Antigen binding proteins

Abstract

The invention provides antigen binding proteins, nucleic acid constructs encoding the antigen binding proteins, vectors comprising the nucleic acid constructs, host cells comprising the vectors and the nucleic acid constructs, and kits for preparing the antigen binding proteins.

Description

[0001] This application claims the benefit of U.S. Provisional Application No. 62 / 819,753, filed on March 18, 2019, which is incorporated herein by reference in its entirety. Background Art

[0002] Multivalent forms of antibody fragments are useful in analyses requiring higher affinity because higher valence states increase analytical sensitivity. Monomeric antibody fragments are usually produced prior to conversion to multivalent forms in order to determine intrinsic affinity. Conversion of monomeric antibody fragments to higher valence states and subsequent production requires several steps, a tedious process that takes several weeks. These process steps are repeated for different valence states. Protein Link

[0003] There are several techniques that allow the covalent coupling of polypeptides at specific predetermined sites. One example is the sortase system (Schmohl et al., 2014), 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, the two modified polypeptides are fused together. Other enzymatic protein ligase systems are butterfly pea viscose (Nguyen et al., 2014) or peptidyl ligase (Toplak et al., 2016).

[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. When such free cysteine-containing polypeptides are mixed under oxidative conditions, they will form disulfide bonds. However, the problem with this type of system is the synthesis of many byproducts and the instability of disulfide bonds under reducing conditions.

[0005] Another example is the SpyTag / SpyCatcher (Reddington et al., 2015) system. Here, the concept of spontaneous isopeptide formation in naturally occurring proteins is used to covalently link one polypeptide to another. A domain of the Streptococcus pyogenes protein FbaB that contains such an isopeptide bond is divided into two parts. One part is SpyTag (SEQ ID NO: 1), which is a 13 amino acid peptide that contains a part of the autocatalytic center (e.g., an aspartic acid). The other part is SpyCatcher (SEQ ID NO: 2), which is a 116 amino acid protein domain that contains another part of the center (e.g., lysine) catalyzed by a nearby glutamic acid or aspartic acid residue. Mixing the two polypeptides restores the autocatalytic center and leads to the formation of an isopeptide bond, thereby covalently linking SpyTag to SpyCatcher (Zakeri et al., 2012). Further engineering resulted in a shortened version of SpyCatcher with only 84 amino acids (SEQ ID NO: 3), as well as optimized versions SpyTag002 (SEQ ID NO: 4) and SpyCatcher002 (SEQ ID NO: 5) with accelerated reactions (Li et al., 2014 and Keeble et al., 2017); the entire texts of which are hereby incorporated by reference. Further engineering resulted in another optimized version, namely SpyTag003 (SEQ ID NO: 22) and SpyCatcher003 (SEQ ID NO: 23), whose reactions are close to the diffusion limit (Keeble et al., 2019), are hereby incorporated by reference in their entirety. Another improvement to this system is the invention of SpyLigase (Fierer et al., 2014), which is 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 that contains glutamic acid residues that induce or catalyze the formation of isopeptide bonds between aspartic acid residues and lysine residues in SpyTag and K-tag, respectively.

[0006] Applications of such systems include protein stabilization by cyclization, vaccine generation, protein multimerization by integrating streptavidin / biotin with SpyTag / SpyCatcher (Reddington et al., 2015), affibody and Fab multimerization (Fierer et al., 2014), antibody generation from modules (Alam et al., 2017), and the generation of antibody-drug conjugates (Siegmund et al., 2016), as well as the generation of bispecific antibodies (Yumura et al., 2017). A similar system was developed using the adhesin RrgA of Streptococcus pneumoniae, called SnoopTag / SnoopCatcher (Veggiani et al., 2016), and later the SnoopLigase system was developed (Buldun et al., 2018). The SnoopTag / SnoopCatcher technology is incorporated herein by reference in its entirety. Another system using the Streptococcus pyogenes pilus subunit Spy0128, called Isopeptag / Split Spy0128, has also been developed (Abe et al., 2013). In addition, another system derived from the fibronectin-binding protein of Streptococcus dysgalactiae, called SdyTag / SdyCatcherDANG short, has also been developed (Tan et al., 2016). Isopeptag / SplitSpy0128 and SdyTag / SdyCatcherDANG short technology as its whole merged. Summary of the invention

[0007] The present invention provides antigen-binding proteins, nucleic acid constructs encoding antigen-binding proteins, vectors containing nucleic acid constructs, and host cells containing vectors and nucleic acid constructs. A kit containing components for preparing antigen-binding proteins is also provided.

[0008] In one embodiment, the antigen binding protein comprises two or more first Fabs, each fragment comprising a first binding motif, and a first fusion protein comprising two or more second binding motifs. The binding motif connected to the first Fab and the two or more second binding motifs forming the fusion protein can be optionally connected by one or more linker sequences. The first binding motif of the two or more first Fabs is covalently coupled to the two or more second binding motifs through protein connection. In some embodiments, the first fusion protein is a dimer or polymer of the second binding motif, which is optionally connected 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 to 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 to 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 to 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 to SEQ ID NO:1, 4, 6, 8, 10, 13 or 22.

[0009] In some embodiments, the first fusion protein further comprises a third binding motif, and the polypeptide comprising the fourth binding motif can be covalently coupled thereto via protein linkage. In some embodiments, the polypeptide is an enzyme, a fluorescent protein, an effector protein, or another antigen binding fragment. The third binding motif can be optionally connected to the fusion protein via one or more linkers. The fourth binding motif can be optionally connected to the polypeptide via one or more linkers.

[0010] In certain embodiments, the antigen binding protein comprises one or more first antigen binding fragments, each fragment comprising a first binding motif, a first fusion protein comprising one or more second binding motifs, the second binding motif being connected to one or more third binding motifs via a linker sequence, and one or more second antigen binding fragments, each comprising a fourth binding motif. The first binding motif can be covalently coupled to the second binding motif via a protein connection, and the third binding motif can be covalently coupled to the fourth binding motif via a protein connection. In some embodiments, the antigen binding protein is bispecific, bispecific and dimeric, or bispecific and multimeric.

[0011] In some embodiments in which the antigen binding protein has a 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 thereto, the second binding motif comprises SEQ ID NO: 2, 3, 5, 9, 12, or 23, or a sequence having at least 60% sequence identity thereto, the third binding motif comprises SEQ ID NO: 6, 10, or 13, or a sequence having at least 60% sequence identity thereto, and the fourth binding motif comprises SEQ ID NO: 7, 11, or 14, or a sequence having at least 60% sequence identity thereto. In other alternative embodiments of the antigen binding protein having a 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 to SEQ ID NO: 2, 3, 5, 9, 12 or 23, and the second binding motif comprises SEQ ID NO: 1, 4, 8 or 22 or a sequence having at least 60% sequence identity to a sequence of 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 to SEQ ID NO: 7, 11 or 14, and the fourth 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.

[0012] In embodiments having a 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.

[0013] In certain embodiments, one or more binding motifs are located at the C-terminus, N-terminus, or embedded in 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 sequential 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.

[0014] In some embodiments, the antigen binding protein may further comprise a detectable label (eg, a fluorophore, a fluorescent protein, biotin, or an enzyme).

[0015] In some embodiments, the linker sequence is 1-5 amino acids having the sequence GGGGS.

[0016] In some embodiments, the antigen binding protein may comprise a third binding motif connected to a first fusion protein having two or more second binding motifs by a linker sequence and a polypeptide having a fourth binding motif (e.g., a protein or protein fragment having additional functions). In some embodiments, the fourth binding motif comprises a sortase recognition domain and the third motif comprises a sortase bridging domain. In other embodiments, the fourth binding motif comprises a sortase recognition domain and the third motif comprises a sortase bridging domain. In some embodiments, the sortase recognition domain comprises the following 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, in the range of 0-5 or 0-10, or any integer up to 100, NPX1TX2 (SEQ ID NO:21), wherein X1 is glutamine or lysine; X2 is asparagine or glycine; N is asparagine; P is proline and T is threonine, and the sortase bridging domain comprises: Gly, (Gly) 2 ,(Gly) 3 ,(Gly) 4 , or (Gly) x , where x is an integer from 1 to 20.

[0017] Also provided are nucleic acid constructs encoding antigen binding proteins. Also provided are vectors comprising nucleic acid constructs. Also provided are host cells comprising vectors. Also provided are kits comprising components for preparing antigen binding proteins. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 A scheme for preparing an antigen-binding protein by protein connection according to a first embodiment is described. In this embodiment, an antigen-binding fragment (e.g., Fab) comprising a first binding motif (e.g., SpyTag) is connected to a multimeric second binding motif (e.g., dimeric SpyCatcher or "BiCatcher"). The antigen-binding protein produced is a dimer (e.g., Fab dimer) or a multimer.

[0019] Figure 2The scheme of preparing antigen-binding proteins by protein connection according to the second embodiment is described. In this embodiment, an antigen-binding fragment (e.g., Fab) comprising a first binding motif (e.g., SpyTag) is connected to a multimeric binding motif having a second binding motif (e.g., dimeric SpyCatcher) that is dimerized or multimerized and connected to a third binding motif (e.g., SnoopCatcher). The polypeptide has a fourth binding motif (e.g., SnoopTag), and the fourth binding motif is connected to the third binding motif. The first binding motif-second binding motif pair is orthogonal to the third binding motif-fourth binding motif pair. The resulting antigen-binding protein is a dimer or multimer of a connection polypeptide (e.g., a fluorescent protein, an enzyme, an effector protein, or an antigen-binding fragment) with additional functions.

[0020] Figure 3 The scheme of preparing antigen-binding proteins by protein connection according to the third embodiment is described. In this embodiment, a first antigen-binding fragment (e.g., Fab) comprising a first binding motif (e.g., SpyTag) is connected to a second binding motif (e.g., SpyCatcher) connected to a third binding motif (e.g., SnoopCatcher). A second antigen-binding fragment with different specificity has a fourth binding motif (e.g., SnoopTag), and the fourth binding motif is connected to the third binding motif. The first binding motif-second binding motif is orthogonal to the third binding motif-fourth binding motif. The antigen-binding protein produced is a bispecific dimer (e.g., bispecific Fab).

[0021] Figure 4 The scheme of preparing antigen-binding proteins by protein connection according to the fourth embodiment is described.In this embodiment, the first Fab (e.g., Fab) comprising the first binding motif (e.g., SpyTag) is connected to a multimeric binding motif having a second binding motif (e.g., dimeric SpyCatcher) connected to a dimeric or multimeric third binding motif (e.g., dimeric SnoopCatcher).The second Fab with different specificities has a fourth binding motif (e.g., SnoopTag), and the fourth binding motif is connected to the third binding motif.The first binding motif-second binding motif is orthogonal to the third binding motif-fourth binding motif.The antigen-binding proteins produced are bispecific dimers (e.g., bispecific dimeric Fab).

[0022] Figure 5 Shown are images of SDS-PAGE gels of various expressed and purified BiCatcher molecules (dimeric long or short SpyCatcher domains) described in Example 1.

[0023] Figure 6An image of an SDS-PAGE gel of the ligation reaction products of various Fab SpyTags and different BiCatchers described in Example 3 is shown.

[0024] Figure 7 The images of SDS-PAGE gels of the ligation reaction products of Fab-FLAG-SpyTag2-His and BiCatcher2 at different time points described in Example 3 are shown.

[0025] Figure 8 Shows the Figure 1 Western blot of monovalent FabSpyTag antibody fragments compared to dimeric Fab dimerized according to the protocol described in Example 4. Fab directed against human HSPA5 contained in HKB11 mammalian cell lysate loaded on the gel. The bivalent Fab has a higher affinity, which results in a significant increase in sensitivity.

[0026] Fig. 9 Shown are the results of an ELISA titration experiment as described in Example 7, wherein according to Figure 1 In the scheme, the Fab-SpyTag antibody was conjugated to seven different cysteine-containing BiCatcher molecules and labeled with biotin. Detection was performed with streptavidin-HRP. The biotinylated IgG1 form of the antibody was used as a benchmark.

[0027] Fig.10 Shown are the results of an ELISA titration experiment as described in Example 7, in which the Fab-SpyTag antibody was conjugated to seven different cysteine-containing BiCatcher molecules labeled with HRP. The antibody was labeled with HRP in IgG1 format as a benchmark.

[0028] Fig.11 The results of an ELISA titration experiment as described in Example 7 are shown, wherein the Fab-SpyTag was coupled to a BiCatcher molecule with 3 cysteine ​​residues for biotin labeling. Neutravidin-HRP was used for detection. The biotinylated IgG1 form of the antibody was used as a benchmark.

