Antigen binding fragments conjugated to multiple fc isotypes and subclasses

Abstract

To provide a plurality of full-length antibodies.SOLUTION: Each full-length antibody comprises an antigen binding fragment specifically binding to a unique antigen, comprises a first binding motif at the C-terminus, and an Fc fragment belonging to a unique combination of a species, an isotype and a subclass, and comprises a second binding motif at the N-terminus, where the first binding motif and the second binding motif for each antibody are covalently conjugated to each other via protein ligation. Assays for detecting a plurality of antigens in a sample by contacting the sample with the plurality of full-length antibodies are also provided. Further provided are nucleic acid constructs encoding the plurality of full-length antibodies.SELECTED DRAWING: None

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Application No. 62 / 819,748, filed March 18, 2019, the contents of which are incorporated by reference. [Background technology]

[0002] Antibodies isolated from libraries, such as PCR-derived, semi-synthetic, or fully synthetic libraries, by in vitro selection techniques, including phage display, bacterial display, or ribosome display, are typically expressed as antigen-binding fragments (e.g., scFv or Fab). This is because bacterial expression systems typically do not allow the expression of functional full-length antibodies. An additional reason for generating antibody libraries containing antigen-binding fragments is the selection of desirable antibodies based on their intrinsic binding affinity, rather than avidity caused by the bivalency of the antibody. After a typical selection experiment (e.g., panning) to identify the desired antigen-binding fragments, the enriched pool of genes encoding the desired antibodies is subcloned into a bacterial expression vector for further analysis.

[0003] Antigen-binding fragments isolated from such libraries often need to be subsequently converted into full-length antibodies of a different isotype and subclass, e.g., human IgG1-4, IgA, IgE, or IgM, or into antibodies with Fc regions from different species, e.g., mouse, rat, rabbit, goat, or chicken. Full-length antibodies containing the desired antigen-binding fragment are produced for the specific practical use of the antigen-binding fragment.

[0004] For example, full-length antibodies are used as positive controls or calibrators in diagnostic assays in which patient samples are assayed for the presence of antibodies against a given target. This is often done in the diagnosis of infectious or autoimmune diseases. In these situations, the positive control antibody should contain an Fc fragment of the antibody to be detected, e.g., IgG1, IgE, or IgM, to allow an isotype- and subclass-specific anti-Fc detection reagent to bind to the control antibody. Alternatively, a full-length antibody with an Fc fragment from a given species and a given isotype can be used in combination with other antibodies from different species or with different isotypes in the same experiment, i.e., multiplexed, e.g., in Western blotting or IHC experiments, when species- or isotype-specific secondary antibodies are used for detection.

[0005] Conversion of antigen-binding fragments into full-length antibodies and their subsequent production is a laborious process consisting of several steps, typically taking several weeks. First, genes encoding antibody variable domains are typically resynthesized to incorporate the codon usage for the mammalian expression system used for antibody production. Potential glycosylation sites sometimes exist in the CDR regions of the selected antigen-binding fragment and need to be removed by site-directed mutagenesis before expression in eukaryotic cells. Such mutations in the CDR regions alter antibody affinity or specificity. Second, the synthesized variable heavy (VH) and variable light (VL) gene fragments are cloned into a mammalian expression vector containing the necessary gene fragments encoding antibody constant regions (e.g., CL and CH1-hinge CH2-CH3 for IgG1). Third, plasmid DNA is prepared and used to transfect an appropriate mammalian cell line. Fourth, the transfected cell line is grown in culture for several days until the antibody-containing supernatant can be harvested. Fifth, the antibody is purified from the cell culture supernatant. If the same antigen-binding site is required as a full-length antibody with several isotypes and subclasses (e.g., IgG1 and IgG2) or with Fc fragments from several species, the cloning and expression steps must be repeated for each type.

[0006] Protein Ligation Several techniques allow for the covalent attachment of polypeptides at specific, predetermined sites. One example is the sortase system (Non-Patent Document 3), whereby 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 polypeptide (or vice versa). In the presence of a sortase enzyme, the two modified polypeptides are fused together. Other enzymatic protein ligase systems are based on butelases (Non-Patent Document 19) or peptiligases (Non-Patent Document 28).

[0007] Another example is the in-frame addition of one or more cysteine-encoding nucleotides to the C- or N-termini of two polypeptides. When such free cysteine-containing polypeptides are mixed under oxidizing conditions, they form disulfide bridges. However, such systems suffer from the synthesis of many by-products and the instability of disulfide bridges under reducing conditions.

[0008] A third example is the so-called SpyTag / SpyCatcher system (Non-Patent Document 22). The concept of spontaneous isopeptide formation in naturally occurring proteins has been used to covalently link one polypeptide to another. Streptococcus pyogenes The domain derived from the protein FbaB is divided into two parts. One part, SpyTag, is a 13-amino acid peptide containing part of the autocatalytic center. The other part, SpyCatcher, is a 116-amino acid protein domain containing the other part of the center. Mixing these two polypeptides restores the autocatalytic center and leads to the formation of an isopeptide bond, thereby covalently linking SpyTag (SEQ ID NO: 7) to SpyCatcher (SEQ ID NO: 8) (see Non-Patent Document 32). Further engineering yielded the short SpyCatcher (SEQ ID NO: 9) with only 84 amino acids, the optimized SpyTag002 (SEQ ID NO: 34) and SpyCatcher002 (SEQ ID NO: 28) (Non-Patent Document 16 and Non-Patent Document 13), and the fast-acting SpyTag003 (SEQ ID NO: 43) and SpyCatcher003 (SEQ ID NO: 44) (Non-Patent Document 14), which are incorporated herein by reference in their entirety. A further modification of this system was the invention of SpyLigase (11), which was achieved by splitting the FbaB domain into three parts: SpyTag, K-tag, and SpyLigase. Both SpyTag and K-tag are short peptides that are covalently fused by the addition of SpyLigase.

[0009] Applications of such systems include protein stabilization by cyclization, vaccine production, protein multimerization by incorporating SpyTag / SpyCatcher and streptavidin / biotin (Non-Patent Document 22), affibody and Fab multimerization (Non-Patent Document 11), generation of antibodies from modules (Non-Patent Document 2), production of antibody-drug conjugates (Non-Patent Document 25), and generation of bispecific antibodies (Non-Patent Document 31). Streptococcus pneumoniae A similar system using the adhesin RrgA from the bacterium was developed and called SnoopTag / SnoopCatcher (Non-Patent Document 29), and later the SnoopLigase system was developed (Non-Patent Document 5). The SnoopTag (SEQ ID NO: 35) / SnoopCatcher (SEQ ID NO: 36) technology is incorporated herein by reference in its entirety. [Prior art documents] [Patent documents]

[0010] [Patent Document 1] U.S. Patent No. 9,547,003 [Patent Document 2] International Publication No. 2016 / 193746 [Patent Document 3] International Publication No. 2017 / 058114 [Patent Document 3] International Publication No. 2013 / 045632 [Non-patent literature]

[0011] [Non-Patent Document 1] 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. [Non-patent document 2] Alam et al., 2017, Synthetic Modular Antibody Construction Using the SpyTag / SpyCatcher Protein Ligase System. Chembiochem. 18(22), 2217-2221.

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[0012] Certain embodiments of the present invention provide full-length antibodies comprising an antigen-binding fragment comprising a first binding motif at the C-terminus and an Fc fragment comprising a second binding motif at the N-terminus (the term "FcCatcher" is used in the Examples section to describe this construct), wherein the first binding motif and the second binding motif can be covalently linked to each other via protein ligation, and wherein the antigen-binding fragment and Fc fragment are derived from different species. Certain embodiments of the present invention also provide a plurality of full-length antibodies, each comprising an antigen-binding fragment that specifically binds to a unique antigen, comprising the first binding motif and an Fc fragment belonging to a unique combination of species, isotype, and subclass at the C-terminus, and the second binding motif at the N-terminus, wherein the first binding motif and the second binding motif for each antibody are covalently linked to each other via protein ligation.

[0013] Certain embodiments of the present invention provide multiple Fc fragments, each containing a binding motif at its N-terminus that is covalently linked via proteolytic ligation to a suitable antigen-binding fragment bearing another binding motif at its C-terminus. Within the multiple Fc fragments, each Fc fragment belongs to a unique combination of species, isotype, and / or subclass. Such multiple Fc fragments can be used to generate full-length antibodies or full-length antibody-like structures via proteolytic ligation of Fc fragments and antigen-binding fragments, each containing the appropriate binding motif. The resulting population of full-length antibodies can be utilized in a variety of applications, such as multiplexed immunoassays.

[0014] The first and second binding motifs that promote the formation of a covalent bond between the Fc fragment and the antigen-binding fragment include SpyTag sequences, SpyCatcher sequences, SnoopTag sequences, SnoopCatcher sequences, Isopeptag / Split Spy0128, SdyTag / SdyCatcherDANG short, SpyLigase, SnoopLigase, sortase motifs, peptilase substrates, and peptiligase substrates. Assays for detecting multiple antigens in a sample by contacting the sample with multiple full-length antibodies are also provided. Additionally, nucleic acid constructs encoding multiple full-length antibodies are provided. [Brief explanation of the drawings]

[0015]

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[0016] The present invention employs protein ligation to avoid the steps currently required for producing full-length antibodies from antigen-binding fragments. The present invention provides modified Fc fragments equipped with motifs that enable site-specific protein conjugation. Such modified Fc fragments are generated from Fc sequence information derived from various species and isotypes and subclasses, such as human IgG1, mouse IgG2a, or rabbit IgG. Prior to conjugation with the antigen-binding fragment, the modified Fc fragment can also be conjugated to a suitable label, such as a fluorescent dye or a detection enzyme such as HRP.

[0017] Thus, certain embodiments of the present invention provide full-length antibodies comprising an antigen-binding fragment containing a first binding motif at its C-terminus and an Fc fragment containing a second binding motif at its N-terminus, where the first and second binding motifs are covalently linked to each other via protein ligation. To produce such full-length antibodies, the antigen-binding fragment is engineered to have the first binding motif at its C-terminus, and the Fc fragment is engineered to have the second binding motif at its N-terminus. The antigen-binding fragment-first binding motif fusion protein is mixed with the Fc fragment-second binding motif fusion protein under appropriate conditions to promote protein ligation of the first and second binding motifs to produce the full-length antibody. The full-length antibodies produced in this manner can be further purified. For example, the reaction may produce antibodies containing only one Fab attached to the Fc. Such by-products can be removed by size-exclusion or affinity chromatography, for example, using a protein A column or a column that specifically binds to a tag incorporated into the Fc or Fab fragment.

[0018] Typically, the antigen-binding fragment is obtained from a first species, and the Fc fragment is obtained from a second species different from the first species. For example, if the antigen-binding fragment is obtained from a human antibody, the Fc fragment can be obtained from a mouse antibody. Any combination of different species can be used. In a preferred embodiment, the antigen-binding fragment attached to the Fc fragment binds to the same epitope. The antigen-binding fragment and the Fc fragment can be derived from humans, non-human primates, rodents such as mice and rats, rabbits, hamsters, goats, sheep, cows, pigs, horses, dogs, cats, and camels. Additional species that can be used in the present invention are known in the art, and such embodiments are within the scope of the present invention.

[0019] In certain embodiments, the present invention provides multiple full-length antibodies that can be used in the same assay reaction, i.e., for multiplexing. Accordingly, such embodiments provide multiple full-length antibodies, each of which comprises an antigen-binding fragment comprising a first binding motif at its C-terminus and an Fc fragment comprising a second binding motif at its N-terminus, where the first binding motif and the second binding motif are covalently linked to each other via protein ligation. Furthermore, within the set of multiple full-length antibodies, each antigen-binding fragment specifically binds to a unique antigen, and each Fc fragment belongs to a unique species, isotype, and subclass. In such embodiments, the antigen-binding fragments and Fc fragments are heterologous (derived from different human or non-human animal species, e.g., a human antigen-binding fragment covalently linked to a mouse Fc fragment via a binding motif) or homologous (derived from the same species, e.g., a human antigen-binding fragment covalently linked to a human Fc fragment via a binding motif).

[0020] In further embodiments, the present invention provides an antigen-binding fragment and an Fc fragment, which are combined to form a full-length antibody. In certain embodiments, the antigen-binding fragment comprises a first binding motif at the C-terminus and the Fc fragment comprises a second binding motif at the N-terminus, and the first and second binding motifs are covalently linked to each other via protein ligation when contacted with each other under appropriate conditions. The antigen-binding fragment and / or the Fc fragment are conjugated to a detectable label, particularly an optical label.

[0021] To produce a full-length antibody, an antigen-binding fragment containing a first binding motif at its C-terminus is mixed with an Fc fragment containing a second binding motif at its N-terminus. This mixing is carried out under appropriate conditions to promote protein ligation of the first and second binding motifs. The full-length antibody thus produced can be further purified. For example, the reaction produces an antibody containing only one Fab attached to the Fc. Such by-products can be removed by size-exclusion or affinity chromatography, for example, using a protein A column or a column that specifically binds to the tag introduced into the Fc or Fab fragment.

[0022] Also provided are kits for producing full-length antibodies. Such kits can include an antigen-binding fragment comprising a first binding motif at the C-terminus and an Fc fragment comprising a second binding motif at the N-terminus. The antigen-binding fragment and / or the Fc fragment are conjugated to a detectable label, particularly an optical label. Thus, in some embodiments, the kit includes two or more of the following components: 1. An antigen-binding fragment comprising a first binding motif at the C-terminus and optionally comprising a detectable label (e.g., biotin, HRP, or a fluorophore); 2. An Fc fragment containing a second binding motif at the N-terminus and optionally containing a detectable label (e.g., biotin, HRP, or a fluorophore), and / or 3. A nucleic acid construct comprising a polynucleotide sequence encoding a peptide as defined in 1 and / or 2, the first binding motif and the second binding motif are capable of being covalently linked to each other via protein ligation.

[0023] The kit user can mix the antigen-binding fragment and the Fc fragment under appropriate conditions in which they interact with each other through protein ligation, either spontaneously or with the aid of an enzyme, to form a covalent bond. The kit user can also express any peptide in a suitable host using a nucleic acid construct comprising a polynucleotide sequence encoding the antigen-binding fragment and / or the Fc fragment.

[0024] Each component of the kit can be provided in liquid form (e.g., as a solution) to be reconstituted with, e.g., a buffer solution, before use, or as a solid (e.g., a powder). In some embodiments, the kit further comprises instructions for ligating one or more binding pairs.

[0025] The term "label" or "detectable label" refers to a composition detectable by spectroscopic, photochemical, biochemical, immunochemical, chemical, or other physical means. For example, useful labels include fluorescent dyes (fluorophores), fluorescence quenchers, luminescent agents, electron-dense reagents, enzymes (e.g., commonly used in ELISA), biotin, digoxigenin, 32 Included are P and other isotopes, haptens, proteins, nucleic acids, or other substances that are made detectable, for example, by incorporating the label into an oligonucleotide or peptide. The term also includes combinations of single labeling agents, for example, combinations of fluorophores, that provide a unique detectable signature at a particular wavelength or combination of wavelengths.

[0026] Examples of detectable labels include, but are not limited to, fluorophores, fluorescent proteins such as green fluorescent protein (GFP), biotin, enzymes such as horseradish peroxidase (HRP) or other peroxidases, alkaline phosphatase, luciferase, and split fluorescent proteins (e.g., split GFP) or enzymes (e.g., NanoLuc® Binary Technology from Promega). Fluorescent materials include Alexa dyes (e.g., Alexa350, Alexa488, etc.), AMCA, BODIPY630 / 650, BODIPY650 / 665, BODIPY-FL, BODIPY-R6G, BODIPY-TMR, BODIPY-TRX, Cascade Blue, Cy2, Cy3, Cy5, Cy5.5, Cy7, Cy7.5, and Dylight dyes (Dylight405, Dylight488, Dylight549, Dylight550, Dylight649, Dylight680, Dylight7). 50, 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 dyes (e.g., Starbright Blue 520, Starbright Blue 700), TAMRA, TET, tetramethylrhodamine, Texas Red, and TRITC are examples, but are not limited to these.

[0027] As used herein, the phrase "antigen-binding fragment" refers to an antigen-binding portion of an antibody, such as a Fab. Other antigen-binding fragments include variable fragments (Fv), single-chain variable fragments (scFv), or variable domains of heavy-chain antibodies (VHH). Further examples of antigen-binding fragments include monovalent forms of antigen-binding fragments that contain the antigen-binding site, such as single-chain Fab fragments (scFab), single-domain antibodies (sdAbs), Shark variable novel antigen receptors (VNAR), or variable lymphocyte receptors (VLR). In addition, binding reagents derived from non-antibody scaffolds, such as affimers, affibodies, darpins, anticalins, and monobodies, are also considered "antigen-binding fragments." Further examples of antigen-binding fragments are known in the art, and the use of such antigen-binding fragments is within the scope of the present invention.

[0028] The phrase "each Fc fragment belongs to a unique combination of species, isotype, and subclass" indicates that the Fc fragment of a full-length antibody in the set of full-length antibodies belongs to a specific combination of species, isotype, subclass, and allotype, and no other Fc fragment from the full-length antibodies has the same combination of species, isotype, and subclass. Thus, if the set of full-length antibodies includes 50 full-length antibodies, each of the 50 antibodies will have an Fc fragment that belongs to a different combination of species, isotype, subclass, or allotype compared to the remaining 49 Fc fragments.

