Antigen-binding protein

Abstract

Antigen-binding proteins, nucleic acid constructs encoding antigen-binding proteins, vectors containing nucleic acid constructs, and host cells containing the vectors and nucleic acid constructs, as well as kits for producing antigen-binding proteins, are provided.

Description

Technical Field

[0001] Cross-reference to related applications This application claims priority to U.S. Provisional Application No. 62 / 819,753, filed Mar. 18, 2019, the contents of which are incorporated by reference herein.

Background Art

[0002] Multivalent formats of antibody fragments are useful in assays where high avidity is desired because the high valency increases assay sensitivity. Monomeric antibody fragments are typically generated prior to conversion to a multivalent format in order to enable determination of the intrinsic affinity. Conversion of monomeric antibody fragments to higher valencies and subsequent production involves several steps and is a cumbersome process that takes several weeks. These processing steps are repeated for different valencies.

[0003] Protein ligation Some techniques enable covalent bonding of polypeptides at specific predetermined sites. One example is the sortase system (Non-Patent Document 11), where a short peptide (sorting motif) is genetically fused to the C-terminus of one polypeptide and two glycine residues are genetically fused to the N-terminus of a second peptide (or vice versa). In the presence of the sortase enzyme, the two modified polypeptides are fused together. Other enzyme protein ligase systems are butelase (Non-Patent Document 9) or peptiligase (Non-Patent Document 14).

[0004] Another example is the in-frame addition of nucleotides encoding one or more cysteines to the C-terminus or N-terminus of a polypeptide. Mixing such free cysteine-containing polypeptides under oxidizing conditions forms disulfide bridges. However, such systems have problems with the synthesis of many by-products and the instability of disulfide bridges under reducing conditions.

[0005] Another example is the SpyTag / SpyCatcher (Non-Patent Document 10) system. Here, the concept of spontaneous isopeptide formation in naturally occurring proteins has been used to covalently link one polypeptide to another. Proteins containing such isopeptide bonds Streptococcus pyogenes The domain derived from protein FbaB is divided into two parts. One part, SpyTag (SEQ ID NO: 1), is a 13-amino acid peptide containing a part of the autocatalytic center (e.g., aspartic acid). The other part, SpyCatcher (SEQ ID NO: 2), is a 116-amino acid protein domain containing another part of the center (e.g., lysine) that is facilitated by a neighboring glutamic acid or aspartic acid. When these two polypeptides are mixed, the autocatalytic center is restored, leading to the formation of an isopeptide bond, thereby covalently linking SpyTag to SpyCatcher (Non-Patent Document 17). Through further engineering, a short version of SpyCatcher with only 84 amino acids (SEQ ID NO: 3), SpyTag002 (SEQ ID NO: 4), and SpyCatcher002 (SEQ ID NO: 5) that promote the reaction are obtained (Non-Patent Documents 8 and 6, both of which are incorporated herein by reference in their entirety). Through further engineering, another optimized version, SpyTag003 (SEQ ID NO: 22) and SpyCatcher003 (SEQ ID NO: 23), with a reaction approaching the diffusion limit are obtained (Non-Patent Document 7, which is incorporated herein by reference in its entirety). A further modification of this system is the invention of SpyLigase (Non-Patent Document 5), which was achieved by dividing the FbaB domain into three parts, SpyTag (SEQ ID NO: 1), K-Tag (SEQ ID NO: 12), and SpyLigase. SpyLigase is a fragment of the FbaB domain containing a glutamic acid residue that induces or catalyzes the formation of an isopeptide bond between the aspartic acid residue and the lysine residue in SpyTag and the K-tag, respectively.

[0006] The applications of such systems include protein stabilization by cyclization, vaccine production, protein multimerization by integrating SpyTag / SpyCatcher and streptavidin / biotin (Non-Patent Document 10), afibody and Fab multimerization (Non-Patent Document 5), antibody production from modules (Non-Patent Document 2), preparation of antibody-drug conjugates (Non-Patent Document 12), and production of bispecific antibodies (Non-Patent Document 16). Streptococcus pneumoniae A similar system using the adhesin RrgA derived therefrom has been developed, which is called SnoopTag / SnoopCatcher (Non-Patent Document 15) and led to the subsequent development of the SnoopLigase system (Non-Patent Document 4). The entire SnoopTag / SnoopCatcher technology is incorporated herein by reference. In the subunit Spy0128 Streptcoccus pyogenes Another system using the pile has also been developed, which is called Isopeptag / Split Spy0128 (Non-Patent Document 1). Streptococcus dysgalactiae Still other systems derived from fibronectin-binding proteins have been developed, which are called SdyTag / SdyCatcherDANG short (Non-Patent Document 13). The entire Isopeptag / Split Spy0128 and SdyTag / SdyCatcherDANG short technologies are incorporated herein by reference.

Prior Art Documents

Patent Documents

[0007]

Patent Document 1

Patent Document 2

Patent Document 3

Non-Patent Documents

[0008]

Non-Patent Document 1

Non-Patent Document 7

Non-Patent Document 8

Non-Patent Document 9

Non-Patent Document 10

Non-Patent Document 11

Non-Patent Document 12

Non-Patent Document 13

Non-Patent Document 14

Non-Patent Document 15

Non-Patent Document 16

Non-Patent Document 17

Summary of the Invention

Means for Solving the Problems

[0009] An antigen-binding protein, a nucleic acid construct encoding the antigen-binding protein, a vector containing the nucleic acid construct, and a host cell containing the vector and the nucleic acid construct are provided. A kit containing components for producing the antigen-binding protein is also provided.

[0010] In one embodiment, the antigen-binding protein comprises two or more first antigen-binding fragments each comprising a first binding motif and a first fusion protein comprising two or more second binding motifs. The binding motifs that bind to the first antigen-binding fragments and the two or more second binding motifs that form the fusion protein may optionally be linked by one or more linker sequences. The first binding motifs of the two or more first antigen-binding fragments are covalently bonded to the two or more second binding motifs via protein ligation. In some embodiments, the first fusion protein is optionally a dimer or multimer of second binding motifs linked by a linker sequence. In some embodiments, the first binding motif comprises SEQ ID NO: 1, 4, 6, 8, 10, 13, or 22, or a sequence having at least 60% sequence identity with SEQ ID NO: 1, 4, 6, 8, 10, 13, or 22, and the second binding motif comprises SEQ ID NO: 2, 3, 5, 7, 9, 11, 12, 14, or 23, or a sequence having at least 60% sequence identity with SEQ ID NO: 2, 3, 5, 7, 9, 11, 12, 14, or 23. In other embodiments, the first binding motif comprises SEQ ID NO: 2, 3, 5, 7, 9, 11, 12, 14, or 23, or a sequence having at least 60% sequence identity with SEQ ID NO: 2, 3, 5, 7, 9, 11, 12, 14, or 23, and the second binding motif comprises SEQ ID NO: 1, 4, 6, 8, 10, 13, or 22, or a sequence having at least 60% sequence identity with SEQ ID NO: 1, 4, 6, 8, 10, 13, or 22.

[0011] In some embodiments, the first fusion protein further comprises a third binding motif to which a polypeptide comprising a fourth binding motif is covalently bonded via protein ligation. In some embodiments, the polypeptide is an enzyme, a fluorescent protein, an effector protein, or another antigen-binding fragment. The third binding motif is optionally linked to the fusion protein via one or more linkers. The fourth binding motif is optionally linked to the polypeptide by one or more linkers.

[0012] In certain embodiments, the antigen-binding protein comprises one or more first antigen-binding fragments each comprising a first binding motif, a first fusion protein comprising one or more second binding motifs each comprising a third binding motif linked to one or more third binding motifs by a linker sequence, and one or more second antigen-binding fragments each comprising a fourth binding motif. The first binding motif is covalently bound to the second binding motif via protein ligation, and the third binding motif is covalently bound to the fourth binding motif via protein ligation. In some embodiments, the antigen-binding protein is bispecific, bispecific and dimeric, or bispecific and multimeric.

[0013] In some embodiments of an antigen-binding protein having first, second, third, and fourth binding motifs, the first binding motif comprises SEQ ID NO: 1, 4, 8, or 22, or a sequence having at least 60% sequence identity with SEQ ID NO: 1, 4, 8, or 22; the second binding motif comprises SEQ ID NO: 2, 3, 5, 9, 12, or 23, or a sequence having at least 60% sequence identity with SEQ ID NO: 2, 3, 5, 9, 12, or 23. The third binding motif comprises SEQ ID NO: 6, 10, or 13, or a sequence having at least 60% sequence identity with SEQ ID NO: 6, 10, or 13; the fourth binding motif comprises SEQ ID NO: 7, 11, or 14, or a sequence having at least 60% sequence identity with SEQ ID NO: 7, 11, or 14. In other alternative embodiments of an antigen-binding protein having first, second, third, and fourth binding motifs, the first binding motif comprises SEQ ID NO: 2, 3, 5, 9, 12 or 23, or a sequence having at least 60% sequence identity with SEQ ID NO: 2, 3, 5, 9, 12 or 23; the second binding motif comprises SEQ ID NO: 1, 4, 8 or 22, or a sequence having at least 60% sequence identity with SEQ ID NO: 1, 4, 8 or 22. The third binding motif comprises SEQ ID NO: 7, 11, or 14, or a sequence having at least 60% sequence identity with SEQ ID NO: 7, 11, or 14; the fourth binding motif comprises SEQ ID NO: 6, 10, or 13, or a sequence having at least 60% sequence identity with SEQ ID NO: 6, 10, or 13.

[0014] In an embodiment having first, second, third, and fourth binding motifs, the first binding motif - second binding motif pair is orthogonal to the third binding motif - fourth binding motif pair.

[0015] In certain embodiments, one or more binding motifs are located at the C-terminus, N-terminus, or embedded within the amino acid sequence of the first and / or second antigen-binding fragment, fusion protein, or polypeptide. In some embodiments, one or more binding motifs in the fusion protein are in a continuous or random order. In some embodiments, the antigen-binding protein further comprises a purification tag at the C-terminus or N-terminus of the fusion protein.

[0016] In some embodiments, the antigen-binding protein may further comprise a detectable label (e.g., a phosphor, fluorescent protein, biotin, or enzyme).

[0017] In some embodiments, the linker sequence is 1 to 5 amino acids having the sequence GGGGS.

[0018] In some embodiments, the antigen-binding protein may comprise a third binding motif linked by a linker sequence to a first fusion protein having two or more second binding motifs and a polypeptide having a fourth binding motif (e.g., a protein or protein fragment having additional functionality). In some embodiments, the fourth binding motif comprises a sortase recognition domain and the third motif comprises a sortase crosslinking domain. Alternative embodiments are those in which the fourth binding motif comprises a sortase recognition domain and the third binding motif comprises a sortase crosslinking domain. In some embodiments, the sortase recognition domain has the amino acid sequence LPTGAA (SEQ ID NO: 15), LPTGGG (SEQ ID NO: 16), LPKTGG (SEQ ID NO: 17), LPETG (SEQ ID NO: 18), LPXTG (SEQ ID NO: 19), or LPXTG(X)n (SEQ ID NO: 20) (X is any amino acid, n is an integer from 0, 1, 2, 3, 4, 5, 7, 8, 9, 10, any integer from 0 to 5, or any integer from 0 to 10, or any integer up to 100), NPX1TX2 (SEQ ID NO: 21) (X1 is glutamine or lysine, X2 is asparagine or glycine, N is asparagine, P is proline, T is threonine, and the sortase crosslinking domain comprises Gly, (Gly)2, (Gly)3, (Gly)4, or (Gly) x including (x is an integer from 1 to 20).

[0019] Also provided are nucleic acid constructs encoding the antigen-binding protein. Vectors comprising the nucleic acid constructs are also provided. Host cells comprising the vectors are also provided. Kits comprising components for making the antigen-binding protein are also provided.

Brief Description of the Drawings

[0020]

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[0021] Provided are an antigen-binding protein, a nucleic acid construct encoding the antigen-binding protein, a vector containing the nucleic acid construct, a host cell containing the vector and the nucleic acid construct, and a kit for producing the antigen-binding protein. The antigen-binding proteins are each covalently linked to a dual or multivalent affinity binding reagent to create either a monospecific (recognizing and binding to one antigen) or bispecific multivalent (i.e., binding to two different antigens or two different epitopes on the same antigen) construct. Multimeric antigen-binding proteins have a higher avidity than monomers, contain additional functional groups, are bispecific, or any combination thereof. Multimeric antigen-binding proteins are produced by protein ligation, which circumvents the genetic engineering steps currently required to create such binding reagents. Multivalency increases the sensitivity of the antigen-binding protein, which is a useful feature in some enzyme-linked immunosorbent assays, in applications such as Western blotting, flow cytometry, immunohistochemistry, and for therapeutic use. Bispecificity is useful for increasing target specificity, target affinity, or for binding to two different antigens simultaneously. Bispecific antigen-binding proteins are also of increasing significance as therapeutic agents. The addition of a second function by protein ligation is useful for the preparation of labeled antigen-binding proteins for applications such as Western blotting, flow cytometry, immunohistochemistry, enzyme-linked immunosorbent assays, for directed immobilization, or for other applications where a second function is required, such as the production of antibody-enzyme fusion proteins for cancer therapy.

[0022] **Definitions** Unless otherwise indicated, the following terms used in this application, including the specification and claims, have the definitions given below. As used in this specification and the appended claims, the singular forms include plural referents unless the content clearly dictates otherwise.

