Fusion protein construct

Abstract

This invention provides a cost-effective method for preparing high molecular weight next-generation antibody therapeutics, enabling the creation of diverse libraries of antibody-like drugs with altered antibody titers, geometric shapes, effector functions, and stoichiometry through the combination of a relatively small number of building blocks. [Solution] One aspect of the present disclosure provides a construct comprising a first fusion protein, a second fusion protein, and a linker, wherein the first fusion protein and the second fusion protein each comprise an affinity reagent and a reactive enzyme, and the linker comprises a first functional group specific to irreversibly inhibit the reactive enzyme of the first fusion protein at its first terminus and a second functional group specific to irreversibly inhibit the reactive enzyme of the second fusion protein at its second terminus.

Description

[Technical Field]

[0001] This disclosure relates, in general terms, to a fusion protein construct and a method for preparing and using the same. More specifically, this disclosure relates to a precisely defined fusion protein construct comprising at least a first fusion protein and a second fusion protein coupled by a linker prepared by modular synthesis. [Background technology]

[0002] Biological drugs, specifically monoclonal antibodies (mAbs) and mAbs incorporating antibody fragments, are of interest to the pharmaceutical industry due to their ability to specifically target disease-related proteins in circulation or on the cell surface. Next-generation antibody therapies, demonstrating improved therapeutic capabilities, have been created by using a format that conjugates effector molecules that increase antibody titer, cytotoxicity, and half-life, directing the immune system towards the target to be eliminated. However, these molecules are often difficult to produce in high yield and with the synthetic flexibility that allows for systematic optimization of desired functions.

[0003] For example, current antibody formats with extended functionality feature multiple domains that must be expressed simultaneously, and therefore must be cleverly optimized in terms of stability or function. Current chemical methods require the manipulation of reactive amino acids or the use of existing reactive amino acid side chains to conjugate the payload to the antibody scaffold. Often, the products of these reactions result in heterogeneous product populations. Such direct amino acid modifications may be unstable to the pharmacological properties of the parent molecule or may cause adverse effects (e.g., increased hydrophobicity, aggregation, immunogenicity, etc.), so considerable effort must be made to select the appropriate site for payload conjugation.

[0004] Furthermore, current antibody technologies do not enable scaffold products with systematically controlled geometric shapes (e.g., cyclic molecules, molecules with variable distances between domains), orientation, stoichiometry, and antibody titers beyond those made possible by classical polypeptide manipulation. This limitation of production technology imposes significant constraints on the therapeutic space that current formats can investigate. Current protein engineering methods are limited to the intrinsic repertoire of natural polypeptide folding / peptide linkers to obtain the desired construct. Therefore, it is not possible to prepare non-natural formats that may exhibit higher efficacy using conventional engineering methods. In addition, due to toxicity constraints or metabolic characteristics of the expression host, some effector molecules cannot be produced collectively and effectively in a single culture system. Moreover, as molecular complexity increases, the precise folding, assembly, and purification of larger molecules become more difficult, thereby reducing yields and increasing costs.

[0005] Furthermore, the industrial production of antibody molecules generally requires the expression of antibodies or antibody-like fragments in eukaryotic hosts that are less robust than those in prokaryotic hosts, resulting in lower product yields compared to prokaryotic hosts. Consequently, the cost of processing antibodies or antibody-like fragments that can be expressed in prokaryotic hosts is higher.

[0006] Therefore, there is a need in the art for a cost-effective method for preparing high molecular weight next-generation antibody therapeutics that simply allows for the creation of diverse libraries of antibody-like drugs with altered titers, geometric shapes, effector functions, and stoichiometry by combining a relatively small number of building blocks. [Overview of the project] [Means for solving the problem]

[0007] One aspect of the present disclosure provides a construct comprising a first fusion protein, a second fusion protein, and a linker, wherein the first fusion protein and the second fusion protein each comprise an affinity reagent and a reactive enzyme, and the linker comprises a first functional group specific to irreversibly inhibit the reactive enzyme of the first fusion protein at its first terminus and a second functional group specific to irreversibly inhibit the reactive enzyme of the second fusion protein at its second terminus.

[0008] Another aspect of the present disclosure provides a method comprising: (a) contacting a first fusion protein comprising an affinity reagent and a reactive enzyme with a linker having a functional group specific to irreversibly inhibit the first fusion protein reactive enzyme at its first terminus, thereby coupling the first fusion protein and the linker at its first terminus; and (b) contacting a second fusion protein comprising an affinity reagent and a reactive enzyme with a second terminus of a linker, wherein the second terminus of the linker has a functional group specific to irreversibly inhibit the second fusion protein reactive enzyme, thereby coupling the second fusion protein and the linker at its second terminus.

[0009] Another aspect of this disclosure provides a construct prepared by the methods of this disclosure.

[0010] Another aspect of the Disclosure provides a method comprising administering the Construct of the Disclosure to a patient who requires the administration of the Construct of the Disclosure. Other aspects of the Disclosure provide the use of the Construct of the Disclosure as a pharmaceutical and / or diagnostic agent.

