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Ep 2402438 A2 (19) & (11) EP 2 402 438 A2 (12) EUROPEAN PATENT APPLICATION (43) Date of publication: (51) Int Cl.: 04.01.2012 Bulletin 2012/01 C12N 9/64 (2006.01) C12N 15/10 (2006.01) C12N 9/50 (2006.01) C12N 9/72 (2006.01) (21) Application number: 11173353.1 (22) Date of filing: 05.07.2007 (84) Designated Contracting States: (72) Inventor: MADISON, Edwin AT BE BG CH CY CZ DE DK EE ES FI FR GB GR San Francisco, CA California 94121 (US) HU IE IS IT LI LT LU LV MC MT NL PL PT RO SE SI SK TR (74) Representative: Baldock, Sharon Claire Designated Extension States: Boult Wade Tennant AL BA HR MK RS Verulam Gardens 70 Gray’s Inn Road (30) Priority: 05.07.2006 US 818804 P London WC1X 8BT (GB) 05.07.2006 US 818910 P Remarks: (62) Document number(s) of the earlier application(s) in •Thecomplete document including Reference Tables accordance with Art. 76 EPC: and the Sequence Listing can be downloaded from 07861330.4 / 2 046 951 the EPO website •This application was filed on 08-07-2011 as a (71) Applicants: divisional application to the application mentioned • Catalyst Biosciences, Inc. under INID code 62. South San Francisco, CA 94080 (US) • Torrey Pines Institute For Molecular Studies San Diego, CA 92121 (US) (54) Protease screening methods and proteases identified thereby (57) Methods for identifying modified proteases with modified substrate specificity or other properties are pro- vided. The methods screen candidate and modified pro- teases by contacting them with a substrate, such as a serpin, an alpha macro globulins or a p35 family protein or modified serpins and modified p35 family members or modified alpha macroglobulins, that, upon cleavage of the substrate, traps the protease by forming a stable com- plex. Also provided are modified proteases EP 2 402 438 A2 Printed by Jouve, 75001 PARIS (FR) EP 2 402 438 A2 Description RELATED APPLICATIONS 5 [0001] Benefit of priority is claimed to U.S. Provisional Application Serial No. 60/818,804, to Edwin Madison, entitled "Protease Screening Methods and Proteases Identified Thereby," filed July 05, 2006, and to U.S. Provisional Application Serial No. 60/818,910, to Edwin Madison, entitled "Modified Urinary- Plasminogen Activator (u-PA) Proteases," filed July 05, 2006. Where permitted, the subject matter of the above-noted applications are incorporated by reference in its entirety. [0002] This application is related to U.S. Application Serial No. 11/825,627 to Edwin Madison, entitled "Protease 10 Screening Methods and Proteases Identified Thereby," filed July 05, 2007, which claims priority to U.S. Provisional Application Serial No. 60/818,804 and to U.S. Provisional Application Serial No. 60/818,910. [0003] This application also is related to U.S. Application Serial No. 10/677,977, filed October 02, 2003 and published as U.S. Application No. US- 2004-0146938 on July 29, 2004, entitled " Methods of Generating a nd Screening for Proteases with Altered Specificity" to J Nguyen, C Thanos, S Waugh-Ruggles, and C Craik, and to corresponding published 15 International PCT Application No. WO2004/031733, published April 15, 2004, which claim benefit to U.S. Provisional Application Serial No. 60/415,388 filed October 02, 2002. [0004] This application also is related to U.S. Application Serial No. 11/104,110, filed April 12, 2005 and published as U.S. Application No. US- 2006-0002916 on January 05, 2006, entitled " Cleavage of VEGF and VEGF Receptor by Wild- Type and Mutant MTSP-1 " to J Nguyen and S Waugh-Ruggles, and to corresponding published International PCT 20 Application No. WO2005/110453, published November 24, 2005, which claim benefit to U.S. Provisional Application Serial No. 60/561,720 filed April 12, 2004. [0005] This application also is related to U.S. Application Serial No. 11/104,111, filed April 12, 2005 and published as U.S. Application No. US-2006-0024289 on February 02, 2006, entitled Cleavage" of YEGF and VEGF Receptor by Wild-Type and Mutant Proteases " to J Nguyen and S Waugh-Ruggles, and to corresponding published International 25 PCT Application No. WO2005/100556, published October 27, 2005, which claim benefit to U.S. Provisional Application Serial No. 60/561,671 filed April 12, 2004. [0006] This application also is related to U.S. Provisional Application Serial No. 60/729,817 filed October 21, 2005, entitled "Modified Proteases that Inhibit Complement Activation" to Edwin L. Madison. This application also is related to U.S. Application Serial No. 11/584,776 filed October 20, 2006, entitled " Modified Proteases that Inhibit Complement 30 Activation" to Edwin L. Madison, Jack Nguyen, Sandra Waugh Ruggles and Christopher Thanos, and to corresponding published International PCT Application No. WO2007/047995, published April 26, 2007, which each claim benefit to U.S. Provisional Application No. 60/729,817. [0007] Where permitted, the subject matter of each of the above- noted related applications is incorporated by reference in its entirety. 