Fusion protein construct

Abstract

To provide a cost-effective method of preparing next generation antibody therapeutics with high molecular weights that allows creation of diverse libraries of antibody-like drugs with altered valences, geometries, effector functions and stoichiometry simply by combinations of a comparatively small number of building blocks.SOLUTION: One aspect of the disclosure provides constructs comprising a first fusion protein, a second fusion protein, and a linker. The first fusion protein and the second fusion protein each include an affinity reagent and a reactive enzyme. The linker includes a first functional group specific for irreversibly inhibiting the first fusion protein reactive enzyme at a first terminus, and a second functional group specific for irreversibly inhibiting the second fusion protein reactive enzyme at a second terminus.SELECTED DRAWING: None

Description

[Technical field]

[0001] The present disclosure relates generally to fusion protein constructs and methods for their preparation and use. More specifically, the present disclosure relates to precisely defined fusion protein constructs 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 in the pharmaceutical industry due to their ability to specifically target disease-related proteins in the circulation or on the cell surface. By using formats that increase antibody titers, conjugate effector molecules that increase cytotoxicity and half-life, and direct the immune system to targets for elimination, the next generation of antibody therapeutics has been created, one that shows improved therapeutic potential. However, these molecules are often difficult to produce with high yields and 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 simultaneously expressed and must be expertly optimized for stability or function. Current chemical methods require the manipulation of reactive amino acids or the use of existing reactive amino acid side chains to attach payloads to the antibody scaffold. In many cases, the products of these reactions result in a heterogeneous population of products. Such direct amino acid modifications may not be stable or may even cause adverse effects (e.g., increased hydrophobicity, aggregation, immunogenicity, etc.) on the pharmacological properties of the parent molecule, so great efforts must be made to select the appropriate site for attaching the payload.

[0004] Furthermore, current antibody technology does not allow for the production of scaffolds whose geometry (e.g., cyclic molecules, molecules with variable distances between domains, etc.), orientation, stoichiometry, and titers can be systematically controlled outside of what is possible through classical polypeptide engineering. This limitation of production technology places significant constraints on the therapeutic space that current formats can explore. Current protein engineering methods are limited to the inherent repertoire of natural polypeptide folds / peptide linkers to obtain the desired constructs. Therefore, non-natural formats that may exhibit higher efficacy cannot be prepared using conventional engineering methods. Furthermore, some effector molecules cannot be effectively produced en masse in one culture system due to toxicity constraints or metabolic properties of the expression host. In addition, as the complexity of the molecules increases, the correct folding, assembly, and purification of larger molecules becomes more difficult, thereby reducing yields and increasing costs.

[0005] Furthermore, industrial production of antibody molecules generally requires expression of antibodies or antibody-like fragments in eukaryotic hosts which are less robust than prokaryotic hosts and result in reduced product yields compared to prokaryotic hosts, resulting in higher engineering costs for antibodies or antibody-like fragments that can be expressed in prokaryotic hosts.

[0006] Thus, there is a need in the art for cost-effective methods of preparing next-generation antibody therapeutics with high molecular weights that allow for the creation of diverse libraries of antibody-like drugs with altered titers, geometries, effector functions and stoichiometries simply by combining a relatively small number of building blocks. Summary of the Invention [Means for solving the problem]

[0007] One embodiment 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 at a first end specific for irreversibly inhibiting the first fusion protein reactive enzyme and a second functional group at a second end specific for irreversibly inhibiting the second fusion protein reactive enzyme.

[0008] 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 a first end, thereby coupling the first fusion protein and the linker to the first end; and (b) contacting a second fusion protein comprising an affinity reagent and a reactive enzyme with a second end of the linker, the second end of the linker comprising a functional group specific for irreversibly inhibiting the second fusion protein-reactive enzyme, thereby coupling the second fusion protein and the linker to the second end.