[0011] Further aspects and advantages will become apparent to those skilled in the art from the consideration of the following detailed description in conjunction with the drawings. While the construct and the methods for manufacturing and using the same may be subject to various embodiments, the following description includes specific embodiments, with the understanding that this disclosure is illustrative and not intended to limit the invention to the specific embodiments described herein. In certain embodiments, for example, the following are provided: (Item 1) It is a construct, It comprises a first fusion protein, a second fusion protein, and a linker. The first fusion protein and the second fusion protein each comprise an affinity reagent and a reactive enzyme, A construct wherein the linker includes a first functional group at its first terminus that is specific to irreversibly inhibit the first fusion protein-reactive enzyme, and a second functional group at its second terminus that is specific to irreversibly inhibit the second fusion protein-reactive enzyme. (Item 2) The construct described in item 1, wherein the first fusion protein-reactive enzyme and the second fusion protein-reactive enzyme are different. (Item 3) The construct described in item 1, wherein the first fusion protein-reactive enzyme and the second fusion protein-reactive enzyme are the same. (Item 4) A construct according to any one of items 1 to 3, wherein the first fusion protein affinity reagent and the second fusion protein affinity reagent are different. (Item 5) A construct according to any one of items 1 to 3, wherein the first fusion protein affinity reagent and the second fusion protein affinity reagent are the same. (Item 6) A construct as described in any one of items 1 to 5, wherein each affinity reagent is independently selected from the group consisting of antibodies or their fragments, small molecules, monobodies, proteins, and combinations thereof. (Item 7) The construct according to item 6, wherein the affinity reagent is an antibody or a fragment thereof. (Item 8) The antibody or fragment thereof has a light chain variable domain (V L ), light chain constant domain (C L ), heavy chain variable domain (VH ), heavy chain constant domain (C H 1) A construct as described in item 7, selected from the group consisting of combinations thereof. (Item 9) The construct according to item 7 or 8, wherein the first fusion protein antibody or fragment thereof is a chimeric antibody, a human antibody, and a humanized antibody. (Item 10) The construct according to any one of items 7 to 9, wherein the antibody or fragment thereof comprises trastuzumab or a fragment thereof. (Item 11) The antibody or fragment thereof is adalimumab, alemtuzumab, alsitumomab, cetuximab, trastuzumab, imusilomab, capromab, infliximab, absiximab, rituximab, basiliximab, palivizumab, nofetumomab, omalizumab, daclizumab, ibritumomab, tiuxetan, muromomab, edrecolomab, gemtuzumab, ozogamicin, golimumab, certolizumab, eculizumab, uste A construct described in any one of items 7-10, selected from the group consisting of mumab, panitumumab, tositumomab, bevacizumab, laxibakumab, tocilizumab, brentuximab, ofatumumab, belimumab, ramucirumab, vedolizumab, obinutuzumab, pembrolizumab, ranibizumab, pentuzumab, denosumab, catumakisomab, golimumab, siltuximab, natalizumab, panitumumab, and denosumab. (Item 12) The construct according to item 6, wherein the affinity reagent is a small molecule. (Item 13) The construct described in item 12, wherein the small molecule is a drug. (Item 14) The aforementioned small molecules include aldresleukin, alendronate, alphaferon, alitretinoin, allopurinol, alloprim, aloxy, altretamine, aminoglutethimide, L-type asparaginase, amiphostin, amrubicin, amsacrin, anastrozole, anzumet, alanesp, algravin, arsenic trioxide, aromasin, 5-azacitidine, azathioprine, BCG or Tys BCG, bestatin, betamethasone acetate, betamethasone sodium phosphate, bexarotene, bleomycin sulfate, proxuridine, and bortezomi Bleomycin, Busulfan, Calcitonin, Campus, Capecitabine, Carboplatin, Carmustine, Casodex, Cephezone, Sermoloukin, Serubidin, Chlorambucil, Cisplatin, Collaspase, Cladribine, Clodronate, Cyclophosphamide, Cytarabine, Dacarbazine, Dactinomycin, Daunoxosome, Decadron, Decadron Phosphate, Delestrogen, Denileukin Difutitox, Depo-Medrol, Deslorerin, Dexarazoxane, Daunorubicin, Diethylstilbestrol, 2',2'- Difluorodeoxycytidine, Diflucan, Docetaxel, Doxifluridine, Doxorubicin, Dronabinol, DW-166HC, Eliguard, Elitech, Ellens, Emend, Epirubicin, Epoetin-Alpha, Epogen, Eptaplatin, Ergamisol, Estrasse, Estradiol, Estramustine Sodium Phosphate, Ethinylestradiol, Ethiol, Etidronic Acid, Etopophos, Etoposide, Fadrozol, Farston, Filgrastim, Finasteride, Froxuridine, Flucona Zol, fludarabine, fludarabine phosphate, 5-fluorodeoxyuridine, 5-fluorodeoxyuridine monophosphate, 5-fluorouracil (5-FU), fluoxymesterone, flutamide, hexamethylmelamine, formestan, fosteabine, hotemustine, fulvestrant, gammaguard, gemcitabine, gemtuzumab, gleevec, gliadel, goserelin, granisetron hydrochloride, histrelin, hicamtin, hydrocolton, erythro-hydroxosinyl adenine, hydroxyurea,Hydroxyprogesterone Caproate, Ibritumomab tiuxetan, Idarubicin, Ifosfamide, Interferon-Alpha, Interferon-Alpha-2, Interferon-Alpha-2α, Interferon-Alpha-2β, Interferon-Alpha-n1, Interferon-Alpha-n3, Interferon-Beta, Interferon-Gamma-1α, Interleukin-2, Intron A, Iressa, Irinotecan, Quitril, Lentinan sulfate, Letrozole, Leucovorin, Leuprolide, Leuprolide acetate, Levamisole, Levoformic acid (acid) Calcium salt, Levotroid, Levoxil, Lomustine, Ronidamin, Marinol, Mechloretamine, Mecobalamin, Medroxyprogesterone acetate, Megestrol acetate, Melphalan, Menest, 6-Mercaptopurine, Mesna, Methotrexate, Metobicus, Miltefosine, Minocycline, Mitomycin C, Mitotan, Mitoxantrone, Modrenal, Myoseto, Neda Platin, Neurasta, Newmega, Newpogen, Niltamide, Nolvadex, NSC-631570, OCT-43, Octreotide, Ondansetron Hydrochloride, Olapred, Oxaliplatin, Paclitaxel, Pediapred, Pegaspargase, Pegasys, Pentostatin, N-Phosphonoacetyl L-Aspartate (PALA), Picibanil, Pilocarpine Hydrochloride, Pirarubi Syn, Plicamycin, Porfimer sodium, Prednimustine, Prednisolone, Prednisone, Premarin, Procarbazine, Procrit, Larcitrexed, Levif, Rhenium-186 etidronate, Rituximab, Loferon A, Lomultide, Salagen, Sandostatin, Salglamostim, Semustine, Schizofran, Sobuzoxan, Solu-Medrol, Streptozocin, Strontium Um-89 chloride, Synthroid, Tamoxifen, Tamsulosin, Tasonelmin, Tastractone, Taxotel, Teseloquin, Temozolomide, Teniposide, Testosterone propionate, Testored, Thioguanine, Thiotepa, Thyrotropin, Childronic acid, Topotecan, Toremifene, Tositumomab, Tastuzumab, Theosulfan, Treinoin, Trexaol, Trimethylmelamine,Trimethrexate, Triptorelin acetate, Triptorelin pamoate, UFT, Uridine, Barrubicin, Vesnarinone, Vinblastine, Vincristine, Vindesine, Vinorelbine, Biruzinine, Zincard, Zinostatin-Stimalamel, Zofran, ABI-007, Acorbifen, Actimun, Afinitac, Aminopterin, Alzoxifen, Asoprisnil, Atamestan, Atrasentan, Avastin, BAY43-9006 (Sorafe Nib), CCI-779, CDC-501, Celebrex, Cetuximab, Cristinator, Cyproterone acetate, Decitabine, DN-101, Doxorubicin-MTC, dSLIM, Dutasteride, Edtecalin, Eflornithine, Exatecan, Fenretinide, Histamine dihydrochloride, Histreline hydrogel implant, Holmium-166 DOTMP, Ibandronate, Interferon-gamma, Interferon-PEG, Ixabepylone Keyhole Limpet Hemocyanin, L-651582, Lanreotide, Lasofoxifen, Libra, Ronafarnib, Miproxifen, Minodronic Acid, MS-209, Liposome MTP-PE, MX-6, Nafarelin, Nemorubicin, Neovastat, Noratexed, Oblimersen, Onco-TCS, Osidem, Paclitaxel Polyglutamimate, Pamidronate Disodium, PN-401, QS-21, Quazepam, R-1549, Raloxifen, Constructs as described in item 13, selected from the group consisting of lampirunas, 13-cis-retinic acid, sataraplatin, theocalcitol, T-138067, tarceva, taxoplexin, thymosin-alpha-1, thiazophrine, tipifarnib, tirapazamine, TLK-286, toremifene, transMID-107R, valspodar, vapreotide, batalanib, verteporfin, vinflunin, Z-100, zoledronic acid, and combinations thereof. (Item 15) The construct according to item 6, wherein the affinity reagent is a monobody. (Item 16) The construct described in item 6, wherein the affinity reagent is a protein. (Item 17) The construct according to item 16, wherein the protein comprises at least one non-natural amino acid. (Item 18) The construct according to item 16 or 17, wherein the protein is a protein in a phage. (Item 19) The construct according to item 16 or 17, wherein the protein is a therapeutic protein. (Item 20) The construct according to item 6, wherein the affinity reagent is a synthetic antibody domain. (Item 21) The affinity reagent is selected from the group consisting of the designed ankyrin repeat protein (DARPin), HEL4 Vh domain (Predator), the Z domain of Staphylococcus protein A (Affibody), the archaeal "7kDa DNA binding agent" protein family (Affitin), carbohydrate binding molecules (CBD domain), cystine notch miniprotein (knottin), fibronectin type III domain (monobody, Adnectin), γ-B crystallin (Affilin), cystatin (Affimer), triple helix coiled-coil domain (Alphabody), lipocalin domain (Anticalin), A domains of various membrane receptors (Avimer), Fyn's SH3 domain (Fynomers), Kunitz domain peptides, and combinations thereof, as described in any one of items 1 to 5. (Item 22) The linker may be polyoxazoline, polyacrylomorpholine, polyvinylpyrrolidone, polyphosphazene, polyethylene-com-maleic anhydride, polystyrene-com-maleic anhydride, poly(1-hydroxymethylethylene hydroxymethylformal) ("PHF"), polyhydroxyalkyl acrylate, 2-methacryloyloxy-2'-ethyltrimethylammonium phosphate ("MPC"), or a structure selected from the following: [ka] And, During the ceremony, m is between 0 and 10. n is between 1 and 100. Each p is independently 0, 1, 2, 3, or 4. q is 0, 1, or 2. r is either 1 or 2, E is NH or CHR 10 And, G is O, CH2, CHOH, CHNH2, CHCOOH, or CHSO3H. R 10 These are OH, NH2, or COOH. Each R 11 The construct is independently H, OH, NH2, or COOH, as described in any one of items 1 through 21. (Item 23) Each reactive enzyme is a cutinase, SnapTag, HaloTag, type I DNA topoisomerase relaxase domain, β-lactamase, glycosidase, matrix metalloproteinase, cytoplasmic protein tyrosine kinase domain, alkaline phosphatase, protein-tyrosine-phsophatase, 23S rRNA (adenine(2503)-C(2))-methyltransferase variant, glucosidase, N-6 adenine-specific DNA methyltransferase. Constructs described in any one of items 1 to 22, independently selected from the group consisting of lases, N(4)-cytosine-specific DNA methylase, DNA(cytosine-5-)-methyltransferase, variants of haloalkane dehalogenases, HNH endonucleases, nickel endonucleases, gelatinase B, gelatinase A, stromericin, fatty acid amide hydrolases, esterases, cytochrome P450, methionine aminopeptidases, and combinations thereof. (Item 24) The construct according to any one of items 1 to 23, wherein the first fusion protein-reactive enzyme comprises cutinase, and the first terminal functional group comprises p-nitrophenylphosphonate. (Item 25) The first fusion protein-reactive enzyme contains a SnapTag, and the first terminal functional group is O 6 - A construct containing benzyl guanine, as described in any one of items 1 through 23. (Item 26) The construct according to any one of items 1 to 23, wherein the first fusion protein-reactive enzyme comprises a HaloTag, and the first terminal functional group comprises an α-chloroalkane. (Item 27) The construct according to any one of items 1 to 23, wherein the first fusion protein-reactive enzyme comprises a beta-lactamase, and the functional group at the first terminus comprises clavulanic acid or a derivative thereof. (Item 28) The construct according to any one of items 1 to 23, wherein the first fusion protein-reactive enzyme comprises a glycosidase, and the first terminal functional group comprises an aglycone or a derivative thereof. (Item 29) The construct according to any one of items 1 to 23, wherein the first fusion protein-reactive enzyme comprises a matrix metalloproteinase, and the functional group at the first terminus comprises hydroxamic acid-benzophenone or a derivative thereof. (Item 30) The construct according to any one of items 1 to 23, wherein the first fusion protein-reactive enzyme comprises a relaxase domain of type I DNA topoisomerase, and the first terminal functional group comprises a congeneral oriT oligonucleotide sequence. (Item 31) The construct according to any one of items 1 to 23, wherein the first fusion protein-reactive enzyme comprises a cytoplasmic protein tyrosine kinase domain, and the first terminal functional group comprises a cysteine-reactive ATP-binding site inhibitor. (Item 32) The construct according to any one of items 1 to 23, wherein the first fusion protein-reactive enzyme comprises an alkaline phosphatase, and the functional group at the first terminus comprises a functional group selected from the group consisting of quinone methides, α-halophosphonic acids, precursors thereof, derivatives thereof, and combinations thereof. (Item 33) The construct according to any one of items 1 to 23, wherein the first fusion protein-reactive enzyme comprises a protein-tyrosine-phosphatase, and the functional group at the first terminus comprises a functional group selected from the group consisting of formylchromone, α-halophosphonic acid, precursors thereof, derivatives thereof, and combinations thereof. (Item 34) The construct according to any one of items 1 to 23, wherein the first fusion protein-reactive enzyme comprises a variant of 23S rRNA (adenine(2503)-C(2))-methyltransferase, and the first terminal functional group comprises a congener RNA sequence. (Item 35) The construct according to any one of items 1 to 23, wherein the first fusion protein-reactive enzyme comprises a glucosidase, and the first terminal functional group comprises an aglycone or a derivative thereof. (Item 36) The construct according to any one of items 1 to 23, wherein the first fusion protein-reactive enzyme comprises an N-6 adenine-specific DNA methylase, and the functional group at the first terminus comprises adenosine or a derivative thereof. (Item 37) The construct according to any one of items 1 to 23, wherein the first fusion protein-reactive enzyme comprises an N(4)-cytosine-specific DNA methylase, and the functional group at the first terminus comprises cytosine or a derivative thereof. (Item 38) The construct according to any one of items 1 to 23, wherein the first fusion protein-reactive enzyme comprises DNA(cytosine-5-)-methyltransferase, and the functional group at the first terminus comprises cytosine or a derivative thereof. (Item 39) The construct according to any one of items 1 to 23, wherein the first fusion protein-reactive enzyme comprises a variant of a haloalkane dehalogenase, and the first terminal functional group comprises a functional group selected from the group consisting of haloalkanes, haloaromatic compounds, or derivatives thereof. (Item 40) The construct according to any one of items 1 to 23, wherein the first fusion protein-reactive enzyme comprises an HNH endonuclease, and the first terminal functional group comprises a congeneral DNA nicking site. (Item 41) The construct according to any one of items 1 to 23, wherein the first fusion protein-reactive enzyme comprises a nickeling endonuclease, and the functional group at the first terminus comprises a congeneral DNA nickeling site. (Item 42) The construct according to any one of items 1 to 23, wherein the first fusion protein-reactive enzyme comprises gelatinase B, and the first terminal functional group comprises a functional group selected from the group consisting of thiirane, hydroxamic acid, derivatives thereof, and combinations thereof. (Item 43) The construct according to any one of items 1 to 23, wherein the first fusion protein-reactive enzyme comprises gelatinase A, and the first terminal functional group comprises a functional group selected from the group consisting of thiirane, hydroxamic acid, derivatives thereof, and combinations thereof. (Item 44) The construct according to any one of items 1 to 23, wherein the first fusion protein-reactive enzyme comprises stromelicin, and the first terminal functional group comprises a functional group selected from the group consisting of thiirane, hydroxamic acid, derivatives thereof, and combinations thereof. (Item 45) The construct according to any one of items 1 to 23, wherein the first fusion protein-reactive enzyme comprises a fatty acid amide hydrolase, and the first terminal functional group comprises an α-ketoxazole inhibitor or a derivative thereof. (Item 46) The construct according to any one of items 1 to 23, wherein the first fusion protein-reactive enzyme contains an esterase, and the functional group at the first end contains a functional group selected from the group consisting of phosphonates, carbamates, derivatives thereof, and combinations thereof. (Item 47) The construct according to any one of items 1 to 23, wherein the first fusion protein-reactive enzyme contains cytochrome P450, and the functional group at the first end contains a functional group selected from the group consisting of electrophilic steroids, aromatic alkynes, derivatives thereof, and combinations thereof. (Item 48) The construct according to any one of items 1 to 23, wherein the first fusion protein-reactive enzyme contains methionine aminopeptidase, and the functional group at the first end contains veloranib or a derivative thereof. (Item 49) The construct according to any one of items 1 to 48, wherein the second fusion protein-reactive enzyme contains cutinase, and the functional group at the second end contains p-nitrophenylphosphonate. (Item 50) The construct according to any one of items 1 to 48, wherein the second fusion protein-reactive enzyme contains SnapTag, and the functional group at the second end contains O 6 -benzylguanine. (Item 51) The construct according to any one of items 1 to 48, wherein the second fusion protein-reactive enzyme contains HaloTag, and the functional group at the second end contains an α-chloroalkane. (Item 52) The construct according to any one of items 1 to 48, wherein the second fusion protein-reactive enzyme contains beta-lactamase, and the functional group at the second end contains clavulanic acid or a derivative thereof. (Item 53) The construct according to any one of items 1 to 48, wherein the second fusion protein-reactive enzyme contains glycosidase, and the functional group at the second end contains an aglycone or a derivative thereof. (Item 54) The construct according to any one of items 1 to 48, wherein the second fusion protein-reactive enzyme comprises a matrix metalloproteinase, and the functional group at the second terminus comprises hydroxamic acid-benzophenone or a derivative thereof. (Item 55) The construct according to any one of items 1 to 48, wherein the second fusion protein-reactive enzyme comprises a relaxase domain of type I DNA topoisomerase, and the functional group at the second terminus comprises a homologous oriT oligonucleotide sequence. (Item 56) The construct according to any one of items 1 to 48, wherein the second fusion protein-reactive enzyme comprises a cytoplasmic protein tyrosine kinase domain, and the functional group at the second terminus comprises a cysteine-reactive ATP-binding site inhibitor. (Item 57) The construct according to any one of items 1 to 48, wherein the second fusion protein-reactive enzyme comprises an alkaline phosphatase, and the functional group at the second terminus comprises a functional group selected from the group consisting of quinone methides, α-halophosphonic acids, their precursors, their derivatives, and combinations thereof. (Item 58) The construct according to any one of items 1 to 48, wherein the second fusion protein-reactive enzyme comprises a protein-tyrosine-phosphatase, and the functional group at the second terminus comprises a functional group selected from the group consisting of formylchromone, α-halophosphonic acid, precursors thereof, derivatives thereof, and combinations thereof. (Item 59) The construct according to any one of items 1 to 48, wherein the second fusion protein-reactive enzyme comprises a variant of 23S rRNA (adenine(2503)-C(2))-methyltransferase, and the functional group at the second terminus comprises a congener RNA sequence. (Item 60) The construct according to any one of items 1 to 48, wherein the second fusion protein-reactive enzyme comprises a glucosidase, and the functional group at the second terminus comprises an aglycone or a derivative thereof. (Item 61) The construct according to any one of items 1 to 48, wherein the second fusion protein-reactive enzyme comprises an N-6 adenine-specific DNA methylase, and the functional group at the second terminus comprises adenosine or a derivative thereof. (Item 62) The construct according to any one of items 1 to 48, wherein the second fusion protein-reactive enzyme comprises an N(4)-cytosine-specific DNA methylase, and the functional group at the second terminus comprises cytosine or a derivative thereof. (Item 63) The construct according to any one of items 1 to 48, wherein the second fusion protein-reactive enzyme comprises DNA(cytosine-5-)-methyltransferase, and the functional group at the second terminus comprises cytosine or a derivative thereof. (Item 64) The construct according to any one of items 1 to 48, wherein the second fusion protein-reactive enzyme comprises a variant of a haloalkane dehalogenase, and the functional group at the second terminus comprises a functional group selected from the group consisting of haloalkanes, haloaromatic compounds, or derivatives thereof. (Item 65) The construct according to any one of items 1 to 48, wherein the second fusion protein-reactive enzyme comprises an HNH endonuclease, and the functional group at the second terminus comprises a congeneral DNA-nicking site. (Item 66) The construct according to any one of items 1 to 48, wherein the second fusion protein-reactive enzyme comprises a nickeling endonuclease, and the functional group at the second terminus comprises a congeneral DNA nickeling site. (Item 67) The construct according to any one of items 1 to 48, wherein the second fusion protein-reactive enzyme comprises gelatinase B, and the functional group at the second terminus comprises a functional group selected from the group consisting of thiirane, hydroxamic acid, derivatives thereof, and combinations thereof. (Item 68) The construct according to any one of items 1 to 48, wherein the second fusion protein-reactive enzyme comprises gelatinase A, and the functional group at the second terminus comprises a functional group selected from the group consisting of thiirane, hydroxamic acid, derivatives thereof, and combinations thereof. (Item 69) The construct according to any one of items 1 to 48, wherein the second fusion protein-reactive enzyme comprises stromelicin, and the functional group at the second terminus comprises a functional group selected from the group consisting of thiirane, hydroxamic acid, derivatives thereof, and combinations thereof. (Item 70) The construct according to any one of items 1 to 48, wherein the second fusion protein-reactive enzyme comprises a fatty acid amide hydrolase, and the functional group at the second terminus comprises an α-ketoxazole inhibitor or a derivative thereof. (Item 71) The construct according to any one of items 1 to 48, wherein the second fusion protein-reactive enzyme comprises an esterase, and the functional group at the second terminus comprises a functional group selected from the group consisting of phosphonates, carbamates, derivatives thereof, and combinations thereof. (Item 72) The construct according to any one of items 1 to 48, wherein the second fusion protein-reactive enzyme comprises cytochrome P450, and the functional group at the second terminus comprises a functional group selected from the group consisting of electrophiles, aromatic alkynes, derivatives thereof, and combinations thereof. (Item 73) The construct according to any one of items 1 to 48, wherein the second fusion protein-reactive enzyme comprises methionine aminopeptidase, and the functional group at the second terminus comprises veroranib or a derivative thereof. (Item 74) It is a method, (a) Contacting a first fusion protein comprising an affinity reagent and a reactive enzyme with a linker having a functional group specific to irreversibly inhibit the first fusion protein reactive enzyme at its first terminus, thereby coupling the first fusion protein and the linker at the first terminus, (b) A method comprising contacting a second fusion protein comprising an affinity reagent and a reactive enzyme with the second end of the linker, wherein the second end of the linker comprises a functional group specific to irreversibly inhibit the second fusion protein reactive enzyme, thereby coupling the second fusion protein and the linker to the second end. (Item 75) The method according to item 74, wherein steps (a) and (b) are performed sequentially. (Item 76) The method according to item 74, wherein steps (a) and (b) are pre-formed simultaneously. (Item 77) The method according to any one of items 74 to 76, wherein the first fusion protein-reactive enzyme and the second fusion protein-reactive enzyme are different. (Item 78) The method according to any one of items 74 to 76, wherein the first fusion protein-reactive enzyme and the second fusion protein-reactive enzyme are the same. (Item 79) The method according to any one of items 74 to 78, wherein the first fusion protein affinity reagent and the second fusion protein affinity reagent are different. (Item 80) The method according to any one of items 74 to 78, wherein the first fusion protein affinity reagent and the second fusion protein affinity reagent are the same. (Item 81) The method according to any one of items 74-80, wherein each affinity reagent is independently selected from the group consisting of antibodies or their fragments, small molecules, monobodies, proteins, and combinations thereof. (Item 82) The method according to item 81, wherein the affinity reagent is an antibody or a fragment thereof. (Item 83) The antibody or fragment thereof has a light chain variable domain (V L ), light chain constant domain (C L ), heavy chain variable domain (V H ), heavy chain constant domain (C H 1) The method described in item 82, selected from the group consisting of combinations thereof. (Item 84) The method according to item 82 or 83, wherein the first fusion protein antibody or fragment thereof is a chimeric antibody, a human antibody, and a humanized antibody. (Item 85) The method according to any one of items 82 to 84, wherein the antibody or fragment thereof comprises trastuzumab or a fragment thereof. (Item 86) The antibody or fragment thereof is adalimumab, alemtuzumab, alsitumomab, cetuximab, trastuzumab, imusilomab, capromab, infliximab, absiximab, rituximab, basiliximab, palivizumab, nofetumomab, omalizumab, daclizumab, ibritumomab, tiuxetan, muromomab, edrecolomab, gemtuzumab, ozogamicin, golimumab, certolizumab, eculizumab, u The method according to any one of items 82 to 85, selected from the group consisting of tekimmubab, panitumumab, tositumomab, bevacizumab, laxibakumab, tocilizumab, brentuximab, ofatumumab, belimumab, ramucirumab, vedolizumab, obinutuzumab, pembrolizumab, ranibizumab, pentuzumab, denosumab, catumakisomab, golimumab, siltuximab, natalizumab, panitumumab, and denosumab. (Item 87) The method according to item 81, wherein the affinity reagent is a small molecule. (Item 88) The method according to item 87, wherein the small molecule is selected from the group consisting of drugs. (Project 89) The aforementioned small molecules include aldresleukin, alendronate, alphaferon, alitretinoin, allopurinol, alloprim, aloxy, altretamine, aminoglutethimide, L-type asparaginase, amiphostin, amrubicin, amsacrin, anastrozole, anzmet, alanesp, algravin, arsenic trioxide, aromasin, 5-azacitidine, azathioprine, BCG or Tys-BCG, bestatin, betamethasone acetate, betamethasone sodium phosphate, bexarotene, bleomycin sulfate, proxuridine, and voltene. Zomib, bleomycin, busulfan, calcitonin, campus, capecitabine, carboplatin, carmustine, casodex, cephezon, cermoleukin, serubidin, chlorambucil, cisplatin, colaspase, cladribine, clodronate, cyclophosphamide, cytarabine, dacarbazine, dactinomycin, daunoxosome, decadron, decadron phosphate, delestrogen, denileukin difutitox, depo-medrol, deslorerin, dexarazoxane, daunorubicin, diethylstilbestrol, 2',2' -Difluorodeoxycytidine, Diflucan, Docetaxel, Doxifluridine, Doxorubicin, Dronabinol, DW-166HC, Eliguard, Elitech, Ellens, Emend, Epirubicin, Epoetin-Alpha, Epogen, Eptaplatin, Ergamisol, Estrasse, Estradiol, Estramustine Sodium Phosphate, Ethinylestradiol, Ethiol, Etidronic Acid, Etopophos, Etoposide, Fadrozol, Farston, Filgrastim, Finasteride, Frigrastim, Floroxuridine, Fluco Nazol, fludarabine, fludarabine phosphate, 5-fluorodeoxyuridine, 5-fluorodeoxyuridine monophosphate, 5-fluorouracil (5-FU), fluoxymesterone, flutamide, hexamethylmelamine, formestan, fosteabine, hotemustine, fulvestrant, gammaguard, gemcitabine, gemtuzumab, gleevec, gliadel, goserelin, granisetron hydrochloride, histrelin, hicamtin, hydrocolton, erythro-hydroxynonyladenine, hydroxyurea,Hydroxyprogesterone Caproate, Ibritumomab tiuxetan, Idarubicin, Ifosfamide, Interferon-Alpha, Interferon-Alpha-2, Interferon-Alpha-2α, Interferon-Alpha-2β, Interferon-Alpha-n1, Interferon-Alpha-n3, Interferon-Beta, Interferon-Gamma-1α, Interleukin-2, Intron A, Iressa, Irinotecan, Quitril, Lentinan sulfate, Letrozole, Leucovorin, Leuprolide, Leuprolide acetate, Levamisole, Levoformic acid (acid) Calcium salt, Levotroid, Levoxil, Lomustine, Ronidamin, Marinol, Mechloretamine, Mecobalamin, Medroxyprogesterone acetate, Megestrol acetate, Melphalan, Menest, 6-Mercaptopurine, Mesna, Methotrexate, Metobicus, Miltefosine, Minocycline, Mitomycin C, Mitotan, Mitoxantrone, Modrenal, Myoseto, Neda Platin, Neurasta, Newmega, Newpogen, Niltamide, Nolvadex, NSC-631570, OCT-43, Octreotide, Ondansetron Hydrochloride, Olapred, Oxaliplatin, Paclitaxel, Pediapred, Pegaspargase, Pegasys, Pentostatin, N-Phosphonoacetyl L-Aspartate (PALA), Picibanil, Pilocarpine Hydrochloride, Pirarubi Syn, Plicamycin, Porfimer sodium, Prednimustine, Prednisolone, Prednisone, Premarin, Procarbazine, Procrit, Larcitrexed, Levif, Rhenium-186 etidronate, Rituximab, Loferon A, Lomultide, Salagen, Sandostatin, Salglamostim, Semustine, Schizofran, Sobuzoxan, Solu-Medrol, Streptozocin, Strontium Um-89 chloride, Synthroid, Tamoxifen, Tamsulosin, Tasonelmin, Tastractone, Taxotel, Teseloquin, Temozolomide, Teniposide, Testosterone propionate, Testored, Thioguanine, Thiotepa, Thyrotropin, Childronic acid, Topotecan, Toremifene, Tositumomab, Tastuzumab, Theosulfan, Treinoin, Trexaol, Trimethylmelamine,Trimethrexate, Triptorelin acetate, Triptorelin pamoate, UFI, Uridine, Barrubicin, Vesnarinone, Vinblastine, Vincristine, Vindesine, Vinorelbine, Biruzinine, Zincard, Zinostatin-Stimalamel, Zofran, ABI-007, Acorbifen, Actimun, Afinitac, Aminopterin, Alzoxifen, Asoprisnil, Atamestan, Atrasentan, Avastin, BAY43-9006 (Solaf Enib, CCI-779, CDC-501, Celebrex, Cetuximab, Cristinator, Cyproterone acetate, Decitabine, DN-101, Doxorubicin-MTC, dSLIM, Dutasteride, Edtecalin, Eflornithine, Exatecan, Fenretinide, Histamine dihydrochloride, Histreline hydrogel implant, Holmium-166 DOTMP, Ibandronate, Interferon-gamma, Interferon-PEG, Ixavepi Ron, Keyhole Limpet Hemocyanin, L-651582, Lanreotide, Lasofoxifen, Libra, Ronafarnib, Miproxifen, Minodronate, MS-209, Liposome MTP-PE, MX-6, Nafarelin, Nemorubicin, Neovastat, Noratexed, Oblimersen, Onco-TCS, Osidem, Paclitaxel Polyglutamimate, Pamidronate Disodium, PN-401, QS-21, Quazepam, R-1549, Raloxi The method according to item 88, selected from the group consisting of fen, lampirunas, 13-cis-retinic acid, satoraplatin, theocalcitol, T-138067, tarceva, taxoplexin, thymosin-alpha-1, thiazophrine, tipifarnib, tirapazamine, TLK-286, toremifene, transMID-107R, valspodar, vapreotide, batalanib, verteporfin, vinflunin, Z-100, zoledronic acid, and combinations thereof. (Item 90) The method according to item 81, wherein the affinity reagent is a monobody. (Item 91) The method according to item 81, wherein the affinity reagent is a protein. (Item 92) The method according to item 91, wherein the protein comprises at least one non-natural amino acid. (Item 93) The method according to item 91 or 92, wherein the protein is a protein in a phage. (Item 94) The method according to item 91 or 92, wherein the protein is a therapeutic agent. (Item 95) The method according to item 81, wherein the affinity reagent is a synthetic antibody domain. (Item 96) The method according to any one of items 74 to 80, wherein the affinity reagent is selected from the group consisting of the designed ankyrin repeat protein (DARPin), HEL4 Vh domain (Predator), the Z domain of Staphylococcus protein A (Affibody), the archaeal "7kDa DNA binding" protein family (Affitin), carbohydrate binding molecules (CBD domain), cystine notch miniprotein (knottin), fibronectin type III domain (monobody, Adnectin), γ-B crystallin (Affilin), cystatin (Affimer), triple helix coiled-coil domain (Alphabody), lipocalin domain (Anticalin), A domains of various membrane receptors (Avimer), SH3 domains of Fyn (Fynomers), Kunitz domain peptides, and combinations thereof. (Item 97) The linker may be polyoxazoline, polyacrylomorpholine, polyvinylpyrrolidone, polyphosphazene, polyethylene-com-maleic anhydride, polystyrene-com-maleic anhydride, poly(1-hydroxymethylethylene hydroxymethylformal) ("PHF"), polyhydroxyalkyl acrylate, 2-methacryloyloxy-2'-ethyltrimethylammonium phosphate ("MPC"), or a structure selected from the following: [ka] And, During the ceremony, m is between 0 and 10. n is between 1 and 100. Each p is independently 0, 1, 2, 3, or 4. q is 0, 1, or 2. r is either 1 or 2, E is NH or CHR 10 And, G is O, CH2, CHOH, CHNH2, CHCOOH, or CHSO3H. R 10 It is OH, NH2, or COOH. Each R 11 The method according to any one of items 74-96, wherein is independently H, OH, NH2, or COOH. (Item 98) Each reactive enzyme is a cutinase, SnapTag, HaloTag, type I DNA topoisomerase relaxase domain, beta-lactamase, glycosidase, matrix metalloproteinase, cytoplasmic protein tyrosine kinase domain, alkaline phosphatase, protein-tyrosine-phsophatase, 23S rRNA (adenine(2503)-C(2))-methyltransferase variant, glucosidase, N-6 adenine-specific DN The method described in any one of items 74 to 97, independently selected from the group consisting of A methylase, N(4)-cytosine-specific DNA methylase, DNA(cytosine-5-)-methyltransferase, variants of haloalkane dehalogenase, HNH endonuclease, nickel endonuclease, gelatinase B, gelatinase A, stromericin, fatty acid amide hydrolase, esterase, cytochrome P450, methionine aminopeptidase, and combinations thereof. (Item 99) The method according to any one of items 74 to 98, wherein the first fusion protein-reactive enzyme comprises cutinase, and the first terminal functional group comprises p-nitrophenylphosphonate. (Item 100) The first fusion protein-reactive enzyme contains a SnapTag, and the first terminal functional group is O 6-A method described in any one of items 74-98, including benzyl guanine. (Item 101) The method according to any one of items 74 to 98, wherein the first fusion protein-reactive enzyme comprises a HaloTag and the first terminal functional group comprises an α-chloroalkane. (Item 102) The method according to any one of items 74 to 98, wherein the first fusion protein-reactive enzyme comprises a beta-lactamase, and the first terminal functional group comprises clavulanic acid or a derivative thereof. (Item 103) The method according to any one of items 74 to 98, wherein the first fusion protein-reactive enzyme comprises a glycosidase, and the first terminal functional group comprises an aglycone or a derivative thereof. (Item 104) The method according to any one of items 74 to 98, wherein the first fusion protein-reactive enzyme comprises a matrix metalloproteinase, and the functional group at the first terminus comprises hydroxamic acid-benzophenone or a derivative thereof. (Item 105) The method according to any one of items 74 to 98, wherein the first fusion protein-reactive enzyme comprises a relaxase domain of type I DNA topoisomerase, and the first terminal functional group comprises a congeneral oriT oligonucleotide sequence. (Item 106) The method according to any one of items 74 to 98, wherein the first fusion protein-reactive enzyme comprises a cytoplasmic protein tyrosine kinase domain, and the functional group at the first terminus comprises a cysteine-reactive ATP-binding site inhibitor. (Item 107) The method according to any one of items 74 to 98, wherein the first fusion protein-reactive enzyme comprises an alkaline phosphatase, and the functional group at the first terminus comprises a functional group selected from the group consisting of quinone methides, α-halophosphonic acids, their precursors, and combinations thereof. (Item 108) The method according to any one of items 74 to 98, wherein the first fusion protein-reactive enzyme comprises a protein-tyrosine-phosphatase, and the functional group at the first terminus comprises formylchromone, α-halophosphonic acid, precursors thereof, derivatives thereof, and combinations thereof. (Item 109) The method according to any one of items 74 to 98, wherein the first fusion protein-reactive enzyme comprises a variant of 23S rRNA(adenine(2503)-C(2))-methyltransferase, and the first terminal functional group comprises a congener RNA sequence. (Item 110) The method according to any one of items 74 to 98, wherein the first fusion protein-reactive enzyme comprises a glucosidase, and the first terminal functional group comprises an aglycone or a derivative thereof. (Item 111) The method according to any one of items 74 to 98, wherein the first fusion protein-reactive enzyme comprises an N-6 adenine-specific DNA methylase, and the functional group at the first terminus comprises adenosine or a derivative thereof. (Item 112) The method according to any one of items 74 to 98, wherein the first fusion protein-reactive enzyme comprises an N(4)-cytosine-specific DNA methylase, and the functional group at the first terminus comprises cytosine or a derivative thereof. (Item 113) The method according to any one of items 74 to 98, wherein the first fusion protein-reactive enzyme comprises DNA(cytosine-5-)-methyltransferase, and the functional group at the first terminus comprises cytosine or a derivative thereof. (Item 114) The method according to any one of items 74 to 98, wherein the first fusion protein-reactive enzyme comprises a variant of a haloalkane dehalogenase, and the first terminal functional group comprises a functional group selected from the group consisting of haloalkanes, haloaromatic compounds, or derivatives thereof. (Item 115) The method according to any one of items 74 to 98, wherein the first fusion protein-reactive enzyme comprises an HNH endonuclease, and the functional group at the first terminus comprises a nicking site of congener DNA. (Item 116) The method according to any one of items 74 to 98, wherein the first fusion protein-reactive enzyme comprises a nickeling endonuclease, and the functional group at the first terminus comprises a congeneral DNA nickeling site. (Item 117) The method according to any one of items 74 to 98, wherein the first fusion protein-reactive enzyme comprises gelatinase B, and the functional group at the first terminus comprises a functional group selected from the group consisting of thiirane, hydroxamic acid, derivatives thereof, and combinations thereof. (Item 118) The method according to any one of items 74 to 98, wherein the first fusion protein-reactive enzyme comprises gelatinase A, and the first terminal functional group comprises a functional group selected from the group consisting of thiirane, hydroxamic acid, derivatives thereof, and combinations thereof. (Item 119) The method according to any one of items 74 to 98, wherein the first fusion protein-reactive enzyme comprises stromelicin, and the functional group at the first terminus comprises a functional group selected from the group consisting of thiirane, hydroxamic acid, derivatives thereof, and combinations thereof. (Item 120) The method according to any one of items 74 to 98, wherein the first fusion protein-reactive enzyme comprises a fatty acid amide hydrolase, and the functional group at the first terminus comprises an α-ketoxazole inhibitor or a derivative thereof. (Item 121) The method according to any one of items 74 to 98, wherein the first fusion protein-reactive enzyme comprises an esterase, and the functional group at the first terminus comprises a functional group selected from the group consisting of phosphonates, carbamates, derivatives thereof, and combinations thereof. (Item 122) The method according to any one of items 74 to 98, wherein the first fusion protein-reactive enzyme comprises cytochrome P450, and the functional group at the first terminus comprises a functional group selected from the group consisting of electrophiles, aromatic alkynes, derivatives thereof, and combinations thereof. (Item 123) The method according to any one of items 74 to 98, wherein the first fusion protein-reactive enzyme comprises methionine aminopeptidase, and the first terminal functional group comprises veroranib or a derivative thereof. (Item 124) The method according to any one of items 74 to 124, wherein the second fusion protein-reactive enzyme comprises cutinase, and the functional group at the second terminus comprises p-nitrophenylphosphonate. (Item 125) The second fusion protein-reactive enzyme contains a SnapTag, and the functional group at the second terminus is O 6 - A method described in any one of items 74 to 124, comprising benzyl guanine. (Item 126) The method according to any one of items 74 to 124, wherein the second fusion protein-reactive enzyme comprises a HaloTag, and the functional group at the second terminus comprises an α-chloroalkane. (Item 127) The method according to any one of items 74 to 124, wherein the second fusion protein-reactive enzyme comprises a beta-lactamase, and the functional group at the second terminus comprises clavulanic acid or a derivative thereof. (Item 128) The method according to any one of items 74 to 124, wherein the second fusion protein-reactive enzyme comprises a glycosidase, and the functional group at the second terminus comprises an aglycone or a derivative thereof. (Item 129) The method according to any one of items 74 to 124, wherein the second fusion protein-reactive enzyme comprises a matrix metalloproteinase, and the functional group at the second terminus comprises hydroxamic acid-benzophenone or a derivative thereof. (Item 130) The method according to any one of items 74 to 124, wherein the second fusion protein-reactive enzyme comprises a relaxase domain of type I DNA topoisomerase, and the functional group at the second terminus comprises a congeneral oriT oligonucleotide sequence. (Item 131) The method according to any one of items 74 to 124, wherein the second fusion protein-reactive enzyme comprises a cytoplasmic protein tyrosine kinase domain, and the functional group at the second terminus comprises a cysteine-reactive ATP-binding site inhibitor. (Item 132) The method according to any one of items 74 to 124, wherein the second fusion protein-reactive enzyme comprises an alkaline phosphatase, and the functional group at the second terminus comprises a functional group selected from the group consisting of quinone methides, α-halophosphonic acids, their precursors, and combinations thereof. (Item 133) The method according to any one of items 74 to 124, wherein the second fusion protein-reactive enzyme comprises a protein-tyrosine-phosphatase, and the functional group at the second terminus comprises formylchromone, α-halophosphonic acid, precursors thereof, derivatives thereof, and combinations thereof. (Item 134) The method according to any one of items 74 to 124, wherein the second fusion protein-reactive enzyme comprises a variant of 23S rRNA (adenine(2503)-C(2))-methyltransferase, and the functional group at the second terminus comprises a congener RNA sequence. (Item 135) The method according to any one of items 74 to 124, wherein the second fusion protein-reactive enzyme comprises a glucosidase, and the functional group at the second terminus comprises an aglycone or a derivative thereof. (Item 136) The method according to any one of items 74 to 124, wherein the second fusion protein-reactive enzyme comprises an N-6 adenine-specific DNA methylase, and the functional group at the second terminus comprises adenosine or a derivative thereof. (Item 137) The method according to any one of items 74 to 124, wherein the second fusion protein-reactive enzyme comprises an N(4)-cytosine-specific DNA methylase, and the functional group at the second terminus comprises cytosine or a derivative thereof. (Item 138) The method according to any one of items 74 to 124, wherein the second fusion protein-reactive enzyme comprises DNA(cytosine-5-)-methyltransferase, and the functional group at the second terminus comprises cytosine or a derivative thereof. (Item 139) The method according to any one of items 74 to 124, wherein the second fusion protein-reactive enzyme comprises a variant of a haloalkane dehalogenase, and the functional group at the second terminus comprises a functional group selected from the group consisting of haloalkanes, haloaromatic compounds, or derivatives thereof. (Item 140) The method according to any one of items 74 to 124, wherein the second fusion protein-reactive enzyme comprises an HNH endonuclease, and the functional group at the second terminus comprises a congeneral DNA nicking site. (Item 141) The method according to any one of items 74 to 124, wherein the second fusion protein-reactive enzyme comprises a nickeling endonuclease, and the functional group at the second terminus comprises a congeneral DNA nickeling site. (Item 142) The method according to any one of items 74 to 124, wherein the second fusion protein-reactive enzyme comprises gelatinase B, and the functional group at the second terminus comprises a functional group selected from the group consisting of thiirane, hydroxamic acid, derivatives thereof, and combinations thereof. (Item 143) The method according to any one of items 74 to 124, wherein the second fusion protein-reactive enzyme comprises gelatinase A, and the functional group at the second terminus comprises a functional group selected from the group consisting of thiirane, hydroxamic acid, derivatives thereof, and combinations thereof. (Item 144) The method according to any one of items 74 to 124, wherein the second fusion protein-reactive enzyme comprises stromelicin, and the functional group at the second terminus comprises a functional group selected from the group consisting of thiirane, hydroxamic acid, derivatives thereof, and combinations thereof. (Item 145) The method according to any one of items 74 to 124, wherein the second fusion protein-reactive enzyme comprises a fatty acid amide hydrolase, and the functional group at the second terminus comprises an α-ketoxazole inhibitor or a derivative thereof. (Item 146) The method according to any one of items 74 to 124, wherein the second fusion protein-reactive enzyme comprises an esterase, and the functional group at the second terminus comprises a functional group selected from the group consisting of phosphonates, carbamates, derivatives thereof, and combinations thereof. (Item 147) The method according to any one of items 74 to 124, wherein the second fusion protein-reactive enzyme comprises cytochrome P450, and the functional group at the second terminus comprises a functional group selected from the group consisting of electrophiles, aromatic alkynes, derivatives thereof, and combinations thereof. (Item 148) The method according to any one of items 74 to 124, wherein the second fusion protein-reactive enzyme comprises methionine aminopeptidase, and the functional group at the second terminus comprises veroranib or a derivative thereof. (Item 149) A construct prepared by any one of items 74-148. (Item 150) A method comprising administering a construct described in any one of items 1 to 73 or 149 to a patient requiring the said construct. (Item 151) Use of the said construct as a drug for patients requiring the construct described in any one of items 1-73 or 149. (Item 152) The patient has breast cancer, and at least one antibody or fragment of the construct comprises trastuzumab or a fragment thereof, as described or used in item 150 or 151. (Item 153) The patient has pulmonary anthrax, and at least one antibody or fragment of the construct comprises laxibacumab or a fragment thereof, as described or used in item 150 or 151. (Item 154) The method or use described in item 150 or 151, wherein the patient has rheumatoid arthritis or systemic juvenile idiopathic arthritis, and at least one antibody or fragment of the construct comprises tocilizumab or a fragment thereof. (Item 155) The patient has Hodgkin lymphoma or systemic anaplastic large cell lymphoma, and at least one antibody or fragment of the construct comprises brentuximab or a fragment thereof, as described or used in item 150 or 151. (Item 156) The patient suffers from a condition selected from the group consisting of chronic lymphocytic leukemia, follicular non-Hodgkin lymphoma, diffuse large B-cell lymphoma, rheumatoid arthritis, and relapsing flaccid multiple sclerosis, and at least one antibody or fragment of the construct comprises ofatumumab or a fragment thereof, the method or use described in item 150 or 151. (Item 157) The patient has systemic lupus erythematosus, and at least one antibody or fragment of the construct comprises belimumab or a fragment thereof, as described or used in item 150 or 151. (Item 158) The patient has adenocarcinoma of the stomach or gastroesophageal junction or metastatic non-small cell lung cancer, and at least one antibody or fragment of the construct comprises ramucirumab or a fragment thereof, as described or used in item 150 or 151. (Item 159) The method or use described in item 150 or 151, wherein the patient has ulcerative colitis or Crohn's disease, and at least one antibody or fragment of the construct comprises vedolizumab or a fragment thereof. (Item 160) The patient has chronic lymphocytic leukemia or follicular lymphoma, and at least one antibody or fragment of the construct comprises obinutuzumab or a fragment thereof, as described or used in item 150 or 151. (Item 161) The patient has melanoma or metastatic non-small cell lung cancer, and at least one antibody or fragment of the construct comprises pembrolizumab or a fragment thereof, as described or used in item 150 or 151. (Item 162) The method or use described in item 150 or 151, wherein the patient has macular degeneration and at least one antibody or fragment of the construct comprises ranibizumab or a fragment thereof. (Item 163) The patient has breast cancer, and at least one antibody or fragment of the construct comprises pertuzumab or a fragment thereof, as described or used in item 150 or 151. (Item 164) The patient suffers from a condition selected from the group consisting of osteoporosis, treatment-induced bone regression, bone metastases, and giant cell tumor of bone, and at least one antibody or fragment of the construct comprises denosumab or a fragment thereof, the method or use described in item 150 or 151. (Item 165) The patient is suffering from malignant ascites, and at least one antibody or fragment of the construct comprises catumakisomab or a fragment thereof, as described or used in item 150 or 151. (Item 166) The patient suffers from a condition selected from the group consisting of rheumatoid arthritis, psoriatic arthritis, ankylosing spondylitis, and ulcerative colitis, and at least one antibody or fragment of the construct comprises golimumab or a fragment thereof, as described or used in item 150 or 151. (Item 167) The patient has a condition selected from the group consisting of metastatic renal cell carcinoma, prostate cancer, ovarian cancer, non-Hodgkin lymphoma, multiple myeloma, and Castleman disease, and at least one antibody or fragment of the construct comprises siltuximab or a fragment thereof, as described or used in item 150 or 151. (Item 168) The patient suffers from multiple sclerosis or Crohn's disease, and at least one antibody or fragment of the construct comprises natalizumab or a fragment thereof, as described or used in item 150 or 151. (Item 169) The method or use described in item 150 or 151, wherein the patient has colorectal cancer and at least one antibody or fragment of the construct comprises panitumumab or a fragment thereof. (Item 170) The patient suffers from a condition selected from the group consisting of osteoporosis, treatment-induced bone regression, bone metastases, and giant cell tumor of bone, and at least one antibody or fragment of the construct comprises denosumab or a fragment thereof, as described or used in item 150 or 151. (Item 171) Use of any one of the constructs described in items 1-73 or 149 as a diagnostic agent. [Brief explanation of the drawing]

