Protein-active agent conjugates and methods of use

Abstract

The invention provides methods for preparing an antibody-active agent conjugate. The conjugate comprises an antibody, a cysteine residue of an amino acid motif that can be recognized by an isoprenoid transferase located at or after the carboxy-terminus of the antibody, an isoprenoid unit operably linked to the cysteine residue, and an active agen. The invention also provides a composition comprising the antibody-active agent conjugate prepared by the methods.

1 - 6 . (canceled) 
     
     
         7 . A method of treating a disease in a subject, comprising administering to the subject an antibody active agent conjugate comprising:
 (a) an antibody;   (b) at least one amino acid motif directly or indirectly linked to a carboxy terminus of the antibody, wherein the amino acid motif is recognizable by an isoprenoid transferase;   (c) an isosubstrate directly linked to a cysteine moiety of the at least one amino acid motif, wherein the isosubstrate contains at least one isoprenoid unit and is recognizable by the isoprenoid transferase; and   (d) at least one active agent directly or indirectly linked to the isosubstrate.   
     
     
         8 . The method of  claim 7 , wherein the antibody is an immunoglobulin molecule. 
     
     
         9 . The method of  claim 7 , wherein the antibody is an intact polyclonal antibody, an intact monoclonal antibody, an antibody fragment, an antibody analog, a single chain Fv (scFv) mutant, a multispecific antibody, a bispecific antibody, a chimeric antibody, a humanized antibody, a human antibody, or a fusion protein comprising an antigen determination portion of an antibody. 
     
     
         10 . The method of  claim 7 , wherein the antibody is Muromonab-CD3, Abciximab, Rituximab, Daclizumab, Palivizumab, Infliximab, Trastuzumab, Basiliximab, Gemtuzumab, Alemtuzumab, Ibritumomab, Adalimumab, Omalizumab, Efalizumab, Tositumomob-I131, Cetuximab, Bevacizumab, Natalizumab, Ranibizumab, Panitumumab, Eculizumab, Certolizumab, Golimumab, Ustekinumab, Briakinumab, Belimumab, anti-CD20, Canakinumab, Tocilizumab, Atlizumab, Mepolizumab, Pertuzumab, Ofatumumab, Tremelimumab, Ticilimumab, Ipilimumab, anti-CD80, Inotuzumab, Zalutumumab, Zanolimumab, Otelixizumab, Catumaxomab, anti-EpCAM antibody, Adecatumomab, Pregovomab, anti-di sialoganglioside antibody, chimeric anti-carbonic anhydrase IV antibody, Denosumab, Bapineuzumab, Motavizumab, Raxibacumab, Ocaratuzumab, Veltuzumab, or an antibody directed against c-Met. 
     
     
         11 . The method of  claim 7 , wherein the antibody comprises at least one light chain. 
     
     
         12 . The method of  claim 11 , wherein the amino acid motif is directly or indirectly linked to the carboxy-terminus of a light chain of the antibody. 
     
     
         13 . The method of  claim 7 , wherein the antibody comprises at least one heavy chain. 
     
     
         14 . The method of  claim 13 , wherein the amino acid motif is directly or indirectly linked to the carboxy-terminus of a heavy chain of the antibody. 
     
     
         15 . The method of  claim 7 , wherein the antibody comprises two light chains and two heavy chains. 
     
     
         16 . The method of  claim 7 , wherein the at least one amino acid motif is CAAX, XXCC, XCXC, or CXX, wherein C represents cysteine, A represents an aliphatic amino acid, and X represents an amino acid that determines a substrate specificity of the isoprenoid transferase. 
     
     
         17 . The method of  claim 7 , wherein the at least one amino acid motif is indirectly linked to the antibody via a spacer group. 
     
     
         18 . The method of  claim 7 , wherein the isosubstrate is indirectly linked to the at least one active agent via at least one linker. 
     
     
         19 . The method of  claim 18 , wherein the linker is a linear linker. 
     
     
         20 . The method of  claim 19 , wherein the linear linker is directly linked to at least one active agent. 
     