[0029] Typically, a given species produces several types of antibodies. For example, humans or mice can produce five antibody heavy chain-related isotypes: IgA, IgD, IgE, IgG, and IgM. Each isotype may further include several subclasses. For example, human IgG has four subclasses: IgG1, IgG2, IgG3, and IgG4. Thus, a species contains multiple antibody isotypes and several subclasses within each isotype. One skilled in the art can identify and select appropriate isotype subclasses from an appropriate species for use in the present invention. For example, a set of five full-length antibodies includes five Fc fragments, e.g., human IgG1, human IgG2, human IgA, mouse IgG3, and mouse IgE. Furthermore, a particular subclass (e.g., IgG1) includes numerous allotypes, which are variants of this subclass in the gene pool of a species. For example, the human IgG1 subclass contains the allotypes G1m(za), G1m(f), G1m(fa), G1m(zax), and G1m(zav), which are distinguished by their amino acid sequences. Within IgG1, most amino acid differences are located in the CH3 domain. Fc fragments with different allotypes can be used in the present invention, for example, when the reagent used to detect the antibody is allotype-specific.

[0030] As described above, a set of full-length antibodies is used in a multiplex assay, i.e., multiple full-length antibodies are used to detect multiple antigens in an assay, such that within the multiple full-length antibodies, each antigen-binding fragment specifically binds to a unique antigen, and each Fc region is detected using a species-specific, isotype-specific, subclass-specific, or allotype-specific auxiliary reagent.

[0031] The phrase "each antigen-binding fragment specifically binds to a unique antigen" indicates that an antigen-binding fragment of a full-length antibody in a set of full-length antibodies specifically binds to a particular antigen, and other full-length antibodies from the set do not bind to the same antigen. Thus, if the set of full-length antibodies includes 50 full-length antibodies, each of the 50 antibodies specifically binds to a different antigen compared to the remaining 49 full-length antibodies.

[0032] Thus, in a specific embodiment, the present invention provides a plurality of full-length antibodies, each of which is conjugated to a unique label. In this embodiment, the Fc fragments are identical for the plurality of full-length antibodies, and antigen- (or epitope-) specific antibodies are distinguished by unique labels. Thus, a full-length antibody within the plurality of full-length antibodies is conjugated to a label, and other full-length antibodies from the plurality do not have the same label or specificity for the same antigen (or epitope). Thus, if a set of full-length antibodies includes 50 full-length antibodies, each of the 50 antibodies has a different label and specificity for a different antigen (or epitope). The presence of a unique label for each full-length antibody facilitates quantification of the unique Fc fragment, i.e., the unique antigen to which the full-length antibody binds. In one specific embodiment, the label is a unique bead. For example, a unique bead combination is provided by the Bio-Plex® multiplex immunoassay system, which allows multiplexing of up to 100 different assays within a single sample. The use of different colored beads allows for the simultaneous multiplex detection of many full-length antibodies, ie, many antigens in the same sample.

[0033] In a further embodiment, the invention provides a plurality of full-length antibodies, each of which has an Fc fragment that allows for differentiation of the antibodies based on the Fc fragment by using secondary antibodies specific for that unique Fc fragment. Thus, in a multiplex reaction, different secondary antibodies can be used to distinguish between different Fc fragments, and thus different antigens recognized by different antibodies.

[0034] Unique Fc fragments in multiple full-length antibodies can be detected by secondary antibodies directed against the unique Fc fragments. Accordingly, certain embodiments of the invention further include multiple secondary antibodies directed against the multiple unique Fc fragments, each secondary antibody specifically binding to a unique Fc fragment from a particular species, subtype, and subclass (and potentially, allotype). Each secondary antibody is conjugated to a unique detectable label, and the unique label of each secondary antibody facilitates quantification of the unique Fc fragments, i.e., the unique antigen to which the full-length antibody binds.

[0035] Alternatively, certain embodiments of the invention further comprise multiple secondary antibodies directed against multiple unique Fc fragments, each secondary antibody specifically binding to a unique Fc fragment from a particular species, subtype and subclass, and each secondary antibody binding to a unique bead.

[0036] An example of a unique bead combination is the Bio-Plex® multiplex immunoassay system. This technology allows for multiplex immunoassays in which one secondary antibody against a unique Fc fragment is attached to a set of beads having the same color, and such secondary antibodies attached to a unique set of beads are visualized, for example, by using a detectable label. The use of beads of different colors allows for simultaneous multiplex detection of many antigens in the same sample.

[0037] In certain embodiments, the present invention provides multiple Fc fragments containing a binding motif at each N-terminus. Such multiple Fc fragments can be used to produce a customizable set of full-length antibodies that bind to a single antigen or multiple antigens of interest. For example, the multiple antigen-binding fragments can then be expressed with an appropriate binding motif at each C-terminus, and these antigen-binding fragments can be mixed with appropriate Fc fragments to produce multiple customized full-length antibodies. Thus, certain embodiments of the present invention provide multiple Fc fragments, each containing a binding motif at its N-terminus, which is covalently linked to an antigen-binding fragment bearing the appropriate binding motif via protein ligation. Furthermore, within the multiple Fc fragments, each Fc fragment belongs to the same species, isotype, and subclass, or is unique (e.g., unique species, isotype, or subclass).

[0038] Certain embodiments of the present invention provide multiple antigen-binding fragments, each fused to a first binding motif at its C-terminus, each specifically binding to the same antigen but recognizing different epitopes of the antigen and / or having different antigen-binding affinities. When the first binding motifs from the multiple antigen-binding motifs contact each other, either naturally or with the aid of an enzyme, they form a covalent bond with a second binding motif present at the N-terminus of the Fc fragment. This embodiment provides a "synthetic polyclonal antibody preparation," i.e., a preparation of antibodies that specifically bind to the same antigen, but each antibody in the preparation recognizes a different epitope and / or has a different binding affinity. As described above, each Fc fragment belongs to the same species, isotype, and subclass, or is unique (e.g., a unique species, isotype, or subclass).

[0039] A further embodiment of the present invention provides a method for producing a plurality of full-length monoclonal antibodies. Such a method comprises contacting an antigen-binding fragment from a plurality of antigen-binding fragments with an Fc fragment from a plurality of Fc fragments, wherein each of the plurality of antibody-discovery fragments comprises a first binding motif at its C-terminus and each of the plurality of Fc fragments comprises a second binding motif at its N-terminus. The conditions for contacting the antigen-binding fragment and the Fc fragment are such that a covalent bond is formed between the first binding motif and the second binding motif. Thus, a plurality of full-length antibodies is produced, each antibody comprising an antigen-binding fragment comprising the first binding motif at its C-terminus and an Fc fragment comprising the second binding motif at its N-terminus.

[0040] As mentioned above, several techniques allow for the covalent attachment of polypeptides at specific predetermined sites via "protein ligation." As used herein, the term "protein ligation" refers to the covalent attachment of a first binding motif of a first protein to a second binding motif of a second protein, which occurs when the first and second binding motifs contact each other and a covalent bond forms spontaneously under appropriate conditions or with the aid of an enzyme. Typically, the first binding motif is present at the C-terminus of the first protein, and the second binding motif is present at the N-terminus of the second protein. Usually, the first protein is expressed as a fusion protein with the first binding motif at its C-terminus, and the second protein is expressed as a fusion protein with the second binding motif at its N-terminus.

[0041] The term "first binding motif" refers to a peptide sequence that is attached to the C-terminus of an antigen-binding fragment and promotes covalent bond formation via proteolytic ligation to a second binding motif present at the N-terminus of the Fc fragment. Similarly, the term "second binding motif" refers to a peptide sequence that is attached to the N-terminus of an Fc fragment and promotes covalent bond formation via proteolytic ligation to a first binding motif present at the C-terminus of the antigen-binding fragment.

[0042] As noted above, "protein ligation" refers to the spontaneous or enzymatic formation of a covalent bond between a first binding motif and a second binding motif when these motifs are brought into contact with one another. Also, as described throughout this disclosure, protein ligation occurs between specific combinations of peptide sequences, such as SpyTag and SpyCatcher, SnoopTag and SnoopCatcher, a sortase recognition domain and a sortase bridge domain, a buterase recognition motif and the amino terminus of another polypeptide, SpyTag002 (SEQ ID NO:34) and SpyCatcher002 (SEQ ID NO:28), SpyTag (SEQ ID NO:29) and K-Tag (SEQ ID NO:33), SpyTag003 (SEQ ID NO:43) and SpyCatcher003 (SEQ ID NO:44), etc.

[0043] Thus, to produce a full-length antibody of the present invention, a first binding motif present at the C-terminus of the antigen-binding fragment can be covalently linked to a second binding motif at the N-terminus of the Fc fragment via proteolytic ligation. For example, if the first binding motif present at the C-terminus of the antigen-binding fragment is SpyTag, or its improved version SpyTag002 or SpyTag003, the corresponding second binding motif present at the N-terminus of the Fc fragment is SpyCatcher, or its improved version SpyCatcher002 or SpyCatcher003. Alternatively, if the first binding motif present at the C-terminus of the antigen-binding fragment is SpyCatcher, or its improved version SpyCatcher002 or SpyCatcher003, the corresponding second binding motif present at the N-terminus of the Fc fragment is SpyTag, or its improved version SpyTag002 (SEQ ID NO: 34) or SpyTag003 (SEQ ID NO: 43).

[0044] Similarly, if the first binding motif present at the C-terminus of the antigen-binding fragment is SnoopTag (SEQ ID NO: 35) or a sequence at least 70% identical to SEQ ID NO: 35, the corresponding second binding motif present at the N-terminus of the Fc fragment is SnoopCatcher (SEQ ID NO: 36) or a sequence at least 50% identical to SEQ ID NO: 36. Alternatively, if the first binding motif present at the C-terminus of the antigen-binding fragment is SnoopCatcher (SEQ ID NO: 36) or a sequence at least 50% identical to SEQ ID NO: 36, the corresponding second binding motif present at the N-terminus of the Fc fragment is SnoopTag (SEQ ID NO: 35) or a sequence at least 70% identical to SEQ ID NO: 35.

[0045] Furthermore, in the case of enzymatic protein ligation, if the first binding motif present at the C-terminus of the antigen-binding fragment is a SpyTag, the corresponding second binding motif present at the N-terminus of the Fc fragment is a K-Tag. Alternatively, if the first binding motif present at the C-terminus of the antigen-binding fragment is a K-Tag, the corresponding second binding motif present at the N-terminus of the Fc fragment is a SpyTag. In both cases, SpyLigase is required to catalyze isopeptide bond formation between the two tags.

[0046] As such, the first binding motif present at the C-terminus of the antigen-binding motif and the second binding motif present at the N-terminus of the Fc fragment are selected such that the two motifs interact with each other via protein ligation, either spontaneously or enzymatically assisted, to form a covalent bond.

[0047] Expression of these proteins can be achieved in prokaryotic cells, e.g. Escherichia coli or in suitable host cells, including eukaryotic cells, such as yeast or CHO cells. Suitable techniques for expression of fusion proteins are known to those skilled in the art, and such embodiments are within the scope of the present invention.

[0048] Thus, a protein ligation system involves adding a first binding motif to a first protein, adding a second binding motif to a second protein, and covalently linking the first and second proteins via a covalent bond between the first and second binding motifs. Such covalent binding is autocatalytic, i.e., catalyzed by the interaction between the first and second binding motifs. Covalent binding can also be achieved by an enzyme that catalyzes such a binding reaction.

[0049] Protein ligation systems include, but are not limited to, sortase systems, butelase systems, peptiligase systems, cysteine-mediated disulfide bridge formation, SpyTag / SpyCatcher systems, SpyTag with short SpyCatcher, SpyTag002 / SpyCatcher002 or SpyTag003 / SpyCatcher003 systems, accelerated reaction SpyTag / K-tag / SpyLigase systems, and SnoopTag / SnoopCatcher systems. Further examples of protein ligation systems will be known to those skilled in the art, and such embodiments are within the scope of the present invention.

[0050] Thus, the antigen-binding fragment is produced as a fusion protein with a first binding motif fused to the C-terminus of a protein ligation system, and the Fc fragment is produced as a fusion protein with a second binding motif fused to the N-terminus of the protein ligation system. The antigen-binding fragment fusion protein and the Fc fragment fusion protein are mixed together (in the presence of a suitable enzyme, as appropriate, in the case of enzymatic protein ligation) to produce an artificial full-length antibody containing the C-terminal first binding motif of the antigen-binding fragment fusion protein covalently linked to the N-terminal second binding motif of the Fc fusion protein.

[0051] Any of the protein ligation systems described above or known in the art can be used to produce full-length antibodies, and certain such protein ligation systems are described below.

[0052] SpyTag / SpyCatcher Components of the SpyTag / SpyCatcher system are disclosed in U.S. Patent No. 6,277,629 (the disclosure of which is incorporated herein by reference in its entirety). In this regard, the peptide tags and binding partners disclosed in U.S. Patent No. 6,277,629 are used as binding motifs in the present invention. Accordingly, binding motifs suitable for use in the present invention can be derived from SEQ ID NOs: 1, 3, 5, or 6 and can be any length, e.g., about 5 to 50 amino acids in length (e.g., about 10, 20, 30, 40, or 50 amino acids in length), or longer. Examples of first and second binding motifs for the SpyTag / SpyCatcher system are shown in Table 1.

[0053] The binding motif is fused to the N-terminus of the Fc fragment or the C-terminus of the antigen-binding fragment. In particular, a spacer sequence (e.g., a glycine / serine-rich spacer) flanks the binding motif to enhance its accessibility for reaction. Obviously, the first and second binding motifs can be interchanged on one of the antibody fragments (e.g., the first binding motif is fused to the N-terminus of the Fc fragment and the second binding motif is fused to the antigen-binding fragment at its C-terminus, or the second binding motif is fused to the N-terminus of the Fc fragment and the first binding motif is fused to the antigen-binding fragment at its C-terminus).

[0054] Thus, in certain embodiments, the first binding motif comprises residues 302-308 of the sequence set forth in SEQ ID NO:1 or SEQ ID NO:25 or SEQ ID NO:27, or a sequence having at least 50% identity to SEQ ID NO:1 or 25 or 27, and the first binding motif is less than 50 amino acids in length. In certain embodiments, the first binding motif has at least about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, or about 95% identity to SEQ ID NO:1 and is less than 50 amino acids in length. More specifically, the first binding motif comprises residues 302-308, 301-308, 300-308, 299-308, 298-308, 297-308, 296-308, 295-308, 294-308, 293-308, 292-308, 291-308, or 290-308 of SEQ ID NO: 1, or a sequence having at least about 50%-95% identity thereto. Preferably, the first binding motif comprises a reactive asparagine at position 303 of SEQ ID NO: 1, i.e., this residue is preferably unchanged. Furthermore, the first binding motif may be a fragment of SEQ ID NO: 1 or 25; in a preferred embodiment, the first binding motif is less than 50 amino acids and comprises residues 293-308 of the sequence shown in SEQ ID NO: 1, or a sequence having at least 50% identity thereto. The first binding motif preferably comprises fewer than 50 amino acids. Thus, the first binding motif does not comprise the sequence of SEQ ID NO: 1, but only a specific fragment thereof, or a sequence having at least 50% identity, e.g., 75, 80, 85, 90, or 95% identity to such a specific fragment. Other embodiments utilize SEQ ID NO: 25, or a sequence having at least 50% sequence identity thereto, as the binding motif.

[0055] When the first binding motif is any of the sequences listed in the preceding paragraph, the second binding motif comprises or consists of residues 31-291 of the sequence set forth in SEQ ID NO:1 or SEQ ID NO:26 or SEQ ID NO:28, or a sequence having at least 50% identity thereto, e.g., 75, 80, 85, 90, 95, 96, 97, 98 or 9 ... to residues 32-291 of SEQ ID NO:1 or SEQ ID NO:26 or SEQ ID NO:28. Specifically excluded is the complete sequence set forth in SEQ ID NO:1, although the second binding motif preferably includes a reactive lysine corresponding to position 179 of SEQ ID NO:1. In particular, the second binding motif comprises residues 31-292, 31-293, 31-294, 31-295, 31-296, 31-297, 31-298, 31-299, 31-300, 31-301 or 31-302 of the sequence shown in SEQ ID NO: 1, or a sequence having at least 70% identity thereto, excluding the sequence of SEQ ID NO: 1.