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

[0010] Another aspect of the disclosure provides a method comprising administering a construct of the disclosure to a patient in need thereof. Another aspect of the disclosure provides the use of a 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 consideration of the following detailed description in conjunction with the drawings. While the present constructs and methods of making and using same may be susceptible to embodiment in a variety of forms, the following description includes specific embodiments, with the understanding that the disclosure is illustrative and is not intended to limit the invention to the specific embodiments described herein. In certain embodiments, for example, the following are provided: (Item 1) 1. A construct comprising: 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 comprises a first functional group at a first end specific for irreversibly inhibiting the first fusion protein-reactive enzyme and a second functional group at a second end specific for irreversibly inhibiting the second fusion protein-reactive enzyme. (Item 2) 2. The construct of item 1, wherein the first fusion protein-reactive enzyme and the second fusion protein-reactive enzyme are different. (Item 3) 2. The construct of item 1, wherein the first fusion protein-reactive enzyme and the second fusion protein-reactive enzyme are the same. (Item 4) 4. The 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) 4. The 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) 6. The construct of any one of items 1 to 5, wherein each affinity reagent is independently selected from the group consisting of an antibody or fragment thereof, a small molecule, a monobody, a protein, and combinations thereof. (Item 7) 7. The construct of item 6, wherein the affinity reagent is an antibody or a fragment thereof. (Item 8) The antibody or fragment thereof comprises a light chain variable domain (V L ), light chain constant domain (C L ), heavy chain variable domain (VH ), heavy chain constant domain (C H 8. The construct according to item 7, wherein the construct is selected from the group consisting of: (Item 9) 9. The construct of item 7 or 8, wherein the first fusion protein antibody or fragment thereof is a chimeric antibody, a human antibody, or a humanized antibody. (Item 10) 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 selected from the group consisting of adalimumab, alemtuzumab, arcitumomab, cetuximab, trastuzumab, imciromab, capromab, infliximab, abciximab, rituximab, basiliximab, palivizumab, nofetumomab, omalizumab, daclizumab, ibritumomab tiuxetan, muromomab, edrecolomab gemtuzumab ozogamicin, golimumab, certolizumab, eculizumab, ustecitima, 11. The construct according to any one of items 7 to 10, wherein the construct is selected from the group consisting of: tumab, panitumumab, tositumomab, bevacizumab, raxibacumab, tocilizumab, brentuximab, ofatumumab, belimumab, ramucirumab, vedolizumab, obinutuzumab, pembrolizumab, ranibizumab, pentuzumab, denosumab, catumaxomab, golimumab, siltuximab, natalizumab, panitumumab, and denosumab. (Item 12) 7. The construct of item 6, wherein the affinity reagent is a small molecule. (Item 13) 13. The construct of item 12, wherein the small molecule is a drug. (Item 14) The small molecule is selected from the group consisting of aldesleukin, alendronate, alphaferon, alitretinoin, allopurinol, alloprim, aloxi, altretamine, aminoglutethimide, L-type asparaginase, amifostine, amrubicin, amsacrine, anastrozole, anzumet, aranesp, aruglavin, arsenic trioxide, aromasin, 5-azacytidine, azathioprine, BCG or Tice BCG, bestatin, betamethasone acetate, betamethasone sodium phosphate, bexarotene, bleomycin sulfate, broxuridine, bortezomib, bu, bleomycin, busulfan, calcitonin, campath, capecitabine, carboplatin, carmustine, casodex, cefesone, cermoleukin, cerbizin, chlorambucil, cisplatin, colspase, cladribine, clodronic acid, cyclophosphamide, cytarabine, dacarbazine, dactinomycin, daunoxome, decadron, decadron phosphate, delestrogen, denileukin diftitox, depomedrol, deslorelin, dexalazoxane, daunorubicin, diethylstilbestrol, 2',2'- Difluorodeoxycytidine, Diflucan, Docetaxel, Doxifluridine, Doxorubicin, Dronabinol, DW-166HC, Eligard, Erytech, Elence, Emend, Epirubicin, Epoetin-alpha, Epogen, Eptaplatin, Ergamisole, Estrace, Estradiol, Estramustine sodium phosphate, Ethinyl estradiol, Ethiol, Etidronic acid, Etopofos, Etoposide, Fadrozole, Falston, Filgrastim, Finasteride, Frigrastim, Floxuridine, Flucona zole, fludarabine, fludarabine phosphate, 5-fluorodeoxyuridine, 5-fluorodeoxyuridine monophosphate, 5-fluorouracil (5-FU), fluoxymesterone, flutamide, hexamethylmelamine, formestane, fosteabine, fotemustine, fulvestrant, gammaguard, gemcitabine, gemtuzumab, Gleevec, Gliadel, goserelin, granisetron hydrochloride, histrelin, hicamtine, hydrocortone, erythro-hydroxocinoyl 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, Kytril, lentinan sulfate, letrozole, leucovorin, leuprolide, leuprolide acetate, levamisole, levofolic acid acid) calcium salt, levothroid, levoxyl, lomustine, lonidamine, marinol, mechlorethamine, mecobalamin, medroxyprogesterone acetate, megestrol acetate, melphalan, menest, 6-mercaptopurine, mesna, methotrexate, metobic, miltefosine, minocycline, mitomycin C, mitotane, mitoxantrone, modrenal, myoset, neda Platinum, Neulasta, Neumega, Neupogen, Nilutamide, Nolvadex, NSC-631570, OCT-43, Octreotide, Ondansetron hydrochloride, Orapred, Oxaliplatin, Paclitaxel, Pediapred, Pegaspargase, Pegasys, Pentostatin, N-phosphonoacetyl L-aspartic acid (PALA), Picibanil, Pilocarpine hydrochloride, Pirarubi Syn, plicamycin, porfimer sodium, prednimustine, prednisolone, prednisone, Premarin, procarbazine, Procrit, raltitrexed, Rebif, rhenium-186 etidronate, rituximab, Roferon A, romurtide, Salagen, Sandostatin, sargramostim, semustine, sizofuran, sobuzoxane, Solumedrol, streptozocin, strontium Um-89 chloride, Synthroid, Tamoxifen, Tamsulosin, Tasonermin, Tastractone, Taxotere, Teceleukin, Temozolomide, Teniposide, Testosterone Propionate, Testred, Thioguanine, Thiotepa, Thyrotropin, Tiludronic Acid, Topotecan, Toremifene, Tositumomab, Tastuzumab, Teosulfan, Treinoin, Trexaol, Trimethylmelamine,Trimetrexate, Triptorelin acetate, Triptorelin pamoate, UFT, Uridine, Valrubicin, Vesnarinone, Vinblastine, Vincristine, Vindesine, Vinorelbine, Virulizine, Zincard, Zinostatin-stimaramel, Zofran, ABI-007, Acolbifene, Actimun, Afinitak, Aminopterin, Arzoxifene, Asoprisnil, Atamestane, Atrasentan, Avastin, BAY43-9006 (Sorafen) nib), CCI-779, CDC-501, Celebrex, cetuximab, crisnatol, cyproterone acetate, decitabine, DN-101, doxorubicin-MTC, dSLIM, dutasteride, edotecarin, eflornithine, exatecan, fenretinide, histamine dihydrochloride, histrelin hydrogel implant, holmium-166 DOTMP, ibandronate, interferon-gamma, interferon-PEG, ixabepilone , Keyhole Limpet Hemocyanin, L-651582, Lanreotide, Lasofoxifene, Libra, Lonafarnib, Miproxifene, Minodronic Acid, MS-209, Liposomal MTP-PE, MX-6, Nafarelin, Nemorubicin, Neovastat, Nolatrexed, Oblimersen, Onco-TCS, Osidem, Paclitaxel Polyglutamimate, Pamidronate Disodium, PN-401, QS-21, Quazepam, R-1549, Raloxifene, 14. The construct according to item 13, which is selected from the group consisting of: ranpirnas, 13-cis-retinoic acid, satraplatin, seocalcitol, T-138067, tarceva, taxoplexin, thymosin-alpha-1, tiazofurin, tipifarnib, tirapazamine, TLK-286, toremifene, transMID-107R, valspodar, vapreotide, vatalanib, verteporfin, vinflunine, Z-100, zoledronic acid, and combinations thereof. (Item 15) 7. The construct of item 6, wherein the affinity reagent is a monobody. (Item 16) 7. The construct of item 6, wherein the affinity reagent is a protein. (Item 17) 17. The construct of item 16, wherein the protein comprises at least one unnatural amino acid. (Item 18) 18. The construct according to item 16 or 17, wherein the protein is a protein in a phage. (Item 19) 18. The construct according to item 16 or 17, wherein the protein is a therapeutic protein. (Item 20) 7. The construct of item 6, wherein the affinity reagent is a synthetic antibody domain. (Item 21) 6. The construct according to any one of items 1 to 5, wherein the affinity reagent is selected from the group consisting of designed ankyrin repeat proteins (DARPins), HEL4 Vh domains (Predator), Z domains of Staphylococcal protein A (Affibodies), Archaeal "7 kDa DNA binders" protein family (Affitins), carbohydrate binding molecules (CBD domains), cystine-knot mini-proteins (knottins), fibronectin type III domains (monobodies, Adnectins), gamma-B-crystallin (Affilins), cystatins (Affimers), triple helix coiled-coil domains (Alphabodies), lipocalin domains (Anticalins), A domains of various membrane receptors (Avimers), SH3 domains of Fyn (Fynomers), Kunitz domain peptides, and combinations thereof. (Item 22) The linker is a polyoxazoline, a polyacrylomorpholine, a polyvinylpyrrolidone, a polyphosphazene, a polyethylene-co-maleic anhydride, a polystyrene-co-maleic anhydride, a poly(1-hydroxymethylethylene hydroxymethyl formal) ("PHF"), a polyhydroxyalkyl acrylate, a 2-methacryloyloxy-2'-ethyltrimethylammonium phosphate ("MPC"), or a structure selected from the following: [ka] and During the ceremony, m is 0 to 10; n is 1 to 100; each p is independently 0, 1, 2, 3, or 4; q is 0, 1 or 2; r is 1 or 2; E is NH or CHR 10 and G is O, CH2, CHOH, CHNH2, CHCOOH or CHSO3H; R 10 is OH, NH2 or COOH, Each R 11 is independently H, OH, NH2 or COOH. (Item 23) The reactive enzymes were cutinases, SnapTag, HaloTag, relaxase domains of type I DNA topoisomerases, β-lactamases, glycosidases, matrix metalloproteinases, cytoplasmic protein tyrosine kinase domains, alkaline phosphatases, protein-tyrosine fusophatases, mutants of 23S rRNA (adenine(2503)-C(2))-methyltransferases, glucosidases, and N-6 adenine-specific DNA methyltransferases. 24. The construct according to any one of claims 1 to 22, wherein the endonuclease is independently selected from the group consisting of an enzyme, an N(4)-cytosine-specific DNA methylase, a DNA (cytosine-5-)-methyltransferase, a mutant of a haloalkane dehalogenase, an HNH endonuclease, a nicking endonuclease, a gelatinase B, a gelatinase A, a stromelysin, a fatty acid amide hydrolase, an esterase, a cytochrome P450, a methionine aminopeptidase, and a combination thereof. 24. The construct of any one of items 1 to 23, wherein the first fusion protein-reactive enzyme comprises cutinase and the functional group at the first terminus comprises p-nitrophenylphosphonate. (Item 25) the first fusion protein-reactive enzyme comprises a SnapTag, and the functional group at the first terminus is O 6 24. The construct according to any one of items 1 to 23, comprising benzylguanine. (Item 26) 24. The construct of any one of items 1 to 23, wherein the first fusion protein-reactive enzyme comprises a HaloTag and the functional group at the first terminus comprises an α-chloroalkane. (Item 27) 24. The construct of 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 end comprises clavulanic acid or a derivative thereof. (Item 28) 24. The construct according to any one of items 1 to 23, wherein the first fusion protein-reactive enzyme comprises a glycosidase and the functional group at the first terminus comprises an aglycone or a derivative thereof. (Item 29) 24. The construct of 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 a hydroxamic acid-benzophenone or a derivative thereof. (Item 30) 24. The construct according to any one of items 1 to 23, wherein the first fusion protein-reactive enzyme comprises a relaxase domain of a type I DNA topoisomerase and the functional group at the first end comprises a cognate oriT oligonucleotide sequence. (Item 31) 24. The construct of any one of items 1 to 23, wherein the first fusion protein-reactive enzyme comprises a cytoplasmic protein tyrosine kinase domain and the functional group at the first end comprises a cysteine-reactive ATP binding site inhibitor. (Item 32) 24. The construct of any one of items 1 to 23, wherein the first fusion protein-reactive enzyme comprises 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) 24. 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 end comprises a functional group selected from the group consisting of formylchromone, α-halophosphonic acid, precursors thereof, derivatives thereof, and combinations thereof. (Item 34) 24. The construct of any one of items 1 to 23, wherein the first fusion protein-reactive enzyme comprises a mutant of 23SrRNA (adenine(2503)-C(2))-methyltransferase and the functional group at the first end comprises a cognate RNA sequence. (Item 35) 24. The construct according to any one of items 1 to 23, wherein the first fusion protein-reactive enzyme comprises a glucosidase and the functional group at the first terminus comprises an aglycone or a derivative thereof. (Item 36) 24. The construct of 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 end comprises adenosine or a derivative thereof. (Item 37) 24. The construct of 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 end comprises cytosine or a derivative thereof. (Item 38) 24. The construct according to any one of items 1 to 23, wherein the first fusion protein-reactive enzyme comprises a DNA (cytosine-5-)-methyltransferase and the functional group at the first end comprises a cytosine or a derivative thereof. (Item 39) 24. The construct of any one of items 1 to 23, wherein the first fusion protein-reactive enzyme comprises a mutant of a haloalkane dehalogenase, and the functional group at the first terminus comprises a functional group selected from the group consisting of a haloalkane, a haloaromatic compound, or a derivative thereof. (Item 40) 24. The construct of any one of items 1 to 23, wherein the first fusion protein-reactive enzyme comprises an HNH endonuclease and the functional group at the first end comprises a cognate DNA nicking site. (Item 41) 24. The construct of any one of items 1 to 23, wherein the first fusion protein-reactive enzyme comprises a nicking endonuclease and the functional group at the first end comprises a cognate DNA nicking site. (Item 42) 24. The construct according to any one of items 1 to 23, wherein the first fusion protein-reactive enzyme comprises gelatinase B, and the functional group at the first end comprises a functional group selected from the group consisting of thiirane, hydroxamic acid, derivatives thereof, and combinations thereof. (Item 43) 24. The construct of any one of items 1 to 23, wherein the first fusion protein-reactive enzyme comprises gelatinase A and the functional group at the first end comprises a functional group selected from the group consisting of thiirane, hydroxamic acid, derivatives thereof, and combinations thereof. (Item 44) 24. The construct of any one of items 1 to 23, wherein the first fusion protein-reactive enzyme comprises stromelysin and the functional group at the first end comprises a functional group selected from the group consisting of thiirane, hydroxamic acid, derivatives thereof, and combinations thereof. (Item 45) 24. 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 functional group at the first terminus comprises an α-ketoxazole inhibitor or a derivative thereof. (Item 46) 24. The construct of any one of items 1 to 23, 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 phosphonate, carbamate, derivatives thereof, and combinations thereof. (Item 47) 24. The construct of any one of items 1 to 23, wherein the first fusion protein-reactive enzyme comprises a cytochrome P450 and the functional group at the first terminus comprises a functional group selected from the group consisting of electrophilic steroids, aromatic alkynes, derivatives thereof, and combinations thereof. (Item 48) 24. The construct of any one of items 1 to 23, wherein the first fusion protein-reactive enzyme comprises methionine aminopeptidase and the functional group at the first terminus comprises beloranib or a derivative thereof. (Item 49) 49. The construct of any one of items 1 to 48, wherein the second fusion protein-reactive enzyme comprises cutinase and the functional group at the second end comprises p-nitrophenylphosphonate. (Item 50) the second fusion protein-reactive enzyme comprises a SnapTag, and the functional group at the second end is O 6 49. The construct according to any one of items 1 to 48, comprising benzylguanine. (Item 51) 49. The construct of any one of items 1 to 48, wherein the second fusion protein-reactive enzyme comprises a HaloTag and the functional group at the second end comprises an α-chloroalkane. (Item 52) 49. The construct of any one of items 1 to 48, wherein the second fusion protein-reactive enzyme comprises a beta-lactamase and the functional group at the second end comprises clavulanic acid or a derivative thereof. (Item 53) 49. The construct of any one of items 1 to 48, 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 54) 49. The construct of 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 end comprises a hydroxamic acid-benzophenone or a derivative thereof. (Item 55) 49. The construct according to any one of items 1 to 48, wherein the second fusion protein-reactive enzyme comprises a relaxase domain of a type I DNA topoisomerase and the functional group at the second end comprises a cognate oriT oligonucleotide sequence. (Item 56) 49. The construct of 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 end comprises a cysteine-reactive ATP binding site inhibitor. (Item 57) 49. The construct of any one of items 1 to 48, wherein the second fusion protein-reactive enzyme comprises alkaline phosphatase and the functional group at the second end comprises a functional group selected from the group consisting of quinone methides, α-halophosphonic acids, precursors thereof, derivatives thereof, and combinations thereof. (Item 58) 49. The construct of 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 end comprises a functional group selected from the group consisting of formylchromone, α-halophosphonic acid, precursors thereof, derivatives thereof, and combinations thereof. (Item 59) 49. The construct of any one of items 1 to 48, wherein the second fusion protein-reactive enzyme comprises a mutant of 23SrRNA (Adenine(2503)-C(2))-methyltransferase and the functional group at the second end comprises a cognate RNA sequence. (Item 60) 49. The construct of 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) 49. The construct of 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 end comprises adenosine or a derivative thereof. (Item 62) 49. The construct of 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 end comprises cytosine or a derivative thereof. (Item 63) 49. The construct according to any one of items 1 to 48, wherein the second fusion protein-reactive enzyme comprises a DNA (cytosine-5-)-methyltransferase and the functional group at the second end comprises a cytosine or a derivative thereof. (Item 64) 49. The construct of any one of items 1 to 48, wherein the second fusion protein-reactive enzyme comprises a mutant of a haloalkane dehalogenase and the functional group at the second end comprises a functional group selected from the group consisting of a haloalkane, a haloaromatic compound, or a derivative thereof. (Item 65) 49. The construct of 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 end comprises a cognate DNA nicking site. (Item 66) 49. The construct of any one of items 1 to 48, wherein the second fusion protein-reactive enzyme comprises a nicking endonuclease and the functional group at the second end comprises a cognate DNA nicking site. (Item 67) 49. The construct of any one of items 1 to 48, wherein the second fusion protein-reactive enzyme comprises gelatinase B and the functional group at the second end comprises a functional group selected from the group consisting of thiirane, hydroxamic acid, derivatives thereof, and combinations thereof. (Item 68) 49. The construct of any one of items 1 to 48, wherein the second fusion protein-reactive enzyme comprises gelatinase A and the functional group at the second end comprises a functional group selected from the group consisting of thiirane, hydroxamic acid, derivatives thereof, and combinations thereof. (Item 69) 49. The construct of any one of items 1 to 48, wherein the second fusion protein-reactive enzyme comprises stromelysin and the functional group at the second end comprises a functional group selected from the group consisting of thiirane, hydroxamic acid, derivatives thereof, and combinations thereof. (Item 70) 49. The construct of 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 end comprises an α-ketoxazole inhibitor or a derivative thereof. (Item 71) 49. The construct of any one of items 1 to 48, wherein the second fusion protein-reactive enzyme comprises an esterase and the functional group at the second end comprises a functional group selected from the group consisting of phosphonate, carbamate, derivatives thereof, and combinations thereof. (Item 72) 49. The construct according to any one of items 1 to 48, wherein the second fusion protein-reactive enzyme comprises a cytochrome P450, and the functional group at the second end comprises a functional group selected from the group consisting of electrophilic steroids, aromatic alkynes, derivatives thereof, and combinations thereof. (Item 73) 49. The construct of any one of items 1 to 48, wherein the second fusion protein-reactive enzyme comprises methionine aminopeptidase and the functional group at the second end comprises beloranib or a derivative thereof. (Item 74) 1. 