     
         21 . The method of  claim 18 , wherein the linker is a linker having branches. 
     
     
         22 . The method of  claim 21 , wherein one or more of the branches are directly linked to at least one of the active agents. 
     
     
         23 . The method of  claim 22 , wherein at least two of the branches are directly linked to different active agents. 
     
     
         24 . The method of  claim 18 , wherein the linker is a cleavable linker. 
     
     
         25 . The method of  claim 24 , wherein the linker is a chemically cleavable linker, an enzymatically cleavable linker, a hydrolysable linker, or a combination thereof. 
     
     
         26 . The method of  claim 25 , wherein the linker is an enzymatically cleavable linker that contains a peptide that can be cleaved by cathepsin B or a glucuronide that can be cleaved by β-glucuronidase. 
     
     
         27 . The method of  claim 7 , wherein the conjugate comprises at least one active agent selected from:
 (a) erlotinib, bortezomib, fulvestrant, sutent, letrozole, imatinib mesylate, PTK787/ZK 222584, oxaliplatin, 5-fluorouracil, leucovorin, rapamycin, lapatinib, lonafarnib, sorafenib, gefitinib, Tyrphostin AG1478, an inhibitor of EGFR tyrosine kinase activity, thiotepa, cyclophosphamide, busulfan, improsulfan, piposulfan, benzodopa, carboquone, meturedopa, uredopa ethylenimine, altretamine, triethylenemelamine, triethylene phosphoramide, triethylene thiophosphoramide, trimethylol melamine, bullatacin, bullatacinone, camptothecin topotecan, bryostatin, callystatin, adozelesin, carzelesin, bizelesin, cryptophycin 1, cryptophycin 8, dolastatin, duocarmycin, KW-2189, CB1-TM1, eleutherobin, pancratistatin, sarcodictyin, spongistatin, chlorambucil, chlornaphazine, cyclophosphamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, novembichin, phenesterine, prednimustine, trofosfamide, uracil mustard, carmustine, chlorozotocin, fotemustine, lomustine, nimustine, calicheamycin, calicheamycin gamma 1, calicheamycin omega 1, dynemicin, dynemicin A, clodronate, esperamicin, neocarzinostatin chromophore, aclacinomysins, actinomycin, anthramycin, azaserine, bleomycins, cactinomycin, carabicin, carninomycin, carzinophilin, chromomycins, dactinomycin, daunorubicin, 6-diazo-5-oxo-L-norleucine, doxorubicin, morpholino-doxorubicin, cyanomorpholino-doxorubicin, 2-pyrrolino-doxorubucin, liposomal doxorubicin, deoxydoxorubicin, epirubicin, esorubicin, marcellomycin, mitomycin C, mycophenolic acid, nogalamycin, olivomycins, peplomycin, potfiromycin, puromycin, quelamycin, rodorubicin, streptomigrin, streptozocin, tubercidin, ubenimex, zinostatin, zorubicin, 5-fluorouracil, denopterin, methotrexate, pteropterin, trimetrexate, fludarabine, 6-mercaptopurine, thiamiprine, tioguanine, ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine, calusterone, dromostanolone propionate, epitiostanol, mepitiostane, testolactone, aminoglutethimide, mitotane, trilostane, folinic acid, aceglatone, aldophosphamide glycoside, aminolevulinic acid, eniluracil, amsacrine, bestrabucil, bisantrene, edatraxate, demecolcine, diaziquone, elfornithine, elliptinium acetate, etoglucid, gallium nitrate, hydroxyurea, lentinan, lonidainine, maytansine, ansamitocins, mitoguazone, mitoxantrone, mopidanmol, nitraerine, pentostatin, phenamet, pirarubicin, losoxantrone, 2-ethylhydrazide, procarbazine, polysaccharide-k, razoxane, rhizoxin, sizofiran, spirogermanium, tenuazonic acid, triaziquone, 