35 Incorporation by reference of Sequence Listing provided on compact discs [0008] An electronic version on compact disc (CD-R) of the Sequence Listing is filed herewith in four copies (labeled COPY 1, COPY 2, COPY 3, and CRF), the contents of which are incorporated by reference in their entirety. The computer- 40 readable file on each of the aforementioned compact discs, created on July 05, 2007, is identical, 1,809 kilobytes in size, and titled 4902SEQ.PC1.txt. FIELD OF THE INVENTION 45 [0009] Methods for identifying modified proteases with modified substrate specificity or other properties are provided. The methods screen candidate and modified proteases by contacting them with a substrate that traps them upon cleavage of the substrate. BACKGROUND 50 [0010] Proteases are protein-degrading enzymes. Because proteases can specifically interact with and inactivate or activate a target protein, they have been employed as therapeutics. Naturally-occurring proteases often are not optimal therapeutics since they do not exhibit the specificity, stability and/or catalytic activity that renders them suitable as biotherapeutics (see, e.g., Fernandez-Gacio et al. (2003) Trends in Biotech. 21: 408-414). Among properties of thera- 55 peutics that are important are lack of immunogenicity or reduced immunogenicity; specificity for a target molecule, and limited side-effects. Naturally-occurring proteases generally are deficient in one or more of these properties. [0011] Attempts have been made to engineer proteases with improved properties. Among these approaches include 1) rational design, which requires information about the structure, catalytic mechanisms, and molecular modeling of a 2 EP 2 402 438 A2 protease; and 2) directed evolution, which is a process that involves the generation of a diverse mutant repertoire for a protease, and selection of those mutants that exhibit a desired characteristic (Bertschinger et al. (2005) in Phage display in Biotech. and Drug Discovery (Sidhu s, ed), pp. 461-491). For the former approach, a lack of information regarding the structure-function relationship of proteases limits the ability to rationally design mutations for most proteases. Directed 5 evolution methodologies have been employed with limited success. [0012] Screening for improved protease activity often leads to a loss of substrate selectivity and vice versa. An optimal therapeutic protease should exhibit a high specificity for a target substrate and a high catalytic efficiency. Because of the limited effectiveness of available methods to select for proteases with optimized specificity and optimized activity, there remains a need to develop alternate methods of protease selection. Accordingly, it is among the objects herein to 10 provide methods for selection of proteases or mutant proteases with desired substrate specificities and activities. SUMMARY [0013] Provided are methods for selection or identification of proteases or mutant proteases or catalytically active 15 portions thereof with desired or predetermined substrate specificities and activities. In particular, provided herein are protease screening methods that identify proteases that have an altered, improved, or optimized or otherwise altered substrate specificity and/or activity for a target substrate or substrates. The methods can be used, for example, to screen for proteases that have an altered substrate specificity and/or activity for a target substrate involved in the etiology of a disease or disorder. By virtue of the altered, typically increased, specificity and/or activity for a target substrate, the 20 proteases identified or selected in the methods provided herein are candidates for use as reagents or therapeutics in the treatment of diseases or conditions for which the target substrate is involved. In practicing the methods provided herein, a collection of proteases or catalytically active portion thereof is contacted with a protease trap polypeptide resulting in the formation of stable complexes of the protease trap polypeptide with proteases or catalytically active portion thereof in the collection. In some examples, the protease trap polypeptide is modified to be cleaved by a protease 25 having a predetermined substrate specificity and/or activity for a target substrate, for example, a target substrate involved in a disease or disorder. The method can further comprise screening the complexes for substrate specificity for the cleavage sequence of the target substrate. In such examples, the identified or selected protease has an altered activity and/or specificity towards the target substrate. In one example, the stable complex is formed by covalent linkage of a selected protease with a protease trap polypeptide. The selected proteases or catalytically active portion thereof are 30 identified or selected from the complex in the methods provided herein. The methods provided herein can further include the step of separating the complexed proteases from the uncomplexed protease members of the collection. In one example, the protease trap polypeptide is labeled for detection or separation and separation is effected by capture of complexes containing the detectable protease trap polypeptide and the protease or catalytically active portion thereof.
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