US009 161988B2

(12) United States Patent (10) Patent No.: US 9,161,988 B2 Castaigne et al. (45) Date of Patent: *Oct. 20, 2015

(54) MULTIMERIC CONJUGATES AND 5,578,451 A 1 1/1996 Nishimoto USES THEREOF 5,627,270 A 5/1997 Kahne et al. RE35,524 E 6/1997 Saulnier et al. 5,683,694 A 11/1997 Bagshawe et al. (75) Inventors: Jean-Paul Castaigne, Mont-Royal 5,780,265 A 7, 1998 E. et al. (CA); Michel Demeule, Beaconsfield 5,807,980 A 9/1998 Lasters et al. (CA); Christian Che, Longueuil (CA); 5,869,045 A 2f1999 Hellstrom et al. Carine Thiot, Montreal (CA); 5,922,754 A 7/1999 Burchett et al. 5.948,750 A 9/1999 Garsky et al. Cathene Gagnon, Montral-Nord 5.948,888 A 9/1999 de la Monte et al. (CA); Betty Lawrence, Bolton (CA) 5,955,444 A 9, 1999 Ingram et al. 5,962,266 A 10, 1999 White et al. (73) Assignee: Angiochem Inc., Montreal (CA) 5,981,564 A 1 1/1999 Pagé et al. 6,093,692 A 7/2000 Shen et al. (*) Notice: Subject to any disclaimer, the term of this 3396 f g3. Sist et al. past listdoyed under 35 6,245,359k - J. B1 6/2001 MilsteinaIavy et al. M YW- 6,306,993 B1 10/2001 Rothbard et al. This patent is Subject to a terminal dis- 6,316,0246,310,039 B1 110/2001 1/2001 AllenKratz et al. claimer. 6,348,207 B1 2/2002 Milstein et al. 6,376,648 B2 4/2002 White et al. (21) Appl. No.: 13/382,069 6,391.305 B1 5/2002 Feng et al. 6,469,047 B1 10/2002 Jackson et al.

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Related U.S. Application Data U.S. Appl. No. 61/138,375, Beliveau et al Author manuscript of Howes et al., “Rapid induction of therapeutic (60) Provisional application No. 61/222,785, filed on Jul. 2, hypothermia using convective-immersion Surface cooling: Safety, 2009, provisional application No. 61/252,024, filed on efficacy and outcomes.” published in final edited form as: Resusci Oct. 15, 2009. tation. 81 (4):388-392 (2010); (13 pages). Belkin et al., “Matrix-dependent proteolysis of surface 51) Int.nt. CC. transglutaminase by membrane-type metalloproteinase regulates A6 IK38/10 (2006.01) cancer cell adhesion and locomotion.” J Biol Chem. 276(21): 18415 A6 IK 47/48 (2006.01) 18422 (2001). (52) U.S. Cl. Boado et al., “GDNF fusion for targeted-drug delivery across CPC ...... A61K47/48246 (2013.01) the human blood-brain barrier." Biotechnol Bioeng, 100(2):387-96 (58) Field of ClassificationO A O Search ( 2008). CPC ...... A61K 47/48246 (Continued) See application file for complete search history. Primary Examiner — Karlheinz, R Skowronek (56) References Cited Assistant Examiner — Lianko Garyu U.S. PATENT DOCUMENTS (74) Attorney, Agent, or Firm — Clark & Elbing LLP 4,671,958 A 6, 1987 Rodwell et al. (57) ABSTRACT 4,801,575 A 1/1989 Pardridge 4.902,505 A 2/1990 Pardridge et al. The present invention relates to multimeric (e.g., dimeric, 4.942,184 A 7/1990 Haugwitz et al. trimeric) forms of peptide vectors that are capable of crossing 5,028,697 A 7, 1991 Johnson et al. 5,041,424 A 8, 1991 Saulnier et al. the blood-brain barrier (BBB) or efficiently entering particu 5,118,668 A 6, 1992 Auerswald et al. lar cell types. These multimeric peptide vectors, when con 5,126,249 A 6, 1992 Becker et al. jugated to agents (e.g., therapeutic agents) are capable of 5,169,933 A 12, 1992 Anderson et al. transporting the agents across the BBB or into particular cell 5,204,354 A 4, 1993 Chakravarty et al. 5,223,409 A 6, 1993 Ladner et al. types. These compounds are therefore particularly useful in 5,258.499 A 11/1993 Konigsberg et al. the treatment of neurological diseases. 5,362,831 A 1 1/1994 Mongelli et al. 5,442,043 A 8, 1995 Fukuta et al. 14 Claims, 12 Drawing Sheets US 9,161,988 B2 Page 2

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Inters. Chromatogram 1.TMEA-(AN2) before purification (282 mg of crude) mAU 500 UV spectrum at 229 nm 400 S.E. 3 Mass 7596 3OO Trimer TMEA-(AN2)3 200 Mass 2789 3 Monomer TMEA-AN2 100

O OO O5 10 1.5 2.O 2.5 Time min intens. Chromatogram 2.TMEA-(AN2) after purification

mAU). 1

1OOO mmM 800 600 400 119 mg recovered 200 > 98% purity O O.O O5 1.O. 1.5 2.O. 2.5 Time min)

RT Area i min. Area Int. Type Intens. S/N Chromatogram Max. m/z. Frac,' ().93 54.8 Manual 155 550.4 UW Chromatogram, 869.6245 100.0 229 mm. 2. 0.94 m.a. Average ... . O39.7438 Spectrum

Figure 1 U.S. Patent Oct. 20, 2015 Sheet 2 of 12 US 9,161,988 B2

Chromatogram 3.Purification of TMEA-(AN2) on Waters PreplC4000 System X-bridge Prep Column Pheny OBD 19 x 150 mm

5 10 15, 20 25 30 35 min

Figure 2 U.S. Patent Oct. 20, 2015 Sheet 3 of 12 US 9,161,988 B2

Chromatogram 4.SATP-AN2-AN2 before purification (220 mg) Intens. mAU UV spectrum at 229n 6OO

400

200

0.5 1.O 1.5 2.0 2.5 3.0 3.5 Time min) Chromatogram 5.SATP-AN2-AN2 after purification

50 UV spectrum at 229nm O 000025s------osotors 100 125 1.50 1.75 2.00Time (min)

RT Area it min Area Int. e Intens. S/N Chromato Max, m/z. Frag.9% 1.04 9153.6 Manual 2583 1248.2 UV Chromatogram, 229 713.3567 100.0

Figure 3 U.S. Patent Oct. 20, 2015 Sheet 4 of 12 US 9,161,988 B2

Chromatogram 6. Purification of SATPAN2-AN2 on Waters PrepC4000 System Xbridge Prep Column Pheny OBD 19 x 150 mm

ADC1A, 229 nm (SICHACT36-30.D) . . . . . mAU - 38%%B a?- 40% B UV spectrum at 229mm 17500 : | 15000 pMSo 12500 1OOOO 7500 Mixed 5000 : fractions . 2500:O +H, ------4 Siristyish 5 10 15 20 25 30 35 in

Figure 4 U.S. Patent Oct. 20, 2015 Sheet 5 of 12 US 9,161,988 B2

Dimerization of Angiopep-l

TFFYGGCRGKRNNFKTEEY + TFFYGGCRGKRNNFKTEEY Angiopep-1 Angiopep-1

2 hrs at 37°C PBS, pH 8.5

TFFYGGCRGKRNNFKTEEY

TFFYGGCRGKRNNFKTEEY Angiopep-1 dimer

Figure 5 U.S. Patent Oct. 20, 2015 Sheet 6 of 12 US 9,161,988 B2

Wr to zo so ado s Concentration (nM)

Figure 6 U.S. Patent Oct. 20, 2015 Sheet 7 of 12 US 9,161,988 B2

Angiopep-1 dimer e Argopep-2

SW so sox to Coo 12so Concentration (nm)

Figure 7 U.S. Patent Oct. 20, 2015 Sheet 8 of 12 US 9,161,988 B2

Brain 50 nM

35

30

An2 Recomb Trimeric Dimeric Recomb Lean mice DiO mice

Figure 8 U.S. Patent Oct. 20, 2015 Sheet 9 of 12 US 9,161,988 B2

His-Gly-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Leu-Ser-Lys-Gln-Met-Glu-Glu-Glu Ala-Val-Arg-Leu-Phe-Ile-Glu-Trp-Leu-Lys-Asn-Gly-Gly-Pro-Ser-Ser-Gly Ala-Pro-Pro-Pro-Lys39)

Figure 9A

U.S. Patent Oct. 20, 2015 Sheet 11 of 12 US 9,161,988 B2

Exendin-4 (Phoenix Pharm.) C3 C6 Exen-4-An2 C11 40 Exen-4-S4 Exen-An2-An2 2O30

1 O

Brain Capillaries Parenchyma

Figure 10 U.S. Patent Oct. 20, 2015 Sheet 12 of 12 US 9,161,988 B2

- Ctrl -a- Exendin-4 (2 g/kg) -- Exen-An2-An2 (4.5 ug/kg)

140 Š 120 s : E 100-s 2. 8O 60 9.g 40 20 O O 3. SO 9. Time (min)