[0012] [Figure 1] This shows the trastuzumab F(ab)(TFab) fusion gene / protein construct. [Figure 2]The SDS-PAGE gels of purified TFab-cutinase and TFab-SnapTag fusions are shown. [Figure 3] The SDS-PAGE gel of the divalent construct is shown. [Figure 4] Cell viability assay data for trastuzumab and the fusion protein construct B-13 are shown. [Figure 5] This is a time-course plot of serum concentrations of the B-13 construct in mice. [Figure 6] The results of B-13 administration to mice with BT474 tumor xenografts are shown. A) Progression of tumor volume in animals treated with 2.5 and 5 mg / kg of B13 daily on a vehicle for 4 weeks, and with 2.5 mg / kg of doxorubicin (dox) twice weekly. After 28 days, there was a significant difference in tumor volume between the vehicle vs. doxorubicin group (p=.0002) and the vehicle vs. two B13 treatment groups (p=.0001). B) Measurement of total body weight of animals in the four treatment cohorts. There was a significant difference in total body weight between the vehicle and doxorubicin (p=.0116), but no significant difference between the vehicle and B13 treatment at 2.5 mg / kg (p=.2237) or 5 mg / kg (p=.4297). C) Organ weight of animals in the four treatment cohorts. There was no significant difference in organ weight between the groups (p=0.9493). The p-value was calculated using 2-way ANOVA in GraphPad 7.0. [Modes for carrying out the invention]

