Epitope tags recognized by specific binding agents

Abstract

The present invention provides peptides useful as epitope tags that can be fused to a polypeptide of interest, as well as antibodies that specifically bind to these peptides, which can be used to detect, immobilize, isolate, or purify molecules conjugated to such peptides and / or antibodies.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to European Patent Application Publication No. 18193663.4, filed September 11, 2018, and European Patent Application Publication No. 19160485.9, filed March 4, 2019, the contents of which are incorporated herein by reference in their entireties for all purposes.

[0002] FIELD OF THEINVENTION The present invention provides peptides useful as epitope tags that can be fused to a polypeptide of interest, as well as antibodies that specifically bind to these peptides, which can be used to detect, immobilize, isolate, or purify molecules conjugated to such peptides and / or antibodies. [Background technology]

[0003] background Epitope tags play an important role in virtually every aspect of life science. They are utilized in biotechnology applications, for example, to facilitate recombinant protein expression and purification (Waugh, DS Making the most of affinity tags. Trends Biotechnol 23, 316-320(2005)). In cell biology, epitope tags are often utilized to monitor the biosynthesis or topology of a given protein of interest (POI) (Nooh, MM & Bahouth, SW Visualization and quantification of GPCR trafficking in mammalian cells by confocal microscopy. Methods Cell Biol. 142, 67-78(2017) (Non-Patent Document 2); Kocaoglu, O. & Carlson, EE Progress and prospects for small-molecule probes of bacterial imaging. Nat Chem Biol 12, 472-478(2016) (Non-Patent Document 3)). Tags also continue to be a tool for immunoprecipitation of protein complexes studied using mass spectrometry techniques (Shi, Y. et al. A strategy for dissecting the architectures of native macromolecular assemblies. Nat Methods 12, 1135-1138 (2015) (Non-Patent Document 4); Smits, AH & Vermeulen, M. Characterizing Protein-Protein Interactions Using Mass Spectrometry: Challenges and Opportunities. Trends Biotechnol 34, 825-834 (2016) (Non-Patent Document 5)).Over the years, at least a dozen different tags have evolved, providing researchers with multiple tools for most scientific situations (Waugh, DS Making the most of affinity tags. Trends Biotechnol 23, 316-320 (2005); Brizzard, B. Epitope tagging. BioTechniques 44, 693-695 (2008)). A given tag, however, may work exceptionally well in certain applications while failing altogether in others. As a result, most researchers rely on a variety of tags to cover the range of applications required.

[0004] No truly universal tag appears to exist to date, one explanation for which may be that most tags were discovered as a by-product during screening of binding agents (typically monoclonal antibodies) against naturally occurring proteins. This has been demonstrated by, for example, the c-myc tag (Evan, GI, Lewis, GK, Ramsay, G. & Bishop, JM Isolation of monoclonal antibodies specific for human c-myc proto-oncogene product. Mol Cell Biol 5, 3610-3616 (1985) (Non-Patent Document 7)), 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) (Non-Patent Document 8)), or the Spot tag (registered trademark) (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 (Non-Patent Document 9); Braun, MB et al. Peptides in headlock-a novel high-affinity and versatile peptide-binding nanobody for proteomics and microscopy. Sci Rep 6,19211(2016) (Non-Patent Document 10). Typically, tags are the smallest peptides that are efficiently recognized by the respective binders from the outset. As a result, the properties of such tags are largely contingent, depending on the binder selected, and generally cannot be retuned to the needs and conditions of a particular experiment. On the other hand, some tags have been rationally designed for certain applications.For example, the His tag is ideally suited for the initial crude purification of recombinantly expressed proteins on metal ion chelating resins, which is what the His tag was developed for (Hochuli, E., Dobeli, H. & Schacher, A. New metal chelate adsorbent selective for proteins and peptides containing neighbouring histidine residues. J. Chromatogr. 411, 177-184 (1987) (Non-Patent Document 11)). However, due to a lack of high affinity binders, the His tag has not been widely used for microscopic applications in cell biology to date.

[0005] It is an object of the present invention to provide improved epitope tags and specific binding molecules thereto. [Prior art documents] [Non-patent literature]

[0006] [Non-Patent Document 1] Waugh, DS Making the most of affinity tags. Trends Biotechnol 23, 316-320(2005) [Non-Patent Document 2] Nooh,MM&Bahouth,SW Visualization and quantification of GPCR trafficking in mammalian cells by confocal microscopy. Methods Cell Biol. 142,67-78(2017) [Non-Patent Document 3] Kocaoglu, O. & Carlson, EE Progress and prospects for small-molecule probes of bacterial imaging. Nat Chem Biol 12,472-478(2016) [Non-Patent Document 4] Shi,Y. et al. A strategy for dissecting the architectures of native macromolecular assemblies. Nat Methods 12,1135-1138(2015) [Non-Patent Document 5] Smits, AH & Vermeulen, M. Characterizing Protein-Protein Interactions Using Mass Spectrometry:Challenges and Opportunities. Trends Biotechnol 34,825-834(2016) [Non-Patent Document 6] Brizzard, B. Epitope tagging. BioTechniques 44,693-695(2008) [Non-Patent Document 7] Evan,GI,Lewis,GK,Ramsay,G.&Bishop,JM Isolation of monoclonal antibodies specific for human c-myc proto-oncogene product. Mol Cell Biol 5,3610-3616(1985) [Non-Patent Document 8] 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) [Non-Patent Document 9] 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 [Non-Patent Document 10] Braun,MB et al. Peptides in headlock-a novel high-affinity and versatile peptide-binding nanobody for proteomics and microscopy. Sci Rep 6,19211(2016) [Non-Patent Document 11] Hochuli, E., Dobeli, H. & Schacher, A. New metal chelate adsorbent selective for proteins and peptides containing neighboring histidine residues. J. Chromatogr. 411,177-184(1987) Summary of the Invention

[0007] (a) comprising the sequence X1-X2-X3-X4-X5-X6-X7-X8-X9-RL-X12-X13 (SEQ ID NO:01), 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, 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; X13 is E or K or P or S or A or D or no amino acid; A peptide, (b) a polypeptide; The present invention relates to a fusion protein comprising:

[0008] The present invention also relates to an antibody that specifically binds to a peptide contained in the fusion protein of the present invention.

[0009] The present invention also relates to a fusion protein comprising a peptide to which the antibody of the present invention binds.

[0010] The present invention also relates to a complex comprising the fusion protein of the present invention and the antibody of the present invention.

[0011] The present invention also relates to a nucleic acid encoding a fusion protein according to the invention or encoding an antibody according to the invention.

[0012] The present invention also relates to a vector comprising a nucleic acid of the invention.

[0013] The present invention also relates to a host cell which contains a nucleic acid of the invention or a vector of the invention, or which expresses a fusion protein of the invention or an antibody of the invention.

[0014] The present invention also relates to the use of the antibodies of the present invention for the detection, immobilization, isolation or purification of the fusion proteins of the present invention.

[0015] The present invention also relates to a method for detecting a fusion protein of the present invention, comprising the step of contacting the fusion protein of the present invention with an antibody of the present invention.

[0016] The invention also relates to a method of isolating a fusion protein of the invention comprising the step of contacting the fusion protein with an antibody of the invention. When the fusion protein of the invention comprises an antibody portion, the invention also relates to a method of isolating a specific target of the antibody portion.

[0017] The present invention also relates to kits comprising a nucleic acid or a nucleic acid expression construct encoding a peptide comprised in a fusion protein of the invention and optionally an antibody of the invention. [The present invention 1001] (a) comprising the sequence X1-X2-X3-X4-X5-X6-X7-X8-X9-RL-X12-X13 (SEQ ID NO:01), 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, 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; X13 is E or K or P or S or A or D or no amino acid; A peptide, (b) a polypeptide; A fusion protein comprising: [The present invention 1002] The fusion protein of the present invention 1001, wherein said peptide is fused to the N-terminus or C-terminus of said polypeptide, or at a position between said N-terminus and said C-terminus. [The present invention 1003] The fusion protein of any of claims 1001 and 1002, wherein the peptide is an epitope tag. [The present invention 1004] Any of the fusion proteins of the invention, wherein the polypeptide has a stable folding independent of the peptide. [The present invention 1005] Any of the fusion proteins of the invention, wherein the peptide has an alpha-helical secondary structure. [The present invention 1006] Any of the fusion proteins of the invention, wherein the peptide specifically binds to a Camelidae VHH domain comprising the CDR sequences GVTISALNAMAMG (SEQ ID NO:115), AVSERGNAM (SEQ ID NO:116), and LEDRVDSFHDY (SEQ ID NO:117). [The present invention 1007] Any of the fusion proteins of the invention further comprising one or more linkers linking the peptide and the polypeptide. [The present invention 1008] Any of the fusion proteins of the invention, wherein the polypeptide comprises at least one protein domain. [The present invention 1009] The fusion protein of claim 10, wherein said peptide is fused to said polypeptide at a position outside said at least one protein domain. [The present invention 1010] Any of the fusion proteins of the invention, wherein the polypeptide is a globular protein, a membrane protein, a fibrous protein, or a non-naturally folded protein, or a subunit of a globular protein, a membrane protein, a fibrous protein, or a non-naturally folded protein. [The present invention 1011] Any of the fusion proteins of the invention, wherein the peptide has a length of 8 to 25 amino acids. [The present invention 1012] Any of the fusion proteins of the invention, wherein the polypeptide has 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 present invention 1013] the peptide comprises the sequence X1-X2-LE-X5-E-X7-RRRL-X12-X13 (SEQ ID NO:02); X1 is G or S or P or T; X2 is R, 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; X13 is P or A or S or A or D or E or no amino acid; Any of the fusion proteins of the present invention. [The present invention 1014] the peptide comprises an N-terminus designated X1, which is amino acid Xa-Xb; Xa is D or S or G or M or P or no amino acid; Xb is S or D or P or M or R or G or no amino acid; Any of the fusion proteins of the present invention. [The present invention 1015] the peptide comprises a C-terminus of X13, which is amino acid Xy-Xz; Xy is G or S or P or D or A or E or K or no amino acid; Xz is S or P or no amino acid, Any of the fusion proteins of the present invention. [The present invention 1016] the peptide comprises a C-terminus of X13, which is amino acid Xy-Xz; Xy is G or P or A or E or K or S or D or no amino acid; Xz is P or S or no amino acid, A fusion protein of the present invention 1013 or 1014. [The present invention 1017] the peptide comprises the sequence X1-X2-LE-X5-ELRRRL-X12-X13 (SEQ ID NO:03); X1 is S or T; X2 is R or G, X5 is E or Q, X12 is T, D, or E; X13 is A or D or E or no amino acid; Any of the fusion proteins of the present invention. [The present invention 1018] the peptide comprises the sequence SRLEEELRRRLTE (SEQ ID NO:04) or a variant thereof; When compared to (SEQ ID NO:04), the variant has one to five mutations selected from the group consisting of S1→T, R2→G, E5→Q, T12→D, T12→E, E13→A, E13→D, and a deletion of E13. Any of the fusion proteins of the present invention. [The present invention 1019] When compared to SEQ ID NO:04, the variant has the following mutation: (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, T12→D, and E13→A; (f) R2→G, E5→Q, 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) E13 deletion having The fusion protein of the present invention. [The present invention 1020] the peptide comprises an N-terminus designated X1, which is amino acid Xa-Xb; Xa is S or G or M or P or no amino acid; Xb is R or G or S or P or M or no amino acid; A fusion protein according to any one of claims 1017 to 1019 of the present invention. [The present invention 1021] The fusion protein of the present invention 1020, wherein Xa-Xb is selected from the group consisting of P, MP, GR, PG, PS, SP, GP, SP, M, and MS, and is preferably P or MP. [The present invention 1022] the peptide comprises a C-terminus of X13, which is amino acid Xy-Xz; Xy is P or D or A or no amino acid; Xz is P or S or no amino acid, A fusion protein according to any one of claims 1017 to 1021 of the present invention. [The present invention 1023] The fusion protein of the present invention, wherein Xy-Xz is selected from the group consisting of no amino acids, P, DP, A, and AS, preferably no amino acids or P. [The present invention 1024] The peptide is (a) MP and P, (b) P and P, and (c) No P or amino acids The combinations of Xa-Xb and Xy-Xz are selected from the group consisting of: A fusion protein according to any one of claims 1017 to 1023 of the present invention. [The present invention 1025] The peptide is TIFF0007623006000001.tif187128 The sequence is selected from the group consisting of Any of the fusion proteins of the present invention. [The present invention 1026] the peptide comprises the sequence X1-X2-LE-X5-E-X7-RRRL-X12-X13 (SEQ ID NO:31), X1 is G, 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; X13 is P or A or S or no amino acid; A fusion protein according to any one of claims 1001 to 1016. [The present invention 1027] the peptide comprises the sequence GRLEEELRRRLS (SEQ ID NO:32) or a variant thereof; When compared to (SEQ ID NO:32), the variant has one to six mutations selected from the group consisting of G1→S, G1→P, R2→G, E5→Q, L7→I, S12→T, S12→P, S12→A, an addition of P13, an addition of A13, and an addition of S13. Any of the fusion proteins of the present invention. [The present invention 1028] When compared to SEQ ID NO:32, the variant has the following mutation: (a) Addition of G1 → S, R2 → G, E5 → Q, and P13; (b) addition of R2→G, E5→Q, S12→T, and A13; (c) addition of G1→P, R2→G, E5→Q, S12→T, and A13; (d) addition of G1 → S, R2 → G, E5 → Q, S12 → T, and P13; (e) Addition of G1 → S, R2 → G, S12 → T, and A13; (f) G1→S, R2→G, E5→Q, and S12→T; (g) Addition of G1 → S, R2 → G, E5 → Q, S12 → T, and A13; (h) Addition of G1 → S and S12 → P, and P13; (i) addition of G1 → S, R2 → G, E5 → Q, S12 → T, and P13; (j) E5→Q L7→I and addition of P13; (k) the addition of P13, or (l) S12 → A having A fusion protein of the present invention. [The present invention 1029] the peptide comprises an N-terminus designated X1, which is amino acid Xa-Xb; Xa is M or S or P or D or G or no amino acid; Xb is S or D or P or no amino acid; A fusion protein according to any one of claims 1026 to 1028. [The present invention 1030] The fusion protein of the present invention, wherein Xa-Xb is selected from the group consisting of MS, SD, PD, PS, DS, SP, and GP, and is preferably MS. [The present invention 1031] the peptide comprises a C-terminus of X13, which is amino acid Xy-Xz; Xy is G or P or A or E or K or S or no amino acid; Xz is P or S or no amino acid, A fusion protein according to any one of claims 1026 to 1030. [The present invention 1032] The fusion protein of the present invention 1031, wherein Xy-Xz is selected from the group consisting of no amino acids, G, P, A, EP, AS, K, and S, preferably no amino acids. [The present invention 1033] The fusion protein according to any one of claims 1026 to 1032, wherein the peptide comprises combinations of Xa-Xb and Xy-Xz, each of which is an MS and has no amino acid. [The present invention 1034] The peptide is TIFF0007623006000002.tif121128 The sequence is selected from the group consisting of Any of the fusion proteins of the present invention. [The present invention 1035] the peptide comprises the sequence X1-X2-LE-X5-ELRRRL-X12-X13 (SEQ ID NO:49); X1 is S, G or P; X2 is R, G or P; X5 is E or Q, X12 is S, T, D, or E; X13 is P or A or D or no amino acid; A fusion protein according to any one of claims 1001 to 1016. [The present invention 1036] the peptide comprises an N-terminus designated X1, which is amino acid Xa-Xb; Xa is P or D or S or G or no amino acid; Xb is D or S or P or no amino acid; A fusion protein of the present invention. [The present invention 1037] the peptide comprises a C-terminus of X13, which is amino acid Xy-Xz; Xy is G or P or E or D or S or no amino acid; Xz is P or no amino acid, A fusion protein of the present invention 1035 or 1036. [The present invention 1038] The peptide is TIFF0007623006000003.tif121128 The sequence is selected from the group consisting of Any of the fusion proteins of the present invention. [The present invention 1039] the peptide comprises the sequence X1-X2-X3-X4-X5-X6-X7-X8-X9-RL-X12-X13 (SEQ ID NO:61); 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, 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; X13 is K or P or S or no amino acid, A fusion protein according to any one of claims 1001 to 1016. [The present invention 1040] the peptide comprises an N-terminus designated X1, which is amino acid Xa-Xb; Xa is D or S or G or M or no amino acid; Xb is S or D or P or M or no amino acid; A fusion protein of the present invention. [The present invention 1041] the peptide comprises a C-terminus of X13, which is amino acid Xy-Xz; Xy is G or S or P or no amino acid; Xz is S or no amino acid, A fusion protein of the present invention 1039 or 1040. [The present invention 1042] The peptide is TIFF0007623006000004.tif21688 The sequence is selected from the group consisting of Any of the fusion proteins of the present invention. [The present invention 1043] Any of the fusion proteins of the present invention, complexed with a binding partner that specifically binds to the peptide contained in the fusion protein. [The present invention 1044] Any of the fusion proteins of the invention comprising an antibody. [The present invention 1045] An antibody that specifically binds to the peptide contained in the fusion protein of any one of the present inventions 1001 to 1044. [The present invention 1046] The antibody of the present invention 1045, which is a monovalent antibody. [The present invention 1047] The antibody of the invention 1045 or 1046 which is a single domain antibody. [The present invention 1048] Any of the antibodies of the present invention 1045 to 1047, which contain a Camelidae VHH domain. [The present invention 1049] The CDR1 sequence GVTISALNAMAMG (SEQ ID NO: 115) or a sequence having one or two mutations thereto; The CDR2 sequence AVSERGNAM (SEQ ID NO: 116) or a sequence having one or two mutations thereto, and CDR3 sequence LEDRVDSFHDY (SEQ ID NO: 117) or a sequence having one or two mutations thereto The antibody of any one of 1045 to 1048 of the present invention. [The present invention 1050] The CDR1 sequence GVTISALNAMAMG (SEQ ID NO: 118) or a sequence having one or two mutations thereto; The CDR2 sequence AVSSRGNAM (SEQ ID NO: 119) or a sequence having one or two mutations thereto, and CDR3 sequence LEDRVDSFHDY (SEQ ID NO: 120) or a sequence having one or two mutations thereto The antibody of any one of 1045 to 1048 of the present invention. [The present invention 1051] CDR1 sequence GVTVSALNAMAMG (SEQ ID NO: 121) or a sequence having one or two mutations thereto; The CDR2 sequence AVSERGNAM (SEQ ID NO: 122) or a sequence having one or two mutations thereto, and CDR3 sequence LEDRVDSFHDY (SEQ ID NO: 123) or a sequence having one or two mutations thereto The antibody of any one of 1045 to 1048 of the present invention. [The present invention 1052] Any of the antibodies of 1049 to 1051, wherein E or S at amino acid position 4 of CDR2 is mutated to G, A, L, I, S, T, V, C, M, D, N, E, Q, F, Y, H, W, K, R, or P, preferably to D, N, or H. [The present invention 1053] FR1 sequence TIFF0007623006000005.tif4128 And X 1 is Q, V, E, or L, and X 2 is Q or P, and X 3 is L or M; or A sequence having at least 80%, 85%, 90%, or 95% sequence identity with said sequence. The antibody of any one of 1049 to 1052 of the present invention. [The present invention 1054] FR1 sequence TIFF0007623006000006.tif4128 Or any of the antibodies of the present invention, 1049 to 1052, comprising a sequence having at least 80%, 85%, 90%, or 95% sequence identity with said sequence. [The present invention 1055] FR2 sequence WYRQX 1 PGEX 2 RVMVA And X 1 is A or R, and X 2 is R or E; or A sequence having at least 80%, 85%, 90%, or 95% sequence identity with said sequence. The antibody of any one of 1049 to 1054 of the present invention. [The present invention 1056] The antibody of any one of claims 1049 to 1054, comprising the FR2 sequence WYRQAPGERRVMVA (SEQ ID NO:127) or a sequence having at least 80%, 85%, 90%, or 95% sequence identity thereto. [The present invention 1057] The antibody of any one of claims 1049 to 1054, comprising the FR2 sequence WYRQAPGEERVMVA (SEQ ID NO: 128) or a sequence having at least 80%, 85%, 90%, or 95% sequence identity thereto. [The present invention 1058] FR3 sequence TIFF0007623006000007.tif4128 And X 1 is K or Q, and X 2 is T or M; or A sequence having at least 80%, 85%, 90%, or 95% sequence identity with said sequence. The antibody of any one of claims 1049 to 1057. [The present invention 1059] FR3 sequence TIFF0007623006000008.tif4151 Or any one of the antibodies 1049 to 1057 of the present invention, which comprises a sequence having at least 80%, 85%, 90%, or 95% sequence identity with said sequence. [The present invention 1060] FR4 sequence WGQGX 1 QVTVSS And X 1 is T or I; or A sequence having at least 80%, 85%, 90%, or 95% sequence identity with said sequence. The antibody of any one of 1049 to 1059 of the present invention. [The present invention 1061] Any of the antibodies of the present invention 1049 to 1059, comprising the FR4 sequence WGQGTQVTVSS (SEQ ID NO: 132) or a sequence having at least 80%, 85%, 90%, or 95% sequence identity thereto. [The present invention 1062] VHH sequences TIFF0007623006000009.tif41150TIFF0007623006000010.tif238150 Or any one of the antibodies of the present invention 1049 to 1061, comprising a sequence having at least 80%, 85%, 90%, or 95% sequence identity with any one of said sequences. [The present invention 1063] CDR1 sequence GTMSAINALN (SEQ ID NO: 143) or a sequence having one or two mutations thereto; The CDR2 sequence AITDNGNAH (SEQ ID NO: 144) or a sequence having one or two mutations thereto, and CDR3 sequence LEEEKLGVWVDY (SEQ ID NO:145) or a sequence having one or two mutations thereto The antibody of any one of 1045 to 1048 of the present invention. [The present invention 1064] CDR1 sequence GTMSAINALN (SEQ ID NO: 146) or a sequence having one or two mutations thereto; The CDR2 sequence AITDNGNAH (SEQ ID NO: 147) or a sequence having one or two mutations thereto, and CDR3 sequence LEEKLGAWVDY (SEQ ID NO:148) or a sequence having one or two mutations thereto The antibody of any one of 1045 to 1048 of the present invention. [The present invention 1065] CDR1 sequence GTMSAINALN (SEQ ID NO: 149) or a sequence having one or two mutations thereto; The CDR2 sequence AITDNGNAH (SEQ ID NO: 150) or a sequence having one or two mutations thereto, and CDR3 sequence LEKEKLGVWVDY (SEQ ID NO:151) or a sequence having one or two mutations thereto The antibody of any one of 1045 to 1048 of the present invention. [The present invention 1066] FR1 sequence TIFF0007623006000011.tif4128 And X 1 is V or L; or A sequence having at least 80%, 85%, 90%, or 95% sequence identity with said sequence. The antibody of any one of claims 1063 to 1065 of the present invention. [The present invention 1067] FR2 sequence WYRQX 1 PGKERKMVA And X 1 is P or A; or A sequence having at least 80%, 85%, 90%, or 95% sequence identity with said sequence. The antibody of any one of claims 1063 to 1066 of the present invention. [The present invention 1068] FR3 sequence TIFF0007623006000012.tif4128 And X 1 is K or E; or A sequence having at least 80%, 85%, 90%, or 95% sequence identity with said sequence. The antibody of any one of claims 1063 to 1067. [The present invention 1069] The antibody of any one of claims 1063 to 1068, comprising the FR4 sequence WGQGTQVTVSS (SEQ ID NO:155) or a sequence having at least 80%, 85%, 90%, or 95% sequence identity thereto. [The present invention 1070] VHH sequences TIFF0007623006000013.tif63150 Or any one of the antibodies of the present invention 1063 to 1069, comprising a sequence having at least 80%, 85%, 90%, or 95% sequence identity with any one of said sequences. [The present invention 1071] The antibody of any one of claims 1045 to 1070, which is conjugated to a detectable label. [The present invention 1072] The antibody of the present invention, wherein said detectable label is a fluorescent label. [The present invention 1073] The antibody of the present invention, wherein said detectable label is an affinity tag. [The present invention 1074] The antibody of the present invention 1071 or 1073, 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. [The present invention 1075] The antibody of any one of 1045 to 1074 of the present invention, which is conjugated to a solid support. [The present invention 1076] The antibody of any one of claims 1045 to 1075 of the present invention, which forms a complex with an epitope to which the antibody specifically binds. [The present invention 1077] A fusion protein comprising a peptide to which any one of the antibodies 1045 to 1076 of the present invention binds. [The present invention 1078] (a) a fusion protein; (b) Antibody and Including, The fusion protein is any one of the fusion proteins 1001 to 1044 and 1077 of the present invention, and / or the antibody is any one of the antibodies 1045 to 1076 of the present invention. Complex. [The present invention 1079] A nucleic acid encoding any one of the fusion proteins of the present inventions 1001 to 1044 and 1077 or any one of the antibodies of the present inventions 1045 to 1076. [The present invention 1080] A vector comprising the nucleic acid of the present invention. [The present invention 1081] A host cell comprising the nucleic acid of the present invention 1079 or the vector of the present invention 1080, or expressing the fusion protein of any of the present inventions 1001 to 1044 and 1077, or the antibody of any of the present inventions 1045 to 1076. [The present invention 1082] Use of any of the peptides of the present inventions 1001 to 1044 and 1077 as an epitope tag for a fusion protein of any of the present inventions 1001 to 1045 and 1076, or for detection, immobilization, isolation, or purification of said fusion protein. [The present invention 1083] Use of any one of the antibodies according to the present invention, Nos. 1045 to 1076, for detection, immobilization, isolation, or purification of a fusion protein according to any one of the antibodies according to the present invention, Nos. 1001 to 1044 and 1077. [The present invention 1084] A method for detecting a fusion protein of any one of the antibodies of the present inventions 1001 to 1044 and 1077, comprising the step of contacting the fusion protein with an antibody of any one of the antibodies of the present inventions 1071 to 1074. [The present invention 1085] The method of claim 1084, further comprising the step of contacting said fusion protein and said antibody with a specific binding partner for a detectable label contained in said antibody. [The present invention 1086] The method of claim 1085, wherein said specific binding partner is conjugated to a second detectable label. [The present invention 1087] The method of claim 1086, wherein said second detectable label is a fluorescent label. [The present invention 1088] The method of any one of claims 1084 to 1087, further comprising the step of detecting said detectable label or said second detectable label. [The present invention 1089] The method according to any one of claims 1084 to 1088, further comprising the step of expressing the fusion protein before contacting the fusion protein with the antibody. [The present invention 1090] The method of any of claims 1084 to 1089, wherein detecting said fusion protein comprises determining the presence, subcellular localization, or amount of said fusion protein. [The present invention 1091] A method for isolating a fusion protein of any one of the present inventions 1001 to 1044 and 1077, or isolating a specific target of an antibody moiety contained in the fusion protein of the present invention 1044, comprising a step of contacting the fusion protein with an antibody of any one of the present inventions 1045 to 1076. [The present invention 1092] The method of claim 1091, wherein said antibody is attached to a solid support before, during, or after binding to said fusion protein. [The present invention 1093] The method of claim 1091 or 1092, further comprising the step of eluting said fusion protein. [The present invention 1094] The method of claim 1093, wherein said elution is effected by contacting the complex of said fusion protein and said antibody with a substance that competes with said fusion protein for binding to the antibody. [The present invention 1095] The method of the present invention 1094, wherein the substance is any one of the peptides of the present invention 1001 to 1043 or 1076. [The present invention 1096] The method of any of claims 1091 to 1095, further comprising the step of contacting the fusion protein of claim 1044 with a specific target of said antibody moiety contained in the fusion protein. [The present invention 1097] The method of claim 1096, wherein said specific target is a cell surface receptor. [The present invention 1098] The method according to any one of claims 1091 to 1097, further comprising a washing step. [This invention 1099] A nucleic acid or a nucleic acid expression construct encoding any one of the peptides of the present invention 1001 to 1044 and 1077, and optionally encoding any one of the antibodies of the present invention 1045 to 1076. Including the kit. [Brief description of the drawings]