[0023] "Antibody" refers to an immunoglobulin, complex (e.g., fusion), or fragment form thereof. The term includes polyclonal or monoclonal antibodies of classes IgA, IgD, IgE, IgG, and IgM from an antibody-producing cell line or from an in vitro antibody library, including natural or genetically modified or synthetic forms such as humanized, human, single-chain, chimeric, synthetic, recombinant, hybrid, mutant, grafted, and other in vitro-produced antibodies, but is not limited thereto. "Antibody" also includes, but is not limited to, complex forms including fusion proteins having an immunoglobulin portion.

[0024] As used herein, the phrase "antigen-binding fragment" refers to a protein that includes the antigen-binding portion of an antibody such as Fab. Other antigen-binding fragments include variable fragment (Fv), disulfide-stabilized Fv fragment (dsFv), single-chain variable fragment (scFv), or single-chain Fab fragment (scFab). Further examples of antigen-binding fragments include monovalent forms of antigen-binding fragments that include the variable domain of a heavy-chain antibody (VHH), single-domain antibody (sdAb), or shark variable new antigen receptor (VNAR). Additionally, non-antibody scaffolds such as variable lymphocyte receptor (VLR), affimer, affibody, darpin, anticalin, monobody, avimer, fibronomer, affilin, or antigen-binding peptide can also be considered "antigen-binding fragments".

[0025] The term "binding motif" relates to a protein sequence that attaches to a polypeptide and enables the formation of a covalent bond to another polypeptide. Binding motifs include, but are not limited to, SpyTag sequences (including SpyTag002 and SpyTag003), SpyCatcher sequences (including SpyCatcher short, SpyCatcher002, and SpyCatcher003), SnoopTag sequences, and SnoopCatcher sequences. The binding motif can be fused to the N-terminus, C-terminus, or embedded within the polypeptide. One or more linker sequences (e.g., glycine / serine-rich linker) are adjacent to the binding motif to enhance proximity for the reaction or enhance the flexibility of the fusion polypeptide. In some embodiments, one or more linker sequences are adjacent to both the N-terminus and C-terminus of the binding motif to enhance proximity for the reaction or enhance the flexibility of the fusion polypeptide (e.g., in the case of a fusion protein containing a multimeric binding motif). The phrase "linked by a linker sequence" as used herein permits the use of one or more linker sequences to link two or more binding motifs, a binding motif and a polypeptide, or a binding motif and an antigen-binding fragment. When multiple linker sequences are used to bind a binding motif, to bind an antigen-binding fragment to a binding motif, or to bind a polypeptide and a binding motif, the linker sequences can be the same or different.

[0026] The term "prokaryotic system" refers to prokaryotic cells such as bacterial cells, prokaryotic phages, or bacterial spores. The term "eukaryotic system" refers to eukaryotic cells including animal, plant, fungal, and protist cells, as well as eukaryotic viruses such as retroviruses, adenoviruses, and baculoviruses. Prokaryotic and eukaryotic systems are collectively referred to as "expression systems".

[0027] The term "expression cassette" is used herein to refer to a functional unit constructed in a vector for the purpose of expressing a recombinant polypeptide having a binding motif. The expression cassette contains one or more promoters, transcription terminator sequences, one or more ribosome binding sites, and DNA encoding a fusion protein. Depending on the expression system (e.g., enhancers and polyadenylation signals of eukaryotic expression systems), other genetic components can be added to the expression cassette.

[0028] As used herein, the term "vector" preferably refers to a nucleic acid molecule that self-replicates within a cell and transcribes an inserted nucleic acid molecule into and / or between host cells. Typically, a vector is a circular DNA containing an origin of replication, a selectable marker, and / or a viral packaging signal, as well as other regulatory elements. The terms vector, vector DNA, plasmid DNA, and phagemid DNA are used interchangeably in the description of the present invention. This term includes vectors that mainly function for the insertion of DNA or RNA into cells, replication vectors that mainly function for the replication of DNA or RNA, and expression vectors that function for the transcription and / or translation of DNA or RNA. It also includes vectors that provide two or more of the above functions.

[0029] The term "expression vector" as used herein is a polynucleotide that, when introduced into a suitable host cell, directs the transcription and translation of one or more polypeptides under suitable conditions. The term "expression vector" refers to a vector that directs the expression of a polypeptide fused in-frame with a binding motif.

[0030] As used herein, the terms "polynucleotide", "nucleic acid", and "oligonucleotide" are used interchangeably. They refer to a polymeric form of nucleotides of any length, either deoxyribonucleotides or ribonucleotides, or analogs thereof. Examples of polynucleotides include, but are not limited to, the coding or non-coding regions of genes or gene fragments, loci defined from linkage analysis, exons, introns, messenger RNA (mRNA), transfer RNA, ribosomal RNA, ribozymes, cDNA, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, isolated DNA of any sequence, isolated RNA of any sequence, nucleic acid probes, and primers. Polynucleotides may include modified nucleotides such as methylated nucleotides and nucleotide analogs. When present, modifications to the nucleotide structure may be made before or after assembly of the nucleotide polymer.

[0031] As used herein, the term "amino acid" refers to natural and / or non-natural or synthetic amino acids, both D and L optical isomers, amino acid analogs, and peptidomimetics.

[0032] As used herein, the terms "polypeptide", "peptide", and "protein" refer to a polymer of amino acids of any length and are used interchangeably herein.

[0033] As used herein, the term "host cell" includes individual cells or cell cultures that can serve as, or have served as, a recipient for the disclosed expression constructs. Host cells include the progeny of a single host cell. The progeny may not necessarily be identical to the original parent cell due to natural, accidental, or intentional mutations.

[0034] Two nucleic acid sequences or polypeptides are said to be "identical" if the sequences of each nucleotide or amino acid residue in the two sequences are the same when aligned for maximum correspondence as described below. The term "identical" or percent "identity" in the context of two or more nucleic acid or polypeptide sequences refers to two or more sequences or subsequences that are the same, or two or more sequences or subsequences that have a specified percentage of amino acid residues or nucleotides that are the same when compared and aligned for maximum correspondence over a comparison window, as measured using one of the following sequence comparison algorithms or by manual alignment and visual inspection. When percent sequence identity is used in reference to a protein or peptide, residue positions that are not identical often differ by conservative amino acid substitutions, where an amino acid residue is substituted for another amino acid residue having similar chemical properties (e.g., charge or hydrophobicity), such that the functional properties of the molecule are not changed. When sequences differ by conservative substitutions, the percent sequence identity is adjusted upward to correct for the conservative nature of the substitution. Means for making this adjustment are well known to those of skill in the art. Typically, this involves scoring conservative substitutions as partial mismatches rather than complete mismatches, thereby increasing the percentage of sequence identity. Thus, for example, if a score of 1 is assigned to identical amino acids and a score of 0 is assigned to non-conservative substitutions, a score between 0 and 1 is assigned to conservative substitutions. Scoring of conservative substitutions is calculated, for example, according to the algorithm of Meyers & Miller, Computer Applic. Biol. Sci. 4:11-17 (1988), implemented in the program PC / GENE (Intelligenetics, Mountain View, CA, USA).

[0035] Sequences are "substantially identical" to each other when, aligned for maximum correspondence over a comparison window, they have the same specific percentage of nucleotide or amino acid residues (e.g., at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity over a specified region, or, if no region is specified, over the entire specified sequence).

[0036] For sequence comparison, typically one sequence acts as a reference sequence to which the test sequence is compared. When using a sequence comparison algorithm, the test and reference sequences are input into a computer, subsequence coordinates are designated, and, if necessary, sequence algorithm program parameters are designated. Either the default program parameters or alternative parameters can be used. The sequence comparison algorithm then calculates the percent sequence identity of the test sequence relative to the reference sequence based on the program parameters.

[0037] The "comparison window" used herein includes reference to any one segment of a number of contiguous positions selected from the group consisting of from 10 to 600, about 10 to about 300, about 10 to about 150, and the sequences are compared to the reference sequence for the same number of contiguous positions after the two sequences are optimally aligned. The comparison window can also be the entire length of either the reference or test sequence.

[0038] The percent sequence identity and sequence similarity can be determined using the BLAST 2.0 algorithm described in Altschul et al. (J. Mol. Biol. 215:403-10, 1990). Software for performing BLAST 2.0 analysis is publicly available through the National Center for Biotechnology Information (see Worldwide Website: ncbi.nlm.nih.gov / ). This algorithm involves first identifying high-scoring sequence pairs (HSPs) by identifying short words of length W in the query sequence, which either match or satisfy some positive-valued threshold score T when aligned with words of the same length in the database sequence. T is referred to as the neighborhood word score threshold (Altschul et al., supra). These initial neighborhood word hits serve as seeds to initiate a search for longer HSPs that contain them. The word hits are extended in both directions along each sequence as long as the cumulative alignment score increases. The extension of the word hits in each direction stops when the cumulative alignment score drops from its maximum achieved value by an amount X, or when the cumulative score goes below zero due to the accumulation of one or more negative-scoring residue alignments, or when the end of either sequence is reached. The BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment. The BLAST program, by default, uses a word length (W) of 11, the BLOSUM62 scoring matrix (see Henikoff & Henikoff, Proc Natl Acad Sci USA 89:10915 (1989)), an alignment (B) of 50, an expectation value (E) of 10, M = 5, N = -4, and a comparison of both strands.

[0039] The BLAST algorithm also performs a statistical analysis of the similarity between two sequences (see, e.g., Karlin & Altschul, Proc. Nat'l Acad. Sci. USA 90:5873-5787 (1993)). One measure of similarity provided by the BLAST algorithm is the minimum total probability (P(N)), which indicates the probability that a matching between two nucleotide sequences or amino acid sequences occurs by chance. For example, if the minimum total probability in a comparison between a test nucleic acid and a reference nucleic acid is less than about 0.2, more preferably less than about 0.01, and most preferably less than about 0.001, the nucleic acid is considered to be similar to the reference sequence.

[0040] The term "label" or "detectable label" refers to a composition detectable by spectroscopic, photochemical, biochemical, immunochemical, chemical, or other physical means. For example, useful labels include fluorescent dyes (fluorophores), fluorescence quenchers, chemiluminescent agents, high electron density reagents, enzymes (such as those commonly used in ELISA), biotin, digoxigenin, 32 P and other isotopes, haptens, proteins, nucleic acids, or other substances that can be made detectable, for example, by incorporating the label into an oligonucleotide or peptide. The term includes combinations of a single labeling agent, for example, combinations of phosphors that provide a unique detectable signature at a particular wavelength or combination of wavelengths.

[0041] antigen-binding protein In one embodiment, the antigen-binding protein comprises two or more first antigen-binding fragments each comprising a first binding motif and a first fusion protein comprising two or more second binding motifs linked by a linker sequence (see, e.g., “Bicatcher” such as SpyCatcher-SpyCatcher or “Multicatcher”, FIG. 1). The first binding motifs of the two or more first antigen-binding fragments are covalently bound to the two or more second binding motifs via protein ligation. To generate the antigen-binding protein, a first antigen-binding fragment is generated using the first binding motif, a first fusion protein comprising two or more second binding motifs linked by a linker sequence is generated, and the antigen-binding fragment-first binding motif protein is mixed with the first fusion protein comprising two or more second binding motifs under conditions appropriate to facilitate protein ligation of the first and second binding motifs. In some embodiments, the first fusion protein is a dimer. In some embodiments, the antigen-binding protein is a Fab dimer or multimer. In this embodiment of the antigen-binding protein, the first binding motif comprises SEQ ID NO: 1, 4, 6, 8, 10, 13, or 22, or a sequence having at least 60% sequence identity with SEQ ID NO: 1, 4, 6, 8, 10, 13, or 22, and the second binding motif comprises SEQ ID NO: 2, 3, 5, 7, 9, 11, 12, 14, or 23, or a sequence having at least 60% sequence identity with SEQ ID NO: 2, 3, 5, 7, 9, 11, 12, 14, or 23. The first and second binding motifs are selected such that the two binding motifs form a pair of reactive or cognate binding motifs with each other. For example, when the first binding motif is SEQ ID NO: 1 (SpyTag), the second binding motif is SEQ ID NO: 2, 3, 5, or 23 (SpyCatcher, SpyCatcher short, SpyCatcher002, or SpyCatcher003), and since the SpyTag / SpyCatcher system is orthogonal to the SnoopTag / SnoopCatcher system, it is not SEQ ID NO: 7 or 9 (SnoopCatcher or SplitSpy128).It should also be noted that the components of the pair of mutually reactive motifs or the pair of cognate binding motifs are exchangeable on the fusion protein and the antigen-binding fragment (for example, according to one embodiment, an antigen-binding fragment containing SpyTag and a fusion protein containing SpyCatcher, SpyCatcher short, SpyCatcher002 or SpyCatcher003 are provided, and according to a modified embodiment, an antigen-binding fragment containing SpyCatcher, SpyCatcher short, SpyCatcher002 or SpyCatcher003 and a fusion protein containing SpyTag are provided). As used herein, the term "orthogonal" refers to a pair of binding motifs that are non-reactive or non-cognate with each other (i.e., SpyTag and SpyCatcher cannot react with either SnoopCatcher or SnoopTag to form an isopeptide bond). Exemplary first and second binding motifs for Bicatcher and Multicatcher are shown in Table 1.