[0013] This specification provides constructs, methods for constructing constructs, and methods for using constructs. A construct comprises a first fusion protein, a second fusion protein, and a linker, wherein the first and second fusion proteins each comprise an affinity reagent and a reactive enzyme, and the linker comprises a first functional group specific to irreversibly inhibit the first fusion protein reactive enzyme at its first terminus and a second functional group specific to irreversibly inhibit the second fusion protein reactive enzyme at its second terminus. In some embodiments, the first fusion protein reactive enzyme and the second fusion protein reactive enzyme are different. In some embodiments, the first fusion protein reactive enzyme and the second fusion protein reactive enzyme are the same. In some embodiments, the first fusion protein affinity reagent and the second fusion protein affinity reagent are different. In some embodiments, the first fusion protein affinity reagent and the second fusion protein affinity reagent are the same. In some cases, in some embodiments, each affinity reagent is independently selected from the group consisting of antibodies or their fragments, small molecules, monobodies, proteins, and combinations thereof.

[0014] Constructing a fusion protein of affinity reagents and reactive enzymes The disclosed construct comprises a fusion protein of an affinity reagent and a reactive enzyme linked via a linker.

[0015] As used herein and unless otherwise specified, “affinity reagent” refers to a portion that exhibits affinity for a desired target, such as the ability to bind to a target epitope in a therapeutic system, and / or the ability to bind to or recognize an analyte in a biosensor system. Examples of affinity reagents include, but are not limited to, antibodies or their fragments, small molecules, monobodies, and proteins.

[0016] As used herein and unless otherwise specified, “reactive enzyme” refers to an enzyme containing an active site residue that can be coupled to a functional group that is specific to irreversibly inhibit the active site residue.

[0017] As used herein and unless otherwise specified, “protein” refers to natural polypeptides, polypeptides containing natural and / or non-natural amino acids, proteins in phages, therapeutic proteins, and antibody domains, whether synthetic or naturally derived.

[0018] As used herein, “polypeptide” refers to a polymer composed of amino acid residues. Polypeptides are understood in the art and include, but are not limited to, antibodies, enzymes, structural polypeptides, and hormones. The polypeptides of this disclosure may be natural or non-natural.

[0019] Natural polypeptides include, but are not limited to, biologically active polypeptides (including antibodies) that exist in nature or can be produced in naturally occurring forms by, for example, chemical synthesis or recombinant expression techniques. Natural polypeptides also include lipoproteins and post-translational modified proteins, such as, but not limited to, glycosylated proteins.

[0020] The antibodies intended for use in the methods and compositions disclosed herein include, but are not limited to, antibodies that recognize and associate with a target molecule either in vivo or in vitro.

[0021] The structural polypeptides intended by this disclosure include, but are not limited to, actin, tubulin, collagen, elastin, myosin, kinesin, and dynein.

[0022] The non-natural polypeptides contemplated in this disclosure include, but are not limited to, synthetic polypeptides, as well as fragments, analogs, and variants of natural or non-natural polypeptides as defined herein. Non-natural polypeptides also include proteins or proteinaceous materials having D-type amino acids, modified, derivatized, or non-natural amino acids as part of their structure in D or L configurations and / or peptide-mimicking units. The term "protein" typically refers to a large polypeptide. The term "peptide" typically refers to a short polypeptide.

[0023] Non-natural polypeptides are prepared, for example, using an automated polypeptide synthesizer, or alternatively, using recombinant expression techniques that utilize modified polynucleotides encoding the desired polypeptide.

[0024] As used herein, the term "fragment" of a polypeptide means any portion of a polypeptide or protein that is smaller than the full-length polypeptide or protein expression product.

[0025] As used herein, “analog” refers to any of two or more polypeptides that are substantially similar in structure to the whole molecule or any of its fragments, and that have the same biological activity, but may have varying degrees of activity. Analogs differ in the composition of their amino acid sequence based on one or more mutations, which involve the substitution, deletion, insertion, and / or addition of one or more amino acids to other amino acids. Substitutions may be conserved or non-conservative based on the physicochemical or functional relationship between the amino acid being substituted and the amino acid that replaces it.

[0026] As used herein, “mutant” refers to a polypeptide, protein, or analogue thereof that has been modified to include an additional chemical part that is not normally part of the molecule. Such parts can modulate the solubility, absorption, and / or biological half-life of a molecule, for example, but not limited to the following. Parts that can mediate such effects are disclosed in Remington's Pharmaceutical Sciences (1980). Procedures for coupling such parts to molecules are well known in the art.

[0027] Antibodies and their fragments or derivatives are intended, including but not limited to Fab' fragments, F(ab)2 fragments, Fv fragments, Fc fragments, one or more complementarity-determining region (CDR) fragments, individual heavy chains, individual light chains, dimeric heavy and light chains (in contrast to heterotetrameric heavy and light chains found in untreated antibodies), single-chain antibodies (scAb), humanized antibodies (and antibodies modified in the manner of humanized antibodies, but the resulting antibodies are more closely similar to antibodies of non-human species), chelated recombinant antibodies (CRAB), bispecific and multispecific antibodies, and other antibody derivatives or fragments known in the art.

[0028] In the embodiments described above, at least one antibody or fragment of the construct includes trastuzumab or its fragment, laxibakamab or its fragment, tocilizumab or its fragment, brentuximab or its fragment, ofatumumab or its fragment, belimumab or its fragment, ramucirumab or its fragment, vedolizumab or its fragment, obinutuzumab or its fragment, pembrolizumab or its fragment, ranibizumab or its fragment, pertuzumab or its fragment, denosumab or its fragment, catumakisomab or its fragment, golimumab or its fragment, siltuximab or its fragment, natalizumab or its fragment, panitumumab or its fragment, denosumab or its fragment, and combinations thereof.