[0029] Fig.12 Shown are images of SDS-PAGE gels of various expressed and purified SpyCatcher fusions, i.e. 3 or more SpyCatcher domains linked to each other via linker sequences at the genetic level (MultiCatcher molecules), and the ligation products of these MultiCatchers with Fab SpyTags, as described in Example 9.

[0030] Fig.13Western blot showing SpyCatcher-linked Fabs. Fabs were coupled to SpyCatcher, BiCatcher, Tri-Tetra- or PentaCatcher as described in Example 10. DETAILED DESCRIPTION

[0031] The present invention provides antigen-binding proteins, nucleic acid constructs encoding antigen-binding proteins, vectors comprising nucleic acid constructs, host cells comprising vectors and nucleic acid constructs, and kits for preparing antigen-binding proteins. Antigen-binding proteins are covalently linked to dimer or polymer affinity binding agents to form monospecific (recognize and bind to one antigen) or bispecific multivalent (i.e., bind to two different antigens or two different epitopes on the same antigen) constructs, respectively. The multimeric antigen-binding proteins may have a higher valence than monomers, contain additional functions, or be bispecific, or a combination thereof. Multimeric antigen-binding proteins are prepared by protein connection, which bypasses the genetic engineering steps currently required for preparing such binding agents. Multivalency increases the sensitivity of antigen-binding proteins, which is a useful feature in Western blotting, flow cytometry, immunohistochemistry, certain enzyme-linked immunosorbent assays, and therapeutic applications. Bispecificity helps to improve target specificity, target affinity, or simultaneously bind to two different antigens. Bispecific antigen-binding proteins are also becoming increasingly important as therapeutic drugs. Adding a second functionality by protein conjugation can be used to prepare labeled antigen binding proteins for use in applications such as Western blotting, flow cytometry, immunohistochemistry, enzyme-linked immunosorbent assays, targeted immobilization, or other applications requiring a second functionality, such as creating antibody-enzyme fusion proteins for cancer therapy. definition

[0032] Unless otherwise indicated, the following terms used in this application (including the specification and claims) have the following definitions. As used in this specification and the appended claims, the singular forms "a", "an", and "the" include plural referents unless the context clearly dictates otherwise.

[0033] "Antibody" refers to an immunoglobulin, complex (such as a fusion) or fragment thereof. The term includes, but is not limited to, polyclonal or monoclonal antibodies of the IgA, IgD, IgE, IgG and IgM classes derived from antibody-producing cell lines or from in vitro antibody libraries, including natural forms or genetically modified forms, such as humanized, human, single chain, chimeric, synthetic, recombinant, hybrid, mutated, grafted and in vitro generated antibodies. "Antibody" also includes complex forms, including but not limited to fusion proteins with immunoglobulin portions.

[0034] As used herein, the phrase "antigen binding fragment" refers to a protein comprising an antigen binding portion of an antibody (e.g., Fab). Other antigen binding fragments include variable fragments (Fv), disulfide-stabilized Fv fragments (dsFv), single-chain variable fragments (scFv), or single-chain Fab fragments (scFab). Further examples of antigen binding fragments include antigen binding fragments in monovalent form comprising an antigen binding site, which antigen binding site includes a heavy chain antibody variable domain (VHH), a single domain antibody (SDAB), or a shark variable new antigen receptor (VNAR). In addition, non-antibody scaffolds, such as variable lymphocyte receptors (VLRs), affimers, affibodies, DARPs, darpins, anticalins, monomers, avimers, Fyonomers, affilins, or antigen binding peptides, may also be considered "antigen binding fragments".

[0035] The term "binding motif" refers to a protein sequence that is connected to a polypeptide and can form a covalent bond with another polypeptide. Non-limiting examples of binding motifs include 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, the C-terminus, or embedded in the polypeptide. One or more linker sequences (e.g., a linker rich in glycine / serine) may be located on both sides of the binding motif to enhance the accessibility of the reaction or enhance the flexibility of the fusion polypeptide. In some embodiments, one or more linker sequences flank the N-terminus and C-terminus of the binding motif to enhance the accessibility of the reaction or enhance the flexibility of the fusion polypeptide (e.g., in the case of a fusion protein comprising a multimeric binding motif). As used herein, the phrase "connected by a linker sequence" allows the use of one or more linker sequences to connect two or more binding motifs, binding motifs and polypeptides or binding motifs and antigen binding fragments. Where multiple linker sequences are used to join a binding motif, to join an antigen binding fragment to a binding motif, or to join a polypeptide and a binding motif, the linker sequences may be the same or different.

[0036] The term "prokaryotic system" refers to prokaryotic cells, such as bacterial cells or prokaryotic bacteriophages or bacterial spores. The term "eukaryotic system" refers to eukaryotic cells, including cells of animals, plants, fungi and protists, as well as eukaryotic viruses such as retroviruses, adenoviruses, baculoviruses, etc. Prokaryotic and eukaryotic systems are collectively referred to as "expression systems."

[0037] The term "expression cassette" refers to a functional unit constructed in a vector for expressing a recombinant polypeptide with a binding motif. The expression cassette includes a promoter, a transcription terminator sequence, a ribosome binding site, and a DNA encoding a fusion protein. Depending on the expression system (e.g., enhancers and polyadenylation signals for eukaryotic expression systems), other genetic components may be added to the expression cassette.

[0038] As used herein, the term "vector" refers to a nucleic acid molecule, preferably self-replicating in a cell, which transfers the inserted nucleic acid molecule into a host cell and / or between host cells. Typically, a vector is a circular DNA comprising a replication origin, a selection marker and / or a viral packaging signal and other regulatory elements. In the description of the present invention, vectors, vector DNA, plasmid DNA, and phage DNA are interchangeable terms. The term includes vectors that primarily function to insert DNA or RNA into a cell, replication vectors that primarily function to replicate DNA or RNA, and expression vectors that function to transcribe and / or translate DNA or RNA. Also included are vectors that provide more than one of the above functions.

[0039] As used herein, the term "expression vector" is a polynucleotide that, when introduced into an appropriate host cell, results in the transcription and translation of one or more polypeptides under appropriate conditions. The term "expression vector" refers to a vector that directs the expression of a polypeptide fused in frame with a binding motif.

[0040] As used herein, the terms "polynucleotide," "nucleic acid," and "oligonucleotide" are used interchangeably. They refer to a polymeric form of nucleotides of any length, whether deoxyribonucleotides or ribonucleotides or their analogs. The following are non-limiting examples of polynucleotides: coding or non-coding regions of genes or gene fragments, loci defined by 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. If present, modifications to the nucleotide structure may be imparted before or after the assembly of the nucleotide polymer.

[0041] As used herein, the term "amino acid" refers to natural and / or unnatural or synthetic amino acids, D or L optical isomers, amino acid analogs and peptidomimetics.

[0042] As used herein, the terms "polypeptide," "peptide," and "protein" are used interchangeably herein to refer to polymers of amino acids of any length.

[0043] As used herein, the term "host cell" includes a single cell or cell culture that can be or has been a recipient of a disclosed expression construct. Host cells include progeny of a single host cell. Progeny may not necessarily be identical to the original parent cell due to natural, accidental or intentional mutations.

[0044] When the maximum correspondence is compared as described below, two nucleic acid sequences or polypeptides are referred to as "identical" if the sequence of nucleotides or amino acid residues in the two sequences is respectively the same. The term "identical" or "identity" percentage in the context of two or more nucleic acid or polypeptide sequences refers to two or more sequences or subsequences of identical or specific percentages of identical amino acid residues or nucleotides when compared and compared with respect to the maximum correspondence of the comparison window, such as using one of the following sequence comparison algorithms or by manual comparison and visual measurement. When the percentage of sequence identity relates to the use of proteins and peptides, it is believed that different residue positions are usually different due to conservative amino acid substitutions, wherein the amino acid residues are replaced by other amino acid residues with similar chemical properties (such as charge or hydrophobicity), thus not changing the functional properties of the molecule. When the sequences differ due to conservative substitutions, the percentage of sequence identity can be adjusted upward to correct according to the conservative nature of the substitution. Methods for making this adjustment are well known to those skilled in the art. It is generally included to score conservative substitutions as part of the score rather than complete mismatches, thereby increasing the percentage of sequence identity. Thus, for example, identical amino acids are scored as 1, non-conservative substitutions are scored as 0, and conservative substitutions are scored as 0-1. Scoring for conservative substitutions is calculated, for example, according to the algorithm of Meyers and Miller, Computer Applic. Biol. Sci. 4: 11-17 (1988), for example, as implemented in the program PC / GENE (Intelligenetics, Mountain View, California, USA).

[0045] Sequences are "substantially identical" to each other if they have a specified percentage of nucleotide or amino acid residues that are identical (e.g., at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identical over a specified region or over the entire specified region if a region is not specifically specified) when compared and aligned for maximum correspondence over a comparison window.

[0046] For sequence comparison, generally a sequence is used as a reference sequence compared with a test sequence. When using a sequence comparison algorithm, the test and reference sequences are input into a computer, and if necessary, subsequence coordinates are specified, and sequence algorithm program parameters are specified. Default program parameters can be used, or other parameters can be specified. Then, the sequence comparison algorithm calculates the sequence identity percentage of the test sequence relative to the reference sequence based on the program parameters.

[0047] As used herein, a "comparison window" includes a reference to a segment of any one of a plurality of contiguous positions selected from 10 to 600, about 10 to about 300, about 10 to about 150, wherein after the two sequences are optimally aligned, a sequence can be compared to a reference sequence of the same number of consecutive positions. The comparison window can also be the entire length of a reference or test sequence.

[0048] Percent sequence identity and sequence similarity can be determined using the BLAST 2.0 algorithm, which is described in Altschul et al. (J. Mol. Biol. 215: 403-10, 1990). Software for performing BLAST 2.0 analysis is publicly available from the National Center for Biotechnology Information (see World Wide Web address: 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 that match or satisfy some positive threshold score T when aligned with words of the same length in the database sequence. T is called the neighboring word score threshold (Altschul et al., supra). These initial neighboring word hits are used as seeds to initiate searches in order to find longer HSPs containing them. The word hits are extended in both directions along each sequence as far as the cumulative alignment score can be improved. Extension of the word hits in each direction is terminated when: the cumulative alignment score falls by X from its maximum achieved value; the cumulative score goes to zero or below due to the accumulation of one or more negative scoring residue alignments; or 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 uses as defaults: word length (W) 11, BLOSUM62 scoring matrix (see Henikoff and Henikoff, Proc. Natl. Acad. Sci. USA 89:10915 (1989)) alignments (B) 50, expectation (E) 10, M=5, N=-4, and comparison of both strands.

[0049] The BLAST algorithm also performs a statistical analysis of the similarity between two sequences (see, e.g., Karlin and Altschul, Proc. Nat'l. Acad. Sci. USA 90:5873-5787 (1993)). One measure of similarity provided by the BLAST algorithm is the smallest sum probability (P(N)), which indicates the probability that a match between two nucleotide or amino acid sequences will occur by chance. For example, if the smallest sum probability when comparing a test nucleic acid to 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, then the nucleic acid is considered similar to the reference sequence.

