USOO9545449B2 (12) United States Patent (10) Patent No.: US 9,545.449 B2 Krantz (45) Date of Patent: Jan. 17, 2017 (54) SITE-SPECIFIC LABELING AND TARGETED 8,030.459 B2 10/2011 Papisov et al. DELIVERY OF PROTEINS FOR THE 8,927.485 B2 1/2015 Krantz et al. 2003/0215877 A1 11/2003 Love et al. TREATMENT OF CANCER 2005, 00792O8 A1 4/2005 Albani 2007, 0123465 A1 5/2007 Adermann et al. (71) Applicant: Advanced Proteome Therapeutics 2010.0099649 A1* 4/2010 Krantz et al. ................. 514f131 Inc., Boston, MA (US) 2011 0002978 A1 1/2011 Harrison 2011/0263832 A1 10/2011 Krantz et al. (72) Inventor: Alexander Krantz, Boston, MA (US) 2013,0165382 A1 6/2013 Krantz et al. (73) Assignee: Advanced Proteone Therapeutics Inc., FOREIGN PATENT DOCUMENTS Boston, MA (US) WO WO-89/11867 A1 12/1989 WO WO-02/42427 A2 5, 2002 (*) Notice: Subject to any disclaimer, the term of this WO WO-O2/O87497 A2 11/2002 patent is extended or adjusted under 35 WO WO-03/093478 A1 11, 2003 U.S.C. 154(b) by 0 days. WO WO-2007 112362 A2 10, 2007 WO WO-2010, 140886 A1 12/2010 (21) Appl. No.: 14/400,190 WO WO-2011, 153250 A2 12/2011 (22) PCT Filed: May 13, 2013 OTHER PUBLICATIONS (86). PCT No.: PCT/US2O13/040823 Yu et al. Site-specific crosslinking of annexin proteins by 1.4- benzoquinone: a novel crosslinker for the formation of protein S 371 (c)(1), dimers and diverse protein conjugates (Org. Biomol. Chem..., 2012, (2) Date: Nov. 10, 2014 10, 4500).* Blanco-Canosa et al., “An efficient Fmoc-SPPS approach for the (87) PCT Pub. No.: WO2013/170272 generation of thioester peptide precursors for use in native chemical PCT Pub. Date: Nov. 14, 2013 ligation.” Angew Chem Int Ed Engl. 47(36):6851-5 (2008) (10 pages). (65) Prior Publication Data Brandt et al., "Covalent attachment of proteins to polysaccharide carriers by means of benzoquinone.” Biochim Biophys Acta. US 2015/02O2314 A1 Jul. 23, 2015 386(1): 196-202 (1975). Deady et al., “Synthesis and antitumor activity of Some indeno1,2- Related U.S. Application Data bduinoline-based bis carboxamides.” Bioorg Med Chem. 8(5):977 84 (2000). (60) Provisional application No. 61/849,034, filed on Jan. Dixon, “N-terminal modification of proteins—A review.” J Protein 17, 2013, provisional application No. 61/848,601, Chem. 3(1):99-108 (1984). filed on Jan. 7, 2013, provisional application No. Gentle et al., “Direct production of proteins with N-terminal cys 61/741,984, filed on Aug. 1, 2012, provisional teine for site-specific conjugation.” Bioconjug Chem. 15(3):658-63 application No. 61/688,308, filed on May 11, 2012. (2004). Hauser et al., “Expressed protein ligation using an N-terminal (51) Int. Cl. cysteine containing fragment generated in vivo from a pelB fusion A6 IK 38/00 (2006.01) protein.” Protein Expr Purif. 54(2):227-33 (2007) (13 pages). A6 IK 47/48 (2006.01) International Preliminary Report on Patentability and Written Opin A6 IK 38/17 (2006.01) ion for International Patent Application No. PCT/US2013/040823, A6 IK 38/38 (2006.01) issued Nov. 11, 2014 (15 pages). A6 IK 38/40 (2006.01) (Continued) A6 IK 45/06 (2006.01) C07K I4/435 (2006.01) (52) U.S. Cl. Primary Examiner — James H Alstrum Acevedo CPC ......... A61K 47/48246 (2013.01); A61K 38/17 Assistant Examiner — Sergio Coffa (2013.01); A61K 38/38 (2013.01); A61K 38/40 (74) Attorney, Agent, or Firm — Clark & Elbing LLP (2013.01); A61K 45/06 (2013.01); C07K 14/435 (2013.01) (58) Field of Classification Search (57) ABSTRACT None The present invention relates to the formation of protein See application file for complete search history. conjugates from proteins chemically modified for linkage to (56) References Cited (1) anticancer drug pharmacophores, (2) ligands to biomark ers on cancer cell Surfaces, (3) and/or another protein U.S. PATENT DOCUMENTS molecule. It provides and specifies new compositions, meth 5,900.404 A 5/1999 Gegg et al. ods and combinations for tumor, and tumor vasculature 6,017,876 A 1/2000 Gegg et al. targeting and cancer treatment. 6,204.247 B1 3/2001 Gegg et al. 6,326,468 B1 12/2001 Canne et al. 6.420,340 B2 7/2002 Gegg et al. 16 Claims, No Drawings US 9,545.449 B2 Page 2 (56) References Cited Radic et al., “Probing gorge dimensions of cholinesterases by freeze-frame click chemistry.” Chem Biol Interact. 175(1-3):161-5 (2008) (10 pages). OTHER PUBLICATIONS Rice et al., “Inhibitors of HIV nucleocapsid protein zinc fingers as candidates for the treatment of AIDS,” Science. 270(5239): 1194-7 International Search Report for International Patent Application No. (1995). PCT/US 13/40823, mailed Dec. 16, 2013 (8 pages). Selden et al., “Cross-linked dimers with nucleating activity in actin Johnson et al., “Insights into the mechanism and catalysis of the prepared from muscle acetone powder,” Biochemistry. 