[0056] Alternatively, in certain embodiments, the second binding motif may comprise residues 302-308 of the sequence set forth in SEQ ID NO:1 or SEQ ID NO:25 or SEQ ID NO:27, or a sequence having at least 50% identity to residues 302-308 of SEQ ID NO:1, SEQ ID NO:1 or SEQ ID NO:25 or SEQ ID NO:27, and the length of the second binding motif is less than 50 amino acids. In certain embodiments, the second binding motif has at least about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, or about 95% identity to SEQ ID NO:1 and is less than 50 amino acids in length. More specifically, the second binding motif comprises residues 302-308, 301-308, 300-308, 299-308, 298-308, 297-308, 296-308, 295-308, 294-308, 293-308, 292-308, 291-308, or 290-308 of SEQ ID NO: 1, or a sequence having at least about 50%-95% identity thereto. Preferably, the second binding motif comprises a reactive asparagine at position 303 of SEQ ID NO: 1, i.e., this residue is preferably unchanged. Furthermore, the second binding motif may be a fragment of SEQ ID NO: 1 or 25; in a preferred embodiment, the second binding motif is less than 50 amino acids and comprises residues 293-308 of the sequence shown in SEQ ID NO: 1, or a sequence having at least 50% identity thereto. The second binding motif preferably comprises fewer than 50 amino acids. Thus, the second binding motif does not comprise the sequence of SEQ ID NO: 1, but only a specific fragment thereof, or a sequence having at least 50% identity, e.g., 75, 80, 85, 90, or 95% identity to such a specific fragment. Other embodiments utilize SEQ ID NO: 25, or a sequence having at least 50% sequence identity thereto, as the binding motif.

[0057] When the second binding motif is one of the sequences listed in the preceding paragraph, the first binding motif comprises or consists of residues 31-291 of the sequence set forth in SEQ ID NO:1 or SEQ ID NO:26 or SEQ ID NO:28, or a sequence having at least 50% identity thereto, e.g., 75, 80, 85, 90, 95, 96, 97, 98 or 9 ... to residues 32-291 of SEQ ID NO:1 or SEQ ID NO:26 or SEQ ID NO:28. Specifically excluded is the complete sequence set forth in SEQ ID NO:1, although the first binding motif preferably includes a reactive lysine corresponding to position 179 of SEQ ID NO:1. In particular, the first binding motif comprises residues 31-292, 31-293, 31-294, 31-295, 31-296, 31-297, 31-298, 31-299, 31-300, 31-301 or 31-302 of the sequence shown in SEQ ID NO: 1, or a sequence having at least 70% identity thereto, excluding the sequence of SEQ ID NO: 1.

[0058] In certain embodiments, the first binding motif comprises SEQ ID NO:7 or a sequence having at least 70% identity to SEQ ID NO:7, and the second binding motif comprises SEQ ID NO:8 or a sequence having at least 50% identity to SEQ ID NO:8. Alternatively, the first binding motif comprises SEQ ID NO:8 or a sequence having at least 50% identity to SEQ ID NO:8, and the second binding motif comprises SEQ ID NO:7 or a sequence having at least 70% identity to SEQ ID NO:7.

[0059] In a further embodiment, the first binding motif comprises SEQ ID NO:7 or a sequence having at least 70% identity to SEQ ID NO:7, and the second binding motif comprises SEQ ID NO:9 or a sequence having at least 50% identity to SEQ ID NO:9. Alternatively, the first binding motif comprises SEQ ID NO:9 or a sequence having at least 50% identity to SEQ ID NO:9, and the second binding motif comprises SEQ ID NO:7 or a sequence having at least 70% identity to SEQ ID NO:7.

[0060] In a further embodiment, the first binding motif comprises SEQ ID NO: 34, or a sequence having at least 70% identity to SEQ ID NO: 34, and the second binding motif comprises SEQ ID NO: 28, or a sequence having at least 50% identity to SEQ ID NO: 28. Alternatively, the first binding motif comprises SEQ ID NO: 28, or a sequence having at least 50% identity to SEQ ID NO: 28, and the second binding motif comprises SEQ ID NO: 34, or a sequence having at least 70% identity to SEQ ID NO: 34.

[0061] SpyTag002 (SEQ ID NO: 34) reacts with SpyCatcher002 (SEQ ID NO: 28), SpyCatcher (SEQ ID NO: 8), SpyCatcher with only 84 amino acids (SpyCatcher short, SEQ ID NO: 9), and SpyCatcher003 (SEQ ID NO: 44). Thus, in some embodiments, one system, i.e. S.pyogenes Mutations in the binding motifs can be exchanged within the derived CnaB2 domain. For example, the first binding motif comprises SEQ ID NO: 34 or a sequence having at least 70% identity to SEQ ID NO: 34, and the second binding motif comprises SEQ ID NO: 28, 8, 9, or 44 or a sequence having at least 50% identity to SEQ ID NO: 28, 8, 9, or 44. Alternatively, the first binding motif comprises SEQ ID NO: 28, 8, 9, or 44 or a sequence having at least 50% identity to SEQ ID NO: 28, 8, 9, or 44, and the second binding motif comprises SEQ ID NO: 34 or a sequence having at least 70% identity to SEQ ID NO: 34.

[0062] In a further embodiment, the first binding motif comprises SEQ ID NO: 43 (SpyTag003), or a sequence having at least 70% identity to SEQ ID NO: 43, and the second binding motif comprises SEQ ID NO: 44 (SpyCatcher003), or a sequence having at least 50% identity to SEQ ID NO: 44. Alternatively, the first binding motif comprises SEQ ID NO: 44, or a sequence having at least 50% identity to SEQ ID NO: 44, and the second binding motif comprises SEQ ID NO: 43, or a sequence having at least 70% identity to SEQ ID NO: 43.

[0063] Furthermore, a binding motif may be designed from the major pilin protein SpyO128 using an alternative isopeptide bond at the N-terminus. SpyO128 is a major pilin protein SpyO128, which has the amino acid sequence shown in SEQ ID NO: 1 and is encoded by the nucleotide sequence shown in SEQ ID NO: 2. The use of SpyO128 for protein ligation is described in Non-Patent Document 1, the reference of which is incorporated herein in its entirety. Thus, a binding motif is designed or obtained from an N-terminal fragment of an isopeptide protein, with the remaining truncated or overlapping protein fragment constituting the other binding motif. The reactive lysine involved in the isopeptide bond at the N-terminus is found at position 36 of SEQ ID NO: 1, and the reactive asparagine involved in the isopeptide bond is found at position 168 of SEQ ID NO: 1.

[0064] Thus, in a preferred embodiment, one of the binding motifs contains a reactive lysine residue, and the other binding motif contains a reactive glutamic acid, aspartic acid, or asparagine. In particular, in certain embodiments, the first binding motif comprises residues 31-40 of the sequence shown in SEQ ID NO: 1, or a sequence having at least 70% identity thereto and less than 50 amino acids in length. Meanwhile, the second binding motif comprises residues 37-304 of the sequence shown in SEQ ID NO: 1, or a sequence having at least 70% identity thereto, excluding the sequence shown in SEQ ID NO: 1. Alternatively, in certain embodiments, the second binding motif comprises residues 31-40 of the sequence shown in SEQ ID NO: 1, or a sequence having at least 70% identity thereto and less than 50 amino acids in length. Meanwhile, the first binding motif comprises residues 37-304 of the sequence shown in SEQ ID NO: 1, or a sequence having at least 70% identity thereto, excluding the sequence shown in SEQ ID NO: 1. Preferably, the reactive residues in the binding motifs are not mutated.

[0065] In further embodiments, the first binding motif comprises residues 179-184, e.g., 173-185, of the sequence set forth in SEQ ID NO: 3, or a sequence having at least 50% identity thereto, and is less than 50 amino acids in length. In such embodiments, the second binding motif comprises residues 191-317, e.g., 186-318, of SEQ ID NO: 3, or a sequence having at least 50% identity thereto, excluding SEQ ID NO: 3. Alternatively, in further embodiments, the second binding motif comprises residues 179-184, e.g., 173-185, of the sequence set forth in SEQ ID NO: 3, or a sequence having at least 50% identity thereto, and is less than 50 amino acids in length. In such embodiments, the first binding motif comprises residues 191-317, e.g., 186-318, of SEQ ID NO: 3, or a sequence having at least 50% identity thereto, excluding SEQ ID NO: 3. Specifically excluded as a binding motif is the full-length sequence of SEQ ID NO: 3.

[0066] In a further embodiment, the first binding motif comprises an asparagine at position 266 (or a sequence having at least 50% identity thereto), and the second binding motif comprises a fragment of SEQ ID NO: 5 or a sequence having at least 50% sequence identity thereto, comprising a lysine residue at position 149 but not an asparagine at position 266. Alternatively, in a further embodiment, the second binding motif comprises an asparagine at position 266 (or a sequence having at least 50% identity thereto), and the first binding motif comprises a fragment of SEQ ID NO: 5 or a sequence having at least 50% sequence identity thereto, comprising a lysine residue at position 149 but not an asparagine at position 266. Preferably, neither of the binding motifs comprises SEQ ID NO: 5.

[0067] In a further embodiment, the first binding motif comprises a fragment of SEQ ID NO:6 comprising an aspartic acid residue at position 101 (or a sequence at least 70% identical thereto), and the second binding motif comprises a fragment of SEQ ID NO:6 comprising a reactive lysine at position 15 (or a sequence at least 50% identical thereto). Alternatively, in a further embodiment, the second binding motif comprises a fragment of SEQ ID NO:6 comprising an aspartic acid residue at position 101 (or a sequence at least 70% identical thereto), and the first binding motif comprises a fragment of SEQ ID NO:6 comprising a reactive lysine at position 15 (or a sequence at least 50% identical thereto). None of these binding motifs comprises SEQ ID NO:6.

[0068] Another embodiment provides a first binding motif comprising SEQ ID NO:25 or SEQ ID NO:27, or a sequence having at least 50% identity to SEQ ID NO:25 or 27, wherein the binding motif is 15 to 40 or 50 amino acids in length. In certain embodiments, the first binding motif has at least about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, or about 95% identity to SEQ ID NO:25 or 27 and is less than 50 amino acids in length. Preferably, the first binding motif comprises the reactive aspartic acid at position 8 of SEQ ID NO:27, i.e., this residue is preferably unchanged.

[0069] When the first binding motif is any of the sequences listed in the previous paragraph, the second binding motif comprises or consists of SEQ ID NO: 26, or a sequence having at least 50% identity to SEQ ID NO: 26, e.g., 75, 80, 85, 90, 95, 96, 97, 98 or 99% identity to SEQ ID NO: 26 or SEQ ID NO: 28, or a sequence having at least 50% identity to SEQ ID NO: 28, e.g., 75, 80, 85, 90, 95, 96, 97, 98 or 99% identity to SEQ ID NO: 28. Variants have at least 50% sequence identity and retain a lysine at position 57 of SEQ ID NO: 26.

[0070] Further embodiments provide a second binding motif comprising SEQ ID NO:25 or SEQ ID NO:27, or a sequence having at least 50% identity to SEQ ID NO:25 or 27, wherein the binding motif is 15 to 40 or 50 amino acids in length. In certain embodiments, the second binding motif has at least about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, or about 95% identity to SEQ ID NO:25 or 27 and is less than 50 amino acids in length. Preferably, the second binding motif comprises the reactive aspartic acid at position 8 of SEQ ID NO:27, i.e., this residue is preferably unchanged.

[0071] When the second binding motif is any of the sequences listed in the previous paragraph, the first binding motif comprises or consists of SEQ ID NO: 26, or a sequence having at least 50% identity thereto, for example a sequence having 75, 80, 85, 90, 95, 96, 97, 98 or 99% identity to SEQ ID NO: 26 or SEQ ID NO: 28, or a sequence having at least 50% identity thereto, for example a sequence having 75, 80, 85, 90, 95, 96, 97, 98 or 99% identity to SEQ ID NO: 28. Variants have at least 50% sequence identity and retain a lysine at position 57 of SEQ ID NO: 26.

[0072] In certain embodiments, the first binding motif comprises SEQ ID NO: 39 or a sequence having at least 70% identity thereto, while the second binding motif comprises SEQ ID NO: 40 or a sequence having at least 50% identity thereto. Alternatively, the first binding motif comprises SEQ ID NO: 40 or a sequence having at least 50% identity thereto, while the second binding motif comprises SEQ ID NO: 39 or a sequence having at least 70% identity thereto.

[0073] SpyLigase / SnoopLigase Alternatively, the Fc fragment is conjugated to the antigen-binding fragment using the system described in Patent Document 2 and Non-Patent Document 29 (each of which is incorporated herein by reference in its entirety). In such embodiments, binding motifs are added to the Fc fragment and antigen-binding fragment, optionally via a linker sequence such as a glycine / serine-rich spacer. These binding motifs are then ligated by a ligase. In some embodiments, each of the first and second binding motifs can have a length of 6 to 50 amino acids, e.g., 7 to 45, 8 to 40, 9 to 35, 10 to 30, or 11 to 25 amino acids, and can comprise or consist of, for example, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acids. In other embodiments, each of the first and second binding motifs is about 20 to 300 amino acids in length (e.g., about 10, 20, 30, 40, 50, 60, 70, etc. amino acids). In some embodiments, the peptide ligase is 50 to 300 amino acids in length, e.g., 60 to 250, 70 to 225, 80 to 200 amino acids in length, e.g., comprises or consists of 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, or 200 amino acids, so long as it meets the definition of a ligase set forth below.

[0074] In certain embodiments, the SpyLigase system of protein ligation is used. In such embodiments, an antigen-binding fragment-SpyTag fusion protein is produced as described above. The Fc fragment is not fused to SpyCatcher, but is fused at its N-terminus to a 10-amino acid K-Tag (ATHIKFSKRD, SEQ ID NO: 33), optionally with one or more linkers as described above. The purified antigen-binding fragment-SpyTag and purified K-Tag-Fc fragment fusion protein are mixed in the presence of SpyLigase to form a covalent bond between the two molecules. Thus, in certain such embodiments, a first binding motif comprising the SpyTag is present at the C-terminus of the antigen-binding fragment, and a second binding motif comprising the K-Tag (SEQ ID NO: 33) is present at the N-terminus of the Fc fragment. Alternatively, a first binding motif comprising the K-Tag (SEQ ID NO: 33) is present at the C-terminus of the antigen-binding fragment, and a second binding motif comprising the SpyTag is present at the N-terminus of the Fc fragment.

[0075] An advantage of these embodiments is the shorter K-tag compared to SpyCatcher, resulting in antibodies that are more similar to natural antibodies. For example, when the natural "hinge linker" is used, only 23 "unnatural" amino acids are present between the Fab and Fc portions in the artificial full-length antibody.

[0076] SnoopTagJr / DogTag / SnoopLigase Furthermore, the binding motif may be, as described by [5]: S.pneumoniaeThe RrgA protein of Streptococcus aureus (Streptococcus aureus) is designed from the RrgA protein of Streptococcus aureus (Streptococcus aureus) in SEQ ID NO: 37. The reference to Non-Patent Document 5 is incorporated by reference in its entirety. Thus, in certain embodiments, the first binding motif comprises SEQ ID NO: 37 or a sequence having at least 70% identity thereto, while the second binding motif comprises SEQ ID NO: 38 or a sequence having at least 70% identity thereto. Alternatively, the first binding motif comprises SEQ ID NO: 38 or a sequence having at least 70% identity thereto, while the second binding motif comprises SEQ ID NO: 37 or a sequence having at least 70% identity thereto. A Snoop Ligase can be provided that promotes the formation of bonds between the first binding motif and the second binding motif.

[0077] In certain embodiments, the pair of first and second binding motifs is derived from any suitable isopeptide protein. For example, each of the first and second binding motifs is derived from the major pilin protein SpyO128, which has the amino acid sequence set forth in SEQ ID NO:1 and is encoded by the nucleotide sequence set forth in SEQ ID NO:2. Two isopeptide bonds are formed in the protein. One isopeptide bond is formed between the lysine at position 179 of SEQ ID NO:1 and the asparagine at position 303 (the reactive residue) of SEQ ID NO:1. The glutamic acid residue that induces the naturally occurring isopeptide bond is found at position 258 of SEQ ID NO:1. Thus, a pair of binding motifs expressed from the isopeptide protein set forth in SEQ ID NO:1 preferably includes a first binding motif comprising a fragment of the protein that includes the reactive asparagine at position 303 and a second binding motif comprising a fragment of the protein that includes the reactive lysine at position 179. Alternatively, the pair of binding motifs expressed from the isopeptide protein set forth in SEQ ID NO: 1 preferably comprises a second binding motif comprising a fragment of the protein comprising a reactive asparagine at position 303, and a first binding motif comprising a fragment of the protein comprising a reactive lysine at position 179. In such an embodiment, a fragment of the protein comprising a glutamic acid residue at position 258 is provided separately, i.e., as a peptide ligase that forms an isopeptide bond.

[0078] Another isopeptide bond in the major pilin protein SpyO128 occurs between the lysine residue at position 36 of SEQ ID NO:1 and the asparagine residue at position 168 of SEQ ID NO:1. The glutamic acid residue that induces isopeptide formation is found at position 117 of SEQ ID NO:1. Thus, a pair of binding motifs expressed from the isopeptide protein set forth in SEQ ID NO:1 preferably comprises a first binding motif comprising a fragment of the protein containing the reactive lysine residue at position 36, and a second binding motif comprising a fragment of the protein containing the reactive asparagine at position 168. Alternatively, a pair of binding motifs expressed from the isopeptide protein set forth in SEQ ID NO:1 preferably comprises a second binding motif comprising a fragment of the protein containing the reactive lysine residue at position 36, and a first binding motif comprising a fragment of the protein containing the reactive asparagine at position 168. In such an embodiment, a fragment of the protein containing a glutamic acid residue at position 117 is provided separately as a peptide ligase.