[0042] [Table 1]

[0043] In some embodiments, the antigen-binding protein further comprises a polypeptide (e.g., a protein or protein fragment having additional functionality) comprising a third binding motif and a fourth binding motif linked to a first fusion protein by a linker sequence (Figure 2). The third binding motif is linked by the linker sequence to the N-terminus or C-terminus of the first fusion protein (e.g., "Bicatcher-SnoopCatcher" (SpyCatcher-SpyCatcher-SnoopCatcher) or "SnoopCatcher-Bicatcher" (SnoopCatcher-Spycatcher-Spycatcher)), or between the second binding motifs of the first fusion protein (e.g., Spycatcher-SnoopCatcher-Spycatcher). The third binding motif is covalently linked to the fourth binding motif via protein ligation. In this embodiment, the first binding motif-second binding motif pair is orthogonal to the third binding motif-fourth binding motif pair. To generate the antigen-binding protein, a first antigen-binding fragment is generated using the first binding motif, a first fusion protein is generated using the second and third binding motifs, and the polypeptide is generated using the fourth binding motif. In certain embodiments, the first antigen-binding fragment comprising the first binding motif, the first fusion protein comprising the second and third binding motifs, and the polypeptide comprising the fourth binding motif are mixed under appropriate conditions to promote protein ligation of the first binding motif to the second binding motif and the third binding motif to the fourth binding motif. In some embodiments, the first antigen-binding fragment comprising the first binding motif and the first fusion protein comprising the second and third binding motifs are mixed under appropriate conditions to promote protein ligation of the first binding motif to the second binding motif. The polypeptide comprising the fourth binding motif is then added to the mixture of the ligated first binding motif and second binding motif under appropriate conditions to promote protein ligation of the third binding motif to the fourth binding motif, or vice versa.In certain embodiments, the polypeptide is an enzyme, a fluorescent protein, an effector protein, an antigen-binding fragment, or any polypeptide used to detect the binding of an antigen-binding protein to a target. In some embodiments, the antigen-binding protein is a dimeric Fab conjugated to an additional protein or protein fragment.

[0044] In one embodiment, the antigen-binding protein comprises one or more first antigen-binding fragments each comprising a first binding motif, a first fusion protein comprising one or more second binding motifs linked by a linker sequence to one or more third binding motifs, and one or more second antigen-binding fragments each comprising a fourth binding motif. The first binding motif is covalently bound to the second binding motif, the third binding motif is covalently bound to the fourth binding motif, and the first binding motif-second binding motif pair is orthogonal to the third binding motif-fourth binding motif pair. The paired motifs are covalently bound to each other by protein ligation. In some embodiments, the second antigen-binding fragment has a different specificity than the first antigen-binding fragment, i.e., the second antigen-binding fragment recognizes a different antigen or a different epitope on the same antigen than the first antigen-binding fragment. The antigen-binding protein can be bispecific (e.g., "Heterocatcher" or SpyCatcher-SnoopCatcher, Figure 3), bispecific and dimeric (e.g., "Heterobicatcher" or SpyCatcher-SpyCatcher-SnoopCatcher-SnoopCatcher, Figure 4), or bispecific and multimeric (Figure 4). "Heterobicatcher" can have binding motifs to each other in a random or sequential order, such as, for example, SpyCatcher-SpyCatcher-SnoopCatcher-SnoopCatcher, SnoopCatcher-SnoopCatcher-SpyCatcher-SpyCatcher, SpyCatcher-SnoopCatcher-SpyCatcher-SnoopCatcher, SpyCatcher-SnoopCatcher-SnoopCatcher-SpyCatcher, or SnoopCatcher-SpyCatcher-SpyCatcher-SnoopCatcher. In certain embodiments, the antigen-binding protein is a bispecific Fab comprising two antigen-binding fragments each having a different specificity.In some embodiments, the antigen-binding protein comprises four antigen-binding fragments and is a bispecific dimer Fab that binds to two different antigens or different epitopes on the same antigen. In some embodiments, the antigen-binding protein is a bispecific and multimeric Fab that comprises four or more antigen-binding fragments and two specificities (e.g., the antigen-binding fragments are specific for two different antigens or for two different epitopes on the same antigen). To generate the antigen-binding protein, a first antigen-binding fragment is generated using a first binding motif, a first fusion protein is generated using the second and third binding motifs, and a second antigen-binding fragment is generated using a fourth binding motif. In certain embodiments, the first antigen-binding fragment comprising the first binding motif, the first fusion protein comprising the second and third binding motifs, and the polypeptide comprising the fourth binding motif are mixed under appropriate conditions to facilitate protein ligation of the first binding motif to the second binding motif and the third binding motif to the fourth binding motif. In some embodiments, the first antigen-binding fragment comprising the first binding motif and the first fusion protein comprising the second and third binding motifs are mixed under appropriate conditions to facilitate protein ligation of the first binding motif to the second binding motif. Then, the polypeptide comprising the fourth binding motif is added to the mixture of the ligated first binding motif and the second binding motif under appropriate conditions to facilitate protein ligation of the third binding motif to the fourth binding motif, or vice versa. In some embodiments, the antigen-binding protein is a bispecific antigen-binding fragment such as Fab or a heterodimer.

[0045] In some embodiments, one or more of the binding motifs are located at the C-terminus, N-terminus of the first and / or second antigen-binding fragment, fusion protein, or polypeptide comprising a second function, or are embedded within its amino acid sequence. In certain embodiments, one or more of the binding motifs in the fusion protein are in a continuous or random order. In certain embodiments, the antigen-binding fragment, fusion protein and / or antigen-binding protein further comprise a purification tag at their N-terminus or C-terminus.

[0046] In embodiments of an antigen-binding protein having first, second, third, and fourth binding motifs, the first binding motif comprises SEQ ID NO: 1, 4, 8, or 22, or a sequence having at least 60% sequence identity with SEQ ID NO: 1, 4, 8, or 22, the second binding motif comprises SEQ ID NO: 2, 3, 5, 9, 12, or 23, or a sequence having at least 60% sequence identity with SEQ ID NO: 2, 3, 5, 9, 12, or 23. The third binding motif comprises SEQ ID NO: 6, 10 or 13, or a sequence having at least 60% sequence identity to SEQ ID NO: 6, 10 or 13, and the fourth binding motif comprises SEQ ID NO: 7, 11 or 14, or a sequence having at least 60% sequence identity to SEQ ID NO: 7, 11 or 14. In these embodiments, the first binding motif - second binding motif pair is orthogonal to the third binding motif - fourth binding motif pair. In some embodiments, the first binding motif is covalently linked to the second binding motif, and / or the third binding motif is covalently linked to the fourth discovery motif, with the help of a naturally occurring or enzyme (e.g., ligase). As described in paragraph

[0037] , the pair of cross-reactive motifs or cognate binding motif pairs are exchanged / reversed on the fusion protein and antigen-binding fragment (or polypeptide) provided that one component of the cognate binding motif pair is provided by the antigen-binding fragment or polypeptide and the second component of the cognate binding motif pair is provided by the fusion protein (and vice versa). Exemplary binding motifs of embodiments having first, second, third, and fourth binding motifs are shown in Table 2.

[0047]

Table 2

[0048] In various embodiments, the antigen-binding protein can further include a detectable label. Examples of detectable labels include, but are not limited to, phosphors, fluorescent proteins such as green fluorescent protein (GFP), biotin, enzymes such as horseradish peroxidase (HRP) or other peroxidases, alkaline phosphatase, luciferase, and split fluorescent proteins (e.g., split GFP) or enzymes (e.g., NanoLuc® Binary Technology from Promega). Exemplary phosphors include Alexa dyes (e.g., Alexa350, Alexa488, etc.), AMCA, BODIPY630 / 650, BODIPY650 / 665, BODIPY-FL, BODIPY-R6G, BODIPY-TMR, BODIPY-TRX, cascade blue, Cy2, Cy3, Cy5, Cy5.5, Cy7, Cy7.5, Dylight dyes (Dylight405, Dylight488, Dylight549, Dylight550, Dylight649, Dylight680, Dylight750, Dylight800), 6-FAM, fluorescein, FITC, HEX, 6-JOE, Oregon Green 488, Oregon Green 500, Oregon Green 514, Pacific Blue, REG, rhodamine green, rhodamine red, ROX, R-phycoerythrin (R-PE), Starbright Blue dyes (e.g., Starbright Blue 520, Starbright Blue 700), TAMRA, TET, tetramethylrhodamine, Texas Red, and TRITC, but are not limited thereto.

[0049] As used herein, "linker sequence" or "linker" refers to a peptide or polypeptide comprising two or more amino acid residues linked by peptide bonds that provides increased rotational freedom to two linked polypeptides compared to what the two linked polypeptides would have in the absence of the linker, such that each component of the fusion protein can interact with the target of interest without interference. Generally, these linkers are mixtures of glycine and serine such as -(GGGS)n- (where n is 1, 2, 3, 4, or 5). Other suitable peptide / polypeptide linker sequences optionally include peptides or polypeptides that are either naturally occurring or non-naturally occurring. The peptide linker sequence is at least two amino acids in length. Optionally, the peptide or polypeptide domain is a flexible peptide or polypeptide. Examples of flexible peptides / polypeptides include, but are not limited to, the amino acid sequences Gly-Ser, Ala-Ser, Gly-Ser, Gly4-Ser, (Gly4-Ser)2, (Gly4-Ser)3, (Gly4-Ser)4, (Gly4-Ser)Gly4, 2-Gly-Ala-Gly-SerGly4-Ser, Gly-(Gly4-Ser)2, Gly4-Ser-Gly, Gly-Ser3, Gly-SerGly4-Ser. Other suitable peptide linker domains optionally include the TEV linker ENLYFQG, a linear epitope recognized by tobacco etch virus protease. Examples of peptides / polypeptides include, but are not limited to, GSENLYFQGSG. Other suitable peptide linker sequences include Ala-(Glu-Ala-Ala-Lys) nIt includes a helix-forming linker such as -Ala (n = 1 - 5). In some embodiments, the linker sequence is a GAP (Gly Ala Pro) sequence. In some embodiments, a sequence of 1 to 50 amino acid residues can be used as the linker. In some embodiments, such linkers are soluble, flexible, and protease-resistant (i.e., expression of the polypeptide with the linker in the host cell occurs without cleavage of the linker by proteases). As described above, the phrase "linked by a linker sequence" enables the use of one or more linker sequences to link two or more binding motifs, a binding motif and a polypeptide, or a binding motif and an antigen-binding fragment. When multiple linker sequences are used to link binding motifs, to link an antigen-binding fragment to a binding motif, or to link a polypeptide and a binding motif, the linker sequences can be the same or different.

[0050] In some embodiments of an antigen-binding protein having first, second, third, and fourth binding motifs, sortase-mediated ligation is used to ligate the binding motifs. Sortase-mediated ligation for site-specific modification of proteins is described in Schmohl et al. (2014), which is hereby incorporated by reference in its entirety. The sortase system uses a sortase enzyme and a sortase recognition and cross-linking domain. In embodiments of the antigen-binding protein, the sortase recognition domain and the cross-linking domain are considered binding motifs. Sortase is a transpeptidase produced by Gram-positive bacteria that covalently attaches cell surface proteins to the cell wall. Staphylococcus aureusSortase A (SrtA) cleaves a short C-terminal recognition motif (LPXTG (SEQ ID NO: 19)) (referred to herein as the sortase recognition domain). The sortase recognition domain is a sortase A recognition domain or a sortase B recognition domain. The sortase A recognition domain includes the amino acid sequences LPTGAA (SEQ ID NO: 15), LPTGGG (SEQ ID NO: 16), LPKTGG (SEQ ID NO: 17), LPETG (SEQ ID NO: 18), LPXTG (SEQ ID NO: 19) or LPXTG(X) n (SEQ ID NO: 20), where X is any amino acid and n is any integer from 0, 1, 2, 3, 4, 5, 7, 8, 9, 10, 0 to 5 or 0 to 10, or up to 100. The sortase B recognition domain includes the amino acid sequence NPX1TX2 (SEQ ID NO: 21), where X1 is glutamine or lysine, X2 is asparagine or glycine, N is asparagine, P is proline, and T is threonine.

[0051] The sortase A and B cross-linking domains contain one or more glycine residues at the N-terminus of the peptide. In certain embodiments, the one or more glycine residues may optionally be Gly, (Gly)2, (Gly)3, (Gly)4, or (Gly) x where x is an integer from 1 to 20.

[0052] In some embodiments of an antigen-binding protein comprising a third binding motif and a fourth binding motif, the fourth binding motif comprises a sortase A or B recognition domain and the third motif comprises a sortase A or B cross-linking domain. The sortase A or B recognition domain is optionally fused to the polypeptide at the C-terminus via a glycine / serine-rich linker, and the sortase A or B cross-linking domain is optionally fused to the N-terminus of the first fusion protein via a glycine / serine-rich linker.

[0053] In some embodiments of the antigen-binding protein comprising the third binding motif and the fourth binding motif, the third binding motif comprises a sortase A or B recognition domain, and the fourth binding motif comprises a sortase A or B crosslinking domain. The sortase A or B recognition domain is optionally fused to the C-terminal antigen-binding fragment via a glycine / serine-rich linker, and the sortase A or B crosslinking domain is optionally fused to the second binding motif via a glycine / serine-rich linker.

[0054] Accordingly, in certain embodiments of the antigen-binding protein having the first, second, third, and fourth binding motifs, the fourth binding motif is an amino acid sequence LPTGAA (SEQ ID NO: 15), LPTGG (SEQ ID NO: 17), LPETG (SEQ ID NO: 18), LPXTG (SEQ ID NO: 19), or LPXTG(X)n (SEQ ID NO: 20) (wherein X is any amino acid and n is an integer in the range of 0, 1, 2, 3, 4, 5, 7, 8, 9, 10, or any integer from 0 to 5), NPX1TX2 (SEQ ID NO: 21) (wherein X1 is glutamine or lysine, X2 is asparagine, P is proline, and T is threonine), comprising or consisting of a sortase recognition domain, and the third binding motif is Gly, (Gly)2, (Gly)3, (Gly)4, or (Gly) x (wherein x is an integer from 1 to 20), comprising or consisting of a sortase crosslinking domain.