[0029] In several embodiments, the linker is a structure selected from polyoxazoline, polyacrylomorpholine, polyvinylpyrrolidone, polyphosphazene, polyethylene-com-maleic anhydride, polystyrene-com-maleic anhydride, poly(1-hydroxymethylethylene hydroxymethylformal) ("PHF"), polyhydroxyalkyl acrylate, 2-methylacryloyloxy-2'-ethyltrimethylammonium phosphate ("MPC"), or the following: [ka] And, In the formula, m is between 0 and 10, n is between 1 and 100, each p is independently 0, 1, 2, 3, or 4, q is 0, 1, or 2, r is 1 or 2, and E is NH or CHR 10 G is O, CH2, CHOH, CHNH2, CHCOOH, or CHSO3H, and R 10 OH, NH2, or COOH, and each R 11 These are independently H, OH, NH2, or COOH.

[0030] Each reactive enzyme is independently selected from the group consisting of cutinase, SnapTag, HaloTag, relaxase domain of type I DNA topoisomerase, beta-lactamase, glycosidase, matrix metalloproteinase, cytoplasmic protein tyrosine kinase domain, alkaline phosphatase, protein-tyrosine phosphatase, variant of 23S rRNA (adenine(2503)-C(2))-methyltransferase, glucosidase, N-6 adenine-specific DNA methylase, N(4)-cytosine-specific DNA methylase, DNA(cytosine-5-)-methyltransferase, variant of haloalkane dehalogenase, HNH endonuclease, nickel endonuclease, gelatinase B, gelatinase A, stromericin, fatty acid amide hydrolase, esterase, cytochrome P450, methionine aminopeptidase, and combinations thereof. In several embodiments, each reactive enzyme is a cutinase, a haloalkane dehydrogenase HaloTag, O 6 -Alkylguanine DNA alkyltransferase variants, SnapTag, type I DNA topoisomerase relaxase domain, beta-lactamase, glycosidase, matrix metalloproteinase, and combinations thereof are independently selected from the group. In several embodiments, each reactive enzyme is cutinase, haloalkane dehydrogenase HaloTag, O 6 - Independently selected from the group consisting of alkylguanine DNA alkyltransferase variants, SnapTag, and combinations thereof.

[0031] In several embodiments, the first fusion protein-reactive enzyme and the second fusion protein-reactive enzyme are different. In several embodiments, the first fusion protein-reactive enzyme and the second fusion protein-reactive enzyme are the same. In several embodiments, the first fusion protein affinity reagent and the second fusion protein affinity reagent are different. In several embodiments, the first fusion protein affinity reagent and the second fusion protein affinity reagent are the same. In some cases, in several embodiments, each affinity reagent is independently selected from the group consisting of antibodies or their fragments, small molecules, monobodies, proteins, and combinations thereof.

[0032] In several embodiments, fusion proteins of affinity reagents and reactive enzymes are formed by conventional synthetic chemical techniques for conjugating the reactive enzyme to the affinity reagent. In some cases, if the affinity reagent comprises a protein or antibody, the fusion protein is produced by expressing polynucleotide sequences encoding the affinity reagent and the reactive enzyme. In some embodiments, the disclosure provides a vector comprising a polynucleotide ligated to a promoter. In further embodiments, the disclosure provides a host cell comprising the vector. In some embodiments, the host cell is an Escherichia coli cell. In further embodiments, the host cell is a mammalian cell. In relevant embodiments, the host cell is a Chinese hamster ovary (CHO) cell. In further embodiments, the host cell is a yeast cell. In further embodiments, a method for producing the fusion protein disclosed herein is provided, the method comprising the step of culturing the host cell of the disclosure under conditions suitable for inducing polypeptide expression. In relevant embodiments, the polypeptide is isolated.

[0033] Affinity reagent The constructs of this disclosure include fusion proteins containing affinity reagents that provide functionality to the fusion proteins. Thus, constructs comprising a first fusion protein and a second fusion protein having precisely defined structures and functions can be assembled. Examples of functionality conferred to fusion proteins via affinity reagents include, but are not limited to, enhanced targeting of disease-related proteins in circulation or on the cell surface, enhanced cytotoxicity, immunomodulation, drug delivery, use as a contrast agent, use as a diagnostic agent, or a combination thereof.

[0034] The first affinity reagent and the second affinity reagent may be the same or different. Each affinity reagent can be independently selected from the group consisting of antibodies or their fragments, small molecules, monobodies, proteins, and combinations thereof.

[0035] In several embodiments, the affinity reagent comprises an antibody or a fragment thereof. The antibody or fragment comprises a light chain variable domain (V L ), light chain constant domain (C L ), heavy chain variable domain (V H ), heavy chain constant domain (C H1) and combinations thereof can be selected from the group. The antibody or fragment may be a chimeric antibody, a human antibody, or a humanized antibody. Examples of antibodies or fragments include, but are not limited to, trastuzumab or fragment, laxibakamab or fragment, tocilizumab or fragment, brentuximab or fragment, ofatumumab or fragment, belimumab or fragment, ramucirumab or fragment, vedolizumab or fragment, obinutuzumab or fragment, pembrolizumab or fragment, ranibizumab or fragment, pertuzumab or fragment, denosumab or fragment, catumakisomab or fragment, golimumab or fragment, siltuximab or fragment, natalizumab or fragment, panitumumab or fragment, and denosumab or fragment.

[0036] In several embodiments, the affinity reagent includes small molecules. The small molecules may be drugs. Examples of small molecule drugs include aldesleukin, alendronate, alphaferon, alitretinoin, allopurinol, alloprim, aloxy, altretamine, aminoglutethimide, L-type asparaginase, amiphostin, amrubicin, amsacrin, anastrozole, anzmet, alanesp, algravin, arsenic trioxide, aromasin, 5-azacitidine, azathioprine, BCG or Tys BCG, bestatin, betamethasone acetate, betamethasone sodium phosphate, bexarotene, bleomycin sulfate, bloc Suridine, Bortezomib, Bleomycin, Busulfan, Calcitonin, Campus, Capecitabine, Carboplatin, Carmustine, Casodex, Cephezone, Sermoloukin, Serubidin, Chlorambucil, Cisplatin, Collaspase, Cladribine, Clodronate, Cyclophosphamide, Cytarabine, Dacarbazine, Dactinomycin, Daunoxosome, Decadron, Decadron Phosphate, Delestrogen, Denileukin Difutitex, Depo-Medrol, Deslorerin, Dexarazoxane, Daunorubicin, Diethyl Stilbestrol, 2',2'-difluorodeoxycytidine, Diflucan, Docetaxel, Doxifluridine, Doxorubicin, Dronabinol, DW-166HC, Eliguard, Elitech, Ellens, Emend, Epirubicin, Epoetin-Alpha, Epogen, Eptaplatin, Ergamisol, Estrasse, Estradiol, Estramustine Sodium Phosphate, Ethinylestradiol, Ethiol, Etidronic Acid, Etopophos, Etoposide, Fadrozol, Farston, Filgrastim, Finasteride Frigrastim, floxuridine, fluconazole, fludarabine, fludarabine phosphate, 5-fluorodeoxyuridine, 5-fluorodeoxyuridine monophosphate, 5-fluorouracil (5-FU), fluoxymesterone, flutamide, hexamethylmelamine, formestan, fosteabine, hotemustine, fulvestrant, gammaguard, gemcitabine, gemtuzumab, gleevec, gliadel, goserelin, granisetron, hydrochloride, histrelin, hicamtin, hydrocolton,Erythro-hydroxynonyladenine, hydroxyurea, hydroxyprogesterone caproate, ibritumomab tiuxetan, idarubicin, ifosfamide, interferon-alpha, interferon-alpha-2, interferon-alpha-2α, interferon-alpha-2β, interferon-alpha-n1, interferon-alpha-n3, interferon-beta, interferon-gamma-1α, interleukin-2, intron A, Iressa, irinotecan, citril, lentinan sulfate, letrozole, leucovorin, leuprolide, leuprolide acetate, levamisol, levofolic acid Calcium acid salt, Levotroid, Levoxil, Lomustine, Ronidamin, Marinol, Mechloretamine, Mecobalamin, Medroxyprogesterone acetate, Megestrol acetate, Melphalan, Menest, 6-Mercaptopurine, Mesna, Methotrexate, Metobicus, Miltefosine, Minocycline, Mitomycin C, Mitotan, Mitoxantrone, Modrenal, Myoseto, Nedaplatin, Neulasta, Newmega, Newpogen, Niltamide, Nolvadex, NSC-631570, OCT-43, Octreotide, Ondansetron hydrochloride, Olapred, Oxaliplatin, Paclitaxel, Pediapred, Pegaspargase, Pegasis, Pentostatin, N-phosphonoacetyl L-aspartate (PALA), Picibanil, Pillocarpine hydrochloride Chloride, pirarubicin, plicamycin, porfimer sodium, prednimustine, prednisolone, prednisone, premarin, procarbazine, procrit, larcitrexed, levif, rhenium-186 etidronate, rituximab, loferon A, romultide, salagen, sandostatin, salglamostim, semustine, schizofran, sobuzoxan, solu-medrol, s Treptozosin, Strontium-89 Chloride, Synthroid, Tamoxifen, Tamsulosin, Tasonelmin, Tastractone, Taxotene, Teseloykin, Temozolomide, Teniposide, Testosterone Propionate, Testored, Thioguanine, Thiotepa, Thyrotropin, Childronic Acid, Topotecan, Toremifene, Tositumomab, Tastuzumab, Theosulfan, Treinoin,Trexaol, Trimethylmelamine, Trimethrexate, Triptorelin acetate, Triptorelin pamoate, UFT, Uridine, Barrubicin, Vesnarinone, Vinblastine, Vincristine, Vindesine, Vinorelbine, Biruzinine, Zincard, Dinostatin-Stimalamel, Zofran, ABI-007, Acorbifen, Actimun, Afinitac, Aminopterin, Alzoxifen, Asoprisnil, Atamestan, Atrasentan, Avastin BAY43-9006 (Sorafenib), CCI-779, CDC-501, Celebrex, Cetuximab, Cristinator, Cyproterone acetate, Decitabine, DN-101, Doxorubicin-MTC, dSLIM, Dutasteride, Edtecalin, Eflornithine, Exatecan, Fenretinide, Histamine dihydrochloride, Histrelin hydrogel implant, Holmium-166 DOTMP, Ibandronate, Interferon-gamma, Interferon Lon-PEG, Ixabepylone, Keyhole Limpet Hemocyanin, L-651582, Lanreotide, Lasofoxifen, Libra, Ronafarnib, Miproxyfen, Minodronate, MS-209, Liposome MTP-PE, MX-6, Nafarelin, Nemorubicin, Neovastat, Noratexed, Oblimersen, Onco-TCS, Osidem, Paclitaxel Polyglutamimate, Pamidronate Disodium, PN-401, QS-21, Quazepam, R-1 Examples include, but are not limited to, 549, raloxifene, lampirunas, 13-cis-retinic acid, satoraplatin, theocalcitol, T-138067, tarceva, taxoplexin, thymosin-alpha-1, thiazophrine, tipifarnib, tirapazamin, TLK-286, toremifene, transMID-107R, valspodar, vapreotide, batalanib, verteporfin, vinflunin, Z-100, zoledronic acid, and combinations thereof.

[0037] In several embodiments, the affinity reagent comprises a protein. In several embodiments, the protein is selected from the group consisting of natural proteins, natural amino acids, unnatural amino acids, proteins in phages, therapeutic proteins, synthetic antibody domains, and combinations thereof. In several embodiments, the protein is selected from the group consisting of unnatural amino acids, proteins in phages, therapeutic proteins, synthetic antibody domains, and combinations thereof.

[0038] In several embodiments, affinity reagents are selected from the group consisting of designed ankyrin repeat proteins (DARPin), HEL4 Vh domains (Predator), the Z domain of Staphylococcus protein A (Affibody), the archaeal "7kDa DNA binding" protein family (Affitin), carbohydrate binding molecules (CBD domain), cystine notch miniproteins (knottin), fibronectin type III domains (monobody, Adnectin), γ-B crystallin (Affilin), cystatin (Affimer), triple helix coiled-coil domains (Alphabody), lipocalin domains (Anticalin), A domains of various membrane receptors (Avimer), SH3 domains of Fyn (Fynomers), Kunitz domain peptides, and combinations thereof.

[0039] Reactive enzymes The fusion proteins of this disclosure contain a reactive enzyme that can be coupled to a functional group specific to irreversibly inhibit the reactive enzyme present on the linker. Thus, a construct comprising a first fusion protein and a second fusion protein having precisely defined structures can be assembled using a modular approach that employs a synthetic linker that selectively couples to the active site residues of the fusion protein reactive enzyme.

[0040] The first fusion protein-reactive enzyme and the second fusion protein-reactive enzyme may be the same or different. Each reactive enzyme can be independently selected from the group consisting of cutinase, SnapTag, HaloTag, relaxase domain of type I DNA topoisomerase, beta-lactamase, glycosidase, matrix metalloproteinase, cytoplasmic protein tyrosine kinase domain, alkaline phosphatase, protein-tyrosine-phsophatase, variants of 23S rRNA (adenine(2503)-C(2))-methyltransferase, glucosidase, N-6 adenine-specific DNA methylase, N(4)-cytosine-specific DNA methylase, DNA(cytosine-5-)-methyltransferase, variants of haloalkane dehalogenase, HNH endonuclease, nickel endonuclease, gelatinase B, gelatinase A, stromericin, fatty acid amide hydrolase, esterase, cytochrome P450, methionine aminopeptidase, and combinations thereof.

[0041] In multiple embodiments, each reactive enzyme is independently selected from the group consisting of cutinase, HaloTag, SnapTag, the relaxase domain of type I DNA topoisomerase, beta-lactamase, glycosidase, matrix metalloproteinase, and combinations thereof.

[0042] In several embodiments, the reactive enzyme is the mobility class (MOB) relaxase domain of type I DNA topoisomerase. The MOB relaxase domain of type I DNA topoisomerase is an enzyme that covalently binds to a small, congeneral transfer origin (oriT) DNA sequence located within a mobile plasmid that also encodes a relaxase protein. Such an enzyme cleaves the oriT DNA sequence at specific nucleotide residues and covalently binds to the oriT DNA sequence at the cleavage site. The MOB relaxase domain of type I DNA topoisomerase can be characterized by a family, with each family member having similar structure and mechanism. An example of a known MOB relaxase domain of the type I DNA topoisomerase family is MOB F MOB H MOB Q MOB C MOB P , and MOB V Examples include: The OriT sequence for each member of the relaxase family is specific to the mobile plasmid from which each relaxase protein sequence originates. Known examples of relaxase families and their associated plasmids include MOB F Family: pF, pR100, pR388, pWWO, pMFLV02, pREB5, pREC1, and pNAC3; MOB H Family: pR27, pCAR1, pSXT, pIP1202, pMOL28, and pPNAP01; MOB C Family: pCloDF13, p23023, pCRY, pAD1, pSKU146.2, pLM7, and pSt0;MOB Q Family: pRSF1010, pPRO2, pTi, pAt, pGOX3, pWCFS103, pBM300, pCAUL01, pTB3, pKJ50, and pRF;MOB PFamily: pRP4, pEST4011, pBI1063, pR64, pET46, pACRY04, pRAS3, pVirD2pTi, pColE1, pRA3, pVT745, pCP13, pFMC, pRP4, pR64, pK, pRAY, pCH4, pET35, pNP40, pCF10, pVir, pBOT3502, pMD136, pCIZ2, pS194, pSK639, and pRJ9; and MOB V The family includes pMV158, pE194, pUB110, pBBR1, pTn5520, pTn4555, pIncU, and pIncX, where the subscript "p" represents a plasmid.