Figure 11 US 9, 161988 B2 1. MULTIMERIC PEPTIDE CONUGATES AND USES THEREOF - ). CROSS-REFERENCE TO RELATED V APPLICATIONS X-A-X-A), A. This Application is a U.S. National Stage Application of international patent application PCT/CA2010/001014, filed Yx-) Jun. 30, 2010, which, in turn, claims benefit of U.S. Provi sional Application No. 61/222,785, filed on Jul. 2, 2009, and 10 where A, A, each A, each A, and each A are, indepen U.S. Provisional Application No. 61/252,024, filed on Oct. dently, peptide vector, e.g., any described herein Such as one 15, 2009. that includes a sequence Substantially identical to a sequence BACKGROUND OF THE INVENTION selected from the group consisting of SEQID NOS:1-105 and 15 107-117 or a functional fragment thereof; A is a peptide The invention relates to compounds including dimeric or vector including a sequence Substantially identical to a multimeric peptide vectors and uses of Such compounds. sequence selected from the group consisting of SEQID NOS: The brain is shielded against potentially toxic Substances 1-105 and 107-117 or a functional fragment thereof or is by the presence of two barrier systems: the blood-brain bar absent; X, each X7, each X, and each X are, independently, rier (BBB) and the blood-cerebrospinal fluid barrier (BC linkers that join peptide vectors; m, n, and pare, indepen SFB). The BBB is considered to be the major route for the dently, 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10; q is an integer from 4 uptake of serum ligands since its Surface area is approxi to m+3; ris an integer from m+4 to m--n +3; and S is an integer mately 5000-fold greater than that of BCSFB. The brain from m+n+4 to m--n +p+3. endothelium, which constitutes the BBB, represents the Any of the above compounds may be conjugated to one or major obstacle for the use of potential drugs against many 25 more agents (e.g., any described herein), through one or more disorders of the CNS. As a general rule, only small lipophilic linkers or through one or more peptide vectors. molecules may pass across the BBB, i.e., from circulating In another aspect, the invention features a compound of systemic blood to brain. Many drugs that have a larger size or including the formula: higher hydrophobicity show promising results in animal stud ies for treating CNS disorders. Thus, peptide and protein 30 therapeutics are generally excluded from transport from blood to brain, owing to the negligible permeability of the X brain capillary endothelial wall to these drugs. A11 NA2 Therapy of brain diseases can be impaired by the inability of otherwise effective therapeutic agents to cross the BBB. 35 where A, X, and A are as described above and B is an agent Thus, new strategies for transporting agents into the brain are and is conjugated to the linker X. The invention also features desired. a compound including the formula: SUMMARY OF THE INVENTION 40 We have now developed compounds containing dimeric or multimeric peptide vectors that are capable of crossing the blood-brain barrier (BBB) or entering particular cell types 11N1 n1n B2 (e.g., liver, lung, spleen, kidney, and muscle) with enhanced efficiency. When these compounds are joined with (e.g., con 45 where A', X, and A areas described above; B' is an agent, Bf jugated to) one or more agents, efficiency of transport across and Bare, independently, agents or are absent, Y and Y are, the BBB or into particular cell types is likewise enhanced. independently, linkers joining A' to B' and A to B, respec Accordingly, the present invention features multimeric pep tively, where Y is absent if B is absent. The compound may tide vectors optionally conjugated to an agent (e.g., a thera include the formula: peutic agent), and use of Such compounds in treatment and 50 diagnosis of disease. In a first aspect, the invention features a compound includ X ing the formula: 1NA11 NA2. A-CX-A”), 55 In another aspect, the invention features a compound where n is or is at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10; m is an including (a) at least two peptide vectors, where each peptide integer from 2 to n+1: A' and each A" are, independently, a vector independently includes an amino acid sequence Sub peptide vector, e.g., any described herein such as one that stantially identical to a sequence selected from the group includes an amino acid sequence Substantially identical to a consisting of SEQ ID NOS:1-105 and 107-117, where the sequence selected from the group consisting of SEQID NOS: 60 peptide vectors are joined by a linker, and (b) an agent con 1-105 and 107-117 or a functional fragment thereof; and each jugated to at least one of the peptide vectors or to the linker. X" is, independently, a linker joined to the adjacent peptide Any of the above compounds may include at least one of vectors. The compound may include the formula: the peptide vectors including an amino acid sequence at least 70%, 80%, 85%, 90%. 95%, or 100% identical to a sequence 65 selected from the group consisting of SEQID NO: 1-105 and In another aspect, the invention features a compound 107-117 (e.g., Angiopep-1 (SEQ ID NO:67), Angiopep-2 including the formula: (SEQIDNO:97), cys-Angiopep-2 (SEQID NO:113), Angio US 9, 161988 B2 3 4 pep-2-cys (SEQID NO:114), and reversed Angiopep-2 (SEQ GSK933776A (GlaxoSmithKline). In a specific embodi ID NO:117)). In compounds including an agent, the agent can ment, the compound has the structure shown in FIG.9A, or a be a therapeutic agent (e.g., any described herein, Such as an pharmaceutically acceptable salt thereof (e.g., a TFA salt). agent selected from the group consisting of an anticancer The compounds of the invention (e.g., those described agent, a therapeutic nucleic acid, a GLP-1 agonist, leptin or a above and in the detailed description) can be used in the leptin analog, neurotensin or a neurotensin analog, glial-de treatment of disease and conditions. Such methods are as rived neurotrophic factor (GDNF) or a GDNF analog, brain follows. derived neurotrophic factor (BDNF) or a BDNF analog), or The invention also features a method of treating or treating an antibody. The anticancer agent may be paclitaxel (Taxol), prophylactically a Subject having a cancer. The method vinblastine, Vincristine, etoposide, doxorubicin, cyclophos 10 includes administering to the patient a compound including phamide, docetaxel (TaxotereR), melphalan, and chloram an anticancer agent or a RNAi capable of inhibiting a gene bucil, abarelix, aldesleukin, alemtuzumab, allitertinoin, whose expression is associated with or causes cancer (e.g., allopurinol, altretamine, amifostine, anakinra, anastroZole, EGFR or VEGF). The cancer may be selected from the group arsenic trioxide, asparaginase, azacitidine, BCG Live, bev consisting of brain cancer, hepatocellular carcinoma, breast acuzimab, bexarotene, bleomycin, bleomycin, bortezombi, 15 cancer, cancers of the head and neck including various lym bortezomib, buSulfan, buSulfan, calusterone, capecitabine, phomas Such as mantle cell lymphoma, non-Hodgkin’s lym carboplatin, carmustine, celecoxib, cetuximab, cisplatin, phoma, adenoma, squamous cell carcinoma, laryngeal carci cladribine, clofarabine, cytarabine, dacarbazine, dactinomy noma, cancers of the retina, cancers of the esophagus, cin, actinomycin D, dalteparin, darbepoetin alfa, dasatinib, multiple myeloma, ovarian cancer, uterine cancer, melanoma, daunorubicin, daunomycin, decitabine, denileukin, denileu colorectal cancer, bladder cancer, prostate cancer, lung cancer kin diftitox, dexraZOxane, dromostanolone propionate, eculi (including non-Small cell lung carcinoma), pancreatic cancer, Zumab, epirubicin , epoetin alfa, erlotinib, estramustine, cervical cancer, head and neck cancer, skin cancers, nasopha exemestane, fentany, filgrastim, floXuridine, fludarabine, ryngeal carcinoma, liposarcoma, epithelial carcinoma, renal fluorouracil, 5-FU, fulvestrant, gefitinib, gemcitabine, gem cell carcinoma, gallbladder adenocarcinoma, parotid adeno tuZumab ozogamicin, goserelin, histrelin, hydroxyurea, ibri 25 carcinoma, endometrial sarcoma, and multidrug resistant tumomab tiuxetan, idarubicin, ifosfamide, imatinib, Inter cancers. The brain cancer may be selected from the group feron alfa-2b, irinotecan, lapatinib ditosylate, lenalidomide, consisting of astrocytoma, pilocytic astrocytoma, dysem letrozole, leucovorin, leuprolide, levamisole, lomustine, bryoplastic neuroepithelial tumor, oligodendrogliomas, CCNU, meclorethamine (nitrogen mustard), megestrol, mer ependymoma, glioblastoma multiforme, mixed gliomas, oli captopurine (6-MP), mesna, methotrexate, methoXSalen, 30 goastrocytomas, medulloblastoma, retinoblastoma, neuro mitomycin C, mitotane, mitoxantrone, nandrolone phenpro blastoma, germinoma, and teratoma. pionate, nelarabine, nofetumomab, oprelvekin, oxaliplatin, The invention also features a method of treating or treating palifermin, pamidronate, panitumumab, pegademase, pegas prophylactically a subject having a metabolic disorder by pargase, pegfilgrastim, peginterferon alfa-2b, pemeterxed, administering a compound including GLP-1 agonist, leptin, a pentostatin, pipobroman, plicamycin (mithramycin), por 35 leptin analog, neurotensin, or a neurotensin analog in an fimer, procarbazine, quinacrine, rasburicase, rituximab, sar amount sufficient to treat the disorder. The metabolic disorder gramostim, Sorafenib, Streptozocin, Sunitinib, talc, tamox may be diabetes (e.g., type I or type II diabetes), obesity, ifen, temozolomide, teniposide (VM-26), testolactone, diabetes as a consequence of obesity, hyperglycemia, dyslipi thalidomide, thioguanine (6-TG), thiotepa, thiotepa, thiotepa, demia, hypertriglyceridemia, syndrome X, insulin resistance, topotecan, toremifene, Tositumomab/I-131 (tositumomab), 40 impaired glucose tolerance (IGT), diabetic dyslipidemia, trastuzumab, trastuzumab, tretinoin (ATRA), uracil mustard, hyperlipidemia, a cardiovascular disease, or hypertension. Vairubicin, vinorelbine, Vorinostat, Zoledronate, and The invention also features a method of reducing food Zoledronic acid; or a pharmaceutically acceptable salt intake by, or reducing body weight of a subject by adminis thereof. In particular embodiments, the anticancer agent is tering a compound including GLP-1 agonist, leptin, or a paclitaxel, etoposide, or doxorubicin, or an analog thereof. 45 leptin analog to a Subject in an amount Sufficient to reduce The agent may be an RNAi agent (e.g., any RNAi agent food intake or reduce body weight. The subject may be over described herein such as an RNAi agent is capable of silenc weight, obese, or bulimic. ing EGFR or VEGF expression). The agent may be a GLP-1 The invention also features a method of treating or treating agonist (e.g., any described herein, Such as exendin-4 (SEQ prophylactically a disorder selected from the group consist ID NO: 132), or an analog or fragment thereofhaving GLP-1 50 ing of anxiety, movement disorder, aggression, psychosis, agonist activity, exendin-4 (SEQID NO: 132), Lyslexen seizures, panic attacks, hysteria, sleep disorders, Alzheimer's din-4 (SEQID NO: 134), or Cys'exendin-4 (SEQID NO: disease, and Parkinson's disease by administering a com 133)). The agent may be leptin or a leptin analog (e.g., any pound including a GLP-1 agonist to a subject in an amount described herein, such as leptin or leptin analog is full-length sufficient to treat or prevent the disorder. human leptin, mature human leptin (amino acids 22-167 of 55 The invention also features a method of increasing neuro the full length human leptin), or leptino. The agent may genesis in a Subject by administering to the Subject and effec be neurotensin or a neurotensin analog (e.g., any described tive amount of a compound including a GLP-1 agonist to the herein, Such as human neurotensin, human neurotensin(8- subject. The subject may be suffering from Parkinson's Dis 13), or pELYENKPRRPYIL-OH, where pE represents L-py ease, Alzheimer's Disease, Huntington's Disease, ALS, roglutamic acid). The agent may be GDNF, BDNF, or an 60 stroke, ADD, or a neuropsychiatric syndrome. The increase in analog thereof (e.g., a full length GDNF or BDNF sequence neurogenesis may improve learning or enhances neuropro or a mature form of GDNF or BDNF or is human tection in the Subject. GDNF'''). The antibody may be a monoclonal antibody The invention also features a method for converting liver Such as an antibody directed against the amyloid-?protein. stem/progenitor cells into functional pancreatic cells; pre The antibody may be selected from the group consisting of 65 venting beta-cell deterioration and stimulation of beta-cell R1450 (Roche), bapineuzumab, Solanezumab (LY2062430; proliferation; treating obesity; Suppressing appetite and Eli Lilly), BAN2401, PF-04360365 (Pfizer), and inducing Satiety; treating irritable bowel syndrome; reducing US 9, 161988 B2 5 6 the morbidity and/or mortality associated with myocardial The invention also features a method of treating or treating infarction and stroke; treating acute coronary syndrome char prophylactically a neurological disorder in a Subject, the acterized by an absence of Q-wave myocardial infarction; method including administering to the Subject in an amount attenuating post-Surgical catabolic changes; treating hiber Sufficient to treat or prevent the disorder (e.g., Schizophrenia). nating myocardium or diabetic cardiomyopathy; Suppressing The invention also features a method of treating or treating plasma blood levels of norepinepherine; increasing urinary prophylactically a Subject having a neurodegenerative disor Sodium excretion, decreasing urinary potassium concentra der, the method including administering to the Subject an tion; treating conditions or disorders associated with toxic effective amount of a compound including GDNF, BDNF, or hypervolemia, renal failure, congestive failure, neph an analog thereof. The neurodegenerative disorder may be 10 selected from the group consisting of a polyglutamine expan rotic syndrome, cirrhosis, pulmonary edema, and hyperten sion disorder, fragile X syndrome, fragile XE mental retarda sion; inducing an inotropic response and increasing cardiac tion, Friedreich's ataxia, myotonic dystrophy, spinocerebel contractility; treating polycystic syndrome; treating lar ataxia type 8, and spinocerebellar ataxia type 12, respiratory distress; improving nutrition via a non-alimentary Alexander disease, Alper's disease, Alzheimer's disease, route, i.e., via intravenous, Subcutaneous, intramuscular, 15 amyotrophic lateral Sclerosis (ALS), ataxia telangiectasia, peritoneal, or other injection or infusion; treating nephropa Batten disease (Spielmeyer-Vogt-Sjogren-Batten disease), thy; treating left ventricular systolic dysfunction (e.g., with Canavan disease, Cockayne syndrome, corticobasal degen abnormal left ventricular ejection fraction); inhibiting antro eration, Creutzfeldt-Jakob disease, ischemia stroke, Krabbe duodenal motility (e.g., for the treatment or prevention of disease, Lewy body dementia, multiple Sclerosis, multiple gastrointestinal disorders such as diarrhea, postoperative system atrophy, Parkinson's disease, Pelizaeus-Merzbacher dumping syndrome and irritable bowel syndrome, and as disease, Pick's disease, primary lateral Sclerosis, RefSum's premedication in endoscopic procedures; treating critical ill disease, Sandhoff disease, Schilder's disease, spinal cord ness polyneuropathy (CIPN) and systemic inflammatory injury, spinal muscular atrophy, Steele-Richardson-Olsze response syndrome (SIRS; modulating triglyceride levels and wski disease, and Tabes dorsalis. In certain embodiments, the treating dyslipidemia; treating organ tissue injury caused by 25 polyglutamine repeat disease is Huntington's disease (HD), reperfusion of blood flow following ischemia; or treating dentatorubropallidoluysian atrophy, Kennedy's disease (also coronary heart disease risk factor (CHDRF) syndrome in a referred to as spinobulbar muscular atrophy), or a spinocer Subject by administering and effective amount of a compound ebellar ataxia selected from the group consisting of type 1, including a GLP-1 agonist to the Subject. type 2, type 3 (Machado-Joseph disease), type 6, type 7, and The invention also features a method of increasing GLP-1 30 type 17). receptor activity in a subject by administering a compound The invention also features a method of treating a subject including a GLP-1 agonist to a subject in an amount sufficient having a neuronal damage, the method including administer to increase GLP-1 receptor activity. ing to the Subject an effective amount of a compound includ The invention also features a method of reducing body ing GDNF, BDNF, or an analog thereof. The neuronal dam temperature of a Subject, the method including administering 35 age may be caused by an ischemic stroke, a hemorrhagic a compound including neurotensin or a neurotensin analog in stroke, or a spinal cord injury. a sufficient amount to reduce body temperature. The subject The invention also features a method of treating a subject may be suffering from or has suffered from cerebralischemia, having depression or Schizophrenia, the method including cardiac ischemia, or a nerve injury. The nerve injury may be administering to the Subject an effective amount of a com a spinal cord injury. 40 pound including GDNF, BDNF, or an analog thereof. The invention also features a method of treating pain or The invention also features a method of treating a subject prophylactically treating pain in a Subject, the method includ having a disease related to the amyloid-?protein (e.g., Alzhe ing administering a compound of including neurotensin or a imer's disease or cerebral amyloid angiopathy) by adminis neurotensin analog in an amount Sufficient to treat the pain. tering to said patient an effective amount of a therapeutic The pain may be an acute pain selected from the group con 45 antibody (e.g., an antibody that specifically binds amyloid-f sisting of mechanical pain, heat pain, cold pain, ischemic or a fragment thereof). pain, and chemical-induced pain. The pain may be peripheral In any of the above methods, the Subject may be a human. or central neuropathic pain, inflammatory pain, migraine In the treatment methods of the invention, in certain related pain, headache-related pain, irritable bowel syn embodiments, the compound is administered at a lower (e.g., drome-related pain, fibromyalgia-related pain, arthritic pain, 50 less than 95%, 75%, 60%, 50%, 40%, 30%, 25%, 10%, 5%, or skeletal pain, joint pain, gastrointestinal pain, muscle pain, 1%) equivalent dosage as compared to the recommended angina pain, facial pain, pelvic pain, claudication, postopera dosage of the unconjugated agent. In other embodiments, the tive pain, post traumatic pain, tension-type headache, obstet compound is administered at a higher (1.5.x, 2x, 2.5x, 3.0x. ric pain, gynecological pain, or chemotherapy-induced pain. 5x, 8x. 10x. 15x, 20x, 25x) equivalent dosage than a dosage The invention also features a method of treating or treating 55 recommended for the unconjugated agent. prophylactically a Subject having a psychotic disorder (e.g., In any of the above aspects, the peptide vector may be a Schizophrenia), the method including administering a com polypeptide Substantially identical to any of the sequences set pound including neurotensin or a neurotensin analog in an Table 1, or a fragment thereof. In certain embodiments, the amount sufficient to treat the disorder. peptide vector has a sequence of Angiopep-1 (SEQ ID The invention also features a method of treating drug 60 NO:67), Angiopep-2 (SEQID NO:97), Angiopep-3 (SEQID addiction or drug abuse in a subject, the method including NO:107), Angiopep-4a (SEQ ID NO:108), Angiopep-4b administering to the Subject a compound including neuro (SEQ ID NO:109), Angiopep-5 (SEQ ID NO:110), Angio tensin or a neurotensin analog in an amount Sufficient to treat pep-6 (SEQID NO:111), Angiopep-7 (SEQ ID NO:112) or the addiction or abuse. The drug may be a psychoStimulant reversed Angiopep-2 (SEQID NO:117)). The peptide vector (e.g., amphetamine, methamphetamine, 3.4-methylene 65 or compound of the invention may be efficiently transported dioxymethamphetamine, nicotine, cocaine, methylpheni into a particular cell type (e.g., any one, two, three, four, or date, and arecoline) five of liver, lung, kidney, spleen, and muscle) or may cross

US 9, 161988 B2 9 10 TABLE 1. - Continued TABLE 1 - continued Exemplary Peptide Vectors Exemplary Peptide Vectors

SEQ ID SEQ ID NO: NO :

68 K. F. F. Y. G. G. C. R. G. K R N N F K. T E E Y O4 G L C Q T F W Y G G C R A K R N N F K S A. E 69 T F Y Y G. G. C. R. G. K R N N Y K. T E E Y O5 L C Q T F W Y G G C E A K R N N F K S A. 70 T. F. F. Y. G. G. S. R. G. K R N N F K. T E E Y 10 Of T F. F. Y. G. G. S R G K R N N F K. T E E Y 71 C T E F Y G. C. C. R. G. K R N N F K. T E E Y O8 R. F. F. Y. G. G. S R G K R N N F K. T E E Y 72 T E F Y G. G. C. R. G. K R N N F K. T E E Y C O9 R. F. F. Y. G. G. S R G K R N N F K. T E E Y 73 C T E F Y G. S C R. G. K R N N F K. T E E Y 15 10 R. F. F. Y. G. G. S R G K R N N F R T E E Y 74 T F. F. Y. G. G. S. R. G. K R N N F K. T E E Y C 11 T. F. F. Y. G. G. S R G K R N N F R T E E Y 7s P F. F. Y. G. G. C. R. G. K R N N F K. T E E Y 12 T F. F. Y. G. G. S. R. G. R. R. N. N. F. R T E E Y 76. T E F Y G. G. C. R. G. K R N N F K T K E Y 13 C T F. F. Y. G. G. S. R. G. K R N N F K. T E E Y 77 T E F Y G. G. K. R. G. K R N N F K. T E E Y 14 T F. F. Y. G. G. S R G K R N N F K. T E E Y C 78 T E F Y G. G. C. R. G. K R N N F K. T. K R Y 15 C T F. F. Y. G. G. S. R. G. R. R. N. N. F. R T E E Y 79 T F. F. Y. G. G. K. R. G. K R N N F K. T. A. E. Y 25 16 T F. F. Y. G. G. S. R. G. R. R. N. N. F. R T E E Y C 80 T. F. F. Y. G. G. K. R. G. K R N N F K. T. A. G. Y 17 Y E E T K F. N. N. R. K. G. R. S. G. G Y F. F. T 81 T. F. F. Y. G. G. K. R. G. K R N N F K. R. E. K. Y Polypeptides Nos. 5, 67, 76, and 91, include the sequences of 82 T F. F. Y. G. G. K. R. G. K R N N F K. R. A. K. Y SEQ ID NOS: 5, 67, 76, and 91, respectively, and are amidated at the C-terminus. 30 Polypeptides Nos. 107, 109, and 110 include the sequences of SEQ 83 T. F. F. Y. G. G. C. L. G. N. R. N. N. F. K. T E E Y ID NOS: 97, 109, and 110, respectively, and are acetylated at the N-terminus. 84 T F. F. Y. G. C. G. R. G. K R N N F K. T E E Y In any of the above aspects, the peptide vector may include 85 T. F. F. Y. G. G. R. C. G. K R N N F K. T E E Y an amino acid sequence having the formula: 35 86 T F. F. Y. G. G. C. L. G. N. G. N N F D T E E E