[0055] Methods for generating antigen-binding proteins are also provided. In one embodiment, the method comprises contacting two or more first antigen-binding fragments, each comprising a first binding motif, with a first fusion protein comprising two or more second binding motifs linked by a linker sequence. The conditions for contacting the two or more first antigen-binding fragments with the first fusion protein are such that a covalent bond is formed between the first binding motif and the second binding motif via protein ligation. In embodiments where the first fusion protein further comprises a third binding motif and the antigen-binding protein further comprises a polypeptide comprising a fourth binding motif, after ligating the first binding motif to the second binding motif, the first fusion protein comprising the third binding motif is contacted with a polypeptide comprising the fourth binding motif. The contacting is performed under conditions that allow the third binding motif to undergo protein ligation to the fourth binding motif, either spontaneously or with the aid of an enzyme.

[0056] In some embodiments, the method for preparing an antigen-binding protein comprises contacting one or more first antigen-binding fragments, each comprising a first binding motif, with a first fusion protein comprising one or more second binding motifs linked by a linker sequence to one or more third binding motifs to form the antigen-binding protein. The contacting is performed under conditions that allow the first binding motif to covalently bond to the second binding motif via protein ligation, either spontaneously or with the aid of an enzyme. After ligating the first binding motif to the second binding motif, one or more second antigen-binding fragments, each comprising a fourth binding motif, are contacted with the ligated first antigen-binding fragment - first fusion protein under conditions that also allow the third binding motif to covalently bond to the fourth binding motif via protein ligation, either spontaneously or with the aid of an enzyme.

[0057] In a method for generating an antigen-binding protein having first, second, third, and fourth binding motifs, the first binding motif-second binding motif pair is orthogonal to the third binding motif-fourth binding motif pair.

[0058] As used herein, the term "protein ligation" refers to site-specific covalent bond formation, either spontaneous or with the aid of an enzyme, between the first and second binding motifs and between the third and fourth binding motifs when the first and second or third and fourth motifs come into contact with each other. Also, as described in the present disclosure, protein ligation occurs between specific combinations of binding motifs, for example, between SpyTag (SEQ ID NO: 1), SpyTag002 (SEQ ID NO: 4) or SpyTag003 (SEQ ID NO: 22), and SpyCatcher (SEQ ID NO: 2), SpyCatcher short (SEQ ID NO: 3), SpyCatcher002 (SEQ ID NO: 5) or SpyCatcher003 (SEQ ID NO: 23), between SnoopTag (SEQ ID NO: 6) and SnoopCatcher (SEQ ID NO: 7), between Isopeptag (SEQ ID NO: 8) and Split Spy0128 (SEQ ID NO: 9), between SpyTag (SEQ ID NO: 10) and SdyCatcherDANGshort (SEQ ID NO: 11), between SpyTag and K-Tag (SEQ ID NO: 12), between SnoopTagJr (SEQ ID NO: 13) and DogTag (SEQ ID NO: 14), between the sortase recognition domain (SEQ ID NOs: 15-21) and the sortase bridging domain (Gly, (Gly)2, (Gly)3, (Gly)4, or (Gly) x (wherein x is an integer from 1 to 20), and between the butelase recognition motif (Asn-His-Val or Asp-His-Val) and the amino terminus of another polypeptide.

[0059] Thus, to generate an antigen-binding protein, a first binding motif present in a first antigen-binding fragment (e.g., embedded in the C-terminus, N-terminus, or amino acid sequence) can form a covalent bond via protein ligation to a second binding motif in the first fusion protein. For example, if the first binding motif present in the first antigen-binding fragment is SpyTag, SpyTag002, or SpyTag003, the corresponding second binding motif is SpyCatcher, SpyCatcher002, or SpyCatcher003 present in the first fusion protein. Alternatively, if the first binding motif present in the first antigen-binding fragment is SpyCatcher, SpyCatcher002, or SpyCatcher003, the corresponding second binding motif is SpyTag, SpyTag002, or SpyTag003 present in the first fusion protein.

[0060] Similarly, if the third binding motif in the first fusion protein is SnoopCatcher, the corresponding fourth binding motif (e.g., embedded in the C-terminus, N-terminus, or amino acid sequence) present in the second antigen-binding fragment is SnoopTag. Alternatively, if the third binding motif in the first fusion protein is SnoopTag, the corresponding fourth binding motif present in the second antigen-binding fragment is SnoopCatcher.

[0061] Furthermore, if the first binding motif present in the first antigen-binding fragment is a Spy tag, the corresponding second binding motif in the first fusion protein is a K-Tag. Alternatively, if the first binding motif present in the first antigen-binding fragment is a K-Tag, the corresponding second binding motif in the first fusion protein is SpyTag. In both cases, SpyLigase is required to catalyze the formation of an isopeptide bond between the two motifs.

[0062] Similarly, when the third binding motif is the DogTag in the first fusion protein, the corresponding fourth binding motif present in the second antigen-binding fragment is the SnoopTagJr. Alternatively, when the third binding motif is the SnoopTagJr in the first fusion protein, the corresponding fourth binding motif present in the second antigen-binding fragment is the DogTag. In both cases, SnoopLigase is required to catalyze the formation of an isopeptide bond between the two motifs.

[0063] Thus, pairs of motifs (i.e., the first binding motif paired with the second binding motif and the third binding motif paired with the fourth binding motif) are selected such that the two motifs interact with each other via protein ligation to form a covalent bond either spontaneously or with the help of an enzyme. The two pairs of motifs are also selected such that the pairs do not interact with each other or are orthogonal, i.e., the SpyTag / SpyCatcher system components do not interact with the SnoopTag / SnoopCatcher system components.

[0064] Expression of the proteins having the binding motifs can be Escherichia coli performed in suitable host cells including prokaryotic cells such as E. coli, or yeast, or mammalian cells, i.e., eukaryotic cells such as CHO cells. In certain embodiments, the disclosed proteins comprising various binding motifs are produced in protease-deficient prokaryotic cells such as protease-deficient E. coli. In various embodiments, the protease-deficient prokaryotic cells (e.g., protease-deficient E. coli cells) are periplasm protease-deficient.

[0065] Accordingly, a protein ligation system for generating an antigen-binding protein is provided, which includes adding a first binding motif to an antigen-binding fragment, adding two or more second binding motifs to each other via a linker to produce a fusion protein, and covalently bonding the antigen-binding fragment containing the first binding motif and the fusion protein containing the second binding motif via protein ligation between the first binding motif and the second binding motif. A third binding motif or a multimeric third binding motif can be added to the fusion protein containing the second binding motif via a linker, and a fourth binding motif can be added to a fourth polypeptide such as an enzyme, a fluorescent protein, an effector protein, or another antigen-binding fragment. The fusion protein containing the second and third binding motifs can be covalently bonded to the antigen-binding fragment containing the first binding motif and the fourth polypeptide via protein ligation between the first and second binding motifs and the third and fourth binding motifs to form an antigen-binding protein.

[0066] Examples of the protein ligation system include, but are not limited to, the SpyTag / SpyCatcher system, SpyTag with a shorter version of SpyCatcher, the SpyTag002 / SpyCatcher002 and SpyTag003 / SpyCatcher003 systems with accelerated reactions, the SpyTag / K-tag / SpyLigase system, the Isopeptag / Split Spy0128 system, the SnoopTag / SnoopCatcher system, the SdyTag / SdyCatcher system, and the SnoopTagJr / DogTag / SnoopLigase system.

[0067] Accordingly, a first antigen-binding fragment comprising an antigen-binding fragment is generated, capable of forming a covalent bond via a first binding motif and protein ligation, and as described in paragraph

[0062] , a fusion protein comprising a multimer (e.g., two or more) second binding motif that is one component of a protein ligation system is generated. The first antigen-binding fragment comprising the first binding motif can be mixed with the fusion protein comprising the multimer second binding motif to generate an antigen-binding protein. A linker is optionally used to bind together the multimer second binding motifs in the fusion protein. In some embodiments, the fusion protein further comprises one or more third binding motifs of a second protein ligation system. A linker is optionally used to incorporate one or more third binding motifs into the fusion protein. A polypeptide comprising a fourth binding motif or a second antigen-binding fragment comprising the fourth binding motif (of the second protein ligation system) is generated. The first antigen-binding fragment comprising the first binding motif is mixed with the fusion protein comprising the multimer second binding motif and one or more third binding motifs under conditions where an antigen-binding protein is generated by protein ligation. In certain embodiments, the polypeptide comprising the fourth binding motif or the second antigen-binding fragment comprising the fourth binding motif and the first antigen-binding fragment comprising the first binding motif are mixed with the fusion protein comprising the multimer second binding motif and one or more third binding motifs under conditions where an antigen-binding protein can be generated by protein ligation. Alternative embodiments provide a fusion protein comprising at least one second binding motif and at least one third binding motif, optionally linked by one or more linker sequences, used in the methods described above to generate an antigen-binding protein.

[0068] To generate an antigen-binding protein, any of the protein ligation systems described above or known in the art can be used.

[0069] For example, in certain embodiments, as a first binding motif, a first antigen-binding fragment comprising SpyTag (e.g., embedded in the C-terminus, N-terminus, or amino acid sequence) is generated, and as a second binding motif, a fusion protein comprising a multimeric second binding motif is generated using SpyCatcher. The first antigen-binding fragment - first binding motif fusion protein and the multimeric second binding motif fusion protein are mixed with each other under conditions in which an antigen-binding protein is generated by protein ligation. Alternatively, as a first binding motif, an antigen-binding fragment comprising SpyCatcher (instead of SpyTag), and as a second binding motif, a fusion protein comprising two or more SpyTags (instead of SpyCatcher) can be generated. The antigen-binding fragment - first binding motif fusion protein and the multimeric fusion protein are mixed with each other under conditions in which an antigen-binding fragment is generated by protein ligation.

[0070] In some embodiments, as a first binding motif, a first antigen-binding fragment comprising a SpyTag is generated, and one or more second binding motifs linked to one or more third binding motifs are generated as a fusion protein of SpyCatcher as the second binding motif and SnoopCatcher as the third binding motif (SpyCatcher and SnoopCatcher are linked in any order). As a fourth binding motif, a polypeptide comprising a SnoopTag or a second antigen-binding fragment comprising a SnoopTag (e.g., embedded in the C-terminus, N-terminus, or amino acid sequence) is generated. The first antigen-binding fragment, the fusion protein, and the second antigen-binding fragment (or polypeptide) are mixed with each other under conditions that allow an antigen-binding protein comprising the first antigen-binding fragment comprising a SpyTag ligated to the SpyCatcher (second binding motif) of the fusion protein and the second antigen-binding fragment (or polypeptide) comprising a SnoopTag ligated to the SnoopCatcher (third binding motif) of the fusion protein to be generated by protein ligation. In certain embodiments, a first antigen-binding fragment comprising a SpyTag (as the first binding motif) is generated, and a fusion protein comprising one or more SpyCatchers as the second binding motif and one or more SnoopTags as the third binding motif in a random or sequential order is generated, and a second antigen-binding fragment (having the same or different specificity as the first antigen-binding fragment) comprising a SnoopCatcher as the fourth binding motif is generated. The first antigen-binding fragment comprising a SpyTag as the first binding motif and the multimeric SpyCatcher n -SnoopTag m The second antigen-binding fragment comprising the fusion protein (where n and m are greater than or equal to 1) and a SnoopCatcher as the fourth binding motif is SpyCatcher n -SnoopTag mA first antigen-binding fragment comprising a SpyTag ligated to SpyCatcher of the fusion protein, and SpyCatcher n -SnoopTag m A second antigen-binding fragment comprising SnoopCatcher ligated to SnoopTag of the fusion protein, are mixed under conditions that allow an antigen-binding protein comprising them to be generated by protein ligation. Similarly, a first antigen-binding fragment comprising SpyCatcher and SpyTag n -SnoopTag m A second antigen-binding fragment comprising a fusion protein (where n and m are 1 or more) and SnoopCatcher, are such that SpyTag n -SnoopTag m A first antigen-binding fragment comprising SpyCatcher ligated to SpyTag of the fusion protein, and SpyTag n -SnoopTag m A second antigen-binding fragment comprising SnoopCatcher ligated to SnoopTag of the fusion protein, are generated and can be mixed with each other under conditions that allow an antigen-binding protein comprising them to be generated by protein ligation. Also, a first antigen-binding fragment comprising SpyCatcher and SpyTag n -SnoopCatcher m A second antigen-binding fragment comprising a fusion protein (where n and m are 1 or more) and SnoopTag, are such that SpyTag n -SnoopCatcher m A first antigen-binding fragment comprising SpyCatcher ligated to SpyTag of the fusion protein, SpyTag-SnoopCatcher mThe fusion protein can be produced under conditions in which an antigen-binding protein comprising a SnoopTag ligated to the SnoopCatcher of the fusion protein and a second antigen-binding fragment comprising the SnoopTag can be produced by protein ligation and mixed together. The SpyTags, SpyCatchers, SnoopTags, SnoopCatcher sequences, as well as the linker sequences described above, are also included in these embodiments.