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 a first end, thereby coupling the first fusion protein and the linker to the first end; (b) contacting a second fusion protein comprising an affinity reagent and a reactive enzyme with a second end of the linker, wherein the second end 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 to the second end. (Item 75) Item 75. The method according to item 74, wherein steps (a) and (b) are carried out sequentially. (Item 76) Item 75. The method of item 74, wherein steps (a) and (b) are preformed simultaneously. (Item 77) 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) 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 the same. (Item 79) 79. The method of 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) 79. The method of 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) 81. The method of any one of items 74 to 80, wherein each affinity reagent is independently selected from the group consisting of an antibody or fragment thereof, a small molecule, a monobody, a protein, and combinations thereof. (Item 82) 82. The method of claim 81, wherein the affinity reagent is an antibody or a fragment thereof. (Item 83) The antibody or fragment thereof 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 H 83. The method of claim 82, wherein the compound is selected from the group consisting of: 1), 2), and combinations thereof. (Item 84) 84. The method of claim 82 or 83, wherein the first fusion protein antibody or fragment thereof is a chimeric antibody, a human antibody, or a humanized antibody. (Item 85) 85. The method of 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 selected from the group consisting of adalimumab, alemtuzumab, arcitumomab, cetuximab, trastuzumab, imciromab, capromab, infliximab, abciximab, rituximab, basiliximab, palivizumab, nofetumomab, omalizumab, daclizumab, ibritumomab tiuxetan, muromomab, edrecolomab gemtuzumab ozogamicin, golimumab, certolizumab, eculizumab, usu 86. The method of any one of items 82 to 85, wherein the antibody is selected from the group consisting of tekimumab, panitumumab, tositumomab, bevacizumab, raxibacumab, tocilizumab, brentuximab, ofatumumab, belimumab, ramucirumab, vedolizumab, obinutuzumab, pembrolizumab, ranibizumab, pentuzumab, denosumab, catumaxomab, golimumab, siltuximab, natalizumab, panitumumab, and denosumab. (Item 87) 82. The method of claim 81, wherein the affinity reagent is a small molecule. (Item 88) 88. The method of claim 87, wherein the small molecule is selected from the group consisting of drugs. (Item 89) The small molecule is selected from the group consisting of aldesleukin, alendronate, alphaferon, alitretinoin, allopurinol, alloprim, aloxi, altretamine, aminoglutethimide, L-type asparaginase, amifostine, amrubicin, amsacrine, anastrozole, anzumet, aranesp, aruglavin, arsenic trioxide, aromasin, 5-azacytidine, azathioprine, BCG or Tice-BCG, bestatin, betamethasone acetate, betamethasone sodium phosphate, bexarotene, bleomycin sulfate, broxuridine, bortezomib, bromocriptine ... Zomib, bleomycin, busulfan, calcitonin, campath, capecitabine, carboplatin, carmustine, casodex, cefesone, cermoleukin, cerbizin, chlorambucil, cisplatin, colspase, cladribine, clodronic acid, cyclophosphamide, cytarabine, dacarbazine, dactinomycin, daunoxome, decadron, decadron phosphate, delestrogen, denileukin diftitox, depomedrol, deslorelin, dexalazoxane, daunorubicin, diethylstilbestrol, 2',2' -Difluorodeoxycytidine, Diflucan, Docetaxel, Doxifluridine, Doxorubicin, Dronabinol, DW-166HC, Eligard, Erytech, Elence, Emend, Epirubicin, Epoetin-alpha, Epogen, Eptaplatin, Ergamisole, Estrace, Estradiol, Estramustine sodium phosphate, Ethinyl estradiol, Ethiol, Etidronic acid, Etopofos, Etoposide, Fadrozole, Falston, Filgrastim, Finasteride, Frigrastim, Floxuridine, Fluco Nazole, fludarabine, fludarabine phosphate, 5-fluorodeoxyuridine, 5-fluorodeoxyuridine monophosphate, 5-fluorouracil (5-FU), fluoxymesterone, flutamide, hexamethylmelamine, formestane, fosteabine, fotemustine, fulvestrant, gammaguard, gemcitabine, gemtuzumab, Gleevec, Gliadel, goserelin, granisetron hydrochloride, histrelin, hicamtine, hydrocortone, 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, Kytril, lentinan sulfate, letrozole, leucovorin, leuprolide, leuprolide acetate, levamisole, levofolic acid acid) calcium salt, levothroid, levoxyl, lomustine, lonidamine, marinol, mechlorethamine, mecobalamin, medroxyprogesterone acetate, megestrol acetate, melphalan, menest, 6-mercaptopurine, mesna, methotrexate, metobic, miltefosine, minocycline, mitomycin C, mitotane, mitoxantrone, modrenal, myoset, neda Platinum, Neulasta, Neumega, Neupogen, Nilutamide, Nolvadex, NSC-631570, OCT-43, Octreotide, Ondansetron hydrochloride, Orapred, Oxaliplatin, Paclitaxel, Pediapred, Pegaspargase, Pegasys, Pentostatin, N-phosphonoacetyl L-aspartic acid (PALA), Picibanil, Pilocarpine hydrochloride, Pirarubi Syn, plicamycin, porfimer sodium, prednimustine, prednisolone, prednisone, Premarin, procarbazine, Procrit, raltitrexed, Rebif, rhenium-186 etidronate, rituximab, Roferon A, romurtide, Salagen, Sandostatin, sargramostim, semustine, sizofuran, sobuzoxane, Solumedrol, streptozocin, strontium Um-89 chloride, Synthroid, Tamoxifen, Tamsulosin, Tasonermin, Tastractone, Taxotere, Teceleukin, Temozolomide, Teniposide, Testosterone Propionate, Testred, Thioguanine, Thiotepa, Thyrotropin, Tiludronic Acid, Topotecan, Toremifene, Tositumomab, Tastuzumab, Teosulfan, Treinoin, Trexaol, Trimethylmelamine,Trimetrexate, Triptorelin acetate, Triptorelin pamoate, UFI, Uridine, Valrubicin, Vesnarinone, Vinblastine, Vincristine, Vindesine, Vinorelbine, Virulizine, Zincard, Zinostatin-stimaramel, Zofran, ABI-007, Acolbifene, Actimun, Afinitak, Aminopterin, Arzoxifene, Asoprisnil, Atamestane, Atrasentan, Avastin, BAY43-9006 (Soraf cerebrospinal fluid), CCI-779, CDC-501, Celebrex, cetuximab, crisnatol, cyproterone acetate, decitabine, DN-101, doxorubicin-MTC, dSLIM, dutasteride, edotecarin, eflornithine, exatecan, fenretinide, histamine dihydrochloride, histrelin hydrogel implant, holmium-166 DOTMP, ibandronate, interferon-gamma, interferon-PEG, ixabepi Ron, keyhole limpet hemocyanin, L-651582, lanreotide, lasofoxifene, Libra, lonafarnib, miproxifene, minodronic acid, MS-209, liposomal MTP-PE, MX-6, nafarelin, nemorubicin, neovastat, nolatrexed, oblimersen, Onco-TCS, Osidem, paclitaxel polyglutamimate, disodium pamidronate, PN-401, QS-21, quazepam, R-1549, raloxifene 89. The method of claim 88, wherein the medicament is selected from the group consisting of fen, ranpirnas, 13-cis-retinoic acid, satraplatin, seocalcitol, T-138067, tarceva, taxoplexin, thymosin-alpha-1, tiazofurin, tipifarnib, tirapazamine, TLK-286, toremifene, trans-MID-107R, valspodar, vapreotide, vatalanib, verteporfin, vinflunine, Z-100, zoledronic acid, and combinations thereof. (Item 90) 82. The method of item 81, wherein the affinity reagent is a monobody. (Item 91) 82. The method of claim 81, wherein the affinity reagent is a protein. (Item 92) 92. The method of claim 91, wherein the protein comprises at least one unnatural amino acid. (Item 93) Item 94. The method according to item 91 or 92, wherein the protein is a protein in a phage. 93. The method of claim 91 or 92, wherein the protein is a therapeutic agent. (Item 95) 82. The method of claim 81, wherein the affinity reagent is a synthetic antibody domain. (Item 96) 81. The method of any one of items 74 to 80, wherein the affinity reagent is selected from the group consisting of designed ankyrin repeat proteins (DARPins), HEL4 Vh domains (Predator), Z domains of Staphylococcus aureus protein A (Affibodies), Archaeal "7 kDa DNA binders" protein family (Affitins), carbohydrate binding molecules (CBD domains), cystine knot mini-proteins (knottins), fibronectin type III domains (monobodies, Adnectins), gamma-B-crystallin (Affilins), cystatins (Affimers), triple helix coiled-coil domains (Alphabodies), lipocalin domains (Anticalins), A domains of various membrane receptors (Avimers), SH3 domains of Fyn (Fynomers), Kunitz domain peptides, and combinations thereof. (Item 97) The linker is a polyoxazoline, a polyacrylomorpholine, a polyvinylpyrrolidone, a polyphosphazene, a polyethylene-co-maleic anhydride, a polystyrene-co-maleic anhydride, a poly(1-hydroxymethylethylene hydroxymethyl formal) ("PHF"), a polyhydroxyalkyl acrylate, a 2-methacryloyloxy-2'-ethyltrimethylammonium phosphate ("MPC"), or a structure selected from the following: [ka] and During the ceremony, m is 0 to 10; n is 1 to 100; each p is independently 0, 1, 2, 3, or 4; q is 0, 1 or 2; r is 1 or 2; E is NH or CHR 10 and G is O, CH2, CHOH, CHNH2, CHCOOH or CHSO3H; R 10 is OH, NH2 or COOH. Each R 11 is independently H, OH, NH2 or COOH. (Item 98) Each reactive enzyme is a 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, mutant 23S rRNA (adenine(2503)-C(2))-methyltransferase, glucosidase, N-6 adenine-specific DNA 98. The method of any one of items 74 to 97, wherein the cytosine-specific DNA methylase is independently selected from the group consisting of A methylase, N(4)-cytosine-specific DNA methylase, DNA (cytosine-5-)-methyltransferase, a mutant of haloalkane dehalogenase, HNH endonuclease, a nicking endonuclease, gelatinase B, gelatinase A, stromelysin, fatty acid amide hydrolase, esterase, cytochrome P450, methionine aminopeptidase, and combinations thereof. (Item 99) 99. The method of any one of items 74 to 98, wherein the first fusion protein-reactive enzyme comprises cutinase and the functional group at the first terminus comprises p-nitrophenylphosphonate. (Item 100) the first fusion protein-reactive enzyme comprises a SnapTag, and the functional group at the first terminus is O 6- The method according to any one of items 74 to 98, comprising benzylguanine. (Item 101) 99. The method of any one of items 74 to 98, wherein the first fusion protein-reactive enzyme comprises a HaloTag and the functional group at the first terminus comprises an α-chloroalkane. (Item 102) 99. The method of any one of items 74 to 98, 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 103) 99. The method of any one of items 74 to 98, wherein the first fusion protein-reactive enzyme comprises a glycosidase and the functional group at the first terminus comprises an aglycone or a derivative thereof. (Item 104) 99. The method of 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 a hydroxamic acid-benzophenone or a derivative thereof. (Item 105) 99. The method of any one of items 74 to 98, wherein the first fusion protein-reactive enzyme comprises a relaxase domain of a type I DNA topoisomerase and the functional group at the first end comprises a cognate oriT oligonucleotide sequence. (Item 106) 99. The method of 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 end comprises a cysteine-reactive ATP binding site inhibitor. (Item 107) 99. The method of any one of items 74 to 98, wherein the first fusion protein-reactive enzyme comprises 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, and combinations thereof. (Item 108) 99. The method of 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) 99. The method of any one of items 74 to 98, wherein the first fusion protein-reactive enzyme comprises a mutant of 23S rRNA (adenine (2503)-C(2))-methyltransferase and the functional group at the first end comprises a cognate RNA sequence. (Item 110) 99. The method of any one of items 74 to 98, wherein the first fusion protein-reactive enzyme comprises a glucosidase and the functional group at the first terminus comprises an aglycone or a derivative thereof. (Item 111) 99. The method of 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 end comprises adenosine or a derivative thereof. (Item 112) 99. The method of 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 end comprises cytosine or a derivative thereof. (Item 113) 99. The method according to any one of items 74 to 98, wherein the first fusion protein-reactive enzyme comprises a DNA (cytosine-5-)-methyltransferase and the functional group at the first end comprises cytosine or a derivative thereof. (Item 114) 99. The method of any one of items 74 to 98, wherein the first fusion protein-reactive enzyme comprises a mutant of a haloalkane dehalogenase, and the functional group at the first terminus comprises a functional group selected from the group consisting of a haloalkane, a haloaromatic compound, or a derivative thereof. (Item 115) 99. The method of 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 end comprises a nicking site for cognate DNA. (Item 116) 99. The method of any one of items 74 to 98, wherein the first fusion protein-reactive enzyme comprises a nicking endonuclease and the functional group at the first end comprises a cognate DNA nicking site. (Item 117) 99. The method of any one of items 74 to 98, wherein the first fusion protein-reactive enzyme comprises gelatinase B, and the functional group at the first end comprises a functional group selected from the group consisting of thiirane, hydroxamic acid, derivatives thereof, and combinations thereof. (Item 118) 99. The method according to any one of items 74 to 98, wherein the first fusion protein-reactive enzyme comprises gelatinase A and the functional group at the first end comprises a functional group selected from the group consisting of thiirane, hydroxamic acid, derivatives thereof, and combinations thereof. (Item 119) 99. The method of any one of items 74 to 98, wherein the first fusion protein-reactive enzyme comprises stromelysin, and the functional group at the first end comprises a functional group selected from the group consisting of thiirane, hydroxamic acid, derivatives thereof, and combinations thereof. (Item 120) 99. 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) 99. The method of 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 phosphonate, carbamate, derivatives thereof, and combinations thereof. (Item 122) 99. The method according to any one of items 74 to 98, wherein the first fusion protein-reactive enzyme comprises a cytochrome P450, and the functional group at the first end comprises a functional group selected from the group consisting of electrophilic steroids, aromatic alkynes, derivatives thereof, and combinations thereof. (Item 123) 99. The method of any one of items 74 to 98, wherein the first fusion protein-reactive enzyme comprises methionine aminopeptidase and the functional group at the first terminus comprises beloranib or a derivative thereof. (Item 124) 125. The method of 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 comprises a SnapTag, and the functional group at the second end is O 6 - The method according to any one of items 74 to 124, comprising benzylguanine. (Item 126) 125. The method of 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) 125. The method of 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 end comprises clavulanic acid or a derivative thereof. (Item 128) 125. The method of 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) 125. The method of 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 end comprises a hydroxamic acid-benzophenone or a derivative thereof. (Item 130) 125. The method of any one of items 74 to 124, wherein the second fusion protein-reactive enzyme comprises a relaxase domain of a type I DNA topoisomerase and the functional group at the second end comprises a cognate oriT oligonucleotide sequence. (Item 131) 125. The method of 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 end comprises a cysteine-reactive ATP binding site inhibitor. (Item 132) 125. The method of any one of items 74 to 124, wherein the second fusion protein-reactive enzyme comprises alkaline phosphatase and the functional group at the second end comprises a functional group selected from the group consisting of quinone methides, α-halophosphonic acids, precursors thereof, and combinations thereof. (Item 133) 125. The method of 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) 125. The method of any one of items 74 to 124, wherein the second fusion protein-reactive enzyme comprises a mutant of 23SrRNA(adenine(2503)-C(2))-methyltransferase and the functional group at the second end comprises a cognate RNA sequence. (Item 135) 125. The method of 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) 125. The method of 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 end comprises adenosine or a derivative thereof. (Item 137) 125. The method of 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 end comprises cytosine or a derivative thereof. (Item 138) 125. The method according to any one of items 74 to 124, wherein the second fusion protein-reactive enzyme comprises a DNA (cytosine-5-)-methyltransferase and the functional group at the second end comprises cytosine or a derivative thereof. (Item 139) 125. The method of any one of items 74 to 124, wherein the second fusion protein-reactive enzyme comprises a mutant of a haloalkane dehalogenase, and the functional group at the second end comprises a functional group selected from the group consisting of a haloalkane, a haloaromatic compound, or a derivative thereof. (Item 140) 125. The method of 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 end comprises a cognate DNA nicking site. (Item 141) 125. The method of any one of items 74 to 124, wherein the second fusion protein-reactive enzyme comprises a nicking endonuclease and the functional group at the second end comprises a cognate DNA nicking site. (Item 142) 125. The method of any one of items 74 to 124, wherein the second fusion protein-reactive enzyme comprises gelatinase B, and the functional group at the second end comprises a functional group selected from the group consisting of thiirane, hydroxamic acid, derivatives thereof, and combinations thereof. (Item 143) 125. 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 end comprises a functional group selected from the group consisting of thiirane, hydroxamic acid, derivatives thereof, and combinations thereof. (Item 144) 125. The method of any one of items 74 to 124, wherein the second fusion protein-reactive enzyme comprises stromelysin and the functional group at the second end comprises a functional group selected from the group consisting of thiirane, hydroxamic acid, derivatives thereof, and combinations thereof. (Item 145) 125. The method of 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 end comprises an α-ketoxazole inhibitor or a derivative thereof. (Item 146) 125. The method of any one of items 74 to 124, wherein the second fusion protein-reactive enzyme comprises an esterase and the functional group at the second end comprises a functional group selected from the group consisting of phosphonate, carbamate, derivatives thereof, and combinations thereof. (Item 147) 125. The method according to any one of items 74 to 124, wherein the second fusion protein-reactive enzyme comprises a cytochrome P450, and the functional group at the second end comprises a functional group selected from the group consisting of electrophilic steroids, aromatic alkynes, derivatives thereof, and combinations thereof. (Item 148) 125. The method of any one of items 74 to 124, wherein the second fusion protein-reactive enzyme comprises methionine aminopeptidase and the functional group at the second end comprises beloranib or a derivative thereof. (Item 149) A construct prepared by the method according to any one of items 74 to 148. (Item 150) 150. A method comprising administering the construct according to any one of items 1 to 73 or 149 to a patient in need of said construct. (Item 151) 20. Use of the construct according to any one of items 1 to 73 or 149 as a medicament for a patient in need thereof. (Item 152) 152. The method or use according to item 150 or 151, wherein the patient is suffering from breast cancer and at least one antibody or fragment thereof of the construct comprises trastuzumab or a fragment thereof. (Item 153) 152. The method or use according to item 150 or 151, wherein the patient is suffering from pulmonary anthrax and at least one antibody or fragment thereof of the construct comprises raxibacumab or a fragment thereof. (Item 154) 152. The method or use according to item 150 or 151, wherein the patient suffers from rheumatoid arthritis or systemic juvenile idiopathic arthritis and at least one antibody or fragment thereof of the construct comprises tocilizumab or a fragment thereof. (Item 155) 152. The method or use according to item 150 or 151, wherein the patient is suffering from Hodgkin's lymphoma or systemic anaplastic large cell lymphoma and at least one antibody or fragment thereof of the construct comprises brentuximab or a fragment thereof. (Item 156) 152. The method or use according to item 150 or 151, wherein the patient is suffering from a condition selected from the group consisting of chronic lymphocytic leukemia, follicular non-Hodgkin's lymphoma, diffuse large B-cell lymphoma, rheumatoid arthritis, and relapsing remitting multiple sclerosis, and wherein at least one antibody or fragment thereof of the construct comprises ofatumumab or a fragment thereof. (Item 157) 152. The method or use according to item 150 or 151, wherein the patient is suffering from systemic lupus erythematosus and at least one antibody or fragment thereof of the construct comprises belimumab or a fragment thereof. (Item 158) 152. The method or use according to item 150 or 151, wherein the patient is suffering from adenocarcinoma of the stomach or gastroesophageal junction or metastatic non-small cell lung cancer, and wherein at least one antibody or fragment thereof of the construct comprises ramucirumab or a fragment thereof. (Item 159) 152. The method or use according to item 150 or 151, wherein the patient is suffering from ulcerative colitis or Crohn's disease and at least one antibody or fragment thereof of the construct comprises vedolizumab or a fragment thereof. (Item 160) 152. The method or use according to item 150 or 151, wherein the patient is suffering from chronic lymphocytic leukemia or follicular lymphoma and at least one antibody or fragment thereof of the construct comprises obinutuzumab or a fragment thereof. (Item 161) 152. The method or use according to item 150 or 151, wherein the patient is suffering from melanoma or metastatic non-small cell lung cancer and at least one antibody or fragment thereof of the construct comprises pembrolizumab or a fragment thereof. (Item 162) 152. The method or use according to item 150 or 151, wherein the patient is suffering from macular degeneration and at least one antibody or fragment thereof of the construct comprises ranibizumab or a fragment thereof. (Item 163) 152. The method or use according to item 150 or 151, wherein the patient is suffering from breast cancer and at least one antibody or fragment thereof of the construct comprises pertuzumab or a fragment thereof. (Item 164) 152. The method or use according to item 150 or 151, wherein the patient is suffering from a condition selected from the group consisting of osteoporosis, treatment-induced bone loss, bone metastases, and giant cell tumor of bone, and wherein at least one antibody or fragment of the construct comprises denosumab or a fragment thereof. (Item 165) 152. The method or use according to item 150 or 151, wherein the patient is suffering from malignant ascites and at least one antibody or fragment thereof of the construct comprises catumaxomab or a fragment thereof. (Item 166) 152. The method or use according to item 150 or 151, wherein the patient is suffering from a condition selected from the group consisting of rheumatoid arthritis, psoriatic arthritis, ankylosing spondylitis, and ulcerative colitis, and wherein at least one antibody or fragment thereof of the construct comprises golimumab or a fragment thereof. (Item 167) 152. The method or use according to item 150 or 151, wherein the patient is suffering from a condition selected from the group consisting of metastatic renal cell carcinoma, prostate cancer, ovarian cancer, non-Hodgkin's lymphoma, multiple myeloma, and Castleman's disease, and wherein at least one antibody or fragment thereof of the construct comprises siltuximab or a fragment thereof. (Item 168) 152. The method or use according to item 150 or 151, wherein the patient suffers from multiple sclerosis or Crohn's disease and wherein at least one antibody or fragment thereof of the construct comprises natalizumab or a fragment thereof. (Item 169) 152. The method or use according to item 150 or 151, wherein the patient is suffering from colorectal cancer and at least one antibody or fragment thereof of the construct comprises panitumumab or a fragment thereof. (Item 170) 152. The method or use according to item 150 or 151, wherein the patient is suffering from a condition selected from the group consisting of osteoporosis, treatment-induced bone loss, bone metastases, and giant cell tumor of bone, and wherein at least one antibody or fragment thereof of the construct comprises denosumab or a fragment thereof. (Item 171) 150. Use of a construct according to any one of items 1 to 73 or 149 as a diagnostic agent. [Brief description of the drawings]

