US0093 03076B2 (12) United States Patent (10) Patent No.: US 9,303,076 B2 Brinkmann et al. (45) Date of Patent: Apr. 5, 2016 (54) CELL-PENETRATING PEPTIDES AND USES WO 2007/069068 A2 6/2007 THEREOF WO 2011 OO3557 A1 1/2011 WO 2011 OO3557 A8 1/2011 (71) Applicant: Hoffmann-La Roche Inc., Little Falls, WO 2011 003780 A1 1, 2011 NJ (US) OTHER PUBLICATIONS UniProtKB U6D6M6 (U6D6M6 NEOVI).* (72) Inventors: Ulrich Brinkmann, Weilheim (DE): UniProtKB B4DU57 (B4DU57 HUMAN).* Alexander Haas, Munich (DE); Daniela UniProtKB Q99748 (NRTN HUMAN).* Maisel, Penzberg (DE) Decision to Grant Patent issued in Japanese Patent Application No. 2013-5 14688, dated Mar. 26, 2015, in 3 pages. (73) Assignee: HOFFMANN-LA ROCHE INC., Haas et al., “Human-protein-derived peptides for intracellular deliv Nutley, NJ (US) ery of biomolecules”, Biochem. J., 442:583-593 (2012). PCT/EP2011/059853 International Search Report., pp. 1-8 (Feb. 23, 2012). (*) Notice: Subject to any disclaimer, the term of this El-Andaloussi et al., "Cargo-dependent cytotoxicity and delivery patent is extended or adjusted under 35 efficacy of cell-penetrating peptides: a comparative study Biochem. U.S.C. 154(b) by 0 days. J. 407:285-292 (2007). Examination Report issued in the corresponding European Patent (21) Appl. No.: 14/706,645 Application No. 11729276.3, pp. 1-7 (Issued Apr. 2, 2014). Fonseca, S.B. et al., “Recent advances in the use of cell-penetrating (22) Filed: May 7, 2015 peptides for medical and biological applications' Advanced Drug Delivery Reviews 61 (11):953-964 (Sep. 30, 2009). (65) Prior Publication Data Further Examination Report issued in New Zealand Patent Applica tion No. 603732, issued on Jan. 12, 2015 (in 3 pages) (Jan. 12, 2015). US 2015/0239947 A1 Aug. 27, 2015 Green, M. et al., “Autonomous functional domains of chemically Synthesized human immunodeficiency virus tat trans-activator pro Related U.S. Application Data tein' Cell 55(6): 1179-1188 (Dec. 23, 1988). GSP:AEV74707, (Human cathelin-like protein, CAP18/LL-37 pro (63) Continuation of application No. 13/712,088, filed on tein fragment (aa: 132-170)), pp. 1 May 31, 2007. Dec. 12, 2012, which is a continuation of application Haas et al., “Human-protein-derived peptides for intracellular deliv No. PCT/EP2011/059853, filed on Jun. 14, 2011. ery of biomolecules' Biochem. J. 442:583-593 (2012). Heitz, F. et al., “Twenty years of cell-penetrating peptides: from (30) Foreign Application Priority Data molecular mechanisms to therapeutics' British Journal of Pharma cology 157(2): 195-206 (Mar. 20, 2009). Jun. 14, 2010 (EP) ..................................... 10165793 Office Action issued in the corresponding Chinese Patent Application Dec. 15, 2010 (EP) ..................................... 10195278 No. 20118.0029464.X (Issued Feb. 17, 2014), with English Transla tion in 9 pages., pp. 1-10 (Issued Feb. 17, 2014). (51) Int. Cl. Snyder, E.L. et al., “Recent advances in the use of protein transduc C07K I4/47 (2006.01) tion domains for the delivery of peptides, proteins and nucleic acids C07K I4/00 (2006.01) in vivo” Expert Opinion on Drug Delivery 2(1):43-51 (Jan. 1, 2005). Uniprot. ASM3B HUMAN, B4DEC6 HUMAN. (Last Modified C07K L/107 (2006.01) May 14, 2014). C07K I4/435 (2006.01) 2nd Office Action issued in Chinese Patent Application No. A6 IK 47/48 (2006.01) 20118.0029464.X. dated Jan. 4, 2015, with English Translation (total (52) U.S. Cl. in 9 pages). CPC ........... C07K 14/47 (2013.01); A61K 47/48323 Altschul et al., “Basic local alignment search tool” J. Mol. Biol. (2013.01); C07K I/1075 (2013.01); C07K 215:403-410 (1990). 