2,2′,2″-trichlorotriethylamine, T-2 toxin, verracurin A, roridin A, anguidine, urethane, vindesine, dacarbazine, mannomustine, mitobronitol, mitolactol, pipobroman, gacytosine, arabinoside (‘Ara-C’), cyclophosphamide, thiotepa, paclitaxel, doxetaxel, chlorambucil, gemcitabine, 6-thioguanine, mercaptopurine, cisplatin, carboplatin, vinblastine, platinum, etoposide, ifosfamide, mitoxantrone, vincristine, vinorelbine, novantrone, teniposide, edatrexate, daunomycin, aminopterin, ibandronate, CPT-11, topoisomerase inhibitor RFS 2000, difluoromethylornithine (DFMO), retinoic acid, capecitabine, or pharmaceutically acceptable salts, solvates, or acids of any of the foregoing;   (b) monokine, a lymphokine, a traditional polypeptide hormone, a growth hormone, human growth hormone, N-methionyl human growth hormone, bovine growth hormone, parathyroid hormone, thyroxine, insulin, proinsulin, relaxin, prorelaxin, a glycoprotein hormone, follicle stimulating hormone (FSH), thyroid stimulating hormone (TSH), luteinizing hormone (LH), hepatic growth factor fibroblast growth factor, prolactin, placental lactogen, tumor necrosis factor-α, tumor necrosis factor-β, mullerian-inhibiting substance, mouse gonadotropin-associated peptide, inhibin, activin, vascular endothelial growth factor, integrin, thrombopoietin (TPO), a nerve growth factor, NGF-β, platelet-growth factor, a transforming growth factor (TGF), TGF-α, TGF-β, insulin-like growth factor-I, insulin-like growth factor-II, erythropoietin (EPO), an osteoinductive factor, an interferon, interferon-α, interferon-β, interferon-γ, a colony stimulating factor (CSF), macrophage-CSF (M-CSF), granulocyte-macrophage-CSF (GM-CSF), granulocyte-CSF (G-CSF), an interleukin (IL), IL-1, IL-1α, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, a tumor necrosis factor, TNF-α, TNF-β, a polypeptide factor, LIF, kit ligand (KL), or a combination of any of the foregoing;   (c) diphtheria toxin, botulinum toxin, tetanus toxin, dysentery toxin, cholera toxin, α-amanitin, pyrrolobenzodiazepin derivatives, tetrodotoxin, brevetoxin, ciguatoxin, ricin, AM toxin, auristatin, tubulysin, geldanamycin, maytansinoid, calicheamycin, daunomycin, doxorubicin, methotrexate, vindesine, SG2285, dolastatin, auristatin, cryptophycin, camptothecin, a rhizoxin derivative, duocarmycin, an enediyne antibiotic, esperamicin, epothilone, a toxoid, or a combination of any of the foregoing;   (d) an affinity ligand, wherein the affinity ligand is a substrate, an inhibitor, a stimulating agent, a neurotransmitter, a radioisotope, or a combination of any of the foregoing;   (e) an immunomodulatory compound, an anti-cancer agent, an anti-viral agent, an anti-bacterial agent, an anti-fungal agent, and an anti-parasitic agent, or a combination of any of the foregoing;   (f) tamoxifen, raloxifene, droloxifene, 4-hydroxytamoxifen, trioxifene, keoxifene, LY117018, onapristone, or toremifene;   (g) 4(5)-imidazoles, aminoglutethimide, megestrol acetate, exemestane, letrozole, or anastrozole;   (h) flutamide, nilutamide, bicalutamide, leuprolide, goserelin, or troxacitabine;   (i) an aromatase inhibitor;   (j) a protein kinase inhibitor;   (k) a lipid kinase inhibitor;   (l) an antisense oligonucleotide;   (m) a ribozyme;   (n) a vaccine; or   (o) an anti-angiogenic agent.   
     
     
         28 . The method of  claim 7 , wherein the at least one active agent is linked to an antibody-linker conjugate selected from trastuzumab-LC-G 7 CVIM-BG or trastuzumab-LC-G 7 CVIM-VC.