87 T F O Y G G C R G K R N N F K T E E Y where each of X1-X19 (e.g., X1-X6, X8, X9, X11-X14, and 88 Y IN K E F. G. T. F. N. T. K. G. C E R G Y R. F X16-X19) is, independently, any amino acid (e.g., a naturally 89 R. F. K. Y. G. G. C. L. G. N. M. N. N. F. E. T L E E 40 occurring amino acid Such as Ala, Arg, ASn, Asp, Cys, Gln, Glu, Gly. His, Ile, Leu, Lys, Met, Phe, Pro, Ser. Thr, Trp, Tyr, 9 O R. F. K. Y. G. G. C. L. G. N. K. N. N. F. L. R. L. K. Y and Val) or absent and at least one (e.g., 2 or 3) of X1, X10, and X15 is . In some embodiments, X7 is Seror Cys: 91 R F K. Y. G. G. C. L. G. N. K. N. N. Y. L. R. L. K. Y or X10 and X15 each are independently Arg or Lys. In some 92 K T K R K R K K Q R V K I AY E E I F. K. N. Y 45 embodiments, the residues from X1 through X19, inclusive, are substantially identical to any of the amino acid sequences 93 K T K R K R K K Q R V K I AY of any one of SEQID NOS:1-105 and 107-117 (e.g., Angio pep-1, Angiopep-2. Angiopep-3, Angiopep-4a, Angiopep-4b. 94 R G G R L S Y S R R F S T S T G R Angiopep-5, Angiopep-6, Angiopep-7, and reversed Angio 95 R. R. L. S. Y S R R. R. F 50 pep-2). In some embodiments, at least one (e.g., 2, 3, 4, or 5) of the amino acids X1-X19 is Arg. In some embodiments, the 96 R O I K I W F O N R. R. M K W K. K. polypeptide has one or more additional residues at 97 T F. F. Y. G. G. S. R. G. K R N N F K. T E E Y the N-terminal of the polypeptide, the C-terminal of the polypeptide, or both. 98 M R P D F C L E P P Y T G P C W. A. R. I 55 I R Y F Y IN A K A G L C Q T F W Y G G In certain embodiments of any of the above aspects, the C R A K R N N F K S A E D C M R T C G G. A. peptide vector or a peptide therapeutic described herein is modified (e.g., as described herein). The peptide or polypep 99 T E F Y G. G. C. R. G. K R N N F K T K E Y tide may be amidated, acetylated, or both. Such modifications 1 OO R. F. K. Y. G. G. C. L. G. N. K. N. N. Y. L. R. L. K. Y may be at the amino or carboxy terminus of the polypeptide. 60 The peptide or polypeptide may also include peptidomimet 101 T. F. F. Y. G. G. C. R. A. K R N N F K. R. A. K. Y ics (e.g., those described herein) of any of the polypeptides described herein. 1 O2 N A K A G L C Q T F W Y G G C L A K R N N F In certain embodiments, the peptide vector or a peptide E S A E D C M R T C G G. A. therapeutic described herein has an amino acid sequence 103 Y G. G. C. R. A. K R N N F K S A E D C M R T C G 65 described herein with at least one amino acid substitution (e.g., 2,3,4,5,6,7,8,9, 10, 11, or 12 substitutions), insertion, or deletion or is Substantially identical to an amino acid US 9, 161988 B2 11 12 sequence described herein. The peptide or polypeptide may By “therapeutic agent' is meant an agent that is capable of contain, for example, 1 to 12, 1 to 10, 1 to 5, or 1 to 3 amino being used in the treatment or prophylactic treatment of a acid substitutions, for example, 1 to 10 (e.g., to 9, 8, 7, 6, 5, 4, disease or condition. 3, 2) amino acid Substitutions. The amino acid substitution(s) By "RNAi agent' is meant any agent or compound that may be conservative or non-conservative. For example, the 5 exerts a gene silencing effect by way of an RNA interference peptide vector may have an arginine at one, two, or three of pathway. RNAi agents include any nucleic acid molecules the positions corresponding to positions 1, 10, and 15 of the that are capable of mediating sequence-specific RNAi, for amino acid sequence of any of SEQID NO:1, Angiopep-1, example, a short interfering RNA (siRNA), double-stranded Angiopep-2. Angiopep-3, Angiopep-4a, Angiopep-4b. RNA (dsRNA), microRNA (miRNA), short hairpin RNA Angiopep-5, Angiopep-6, Angiopep-7, and reversed Angio 10 (shRNA), short interfering oligonucleotide, short interfering pep-2. In certain embodiments, the BDNF, GDNF, or related nucleic acid, short interfering modified oligonucleotide, molecule may have a cysteine or lysine Substitution or addi chemically-modified siRNA, and post-transcriptional gene tion at any position (e.g., a lysine Substitution at the N- or silencing RNA (ptgsRNA). C-terminal position). 15 By “treating a disease, disorder, or condition in a subject In any of the above aspects, the compound may specifically is meant reducing at least one symptom of the disease, disor exclude a polypeptide including or consisting of any of SEQ der, or condition by administrating a therapeutic agent to the ID NOS: 1-105 and 107-117 (e.g., Angiopep-1, Angiopep-2. Subject. Angiopep-3, Angiopep-4a, Angiopep-4b, Angiopep-5, By “treating prophylactically a disease, disorder, or con Angiopep-6, Angiopep-7, and reversed Angiopep-2). In some dition in a subject is meant reducing the frequency of occur embodiments, the polypeptides and compounds of the inven rence or severity of (e.g., preventing) a disease, disorder or tion exclude the polypeptides of SEQID NOS:102, 103, 104, condition by administering to the Subject a therapeutic agent and 105. to the Subject prior to the appearance of a disease symptom or By "fragment' is meant a portion of a full-length amino symptoms. acid or nucleic acid sequence (e.g., any sequence described 25 By "subject' is meant a human or non-human animal (e.g., herein). Fragments may include at least 4, 5, 6, 8, 10, 15, 20, a mammal). 25, 30, 35, 40, 45, 50, 60, 70, 75, 80,90, 100,125, 150, 200, By "equivalent dosage' is meant the amount of a com 500, 1000, 1500, 2000, or 5000 amino acids or nucleic acids pound of the invention required to achieve the same molar of the full length sequence. A fragment may retain at least one amount of agent in the compound of the invention, as com of the biological activities of the full length protein. 30 pared to the unconjugated molecule. By "substantially identical” is meant a polypeptide or By a polypeptide which is “efficiently transported across nucleic acid exhibiting at least 35%, 40%, 50%, 55%, 60%, the BBB is meant a polypeptide that is able to cross the BBB 65%, 70%, 75%, 85%, 90%, 95%, or even 99% identity to a at least as efficiently as Angiopep-6 (i.e., greater than 38.5% reference amino acid or nucleic acid sequence. For polypep that of Angiopep-1 (250 nM) in the in situ brain perfusion tides, the length of comparison sequences will generally beat 35 assay described in U.S. patent application Ser. No. 1 1/807, least 4 (e.g., at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17. 597, filed May 29, 2007, hereby incorporated by reference). 18, 19, 20, 25, 50, or 100) amino acids. For nucleic acids, the Accordingly, a polypeptide which is “not efficiently trans length of comparison sequences will generally be at least 60 ported across the BBB is transported to the brain at lower nucleotides, preferably at least 90 nucleotides, and more pref 40 levels (e.g., transported less efficiently than Angiopep-6). erably at least 120 nucleotides, or full length. It is to be By a polypeptide or compound which is “efficiently trans understood herein that gaps may be found between the amino ported to a particular cell type' is meant that the polypeptide acids of sequences that are identical or similar to amino acids or compound is able to accumulate (e.g., either due to of the original polypeptide. The gaps may include no amino increased transport into the cell, decreased efflux from the acids, one or more amino acids that are not identical or similar 45 cell, or a combination thereof) in that cell type to at least a to the original polypeptide. Percent identity may be deter 10% (e.g., 25%, 50%, 100%, 200%, 500%. 1,000%, 5,000%, mined, for example, with n algorithm GAP, BESTFIT, or or 10,000%) greater extent than either a control substance, or, FASTA in the Wisconsin Genetics Software Package Release in the case of a conjugate, as compared to the unconjugated 7.0, using default gap weights. agent. Such activities are described in detail in International By "peptide vector” is meanta compound or molecule such 50 Application Publication No. WO 2007/009229, hereby incor as a polypeptide or a peptidomimetic that can be transported porated by reference. into a particular cell type (e.g., liver, lungs, kidney, spleen, or Other features and advantages of the invention will be muscle) or across the BBB. The vector may be attached to apparent from the following Detailed Description, the draw (covalently or not) or conjugated to an agent and thereby may ings, and the claims. be able to transport the agent into a particular cell type or 55 across the BBB. In certain embodiments, the vector may bind BRIEF DESCRIPTION OF THE DRAWINGS to receptors present on cancer cells or brain endothelial cells and thereby be transported into the cancer cell or across the FIG. 1 is a set of graphs showing the TMEA-(Angiopep-2) BBB by transcytosis. The vector may be a molecule for which conjugate before (Chromatogram 1) and after (Chromato high levels of transendothelial transport may be obtained, 60 gram 2) purification. without affecting the cellor BBB integrity. The vector may be FIG. 2 is a graph showing purification of the TMEA-(An a polypeptide or a peptidomimetic and may be naturally giopep-2) conjugate. occurring or produced by chemical synthesis or recombinant FIG. 3 is a set of graphs showing the SATP-Angiopep-2- genetic technology. Angiopep-2 conjugate before (Chromatogram 4) and after By "agent' is meant any compound having at least one 65 (Chromatogram 5) purification. biological activity. Agents include both diagnostic and thera FIG. 4 is a graph showing purification of the SATP-Angio peutic agents. pep-2-Angiopep-2 conjugate. US 9, 161988 B2 13 14 FIG. 5 is a schematic diagram showing formation of the Multimeric Peptide Vectors Angiopep-1 (SEQID NO: 67)dimmer formed through disul The compounds of the invention feature a multimeric (e.g., fide bonds. dimeric) form of the peptide vectors described herein. The FIG. 6 is a graph showing apparent Volume of parenchyma peptide vectors are joined by a chemical bond either directly distribution measured by an in situ brain perfusion assay for 5 (e.g., a covalent bond such as a disulfide or a peptide bond) or the Angiopep-1 dimer and Angiopep-2. indirectly (e.g., through a linker Such as those described FIG. 7 is a graph showing parenchymal uptake (volume of herein). Exemplary multimeric peptides are described below. parenchyma transformed to pmol uptake) using an in situ Peptides Joined by Linkers brain perfusion assay for the Angiopep-1 dimer and Angio In some embodiments, the peptide vectors described herein pep-2. 10 are joined by a chemical linker. Such chemical linkers are FIG. 8 is a graph showing uptake of Angiopep-2 monomers known in the art and are described herein. Any appropriate (synthetic and recombinant) as well as Angiopep-2 dimers linker can be used to produce a multimer of the invention. and trimers in lean mice at 50 nM concentration using the in Exemplary chemical linkers include those described below. situ brain perfusion assay. A comparison using recombinant 15 In certain embodiments, the multimeric peptide vector is a Angiopep-2 in diet-induced obese (DIO) mice is also shown. dimer having the formula: FIG.9A is a schematic diagram showing the structure of an Exendin-4-Angiopep-2 dimmer conjugate (EX4(Lys39 (MHA))-AN2-AN2). The compound has the structure where A' and A are each, independently, a peptide vector HGEGTFTSDLSKQMEEEAVR (e.g., any peptide vector described herein) and X is a linker. LFIEWLKNGGPSSGAPPPK (SEQ ID NO: 134)-(MHA)- The linker may be any linker described herein. In particular TFFYGGSRGKRNNFKTEEYC-(MPA)- embodiments, the linker contains a maleimido moiety and TFFYGGSRGKRNNFKTEEY (SEQ ID NO: 178)-OH, binds to a cysteine present in the peptide vector (e.g., a pep where MHA is maleimidohexanoic acid and MPA is male tide vector to which an N-terminal or C-terminal cysteine imido propionic acid. 25 FIG. 9B is a schematic structure of an Exendin-4- residue has been added). scramble-Angiopep-2 (Ex4(Cys32)-ANS4 (N-Term) or In other embodiments, the multimeric peptide vector has or Exen-S4) that was used a control. This compound has the includes a formula selected from the group consisting of Structure HGEGTFTSDLSKQMEEEAVR 30 LFIEWLKNGGPCSGAPPPS-(MHA)-GYKGERYRG A3 FKETNFNTES-OH (SEQ ID NO: 123), where MHA is maleimidohexanoic acid. X X XP FIG. 10 is a graph showing the ability of, in order from left A11 YA2 and A11 (a- s to right, Exendin-4, Exendin-4-Angiopep-2 conjugates C3, 35 C6, and C11 (where the number indicates the length of the where A, A, A, A", and each A are, independently, a carbon chain connecting the Angiopep-2 and Exendin-4, as peptide vector (e.g., any peptide vector described herein); X, described in U.S. Provisional Application No. 61/105,618, X', and each X are, independently, a linker (e.g., any linker filed Oct. 15, 2008), Exen-S4, and Exendin-4 when conju described herein) that joins together two peptide vectors; n is gated to a dimeric form of Angiopep-2, to cross the BBB. 40 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10; m is n+2; and p is an integer FIG. 11 is a graph showing the ability of Exendin-4 and from 2 to n+1. In particular embodiments, n is 1, and the Exen-An2-An2 to reduce glycemia in mice as compared to a compound has the formula: control.