[0079] The isopeptide bond is located at the lysine residue at position 181 of SEQ ID NO: 3 (ACE19, E.faecalis The binding occurs between the nucleotide sequence of the adhesin protein (domain of the adhesin protein derived from SEQ ID NO: 3) and the asparagine residue at position 294 of SEQ ID NO: 3. Binding is induced by the aspartic acid residue at position 213 of SEQ ID NO: 3. Thus, a pair of binding motifs expressed from the isopeptide protein set forth in SEQ ID NO: 3 preferably comprises a first binding motif comprising a fragment of the protein comprising a reactive asparagine residue at position 294, and a second binding motif comprising a fragment of the protein comprising a reactive lysine residue at position 181. Alternatively, a pair of binding motifs expressed from the isopeptide protein set forth in SEQ ID NO: 3 preferably comprises a second binding motif comprising a fragment of the protein comprising a reactive asparagine residue at position 294, and a first binding motif comprising a fragment of the protein comprising a reactive lysine residue at position 181. In such an embodiment, the fragment of the protein comprising the aspartic acid residue at position 213 is provided separately as a peptide ligase.

[0080] having the amino acid sequence shown in SEQ ID NO: 10 S.aureus Collagen-binding domains derived from SEQ ID NO: 10 can also be used. The isopeptide bond occurs between the lysine at position 176 of SEQ ID NO: 10 and the asparagine at position 308 of SEQ ID NO: 10. The aspartic acid residue inducing the isopeptide bond is at position 209 of SEQ ID NO: 10. Thus, a pair of binding motifs expressed from the isopeptide protein set forth in SEQ ID NO: 10 preferably comprises a first binding motif comprising a fragment of the protein containing the reactive lysine at position 176 and a second binding motif comprising a fragment of the protein containing the reactive asparagine at position 308. Alternatively, a pair of binding motifs expressed from the isopeptide protein set forth in SEQ ID NO: 10 preferably comprises a second binding motif comprising a fragment of the protein containing the reactive lysine at position 176 and a first binding motif comprising a fragment of the protein containing the reactive asparagine at position 308. In such an embodiment, a fragment of the protein containing an aspartic acid residue at position 209 can be provided separately as a peptide ligase.

[0081] Streptococcus pyogenesFbaB from SEQ ID NO:11 can also be used to provide a binding motif and comprises the domain CnaB2, which has the amino acid sequence set forth in SEQ ID NO:11 and is encoded by the nucleotide sequence set forth in SEQ ID NO:12. The isopeptide bond in the CnaB2 domain is formed between the lysine at position 15 of SEQ ID NO:11 and the aspartic acid residue at position 101 of SEQ ID NO:11. The glutamic acid residue inducing the isopeptide bond is at position 61 of SEQ ID NO:11. Thus, a pair of binding motifs expressed from the isopeptide protein set forth in SEQ ID NO:11 preferably comprises a first binding motif comprising a fragment of the protein containing the reactive lysine at position 15, and a second binding motif comprising a fragment of the protein containing the reactive aspartic acid at position 101. Alternatively, a pair of binding motifs expressed from the isopeptide protein set forth in SEQ ID NO:11 preferably comprises a second binding motif comprising a fragment of the protein containing the reactive lysine at position 15, and a first binding motif comprising a fragment of the protein containing the reactive aspartic acid at position 101. In such an embodiment, a fragment of the protein comprising a glutamic acid residue at position 61 is provided separately as a peptide ligase.

[0082] The RrgA protein Streptococcus pneumoniaeThe adhesin protein is derived from SEQ ID NO: 13, which has the amino acid sequence set forth in SEQ ID NO: 13 and is encoded by the nucleotide sequence set forth in SEQ ID NO: 14. An isopeptide bond is formed between the lysine at position 742 of SEQ ID NO: 13 and the asparagine at position 854 of SEQ ID NO: 13. This bond is induced by the glutamic acid residue at position 803 of SEQ ID NO: 13. Thus, a pair of binding motifs expressed from the isopeptide protein set forth in SEQ ID NO: 13 preferably comprises a first binding motif comprising a fragment of the protein containing the reactive asparagine at position 854, and a second binding motif comprising a fragment of the protein containing the reactive lysine at position 742. Alternatively, a pair of binding motifs expressed from the isopeptide protein set forth in SEQ ID NO: 13 preferably comprises a second binding motif comprising a fragment of the protein containing the reactive asparagine at position 854, and a first binding motif comprising a fragment of the protein containing the reactive lysine at position 742. In such an embodiment, the protein fragment containing the glutamic acid residue at position 803 is separately provided as a peptide ligase as defined above.

[0083] PsCs proteins are Streptococcus intermedius a fragment of the por secretion system C-terminal sorting domain protein derived from SEQ ID NO: 15, which has the amino acid sequence set forth in SEQ ID NO: 15 and is encoded by the nucleotide sequence set forth in SEQ ID NO: 16. An isopeptide bond is formed between the lysine at position 405 of SEQ ID NO: 15 and the aspartate at position 496 of SEQ ID NO: 15. Thus, a pair of binding motifs expressed from the isopeptide protein set forth in SEQ ID NO: 15 preferably comprises a first binding motif comprising a fragment of the protein that includes the reactive aspartate at position 496, and a second binding motif comprising a fragment of the protein that includes the reactive lysine at position 405. Alternatively, a pair of binding motifs expressed from the isopeptide protein set forth in SEQ ID NO: 15 preferably comprises a first binding motif comprising a fragment of the protein that includes the reactive aspartate at position 496, and a second binding motif comprising a fragment of the protein that includes the reactive lysine at position 405.

[0084] In various embodiments, the first and second binding motifs are derived from an isopeptide protein comprising the amino acid sequence set forth in any one of SEQ ID NOs: 21 or 23 or 25 or 27, or a protein having at least 70% sequence identity to the amino acid sequence set forth in any one of SEQ ID NOs: 21 or 23 or 25 or 27. In some embodiments, the isopeptide protein sequence is at least 75, 80, 85, 90, 95, 96, 97, 98, 99 or 100% identical to the sequence to which it is being compared (SEQ ID NO: 21 or 23 or 25 or 27).

[0085] SnoopTagJr / DogTag / SnoopLigase Furthermore, the binding motif may be, as described by [5]: S.pneumoniae The snoop ligase is designed from the RrgA protein of the RrgA gene. Reference 5 is incorporated in its entirety by reference. Thus, in certain embodiments, the first binding motif comprises SEQ ID NO: 37 or a sequence having at least 70% identity thereto, while the second binding motif comprises SEQ ID NO: 38 or a sequence having at least 70% identity thereto. Alternatively, the first binding motif comprises SEQ ID NO: 38 or a sequence having at least 70% identity thereto, while the second binding motif comprises SEQ ID NO: 37 or a sequence having at least 70% identity thereto. A snoop ligase can be provided that promotes the formation of a bond between the first binding motif and the second binding motif.

[0086] SdyTag / SdyCatcher (DANG short) In a further embodiment, the binding motif is S.dysgalactiaeThe fibronectin binding motif is designed from the fibronectin-binding protein of SEQ ID NO: 41. Such protein ligation is described in Non-Patent Document 26, which is incorporated by reference in its entirety. Thus, in certain embodiments, the first binding motif comprises SEQ ID NO: 41 or a sequence having at least 70% identity thereto, while the second binding motif comprises SEQ ID NO: 42 or a sequence having at least 50% identity thereto. Alternatively, the first binding motif comprises SEQ ID NO: 42 or a sequence having at least 50% identity thereto, while the second binding motif comprises SEQ ID NO: 41 or a sequence having at least 70% identity thereto.

[0087] Sortase Another means for linking the Fc fragment to the antigen-binding fragment involves the use of sortase enzymes, sortase recognition domains, and bridging domains. (2003) describes sortase-mediated ligation for site-specific modification of proteins, which is incorporated herein by reference in its entirety. In this aspect of the invention, the sortase recognition domain and bridging domain are considered binding motifs. Sortases are transpeptidases produced by Gram-positive bacteria that 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: 17)), referred to herein as the sortase recognition domain. The sortase recognition domain is a sortase A recognition domain or a sortase B recognition domain. In certain embodiments, the sortase recognition domain has the amino acid sequence: LPTGAA (SEQ ID NO: 18), LPTGGG (SEQ ID NO: 19), LPKTGG (SEQ ID NO: 20), LPETG (SEQ ID NO: 21), LPXTG (SEQ ID NO: 22), or LPXTG(X) n (SEQ ID NO: 23), wherein X is any amino acid, and n is 0, 1, 2, 3, 4, 5, 7, 8, 9, 10, 0-5, or 0-10, or any integer up to 100.

[0088] The sortase A bridge domain comprises one or more glycine residues at one of its termini. In certain embodiments, the one or more glycine residues are optionally selected from the group consisting of Gly, (Gly), (Gly), (Gly), (Gly), or (Gly). x where x is an integer between 1 and 20. The sortase A recognition domain can be fused to the antigen-binding fragment at the C-terminus, optionally via a glycine / serine-rich spacer, and the sortase A bridging domain can be fused to the Fc fragment at the N-terminus, optionally via a glycine / serine-rich spacer.

[0089] Thus, in certain embodiments, the first binding domain fused at its C-terminus to the antigen-binding fragment has the amino acid sequence LPTGAA (SEQ ID NO: 18), LPTGGG (SEQ ID NO: 19), LPKTGG (SEQ ID NO: 20), LPETG (SEQ ID NO: 21), LPXTG (SEQ ID NO: 22), or LPXTG(X) n (SEQ ID NO: 23) (wherein X is any amino acid and n is 0, 1, 2, 3, 4, 5, 7, 8, 9, 10, 0-5 or 0-10, or any integer up to 100), and the second binding domain fused at its N-terminus to an Fc fragment comprises a sortase A recognition domain comprising or consisting of Gly, (Gly)2, (Gly)3, (Gly)4, or (Gly) X (wherein x is an integer from 1 to 20).

[0090] The sortase B recognition domain comprises the amino acid sequence NPX1TX2 (SEQ ID NO: 24), where X1 is glutamine or lysine, X2 is asparagine or glycine, N is asparagine, P is proline, and T is threonine. The sortase B bridging domain comprises one or more glycine residues at one of its termini. In certain embodiments, the one or more glycine residues are optionally selected from the group consisting of Gly, (Gly)2, (Gly)3, (Gly)4, or (Gly) xwhere x is an integer between 1 and 20. The sortase B recognition domain can be fused to the antigen-binding fragment at the C-terminus, optionally via a glycine / serine-rich spacer, and the sortase B bridging domain can be fused to the Fc fragment at the N-terminus, optionally via a glycine / serine-rich spacer.

[0091] Thus, in certain embodiments, the first binding domain fused to the C-terminal antigen-binding fragment comprises a sortase recognition domain comprising or consisting of the amino acid sequence NPX1TX2 (SEQ ID NO: 24) (X1 is glutamine or lysine, X2 is asparagine or glycine, N is asparagine, P is proline, and T is threonine), and the second binding domain fused to the N-terminal Fc fragment comprises a sortase recognition domain comprising or consisting of Gly, (Gly)2, (Gly)3, (Gly)4, or (Gly) x (wherein x is an integer of 1 to 20).

[0092] Buterase Yet another means for linking an Fc fragment to an antigen-binding fragment involves the use of butelase 1 to form a peptide bond between a butelase recognition motif (Asx is Asn or Asp) and the amino terminus of another polypeptide. In this case, the Asx-His-Val motif is fused in frame to the antigen-binding fragment, optionally via a glycine / serine-rich spacer. Butelase can then be used to form a peptide bond between the Asx-His-Val motif and the N-terminal amino acid of the Fc fragment. Patent Document 3 discloses methods and materials for butelase-mediated peptide ligation and is incorporated herein by reference in its entirety.

[0093] Thus, in a specific embodiment, the first binding domain fused at its C-terminus to the antigen-binding fragment comprises a butyrate recognition domain comprising or consisting of the amino acid sequence Asx-His-Val (where Asx is Asn or Asp), and the second binding domain comprises the N-terminal amino acids of the Fc fragment.

[0094] Split intein Another method for linking an Fc fragment to an antigen-binding fragment involves the use of split inteins. Inteins exist as two fragments encoded by two separately transcribed and translated genes. These so-called split inteins self-associate and catalyze protein splicing activity in trans. Split inteins have been identified in a variety of cyanobacteria and archaea (Non-Patent Document 6, Non-Patent Document 9, Non-Patent Document 10, Non-Patent Document 17, Non-Patent Document 30, and Non-Patent Document 33, the disclosures of which are incorporated herein by reference in their entireties). Non-Patent Document 27 and Patent Document 4 (each of which is also incorporated herein by reference in its entirety) also disclose the use of split inteins used to fuse an antigen-binding fragment and an Fc fragment.

[0095] Thus, in certain embodiments, the first binding domain comprises a first split intein, the second binding domain comprises a second split intein, and the first and second split inteins combine to form a catalytically competent enzyme that then catalyzes its own excision and ligation of its flanking sequences.

[0096] Any of the protein ligation systems described above, or others known in the art, can be used to design the antigen-binding fragments and binding motifs fused to the Fc fragment to produce the full-length antibodies of the invention.

[0097] For example, in certain embodiments, an antigen-binding fragment is produced as a fusion protein with a C-terminally fused SpyTag as a first binding motif, and an Fc fragment is produced as a fusion protein with an N-terminally fused SpyCatcher as a second binding motif. The antigen-binding fragment-first binding motif fusion protein and the Fc fragment-second binding motif fusion protein can be mixed together to produce an artificial full-length antibody containing an antigen-binding fragment with a C-terminal SpyTag attached to the N-terminal SpyCatcher of the Fc fragment fusion protein.

[0098] The antigen-binding fragment-SpyTag fusion protein can be prepared by using a vector that adds SpyTag to the C-terminus of the antigen-binding fragment, e.g., Escherichia coli The antigen-binding fragment is produced by expressing a gene encoding the antigen-binding fragment in a vector. A second tag, e.g., a His tag, is added before or after the SpyTag for purification of the antigen-binding fragment-SpyTag fusion protein by affinity chromatography. In a specific embodiment, the SpyTag has the sequence AHIVMVDAYKPTK (SEQ ID NO: 29) or AHIVMVDAYK (SEQ ID NO: 30).

[0099] SpyCatcher-Fc fusion proteins are separately produced by expressing an Fc fragment, such as a human IgG1 Fc fragment, in mammalian expression host cells, such as a CHO cell line or an HEK293 cell line. In the vector used to express the Fc, a gene fragment encoding the heavy chain Fc (CH2-CH3), with or without the hinge region connecting CH1 and CH2 in a native antibody, precedes the SpyCatcher domain, either a 116-amino acid domain (SEQ ID NO: 8) (see Non-Patent Document 16) or a truncated 84-amino acid (SEQ ID NO: 9) version (see Non-Patent Document 16). The region between the SpyCatcher and CH2-CH3 domains contains a peptide that acts as a spacer between SpyCatcher and the Fc fragment, providing additional flexibility. For example, such spacer peptides can be the natural antibody hinge region (e.g., human IgG1: EPKSCDKTHTCPPCP (SEQ ID NO: 31)) or linker peptides, such as peptides containing one or more of the five-amino acid GGGS (SEQ ID NO: 32) sequence motifs, which are known to be both flexible and soluble, or a combination thereof. The Fc fragment fusion protein construct is preceded by a signal sequence that enables extracellular transport of the resulting Fc fragment fusion protein. SpyCatcher-Fc fusion proteins are purified by standard affinity chromatography, for example, using protein A. Antigen-binding fragment-SpyTag fusion proteins can be mixed with SpyCatcher-Fc fusion proteins in the appropriate stoichiometry to generate artificial full-length antibodies. For example, Fab-SpyTag and SpyCatcher-Fc fusions can be mixed in a stoichiometry of two Fab-SpyTag molecules per SpyCatcher-Fc molecule to generate full-length artificial antibodies. Other stoichiometries, such as an excess of Fab-SpyTag, can also be used to increase product yield. Subsequent purification steps can be added to remove excess reactants.

[0100] Appropriate conditions, such as buffer conditions, pH, temperature, and the presence of detergent, are provided for optimal binding via the SpyTag / SpyCatcher system. In one embodiment, the half-time of reaction with each partner at 10 μM at 25°C and pH 7.0 was determined to be 74 seconds (32). The artificial full-length antibodies thus produced can be used directly or further purified before use. Such purification can be achieved by size-exclusion or affinity chromatography using immobilized antibodies that specifically bind to the intact FbaB domain but not to SpyTag or SpyCatcher.

[0101] In certain embodiments, conjugation is carried out in the presence of excess Fab-SpyTag to drive the reaction toward the formation of full-length antibodies. The resulting full-length antibodies are then purified to remove excess Fab-SpyTag, for example, using a Protein A binding matrix or another binding interaction (e.g., His-tag or Strep-tag®). Such tags can be introduced into the Fc, for example, at the Fc C-terminus.

[0102] Because certain Fab frameworks, e.g., Fabs containing VHs derived from the VH3 germline family, also bind Protein A, Fc-SpyCatcher fusions containing C-terminal purification tags (e.g., His-Tag or Strep-tag) can be used for purification to avoid contamination with Protein A-binding Fab fragments.

