115 120 125 Pro Gln Phe Glu Lys Gly Gly Gly Ser Gly Gly Ser Ser Gly Gly Ser 130 135 140 Ala Trp Ser His Pro Gln Phe Glu Lys Gly Ser Gly Ser Ala Glu Ser 145 150 155 160 Glu Ala Ala Ser Ser Thr Met Ile Lys Val Lys Thr Leu Thr Gly Lys 165 170 175 Glu Ile Glu Ile Asp Ile Glu Pro Thr Asp Thr Ile Asp Arg Ile Lys 180 185 190 Glu Arg Val Glu Glu Lys Glu Gly Ile Pro Pro Val Gln Gln Arg Leu 195 200 205 Ile Tyr Ala Gly Lys Gln Leu Ala Asp Asp Lys Thr Ala Lys Asp Tyr 210 215 220 Asn Ile Glu Gly Gly Ser Val Leu His Leu Val Leu Ala Leu Arg Gly 225 230 235 240 Gly Ala Thr Gly Thr Ala Ser Thr Arg Leu Glu Glu Glu Leu Arg Arg 245 250 255 Arg Leu Ala Ser 260

Claims

1. A fusion protein, the fusion protein comprising: (a) A peptide, and the sequence of the peptide is: SRLEEELRRRLTE (SEQ ID NO: 04); MPSRLEEELRRRLTEP (SEQ ID NO: 05); PSRLEEELRRRLTEP (SEQ ID NO: 06); or PSRLEEELRRRLTE (SEQ ID NO: 07); (b) And a polypeptide; Among them, The peptide is an epitope tag.

2. The fusion protein according to claim 1, wherein, The sequence of the peptide is: SRLEEELRRRLTE (SEQ ID NO: 04).

3. The fusion protein according to claim 1, wherein, The sequence of the peptide is: MPSRLEEELRRRLTEP (SEQ ID NO: 05); PSRLEEELRRRLTEP (SEQ ID NO: 06); or PSRLEEELRRRLTE (SEQ ID NO: 07).

4. The fusion protein according to claim 1, wherein, The peptide is fused to the N-terminus or C-terminus of the polypeptide or at a position between the N-terminus and C-terminus of the polypeptide.

5. The fusion protein according to claim 1, wherein, The polypeptide has a stable fold independent of the peptide.

6. The fusion protein according to claim 1, wherein, The peptide has an α-helical secondary structure.

7. The fusion protein according to claim 1, wherein, The peptide specifically binds to a camel VHH domain comprising the CDR sequences GVTISALNAMAMG (SEQ ID NO: 115), AVSERGNAM (SEQ ID NO: 116), and LEDRVDSFHDY (SEQ ID NO: 117).

8. The fusion protein according to claim 1, the fusion protein further comprising one or more linkers connecting the peptide and the polypeptide.

9. The fusion protein according to claim 1, wherein, The polypeptide comprises at least one protein domain.

10. The fusion protein according to claim 9, wherein, The peptide is fused to the polypeptide at a position outside the at least one protein domain.

11. The fusion protein according to claim 1, wherein, The polypeptide is a globular protein, a membrane protein, a fibrous protein, or a naturally unfolded protein, or a subunit of a globular protein, a membrane protein, a fibrous protein, or a naturally unfolded protein.

12. The fusion protein according to claim 1, wherein, The polypeptide has a length of at least 3 amino acids.

13. The fusion protein according to claim 12, wherein, The polypeptide has a length of at least 4 amino acids.

14. The fusion protein according to claim 13, wherein, The polypeptide has a length of at least 5 amino acids.

15. The fusion protein according to claim 14, wherein, The polypeptide has a length of at least 6 amino acids.

16. The fusion protein according to claim 15, wherein, The polypeptide has a length of at least 7 amino acids.

17. The fusion protein according to claim 16, wherein, The polypeptide has a length of at least 8 amino acids.

18. The fusion protein according to claim 17, wherein, The polypeptide has a length of at least 9 amino acids. The fusion protein according to claim 18, wherein, The polypeptide has a length of at least 10 amino acids. The fusion protein according to claim 19, wherein, The polypeptide has a length of at least 15 amino acids.