DETAILED DESCRIPTION 45 X X2 A11 NA21NA We have now developed multimeric forms of peptide vec tors that are able to cross the blood-brain barrier (BBB) or are Higher order multimers can also be described by the for able to enter particular cell types (e.g., liver, spleen, kidney, mula: muscle, ovary) with enhanced efficiency. These multimeric 50 forms, when conjugated to a therapeutic agent, can transport the agent across the BBB or into particular cells. In some cases, the multimeric (e.g., dimeric) form of the peptide vec Xi-Al tor is capable of crossing the BBB or entering particular cell V -). types more efficiently, and in certain cases as described 55 X-A2--X-A) herein, far more efficiently, than the monomeric form of the peptide vector. This increased efficiency in transport may A. allow for lower dosages of the therapeutic as compared either to the unconjugated agent or to the agent conjugated to a fx-A), monomeric form of the peptide vector. In other cases, by 60 directing the agent more efficiently to its target tissue(s), the where A, A, each A7, each A, and each A are, indepen compounds of the invention may administered in higher dos dently, peptide vectors (e.g., any of those described herein); ages than either the unconjugated agent or the agent conju A is a peptide vector or is absent; X, each X7, each X, and gated to a monomeric form of the peptide vector, as the each X are, independently, linkers that join peptide vectors; greater targeting efficiency can reduce side effects. Com 65 m, n, and pare each, independently, 0, 1, 2, 3, 4, 5, 6, 7, 8, 9. pounds including Such multimers and their use in treatment of or 10; q is an integer from 4 to m+3; ris an integer from m+4 disease are described in detail below. to m--n +3; and S is an integer from m+n+4 to m+n+p+3. US 9, 161988 B2 15 16 Fusion Protein Multimers is reactive towards amines and adds protected Sulfhydryls In other embodiments, the multimeric peptide is in the groups), and BMOE (bis-maleimidoethane). form of a fusion protein. The fusion protein may contain 2, 3, To form covalent bonds, one can use as a chemically reac 4, 5, or more peptide vectors, either joined directly by a tive group a wide variety of active carboxyl groups (e.g., peptide bond, or through peptide linkers. In one example, esters) where the hydroxyl moiety is physiologically accept fusion protein dimers are described by the formula: able at the levels required to modify the peptide. Particular agents include N-hydroxysuccinimide (NHS), N-hydroxy sulfosuccinimide (sulfo-NHS), maleimide-benzoyl-succin where A' and Aare, independently, a peptide vector (e.g., an imide (MBS), gamma-maleimido-butyryloxy Succinimide amino acid sequence Substantially identical to a sequence 10 ester (GMBS), maleimido propionic acid (MPA), maleimido selected from the group consisting of SEQID NO: 1-105 and hexanoic acid (MHA), and maleimido undecanoic acid 107-117, or a functional fragment thereof) and X is either (a) (MUA). a peptide bond that joins A" and A’ or (b) one or more amino Primary amines are the principal targets for NHS esters. acids joined to A' and Abypeptide bonds. In certain embodi Accessible O.-amine groups present on the N-termini of pro ments, the peptide is a single amino acid (e.g., a naturally 15 teins and the 8-amine of lysine react with NHS esters. Thus, occurring amino acid), a flexible linker, a rigid linker, or an compounds of the invention can include a linker having a a-helical linker. Exemplary peptide linkers that can be used in NHS ester conjugated to an N-terminal amino of a peptide or the invention are described in the section entitled “peptide to an e-amine of lysine. An amide bond is formed when the linkers” below. In certain embodiments A and A are the NHS ester conjugation reaction reacts with primary amines same peptide vector. releasing N-hydroxySuccinimide. These Succinimide con Fusion protein multimers can be described by the formula: taining reactive groups are herein referred to as Succinimidyl groups. In certain embodiments of the invention, the func A-CX-A"), tional group on the protein will be a thiol group and the where n is or is at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10; m is an chemically reactive group will be a maleimido-containing integer from 2 to n+1: A' and each A" are, independently, a 25 group such as gamma-maleimide-butrylamide (GMBA or peptide vector (e.g., an amino acid sequence Substantially MPA). Such maleimide containing groups are referred to identical to a sequence selected from the group consisting of herein as maleido groups. SEQ ID NO:1-105 and 107-117, or a functional fragment The maleimido group is most selective for sulfhydryl thereof); and each X" is, independently, either (a) a peptide groups on peptides when the pH of the reaction mixture is bond that joins A" and A’ or (b) one or more amino acids 30 6.5-7.4. At pH 7.0, the rate of reaction of maleimido groups joined to the adjacent peptide vector (A' or A") by peptide with Sulfhydryls (e.g., thiol groups on proteins such as serum bonds. albuminor IgG) is 1000-fold faster than with amines. Thus, a The peptide vectors forming the multimer, in certain stable thioether linkage between the maleimido group and the embodiments, may each be fewer than 100, 50, 40,35, 30, 25, Sulfhydryl can be formed. Accordingly, a compound of the 24, 23, 22, 21, 20, 19, 18, 17, 16, or 15 amino acids in length. 35 invention can include a linker having a maleimido group The fusion protein may be fewer than 1,000, 500, 250, 150, conjugated to a Sulfhydryl group of a peptide vector or of an 100, 90, 80, 75, 70, 65, 60, 55, 50, 45, 40, or 35 amino acids agent. in length. Amine-to-amine linkers include NHS esters and imi Linkers doesters. Exemplary NHS esters are DSG (disuccinimidyl The peptide vectors may be bound to each other or to a 40 glutarate), DSS (disuccinimidyl suberate), BS (bis sulfosuc therapeutic agent either directly (e.g., through a covalent cinimidylsuberate), TSAT (tris-succinimidyl aminotriac bond Such as a peptide bond) or may be bound through a etate), variants of bis-succinimide ester-activated compounds linker. Linkers include chemical linking agents (e.g., cleav that include a polyethylene glycol spacer such as BS(PEG), able linkers) and peptides. Any of the linkers described below where n is 1-20 (e.g., BS(PEG) and BS(PEG)), DSP may be used in the compounds of the invention. 45 (Dithiobis succinimidyl propionate), DTSSP (3,3'-dithiobis Chemical Linking Agents sulfosuccinimidylpropionate), DST (disuccinimidyl tart In some embodiments, the linker is a chemical linking arate), BSOCOES (bis 2-(succinimidooxycarbonyloxy) agent. The peptide vector may be conjugated through sulfhy ethylsulfone), EGS (ethylene glycol bis dryl groups, amino groups (amines), or any appropriate reac succinimidylsuccinate), and sulfo-EGS (ethylene glycol bis tive group. Homobifunctional and heterobifunctional cross 50 sulfosuccinimidylsuccinate). Imidoesters include DMA linkers (conjugation agents) are available from many (dimethyl adipimidate.2 HCl), DMP (dimethyl commercial Sources. Sites available for cross-linking may be pimelimidate.2HCl). DMS (dimethyl suberimidate.2HCl). found on the peptides and agents described herein. The cross and DTBP (dimethyl 3,3'-dithiobispropionimidate.2HCl). linker may comprise a flexible arm, e.g., 2, 3, 4, 5, 6, 7, 8, 9. Other amine-to-amine linkers include DFDNB (1.5-difluoro 10, 11, 12, 13, 14, or 15 carbon atoms. Exemplary cross 55 2,4-dinitrobenzene) and THPP (B-tris(hydroxymethyl)phos linkers include BS (Bis(sulfosuccinimidyl)suberatel: BS is phinopropionic acid (betaine)). a homobifunctional N-hydroxysuccinimide ester that targets The linker may be a sulfhydryl-to-sulfhydry linker. Such accessible primary amines), NHS/EDC (N-hydroxysuccin linkers include maleimides and pyridyldithiols. Exemplary imide and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide: maleimides include BMOE (bis-maleimidoethane), BMB NHS/EDC allows for the conjugation of primary amine 60 (1,4-bismaleimidobutane), BMH (bismaleimidohexane), groups with carboxyl groups), sulfo-EMCS (IN-e-maleimi TMEA (tris(2-maleimidoethylamine), BM(PEG)2 1.8-bis docaproic acid hydrazide; sulfo-EMCS are heterobifunc maleimidodiethyleneglycol) or BM(PEG), where n is 1 to 20 tional reactive groups (maleimide and NHS-ester) that are (e.g., 2 or 3), BMDB (1.4 bismaleimidyl-2,3-dihydroxybu reactive toward Sulfhydryl and amino groups), hydrazide tane), and DTME (dithio-bismaleimidoethane). Exemplary (most proteins contain exposed carbohydrates and hydrazide 65 pyridyldithiols include DPDPB (1,4-di-3'-(2-py is a useful reagent for linking carboxyl groups to primary ridyldithio)-propionamidobutane). Other sulfhydryl linkers amines), SATA (N-succinimidyl-S-acetylthioacetate; SATA include HBVS (1,6-hexane-bis-Vinylsulfone). US 9, 161988 B2 17 18 The linker may be an amine-to-sulfhydryl linker, which propionyl hydrazide)). Exemplary carbohydrate-to includes NHS ester/maliemide compounds. Examples of nonselective linkers includehydraZide/aryl azide compounds these compounds are AMAS (N-(C.-maleimidoacetoxy)suc (e.g., ABH (p-azidobenzoyl hydrazide)). Exemplary cinimide ester), BMPS(N-B-maleimidopropyloxysuccin hydroxyl-to-sulfhydryl linkers include isocyanate/maleimide imide ester), GMBS (N-y-maleimidobutyryloxysuccinim compounds (e.g., (N-p-maleimidophenylisocyanate)). ide ester), sulfo-GMBS (N-y-maleimidobutyryloxy Exemplary amine-to-DNA linkers include NHS ester/psor sulfosuccinimide ester), MBS (m-maleimidobenzoyl-N- alen compounds (e.g., SPB (Succinimidyl-4-(psoralen-8- hydroxySuccinimide ester), Sulfo-MBS yloxy)-butyrate)). (m-maleimidobenzoyl-N-hydroxysulfosuccinimide ester), In other embodiments, the linker is a trifunctional, tet SMCC (succinimidyl 4-N-maleimidomethylcyclohexane 10 rafunctional, or greater linking agent. Exemplary trifunc 1-carboxylate), sulfo-SMCC (Sulfosuccinimidyl 4-N-male tional linkers include TMEA, THPP TSAT, LC-TSAT (tris imidomethylcyclohexane-1-carboxylate), EMCS (IN-e-ma Succinimidyl (6-aminocaproyl)aminotriacetate), tris leimidocaproyloxysuccinimide ester), Sulfo-EMCS (IN-e- succinimidyl-1,3,5-benzenetricarboxylate, MDSI maleimidocaproyloxysulfosuccinimide ester), SMPB (maleimido-3,5-disuccinimidyl isophthalate), SDMB (suc (succinimidyl 4-p-maleimidophenylbutyrate), sulfo-SMPB 15 cinimidyl-3,5-dimaleimidophenylbenzoate, Mal-4 (tetrakis (sulfosuccinimidyl 4-p-maleimidophenylbutyrate), SMPH (3-maleimidopropyl)pentaerythritol, NHS-4 (tetrakis-(N- (Succinimidyl-6-B-maleimidopropionamidohexanoate), Succinimidylcarboxypropyl)pentaerythritol)). LC-SMCC (succinimidyl-4-N-maleimidomethylcyclohex ane-1-carboxy-6-amidocaproate), sulfo-KMUS (N-K-ma TMEA has the structure: leimidoundecanoyloxysulfosuccinimide ester), SM(PEG), (Succinimidyl-(N-maleimidopropionamido polyethyleneglycol) ester), where n is 1 to 30 (e.g., 2, 4, 6, 8, 12, or 24), SPDP(N-succinimidyl 3-(2-pyridyldithio)-propi onate), LC-SPDP (succinimidyl 6-(3-2-pyridyldithio-pro pionamido)hexanoate), sulfo-LC-SPDP (sulfosuccinimidyl 6-(3'-(2-pyridyldithio-propionamido)hexanoate), SMPT 25 (4-Succinimidyloxycarbonyl-O-methyl-O-2-pyridyldithio toluene), Sulfo-LC-SMPT (4-sulfosuccinimidyl-6-O-me thyl-O-(2-pyridyldithio)toluamidohexanoate), SIA (N-suc cinimidyl iodoacetate), SBAP (Succinimidyl 3-bromoacetamidopropionate), SIAB (N-succinimidyl-4- 30 iodoacetylaminobenzoate), and sulfo-SIAB (N-sulfosuccin imidyl4-iodoacetylaminobenzoate). In other embodiments, the linker is an amino-to-nonselec tive linker. Examples of such linkers include NHS ester/aryl TMEA, through its maleimide groups, can react with sulfhy azide and NHS ester/diazirine linkers. NHS ester/aryl azide dryl groups (e.g., through cysteine amino acid side chains). linkers include NHS-ASA (N-hydroxysuccinimidyl-4-azi 35 THPP has the structure: dosalicylic acid), ANB-NOS(N-5-azido-2-nitrobenzoylox y succinimide), sulfo-HSAB (N-hydroxysulfosuccinimidyl 4-azidobenzoate), sulfo-NHS-LC-ASA (sulfosuccinimidyl HO 4-azidosalicylamidohexanoate), SANPAH (N-succinimidyl-6-(4-azido-2'-nitrophenylamino)hex 40 anoate), sulfo-SANPAH (N-sulfosuccinimidyl-6-(4-azido 2'-nitrophenylamino)hexanoate), sulfo-SFAD (sulfosuccin imidyl-(perfluoroazidobenzamido)-ethyl-1,3'- / t-r dithioproprionate), sulfo-SAND (sulfosuecinimidyl-2-(m- azido-o-nitrobenzamido)-ethyl-1,3'-proprionate), and Sulfo 45 The hydroxyl groups and carboxy group of THPP can react SAED (sulfosuccinimidyl 2-7-amino-4-methylcoumarin-3- with primary or secondary amines. acetamido)ethyl-1,3'dithiopropionate). NHS ester/diazirine Linkers are also described in U.S. Pat. No. 4,680,338 hav linkers include SDA (succinimidyl 4,4'-azipentanoate), LC ing the formula Y=C=N-Q-A-C(O)—Z, where Q is a SDA (Succinimidyl 6-(4,4'-azipentanamido)hexanoate), homoaromatic or heteroaromatic ring system; A is a single SDAD (succinimidyl 2-(4,4'-azipentanamido)ethyl)-1,3'- 50 bond or an unsubstituted or Substituted divalent Clso bridg dithioproprionate), sulfo-SDA (sulfosuccinimidyl 4,4'-azi ing group, Y is O or S; and Z is Cl, Br, I, N,N-succinimidy pentanoate), sulfo-LC-SDA (sulfosuccinimidyl 6-(4,4'-azi loxy, imidazolyl, 1-benzotriazolyloxy, OAr where Ar is an pentanamido)hexanoate), and Sulfo-SDAD electron-deficient activating aryl group, or OC(O)R where R (SulfoSuccinimidyl 2-(4,4'-azipentanamidolethyl)-1,3'- is -A-Q-N=C=Y or C-20 tertiary-alkyl. dithioproprionate). 55 Linkers are also described in U.S. Pat. No. 5,306,809, Exemplary amine-to-carboxyl linkers include carbodiim which describes linkers having the formula ide compounds (e.g., DCC(N,N-dicyclohexylcarbodimide) and EDC (1-ethyl-3-3-dimethylaminopropylcarbodiim ide)). Exemplary sulfhydryl-to-nonselective linkers include O pyridyldithiol/aryl azide compounds (e.g., APDP((N-4-(p- R azidosalicylamido)butyl-3'-(2-pyridyldithio)propiona 60 mide)). Exemplary sulfhydryl-to-carbohydrate linkers R O include maleimide/hydrazide compounds (e.g., BMPH(N- n N B-maleimidopropionic acid hydrazide), EMCH (N-e-male imidocaproic acid hydrazide), MPBH 4-(4-N-maleimi l 3 O dophenyl)butyric acid hydrazide), and KMUH(N-K- 65 R1 maleimidoundecanoic acid hydrazide)) and pyridyldithiol/ hydrazide compounds (e.g., PDPH (3-(2-pyridyldithio) US 9, 161988 B2 19 20 where R is H. C. alkyl, Calkenyl, Caryl or aralkyl or Asp (e.g., which forms an amide bond with the e-amino group these coupled with a divalent organic —O— —S—, or of Lys and anotheramide bond with a carboxyl group present in the substituent), that is, the substituent is a N-acylated lysine residue. In other embodiments, the peptide linker is a branched i polypeptide. Exemplary branched peptide linkers are 1N, described in U.S. Pat. No. 6,759,509. Such linkers includes those of the formula: where R is C alkyl, linking moiety; R is H. C. alkyl, 10 C-2 aryl, or C-2 aralkyl, R is

O O O S us N- N- N 15 S O S where A is a thiol acceptor; W is a bridging moiety; c is an integer of 0 to 1; a is an integer of 2 to 12: Q is O. NH, or N-lower alkyl; p is an integer of 0 or 1; d is an integer of 0 or N- N-H NH 1: E is a polyvalent atom; each b is an integer of 1 to 10; each X is of the formula: or another chemical structure which is able to delocalize the lone pair electrons of the adjacent nitrogen and R is a pendant reactive group capable of linking R to a peptide vector or to where Y is two amino acid residues in the L form; Z is one or an agent. 25 two amino acid residues; m is an integer of 0 or 1: G is a Amino Acid and Peptide Linkers self-immolative spacer; and n is a integer of 0 or 1, provided In other embodiments, the linker includes at least one that when n is 0 then —Y Z is Ala-Leu-Ala-Leu or Gly amino acid (e.g., a peptide of at least 2, 3, 4, 5, 6, 7, 10, 15, 20, Phe-Leu-Gly; or each X is of the formula: 25, 40, or 50 amino acids). In certain embodiments, the linker is a single amino acid (e.g., any naturally occurring amino 30 acid Such as CyS). In other embodiments, a -rich pep tide such as a peptide having the sequence Gly-Gly-Gly Gly-Ser (SEQID NO: 124) where n is 1, 2, 3, 4, 5 or 6 is used, as described in U.S. Pat. No. 7,271,149. In other embodiments, a -rich peptide linker is used, as 35 described in U.S. Pat. No. 5,525,491. Serine rich peptide linkers include those of the formula X-X-X-X-Gly (SEQ where each X is of the formula –CO Y Z-G, and ID NO: 125) where up to two of the X are Thr, and the where Y, Z, Q, E, G, m, d. p., a, b, and n are as defined above; remaining Xare Ser, and y is 1 to 5 (e.g., Ser-Ser-Ser-Ser-Gly, or each X is of the formula: (SEQID NO: 126) where y is greater than 1). In some cases, 40 the linker is a single amino acid (e.g., any amino acid, such as Gly or Cys). Amino acid linkers may be selected for flexibility (e.g., flexible or rigid) or may be selected on the basis of charge M (e.g., positive, negative, or neutral). Flexible linkers typically 45 include those with Gly resides (e.g., Gly-Gly-Gly-Gly-Ser, (SEQID NO:124) where n is 1,2,3,4, 5 or 6). Other linkers where each X* is of the formula –CO Y Z-G, and include rigid linkers (e.g., PAPAP (SEQ ID NO: 127) and where Y, Z, G, Q, E, m, d. p., a, b, and n are as defined above; (PT)P (SEQID NO: 128), where n is 2, 3, 4, 5, 6, or 7) and or each X* is of the formula: C.-helical linkers (e.g., A(EAAAK), A (SEQ ID NO : 129), 50 where n is 1, 2, 3, 4, or 5). Examples of Suitable linkers are Succinic acid, Lys, Glu, and Asp, or a such as Gly-Lys. When the linker is Succinic acid, one carboxyl group thereof may forman amide bond with an amino group of the amino acid residue, and the 55 other carboxyl group thereof may, for example, form an amide bond with an amino group of the peptide or Substituent. where each X is of the formula –CO Y Z-G, and When the linker is Lys, Glu, or Asp, the carboxyl group wherein Y, Z, G, Q.E. m., d. p., a, b, and n areas defined above; thereof may form an amide bond with an amino group of the or each X is of the formula amino acid residue, and the amino group thereof may, for 60 example, form an amide bond with a carboxyl group of the substituent. When Lys is used as the linker, a further linker may be inserted between the e-amino group of Lys and the substituent. In one particular embodiment, the further linker M is succinic acid, which can form an amide bond with the 65 (CH)-X e-amino group of Lys and with an amino group present in the substituent. In one embodiment, the further linker is Glu or US 9, 161988 B2 21 22 where each X* is of the formula –CO Y Z-G, and the bond —S CH-. Preferably, the thiol acceptor is a where Y, Z, G, Q, E, m, d, p, a, b, and n are as defined above. Michael Addition acceptor. A representative Michael Addi The branched linker may employ an intermediate self tion acceptor of this invention has the formula immolative spacer moiety (G), which covalently links together the agent or peptide vector and the branched peptide linker. A self-immolative spacer can be a bifunctional chemi cal moiety capable of covalently linking together two chemi cal moieties and releasing one of said spaced chemical moi eties from the tripartate molecule by means of enzymatic cleavage (e.g., any appropriate linker described herein. In 10 certain embodiments, G is a self-immolative spacer moiety which spaces and covalently links together the agent or pep O tide vector and the peptide linker, where the spacer is linked to the peptide vector or agent via the T moiety (as used in the After linkage the thiol group of the ligand, the Michael Addi following formulas “T” represents a nucleophilic atom which tion acceptor becomes a Michael Addition adduct, e.g., is already contained in the agent or peptide vector), and which 15 may be represented by