[0012] [Figure 1] 1 shows the trastuzumab F(ab) (TFab) fusion gene / protein construct. [Diagram 2]An SDS-PAGE gel of purified TFab-cutinase and TFab-SnapTag fusions is shown. [Diagram 3] An SDS-PAGE gel of the bivalent constructs is shown. [Figure 4] 1 shows cell viability assay data for trastuzumab and fusion protein construct B-13. [Diagram 5] 1 is a plot of serum concentrations of B-13 constructs over time in mice. [Figure 6] Results of the effect of B-13 administration on mice bearing BT474 tumor xenografts are shown. A) Tumor volume progression in animals treated with vehicle over 4 weeks, B13 at 2.5 and 5 mg / kg daily, and doxorubicin (dox) at 2.5 mg / kg twice weekly. After 28 days, there is a significant difference in tumor volume between vehicle vs. doxorubicin (p=.0002) and vehicle vs. the two B13 treatment groups (p=.0001). B) Total body weight measurements of animals in the four treatment cohorts. There was a significant difference in total body weight between vehicle and doxorubicin (p=.0116), but not between vehicle and B13 treatment at 2.5 mg / kg (p=.2237) or 5 mg / kg (p=.4297). C) Organ weights of animals in the four treatment cohorts. There were no significant differences in organ weights between groups (p=0.9493). P values ​​were calculated using two-way ANOVA in Graph Pad 7.0. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0013] Provided herein are constructs and methods of making and using the constructs. The constructs include a first fusion protein, a second fusion protein, and a linker, the first fusion protein and the second fusion protein each including an affinity reagent and a reactive enzyme, the linker including a first functional group at a first end specific for irreversibly inhibiting the first fusion protein reactive enzyme and a second functional group at a second end specific for irreversibly inhibiting the second fusion protein reactive enzyme. 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 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. Optionally, in some embodiments, each affinity reagent is independently selected from the group consisting of an antibody or fragment thereof, a small molecule, a monobody, a protein, and combinations thereof.

[0014] Constructing fusion proteins with affinity reagents and reactive enzymes The disclosed constructs include a fusion protein of an affinity reagent and a reactive enzyme linked via a linker.

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

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

[0017] As used herein, and unless otherwise specified, "protein" refers to naturally occurring polypeptides, polypeptides containing natural and / or unnatural amino acids, proteins in phages, therapeutic proteins, and antibody domains, either synthetic or naturally occurring.

[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. Polypeptides of the present disclosure can be either natural or non-natural.

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

[0020] Antibodies contemplated for use in the methods and compositions of the present disclosure include, but are not limited to, antibodies that recognize and associate with a target molecule either in vivo or in vitro.

[0021] Structural polypeptides contemplated by the present disclosure include, but are not limited to, actin, tubulin, collagen, elastin, myosin, kinesin, and dynein.