[0018] [Figure 1A] Figure 1: Interaction of ALFAST and ALFAPE with NbALFAST. A: Schematic diagram of proteins used in ALFA binding assay. In this figure, the ALFA tag used in shGFP2 fusions can be either ALFAST (SEQ ID NO:05-07) or ALFAPE (SEQ ID NO:33). [Figure 1B]B: 20 ​​μl of ALFA SelectorST resin displaying NbALFAST (SEQ ID NO:133) was saturated with GFP variants (shGFP2, Frey and Gorlich; Cell. 2018 Jun 28;174(1):202-217.e9.doi:10.1016 / j.cell. 2018.05.045) fused to ALFAST or ALFAPE at different positions (intermediate (left), N-terminal (center), C-terminal (right)). After 4 washes with PBS, beads were suspended in 10x more PBS containing 200 μM ALFAST peptide (Ac-PSRLEEELRRRLTEP-Amide, SEQ ID NO:179) and mixed gently at room temperature. Specific elution from the beads was quantified using GFP fluorescence emitted in the supernatant at the indicated time points. The mean fluorescence values ​​from three independent experiments performed in parallel and the standard deviation for each time point are shown. Efficient peptide elution of ALFAPE fusions was observed already after 20-30 min at room temperature. In contrast, all GFP variants fused to ALFAST remained tightly bound to the resin. It should be noted that the elution kinetics was largely independent of the localization of each ALFA tag variant within the fusion protein. [Figure 1C] C: 10 μl of ALFA SelectorST resin saturated with ALFAST-shGFP2 (top) or ALFAPE-shGFP2 (bottom) was transferred into 8-well PCR strips. After removing residual liquid, the beads were incubated with 100 μl of the indicated material for 60 min at room temperature. Pictures were taken after beads had settled. Asterisks (*) indicate partial or complete loss of GFP fluorescence. [Figure 2A]Figure 2: Pull-down of ALFA-tagged target proteins and ALFA-tagged protein complexes from complex lysates using ALFA SelectorST. A: shGFP2 fused to ALFAST (SEQ ID NO:07; ALFAST-sfGFP2, left) or ALFAPE (SEQ ID NO:33; ALFAPE-GFP, right) at the N-terminus. Proteins were overexpressed in E. coli and purified by nickel affinity chromatography via their C-terminal His6-tags, followed by gel filtration on a Superdex75 size-exclusion column. [Figure 2B]B+C: To obtain a defined input material for one-step affinity purification using ALFA SelectorST, mock extracts of E. coli (B) or HeLa cells (C) were mixed with 3 μM of the respective substrate. Mock lysates served as specificity controls. 1 mL of each lysate / substrate mixture was incubated with 25 μl of ALFA-SelectorST containing NbALFAST (SEQ ID NO:133) for 1 h at 4°C. After 4 washes with 1 mL of PBS, bound proteins were eluted twice with 25 μl of 200 μM ALFAST peptide (SEQ ID NO:179) in PBS for 10 min at room temperature. Proteins remaining on the beads were then eluted with SDS sample buffer. 0.5 μL (B) or 1.5 μL (C) of the input and unbound fractions were analyzed by SDS-PAGE (12%) and Coomassie staining. The elution fractions shown correspond to material eluted from 1 μl of ALFA-SelectorST resin. It should be noted that proteins tagged with either the ALFAST or ALFAPE tag can be specifically pulled down under native conditions using ALFA-SelectorST. Highly efficient and specific elution of proteins fused with the ALFAPE tag can be achieved under native conditions by competition with the ALFAST free peptide. It should also be noted that proteins extracted from both lysates using ALFA-SelectorST resin contain significantly fewer impurities than the respective substrate proteins purified by conventional two-step chromatography. [Figure 2C] See legend to Figure 2B. [Figure 2D]D: Left: Schematic representation of the YfgM-PpiD complex. Right: Untagged (-) (SEQ ID NO:198) or C-terminally ALFAPE-tagged YfgM (+) (SEQ ID NO:197) were expressed in yfgM deletion dye. Membrane protein complexes were solubilized using 1% DDM of total lysate. YfgM-ALFAPE-containing complexes were purified in a single step using ALFA SelectorST affinity resin containing the nanobody of SEQ ID NO:133. Serum raised against the YfgM-PpiD complex recognized both PpiD and YfgM in the input fraction. ALFA SelectorST specifically immunoprecipitated the native protein complex of YfgM-ALFAPE and its interacting partner PpiD. [Figure 3A] Figure 3: Nanobody-based detection of ALFAST-tagged proteins in immunofluorescence applications. A: Schematic representation of NbALFAST with ALFAST tag (left) or ALFAPE tag (right). The ALFA tag sequences used for tagging at different positions are shown (N-terminal ALFAST tag: SEQ ID NO:05, intermediate position ALFAST tag: SEQ ID NO:06, C-terminal ALFAST tag: SEQ ID NO:07). [Figure 3B] B: Sequence of NbALFAST (NbALFA clone 1G5, SEQ ID NO: 133). Grey boxes indicate CDRs 1-3 (AbM definition, SEQ ID NO: 115-117). [Figure 3C] C: COS-7 cells transfected with Tom70-EGFP-ALFAST (top) or Tom70-EGFP-ALFAPE (bottom) were fixed with 4% paraformaldehyde. Staining with NbALFAST conjugated to AbberiorStar635P (FluoTag-X2 anti-ALFA AbberiorStar635P) was performed after permeabilization. 1st row: FluoTag-X2 anti-ALFA, 2nd row: target detection using EGFP autofluorescence, 3rd row: overlay including DAPI staining, 4th row: schematic representation of target proteins detected by fluorescently labeled NbALFAST. All scale bars: 20 μm. [Figure 3D]D: N-terminally ALFAST-tagged vimentin (top row) or N-terminally ALFAPE-tagged vimentin (bottom row) were detected with FluoTag-X2 anti-ALFA AbberiorStar635P after fixation with 4% paraformaldehyde (PFA), 2% glutaraldehyde (GA), or 100% methanol (MeOH). Right column: Schematic representation of ALFA-tagged vimentin detected by fluorescently labeled NbALFAST. [Figure 3E] E: Intracellular antibody-based detection of ALFAST-tagged proteins. COS-7 cells were co-transfected with NbALFAST-mScarlet-I fusions and ALFAST-tagged target proteins. Target proteins were detected via immunofluorescence using EGFP fluorescence (for TOM70-EGFP-ALFAST) or FluoTag anti-ALFA AbberiorStar635P (for ALFAST-FLAG-Vimentin). In parallel, NbALFAST-mScarlet-I was detected by red mScarlet-I fluorescence. Note the excellent colocalization between the target proteins (left column) and the mScarlet-I signal (middle column). [Figure 4] ALFA-tagged proteins can be detected by fluorescently labeled NbALFAST, regardless of their localization within the fusion protein. COS-7 cells were transfected with constructs encoding proteins with ALFA tags fused to their N-terminus (ALFAST-FLAG-vimentin or ALFAPE-FLAG-vimentin, A) or between individual protein domains (EGFP-ALFAST-myc-TM, B). Cells were fixed with 4% PFA and stained as indicated. For A and B, cells were permeabilized with 0.1% TritonX-100, for C, cells were stained under conditions that did not permeabilize the cells. TM: transmembrane domain. Schematic diagram shows the topology of the substrate and its detection by fluorescently labeled NbALFAST (FluoTag-X2 anti-ALFA). [Diagram 5]GFP fused to the ALFAST tag at the N- or C-terminus shows normal subcellular localization. 3T3 cells were transiently transfected with EGFP fusions with the ALFAST tag at the N- or C-terminus. Untagged EGFP from pEGFP-N1 served as a control. The localization of each EGFP variant was analyzed in 6–7 individual images for each construct. In total, 120–130 cells were imaged per construct and analyzed for EGFP localization. In general, each EGFP construct showed a distribution across the cytosol and nucleus. Cells were classified into three groups ("slightly nuclear", "uniform", and "other") according to the observed nucleocytoplasmic localization of EGFP. Standard deviations were estimated from the values ​​obtained from each image. There was no statistically significant difference in localization between tagged and untagged EGFP variants (Student's t-test). [Figure 6]Western blot and dot blot detection of ALFA-tagged target proteins using fluorescently labeled NbALFAST (FluoTag-X2 anti-ALFA). A: COS-7 cells transfected with ALFAST-FLAG-vimentin or ALFAPE-FLAG-vimentin were lysed in SDS buffer. Cells transfected with an irrelevant plasmid served as control. Lysates corresponding to the same number of cells were analyzed by SDS-PAGE and Western blotting. Vimentin fusion proteins were visualized using NbALFAST (FluoTag-X2 anti-ALFA IRDye800) conjugated with IRDye800. Tubulin served as a loading control and was detected by mouse anti-tubulin followed by FluoTag-X2 anti-mouse conjugated with IRDye680. Complete lanes are shown in Figure 7A. B: Schematic representation of recombinant E. coli maltose binding protein (MBP) with multiple epitope tags (FLAG, HA, myc, and ALFAST) used in the experiments shown in C and D. C: A dilution series of the proteins depicted in B was spotted onto a nitrocellulose membrane. Pre-made monoclonal antibodies (anti-FLAG M2-Sigma #F1804, anti-myc 9E10 SynapticSystems #343 011, anti-HA F-7-SantaCruz #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 ALFAST tag was detected utilizing FluoTag-X2 anti-ALFA directly conjugated to IRDye800CW. Nanobodies and all primary antibodies were used at a final concentration of 2.7 nM, which is sufficient for the range recommended by the supplier. The results of all experiments including internal standards are shown in Figure 7B. D: Quantification of the signals obtained in C is shown in a log-log plot. The line represents a linear fit to the values ​​obtained. Even without signal amplification by a secondary antibody, the signals obtained with NbALFAST were 3- to 10-fold stronger than with ready-made reagents recognizing the epitope tag. At the same time, detection with NbALFAST was 10-fold more sensitive and showed excellent linearity over about three orders of magnitude. [Figure 7]Sensitive Western blot and dot blot detection of ALFA-tagged target proteins using fluorescently labeled NbALFAST (FluoTag-X2 anti-ALFA). A: Same experiment as shown in Figure 6A, but now the complete lane is shown. Note that without any vector encoding the ALFAST-tagged protein, only a very minor band (*) can be detected using fluorescently labeled NbALFA (FluoTag-X2 anti-ALFA). B: Same experiment as shown in Figure 6C. In addition to the data presented in Figure 6C, detection of MBP by a rabbit polyclonal serum recognizing MBP (SynapticSystems) in combination with anti-rabbit IgG IRDye680RD (Li-Cor #925-68071) is shown as an internal loading standard. For the overlay, the MBP signal is shown in red and the signal corresponding to the epitope tag in green. [Figure 8]Interaction of ALFAST-tagged proteins with ALFA SelectorST and ALFA SelectorPE resins. A: Schematic of ALFA Selector resin with bound shGFP2-ALFAST. In this schematic, the ALFA Selector resin can be ALFA SelectorST or ALFA SelectorPE. B and C: Peptide elution from NbALFA-conjugated affinity resin. Agarose-based resin conjugated with NbALFAST (SEQ ID NO:133, ALFA SelectorST, left) or NbALFAPE variant (SEQ ID NO:134, ALFA SelectorPE, right) was loaded with shGFP2 with an ALFAST tag at its C-terminus. To estimate the off-rate, the resin was suspended in PBS containing an excess of ALFAST-release peptide and incubated at 25°C. A control reaction was performed without peptide. At the time points indicated in the figure, shGFP2 released from the resin was quantified. B shows the mean fluorescence readings of three experiments for each time point as well as the standard deviation. The lines represent fits with a single exponential function. Efficient peptide elution of shGFP2-ALFAST from ALFA SelectorPE was observed already after 15-20 min at room temperature. In contrast, peptide elution from ALFA SelectorST was inefficient even after extended incubation. During elution, in the absence of ALFAST free peptide, the ALFAST-tagged target protein remained tightly bound to both resins. Pictures were taken under UV illumination 3 h after elution (C). D: Resistance to stringent washing steps. The ALFA Selector variants described in B were loaded with either ALFAST-shGFP2 or shGFP2-ALFAST and incubated with 10-fold volume of the indicated material for 1 h at 25 °C with shaking. Pictures were taken under UV illumination after the beads had settled without further washing steps. E: Resistance to non-physiological pH. As in D, but now after 30 min of incubation at the pH indicated in the figure, the resin was washed to remove unbound material. Photographs were taken after re-equilibration in PBS to restore GFP fluorescence. [Figure 9-1]Figure 9: Pull-down of ALFAST-tagged target proteins and ALFAST-tagged protein complexes from complex lysates using ALFA SelectorST and ALFA SelectorPE. A: Input proteins used in the experiment are listed in (B and C). B and C: One-step affinity purification results using ALFA Selector resin. E. coli (A) or HeLa (B) lysates mixed with 3 μM purified ALFAST-tagged shGFP2 (A) were incubated with ALFA SelectorST, ALFA SelectorPE, or a similar resin without immobilized sdAb (Selector control). After washing with PBS, the resin was incubated with 200 μM ALFAST peptide for 20 min. Proteins remaining on the beads were eluted with SDS sample buffer. Fractions analyzed by SDS-PAGE and Coomassie staining are shown in the figures. Eluate fractions corresponding to material eluted from 1 μl of resin are shown. [Figure 9-2]D: Pull-down of native E. coli YfgM-PpiD inner membrane protein complex using ALFA SelectorPE. Left: Schematic representation of the YfgM-PpiD membrane protein complex. Right: yfgM deletion strains were supplemented with either C-terminally ALFAST-tagged YfgM (left panel) or untagged YfgM (right panel, control reaction) expressed from a low copy vector. Membrane protein complexes were solubilized from total lysates using DDM. Complexes containing YfgM-ALFAST were purified in a single step using ALFA SelectorPE affinity resin and eluted under native conditions using 200 μM ALFAST peptide. Samples representing 1 / 800 of the input and unbound material or 1 / 80 of the eluate fraction were resolved by SDS page and analyzed by Western blotting. Rabbit serum raised against the YfgM-PpiD complex (Gotzke 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 SelectorPE specifically immunoprecipitated the native protein complex containing ALFAST-tagged YfgM and its interacting partner PpiD. In the control reaction (YfgM without ALFAST tag), both proteins were absent in the eluate. [Figure 10]Peptide elution of ALFAST-tagged GFP from ALFA Selector resin. 20 μl of ALFA SelectorST (presenting NbALFAST, SEQ ID NO:133) or ALFA SelectorPE (presenting NbALFAPE, SEQ ID NO:134) were loaded with shGFP2-ALFAST (A), bdSUMO-ALFAST-shGFP2 (B), or ALFAST-shGFP2 (C). After washing with PBS, the beads were suspended in 10x more PBS containing 200 μM ALFAST free peptide and mixed gently at 25° C. Control reactions were performed without peptide. Specific elution from the beads was quantified using GFP fluorescence emitted into the supernatant at the indicated time points. The average fluorescence readings of three experiments for each time point are shown as well as the standard deviation. The lines represent the fits with a single exponential function. Only half-times of peptide elution from ALFA SelectorPE are presented. For all substrate proteins, peptide elution from ALFA SelectorST was poor even after extended incubation. In the absence of ALFAST peptide, ALFAST-tagged target proteins remained tightly bound to both resins. Figure 10A reproduces the data shown in Figure 8B and is repeated here to allow direct comparison. Left panel: Schematic diagram showing the experimental set-up. The ALFA Selector resin can be either ALFA SelectorST or ALFA SelectorPE. Center panel: Experiment performed with ALFA SelectorST. Right panel: Experiment performed with ALFA SelectorPE. [Figure 11]X-ray structure analysis of NbALFAST bound to the ALFAST peptide. A-C: Diagrams of the NbALFAST-ALFAST peptide structure. A: N-terminal view of the ALFAST peptide. B: Side view of the ALFAST peptide. C: C-terminal view of the ALFAST peptide. NbALFAST is shown in light grey with side chains as lines. Residues contacting the ALFAST peptide are shown as sticks. The ALFAST peptide is depicted in dark grey with side chains as sticks. The ALFAST peptide is used with an acetylated N-terminus and an amidated C-terminus (SEQ ID NO:179). D: NbALFAST (SEQ ID NO:133) sequence. Boxes indicate CDRs 1-3 (SEQ ID NO:115-117) as in Figure 3B. Residues directly contacting the ALFAST peptide are boxed. Residues in filled boxes were mutated to reduce affinity for the ALFAST peptide. [Figure 12] Naive lymphocyte isolation using an ALFA-tagged nanobody that recognizes CD62L. Total human PBMCs were left untreated (pre-sorting) or isolated using ALFA SelectorPE resin loaded with an ALFA-tagged anti-human CD62L nanobody (post-sorting). A schematic of the affinity purification method is shown in (a). Cells were stained with anti-CD62L antibody and analyzed by flow cytometric analysis (b). The same cells as in (b) were stained with antibodies against CD3, CD19, and CD62L and analyzed by flow cytometric analysis (c). A forward-scattering / side-scattering gate was set for lymphocytes for all analyses. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0019] Detailed Description To overcome some of the shortcomings of the state of the art, the inventors of the present application have created small epitope tags recognized by high affinity nanobodies. Such a system allows the analysis of a protein's function in multiple aspects, including but not limited to the analysis of its localization, its interaction partners by purification from lysates, or in vivo manipulation, including induction of protein mislocalization or depletion, using a minimal set of recombinant constructs and cell lines, without unintentionally interfering with the physiological function of the protein of interest.