25 where L is an agent or peptide vector. where T is O, N or S:-HN R' COT, where T is O, Nor The bridging group “W' is a bifunctional chemical moiety S, and R' is Cls alkyl: capable of covalently linking together two spaced chemical moieties into a stable tripartate molecule. Examples of bridg ing groups are described in S. S. Wong, Chemistry of Protein T 30 Conjugation and Crosslinking. CRC Press, Florida, (1991); and G. E. Means and R. E. Feeney, Bioconjugate Chemistry, N COOR, vol. 1, pp. 2-12. (1990), the disclosures of which are incor H porated herein by reference. W can covalently link the thiol where T is O, N, or S, and R is H or Cs alkyl: acceptor to a keto moiety. An exemplary a bridging group has 35 the formula—(CH2), (Z)-(CH2), , where fis0 to 10;h is 0 to 10; g is 0 or 1, provided that wheng is 0, then f--his 1 to 10; Z is S. O. NH, SO, phenyl, naphthyl, a polyethylene glycol, a cycloaliphatic hydrocarbon ring containing 3 to 10 carbonatoms, or a heteroaromatic hydrocarbon ring contain ing 3 to 6 carbonatoms and 1 or 2 heteroatoms selected from where T is O, N or S; or O, N, or S. Preferred cycloaliphatic moieties include cyclo propyl, cyclobutyl, cyclopentyl, cyclohexyl, and the like. Pre ferred heteroaromatic moieties include pyridyl, polyethlene COT glycol (1-20 repeating units), furanyl, pyranyl, pyrimidinyl, - OCO 45 pyrazinyl, pyridazinyl, oxazinyl, pyrrolyl, thiazolyl, mor pholinyl, and the like. In the bridging group, it is preferred that wheng is 0, f--his an integer of 2 to 6 (e.g., 2 to 4 such as 2). When g is 1, it is preferred that f is 0, 1 or 2; and that his 0, 1 or 2. Preferred bridging groups coupled to thiol acceptors 50 are shown in the Pierce Catalog, pp. E-12, E-13, E-14, E-15, where T is O, N, or S. Preferred Gs include PABC (p-ami E-16, and E-17 (1992). nobenzyl-carbamoyl), GABA (y-aminobutyric acid), C.C.- Joining of the Peptide Vector Multimer to an Agent dimethyl GABA, and B.B-dimethyl GABA. In addition to the multimeric peptide vectors described In the branched linker, the thiol acceptor 'A' is linked to a above, the invention features compounds where the multim peptide vector or agent by a sulfur atom derived from the eric peptide vector is joined (e.g., by a covalent bond) to one peptide vector or agent. The thiol acceptor can be, for 55 or more agents (e.g., a diagnostic ortherapeutic agent, such as example, an O-substituted acetyl group. Such a group has the any of those described herein). The agent may be joined to the formula: peptide vector directly through a covalent bond Such as a peptide bond or disulfide bond, or may be joined to the pep tide vector through a linker (e.g., any linker described herein). O 60 The agent may be joined to the peptide vector through any appropriate reactive moiety on the vector, e.g., through a primary amine such as an N-terminal amine or a e-amino sus, group on a lysine side chain, through a thiol bond (e.g., through a cysteine side chain), or through a carboxyl group where Y is a leaving group Such as Cl, Br, I, mesylate, tosy 65 (e.g., a C-terminal carboxyl group or a or late, and the like. If the thiol acceptor is an alpha-substituted side chain). In embodiments where the agent is acetyl group, the thiol adduct after linkage to the ligand forms a peptide or polypeptide, the agent may be joined to the US 9, 161988 B2 23 24 peptide vector by a peptide bond (e.g., produced synthetically Trimeric Conjugates or recombinantly as a fusion protein). Compounds of the invention can also include a trimeric Dimeric Conjugates peptide vector. Where the trimeric peptide vector is joined to Compounds including an agent and dimeric peptide vector a single agent through one of the peptide vectors, the com can be conjugated either through the peptide vector portion of 5 pound can have one of the following formulas: the molecule or through the linker portion of the molecule. Compounds of the invention in which the agent is joined (e.g., through a linker where the linker is a chemical linker, Y X X2 X X2 peptide, or a covalent bond such as a peptide bond) to the 11 NAI1 N21 na: A1 N21 ns peptide vector can be represented by the formula: 10 B11 Yl A2 Y X B1 NA11 NA2 -XN-YN A 15 Al A3 Bl, and A-XI-B V 3 where A' and A are each, independently, peptide vectors A (e.g., any described herein); X is a linker (e.g., chemical linker, peptide, or covalent bond) that joins A" and A; B' is an where A, A, and A are each, independently, a peptide agent; and Y is a linker that joins B and A'. In certain vector (e.g., any described herein); X' and X’ are linkers; B' embodiments, two or more (e.g., 3, 4, 5, 6, 7, 8, 9, or 10) 20 is an agent; and Y is a linker that joins B to a peptide vector agents are joined to one or both of the peptide vectors. Such (e.g., A', A, and A) or to the linker X". compounds can be represented by the formula: In other embodiments, the trimeric peptide vector is con jugated to one or more than one agent. Such conjugation can 25 be through either the peptide vector, or through the linker(s). Such compounds can include one of the following formulas: where A, A, and Xare as defined above; m is 1,2,3,4,5,6, B2 7,8,9, or 10; n is 0,1,2,3,4,5,6,7,8,9, or 10; p is an integer 30 Y from 1 to m; q is an integer from m+1 to m+n, each B and 1 in 1 NA3 B' , each B7 are, independently, an agent (e.g., any described herein); and each Y and each Y are, independently, a linker Yq that joins each B or each B" to A' or A, respectively. (1 )iii. In other embodiments, the agent is joined (e.g., through a 35 covalent bond or a chemical linker such as those described where A, A, and Aare peptide vectors: n,m, andjare 0, 1, herein) to the dimer through the linker that joins the peptide 2, 3, 4, 5, 6, 7, 8, 9, or 10: Each B, each B7, and each Bare, vectors forming the dimer. Such compounds can have the independently, agents (e.g., any agent described herein); Bf formula: and Bare, independently, agents or are absent; X" is a linker 40 joining A', A, and B, if present; X is a linkerjoining A, A, and B', if present. In certain embodiments, at least one of n, B m, or j is at least one, B is present, or B' is present. In other X embodiments, at least two (e.g., at least 3, 4, 5, 6, 7, 8, 9, 10. A11 NA2 15, 20, 25, or 30) of B, B7, B, B, and B are present. 45 Higher Order Multimer Conjugates where A' and A are peptide vectors (e.g., any described The compounds of the invention can also include peptide herein); B is an agent; and X is a linker that joins A, A, and multimers of a higher order (e.g., quatromers, pentomers, B. etc.). Such multimers can be described by the formula: In other embodiments, agents can be joined to both the linker and a peptide vector. Such compounds can be repre- 50 sented by the formula: (-) Al B2 V 2 55 X-A --X-A) (y Y ')NA11 X NA2 e' n B' ), A3 where A' and Aare, independently, peptide vectors; B5 is an N-) agent or is absent; m is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10; n is 0, 60 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10; p is an integer from 1 to m; q is where A, A, each A7, each A, and each A are, indepen an integer from m+1 to m+n. Each B and B7 is, indepen dently, peptide vectors; A is a peptide vector or is absent; X, dently, an agent (e.g., any described herein); and each Y and each X, X, and X are, independently, linkers that join pep Y is, independently, a linker that joins each B or each B" to tide vectors; m, n, and pare each, independently, 0, 1, 2, 3, 4, A' or A, respectively, where at least one (e.g., at least two) of 65 5, 6,7,8,9, or 10, q is an integer from 4 to m+3; ris an integer the following is true (i) B1 is present; (ii) m is at least 1; and from m+4 to m+n+3; and S is an integer from m+n+4 to (iii) n is at least 1. m+n+p-3. One or more agents can be joined to either the US 9, 161988 B2 25 26 linkers (X, any X, X", or X) or the peptide vectors (A', A, atcc.gcggaagactgcatgc gtacttgcgg tggtgcttag: SEQID NO:6; A, each A7, each A, and each A) of this formula in order to Genbank accession No. X04666). Other examples of aproti form higher order multimer conjugates. nin analogs may be found by performing a protein BLAST Peptide Vectors (Genbank: www.ncbi.nlm.nih.gov/BLAST/) using the syn The compounds of the invention can feature any of 5 thetic aprotinin sequence (or portion thereof) disclosed in polypeptides described herein, for example, any of the pep International Application No. PCT/CA2004/000011. Exem tides described in Table 1 (e.g., Angiopep-1, Angiopep-2. plary aprotinin analogs are also found under accession Nos. Angiopep-7, or reversed Angiopep-2), or a fragment or ana CAA37967 (GI:58.005) and 1405218C (GI:3604747). log thereof. In certain embodiments, the polypeptide may Agents have at least 35%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 10 Any therapeutic or diagnostic agent may be conjugated to 99%, or even 100% identity to a polypeptide described a multimer (e.g., a dimer) of the invention. Such agents may herein. The polypeptide may have one or more (e.g., 2, 3, 4, 5, be chemically conjugated to one or more of the peptide vec 6,7,8,9, 10, 11, 12, 13, 14, or 15) substitutions relative to one tors, or may be conjugated to a linker that joins two (or more) of the sequences described herein. Other modifications are of the peptide vectors (e.g., a trifunetional linker). Agents of described in greater detail below. 15 particular interest include anticancer agents (e.g., paclitaxel, The invention also features fragments of these polypep etoposide, doxorubicin and analogs thereof), RNAi agents, tides (e.g., a functional fragment). In certain embodiments, and peptide and polypeptide therapeutics (e.g., GLP-1 ago the fragments are capable of efficiently being transported to nists, neurotensin and neurotensin receptor agonists, leptin or accumulating in a particular cell type (e.g., liver, eye, lung, and OB receptor agonists, GDNF, BDNF, and analogs kidney, or spleen) or are efficiently transported across the thereof). BBB. Truncations of the polypeptide may be 1,2,3,4,5,6,7, In certain embodiments, the agent is a small molecule drug, 8, 9, 10, 11, 12, or more amino acids from either the N-ter an antibiotic, a medicine, a detectable label, a protein (e.g., an minus of the polypeptide, the C-terminus of the polypeptide, enzyme), protein-based compound (e.g., a protein complex or a combination thereof. Other fragments include sequences comprising one or polypeptide chain) and a peptide or where internal portions of the polypeptide are deleted. 25 polypeptide. Exemplary peptide and polypeptide therapeu Additional polypeptides may be identified by using one of tics that can be used in the present invention are described, for the assays or methods described herein. For example, a can example in U.S. Provisional Application No. 61/200,947, didate polypeptide may be produced by conventional peptide filed Dec. 5, 2008, which is hereby incorporated by reference. synthesis, conjugated with paclitaxel and administered to a The agent may be more particularly, a molecule which is laboratory animal. A biologically-active polypeptide conju 30 active at the level of the central nervous system. The agent gate may be identified, for example, based on its ability to may be any agent for treating or detecting a neurological increase survival of an animal injected with tumor cells and disease. treated with the conjugate as compared to a control which has The agent may be a small molecule drug, an antibiotic, a not been treated with a conjugate (e.g., treated with the medicine, a detectable label, a protein (e.g., an enzyme), unconjugated agent). For example, a biologically active 35 protein-based compound (e.g., a protein complex comprising polypeptide may be identified based on its location in the one or polypeptide chain) and a peptide or polypeptide. The parenchyma in an in situ cerebral perfusion assay. agent may be more particularly, a molecule that is active at the Assays to determine accumulation in other tissues may be level of the central nervous system. The agent may be any performed as well. Labeled conjugates of a polypeptide can agent for treating or detecting a neurological disease. be administered to an animal, and accumulation in different 40 The detectable label may be a radioimaging agent. Other organs can be measured. For example, a polypeptide conju label include an isotope, a fluorescent label (e.g., rhodamine), gated to a detectable label (e.g., a near-IR fluorescence spec a reporter molecule (e.g., biotin), etc. Other examples of troscopy label such as Cy5.5) allows live in vivo visualiza detectable labels include, for example, a green fluorescent tion. Such a polypeptide can be administered to an animal, protein, biotin, a histag protein and B-galactosidase. and the presence of the polypeptide in an organ can be 45 Protein or protein-based compound which may be conju detected, thus allowing determination of the rate and amount gated to a multimer of the invention include an antibody, an of accumulation of the polypeptide in the desired organ. In antibody fragment (e.g., an antibody binding fragment Such other embodiments, the polypeptide can be labelled with a as Fv fragment, F(ab), F(ab) and Fab and the like), a pep radioactive isotope (e.g., 'I). The polypeptide is then tidic- or protein-based drug (e.g., a positive pharmacological administered to an animal. After a period of time, the animal 50 modulator (agonist) oran pharmacological inhibitor (antago is sacrificed and the organs are extracted. The amount of nist)). Other examples of agents are cellular toxins (e.g., radioisotope in each organ can then be measured using any monomethyl auristatin E (MMAE), toxins from bacteria means known in the art. By comparing the amount of a endotoxins and eXotoxins; diphtheria toxins, botunilum tox labeled candidate polypeptide in a particular organ relative to ins, tetanus toxins, perussis toxins, staphylococcus enterotox the amount of a labeled control polypeptide, the ability of the 55 ins, toxin shock syndrome toxin TSST-1, adenylate cyclase candidate polypeptide to access and accumulate in a particu toxin, shiga toxin, cholera enterotoxin, and others) and anti lar tissue can be ascertained. Appropriate negative controls angiogenic compounds (endostatin, catechins, nutriceuticals, include any peptide or polypeptide known not to be efficiently chemokine IP-10, inhibitors of matrix metalloproteinase transported into aparticular cell type (e.g., a peptide related to (MMPIs), anastellin, vironectin, antithrombin, tyrosine Angiopep that does not cross the BBB, or any other peptide). 60 kinase inhibitors, VEGF inhibitors, antibodies against recep Additional sequences are described in U.S. Pat. No. 5,807, tor, trastuzumab (HerceptinR), Bevacizumab (AvastinR), 980 (e.g., SEQID NO:102 herein), U.S. Pat. No. 5,780,265 and panitumumab and others). (e.g., SEQ ID NO:103), U.S. Pat. No. 5,118,668 (e.g., SEQ Particular agents are described in greater detail below. ID NO:105). An exemplary nucleotide sequence encoding an Anticancer Agents aprotinin analog atgagaccag atttctgcct cgagcc.gccg. 65 Also in accordance with the present invention, the agent tacactgggCCCtgcaaagCtcgtatCatC Cgttacttct acaatgcaaaggcag may be an anticancer drug. An anticancer drug encompassed gCCtgttgtcagacct tcgtatacgg CggCtgcagagctaagcgta acaacttcaa by the present invention may include, for example, a drug US 9, 161988 B2 27 28 having a group allowing its conjugation to the carrier of the or 3 C-C alkyl, C-C alkoxy, halo, C-C alkylthio, trifluo invention. Particular anticancer drugs include those selected romethyl, C-C dialkylamino, hydroxy or nitro, NHC(O) from the group consisting of paclitaxel (Taxol), vinblastine, C(CH)—CHCH, -NHC(O)CC(CH), NHC(O)OCH, Vincristine, etoposide, doxorubicin, cyclophosphamide, doc phenyl, —NH, -NHSO-4-methylphenyl, - NHC(O) etaxel (TaxotereR), melphalan, and chlorambucil; pharma (CH),COOH, -NHC(O)-4-(SOH)phenyl, -OH, -NHC ceutically acceptable salts thereof; or a combination thereof. (O)-1-adamantyl, - NHC(O)O-3-tetrahydrofuranyl, - NHC In particular embodiments, the anticancer agent is paclitaxel, (O)O-4-tetrahydropyranyl, NHC(O)CHC(CH), etoposide, or doxorubicin; a pharmaceutically acceptable salt - NHC(O)C(CH), -NHC(O)OC-C alkyl, - NHC(O) thereof, or a derivative thereof. NHC-C alkyl, NHC(O)NHPh, NHC(O)NHPhsubsti Other exemplary agents include abarelix, aldesleukin, ale 10 tuted with one, 2, or 3 C-C alkyl, C-C alkoxy, halo, C-C, mtuzumab, allitertinoin, allopurinol, altretamine, amifostine, alkylthio, trifluoromethyl, C-C dialkylamino, or nitro, anakinra, anastroZole, arsenic trioxide, asparaginase, azaciti —NHC(O)C-C cycloalkyl, - NHC(O)C(CH2CH)2CH, dine, BCG Live, bevacuzimab, bexarotene, bleomycin, bleo NHC(O)C(CH),CHCl, -NHC(O)C(CH),CHCH mycin, bortezombi, bortezomib, busulfan, busulfan, caluster phthalimido, NHC(O)-1-phenyl-1-cyclopentyl, - NHC one, capecitabine, carboplatin, carmustine, celecoxib, 15 (O)-1-methyl-1-cyclohexyl, NHC(S)NHC(CH), cetuximab, cisplatin, cladribine, clofarabine, cytarabine, dac - NHC(O)NHCC(CH), or NHC(O)NHPh; R is selected arbazine, dactinomycin, actinomycin D. dalteparin (e.g., from the group consisting of —H, NHC(O)phenyl or Sodium), darbepoetin alfa, dasatinib, daunorubicin, dauno —NHC(O)OC(CH), with the overall proviso that one of R mycin, decitabine, denileukin, denileukin diftitox, dexraZOX and R is —H but R, and R are not both —H; R is —H or ane, dromoStanolone propionate, eculizumab, epirubicin selected from the group consisting of —OH, - OAc (—OC (e.g., HCl), epoetin alfa, erlotinib, estramustine, exemestane, (O)CH), OC(O)CCHC(Cl), —OCOCHCH, NH." fentanyl (e.g., citrate), filgrastim, floXuridine, fludarabine, HCOO, —NHC(O)phenyl, —NHC(O)OC(CH), fluorouracil, 5-FU, fulvestrant, gefitinib, gemcitabine (e.g., —OCOCHCH COOH and pharmaceutically acceptable HCl), gemtuzumab ozogamicin, goserelin (e.g., acetate), his salts thereof, —OCO(CH2)COOH and pharmaceutically trelin (e.g., acetate), hydroxyurea, ibritumomab tiuxetan, ida acceptable salts thereof, and —OC(O)—Z C(O) R' rubicin, ifosfamide, imatinib (e.g., mesylate), Interferon alfa 25 where Z is ethylene (—CHCH), propylene 2b, irinotecan, lapatinib ditosylate, lenalidomide, letrozole, (—CHCHCH ), —CH=CH-, 1.2-cyclohexane or 1.2- leucovorin, leuprolide (e.g., acetate), levamisole, lomustine, phenylene, R' s —OH, -OH base, —NR'R' —OR', CCNU, meclorethamine (nitrogen mustard), megestrol, mer —SR's, OCHC(O)NR'R' where R', is - H or -CH, captopurine (6-MP), mesna, methotrexate, methoXSalen, R's s—(CH)NR'R'', or (CH)NR'R'R'sX where n is mitomycin C, mitotane, mitoxantrone, nandrolone phenpro 30 1-3, R is —H or —C-C alkyl, R's is —H. —C-C alkyl, pionate, nelarabine, nofetumomab, oprelvekin, oxaliplatin, benzyl, hydroxyethyl, —CH2COH or dimethylaminoethyl, palifermin, pamidronate, panitumumab, pegademase, pegas R" and R', are —CH, —CHCH, benzyl or R' and R', pargase, pegfilgrastim, peginterferon alfa-2b, pemeterxed together with the nitrogen of NR'R'', form a pyrrolidino, (e.g., disodium), pentostatin, pipobroman, plicamycin (mith piperidino, morpholino, or N-methylpiperizino group; R's S ramycin), porfimer (e.g., Sodium), procarbazine, quinacrine, 35 —CH, —CHCH or benzyl, X is halide, and base is NH, rasburicase, rituximab, SargramoStim, Sorafenib, StreptoZo (HOCH).N.N(CH), CHN(CH), NH, NH. (CH)NH, cin, Sunitinib (e.g., maleate), talc, tamoxifen, temozolomide, N-methylglucamine, NaOH or KOH. —OC(O)(CH), teniposide (VM-26), testolactone, thalidomide, thioguanine NRR where n is 1-3, R is - Hor-C-C, alkyl and R is (6-TG), thiotepa, thiotepa, thiotepa, topotecan (e.g., hel), -H or - C-C alkyl, - OC(O)CH(R")NH where R" is toremifene, Tositumomab/1-131 (tositumomab), trastu selected from the group consisting of H. —CH. —CHCH Zumab, trastuzumab, tretinoin (ATRA), uracil mustard, Vai 40 (CH), —CH(CH)CHCH. —CH(CH), —CH phenyl, rubicin, Vinorelbine, Vorinostat, Zoledronate, and Zoledronic —(CH)NH, -CHCHCOON, -(CH)NHC(=NH) acid. NH, the residue of the amino acid . —OC(O) Paclitaxel Derivatives CH=CH, C(O)CHCHC(O)NHCHCHSOY", In certain embodiments, the agent is a derivative of pacli OC(O)CHCHC(O)NHCHCHCHSOY" whereinYi" taxel. Structural analogs of paclitaxel are disclosed in U.S. 45 is Na" or N(Bu) —OC(O)CHCHC(O)OCHCH-OH: Rs Pat. No. 6,911,549 and can be described by the formula: is —H or —OH, with the overall proviso that when Rs s —OH. R. is - Hand with the further proviso that when Rs is —H. R. is not —H; R is —H:—H when R, is C.-R7:B-R7.