[0043] Linker The linker comprises at least a first and a second terminus. The linker further comprises a first functional group at the first terminus that is specific to irreversibly inhibit the fusion protein. The linker further comprises a second functional group at the second terminus that is specific to irreversibly inhibit the fusion protein.

[0044] In several embodiments, the linker is nitrogen-containing C 3~7 The linker further includes heterocyclic compounds, polyoxazolines, polyacrylomorpholines, polyvinylpyrrolidone, polyphosphazenes, polyethylene-com-maleic anhydride, polystyrene-com-maleic anhydride, poly(1-hydroxymethylethylene hydroxymethyl formal) ("PHF"), polyhydroxyalkyl acrylates (e.g., 2-hydroxyethyl methacrylate), 2-methiacryloyloxy-2'-ethyltrimethylammonium phosphate ("MPC"), or combinations thereof. For example, the linker may contain polyoxazolines, polyacrylomorpholines, polyvinylpyrrolidone, polyphosphazenes, polyethylene-com-maleic anhydride, polystyrene-com-maleic anhydride, PHF, polyhydroxyalkyl acrylates, or MPC.

[0045] In some embodiments, the linker is [ka] Includes a group selected from. During the ceremony, m is between 0 and 10. n is between 1 and 100. Each p is independently 0, 1, 2, 3, or 4. q is 0, 1, or 2, r is either 1 or 2, E is O, NH or CHR 10 And, G is O, CH2, CHOH, CHNH2, CHCOOH, or CHSO3H. R 10 is OH, NH2 or COOH, and Each R 11 These are independently H, OH, NH2, or COOH.

[0046] In some embodiments, the linker is [ka] Includes. In some embodiments, E is O. In various embodiments, E is NH. In some embodiments, E is CHR 10 For example, E can include CHOH, CHNH2, CHCOOH, or CHSO3H. In these embodiments, m can be 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. For example, m can be 1, 2, 3, 4, or 5. In some embodiments, m is 1, 2, or 3. In various embodiments, n can be 1-50, 1-40, 1-30, 1-20, 1-25, 1-20, 1-15, 1-10, or 1-5.

[0047] In some embodiments, the linker is [ka] This includes. In some cases, p is 1, 2, or 3 (e.g., 1 or 2). For example, the linker may contain aspartic acid, glutamic acid, or gamma-glutamic acid. In various embodiments, n can be 1-50, 1-40, 1-30, 1-20, 1-25, 1-20, 1-15, 1-10, or 1-5.

[0048] In some embodiments, the linker is [ka] This includes the following. In some cases, p is 0, 1, 2, or 3 (e.g., 1 or 2). In various embodiments, n can be 1-50, 1-40, 1-30, 1-20, 1-25, 1-20, 1-15, 1-10, or 1-5.

[0049] In some embodiments, the linker is [ka] This includes. In some embodiments, G is O. In some embodiments where G is O, each R 11 G can independently be H or OH. In some embodiments, G is CH2. In some embodiments where G is CH2, each R 11 R can be independently H or OH, provided that at least one R 11 The condition is that is OH. In multiple embodiments, each R 11 is OH. In various embodiments where G is CH2, each R 11 can be independently H or NH2, provided that at least one R 11 The condition is that is NH2. In various embodiments where G is CH2, each R 11 R can be independently H or COOH, provided that at least one R 11 The condition is that G is COOH. In some embodiments, G is CHOH. In some embodiments where G is CHOH, each R11 R can be independently H or OH. In some embodiments, each R 11 is OH. In some embodiments, G is CHNH2. In some embodiments where G is CHNH2, each R 11 G can be independently H or NH2. In some embodiments, G is CHCOOH. In some embodiments where G is CHCOOH, each R 11 R can be independently H or COOH, provided that at least one R 11 The condition is that is COOH. In these embodiments, n can be 1-50, 1-40, 1-30, 1-20, 1-25, 1-20, 1-15, 1-10, or 1-5.

[0050] In several embodiments, the linker is nitrogen-containing C 3~7 These include heterocyclic compounds (e.g., pyrrolidine, piperidine, piperazine), polyalkylenediamines (e.g., polyethylenediamine, polypropylenediamine, polybutylenediamine), negatively charged amino acids (e.g., aspartic acid, glutamic acid, gamma-glutamic acid), or sugars (e.g., monosaccharides, polysaccharides, inositol, or polysialic acid).

[0051] In several embodiments, the linker comprises a polyalkylene oxide. The polyalkylene oxide may be linear or branched. For example, the linker may include polyethylene glycol (e.g., PEG40, PEG100, PEG150, PEG200, PEG300, PEG2000), polypropylene glycol, or polybutylene glycol. The polyalkylene oxide may have a molecular weight of about 30 to about 5000 (e.g., about 30 to 4500, about 30 to 4000, about 30 to 3500, about 30 to 3000, about 30 to 2500, about 30 to 2000, about 30 to 1500, about 30 to 1000, about 46 to 1000, about 46 to 750, about 46 to 500, about 46 to 250, or about 46 to 100). Polyethylene oxide spacers, with and without reactive functional groups at one or both of their ends, are well known in the art and are commercially available, for example, through Quanta Biodesign, PierceNet, and SigmaAldrich.

[0052] Through the functional groups at the first and second terminals, the linker reacts with the fusion protein-reactive enzyme site-specifically, in high yield, and with rapid kinetics under mild conditions to provide at least a bivalent antibody construct. The functional groups at the linker terminals may be any functional groups that irreversibly inhibit the reactive enzyme.

[0053] Examples of functional groups include p-nitrophenylphosphonate, O 6Examples include benzylguanine, α-haloalkanes or their derivatives, haloaromatic compounds or their derivatives, beta-lactams or their derivatives such as clavulanic acid or its derivatives, aglycones or their derivatives, hydroxamic acid-benzophenone or its derivatives, congeneral oriT oligonucleotide sequences, cysteine-reactive ATP binding site inhibitors, quinone methides or their derivatives, α-halophosphonic acids or their precursors or derivatives, formylchromone or its derivatives, congeneral RNA sequences, adenosine or its derivatives, cytosine or its derivatives, congeneral DNA nicking sites, thiran or its derivatives, hydroxamic acid or its derivatives, α-ketoxazole inhibitors or their derivatives, electrophiles, phosphonates, carbamates, aromatic alkynes, veroranib or its derivatives, and combinations thereof.

[0054] In several embodiments, the functional group is p-nitrophenylphosphonate, O 6 -Selected from the group consisting of benzylguanine, α-haloalkanes or their derivatives, haloaromatic compounds or their derivatives, beta-lactams or their derivatives such as clavulanic acid or its derivatives, hydroxamic acid-benzophenone or its derivatives, congeneral oriT oligonucleotide sequences, and combinations thereof. In some embodiments, the functional group is p-nitrophenylphosphonate, O 6 - Selected from the group consisting of benzylguanine, α-haloalkanes or their derivatives, haloaromatic compounds or their derivatives, and combinations thereof.

[0055] In several embodiments, the functional group is a congeneral oriT oligonucleotide sequence. In an improved version of this embodiment, the congeneral oriT oligonucleotide sequence is selected from the group consisting of 5'-TTTGCGTAGTGTGTGGTGCTTT-3' (SEQ ID NO: 1), 5'-TTTGCGTGGGGTGTGGTGCTTT-3' (SEQ ID NO: 2), 5'-TTTGCGTAGGGTGTGGTGCTTT-3' (SEQ ID NO: 3), 5'-CGCGCACCGAAAGGTGCGTATTGTCTATAGCCCAGATTTAAGGA-3' (SEQ ID NO: 4), 5'-CCATTTCTCGAAGAGAAACCGGTAAATGCGCCCT-3' (SEQ ID NO: 5), 5'-CACACACTTTATGAATATAAAGTATAGTGTTATACTTTA-3' (SEQ ID NO: 6), 5'ACGTTTCTGAACGAAGTGAAGAAACGTCTAAGTGCGCCCT-3' (SEQ ID NO: 7), and combinations thereof.

[0056] In several embodiments, the fusion protein-reactive enzyme is the relaxase domain of type I DNA topoisomerase, and the functional group is a congenerate oriT oligonucleotide sequence. In several embodiments, the type I DNA topoisomerase relaxase domain (identified by a mobile plasmid for each relaxase protein sequence) / congenerate oriT oligonucleotide sequence pair is pR100 / 5'-TTTGCGTAGTGTGTGGTGCTTT-3' (SEQ ID NO: 1), pF / 5'-TTTGCGTGGGGTGTGGTGCTTT-3' (SEQ ID NO: 2), TraI P307The following are selected from the group consisting of / 5'-TTTGCGTAGGGTGTGGTGCTTT-3' (SEQ ID NO: 3), pR388 / 5'-CGCGCACCGAAAGGTGCGTATTGTCTATAGCCCAGATTTAAGGA-3' (SEQ ID NO: 4), pR1162 / 5'-CCATTTCTCGAAGAGAAACCGGTAAATGCGCCCT-3' (SEQ ID NO: 5), pMobMN199 / 5'-CACACACTTTATGAATATAAAGTATAGTGTTATACTTTA-3' (SEQ ID NO: 6), pSC101 / 5'ACGTTTCTGAACGAAGTGAAGAAACGTCTAAGTGCGCCCT-3' (SEQ ID NO: 7), and combinations thereof.

[0057] In several embodiments, the first fusion protein-reactive enzyme comprises cutinase, and the first terminal functional group comprises p-nitrophenylphosphonate.

[0058] In several embodiments, the first fusion protein-reactive enzyme comprises a SnapTag, and the first terminal functional group is O 6 -Contains benzylguanine.

[0059] In several embodiments, the first fusion protein-reactive enzyme comprises a HaloTag, and the first terminal functional group comprises an α-chloroalkane.

[0060] In several embodiments, the first fusion protein-reactive enzyme comprises a beta-lactamase, and the first terminal functional group comprises a beta-lactam or a derivative thereof.

[0061] In several embodiments, the first fusion protein-reactive enzyme comprises a glycosidase, and the first terminal functional group comprises an aglycone or a derivative thereof.

[0062] In several embodiments, the first fusion protein-reactive enzyme comprises a matrix metalloproteinase, and the first terminal functional group comprises hydroxamic acid-benzophenone or a derivative thereof.

[0063] In several embodiments, the first fusion protein-reactive enzyme comprises a relaxase domain of type I DNA topoisomerase, and the first terminal functional group comprises a congeneral oriT oligonucleotide sequence.

[0064] In several embodiments, the first fusion protein-reactive enzyme comprises a cytoplasmic protein tyrosine kinase domain, and the first terminal functional group comprises a cysteine-reactive ATP-binding site inhibitor.

[0065] In several embodiments, the first fusion protein-reactive enzyme comprises an alkaline phosphatase, the first terminal functional group comprising a functional group selected from the group consisting of quinone methides, α-halophosphonic acids, their precursors, their derivatives, and combinations thereof.

[0066] In several embodiments, the first fusion protein-reactive enzyme comprises a protein-tyrosine-phosphatase, wherein the first terminal functional group comprises a functional group selected from the group consisting of formilchromone, α-halophosphonic acid, their precursors, their derivatives, and combinations thereof.

[0067] In several embodiments, the first fusion protein-reactive enzyme comprises a variant of 23S rRNA(adenine(2503)-C(2))-methyltransferase, and the first terminal functional group comprises a congeneral RNA sequence.

[0068] In several embodiments, the first fusion protein-reactive enzyme comprises a glucosidase, and the first terminal functional group comprises an aglycone or a derivative thereof.

[0069] In several embodiments, the first fusion protein-reactive enzyme comprises an N-6 adenine-specific DNA methylase, and the first terminal functional group comprises adenosine or a derivative thereof.

[0070] In several embodiments, the first fusion protein-reactive enzyme comprises an N(4)-cytosine-specific DNA methylase, and the first terminal functional group comprises cytosine or a derivative thereof.

[0071] In several embodiments, the first fusion protein-reactive enzyme comprises DNA(cytosine-5-)-methyltransferase, and the first terminal functional group comprises cytosine or a derivative thereof.

[0072] In several embodiments, the first fusion protein-reactive enzyme comprises a variant of a haloalkane dehalogenase, the first terminal functional group comprising a functional group selected from the group consisting of haloalkanes, haloaromatic compounds, or derivatives thereof.

[0073] In several embodiments, the first fusion protein-reactive enzyme comprises an HNH endonuclease, and the first terminal functional group comprises a cognate DNA-nicking site.

[0074] In several embodiments, the first fusion protein-reactive enzyme comprises a nickeling endonuclease, the first terminal functional group comprising a cognate DNA nickeling site.

[0075] In several embodiments, the first fusion protein-reactive enzyme comprises gelatinase B, and the first terminal functional group comprises a functional group selected from the group consisting of thiirane, hydroxamic acid, derivatives thereof, and combinations thereof.

[0076] In several embodiments, the first fusion protein-reactive enzyme comprises gelatinase A, and the first terminal functional group comprises a functional group selected from the group consisting of thiirane, hydroxamic acid, derivatives thereof, and combinations thereof.

[0077] In several embodiments, the first fusion protein-reactive enzyme comprises stromelicin, and the first terminal functional group comprises a functional group selected from the group consisting of thiirane, hydroxamic acid, derivatives thereof, and combinations thereof.

[0078] In several embodiments, the first fusion protein-reactive enzyme comprises a fatty acid amide hydrolase, and the first terminal functional group comprises an α-ketoxazole inhibitor or a derivative thereof.

[0079] In several embodiments, the first fusion protein-reactive enzyme comprises an esterase, and the first terminal functional group comprises a functional group selected from the group consisting of phosphonates, carbamates, derivatives thereof, and combinations thereof.

[0080] In several embodiments, the first fusion protein-reactive enzyme comprises a cytochrome P450, and the first terminal functional group comprises a functional group selected from the group consisting of electrophiles, aromatic alkynes, derivatives thereof, and combinations thereof.

[0081] In several embodiments, the first fusion protein-reactive enzyme comprises methionine aminopeptidase, and the first terminal functional group comprises veroranib or a derivative thereof.

[0082] In several embodiments, the second fusion protein-reactive enzyme comprises cutinase, and the second terminal functional group comprises p-nitrophenylphosphonate.

[0083] In several embodiments, the second fusion protein-reactive enzyme includes a SnapTag, and the second terminal functional group is O 6 -Contains benzylguanine.

[0084] In several embodiments, the second fusion protein-reactive enzyme includes a HaloTag, and the second terminal functional group includes an α-chloroalkane.

[0085] In several embodiments, the second fusion protein-reactive enzyme comprises a beta-lactamase, and the second terminal functional group comprises a beta-lactam or a derivative thereof.

[0086] In several embodiments, the second fusion protein-reactive enzyme comprises a glycosidase, and the second terminal functional group comprises an aglycone or a derivative thereof.

[0087] In several embodiments, the second fusion protein-reactive enzyme comprises a matrix metalloproteinase, the second terminal functional group comprising hydroxamic acid-benzophenone or a derivative thereof.

[0088] In several embodiments, the second fusion protein-reactive enzyme comprises the relaxase domain of a type I DNA topoisomerase, and the second terminal functional group comprises a cognate oriT oligonucleotide sequence.

[0089] In several embodiments, the second fusion protein-reactive enzyme comprises a cytoplasmic protein tyrosine kinase domain, and the second terminal functional group comprises a cysteine-reactive ATP-binding site inhibitor.

[0090] In several embodiments, the second fusion protein-reactive enzyme comprises an alkaline phosphatase, and the second terminal functional group comprises a functional group selected from the group consisting of quinone methides, α-halophosphonic acids, their precursors, their derivatives, and combinations thereof.