[0050] The term "label" or "detectable marker" 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, luminescent agents, high electron density reagents, enzymes (e.g., enzymes commonly used in ELISA), biotin, digoxigenin, 32 P and other radioisotopes, haptens, proteins, nucleic acids or other substances that can be detected, for example, by incorporating the label into an oligonucleotide or peptide. The term includes combinations of single labeling agents, for example, combinations of fluorophores that provide unique detectable characteristics (e.g., at a specific wavelength or combination of wavelengths). Antigen Binding Protein

[0051] In one embodiment, the antigen binding protein comprises two or more first antigen binding fragments, each fragment comprising a first binding motif, and comprising two or more second binding motifs (e.g., "Bicatcher" such as SpyCatcher-SpyCatcher or "Multicatcher"; see Figure 1) is connected by a linker sequence. The first binding motif of two or more first Fabs is covalently coupled to two or more second binding motifs through protein connection. In order to produce an antigen binding protein, a first Fab with a first binding motif is produced, a first fusion protein comprising two or more second binding motifs connected by a linker sequence is produced, and under appropriate conditions, the Fab-first binding motif protein is mixed with the first fusion protein comprising two or more second binding motifs to promote protein connection of the first binding motif and the second binding motif. In some embodiments, the first fusion protein is a dimer. In some embodiments, the antigen binding protein is a Fab dimer or polymer. In an 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 thereto, 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 thereto. The first and second binding motifs are selected such that the two binding motifs form a mutually reactive or cognate binding motif pair. For example, if the first binding motif is SEQ ID NO: 1 (SpyTag), the second binding motif can be SEQ ID NO: 2, 3, 5 or 23 (SpyCatcher, SpyCatcher short, SpyCatcher002 or SpyCatcher003), but not SEQ ID NO: 7 or 9 (SnoopCatcher or SplitSpy128) because the SpyTag / SpyCatcher system is orthogonal to the SnoopTag / SnoopCatcher system. It should also be noted that the components of mutually reactive motif pairs or homologous binding motif pairs can be interchanged on fusion proteins and antigen binding fragments (e.g., one embodiment provides an antigen binding fragment comprising SpyTag and a fusion protein comprising SpyCatcher, SpyCatcher short, SpyCatcher 002, or SpyCatcher003, and another embodiment provides an antigen binding fragment comprising SpyCatcher, SpyCatcher short, SpyCatcher 002, or SpyCatcher 003 and a fusion protein comprising SpyTag). As used herein, the term "orthogonal" refers to mutually non-reactive or non-homologous binding motif pairs (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 embodiments are provided in Table 1. Table 1

[0052] In some embodiments, the antigen binding protein may further comprise a third binding motif connected to the first fusion protein via a linker sequence and a polypeptide having a fourth binding motif (e.g., a protein or protein fragment having additional functions) ( Figure 2 The third binding motif can be linked to the first fusion protein via a linker sequence (e.g., "Bicatcher-SnoopCatcher" (SpyCatcher-SpyCatcher-SnoopCatcher) or "SnoopCatcher-Bicatcher" The third binding motif is covalently coupled to the fourth binding motif via a protein connection. In this embodiment, the first binding motif-second binding motif pair is orthogonal to the third binding motif-fourth binding motif pair. In order to produce an antigen-binding protein, a first antigen-binding fragment is produced with the first binding motif, a first fusion protein is produced with the second and third binding motifs, and a polypeptide is produced with the fourth binding motif. In certain embodiments, a first antigen-binding fragment comprising a first binding motif, a first fusion protein comprising a second and third binding motifs, and a polypeptide comprising a fourth binding motif are mixed under appropriate conditions to promote the connection of the first binding motif to the second binding motif and the third binding motif to the fourth binding motif. In some embodiments, a first antigen-binding fragment comprising a first binding motif and a first fusion protein comprising a second and third binding motifs are mixed under appropriate conditions to promote the connection of the first binding motif to the protein of the second binding motif. Then, under appropriate conditions, a polypeptide comprising a fourth binding motif is added to the mixture having the first and second binding motifs connected to promote the third binding motif to be connected to the protein of 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 that can be used to detect the binding of the antigen binding protein to the target. In some embodiments, the antigen binding protein is a dimeric Fab coupled to an additional protein or protein fragment.

[0053] In one embodiment, the antigen binding protein comprises one or more first antigen binding fragments, each fragment comprising a first binding motif, a first fusion protein comprising one or more second binding motifs, the second binding motif being connected to one or more third binding motifs via a linker sequence, and one or more second antigen binding fragments, each comprising a fourth binding motif. The first binding motif is covalently coupled to the second binding motif, the third binding motif is covalently coupled 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 coupled to each other via protein connections. In some embodiments, the second antigen binding fragment has a different specificity from the first antigen binding fragment, that is, the second antigen binding fragment recognizes a different antigen from the first antigen binding fragment or a different epitope on the same antigen. The antigen binding protein can be bispecific (e.g., "Heterocatcher" or SpyCatcher-SnoopCatcher; Figure 3 ), bispecific and dimer (e.g., "Heterocatcher" or SpyCatcher-SpyCatcher-SnoopCatcher-SnoopCatcher; Figure 4 ) or bispecific and multimeric ( Figure 4). "Heterobicatcher" may have binding motifs randomly or sequentially linked to each other, such as SpyCatcher-SpyCatcher-SnoopCatcher-SnoopCatcher, SnoopCatcher-SnoopCatcher-SpyCatcher-SpyCatcher, SpyCatcher-SnoopCatcher-SpyCatcher-SnoopCatcher, SpyCatcher-SnoopCatcher-SpyCatcher-SnoopCatcher, SpyCatcher-SnoopCatcher-SpyCatcher-SnoopCatcher, or SnoopCatcher-SpyCatcher-SpyCatcher-SpyCatcher-SnoopCatcher. In certain embodiments, the antigen binding protein is a bispecific Fab comprising two antigen binding fragments, each fragment having a different specificity. In some embodiments, the antigen binding protein is a bispecific dimeric Fab, which comprises four antigen binding fragments and binds to two different antigens or different epitopes on the same antigen. In some embodiments, the antigen binding protein is a bispecific multi-Fab comprising four or more antigen binding fragments and two specificities (for example, the antigen binding fragment has specificity for two different antigens or two different epitopes on the same antigen). In order to produce an antigen binding protein, a first antigen binding fragment is produced with a first binding motif, a first fusion protein is produced with a second and third binding motif, and a second antigen binding fragment is produced with a fourth binding motif. In certain embodiments, a first antigen binding fragment comprising a first binding motif, a first fusion protein comprising a second and third binding motif, and a polypeptide comprising a fourth binding motif are mixed under appropriate conditions to promote the first binding motif to be connected to the second binding motif, and the third binding motif to be connected to the protein of the fourth binding motif. In some embodiments, a first antigen binding fragment comprising a first binding motif and a first fusion protein comprising a second and third binding motif are mixed under appropriate conditions to promote the first binding motif to be connected to the protein of the second binding motif. Then, a polypeptide comprising a fourth binding motif is added to a mixture of the first binding motif and the second binding motif connected under appropriate conditions to promote the third binding motif to be connected to the protein of the fourth binding motif, or vice versa. In some embodiments, the antigen binding protein is a bispecific antigen binding fragment, such as a Fab or a heterodimer.

[0054] In some embodiments, one or more binding motifs are located at the C-terminus, N-terminus, or embedded in the amino acid sequence of the first and / or second antigen binding fragment, fusion protein, or polypeptide containing the second function. In certain embodiments, one or more binding motifs in the fusion protein are sequential or random order. In certain embodiments, the antigen binding fragment, fusion protein, and / or antigen binding protein further comprises a purification tag at its N-terminus or C-terminus.

[0055] In embodiments where the antigen binding protein has a 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 thereto, the second binding motif comprises SEQ ID NO: 2, 3, 5, 9, 12, or 23, or a sequence having at least 60% sequence identity thereto, the third binding motif comprises SEQ ID NO: 6, 10, or 13, or a sequence having at least 60% sequence identity thereto, and the fourth binding motif comprises SEQ ID NO: 7, 11, or 14, or a sequence having at least 60% sequence identity thereto. 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 coupled to the second binding motif and / or the third binding motif is covalently coupled to the fourth binding motif spontaneously or with the aid of an enzyme (e.g., a ligase). As described in paragraph

[0047] , mutually reactive motif pairs or homologous binding motif pairs can be interchanged / flipped on fusion proteins and Fabs (or polypeptides), provided that one component of the homologous binding motif pair is provided by the Fab or polypeptide and the second component of the homologous binding motif pair is provided by the fusion protein, or vice versa. Exemplary binding motifs of embodiments having first, second, third, and fourth binding motifs are provided in Table 2. Table 2

[0056] In various embodiments, the antigen binding protein may further comprise a detectable label. Exemplary detectable labels include, but are not limited to, fluorophores, fluorescent proteins (e.g., green fluorescent protein (GFP), biotin, enzymes (e.g., horseradish peroxidase (HRP) or other peroxidases, alkaline phosphatase, luciferase), and split fluorescent proteins (e.g., split GFP) or enzymes (e.g., Promega's Binary Technology). Exemplary fluorophores include, but are not limited to, Alexa dyes (e.g., Alexa 350, Alexa 430, Alexa 488, etc.), AMCA, BODIPY 630 / 650, BODIPY 650 / 665, BODIPY-FL, BODIPY-R6G, BODIPY-TMR, BODIPY-TRX, Cascade Blue, Cy2, Cy3, Cy5, Cy5.5, Cy7, Cy7.5, Dylight dyes (Dylight 405, Dylight 488, Dylight 549, Dylight 550, Dylight 649, Dylight 680, Dylight 750, Dylight 800), 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 dye (e.g. Starbright Blue 520, Starbright Blue 700), TAMRA, TET, Tetramethylrhodamine, Texas Red and TRITC.

[0057] "Linker sequence" or "linker" herein refers to a peptide or polypeptide containing two or more amino acid residues connected by peptide bonds, which provides greater rotational freedom for the two polypeptides connected thereby than the two polypeptides connected without a linker. This rotational freedom allows each component of the fusion protein to interact with its intended target without hindrance. Usually these linkers can be a mixture of glycine and serine, for example - (GGGS) n -, wherein n is 1, 2, 3, 4 or 5. Other suitable peptide / polypeptide linker sequences optionally include naturally occurring or non-naturally occurring peptides or polypeptides. The peptide linker sequence is at least 2 amino acids in length. Optionally, the peptide or polypeptide domain is a flexible peptide or polypeptide. Exemplary flexible peptides / polypeptides include, but are not limited to, the amino acid sequences Gly-Ser, Gly-Ser-Gly-Ser, Ala-Ser, Gly-Gly-Gly-Ser, Gly 4 -Ser,(Gly 4 -Ser) 2 ,(Gly 4 -Ser) 3 ,(Gly 4 -Ser) 4 ,(Gly 4 -Ser) 2-Gly-Ala-Gly-Ser-Gly 4 -Ser,Gly-(Gly 4 -Ser) 2 ,Gly 4 -Ser-Gly,Gly-Ser-Gly 2 and Gly-Ser-Gly 2 -Ser. Other suitable peptide linker domains optionally include the TEV linker ENLYFQG, a linear epitope recognized by tobacco mosaic virus protease. Exemplary peptides / polypeptides include, but are not limited to, GSENLYFQGSG. Other suitable peptide linker sequences include helical linkers, such as Ala-(Glu-Ala-Ala-Ala-Lys) n -Ala (n = 1-5). In some embodiments, the linker sequence is a GAP (Gly AlaPro) sequence. In some embodiments, a sequence of 1 to 50 amino acid residues can be used as a linker. In some embodiments, the linker is soluble, flexible, and protease-resistant (i.e., a polypeptide with a linker is expressed in a host cell, and the linker is not cleaved by a protease). As described above, the phrase "connected by a linker sequence" allows the use of one or more linker sequences to connect two or more binding motifs, binding motifs and polypeptides, or binding motifs and antigen-binding fragments. In the case where multiple linker sequences are used to connect binding motifs, connect antigen-binding fragments to binding motifs, or connect polypeptides and binding motifs, the linker sequences may be the same or different.

[0058] In some embodiments of the antigen binding protein having the first, second, third and fourth binding motifs, the binding motifs are linked using a sortase-mediated ligation. Schmohl et al. (2014) discusses sortase-mediated ligation for site-specific modification of proteins, which is now incorporated herein by reference in its entirety. The sortase system uses a sortase and a sortase recognition and bridging domain. In embodiments of the antigen binding protein, the sortase recognition and bridging domains are considered to be binding motifs. Sortase is a transpeptidase produced by Gram-positive bacteria to covalently anchor cell surface proteins to the cell wall. Staphylococcus aureus sortase A (SrtA) cleaves a short C-terminal recognition motif (LPXTG (SEQ ID NO: 19)) (referred to herein as a sortase recognition domain). The sortase recognition domain is a sortase A recognition domain or a sortase B recognition domain. The sortase A recognition domain comprises or consists of the following 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 and n is 0, 1, 2, 3, 4, 5, 7, 8, 9, 10, in the range of 0-5 or 0-10, or any integer not exceeding 100. The sortase B recognition domain comprises the amino acid sequence NPX1TX2 (SEQ ID NO: 21), wherein X1 is glutamine or lysine; X2 is asparagine or glycine; N is asparagine; P is proline and T is threonine.

[0059] The sortase A and B bridging domains contain one or more glycine residues at the N-terminus of the peptide. In certain embodiments, the one or more glycine residues may be optionally: Gly, (Gly) 2 , (Gly) 3 , (Gly) 4 , or (Gly) x , where x is an integer from 1 to 20.

[0060] In some embodiments of the 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 bridging domain. The sortase A or B recognition domain may be optionally fused to the polypeptide at the C-terminus via a glycine / serine-rich linker, and the sortase A or B bridging domain may be optionally fused to the N-terminus of the first fusion protein via a glycine / serine-rich linker.

[0061] In some embodiments of the antigen binding protein comprising a third binding motif and a 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 bridging domain. The sortase A or B recognition domain may be optionally fused to the antigen binding fragment at the C-terminus via a glycine / serine-rich linker, and the sortase A or B bridging domain may be optionally fused to the second binding motif via a glycine / serine-rich linker.