R10O O where one of R, and R is —H and the other of R, and 50 R, s —X where X is halo and Rs S —CH; R is —H:—H when R, is C.-H. B-R where R and Rs are taken together to form a cyclopropyl ring; Ro is —H or —C(O)CH; and pharmaceutically acceptable salts thereof when the com pound contains either an acidic or basic functional group. Particular paclitaxel analogs include ((azidophenyl)ure 55 ido)taxoid, (2O,5C,7f8.9C, 103,13C)-5,10,13,20-tetraacetox ytax-11-ene-2.7.9-triol, (2C.5C,9C.10B)-2,9,10-triacetoxy 5-(B-D-glucopyranosyl)oxy)-3.11-cyclotax-11-en-13-one, 13-hydroxybaccatin I, 1,7-dihydroxytaxinine, 1-acety-5.7, 10-deacetyl-baccatin I, 1-dehydroxybaccatin VI, 1-hydroxy where R is selected from the group consisting of —CH: 60 2-deacetoxy-5-decinnamoyl-taxinine j, 1-hydroxy-7.9-dide —CHs, or phenyl Substituted with one, 2 or 3 C-C alkyl, acetylbaccatin I, 1-hydroxybaccatin I, 10-acetyl-4- C-C alkoxy, halo, C-C alkylthio, trifluoromethyl, C-C, deacetyltaxotere, 10-deacetoxypaclitaxel, 10-Deacetyl dialkylamino, hydroxyl, or nitro; and -2-furyl, 2-thienyl, baccatin III dimethyl sulfoxide disolvate, 10-deacetyl-10-(3- 1-naphthyl 2-naphthyl or 3.4-methylenedioxyphenyl; R is aminobenzoyl)paclitaxel, 10-deacetyl-10-(7-(diethylamino) selected from the group consisting of —H, NHC(O)H, 65 coumarin-3-carbonyl)paclitaxel, 10-deacetyl-9-dihydro —NHC(O)C-C alkyl (preferably NHC(O)C-C alkyl). taxol. 10-deacetylbaccatine III, 10-deacetylpaclitaxel, —NHC(O)phenyl, - NHC(O)phenyl substituted with one, 2. 10-deacetyltaxinine, 10-deacetyltaxol. 10-deoxy-10-C-mor

US 9, 161988 B2 31 32 Provisional Application Nos. 61/105,654, filed Oct. 15, 2008, -continued and 61/171,010, filed Apr. 20, 2009. Other derivatives of etoposide include etoposide phosphate (ETOPOPHOSR), where the phenolic-OHis replaced with —OP(O)(OH), or any pharmaceutically acceptable salt 5 thereof (e.g., —OP(O)(ONa)). Etoposide phosphate has improved water Solubility compared to etoposide. Other etoposide derivatives include those where the phe nolic —OH is replaced with an acyloxy group (e.g., —OC 10 (O)Rs, as described herein) such as the following compound:

15

NK 611

25 Still other podophyllotoxin derivatives suitable for use in the invention are described in U.S. Pat. Nos. 4,567,253; 4,609,644; 4,900,814; 4,958,010; 5,489,698; 5,536,847: 5,571,914; 6,051,721; 6,107,284; 6,475,486; 6,610,299; 30 6,878,746; 6,894,075; 7,087,641; 7,176,236; 7,241,595; 7,342,114; and 7,378.4.19; and in U.S. Patent Publication N1 CH3 Nos. 20030064482, 20030162722, 20040044058, 20060148728, and 20070249651, each of which is hereby CH3 35 incorporated by reference. (“etoposide 4'-dimethylglycine' or “etoposide.) Doxorubicin Derivatives In some embodiments, the anti-cancer agent is doxorubicin (hydroxydaunorubicin or Adriamycin R) or a doxorubicin These acylated etoposide derivatives can also show improved 40 water solubility relative to etoposide when covalently derivative such as epirubicin (Ellence(R) or Pharmorubicie?R). attached to any of the polypeptides described herein. The structures of these exemplary compounds are shown Other exemplary podophyllotoxin derivatives include teni below. Doxorubicin and doxorubicin derivatives can be poside and NK611. covalently attached to an amino acid in any of the polypep 45 tides described herein through a hydrolyzable covalent linker bonded to, for example, the 14-hydroxyl group.

OH 65 TENIPOSIDE Doxorubicin US 9, 161988 B2 33 34

-continued Nucleic acids include any type known in the art, such as double and single-stranded DNA and RNA molecules of any length, conformation, charge, or shape (i.e., linear, concate mer, circular (e.g., a plasmid), nicked circular, coiled, Super coiled, or charged. Additionally, the nucleic acid can contain 5' and 3' terminal modifications and include blunt and over hanging nucleotides at these termini, or combinations O thereof. In certain embodiments of the invention the nucleic acid is or encodes an RNA interference sequence (e.g., an 10 siRNA, shRNA, miRNA, or dsRNA nucleotide sequence) that can silence a targeted gene product. The nucleic acid can be, for example, a DNA molecule, an RNA molecule, or a modified form thereof. NH2 Exemplary RNAi targets include growth factors (e.g., epi epirubicin dermal growth factor (EGF), vascular endothelial growth fac 15 tor (VEGF), transforming growth factor-fi (TGF-B)), growth Doxorubicin derivatives can be described generally by the factor receptors, including receptor tyrosine kinases (e.g., following formula: EGF receptor (EGFR), including Her2/neu (ErbB), VEGF receptor (VEGFR), platelet-derived growth factor receptor (II) R22 (PDGFR), cytokines, chemokines, kinases, including cyto O O1 O plasmic tyrosine and serine/ kinases (e.g., focal adhesion kinase, cyclin-dependent kinase, SRC kinases, Syk O-R-1, ZAP70 kinases, BTK kinases, RAF kinase, MAP kinases (including ERIC), and Wnt kinases), phosphatases, regula tory GTPases (e.g., Ras protein), transcription factors (e.g., 25 MYC), and hormone receptors (e.g., and H estrogen receptor), anti-apoptotic molecules (e.g., Survivin, R24X5 O -O O O X4R20 Bcl-2, Bcl-XL), oncogenes (e.g., tumor Suppressor regulators R23 Such as mdm2), enzymes (e.g., Superoxide dismutase 1 (SOD-1), C. B (BACE), and Y secretases, alpha-L-idu 30 ronidase, iduronate Sulfatase, heparan N-sulfatase, alpha-N- R17X X3R19 acetylglucosaminidase, acetyl-CoAlpha-glucosaminide XR18 acetyltransferase, N-acetylglucosamine 6-sulfatase, N-acetylgalactosamine 4-sulfatase, beta-galactosidase, sph where eachX, X, X, X, and Xs is selected, independently, ingomyelinase, glucocerebrosidase, alpha-galactosidase-A, from a covalent bond, O, or NRs; each R7, Rs. Rio, Ro, ceramidase, galactosylceramidase, arylsulfatase A, aspartoa Ro R. R. R. R. and Rs. is selected, independently, 35 cylase, phytanoyl-CoA hydroxylase, peroxin-7, beta-hex from H., optionally Substituted C. alkyl, optionally Substi osaminidase A, aspartylglucosaminidase, fucosidase, and tuted C- alkenyl, optionally substituted C- alkynyl. alpha-mannosidase, Sialidase), and other proteins (e.g., Hun optionally substituted cycloalkyl, optionally substituted het tingtin (Htt protein), amyloid precursor protein (APP), sort erocyclyl, or is a hydrolyzable linker Y as defined herein. ing nexins (including SNX6), C-synuclein, LINGO-1, Nogo When a compound of Formula (II) is attached to any of the 40 A, and Nogo receptor 1 (NgR-1)), and glial fibrillary acidic polypeptides described herein, one of R7, Rs. Rio, Ro Ro, protein. Table 2 illustrates the relationship between exem R2, R22, R2, R2, and R2s is Y. In certain embodiments, R21 plary RNAi targets and diseases. is Y. Exemplary RNAi sequences to silence EGFR are SEQID Other doxorubicin derivatives can be found in U.S. Pat. NO:117 (GGAGCUGCCCAUGAGAAAU) and SEQ ID Nos. 4,098,884, 4,301277, 4,314,054, 4,464,529,4,585,859, 45 NO:118 (AUUUCUCAUGGGCAGCUCC). Likewise, 4,672,057, 4,684,629, 4,826,964, 5,200,513, 5,304,687, VEGF can be silenced with an RNAi molecule having the 5,594,158, 5,625,043, and 5,874,412, each of which is hereby sequence, for example, set forth in SEQID NO:119 (GGAG incorporated by reference. TACCCTGATGAGATC). Additional RNAi sequences for Nucleic Acids use in the agents of the invention may be either commercially The multimeric peptide vectors may be conjugated to any 50 available (e.g., Dharmacon, Ambion) or the practitioner may nucleic acid, including expression vectors (e.g., a plasmid) use one of several publicly available software tools for the and therapeutic nucleic acids (e.g., RNAi agents). The construction of viable RNAi sequences (e.g., The siRNA expression vector may encode a polypeptide (e.g., a therapeu Selection Server, maintained by MIT/Whitehead; available tic polypeptide Such as an interferon, a therapeutic cytokine at: http://iura.wi.mit.edu/bioc/siRNAext/). Examples of dis (e.g., IL-12), or FGF-2) or may encode a therapeutic nucleic eases or conditions, and RNAi target that may be useful in acid (e.g., an RNAi agent such as those described herein). treatment of such diseases, are shown in Table 3. TABLE 2 Exemplary Diseases and Target Molecules Diseasef Condition RNAi Target Molecules Cancer Glioblastoma Epidermal growth factor receptor (EGFR), Vascular endothelial growth factor (VEGF) Glioma EGFR, VEGF Astrocytoma EGFR, VEGF US 9, 161988 B2 35 36 TABLE 2-continued Exemplary Diseases and Target Molecules Diseasef Condition RNAi Target Molecules Neuroblastoma EGFR, VEGF Lung cancer EGFR, VEGF Breast cancer EGFR, VEGF Hepatocellular carcinoma EGFR, VEGF Neurodegenerative Disease Huntington's disease Huntingtin (Htt) Parkinson's disease Alpha-synuclein Alzheimer's disease Amyloid precursor protein (APP), Presenilin-1 or -2, Apollipoprotein E (ApoE) Amyotropic lateral Schlerosis Superoxide dismutase 1 (SOD-1) Multiple schlerosis Sorting nexin-6 (SNX6), LINGO-1, Nogo-A, NgR-1, APP Lysosomal Storage Disease MPS-I (Hurler, Scheie diseases) Alpha-L-iduronidase MPS-II (Hunter syndrome) Iduronate Sulfatase MPS-IIIA (Sanfilippo syndrome A) Heparan N-sulfatase MPS-IIIB (Sanfilippo syndrome B) Alpha-N-acetylglucosaminidase MPS-IIIC (Sanfilippo syndrome C) Acetyl-CoAlpha-glucosaminide acetyltransferase MPS-IIID (Sanfilippo syndrome D) N-acetylglucosamine 6-sulfatase MPS-VI (Maroteaux-Lamy syndrome) N-acetylgalactosamine 4-sulfatase MPS-VII (Sly syndrome) Beta-glucuronidase Niemann-Pick disease Sphingomyelinase Gaucher's disease Glucocerebrosidase Fabry disease Alpha-galactosidase-A Farber's disease Ceramidase Krabbé disease Galactosylceramidase Metachromatic leukodystrophy Arylsulfatase A Alexander disease Glial fibrillary acidic protein Canavan disease Aspartoacylase RefSum's disease Phytanoyl-CoA hydroxylase or peroxin-7 GM1 gangliosidoses Beta-galactosidase GM2 gangliosidoses (e.g., Tay-Sachs, Beta-hexosaminidase A Sandhoff diseases) Aspartylglucosaminuria Aspartylglucosaminidase (AGA). Fucosidosis Fucosidase Mannosidosis Alpha-mannosidase Mucolipodosis (sialidosis Sialidase