[0022] The non-natural polypeptides contemplated by the present 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 substances that have D-amino acids, modified, derivatized or non-natural amino acids in D or L configurations and / or peptidomimetic units as part of their structure.The term "protein" typically refers to large polypeptides.The term "peptide" typically refers to short polypeptides.

[0023] Non-naturally occurring polypeptides are prepared, for example, by use of an automated polypeptide synthesizer or, alternatively, by recombinant expression techniques using a modified polynucleotide encoding the desired polypeptide.

[0024] As used herein, a "fragment" of a polypeptide is meant to refer to 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 either the entire molecule or its fragments and have the same biological activity, but may have varying degrees of activity. Analogs differ in their amino acid sequence composition based on one or more mutations, including the substitution, deletion, insertion and / or addition of one or more amino acids for other amino acids. Substitutions may be conservative or non-conservative, based on the physicochemical or functional relatedness of the amino acid being replaced and the amino acid replacing it.

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

[0027] Antibodies and fragments or derivatives thereof are contemplated, 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 (as opposed to the heterotetrameric heavy and light chains found in intact antibodies), single chain antibodies (scAbs), humanized antibodies (and antibodies modified in the manner of humanized antibodies, but where the resulting antibody more closely resembles the antibody of the non-human species), chelating recombinant antibodies (CRABs), bispecific and multispecific antibodies, and other antibody derivatives or fragments known in the art.

[0028] In an embodiment of the preceding aspect, at least one antibody or fragment thereof of the construct comprises trastuzumab or a fragment thereof, racivacaumab 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.

[0029] In embodiments, the linker is a polyoxazoline, a polyacrylomorpholine, a polyvinylpyrrolidone, a polyphosphazene, a polyethylene-co-maleic anhydride, a polystyrene-co-maleic anhydride, a poly(1-hydroxymethylethylene hydroxymethyl formal) ("PHF"), a polyhydroxyalkyl acrylate, 2-methylacryloyloxy-2'-ethyltrimethylammonium phosphate ("MPC"), or a structure selected from the following: [ka] And, wherein m is 0 to 10, n is 1 to 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, R 10 is OH, NH2 or COOH, and each R 11 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, mutant 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, mutant of haloalkane dehalogenase, HNH endonuclease, nicking endonuclease, gelatinase B, gelatinase A, stromelysin, fatty acid amide hydrolase, esterase, cytochrome P450, methionine aminopeptidase, and combinations thereof. In some embodiments, each reactive enzyme is a cutinase, a haloalkane dehydrogenase, HaloTag, O 6 In some embodiments, each reactive enzyme is independently selected from the group consisting of cutinase, haloalkane dehydrogenase HaloTag, O-alkylguanine DNA alkyltransferase mutant, SnapTag, relaxase domain of type I DNA topoisomerase, beta-lactamase, glycosidase, matrix metalloproteinase, and combinations thereof. 6 -alkylguanine DNA alkyltransferase mutant, SnapTag, and combinations thereof.

[0031] 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. Optionally, in some embodiments, each affinity reagent is independently selected from the group consisting of an antibody or fragment thereof, a small molecule, a monobody, a protein, and combinations thereof.

[0032] In embodiments, a fusion protein of the affinity reagent and the reactive enzyme is formed by conventional synthetic chemistry techniques for attaching the reactive enzyme to the affinity reagent. In some cases, when the affinity reagent comprises a protein or an antibody, the fusion protein is produced by expressing a polynucleotide sequence encoding the affinity reagent and the reactive enzyme. In some aspects, the disclosure provides a vector comprising a polynucleotide operably linked to a promoter. In a further aspect, the disclosure provides a host cell comprising the vector. In some embodiments, the host cell is an Escherichia coli cell. In a further embodiment, the host cell is a mammalian cell. In a related embodiment, the host cell is a Chinese Hamster Ovary (CHO) cell. In yet a further embodiment, the host cell is a yeast cell. In a further aspect, a method of producing a fusion protein disclosed herein is provided, the method comprising culturing a host cell of the disclosure under conditions suitable to induce expression of the polypeptide. In a related embodiment, the polypeptide is isolated.

[0033] Affinity Reagents The construct of the present disclosure comprises a fusion protein that comprises an affinity reagent that provides functionality to the fusion protein. Thus, a construct can be assembled that comprises a first fusion protein and a second fusion protein with precisely defined structure and function. Examples of functionality that can be imparted to the fusion protein via the affinity reagent 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 an imaging agent, use as a diagnostic agent, or combinations thereof.

[0034] The first affinity reagent and the second affinity reagent can be the same or different. Each affinity reagent can be independently selected from the group consisting of an antibody or fragment thereof, a small molecule, a monobody, a protein, and combinations thereof.

[0035] In some embodiments, the affinity reagent comprises an antibody or a fragment thereof. The antibody or fragment thereof 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. The antibody or fragment thereof can be a chimeric antibody, a human antibody, and a humanized antibody. Examples of antibodies or fragments thereof include, but are not limited to, trastuzumab or fragments thereof, racivacaumab or fragments thereof, tocilizumab or fragments thereof, brentuximab or fragments thereof, ofatumumab or fragments thereof, belimumab or fragments thereof, ramucirumab or fragments thereof, vedolizumab or fragments thereof, obinutuzumab or fragments thereof, pembrolizumab or fragments thereof, ranibizumab or fragments thereof, pertuzumab or fragments thereof, denosumab or fragments thereof, catumaxomab or fragments thereof, golimumab or fragments thereof, siltuximab or fragments thereof, natalizumab or fragments thereof, panitumumab or fragments thereof, and denosumab or fragments thereof.

[0036] In some embodiments, the affinity reagent comprises a small molecule. The small molecule can be a drug. Examples of small molecule drugs include aldesleukin, alendronate, alphaferon, alitretinoin, allopurinol, alloprim, aloxi, altretamine, aminoglutethimide, L-type asparaginase, amifostine, amrubicin, amsacrine, anastrozole, anzumet, aranesp, aruglavin, arsenic trioxide, aromasin, 5-azacytidine, azathioprine, BCG or Tice BCG, bestatin, betamethasone acetate, betamethasone sodium phosphate, bexarotene, bleomycin sulfate, bromocriptine ... Suridine, bortezomib, bleomycin, busulfan, calcitonin, campath, capecitabine, carboplatin, carmustine, casodex, cefezone, cermoleukin, cerbizin, chlorambucil, cisplatin, colspase, cladribine, clodronic acid, cyclophosphamide, cytarabine, dacarbazine, dactinomycin, daunoxome, decadron, decadron phosphate, delestrogen, denileukin diftitox, depomedrol, deslorelin, dexalazoxane, daunorubicin, diethyl Stilbestrol, 2',2'-difluorodeoxycytidine, Diflucan, docetaxel, doxifluridine, doxorubicin, dronabinol, DW-166HC, Eligard, Erytech, Elence, Emend, epirubicin, epoetin-alpha, epogen, eptaplatin, Ergamisole, estrase, estradiol, estramustine sodium phosphate, ethinyl estradiol, ethiol, etidronic acid, etopophos, etoposide, fadrozole, falston, filgrastim, finasteride, Frigrastim, floxuridine, fluconazole, fludarabine, fludarabine phosphate, 5-fluorodeoxyuridine, 5-fluorodeoxyuridine monophosphate, 5-fluorouracil (5-FU), fluoxymesterone, flutamide, hexamethylmelamine, formestane, fosteabine, fotemustine, fulvestrant, gammaguard, gemcitabine, gemtuzumab, Gleevec, Gliadel, goserelin, granisetron, hydrochloric acid, histrelin, hicamucine, hydrocortone,Erythro-hydroxynonyl 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, Kytril, lentinan sulfate, letrozole, leucovorin, leuprolide, leuprolide acetate, levamisole, levofolic acid acid) calcium salt, levothroid, levoxyl, lomustine, lonidamine, marinol, mechlorethamine, mecobalamin, medroxyprogesterone acetate, megestrol acetate, melphalan, menest, 6-mercaptopurine, mesna, methotrexate, metobic, miltefosine, minocycline, mitomycin C, mitotane, mitoxantrone, modrenal, myoset, nedaplatin, neulasta, neumega, neupogen, nilutamide, nolvadex, NSC-631570, OCT-43, octreotide, ondansetron hydrochloride, orapred, oxaliplatin, paclitaxel, pediapred, pegaspargase, pegasys, pentostatin, N-phosphonoacetyl L-aspartic acid (PALA), picibanil, pilocarpine hydrochloride Chloride, pirarubicin, plicamycin, porfimer sodium, prednimustine, prednisolone, prednisone, premarin, procarbazine, procrit, raltitrexed, Rebif, rhenium-186 etidronate, rituximab, Roferon A, romurtide, Salagen, Sandostatin, sargramostim, semustine, sizofuran, sobuzoxane, solumedrol, s Treptozocin, strontium-89 chloride, synthroid, tamoxifen, tamsulosin, tasonermin, tastractone, taxotere, teceleukin, temozolomide, teniposide, testosterone propionate, testred, thioguanine, thiotepa, thyrotropin, tiludronic acid, topotecan, toremifene, tositumomab, tastuzumab, teosulfan, trenoin,Trexaol, trimethylmelamine, trimetrexate, triptorelin acetate, triptorelin pamoate, UFT, uridine, valrubicin, vesnarinone, vinblastine, vincristine, vindesine, vinorelbine, virulidzin, zincard, zinostatin-stimaramel, zofran, ABI-007, acolbifene, actimun, afinitak, aminopterin, arzoxifene, asoprisnil, atamestane, atrasentan, avastin , BAY43-9006 (sorafenib), CCI-779, CDC-501, Celebrex, cetuximab, crisnatol, cyproterone acetate, decitabine, DN-101, doxorubicin-MTC, dSLIM, dutasteride, edotecarin, eflornithine, exatecan, fenretinide, histamine dihydrochloride, histrelin hydrogel implant, holmium 166 DOTMP, ibandronate, interferon-gamma, interferon Lon-PEG, ixabepilone, keyhole limpet hemocyanin, L-651582, lanreotide, lasofoxifene, Libra, lonafarnib, miproxifene, minodronic acid, MS-209, liposomal MTP-PE, MX-6, nafarelin, nemorubicin, neovastat, nolatrexed, oblimersen, Onco-TCS, Osidem, paclitaxel polyglutamimate, pamidronate disodium, PN-401, QS-21, quazepam, R-1 549, raloxifene, ranpirnas, 13-cis-retinoic acid, satraplatin, seocalcitol, T-138067, tarceva, taxoplexin, thymosin-alpha-1, tiazofurin, tipifarnib, tirapazamine, TLK-286, toremifene, trans-MID-107R, valspodar, vapreotide, vatalanib, verteporfin, vinflunine, Z-100, zoledronic acid and combinations thereof.

[0037] In some embodiments, the affinity reagent comprises a protein. In some embodiments, the protein is selected from the group consisting of a natural protein, a natural amino acid, a non-natural amino acid, a protein in a phage, a therapeutic protein, a synthetic antibody domain, and combinations thereof. In some embodiments, the protein is selected from the group consisting of a non-natural amino acid, a protein in a phage, a therapeutic protein, a synthetic antibody domain, and combinations thereof.

[0038] In some embodiments, the affinity reagent is selected from the group consisting of designed ankyrin repeat proteins (DARPins), HEL4 Vh domains (Predator), the Z domain of Staphylococcal protein A (Affibody), the archaeal "7 kDa DNA binder" protein family (Affitins), carbohydrate binding molecules (CBD domains), cystine knot miniproteins (knottins), fibronectin type III domains (monobodies, Adnectins), gamma-B-crystallin (Affilins), cystatins (Affimers), triple helix coiled-coil domains (Alphabodies), lipocalin domains (Anticalins), A domains of various membrane receptors (Avimers), the SH3 domain of Fyn (Fynomers), Kunitz domain peptides, and combinations thereof.

[0039] Reactive enzymes The fusion proteins of the present disclosure contain a reactive enzyme that can be coupled to a specific functional group present on the linker to irreversibly inhibit the reactive enzyme. Thus, constructs containing a first fusion protein and a second fusion protein with precisely defined structures can be assembled using a modular approach that uses synthetic linkers that selectively couple to active site residues of the fusion protein reactive enzyme.

[0040] The first fusion protein-reactive enzyme and the second fusion protein-reactive enzyme can 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, mutant 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, mutant of haloalkane dehalogenase, HNH endonuclease, nicking endonuclease, gelatinase B, gelatinase A, stromelysin, fatty acid amide hydrolase, esterase, cytochrome P450, methionine aminopeptidase, and combinations thereof.