[0020] In view of the limitations of current epitope tags, the inventors of this application decided to tackle this problem and seek out the characteristics that an ideal epitope tag system should have. A truly versatile tag should be small to minimize potential side effects (Kocaoglu, O. & Carlson, E. E. Progress and prospects for small-molecule probes of bacterial imaging. Nat Chem Biol 12, 472-478 (2016)). To minimize artificial multimerization of tagged proteins, the tag is preferably monomeric. Also, to avoid the addition of a net charge to the tagged protein, the tag should be electroneutral and at the same time soluble (Esposito, D. & 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 tagged protein (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 eukaryotic and prokaryotic host organisms and resistant to proteolysis. Ideally, the tag should be resistant to fixation and its sequence should be absent in common model organisms to avoid unintended detection of endogenous host proteins.

[0021] Like an ideal epitope tag, its corresponding binder should also have several features to make it ideal for tag detection. For example, the binder should be small for easy access to crowded areas and provide the highest binding affinity for various applications. Current advanced applications (e.g., in vivo live imaging) require specific, genetically accessible probes with high affinity for the tag, which should be able to fold autonomously in vivo in various host organisms. For biochemical applications, however, the preferred binder should preferentially have moderate affinity to allow competitive elution of immunoprecipitated substances under native conditions. When evaluating the commonly used epitope tags that exist so far, the state-of-the-art applicants will eventually have to sacrifice at least one of the features that have been described (see Table 1 below). To create an epitope tag with ultimate versatility that meets all the boundary conditions that have been described, the inventors of this application recognized that the most straightforward approach is to design it de novo.

[0022] Table 1. Characteristics of common epitope tag systems TIFF0007623006000014.tif174153TIFF0007623006000015.tif114153n.a.:Not applicable nd: No data mAb: Monoclonal antibody sdAb: single domain antibody 1For optimal performance, often multiple tags are used in parallel. (Hernan, R., Heuermann, K. & Brizzard, B. Multiple epitope tagging of expressed proteins for enhanced detection. BioTechniques 28,789-793(2000); Ross-Macdonald, P., Sheehan, A., Roeder, GS & 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,RA&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)) 2 The binding agent also recognizes biotinylated proteins. 3 The binding agent recognizes endogenous proteins that have multiple accessible histidines. 4 Depending on the chelate and poly-His tag used. 5 The binding agent also recognizes endogenous β-catenin. 6For high-profile imaging applications, the binder needs to be dimerized. (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) 7 Immobilization with amine-reactive fixatives and crosslinkers. Sequence-derived. TIFF0007623006000016.tif199161

[0023] With this express goal in mind, the inventors of the present application have designed the epitope tags described herein, which preferably consist of between about 8 and 25 amino acids and are collectively referred to as ALFA tags.

[0024] The inventors of this application decided to take a novel approach to generate new epitope tags. The epitope tag according to the invention is a small, monomeric epitope tag, preferably with a minimum size of 15 aa or less. The sequence is preferably uncharged and hydrophilic at physiological pH, and most preferably has no residues that are susceptible to modification by amine-reactive fixatives and cross-linkers. Its size is in contrast to larger tags, such as FLAG tag trimers, or even larger fluorescent proteins, which are typically used 3x in series to increase binding strength. Furthermore, the epitope tag of the present invention has no counterpart in eukaryotic or prokaryotic sequence databanks, minimizing the risk of cross-linking with native structures. This is in contrast to the SPOT-tag® described in WO 2017 / 085086 A1.

[0025] A further advantage of the epitope tag of the present invention is that, unlike e.g. the Myc tag, it is compatible with common amine-reactive fixatives (paraformaldehyde (PFA), glutaraldehyde (GA)). It is also compatible with methanol fixation. A further advantage of the epitope tag of the present invention is that it is not limited in terms of location (N, C, or interprotein) unlike the EPEA tag described in WO 2011 / 147890 A1.

[0026] Without wishing to be bound by theory, it is believed that the epitope tag according to the present invention forms a stable alpha-helical structure. It is believed that certain antibodies, such as single domain antibodies (sdAbs) or nanobodies, prefer a well-defined three-dimensional surface for binding, and therefore forming a stable alpha-helix is ​​believed to be more advantageous than a differently folded, unfolded, or unstable folded structure. Historically, it has been very difficult to generate single domain antibodies that bind to native unfolded peptides, resulting in only sdAbs with weak to moderate binding affinity. The inventors of the present application therefore focused on providing an epitope tag that is believed to form a compact alpha-helix that stably folds in solution, as this structure is the smallest entity that forms a stable secondary structure as a monomer. Without wishing to be bound by theory, it is believed that the alpha-helical structure undergoes efficient spontaneous refolding, even after exposure to harsh chemical treatments. Furthermore, it is believed that due to its helical structure, the tag is smaller than most amorphous linear epitope tags. In addition, the epitope tags of the present invention can be placed at the N-terminus or C-terminus of a target protein, or even between two folded protein domains, without compromising proper targeting and folding of the target protein.

[0027] Finding the ideal binder or binders has however proven challenging: although conventional antibodies would indeed meet most of the requirements, their large size makes them suboptimal for current super-resolution microscopy (Fornasiero, EF & 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 living intracellular targets. The inventors of the present application therefore 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 fulfill the set criteria. A novel in-house selection method for this, called "Celline", enabled the inventors to generate alpaca-derived sdAbs with extremely high affinity in a timely manner. The antibodies of the present invention in combination with an epitope tag are expressed as ALFA ST It proved to be ideal for imaging and intracellular detection of tagged target proteins, allowing efficient and clean immunoprecipitation.

[0028] The present invention also provides high affinity antibodies for the epitope tags of the present invention. Some of the antibodies are monovalent sdAb-based binders. The monovalent binding is in contrast to epitope tags that are bound by conventional antibodies. Although SPOT Tags are bound by sdAbs, for some applications of SPOT Tags, SPOT Tag-binding sdAbs are employed as dimers to increase avidity. The use of monovalent antibodies has the advantage that clustering can be prevented. Thanks to the alpha-helical structure of the epitope tag, the inventors of the present invention have been able to achieve a K in the range of about 10 pM, which is about 1000-fold higher affinity than comparable epitope tag / sdAb systems, for example as described in WO 2017 / 085086 A1 or WO 2011 / 147890 A1. d It is further believed that it would be possible to generate single domain antibodies that bind to the epitope tags of the present invention.

[0029] For some of the epitope tags of the present invention, such as SEQ ID NOs:05-07, it has been virtually impossible to efficiently separate the antibody, including SEQ ID NO:133, from the tag under native conditions. In some instances, this will limit the use of SEQ ID NOs:05-07 for the purification of native proteins and their interacting partners. By solving the crystal structure of a high affinity complex of the antibody of SEQ ID NO:133 and the peptide of SEQ ID NO:179, the inventors have been able to map the interacting determinants in detail and to characterize the ALFA binding sites under native conditions. ST It has become possible to design new versions of single domain antibodies that allow for competitive elution of tagged target proteins with their interaction partners. An exemplary antibody for this purpose comprises the amino acid sequence of SEQ ID NO:134.

[0030] The epitope tag and antibody system presented herein is suitable for an exceptionally broad range of applications, spanning from biotechnology to cell biology, such that a single tag can simultaneously replace a large variety of conventional epitope tags.

[0031] Thus, the present invention provides (a) comprising the sequence of X1-X2-X3-X4-X5-X6-X7-X8-X9-RL-X12-X13 (SEQ ID NO:01); 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, I, or V; X8 is R or A or Q or E; X9 is R, A, Q, or E; X12 is S or T or D or E or P or A or no amino acid; X13 is E or K or P or S or A or D or no amino acid; A peptide, (b) a polypeptide; In the fusion protein of the present invention, a peptide may be used as an epitope tag.

[0032] The term "peptide" as used herein refers to a linear series of amino acids, preferably connected to each other by peptide bonds between the α-amino and carboxy groups of adjacent residues. The term "amino acid" as used herein refers to either natural and / or unnatural amino acids or synthetic amino acids, including glycine and both D and L optical isomers, as well as amino acid analogs and peptidomimetics, with proteinogenic amino acids being preferred. A "proteinogenic amino acid" is an amino acid that can be biosynthetically incorporated into a protein during protein synthesis. Currently, there are 22 genetically encoded (proteinogenic) amino acids, 20 of which exist in the standard genetic code, and an additional two can be incorporated by specialized protein synthesis machinery. The term "peptide" as used herein preferably includes up to about 50 amino acids.

[0033] The term "polypeptide" as used herein generally refers to a peptide having at least about 30 amino acids, at least about 40 amino acids, or at least about 50 amino acids. The term "protein" as used herein includes one or more polypeptides.

[0034] The term "fusion protein" as used herein refers to a polypeptide or protein that comprises two or more subunits. At least one of the subunits is preferably a protein or polypeptide, and at least one of the subunits is preferably a peptide. In the fusion protein, the subunits can be linked by covalent or non-covalent bonds. Preferably, the fusion protein is a translational fusion between two or more subunits. The translational fusion can occur by genetically engineering the coding nucleotide sequence for one subunit in a reading frame with the coding nucleotide sequence for a further subunit. The subunits can be interspersed by linkers.

[0035] If one or more of the subunits are part of a protein (complex) consisting of more than one polypeptide chain, the term "fusion protein" can also refer to the protein including the fused sequence and all other polypeptide chain(s) of that protein (complex).

[0036] As used herein, an "epitope tag" refers to a stretch of amino acids against which a specific antibody or proteinaceous molecule with antibody-like function can be produced. Such epitope tags allow for detailed identification and / or tracking of tagged polypeptides or proteins that may be present in an organism or in cultured cells. Detection of tagged molecules can be achieved using a number of different techniques. Examples of such techniques include immunohistochemistry, immunoprecipitation, flow cytometry, immunofluorescence microscopy, electron microscopy, ELISA, immunoblotting ("Western blotting"), and affinity chromatography. Epitope tags add a known epitope (antibody binding site) to a polypeptide of interest to allow binding of a known and often high affinity antibody. Epitope tags can also be used for isolation and / or purification of tagged molecules, for example, by pull-down procedures.

[0037] In the fusion protein of the present invention, the peptide or 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 a specific example, the peptide can be fused between two domains of the polypeptide.

[0038] The polypeptide contained in the fusion protein can have a stable folding that is independent of the presence or absence of the peptide, meaning that the peptide preferably does not change or interfere with the native structure of the polypeptide.

[0039] The peptide itself preferably has an alpha helical structure. ALFA, which contains the sequence Ac-PSRLEEELRRRLTEP-amide (SEQ ID NO:179). ST Crystal structure analysis of an antibody having the sequence of SEQ ID NO:133 in complex with the peptide shows that the epitope tag binds to the nanobody as a stably folded alpha-helix. This structure is believed to explain the unusually tight binding. "Alpha-helical structure" refers herein to a secondary structure in the form of an alpha-helix. The alpha-helical secondary structure of the peptide is preferably independent of the fusion partner. This means that the peptide is preferably capable of forming an alpha-helical secondary structure in physiological buffers, both when in the form of an isolated peptide and when the peptide is part of a fusion protein.

[0040] This peptide may be specifically recognized by a Camelidae VHH domain containing the CDR sequences GVTISALNAMAMG (SEQ ID NO:115), AVSERGNAM (SEQ ID NO:116), and LEDRVDSFHDY (SEQ ID NO:117). This peptide may also be specifically recognized by other antibodies described herein.

[0041] In the fusion protein of the present invention, the peptide may be fused to the polypeptide either by direct fusion or through a linker. A "linker" as used herein connects two or more subunits of a fusion protein as described herein to each other. The bridge may be covalent. A preferred covalent bridge is via a peptide bond, such as a peptide bond between amino acids. A preferred linker is a peptide linker. The linker preferably comprises 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 polymer (PAS) linkers. The GS linker may be a (G4S)3 linker as described in SEQ ID NO:159.

[0042] The polypeptides contained in the fusion proteins of the invention can comprise at least one protein domain. A "protein domain" as used herein refers to a (tertiary) structure that is part of a given protein sequence and can function and / or exist independently from the rest of the protein chain. Protein domains preferably form compact three-dimensional structures and are often independently stable and can fold. Protein domains further form functional units. The polypeptides contained in the fusion proteins can comprise more than one protein domain, such as two, three, four or even more protein domains. A preferred location for this peptide can be outside the protein domain. It can be at the N-terminus or C-terminus of at least one protein domain of the polypeptide, or between two protein domains of the polypeptide. The polypeptides contained in the fusion proteins of the invention can be globular, membrane, fibrous or non-naturally folded proteins, or subunits or domains of globular, membrane, fibrous or non-naturally folded proteins.

[0043] A peptide included in a fusion protein of the 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. A polypeptide included in a fusion protein of the 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 naturally occurring polypeptide or protein in a cell that expresses a fusion protein of the invention.

[0044] The fusion protein of the invention can comprise a peptide comprising the sequence X1-X2-LE-X5-E-X7-RRRL-X12-X13 (SEQ ID NO:02), 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 X13 is P or A or S or A or D or E or no amino acids. This sequence defines the core structure of the peptide and can further include 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 usually do not necessarily affect the secondary structure of the peptide or the specific binding of the peptide to a specific antibody against the peptide, but can serve as a linker structure within the fusion protein. Thus, the type and number of additional amino acids may depend on the position of the peptide within the fusion protein, and may vary depending on whether the peptide is located at the N-terminus, C-terminus, or anywhere within the polypeptide. A peptide comprising the sequence of SEQ ID NO:02 has a T of at least about 2 minutes for dissociation from a single domain antibody comprising the sequence of SEQ ID NO:133, e.g., when measured in an assay essentially as described in Example 1. 1 / 2 The peptide can have a K of about 30 nM or less for binding to a single domain antibody comprising the sequence of SEQ ID NO:133. d may have the following structure:

[0045] A peptide included in a fusion protein of the invention can include an N-terminus of X1, which is amino acids Xa-Xb, where Xa is D or S or G or M or P or no amino acids, and Xb is S or D or P or M or R or G or no amino acids.

[0046] A peptide included in a fusion protein of the invention can include a C-terminus of X13 that is amino acids Xy-Xz, where Xy is G or S or P or D or A or E or K or no amino acids and Xz is S or P or no amino acids.

[0047] Fusion proteins of the invention may include peptides comprising the sequence X1-X2-LE-X5-ELRRRL-X12-X13 (SEQ ID NO:03) 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 X13 is A or D or E or no amino acids. Such peptides may have a T1 / 2 of at least about 100 minutes for dissociation from a single domain antibody comprising the sequence of SEQ ID NO:133, e.g., as measured in an assay essentially as described in Example 1. The peptide may have a K of about 1 nM or less for binding to a single domain antibody comprising the sequence of SEQ ID NO:133. d Such peptides can have the sequence SRLEEELRRRLTE (SEQ ID NO:04) or a variant thereof, where compared to (SEQ ID NO:04), the variant has one to five mutations selected from the group consisting of S1→T, R2→G, E5→Q, T12→D, and T12→E, E13→A, E13→D, and a deletion of E13. Compared to SEQ ID NO:04, this variant can have the following mutations: (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) a deletion of E13.