[0091] In several embodiments, the second fusion protein-reactive enzyme comprises a protein-tyrosine-phosphatase, wherein the second terminal functional group comprises a functional group selected from the group consisting of formylchromone, α-halophosphonic acid, their precursors, their derivatives, and combinations thereof.

[0092] In several embodiments, the second fusion protein-reactive enzyme comprises a variant of 23S rRNA(adenine(2503)-C(2))-methyltransferase, and the second terminal functional group comprises a congener RNA sequence.

[0093] In several embodiments, the second fusion protein-reactive enzyme comprises a glucosidase, and the second terminal functional group comprises an aglycone or a derivative thereof.

[0094] In several embodiments, the second fusion protein-reactive enzyme comprises an N-6 adenine-specific DNA methylase, and the second terminal functional group comprises adenosine or a derivative thereof.

[0095] In several embodiments, the second fusion protein-reactive enzyme comprises an N(4)-cytosine-specific DNA methylase, and the functional group at the second terminus comprises cytosine or a derivative thereof.

[0096] In several embodiments, the second fusion protein-reactive enzyme comprises DNA(cytosine-5-)-methyltransferase, and the second terminal functional group comprises cytosine or a derivative thereof.

[0097] In several embodiments, the second fusion protein-reactive enzyme comprises a variant of a haloalkane dehalogenase, wherein the second terminal functional group comprises a functional group selected from the group consisting of haloalkanes, haloaromatic compounds, or derivatives thereof.

[0098] In several embodiments, the second fusion protein-reactive enzyme comprises an HNH endonuclease, and the second terminal functional group comprises a cognate DNA-nicking site.

[0099] In several embodiments, the second fusion protein-reactive enzyme comprises a nickeling endonuclease, the second terminal functional group comprising a cognate DNA nickeling site.

[0100] In several embodiments, the second fusion protein-reactive enzyme comprises gelatinase B, and the second terminal functional group comprises a functional group selected from the group consisting of thiirane, hydroxamic acid, derivatives thereof, and combinations thereof.

[0101] In multiple embodiments, the second fusion protein-reactive enzyme comprises gelatinase A, and the functional group at the second terminus comprises a functional group selected from the group consisting of thiirane, hydroxamic acid, derivatives thereof, and combinations thereof.

[0102] In multiple embodiments, the second fusion protein-reactive enzyme comprises stromelysin, and the functional group at the second terminus comprises a functional group selected from the group consisting of thiirane, hydroxamic acid, derivatives thereof, and combinations thereof.

[0103] In multiple embodiments, the second fusion protein-reactive enzyme comprises fatty acid amide hydrolase, and the functional group at the second terminus comprises an α-ketoxazole inhibitor or a derivative thereof.

[0104] In multiple embodiments, the second fusion protein-reactive enzyme comprises esterase, and the functional group at the second terminus comprises a functional group selected from the group consisting of phosphonate, carbamate, derivatives thereof, and combinations thereof.

[0105] In multiple embodiments, the second fusion protein-reactive enzyme comprises cytochrome P450, and the functional group at the second terminus comprises a functional group selected from the group consisting of electrophilic steroids, aromatic alkynes, derivatives thereof, and combinations thereof.

[0106] In multiple embodiments, the second fusion protein-reactive enzyme comprises methionine aminopeptidase, and the functional group at the second terminus comprises veloranib or a derivative thereof.

[0107] Method for preparing a construct Another aspect of the disclosure provides a method comprising: (a) contacting a first fusion protein comprising an affinity reagent and a reactive enzyme with a linker comprising a functional group specific for irreversibly inhibiting the first fusion protein reactive enzyme at the first terminus, thereby coupling the first fusion protein and the linker at the first terminus; and (b) contacting a second fusion protein comprising an affinity reagent and a reactive enzyme with the second terminus of the linker, wherein the second terminus of the linker comprises a functional group specific for irreversibly inhibiting the second fusion protein reactive enzyme, thereby coupling the second fusion protein and the linker at the second terminus.

[0108] As used herein and unless otherwise specified, "coupled," "coupling," or "couples" encompasses covalent bond formation, e.g., via covalent bond formation, a fusion protein irreversibly associates with a functional group at a linker terminus.

[0109] The methods disclosed herein use a modular format with small molecule linkers having functional groups at their termini that react site specifically with reactive enzymes to specifically bind a fusion protein comprising an affinity reagent and a reactive enzyme to the reactive enzyme using rapid kinetics under mild reaction conditions.

[0110] The methods disclosed herein offer one or more advantages, for example, providing a means by which a very diverse population of therapeutic constructs can be generated in a modular manner. In addition, the preparation of antibody formats with extended functionality is straightforward when using the constructs disclosed herein, as there is no need to directly manipulate / functionalize reactive amino acids on the antibody scaffold. Furthermore, the mild, rapid, and site-specific nature of the coupling of the fusion protein and linker allows for the detachment of diverse effector molecules from the antibody-like fragment in high yield and with precise stoichiometry. Moreover, molecules that cannot be expressed in homologous hosts with antibody fragments, such as toxins or glycosylated proteins, can be expressed in various organisms and bound to the construct in a modular manner. Modular synthesis enables the creation of molecules that are too large or too complex to prepare using standard protein engineering / expression methods, although such methods require the expression of polypeptides as one or more chains in culture.

[0111] A chemically bonded modular approach enables an efficient method for generating precisely defined protein constructs with variable stoichiometry, orientation, and geometric structure. These attributes can be systematically altered by encoding diversity in the linker and / or by altering the order in which fusion proteins are bound to the linker. Current protein engineering methods are limited to the intrinsic repertoire of native polypeptide folding / peptide linkers to achieve the desired construct. Therefore, it is not possible to prepare non-native formats that may exhibit greater efficacy using conventional engineering methods.

[0112] Finally, the expression of smaller antibody-like fragments enables expression in prokaryotic hosts in addition to the eukaryotic hosts typically used in the industrial production of antibody molecules. The robustness of prokaryotes is well known to enable lower-cost production platforms, in addition to product yields that are often five times higher than those of eukaryotic expression systems.

[0113] Another aspect of this disclosure provides a construct prepared by the methods of this disclosure.

[0114] In some embodiments, steps (a) and (b) are performed sequentially (i.e., stepwise). In some embodiments, steps (a) and (b) are performed simultaneously (i.e., a one-pot reaction). Each affinity reagent and reactive enzyme can be selected from any of the affinity reagents and reactive enzymes disclosed herein. The linker, which includes a first-terminal functional group and a second-terminal functional group, can be selected from any of the linkers and functional groups disclosed herein.

[0115] In several embodiments, the first fusion protein-reactive enzyme and the second fusion protein-reactive enzyme are the same, and the functional group specific to irreversibly inhibit the first fusion protein-reactive enzyme at the first end of the linker is the same as the functional group specific to irreversibly inhibit the second fusion protein-reactive enzyme at the second end of the linker, and steps (a) and (b) are performed simultaneously.

[0116] In several embodiments, the first fusion protein-reactive enzyme and the second fusion protein-reactive enzyme are the same, and the functional group specific to irreversibly inhibit the first fusion protein-reactive enzyme at the first end of the linker is different from the functional group specific to irreversibly inhibit the second fusion protein-reactive enzyme at the second end of the linker, and steps (a) and (b) are performed simultaneously.

[0117] In several embodiments, the first fusion protein-reactive enzyme and the second fusion protein-reactive enzyme are different, and the functional group specific to irreversibly inhibit the first fusion protein-reactive enzyme at the first end of the linker is the same as the functional group specific to irreversibly inhibit the second fusion protein-reactive enzyme at the second end of the linker, and steps (a) and (b) are performed sequentially.

[0118] In several embodiments, the first fusion protein-reactive enzyme and the second fusion protein-reactive enzyme are different, and the functional group specific to irreversibly inhibit the first fusion protein-reactive enzyme at the first end of the linker is different from the functional group specific to irreversibly inhibit the second fusion protein-reactive enzyme at the second end of the linker, so that steps (a) and (b) are performed simultaneously.

[0119] In several embodiments, the first fusion protein-reactive enzyme and the second fusion protein-reactive enzyme are different, and a functional group specific to irreversibly inhibit the first fusion protein-reactive enzyme at the first end of the linker is different from a functional group specific to irreversibly inhibit the second fusion protein-reactive enzyme at the second end of the linker, so that steps (a) and (b) are performed simultaneously.

[0120] In several embodiments, the first fusion protein-reactive enzyme and the second fusion protein-reactive enzyme are different, and the functional group specific to irreversibly inhibit the first fusion protein-reactive enzyme at the first end of the linker is different from the functional group specific to irreversibly inhibit the second fusion protein-reactive enzyme at the second end of the linker, and steps (a) and (b) are performed sequentially.

[0121] The methods disclosed herein enable the scalable preparation of molecules having molecular weights greater than approximately 100 kDa, 150 kDa, 200 kDa, 250 kDa, and / or 300 kDa, and / or provide one or more advantages, such as the creation of diverse libraries of antibody-like drugs having altered valence, geometric configuration, effector function, and stoichiometry, by combining a relatively small number of construction blocks.

[0122] Steps (a) and (b) can be carried out in some aqueous solution buffered to physiological pH, for example, a phosphate buffer solution (PBS) at pH 7.4. The concentrations of the first fusion protein, the second fusion protein, and the linker can generally be any concentrations. The concentrations are typically chosen so that the first fusion protein, the second fusion protein, and the linker dissolve completely in the selected solvent without forming a saturated solution. If the linker contains only functional groups specific to irreversibly inhibit the enzyme at its first and second terminals, the relative concentrations of the first fusion protein and the linker and the second fusion protein are at least about 1:1:1, providing one functional group per fusion protein that is specific to irreversibly inhibiting the enzyme linker terminal.

[0123] In embodiments, steps (a) and (b) are carried out at ambient temperature. The temperature should not be high enough to denature any proteins present in the fusion protein.

[0124] In several embodiments, the construct can be prepared on a solid support. In several embodiments, one or more components of step (a) and / or (b) can be provided on a solid support. Suitable solid supports are well known in the art.

[0125] How to use constructs Another aspect of the Disclosure provides a method comprising administering the construct to a patient requiring the construct of the Disclosure. Yet another aspect of the Disclosure provides the use of the construct of the Disclosure as a drug for a patient requiring the construct of the Disclosure. Another aspect of the Disclosure provides the use of the construct of the Disclosure as a diagnostic agent.

[0126] In multiple embodiments, the patient is selected from the group consisting of breast cancer, pulmonary anthrax, rheumatoid arthritis, systemic juvenile idiopathic arthritis, Hodgkin lymphoma, chronic lymphocytic leukemia, follicular non-Hodgkin lymphoma, diffuse large B-cell lymphoma, relapsing remitting multiple sclerosis, systemic lupus erythematosus, adenocarcinoma of the stomach or gastroesophageal junction, metastatic non-small cell lung cancer, ulcerative colitis, Crohn's disease, melanoma, macular degeneration, osteoporosis, treatment-induced bone loss, bone metastasis, giant cell tumor of bone, malignant ascites, psoriatic arthritis, ankylosing spondylitis, metastatic renal cell carcinoma, prostate cancer, ovarian cancer, multiple myeloma, Castleman disease, colorectal cancer, and combinations thereof.

[0127] As used herein and unless otherwise specified, "disease" and "disorder" are used interchangeably.

[0128] In multiple embodiments, at least one antibody or fragment thereof of the construct includes, but is not limited to, trastuzumab or a fragment thereof, lacutamab or a fragment thereof, tocilizumab or a fragment thereof, brentuximab or a fragment thereof, ofatumumab or a fragment thereof, belimumab or a fragment thereof, ramucirumab or a fragment thereof, vedolizumab or a fragment thereof, obinutuzumab or a fragment thereof, pembrolizumab or a fragment thereof, ranibizumab or a fragment thereof, pertuzumab or a fragment thereof, denosumab or a fragment thereof, catumaxomab or a fragment thereof, golimumab or a fragment thereof, siltuximab or a fragment thereof, natalizumab or a fragment thereof, panitumumab or a fragment thereof, denosumab or a fragment thereof, and combinations thereof.

[0129] In multiple embodiments, the patient has breast cancer and at least one antibody or fragment thereof of the construct includes trastuzumab or a fragment thereof.

[0130] In several embodiments, the patient has pulmonary anthrax, and at least one antibody or fragment of the construct comprises laxibakamab or a fragment thereof.

[0131] In several embodiments, the patient suffers from rheumatoid arthritis or systemic juvenile idiopathic arthritis, and at least one antibody or fragment of the construct comprises tocilizumab or a fragment thereof.

[0132] In several embodiments, the patient has Hodgkin lymphoma or systemic anaplastic large cell lymphoma, and at least one antibody or fragment of the construct comprises brentuximab or a fragment thereof.

[0133] In several embodiments, the patient suffers from a condition selected from the group consisting of chronic lymphocytic leukemia, follicular non-Hodgkin lymphoma, diffuse large B-cell lymphoma, rheumatoid arthritis, and relapsing flaccid multiple sclerosis, and at least one antibody or fragment of the construct comprises ofatumumab or a fragment thereof.

[0134] In several embodiments, the patient has systemic lupus erythematosus, and at least one antibody or fragment of the construct comprises belimumab or a fragment thereof.

[0135] In several embodiments, the patient has adenocarcinoma of the stomach or gastroesophageal junction or metastatic non-small cell lung cancer, and at least one antibody or fragment of the construct comprises ramucirumab or a fragment thereof.

[0136] In several embodiments, the patient has ulcerative colitis or Crohn's disease, and at least one antibody or fragment of the construct comprises vedolizumab or a fragment thereof.

[0137] In several embodiments, the patient has chronic lymphocytic leukemia or follicular lymphoma, and at least one antibody or fragment of the construct comprises obinutuzumab or a fragment thereof.

[0138] In several embodiments, the patient has melanoma or metastatic non-small cell lung cancer, and at least one antibody or fragment of the construct comprises pembrolizumab or a fragment thereof.

[0139] In several embodiments, the patient suffers from macular degeneration, and at least one antibody or fragment of the construct comprises ranibizumab or a fragment thereof.

[0140] In several embodiments, the patient has breast cancer, and at least one antibody or fragment of the construct comprises pertuzumab or a fragment thereof.

[0141] In several embodiments, the patient suffers from a condition selected from the group consisting of osteoporosis, treatment-induced bone regression, bone metastases, and giant cell tumor of bone, and at least one antibody or fragment of the construct comprises denosumab or a fragment thereof.

[0142] In several embodiments, the patient suffers from malignant ascites, and at least one antibody or fragment of the construct comprises catumakisomab or a fragment thereof.

[0143] In several embodiments, the patient suffers from a condition selected from the group consisting of rheumatoid arthritis, psoriatic arthritis, ankylosing spondylitis, and ulcerative colitis, and at least one antibody or fragment of the construct comprises golimumab or a fragment thereof.

[0144] In several embodiments, the patient suffers from a condition selected from the group consisting of metastatic renal cell carcinoma, prostate cancer, ovarian cancer, non-Hodgkin lymphoma, multiple myeloma, and Castleman disease, and at least one antibody or fragment of the construct comprises siltuximab or a fragment thereof.

[0145] In several embodiments, the patient has multiple sclerosis or Crohn's disease, and at least one antibody or fragment of the construct comprises natalizumab or a fragment thereof.

[0146] In several embodiments, the patient has colorectal cancer, and at least one antibody or fragment of the construct comprises panitumumab or a fragment thereof.

[0147] In several embodiments, the patient suffers from a condition selected from the group consisting of osteoporosis, treatment-induced bone regression, bone metastases, and giant cell tumor of bone, and at least one antibody or fragment of the construct comprises denosumab or a fragment thereof.