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

[0042] In some embodiments, the reactive enzyme is a 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 cognate origin of transfer (oriT) DNA sequence located in a mobile plasmid that also encodes a relaxase protein. Such an enzyme cleaves the oriT DNA sequence at a specific nucleotide residue and covalently binds to the oriT DNA sequence at the cleavage point. The MOB relaxase domain of type I DNA topoisomerase can be characterized by families, with each family member having a similar structure and mechanism. Examples of known MOB relaxase domains of the type I DNA topoisomerase family include the MOB relaxase domains of type I DNA topoisomerase, ... F , M.O.B. H , M.O.B. Q , M.O.B. C , M.O.B. P , and M.O.B. V The OriT sequence for each relaxase family member is specific to the mobile plasmid from which each relaxase protein sequence is derived. 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 Family: pMV158, pE194, pUB110, pBBR1, pTn5520, pTn4555, pIncU, and pIncX, where the subscript "p" denotes plasmid.

[0043] Linker The linker comprises at least a first end and a second end. The linker further comprises a first functional group at the first end specific for irreversibly inhibiting the fusion protein. The linker further comprises a second functional group at the second end specific for irreversibly inhibiting the fusion protein.

[0044] In some embodiments, the linker is a nitrogen-containing C 3~7 The linker may further comprise a heterocycle, a polyoxazoline, a polyacrylomorpholine, a polyvinylpyrrolidone, a polyphosphazene, a polyethylene-co-maleic anhydride, a polystyrene-co-maleic anhydride, a poly(1-hydroxymethylethylene hydroxymethyl formal) ("PHF"), a polyhydroxyalkyl acrylate (e.g., 2-hydroxyethyl methacrylate), a 2-methyacryloyloxy-2'-ethyltrimethylammonium phosphate ("MPC"), or a combination thereof. For example, the linker may comprise a polyoxazoline, a polyacrylomorpholine, a polyvinylpyrrolidone, a polyphosphazene, a polyethylene-co-maleic anhydride, a polystyrene-co-maleic anhydride, a PHF, a polyhydroxyalkyl acrylate, or a MPC.

[0045] In embodiments, the linker is [ka] The present invention includes a group selected from the group consisting of During the ceremony, m is 0 to 10; n is 1 to 100; each p is independently 0, 1, 2, 3, or 4; q is 0, 1, or 2; r is 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 is independently H, OH, NH2, or COOH.

[0046] In some embodiments, the linker is [ka] 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 to 50, 1 to 40, 1 to 30, 1 to 20, 1 to 25, 1 to 20, 1 to 15, 1 to 10, or 1 to 5.

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

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

[0049] In some embodiments, the linker is [ka] In some embodiments, G is O. In some embodiments where G is O, each R 11 can independently be H or OH. In embodiments, G is CH2. In embodiments where G is CH2, each R 11 can independently be H or OH, provided that at least one R 11 is OH. In some embodiments, each R 11 In various embodiments where G is CH2, each R 11 can be independently H or NH2, provided that at least one R 11 is NH2. In various embodiments where G is CH2, each R 11 can be independently H or COOH, provided that at least one R 11 is COOH. In embodiments, G is CHOH. In embodiments where G is CHOH, each R11 may independently be H or OH. In some embodiments, each R 11 is OH. In some embodiments, G is CHNH. In some embodiments where G is CHNH, each R 11 can independently be H or NH. In some embodiments, G is CHCOOH. In some embodiments where G is CHCOOH, each R 11 can be independently H or COOH, provided that at least one R 11 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 some embodiments, the linker is a nitrogen-containing C 3~7 They include heterocycles (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 some embodiments, the linker comprises a polyalkylene oxide. The polyalkylene oxide may be linear or branched. For example, the linker may comprise 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 termini, 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 termini, the linker reacts with the fusion protein-reactive enzyme site-specifically, in high yield and with rapid kinetics under mild conditions to provide an at least bivalent antibody construct. The functional groups at the linker termini can be any functional group that irreversibly inhibits the reactive enzyme.

[0053] Examples of functional groups include p-nitrophenylphosphonate, O 6-benzylguanine, α-haloalkanes or derivatives thereof, haloaromatic compounds or derivatives thereof, beta-lactams or derivatives thereof such as clavulanic acid or derivatives thereof, aglycones or derivatives thereof, hydroxamic acid-benzophenones or derivatives thereof, cognate oriT oligonucleotide sequences, cysteine ​​reactive ATP binding site inhibitors, quinone methides or derivatives thereof, α-halophosphonic acids or precursors or derivatives thereof, formylchromones or derivatives thereof, cognate RNA sequences, adenosine or derivatives thereof, cytosine or derivatives thereof, cognate DNA nicking sites, thiirane or derivatives thereof, hydroxamic acids or derivatives thereof, α-ketoxazole inhibitors or derivatives thereof, electrophilic steroids, phosphonates, carbamates, aromatic alkynes, verolanib or derivatives thereof, and combinations thereof.

[0054] In embodiments, the functional group is p-nitrophenylphosphonate, O 6 In some embodiments, the functional group is selected from the group consisting of p-nitrophenylphosphonate, O-benzylguanine, α-haloalkane or derivative thereof, haloaromatic compound or derivative thereof, beta-lactam or derivative thereof such as clavulanic acid or derivative thereof, hydroxamic acid-benzophenone or derivative thereof, cognate oriT oligonucleotide sequence, and combinations thereof. 6 -benzylguanine, an α-haloalkane or derivative thereof, a haloaromatic compound or derivative thereof, and combinations thereof.

[0055] In some embodiments, the functional group is a cognate oriT oligonucleotide sequence. In a refinement of this embodiment, the cognate 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 some embodiments, the fusion protein reactive enzyme is a relaxase domain of a type I DNA topoisomerase and the functional group is a cognate oriT oligonucleotide sequence. In some embodiments, the relaxase domain of a type I DNA topoisomerase (specified by a mobilizable plasmid for each relaxase protein sequence) / cognate oriT oligonucleotide sequence pair is pR100 / 5'-TTTGCGTAGTGTGTGGTGCTTT-3' (SEQ ID NO: 1), pF / 5'-TTTGCGTGGGGTGTGGTGCTTT-3' (SEQ ID NO: 2), TraI P307 / 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 embodiments, the first fusion protein reactive enzyme comprises a cutinase and the first terminal functional group comprises p-nitrophenylphosphonate.

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

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

[0060] In 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 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 embodiments, the first fusion protein-reactive enzyme comprises a matrix metalloproteinase and the first terminal functional group comprises a hydroxamic acid-benzophenone or a derivative thereof.

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

[0064] In 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 embodiments, the first fusion protein-reactive enzyme comprises alkaline phosphatase and the first terminal functional group comprises a functional group selected from the group consisting of a quinone methide, an α-halophosphonic acid, precursors thereof, derivatives thereof, and combinations thereof.

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

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

[0068] In 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 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 some 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 embodiments, the first fusion protein-reactive enzyme comprises a DNA (cytosine-5-)-methyltransferase and the first terminal functional group comprises a cytosine or a derivative thereof.

[0072] In embodiments, the first fusion protein-reactive enzyme comprises a mutant of a haloalkane dehalogenase and the first terminal functional group comprises a functional group selected from the group consisting of a haloalkane, a haloaromatic compound, or a derivative thereof.

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

[0074] In embodiments, the first fusion protein-reactive enzyme comprises a nicking endonuclease and the functional group at the first end comprises a cognate DNA nicking site.

[0075] In embodiments, 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.

[0076] In embodiments, the first fusion protein-reactive enzyme comprises gelatinase A 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.

[0077] In embodiments, the first fusion protein-reactive enzyme comprises stromelysin 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.

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

[0079] In 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 a phosphonate, a carbamate, derivatives thereof, and combinations thereof.

[0080] In 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 electrophilic steroids, aromatic alkynes, derivatives thereof, and combinations thereof.

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

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

[0083] In some embodiments, the second fusion protein reactive enzyme comprises a SnapTag and the functional group at the second terminus is O 6 -Contains benzylguanine.

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

[0085] In 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 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 embodiments, the second fusion protein-reactive enzyme comprises a matrix metalloproteinase and the second terminal functional group comprises a hydroxamic acid-benzophenone or a derivative thereof.

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

[0089] In 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 embodiments, the second fusion protein-reactive enzyme comprises alkaline phosphatase and the second terminal functional group comprises a functional group selected from the group consisting of a quinone methide, an α-halophosphonic acid, precursors thereof, derivatives thereof, and combinations thereof.

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

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

[0093] In 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 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 some embodiments, the second fusion protein-reactive enzyme comprises an N(4)-cytosine-specific DNA methylase and the second terminal functional group comprises cytosine or a derivative thereof.

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

[0097] In embodiments, the second fusion protein-reactive enzyme comprises a mutant of a haloalkane dehalogenase and the second terminal functional group comprises a functional group selected from the group consisting of a haloalkane, a haloaromatic compound, or a derivative thereof.

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

[0099] In embodiments, the second fusion protein-reactive enzyme comprises a nicking endonuclease and the functional group at the second end comprises a cognate DNA nicking site.

[0100] In embodiments, 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.

[0101] In 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 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 embodiments, the second fusion protein reactive enzyme comprises a fatty acid amide hydrolase and the second terminal functional group comprises an alpha-ketoxazole inhibitor or a derivative thereof.

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

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

[0106] In embodiments, the second fusion protein reactive enzyme comprises a methionine aminopeptidase and the second terminal functional group comprises beloranib or a derivative thereof.

[0107] Methods for preparing constructs 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 a first end, thereby coupling the first fusion protein and the linker at the first end; and (b) contacting a second fusion protein comprising an affinity reagent and a reactive enzyme with a second end of the linker, wherein the second end 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 end.

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

[0109] The method disclosed herein specifically couples a fusion protein comprising an affinity reagent and a reactive enzyme to the reactive enzyme in a modular manner using a terminally functionalized small molecule linker that reacts with the reactive enzyme in a site-specific manner with rapid kinetics under mild reaction conditions.

[0110] The methods disclosed herein provide one or more advantages, for example, providing a means by which a highly diverse population of therapeutic constructs can be generated in a modular fashion. In addition, the preparation of antibody formats with extended functionality is straightforward with the disclosed constructs, since reactive amino acids do not need to be directly engineered / functionalized on the antibody scaffold. Furthermore, the mild, rapid, and site-specific nature of the coupling of the fusion protein with the linker allows a wide variety of effector molecules to be attached to the antibody fragment in high yield and with precise stoichiometry, away from the antibody-like fragment. Furthermore, molecules that cannot be expressed in the homologous host with the antibody fragment, such as toxins or glycosylated proteins, can be expressed in various organisms and attached to the construct in a modular fashion. Modular synthesis allows the creation of molecules that are too large or complex to be prepared using standard protein engineering / expression, but such methods require the polypeptide to be expressed as one or more chains in culture.

[0111] Chemical coupling modular approaches allow efficient methods to generate precisely defined protein constructs of variable stoichiometry, orientation, and geometry. These attributes can be systematically varied by coding diversity into the linker and / or by varying the order of attachment of fusion proteins to the linker. Current protein engineering methods are limited to the inherent repertoire of natural polypeptide folds / peptide linkers to arrive at the desired construct. Therefore, non-natural formats that may exhibit greater efficacy cannot be prepared using conventional engineering methods.

[0112] Finally, expression of smaller antibody-like fragments allows 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 allow for lower cost manufacturing platforms, in addition to product yields that are often five times higher than eukaryotic expression systems.

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

[0114] In some embodiments, steps (a) and (b) are carried out sequentially (i.e., stepwise). In some embodiments, steps (a) and (b) are carried out 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 comprising a functional group at a first end and a functional group at a second end can be selected from any of the linkers and functional groups disclosed herein.

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

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

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

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

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

[0120] In embodiments, the first fusion protein-reactive enzyme and the second fusion protein-reactive enzyme are different, the functional group specific for irreversibly inhibiting the first fusion protein-reactive enzyme at the first end of the linker is different from the functional group specific for irreversibly inhibiting 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 offer one or more advantages, such as allowing for the scalable preparation of molecules having molecular weights greater than about 100 kDa, about 150 kDa, about 200 kDa, about 250 kDa and / or about 300 kDa, and / or the generation of diverse libraries of antibody-like drugs with altered valencies, geometries, effector functions, and stoichiometries by simply combining a relatively small number of building blocks.