[0062] Thus, in certain embodiments of the antigen binding protein having a first, second, third and fourth binding motifs, the fourth binding motif comprises a sortase recognition domain comprising or consisting of the following 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 and n is 0, 1, 2, 3, 4, 5, 7, 8, 9, 10, in the range of 0-5 or 0-10, or any integer 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 and T is threonine, and the sortase bridging domain comprises: Gly, (Gly) 2 ,(Gly) 3 ,(Gly) 4 , or (Gly) x , where x is an integer from 1 to 20.

[0063] Also provided is a method for producing an antigen binding protein. In one embodiment, the method comprises contacting two or more first antigen binding fragments (each fragment comprising a first binding motif) with a first fusion protein, the first fusion protein comprising two or more second binding motifs connected by a linker sequence. The conditions for contacting the two or more first antigen binding fragments and the first fusion protein are such that a covalent bond is formed between the first binding motif and the second binding motif by protein connection. After the first binding motif is connected to the second binding motif, in an embodiment where the first fusion protein further comprises a third binding motif and the antigen binding protein further comprises a polypeptide having a fourth binding motif, the first fusion protein comprising the third binding motif is contacted with a polypeptide comprising the fourth binding motif. The contact is performed under conditions that also allow the third binding motif to allow the protein to connect to the fourth binding motif spontaneously or with the help of an enzyme.

[0064] In some embodiments, the method of preparing an antigen binding protein comprises contacting one or more first antigen binding fragments (each fragment comprising a first binding motif) with a first fusion protein, the first fusion protein comprising one or more second binding motifs, the second binding motif being connected to one or more third binding motifs via a linker sequence to form an antigen binding protein. The contact is performed under conditions that allow the first binding motif to be covalently coupled to the second binding motif by protein linkage, either spontaneously or with the aid of an enzyme. After the first binding motif is linked to the second binding motif, one or more second antigen binding fragments (each fragment comprising a fourth binding motif) are contacted with the linked first antigen binding fragment-first fusion protein under conditions that also allow the third binding motif to be covalently coupled to the fourth binding motif by protein linkage (spontaneously or with the aid of an enzyme).

[0065] In the method of producing an antigen binding protein having a 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.

[0066] As used herein, the term "protein ligation" refers to the formation of site-specific covalent bonds between the first and second binding motifs and between the third and fourth binding motifs, either spontaneously or with the aid of an enzyme, when the first and second or the third and fourth motifs come into contact with each other. As also described in the present disclosure, protein ligation occurs between specific combinations of binding motifs, such as 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), SnoopTag (SEQ ID NO: 6) and SnoopCatcher (SEQ ID NO: 7), Isopeptag (SEQ ID NO: 8) and Split Spy0128 (SEQ ID NO: 9), SdyTag (SEQ ID NO: 10) and between SdyCatcherDANG short (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 NO: 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 a butterfly pea mucin recognition motif (Asn-His-Val or Asp-His-Val) and the amino terminus of another polypeptide.

[0067] Thus, to generate an antigen binding protein, a first binding motif present in the first antigen binding fragment (e.g., at the C-terminus, N-terminus, or embedded in an amino acid sequence) is capable of forming a covalent bond with a second binding motif in the first fusion protein via protein linkage. 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 in the first fusion protein.

[0068] Similarly, if the third binding motif in the first fusion protein is SnoopCatcher, the corresponding fourth binding motif present in the second antigen binding fragment (e.g., at the C-terminus, N-terminus, or embedded in the amino acid sequence) 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.

[0069] In addition, if the first binding motif present in the first antigen binding fragment is SpyTag, the corresponding second binding motif in the first fusion protein can be K-Tag. Alternatively, if the first binding motif present in the first antigen binding fragment is K-Tag, the corresponding second binding motif in the first fusion protein can be SpyTag. In both cases, SpyLigase is required to catalyze the formation of the isopeptide bond between the two motifs.

[0070] Similarly, if the third binding motif in the first fusion protein is DogTag, the corresponding fourth binding motif present in the second antigen binding fragment is SnoopTag Jr. 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 DogTag. In both cases, SnoopLigase is required to catalyze the formation of the isopeptide bond between the two motifs.

[0071] Therefore, the pairs of motifs (i.e., the first binding motif is paired with the second binding motif, and the third binding motif is paired with the fourth binding motif) are selected so that the two motifs interact with each other through protein ligation, forming a covalent bond spontaneously or with the help of an enzyme. The two pairs of motifs are also selected so that the two pairs of motifs do not interact or are orthogonal, i.e., the SpyTag / SpyCatcher system components do not interact with the SnoopTag / Snooptacher system components.

[0072] The expression of the above-mentioned proteins with binding motifs can be carried out in appropriate host cells, including prokaryotes, such as E. coli, or eukaryotic cells, such as yeast or mammalian cells, such as CHO cells. In certain embodiments, the disclosed proteins comprising various binding motifs are produced in protease-deficient prokaryotes (e.g., protease-deficient E. coli). In various embodiments, the protease-deficient prokaryotic cells (e.g., protease-deficient E. coli cells) are periplasmic protease-deficient.

[0073] Therefore, a protein connection system for producing an antigen binding protein is provided, which includes connecting a first binding motif to an antigen binding fragment, connecting two or more second binding motifs to each other through a linker to produce a fusion protein, and covalently connecting an antigen binding fragment comprising the first binding motif and a fusion protein comprising the second binding motif through a protein connection between the first binding motif and the second binding motif. The third binding motif or the multimeric third binding motif can be connected to a fusion protein comprising the second binding motif through a linker, and the fourth binding motif can be connected to a fourth polypeptide, such as an enzyme, a fluorescent protein, an effector protein, or another antigen binding fragment. The fusion protein comprising the second and third binding motifs can be covalently connected to an antigen binding fragment comprising the first binding motif and the fourth polypeptide through a protein connection between the first and second binding motifs and the third and fourth binding motifs to form an antigen binding protein.

[0074] Non-limiting examples of protein ligation systems include the SpyTag / SpyCatcher system, shorter versions of SpyTag and 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.

[0075] Thus, a first antigen binding fragment comprising an antigen binding fragment and a first binding motif is produced, and a fusion protein comprising a multimeric (e.g., two or more) second binding motif is produced, which can be connected by proteins to form a covalent bond, and can be a component of a protein connection system discussed in paragraph

[0070] . The first antigen binding fragment comprising the first binding motif can be mixed with a fusion protein comprising a multimeric second binding motif to produce an antigen binding protein. The multimeric second binding motif can be optionally connected together in the fusion protein with a linker. In some embodiments, the fusion protein can further comprise one or more third binding motifs of the second protein connection system. One or more third binding motifs can be optionally incorporated into the fusion protein with a linker. A polypeptide comprising a fourth binding motif or a second antigen binding fragment comprising a fourth binding motif (of the second protein connection system) is prepared. Under conditions that allow protein connection to produce an antigen binding protein, the first antigen binding fragment comprising the first binding motif can be mixed with a fusion protein comprising a multimeric second binding motif and one or more third binding motifs. In certain embodiments, a polypeptide comprising a fourth binding motif or a second antigen-binding fragment comprising a fourth binding motif and a first antigen-binding fragment comprising a first binding motif can be mixed with a fusion protein comprising a multimeric second binding motif and one or more third binding motifs under conditions that allow protein ligation to produce an antigen-binding protein. An alternative embodiment provides 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, which can be used in the above method to produce an antigen-binding protein.

[0076] Any protein ligation system described above or known in the art can be used to produce antigen binding proteins.

[0077] For example, in some embodiments, a first antigen binding fragment comprising SpyTag (e.g., at the C-terminus, N-terminus, or embedded in an amino acid sequence) as a first binding motif is produced, and a fusion protein comprising a multimeric second binding motif is produced, wherein SpyCatcher is used as a second binding motif. Under conditions that allow protein connection to produce antigen binding proteins, the first antigen binding fragment-first binding motif fusion protein can be mixed with the multimeric second binding motif fusion protein. Alternatively, an antigen binding fragment comprising SpyCatcher (instead of SpyTag) as a first binding motif and a fusion protein comprising two or more SpyTags (instead of SpyCatcher) as a second binding motif can be produced. Under conditions that allow protein connection to produce antigen binding proteins, the antigen binding fragment-first binding motif fusion protein can be mixed with the multimeric fusion protein.

[0078] In some embodiments, a first antigen binding fragment as a fusion protein comprising a SpyTag as a first binding motif, one or more second binding motifs connected to one or more third binding motifs, wherein SpyCatcher is a second binding motif, SnoopCatcher is a third binding motif (SpyCatcher and SnoopCatcher are connected in any order), and a polypeptide comprising SnoopTag or comprising a second antigen binding fragment of SnoopTag (e.g., at the C-terminus, N-terminus, or embedded in an amino acid sequence) as a fourth binding motif is produced. The first antigen binding fragment, the fusion protein, and the second antigen binding fragment (or polypeptide) can be mixed with each other under conditions that allow protein connection to produce an antigen binding protein, which comprises a first antigen binding fragment comprising a SpyTag connected to a SpyCatcher (second binding motif) of a fusion protein, and the second antigen binding fragment (or polypeptide) comprising a SnoopTag connected to a SnoopCatcher (third binding motif) of the fusion protein. In certain embodiments, a first antigen-binding fragment comprising a SpyTag (as a first binding motif) is generated, a fusion protein comprising one or more SpyCatchers (as a second binding motif) and one or more SnoopTags (as a third binding motif) is generated, either randomly or sequentially, and a second antigen-binding fragment (having the same or different specificity as the first antigen-binding fragment) comprising a SnoopCatcher as a fourth binding motif is generated. n -SnoopTag m The fusion protein (where n and m are 1 or greater) and the second antigen-binding fragment comprising SnoopCatcher as the fourth binding motif can be mixed with each other under conditions that allow protein ligation to produce an antigen-binding protein containing the first antigen-binding fragment and the second antigen-binding fragment, wherein the first antigen-binding fragment comprises a second antigen-binding fragment linked to SpyCatcher. n -SnoopTag m The SnoopCatcher-SpyTag fusion protein, the second antigen binding fragment comprises a n -SnoopTag m Similarly, a first antigen-binding fragment comprising SpyCatcher, SpyTag, and SnoopCatcher can be generated. n -SnoopTag mA fusion protein (where n and m are 1 or greater), and a second antigen-binding fragment comprising SnoopCatcher, and can be mixed with each other under conditions that allow protein ligation to produce an antigen-binding protein containing the first antigen-binding fragment and the second antigen-binding fragment, wherein the first antigen-binding fragment comprises a second antigen-binding fragment linked to SpyTag n -SnoopTag m The second antigen binding fragment comprises a fusion protein of SpyTag and SpyCatcher, and is linked to SpyTag n -SnoopTag m In addition, a first antigen-binding fragment comprising SpyCatcher, SpyTag, and SnoopCatcher can be generated. n -SnoopCatcher m A fusion protein (where n and m are 1 or greater), and a second antigen-binding fragment comprising SnoopTag, and can be mixed with each other under conditions that allow protein ligation to produce an antigen-binding protein containing the first antigen-binding fragment and the second antigen-binding fragment, wherein the first antigen-binding fragment comprises a second antigen-binding fragment linked to SpyTag n -SnoopTag m The second antigen binding fragment comprises a fusion protein of SpyTag and SpyCatcher, and is linked to SpyTag n -SnoopCatcher m SnoopCatcher SnoopTag of the fusion protein. In these embodiments, the sequences of SpyTag, SpyCatcher, SnoopTag, SnoopCatcher and linker sequences discussed above may also be included.

[0079] Antigen binding fragments comprising SpyTag or SnoopTag can be produced by expressing a gene encoding an antigen binding fragment in Escherichia coli using a vector that adds SpyTag or SnoopTag to the C-terminus, N-terminus, or embeds the SpyTag or SnoopTag into its amino acid sequence. A second tag, such as a His tag, can be added to purify the antigen binding fragment containing SpyTag or SnoopTag by affinity chromatography. Antigen binding fragments containing SpyTag or SnoopTag can also be purified without using a second tag. In certain embodiments, SpyTag has a sequence of AHIVMVDAYKPTK (SEQ ID NO: 1) or VPTIVMVDDAYKRYK (SEQ ID NO: 4). In some embodiments, SnoopTag has a sequence of KLGDIEFIKVNK (SEQ ID NO: 6).

