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(12) Patent Application Publication (10) Pub. No.: US 2004/0081648A1 Afeyan Et Al

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(12) Patent Application Publication (10) Pub. No.: US 2004/0081648A1 Afeyan Et Al US 2004.008 1648A1 (19) United States (12) Patent Application Publication (10) Pub. No.: US 2004/0081648A1 Afeyan et al. (43) Pub. Date: Apr. 29, 2004 (54) ADZYMES AND USES THEREOF Publication Classification (76) Inventors: Noubar B. Afeyan, Lexington, MA (51) Int. Cl." ............................. A61K 38/48; C12N 9/64 (US); Frank D. Lee, Chestnut Hill, MA (52) U.S. Cl. ......................................... 424/94.63; 435/226 (US); Gordon G. Wong, Brookline, MA (US); Ruchira Das Gupta, Auburndale, MA (US); Brian Baynes, (57) ABSTRACT Somerville, MA (US) Disclosed is a family of novel protein constructs, useful as Correspondence Address: drugs and for other purposes, termed “adzymes, comprising ROPES & GRAY LLP an address moiety and a catalytic domain. In Some types of disclosed adzymes, the address binds with a binding site on ONE INTERNATIONAL PLACE or in functional proximity to a targeted biomolecule, e.g., an BOSTON, MA 02110-2624 (US) extracellular targeted biomolecule, and is disposed adjacent (21) Appl. No.: 10/650,592 the catalytic domain So that its affinity Serves to confer a new Specificity to the catalytic domain by increasing the effective (22) Filed: Aug. 27, 2003 local concentration of the target in the vicinity of the catalytic domain. The present invention also provides phar Related U.S. Application Data maceutical compositions comprising these adzymes, meth ods of making adzymes, DNA's encoding adzymes or parts (60) Provisional application No. 60/406,517, filed on Aug. thereof, and methods of using adzymes, Such as for treating 27, 2002. Provisional application No. 60/423,754, human Subjects Suffering from a disease, Such as a disease filed on Nov. 5, 2002. Provisional application No. asSociated with a Soluble or membrane bound molecule, e.g., 60/430,001, filed on Nov. 27, 2002. an allergic or inflammatory disease. Patent Application Publication Apr. 29, 2004 Sheet 1 of 19 US 2004/008 1648A1 A 3 ADO CAT Ald is 6A I AD d as | a do CF Add CA G A = JF (3 add Cer A D D Uear T J O t . AK - Car Patent Application Publication Apr. 29, 2004 Sheet 2 of 19 US 2004/008 1648A1 AV2 yaaZealay Active S/7A SA6s.7A7a erre seasTaafe bipole at 6 up/pp.A44tleA 6.7 AD . Blaid two ra. avoiwa st7Af Ab1" VA A/6. 2. A fig, 2 (3 gassrATE 7 substate AGE7 site A-se bayPala 1(ARGET elp? A feAA. atmplate, s/7 A G 24 Patent Application Publication Apr. 29, 2004 Sheet 3 of 19 US 2004/008 1648A1 7AGF7 Seesaare A-Meeaaya Sayf CD Staszaga TAGEf firf gupatul.46 as s s BModiv, a. S4 so ar wav4 swiri fle, a a? G 2 A Patent Application Publication Apr. 29, 2004 Sheet 4 of 19 US 2004/008 1648A1 subsrAA7 6 rew full GS72Alfa 1AKA67 // 7.64 able ADWe st 7 Set - givipes sa? a AD S/AlA. Meef Cal E t - tworror S F163A (6) Alez. 3 3 7AR6A7 Sdesstre Maeace-f Patent Application Publication Apr. 29, 2004 Sheet 5 of 19 US 2004/008 1648A1 SatALL MoléCee Le 2)/ Ayreffer? - te, 3 C 9 elf 3/wdm 6 417A A/6. 3P sosrite AA6 7A?eer Best &fears /2t24Afana AMN Ital d A D 8. twbhag is 175 S. stre 2 two eye C. C.GS Fig. 3e TARGE 2. AbMAt ACAaf st SeSIAE 5360/We ra Patent Application Publication Apr. 29, 2004 Sheet 6 of 19 US 2004/008 1648A1 F1G. A Patent Application Publication Apr. 29, 2004 Sheet 7 of 19 US 2004/008 1648A1 Enzyme: prethrombin A) Address: ScFvOHA Model ADZYME: prethrombin-(GGGGS)3-scFvOHA Patent Application Publication Apr. 29, 2004 Sheet 8 of 19 US 2004/008 1648A1 Fig. 5 Panel A Panel B 7 8 9 10 KDa Patent Application Publication Apr. 29, 2004 Sheet 9 of 19 US 2004/008 1648A1 Fig. 6 Patent Application Publication Apr. 29, 2004 Sheet 10 of 19 US 2004/0081648A1 Fig. 7 - A -1 ul Adzyme-post cleanup -0-3.3 nM Sigma Thrombin -Yé-1 ul Adzyme - no activation SSSSSSSSS S S S S S rS Time in minutes Patent Application Publication Apr. 29, 2004 Sheet 11 of 19 US 2004/0081648A1 Fig. 8 S --Adzyne-S1 O q -A-Thrombin-S1 -- Adzyme-S2 O l O -A-Thrombin-S2 O C -e-Unactivated Adzyme-St 1.5 time in minutes Patent Application Publication Apr. 29, 2004 Sheet 12 of 19 US 2004/0081648A1 Fig. 9 -o-Adzyme E --Thrombin O ve -A-Thrombin + ScFvaHA 2 D c) -o-ScFWahA O g Time in minutes Patent Application Publication Apr. 29, 2004 Sheet 13 of 19 US 2004/008 1648A1 Fig. 10 100 1 OOOO 1OOOOOO 10OOOOOOO bioactive TNF(pg/ml) Patent Application Publication Apr. 29, 2004 Sheet 14 of 19 US 2004/0081648A1 Fig.11 (LDS-TNF Binding to Captured p55 3Control Binding to Captured p55 E C N O % soluble TNFRp55 supernatant Patent Application Publication Apr. 29, 2004 Sheet 15 of 19 US 2004/008 1648A1 Fig. 12 Patent Application Publication Apr. 29, 2004 