[0122] Steps (a) and (b) can be carried out in any aqueous solution buffered at physiological pH, for example, phosphate buffered saline (PBS) at pH 7.4. The concentrations of the first fusion protein, the second fusion protein, and the linker can generally be any concentration. The concentrations are typically selected so that the first fusion protein, the second fusion protein, and the linker are completely soluble in the selected solvent without forming a saturated solution. When the linker contains only functional groups specific for irreversibly inhibiting the enzyme at the first and second ends, the relative concentrations of the first fusion protein, the linker, and the second fusion protein are at least about 1:1:1, providing one functional group specific for irreversibly inhibiting the enzyme linker end per fusion protein.

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

[0124] In some embodiments, the construct can be prepared on a solid support. In some 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 of the disclosure to a patient in need thereof. Yet another aspect of the disclosure provides the use of the construct of the disclosure as a medicament for a patient in need thereof. Another aspect of the disclosure provides the use of the construct of the disclosure as a diagnostic agent.

[0126] In some embodiments, the patient is selected from the group consisting of breast cancer, pulmonary anthrax, rheumatoid arthritis, systemic juvenile idiopathic arthritis, Hodgkin's lymphoma, chronic lymphocytic leukemia, follicular non-Hodgkin's 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 metastases, giant cell tumor of bone, malignant ascites, psoriatic arthritis, ankylosing spondylitis, metastatic renal cell carcinoma, prostate cancer, ovarian cancer, multiple myeloma, Castleman's disease, colorectal cancer, and combinations thereof.

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

[0128] In embodiments, at least one antibody or fragment thereof of the construct includes, but is not limited to, trastuzumab or a fragment thereof, racivacaumab 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 embodiments, the patient is afflicted with breast cancer and at least one antibody or fragment thereof of the construct comprises trastuzumab or a fragment thereof.

[0130] In embodiments, the patient is suffering from pulmonary anthrax and at least one antibody or fragment thereof of the construct comprises racivacaumab or a fragment thereof.

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

[0148] The constructs, methods, and uses according to the present disclosure can be better understood in view of the following examples, which are intended merely to illustrate the constructs and are not meant in any way to limit their scope. EXAMPLES

[0149] Example 1: Gene / protein expression vector construction for F(ab) domain derived from trastuzumab The F(ab) domain (light chain variable V) from the monoclonal antibody trastuzumab (hereafter referred to as "TFab(s)") L -Light chain constant kappa chain C κ , and the heavy chain variable domain V H -heavy chain constant domain C HThe gene encoding 1) was synthesized with codon optimization for E. coli by Genscript. The following eight bicistronic 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 NOs: 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 NOs: 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 NOs: 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 NOs: 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' (SEQ ID NOs: 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' (SEQ ID NOs: 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' (SEQ ID NOs: 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' (SEQ ID NOs: 22 and 23)

[0150] Figure 1 shows trastuzumab F(ab) (TFab) fusion gene / protein constructs. 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 Vectors encoding the eight TFab fusion proteins from Example 1 were transformed into Shuffle E. coli (New England Biolabs). Expression cultures containing growth medium (500 mL x 2 x YT), carbenicillin (200 μg / mL) and spectinomycin (50 μg / mL) were inoculated with overnight cultures (5 mL) of cell stocks carrying the appropriate expression cassettes. Cultures were grown at 30°C with shaking at 250 rpm until they reached an optical dispersion (OD) of approximately 0.8. At this point, anhydrotetracycline (aTc) was added to the cultures at a final concentration of 200 ng / mL to induce protein expression. Cultures were maintained at 24°C with shaking at 250 rpm for 14 hours during the expression phase.

[0152] After expression, cells were harvested via centrifugation and lysed via chemical disruption using 35 mL of CelLytic B (Sigma Aldrich) containing DNase I and EDTA-free protease inhibitors (Roche). The cell lysate was centrifuged to remove debris and loaded onto a column containing cobalt IMAC resin (bed volume 3 mL). The lysate was incubated on the beads for 1 h with agitation at 4° C. After this period, the beads were washed extensively with phosphate-buffered saline (PBS) and then the product was eluted with an imidazole-containing buffer (20 mL). The protein eluate was then first purified on a Protein L column (GE Healthcare) followed by size-exclusion chromatography (SEC) on a Superdex 200 column (GE Healthcare) via a fast protein liquid chromatography (FPLC) instrument. All column purifications via FPLC were performed according to the manufacturer's suggested protocol. Fractions containing pure protein product were pooled and concentrated with a centrifugal concentrator before ligation reactions were performed.

[0153] Figure 2 shows a 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. HLanes 3 and 4 show a band close to the calculated molecular weight of the fusion. H The bands derived from the fusion are shown. Lanes 5 and 6, and 7 and 8, respectively, show the C-terminal C K and C H1 The bands derived from the fusion are indicated.

[0154] Thus, Examples 1 and 2 demonstrate the preparation of a fusion protein of the present 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 with a SnapTag-TFab fusion protein [ka] p-nitrophenylphosphonate at the first end and O 6 A linker according to the present disclosure comprising -benzylguanine, ethylene glycol, and amide units was prepared according to the synthetic scheme shown below.

[0156] 1-Amino-N-(4-(((2-amino-9H-purin-6-yl)oxy)methyl)benzyl)-3,6,9,12,15,18-hexaoxaheneicosanoic acid (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 was added DIC (149 mg, 183 μL, 1.18 mmol) followed by NHS (136 mg, 1.18 mmol) and the reaction was allowed to stir overnight at room temperature. The solvent was removed on a rotary evaporator and the resulting yellow oil was resuspended in 5 mL of DMF. To this solution was added (4-aminomethyl)-O 6-benzylguanine (266 mg, 0.99 mmol) was added and the reaction was allowed to stir at room temperature for 2 days. After this period, DMF was removed from the reaction under a stream of N2 overnight. The resulting orange oil was then treated with 5 mL of 20% piperidine in MeOH and allowed to stir at room temperature for 1 h. The solvent was removed using a rotary evaporator and the resulting orange oil was resuspended in 10 mL of HO. The solution was then centrifuged to pellet any insoluble material and the clarified supernatant was purified by reverse phase C purification via a Waters Delta 400 HPLC. 18 The column was applied in five separate portions. Elution was performed with a linear gradient of 75% ACN in deionized ultrafiltered water (DIUF) + 0.01% TFA over 60 min at a flow rate of 10 mL / min with DIUF + 0.01% TFA as the mobile phase. Fractions from 20 to 35 min were then analyzed by MALDI-MS. Pure fractions were pooled and lyophilized to give an amber oil (424 mg, 71%).

[0157] 1-(4-4-(((2-amino-9H-purin-6-yl)oxy)methyl)phenyl)-3,25-dioxo-6,9,12,15,18,21-hexaoxa-2,24-diazaoctacosan-28-oic 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 the solution. When the reaction was complete (approximately 30 min as monitored by MALDI-MS), the solvent was removed on a rotary evaporator. The resulting amber oil was then purified via silica gel chromatography using 10:1 CH2Cl2:MeOH to remove impurities and then eluted with MeOH to give a gummy amber solid (42 mg, 72%).

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

[0159] Ethyl (4-nitrophenyl) (1-(4-(((2-amino-9H-purin-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 was added PyBOP (34 mg, 58 μmol) followed by N-methylmorpholine (5.6 mg, 6.1 μL, 55 μmol) and the reaction was allowed to stir at room temperature for 30 min. 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 was allowed to stir overnight (approximately 16 hours). After this period, the mixture was diluted to 5 mL with DIUF and the mixture was centrifuged to remove insoluble material. The clarified supernatant was then purified by C chromatography via a Waters Delta 4000 HPLC. 18 The product was injected onto a semi-prep scale column and purified at a flow rate of 10 mL / min using a linear gradient of 75% ACN in water + 0.1% TFA in DIUF + 0.1% TFA as the mobile phase over 70 min. Fractions were collected at a rate of 1 / min. Fractions from 25-40 min were then analyzed by MALDI. Those containing pure product were pooled, frozen at -80°C, and lyophilized to give a yellow oil. This oil was then subjected to one further round of HPLC purification using the procedure described above. Pure fractions from this run were pooled, frozen, and lyophilized to give a pale yellow oil (11 mg, 17%).

[0160] Thus, Example 3 illustrates the preparation of a linker of the present disclosure that includes a first functional group specific for irreversibly inhibiting a first fusion protein-reactive enzyme at a first end and a second functional group specific for irreversibly inhibiting a second fusion protein-reactive enzyme at a second end.

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

[0162] Figure 5 shows an SDS-PAGE gel of the prepared constructs. From a pool of 8 TFabs and one linker, 16 bivalent analog constructs can be generated.

[0163] Thus, Example 4 illustrates the preparation of a library of constructs of the present disclosure comprising a first fusion protein, a second fusion protein, and a linker, where 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 for irreversibly inhibiting the first fusion protein reactive enzyme at a first end and a second functional group specific for irreversibly inhibiting the second fusion protein reactive enzyme at a second end.

[0164] Example 5: Cell growth inhibition assay Trastuzumab is employed in the treatment of 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 established protocols and treated for 96 hours with multiple doses of trastuzumab or one of the bivalent trastuzumab analogs prepared in Example 4 at concentrations ranging from 4 pM to 4 μM. Viability after this period was measured using the Alamar blue assay (Life Technologies). Viability was calculated as a percentage value relative to the untreated control.

[0165] From these experiments, one construct, designated 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 with HER2(-)AT-431 cells showing no growth inhibition against trastuzumab or B-13.

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

[0167] Example 6: In vivo pharmacokinetics of Di-TFab constructs B-13 constructs (2 mg / mL) were administered as an intravenous bolus to SCID beige mice for a total dose of 10 mg / kg. Blood samples (approximately 200 μL) were collected from the tail vein at various time points after administration. A total of three mice (n=3) were treated and sampled for each time point. An ELISA method was used to measure the concentration of B13 in serum via standard curve comparison. These data were plotted and fitted to a two-compartment (biexponential) pharmacokinetic model, as shown in Figure 5, to obtain a mean serum B13 concentration of 0.22 h. -1 An elimination rate constant of 0.01 and a half-life of 3.1 hours were obtained.

[0168] Thus, Example 6 provides in vivo uses of the fusion protein constructs of the present disclosure, as well as pharmacokinetic data for the constructs of the present disclosure.

[0169] Example 7 - Effect of B-13 Administration on Mice Bearing BT474 Tumor Xenografts Eight-week-old female SCID beige mice were obtained from Charles River Laboratories and implanted with 0.025 mg, 90-day release 17β-estradiol pellets (Innovative Research of America). After 2 days, 2 × 10 6 BT474 cells were resuspended in 100 μl of PBS and inoculated orthotopically into the mammary fat pad in a 1:1 mixture with Matrigel. Tumors grew to approximately 150 mm 3 Once tumor volumes reached 100-200 mg / kg, 20 animals were randomly assigned to 4 treatment cohorts (n=5) with equal mean tumor volumes. In one cohort, B13 was administered by intraperitoneal (IP) injection 5 times per week (Monday-Friday) for 4 weeks for a total of 20 injections of 5 mg / kg. -1 In the first 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 via IP injection twice weekly at 2.5 mg / kg for a total of eight doses over four weeks. The control cohort was injected five times weekly with vehicle (PBS + 0.004% (w / w) polysorbate 20). Both B13 and dox solutions were formulated in the same vehicle. B13 samples contained less than 1.0 endotoxin units (EU) / mL as determined by Limulus amebocyte lysate (LAL) assay. Tumor dimensions were measured sequentially every two days and were calculated using the formula V = (L × W 2 The volume was calculated using the formula: V=volume, L=length, W=width. The results are shown in FIG.

[0170] B13 shows significant antitumor activity in the BT474 mouse xenograft model. Furthermore, B13 shows no outward signs of toxicity at the two doses used in this study. This is in contrast to dox, where treatment cohorts showed significant weight loss.

[0171] The foregoing description is given for clarity of understanding only, and no unnecessary limitations should be understood therefrom, since modifications within the scope of the invention may be apparent to those skilled in the art.

Claims

1. A construct comprising: a first fusion protein, a second fusion protein, and a linker; the first fusion protein comprises a first reactive enzyme and a first affinity reagent, and the second fusion protein comprises a second reactive enzyme and a second affinity reagent; the linker comprises a first functional group at a first end specific for irreversibly inhibiting the first reactive enzyme and a second functional group at a second end specific for irreversibly inhibiting the second reactive enzyme; wherein at least one of the first or second affinity reagents comprises an antibody or fragment thereof that binds to the same target epitope as an antibody; A construct, wherein the first and second reactive enzymes are independently selected from cutinase, HaloTag, and SnapTag, the first reactive enzyme is different from the second reactive enzyme, the first functional group is different from the second functional group, the first functional group is coupled to the first reactive enzyme and the second functional group is coupled to the second reactive enzyme, such that the first fusion protein and the second fusion protein are linked in the form of a construct.