1/1077 (2013.01); C07K 14/00 (2013.01); Lipman et al., “Rapid and sensitive protein similarity searches' Sci ence 227:1435-1441 (1985). C07K 14/435 (2013.01) Smith et al., “Identification of common molecular subsequences' J. (58) Field of Classification Search Mol. Biol. 147: 195-197 (1981). None See application file for complete search history. * cited by examiner (56) References Cited Primary Examiner — Amber D Steele U.S. PATENT DOCUMENTS Assistant Examiner — Schuyler Milton (74) Attorney, Agent, or Firm — Scott L. Ausenhus 7,015,311 B1 3/2006 Johnson et al. 7,067.473 B1 6, 2006 Masure (57) ABSTRACT 7,173,007 B1 2/2007 Zaiou et al. The present invention relates to the identification and func FOREIGN PATENT DOCUMENTS tional characterization of human cell-penetrating peptides (CPPs) and their use; in particular as transfection vehicles. WO 97.08.196 A1 3, 1997 WO O3 (106491 A2 12/2003 12 Claims, 16 Drawing Sheets U.S. Patent Apr. 5, 2016 Sheet 1 of 16 US 9,303,076 B2 All known human proteins AllAl human proteins 19929 non-redundant sequences as of Protein id 11.07.2008, ID, sequence, Name description, GO annotation Description Go annotation Sequence Sliding window All human protein derived peptides Peptide ID All 30 merpeptides in Protein ID all known human proteins Position in protein 10,459,557 # (R, H, K) D 30 mer sequences inked to protein D # Cys P P Hydrophobicity Sequence analysis Go annotation Sequence step Filter # Rationale for filter 2 (HKR)>= 10 227,307 Fits search pattern 3 2 + GO extracellular' Visible, exposed 4 3 + EP DE 12.53 1000 Poly-cation effects? 5 3 - Phob >25.4 1000 Membrane effects? 6 Intersect 4 and 5 91 C Combined effects 6 + nonred. Prot) 29 Combine clusters 8 3+ similarity to TATY 6 135 CPP prediction 9 8 + nonred. Prot) Combine clusters 10 3 + nonred. Prot) 583 Combine clusters 11 10+ subj. Description Membranellipids/bactericidal U.S. Patent Apr. 5, 2016 Sheet 2 of 16 US 9,303,076 B2 50 -5 O Legend: + 500 random peptides O non-functional & non-toxic peptides (3 toxic peptides (S functional & toxic peptides O functional peptides D Control peptides Fic. 2 U.S. Patent Apr. 5, 2016 Sheet 3 of 16 US 9,303,076 B2 -----Transfection- +++ TA T o ASM3B REW 810xArg INF7 a CPXM2 0 Protamine o NRTN oFGF12 o GSO CU025 OCPXM FAL o CD026 O Apoll cons 2 o BPIL3(38aa) o BPIL3(30aa) -180 -160 -140 -120 -100 U.S. Patent Apr. 5, 2016 Sheet 4 of 16 US 9,303,076 B2 Fig. 4 WNT16. AHA1GAPDH WNT16. Viability 140 120 100 80 60 40 20 1, M 5uM 10M 20 M 1M 5M 10u M 20 M U.S. Patent Apr. 5, 2016 Sheet 5 of 16 US 9,303,076 B2 Fig. 5 A BPIL3 Aha 1 GAPDH BPIL3-Viability 600 12 500 400 0.8 300 0.6 200 0.4 100 0.2 O O Sree 1.M 5uM 10u M 20 M tu M 5uM 10u M. 20 M 120 BP3 - Aha1 120 BPI 3- GAPDH 100 100 80 80 60 60 40 40 20 20 O O 14 M 5uM 10u M 20 M 1M 5uM 10M 20 M B FA - Aha1/GAPDH FALL - Viability 120 1.2 100 1 80 0.8 60 0,6 40 0.4 20 0.2 O O 1M 5uM 10u M 201M 1M 5M 10M 20 M 120 FA - Aha1 FAL - GAPDH g 120100 60 80 60 20 20 O O 1M 5uM 10u M 20, M 1M 5uM 10M 20 M --Aha --O-- Luc U.S. Patent Apr. 5, 2016 Sheet 6 of 16 US 9,303,076 B2 Fig. 6 CU025. Aha 1 GAPDH CU025 - Viability 120 100 80 60 40 20 1u M 5uM 10u M 20 M 1. M 5M 10M 20 M -- Aha? --O-- Luc U.S. Patent Apr. 5, 2016 Sheet 7 of 16 US 9,303,076 B2 Poly-Arg-Aha IGAPDH 140 120 - - --O-- a--- 10080 's 60 40 20 O 1u M 5uM 10u M 20 M 1M 5uM 10M 20 M B 120 CPXM2 - Aha 1 GAPDH 2 CPXM2. Viability 1.8 100 1.6 80 33 60 o: 40 66 2. 0.40.2 1M 5uM 10 M20M 1M' 5uM" to M' 20M C ASM3B. Aha GAPDH ASM3B. Viability 20 2 100 1 80 0.8 60 0.6 40 0.4 20 0.2 O 1H M 5uM 101 M 20 M 0 11M 5uM 10M 201M D 120 NRTN. Ahaf IGAPDH NRTN. Viability 1.4 100 1.2 80 o: --SS . 0.6t No 0.4 20 0.2 O O 1 M 5uM 10p M. 20 M 1a M 51M 10M 20, M 120 NRTN. Aha1 NRTN - GAPDH 100 80 60 40 20 1M 5uM 10u M 20 M 1, M 5aM 10 M. 20 M U.S. Patent Apr. 5, 2016 Sheet 8 of 16 US 9,303,076 B2 8 s vent en se S s A if sk 4 a A4 R w reas re vary area va wars se vers very A & U.S. Patent Apr. 5, 2016 Sheet 9 of 16 US 9,303,076 B2 Fig. 9 NRTN - Eg51 GAPDH NRTN - Cytotoxicity 1M 5u M 10u M 20 M 1M 5uM 10u M 20 M NRTN. Eg5 NRN GAPDH 140 140 120 120 100 100 80 80 60 60 40 40 20 20 1H M 5u M 10u M 20 M 1M 5uM 10 M. 20 M -- Eg5 --O-- Luc U.S. Patent Apr. 5, 2016 Sheet 11 of 16 US 9,303,076 B2 25%TFE 220 240 260 nm) TAT -- re-rrs U.S. Patent Apr. 5, 2016 Sheet 12 of 16 US 9,303,076 B2 i O i O 2 O 2 O O 0 100 10 102 103 100 101 102 to FITC-A FITC-A ViabieFTC-A subset ViableFC-A Subset Geom.