[0048] A peptide comprising the sequence of SEQ ID NO:03 as a core structure can comprise an N-terminus of X1, which is amino acids Xa-Xb, where Xa is S or G or M or P or no amino acids, and Xb is R or G or S or P or M or no amino acids. Xa-Xb can be selected from the group consisting of P, MP, GR, PG, PS, SP, GP, SP, M, and MS, and can preferably be P or MP. Such peptides can also comprise a C-terminus of X13, which is amino acids Xy-Xz, where P or D or A or no amino acids, and Xz is P or S or no amino acids. Xy-Xz can be selected from the group consisting of no amino acids, P, DP, A, and AS, and can preferably be no amino acids or P. The peptide can comprise a combination of Xa-Xb and Xy-Xz selected from the group consisting of (a) MP and P, (b) P and P, and (c) P and no amino acids.

[0049] The peptide contained in the fusion protein of the present invention may have a core structure of SEQ ID NO:03, The sequence may include a sequence selected from the group consisting of: TIFF0007623006000017.tif187128.

[0050] Fusion proteins of the invention may comprise a peptide comprising the sequence X1-X2-LE-X5-E-X7-RRRL-X12-X13 (SEQ ID NO:31) as a 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 peptides may have a T of about 2.5 minutes to about 30 minutes for dissociation from a single domain antibody comprising the sequence of SEQ ID NO:133, e.g., as measured in an assay essentially as described in Example 1. 1 / 2The peptide can have a K of about 3-40 nM or less for binding to a single domain antibody comprising the sequence of SEQ ID NO:133. d may have the following structure:

[0051] Such peptides may comprise the sequence GRLEEELRRRLS (SEQ ID NO:32) or a variant thereof, which has from one to six mutations compared to (SEQ ID NO:32) selected from the group consisting of G1→S, G1→P, R2→G, E5→Q, L7→I, S12→T, S12→P, and S12→A, an addition of P13, an addition of A13, and an addition of S13. Compared with NO:32, the following mutations were observed: (a) G1→S, R2→G, E5→Q, and the addition of P13; (b) R2→G, E5→Q, S12→T, and the addition of A13; (c) G1→P, R2→G, E5→Q, S12→T, and the addition of A13; (d) G1→S, R2→G, E5→Q, S12→T, and the addition of P13; (e) G1→S , R2→G, S12→T, and the addition of A13; (f) G1→S, R2→G, E5→Q, and S12→T; (g) the addition of G1→S, R2→G, E5→Q, S12→T, and A13; (h) the addition of G1→S and S12→P, and the addition of P13; (i) the addition of G1→S, R2→G, E5→Q, S12→T, and P13; (j) the addition of E5→Q L7→I, and the addition of P13; (k) the addition of P13; or (l) S12→A.

[0052] A peptide comprising the sequence of SEQ ID NO:31 as a core structure can comprise an N-terminus of X1, which is amino acids Xa-Xb, where Xa is M or S or P or D or G or no amino acids, and Xb is S or D or P or no amino acids. Xa-Xb can be selected from the group consisting of MS, SD, PD, PS, DS, SP, and GP, and can be preferably MS. Such peptides can also comprise a C-terminus of X13, which is amino acids Xy-Xz, where Xy is G or P or A or E or K or S or no amino acids, and Xz is P or S or no amino acids. Xy-Xz can be selected from the group consisting of no amino acids, G, P, A, EP, AS, K, and S, and can be preferably no amino acids. The peptide can comprise a combination of Xa-Xb and Xy-Xz, which is MS and no amino acids.

[0053] The peptide contained in the fusion protein of the present invention may have a core structure of SEQ ID NO:31, The sequence may include a sequence selected from the group consisting of: TIFF0007623006000018.tif121128.

[0054] Fusion proteins of the invention may include peptides comprising the sequence X1-X2-LE-X5-ELRRRL-X12-X13 (SEQ ID NO:49) as a core structure, where 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 acids. Such peptides may have a T1 / 2 of about 20 minutes to about 100 minutes for dissociation from a single domain antibody comprising the sequence of SEQ ID NO:133, e.g., as measured in an assay essentially as described in Example 1. The peptide may have a K of about 1-5 nM or less for binding to a single domain antibody comprising the sequence of SEQ ID NO:133. d may have the following structure:

[0055] A peptide comprising the sequence of SEQ ID NO:49 as a core structure can comprise an N-terminus of X1, which is amino acids Xa-Xb, where Xa is P or D or S or G or no amino acids, and Xb is D or S or P or no amino acids. Such peptides can also comprise a C-terminus of X13, which is amino acids Xy-Xz, where Xy is G or P or E or D or S or no amino acids, and Xz is P or no amino acids.

[0056] The peptide contained in the fusion protein of the present invention may have a core structure of SEQ ID NO: 49, The sequence may include a sequence selected from the group consisting of: TIFF0007623006000019.tif121128.

[0057] Fusion proteins of the invention include peptides comprising the sequence X1-X2-X3-X4-X5-X6-X7-X8-X9-RL-X12-X13 (SEQ ID NO:61) 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 peptides have a T of about 2 to about 10 minutes for dissociation from a single domain antibody comprising the sequence of SEQ ID NO:133, e.g., as measured in an assay essentially as described in Example 1. 1 / 2 The peptide may have a Kd of about 10-50 nM or less for binding to a single domain antibody comprising the sequence of SEQ ID NO:133.

[0058] A peptide comprising the sequence of SEQ ID NO:61 as a core structure can comprise an N-terminus of X1, which is amino acids Xa-Xb, where Xa is D or S or G or M or no amino acids, and Xb is S or D or P or M or no amino acids. Such peptides can also comprise a C-terminus of X13, which is amino acids Xy-Xz, where Xy is G or S or P or no amino acids, and Xz is S or no amino acids.

[0059] The peptide contained in the fusion protein of the present invention may have a core structure of SEQ ID NO:61, The sequence may include a sequence selected from the group consisting of: TIFF0007623006000020.tif21690.

[0060] The fusion proteins 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 as disclosed herein.

[0061] The fusion protein of the present invention can include 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 include or be a structure on the cell surface, such as a cell surface receptor. A preferred target is CD62L.

[0062] The present invention further provides an antibody that specifically binds to a peptide comprised in a 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 Camelidae VHH domain. In a preferred embodiment, the antibody of the present invention is a single domain antibody, such as a Camelidae single domain antibody.

[0063] The term "antibody" generally refers to a proteinaceous binding molecule with immunoglobulin-like functions. Typical examples of antibodies include, but are not limited to, immunoglobulins and derivatives thereof or functional fragments thereof that still retain binding specificity. Antibody production techniques are well known in the art. The term "antibody" also includes immunoglobulins (Igs) of different classes (i.e., IgA, IgG, IgM, IgD, IgE, IgY, etc.) and subclasses (e.g., IgG1, IgG2, etc.), even if produced recombinantly in a foreign host using techniques known to those skilled in the art. Specific examples of antibodies include full-length immunoglobulins, Fab fragments, F(ab')2, Fv fragments, single-chain Fv fragments (scFv), bispecific antibodies, or domain antibodies (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 antibody, having only one variable domain, which can be a VH or VL, that specifically binds an antigen or epitope independently of other V regions or domains. Particularly preferred single domain antibodies are VHH domains, which are heavy chain antibodies. Such immunoglobulin single variable domains can encompass not only isolated single variable domain antibody polypeptides, but also larger polypeptides that include or consist of one or more monomeric single variable domain antibody polypeptide sequences. It is understood that single domain antibodies can include a VHH domain and a fusion partner, such as a protein tag or peptide tag. The definition of the term "antibody" therefore also includes embodiments such as chimeric, single chain humanized antibodies. The term "antibody" can also include fragments of antibodies.

[0064] A single domain antibody is an antibody whose complementary determining regions are part of a single domain polypeptide. Examples include, but are not limited to, heavy chain antibodies, antibodies naturally devoid of light chains, single domain antibodies derived from conventional four-chain antibodies, engineered antibodies, and single domain scaffolds other than antibody-derived scaffolds. A single domain antibody can be any single domain antibody in the art or in the future. A single domain antibody can be derived from any species, including, but not limited to, mouse, human, camel, llama, goat, rabbit, or cow. According to the present invention, a single domain antibody is preferably an antibody of natural origin known herein as a heavy chain antibody devoid of light chains. Such single domain antibodies are disclosed, for example, in WO 94 / 04678. For reasons of clarity, this variable domain derived from a heavy chain antibody naturally devoid of light chains is known herein as a VHH to distinguish it from conventional VH, which is a four-chain immunoglobulin. Such VHH molecules can be derived from antibodies produced in Camelidae species, such as camel, dromedary, llama, picuna, alpaca, guanaco, etc. Other species outside of Camelidae can also produce heavy chain antibodies naturally devoid of light chains. As a specific example, sharks are known to produce heavy chain antibodies 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.

[0065] A VHH according to the invention and as known to the skilled person is preferably a heavy chain variable domain from an immunoglobulin naturally devoid of light chains, such as an immunoglobulin from Camelidae as described in WO 94 / 04678 (hereafter referred to as a VHH domain or nanobody). VHH molecules are about 10 times smaller than IgG molecules. VHH molecules are very stable as single polypeptides and are resistant to extreme pH and temperature conditions. Furthermore, VHH molecules are highly resistant to the activity of proteases, which is not the case for conventional antibodies. Furthermore, in vitro expression or expression in prokaryotes or eukaryotes, suitable for recombinant protein expression, of VHH produces high yields of properly folded functional VHH. A single domain antibody according to the invention is preferably understood to be a VHH.

[0066] The antibodies according to the invention may have one or more domains with a sequence that has 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 with a domain of a corresponding naturally occurring immunoglobulin naturally devoid of light chains. It should be noted in this regard that the term "about" or "approximately" is used herein to mean within a 20% deviation, such as within a 10% deviation or within a 5% deviation, of a given numerical value or range.

[0067] "Percent sequence identity" with respect to the amino acid sequences disclosed herein is defined as the percentage of amino acid residues in a candidate sequence that are pairwise identical to amino acid residues in a reference sequence, i.e., the percentage of antibody molecules of the present disclosure, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, and without considering any conservative substitutions as part of this sequence identity. Alignment to determine percent amino acid sequence identity can be accomplished using a variety of methods within the skill of the art, such as publicly available computer software, such as BLAST, ALIGN, or Megalign (DNASTAR) software. Those skilled in the art can determine appropriate parameters for measuring alignment, including any algorithms required to achieve maximum alignment over the entire length of the sequences being compared. The same applies to nucleotide sequences disclosed herein.

[0068] The term "variable" refers to the parts of the immunoglobulin domains (i.e., "variable domain(s)") that exhibit variability in their sequence and that are responsible for determining the specificity and binding affinity of a particular antibody. The variability is not evenly distributed throughout the variable domains of an antibody. It is concentrated in subdomains of each of the variable regions of the heavy and light chains. These subdomains are called "hypervariable regions", "HVRs" or "HVs", or "complementarity determining regions" (CDRs). The more conserved (i.e., non-hypervariable) parts of the variable domains are called "framework" regions (FRs). Naturally occurring heavy and light chain variable domains each contain four FR regions, most of which adopt a β-sheet structure and are connected by three hypervariable regions that form loops that connect, or in some cases form part of, this β-sheet structure. The hypervariable regions of each chain are held in close proximity to each other by the FRs and, together with the hypervariable regions of the other chain, contribute to the formation of the antigen-binding site (Kabat et al., see below). Generally, naturally occurring immunoglobulins contain six CDRs (see below), three in the VH (H1, H2, H3) and three in the VL (L1, L2, L3). In naturally occurring immunoglobulins, H3 and L3 show the most diversity of the six CDRs, and H3 in particular is thought to play a unique role in providing immunoglobulins with exquisite specificity. Immunoglobulins naturally lack light chains, but contain three CDRs present in the VHH region. The constant domains are not directly involved in antigen binding, but present various effector functions, such as antibody-dependent cellular cytotoxicity, cell-mediated cytotoxicity, and complement activation.

[0069] Each VHH, VH, and VL has three CDRs and four FRs arranged from the amino terminus (N-terminus) to the carboxy terminus (C-terminus) in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of the heavy and / or light chains contain a binding domain that interacts with an epitope of an antigen. The term "immunoglobulin" can refer to a protein, or antigen-binding portion thereof, that can have two heavy chains without light chains, e.g., an antibody lacking a light chain. An immunoglobulin can also include at least two heavy (H) chains and two light (L) chains, or antigen-binding portions thereof, linked by disulfide bonds.

[0070] An immunoglobulin, as used herein, can be a dimeric glycosylated protein composed of two heavy chains, such as camelid heavy chain only IgG (hcIgG) or shark IgNAR. An immunoglobulin, as used herein, can also be a tetrameric glycosylated protein composed of two light (L) chains of approximately 25 kDa each and two heavy (H) chains of approximately 50 kDa each.

[0071] The term "amino acid" or "amino acid residue", when used in connection with a protein or peptide, typically refers to 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-amino acid residue (Asp or D ... It refers to an alpha amino carboxylic acid having an art-recognized definition, such as an amino acid selected from the group consisting of threonine (Thr or T), L-tryptophan (Trp or W), L-tyrosine (Tyr or Y), and L-valine (Val or V); modified, artificial, or rare amino acids may also be used, if desired, such as taurine, ornithine, selenocysteine, homocystine, hydroxyproline, tiopronine, iodotyrosine, 3-nitrotyrosine, ornithine, citrulline, canavanine, 5-hydroxytryptophan, carnosine, cycloleucine, 3,4-dihydroxyphenylalanine, N-acetylcysteine, prolinol, allylglycine, or acetidine-2-carboxylic acid. In general, amino acids can be grouped according to possessing a nonpolar side chain (e.g., Ala, Cys, Ile, Leu, Met, Phe, Pro, Val), a negatively charged side chain (e.g., Asp, Glu), a positively charged side chain (e.g., Arg, His, Lys), or an uncharged polar side chain (e.g., Asn, Cys, Gln, Gly, His, Met, Phe, Ser, Thr, Trp, and Tyr).

[0072] A target according to the present invention is any substance of biological or chemical origin that can be detected directly or indirectly by the antibody of the present invention. A target can be, for example, a protein, a peptide, a nucleic acid, an oligonucleic acid, a saccharide, a polysaccharide, a glycoprotein. Examples include, but are not limited to, a therapeutic target, a diagnostic target, a receptor, a receptor ligand, a viral coat protein, an immune system protein, a hormone, an enzyme, an antigen, a cell signaling protein, or a fragment thereof. A target can be a naturally occurring protein or a fragment thereof, a homologous sequence thereof, a functional part thereof, or a functional part of a homologous sequence.

[0073] The term "epitope", also known as "antigenic determinant", refers to the portion of an antigen to which an antibody specifically binds to form a complex. Thus, the term "epitope" includes any molecular or protein determinant capable of specifically binding to an immunoglobulin or T-cell receptor. The binding site(s) (paratope) of the antibody molecules described herein can specifically bind / interact with a conformational or continuous epitope, which is unique to the target structure. Epitope determinants usually consist of chemically active surface group molecules, such as amino acids or sugar side chains, and usually have specific three-dimensional structural characteristics as well as specific charge characteristics. Epitope determinants can include chemically active surface group molecules, such as amino acids, sugar side chains, phosphoryl, or sulfonyl, and in some embodiments can have specific three-dimensional structural characteristics and / or specific charge characteristics. With respect to polypeptide antigens, conformational or discontinuous epitopes are characterized by the presence of two or more amino acid residues that are separate in the primary sequence, but assemble into a structure on the surface of the molecule when the polypeptide folds into the native protein / antigen (Sela, M., Science (1969) 166, 1365-1374; Laver, WG, et al. Cell (1990) 61, 553-556). The two or more separate amino acid residues that constitute the epitope can be present on separate sections of one or more polypeptide chains. These residues come together on the surface of the molecule when the polypeptide chain(s) fold into a three-dimensional structure and constitute the epitope. In contrast, a continuous or linear epitope consists of two or more separate amino acid residues that are present on a single linear segment of a polypeptide chain. As a specific example, a "context-dependent" CD3 epitope refers to the conformation of the epitope. These context-dependent epitopes located on the ε chain of CD3 are embedded within the remaining ε chain and can only adopt the correct conformation if they are locked into place by heterodimerization of this chain with either the CD3γ or CD3δ chain.In contrast, a context-independent CD3 epitope can be an N-terminal 1-27 amino acid residue polypeptide or a functional fragment thereof of CD3ε. In general, an epitope can be linear or discontinuous in nature. Thus, as used herein, the term "conformational epitope" refers to a discontinuous epitope formed by the spatial relationship between amino acids of an antigen, rather than by a contiguous sequence of amino acids of the antigen. The term "epitope" also includes antigenic determinants of haptens, known as small molecules that can act as antigens by presenting one or more immunologically recognized epitopes when bound to a larger substance, e.g., a large molecule such as a protein.

[0074] An antibody or antibody molecule / fragment is said to "specifically" bind to an antigen when it recognizes its target antigen within a complex of proteins and / or macromolecules. Typically, an antibody is capable of specifically interacting and / or binding to its target, but not necessarily binding to another epitope or antigen. Antibodies are said to "bind to the same epitope" when they compete with each other so that only one antibody can bind to an epitope at any given time, i.e., one antibody prevents the binding or modification of the other antibody from being affected.

[0075] Typically, binding that is considered specific also refers to binding that is, for example, d in terms of 10 -6 M or higher, it can be said to have high affinity. Specifically, the binding affinity is about 10 -8 From 10 -11 M(K d ) or about 10 -9 From 10 -11 M(K d ) or even higher. Thus, affinities in the picomolar range (9.9x10 -10 M to 10 -12 K of M d) are also encompassed by the present invention. Non-specific binding of the binding site can be reduced, if necessary, by altering the binding conditions without substantially affecting the specific binding.

[0076] The antibody according to the present invention can be an isolated antibody molecule. The term "isolated antibody molecule" as used herein refers to an antibody molecule that has been identified and separated and / or recovered from certain components of its natural environment. Contaminating components of the natural environment are substances that would interfere with the use 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 of the antibody, e.g., greater than 99% by weight, as determined by the Lowry method. In some embodiments, the antibody molecule is purified to a sufficient extent to obtain at least 15 residues of the N-terminus or middle of the amino acid sequence by using a rotating cup sequencer. In some embodiments, the antibody is purified to homogeneity as determined by SDS-PAGE under reducing or non-reducing conditions using Coomassie blue or preferably silver stain. An isolated antibody molecule can, in some embodiments, exist in an exogenous host cell in the absence of one or more elements (s) of the antibody's natural environment. Typically, an isolated antibody is prepared by at least one purification step.

[0077] Unless otherwise specified, the CDR sequences of the present disclosure are according to the AbM definition used in Oxford Molecular's AbM antibody modelling software. See generally, for example, Protein Sequence and Structure Analysis of Antibody Variable Domains. In:Antibody Engineering Lab Manual (Ed.:Duebel, S. and Kontermann, R., Springer-Verlag, Heidelberg). There are other standards for defining CDRs, such as the definition by Maass 2007 (Journal of Immunological Methods 324 (2007) 13-25). Another standard is the Kabat CDR definition, as described in Sequences of Proteins of immunological Interest, US Department of Health and Human Services (1991), eds. Kabat et al. Another criterion for characterizing an antigen-binding site is to refer to the hypervariable loops as described by Chothia (see, e.g., Chothia, et al. (1992); J. MoI. Biol. 227:799-817 and Tomlinson et al. (1995) EMBO J. 14:4628-4638). It is understood that the embodiments described with respect to the AbM CDR definitions can alternatively be implemented using similarly described relationships, such as with respect to the Maass, Kabat, or Chothia definitions.

[0078] The valency of an antibody generally describes the number of antigen-binding sites per molecule of any given antibody or the number of antibody-binding sites per any given antigen. Most antibody molecules and molecules belonging to the immunoglobulin classes IgG, IgA, IgD, and IgE have two antigen-binding sites per molecule. Generally, a monovalent antibody contains one antigen-binding site. Examples of monovalent antibodies include single domain antibodies, VHH domains, single Fab fragments, Fv fragments, scFv fragments, or single VH or VL domains.

[0079] The terms "Fab", "Fab region", "Fab portion", or "Fab fragment" refer to an antibody that is an IgG antibody that is an IgG antibody that is an IgG antibody. H , C H 1. V L , and C L It is understood to define a polypeptide including the C-terminus of the antibody molecule according to the invention. Fab can refer to this isolated region or to this region in the context of an antibody molecule according to the invention, as well as to a full-length immunoglobulin or an immunoglobulin fragment. Typically, the Fab region contains the entire light chain of an antibody. The Fab region can be considered to define the "arm" portion of an immunoglobulin molecule. It contains the portion that binds to an Ig epitope. The Fab region of an immunoglobulin of natural origin 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 resulting from reducing the disulfide bonds of the F(ab')2 portion. As used herein, the terms "Fab", "Fab region", "Fab portion" or "Fab fragment" can further include the hinge region that defines the C-terminus of the antibody arm (see above). This hinge region is the C-terminus of the antibody arm in a full-length immunoglobulin. H This corresponds to the hinge region found at the C-terminus of one domain, where the arms of the antibody molecule can be considered to define a Y. The term hinge region is used in the art because immunoglobulins have some flexibility in this region.