21. The fusion protein according to claim 20, wherein, The polypeptide has a length of at least 20 amino acids.

22. The fusion protein according to claim 21, wherein, The polypeptide has a length of at least 25 amino acids.

23. The fusion protein according to claim 22, wherein, The polypeptide has a length of at least 30 amino acids.

24. The fusion protein according to claim 23, wherein, The polypeptide has a length of at least 40 amino acids.

25. The fusion protein according to claim 24, wherein, The polypeptide has a length of at least 50 amino acids.

26. The fusion protein according to claim 25, wherein, The polypeptide has a length of at least 60 amino acids.

27. The fusion protein according to claim 26, wherein, The polypeptide has a length of at least 70 amino acids.

28. The fusion protein according to claim 27, wherein, The polypeptide has a length of at least 80 amino acids.

29. The fusion protein according to claim 28, wherein, The polypeptide has a length of at least 90 amino acids.

30. The fusion protein according to claim 29, wherein, The polypeptide has a length of at least 100 amino acids.

31. The fusion protein according to claim 1, wherein, The fusion protein complexes with a binding partner that specifically binds to the peptide contained in the fusion protein.

32. The fusion protein according to any one of claims 1-31, wherein, The fusion protein comprises an antibody.

33. A single-domain antibody that specifically binds to the peptide contained in the fusion protein according to any one of claims 1-32, the antibody comprising (a) a CDR1 sequence GVTISALNAMAMG (SEQ ID NO: 115), a CDR2 sequence AVSERGNAM (SEQ ID NO: 116), and a CDR3 sequence LEDRVDSFHDY (SEQ ID NO: 117); or (b) a CDR1 sequence GVTISALNAMAMG (SEQ ID NO: 118), a CDR2 sequence AVSSRGNAM (SEQ ID NO: 119), and a CDR3 sequence LEDRVDSFHDY (SEQ ID NO: 120).

34. The antibody according to claim 33, wherein, The antibody comprises: a CDR1 sequence GVTISALNAMAMG (SEQ ID NO: 115), a CDR2 sequence AVSERGNAM (SEQ ID NO: 116), and a CDR3 sequence LEDRVDSFHDY (SEQ ID NO: 117). The antibody according to claim 33, wherein, The antibody comprises: a CDR1 sequence GVTISALNAMAMG (SEQ ID NO: 118), a CDR2 sequence AVSSRGNAM (SEQ ID NO: 119), and a CDR3 sequence LEDRVDSFHDY (SEQ ID NO: 120).

36. The antibody according to claim 33, the antibody comprising a VHH sequence having at least 80% sequence identity with the following: EVQLQESGGGLVQPGGSLRLSCTASGVTISALNAMAMGWYRQAPGE RRVMVAAVSERGNAMYRESVQGRFTVTRDFTNKMVSLQMDNLKPEDTA VYYCHVLEDRVDSFHDYWGQGTQVTVSS (SEQ ID NO: 133); or EVQLQESGGGLVQPGGSLRLSCTASGVTISALNAMAMGWYRQAPGE ERVMVAAVSSRGNAMYRESVQGRFTVTRDFTNKMVSLQMDNLKPEDTA VYYCHVLEDRVDSFHDYWGQGTQVTVSS (SEQ ID NO: 134).

37. The antibody according to claim 36, the antibody comprising a VHH sequence having at least 85% sequence identity with the following: EVQLQESGGGLVQPGGSLRLSCTASGVTISALNAMAMGWYRQAPGE RRVMVAAVSERGNAMYRESVQGRFTVTRDFTNKMVSLQMDNLKPEDTA VYYCHVLEDRVDSFHDYWGQGTQVTVSS (SEQ ID NO: 133); or EVQLQESGGGLVQPGGSLRLSCTASGVTISALNAMAMGWYRQAPGE ERVMVAAVSSRGNAMYRESVQGRFTVTRDFTNKMVSLQMDNLKPEDTA VYYCHVLEDRVDSFHDYWGQGTQVTVSS (SEQ ID NO: 134).

38. The antibody according to claim 37, wherein the antibody comprises a VHH sequence having at least 90% sequence identity with the following: EVQLQESGGGLVQPGGSLRLSCTASGVTISALNAMAMGWYRQAPGE RRVMVAAVSERGNAMYRESVQGRFTVTRDFTNKMVSLQMDNLKPEDTA VYYCHVLEDRVDSFHDYWGQGTQVTVSS (SEQ ID NO: 133); or EVQLQESGGGLVQPGGSLRLSCTASGVTISALNAMAMGWYRQAPGE ERVMVAAVSSRGNAMYRESVQGRFTVTRDFTNKMVSLQMDNLKPEDTA VYYCHVLEDRVDSFHDYWGQGTQVTVSS (SEQ ID NO: 134).

39. The antibody according to claim 38, wherein the antibody comprises a VHH sequence having at least 95% sequence identity with the following: EVQLQESGGGLVQPGGSLRLSCTASGVTISALNAMAMGWYRQAPGE RRVMVAAVSERGNAMYRESVQGRFTVTRDFTNKMVSLQMDNLKPEDTA VYYCHVLEDRVDSFHDYWGQGTQVTVSS (SEQ ID NO: 133); or EVQLQESGGGLVQPGGSLRLSCTASGVTISALNAMAMGWYRQAPGE ERVMVAAVSSRGNAMYRESVQGRFTVTRDFTNKMVSLQMDNLKPEDTA VYYCHVLEDRVDSFHDYWGQGTQVTVSS (SEQ ID NO: 134).