Sheet 16 of 19 US 2004/0081648A1 Fig. 13 EK-dependent Adzyme Proteolytic Activity o C) (9 C (9 8 O 2 L Patent Application Publication Apr. 29, 2004 Sheet 17 of 19 US 2004/0081648A1 Fig. 14 Normalization of Trypsin Activities 350 300 250 200 -O-Tgn-20-p55 -A-Tgn-3-p55 100 -- sp55 50 O 5 10 15 20 25 30 35 minutes Patent Application Publication Apr. 29, 2004 Sheet 18 of 19 US 2004/0081648A1 Fig. 15. Detection of TNFo. Binding of Adzymes by ELISA Binding (+TNF) D Binding (TNF) Tgn-0-p55FL Tgn-20-p55FL Tgn-3-p55FL p55FL pSECTAG2A Buffer Controt Samples Patent Application Publication Apr. 29, 2004 Sheet 19 of 19 US 2004/0081648A1 Fig. 16 <d { o : or rr - - - - - - - - - - - - - - - - - - - - - - - - - - - - ------ s as as a papa aaabs a so as as sad . - a a sa a -- was as . Time (hrs) US 2004/008 1648 A1 Apr. 29, 2004 ADZYMES AND USES THEREOF “adzymes', as well as methods and compositions related to the use and production of adzymes. Adzymes are chimeric RELATED APPLICATIONS protein constructs that join one or more catalytic domains 0001. This application claims priority to U.S. Provisional with one or more targeting moieties (or “addresses'). A Patent Application Serial No. 60/406,517, filed Aug. 27, catalytic domain of an adzyme has an enzymatically active 2002, U.S. Provisional Patent Application Serial No. 60/423, Site that catalyzes a reaction converting a pre-Selected Sub 754, filed Nov. 5, 2002, and U.S. Provisional Patent Appli Strate (the “target” or “targeted Substrate”) into one or more cation Serial No. 60/430,001, filed Nov. 27, 2002, the entire products, Such as by cleavage, chemical modifications contents of each of which are incorporated herein by refer (transformations) or isomerization. Such products may have CCC. an altered activity relative to the Substrate, optionally having an increased or decreased activity or an activity that is BACKGROUND OF THE INVENTION qualitatively different. 0002 This invention relates to synthetic protein con Structs useful in modulating a variety of targeted molecules 0008. In certain aspects, the invention provides adzymes in situ. In particular aspects, it relates to a family of comprising a catalytic domain and a targeting moiety, constructs employing linked molecular parts which target wherein the catalytic domain catalyzes a chemical reaction and modulate the activity of a biomolecule catalytically to converting a Substrate into one or more products, and induce a therapeutic effect. wherein the targeting moiety reversibly binds to an address Site that is either on the Substrate or in functional proximity 0003. Many diseases are caused by or associated with with the substrate. Preferably, the targeting moiety binds biomolecules, either free in solution in body fluids or reversibly to the address site. Optionally, Said targeting exposed to extracellular body fluids Such as membrane moiety and Said catalytic domain are heterologous with bound proteins and polysaccharides, Such as cytokines or respect to each other. Generally, Said targeting moiety, when growth factors, and it is widely recognized that it is possible provided Separately, binds to the Substrate, and Said catalytic to develop therapies for Such diseases by modulating the domain, when provided Separately, catalyzes the chemical activity of the biomolecule. reaction converting Said Substrate to one or more products. 0004 For example, overproduction of TNF-C. and/or 0009. In certain embodiments, a catalytic domain and a TNF-B is closely linked to the development of many dis targeting domain of the adzyme are joined by a polypeptide eases, including Septic Shock, adult respiratory distress Syn linker to form a fusion protein. A fusion protein may be drome, rheumatoid arthritis, Selective autoimmune disor generated in a variety of ways, including chemical coupling ders, graft-host disease following bone marrow and cotranslation. In a preferred embodiment, the fusion transplantation and cachexia. Other diseases associated with protein is a cotranslational fusion protein encoded by a excessive TNF-C. and/or TNF-B production include hemor recombinant nucleic acid. In certain embodiments the linker rhagic Shock, asthma and post-renal dialysis Syndrome. The for the fusion protein is an unstructured peptide. Optionally, multiplicity of actions of TNF-C. and TNF-B can be ascribed the linker includes one or more repeats of Ser Gly or to the fact that TNF-C. and/or TNF-B actions result in SerGly. In preferred embodiments, the linker is selected to activation of multiple Signal transduction pathways, kinases, provide Steric geometry between Said catalytic domain and transcription factors, as well as an unusually large array of Said targeting moiety Such that Said adzyme is more effective cellular genes.
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