[0080] Contains (SpyCatcher) n (where n is ≥ 2), (SnoopCatcher) n (where n≥2), SpyCatcher n -SnoopCatcher m ,SpyCatcher n -SnoopTag m ,SpyTag n -SnoopCatcher m , and SpyTag n -SnoopTag m (where n and m are 1 or greater) a multimeric fusion protein (i.e., a multimeric binding motif) can be produced by expressing a gene encoding a multimeric binding motif in, for example, Escherichia coli, wherein the multimeric binding motif is connected by one or more joints. A tag such as a His tag can also be added to the N- or C-terminus of the multimeric fusion protein to facilitate purification of the fusion protein by affinity chromatography. A protease cleavage site, such as a TEV protease site, 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. A first antigen binding fragment comprising a SpyTag motif and / or a second antigen binding motif (or polypeptide) comprising a SnoopTag binding motif is mixed with a multimeric fusion protein in an appropriate stoichiometric ratio to produce an antigen binding protein. For example, a Fab-SpyTag antigen binding fragment and a SpyCatcher-SpyCatcher fusion protein can be mixed in a stoichiometric ratio of each SpyCatcher-SpyCatcher molecule to 2 Fab-SpyTag molecules to produce a dimeric antigen binding protein.

[0081] Through the SpyTag / SpyCatcher and SnoopTag / SnoopCatcher systems, appropriate conditions such as buffer conditions, pH, temperature and the presence of detergents can be provided to achieve optimal coupling. The artificial antigen-binding protein thus produced can be used as is or further purified before use. Such purification can be performed by size exclusion chromatography, affinity chromatography or other chromatography or other separation techniques known in the art.

[0082] In certain embodiments, coupling is performed in the presence of excess Fab-SpyTag to drive the reaction toward antigen binding protein formation. The resulting antigen binding protein can be purified to remove excess Fab-SpyTag, for example, using size exclusion chromatography or through a purification tag that has been added to the multimeric fusion protein but not to the Fab-SpyTag protein.

[0083] In embodiments where the antigen binding protein is a dimer connected to a polypeptide having additional functions, a SpyLigase and / or SnoopLigase protein connection system can be used. In such embodiments, an antigen binding fragment comprising a SpyTag motif protein can be produced as described above. For a multimeric binding motif, each of the two or more second binding motifs (SpyCatcher) can be replaced with a 10 amino acid K-Tag motif (SEQ ID NO: 12) in the same orientation, and optionally replaced with one or more linkers as described above. Alternatively, 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 motifs can be mixed in the presence of SpyLigase under conditions that allow SpyTag and K-Tag to be attached. Then, a polypeptide comprising SnoopTagJr is added to the mixture in the presence of SnoopLigase under conditions that allow SnoopTagJr and DogTag to be attached. Therefore, in some such embodiments, the first binding motif comprises SpyTag, the second binding motif comprises two or more K-Tags, the third binding motif comprises DogTag, and the fourth binding motif comprises SnoopTagJr. Alternatively, the first binding motif may comprise K-Tag, the second binding motif may comprise two or more SpyTags, the third binding motif may comprise SnoopTagJr, and the fourth binding motif may comprise Dogtag. Similarly, the first binding motif may comprise SpyTag, the second binding motif may comprise two or more K-Tags, the third binding motif may comprise SnoopTagJr, and the fourth binding motif may comprise Dogtag. Alternatively, the first binding motif may comprise K-Tag, the second binding motif may comprise two or more SpyTags, the third binding motif may comprise DogTag, and the fourth binding motif may comprise SnoopTagJr.

[0084] In some embodiments, the first binding motif comprises a SpyTag, the second binding motif comprises two or more K-Tags, the third binding motif comprises a SnoopCatcher, and the fourth binding motif comprises a SnoopTag. 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 SpyTag and K-Tag to connect. Alternatively, the first binding motif comprises a K-Tag, the second binding motif comprises two or more SpyTags, the third binding motif comprises a SnoopTag, and the fourth binding motif comprises a SnoopCatcher, which can then be mixed in the presence of SpyLigase under conditions that allow homologous binding motif pairs to connect.

[0085] In certain embodiments, the first binding motif includes SpyTag, the second binding motif includes two or more SpyCatchers, the third binding motif includes DogTag, and the fourth binding motif includes SnoopTagJr. The antigen binding fragment-SpyTag motif, the SpyCatcher-SpyCatcher-DogTag multimer binding motif, and the polypeptide-SnoopTagJr motif can be mixed in the presence of SnoopLigase under conditions that allow the DogTag motif and the SnoopTagJr motif to be connected. Alternatively, the first binding motif includes SpyCatcher, the second binding motif includes two or more SpyTags, the third binding motif includes SnoopTagJr, and the fourth binding motif includes DogTag. Similarly, the first binding motif may include SpyTag, the second binding motif may include two or more SpyCatchers, the third binding motif may include SnoopTagJr, and the fourth binding motif may include Dogtag. Alternatively, the first binding motif may include SpyCatcher, the second binding motif may include two or more SpyTags, the third binding motif may include DogTag, and the fourth binding motif may include SnoopTagJr. Nucleic acid construct

[0086] Also provided are nucleic acid constructs encoding the antigen binding fragment fused to the binding motif and nucleic acid constructs encoding the multimeric binding motif. Such nucleic acid can be present in an expression vector in a suitable host cell. As described below, the host cell can be prokaryotic or eukaryotic.

[0087] Thus, in one embodiment, a pair of nucleic acid constructs comprises: 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, which may optionally be connected by a linker sequence, The first binding motif and the second binding motif form a covalent bond by ligation when they come into contact with each other spontaneously or with the aid of an enzyme.

[0088] Typically, the 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, can be fused to the L or H chain. The Fab expression cassette can comprise a bicistronic vector that produces an mRNA encoding both the L chain and the H chain, at least one of which is fused to the binding motif. In addition, both the H and L chains can have a signal peptide to direct their export to the periplasm.

[0089] In some embodiments, the polynucleotide of the second nucleic acid construct further encodes a third binding motif, and the third binding motif is optionally connected to the two or more second binding motifs or between the two or more second binding motifs through a linker sequence. A third nucleic acid construct comprising a polynucleotide sequence encoding a polypeptide fused to a fourth binding motif can also be provided. When the third binding motif contacts the fourth binding motif spontaneously or with the aid of an enzyme, the third binding motif is connected to the fourth binding motif. The first binding motif-second binding motif pair is orthogonal to the third binding motif-fourth binding motif pair.

[0090] 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 fragment fused to a first binding motif, 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 may optionally be connected by a linker disclosed herein. A third nucleic acid construct may also be provided, comprising a polynucleotide sequence encoding one or more second antigen binding fragments each fused to a fourth binding motif. Any or all of the polynucleotides encoding one or more second binding motifs may be located before, after or between any or all of the polynucleotides encoding one or more third binding motifs. The first antigen binding fragment and the second antigen binding fragment may bind to the same or different antigens. The first binding motif and the second binding motif achieve protein connection when they contact each other spontaneously or with the aid of an enzyme. The third binding motif and the fourth binding motif achieve protein connection when they contact each other 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.

[0091] Nucleic acid constructs are usually introduced into various vectors. The vector of the present invention usually contains transcription or translation control sequences required for expressing fusion proteins. Suitable transcription or translation control sequences include but are not limited to replication origin, promoter, enhancer, repressor binding region, transcription start site, ribosome binding site, translation start site and termination site for transcription and translation.

[0092] The origin of replication (commonly referred to as the ori sequence) allows the vector to replicate in a suitable host cell. The choice of ori depends on the type of host cell and / or gene packaging used. When the host cell is a prokaryotic organism, the expression vector usually contains an ori sequence that directs the autonomous replication of the vector in the prokaryotic cell. Preferred prokaryotic ori can direct the replication of the vector in bacterial cells. Non-limiting examples of such ori include pMB1, pUC and other E. coli origins.

[0093] In eukaryotic systems, higher eukaryotic organisms contain multiple DNA replication origins, but the ori sequence has not been clearly determined. Suitable replication origins 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.

[0094] As used herein, "promoter" is a DNA region that can bind RNA polymerase under specific conditions and initiate transcription of the coding region located downstream (3' direction) of the promoter. It can be constitutive or inducible. Typically, the promoter sequence binds to the transcription start site at its 3' end and extends upstream (5' direction) to include the minimum number of bases or elements necessary to initiate transcription at a detectable level above background. The promoter sequence has a transcription start site and a protein binding domain responsible for RNA polymerase binding. Eukaryotic promoters typically, but not always, contain "TATA" boxes and "CAT" boxes.

[0095] The selection of promoter depends to a great extent on the host cell into which the vector is introduced. For prokaryotes, various powerful promoters are known in the art. Preferred promoters are lac promoter, Trc promoter, T7 promoter and pBAD promoter. Usually, in order to obtain expression of exogenous sequences in multiple species, a prokaryotic promoter can be placed directly after a eukaryotic promoter, or placed in an intron sequence downstream of a eukaryotic promoter.

[0096] Suitable promoter sequences for eukaryotic cells include 3-phosphoglycerate kinase or other glycolytic enzymes, such as enolase, 3-phosphoglyceraldehyde dehydrogenase, hexokinase, pyruvate decarboxylase, phosphofructokinase, glucose-6 phosphate isomerase, 3-phosphoglycerate mutant enzyme, pyruvate kinase, trisaccharide phosphate isomerase, phosphoglucose isomerase and glucokinase promoters. Other promoters with the additional advantage of transcription controlled by growth conditions are promoter regions for alcohol dehydrogenase 2, isocytochrome C, acid phosphatase, degradation enzymes associated with nitrogen metabolism and the above-mentioned 3-phosphoglyceraldehyde dehydrogenase, and enzymes responsible for maltose and galactose utilization. Preferred promoters for mammalian cells are SV40 promoter, CMV promoter, β-actin promoter and hybrids thereof. Preferred promoters for yeast cells include but are not limited to GAL 10, GAL I, TEFI in Saccharomyces cerevisiae, and GAP, AOX1 in Pichia pastoris.

[0097] In constructing the vector of the present invention, the terminator sequence combined with the protein coding sequence is also inserted into the 3' end of the sequence to be transcribed, to provide polyadenylation of mRNA and / or transcription termination signal. The terminator sequence preferably comprises one or more transcription termination sequences (e.g., polyadenylation sequence), and can also be extended by comprising additional DNA sequences to further interrupt transcription read-through. The preferred terminator sequence (or termination site) of the present invention has a gene followed by a transcription termination sequence, which is its own termination sequence or a heterologous termination sequence. Examples of such terminator sequences include termination codons coupled to various yeast transcription termination sequences or mammalian polyadenylation sequences, which are known in the art and can be widely available. In the case where the terminator comprises a gene, it may be advantageous to use a gene encoding a detectable or selective marker; thereby providing a means by which the presence and / or absence of a terminator sequence (and the corresponding inactivation and / or activation of the transcription unit) can be detected and / or selected.

[0098] In addition to the above elements, the vector may contain a selective 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 may be carried on another polynucleotide sequence co-introduced into the host cell. Only those host cells into which the selectable gene has been introduced can survive and / or grow under selective conditions. Typical selection genes encode proteins that confer resistance to (a) antibiotics or other toxins such as ampicillin, kanamycin, neomycin, bleomycin, G418, methotrexate, etc.; (b) compensate for auxotrophic deficiencies; or (c) provide key nutrients that cannot be obtained from complex culture media. The choice of a suitable marker gene will depend on the host cell, and suitable genes for different hosts are known in the art.

[0099] In one embodiment, the expression vector is a shuttle vector that can replicate in at least two unrelated host systems. In order to promote this replication, the vector usually contains at least two replication origins, one effective in each host system. Generally, the shuttle vector can replicate in a eukaryotic host system and a prokaryotic host system. This can detect protein expression (expression cell type) in a eukaryotic host and vector amplification (amplification cell type) in a prokaryotic host. Preferably, one replication origin is from SV40 or 2u, and one is from pUC, although any suitable starting point known in the art can be used, as long as it guides the replication of the vector. When the vector is a shuttle vector, the vector preferably includes at least two selection markers, one for expressing cell types and one for amplifying cell types. Any selection marker known in the art or those described herein can be used, as long as it works in the expression system utilized.

[0100] The vector of the present invention can be obtained by using recombinant cloning methods and / or by chemical synthesis. A large amount of recombinant cloning techniques, such as PCR, restriction endonuclease digestion and connection, are well known in the art and do not need to be described in detail at this time. Those skilled in the art can also use the sequence data provided herein or the sequence data in a public or proprietary database to obtain the required vector by any synthetic means available in the art. In addition, suitable sequences can be cut out from various DNA sources using well-known restriction and ligation techniques, and operably integrated with the exogenous sequence to be expressed according to embodiments of the present invention. Reagent test kit

[0101] Also provided are kits for preparing antigen binding proteins. In some embodiments, the kit includes two or more of the following components: 1. a first antigen-binding fragment comprising a first binding motif; and 2. A first fusion protein comprising two or more second binding motifs, optionally linked by a linker sequence and optionally comprising a detectable label (e.g., biotin, HRP or a fluorophore); and / or 3. a first fusion protein comprising two or more second binding motifs, which are optionally connected by a linker sequence, and a third binding motif, which is optionally connected to the two or more second binding motifs by a linker sequence and optionally comprises a detectable label; and / or 4. A polypeptide comprising a fourth binding motif, optionally comprising a detectable label; and / or 5. A fusion protein comprising one or more second binding motifs and one or more third binding motifs, the binding motifs are optionally connected by a linker sequence and optionally comprise 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.