[0103] Alternatively, the antigen-binding fragment can be produced as a fusion protein with a C-terminally fused SpyCatcher as the first binding motif, and the Fc fragment can be produced as a fusion protein with an N-terminally fused SpyTag as the second binding motif. The antigen-binding fragment-first binding motif fusion protein and the Fc fragment-second binding motif fusion protein can be mixed together to produce an artificial full-length antibody containing an antigen-binding fragment with a C-terminal SpyCatcher linked to the N-terminal SpyTag of the Fc fragment fusion protein. Linker sequences derived from SpyTags and SpyCatchers sequences, as well as hinge or other flexible and soluble sequence motifs as described above, are also included in these embodiments.

[0104] In certain embodiments, the SpyLigase system of protein ligation is used. In such embodiments, an antigen-binding fragment-SpyTag fusion protein is produced as described above. The Fc fragment is not fused to SpyCatcher, but is fused at its N-terminus to a 10-amino acid K-Tag (ATHIKFSKRD, SEQ ID NO: 33), optionally with one or more linkers as described above. The purified antigen-binding fragment-SpyTag and purified K-Tag-Fc fragment fusion protein are mixed in the presence of SpyLigase to form a covalent bond between the two molecules. Thus, in certain such embodiments, a first binding motif containing the SpyTag is present at the C-terminus of the antigen-binding fragment, and a second binding motif containing the K-Tag (SEQ ID NO: 33) is present at the N-terminus of the Fc fragment. Alternatively, a first binding motif containing the K-Tag (SEQ ID NO: 33) is present at the C-terminus of the antigen-binding fragment, and a second binding motif containing the SpyTag is present at the N-terminus of the Fc fragment.

[0105] An advantage of these embodiments is that the K-tag is shorter compared to SpyCatcher, resulting in antibodies that are more similar to natural antibodies: for example, when the natural "hinge linker" is used, only 23 "unnatural" amino acids are present between the Fab and Fc portions in the artificial full-length antibody.

[0106] In a further embodiment, instead of the SpyTag / SpyCatcher system, the SnoopTag / SnoopCatcher system (Non-Patent Document 29) is used. Streptococcus pneumoniae It follows the same principle as the SpyTag / SpyCatcher system, except that the D4Ig-like domain of the derived adhesin RrgA is used as the starting point.

[0107] Furthermore, the SpyTag and SnoopTag devices can be combined to produce multispecific antibodies. For example, a polymer of two or more Fc fragments is produced, each of the Fc fragments having a specific second binding motif at its N-terminus, wherein one or more second binding motifs in the polymer of two or more Fc fragments are derived from the SpyTag system, and one or more second binding motifs in the polymer of two or more Fc fragments are derived from the SnoopTag system. Such a polymer of two or more Fc fragments can be contacted with multiple antigen-binding fragments, each specific for a different antigen or epitope, each having a specific first binding motif, wherein one or more first binding motifs in one or more antigen-binding fragments from the multiple antigen-binding fragments are derived from the SpyTag system, and one or more first binding motifs in one or more antigen-binding fragments from the multiple antigen-binding fragments are derived from the SnoopTag system.

[0108] Polymers of two or more Fc fragments are produced by techniques known in the art. Certain such techniques are described in Mekhaiel et al. (2011), Scientific Reports; 1:124 and Czajkowsky et al. (2012), EMBO Mol. Med.; 4(10):1015-1028. Additional techniques for producing polymers having two or more Fc fragments are known in the art, and such embodiments are within the scope of the present invention.

[0109] In yet further embodiments, the sortase system is used to generate full-length artificial antibodies. In such embodiments, the antigen-binding fragments contain the sorting motif LPXTG (SEQ ID NO: 17) at the C-terminus, e.g., the C-terminus of the heavy chain of a Fab fragment. The Fc portion is produced with a GG sequence at the N-terminus, allowing for covalent attachment by addition of a sortase. The sortase-mediated attachment reaction involves the addition of Ca 2+ In these embodiments, when a "hinge linker" is used as a spacer, the number of "unnatural" amino acids between the Fab and Fc fragments is only six. Alternatively, the antigen-binding fragment contains a GG sequence at the C-terminus, and a sorting motif LPXTG (SEQ ID NO: 17) is present at the N-terminus of the Fc fragment. Covalent binding is catalyzed by a sortase.

[0110] In further embodiments, split intein systems (Non-Patent Document 24) or butelase-mediated ligation (Non-Patent Document 20) are used. In these systems, an enzyme formed from the two components of the split intein system catalyzes the formation of a covalent bond between an antigen-binding fragment fusion protein and an Fc fragment fusion protein. Thus, in various embodiments of the present invention, a series of Fc fragment fusion proteins, all equipped with a binding motif at the N-terminus that allows site-specific covalent protein attachment, are produced either by autocatalysis (SpyTag and SnoopTag systems, split intein systems) or by enzyme-mediated catalysis (sortase, SpyLigase, SnoopLigase, butelase). For example, antigen-binding fragments derived from library technology are produced with the corresponding binding motif at the C-terminus. Such fragments contribute to the specificity required for the assay. Any of these antibody fragments can be combined with any of the Fc fragment fusion proteins generated to generate full-length antibodies containing both the specificity for the target and the Fc portion required to allow multiplexing of the assay or use as a control or calibrator in diagnostic assays to measure a patient's antibody titer to a given antigen.

[0111] Multiplex Assays The multiple full-length monoclonal antibodies provided herein are used to distinguish multiple antigens in the same reaction. Accordingly, certain embodiments of the present invention provide methods for determining the levels of multiple antigens in a sample, comprising contacting the sample with multiple full-length antibodies provided herein and quantifying binding between each of the multiple full-length antibodies and their corresponding antigens to determine the level of each of the multiple antigens in the sample.

[0112] Various methods for visualizing and quantifying binding between antibodies and their corresponding antigens are known in the art, and such embodiments are within the scope of the present invention. For example, unique bead combinations are provided by the Bio-Plex® multiplex immunoassay system and are used to quantify binding between each of multiple full-length antibodies and their corresponding antigens. Alternatively, unique label combinations can be used to quantify binding between each of multiple full-length antibodies and their corresponding antigens.

[0113] nucleic acid construct A further aspect of the present invention provides nucleic acid constructs encoding antigen-binding fragments fused to specific binding motifs and Fc fragments fused to corresponding binding motifs. Such nucleic acids are present in expression vectors in suitable host cells. As described below, the host cells can be prokaryotic or eukaryotic.

[0114] Accordingly, certain embodiments of the present invention provide a plurality of pairs of nucleic acid constructs, each pair of nucleic acid constructs comprising: a) a first nucleic acid construct comprising a polynucleotide sequence encoding an antigen-binding fragment fused at its C-terminus to a first binding motif; and b) a second nucleic acid construct comprising a polynucleotide encoding an Fc fragment fused at its N-terminus to a second binding motif. Including, Each antigen-binding fragment specifically binds to a specific antigen, and each Fc fragment belongs to a specific combination of species, isotype, and subclass; The first binding motif and the second binding motif form a covalent bond when brought into contact with one another, either spontaneously or with the aid of an enzyme.

[0115] Typically, the polynucleotide sequence encoding the Fab fused to the first binding motif at its C-terminus encodes two peptides, the light and heavy chains of the Fab. The first binding motif, such as SpyTag, is fused to either the light or heavy chain. Preferably, the first binding motif is fused to the heavy chain. The Fab expression cassette can comprise a bicistronic vector that produces a single mRNA encoding both the light and heavy chains. Additionally, both the heavy and light chains have signal peptides that direct their transport into the periplasm.

[0116] In certain embodiments, the invention provides a plurality of nucleic acid constructs, each nucleic acid construct comprising a polynucleotide sequence encoding an antigen-binding fragment fused at its C-terminus to a first binding motif, wherein each antigen-binding fragment specifically binds to a unique antigen, and wherein the first binding motifs form a covalent bond with a second binding motif upon contacting each other, either spontaneously or with the assistance of an enzyme.

[0117] A further embodiment of the present invention provides a plurality of nucleic acid constructs, each nucleic acid construct comprising a polynucleotide encoding an Fc fragment fused at its N-terminus to a second binding motif, each Fc fragment belonging to a unique combination of species, isotype and subclass, and wherein the second binding motifs form a covalent bond with the first binding motif when contacted with each other, either spontaneously or with the aid of an enzyme.

[0118] The various embodiments of the first and second binding motifs described above in relation to the full-length antibodies of the invention are also applicable to the nucleic acid constructs of the invention.

[0119] The nucleic acid construct is typically present in various vectors. The vectors of the present invention generally contain transcriptional or translational control sequences necessary for expressing a fusion protein comprising an antigen-binding fragment and an Fc fragment. Suitable transcriptional or translational control sequences include, but are not limited to, a replication origin, a promoter, an enhancer, a repressor binding region, a transcription initiation site, a ribosome binding site, a translation initiation site, and a termination site for transcription and translation.

[0120] An origin of replication (commonly referred to as an ori sequence) allows a vector to replicate in an appropriate host cell. The choice of ori depends on the type of host cell and / or genetic package used. When the host cell is prokaryotic, the expression vector typically contains an ori sequence that directs autonomous replication of the vector in the prokaryotic cell. Preferred prokaryotic ori are capable of directing vector replication in bacterial cells. Origins of this class include, but are not limited to, pMB1, pUC, and other E. coli sources.

[0121] In eukaryotic systems, higher eukaryotes have multiple origins of DNA replication, but the ori sequence has not been clearly defined. Replication origins suitable for mammalian vectors are usually derived from eukaryotic viruses. Preferred eukaryotic ori include, but are not limited to, SV40 ori, EBV ori, or HSV ori. Eukaryotic vectors and eukaryotic host cells are typically used to express Fc fragment-binding motif fusion proteins.

[0122] As used herein, a "promoter" is a DNA region that, under certain conditions, is capable of binding RNA polymerase and initiating transcription of a coding region located downstream (3' direction) of the promoter. It may be constitutive or inducible. Generally, a promoter sequence is bounded at its 3' end by a transcription initiation site and extends upstream (5' direction) to include the minimum number of bases or elements required to initiate transcription at a level detectable above background. Within the promoter sequence are transcription initiation sites, as well as protein binding domains responsible for binding RNA polymerase. Eukaryotic promoters often, but not always, contain "TATA" boxes and "CAT" boxes.

[0123] The choice of promoter largely depends on the host cell into which the vector will be introduced. For prokaryotic cells, various robust promoters are known in the art. Preferred promoters are the lac promoter, the Trc promoter, the T7 promoter, and the pBAD promoter. Usually, to obtain expression of a foreign sequence in multiple species, a prokaryotic promoter can be placed immediately after a eukaryotic promoter or within an intron sequence downstream of the eukaryotic promoter.

[0124] Suitable promoter sequences for eukaryotic cells include promoters for 3-phosphoglycerate kinase or other glycolytic enzymes, such as enolase, glyceraldehyde-3-phosphate dehydrogenase, hexokinase, pyruvate decarboxylase, phosphofructokinase, glucose-6-phosphate isomerase, 3-phosphoglycerate mutase, pyruvate kinase, triosephosphate isomerase, phosphoglucose isomerase, and glucokinase. Other promoters with the added advantage of transcription controlled by growth conditions include promoter regions for alcohol dehydrogenase 2, isocytochrome C, acid phosphatase, degradative enzymes involved in nitrogen metabolism, and the aforementioned glyceraldehyde-3-phosphate dehydrogenase, as well as enzymes involved in maltose and galactose utilization. Preferred promoters for mammalian cells are the SV40 promoter, CMV promoter, β-actin promoter, and hybrids thereof. Preferred promoters for yeast cells include, but are not limited to, GAL10, GALI, TEFI in Scerevisiae, and GAP, AOX1 in Pastoris.

[0125] In constructing a subject vector, a termination sequence associated with a protein-coding sequence can also be inserted at the 3' end of the sequence desired to be transcribed to provide polyadenylation and / or transcription termination signals for mRNA. The terminator sequence preferably comprises one or more transcription termination sequences (e.g., polyadenylation sequences) and can also be lengthened by including additional DNA sequences to further disrupt transcription readthrough. Preferred terminator sequences (or termination sites) of the present invention comprise a gene followed by a transcription termination sequence, either its own termination sequence or a heterologous termination sequence. Examples of such termination sequences include a stop codon linked to various yeast transcription termination sequences or mammalian polyadenylation sequences, which are known and widely available in the art. When the terminator comprises a gene, it is advantageous to use a gene encoding a detectable or selectable marker, thereby providing a means for detecting and / or selecting for the presence and / or absence of the terminator sequence (and thus the corresponding inactivation and / or activation of the transcription unit).

[0126] In addition to the above elements, vectors may also contain a selectable marker (e.g., a gene encoding a protein necessary for the survival or growth of a host cell transformed with the vector), provided that such a marker gene is carried on another polynucleotide sequence co-introduced into the host cell. Only those host cells into which the selectable gene has been introduced will survive and / or grow under selective conditions. Typical selection genes encode (a) proteins that confer resistance to antibiotics or other toxins, such as ampicillin, kanamycin, neomycin, zeocin, G418, methotrexate, etc.; (b) proteins that complement auxotrophic deficiencies; or (c) proteins that supply critical nutrients not available from complex media. The choice of the appropriate marker gene will depend on the host cell, and appropriate genes for different hosts are known in the art.

[0127] In one embodiment, the expression vector is a shuttle vector capable of replicating in at least two unrelated host systems. To facilitate such replication, the vector generally contains at least two origins of replication, one effective in each host system. Typically, shuttle vectors are capable of replicating in both eukaryotic and prokaryotic host systems. This allows for detection of protein expression in eukaryotic hosts (expression cell types) and amplification of the vector in prokaryotic hosts (amplification cell types). Preferably, one origin of replication is derived from SV40 or 2u, and one from pUC. However, any suitable origin known in the art can be used, provided it directs vector replication. If the vector is a shuttle vector, it preferably contains at least two selectable markers, one for the expression cell type and one for the amplification cell type. Any selectable marker known in the art or described herein may be used, as long as it functions in the expression system utilized.

[0128] Vectors encompassed by the present invention can be obtained using recombinant cloning methods and / or by chemical synthesis. Numerous recombinant cloning techniques, such as PCR, restriction endonuclease digestion, and ligation, are well known in the art and need not be described in detail herein. One skilled in the art can also use the sequence data provided herein or in public or proprietary databases to obtain desired vectors by any synthetic means available in the art. Furthermore, using well-known restriction and ligation techniques, appropriate sequences can be excised from various DNA sources and incorporated into an operable relationship with the exogenous sequence to be expressed according to the present invention.

[0129] definition As used in this specification and claims, the singular forms "a," "an," and "the" include plural referents unless the content clearly dictates otherwise.

[0130] "Full-length antibody" refers to an antibody-like molecule having at least one antigen-binding fragment and at least one Fc fragment.

[0131] Antigen-binding fragment refers to the antigen-binding portion of an antibody, such as a Fab fragment, variable fragment (Fv), single-chain variable fragment (scFv), single-chain Fab fragment (scFab), or any single domain antibody (sdAb) such as a camelid VHH or shark variable novel antigen receptor (VNAR) domain. It also refers to other proteinaceous affinity reagents or binding scaffolds not derived from the antibody immunoglobulin fold, such as variable lymphocyte receptors (VLRs), affimers, affibodies, darpins, anticalins, and monobodies.

[0132] The Fc fragment is defined as the tail region of an antibody that interacts with Fc receptors and several proteins of the complement system and is detected by a corresponding Fc-specific secondary antibody in an immunoassay. The Fc fragment may or may not contain a hinge sequence at the N-terminus. The Fc fragment typically consists of two or more constant domains that form a soluble homodimer or a higher-order structure of such a homodimer.

[0133] The term "binding motif" refers to a protein sequence that binds to the Fc fragment and the antigen-binding fragment and facilitates the formation of a covalent bond to link the Fc fragment and the antigen-binding fragment to produce a full-length antibody. Binding motifs include, but are not limited to, SpyTag sequences, including SpyTag002 (SEQ ID NO: 34) and SpyTag003 (SEQ ID NO: 43), SpyCatcher sequences, including SpyCatcher002 and SpyCatcher003 sequences, SnoopTag sequences, SnoopCatcher sequences, sortase motifs, butelase substrates, and peptiligase substrates. The binding motif is fused to the Fc fragment at the N-terminus or to the antigen-binding fragment at the C-terminus. Alternatively, the binding motif is fused to the Fc fragment and the antigen-binding fragment at the C-terminus, or to the Fc fragment and the antigen-binding fragment at the C-terminus. Spacer sequences (eg, glycine / serine-rich spacers) flank the binding motifs to enhance accessibility for reaction or to enhance flexibility of the antigen-binding fragment fused to Fc.

[0134] The term "prokaryotic system" refers to prokaryotic cells such as bacterial cells or prokaryotic viruses, prokaryotic phages, or bacterial spores. The term "eukaryotic system" refers to eukaryotic cells, including animal, plant, fungal, and protist cells, and eukaryotic viruses such as retroviruses, adenoviruses, and baculoviruses. Prokaryotic and eukaryotic systems may be collectively referred to as "expression systems."