[0071] The antigen-binding fragment containing SpyTag or SnoopTag is produced by expressing a gene encoding the antigen-binding fragment in E. coli, for example, using a vector in which SpyTag or SnoopTag is added to the C-terminus, N-terminus, or embedded within the amino acid sequence of the antigen-binding fragment. A second tag, such as a His-tag, can be added to purify the antigen-binding fragment containing SpyTag or SnoopTag by affinity chromatography. The antigen-binding fragment containing SpyTag or SnoopTag can also be purified without a second tag. In certain embodiments, the SpyTag has the sequence AHIVMVDAYKPTK (SEQ ID NO: 1) or VPTIVMVDAYKRYK (SEQ ID NO: 4). In some embodiments, the SnoopTag has the sequence KLGDIEFIKVNK (SEQ ID NO: 6).

[0072] (SpyCatcher) n (n is ≧2), (SnoopCatcher) n (n is ≧2), SpyCatcher n -SnoopCatcher m , SpyCatcher n -SnoopTag m , SpyTag n -SnoopCatcher m and SpyTag n -SnoopTag mMultimeric fusion proteins (i.e., multimeric binding motifs) containing (where n and m are 1 or greater) can also be produced by expressing a gene encoding a multimeric binding motif, each of which optionally, for example, Escherichia coli in may be linked by one or more linkers. Tags such as His-tags are also added to the N-terminus or C-terminus of the multimeric fusion protein to facilitate purification of the fusion protein by affinity chromatography. Protease cleavage sites such as TEV protease sites can also be added between the tag (e.g., His-tag) and the binding motif of the fusion protein to allow removal of the tag after affinity chromatography. An antigen-binding protein can be produced by mixing a first antigen-binding fragment containing a SpyTag motif and / or a second antigen-binding motif (or polypeptide) containing a SnoopTag binding motif with the multimeric fusion protein in an appropriate stoichiometry. For example, a Fab-SpyTag antigen-binding fragment and a SpyCatcher-SpyCatcher fusion protein can be mixed in a stoichiometry of two Fab-SpyTag molecules per SpyCatcher-SpyCatcher molecule to produce a dimeric antigen-binding protein.

[0073] For optimal binding by the SpyTag / SpyCatcher and SnoopTag / SnoopCatcher systems, appropriate conditions such as buffer conditions, pH, temperature, and the presence of detergents can be provided. The artificial antigen-binding proteins thus produced can be used as is or further purified prior to use. Such purification can be performed by size exclusion chromatography, affinity chromatography, other chromatography, or other separation techniques known in the art.

[0074] In certain embodiments, the conjugation is performed in the presence of excess Fab-SpyTag to drive the reaction towards formation of the antigen-binding protein. The resulting antigen-binding protein is purified to remove the excess Fab-SpyTag, for example, by using size-exclusion chromatography or by using a purification tag that was added to the multimeric fusion protein but not to the Fab-SpyTag protein.

[0075] In embodiments where the antigen-binding protein is a dimer linked to a polypeptide having an additional function, a SpyLigase and / or SnoopLigase protein ligation system is used. In such embodiments, an antigen-binding fragment comprising a SpyTag motif protein is generated as described above. For the multimeric binding motif, each of two or more second binding motifs (SpyCatcher) is replaced with a 10-amino acid K-Tag motif (SEQ ID NO: 12), in the same orientation, and optionally, with one or more linkers as described above. Alternatively or additionally, the third binding motif (SnoopCatcher) of the multimeric binding motif can be replaced with a 23-amino acid DogTag motif (SEQ ID NO: 14), and the fourth binding motif (SnoopTag) of the polypeptide can be replaced with a 12-amino acid SnoopTagJr motif (SEQ ID NO: 13). The antigen-binding fragment - SpyTag and K-Tag - K-Tag - DogTag multimeric binding motif are mixed in the presence of SpyLigase under conditions that allow ligation of the SpyTag and K-Tag. Then, a polypeptide comprising SnoopTagJr is added to the mixture in the presence of SnoopLigase under conditions that allow ligation of SnoopTagJr and DogTag. Thus, in certain such embodiments, the first binding motif comprises a SpyTag, the second binding motif comprises two or more K-tags, the third binding motif comprises a DogTag, and the fourth binding motif comprises a SnoopTagJr. Alternatively, the first binding motif can comprise a K-Tag, the second binding motif can comprise two or more SpyTags, the third binding motif can comprise a SnoopTagJr, and the fourth binding motif can comprise a DogTag. Similarly, the first binding motif can comprise a SpyTag, the second binding motif can comprise two or more K-Tags, the third binding motif can comprise a SnoopTagJr, and the fourth binding motif can comprise a DogTag.In addition, the first binding motif can include a K-Tag, the second binding motif can include two or more SpyTags, the third binding motif can include a DogTag, and the fourth binding motif can include a SnoopTagJr.

[0076] In some embodiments, the first binding motif includes a SpyTag, the second binding motif includes two or more K-Tags, the third binding motif includes a SnoopCatcher, and the fourth binding motif includes a SnoopTag. The antigen-binding fragment-SpyTag, K-Tag-K-Tag-SnoopCatcher multimeric binding motif, and polypeptide-SnoopTag can be mixed in the presence of SpyLigase under conditions that allow ligation of the SpyTag and K-Tag. Alternatively, the first binding motif includes a K-Tag, the second binding motif includes two or more SpyTags, the third binding motif includes a SnoopTag, and the fourth binding motif includes a SnoopCatcher, and then can be mixed in the presence of SpyLigase under conditions that allow ligation of cognate binding motif pairs.

[0077] In certain embodiments, the first binding motif comprises a SpyTag, the second binding motif comprises two or more SpyCatchers, the third binding motif comprises a DogTag, and the fourth binding motif comprises a SnoopTagJr. The antigen-binding fragment-SpyTag motif, the SpyCatcher-SpyCatcher-DogTag multimeric binding motif, and the polypeptide-SnoopTagJr motif are mixed in the presence of SnoopLigase under conditions that allow ligation of the DogTag motif and the SnoopTagJr motif. Alternatively, the first binding motif comprises a SpyCatcher, the second binding motif comprises two or more SpyTags, the third binding motif comprises a SnoopTagJr, and the fourth binding motif comprises a DogTag. Similarly, the first binding motif can comprise a SpyTag, the second binding motif can comprise two or more SpyCatchers, the third binding motif can comprise a SnoopTagJr, and the fourth binding motif can comprise a DogTag. Also, the first binding motif comprises a SpyCatcher, the second binding motif can comprise two or more SpyTags, the third binding motif can comprise a DogTag, and the fourth binding motif can comprise a SnoopTagJr.

[0078] Nucleic acid construct Also provided are nucleic acid constructs encoding an antigen-binding fragment fused to a binding motif and nucleic acid constructs encoding a multimeric binding motif. Such nucleic acids are present in an expression vector in a suitable host cell. As described below, the host cell can be a prokaryotic cell or a eukaryotic cell.

[0079] Thus, in one embodiment, a pair of nucleic acid constructs is a) a first nucleic acid construct comprising a polynucleotide sequence encoding a first antigen-binding fragment fused to a first binding motif, and b) A second nucleic acid construct comprising a polynucleotide encoding two or more second binding motifs, optionally linked by a linker sequence, the second nucleic acid construct comprising When the first binding motif and the second binding motif are brought into contact with each other spontaneously or with the aid of an enzyme, they form a covalent bond via ligation.

[0080] Typically, a polynucleotide sequence encoding a Fab fused to the first binding motif at the C-terminus encodes two peptides, namely the L-chain and the H-chain of the Fab. The first binding motif, such as SpyTag, is fused to either the L-chain or the H-chain. The Fab expression cassette comprises a bicistronic vector that generates one mRNA encoding both the L-chain and the H-chain, at least one of which is fused to a binding motif. Also, both the H-chain and the L-chain have a signal peptide for directing their transport to the periplasm.

[0081] In certain embodiments, the polynucleotide of the second nucleic acid construct optionally further encodes, by a linker sequence, two or more second binding motifs or a third binding motif linked therebetween. A third nucleic acid construct comprising a polynucleotide sequence encoding a polypeptide fused to a fourth binding motif can also be provided. The third binding motif ligates with the fourth binding motif when brought into contact spontaneously or with the aid of an enzyme. The first binding motif - second binding motif pair is orthogonal to the third binding motif - fourth binding motif pair.

[0082] In certain embodiments, the nucleic acid construct comprises a first nucleic acid construct comprising a polynucleotide sequence encoding one or more first antigen-binding fragments each fused to a first binding motif, and a second nucleic acid construct comprising a polynucleotide sequence encoding one or more second binding motifs and one or more third binding motifs. The second and third binding motifs are optionally linked by a linker as disclosed herein. A third nucleic acid construct can also be provided that comprises a polynucleotide sequence encoding one or more second antigen-binding fragments each fused to a fourth binding motif. The polynucleotide encoding any or all of the one or more second binding motifs is located before, after, or in between the polynucleotide encoding any or all of the one or more third binding motifs. The first antigen-binding fragment and the second antigen-binding fragment bind to the same or different antigens. The first binding motif and the second binding motif are protein ligated when brought into contact with each other either spontaneously or with the aid of an enzyme. The third binding motif and the fourth binding motif are protein ligated when brought into contact with each other either spontaneously or with the aid of an enzyme. The first binding motif-second binding motif pair is orthogonal to the third binding motif-fourth binding motif pair.

[0083] The nucleic acid constructs are typically introduced into a variety of vectors. The vectors of the invention generally include the transcriptional or translational control sequences necessary for the expression of the fusion proteins. Suitable transcriptional or translational control sequences include, but are not limited to, origins of replication, promoters, enhancers, repressor binding regions, transcription start sites, ribosome binding sites, translation start sites, and termination sites for transcription and translation.

[0084] The origin of replication (commonly referred to as the ori sequence) enables the replication of the vector within a suitable host cell. The choice of ori depends on the type of host cell and / or the genetic package being used. When the host cell is a prokaryote, the expression vector typically contains an ori sequence that directs autonomous replication of the vector within the prokaryotic cell. Preferred prokaryotic ori can direct vector replication in bacterial cells. Examples of this class of ori include, but are not limited to, pMB1, pUC, and other E. coli sources.

[0085] In eukaryotic systems, higher eukaryotes have multiple DNA origins of replication, but the ori sequences are not clearly defined. Origins of replication suitable for mammalian vectors are usually derived from eukaryotic viruses. Preferred eukaryotic ori include, but are not limited to, SV40 ori, EBV ori, or HSV ori.

[0086] As used herein, a "promoter" is a DNA region that, under specific conditions, can bind to RNA polymerase and initiate transcription of a coding region located downstream (in the 3' direction) of the promoter. It can be either constitutive or inducible. Generally, the promoter sequence is bound at its 3' end by the transcription start site and extends upstream (in the 5' direction) to include the minimum number of bases or elements necessary to initiate transcription at a detectable level above background. Within the promoter sequence are the transcription start site and protein-binding domains responsible for binding RNA polymerase. Eukaryotic promoters often, but not always, contain a "TATA" box and a "CAT" box.

[0087] The selection of a promoter depends largely on the host cell into which the vector is introduced. For prokaryotic cells, various robust promoters are known in the art. Preferred promoters are the lac promoter, Trc promoter, T7 promoter, and pBAD promoter. Usually, to obtain the expression of foreign sequences in multiple species, a prokaryotic promoter can be placed immediately after a eukaryotic promoter or within an intron sequence downstream of the eukaryotic promoter.

[0088] Suitable promoter sequences for eukaryotic cells include those for 3 - phosphoglycerate kinase, or other glycolytic enzymes such as enolase, glyceraldehyde - 3 - phosphate dehydrogenase, hexokinase, pyruvate decarboxylase, phosphofructokinase, glucose - 6 - phosphate isomerase, 3 - phosphoglycerate mutase, pyruvate kinase, triose phosphate isomerase, phosphoglucose isomerase, and glucokinase. Other promoters with the additional advantage of transcription being controlled by growth conditions are the promoters for alcohol dehydrogenase 2, isocytochrome C, acid phosphatase, catabolic enzymes related to nitrogen metabolism, and the aforementioned glyceraldehyde - 3 - phosphate dehydrogenase, as well as the enzymes responsible for the utilization of maltose and galactose. Preferred promoters for mammalian cells are the SV40 promoter, CMV promoter, β - actin promoter, and their hybrids. Preferred promoters for yeast cells include GAL 10, GAL I, S.cerevisiae TEF1 therein, and P.pastoris GAP, AOX1 therein, but are not limited thereto.

[0089] In the construction of the present vector, the termination sequence associated with the protein coding sequence is also inserted at the 3' end of the sequence to be transcribed in order to provide a polyadenylation and / or transcription termination signal for the mRNA. The terminator sequence preferably contains one or more transcription termination sequences (e.g., polyadenylation sequences) and can be lengthened by including additional DNA sequences to further disrupt transcriptional read-through. The preferred terminator sequence (or termination site) of the present invention has a gene followed by a transcription termination sequence of either its own termination sequence or a heterologous termination sequence. Examples of such termination sequences include stop codons linked to various yeast transcription termination sequences or mammalian polyadenylation sequences known in the art and widely available. When the terminator contains a gene, it is advantageous to use a gene encoding a detectable or selectable marker, thereby providing a means by which the presence and / or absence of the terminator sequence (and thus the corresponding inactivation and / or activation of the transcription unit) can be detected and / or selected.