2. The construct described in claim 1, wherein the first and / or second affinity reagent is selected from the group consisting of an antibody or fragment thereof, a small molecule, a monobody, a protein, and combinations thereof.

3. The construct of claim 1, wherein the first affinity reagent is selected from the group consisting of a light chain variable domain (VL), a light chain constant domain (C L), a heavy chain variable domain (VH), a heavy chain constant domain (C H1), and combinations thereof; and the second affinity reagent is selected from the group consisting of a light chain variable domain (VL), a light chain constant domain (C L), a heavy chain variable domain (VH), a heavy chain constant domain (C H1), and combinations thereof.

4. The first and / or second antibody or fragment thereof is selected from the group consisting of adalimumab, alemtuzumab, arcitumomab, cetuximab, trastuzumab, imciromab, capromab, infliximab, abciximab, rituximab, basiliximab, palivizumab, nofetumomab, omalizumab, daclizumab, ibritumomab tiuxetan, muromomab, edrecolomab, gemtuzumab ozogamicin, golimumab, certolizumab, eculizumab, 4. The construct of claim 1, wherein the IL-16A is selected from the group consisting of IL-16A, IL-16B, IL-16C, IL-16D, IL-16E, IL-16F ...

5. One of the first and / or second affinity reagents comprises a small molecule, the small molecule being a drug, and the drug is selected from the group consisting of aldesleukin, alendronate, alphaferon, alitretinoin, allopurinol, alloprim, aloxi, altretamine, aminoglutethimide, L-type asparaginase, amifostine, amrubicin, amsacrine, anastrozole, anzumet, aranesp, aruglavin, arsenic trioxide, aromasin, 5-azacytidine, azathioprine, BCG or Tice BCG, bestatin, betamethasone acetate, betamethasone phosphate, and the like. Tazone sodium, bexarotene, bleomycin sulfate, broxuridine, bortezomib, bleomycin, busulfan, calcitonin, campath, capecitabine, carboplatin, carmustine, casodex, cefezone, cermoleukin, cerbidine, chlorambucil, cisplatin, colspase, cladribine, clodronate, cyclophosphamide, cytarabine, dacarbazine, dactinomycin, daunoxome, decadron, decadron phosphate, delestrogen, denileukin diftitox, depo-medrol, deslorelin , dexarazoxane, daunorubicin, diethylstilbestrol, 2',2'-difluorodeoxycytidine, diflucan, docetaxel, doxifluridine, doxorubicin, dronabinol, DW-166HC, Eligard, Erytech, Elence, Emend, epirubicin, epoetin-alpha, epogen, eptaplatin, ergamisole, estrase, estradiol, estramustine sodium phosphate, ethinyl estradiol, ethiol, etidronic acid, etopofos, etoposide, fadrozole, falston, f Filgrastim, finasteride, fuligrastim, floxuridine, fluconazole, fludarabine, fludarabine phosphate, 5-fluorodeoxyuridine, 5-fluorodeoxyuridine monophosphate, 5-fluorouracil (5-FU), fluoxymesterone, flutamide, hexamethylmelamine, formestane, fosteabine, fotemustine, fulvestrant, gammaguard, gemcitabine, gemtuzumab, Gleevec, Gliadel, goserelin, granisetron hydrochloride, histrelin, hicamucine,Hydrocortone, erythro-hydroxocinonyl 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, Kytril, lentinan sulfate, letrozole, leucovorin, leuprolide, leuprolide acetate, levamisole, levofolic acid acid) calcium salt, levothroid, levoxyl, lomustine, lonidamine, marinol, mechlorethamine, mecobalamin, medroxyprogesterone acetate, megestrol acetate, melphalan, menest, 6-mercaptopurine, mesna, methotrexate, metobix, miltefosine, minocycline, mitomycin C, mitotane, mitoxantrone, modrenal, myoset, nedaplatin, neulasta, neumega, neupogen, nilutamide, nolvadex, NSC-631570, OCT-43, octreotide, ondansetron hydrochloride, orapred, oxaliplatin, paclitaxel, pediapred, pegaspargase, pegasys, pentostatin, N-phosphonoacetyl L-aspartic acid (PALA), picibanil, pyro Carpin hydrochloride, pirarubicin, plicamycin, porfimer sodium, prednimustine, prednisolone, prednisone, premarin, procarbazine, procrit, raltitrexed, Rebif, rhenium-186 etidronate, rituximab, Roferon A, romurtide, Salagen, sandostatin, sargramostim, semustine, schizofuran, sobuzoxane, solumedrol, streptozocin, strontium-89 chloride, synthroid, tamoxifen, tamsulosin, tasonermin, tastractone, taxotere, teceleukin, temozolomide, teniposide, testosterone propionate, testred, thioguanine, thiotepa, thyrotropin, tiludronic acid, topotecan, toremifene, tositumomab, tastuzumab,Theosulfan, tretinoin, trexaol, trimethylmelamine, trimetrexate, triptorelin acetate, triptorelin pamoate, UFT, uridine, valrubicin, vesnarinone, vinblastine, vincristine, vindesine, vinorelbine, virulidzin, zincard, zinostatin-stimaramel, zofran, ABI-007, acolbifene, actimun, affinitak, aminopterin, arzoxifene, asoprisnil, atamestane, atrace , Avastin, BAY 43-9006 (Sorafenib), CCI-779, CDC-501, Celebrex, Cetuximab, Crisnatol, Cyproterone acetate, Decitabine, DN-101, Doxorubicin-MTC, dSLIM, Dutasteride, Edotecarin, Eflornithine, Exatecan, Fenretinide, Histamine dihydrochloride, Histrelin hydrogel implant, Holmium 166 DOTMP, Ibandronate, Interferon-gamma, Interferon-α, Interferon-β, Interferon-β-gamma ... -feron-PEG, ixabepilone, keyhole limpet hemocyanin, L-651582, lanreotide, lasofoxifene, libra, lonafarnib, miproxifene, minodronic acid, MS-209, liposomal MTP-PE, MX-6, nafarelin, nemorubicin, neovastat, nolatrexed, oblimersen, onco-TCS, ocidem, paclitaxel polyglutamate, disodium pamidronate, PN-401, QS-21, quazepam, R-1549, 3. The construct of claim 2, which is selected from the group consisting of raloxifene, ranpirnas, 13-cis-retinoic acid, satraplatin, seocalcitol, T-138067, tarceva, taxoplexin, thymosin-alpha-1, tiazofurin, tipifarnib, tirapazamine, TLK-286, toremifene, trans-MID-107R, valspodar, vapreotide, vatalanib, verteporfin, vinflunine, Z-100, zoledronic acid, and combinations thereof.

6. A construct described in claim 1 or 2, wherein at least one of the first and / or second affinity reagents is a protein, a protein in a phage or a therapeutic protein comprising at least one unnatural amino acid.

7. A construct described in claim 1 or 2, wherein the first and / or second affinity reagent is a synthetic antibody domain.

8. The construct of claim 1, wherein the first and / or second affinity reagent is selected from the group consisting of designed ankyrin repeat proteins (DARPins), HEL4 Vh domains (Predators), the Z domain of Staphylococcal protein A (Affibodies), the archaeal "7 kDa DNA binder" protein family (Affitins), carbohydrate binding molecules (CBD domains), cystine knot mini-proteins (knottins), fibronectin type III domains (monobodies, Adnectins), gamma-B-crystallin (Affilins), cystatins (Affimers), triple helix coiled-coil domains (Alphabodies), lipocalin domains (Anticalins), A domains of various membrane receptors (Avimers), the SH3 domain of Fyn (Fynomers), Kunitz domain peptides, and combinations thereof.

9. The method of claim 8, wherein the linker is selected from polyoxazoline, polyacrylomorpholine, polyvinylpyrrolidone, polyphosphazene, polyethylene-co-maleic anhydride, polystyrene-co-maleic anhydride, poly(1-hydroxymethylethylene hydroxymethyl formal) ("PHF"), polyhydroxyalkyl acrylate, 2-methacryloyloxy-2'-ethyltrimethylammonium phosphate ("MPC"), or a structure selected from the following: 【Chemistry 1】 and During the ceremony, m is 0 to 10; n is 1 to 100; each p is independently 0, 1, 2, 3, or 4; q is 0, 1 or 2; r is 1 or 2; E is NH or CHR 10 ; G is O, CH2, CHOH, CHNH2, CHCOOH or CHSO3H; R 10 is OH, NH 2 or COOH; The construct of any one of claims 1-3 and 5-8, wherein each R 11 is independently H, OH, NH 2 or COOH.

10. A method comprising: (a) contacting a first fusion protein comprising a first affinity reagent and a first reactive enzyme with a linker comprising a functional group specific for irreversibly inhibiting said first reactive enzyme at a first end, thereby coupling said first fusion protein and said linker at said first end; (b) contacting a second fusion protein comprising a second affinity reagent and a second reactive enzyme with a second end of the linker, the second end of the linker comprising a functional group specific for irreversibly inhibiting the second reactive enzyme, thereby coupling the second fusion protein and the linker at the second end; wherein at least one of the first and second affinity reagents comprises an antibody or fragment thereof that binds to the same target epitope as an antibody; The method, wherein the reactive enzymes of the first and second fusion proteins are independently selected from cutinase, HaloTag, and SnapTag, the reactive enzyme of the first fusion protein is different from the reactive enzyme of the second fusion protein, and the first functional group is different from the second functional group.

11. The method of claim 10, wherein steps (a) and (b) are performed simultaneously.

12. The method described in claim 10 or 11, wherein the first and / or second affinity reagent is selected from the group consisting of an antibody or fragment thereof, a small molecule, a monobody, a protein, and combinations thereof.

13. The method of claim 10 or 11, wherein the first and / or second affinity reagent is selected from the group consisting of designed ankyrin repeat proteins (DARPins), HEL4 Vh domains (Predators), the Z domain of Staphylococcal protein A (Affibodies), the archaeal "7 kDa DNA binder" protein family (Affitins), carbohydrate binding molecules (CBD domains), cystine knot mini-proteins (knottins), fibronectin type III domains (monobodies, Adnectins), gamma-B-crystallin (Affilins), cystatins (Affimers), triple helix coiled-coil domains (Alphabodies), lipocalin domains (Anticalins), A domains of various membrane receptors (Avimers), the SH3 domain of Fyn (Fynomers), Kunitz domain peptides, and combinations thereof.

14. The method of claim 13, wherein the linker is selected from polyoxazoline, polyacrylomorpholine, polyvinylpyrrolidone, polyphosphazene, polyethylene-co-maleic anhydride, polystyrene-co-maleic anhydride, poly(1-hydroxymethylethylene hydroxymethyl formal) ("PHF"), polyhydroxyalkyl acrylate, 2-methacryloyloxy-2'-ethyltrimethylammonium phosphate ("MPC"), or a structure selected from the following: 【Chemistry 2】 and During the ceremony, m is 0 to 10; n is 1 to 100; each p is independently 0, 1, 2, 3, or 4; q is 0, 1 or 2; r is 1 or 2; E is NH or CHR 10 ; G is O, CH2, CHOH, CHNH2, CHCOOH or CHSO3H; R 10 is OH, NH 2 or COOH. The method of any one of claims 10 to 13, wherein each R 11 is independently H, OH, NH 2 or COOH.

15. A construct prepared by a method according to any one of claims 10 to 14.

16. A pharmaceutical agent for treating a patient in need thereof, the patient being afflicted with breast cancer, pulmonary anthrax, rheumatoid arthritis, systemic juvenile idiopathic arthritis, Hodgkin's lymphoma, systemic anaplastic large cell lymphoma, chronic lymphocytic leukemia, non-Hodgkin's lymphoma, diffuse large B-cell lymphoma, multiple sclerosis, systemic lupus erythematosus, gastric or gastroesophageal junction adenocarcinoma, metastatic non-small cell lung cancer, ulcerative colitis, Crohn's disease, follicular lymphoma, melanoma, macular degeneration, osteoporosis, treatment-induced bone loss, bone metastases, giant cell tumor of bone, malignant ascites, psoriatic arthritis, ankylosing spondylitis, metastatic renal cell carcinoma, prostate cancer, ovarian cancer, colorectal cancer, multiple myeloma, or Castleman's disease, the pharmaceutical agent comprising the construct of any one of claims 1 to 9 or 15.

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