39(1):64-74 native chemical ligation reaction.” JAm ChemSoc. 128(20):6640-6 (2000). (2006). Sunde et al., “Improved conditions for the removal of 2-oxoacyl Kalia et al., “Hydrolytic stability of hydrazones and groups from the N-terminus of proteins,” Biochim Biophys Acta. oximes.” Angew Chem Int Ed Engl. 47(39):7523-6 (2008). 1388(1):45-52 (1998). Kent, “Total chemical synthesis of proteins,” Chem Soc Rev. Tam et al., “Orthogonal ligation strategies for peptide and protein.” 38(2):338-51 (2009). Biopolymers. 51(5):311-32 (1999). Kolb et al., “Click chemistry: diverse chemical function from a few Wang et al., “Interaction of a self-assembling peptide with oligo good reactions.” Angew Chem Int Ed. 40(11):2004-21 (2001). nucleotides: complexation and aggregation,” Biophys J. Lo Conte et al., “Photoinduced addition of glycosylthiols to alkynyl 93(7):2477-90 (2007). peptides: use of free-radical thiol-yne coupling for post-translational Wen et al., “Erythropoietin structure-function relationships: high double-glycosylation of peptides,” J Org Chem. 75(13):4644-7 degree of sequence homology among mammals,” Blood. (2010). 82(5): 1507-16 (1993). Loo et al., "Biophysical characterization of zinc ejection from HIV Witus et al., “Identification of highly reactive sequences for PLP nucleocapsid protein by anti-HIV 2,2'-dithiobisbenzamides and mediated bioconjugation using a combinatorial peptide library,” J benzisothiazolones,” J Med Chem. 39(21):4313-20 (1996). Am ChemSoc. 132(47): 16812-7 (2010) (14 pages). Niemeyer et al., “Oligonucleotide-directed self-assembly of pro Extended European Search Report for European Application No. teins: semisynthetic DNA-streptavidin hybrid molecules as con 13787738.7, mailed Jul. 29, 2016 (17 pages). nectors for the generation of macroscopic arrays and the construc Liet al., “Site-specific labeling of annexin V with F-18 for apoptosis tion of Supramolecular bioconjugates. Nucleic Acids Res. imaging.” Bioconjug Chem. 19(8): 1684-8 (2008). 22(25):5530-9 (1994). Green et al., “Quantitative evaluation of the lengths of Nishimura et al., “An efficient chemical method for removing homobifunctional protein cross-linking reagents used as molecular N-terminal extra methionine from recombinant methionylated rulers.” Protein Sci. 10(7): 1293-304 (2001). human growth hormone.” Chem Commun. 1135-6 (1998). Supplemental Partial European Search Report for European Appli Niwa et al., “A flexizyme that selectively charges amino acids cation No. 13787738.7, mailed Mar. 8, 2016 (12 pages). activated by a water-friendly leaving group.” Bioorg Med Chem Lett. 19(14):3892-4 (2009). * cited by examiner US 9,545,449 B2 1. 2 SITE-SPECIFIC LABELING AND TARGETED SUMMARY OF THE INVENTION DELIVERY OF PROTEINS FOR THE TREATMENT OF CANCER In a first aspect, the invention features a pharmaceutical composition including a therapeutic protein having a struc CROSS-REFERENCE TO RELATED ture according to formula (I), APPLICATIONS This application is a national stage of International Patent (I) Application No. PCT/US2013/040823, which claims benefit 10 P --L-T), of U.S. Provisional Application No. 61/688,308, filed May 11, 2012, 61/741,984, filed Aug. 1, 2012, 61/848,601, filed Jan. 7, 2013, and 61/849,034, filed Jan. 17, 2013, each of where which is hereby incorporated by reference in its entirety. Prepresents a monomeric protein or a multimeric protein, where P includes 1, 2, 3, or 4 proteins independently selected 15 FIELD OF THE INVENTION from cell-targeting proteins, cell-binding proteins, cell-traf ficking proteins, and transport proteins, or any combination The present invention relates to the formation of protein thereof; conjugates from proteins chemically modified for linkage to each L independently represents an organic linker group; (1) anticancer drug pharmacophores, (2) ligands to biomark each T independently represents a therapeutic agent, a ers on cancer cell Surfaces, and/or (3) another protein protein, or a ligand to a biomarkers; molecule. It provides and specifies new compositions, meth n is an integer from 0-10; and ods and combinations for tumor, and tumor vasculature where each L-T moiety present is site-specifically targeting and cancer treatment. The present invention also attached to an amino acid residue of P. and specifies fusion proteins that are produced recombinantly 25 where the compound according to formula (I) represents that can be utilized to link anticancer drug pharmacophores at least about 75% of biologically active proteins present in or ligands to biomarkers on cancer cell Surfaces, or as the composition. therapeutics in their own right because of their increased In some embodiments, n is an integer from 1-10. duration of action and T-cell mediated antitumor immunity.