[0135] The term "expression cassette" is used herein to refer to a functional unit constructed in a vector for the purpose of expressing recombinant antigen-binding fragments and Fc fragments. The expression cassette contains one or more promoters, transcription termination sequences, ribosomal binding sites, and a cDNA encoding the fusion protein. Depending on the expression system (e.g., an enhancer and polyadenylation signal for eukaryotic expression systems), other genetic components may be added to the expression cassette.

[0136] As used herein, the term "vector" preferably refers to a self-replicating nucleic acid molecule, which transfers an inserted nucleic acid molecule into and / or between host cells. Typically, a vector is a circular DNA containing an origin of replication, a selection marker, and / or a viral packaging signal, as well as other regulatory elements. The terms vector, vector DNA, plasmid DNA, and phagemid DNA are used interchangeably in describing the present invention. This term includes vectors that function primarily for the insertion of DNA or RNA into cells, replication vectors that function primarily for the replication of DNA or RNA, and expression vectors that function for the transcription and / or translation of DNA or RNA. Also included are vectors that provide two or more of the above functions.

[0137] The term "expression vector" refers to a polynucleotide that, when introduced into an appropriate host cell, is transcribed and translated into a polypeptide. The term "expression vector" refers to a vector that directs the expression of an Fc fragment or antigen-binding fragment of interest fused in-frame with a binding motif.

[0138] As used herein, the terms "polynucleotide," "nucleic acid," and "oligonucleotide" are used interchangeably. They refer to polymeric forms of nucleotides of any length, either deoxyribonucleotides or ribonucleotides, or their analogs. Non-limiting examples of polynucleotides include coding or non-coding regions of a gene or gene fragment, 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 contain modified nucleotides, such as methylated nucleotides and nucleotide analogs. If present, modifications to the nucleotide structure may be imparted before or after assembly of the nucleotide polymer.

[0139] As used herein, the term "amino acid" refers to natural and / or unnatural or synthetic amino acids, glycine and both the D or L optical isomers, amino acid analogs, and peptidomimetics.

[0140] As used herein, the terms "polypeptide," "peptide," and "protein" are used interchangeably herein to refer to polymers of amino acids of any length.

[0141] As used herein, the term "host cell" includes an individual cell or cell culture that is or has been a recipient of the disclosed expression constructs. A host cell includes the progeny of a single host cell. The progeny may not necessarily be completely identical to the original parent cell due to natural, accidental, or deliberate mutations.

[0142] [Table 1] [Table 2]

[0143] Additional Disclosures and Subject Matter Item 1. A full-length antibody comprising an antigen-binding fragment comprising a first binding motif at the C-terminus and an Fc fragment comprising a second binding motif at the N-terminus, wherein the first binding motif and the second binding motif are covalently linked to each other via protein ligation, and wherein, if the antigen-binding fragment and the Fc fragment are obtained from the same species, the Fc fragment is labeled with a detectable label.

[0144] Item 2. The full-length antibody of item 1, wherein the antigen-binding fragment is obtained from a first species and the Fc fragment is obtained from a second species different from the first species.

[0145] Item 3. A plurality of full-length antibodies, each full-length antibody comprising an antigen-binding fragment comprising a first binding motif at the C-terminus and an Fc fragment comprising a second binding motif at the N-terminus, wherein the first binding motif and the second binding motif are covalently linked to each other via protein ligation.

[0146] Item 4. The plurality of full-length antibodies according to Item 3, wherein each antigen-binding fragment specifically binds to a unique antigen and each Fc fragment belongs to a unique combination of species, isotype, and subclass.

[0147] Item 5. The plurality of full length antibodies of Item 3 or 4, wherein each full length antibody is conjugated to a unique label.

[0148] Item 6. The plurality of full-length antibodies of Item 3 or 4, wherein each full-length antibody is bound to a unique bead.

[0149] Item 7: One of the first binding motif and / or the second binding motif comprises the sequence set forth in SEQ ID NO: 1 or 3 or 5 or 6 or 7 or 25 or 27 or 29 or 30 or 34 or 35 or 37 or 39 or 41 or 43, residues 302 to 308 set forth in SEQ ID NO: 1, or a sequence having at least 50% identity to SEQ ID NO: 1 or 3 or 5 or 6 or 7 or 25 or 27 or 29 or 30 or 34 or 35 or 37 or 39 or 41 or 43; or a fragment thereof; and the other binding motif comprises the sequence set forth in SEQ ID NO: 1. 7. The multiple full-length antibodies of any one of items 3 to 6, comprising residues 31 to 291 of the sequence SEQ ID NO: 8 or 9 or 26 or 28 or 33 or 36 or 38 or 40 or 42 or 44, or a sequence having at least 50% identity to SEQ ID NO: 1 or 8 or 9 or 26 or 28 or 33 or 36 or 38 or 40 or 42 or 44; or a fragment thereof, wherein the first binding motif and the second binding motif interact with each other via protein ligation, either spontaneously or with the aid of an enzyme, to form a covalent bond.

[0150] Item 8. The multiple full-length antibodies according to Item 7, wherein the fragments of SEQ ID NO: 1, 3, 5, or 6 comprise about 5 to 50 amino acids.

[0151] Item 9: The plurality of full-length antibodies according to Item 7 or 8, wherein one of the first binding motif and the second binding motif comprises residues 302 to 308, 301 to 308, 300 to 308, 299 to 308, 299 to 308, 298 to 308, 297 to 308, 296 to 308, 295 to 308, 294 to 308, 293 to 308, 292 to 308, 291 to 308, or 290 to 308 of SEQ ID NO: 1, and the first binding motif and the second binding motif interact with each other via protein ligation, spontaneously or with the aid of an enzyme, to form a covalent bond.

[0152] Item 10. The plurality of full-length antibodies according to any one of Items 3 to 6, wherein the first binding motif or the second binding motif comprises a reactive asparagine at position 303 of SEQ ID NO: 1, and the first binding motif and the second binding motif interact to form a covalent bond via protein ligation, either spontaneously or with the aid of an enzyme.

[0153] Item 11: A plurality of full-length antibodies described in any one of Items 3 to 6, wherein one of the first binding motif and the second binding motif comprises a fragment of SEQ ID NO: 1 containing a reactive lysine residue at position 36 of SEQ ID NO: 1, and the other binding motif comprises a fragment of SEQ ID NO: 1 containing a reactive asparagine residue at position 168 of SEQ ID NO: 1, and the first binding motif and the second binding motif interact to form a covalent bond via protein ligation, either spontaneously or with the aid of an enzyme.

[0154] Item 12: A plurality of full-length antibodies described in any one of Items 3 to 6, wherein one of the first binding motif and the second binding motif comprises a fragment of SEQ ID NO: 5 containing a reactive lysine residue at position 149 of SEQ ID NO: 5, and the other binding motif comprises a fragment of SEQ ID NO: 5 containing a reactive asparagine residue at position 266 of SEQ ID NO: 5, and the first binding motif and the second binding motif interact to form a covalent bond via protein ligation, either spontaneously or with the aid of an enzyme.

[0155] Item 13: A plurality of full-length antibodies described in any one of Items 3 to 6, wherein one of the first binding motif and the second binding motif comprises a fragment of SEQ ID NO: 6 that includes a reactive lysine residue at position 15 of SEQ ID NO: 6, and the other binding motif comprises a fragment of SEQ ID NO: 6 that includes a reactive aspartic acid residue at position 101 of SEQ ID NO: 6, and the first binding motif and the second binding motif interact to form a covalent bond via protein ligation, either spontaneously or with the aid of an enzyme.

[0156] Item 14: The plurality of full-length antibodies described in any one of Items 3 to 6, wherein one of the first binding motif and the second binding motif comprises a fragment of SEQ ID NO: 1 containing the reactive asparagine at position 303 of SEQ ID NO: 1, and the other binding motif comprises a fragment of SEQ ID NO: 1 containing the reactive lysine at position 179 of SEQ ID NO: 1, and the first binding motif and the second binding motif interact to form a covalent bond via protein ligation, either spontaneously or with the aid of an enzyme.

[0157] Item 15: The plurality of full-length antibodies described in any one of Items 3 to 6, wherein one of the first binding motif and the second binding motif comprises a fragment of SEQ ID NO: 1 containing a reactive lysine at position 36 of SEQ ID NO: 1, and the other binding motif comprises a fragment of SEQ ID NO: 1 containing a reactive asparagine at position 168 of SEQ ID NO: 1, and the first binding motif and the second binding motif interact with each other via protein ligation, spontaneously or with the aid of an enzyme, to form a covalent bond.

[0158] Item 16: The plurality of full-length antibodies described in any one of Items 3 to 6, wherein one of the first binding motif and the second binding motif comprises a fragment of SEQ ID NO: 3 including a reactive lysine at position 181 of SEQ ID NO: 3, and the other binding motif comprises a fragment of SEQ ID NO: 3 including a reactive asparagine at position 294 of SEQ ID NO: 3, and the first binding motif and the second binding motif interact to form a covalent bond via protein ligation, either spontaneously or with the aid of an enzyme.

[0159] Item 17: The plurality of full-length antibodies according to any one of Items 3 to 6, wherein one of the first binding motif and the second binding motif comprises a fragment of SEQ ID NO: 10 containing a reactive lysine at position 176 of SEQ ID NO: 10, and the other binding motif comprises a fragment of SEQ ID NO: 10 containing a reactive asparagine at position 308 of SEQ ID NO: 10, and the first binding motif and the second binding motif interact with each other via protein ligation, spontaneously or with the aid of an enzyme, to form a covalent bond.

[0160] Item 18: The plurality of full-length antibodies described in any one of Items 3 to 6, wherein one of the first binding motif and the second binding motif comprises a fragment of SEQ ID NO: 11 containing a reactive lysine at position 15 of SEQ ID NO: 11, and the other binding motif comprises a fragment of SEQ ID NO: 11 containing a reactive aspartic acid at position 101 of SEQ ID NO: 11, and the first binding motif and the second binding motif interact to form a covalent bond via protein ligation, either spontaneously or with the aid of an enzyme.

[0161] Item 19: The plurality of full-length antibodies described in any one of Items 3 to 6, wherein one of the first binding motif and the second binding motif comprises a fragment of SEQ ID NO: 13 containing a reactive lysine at position 742 of SEQ ID NO: 13, and the other binding motif comprises a fragment of SEQ ID NO: 13 containing a reactive asparagine at position 854 of SEQ ID NO: 13, and the first binding motif and the second binding motif interact with each other via protein ligation, spontaneously or with the aid of an enzyme, to form a covalent bond.

[0162] Item 20: The plurality of full-length antibodies according to any one of Items 3 to 6, wherein one of the first binding motif and the second binding motif comprises a fragment of SEQ ID NO: 15 that includes a reactive lysine at position 405 of SEQ ID NO: 15, and the other binding motif comprises a fragment of SEQ ID NO: 15 that includes a reactive aspartic acid at position 496 of SEQ ID NO: 15.

[0163] Item 21: The plurality of full-length antibodies according to any one of Items 3 to 6, wherein the first binding motif and / or the second binding motif comprises an isopeptide comprising the amino acid sequence of SEQ ID NO: 21, 23, 25, or 27, or a protein having at least 70% sequence identity to the amino acid sequence set forth in any one of SEQ ID NO: 21, 23, 25, or 27.

[0164] Item 22: The plurality of full-length antibodies according to any one of Items 3 to 6, wherein the first binding motif comprises a sortase recognition domain and the second binding motif comprises a sortase bridge domain.

[0165] Item 23. The sortase recognition domain has the amino acid sequence: LPTGAA (SEQ ID NO: 18), LPTGGG (SEQ ID NO: 19), LPKTGG (SEQ ID NO: 20), LPETG (SEQ ID NO: 21), LPXTG (SEQ ID NO: 22), or LPXTG(X) n(SEQ ID NO: 23), wherein X is any amino acid, and n is 0, 1, 2, 3, 4, 5, 7, 8, 9, 10, any integer in the range of 0 to 5, or 0 to 10, or any integer up to 100; NPX1TX2 (SEQ ID NO: 24), 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, and x is an integer from 1 to 20.

[0166] Item 24: The plurality of full-length monoclonal antibodies according to any one of Items 3 to 6, wherein the first binding motif comprises a buterase recognition domain.

[0167] Item 25. The multiple full-length antibodies described in Item 24, wherein the buterase recognition domain comprises the amino acid sequence Asn-His-Val or Asp-His-Val.

[0168] Item 26: The plurality of full-length antibodies described in any one of Items 3 to 6, wherein the first binding motif and the second binding motif each comprise a split intein, and the first binding motif and the second binding motif interact with each other to form a covalent bond via protein ligation, either spontaneously or with the assistance of an enzyme.

[0169] Item 27. A method for determining the levels of multiple antigens in a sample, comprising contacting the sample with multiple full length antibodies according to any one of Items 3 to 26 and quantifying binding between each of the multiple full length antibodies and their corresponding antigens to determine the presence and levels of the multiple antigens in the sample.

[0170] Item 28: A plurality of pairs of nucleic acid constructs, each pair of nucleic acid constructs comprising: a) a first nucleic acid construct comprising a polynucleotide sequence encoding an antigen-binding fragment fused at its C-terminus to a first binding motif; b) a second nucleic acid construct comprising a polynucleotide encoding an Fc fragment fused at its N-terminus to a second binding motif; Including, each antigen-binding fragment specifically binds to a unique antigen, and each Fc fragment belongs to a unique combination of species, isotype, and subclass; A plurality of paired nucleic acid constructs, wherein the first binding motif and the second binding motif form a covalent bond when contacted with each other, either spontaneously or with the aid of an enzyme.

[0171] Item 29 a) one of the first binding motif and the second binding motif comprises SEQ ID NO: 1 or 3 or 5 or 6 or 7 or 25 or 27 or 29 or 30 or 34 or 35 or 37 or 39 or 41 or 43, residues 302 to 308 of the sequence shown in SEQ ID NO: 1, or a sequence having at least 50% identity to SEQ ID NO: 1 or 3 or 5 or 6 or 7 or 25 or 27 or 29 or 30 or 34 or 35 or 37 or 39 or 41 or 43; or a fragment thereof; and the other binding motif comprises residues 31 to 291 of the sequence shown in SEQ ID NO: 1, SEQ ID NO: 8 or 9 or 26 or 33 or 36 or 38 or 40 or 42 or 44, or a sequence having at least 50% identity to SEQ ID NO: 1 or 8 or 9 or 26 or 28 or 33 or 36 or 38 or 40 or 42 or 44; or a fragment thereof; b) the first binding motif and the second binding motif comprise residues 302 to 308, 301 to 308, 300 to 308, 299 to 308, 298 to 308, 297 to 308, 296 to 308, 295 to 308, 294 to 308, 293 to 308, 292 to 308, 291 to 308, or 290 to 308 of SEQ ID NO: 1, or a sequence having at least about 50% to 95% identity to residues 302 to 308 of SEQ ID NO: 1; c) the first binding motif or the second binding motif comprises a reactive asparagine at position 303 of SEQ ID NO: 1; d) one of the first binding motif and the second binding motif comprises a fragment of SEQ ID NO: 1 containing a reactive lysine residue at position 36 of SEQ ID NO: 1, and the other binding motif comprises a fragment of SEQ ID NO: 1 containing a reactive asparagine residue at position 168 of SEQ ID NO: 1; e) one of the first binding motif and the second binding motif comprises a fragment of SEQ ID NO: 5 containing a reactive lysine residue at position 149 of SEQ ID NO: 5, and the other binding motif comprises a fragment of SEQ ID NO: 5 containing a reactive asparagine residue at position 266 of SEQ ID NO: 5; f) one of the first binding motif and the second binding motif comprises a fragment of SEQ ID NO: 6 containing a reactive lysine residue at position 15 of SEQ ID NO: 6, and the other binding motif comprises a fragment of SEQ ID NO: 6 containing a reactive aspartic acid residue at position 101 of SEQ ID NO: 6; g) one of the first binding motif and the second binding motif comprises a fragment of SEQ ID NO: 1 containing a reactive asparagine at position 303 of SEQ ID NO: 1, and the other binding motif comprises a fragment of SEQ ID NO: 1 containing a reactive lysine at position 179 of SEQ ID NO: 1; h) one of the first binding motif and the second binding motif comprises a fragment of SEQ ID NO: 1 containing a reactive lysine at position 36 of SEQ ID NO: 7, and the other binding motif comprises a fragment of SEQ ID NO: 1 containing a reactive asparagine at position 168 of SEQ ID NO: 1; i) one of the first binding motif and the second binding motif comprises a fragment of SEQ ID NO: 3 containing a reactive lysine at position 181 of SEQ ID NO: 3, and the other binding motif comprises a fragment of SEQ ID NO: 3 containing a reactive asparagine at position 294 of SEQ ID NO: 3; j) one of the first binding motif and the second binding motif comprises a fragment of SEQ ID NO: 10 containing a reactive lysine at position 176 of SEQ ID NO: 10, and the other binding motif comprises a fragment of SEQ ID NO: 10 containing a reactive asparagine at position 308 of SEQ ID NO: 10; k) one of the first binding motif and the second binding motif comprises a fragment of SEQ ID NO: 11 containing a reactive lysine at position 15 of SEQ ID NO: 11, and the other binding motif comprises a fragment of SEQ ID NO: 11 containing a reactive aspartic acid at position 101 of SEQ ID NO: 11; l) one of the first binding motif and the second binding motif comprises a fragment of SEQ ID NO: 13 containing a reactive lysine at position 742 of SEQ ID NO: 13, and the other binding motif comprises a fragment of SEQ ID NO: 13 containing a reactive asparagine at position 854 of SEQ ID NO: 13; m) one of the first binding motif and the second binding motif comprises a fragment of SEQ ID NO: 15 containing a reactive lysine at position 405 of SEQ ID NO: 15, and the other binding motif comprises a fragment of SEQ ID NO: 15 containing a reactive aspartic acid at position 496 of SEQ ID NO: 15; n) the first binding motif and / or the second binding motif comprises an isopeptide comprising the amino acid sequence of SEQ ID NO: 21 or 23 or 25 or 27, or a protein having at least 70% sequence identity to the amino acid sequence set forth in any one of SEQ ID NO: 21 or 23 or 25 or 27; o) the first binding motif comprises a sortase recognition domain and the second binding motif comprises a sortase bridge domain; p) the first binding motif comprises a butelase 1 recognition domain; or q) the first binding motif and the second binding motif each comprise a split intein; 29. A pair of nucleic acid constructs according to item 28, wherein the first binding motif and the second binding motif interact with each other via protein ligation, spontaneously or with the aid of an enzyme, to form a covalent bond.