[0148] The constructs, methods, and uses relating to this disclosure can be better understood in terms of the following embodiments, which are intended merely to illustrate the constructs and are not intended in any way to limit their scope. [Examples]

[0149] Example 1: Construction of a gene / protein expression vector for the F(ab) domain derived from trastuzumab. The F(ab) domain (light chain variable V) derived from the monoclonal antibody trastuzumab (hereinafter referred to as "TFab(s)"). L - Light chain steady kappa chain C κ , and heavy chain variable domain V H - Heavy chain constant domain C HThe gene encoding (1) was synthesized by Genscript using codon optimization for Escherichia coli (E. coli). The following eight two-cistronic fusion proteins were assembled into a tetracycline-inducible expression vector based on pASK-IBA32 (IBA Biosciences) using restriction enzyme-free PCR: 1. "N-terminal Cutinase-V L Fusion" - 5'-RBS1-ATG-Cutinase-(EAAAK)2-V L C K - ** -RBS2-ATG-V H C H 1-LVPRGS-HHHHHH- ** -3’ (SEQ ID NO: 8 and 9) 2. "N-terminal SnapTag-V L Fusion" - 5'-RBS1-ATG-SnapTag-(EAAAK)2-V L C K - ** -RBS2-ATG-V H C H 1-LVPRGS-HHHHHH- ** -3’ (SEQ ID NO: 10 and 11) 3. "C-terminal Cutinase-C K Fusion" - 5'-RBS1-ATG-V L C K -(EAAAK)2-Cutinase- ** -RBS2-ATG-V H C H 1-LVPRGS-HHHHHH- ** -3’ (SEQ ID NO: 12 and 13) 4. "C-terminal SnapTag-C K Fusion" - 5'-RBS1-ATG-V L C K -(EAAAK)2-SnapTag- ** -RBS2-ATG-V H C H 1-LVPRGS-HHHHHH- ** -3’ (SEQ ID NO: 14 and 15) 5. "N-terminal Cutinase-V H Fusion" - 5'-RBS1-ATG-VL C K - ** -RBS2-ATG-cutinase-(EAAAK)2-V H C H 1-LVPRGS-HHHHHH- ** -3' (Sequences 16 and 17) 6. “N-terminal SnapTag-V H Fusion'-5'-RBS1-ATG-V L C K - ** -RBS2-ATG-SnapTag-(EAAAK)2-V H C H 1-LVPRGS-HHHHHH- ** -3' (Sequences 18 and 19) 7. C-terminal cutinase-C H 1 fusion'-5'-RBS1-ATG-V L C K - ** -RBS2-ATG-V H C H 1-(EAAAK)2-cutinase-LVPRGS-HHHHHH- ** -3' (Sequences 20 and 21) 8. “C-terminal SnapTag-C H 1 fusion'-5'-RBS1-ATG-V L C K - ** -RBS2-ATG-V H C H 1-(EAAAK)2-SnapTag-LVPRGS-HHHHHH- ** -3' (Sequences 22 and 23)

[0150] Figure 1 shows the trastuzumab F(ab)(TFab) fusion gene / protein construct. A) C-terminal heavy chain fusion. B) N-terminal heavy chain fusion. C) C-terminal light chain fusion. D) N-terminal light chain fusion.

[0151] Example 2: Protein Expression / Purification The vector encoding the eight TFab fusion proteins from Example 1 was used to transform Shuffle E. coli (New England Biolabs). An expression culture containing growth medium (500 mL × 2 × YT), carbenicillin (200 μg / mL), and spectinomycin (50 μg / mL) was inoculated with a 5 mL overnight culture medium from a cell stock with a suitable expression cassette. The culture was grown at 30°C with shaking at 250 rpm until an optical dispersion (OD) of approximately 0.8 was reached. At this point, anhydrotetracycline (aTc) was added to the culture at a final concentration of 200 ng / mL to induce protein expression. The culture was maintained at 24°C with shaking at 250 rpm for 14 hours during the expression phase.

[0152] After expression, cells were collected by centrifugation and lysed by chemical disruption using 35 mL of CelLytic B (Sigma Aldrich) containing DNase I and an EDTA-free protease inhibitor (Roche). The cell lysates were removed by centrifugation to remove fragments and added to a column containing cobalt IMAC resin (bed volume 3 mL). The lysates were incubated on beads at 4°C with agitation for 1 hour. After this period, the beads were thoroughly washed with phosphate-buffered salt solution (PBS), and the product was eluted with imidazole-containing buffer (20 mL). The protein eluent was then purified first on a protein L column (GE Healthcare) and then purified by size exclusion chromatography (SEC) on a Superdex 200 column (GE Healthcare) via high-performance protein liquid chromatography (FPLC). All FPLC column purifications were performed according to the manufacturer's suggested protocol. The fractions containing the pure protein product were pooled, concentrated using a centrifuge, and then ligated.

[0153] Figure 2 shows the sodium dodecyl sulfate polyacrylamide (SDS-PAGE) gel of purified TFab-cutinase and TFab-SnapTag fusions. Lanes 1 and 2 show the N-terminal V of cutinase (72 kDa) and SnapTag (69 kDa), respectively. HThe bands shown are close to the calculated molecular weight of the fusion. Lanes 3 and 4 show the C-terminal V H This shows the bands derived from the fusion. Lanes 5 and 6, and 7 and 8, respectively, represent the C-terminal C K and C H1 This shows bands originating from the fusion.

[0154] Accordingly, Examples 1 and 2 illustrate preparations of the fusion protein of this disclosure comprising an affinity reagent (TFab) and a reactive enzyme (cutinase and / or SnapTag).

[0155] Example 3: Preparation of a linker for joining a cutinase-TFab fusion protein containing a SnapTag-TFab fusion protein. [ka] The first terminal is p-nitrophenylphosphonate, and the second terminal is O 6 -A linker according to this disclosure, comprising benzyl guanine, ethylene glycol, and an amide unit, was prepared by the synthesis scheme shown below.

[0156] 1-Amino-N-(4-(((2-amino-9H-purine-6-yl)oxy)methyl)benzyl)-3,6,9,12,15,18-hexaoxaheneicosanoic acid-21-amide (1). Fmoc-21-amino-4,7,10,13,16,19-hexaoxaheneicosanoic acid (567 mg, 0.99 mmol) was dissolved in 5 mL of THF. To this solution, DIC (149 mg, 183 μL, 1.18 mmol), followed by NHS (136 mg, 1.18 mmol), was added, and the reaction mixture was stirred overnight at room temperature. The solvent was removed using a rotary evaporator, and the resulting yellow oily substance was resuspended in 5 mL of DMF. (4-aminomethyl)-O 6-Benzyl guanine (266 mg, 0.99 mmol) was added, and the reaction mixture was stirred at room temperature for 2 days. After this period, DMF was removed from the reaction mixture overnight under a stream of N2. The resulting orange oily substance was then treated with 20% piperidine in 5 mL of MeOH and stirred at room temperature for 1 hour. The solvent was removed using a rotary evaporator, and the resulting orange oily substance was resuspended in 10 mL of H2O. This solution was then centrifuged to pelletize the insoluble material, and the clarified supernatant was analyzed via reversed-phase C HPLC using a Waters Delta 400 HPLC. 18 The sample was applied to the column in five separate portions. Elution was performed using a linear gradient of 75% ACN in deionized ultrafiltered water (DIUF) + 0.01% TFA as the mobile phase, at a flow rate of 10 mL / min for 60 minutes. The fractions from 20–35 minutes were then analyzed by MALDI-MS. The pure fraction was pooled and freeze-dried to obtain an amber-colored oily substance (424 mg, 71%).

[0157] 1-(4-4-(((2-amino-9H-purine-6-yl)oxy)methyl)phenyl)-3,25-dioxo-6,9,12,15,18,21-hexaoxa-2,24-diazaoctacosan-28-acid(2). Compound 1 (50 mg, 83 μmol) was dissolved in 2.5 mL of MeOH, and succinic anhydride (8.3 mg, 83 μmol) was added to this solution. When the reaction was complete (monitored by MALDI-MS for approximately 30 minutes), the solvent was removed using a rotary evaporator. The resulting amber-colored oily substance was then purified to remove impurities by silica gel chromatography using 10:1 CH2Cl2:MeOH, and then eluted with MeOH to obtain a rubbery, amber-colored solid (42 mg, 72%).

[0158] For the preparation of intermediate (3), 17-(ethoxy(4-nitrophenoxy)phosphoryl)-3,6,9,12,15-pentaoxyheptadecane-1-aminium chloride, see Modica, JAet., Chembiochem. 2012 Nov 5;13(16):2331-4, which is incorporated herein by reference in its entirety.

[0159] Ethyl(4-nitrophenyl)(1-(4-(((2-amino-9H-purine-6-yl)oxy)methyl)phenyl)-3,25,28-trioxo-6,9,12,15,18,21,32,35,38,41,44-undecaoxa-2,2,24,29-triazahexatetracontan-46-yl)phosphonate (4). Compound 2 (39 mg, 55 μmol) was dissolved in 1 mL of DMF. To this solution, PyBOP (34 mg, 58 μmol), followed by N-methylmorpholine (5.6 mg, 6.1 μL, 55 μmol), was added, and the reaction mixture was stirred at room temperature for 30 minutes. After this period, compound 3 (29.3 mg, 55 μmol) dissolved in 250 μL of DMF was added to the mixture, followed by N-methylmorpholine (11.2 mg, 12.2 μL, 110 μmol), and the reaction mixture was stirred overnight (approximately 16 hours). After this period, the mixture was diluted to 5 mL using DIUF, and the mixture was centrifuged to remove insoluble materials. The clarified supernatant was then subjected to C14 HPLC via Waters Delta 4000. 18 The solution was injected into a semi-prep scale column and purified over 70 minutes at a flow rate of 10 mL / min using a linear gradient of 75% ACN aqueous solution + 0.1% TFA with DIUF + 0.1% TFA as the mobile phase. The fractions were collected at a rate of 1 / min. The fractions from 25–40 minutes were then analyzed by MALDI. The fractions containing the pure product were pooled, frozen at -80°C, and lyophilized to obtain a yellow oil. This oil was then subjected to a further HPLC purification using the procedure described above. The pure fraction from this trial was pooled, frozen, and lyophilized to obtain a pale yellow oil (11 mg, 17%).

[0160] Accordingly, Example 3 demonstrates the preparation of the linker of this disclosure comprising a first functional group specific to irreversibly inhibit a first fusion protein-reactive enzyme at the first terminal and a second functional group specific to irreversibly inhibit a second fusion protein-reactive enzyme at the second terminal.

[0161] Example 4: Assembly of the TFab construct Equal amounts of cutinase-TFab fusion (5 μM) and SnapTag-TFab fusion (5 μM) from Example 2, along with cutinase-SnapTag linker (5 μM) from Example 3, were mixed in PBS (pH 7.4) and reacted for 4 hours. After the reaction, the product was purified by size exclusion chromatography.

[0162] Figure 5 shows the SDS-PAGE gel of the prepared construct. Sixteen divalent analog constructs can be prepared from a pool of eight TFabs and one linker.

[0163] Accordingly, Example 4 demonstrates the preparation of a library of the construct of the present disclosure, comprising a first fusion protein, a second fusion protein, and a linker, wherein the first and second fusion proteins each comprise an affinity reagent and a reactive enzyme, and the linker comprises a first functional group at its first terminus that is specific to irreversibly inhibit the reactive enzyme of the first fusion protein, and a second functional group at its second terminus that is specific to irreversibly inhibit the reactive enzyme of the second fusion protein.

[0164] Example 5: Cell Growth Inhibition Assay Trastuzumab is used to treat HER2+ breast cancer. Two HER2(+++) cell lines (BT474, SKBR3), one HER2(++) cell line (MDA-MB-VII-135), and one control cell line AT-431 (HER2-) were cultured according to an established protocol. Trastuzumab or one of the divalent trastuzumab analogs prepared in Example 4 was administered multiple times at concentrations ranging from 4 pM to 4 μM for 96 hours. Survival rates after this period were measured using the Alamar blue assay (Life Technologies). Survival rates were calculated as a percentage of the untreated control.

[0165] From these experiments, one construct, named B-13, showed the most favorable cell growth inhibition and EC50 value against trastuzumab. Cell viability experiments were repeated with B-13 versus trastuzumab. Figure 4 shows cell viability assay data showing significant cell growth inhibition by trastuzumab and B-13 in various cell lines. A) Growth inhibition of BT474 cells. B) Growth inhibition of SKBR3 cells. C) Growth inhibition of MDA-MB-135-VII cells. D) Control experiment using HER2(-)AT-431 cells that did not show growth inhibition against trastuzumab or B-13.

[0166] Therefore, Example 5 demonstrates how a library of the constructs of this disclosure can be assayed for biological uses, such as cell growth inhibition. Furthermore, Example 5 demonstrates the cytotoxicity of this construct in an in vitro cancer cell viability assay. The method for assembling the fusion protein construct of Example 3 enables the preparation of multifunctional antibody-like therapeutic molecules in a one-pot manner.

[0167] Example 6: In vivo pharmacokinetics of the Di-TFab construct B-13 construct (2 mg / mL) was administered to SCID beige mice as an intravenous bolus, totaling 10 mg / kg. Blood samples (approximately 200 μL) were collected from the tail vein at various time points after administration. A total of 3 mice (n=3) were treated, and samples were collected at each time point. Serum B13 concentrations were measured using ELISA via standard curve comparison. As shown in Figure 5, these data were plotted and fitted to a two-compartment (two-exponential) pharmacokinetic model, and then analyzed at 0.22 hours. -1 The emission rate constant and half-life of 3.1 hours were obtained.

[0168] Accordingly, Example 6 demonstrates the in vivo use of the fusion protein construct of the present disclosure and pharmacokinetic data of the construct of the present disclosure.

[0169] Example 7 - Effect of B-13 administration to mice with BT474 tumor xenografts Eight-week-old female SCID beige mice were obtained from Charles River Laboratories and implanted with 0.025 mg of 17β-estradiol pellets released over 90 days (Innovative Research of America). Two days later, 2 × 10⁶ mice were implanted. 6 The BT474 cells were resuspended in 100 μl of PBS and orthotopically inoculated into a mammary gland fat pad with a 1:1 mixture of BT474 cells and Matrigel. The tumor was approximately 150 mm. 3 Once the tumor volume reached a certain level, 20 animals were randomly assigned to four treatment cohorts (n=5) with equal mean tumor volume. In each cohort, B13 was administered by intraperitoneal (IP) injection five times a week (Monday to Friday) for four weeks, totaling 20 doses of 5 mg kg. -1 In the second cohort, B13 was administered at a dose of 2.5 mg / kg via the same route and frequency. In the third cohort, doxorubicin (dox) was administered by IP injection twice weekly at 2.5 mg / kg for a total of eight doses over four weeks. The control cohort received a vehicle (PBS + 0.004% (w / w) polysorbate 20) five times weekly. Both B13 and dox solutions were prepared in the same vehicle. B13 samples contained endotoxin units (EU) / mL less than 1.0 as determined by the Limulus lysate (LAL) assay. Tumor dimensions were measured sequentially every two days, using the formula V = (L × W 2 The volume was calculated using ) / 2, where V = volume, L = length, and W = width. The results are shown in Figure 6.

[0170] B13 exhibits significant antitumor activity in the BT474 mouse xenograft model. Furthermore, B13 did not show any external signs of toxicity at the two doses used in this study. This is in contrast to dox, which showed significant weight loss in the treatment cohort.

[0171] The foregoing explanation is provided solely for clarity of understanding, and since modifications within the scope of the invention may be obvious to those skilled in the art, no unnecessary limitations should be inferred therefrom.

Claims

[Claim 1] The invention as shown in the drawings.

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