GLP-1 Agonists Val, Leu, pCily, t-BuG or Met; X is Glu or Asp; X is Trp, The multimers described herein can be conjugated to 40 Phe, Tyr, or NaI, X, Xs, X and X, are independently Pro, GLP-1 agonist known in the art. Particular GLP-1 agonists HPro, 3Hyp, 4Hyp, TPro, N-alkylglycine, N-alkyl-pGly or include GLP-1, exendin-4, and analogs thereof. Exemplary N-alkylalanine; X is Ser. Thr, or Tyr; and Z s —OH or analogs are described below. —NH. (e.g., with the proviso that the compound is not exen Exendin-4 and exendin-4 analogs. Exendin-4 and exen 45 din-3 or exindin-4.) din-4 analogs can also be used in the compositions, methods, Preferred N-alkyl groups for N-alkylglycine, N-alkyl-pGly and kits of the invention. The compounds of the invention can and N-alkylalanine include lower alkyl groups (e.g., C. include fragments of the exendin-4 sequence. Exendin-4 has alkyl or C. alkyl). the sequence. In certain embodiments, X is His or Tyr (e.g., His). X can His-Gly-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Leu-Ser-Lys 50 be Gly. X can be Leu, pGly, or Met. X can be Trp or Phe.X. Gln-Met-Glu-Glu-Glu-Ala-Val-Arg-Leu-Phe-Ile can be Phe or NaI; X can be Ile or Val, and X, Xs, X and Glu-Trp-Leu-Lys-Asn-Gly-Gly-Pro-Ser-Ser-Gly X, can be independently selected from Pro, HPro, TPro, or Ala-Pro-Pro-Pro-Ser-NH. (SEQID NO: 132) N-alkylalanine (e.g., where N-alkylalanine has a N-alkyl Particular exendin-4 analogs include those having a cys group of 1 to about 6 carbon atoms). In one aspect, Xs, X. teine substitution (e.g., Cyslexendin-4 (SEQIDNO: 133)) 55 and X, are the same amino acid residue. Xs may be Ser or or a lysine substitution (e.g., Lyslexendin-4 (SEQID NO: Tyr (e.g., Ser). Z can be NH. 134)). In other embodiments, X is His or Tyr (e.g., His); X is Exendin analogs are also described in U.S. Pat. No. 7,157, Gly: X is Phe or NaI; X is Leu, pCily, or Met; X is Phe or 555 and include those of the formula: NaI; X is Ile or Val; X, Xs, X, and X, are indepen 60 dently selected from Pro, HPro, TPro, or N-alkylalanine; and Xs is Ser or Tyr, (e.g., Ser). Z can be —NH. X-Leu-Lys-Asn-Gly-Gly-X-Ser-Ser-Gly-Ala In other embodiments, X is His or Arg; X is Gly: X is Xs-X-X7-X8-Z (SEQID NO: 135) Asp or Glu, X is Phe or napthylalanine; X is Thror Ser; X where X is His, Arg or Tyr; X is Ser, Gly, Ala or Thr; X is is Seror Thr; X, is Asp or Glu; X is Leu or pGly: X is Leu Asp or Glu; X is Phe, Tyr or NaI; X is Thror Ser; X is Ser 65 or pGly: X is Phe or NaI; X is Ile, Val, ort-butyltylglycine; or Thr; X, is Asp or Glu; X is Leu, Ile, Val, pGly or Met; X X2 is Glu or Asp., X is Trp or Phe; X, Xs, X, and X7 is Leu, Ile, pGly, Valor Met; X is Phe, Tyr, or NaI; X is Ile, are independently Pro, HPro, TPro, or N-methylalanine; Xs

US 9, 161988 B2 39 40 one or more substitutions of the wild type sequence (e.g., the GLP-1 analogs also include peptides of the formula: human wild type sequence), or may have other chemical HN X-CO-R, where R is OH, OM, or—NRR: M is modifications. GLP-1 agonists can also be non-peptide com a pharmaceutically acceptable cation or a lower branched or pounds, for example, as described in U.S. Pat. No. 6,927,214. unbranched alkyl group (e.g., C alkyl); R and R are inde Particular analogs include LY548806, CJC-1131, and Lira pendently selected from the group consisting of hydrogen and glutide. a lower branched or unbranched alkyl group (e.g., C alkyl); The GLP-1 analog can be truncated form of GLP-1. The X is a peptide comprising the sequence His-Ala-Glu-Gly GLP-1 peptide may be truncated by 1, 2,3,4,5,6,7,8,9, 10. Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln 11, 12, 13, 15, 20, or more residues from its N-terminus, its Ala-Ala-Lys-Glu-Phe-Ile-Ala-Trp-Leu-Val-Lys-Gly-Arg; C-terminus, or a combination thereof. In certain embodi 10 (SEQ ID NO: 203): NH is the amine group of the amino ments, the truncated GLP-1 analog is the GLP-1 (7-34), GLP terminus of X; and CO is the carbonyl group of the carboxy 1(7-35), GLP-1 (7-36), or GLP-1 (7-37) human peptide or the terminus of X; acid addition salts thereof; and the protected or C-terminal amidated forms thereof. partially protected derivatives thereof. These compounds In other embodiments of the invention, modified forms of may have insulinotropic activity exceeding that of GLP-1 (1- truncated GLP-1 peptides are used. Exemplary analogs are 15 36) or GLP-1(1-37). described in U.S. Pat. No. 5,545,618 and have the amino acid Other GLP-1 analogs are described in U.S. Pat. No. 5,981, Sequence: 488 and have the formula:

His-Ala-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser R-X-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr Tyr-Leu-Glu-Gly-Gln-Ala-Ala-Lys-Glu-Phe-Ile Leu-Y-Gly-Gln-Ala-Ala-Lys-Z-Phe-Ile-Ala-Trp Ala-Trp-Leu-Val-Lys-(Gly)-(Arg)-(Gly) (SEQID Leu-Val-Lys-Gly-Arg-R (SEQID NO: 142) NO: 138) where R is His, D-His, desamino-His, 2-amino-His, B-hy where (Gly), (Arg), and (Gly) are present or absent depending droxy-His, homohistidine, C-fluoromethyl-His, or C.-methyl on indicated chain length, with at least one modification His: X is Met, Asp, Lys, Thr, Leu, Asn., Gln, Phe, Val, or Tyr; selected from the group consisting of (a) Substitution of a 25 Y and Z are independently selected from Glu, Gln, Ala, Thr, neutral amino acid, Arg, or a D form of Lys for Lys at position Ser, and Gly; and R is selected from NH and Gly-OH (e.g., 26 and/or 34 and/or a neutral amino acid, Lys, or a D form of provided that, if R is His, X is Val, Yis Glu, and Z is Glu, then Arg for Arg at position 36; (b) Substitution of an oxidation R is NH). resistant amino acid for Trp at position 31; (c) Substitution Other GLP-1 analogs are described in U.S. Pat. No. 5,512, according to at least one of Tyr for Val at position 16; Lys for 30 549 and have the formula: Ser at position 18; Asp for Glu at position 21; Ser for Gly at R-Ala-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser position 22: Argfor Gln at position 23; Argfor Alaat position Tyr-Leu-Glu-Gly-Gln-Ala-Ala-Xaa-Glu-Phe-Ile 24; and Gln for Lys at position 26; (d) a Substitution compris Ala-Trp-Leu-Val-Lys(R)-Gly-Arg-R (SEQID ing at least one of an alternative Small neutral amino acid for NO: 143) Ala at position 8; an alternative acidic amino acid or neutral 35 where R is 4-imidazopropionyl (des-amino-histidyl), 4-imi amino acid for Glu at position 9; an alternative neutral amino dazoacetyl, or 4-imidazo-C, adimethyl-acetyl; R, which is acid for Gly at position 10; and an alternative acidic amino bound to the side chain of the Lys (e.g., through the eamino acid for Asp at position 15; and (e) Substitution of an alterna group), is Co unbranched acyl or is absent; R is Gly-OH or tive neutral amino acid or the Asp or N-acylated or alkylated NH; and Xaa is Lys or Arg. form of His for His at position 7. With respect to modifica 40 tions (a), (b), (d), and (e), the Substituted amino acids may be Still other GLP-1 analogs are described in U.S. Pat. No. in the D form. The amino acids substituted at position 7 can 7,084.243. In one embodiment, the GLP-1 analog has the also be the N-acylated or N-alkylated amino acids. Exem formula: plary GLP-1 analogs include D-His'IGLP-1 (7-37), Tyr His-Xs-Glu-Gly-X-X-Thr-Ser-Asp-X-Ser-Ser GLP-1 (7-37), N-acetyl-His"IGLP-1 (7-37), N-isopropyl 45 Tyr-Leu-Glu-X22-X-23-X24-Ala-X26-X27-Phe-Ile His IGLP-1 (7-37), D-AlaGLP-1 (7-37), D-GluGLP-1 Ala-X-Leu-X-X-X-X-R (SEQID NO: (7-37), AspIGLP-1 (7-37), D-AspGLP-1 (7-37), 144) D-Phe'GLP-1(7-37), Ser’Arg, Arg, Gln-IGLP-1(7- where Xs is Gly, Ala, Val, Leu, Ile, Ser, or Thr; X is Asp, 37), and SerGln, Tyr', Lys' Asp?IGLP-1 (7-37). Glu, Arg, Thr, Ala, Lys, or His; X is His, Trp, Phe, or Tyr; Other GLP-1 fragments are described in U.S. Pat. No. 50 X is Leu, Ser Thr, Trp. His, Phe, Asp, Val, Tyr, Glu, or Ala; 5,574,008 have the formula: X is Gly, Asp, Glu, Gln, ASn, Lys, Arg, Cys, or Cya; X is His, Asp, Lys, Glu, or Glin; X is Glu, His, Ala, or LyS, X is R-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-Lys-Glu-Phe Asp, Lys, Glu, or His; X, is Ala, Glu, His, Phe, Tyr, Trp, Arg, Ile-Ala-Trp-Leu-Val-X-Gly-Arg-R2 (SEQID or Lys; X is Ala, Glu, Asp, Ser, or His; X is Asp, Arg, Val, NO:181) 55 LyS, Ala, Gly, or Glu, X is Glu, Lys, or Asp, Xs is Thr, Ser, where R is HN: HN-Ser; HN-Val-Ser; HN-Asp-Val-Ser; Lys, Arg, Trp, Tyr, Phe, Asp, Gly, Pro. His, or Glu, X is Arg, HN-Ser-Asp-Val-Ser; HN-Thr-Ser-Asp-Val-Ser; H.N- Glu, or His; R is Lys, Arg, Thr, Ser, Glu, Asp, Trp, Tyr, Phe, Phe-Thr-Ser-Asp-Val-Ser; HN-Thr-Phe-Thr-Ser-Asp-Val His, NH, Gly, Gly-Pro, or Gly-Pro-NH, or is deleted Ser; HN-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser; HN-Glu-Gly (e.g., provided that the polypeptide does not have the Thr-Phe-Thr-Ser-Asp-Val-Ser; or HN-Ala-Glu-Gly-Thr 60 sequence of GLP-1 (7-37)OH or GLP-1 (7-36)-NH and pro Phe-Thr-Ser-Asp-Val-Ser; X is Lys or Arg; and R is NH, vided that the polypeptide is not Gly-GLP-1 (7-37)OH, OH, Gly-NH, or Gly-OH. Gly-GLP-1(7-36)NH, Val-GLP-1 (7-37)OH, Val-GLP-1 Other GLP-1 analogs, described in U.S. Pat. No. 5,118, (7-36)NH, Leu-GLP-1 (7-37)OH, Leu-GLP-1 (7-36)NH, 666, include the sequence His-Ala-Glu-Gly-Thr-Phe-Thr Ile-GLP-1 (7-37)OH, Ile-GLP-1 (7-36)NH, Ser-GLP-1 Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-Lys 65 (7-37)OH, Ser-GLP-1 (7-36)NH, Thr-GLP-1 (7-37)OH, or Glu-Phe-Ile-Ala-Trp-Leu-Val-X (SEQID NO: 139), whereX Thr-GLP-1 (7-36)NH, Ala-Glp-1(7-37)OH, Ala-Glp-1 is Lys, Lys-Gly, or Lys-Gly-Arg. (7-36)NH, Ala-Glp-1(7-37)OH, Ala-Glp-1(7-36)NH,