[0090] , In addition to the above elements, the vector may contain a selectable marker (e.g., a gene encoding a protein necessary for the survival or growth of a host cell transformed with the vector), although such a marker gene is carried on a separate polynucleotide sequence co-introduced into the host cell. Only those host cells into which a selectable gene has been introduced will survive and / or grow under selective conditions. Typical selectable genes encode (a) proteins that confer resistance to antibiotics or other toxins, such as ampicillin, kanamycin, neomycin, zeocin, G418, methotrexate, etc., (b) proteins that complement auxotrophic deficiencies, or (c) proteins that supply essential nutrients not available from complex media. The selection of an appropriate marker gene depends on the host cell, and the appropriate genes for different hosts are known in the art.

[0091] In one embodiment, the expression vector is a shuttle vector that can replicate in at least two unrelated host systems. To facilitate such replication, the vector generally contains at least two origins of replication (one being effective in each host system). Typically, the shuttle vector can replicate in a eukaryotic host system and a prokaryotic host system. This enables the detection of protein expression in a eukaryotic host (expression cell type) and the amplification of the vector in a prokaryotic host (amplification cell type). Preferably, one origin of replication is derived from SV40 or 2u, and one is derived from pUC, but any suitable origin known in the art can be used if it can direct the replication of the vector. When the vector is a shuttle vector, the vector preferably contains at least two selectable markers (one for the expression cell type and one for the amplification cell type). Any selectable marker known in the art or the markers described herein can be used as long as they function in the expression system being utilized.

[0092] The vectors encompassed by the present invention can be obtained using recombinant cloning methods and / or by chemical synthesis. Numerous recombinant cloning techniques such as PCR, restriction endonuclease digestion, and ligation are well known in the art and need not be described in detail herein. One of ordinary skill in the art can also obtain the desired vector by any synthetic means available in the art using the sequence data provided herein or sequence data in public or proprietary databases. Further, well-known restriction and ligation techniques can be used to excise appropriate sequences from various DNA sources and incorporate them in operable relationship with the exogenous sequences to be expressed according to the embodiments described herein.

[0093] Kit Also provided is a kit for producing an antigen-binding protein. In some embodiments, the kit contains two or more of the following components. 1. A first antigen-binding fragment comprising a first binding motif, and 2. Optionally, a first fusion protein comprising two or more second binding motifs, which are linked by a linker sequence and optionally contain a detectable label (e.g., biotin, HRP, or a phosphor), and / or 3. Optionally, a first fusion protein comprising two or more second binding motifs linked by a linker sequence and, optionally, a third binding motif linked to the two or more second binding motifs by a linker sequence and optionally containing a detectable label, and / or, 4. Optionally, a polypeptide comprising a fourth binding motif and optionally containing a detectable label, and / or 5. A fusion protein comprising one or more second binding motifs and one or more third binding motifs, wherein the binding motifs are optionally linked by a linker sequence and optionally contain a detectable label, and / or 6. A second antigen-binding fragment comprising a fourth binding motif, and / or 7. A nucleic acid construct comprising a polynucleotide sequence encoding an antigen-binding fragment, fusion protein, and / or polypeptide as defined in 1-6.

[0094] The kit user can select at least two components of 1-6 above having at least one pair of binding motifs (i.e., a first binding motif - second binding motif pair and / or a third binding motif - fourth binding motif pair) that, when mixed, form a covalent bond through protein ligation, either spontaneously or with the help of an enzyme. When the kit user selects two pairs of binding motifs, the pairs of binding motifs are selected such that the first pair of binding motifs is orthogonal to the second pair of binding motifs, i.e., the first binding motif - second binding motif pair is orthogonal to the third binding motif - fourth binding motif pair. The kit user can also use a nucleic acid construct comprising a polynucleotide sequence encoding an antigen-binding fragment, fusion protein, and / or polypeptide as defined in 1-6 to express any peptide in a suitable host.

[0095] The components are provided in liquid form (e.g., as a solution) or solid (e.g., powder) that is reconstituted with a liquid, such as a buffer, before use. In some embodiments, the kit further includes instructions for ligating one or more binding motif pairs.

[0096] Additional disclosure and subject matter Item 1. Two or more first antigen-binding fragments comprising a first binding motif, and optionally a fusion protein comprising two or more second binding motifs linked by a linker sequence, wherein the first binding motif of the two or more first antigen-binding fragments is covalently bound to the two or more second binding motifs via protein ligation, an antigen-binding protein.

[0097] Item 2. The antigen-binding protein according to item 1, wherein the fusion protein comprising the two or more second binding motifs is linked by a linker sequence.

[0098] Item 3. The fusion protein optionally further comprises a third binding motif bound to the two or more second binding motifs by a linker, and the antigen-binding protein further comprises a polypeptide comprising a fourth binding motif, wherein the third binding motif is covalently bound to the fourth binding motif of the polypeptide via protein ligation, and the first binding motif - second binding motif pair is orthogonal to the third binding motif - fourth binding motif pair, the antigen-binding protein according to item 2.

[0099] Item 4. The polypeptide is an enzyme, a fluorescent protein, an effector protein, or an antigen-binding fragment, the antigen-binding protein according to item 3.

[0100] Item 5. One or more first antigen-binding fragments comprising a first binding motif, Optionally, a fusion protein comprising one or more second binding motifs and one or more third binding motifs linked by a linker, and one or more second antigen-binding fragments comprising a fourth binding motif, wherein the first binding motif is covalently linked to the second binding motif via protein ligation, wherein the third binding motif is covalently linked to the fourth binding motif via protein ligation, An antigen-binding protein in which the first binding motif-second binding motif pair is orthogonal to the third binding motif-fourth binding motif pair.

[0101] Item 6. The antigen-binding protein according to item 5, wherein the antigen-binding protein is bispecific, bispecific and dimer, or bispecific and multimer.

[0102] Item 7. The antigen-binding protein according to any one of the preceding items, further comprising a purification tag at the N-terminus or C-terminus of the fusion protein.

[0103] Item 8. The antigen-binding protein according to any one of the preceding items, wherein one or more binding motifs are located at the amino acid sequence of the first and / or second antigen-binding fragment, at the C-terminus or N-terminus of the fusion protein or polypeptide, or embedded in its amino acid sequence.

[0104] Item 9. The antigen-binding protein according to item 8, wherein the one or more binding motifs in the fusion protein are in a continuous or random order.

[0105] Item 10. The antigen-binding protein according to any one of the preceding items, wherein the linker sequence is 1 to 5 repeats of the sequence GGGS.

[0106] Item 11 a) The first binding motif comprises the sequence of SEQ ID NO: 1, 4, 6, 8, 10, 13, or 22, or a sequence having at least 60% sequence identity with SEQ ID NO: 1, 4, 6, 8, 10, 13, or 22, and the second binding motif comprises a sequence having at least 60% sequence identity with SEQ ID NO: 2, 3, 5, 7, 9, 11, 12, 14, or 23, or a sequence having at least 60% sequence identity with SEQ ID NO: 2, 3, 5, 7, 9, 11, 12, 14, or 23, or b) The first binding motif comprises the sequence of SEQ ID NO: 2, 3, 5, 7, 9, 11, 12, 14, or 23, or a sequence having at least 60% sequence identity with SEQ ID NO: 2, 3, 5, 7, 9, 11, 12, 14, or 23, and the second binding motif comprises the sequence of SEQ ID NO: 1, 4, 6, 8, 10, 13, or 22, or a sequence having at least 60% sequence identity with SEQ ID NO: 1, 4, 6, 8, 10, 13, or 22, the antigen-binding protein according to item 1 or 2.

[0107] Item 12 a) The first binding motif comprises the sequence of SEQ ID NO: 1, 4, 8, or 22, or a sequence having at least 60% sequence identity with SEQ ID NO: 1, 4, 8, or 22, and the second binding motif comprises the sequence of SEQ ID NO: 2, 3, 5, 9, 12, or 23, or a sequence having at least 60% sequence identity with SEQ ID NO: 2, 3, 5, 9, 12, or 23, or b) The first binding motif comprises the sequence of SEQ ID NO: 2, 3, 5, 9, 12, or 23, or a sequence having at least 60% sequence identity with SEQ ID NO: 2, 3, 5, 9, 12, or 23, and the second binding motif comprises the sequence of SEQ ID NO: 1, 4, 8, or 22, or a sequence having at least 60% sequence identity with SEQ ID NO: 1, 4, 8, or 22, the antigen-binding protein according to any one of items 3 to 6.

[0108] Item 13 a) The third binding motif comprises the sequence of SEQ ID NO: 7, 11, or 14, or a sequence having at least 60% sequence identity with the sequence of SEQ ID NO: 7, 11, or 14, and the fourth motif comprises the sequence of SEQ ID NO: 6, 10, or 13, or a sequence having at least 60% sequence identity with the sequence of SEQ ID NO: 6, 10, or 13, or b) The antigen-binding protein according to item 12, wherein the third binding motif comprises the sequence of SEQ ID NO: 6, 10, or 13, or a sequence having at least 60% sequence identity with the sequence of SEQ ID NO: 6, 10, or 13, and the fourth motif comprises the sequence of SEQ ID NO: 7, 11, or 14, or a sequence having at least 60% sequence identity with the sequence of SEQ ID NO: 7, 11, or 14.

[0109] Item 14 a) The first binding motif comprises the sequence of SEQ ID NO: 6, 10, or 13, or a sequence having at least 60% sequence identity with the sequence of SEQ ID NO: 6, 10, or 13, and the second binding motif comprises the sequence of SEQ ID NO: 7, 11, or 14, or a sequence having at least 60% sequence identity with the sequence of SEQ ID NO: 7, 11, or 14, or b) The antigen-binding protein according to any one of items 3 to 6, wherein the first binding motif comprises the sequence of SEQ ID NO: 7, 11, or 14, or a sequence having at least 60% sequence identity with the sequence of SEQ ID NO: 7, 11, or 14, and the second binding motif comprises the sequence of SEQ ID NO: 6, 10, or 13, or a sequence having at least 60% sequence identity with the sequence of SEQ ID NO: 6, 10, or 13.

[0110] Item 15 a) The third binding motif comprises the sequence of SEQ ID NO: 2, 3, 5, 9, 12, or 23, or a sequence having at least 60% sequence identity with the sequence of SEQ ID NO: 2, 3, 5, 9, 12, or 23, and the fourth binding motif comprises the sequence of SEQ ID NO: 1, 4, 8, or 22, or a sequence having at least 60% sequence identity with the sequence of SEQ ID NO: 1, 4, 8, or 22, or b) The third binding motif comprises the sequence of SEQ ID NO: 1, 4, 8, or 22, or a sequence having at least 60% sequence identity with SEQ ID NO: 1, 4, 8, or 22, and the fourth binding motif comprises the sequence of SEQ ID NO: 2, 3, 5, 9, 12, or 23, or a sequence having at least 60% sequence identity with SEQ ID NO: 2, 3, 5, 9, 12, or 23. The antigen-binding protein according to item 14.

[0111] Item 16 The antigen-binding protein according to item 3, wherein the fourth binding motif comprises a sortase recognition domain and the third binding motif comprises a sortase crosslinking domain.

[0112] Item 17 The antigen-binding protein according to item 5, comprising a first antigen-binding fragment containing a first binding motif, a second binding motif linked by a linker sequence to a third binding motif, and a second antigen-binding fragment containing a fourth binding motif, wherein the fourth binding motif comprises a sortase recognition domain and the third binding motif comprises a sortase crosslinking domain.

[0113] Item 18 The sortase recognition domain comprises the amino acid sequence: LPTGAA (SEQ ID NO: 15), LPTGGG (SEQ ID NO: 16), LPKTGG (SEQ ID NO: 17), LPETG (SEQ ID NO: 18), LPXTG (SEQ ID NO: 19) or LPXTG(X)n (SEQ ID NO: 20), wherein X is any amino acid, n is 0, 1, 2, 3, 4, 5, 7, 8, 9, 10, an arbitrary integer from 0 to 5 or 0 to 10, or up to 100, or NPX1TX2 (SEQ ID NO: 21), wherein X1 is glutamine or lysine, X2 is asparagine or glycine, N is asparagine, P is proline, T is threonine. The sortase crosslinking domain comprises Gly, (Gly)2, (Gly)3, (Gly)4, or (Gly) x wherein x is an integer from 1 to 20. The antigen-binding protein according to item 16 or 17.

[0114] Item 19. The antigen-binding protein according to any one of Items 1 to 5, wherein the fusion protein or polypeptide further comprises a detectable label.

[0115] Item 20. The antigen-binding protein according to Item 19, wherein the detectable label is a phosphor, a fluorescent protein, biotin, or an enzyme.

[0116] Item 21. a) A first nucleic acid construct comprising a polynucleotide sequence encoding a first antigen-binding fragment fused to a first binding motif; b) A second nucleic acid construct comprising a polynucleotide sequence encoding a fusion protein comprising two or more second binding motifs optionally linked by a linker sequence, wherein the first binding motif and the second binding motif form a covalent bond via protein ligation when brought into contact with each other either spontaneously or with the aid of an enzyme, a pair of nucleic acid constructs.

[0117] Item 22. The polynucleotide of the second nucleic acid construct further encodes three or more second binding motifs optionally linked by a linker sequence, or between them, a third binding motif optionally linked by a linker sequence, and the pair of nucleic acid constructs further comprises a third nucleic acid construct comprising a polynucleotide sequence encoding a polypeptide fused to a fourth binding motif, wherein the third binding motif and the fourth binding motif form a covalent bond via protein ligation when brought into contact with each other either spontaneously or with the aid of an enzyme, wherein the first binding motif-second binding motif pair is orthogonal to the third binding motif-fourth binding motif pair, the pair of nucleic acid constructs according to Item 21.