[0080] "Fv" or "Fv fragment" refers to a "single-arm" V of an immunoglobulin. L Domains and V H 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 one heavy-chain and one light-chain variable domain in tight, non-covalent association. Single-chain Fv species (scFv) contain the V domain of an immunoglobulin. H Domain and V L The scFv fragments contain domains that exist as a single polypeptide chain covalently linked to each other by flexible peptide linkers. Typically, in scFv fragments, the variable domain of the light chain and the variable domain of the heavy chain are associated in a dimeric structure similar to the fragments of two-chain Fv species. Single-chain Fv fragments can either have the variable domain of the light chain located at the N-terminus of a single polypeptide chain, followed by a linker, and the variable domain of the heavy chain located at the C-terminus of the polypeptide chain, or vice versa, with the variable domain of the heavy chain located at the N-terminus and the variable domain of the light chain at the C-terminus, with a peptide linker in between. The peptide linker can be any flexible linker known in the art, for example made from glycine or serine residues. The V-chain can be made more flexible by introducing disulfide bonds into the conserved framework regions. H Domain and V L It is also possible to additionally stabilize the domain association between the 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). Such scFv fragments are also known as disulfide-stabilized scFv fragments (ds-scFv).

[0081] The term "Fc region" or "Fc fragment" is used herein to delimit the C-terminal region of an immunoglobulin heavy chain, including native sequence Fc regions and variant Fc regions. The Fc portion is involved in the effector functions of the antibody, such as, for example, activation of the complement system and activation of immune effector cells having Fc receptors, such as NK cells. In human IgG molecules, the Fc region is generated by papain cleavage of the N-terminus to Cys226. Although the boundaries of the Fc region of an immunoglobulin heavy chain can vary, the human IgG heavy chain Fc region is usually delimited from the amino acid residue at position Cys226 or Pro230 to its carboxy terminus. The C-terminal lysine of the Fc region (residue 447 according to the EU numbering system) can be removed, for example, during production or purification of the antibody molecule, or by recombinantly engineering the nucleic acid encoding the heavy chain of the antibody molecule. Thus, the composition of intact antibodies can include antibody populations in which all K447 residues have been removed, antibody populations in which none of the K447 residues have been removed, and antibody populations in which some antibodies have K447 residues and some have not. Native sequence Fc regions suitable for use in the antibodies of the invention include mammalian, e.g., human or murine IgG1, IgG2 (IgG2A, IgG2B), IgG3, and IgG4. The Fc region contains two or three constant regions depending on the class of the antibody. In embodiments where the immunoglobulin is an IgG, the Fc region is a C4 constant region. H 2 domain and C H It has three domains.

[0082] The antibodies according to the invention can be produced using any of the known and well established expression systems and recombinant cell culture techniques, for example by expression in bacterial hosts (prokaryotic systems) or in eukaryotic systems such as yeast, fungi, insect cells, or mammalian cells. The antibody molecules of the invention can also be produced in transgenic organisms such as goats and plants. The antibodies can also be produced by chemical synthesis.

[0083] The antibody of the present invention may comprise the CDR1 sequence GVTISALNAMAMG (SEQ ID NO:115) or a sequence having one or two mutations thereto, the CDR2 sequence AVSERGNAM (SEQ ID NO:116) or a sequence having one or two mutations thereto, and the CDR3 sequence LEDRVDSFHDY (SEQ ID NO:117) or a sequence having one or two mutations thereto. The antibody of the present invention may comprise the CDR1 sequence GVTISALNAMAMG (SEQ ID NO:118) or a sequence having one or two mutations thereto, the CDR2 sequence AVSSRGNAM (SEQ ID NO:119) or a sequence having one or two mutations thereto, and the CDR3 sequence. The antibody of the present invention may comprise the CDR1 sequence GVTVSALNAMAMG (SEQ ID NO:121) or a sequence having one or two mutations therein, the CDR2 sequence AVSERGNAM (SEQ ID NO:122) or a sequence having one or two mutations therein, and the CDR3 sequence LEDRVDSFHDY (SEQ ID NO:123) or a sequence having one or two mutations therein. Such an antibody is understood to preferably comprise or consist of a camelid VHH domain, such as a single domain antibody, or an antibody naturally devoid of light chains. It is further understood that an antibody in which one or two mutations have been introduced into one, two or all three CDR sequences is still capable of specifically binding to a peptide comprised in the fusion protein of the present invention, in particular a peptide having the core structure of SEQ ID NO:3, such as any one of the peptides of SEQ ID NO:05-07. In such an antibody of the invention, E or S at amino acid position 4 of CDR2 can 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 to D, N, or H. Such an antibody of the invention can comprise the following FR1 sequence or a sequence having at least 80%, 85%, 90%, or 95% sequence identity thereto: TIFF0007623006000021.tif4128, where X1 is Q, V, E, or L, X2 is Q or P, and X3 is L or M (SEQ ID NO:124). Such an antibody of the invention may have the FR1 sequence Such an antibody of the invention may comprise the following FR2 sequence or a sequence having at least 80%, 85%, 90%, or 95% sequence identity thereto: WYRQX1PGEX2RVMVA wherein X1 is A or R and X2 is R or E (SEQ ID NO:126). 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 thereto. 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 thereto. Such an antibody of the invention may comprise the following FR3 sequence or a sequence having at least 80%, 85%, 90%, or 95% sequence identity thereto: TIFF0007623006000023.tif4128, where X1 is K or Q and X2 is T or M (SEQ ID NO:129). Such an antibody of the invention may have the FR3 sequence Such antibodies of the invention may comprise the following FR4 sequence or a sequence having at least 80%, 85%, 90%, or 95% sequence identity thereto: WGQGX1QVTVSS wherein X1 is T or I (SEQ ID NO:131).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 thereto.

[0084] The antibody of the present invention TIFF0007623006000025.tif172148TIFF0007623006000026.tif107148, or a sequence having at least 80%, 85%, 90%, or 95% sequence identity to any one of these sequences.

[0085] The antibody of the present invention may comprise the CDR1 sequence GTMSAINALN (SEQ ID NO:143) or a sequence having one or two mutations therein, the CDR2 sequence AITDNGNAH (SEQ ID NO:144) or a sequence having one or two mutations therein, and the CDR3 sequence LEEEKLGVWVDY (SEQ ID NO:145) or a sequence having one or two mutations therein. The antibody of the present invention may comprise the CDR1 sequence GTMSAINALN (SEQ ID NO:146) or a sequence having one or two mutations therein, the CDR2 sequence AITDNGNAH (SEQ ID NO:147) or a sequence having one or two mutations therein, and the CDR3 sequence LEEKLGAWVDY (SEQ ID NO:148) or a sequence having one or two mutations therein. The antibody of the present invention may comprise the CDR1 sequence GTMSAINALN (SEQ ID NO:149) or a sequence having one or two mutations therein, the CDR2 sequence AITDNGNAH (SEQ ID NO:150) or a sequence having one or two mutations therein, and the CDR3 sequence LEKEKLGVWVDY (SEQ ID NO:151) or a sequence having one or two mutations therein. Such an antibody is understood to preferably comprise or consist of a camelid VHH domain, such as a single domain antibody, or an antibody naturally devoid of light chains. It is further understood that an antibody in which one or two mutations have been introduced into one, two or all three CDR sequences is still capable of specifically binding to a peptide comprised in the fusion protein of the present invention, in particular a peptide having the core structure of SEQ ID NO:3, such as any one of the peptides of SEQ ID NO:05-07. Such an antibody of the invention may comprise the following FR1 sequence or a sequence having at least 80%, 85%, 90%, or 95% sequence identity thereto: TIFF0007623006000027.tif4128, where X1 is V or L (SEQ ID NO:152). Such antibodies of the invention may comprise the following FR2 sequence or a sequence having at least 80%, 85%, 90%, or 95% sequence identity thereto: WYRQX1PGKERKMVA wherein X1 is P or A (SEQ ID NO:153). Such antibodies of the invention may comprise the following FR3 sequence or a sequence having at least 80%, 85%, 90%, or 95% sequence identity thereto: TIFF0007623006000028.tif4128, where X1 is K or E (SEQ ID NO:154). Such antibodies of the invention may comprise the FR4 sequence WGQGTQVTVSS (SEQ ID NO:155), or a sequence having at least 80%, 85%, 90%, or 95% sequence identity thereto.

[0086] The antibody of the present invention TIFF0007623006000029.tif63148, or a sequence having at least 80%, 85%, 90%, or 95% sequence identity to any one of these sequences.

[0087] The antibody of the present invention can be conjugated to a detectable label. In general, such a "detectable label" can be any suitable chemical substance or enzyme that generates a detectable compound or signal directly or indirectly in a chemical, physical, optical, or enzymatic reaction. For example, a fluorescent or radioactive label can be conjugated to the antibody to generate fluorescence or X-ray as a detectable signal. Alkaline phosphatase, horseradish peroxidase, and β-galactosidase are examples of enzyme labels (and optical labels at the same time) that catalyze the formation of a colored reaction product. In a preferred embodiment, a detectable label refers to a detectable entity that can be used to detect a target of interest in microscopy, immunohistochemistry, or flow cytometry. It is preferred that the label does not adversely affect the characteristics of the antibody to which it is conjugated. Examples of labels include fluorescent labels such as phycoerythrin, allophycocyanin (APC), brilliant violet 421, Alexa Fluor 488, coumarin, or rhodamine, to name just a few. There are many kinds of detectable labels, including fluorescent, chromophore, isotopic, or metal labels, but fluorescent labels are preferred. 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 to a detectable label and detecting the signal of the detectable label. When referring to a fluorescent label, this refers to the detection of the light emitted upon excitation of the fluorescent label. Although not exhaustive, examples of suitable fluorescent labels include "green" emitters (Atto488, Alexa488, Cy2, etc.), "orange" emitters (Atto542, alexa555, Cy3, etc.), "near-infrared red" emitters (Alexa633, Atto 647N, Cy5, etc.), infrared emitters (Atto700, LiCor IRDye700, LiCor IRDye800, etc.), and ultraviolet absorbing fluorescent dyes (Atto390 or Alexa405). The fluorescent label may also be a fluorescent protein, such as GFP, eGFP, YFP, RFP, CFP, BFP, mCherry, or near-infrared fluorescent protein.Examples of suitable chromophore labels, although not exhaustive, include alkaline phosphatase or peroxidase exposed to TMB (3,3',5,5' tetramethylbenzidine), DAB (3,3',4,4' diaminobenzidine), and 4CN (4-chloro-1-naphthol). Also exposed to ABTS (2,2'-azino-di[3-ethyl-benzthiazoline]sulfonic acid), OPD (o-phenylenediamine), and BCIP / NBT (5-bromo-4-chloro-3-indolyl-phosphate / nitroblue tetrazolium). Examples of isotopic labels, although not exhaustive, include 13C, 15N, 19F, 27Al, 11B, 127I, or various lanthanide isotopes. Examples of metal labels, although not exhaustive, include Au, Pd, Pb, Pt, Ag, Hg, and Os. The label can be a direct label, i.e., a label that is directly detectable, or it can be an indirect label, i.e., a label that is an affinity tag (or epitope tag) to which another specific binding partner can specifically bind that is conjugated to another detectable label, such as, for example, a fluorescent or chromophore label. Examples of suitable epitope tags include, but are not limited to, a FLAG tag (sequence: DYKDDDDK, SEQ ID NO:160), a Strep tag (sequence: WSHPQFEK, SEQ ID NO:178), a Myc tag (sequence: EQKLISEEDL, SEQ ID NO:161), a HA tag (sequence: YPYDVPDYA, SEQ ID NO:162), a VSV-G tag (sequence: YTDIEMNRLGK, SEQ ID NO:163), a HSV tag (sequence: QPELAPEDPED, SEQ ID NO:164), a V5 tag (sequence: GKPIPNPLLGLDST, SEQ ID NO:165), a SPOT tag (sequence: PDRVRAVSHWSS, SEQ ID NO:166), a BC2 tag (sequence: PDRKAAVSHWQQ, SEQ ID NO:167), and an EPEA tag (sequence: EPEA, SEQ ID NO:168). The antigen can also be a protein, for example, glutathione-S-transferase (GST), maltose binding protein (MBP), chitin binding protein (CBP) or thioredoxin.The detectable label can also be a nucleic acid, such as an oligonucleotide with a recognition sequence. Such a recognition sequence can be a random sequence. This random sequence can be a barcode sequence incorporated into the nucleic acid molecule and can be used to identify the target molecule conjugated to said nucleic acid. "The antibody of the present invention can be conjugated to a detectable label" also means that the antibody itself is a detectable label. This means 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 a Camelidae VHH domain. Such a specific binding partner can also be conjugated to a detectable label, such as a fluorescent label.

[0088] The antibody of the present invention may be conjugated to a solid support. The term "solid support" in the context of the present invention refers to any type of carrier material that can be used for immobilization of affinity ligands, such as antibodies or portions thereof, and can refer to particulate materials (e.g., beads or granules commonly used in extraction columns) or sheet-like materials (e.g., membranes or filters, glass or plastic slides, microtiter assay plates, dipsticks, capillary packing materials, or the like), planar, pleated, or hollow fiber or tubular. Although not exhaustive, well-known suitable substrates are silica (porous amorphous silica), agarose or polyacrylamide supports, or macroporous polymers. Examples include dextran, collagen, polystyrene, polypropylene, polyvinyl chloride, polyacrylamide, methacrylates, cellulose, calcium alginate, microporous glass, aluminum, titanium and porous ceramics, synthetic polymers and synthetic copolymers, latex, silica, agarose, metals, glass, and carbon. Alternatively, the solid surface may comprise part of a mass-dependent sensor, such as a surface plasmon resonance detector. Advantageously, an array is provided that comprises a plurality of individual affinity ligands, such as antibodies or antibody fragments, capable of specifically binding to the epitope tags of the invention, which affinity ligands are bound or immobilized on a solid surface. The array can be used to capture tagged polypeptides contained in a solution once the solution comes into contact with the immobilized affinity ligands, such as antibodies or antibody fragments. The solid support may also be magnetic or polymeric beads, or a chromatographic stationary phase.

[0089] The antibody of the present invention can form a complex with an epitope to which the antibody specifically binds. Such an epitope may be a peptide contained in the fusion protein of the present invention. The antibody of the present invention can thus form a complex with the fusion protein of the present invention. The present invention therefore encompasses a fusion protein comprising a peptide to which the antibody of the present invention specifically binds.

[0090] The present invention also relates to a conjugate comprising (a) a fusion protein and (b) an antibody, wherein the fusion protein is a fusion protein of the invention and / or the antibody is an antibody of the invention.

[0091] The present invention also relates to a nucleic acid molecule comprising a sequence encoding a fusion protein of the present invention as described herein or a sequence encoding an antibody of the present invention as described herein. The nucleic acid molecule can be a DNA or RNA molecule. The nucleic acid molecule of the present invention can be part of a vector or any other type of cloning or expression vehicle, such as a plasmid, a phagemid, a phage, a baculovirus, a cosmid, or an artificial chromosome. The nucleic acid molecule can allow the expression of a fusion protein or an antibody. The nucleic acid molecule can include sequence elements that contain information about translation and / or translational regulation, and such sequences can be "operably linked" to a nucleotide sequence that codes for a protein. An operable link is a linkage that connects a regulatory sequence element and a sequence to be expressed in a manner that allows gene expression. Although the precise nature of the regulatory regions necessary for gene expression may vary between species, generally these regions include a promoter, which in prokaryotes contains both promoters, i.e., DNA elements that direct the initiation of translation and DNA elements that signal the initiation of translation when transcribed into RNA. Such promoter regions usually include 5' untranslated sequences involved in translation initiation and translation, such as, for example, -35 / -10 boxes and Shine-Dalgarno elements in prokaryotes, and TATA boxes, CAAT sequences, and 5' capping elements in eukaryotes. These regions may also include enhancer or repressor elements, as well as translation signal and leader sequences that direct the native protein to a specific compartment of the host cell.

[0092] Such vehicles described herein may contain the regulatory sequences described herein and the nucleic acid sequences encoding the peptides or proteins described herein, as well as replication and control sequences derived from a species compatible with the host cell used for expression, and a selection marker that confers a selectable phenotype on the transformed or transfected cells. Many suitable cloning vectors are known in the art and are commercially available. Thus, the present invention also relates to vectors that contain the nucleic acid molecules of the present invention.

[0093] Cloning or expression of the nucleic acid molecule or vector of the present invention can be carried out at least in part in vivo using a host cell transformed with the nucleic acid or vector, or a host cell into which the nucleic acid molecule or vector has been introduced by other means, including transduction or gene transfer. Introduction of DNA can be carried out using standard techniques. Thus, the present disclosure is also directed to a host cell containing the nucleic acid molecule or vector as disclosed herein.

[0094] The peptides of the present invention are useful for several applications. These cellular applications can have in common that the peptides are used as epitope tags. Non-limiting examples of applications for which the peptides are useful include, but are not limited to, detection, immobilization, isolation, or purification of the fusion proteins of the present invention. Similarly, the antibodies of the present invention can be used for all applications involving specific binding of epitope tags. The antibodies of the present invention can be used, for example, for detection, immobilization, isolation, or purification of the fusion proteins of the present invention.

[0095] The present invention also relates to a method for detecting the fusion protein of the present invention. The detection can be by optical detection, isotopic detection, or electron microscopy. The method preferably comprises contacting the fusion protein with the antibody of the present invention under conditions that allow the formation of a complex between the peptide contained in the fusion protein and the antibody. The antibody of the present invention preferably has a detectable label as described herein. When the detectable label attached to the antibody is a fluorescent label, a chromophore label, an isotopic label, or a metal label, it is understood that the antibody preferably has a defined number of labels attached to the antibody. The method can comprise detecting the detectable label. The method can comprise expressing the fusion protein of the present invention before contacting the fusion protein with the antibody.

[0096] The term "detection" as used herein includes both direct detection of a target (i.e., the target is detected by a signal derived from the target) and indirect detection of a target (i.e., the target is detected by a signal not derived directly from the target, e.g., by a signal derived from another molecule attached to the target). The term "detection" as used herein further includes both qualitative and quantitative detection. The term "detection" can refer to determining the presence, subcellular localization, or amount of a given molecule or structure, such as a fusion protein of the invention. The fusion protein to be detected, located, and / or quantified can be detected in an intracellular location of the host cell, e.g., in the cell nucleus, in the cell membrane, or in another cell compartment. The fusion protein to be detected and / or quantified can also be detected in a solution containing the tagged polypeptide or tagged protein, e.g., a cell lysate obtained from the host cell or a tissue lysate containing the host cell.

[0097] The term "light" as used herein preferably refers to visible light, but is generally not limited thereto. The term can also refer to infrared, ultraviolet, and other regions of the electromagnetic spectrum.

[0098] As used herein, "isotope detection" refers to the detection of a molecule in which one or more atoms have been replaced with another isotope, generally of detectable variety (i.e., "labeled"). The isotopic label can be detected by multiple means, such as its 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).

[0099] Electron microscopy refers to the method of detection using electron microscopes. Types of electron microscopes include transmission electron microscope (TEM), scanning electron microscope (SEM), reflection electron microscope (REM), scanning transmission electron microscope (STEM), and correlative light and electron microscopy (CLEM).

[0100] In a first step of the detection method, an antibody that specifically binds to a peptide contained in the fusion protein of the invention can be contacted with a sample containing the fusion protein, which can be a solution containing host cells, tissues, a cell lysate of host cells, or any other sample containing the fusion protein, such as the supernatant obtained after centrifugation of a liquid containing host cells, where the host cells can secrete the polypeptide of interest into the liquid or into another sample, such as a body fluid.