[0172] Item 30. A plurality of prokaryotic or eukaryotic host cells, each of the plurality of prokaryotic or eukaryotic host cells comprising one nucleic acid construct from the nucleic acid constructs of Items 28 or 29.

[0173] Item 31. A plurality of Fc fragments, each Fc fragment comprising a unique second binding motif at its N-terminus, each unique second binding motif capable of covalently binding to a unique first binding motif via protein ligation, either spontaneously or with the assistance of an enzyme, and each Fc fragment belonging to a unique combination of species, isotype, and / or subclass.

[0174] Item 32. The multiple Fc fragments of Item 31, wherein each of the Fc fragments is conjugated to a unique label.

[0175] Item 33. The multiple Fc fragments of Item 31, wherein each of the Fc fragments is bound to a unique bead.

[0176] Item 34 i) the unique second binding motif comprises SEQ ID NO: 1 or 3 or 5 or 6 or 7 or 25 or 27 or 29 or 30 or 34 or 35 or 37 or 39 or 41 or 43, residues 302-308 of the sequence set forth in SEQ ID NO: 1, a sequence having at least 50% identity to SEQ ID NO: 1 or 3 or 5 or 6 or 7 or 25 or 27 or 29 or 30 or 34 or 35 or 37 or 39 or 41 or 43, or a fragment thereof, and is capable of covalently binding, spontaneously or via enzymatically assisted protein ligation, to a unique first binding motif comprising residues 31 to 291 of the sequence set forth in SEQ ID NO: 1 or 8 or 9 or 26 or 28 or 33 or 36 or 38 or 40 or 42 or 44, or a sequence having at least 50% identity to SEQ ID NO: 1 or 8 or 9 or 26 or 28 or 33 or 36 or 38 or 40 or 42 or 44; or a fragment thereof; ii) the unique second binding motif comprises residues 31-291 of the sequence set forth in SEQ ID NO: 1 or 8 or 9 or 26 or 28 or 33 or 38 or 40 or 42 or 44, or a sequence having at least 50% identity to SEQ ID NO: 1 or 8 or 9 or 26 or 28 or 33 or 36 or 38 or 40 or 42 or 44, or a fragment thereof, and binds spontaneously or enzymatically via protein ligation to SEQ ID NO: 1 or 3 or 5 or 6 or 7 or 25 or 26 or 28 or 33 or 36 or 38 or 40 or 42 or 44; 34. The Fc fragments according to any one of items 31 to 33, wherein the Fc fragments are capable of covalently binding to a unique first binding motif comprising residues 1 to 7 of SEQ ID NO: 1 or 3 or 5 or 6 or 7 or 25 or 27 or 29 or 30 or 34 or 35 or 37 or 39 or 41 or 43, or residues 302 to 308 of the sequence shown in SEQ ID NO: 1, or a sequence having at least 50% identity to SEQ ID NO: 1 or 3 or 5 or 6 or 7 or 25 or 27 or 29 or 30 or 34 or 35 or 37 or 39 or 41 or 43; or a fragment thereof.

[0177] Item 35. The multiple Fc fragments according to Item 34, wherein the fragments of SEQ ID NO: 1, 3, 5, or 6 contain about 5 to 50 amino acids.

[0178] Item 36: The unique second binding motif comprises residues 302 to 308, 301 to 308, 300 to 308, 299 to 308, 298 to 308, 297 to 308, 296 to 308, 295 to 308, 294 to 308, 293 to 308, 292 to 308, 291 to 308, or 290 to 308 of SEQ ID NO: 1, or a sequence at least about 50% to 95% identical to residues 302 to 308 of SEQ ID NO: 1; 36. The multiple Fc fragments according to item 34 or 35, wherein the unique second binding motifs are capable of covalently binding to the unique first binding motifs via spontaneous or enzymatically assisted protein ligation.

[0179] Item 37. The multiple Fc fragments of any one of Items 31 to 33, wherein the unique second binding motif comprises a reactive asparagine at position 303 of SEQ ID NO: 1 and can be covalently linked to the unique first binding motif via protein ligation, either spontaneously or with the assistance of an enzyme.

[0180] Item 38 i) the unique second binding motif comprises a fragment of SEQ ID NO: 1 comprising a reactive lysine residue at position 36 of SEQ ID NO: 1 and is capable of covalently binding, spontaneously or through enzymatically assisted protein ligation, to a unique first binding motif comprising a fragment of SEQ ID NO: 1 comprising a reactive asparagine at position 168 of SEQ ID NO: 1; or ii) the multiple Fc fragments according to any one of Items 31 to 33, wherein the unique second binding motif comprises a fragment of SEQ ID NO: 1 comprising a reactive asparagine at position 168 of SEQ ID NO: 1 and is capable of covalently binding, spontaneously or with the aid of an enzyme, via protein ligation to a unique first binding motif comprising a fragment of SEQ ID NO: 1 comprising a reactive lysine residue at position 36 of SEQ ID NO: 1.

[0181] Item 39 i) the unique second binding motif comprises a fragment of SEQ ID NO: 5 comprising a reactive lysine residue at position 149 of SEQ ID NO: 5 and is capable of covalently binding, spontaneously or through enzymatically assisted protein ligation, to a unique first binding motif comprising a fragment of SEQ ID NO: 5 comprising a reactive asparagine at position 266 of SEQ ID NO: 5; or ii) the multiple Fc fragments according to any one of items 31 to 33, wherein the unique second binding motif comprises a fragment of SEQ ID NO: 5 comprising a reactive asparagine at position 266 of SEQ ID NO: 5 and can be covalently bound, spontaneously or with the aid of an enzyme, via protein ligation to a unique first binding motif comprising a fragment of SEQ ID NO: 5 comprising a reactive lysine residue at position 149 of SEQ ID NO: 5;

[0182] Item 40 i) the unique second binding motif comprises a fragment of SEQ ID NO: 6 comprising a reactive lysine residue at position 15 of SEQ ID NO: 6 and is capable of covalently binding, spontaneously or through enzymatically assisted protein ligation, to a unique first binding motif comprising a fragment of SEQ ID NO: 6 comprising a reactive aspartic acid at position 101 of SEQ ID NO: 6; or ii) the multiple Fc fragments according to any one of Items 31 to 33, wherein the unique second binding motif comprises a fragment of SEQ ID NO: 6 comprising a reactive aspartic acid at position 101 of SEQ ID NO: 6 and is capable of covalently binding, spontaneously or with the aid of an enzyme, via protein ligation to a unique first binding motif comprising a fragment of SEQ ID NO: 6 comprising a reactive lysine residue at position 15 of SEQ ID NO: 6.

[0183] Item 41 i) the unique second binding motif comprises a fragment of SEQ ID NO: 1 comprising a reactive asparagine at position 303 of SEQ ID NO: 1 and is capable of covalently binding, spontaneously or through enzymatically assisted protein ligation, to a unique first binding motif comprising a fragment of SEQ ID NO: 1 comprising a reactive lysine at position 179 of SEQ ID NO: 1; or ii) the multiple Fc fragments according to any one of Items 31 to 33, wherein the unique second binding motif comprises a fragment of SEQ ID NO: 1 comprising a reactive lysine at position 179 of SEQ ID NO: 1 and is capable of covalently binding, spontaneously or with the aid of an enzyme, via protein ligation to a unique first binding motif comprising a fragment of SEQ ID NO: 1 comprising a reactive asparagine at position 303 of SEQ ID NO: 1.

[0184] Item 42 i) the unique second binding motif comprises a fragment of SEQ ID NO: 1 comprising a reactive lysine at position 36 of SEQ ID NO: 1 and is capable of covalently binding, spontaneously or through enzymatically assisted protein ligation, to a unique first binding motif comprising a fragment of SEQ ID NO: 1 comprising a reactive asparagine at position 168 of SEQ ID NO: 1; or ii) the multiple Fc fragments according to any one of Items 31 to 33, wherein the unique second binding motif comprises a fragment of SEQ ID NO: 1 comprising a reactive asparagine at position 168 of SEQ ID NO: 1 and is capable of covalently binding, spontaneously or with the aid of an enzyme, via protein ligation to a unique first binding motif comprising a fragment of SEQ ID NO: 1 comprising a reactive lysine at position 36 of SEQ ID NO: 1.

[0185] Item 43 i) the unique second binding motif comprises a fragment of SEQ ID NO: 3 comprising a reactive lysine at position 181 of SEQ ID NO: 3 and is capable of covalently binding, spontaneously or through enzymatically assisted protein ligation, to a unique first binding motif comprising a fragment of SEQ ID NO: 3 comprising a reactive asparagine at position 294 of SEQ ID NO: 3; or ii) the unique second binding motif comprises a fragment of SEQ ID NO: 3 comprising a reactive asparagine at position 294 of SEQ ID NO: 3 and is capable of covalently binding, spontaneously or with the aid of an enzyme, via protein ligation to a unique first binding motif comprising a fragment of SEQ ID NO: 3 comprising a reactive lysine at position 181 of SEQ ID NO: 3.

[0186] Item 44 i) the unique second binding motif comprises a fragment of SEQ ID NO: 10 comprising a reactive lysine at position 176 of SEQ ID NO: 10 and is capable of covalently binding, spontaneously or through enzymatically assisted protein ligation, to a unique first binding motif comprising a fragment of SEQ ID NO: 10 comprising a reactive asparagine at position 308 of SEQ ID NO: 10; or ii) the unique second binding motif comprises a fragment of SEQ ID NO: 10 comprising a reactive asparagine at position 308 of SEQ ID NO: 10 and is capable of covalently binding, spontaneously or with the aid of an enzyme, via protein ligation to a unique first binding motif comprising a fragment of SEQ ID NO: 10 comprising a reactive lysine at position 176 of SEQ ID NO: 10.

[0187] Item 45 i) the unique second binding motif comprises a fragment of SEQ ID NO: 11 comprising a reactive lysine at position 15 of SEQ ID NO: 11 and is capable of covalently binding, spontaneously or through enzymatically assisted protein ligation, to a unique first binding motif comprising a fragment of SEQ ID NO: 11 comprising a reactive aspartic acid at position 101 of SEQ ID NO: 11; or ii) the unique second binding motif comprises a fragment of SEQ ID NO: 11 comprising a reactive aspartic acid at position 101 of SEQ ID NO: 11 and is capable of covalently binding, spontaneously or with the aid of an enzyme, via protein ligation to a unique first binding motif comprising a fragment of SEQ ID NO: 11 comprising a reactive lysine at position 15 of SEQ ID NO: 11.

[0188] Item 46 i) the unique second binding motif comprises a fragment of SEQ ID NO: 13 comprising a reactive lysine at position 742 of SEQ ID NO: 13 and is capable of covalently binding, spontaneously or through enzymatically assisted protein ligation, to a unique first binding motif comprising a fragment of SEQ ID NO: 13 comprising a reactive asparagine at position 854 of SEQ ID NO: 13; or ii) the unique second binding motif comprises a fragment of SEQ ID NO: 13 comprising a reactive asparagine at position 854 of SEQ ID NO: 13 and is capable of covalently binding, spontaneously or with the aid of an enzyme, via protein ligation to a unique first binding motif comprising a fragment of SEQ ID NO: 13 comprising a reactive lysine at position 742 of SEQ ID NO: 13.

[0189] Item 47 i) the unique second binding motif comprises a fragment of SEQ ID NO: 15 comprising a reactive lysine at position 405 of SEQ ID NO: 15 and is capable of covalently binding, spontaneously or through enzymatically assisted protein ligation, to a unique first binding motif comprising a fragment of SEQ ID NO: 15 comprising a reactive aspartic acid at position 496 of SEQ ID NO: 15; or ii) the unique second binding motif comprises a fragment of SEQ ID NO: 15 comprising a reactive aspartic acid at position 496 of SEQ ID NO: 15 and is capable of covalently binding, spontaneously or with the aid of an enzyme, via protein ligation to a unique first binding motif comprising a fragment of SEQ ID NO: 15 comprising a reactive lysine at position 405 of SEQ ID NO: 15.

[0190] Item 48. The multiple Fc fragments according to any one of Items 31 to 33, wherein the unique second binding motif comprises an isopeptide comprising the amino acid sequence of SEQ ID NO: 21, 23, 25, or 27, or a protein having at least 70% sequence identity to the amino acid sequence set forth in any one of SEQ ID NOs: 21, 23, 25, or 27.

[0191] Item 49. The multiple Fc fragments of any one of Items 31 to 33, wherein the unique second binding motif comprises a sortase bridge domain and can be covalently linked, spontaneously or via enzymatically assisted protein ligation, to a unique first binding motif comprising a sortase recognition domain.

[0192] Item 50: The sortase recognition domain has the amino acid sequence: LPTGAA (SEQ ID NO: 18), LPTGGG (SEQ ID NO: 19), LPKTGG (SEQ ID NO: 20), LPETG (SEQ ID NO: 21), LPXTG (SEQ ID NO: 22), or LPXTG(X) n50. The multiple Fc fragments of item 49, comprising NPX1TX2 (SEQ ID NO: 24) (X1 is glutamine or lysine, X2 is asparagine or glycine, N is asparagine, P is proline, and T is threonine), wherein the sortase bridge domain comprises Gly, (Gly)2, (Gly)3, (Gly)4, or (Gly)x (x is an integer of 1 to 20).

[0193] Item 51. The multiple Fc fragments of any one of Items 31 to 33, wherein the unique second binding motif comprises a first split intein and can be covalently linked, spontaneously or enzymatically assisted, via protein ligation to a unique first binding motif comprising a second split intein.

[0194] Item 52. A method of producing a plurality of full-length antibodies, each full-length antibody comprising an antigen-binding fragment comprising a unique first binding motif at its C-terminus and an Fc fragment comprising a unique second binding motif at its N-terminus, the method comprising contacting a plurality of Fc fragments of any one of Items 31 to 51 with a plurality of antigen-binding fragments, each antigen-binding fragment comprising a unique first binding motif at its C-terminus, under conditions that allow the unique second binding motif to covalently bond to the unique first binding motif via protein ligation, either spontaneously or with the assistance of an enzyme.

[0195] Item 53 a) an antigen-binding fragment containing a first binding motif at the C-terminus and optionally a first detectable label; and b) an Fc fragment comprising a second binding motif at the N-terminus and optionally a second detectable label; and / or c) a nucleic acid construct comprising an antigen-binding fragment and / or an Fc fragment as defined in items 1 and / or 2. wherein the first binding motif and the second binding motif are capable of being covalently linked to each other via protein ligation.

[0196] Item 54. The kit of Item 53, wherein the first and second detectable labels are, independently of each other, a fluorophore, a fluorescent protein, or an enzyme.

[0197] Item 55 A method for producing a full-length antibody, comprising the steps of: a) an antigen-binding fragment comprising a first binding motif at the C-terminus, and b) an Fc fragment containing a second binding motif at the N-terminus mixing the The method, wherein the first binding motif and the second binding motif are covalently linked to each other via protein ligation upon said mixing.

[0198] Item 56. The method of Item 55, wherein the antigen-binding fragment and / or the Fc fragment comprises a detectable label.

[0199] Item 57. The method of item 56, wherein the detectable label is a fluorophore, a fluorescent protein, or an enzyme.