[0102] The kit user may select at least two components having at least one binding motif pair (i.e., a first binding motif-second binding motif pair and / or a third binding motif-fourth binding motif pair) from 1-6 above, which, when mixed, will form covalent bonds spontaneously or with the aid of an enzyme through protein ligation. If the kit user selects two binding motif pairs, the binding motif pairs are selected so that the first binding motif pair is orthogonal to the second binding motif pair, i.e., the first binding motif-second binding motif pair is orthogonal to the third binding motif-fourth binding motif pair. The kit user may also express any peptide in a suitable host using a nucleic acid construct comprising a polynucleotide sequence encoding an antigen-binding fragment, fusion protein, and / or polypeptide as defined in 1-6.

[0103] Each component can be provided in liquid form (e.g., solution) or in solid form (e.g., powder) prior to use, which is reconstituted with a liquid (e.g., buffer) prior to use. In some embodiments, the kit further comprises instructions for connecting one or more binding motif pairs. Additional disclosures and requested topics

[0104] Item 1. An antigen-binding protein comprising: two or more first antigen-binding fragments comprising a first binding motif; and A fusion protein comprising two or more second binding motifs, optionally connected by a linker sequence, The first binding motifs of two or more first antigen binding fragments are covalently coupled to two or more second binding motifs via a protein linker.

[0105] Item 2. An antigen-binding protein according to Item 1, wherein the fusion protein comprising two or more second binding motifs is connected by a linker sequence.

[0106] Item 3. The antigen-binding protein according to Item 2, wherein the fusion protein further comprises a third binding motif, the third binding motif is optionally connected to the two or more second binding motifs via a linker, and the antigen-binding protein further comprises a polypeptide comprising a fourth binding motif, wherein the third binding motif is covalently coupled to the fourth binding motif of the polypeptide via a protein linkage, and The first binding motif-second binding motif pair is orthogonal to the third binding motif-fourth binding motif pair.

[0107] Item 4. An antigen-binding protein according to Item 3, wherein the polypeptide is an enzyme, a fluorescent protein, an effector protein or an antigen-binding fragment.

[0108] Item 5. An antigen-binding protein comprising: one or more first antigen-binding fragments comprising a first binding motif; A fusion protein comprising one or more second binding motifs and one or more third binding motifs, wherein the one or more second binding motifs and the one or more third binding motifs are optionally connected by a linker; and one or more second antigen-binding fragments comprising a fourth binding motif, wherein the first binding motif is covalently coupled to the second binding motif via a protein linkage, wherein the third binding motif is covalently coupled to the fourth binding motif via a protein linker, The first binding motif-second binding motif pair is orthogonal to the third binding motif-fourth binding motif pair.

[0109] Item 6. The antigen binding protein according to Item 5, wherein the antigen binding protein is bispecific, bispecific and dimeric, or bispecific and multimeric.

[0110] Item 7. The antigen-binding protein according to any of the above items, further comprising a purification tag at the N-terminus or C-terminus of the fusion protein.

[0111] Item 8. An antigen binding protein according to any of the above items, wherein one or more binding motifs are located at the C-terminus, N-terminus or embedded in the amino acid sequence of the first and / or second antigen binding fragment, fusion protein or polypeptide.

[0112] Item 9. An antigen binding protein according to Item 8, wherein one or more binding motifs in the fusion protein are sequential or random.

[0113] Item 10. An antigen binding protein according to any of the above items, wherein the linker sequence is 1-5 repeats of the sequence GGGGS.

[0114] Item 11. An antigen binding protein according to Item 1 or 2, wherein: a) 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 thereto, 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 thereto; or b) 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 to 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 to SEQ ID NO:1, 4, 6, 8, 10, 13 or 22.

[0115] Item 12. An antigen binding protein according to any one of Items 3 to 6, wherein: a) the first binding motif comprises SEQ ID NO: 1, 4, 8 or 22, or a sequence having at least 60% sequence identity thereto, and the second binding motif comprises SEQ ID NO: 2, 3, 5, 9, 12, or 23, or a sequence having at least 60% sequence identity thereto; or b) the first binding motif comprises SEQ ID NO: 2, 3, 5, 9, 12, or 23 or a sequence having at least 60% sequence identity to SEQ ID NO: 2, 3, 5, 9, 12, or 23, and the second binding motif comprises SEQ ID NO: 1, 4, 8, or 22 or a sequence having at least 60% sequence identity to SEQ ID NO: 1, 4, 8, or 22.

[0116] Item 13. The antigen binding protein according to Item 12, wherein: a) the third binding motif comprises SEQ ID NO: 7, 11 or 14, or a sequence having at least 60% sequence identity thereto, and the fourth binding motif comprises SEQ ID NO: 6, 10 or 13, or a sequence having at least 60% sequence identity thereto; or b) the third binding motif comprises SEQ ID NO: 6, 10 or 13 or a sequence having at least 60% sequence identity thereto, and the fourth binding motif comprises SEQ ID NO: 7, 11 or 14 or a sequence having at least 60% sequence identity thereto.

[0117] Item 14. An antigen binding protein according to any one of Items 3 to 6, wherein: a) the first binding motif comprises SEQ ID NO: 6, 10 or 13 or a sequence having at least 60% sequence identity thereto, and the second binding motif comprises SEQ ID NO: 7, 11 or 14 or a sequence having at least 60% sequence identity thereto; or b) the first binding motif comprises SEQ ID NO: 7, 11 or 14 or a sequence having at least 60% sequence identity thereto, and the second binding motif comprises SEQ ID NO: 6, 10 or 13 or a sequence having at least 60% sequence identity thereto.

[0118] Item 15. The antigen binding protein according to Item 14, wherein: a) the third binding motif comprises SEQ ID NO: 2, 3, 5, 9, 12 or 23 or a sequence having at least 60% sequence identity to SEQ ID NO: 2, 3, 5, 9, 12, or 23, and the fourth binding motif comprises SEQ ID NO: 1, 4, 8, or 22 or a sequence having at least 60% sequence identity to SEQ ID NO: 1, 4, 8, or 22; or b) the third binding motif comprises SEQ ID NO: 1, 4, 8, or 22 or a sequence having at least 60% sequence identity to SEQ ID NO: 1, 4, 8, or 22, and the fourth binding motif comprises SEQ ID NO: 2, 3, 5, 9, 12 or 23 or a sequence having at least 60% sequence identity to SEQ ID NO: 2, 3, 5, 9, 12, or 23.

[0119] Item 16. An antigen binding protein according to Item 3, wherein the fourth binding motif comprises a sortase recognition domain and the third motif comprises a sortase bridging domain.

[0120] Item 17. An antigen-binding protein according to Item 5, wherein the antigen-binding protein comprises a first antigen-binding fragment, the first antigen-binding fragment comprises a first binding motif, a second binding motif connected to a third binding motif via a linker sequence, and the second antigen-binding fragment comprises a fourth binding motif, the fourth binding motif comprises a sortase recognition domain, and the third binding motif comprises a sortase bridging domain.

[0121] Item 18. An antigen binding protein according to Item 16 or 17, wherein 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, in the range of 0-5 or 0-10, or any integer up to 100, NPX1TX2 (SEQ ID NO: 21), wherein X1 is glutamine or lysine; X2 is asparagine or glycine; N is asparagine; P is proline and T is threonine, and the sortase bridging domain comprises: Gly, (Gly) 2 , (Gly) 3 , (Gly) 4 , or (Gly) x , where x is an integer from 1 to 20.

[0122] Item 19. An antigen binding protein according to any one of Items 1-5, wherein the fusion protein or polypeptide further comprises a detectable label.

[0123] Item 20. An antigen binding protein according to Item 19, wherein the detectable label is a fluorophore, a fluorescent protein, biotin or an enzyme.

[0124] Item 21. A nucleic acid construct pair, comprising: 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 sequence encoding a fusion protein comprising two or more second binding motifs, which are optionally connected by a linker sequence, The first binding motif and the second binding motif are linked to form a covalent bond via the protein when they come into contact with each other spontaneously or with the aid of an enzyme.

[0125] Item 22. A nucleic acid construct pair according to Item 21, wherein the polynucleotide of the second nucleic acid construct further encodes a third binding motif, and the third binding motif is optionally connected to the two or more second binding motifs or between two or more second binding motifs via a linker sequence, and the nucleic acid construct pair further includes a third nucleic acid construct, which comprises a polynucleotide sequence, and the polynucleotide encodes a polypeptide fused to a fourth binding motif. wherein the third binding motif and the fourth binding motif are linked to form a covalent bond through the protein when they come into contact with each other spontaneously or with the aid of an enzyme, and The first binding motif-second binding motif pair is orthogonal to the third binding motif-fourth binding motif pair.

[0126] Item 23. A nucleic acid construct, comprising: a) a first nucleic acid construct comprising a polynucleotide sequence encoding one or more first antigen-binding fragments fused to a first binding motif; and b) 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, the binding motifs optionally being connected by a linker sequence; and c) a third nucleic acid construct comprising a polynucleotide sequence encoding one or more second antigen-binding fragments fused to a fourth binding motif; wherein the first binding motif and the second binding motif are linked to form a covalent bond through the protein when they come into contact with each other spontaneously or with the aid of an enzyme, wherein the third binding motif and the fourth binding motif are linked to form a covalent bond through the protein when they come into contact with each other spontaneously or with the aid of an enzyme, and The first binding motif-second binding motif pair is orthogonal to the third binding motif-fourth binding motif pair.

[0127] Item 24. The nucleic acid construct pair according to Item 23, wherein the polynucleotides encoding any or all of the one or more second binding motifs may be located before, after or between the polynucleotides encoding any or all of the one or more third binding motifs.

[0128] Item 25. A vector comprising the nucleic acid construct of any one of Items 21 to 24 or 33.

[0129] Item 26. A host cell comprising the nucleic acid construct and / or vector defined in any one of Items 21-25 or 33.

[0130] Item 27. A method for producing an antigen binding protein, the method comprising contacting two or more first antigen binding fragments (each fragment comprising a first binding motif) with a fusion protein comprising two or more second binding motifs, wherein the two or more second binding motifs are optionally connected by a linker sequence, The contacting is performed under conditions that allow the first binding motif to be covalently coupled to the second binding motif via protein linkage, either spontaneously or with the assistance of an enzyme.

[0131] Item 28. The method according to Item 27, wherein: The fusion protein further comprises a third binding motif, and the antigen binding protein further comprises a polypeptide comprising a fourth binding motif; After linking the first binding motif to the second binding motif, contacting the fusion protein comprising the third binding motif with a polypeptide comprising a fourth binding motif; and The contacting is performed under conditions that allow the third binding motif to be covalently coupled to the fourth binding motif, either spontaneously or with the aid of an enzyme, by protein ligation; provided that the first binding motif-second binding motif pair is orthogonal to the third binding motif-fourth binding motif pair.

[0132] Item 29. A method for producing an antigen binding protein, comprising: contacting one or more first antigen-binding fragments (each fragment comprising a first binding motif) with a fusion protein comprising one or more second binding motifs and one or more third binding motifs, the second binding motifs and the third binding motifs being optionally linked by a linker sequence to form an antigen-binding protein, wherein the contacting is performed under conditions that allow the first binding motif to be covalently coupled to the second binding motif by protein ligation (spontaneously or with the aid of an enzyme); After linking the first binding motif to the second binding motif, contacting one or more second antigen binding fragments (each fragment comprising a fourth binding motif) with the antigen binding protein, wherein the contacting is performed under conditions that also allow the third binding motif to be covalently coupled to the fourth binding motif by protein ligation (spontaneously or with the aid of an enzyme); and The first binding motif-second binding motif pair is orthogonal to the third binding motif-fourth binding motif pair.

[0133] Item 30. A kit comprising: a. a first antigen-binding fragment comprising a first binding motif; and b. a first fusion protein comprising two or more second binding motifs connected by a linker sequence; and / or c. a first fusion protein comprising two or more second binding motifs and a third binding motif, wherein the third binding motif is connected to the two or more second binding motifs via 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 connected to one or more third binding motifs via 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 as defined in any one of a-f.

[0134] Item 31. A kit according to Item 30, wherein the first fusion protein, fusion protein or polypeptide further comprises a detectable label.

[0135] Item 32. A kit according to Item 31, wherein the detectable label is a fluorophore, a fluorescent protein, biotin or an enzyme.