[0101] This contacting step is preferably carried out under conditions that allow specific interaction between the antibody and the peptide to which it specifically binds. Such conditions are well known to those skilled in the art. The step of contacting the antibody with its antigen is typically followed by a washing step, and those skilled in the art know when and how to apply the washing step. Upon contact with the sample, the antibody becomes specifically interacting with the fusion protein. This interaction can be detected, monitored and quantified by measuring or observing the reporter signal obtained from the detectable label. For example, if the detectable label is a fluorescent label, fluorescence can be measured and observed upon excitation.

[0102] If the detectable label is an affinity tag (or epitope tag), another specific binding partner conjugated to another detectable label, such as a fluorescent or chromophore label, can specifically bind to this label. In such a case, the 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 conjugated to the antibody of the present invention. The specific binding partner can be labeled with a further detectable label, preferably distinguishable from the first detectable label, such as a fluorescent or chromophore label. The specific binding partner can, however, also be a structure that can be recognized by another specific (labeled) binding reagent. 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 with a detectable label that is preferably distinguishable from the first detectable label, such as a fluorescent label. The method for detecting the fusion protein of the present invention can therefore comprise a step of contacting the fusion protein and the antibody with a specific binding partner for the detectable label contained in the antibody. If a second detectable label is present, the method can include detecting the first and / or second detectable label.

[0103] 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 a fusion protein can comprise, in a first step, contacting the antibody of the present invention with a sample that contains or is suspected to contain the fusion protein of the present invention. In a second step, a (secondary) specific binding partner can be contacted with the sample that contains the fusion protein bound to the antibody of the present invention. If the (secondary) specific binding partner is not conjugated to a detectable label or if the detectable label is an affinity tag, the method can further comprise contacting a further specific binding partner, such as a further antibody that specifically binds to the (secondary) specific binding partner or its detectable label. This further specific binding partner can comprise 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 can be detected or determined by measuring or observing a reporter signal resulting from a detectable label contained in the (secondary) specific binding partner or the further specific binding partner.

[0104] The advantage of the two-step detection method using two types of binding partners is that the tag-specific interaction is decoupled from the actual detection step. This method allows the antibody of the present invention to remain unchanged since it does not require the antibody to contain an additional detectable moiety. In some cases, the detectable label affects the interaction of the antibody of the present invention with the peptide contained in the fusion protein of the present invention, so the specificity or affinity of this method can be improved in some cases when compared to an antibody containing an additional detectable label. Thus, in some cases, the reliability and effectiveness of the detection method can be improved. Furthermore, by using the antibody of the present invention simply as a capture antibody, rather than as a capture and detection antibody, it is possible to separate the capture and detection steps by only determining the presence and amount of the fusion protein. Thus, the first step using the antibody of the present invention is followed by an isolation or enrichment step to obtain the captured fusion protein of interest. A detection step can then be performed on this isolated and / or enriched fusion protein, resulting in an improved reliability of the quantification obtained and an easier detection step operation.

[0105] Suitable biophysical and biomolecular detection methods for qualitatively detecting the interaction of the epitope tag with the antibody include any suitable method known in the art. Such methods include, but are not limited to, methods applied to qualitative or quantitative assays, such as Enzyme-linked Immunosorbent Assay (ELISA), ELISPOT assay, Western blot, or immunoassay. Such methods include, for example, optical, radioactive, or chromatographic methods, preferably when using any of the above-mentioned labels, markers, or linkers, and more preferably, fluorescent detection methods, radioactive detection methods, Coomassie Blue staining, silver staining or other protein staining methods, electron microscopy, staining of tissue sections by immunohistochemistry or by direct or indirect immunofluorescence, etc. Such methods can be applied either with the antibody or with the use of additional tools, such as, for example, the use of secondary binding partners that specifically bind to a portion of the fusion protein, antibody, or complex.

[0106] In some embodiments, the subcellular localization of a tagged polypeptide or protein of interest can also be determined, e.g., individual subcellular structures, such as intermediate filament networks or essential parts of the replication machinery, can be visualized and monitored.

[0107] Detection of fusion proteins can also be performed using the antibodies of the invention that are intracellular antibodies. "Intracellular antibodies" refers herein to antibodies that are located inside a cell to bind to intracellular proteins. This typically requires expression of the antibody in the target cell, since there is no reliable mechanism to get antibodies from the extracellular environment into living cells. After expression, the antibody can remain in the cytoplasm, can have a nuclear localization signal, or can undergo co-translational translocation across the membrane into the lumen of the endoplasmic reticulum, provided that it is retained in the endoplasmic reticulum compartment through the KDEL sequence. The detectable label conjugated to the intracellular antibody can be a proteinaceous label that can be expressed as a fusion protein with the intracellular antibody. Ideally, such a label is optically detectable, such as by fluorescence. The detectable label can thus be a fluorescent protein.

[0108] The present invention also relates to a method for isolating the fusion protein of the present invention, which comprises a step of 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 allowing the binding of the fusion protein to the antibody. This contacting step, also called a capture step, can be performed by contacting a sample, e.g. a solution, containing the fusion protein with the antibody.

[0109] The sample contacted with the tag-specific antibody can be any kind of sample containing the fusion protein of the invention and can be treated to isolate the polypeptide. Preferably, the sample is a solution, such as a host cell lysate or a body fluid, containing the fusion protein of the invention, or a supernatant, such as a supernatant obtainable by centrifugation of a liquid containing host cells capable of containing or expressing the fusion protein of the invention and capable of secreting or transporting the fusion protein of the invention into the liquid.

[0110] The antibody used for isolation and / or purification in the method of the present invention can be used in solution or can be immobilized. To immobilize the antibody, the antibody can be bound to a sample carrier, solid support, or matrix. This immobilization step can be performed before or after the antibody is bound 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 of immobilization can be used without compromising the binding properties.

[0111] If the antibody of the invention is not immobilized to a solid support, the method can include the further step of isolating the complex, e.g., by using a specific binding partner for the complex, such as, for example, a secondary antibody that is specific for the complex, or specific for the antibody, or specific for a detectable label, such as an affinity tag, conjugated to the antibody. The secondary binding partner can be in solution or can be immobilized or immobilized to a solid support.

[0112] In a further optional step following 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.

[0113] Optionally, in a further step, the fusion protein can be eluted to obtain 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 competitive elution using an epitope peptide as described herein in isolated form. The isolated epitope peptide will then compete with the fusion protein binding to the immobilized tag-specific antibody. If the isolated peptide is added in excess concentration, the reaction balance of binding will shift to 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 different, it is preferred that the epitope peptide used for elution has a higher binding affinity with the antibody than the epitope peptide contained in the fusion protein. Additional steps can optionally be added to further purify the liberated polypeptide, such as process steps well known to those of skill in the art.

[0114] The fusion protein can also remain immobilized on a solid support, such as (magnetic) beads, and can be further processed in downstream applications, such as mass spectrometry, without an elution step.

[0115] The fusion protein may include a linker with a cleavage site that can be cleaved by suitable means, such as a protease, to remove the peptide. This allows the polypeptide of the fusion protein to be released from the immobilized antibody and obtain the polypeptide in its native form. For this embodiment, the nucleic acid sequence encoding the fusion protein should not only include a sequence encoding an epitope tag, but also a sequence encoding a linker with a cleavage site, such as a cleavage site recognized by a protease. The release step by enzymatic cleavage may be substituted for or followed by an elution step.

[0116] When the fusion protein of the invention comprises an antibody portion, the invention also contemplates a method for isolating the target of the antibody portion of the invention. In principle, this method can be carried out as described above for the isolation of the fusion protein. This method can include the additional step of contacting the fusion protein with a specific target of the antibody portion contained in the fusion protein. This contacting step can be performed before or after contacting the fusion protein with an antibody that binds to the peptide tag contained in the fusion protein, the latter being preferred. In a preferred method, an antibody specific for the peptide tag is first immobilized on a solid support, the fusion protein is then immobilized via binding with an antibody specific for the peptide tag, and the target of the antibody portion of the fusion protein is then 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 the cell, such as CD62L. The antibody portion of the fusion protein can be specific for a structure on the cell, such as CD62L. The antibody portion of the fusion protein can be a single domain antibody.

[0117] The present invention also contemplates that detection and isolation of the fusion proteins of the present invention can be combined. Thus, the present invention contemplates methods of detection and isolation of the fusion proteins of the present invention, including detection methods of the present invention and isolation and / or purification methods of the present invention.

[0118] Thus, a combination of both methods can be performed by using one antibody against the same fusion protein, conjugated to a detectable label for detection, and another antibody conjugated to a solid support for isolation. Both antibodies can be any of the antibodies of the present invention. This combination can have the advantage that only one tagged fusion protein needs to be produced, and detection and isolation can be performed in the same transformed construct / cell. Sometimes it may be desirable for both antibodies to have the same sequence or at least the same antigen binding site. For example, both antibodies may comprise the CDR1-3 sequence set forth in SEQ ID NO:115-117, or may comprise the sequence set forth 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 both antibodies to have different sequences or different antigen binding sites. The use of different antibodies can have the advantage that tight binders, e.g., antibodies with high affinity (e.g., from about 1 pM to about 1 nM) for the peptides contained in the fusion protein, can be used for detection, while moderate binders, i.e., antibodies with moderate 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 can comprise the CDR1-3 sequences set forth in SEQ ID NOs: 115-117 or the sequence set forth 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 can comprise the CDR1-3 sequences set forth in SEQ ID NOs: 118-120 or the sequence set forth 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 included in the fusion protein can be a peptide having the core sequence of SEQ ID NO:3 or 4 as described herein.

[0119] The combination of both methods can also be performed by using the same antibody and two different detection and purification peptides. This has the advantage that only one antibody needs to be generated, which can be conjugated to a detectable label or solid support depending on the application. When using two different peptides, these peptides preferably have different binding affinities for the antibody. The peptide used for detection can have a high affinity, for example about 1 pM to 1 nM, while the peptide used for isolation can have a medium binding affinity, for example about 1 nM to 500 nM. By using peptides with different binding affinities, it can have the advantage that a (higher) affinity peptide can be used for elution of the fusion protein, when the medium affinity peptide is included in the fusion protein for isolation. Thus, one peptide can be a peptide with a core sequence of SEQ ID NO: 3 or 4 as described herein, while the other peptide can be a peptide with a core sequence of SEQ ID NO: 31 or 32 as described herein. The antibody can be any antibody of the invention. For example, the antibody can comprise a CDR1-3 sequence set forth in SEQ ID NO:115-117, or can comprise a sequence set forth in SEQ ID NO:133, or a sequence having at least 80%, 85%, 90%, or 95% sequence identity to SEQ ID NO:133.

[0120] The combination of both methods can also be performed by using two peptides and two antibodies. The fusion protein of interest can contain a peptide with high affinity for a given antibody. For example, the peptide can be a peptide with a core sequence of SEQ ID NO:3 or 4 as described herein. The antibody for detection can contain a CDR1-3 sequence as described in SEQ ID NO:115-117, or a sequence as described in SEQ ID NO:133 or a sequence with at least 80%, 85%, 90%, or 95% sequence identity with SEQ ID NO:133. The antibody for isolation / purification can contain a CDR1-3 sequence as described in SEQ ID NO:118-120, or a sequence as described in SEQ ID NO:134 or a sequence with at least 80%, 85%, 90%, or 95% sequence identity with SEQ ID NO:134. The peptide used for elution can be one that has a higher affinity for the eluting antibody than a peptide contained in the fusion protein, such as another peptide described herein.

[0121] The present invention further 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 as described herein.

[0122] The present invention also relates to a kit. The kit comprises the components necessary to carry out the method of the present invention. The kit for the detection or purification of a fusion protein may comprise a nucleic acid or a nucleic acid expression construct encoding a peptide / epitope tag as described herein that may be present in the fusion protein. The nucleic acid may comprise sites such as cleavage sites or recombination sites that genetically facilitate fusing the polypeptide with the peptide / epitope tag. The nucleic acid sequence encoding the peptide / epitope tag may be operably linked to a sequence element that contains information regarding translation and / or translational regulation.

[0123] The kits can also include an antibody of the invention, which can optionally be conjugated to a detectable label, preferably an optically detectable label or affinity tag, as described herein. Alternatively, or in addition, the kits can include a detectable moiety that can be conjugated to an antibody of the invention.

[0124] The kits may also contain buffers and reagents necessary for the isolation / purification and / or detection methods of the invention.

[0125] The kits can also include buffers and reagents necessary for introducing the nucleic acids or nucleic acid expression constructs included in the kit into host cells.

[0126] The kit may also comprise at least one (secondary) specific binding partner as described herein or a (further) specific binding partner which specifically binds to this (secondary) specific binding partner as described herein.

[0127] The kit can also include a solid support comprising an antibody of the invention immobilized or attached to the solid support. The kit can also include an isolated peptide as described herein suitable for competitive elution of a fusion protein bound to an antibody of the invention, or other means for elution of the fusion protein, such as a protease.

[0128] Unless otherwise specified, the term "at least" preceding a series of elements is understood to refer to each element in the series. Those skilled in the art will recognize, or be able to ascertain using only routine experimentation, numerous methods that are equivalent to the specific embodiments of the invention described herein. It is intended that the present invention encompasses such equivalent methods.

[0129] The term "and / or" as used herein wherever applicable means "and", "or" and "all or any other combination of the elements to which said term is associated."

[0130] The terms "about" or "approximately" as used herein mean within 20%, preferably within 10%, and more preferably within 5% of a given numerical value or range, but also including explicit numbers, e.g., about 20 includes 20.

[0131] Throughout this specification and the claims which follow, unless the content requires otherwise, it will be understood that the term "comprise," as well as variations such as "comprises" and "comprising," imply the inclusion of a stated integer or step, or group of integers or steps, but not the exclusion of any other integers or steps, or group of integers or steps. As used herein, the term "comprising" can be interchanged with the terms "containing" or "including," or, as sometimes used herein, with the term "having."

[0132] As used herein, "consisting of" excludes any element, step, or ingredient not specified in the claimed element. As used herein, "consisting essentially of" does not exclude materials or steps that do not materially affect the basic novel characteristics of the claim.

[0133] In any instance herein, any of the terms "comprising," "consisting essentially of" and "consisting of" may be replaced with either of the other two terms.

[0134] It is to be understood that this invention is not limited to the particular methodology, procedures, materials, reagents, and substances described herein as such may vary. The terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the scope of the present invention, which is defined solely by the claims.

[0135] All publications cited throughout the text of this specification (including all patents, patent applications, scientific publications, manufacturer's specifications, instruction manuals, etc.) are incorporated herein by reference in their entirety. Nothing herein is to be construed as an admission that the invention is not entitled to antedate its disclosure by virtue of prior invention. In the event that material incorporated by reference conflicts or is inconsistent with this specification, the present specification will take precedence over any such material. EXAMPLES

[0136] Example 1: System In search of a novel epitope tag / binder system, we identified a novel class of epitope tag / binder systems that form stable α-helices in solution, collectively referred to as “ALFAs.” ST We designed peptides with SEQ ID NOs:05-07, called "tags". These peptides have no known counterparts in any eukaryotic model system, are nearly neutral at physiological pH, and do not contain any primary amines that could be targets for common fixatives. The core ALFA tag sequence (Figure 3A, amino acids written in blue) comprises an artificial peptide that has been reported to form stable alpha-helices in solution (Petukhov,Me t al. Design of stable alpha-helices using global sequence optimization. J.Pept.Sci.15,359-365(2009)). The peptides were selected based on the following features: the sequences i) have no known counterparts in any eukaryotic model system, ii) are neutral at physiological pH, and iii) do not contain any primary amines that could be targets for common amine-reactive fixatives and crosslinkers.

[0137] ALFA STA high affinity nanobody against NbALFA was produced in alpacas and selected by a novel nanobody selection method using antigen-specific enriched B cells, the "cell line". The most preferred nanobody (NbALFA) was selected to generate a selective affinity resin. ST The ALFA Selector 1G5 (SEQ ID NO:133) was site-specifically coupled via a flexible linker to an agarose-based resin that is characterized by low non-specific protein binding. ST Resin is made from Escherichia coli extract and ALFA ST Surprisingly, however, ALFA was highly efficient at pulling down the -shGFP2 fusion protein. ST All efforts to elute the bound protein under native conditions by competing with the peptide (Ac-PSRLEEELRRRLTEP-amide as set forth in SEQ ID NO:179) failed. Even after 10 column volumes of 90 min competition with 200 μM peptide, more than 95% of the ALFA ST The -shGFP2 protein remained on the resin (Fig. 1B, center panel, gray line). ST We speculated that the off-rate of the fusion protein was too low to allow efficient competition by the peptide. ST on the other hand, ALFA binds tightly enough to allow efficient pull-down of the target protein. ST Peptides can be eluted from nanobodies using ALFA ST Systematic and rational mutagenesis was performed to identify the mutants. The peptides analyzed are shown in Table 2.

[0138] (Table 2) TIFF0007623006000030.tif160138TIFF0007623006000031.tif245139TIFF0007623006000032.tif245140n.d.: Undecided Position: N: N-terminus, M: between the two folded domains, C: C-terminus

[0139] Generally accepted estimated on-rate for sdAbs is 2*10 5 As a rough estimate based on (1 / (M*sec)), t less than 2 minutes 1 / 2 K exceeds approximately 30 nM d t of 2 to 30 minutes 1 / 2 has a K of approximately 2–30 nM d t of 20 to 100 minutes 1 / 2 The K is about 0.6 to 3 nM. d equivalent to t over 100 min 1 / 2 The K is approximately 1 nM or less. d is equivalent to.

[0140] The present inventors have identified an ALFA mutant (ALFA PE (Peptide Elutable) (SEQ ID NO:33) was found to meet these criteria. PE The fusion protein efficiently binds to the ALFA Selector ST The binding was resistant to stringent washing steps (e.g., up to 3M NaCl, 1M MgSO4, 4M urea, 1% TX-100, and even 100mM DTT, Fig. 1C). However, binding to 200μM ALFA ST By competing with the peptide (SEQ ID NO:179), efficient elution under native conditions could be achieved within 15-20 min at room temperature (Figure 1B). Interestingly, both ALFA tag variants (ALFA ST and ALFA PE ) is NbALFA ST It can be used at either end of the target protein and even between two protein domains with only minor effects on binding to the target protein (Figure 1B).

[0141] Example 2: NbALFA for pull-down of ALFA-tagged target proteins ST Application of ALFA Selector STTo determine the specificity of ALFA, pull-down experiments from complex lysates were performed under physiological conditions. For this purpose, ALFA was previously expressed in E. coli and purified by nickel chelate chromatography followed by size exclusion chromatography. ST or ALFA PE Three micromolar amounts of shGFP2 tagged at the N-terminus with either ALFA-Selector or ALFA-Selector were spiked into E. coli or HeLa lysates prepared in PBS (Frey, S. et al. Surface Properties Determining Passage Rates of Proteins through Nuclear Pores. Cell 174, 202-217.e9(2018)). The purified input proteins are shown in Figure 2A. Both fusion proteins were efficiently transduced into the ALFA-Selector from all lysates. ST As expected from the previous experiment (Figure 1), ALFA PE GFP tagged with 200 μM ALFA ST Under native conditions using peptides, they could be efficiently eluted within 20 minutes, whereas ALFA ST Tagged target proteins required harsher (denaturing) conditions for efficient elution. What was striking was that pull-down from both lysates was highly specific. Even after elution with SDS buffer, the number and intensity of detectable impurities originating from lysate proteins was very low. ALFA PE ALFA-GFP fusion protein ST When eluting with peptide, essentially all detectable bands were the same in the eluate fractions obtained from both lysates. These bands could therefore be due to artifacts produced by the target protein itself (e.g. mature bands or DTT-resistant dimers). Interestingly, especially after peptide elution, the eluate fractions contained significantly less contaminating proteins than the substrate used as input material (which had been purified using two successive chromatographic steps; Figure 2A).