40. The antibody according to claim 33, wherein the antibody comprises the VHH sequence of EVQLQESGGGLVQPGGSLRLSCTASGVTISALNAMAMGWYRQAPGERRV MVAAVSERGNAMYRESVQGRFTVTRDFTNKMVSLQMDNLKPEDTAVYYCHVLEDRVDSFHDYWGQGTQVTVSS (SEQ ID NO: 133).

41. The antibody according to claim 33, wherein the antibody comprises the VHH sequence of EVQLQESGGGLVQPGGSLRLSCTASGVTISALNAMAMGWYRQAPGEERV MVAAVSSRGNAMYRESVQGRFTVTRDFTNKMVSLQMDNLKPEDTAVYYCHVLEDRVDSFHDYWGQGTQVTVSS (SEQ ID NO: 134). The antibody according to claim 33, wherein, The antibody is conjugated to a detectable label.

43. The antibody according to claim 42, wherein, The detectable label is a fluorescent label. The antibody according to claim 42, wherein, The detectable label is an affinity tag. The antibody according to claim 42, wherein, The detectable label is selected from the group consisting of FLAG-tag, Strep-tag, Myc-tag, His-tag, HA-tag, VSV-G-tag, HSV-tag, V5-tag, GST-tag, Spot-tag, BC2-tag, EPEA-tag, maltose binding protein (MBP), chitin binding protein (CBP), thioredoxin, and biotin.

46. The antibody according to any one of claims 33-45, wherein, The antibody is conjugated to a solid support. The antibody according to any one of claims 33-45, wherein, The antibody is complexed with the epitope to which it specifically binds.

48. A complex, comprising: (a) a fusion protein; and (b) an antibody; wherein the fusion protein is the fusion protein according to any one of claims 1-32 and / or wherein the antibody is the antibody according to any one of claims 33-47.

49. A nucleic acid encoding the fusion protein according to any one of claims 1-32 or the antibody according to any one of claims 33-47.

50. A vector comprising the nucleic acid according to claim 49.

51. A host cell comprising the nucleic acid according to claim 49 or the vector according to claim 50 or expressing the fusion protein according to any one of claims 1-32 or the antibody according to any one of claims 33-47, wherein the host cell is not a plant cell.

52. Use of the peptide as defined in any one of claims 1-32 as an epitope tag, or for detecting, immobilizing, separating, or purifying the fusion protein according to any one of claims 1-32.

53. Use of the antibody according to any one of claims 33-47 for detecting, immobilizing, separating, or purifying the fusion protein according to any one of claims 1-32.

54. A method for detecting the fusion protein according to any one of claims 1-32, the method comprising contacting the fusion protein with the antibody according to any one of claims 42-45.

55. The method according to claim 54, the method further comprising contacting the fusion protein and the antibody with a specific binding partner of the detectable label comprised in the antibody.

56. The method according to claim 55, wherein, The specific binding partner is conjugated to a second detectable label.

57. The method according to claim 56, wherein, The second detectable label is a fluorescent label.

58. The method according to claim 54, the method further comprising the step of detecting the detectable label or the second detectable label.

59. The method according to claim 54, the method further comprising expressing the fusion protein prior to contacting the fusion protein with the antibody. The method according to claim 54, wherein, Detecting the fusion protein comprises assaying for the presence, subcellular localization or amount of the fusion protein.

61. A method of isolating a specific target of the fusion protein according to any one of claims 1-32 or the antibody moiety comprised in the fusion protein according to claim 32, the method comprising contacting the fusion protein with an antibody according to any one of claims 33-47. The method according to claim 61, wherein, The antibody is attached to a solid support before, during or after binding to the fusion protein.

63. The method according to claim 61, the method further comprising eluting the fusion protein. The method according to claim 63, wherein, The elution is effected by contacting the complex of the fusion protein and the antibody with a reagent that competes with the fusion protein for binding to the antibody. The method according to claim 64, wherein, The reagent is a peptide as defined in any one of claims 1-31.

66. The method according to claim 61, the method further comprising contacting the fusion protein according to claim 32 and the specific target of the antibody moiety comprised in the fusion protein. The method according to claim 66, wherein, The specific target comprises a cell surface receptor.

68. The method according to claim 61, the method further comprising a washing step.

69. A kit comprising a nucleic acid or nucleic acid expression construct encoding a peptide as defined in any one of claims 1-32, and optionally an antibody according to any one of claims 33-47.

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