[0118] Item 23. a) A first nucleic acid construct comprising a polynucleotide sequence encoding one or more first antigen-binding fragments each fused to a first binding motif; b) Optionally, a second nucleic acid construct comprising a polynucleotide sequence encoding a fusion protein comprising one or more second binding motifs and one or more third binding motifs linked by a linker array, and c) A third nucleic acid construct comprising a polynucleotide sequence encoding one or more second antigen-binding fragments each fused to a fourth binding motif, and wherein the first binding motif and the second binding motif form a covalent bond via protein ligation when brought into contact with each other either spontaneously or with the aid of an enzyme, wherein the third binding motif and the fourth binding motif form a covalent bond via protein ligation when brought into contact with each other either spontaneously or with the aid of an enzyme, A nucleic acid construct, wherein the first binding motif - second binding motif pair is orthogonal to the third binding motif - fourth binding motif pair.

[0119] Item 24. The pair of nucleic acid constructs according to item 23, wherein the polynucleotide encoding any or all of the one or more second binding motifs is located before, after, or in between the polynucleotide encoding any or all of the one or more third binding motifs.

[0120] Item 25. A vector comprising the nucleic acid construct according to any one of items 21 to 24 or 33.

[0121] Item 26. A host cell having the nucleic acid construct and / or vector according to any one of items 21 to 25 or 33.

[0122] Item 27. A method for producing an antigen-binding protein, comprising contacting two or more first antigen-binding fragments each comprising a first binding motif with a fusion protein comprising two or more second binding motifs optionally linked by a linker array, A method, wherein said contacting is carried out under conditions that enable the first binding motif to covalently bind to the second binding motif via protein ligation, either spontaneously or with the aid of an enzyme.

[0123] Item 28 The fusion protein further comprises a third binding motif, and the antigen-binding protein further comprises a polypeptide comprising a fourth binding motif. After ligating the first binding motif to the second binding motif, the fusion protein comprising the third binding motif is contacted with the polypeptide comprising the fourth binding motif. The method according to item 27, wherein said contacting is carried out under conditions that enable the first binding motif-second binding motif pair to covalently bind to the fourth binding motif via protein ligation, either spontaneously or with the aid of an enzyme, provided that the first binding motif-second binding motif pair is orthogonal to the third binding motif-fourth binding motif pair.

[0124] Item 29 comprises contacting one or more first antigen-binding fragments, each comprising a first binding motif, with a fusion protein comprising one or more second binding motifs and one or more third binding motifs, wherein the second and third binding motifs are optionally linked by a linker sequence to form an antigen-binding protein, and said contacting is carried out under conditions that enable the first binding motif to covalently bind to the second binding motif via protein ligation, either spontaneously or with the aid of an enzyme. After linking the first binding motif to the second binding motif, it comprises contacting one or more second antigen-binding fragments, each comprising a fourth binding motif, with the antigen-binding protein, and said contacting is carried out under conditions that enable the third binding motif to covalently bind to the fourth binding motif via protein ligation, either spontaneously or with the aid of an enzyme. A method for producing an antigen-binding protein, wherein the first binding motif-second binding motif pair is orthogonal to the third binding motif-fourth binding motif pair.

[0125] Item 30 a. A first antigen-binding fragment comprising a first binding motif, and b. A first fusion protein comprising two or more second binding motifs linked by a linker sequence, and / or c. A first fusion protein comprising two or more second binding motifs and a third binding motif linked to the two or more second binding motifs by a linker sequence, and / or, d. A polypeptide comprising a fourth binding motif, and / or e. A fusion protein comprising one or more second binding motifs linked to one or more third binding motifs by a linker sequence, and / or f. A second antigen-binding fragment comprising a fourth binding motif, and / or g. A nucleic acid construct comprising a polynucleotide sequence encoding a peptide defined in any one of a to f A kit comprising.

[0126] Item 31 The kit according to Item 30, wherein the first fusion protein, fusion protein, or polypeptide further comprises a detectable label.

[0127] Item 32 The kit according to Item 31, wherein the detectable label is a phosphor, fluorescent protein, biotin, or enzyme.

[0128] Item 33 The nucleic acid construct according to Items 21 to 24, wherein the binding motifs of the fusion protein are linked by one or more linker sequences.

[0129] Item 34 The method according to Items 27 to 29, wherein the binding motifs of the fusion protein are linked by one or more linker sequences.

[0130] Examples The following examples are for illustrative purposes only and are not intended to be limiting. Those skilled in the art can readily recognize various non-essential parameters that can be changed or modified to achieve essentially the same or similar results.

[0131] Example 1 - Construction, Expression, and Purification of BiCatcher BiCatcher was constructed using long (116 amino acids, SEQ ID NO: 1) and short (84 amino acids, SEQ ID NO: 3) SpyCatcher:Bicatcher_1 and Bicatcher_s. Short linker ((GGGS)2,2G) and long linker ((GGGGS)4,4G) sequences were used to link the SpyCatcher subunits. BiCatchers also had a His-tag and a TEV protease cleavage site at their N-terminus. Each of the BiCatcher_1 and BiCatcher_s sequences with short and long linkers was cloned into the pET28a vector. The vector was transformed into E. coli BL21(DE3). The BiCatcher fusion protein was expressed by culturing BL21(DE3) cells in 250 mL of 2×YT broth containing 0.1% glucose and kanamycin. After growing at 37°C for 1 hour, the culture was induced with 0.8 mM IPTG.

[0132] Expression of the fusion protein was allowed to proceed at 30°C for approximately 16 hours. The culture was centrifuged and the cells were frozen at -80°C. The cells were lysed with BugBuster lysis buffer (Millipore-Sigma). The fusion protein was purified using Ni-NTA affinity matrix and the buffer was exchanged to 1×PBS.

[0133] Next, the fusion proteins were analyzed by SDS-PAGE (Figure 5). The Bio-Rad Criterion™ Vertical Electrophoresis Cell was used together with an AnyKD gel and Bio-Rad Precision Plus Protein Standard molecular weight markers. The gel was stained with Coomassie® dye, and protein purity was measured by densitometry. The concentrations and purities of BiCatcher_l and _s with short and long linkers are shown in Table 3 below. The purity of all fusion proteins exceeded 80 percent.

[0134]

Table 3

[0135] The BiCatcher2 fusion proteins were also constructed based on the SpyCatcher2 sequence (SEQ ID NO: 5) and the short linker (GGGS)2. The His-tag and TEV protease cleavage site were used as described for the above constructs. For all SpyCatcher2 constructs, the change from Asn (N) to Asp (D) was incorporated at position 105 to remove the deamidation site. This sequence was cloned into the pET28a vector and transformed into BL21(DE3) cells. Expression and purification were performed as described for BiCatcher_l.

[0136] Example 2 - Construction, Expression, and Purification of Fab-SpyTag A human Fab fragment with a SpyTag and a His-tag at the C-terminus of the heavy chain was constructed by directly fusing the SpyTag to the C-terminus of the CH1 domain, followed by fusing a short linker (GAP) and a hexahistidine-tag. Alternatively, a human Fab fragment with a FLAG-tag, a SpyTag and a His-tag was constructed by using a short linker (EF) between the C-terminus of CH1 and the FLAG-tag, followed by using a linker (GGS) and, in the same manner as the SpyTag, a linker (GAP) and a His-tag. An additional construct with SpyTag2 (SEQ ID NO: 4) instead of the SpyTag was constructed. The light and heavy chains were cloned into a bicistronic bacterial expression vector with a lac promoter. The light and heavy chain genes also have a secretion signal for transport into the periplasm. A vector with the Fab-SpyTag-H construct was transformed into E. coli TG1 F (F- episome minus). The Fab-FLAG-SpyTag-H construct was transformed into a protease-deficient E. coli strain as described in co-pending U.S. Patent Application No. 62 / 819,748 (Periplasmic Fusion Proteins, filed March 18, 2019, docket number BRL.130P). The Fab fragment was expressed by culturing E. coli cells in 250 mL of 2×YT broth containing 0.1% glucose and chloramphenicol. After growing at 37 °C for 1 hour, the culture was induced with 0.8 mM IPTG. The expression was allowed to proceed at 30 °C for about 16 hours. The culture was centrifuged and the cells were frozen at -80 °C. The cells were lysed with BugBuster lysis buffer (Millipore-Sigma). The fusion protein was purified with a Ni-NTA affinity matrix and the buffer was exchanged to 3×PBS.

[0137] Example 3 - Ligation of Fab-SpyTag and BiCatcher The BiCatcher fusion protein from Example 1 and the Fab-FLAG-SpyTag-His fusion protein from Example 2 were ligated to each other by reacting 12 μM of Fab-FLAG-SpyTag-His with 6 μM of each BiCatcher in 1xPBS. The ligation reaction was allowed to proceed for 16 hours at room temperature. For analysis, SDS loading buffer was added and the samples were heated at 95 °C for 5 minutes prior to SDS-PAGE on a 4–20% polyacrylamide gel (Bio-Rad Mini-PROTEAN TGX). The gel image (Figure 6) shows that the Fab-SpyTag reacted with the BiCatcher construct and the Fab heavy chain was almost completely ligated to the BiCatcher.

[0138] The BiCatcher2 fusion protein from Example 1 and the Fab-Flag-SpyTag2-His antibody from Example 2 were ligated to each other by reacting 10 μM Fab with 4 μM BiCatcher2 in PBS. A 25% molar excess of SpyTag2 over the SpyCatcher2 sites (i.e., 2 sites per BiCatcher) was used to achieve complete reaction of all SpyCatcher2 sites. After different time points (30 seconds to 60 minutes), the reaction was stopped by adding SDS loading buffer. After heating at 95 °C for 5 minutes, the samples were loaded onto a 4–20% polyacrylamide gel (Bio-Rad Mini-PROTEAN TGX). The image of the Coomassie-stained gel (Figure 7) shows that BiCatcher2 reacted with the Fab heavy chain and SpyTag2. After 60 minutes, the BiCatcher2 band completely disappeared, indicating completion of the ligation reaction. At the start of the reaction, two products were visible and BiCatcher2 had bound to one Fab and two Fabs. The band for the single coupling product decreased with longer reaction times until only the double ligation product was visible on the gel after 60 minutes.

[0139] Comparison of the assay performance of Example 4 - Fab-SpyTag and BiCatcher To show the performance improvement of bivalent Fabs over monovalent Fabs by avidity, Western blot was performed on HKB11 mammalian cell lysates. The lysates were separated on a Bio-Rad Mini-PROTEAN TGX 4-20% polyacrylamide gel and subsequently blotted onto a PVDF membrane using a Bio-Rad Trans-Blot Turbo transfer system. The membrane was blocked with 5% milk in TBST overnight at 4 °C. A Fab-SpyTag antibody specific for human HSPA5 was used as such (Fab-Spy-H in Figure 8) or dimerized by protein ligation using a BiCatcher with a short linker (BiCatcher 12G+Fab-SpyTag-H in Figure 8). The antibody samples were then diluted in TBST containing 0.5% milk to a concentration equal to 1 μg / ml Fab (equimolar antigen-binding sites in both preparations), and the membrane was incubated with shaking at room temperature for 1 hour. An HRP-conjugated goat anti-human Fab secondary antibody and Western blot clarity ECL substrate were used for detection, and images were taken with a Gel Doc imaging system. Images of both blots were taken with the same exposure time (10 seconds). The bivalent Fabs gave much stronger bands on the Western blot than the monovalent Fabs (Figure 8), clearly showing the sensitivity advantage of the BiCatcher dimerized Fabs.

[0140] Example 5 - Construction, Expression, and Purification of Labeled BiCatcher Labeled BiCatcher fusion proteins were first constructed by modifying the nucleic acid sequence encoding BiCatcher (i.e., SpyCatcher002 (SEQ ID NO: 5)-linker-SpyCatcher002) to incorporate a cysteine residue at one of three different amino acid positions: 1. N-terminus 2. Embedded within the linker between the two SpyCatcher002 subunits 3. C-terminus

[0141] Seven cysteine-containing BiCatcher nucleic acid constructs (cys-BiCatcher constructs) were prepared using various combinations of the above subunit nucleic acid sequences (Table 4) such that one, two, or three cysteines were incorporated into the cys-BiCatcher constructs.

[0142]

Table 4

[0143] Each of the seven cys-BiCatcher constructs was cloned into the pET28a vector. The vector was transformed into E. coli BL21(DE3). The cys-BiCatcher fusion proteins were each expressed by culturing BL21(DE3) cells in 250 mL of 2×YT broth containing 0.1% glucose and kanamycin. After growing at 37°C for 1 hour, the cultures were induced with 0.8 mM IPTG.

[0144] The expression of each cys-BiCatcher fusion protein was allowed to proceed at 30°C for approximately 16 hours. The cultures were centrifuged and the cells were frozen at -80°C. The cells were lysed with BugBuster lysis buffer (Millipore-Sigma). The fusion proteins were purified using Ni-NTA affinity matrix and the buffer was exchanged to 1×PBS.