[0200] Item 58 a) the first binding motif comprises SEQ ID NO: 7 or a sequence having at least 70% identity to SEQ ID NO: 7, and the second binding motif comprises SEQ ID NO: 8, 9, 28, 33, or 44, or a sequence having at least 50% identity to SEQ ID NO: 8, 9, 28, 33, or 44; b) the first binding motif comprises SEQ ID NO: 34, or a sequence having at least 70% identity to SEQ ID NO: 34, and the second binding motif comprises SEQ ID NO: 8, 9, 28, or 44, or a sequence having at least 50% identity to SEQ ID NO: 8, 9, 28, or 44; c) the first binding motif comprises SEQ ID NO: 35 or a sequence having at least 70% identity to SEQ ID NO: 35, and the second binding motif comprises SEQ ID NO: 36 or a sequence having at least 70% identity to SEQ ID NO: 36; d) the first binding motif comprises SEQ ID NO: 37 or a sequence having at least 70% identity to SEQ ID NO: 37, and the second binding motif comprises SEQ ID NO: 38 or a sequence having at least 70% identity to SEQ ID NO: 38; e) the first binding motif comprises SEQ ID NO: 39 or a sequence having at least 70% identity to SEQ ID NO: 39, and the second binding motif comprises SEQ ID NO: 40 or a sequence having at least 50% identity to SEQ ID NO: 40; f) the first binding motif comprises SEQ ID NO: 41 or a sequence having at least 70% identity to SEQ ID NO: 41, and the second binding motif comprises SEQ ID NO: 42 or a sequence having at least 50% identity to SEQ ID NO: 42; g) The plurality of full-length antibodies according to item 7, wherein the first binding motif comprises SEQ ID NO: 43 or a sequence having at least 70% identity to SEQ ID NO: 43, and the second binding motif comprises SEQ ID NO: 8, 9, 28, or 44 or a sequence having at least 50% identity to SEQ ID NO: 8, 9, 28, or 44.

[0201] Item 59 a) one of the first binding motif and the second binding motif comprises SEQ ID NO: 7 or a sequence having at least 70% identity to SEQ ID NO: 7, and the other binding motif comprises SEQ ID NO: 8, 9, 28, 33, or 44, or a sequence having at least 50% identity to SEQ ID NO: 8, 9, 28, 33, or 44; b) one of the first binding motif and the second binding motif comprises SEQ ID NO: 34 or a sequence having at least 70% identity to SEQ ID NO: 34, and the other binding motif comprises SEQ ID NO: 8, 9, 28, or 44 or a sequence having at least 50% identity to SEQ ID NO: 8, 9, 28, or 44; c) one of the first binding motif and the second binding motif comprises SEQ ID NO: 35 or a sequence having at least 70% identity to SEQ ID NO: 35, and the other binding motif comprises SEQ ID NO: 36 or a sequence having at least 70% identity to SEQ ID NO: 36; d) one of the first binding motif and the second binding motif comprises SEQ ID NO: 37 or a sequence having at least 70% identity to SEQ ID NO: 37, and the other binding motif comprises SEQ ID NO: 38 or a sequence having at least 70% identity to SEQ ID NO: 38; e) one of the first binding motif and the second binding motif comprises SEQ ID NO: 39 or a sequence having at least 70% identity to SEQ ID NO: 39, and the other binding motif comprises SEQ ID NO: 40 or a sequence having at least 50% identity to SEQ ID NO: 40; f) one of the first binding motif and the second binding motif comprises SEQ ID NO: 41 or a sequence having at least 70% identity to SEQ ID NO: 41, and the other binding motif comprises SEQ ID NO: 42 or a sequence having at least 50% identity to SEQ ID NO: 42; g) one of the first binding motif and the second binding motif comprises SEQ ID NO: 43 or a sequence having at least 70% identity to SEQ ID NO: 43, and the other binding motif comprises SEQ ID NO: 8, 9, 28, or 44 or a sequence having at least 50% identity to SEQ ID NO: 8, 9, 28, or 44; A pairwise combination of the nucleic acid constructs according to item 29.

[0202] Item 60. The multiple Fc fragments of Item 34, wherein the second binding motif comprises SEQ ID NO: 44 or a sequence having at least 50% identity to SEQ ID NO: 44 and is capable of covalently binding, spontaneously or enzymatically assisted, via protein ligation to a first binding motif comprising SEQ ID NO: 34 or a sequence having at least 50% identity to SEQ ID NO: 34.

[0203] Example The following examples are illustrative only, not limiting. Those of ordinary skill in the art will readily recognize a variety of non-critical parameters that can be changed or modified to yield essentially the same or similar results.

[0204] Example 1 - Construction, expression, and purification of FcCatcher FcCatcher was constructed using SpyCatcher002 (SEQ ID NO: 34) or SpyCatcher003 (SEQ ID NO: 44) and the human IgG1 (hIgG1) Fc domain, genetically fused via a GSSGS linker to the last five amino acids from the hIgG1 hinge region, EPKSS. The last cysteine ​​in the hinge region was replaced with serine. The resulting products are also referred to as hFcCatcher2 (using SpyCatcher002) and hFcCatcher3 (using SpyCatcher003). A sequence encoding a signal peptide for secretion into the culture medium was cloned in front of the SpyCatcher-Fc sequence. These constructs were cloned into the pMAX vector. The resulting plasmids were transfected into the eukaryotic cell line HKB11 (Cho et al., 2002). Transfected cultures were incubated under standard conditions for 3–4 h and fed by adding Bio-Rad's standard feed medium at a 1:1 ratio. Six days after transfection, approximately 200 ml of culture volume containing FcCatchers was harvested by centrifugation to remove cell debris and subsequently sterile filtered. The clear culture supernatant was subjected to one-step affinity chromatography using an FPLC system. The eluted fraction was neutralized, collected, rebuffered in 1x PBS, pH 7.4, and sterile filtered. Concentration was determined by UV 280 nm measurement using a Nanodrop 2000 system, and the molar extinction coefficient was calculated from the construct sequence.

[0205] Similarly, mouse IgG2a-FcCatcher (mFcCatcher) and mouse IgG2a-FcCatcher3 (SpyCatcher003, based on mFcCatcher3) were cloned by fusion to the Fc domain of SpyCatcher (SEQ ID NO: 8) or SpyCatcher003 (SEQ ID NO: 44), as were rabbit IgG FcCatcher (rbFcCatcher) and rabbit IgG-FcCatcher3. These constructs were transfected, expressed, and purified as described above.

[0206] The fusion proteins were then analyzed by SDS-PAGE (Figure 1, lane 2 and Figure 3, lanes 2, 4, and 6). A Bio-Rad Criterion® Vertical Electrophoresis Cell was used with a 4-20% polyacrylamide gel (Bio-Rad Mini-PROTEAN TGX) and Bio-Rad Precision Plus Protein Standard molecular weight marker. The gel was stained with Coomassie® stain, and protein purity was determined by densitometry. The concentrations and purity of the FcCatchers are shown in Table 2 below.

[0207] [Table 3]

[0208] Example 2 - Fab-SpyTag construction, expression, and purification Human Fab fragments bearing a FLAG® tag, SpyTag or SpyTag002, and a His-tag were constructed by using a short linker (sequence EF) between the C-terminus of CH1 and the FLAG-tag, followed by a linker (sequence GGS) and SpyTag or SpyTag002, as well as a linker (sequence GAP) and a His-tag. The light and heavy chains were cloned into a bicistronic bacterial expression vector with a lac promoter. Both the light and heavy chain genes contained secretion signals for transport to the periplasm. Vectors carrying the Fab-FLAG-SpyTag-H or Fab-FLAG-SpyTag2-H constructs were transformed into a protease-deficient E. coli strain as described in co-pending U.S. patent application Ser. No. 62 / 819,748 (Periplasmic Fusion Proteins, filed March 18, 2019, Attorney Docket No. BRL.130P). The Fab fragment was expressed by culturing E. coli cells in 250 mL of 2xYT broth containing 0.1% glucose and chloramphenicol. After 1 hour of growth at 37°C, the culture was induced with 0.8 mM IPTG. Expression proceeded for approximately 16 hours at 30°C. The culture was centrifuged, and the cells were frozen at -80°C. The cells were lysed in BugBuster lysis buffer (Millipore-Sigma). The fusion protein was purified on a Ni-NTA affinity matrix and the buffer was exchanged into PBS.

[0209] Example 3 - Ligation of Fab-SpyTag and FcCatcher The FcCatcher fusion protein from Example 1 and the Fab-FLAG-SpyTag2-His fusion protein from Example 2 were ligated to each other by reacting 10 μM Fab-FLAG-SpyTag2-His with 4 μM of each FcCatcher3 in 1x PBS. A 25% molar excess of SpyTag2 over the FcCatcher3 sites (i.e., two sites per FcCatcher) was used to achieve complete reaction of all SpyTag2 sites. After different time points (30 seconds to 60 minutes), the reaction was stopped by adding SDS loading buffer. After heating at 95°C for 5 minutes, the samples were loaded onto a 4-20% polyacrylamide gel (Bio-Rad Mini-PROTEAN TGX). The Coomassie-stained gel image (Figure 1) shows that FcCatcher3 reacted with SpyTag2 at the Fab heavy chain. After 60 minutes, the FcCatcher3 band completely disappeared, indicating the completion of the ligation reaction. At the beginning of the reaction, two products were visible: FcCatcher3 bound to one Fab and FcCatcher3 bound to two Fabs. The bands of the single-ligated products diminished with increasing reaction time until almost only the double-ligated product was visible on the gel after 60 minutes.

[0210] Example 4 - Comparison of assay performance of Fab-SpyTag2-FcCatcher3 and IgG The similar performance of the Fab-FcCatcher ligation product and IgG was demonstrated by titration ELISA. Maxisorp ELISA plates were coated overnight with 1 μg / ml GFP in PBS. After washing with PBST and blocking with 5% BSA in PBST, a titration of anti-GFP Fab-SpyTag2 ligated to hIgG1-FcSpyCatcher3 in PBST was performed on the plate. For comparison, the same antibody (identical Fab sequence) produced in full-length human IgG1 format was titrated at equimolar concentrations. Detection was performed using HRP-conjugated anti-human Fc (Bio-Rad MCA647P) at a 1:500 dilution in HiSPEC assay diluent and QuantaBlu fluorogenic peroxidase substrate. The results demonstrate that both antibody constructs yield identical assay sensitivity (Figure 2).

[0211] Example 5 - Immunofluorescence multiplexed assay Immunofluorescence staining of U2OS cells with three human Fabs against three different targets (cyclophilin A, vimentin, and Ki-67) was performed. All three Fabs were produced in the Fab-FLAG-SpyTag-His format as described in Example 2 and ligated overnight to hIgG1-FcCatcher, mIgG2a-FcCatcher, and rbIgG-FcCatcher from Example 1, respectively, using a two-fold molar excess of Fab over FcCatcher. Complete ligation was confirmed by loading the reduced products onto an AnykD polyacrylamide gel (Bio-Rad Mini-PROTEAN TGX) along with Bio-Rad Precision Plus Protein Standard molecular weight markers. The FcCatchers reacted completely with the SpyTag on the Fab heavy chain, as shown in the Coomassie-stained gel image of the ligation products (Figure 3).

[0212] For cell staining, 3.75 x 10 4U2OS cells / well were seeded into 12-well chamber slides with removable silicone gaskets (Ibidi). The next day, cells were fixed with 4% paraformaldehyde in PBS, treated with ice-cold methanol, followed by 0.2% Triton X-100, and blocked with 5% BSA in PBST for 48 hours at 4°C. Three Fab-FcCatcher ligation products, one for each FcCatcher species, were mixed and added to the cells in blocking solution at 33 nM for anti-Ki-67 and anti-vimentin, and 167 nM for anti-cyclophilin A, and incubated at room temperature for 3 hours. After washing with PBST, a blocking solution containing a mixture of three anti-IgG secondary antibodies (goat anti-hIgG Fc:Alexa Fluor 594 F(ab') secondary (Jackson ImmunoResearch), goat anti-mIgG (H+L):DyLight488 (Bio-Rad), and donkey anti-rbIgG (H+L):Alexa Fluor 647 (Jackson ImmunoResearch) and DAPI was added to the cells and incubated for 1 hour at room temperature. After washing, the cells were mounted in ProLong Gold antifade reagent (Thermo Fisher), cured overnight at room temperature, and stored at 4°C. All three possible combinations of three monoclonal antibodies and three species were tested. Cells were imaged with a confocal microscope (Zeiss LSM 880 with an EC Plan-Neofluar 40x / 1.30 immersion lens) and analyzed with Image J software. One set of images is shown in Figure 4.

[0213] Example 6 - Flow cytometry multiplexed assay Jurkat cells were stained with anti-CD3 and anti-CD45 antibodies. The antibodies were derived from mouse hybridomas and recombinantly expressed as Fabs with Flag, SpyTag002, and His tags as described in Example 2. Both antibodies were ligated to human and rabbit FcCatcher3. An excess of 25% Fab was used for Fab-FcCatcher ligation, and the incubation time was 1 hour.

[0214] For the assay, 3 × 10 cells were cultured in 20 μL of flow buffer (3% fetal bovine serum in PBS). 4 Jurkat cells were plated in a V-bottom 384-well plate. Fab-FcCatcher3 ligation products were added to the cells at a final concentration of 200 nM for anti-CD3 and 100 nM for anti-CD45 in a final volume of 60 μl. After 1 h of incubation, the cells were washed with flow buffer, and a mixture of Alexa Fluor 488-conjugated anti-human Fc secondary antibody (Jackson ImmunoResearch) and Alexa Fluor 647-conjugated anti-rabbit IgG (H+L) secondary antibody (Jackson ImmunoResearch) was added for 1 h at room temperature. The cells were washed and analyzed using a flow cytometer (IntelliCyt). Analysis of the data showed specific staining for CD3 and CD45 using both antibodies in parallel, but not using the two secondary antibodies alone (Figure 5).

[0215] All patents, patent applications and other published references cited herein are hereby incorporated by reference in their entirety.

Claims

1. 1. A method for determining the levels of multiple antigens in a sample, comprising: contacting the sample with a plurality of full length antibodies comprising different populations of full length antibodies and detecting or quantifying binding between each population of full length antibodies and a corresponding antigen of the population of antibodies to determine the presence of each antigen of the plurality of antigens in the sample or quantify the level of each antigen of the plurality of antigens in the sample; each population of full length antibodies has an antigen-binding fragment that specifically binds to a unique antigen and a unique Fc fragment that belongs to a unique species, isotype, or subclass; the antigen-binding fragment comprises a first binding motif at the C-terminus of the antigen-binding fragment and the native Fc fragment comprises a second binding motif at the N-terminus; the antigen-binding fragment and the unique Fc fragment are covalently linked to each other via a protein ligation between the first binding motif and the second binding motif; a) the first binding motif comprises a sequence having at least 90% sequence identity to SEQ ID NO: 7 and the second binding motif comprises a sequence having at least 90% sequence identity to SEQ ID NO: 8, 9, 28, 33, or 44; b) the first binding motif comprises a sequence having at least 90% sequence identity to SEQ ID NO: 34 and the second binding motif comprises a sequence having at least 90% sequence identity to SEQ ID NO: 8, 9, 28, or 44; or c) the first binding motif comprises a sequence having at least 90% sequence identity to SEQ ID NO: 43, and the second binding motif comprises a sequence having at least 90% sequence identity to SEQ ID NO: 8, 9, 28, or 44; method.

2. each population of full-length antibodies is bound to a unique label or a unique bead; The method of claim 1.

3. the first binding motif comprises SEQ ID NO: 34; the second binding motif comprises SEQ ID NO: 44; The method of claim 1.

4. Each population of full-length antibodies is detected or quantified by a secondary antibody specific for each unique Fc fragment. The method of claim 1.

5. the secondary antibodies are labeled with a unique detectable label that facilitates detection or quantification of each unique Fc fragment, i.e., the unique antigen bound by the full-length antibody; The method of claim 1 or claim 4.

6. a) the first binding motif comprises a sequence having at least 95% sequence identity to SEQ ID NO: 7 and the second binding motif comprises a sequence having at least 90% sequence identity to SEQ ID NO: 8, 9, 28, 33, or 44; b) the first binding motif comprises a sequence having at least 95% sequence identity to SEQ ID NO: 34 and the second binding motif comprises a sequence having at least 90% sequence identity to SEQ ID NO: 8, 9, 28, or 44; or c) the first binding motif comprises a sequence having at least 95% sequence identity to SEQ ID NO: 43, and the second binding motif comprises a sequence having at least 90% sequence identity to SEQ ID NO: 8, 9, 28, or 44; The method according to any one of claims 1 to 5.

7. a) the first binding motif comprises a sequence having at least 95% sequence identity to SEQ ID NO: 7 and the second binding motif comprises a sequence having at least 95% sequence identity to SEQ ID NO: 8, 9, 28, 33, or 44; b) the first binding motif comprises a sequence having at least 95% sequence identity to SEQ ID NO: 34 and the second binding motif comprises a sequence having at least 95% sequence identity to SEQ ID NO: 8, 9, 28, or 44; or c) the first binding motif comprises a sequence having at least 95% sequence identity to SEQ ID NO: 43, and the second binding motif comprises a sequence having at least 95% sequence identity to SEQ ID NO: 8, 9, 28, or 44; The method according to any one of claims 1 to 5.

8. a) the first binding motif comprises SEQ ID NO: 7 and the second binding motif comprises SEQ ID NO: 8, 9, 28, 33, or 44; b) the first binding motif comprises SEQ ID NO: 34 and the second binding motif comprises SEQ ID NO: 8, 9, 28, or 44; or c) the first binding motif comprises SEQ ID NO: 43 and the second binding motif comprises SEQ ID NO: 8, 9, 28, or 44; The method according to any one of claims 1 to 5.

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