[0136] Item 33. A nucleic acid construct according to Items 21-24, wherein the binding motifs of the fusion protein are connected by one or more linker sequences.

[0137] Item 34. The method according to Items 27-29, wherein the binding motifs of the fusion protein are connected by one or more linker sequences. Example

[0138] The following examples are provided for illustration only and are not intended to be limiting. It will be readily apparent to those skilled in the art that there are a number of non-critical parameters that can be varied or modified and yield substantially the same or similar results. Example 1 - BiCatcher construction, expression and purification

[0139] 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. A short linker ((GGGGS) 2 ,2G) and long linker ((GGGGS) 4 ,4G) sequences were used to connect the spyCatcher subunits. BiCatcher also has a His-tag and a TEV protease cleavage site at its N-terminus. Each BiCatcher_l and BiCatcher_s sequence with short and long linkers was cloned into a pET28a vector. The vector was cloned into E. coli BL21(DE3). The BiCatcher fusion protein was expressed by culturing BL21(DE3) cells in 250ml 2xYT broth containing 0.1% glucose and kanamycin. After growing for 1 hour at 37°C, the culture was induced with 0.8mM IPTG.

[0140] Expression of the fusion protein continued for approximately 16 hours at 30°C. 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 into 1xPBS.

[0141] The fusion protein was then analyzed by SDS-PAGE ( Figure 5 ). TMVertical electrophoresis cell was used with AnyKD gel and Bio-Rad Precision Plus Protein Standard molecular weight markers. The protein purity was determined by densitometry. The concentration and purity of BiCatcher_1 and _s with short and long linkers are provided in Table 3 below. The purity of all fusion proteins was higher than 80%. Table 3

[0142] Based on SpyCatcher2 sequence (SEQ ID NO:5) and short linker (GGGGS) 2 A BiCatcher2 fusion protein was also constructed. His tag and TEV protease cleavage site were used as described for the above constructs. For all SpyCatcher2 constructs, an Asn(N) to Asp(D) mutation was incorporated at position 105 to remove the deamidation site. The sequence was cloned into pET28a vector and transformed into BL21(DE3) cells. Expression and purification were performed as described for BiCatcher_1. Example 2—Fab-SpyTag construction, expression and purification

[0143] Human Fab fragments with SpyTag and His tags at the C-terminus of the heavy chain were constructed by directly fusing SpyTag to the C-terminus of the CH1 domain, followed by a short linker (GAP) and a hexahistidine tag. Alternatively, human Fab fragments with FLAG tags, SpyTag and His tags were constructed by using a short linker (EF) between the C-terminus of CH1 and the FLAG tag, followed by a linker (GGS) and SpyTag and a linker (GAP) and His tag. SpyTag2 (SEQ ID NO: 4) instead of SpyTag's additional construct. The light and heavy chains were cloned into a bicistronic bacterial expression vector with a lac promoter. Both the light and heavy chain genes contain secretion signals for transport to the periplasm. The vector with the Fab-SpyTag-H construct was transformed into E. coli TG1 F- (without the F-episome). The Fab-FLAG-SpyTag-H construct was transformed into a protease-deficient E. coli strain as described in co-filed U.S. application 62 / 819,748 (periplasmic fusion protein; filed March 18, 2019; docket number BRL.130P). The Fab fragment was expressed by culturing E. coli cells in 250 ml 2xYT broth containing 0.1% glucose and chloramphenicol. After growing for 1 hour at 37°C, the culture was induced with 0.8 mM IPTG. Expression was allowed to proceed for approximately 16 hours at 30°C. The culture was centrifuged and the cells were frozen at -80°C. Cells were lysed with BugBuster lysis buffer (Millipore-Sigma). Fusion proteins were purified using Ni-NTA affinity matrix and buffer exchanged into 3xPBS. Example 3 - Ligation of Fab-SpyTag and BiCatcher

[0144] The BiCatcher fusion protein from Example 1 and the Fab-FLAG-SpyTag-His fusion protein from Example 2 were linked to each other by reacting 12 μM 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 heated at 95°C for 5 minutes before SDS-PAGE on a 4-20% polyacrylamide gel (Bio-Rad Mini-PROTEAN TGX). Gel image ( Figure 6 ) showed that the Fab-SpyTag reacted with the BiCatcher construct and the Fab heavy chain was almost completely attached to the BiCatcher.

[0145] The BiCatcher2 fusion protein from Example 1 was linked to the Fab-FLAG-SpyTag2-His antibody from Example 2 by reacting 10 μM Fab with 4 μM BiCatcher2 in PBS. A molar amount of SpyTag2 in excess of 25% of 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 on a 4-20% polyacrylamide gel (Bio-Rad Mini-PROTEAN TGX). Image of the Coomassie-stained gel ( Figure 7 ) shows that BiCatcher2 reacts with SpyTag2 at the Fab heavy chain. After 60 minutes, the BiCatcher2 band disappears completely, indicating that the ligation reaction is complete. At the beginning of the reaction, two products can be seen: BiCatcher2 coupled to one Fab and BiCatcher2 coupled to two Fabs. The band of the single coupling product decreases with the extension of the reaction time until only the double-ligation product can be seen on the gel after 60 minutes. Example 4 - Comparison of Fab SpyTag and BiCatcher Assay Performance

[0146] To show that bivalent Fabs outperform monovalent Fabs due to avidity, Western blotting was performed on HKB11 mammalian cell lysates. Lysates were separated on Bio-Rad Mini-PROTEAN TGX 4-20% polyacrylamide gels and then blotted onto PVDF membranes using the Bio-Rad Trans-Blot Turbo transfer system. The membranes were blocked with 5% milk in TBST overnight at 4°C. Fab-SpyTag antibodies specific for human HSPA5 were prepared as described in ( Figure 8 Fab-Spy-H in ) was used as is or by using BiCatcher with a short linker ( Figure 8 The antibody samples were then diluted to a concentration equivalent to 1 μg / ml Fab (equimolar antigen binding sites in both preparations) in TBST with 0.5% milk, and the membranes were incubated on a shaker for 1 h at room temperature. Detection was performed using an HRP-conjugated goat anti-human Fab secondary antibody and Western Blot Clarity ECL substrate, and pictures were taken using the Gel Doc imaging system. Images of both spots were taken at the same contact time (10 seconds). The bands of the bivalent Fab on the Western blot are much stronger than those of the monovalent Fab ( Figure 8 ), clearly showing the sensitivity advantage of the BiCatcher dimerized Fab. Example 5 - Construction, expression and purification of labeled BiCatcher

[0147] The labeled BiCatcher fusion protein was constructed by first modifying the nucleic acid sequence encoding BiCatcher (i.e., SpyCatcher002 (SEQ ID NO: 5)-Linker-SpyCatcher002) to bind a cysteine ​​residue at one of three different amino acid positions: 1. N-terminus 2. Embedded in the linker between two SpyCatcher002 subunits 3. C-end

[0148] Seven cysteine-containing BiCatcher nucleic acid constructs (cys-BiCatcher constructs) were prepared using various combinations of the above subunit nucleic acid sequences (Table 4) to add one, two or three cysteines to the cys-BiCatcher constructs. Table 4

[0149] Seven cys BiCatcher constructs were cloned into pET28a vectors. The vectors were transformed into E. coli BL21 (DE3). Cys-BiCatcher fusion proteins were expressed by culturing BL21 (DE3) cells in 250 ml 2xYT broth containing 0.1% glucose and kanamycin. After growing for 1 hour at 37°C, the cultures were induced with 0.8 mM IPTG.

[0150] Expression of each cys-BiCatcher fusion protein was continued for approximately 16 hours at 30°C. The culture was 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 buffer exchanged into 1xPBS.

[0151] Biotin or HRP is then specifically attached to the sulfhydryl group of cysteine ​​in each cys-BiCatcher fusion protein using maleimide chemistry. In order to biotinylate the fusion protein, each fusion protein in PBS buffer (100mM phosphate, 150mM NaCl, pH 7.0) is reduced by adding 30 equivalents of tris (2-carboxyethyl) phosphine (TCEP) for 30 minutes at room temperature. Then 20 equivalents of biotin-maleimide (stock solution in DMSO) are added to each reaction and cultured at room temperature for 4.5 hours. For HRP labeling, each cys-BiCatcher fusion protein in PBS is reduced with 30 equivalents of TCEP for 30 minutes at room temperature. 6 equivalents of maleimide-activated HRP (Thermo Fisher #31485) are dissolved in PBS and added to each reduced fusion protein. Each reaction is incubated at room temperature for 4 hours and quenched by incubating each Cys residue with 20 times molar excess of N-ethylmaleimide for 30 minutes at room temperature. All labeled fusion proteins were dialyzed against PBS. Example 6 - Ligation of Fab-SpyTag and labeled BiCatcher

[0152] The biotin- or HRP-labeled BiCatcher fusion proteins from Example 5 and the Fab-FLAG-SpyTag-His fusion proteins from Example 2 were ligated to each other by reacting 12 μM Fab-FLAG-SpyTag-His with 6 μM of each BiCatcher in 1×PBS. The ligation reaction was allowed to proceed at room temperature for 16 hours. Example 7 - Performance of Fab-SpyTag coupled to labeled BiCatcher

[0153] The performance of each conjugate from Example 6 was tested in an ELISA. Antigen (alemtuzumab) was coated on Maxisorp ELISA plates at a concentration of 5 μg / ml in PBS at 4°C. The plates were blocked with 5% BSA in PBST (PBS with 0.05% Tween 20) for 1 hour, and then Fab-Bicatcher002-biotin or Fab-Bicatcher002-HRP-conjugates from Example 6 were serially diluted with 5% BSA in PBST and incubated for 1 hour. The ELISA plates containing biotinylated BiCatcher002 conjugates were then incubated with streptavidin-HRP (Bio-Rad #STAR5A, 1:1000 in 5% BSA in PBST) or neutralizing avidin HRP (Thermo Fisher #31030, 1:8000 in 5% BSA in PBST). All plates were detected using QuantaBlu fluorescent detection reagent (Thermo Fisher #15169) (ie, using biotin and HRP-conjugated BiCatcher002).

[0154] Fig. 9 Shown are fluorescence graphs showing the variation of biotinylated cys-BiCatcher antibody concentrations for seven cys-BiCatcher fusion proteins, detected using streptavidin-HRP. Fig.10 Fluorescence curves showing the variation of HRP-labeled cys-BiCatcher antibody concentrations for seven cys-BiCatcher fusion proteins are shown. The results for both labels show that CysBiCatchers with 2 or 3 cysteine ​​residues perform better than CysBiCatchers with only one cysteine, while CysBiCatcher 111 with 3 cysteine ​​residues performs best.

[0155] Fig.11 The fluorescence graph of the change in antibody concentration of a biotinylated cys-BiCatcher fusion protein (CysBiCatcher111) with 3 cysteine ​​amino acid residues was detected using Neutravidin-HRP. In this experiment, the performance of chemically biotinylated IgG was compared with a Fab attached to biotinylated CysBiCatcher111. The analysis was performed as described in

[0113] . The results showed that the performance of CysBiCatcher 111 was superior to the directly biotinylated IgG. Example 8 - MultiCatcher construction, expression and purification

[0156] A MultiCatcher fusion protein consisting of three (TriCatcher2), four (TetraCatcher2) and five (PentaCatcher2) SpyCatcher2 was constructed. SpyCatcher2 (SEQ ID NO: 5) was fused to the 2-mer SpyCatcher2 protein via a short linker ((GGGGS) 2 ) were genetically linked, 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. Example 9 - Attachment of Fab-FLAG-SpyTag2-His to MultiCatcher

[0157] BiCatcher2 from Example 1 and MultiCatcher from Example 8 were linked to the Fab-FLAG-SpyTag2-His antibody from Example 2 by reacting 12 μM SpyCatcher2 sites (non-MultiCatcher molecules) with 14.4 μM Fab in PBS. A 20% excess of SpyTag2 relative to the SpyCatcher2 sites was used to achieve complete reaction of all SpyCatcher2 sites. The reactions were carried out overnight at room temperature. For analysis, 1 μg of each coupling product was loaded onto a 4-20% polyacrylamide gel (Bio-Rad Mini-PROTEAN TGX) after heating at 95°C for 5 minutes. Images of Coomassie stained gels ( Fig.12 ) showed that all SpyCatcher2 sites of MultiCatcher reacted with SpyTag2 on the Fab heavy chain, and the corresponding multi-Fab molecules appeared in the gel. Example 10 - Western Blot Application of MultiCatcher-coupled Fab

[0158] The performance of various MultiCatcher-conjugated Fabs was compared by Western blotting to show that higher valencies lead to increased affinity and detection sensitivity. For each antibody, lanes containing 1.8 μl (lane a) and 0.36 μl (lane b) of total cell lysate from the human HKB11 cell line and Bio-Rad Precision Plus Protein Standard molecular weight markers were loaded onto a non-reducing AnykD polyacrylamide gel (Bio-Rad Mini-PROTEAN TGX). Proteins were transferred to PVDF membranes using the Bio-Rad Trans-Blot Turbo transfer system. The membranes were blocked with 5% milk in TBST overnight at 4°C. Detection was performed using a specific Fab-SpyTag antibody against human GAPDH, which was conjugated to SpyCatcher2, Bi, Tri-Tetra, or PentaCatcher2. For each antibody construct, equimolar amounts based on the Fab fragment (equivalent to 2 μg of unconjugated Fab) were used in TBST containing 5% milk, and the membranes were incubated on a shaker for 1 h at room temperature. Detection was performed using HRP-conjugated goat anti-human Fab secondary antibody and Western Blot Clarity ECL substrate, and pictures were taken using the Gel Doc Imaging System. Images of all spots were taken at the same contact time (1.0 sec). The increased valency of the MultiCatcher improves the sensitivity of Western blot detection ( Fig.13 ). Therefore, the results clearly show that the avidity of MultiCatcher-coupled Fab improves sensitivity.