[0145] Next, maleimide chemistry was used to site-specifically attach biotin or HRP to the thiol groups of cysteines in each cys-BiCatcher fusion protein. To biotinylate the fusion proteins, each fusion protein in PBS buffer (100 mM phosphate, 150 mM NaCl, pH 7.0) was reduced by adding 30 equivalents of tris(2-carboxyethyl)phosphine (TCEP) at room temperature for 30 minutes. Then, 20 equivalents of biotin-maleimide (stock in DMSO) were added to each reaction and incubated at room temperature for 4.5 hours. For HRP labeling, each cys-BiCatcher fusion protein in PBS was reduced with 30 equivalents of TCEP for 30 minutes at room temperature. 6 equivalents of maleimide-activated HRP (Thermo Fisher #31485) were dissolved in PBS and added to each reduced fusion protein. Each reaction was incubated at room temperature for 4 hours and quenched by incubating with 20x molar excess N-ethylmaleimide per Cys residue for 30 minutes at room temperature. All labeled fusion proteins were dialyzed against PBS.

[0146] Example 6 - Ligation of Fab-SpyTag and Labeled BiCatcher The biotin or HRP-labeled BiCatcher fusion proteins from Example 5 and the Fab-FLAG-SpyTag-His fusion protein from Example 2 were ligated to each other by reacting 6 μM of each labeled BiCatcher and 12 μM of Fab-FLAG-SpyTag-His in 1x PBS. The ligation reaction was allowed to proceed at room temperature for 16 hours.

[0147] Example 7 - Performance of Fab-SpyTag Bound to Labeled BiCatcher The performance of each of the conjugates from Example 6 was tested by ELISA. Maxisorp ELISA plates were coated overnight at 4°C with 5 μg / ml of antigen (alemtuzumab) in PBS. The plates were blocked with 5% BSA in PBST (PBS containing 0.05% Tween 20) for 1 hour and then incubated for 1 hour with serial dilutions of the Fab-BiCatcher002-biotin or Fab-BiCatcher002-HRP conjugates from Example 6 in 5% BSA in PBST. Next, the ELISA plates with biotinylated BiCatcher002 conjugates were incubated with streptavidin-HRP (Bio-Rad #STAR5A, 1:1000 in 5% BSA in PBST) or Neutravidin-HRP (Thermo Fisher #31030, 1:8000 in 5% BSA in PBST). Detection was performed for all plates (i.e., biotin- and HRP-conjugated BiCatcher002) using QuantaBlu Fluorescent Detection Reagent (Thermo Fisher #15169).

[0148] Figure 9 shows a plot of fluorescence as a function of antibody concentration of cys-BiCatcher labeled with biotin for each of the seven cys-BiCatcher fusion proteins, using streptavidin-HRP for detection. Figure 10 shows a plot of fluorescence as a function of antibody concentration of cys-BiCatcher labeled with HRP for each of the seven cys-BiCatcher fusion proteins. According to the results for both labels, it can be seen that CysBiCatchers having two or three cysteine residues show better performance than CysBiCatchers having only one cysteine and CysBiCatcher111 (having three cysteine residues).

[0149] Figure 11 is a plot of fluorescence as a function of antibody concentration of cys - BiCatcher (CysBiCatcher111) labeled with biotin for a cys - BiCatcher fusion protein having three cysteine amino acid residues and using Neutravidin - HRP for detection. In this assay, the performance of chemically biotinylated IgG was compared to Fab ligated to biotinylated CysBiCatcher111. The assay was performed as described in item 10. The results show that CysBiCatcher111 functioned better than directly biotinylated IgG.

[0150] Example 8 - Construction, Expression, and Purification of MultiCatcher A MultiCatcher fusion protein consisting of three (TriCatcher2), four (TetraCatcher2), and five (PentaCatcher2) SpyCatcher2s was constructed. SpyCatcher2 (SEQ ID NO: 5) was genetically linked by a short linker ((GGGGS)2), and a His - tag and two extended Strep tags were added to the C - terminus. The sequence was cloned into the pET28a vector and transformed into BL21(DE3) cells. Expression and purification were performed as described in Example 1.

[0151] Example 9 - Ligation of Fab - FLAG - SpyTag2 - His to MultiCatcher The BiCatcher2 from Example 1 and the MultiCatcher from Example 8 were ligated to the Fab-flag-SpyTag2-His antibody from Example 2 by reacting 12 μM of the SpyCatcher2 moiety (not the MultiCatcher molecule) with 14.4 μM of the Fab in PBS. A 20% excess of SpyTag2 over the SpyCatcher2 moiety was used to achieve complete reaction of all SpyCatcher2 moieties. The reaction was carried out overnight at room temperature. For analysis, 1 μg of each coupling product was heated at 95 °C for 5 minutes and then loaded onto a 4–20% polyacrylamide gel (Bio-Rad Mini-PROTEAN TGX). The image of the Coomassie-stained gel (Figure 12) shows that all SpyCatcher2 moieties of the MultiCatchers reacted with SpyTag2 on the Fab heavy chain, and the corresponding multimeric Fab molecules appeared in the gel.

[0152] Example 10 - Western blot application of MultiCatcher-bound Fab When the performance of various MultiCatcher - conjugated Fabs was compared by Western blot, it was shown that high valency resulted in increased avidity and assay sensitivity. For each antibody, one lane with 1.8 μl (lane a) and 0.36 μl (lane b) of total cell lysate from the human HKB11 cell line, respectively, and the Bio - Rad Precision Plus Protein Standard molecular weight marker were loaded onto a non - reducing AnykD polyacrylamide gel (Bio - Rad Mini - PROTEAN TGX). Proteins were blotted onto a PVDF membrane using the Bio - Rad Trans - Blot Turbo transfer system. The membrane was blocked with 5% milk in TBST overnight at 4 °C. A Fab - SpyTag antibody specific for human GAPDH conjugated to either SpyCatcher2, Bi -, Tri -, Tetra -, or PentaCatcher2 was used for detection. For each antibody construct, an equimolar amount based on the Fab fragment (equivalent to 2 μg of unbound Fab) was used in TBST containing 5% milk, and the membrane was incubated on a shaker at room temperature for 1 hour. An HRP - conjugated goat anti - human Fab secondary antibody and Western blot clarity ECL substrate were used for detection, and images were taken with a Gel Doc imaging system. Images of all blots were taken at the same exposure time (1.0 second). The increase in valency by MultiCatchers increased the detection sensitivity in Western blot (Figure 13). Thus, the results clearly show the improvement in sensitivity due to the avidity of the MultiCatcher - conjugated Fabs.

Sequence Listing Free - Text

[0153] All patents, patent applications, and other published reference materials cited herein are hereby incorporated by reference in their entirety. SEQ ID NO: 1 (SpyTag) AHIVMVDAYK PTK SEQ ID NO: 2 (SpyCatcher) GAMVDTLSGL SSEQGQSGDM TIEEDSATHI KFSKRDEDGK ELAGATMELR DSSGKTISTW ISDGQVKDFY LYPGKYTFVE TAAPDGYEVA TAITFTVNEQ GQVTVNGKAT KGDAHI Sequence number 3 (SpyCatcher short) GDSATHIKFS KRDEDGKELA GATMELRDSS GKTISTWISD GQVKDFYLYP GKYTFVETAA PDGYEVATAI TFTVNEQGQV TVNG Sequence number 4 (SpyTag002) VPTIVMVDAY KRYK Sequence number 5 (SpyCatcher002) AMVTTLSGLS GEQGPSGDMT TEEDSATHIK FSKRDEDGRE LAGATMELRD SSGKTISTWI SDGHVKDFYL YPGKYTFVET AAPDGYEVAT AITFTVNEQG QVTVNGEATK GDAHTGSSGS Sequence number 6 (SnoopTag) KLGDIEFIKV NK Sequence number 7 (SnoopCatcher) KPLRGAVFSL QKQHPDYPDI YGAIDQNGTY QNVRTGEDGK LTFKNLSDGK YRLFENSEPA GYKPVQNKPI VAFQIVNGEV RDVTSIVPQD IPATYEFTNG KHYITNEPIP PK Sequence number 8 (Isopeptag) TDKDMTITFT NKKDAE Sequence number 9 (Split Spy0128) ATTVHGETVV NGAKLTVTKN LDLVNSNALI PNTDFTFKIE PDTTVNEDGN KFKGVALNTP MTKVTYTNSD KGGSNTKTAE FDFSEVTFEK PGVYYYKVTE EKIDKVPGVS YDTTSYTVQV HVLWNEEQQK PVATYIVGYK EGSKVPIQFK NSLDSTTLTV KKKVSGTGGD RSKDFNFGLT LKANQYYKAS EKVMIEKTTK GGQAPVQTEA SIDQLYHFTL KDGESIKVTN LPVGVDYVVT EDDYKSEKYT TNVEVSPQDG AVKNIAGNST EQETSTDKDM TI Sequence number 10 (SdyTag) DPIVMIDNDK PIT Sequence number 11 (SdyCatcherDANG short) GRGSSGLSGE TGQSGNTTIE EDSTTHVKFS KRDANGKELA GAMIELRNLS GQTIQSWISD GTVKVFYLMP GTYQFVETAA PEGYELAAPI TFTIDEKGQI WVDS Sequence number 12 (K-Tag) ATHIKFSKRD Sequence number 13 (SnoopTagJr) KLGSIEFIKV NK Sequence number 14 (DogTag) DIPATYEFTN GKHYITNEPI PPK Sequence number 15 (Sortase recognition domain) LPTGAA Sequence number 16 (Sortase recognition domain) LPTGGG Sequence number 17 (Sortase recognition domain) LPKTGG Sequence number 18 (Sortase recognition domain) LPETG Sequence number 19 (Sortase recognition domain) LPXTG (X is any amino acid) Sequence number 20 (Sortase recognition domain) LPXTG(X) n (X is any amino acid, n is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10) SEQ ID NO: 21 (sortase recognition domain) NPX1TX2 (X1 is Q or L, X2 is N or G) SEQ ID NO: 22 (SpyTag003) RGVPHIVMVDAYKRYK SEQ ID NO: 23 (SpyCatcher003) VTTLSGLSGEQGPSGDMTTEEDSATHIKFSKRDEDGRELAGATMELRDSSGKTISTWISDGHVKDFYLYPGKYTFVETAAPDGYEVATPIEFTVNEDGQVTVDGEATEGDAHT

Claims

**Claim 1** two or more first antigen-binding fragments comprising a first binding motif, a fusion protein comprising two or more second binding motifs linked by a linker sequence, wherein the N-terminus of the second binding motif comprises one or more cysteine residues, the linker sequence comprises one or more cysteine residues, and / or the C-terminus of the second binding motif comprises one or more cysteine residues, comprising, the first binding motif of the two or more first antigen-binding fragments is covalently bound to the two or more second binding motifs via protein ligation, the first binding motif comprises SEQ ID NO: 1, 4, or 22, or a sequence having at least 90% sequence identity with SEQ ID NO: 1, 4, or 22, and the second binding motif comprises SEQ ID NO: 2, 3, 5, or 23, or a sequence having at least 90% sequence identity with SEQ ID NO: 2, 3, 5, or 23, an antigen-binding protein. **Claim 2** the fusion protein further comprises a third binding motif bound to the two or more second binding motifs by a linker, and the antigen-binding protein further comprises a polypeptide comprising a fourth binding motif, the third binding motif is covalently bound to the fourth binding motif of the polypeptide via protein ligation, the first binding motif-second binding motif pair is orthogonal to the third binding motif-fourth binding motif pair, the third binding motif comprises SEQ ID NO: 7, 11, or 14, or a sequence having at least 90% sequence identity with SEQ ID NO: 7, 11, or 14, and the fourth motif comprises SEQ ID NO: 6, 10, or 13, or a sequence having at least 90% sequence identity with SEQ ID NO: 6, 10, or 13, or the third binding motif comprises SEQ ID NO: 6, 10, or 13, or a sequence having at least 90% sequence identity with SEQ ID NO: 6, 10, or 13, and the fourth motif comprises SEQ ID NO: 7, 11, or 14, or a sequence having at least 90% sequence identity with SEQ ID NO: 7, 11, or 14, the antigen-binding protein according to claim 1. **Claim 3** One or more binding motifs are located at the C-terminus, N-terminus of the first and / or second antigen-binding fragment, the fusion protein, or the polypeptide, or are embedded within its amino acid sequence. The antigen-binding protein according to claim 1 or 2.

4. The fusion protein or polypeptide further comprises a detectable label. The antigen-binding protein according to any one of claims 1 to 3.

5. The detectable label is a phosphor, a fluorescent protein, biotin, or an enzyme. The antigen-binding protein according to claim 4.

6. a) A first nucleic acid construct comprising a polynucleotide sequence encoding a first antigen-binding fragment fused to the first binding motif according to claim 1; b) A second nucleic acid construct comprising a polynucleotide sequence encoding a fusion protein comprising two or more second binding motifs linked by the linker sequence according to claim 1; comprising When the first binding motif and the second binding motif are brought into contact with each other spontaneously or with the aid of an enzyme, they form a covalent bond via protein ligation. Nucleic acid construct pair.

7. The polynucleotide of the second nucleic acid construct further encodes a third binding motif linked by a linker sequence to or between the two or more second binding motifs, and the nucleic acid construct pair further comprises a third nucleic acid construct comprising a polynucleotide sequence encoding a polypeptide fused to a fourth binding motif. When the third binding motif and the fourth binding motif are brought into contact with each other spontaneously or with the aid of an enzyme, they form a covalent bond via protein ligation. The first binding motif-second binding motif pair is orthogonal to the third binding motif-fourth binding motif pair. The nucleic acid construct pair according to claim 6.

8. The polynucleotide encoding any one or all of the one or more second binding motifs is located before, after, or in between the polynucleotide encoding any one or all of the one or more third binding motifs. The nucleic acid construct pair according to claim 7.

9. A vector comprising the nucleic acid construct pair according to any one of claims 6 to 8.

10. A host cell having the vector according to claim 9.

Images