[0142] Example 3: ALFA PE Tagged target protein and ALFA Selector ST Co-immunoprecipitation with and To confirm whether the ALFA system can be applied to more sensitive co-immunoprecipitation experiments, we attempted to pull down the two-component E. coli inner membrane protein complex YfgM-PpiD (Gotzke, H. et al. YfgM is an ancillary subunit of the SecYEG translocon in Escherichia coli. J Biol Chem 289, 19089-19097(2014)) under native conditions (Fig. 2D). For this purpose, we used either wild-type YfgM or YfgM-ALFA. PE Either the YfgM variant or the YfgM variant was expressed in the yfgMΔ species. To ensure near-physiological expression levels, both YfgM variants were expressed from low-copy plasmids under the control of the endogenous promoter. As input, YfgM-ALFA, prepared in the presence of the mild non-ionic detergent DDM, was used. PE When using total lysate containing ALFA Selector ST was able to pull down the YfgM-PpiD complex specifically and in a detergent-resistant manner. PE The tag was shown to be compatible with the formation of this unstable membrane complex. Importantly, the native non-denatured membrane protein complex was denatured by the addition of 200 μM ALFA. ST Peptides were used to perform ALFA Selector assays under physiological conditions within 20 minutes. ST YfgM and its interacting partner PpiD were identified as ALFA-binding domains present on the (periplasmic) C-terminus of YfgM, as this complex could not be purified from control lysates expressing untagged YfgM. PE Tags via ALFA Selector ST Therefore, ALFA PE The tag is ALFA Selector STTogether with the resin it can be used for the purification of proteins from various sources but is also suitable for the pull-down of challenging (membrane) protein complexes under native conditions.

[0143] Example 4: ALFA by direct immunofluorescence ST Detection of tagged proteins First, fluorescently labeled NbALFA ST We tested whether the method could be applied to the immunodetection of ALFA-tagged proteins in PFA-fixed samples. Indeed, NbALFA conjugated with two fluorophores (FluoTag-X2 anti-ALFA AbberiorStar635P) ST (SEQ ID NO:133) was used to detect specific staining patterns of ALFA in the target protein. ST Tag or ALFA PE Regardless of the location of the tag, the target protein was successfully obtained in mammalian cells. More specifically, the target protein was C-terminally tagged with ALFA (Tom70-EGFP-ALFA ST , Fig. 3C; Tom70-EGFP-ALFA PE , Fig. 3C ), and the N-terminally placed ALFA-tagged target protein (ALFA ST -FLAG-vimentin, Fig. 3D and Fig. 4A; ALFA PE -FLAG-vimentin, Figure 4A), or an ALFA-tagged target protein placed between the folding and transmembrane domains (EGFP-ALFA ST -TM, FIG. 4B) was successfully tested.

[0144] Example 5: ALFA ST Tagged proteins exhibit normal folding, targeting, and multimerization behavior Importantly, all tested target proteins showed their characteristic localization (Tom70-EGFP-ALFA: mitochondrial outer membrane, ALFA-vimentin: characteristic filamentous structures, EGFP-ALFA ST-TM: plasma membrane), demonstrating that the ALFA tag did not interfere with normal folding or proper targeting of the tagged protein. ST -Vimentin and ALFA PE -Proper incorporation of vimentin into characteristic intermediate filament structures (Fig. 3D and Fig. 4A) suggests that the ALFA tag does not interfere with proper filament assembly.

[0145] ALFA ST To further characterize whether the tag affects the subcellular localization of the fusion protein of interest, we introduced N-terminal ALFA into mammalian cells. ST Tag or C-terminal ALFA ST The localization of the cytosolic EGFP tagged with ALFA was statistically analyzed (Figure 5). ST The nucleoplasmic distribution of ALFA-tagged EGFP was indistinguishable from that of untagged EGFP. ST No indication of any atypical association of the fusion protein with cellular compartments (e.g., membranes or organelles) was observed. ST Gel filtration of the tagged EGFP variant confirmed that it was in a monomeric state and ALFA ST This suggests that the tag does not induce multimerization. ST It is concluded that the tag does not generally impair the behavior of the target protein, but it is noted that (as with any other tag) the specific effect on a given target protein must be analyzed on a protein-by-protein basis.

[0146] Example 6: ALFA tags are compatible with common immobilization conditions Immunofluorescence (IF) applications often require optimization of fixation conditions, which can be complicated, especially when proteins requiring different fixation conditions need to be localized within the same sample. In addition, prefabricated epitope tags often contain lysines, potentially making the tags susceptible to modification by amine-reactive fixatives (Table 1). In contrast, the ALFA tag does not contain lysines. Consistent with these considerations, ALFA ST Tags and ALFA PE The tags could be detected after standard fixation with 4% paraformaldehyde (PFA) or precipitative fixation with 100% methanol, and were also resistant to fixation with 2% glutaraldehyde (Figure 3D), thus demonstrating that both ALFA tags are compatible with most standard fixation methods and may also prove useful in electron microscopy applications, where glutaraldehyde is preferred for its ability to preserve structure at the nanoscale.

[0147] Example 7: ALFA ST In vivo detection of tagged proteins Next, the inventors ST We wanted to know whether nanobodies could also be used as intracellular antibodies. Nanobodies expressed in situ in the cytoplasm (or other compartments) of target cells are often utilized 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 within the reducing environment of the cytoplasm of a eukaryotic host cell. To test our nanobodies under such conditions, we used the ALFAST Tagged target protein (ALFA ST -Vimentin or Tom70-EGFP-ALFA ST ) and mScarlet-I fused NbALFA ST (Bindels, DS et al. mScarlet: a bright monomeric red fluorescent protein for cellular imaging. Nat Methods 11, 121-122 (2016)). In fact, in cells co-transfected with both constructs, the mScarlet-I signal was higher than that of the corresponding ALFA. ST It tightly colocalized with the tagged target protein (Figure 3E).

[0148] Example 8: Western Blotting Fluorescently labeled NbALFA with ALFA tag in Western blot analysis ST To test whether it can be detected using ALFA ST Tagged vimentin (Figure 6A and Figure 7A) or ALFA PE Lysates from COS-7 cells transfected with tagged vimentin (Figure 6A) were analyzed. Lysates from cells transfected with an irrelevant plasmid served as a control. After SDS-PAGE and Western blotting, IRDye800CW-labeled NbALFA ST Using (FluoTag-X2 anti-ALFA IRDye800CW), we were able to specifically detect ALFA-tagged vimentin. Only a limited number of inconspicuous non-specific bands were detectable in control lysates lacking ALFA-tagged protein (Figure 7A).

[0149] NbALFA ST To directly compare the performance of the ELISA kit with commonly used monoclonal tools that recognize epitope tags, we used multiple epitope tags (HA, myc, FLAG, and ALFA). ST, Fig. 6B) to maltose binding protein (MBP). Individual detection of each epitope tag using the same concentration of primary antibody (or nanobody) showed significant differences in signal intensity and signal sensitivity (Fig. 6C, Fig. 7B). ST ALFA ST The detection signals obtained by the tags were overall 3-10 times stronger than those obtained by all other epitope tags. This result indicates that detection with monoclonal antibodies (anti-FLAG M2, anti-HA F-7, anti-myc 9E10) involves signal amplification by the use of polyclonal secondary antibodies, whereas ALFA ST The tag is NbALFA ST This is particularly striking because it relies exclusively and directly on the label. ST yielded a strikingly direct signal spanning at least three orders of magnitude (Figure 6D) and was capable of detecting target protein amounts as low as 100 pg, thus reaching a detection limit approximately 10-fold lower than that observed with all other peptide tags.

[0150] Example 9: ALFA using ALFA Selector resin ST Capture of tagged target proteins Next, the inventors ST was site-specifically immobilized onto an agarose-based resin with ultra-low background via a hydrophilic flexible linker. ST A tagged GFP variant was used to analyze binding to nanobody-conjugated resin (shGFP2, Frey, S. et al. Surface Properties Determining Passage Rates of Proteins through Nuclear Pores. Cell 174, 202-217.e9 (2018), Figure 8A, B). As expected, shGFP2-ALFA ST was efficiently and tightly bound to the resulting resin. However, the binding was so strong that a significant excess of free ALFA was produced. STEven when peptide was used, competitive peptide elution from the resin was not possible (Figure 8A, solid black line). Even after competition with 200 μM peptide for 10 column volumes in 60 min, more than 95% of shGFP2-ALFA ST The protein remained on the resin. ST The resin loaded with this is called the "ALFA Selector" (named after the word "Super-Tight"). ST " NbALFA ST -ALFA ST Based on the complex structure, free ALFA ST In the absence of peptide, ALFA ST Weak NbALFA that would stably associate with tagged proteins while allowing for efficient peptide elution ST To identify mutants, we pursued a rational mutagenesis approach. We determined that shGFP2-ALFA ST Immobilized NbALFA that binds tightly to PE Agarose-based resin (ALFA Selector PE ), NbALFA mutant NbALFA PE (Peptide Elution). The target protein remained stably bound to the resin even after more than an hour of washing. However, the target protein was not bound to free ALFA under native conditions. ST Depending on the competition with peptide, the reaction is incubated at room temperature for approximately 15–20 min (t 1 / 2 The elution kinetics were similar for ALFA (Figure 8B and Figure 10A, black solid line). ST This was seen when the tag was placed between the two folded domains (Fig. 10B), whereas the N-terminal ALFA ST The tagged shGFP2 was slightly faster than the ALFA Selector. PE (t 1 / 2 (approximately 50 seconds, Figure 10C). Notably, in the absence of competing peptides, ALFA Selector ST and ALFA Selector PEThere was little spontaneous elution of the total target protein from both (Figure 8B and Figure 10, grey dotted lines).

[0151] Example 10: Interaction of ALFA tagged proteins with ALFA Selectors is compatible with stringent washing. We decided to further characterize the biochemical properties of both ALFA Selector resins. To this end, both resins were loaded with ALFA ST -shGFP2 or shGFP2-ALFA ST Either ALFA Selector 1 or ALFA Selector 2 were loaded and subjected to stringent washing steps (Figure 8D). For all resin and substrate combinations, the interaction was resistant even to stringent washing steps including up to 3 M NaCl, 1 M MgSO4, 2 M guanidinium 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. Some differences under denaturing conditions were observed between the different resin / substrate combinations. For washes with 4 M or 6 M urea, ALFA Selector 1 and ALFA Selector 2 were loaded and subjected to stringent washing steps (Figure 8D). ... PE From ALFA ST -Partial release of shGFP2 and, to a lesser extent, shGFP2-ALFA ST On the other hand, both target proteins were released by ALFA Selector under the same conditions. ST A striking difference was observed after incubation with 0.1% SDS. PE ALFA attached to ST -shGFP2 partially lost fluorescence while bound to the resin, an effect that was not observed with any other resin and substrate combination.

[0152] Example 11: pH resistance In a similar assay, the loaded ALFA Selector resin was washed with buffers adjusted to different pHs (Figure 8E). The interaction was resistant at pH 7.5 to 9.5 and slightly affected at pH 4.5. However, even after neutralization, both ALFA Selector resins remained completely non-fluorescent after washing with 100 mM glycine, pH 2.2. In contrast, the eluted material successfully regained its fluorescence at neutral pH (not shown), and acidic elution with glycine pH 2.2 significantly reduced the fluorescence of the tightly bound ALFA Selector resin. ST This shows that it is possible even from

[0153] Example 12: Affinity evaluation shGFP2-ALFA ST , NbALFA ST and NbALFA PE The affinity of NbALFA for both was determined in solution by microscale thermophoresis (MST). PE has a dissociation constant of about 15 nM, while NbALFA ST The K is approximately 10 pM, which is the lower detection limit of the instrument. d Even shGFP2-ALFA ST These figures are approximately 2x10 5 Assuming an on-rate of 1 / M*sec, this agrees well with the dissociation kinetics observed during peptide elution (Figure 8B), which is commonly observed for nanobody-target interactions.

[0154] Example 13: ALFA from complex lysates ST Pull-down of tagged target proteins To characterize the specificity of our ALFA Selector resin, we performed pull-down experiments from complex lysates under physiological conditions (Figure 9A-C). To this end, E. coli or HeLa lysates prepared in PBS were incubated with 3 μM of purified recombinant ALFA. STThe fusion protein bound specifically to both ALFA Selectors, but not to the control resin without conjugated nanobody ("Selector Control"). As expected from previous experiments, the ALFA Selectors did not bind to the control resin without conjugated nanobody ("Selector Control"). ST -shGFP2 was grown under native conditions with 200 μM ALFA ST Using peptides, ALFA Selector PE In contrast, ALFA Selector ST Successful elution from ALFA Selector was observed only after treatment with SDS sample buffer. Strikingly, pull-down from both E. coli and HeLa lysates was highly specific (Figure 9B and C). PE After peptide elution from ALFA Selector 100 in a single step, all visible bands can be essentially attributed to input proteins, and even in the SDS eluate the number and intensity of detectable impurities of lysate protein origin are very low. PE ALFA obtained by peptide elution from ST -shGFP2 contained significantly fewer contaminants than the protein used to spike into the input lysate (Figure 9A), an observation particularly striking since the input protein had been purified using two successive chromatographic steps.

[0155] Example 14: ALFA Selector PE Resin-based co-immunoprecipitation To confirm whether the ALFA system can be applied to more sensitive co-immunoprecipitation experiments, we attempted to pull down the two-component E. coli inner membrane protein complex YfgM-PpiD (Gotzke, H. et al. YfgM is an ancillary subunit of the SecYEG translocon in Escherichia coli. J Biol Chem 289, 19089-19097(2014)) under native conditions (Figure 9D). For this purpose, we used either wild-type YfgM or YfgM-ALFA. ST Either YfgM or YfgMΔ 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. YfgM-ALFA, prepared in the presence of the mild non-ionic detergent DDM as input, ST When using total lysate containing ALFA Selector PE We were able to pull down the YfgM-PpiD complex specifically and in a detergent-resistant manner. ST The tag was shown to be compatible with the formation of this unstable membrane complex. Importantly, the native non-denatured membrane protein complex was denatured by treatment with 200 μM ALFA. ST Peptides were used to perform ALFA Selector assays under physiological conditions within 20 minutes. PE YfgM and its interacting partner PpiD are likely associated with ALFA, present on the (periplasmic) C-terminus of YfgM, as the complex could not be purified from control lysates expressing untagged YfgM. ST Via tags, ALFA Selector PE Therefore, ALFA ST The tag is ALFA Selector PE Together with the resin it can be used for the purification of proteins from various sources but is also suitable for the pull-down of challenging (membrane) protein complexes under native conditions.

[0156] Example 15: Viable Lymphocyte Isolation Assumed ALFA Selector PE The application of the ALFA system is the specific enrichment of cells under physiological conditions. This may be of particular interest, for example, in the production of chimeric antigen receptor modified T (CAR-T) cells, whose precursors are usually 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 investigate whether the ALFA system can be applied to enrich viable blood cells, human peripheral blood mononuclear cells (PBMCs) were incubated with ALFA Selector ELISA kits preloaded with ALFA-tagged nanobodies that recognize the surface marker CD62L, normally present on naive T cells. PE The cells were then passed through the column (Lefrancois, L. Development, trafficking, and function of memory T-cell subsets. Immunological Reviews (2006). doi:10.1111 / j.0105-2896.2006.00393.x) (Figure 12a). After washing, the bound cells were purified by ALFA ST The peptide was used to elute lymphocytes, which were 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 PBMC served as a control. Using this method, CD62L+ lymphocytes were enriched from 71.8% to 97.7% (Figure 12b). Furthermore, it was confirmed that the majority of cells eluted by the ALFA peptide were CD3 positive T cells, while B cells were a minor population of isolated cells (Figure 12c).

[0157] Consideration Here, we report the development and initial evaluation of the ALFA system, which consists of three ALFA complexes, each with either exceptionally high or moderate affinity. ST Tag, a novel highly versatile epitope tag, and its mutant variants (ALFA PE Tags), and ALFA STThe tags include a series of related single domain antibodies (nanobodies) that recognize the tags. Importantly, the rational approach chosen allowed us to equip the ALFA system with features crucial for generalizability. When selecting the ALFA tag sequence, it was preferred that the tag be small, lysine-free, hydrophilic without shedding any net charge, absent in the proteome of the relevant model organism, but that it would adopt a stable folded form in solution. As a result, it is preferred that the ALFA tag be designed to be highly specific, insensitive to amine-reactive fixatives, generally well tolerated by the tagged target protein, and easily refold after denaturation.

[0158] In contrast to conventional antibodies, nanobodies were preferred as binding agents because they are small, monovalent and robust probes that can be easily modified by genetic means and recombinantly produced in various expression systems. Nanobodies can be site-specifically immobilized or quantitatively fluorescently labeled (Pleiner, T. et al. Nanobodies: site-specific labeling for super-resolution imaging, rapid epitope-mapping and native protein complex isolation. Elife 4, (2015)). ALFA ST Our preferred highly specific nanobody NbALFA, which recognizes the tag ST Nanobodies can therefore be easily used, for example, in direct immunofluorescence assays. The small size of nanobodies (approximately 3-4 nm in diameter) and the well-defined number and location of attached dyes allow the fluorescently labeled NbALFA ST is an ideal tool for high-resolution or quantitative imaging. ST However, various ALFAs expressed in mammalian cells ST We were able to demonstrate that NbALFA interacts faithfully with the tagged target protein. STThe finding is that NbALFA can also fold within the cytoplasm of eukaryotic cells and can therefore be used as an "intracellular antibody" to detect or manipulate ALFA-tagged target proteins in vivo (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); Roder,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 supports the discovery of NbALFA. ST Consistent with our biochemical evidence showing that ALFA is resistant to at least 100 mM DTT at room temperature, the conserved internal disulfide bridge common to all nanobodies is ST For intracellular antibody applications, we have synthesized NbALFA fused to a fluorescent reporter under the control of the CMV or PGK promoter. ST We found that expression of both ALFA and the target protein produced good results with low background. ST For optimal results or detection of tagged target proteins, more complex titration of relative expression levels may be required.

[0159] In general, most nanobodies recognize conformational epitopes on the surface of their target proteins and therefore do not recognize denatured proteins (e.g., in Western blots). STSince NbALFA can be used for highly sensitive detection of target proteins in Western blot applications, the present inventors ST It was possible to show that is excluded from this definition. This fact is consistent with ALFA ST Tags and ALFA PE This suggests that the tag can be efficiently refolded after incorporation into the membrane and removal of SDS. ST Despite its monovalent binding mode, the ALFA system was shown to be significantly superior to existing monoclonal anti-epitope tag tools in terms of absolute signal intensity and detection limit. Similar advantages are expected in other applications such as ELISA or microarray assays that require high sensitivity. Its resistance to amine-reactive fixatives may also allow the future application of the ALFA system in immunoelectron microscopy applications.

[0160] NbALFA ST ALFA ST The affinity for the tag is extraordinarily high. This is ideal for high profile imaging applications and sensitive detection, but prevents elution in a reasonable time frame under physiological conditions, thus limiting biochemical applications. We therefore aimed to reduce the affinity of the nanobody for the substrate without affecting the specificity of the nanobody. We approached this in two separate ways: 1) A broad range of ALFA peptides were selected for low binding avidity. This approach allowed us to identify NbALFA ST binds efficiently to free ALFA ST Efficient elution by peptide competition, ALFA PE I arrived at the tag. 2) ALFA ST NbALFA complexed with peptides ST Based on the crystal structure of NbALFA, we successfully increased the off-rate to a level that allows efficient peptide elution under physiological conditions. ST The mutated nanobody (NbALFA) was specifically mutated when immobilized on agarose resin with low background binding. PE) has proven ideally suited for the native purification of proteins and protein complexes from various lysates under physiological conditions. ST ) ALFA Selector ST may be advantageous for specialized applications requiring stringent washes with up to 6M urea or up to 0.1% SDS, or when extremely low abundant proteins need to be removed from dilute lysates. ST Elution from these media also requires strongly denaturing or acidic elution (eg, 1% SDS or glycine pH 2.2), which are generally not compatible with the native target protein conformation.

[0161] ALFA ST NbALFA bound to peptide ST The structure of NbALFA ST ALFA in the α-helical conformation ST It is shown that the peptide recognizes ALFA due to its adjacent secondary structure. ST To minimize potential effects on tag conformation, core ALFA ST The sequence (SRLEEELRRRLTE, SEQ ID NO:04) was placed between two prolines that act as an "insulator". By utilizing this approach, NbALFA ST and ALFA ST The interaction with the tag is largely independent of the location of the tag within the protein. ST and NbALFA PE and ALFA located at both the N-terminus and C-terminus of the target protein, or even within two protein domains. ST You will recognize the tag.

[0162] Taken together, we have presented here a novel epitope tag system with an exceptionally wide range of applications. The ALFA system allows the identification of a wide variety of different applications including (super-resolution) imaging, in vivo manipulation of proteins, in vitro detection by Western blotting or even native condition pull-down for the detection of specific interaction partners. ST A single transgenic cell line or organism with a tagged target protein is sufficient. ALFA Selector PE could further be applied to selective enrichment of CD62L-positive lymphocytes from PBMC preparations (Figure 12). We believe that this technology can be easily equipped with highly validated recombinant Fab and scFv fragments currently utilized for cell isolation approaches 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), as well as ALFA tags, and can be easily introduced to novel nanobodies that recognize surface markers. Our novel technology can therefore contribute to modern 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 strongly believe that the ALFA system will make an important contribution that will greatly stimulate the scientific community with its wide range of applications.