[0159] All patents, patent applications, and other published references cited in this specification 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 DSSGKTISTWISDGQVKDFY LYPGKYTFVE TAAPDGYEVA TAITFTVNEQ GQVTVNGKAT KGDAHI SEQ ID NO: 3 (SpyCatcher short) <h2 style=";text-align:left;direction:ltr">GDSATHIKFS KRDEDGKELA GATMELRDSS GKTISTWISD GQVKDFYLYP GKYTFVETAAPDGYEVATAI TFTVNEQGQV TVNG<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> SEQ ID NO: 4(SpyTag002)<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> VPTIVMVDAY KRYK<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> SEQ ID NO: 5(SpyCatcher002)<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> AMVTTLSGLS GEQGPSGDMT TEEDSATHIK FSKRDEDGRE LAGATMELRD SSGKTISTWISDGHVKDFYL YPGKYTFVET AAPDGYEVAT AITFTVNEQG QVTVNGEATK GDAHTGSSGS<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> SEQ ID NO: 6(SnoopTag)<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> KLGDIEFIKV NK<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> SEQ ID NO: 7(SnoopCatcher)<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> KPLRGAVFSL QKQHPDYPDI YGAIDQNGTY QNVRTGEDGK LTFKNLSDGK YRLFENSEPAGYKPVQNKPI VAFQIVNGEV RDVTSIVPQD IPATYEFTNG KHYITNEPIP PK<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> SEQ ID NO: 8(Isopeptag)<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> TDKDMTITFT NKKDAE<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> SEQ ID NO: 9(Split Spy0128)<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> NSLDSTTLTV KKKVSGTGGD RSKDFNFGLT LKANQYYKAS EKVMIEKTTKGGQAPVQTEA SIDQLYHFTL KDGESIKVTN LPVGVDYVVT EDDYKSEKYT TNVEVSPQDG AVKNIAGNSTEQETSTDKDM TI SEQ ID NO: 10 (SdyTag) DPIVMIDNDK PIT SEQ ID NO: 11 (SdyCatcherDANG short) GRGSSGLSGE TGQSGNTTIE EDSTTHVKFS KRDANGKELA GAMIELRNLS GQTIQSWISDGTVKVFYLMP GTYQFVETAA PEGYELAAPI TFTIDEKGQI WVDS SEQ ID NO: 12 (K-tag) ATHIKFSKRD SEQ ID NO: 13 (SnoopTagJr) KLGSIEFIKV NK SEQ ID NO: 14 (DogTag) DIPATYEFTN GKHYITNEPIPPK SEQ ID NO: 15 (sortase recognition domain) LPTGAA SEQ ID NO: 16 (sortase recognition domain) LPTGGG SEQ ID NO: 17 (sortase recognition domain) LPKG SEQ ID NO: 18 (sortase recognition domain) LPETG SEQ ID NO: 19 (sortase recognition domain) LPXTG where X is any amino acid SEQ ID NO: 20 (sortase recognition domain) LPXTG(X)n, wherein X is any amino acid and n is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 SEQ ID NO: 21 (sortase recognition domain) NPX1TX2, where X1 is Q or L, and X2 is N or G SEQ ID NO: 22 (SpyTag003) RGVPHIVMVDAYKRYK SEQ ID NO: 23 (SpyCatcher003) VTTLSGLSGEQGPSGDMTTEEDSATHIKFSKRDEDGRELAGATMELRDSSGKTISTWISDGHVKDFYLYPGKYTFVETAAPDGYEVATPIEFTVNEDGQVTVDGEATEGDAHT References: U.S. Patent No. 9,547,003 WO 2016 / 193746 WO 2018 / 053180 Abe, H., Rie, W., Yonemura, H., Yamada, S., Goto, M., and Kamiya, N., (2013). Split Spy0128 as a Potent Scaffold for Protein Cross-Linking and Immobilization. Bioconjugate Chem., 24(2), 242–250. Alam et al., 2017, Synthetic Modular Antibody Construction Using the SpyTag / SpyCatcher Protein Ligase System. Chembiochem. 18(22), 2217-2221. Alam, MK et al., 2018, Site-specific labeling of SpyCatcher. Mol Imaging Biol. https: / / doi.org / 10.1007 / s11307-018-1222-y. Buldun, CM, Jean, J., Bedford, MR, Howarth, M., 2018, SnoopLigase catalyzes peptide-peptide locking and enables solid-phase conjugate isolation. J Am Chem Soc. 140(8), 3008-3018. Fierer, J. O., Veggiani, G., Howarth, M., 2014, SpyLigase peptide-peptide ligation polymerizesaffibodies to enhance magnetic cancer cell capture. Proc Natl Acad Sci USA. 111: E1176-1181. Keeble, AH, Banerjee, A., Ferla, MP, Reddington, SC, Khairil Anuar, INA, Howarth, M., 2017, Evolving accelerated amidation by SpyTag / SpyCatcher to analyze membrane dynamics. Ange, Chem. Int. Ed. 56: 16521-16525 Keeble,AH,Turkki,P.,Stokes,S.,Khairil Anuar,INA,Rahikainen,R., VP, Howarth, M., 2019, Approaching infinite affinity through engineering of peptide–protein interaction. Proc Natl Acad Sci USA. 116: 26526-26533 Li et al., 2014, Structural analysis and optimization of the covalent association between SpyCatcher and a peptide Tag, J Mol Biol. 426(2), 309-17. Nguyen, G.K.T., Wang, S., Qiu, Y., Hemu, X., Lian, Y., Tam, JP. 2014. Butelase 1 is an Asx-specific ligase enabling peptide macrocyclization and synthesis. Nat Chem Biol. 10: 732-738. Reddington, SC, Howarth, M., 2015, Secrets of a covalent interaction for biomaterials and biotechnology: SpyTag and SpyCatcher. Current Opinion in Chemical Biology. 29: 94-99. Schmohl, L., Schwarzer, D., 2014, Sortase-mediated ligations for the site-specific modification of proteins. Current Opinion in Chemical Biology. 22: 122-128 Siegmund et al., 2016, Spontaneous Isopeptide Bond Formation as a Powerful Tool for Engineering Site-Specific Antibody-Drug Conjugates. Scientific Reports. 6, 39291. Tan et al. (2016). Kinetic Controlled Tag-Catcher Interactions for Directed Covalent Protein Assembly. PLoS ONE, 11(10), e0165074. Toplak, A., Nuljens, T., Quaedflieg, P. J. L., Wu, B., Janssen, D. B., 2016. Peptiligase, an enzyme for efficient chemoenzymatic peptide synthesis and cyclization in water. Adv Synth Catal. 358: 32140-32147. Veggiani, G. et al., 2016, Programmable polyproteams built using twin peptide superglues. Proc Natl Acad Sci USA 113: 1202-1207. Yumura, K. et al., 2017, Use of SpyTag / SpyCatcher to construct bispecific antibodies that target two epitopes of a single antigen. J Biochem. 162(3), 203-210. Zakeri, B. et al., 2012, Peptide tag forming a rapid covalent bond to a protein, through engineering abacterial adhesion. Proc Natl Acad Sci USA. 109: E690-697.

Claims

1. An antigen binding protein comprising: two or more first antigen-binding fragments comprising a first binding motif; and A fusion protein comprising two or more second binding motifs that are the same or different and connected by a linker sequence, wherein the fusion protein comprises one to three cysteine ​​residues, wherein the first binding motifs of the two or more first antigen binding fragments are covalently coupled to the two or more second binding motifs via a protein linkage, The first binding motif consists of SEQ ID NO: 1, 4 or 22, The second binding motif consists of SEQ ID NO: 2 (SpyCatcher), SEQ ID NO: 3 (SpyCatcher short), SEQ ID NO: 5 (SpyCatcher002), or SEQ ID NO: 23 (SpyCacther003), and One to three cysteine ​​residues are located at the N-terminus, the C-terminus and / or within the linker sequence.

2. The antigen-binding protein according to claim 1, wherein the fusion protein further comprises a third binding motif, wherein the third binding motif is optionally connected to the two or more second binding motifs connected by a linker or between the two or more second binding motifs connected by a linker through a linker, and the antigen-binding protein further comprises a polypeptide comprising a fourth binding motif, wherein the third binding motif is covalently coupled to the fourth binding motif of the polypeptide via a protein linkage, and wherein the first binding motif-second binding motif pair is orthogonal to the third binding motif-fourth binding motif pair, wherein the third binding motif consists of SEQ ID NO: 7, 11 or 14, The fourth binding motif consists of SEQ ID NO:6, 10 or 13.

3. The antigen binding protein of claim 2, wherein the polypeptide is a fluorescent protein.

4. The antigen binding protein of claim 2, wherein the polypeptide is an effector protein.

5. The antigen binding protein of claim 2, wherein the polypeptide is an antigen binding fragment. The antigen binding protein according to claim 2 , wherein the polypeptide is an enzyme.

7. An antigen binding protein according to any of the preceding claims, wherein one or more binding motifs are located at the C-terminus or N-terminus of the first and / or second antigen binding fragment, fusion protein or polypeptide.

8. The antigen binding protein according to any one of claims 1 to 7, wherein two or more second binding motifs are identical.

9. The antigen binding protein according to any one of claims 1 to 7, wherein two or more second binding motifs are different.

10. The antigen binding protein according to any one of claims 1-9, wherein the fusion protein or polypeptide further comprises a detectable label. The antigen binding protein of claim 10 , wherein the detectable label is a fluorophore.

12. The antigen binding protein of claim 10, wherein the detectable label is a fluorescent protein.

13. The antigen binding protein of claim 10, wherein the detectable label is biotin.

14. The antigen binding protein of claim 10, wherein the detectable label is an enzyme.

15. A nucleic acid construct pair, wherein include: 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 sequence encoding a fusion protein, wherein the fusion protein comprises two or more second binding motifs, wherein the second binding motifs are connected by a linker sequence, and the fusion protein comprises one to three cysteine ​​residues, wherein the first binding motif and the second binding motif are linked to form a covalent bond through the protein when they come into contact with each other spontaneously or with the aid of an enzyme, and The first binding motif consists of SEQ ID NO: 1, 4 or 22, The second binding motif consists of SEQ ID NO: 2 (SpyCatcher), SEQ ID NO: 3 (SpyCatcher short), SEQ ID NO: 5 (SpyCatcher002), or SEQ ID NO: 23 (SpyCacther003).

16. The nucleic acid construct pair according to claim 15, in, The polynucleotide of the second nucleic acid construct further encodes a third binding motif, and the third binding motif is connected to the two or more second binding motifs or between the two or more second binding motifs through a linker sequence, and the nucleic acid construct pair further includes a third nucleic acid construct, which comprises a polynucleotide sequence, and the polynucleotide encodes a polypeptide fused to a fourth binding motif, wherein the third binding motif and the fourth binding motif are linked to form a covalent bond through the protein when they come into contact with each other spontaneously or with the aid of an enzyme, and wherein the first binding motif-second binding motif pair is orthogonal to the third binding motif-fourth binding motif pair, and wherein the third binding motif consists of SEQ ID NO: 7, 11 or 14, and The fourth binding motif consists of SEQ ID NO:6, 10 or 13.

17. The nucleic acid construct pair according to claim 16, wherein two or more second binding motifs are identical.

18. The nucleic acid construct pair according to claim 16, wherein two or more second binding motifs are different.

19. A vector comprising the nucleic acid construct of any one of claims 15-18.

20. A host cell having the nucleic acid construct according to any one of claims 15 to 18 and / or the vector according to claim 19.

Images