[0163] material and method Transfection of 3T3 and COS-7 cells For immunofluorescence experiments, the appropriate plasmids listed in Table 3 were transiently introduced into 3T3 or COS-7 cells using the PolyJet transfection kit (SignaGen) according to the manufacturer's recommendations. Briefly, cells were seeded into 12-well plates for each experiment. The volume was adjusted according to the size of the well. 1 μg of each plasmid was premixed with 38 μl of serum-free medium, followed by the addition of PolyJet transfection reagent diluted in 38 μl of serum-free medium. The suspension was incubated at room temperature for 15 min and then added dropwise to the cells. The cells were incubated at 37°C with 5% CO2 for 24 h. For co-expression experiments, plasmid DNA was premixed at a 1:1 ratio and processed as described above.

[0164] Fixation and staining of COS-7 cells Transiently transfected cells were fixed 24 hours after transfection with either 4% paraformaldehyde (PFA) (w / v) or 2% glutaraldehyde (GA) (v / v) for 30 min at room temperature. Alternatively, fixation was performed in ice-cold methanol at -20°C for 15 min. Cells were blocked and permeabilized with PBS containing 10% normal goat serum (v / v) and 0.1% Triton-X 10 (v / v) for 15 min. Fluorescently labeled NbALFA ST(FluoTag-X2 anti-ALFA AbberiorStar635P, NanoTag Biotechnologies N1502-Ab635P-L) was diluted 1:500 in PBS containing 3% normal goat serum (v / v) and 0.1% Triton-X 100 (v / v). Cells were incubated in this staining solution for 1 hour at room temperature, followed by three 5-minute washes with PBS. DAPI (0.4 μg / ml) was included in one of the PBS washing steps to stain the nuclei. Coverslips were mounted on cover slides using MOWIOL solution, which were dried at 37° C. and imaged using an epifluorescence microscope with a 20× lens (Axio, Zeiss). Cell surface expressed constructs were co-stained with anti-FLAG M2 (primary antibody, Sigma F1804) and FluoTag-X2 anti-mouse IgG Atto488 (secondary nanobody, NanoTag, Inc. N1202-At488-L) diluted 1:1000 and 1:500, respectively, in PBS containing 3% normal goat serum and 0.1% Triton-X 100 (v / v).

[0165] Effect of the ALFA tag on EGFP localization Transiently transfected 3T3 cells were imaged using an epifluorescence microscope equipped with a 40x 1.3 oil lens (Axio, Zeiss). ST -EGFP, pCMV-EGFP-ALFA ST For cells transfected with either pEGFP-N1 or pEGFP-N1, 107–133 cells were imaged in a total of six to seven individual images. For each individual image, cells were grouped according to EGFP localization ("slightly nuclear," "uniform," "other") and counted. The fraction of cells in each group was statistically analyzed using Student's t-test.

[0166] Western blot with COS-7 lysate 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 separated 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, dilution 1:1000), followed by FluoTag-X2 anti-mouse IgG IRDye680 (NanoTag Biotechnologies #N1202, dilution 1:1000) and FluoTag-X2 anti-ALFA IRDye800 (NanoTag Biotechnologies #1502, dilution 1:1000). The membranes were scanned using an Odyssey CLx (Li-COR).

[0167] Sensitivity Assay FLAG tag, HA tag, myc tag, and ALFA ST Serial dilutions of tagged MBP were made in PBS pH 7.4, 0.1 μg / mL BSA, and 1 μl of each dilution was spotted onto a nitrocellulose membrane.

[0168] The membrane was blocked with 5% milk powder in TBS-T and washed. Pre-made monoclonal antibodies (anti-FLAG M2-Sigma #F1804, anti-myc 9E10-SynapticSystems #343 011, anti-HA F-7-SantaCruz #sc-7392) were used in combination with secondary goat anti-mouse IgG IRDye800CW (Li-COR #925-32210, diluted 1:500) to detect the FLAG, myc, and HA tags, respectively. ALFA STTags were detected using IRDye800CW directly conjugated with FluoTag-X2 anti-ALFA (NanoTag Biotechnologies #N1502). All primary antibodies and nanobodies were used at a final concentration of 2.7 nM. Rabbit polyclonal serum recognizing MBP (SynapticSystems) and detection of MBP with anti-rabbit IgG IRDye680RD (Li-COR #925-68071) served as internal loading controls. The membrane was scanned using an Odyssey CLx (Li-COR). Quantification was performed using ImageStudioLight (Li-COR).

[0169] Off-rate assay 20 μl ALFA Selector ST or ALFA Selector PE (NanoTag Biotechnologies) were saturated with each recombinant target protein. After washing four times with PBS, the beads were saturated with 200 μM ALFA ST The beads were suspended in 10x more PBS containing free peptide and mixed at 25°C. Control reactions were performed without peptide. Specific elution from the beads was quantified using GFP fluorescence emitted in the supernatant at the indicated time points (Q-Bit 3.0, Thermo-Fischer Scientific). Three independent experiments were performed in parallel. Means, standard deviations, and exponential fits were calculated using GraphPad Prism 5.0. Photographs were taken with UV illumination using a Nikon D700 with a 105 mm microlens (Nikon).

[0170] Resistant to stringent cleaning and pH Based on the experimental procedure, 10-15 μl of ALFA Selector solution saturated with the indicated ALFA-tagged shGFP2 fusion protein was used. ST or ALFA Selector PEThe resins were washed with PBS and incubated with 100 μl of the indicated substrate for 60 min at room temperature. After sedimentation of the beads, pictures were taken under UV illumination. For pH resistance tests, the same beads were incubated with 150 mM NaCl buffered at different pHs (100 mM Glycin-HCl pH 2.2, 100 mM Na-Acetate pH 4.5, 100 mM Tris-HCl pH 7.5, 100 mM Carbonate pH 9.5) for 30 min at RT. The resins were washed twice with the same buffers. After several equilibrations with PBS, pictures were taken.

[0171] One-Step Affinity Purification Using ALFA Selector Resin To obtain defined input material for pull-down experiments from E. coli or HeLa lysates, mock lysates were mixed with 3 μM of the indicated ALFA-tagged shGFP2 variants. 1 mL of each lysate / substrate mixture was added to 25 μl of ALFA-Selector ST or ALFA Selector PE The resin was incubated with 0.01% sdAb for 1 h at 4°C. Depending on the experimental setup, either a similar resin without immobilized sdAb (Selector control) or a mock lysate without target protein served as specificity controls. After three washes with 600 μL PBS, the resin was transferred to a MiniSpin column (NanoTag Biotechnologies). Excess buffer was removed by centrifugation (3000x g, 30 sek) and 50 μL of 200 μM ALFA was added each time. ST The beads were incubated twice with peptide in PBS for 10 min at room temperature. Protein remaining on the beads was then eluted with SDS sample buffer. Input and unbound fractions of 0.5 μL (E. coli) or 1.5 μL (HeLa) were analyzed by SDS-PAGE (12%) and Coomassie staining. The eluate fractions shown correspond to 1 μl of material eluted from the corresponding resin.

[0172] ALFA Selector PE YfgM pulldown using The yfgM deletion strain was constructed by expressing C-terminal ALFA from a pSC-based low-copy vector under the control of the endogenous promoter. ST The membrane protein complexes were complemented with either tagged or untagged YfgM. Both membrane protein complexes were solubilized from total lysates prepared in buffer LS (50 mM Tris pH 7.5, 300 mM NaCl, 5 mM MgCl2) with 1% DDM within 1 hour on ice (Maddalo, G. et al. Systematic analysis of native membrane protein complexes in Escherichia coli. J Proteome Res 10,1848-1859(2011)). Both lysates were diluted with 20 μl of ALFA Selector 10 ... PE The resin was incubated in a roller drum at 4° C. for 1 hour. After washing with PBS+0.3% DDM, the bound proteins were eluted with 200 μM ALFA ST Elution was performed by incubation twice with 50 μl PBS containing the peptide under native conditions for a time course. Samples corresponding to 1 / 800 input and unbound material or 1 / 80 eluate fractions were separated by SDS PAGE. Analysis was performed by Western blotting using rabbit polyclonal antiserum raised against the YfgM-PpiD complex (Gotzke, 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 luminescence imager (Fuji Film).

[0173] ALFA Selector ST YfgM pulldown using The yfgM deletion strain was constructed by expressing C-terminal ALFA from a pSC-based low-copy vector under the control of the endogenous promoter. PE The membrane protein complexes were complemented with either tagged or untagged YfgM. Both membrane protein complexes were solubilized from total lysates prepared in buffer LS (50 mM Tris pH 7.5, 300 mM NaCl, 5 mM MgCl2) with 1% DDM within 1 hour on ice (Maddalo, G. et al. Systematic analysis of native membrane protein complexes in Escherichia coli. J Proteome Res 10,1848-1859(2011)). Both lysates were diluted with 20 μl of ALFA Selector 10 ... ST The resin was incubated in a roller drum at 4° C. for 1 hour. After washing with PBS+0.3% DDM, the bound proteins were eluted with 200 μM ALFA ST Elution was performed by incubation twice with 50 μl PBS containing the peptide under native conditions for a time course. Samples corresponding to 1 / 800 input and unbound material or 1 / 80 eluate fractions were separated by SDS PAGE. Analysis was performed by Western blotting using rabbit polyclonal antiserum raised against the YfgM-PpiD complex (Gotzke, 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 luminescence imager (Fuji Film).

[0174] Table 3 Plasmids TIFF0007623006000033.tif188163 (a)Gotzke,H. et al. YfgM is an ancillary subunit of the SecYEG translocon in Escherichia coli. J Biol Chem 289,19089-19097(2014)

[0175] E. coli strains Escherichia coli MC4100 ΔyfgM ΔppiD(Gotzke,H. et al. YfgM is an ancillary subunit of the SecYEG translocon in Escherichia coli. J Biol Chem 289,19089-19097(2014))

[0176] (Table 4) Antibodies TIFF0007623006000034.tif105165 (a) Gotzke,H. et al. YfgM is an ancillary subunit of the SecYEG translocon in Escherichia coli. J Biol Chem 289,19089-19097(2014)

[0177] Table 5. Fusion proteins TIFF0007623006000035.tif74163TIFF0007623006000036.tif250163TIFF0007623006000037.tif250163TIFF0007623006000038.tif250163 TIFF0007623006000039.tif250163TIFF0007623006000040.tif250163TIFF0007623006000041.tif250163TIFF0007623006000042.tif45163

[0178] Protein expression and purification All recombinant proteins were expressed from a ColE1-derived expression vector under the control of the Tac promoter, conferring resistance to kanamycin.

[0179] Multiple epitope tags, i.e. ALFA ST -shGFP2, ALFA PE -shGFP2, and TwinStrepTag-bdNEDD8-ALFA min The MBP fusion protein carrying the N-terminal His 14 The proteins were expressed as -bdSUMO fusions. For protein expression, E. coli were grown in Terrific broth (TB) supplemented with 0.3 mM IPTG for 14-16 h at 23 °C. After harvesting, the E. coli were dissolved in 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 nickel(II) chelating beads. After extensive washing, proteins were eluted by on-column cleavage with bdSENP1 as previously described (Frey, S. & Gorlich, D. A new set of highly efficient, tag-cleaving proteases for purifying recombinant proteins. J Chromatogr A 1337, 95-105(2014); Frey, S. & Gorlich, D. Purification of protein complexes of defined subunit stoichiometry using a set of orthogonal, tag-cleaving proteases. J Chromatogr A 1337, 106-115(2014)).

[0180] 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, but elution was performed using 250 mM imidazole in buffer LS.

[0181] For affinity determination and binding studies from the combined lysates, the substrate proteins were further purified by size exclusion chromatography on a Superdex200 10 / 30 column (GE Healthcare).

[0182] Selection of specific sdAb clones by affinity purification of B cell "cell lines" 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 incubated with a saturating amount of TwinStrepTag-bdNEDD8-ALFA. minThe resin was incubated with the fusion protein for 30 min at RT with rotation. Excess bait protein was removed by extensive washing of the resin with B cell isolation buffer. A 100 mL blood sample was obtained from an alpaca immunized with the ALFA peptide fusion and immediately incubated with 5000 IU / mL heparin (Sigma) to prevent clotting. PBMCs were isolated from fresh blood (<4 hours after collection) using Ficoll-Paque PLUS (GE Healthcare). PBMCs were washed three times consecutively with B cell isolation buffer to remove residual serum. PBMCs were passed three times over the loaded T-Catch resin and the resin was washed with 10 column volumes of B cell isolation buffer. Bound B cells were eluted from the resin by incubation with 2 μM NEDP1 for 30 min at RT (Frey, S. & Gorlich, D. A new set of highly efficient, tag-cleaving proteases for purifying recombinant proteins. J Chromatogr A 1337, 95-105 (2014); Frey, S. & Gorlich, D. Purification of protein complexes of defined subunit stoichiometry using a set of orthogonal, tag-cleaving proteases. J Chromatogr A 1337, 106-115 (2014)). sdAb-specific cDNA libraries were amplified from eluted B cells by multistep nested RT-PCR and cloned into bacterial expression vectors. 96 single clones were tested by ELISA for expression of ALFA-reactive sdAbs.

[0183] Preparation of human PBMCs 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 this dilution was placed on top of a CELLPURE Roti-Sep 1077 (Carl Roth) layer in a 50 mL LEUCOSEP tube (Greiner Bio-One) and centrifuged at 800 x g for 20 minutes at room temperature. The PBMC-containing layer was then harvested and washed five times with cold PBS+EDTA to remove platelets.

[0184] Isolation of CD62L-positive lymphocytes Approximately 2 x 10 7 PBMCs were treated with ALFA Selector PE The resin was allowed to pass by gravity flow and then washed extensively with PBS supplemented with 1 mM EDTA and 1% (w / v) bovine serum albumin. Bound cells were then eluted with the same buffer containing 200 μM ALFA peptide.

[0185] The present invention illustratively described herein can be suitably implemented in the absence of any element or elements, or any limitation or limitations not expressly disclosed herein. In addition, the terms and expressions employed herein are used as descriptive terms, not limiting, and in the use of such terms and expressions, there is no intention to exclude any equivalents of the illustrated and described features or portions thereof, but it is to be recognized that various modifications are possible within the scope of the claimed invention. Thus, although the present invention has been explicitly disclosed by exemplary embodiments and optional features, it should be understood that modifications and alterations of the present invention disclosed and exemplified herein can be made by those skilled in the art, and such modifications and alterations are considered to be within the scope of the present invention.

[0186] The invention has been described broadly and generically herein. Each narrower and subgeneric classification falling within the generic disclosure is also contemplated as part of the invention. This includes the generic description of the invention with a condition or negative limitation that excludes any subject matter of the invention from the generic description, regardless of whether the excluded content is expressly set forth herein.

[0187] Other embodiments are within the scope of the following claims. Additionally, where features or aspects of the invention are described in terms of a Markush group, one of skill in the art will recognize that the invention is also described in terms of each element of the Markush group or any subgroup of elements.

Claims

1. (a) MPSRLEEELRRRLTEP (SEQ ID NO: 05); PSRLEEELRRRLTEP (SEQ ID NO: 06); PSRLEEELRRRLTE (SEQ ID NO: 07); and MSGRLEEELRRRLSP (SEQ ID NO: 33) A peptide comprising a sequence selected from the group consisting of: (b) a polypeptide; A fusion protein comprising:

2. The fusion protein of 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 the C-terminus.

3. A single domain antibody that specifically binds to the peptide contained in the fusion protein according to claim 1 or 2, (a) the CDR1 sequence GVTISALNAMAMG (SEQ ID NO:115), the CDR2 sequence AVSERGNAM (SEQ ID NO:116), and the CDR3 sequence LEDRVDSFHDY (SEQ ID NO:117), or (b) CDR1 sequence GVTISALNAMAMG (SEQ ID NO:118), CDR2 sequence AVSSRGNAM (SEQ ID NO:119), and CDR3 sequence LEDRVDSFHDY (SEQ ID NO:120). A single domain antibody comprising:

4. The antibody of claim 3, wherein the antibody comprises the CDR1 sequence GVTISALNAMAMG (SEQ ID NO:115), the CDR2 sequence AVSERGNAM (SEQ ID NO:116), and the CDR3 sequence LEDRVDSFHDY (SEQ ID NO:117).

5. The antibody of claim 3, wherein the antibody comprises the CDR1 sequence GVTISALNAMAMG (SEQ ID NO:118), the CDR2 sequence AVSSRGNAM (SEQ ID NO:119), and the CDR3 sequence LEDRVDSFHDY (SEQ ID NO:120).

6. EVQLQESGGGLVQPGGSLRLSCTASGVTISALNAMAMGWYRQAPGERRVMVAAVSERGNAMYRESVQGRFTVTRDFTNKMVSLQMDNLKPEDTAVYYCHVLEDRVDSFHDYWGQGTQVTVSS (SEQ ID NO: 133), or EVQLQESGGGLVQPGGSLRLSCTASGVTISALNAMAMGWYRQAPGEERVMVAAVSSRGNAMYRESVQGRFTVTRDFTNKMVSLQMDNLKPEDTAVYYCHVLEDRVDSFHDYWGQGTQVTVSS (SEQ ID NO: 134) The antibody of any one of claims 3 to 5, comprising a VHH sequence having at least 90% sequence identity with:

7. EVQLQESGGGLVQPGGSLRLSCTASGVTISALNAMAMGWYRQAPGERRVMVAAVSERGNAMYRESVQGRFTVTRDFTNKMVSLQMDNLKPEDTAVYYCHVLEDRVDSFHDYWGQGTQVTVSS (SEQ ID NO: 133), or EVQLQESGGGLVQPGGSLRLSCTASGVTISALNAMAMGWYRQAPGEERVMVAAVSSRGNAMYRESVQGRFTVTRDFTNKMVSLQMDNLKPEDTAVYYCHVLEDRVDSFHDYWGQGTQVTVSS (SEQ ID NO: 134) The antibody of any one of claims 3 to 6, comprising a VHH sequence having at least 95% sequence identity with:

8. EVQLQESGGGLVQPGGSLRLSCTASGVTISALNAMAMGWYRQAPGERRVMVAAVSERGNAMYRESVQGRFTVTRDFTNKMVSLQMDNLKPEDTAVYYCHVLEDRVDSFHDYWGQGTQVTVSS (SEQ ID NO: 133) The antibody of any one of claims 3 to 4 and 6 to 7, comprising a VHH sequence of:

9. EVQLQESGGGLVQPGGSLRLSCTASGVTISALNAMAMGWYRQAPGEERVMVAAVSSRGNAMYRESVQGRFTVTRDFTNKMVSLQMDNLKPEDTAVYYCHVLEDRVDSFHDYWGQGTQVTVSS (SEQ ID NO: 134) The antibody of any one of claims 3 and 5 to 7, comprising a VHH sequence of:

10. The antibody of any one of claims 3 to 9, which is conjugated to a detectable label, affinity tag, or solid support.

11. (a) a fusion protein; (b) Antibody and Including, The fusion protein is a fusion protein according to claim 1 or 2 and / or the antibody is an antibody according to any one of claims 3 to 10. Complex.

12. A nucleic acid encoding the fusion protein of claim 1 or 2 or the antibody of any one of claims 3 to 10.

13. A vector comprising the nucleic acid of claim 12.

14. A host cell comprising a nucleic acid according to claim 12 or a vector according to claim 13, or expressing a fusion protein according to claim 1 or 2 or an antibody according to any one of claims 3 to 10.

15. 11. A method for detecting a fusion protein according to claim 1 or 2, comprising the step of contacting said fusion protein with an antibody according to any one of claims 3 to 10, wherein said antibody is conjugated to a detectable label.

16. A method for isolating a specific target of a fusion protein according to claim 1 or 2 or an antibody moiety contained in said fusion protein, said method comprising a step of contacting said fusion protein with an antibody according to any one of claims 3 to 10.

17. A nucleic acid or a nucleic acid expression construct encoding a peptide contained in a fusion protein according to claim 1 or 2, and optionally encoding an antibody according to any one of claims 3 to 10. Including the kit.

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