US 9, 161988 B2 43 44 GluGLP-1 (7-36)NH, Thr GlugLP-1 (7-37)OH, alkynyl, halo, hydroxy, mercapto, nitro, cyano, amino, acy Thr.Glu1GLP-1 (7-36)NH, Val His 7GLP-1 (7-37) lamino, azido, guanidino, amidino, carboxyl, carboxamido, OH, Val His 7GLP-1 (7-36)NH, Gly.His 7GLP-1 (7- carboxamido alkyl, formyl, acyl, carboxyl alkyl, alkoxy, ary 37)OH, Gly.His IGLP-1 (7-36)NH Leu. His IGLP-1 loxy, arylalkyloxy, heteroaryloxy, heterocycleoxy, acyloxy, (7-37)OH, Leu. His 7GLP-1 (7-36)NH, Ile.His 7GLP mercapto, mercapto alkyl, mercaptoaryl, mercaptoacyl, halo, 1(7-37)OH, Ile.His 7GLP-1(7-36)NH, Ser. His 7GLP cyano, nitro, azido, amino, guanidino alkyl, guanidino acyl, 1(7-37)OH, Ser. His7GLP-1 (7-36)NH. (Thr, His 7 Sulfonic, Sulfonamido, alkyl Sulfonyl, aryl Sulfonyl or phos GLP-1(7-37)OH, Thr. His IGLP-1 (7-36)NH). phonic group; wherein, the primary or secondary Substi Other GLP-1 analogs described in U.S. Pat. No. 7,101,843 tutents may optionally be bridged by covalent bonds to form have the formula: 10 one or more fused cyclic or heterocyclic systems with each X7-X-Glu-Gly-Thr-Phe-Thr-Ser-Asp-X-Ser-X- other; where, the other substitution at the alpha-carbon of Y Tyr-Leu-Glu-X-Gln-Ala-Xs-Lys-Glu-Phe-Ile may be substituted with H. C. alkyl, aminoalkyl, hydroxy Ala-Trp-Leu-X-Lys-Gly-Arg-X37 (SEQID alkyl or carboxyalkyl; where the other substitution at the NO: 149) alpha-carbon of Z may be substituted with hydrogen, C. wherein: X, is L-His, D-His, desamino-His, 2-amino-His, 15 alkyl, aminoalkyl, hydroxyalkyl, or carboxyalkyl; B-hydroxy-His, homohistidine, C-fluoromethyl-His, or A and B are optionally present, where A is present and A is C.-methyl-His; X is Gly, Ala, Val, Leu, Ile, Ser, or Thr; X is H, an amino acid or peptide containing from about 1-15 Val, Phe, Tyr, or Trp; X is Ser, Tyr, Trp, Phe, Lys, Ile, Leu, amino acid residues, an R group, an R-C(O) (amide) group, or Val; X is Gly, Glu, Asp, or Lys;Xs is Ala, Val, Ile, or Leu: a carbamate group RO—C(O), a urea RRsN C(O), a sul X is Val or Ile; and X, is Gly, NH, or is absent (e.g., fonamido R—SO, or RRSN SO; where R is selected provided that the GLP-1 compound does not have the from the group consisting of hydrogen, C-2 alkyl, Co sequence of GLP-1 (7-37)OH, GLP-1 (7-36)-NH, Gly cycloalkyl, cycloalkylalkyl, heterocyclyl, heterocycloalkyl, GLP-1 (7-37)OH, GlyGLP-1 (7-36)NH ValGLP-1 (7- aryl, heteroaryl, arylalkyl, aryloxyalkyl, heteroarylalkyl, and 37)OH, ValGLP-1 (7-36)NH, LeuGLP-1 (7-37)OH, heteroaryloxyalkyl; R and Rs are each independently LeuCLP-1 (7-36)NH, IleGLP-1 (7-37)OH, Ile IGLP 25 selected from the group consisting of H, alkyl, cycloalkyl, 1(7-36)NH, Ser|GLP-1 (7-37)OH, Ser|GLP-1 (7-36) cycloalkylalkyl, heterocyclyl, heterocycloalkyl, aryl, het NH, ThrGLP-1 (7-37)OH, ThrGLP-1 (7-36)NH, eroaryl, arylalkyl, aryloxyalkyl, heteroarylalkyl, and het Val-Tyr'IGLP-1 (7-37)OH, Val-Tyr'IGLP1(7-36)NH, eroaryloxyalky; where the C-amino group of X is substituted Val.Glu1GLP-1(7-37)OH, Val.Glu1GLP-1(7-36) with H or an alkyl group, said alkyl group may optionally NH, Gly,Glu’IGLP-1 (7-37)OH, Gly,Glu’IGLP-1 (7- 30 form a ring with A; where B is present and B is OR, NRR, 36)NH, Val Asp?IGLP-1 (7-37)OH, Val Asp?IGLP-1 or an amino acid or peptide containing from 1 to 15 amino (7-36)NH, Gly, Asp221GLP-1 (7-37)OH, Gly. Asp? acid residues (e.g., 1 to 10 or 1 to 5) terminating at the GLP-1(7-36)NH, Val, Lys’IGLP-1 (7-37)OH, Val, C-terminus as a carboxamide, Substituted carboxamide, an Lys’GLP-1 (7-36)NH, Gly, Lys?IGLP-1 (7-37)OH, ester, a free carboxylic acid, or an amino-alcohol; where R Gly, Lys’IGLP-1 (7-36)NH Leu,Glu’IGLP-1 (7-37) 35 and R are independently chosen from H. C. alkyl, Co OH, Leut.Glu’IGLP-1 (7-36)NH, Ile.Glu’IGLP-1 (7- cycloalkyl, cycloalkylalkyl, heterocyclyl, heterocycloalkyl, 37)OH, Ile.Glu1GLP-1 (7-36)NH Leu.Asp’IGLP1 aryl, heteroaryl, arylalkyl, aryloxyalkyl, heteroarylalkyl or (7-37)OH, Leu.Asp?IGLP-1 (7-36)NH, Ile.Asp? heteroaryloxyalkyl. GLP-1 (7-37)OH, Ile, Asp?IGLP-1 (7-36)NH Leu, Exemplary substitutions on the C-carbon atoms of Y and Z Lys’IGLP-1 (7-37)OH, Leu.Lys’IGLP-1 (7-36)NH, 40 include heteroarylarylmethyl, arylheteroarylmethyl, and Ile, Lys’IGLP-1 (7-37)OH, Ile.Lys’IGLP-1 (7-36)NH, biphenylmethyl forming biphenylalanine residues, any of SerGlu’IGLP-1(7-37)OH, SerGlu’IGLP-1(7-36) which is also optionally substituted with one or more, hydro NH, Thr.Glu1GLP-1 (7-37)OH, Thr.Glu1GLP-1 (7- gen, alkyl, cycloalkyl, arylalkyl, aryl, heterocyclyl, het 36)NH, Sers Asp’GLP-1 (7-37)OH, Ser, Asp?IGLP-1 eroaryl, alkenyl, alkynyl, halo, hydroxy, mercapto, nitro, (7-36)NH, Thr, Asp?IGLP-1 (7-37)OH, Thr.Asp? 45 cyano, amino, acylamino, azido, guanidino, amidino, car GLP-1 (7-36)NH, Ser Lys’IGLP-1 (7-37)OH, Ser, boxyl, carboxamido, carboxamido alkyl, formyl, acyl, car Lys’GLP-1 (7-36)NH, (Thr, Lys’IGLP-1 (7-37)OH, boxyl alkyl, alkoxy, aryloxy, arylalkyloxy, heteroaryloxy, Thr, Lys’IGLP-1 (7-36)NH Glu2CLP-1 (7-37)OH, heterocycleoxy, acyloxy, mercapto, mercapto alkyl, mercap Glu’IGLP-1 (7-36)NH, Asp’GLP-1 (7-37)OH, Asp? toaryl, mercaptoacyl, halo, cyano, nitro, azido, amino, guani GLP-1 (7-36)NH, Lys’IGLP-1 (7-37)OH, Lys’IGLP-1 50 dino alkyl, guanidino acyl, Sulfonic, Sulfonamido, alkyl Sul (7-36)NH). fonyl, aryl Sulfonyl and phosphonic group. GLP-1 analogs are also described in U.S. Pat. No. 7,238, Other embodiments include isolated polypeptides where 670 and have the structure: the other substitution at the C-carbon of Y is substituted with H. methyl, or ethyl; and where the other substitution at the 55 C-carbon of Z is substituted with H. methyl, or ethyl. where each of X is a naturally or nonnaturally occurring Further embodiments include isolated polypeptides as amino acid residue;Y and Zare amino acid residues; and one described above where X is naturally or non-naturally occur of the substitutions at the C-carbonatoms of Y and Z may each ring amino acid residue in which one of the Substitutions at independently be substituted with a primary substituent the C-carbon is a primary Substituent selected from the group group selected from the group consisting of hydrogen, alkyl, 60 consisting of heterocyclylalkyl, heteroaryl, heteroarylkalkyl cycloalkyl, cycloalkylalkyl, heterocyclylalkyl, arylalkyl and and arylalkyl, said primary Substituent optionally being Sub heteroarylalkyl, heterocyclylalkyl said primary substituent stituted with secondary substituent selected from heteroaryl optionally being Substituted with a secondary Substituent or heterocyclyl; and in which the other substitution at the selected from a cycloalkyl, heterocyclyl, aryl, or heteroaryl C-carbon is Horalkyl, X2 is naturally or nonnaturally occur group; any of said primary or secondary Substituents may 65 ring amino acid residue in which one of the Substitutions at further be substituted with one or more of H, alkyl, the C-carbon is an alkyl or cycloalkyl where the alkyl group cycloalkyl, arylalkyl, aryl, heterocyclyl, heteroaryl, alkenyl, may optionally form a ring with the nitrogen of X, and

US 9, 161988 B2 47 48 In certain embodiments, when B is not present and Z is Other neurotensin analogs include NT64L L-neo-Trp11 OR, NRR, or an amino-alcohol; where R and R are NT(8-13), NT72D D-Lys9,D-neo-Trp11, tert-Leu12INT(9- independently H. C. alkyl, Co cycloalkyl, cycloalkyla 13), NT64D D-neo-Trp.11 INT(8-13), NT73L D-Lys9,L- lkyl, heterocycle, heterocycloalkyl, aryl, heteroaryl, aryla neo-Trp11NT(9-13), NT65L L-neo-Trp11, tert-Leu12NT lkyl, aryloxyalkyl, heteroarylalkyl, or heteroaryloxyalkyl. In 5 (8-13), NT73D D-Lys9, D-neo-Trp.11 INT(9-13), NT65D certain embodiments, X (where applicable), X2, and X are D-neo-Trp11, tert-Leu12INT(8-13), NT74L DAB9,L-neo N—H or N-alkylated, (e.g., N-methylated) amino acid resi Trp11, tert-Leu12INT(9-13), NT66L D-Lys8, L-neo-Trp.11, dues. The polypeptide may be a 10-mer to 15-mer and capable tert-Leu12NT(8-13), NTT4DDAB9, Pro, D-neo-Trp11, tert of binding to and activating the GLP-1 receptor. Leu12INT(9-13), NT66D D-Lys8, D-neo-Trp11, tert The following abbreviations are used above. 10 NaI-naphthylalanine; pGly-pentylglycine; t-BuG=t-butylg Leu12INT(8-13), NT75L DAB8 L-neo-Trp.11 INT(8-13), lycine: TPro-thioproline: HPro-homoproline; NmA=N-me NT67L D-Lys8, L-neo-Trp.11 INT(8-13), NT75D DAB8, D thylalanine; Cya=cysteic acid; Thi? 2-Thienyl-Ala; neo-Trp11NT(8-13), NT67DD-Lys8, D-neo-Trp.11 INT(8- 13), NT76L D-Orn9,L-neo-Trp.11 INT(8-13), NT69L hSer-homoserine; Aib=CL-aminoisobutyric acid; N-methyl-Arg8,L-Lys9 L-neo-Trp11tert-Leu12NT(8-13), Bip-biphenylalanine: Nle=norleucine: Ahx=2-aminohex 15 anoic acid; Nvanorvaline. NT76D (D-Orn9,D-neo-Trp.11 INT(8-13), NT69D N-me Leptin and Leptin Derivatives thyl-Arg8 L-Lys9.D-neo-Trp11tert-Leu12NT(8-13), The compounds of the invention can include leptin and NT71L D-Orn9,L-neo-Trp11, tert-Leu12INT(8-13), NTT1L leptin derivatives. Leptin is an adipokine, and thus the pro N-methyl-Arg8, DAB9 L-neo-Trp11, tert-leu12INT(8-13), teins or peptides used in the invention can include an adipok NTT 7D D-Orn9, D-neo-Trp11, tert-Leu12NT(8-13), ine or an analog thereof. Adipokines include , NT71D N-methyl-Arg8, DAB9, D-neo-Trp11tert-leu12NT leptin, and . include human, mouse, and (8-13), NT78L N-methyl-Arg8.D-Orn9 L-neo-Trp11tert rat adiponectin. include leptin (116-130), leptin (22 Leu12INT(8-13), NT72L D-Lys9,L-neo-Trp11, tert-Leu12 56), leptin (57-92), leptin (93-105), LY396623, meterleptin, NT(9-13), and NT78D N-methyl-Arg8.D-Orn9, D-neo murine leptin analog, pegylated leptin, and methionyl human 25 Trp11tert-Leu12NT(8-13), where neo-Trp is (2-amino-3- leptin. Resistins include human, mouse, and rat resistin. The 1H-indolylpropanoic acid). Other neurotensin analogs leptin may be a cleaved sequence or the full length protein. include Beta-lactotensin (NTR2 selective), JMV-449, and The polypeptide used in the invention may be any of these PD-149 or PD-163 (NTR1 selective; reduced amide bond peptides or proteins or may be substantially identical to any of 8-13 fragment of neurotensin). these peptides or proteins. 30 Other neurotensin analogs include those with modified Neurotensin and Neurotensin Derivatives amino acids (e.g., any of those described herein). The neuro Neurotensin (NT) is a 13 amino acid peptide found in the tensin analog may be selective for NTR1, NTR2, or NTR3 central nervous system and in the gastrointestinal tract. In (e.g., may bind to or activate one of NTR1, NTR2, or NTR3 brain, NT is associated with dopaminergic receptors and at least 2, 5, 10, 50, 100, 500, 1000, 5000, 10,000, 50,000, or other neurotransmitter system. Peripheral NT acts as a para 35 100,000 greater) as compared to at least one of the other NTR crine and endocrine peptide on both the digestive and cardio receptors or both. vascular systems. To exert its biological effects in the brain GDNF and GDNF Derivatives NT has to be injected ordelivered directly to the brain because In certain embodiments, the peptide vector is attached to NT does not cross the BBB and is rapidly degraded by pep GDNF, a GDNF analog, a GDNF fragment, or a modified tidases following systematic administration. Preclinical phar 40 form thereof. In certain embodiments, the GDNF analog is a macological studies, most of which involve direct injection of sequence substantially identical (e.g., at least 60%, 70%, NT into the brain, strongly suggest that an agonist of NT 80%, 85%, 90%. 95%, 98%, 99% identical) to GDNF, a receptors would be clinically useful for the treatment of neu GDNF analog, or to a fragment thereof. ropsychiatric conditions including psychosis, Schizophrenia, GDNF is secreted as a disulfide-linked homodimer, and is Parkinson's disease, pain, and the abuse of psychoStimulants. 45 able to Support Survival of dopaminergic neurons, Purkinje In particular, in various animal studies, intraventricular injec cells, motoneurons, and sympathetic neurons. GDNF analogs tion of NT led to hypothermia and analgesia in antinocicep or fragments having one or more of these activities may be tion experiments. used in the present invention, and activity of such analogs and The peptide therapeutic may be neurotensin or analog fragments can be tested using any means known in the art. thereof. Human neurotensin is a thirteen amino acid peptide 50 Human GDNF is expressed as a 211 amino acid protein having the sequence QLYENKPRRPYIL. Exemplary neuro (isoform 1); a 185 amino acid protein (isoform 2), and a 133 tensin analogs include (VIP-neurotensin) hybrid antagonist, amino acid protein. Mature GDNF is a 134 amino acid acetylneurotensin(8-13), JMV 1193, KK13 peptide, neuro sequence that includes amino acids 118-211 of isoform 1. medin N, neuromedin N precursor, neurotensin(1-10), neu amino acids 92-185 of isoform 2. Isoform 3 includes a trans rotensin (1-11), neurotensin (1-13), neurotensin (1-6), neuro 55 forming growth factor like domain from amino acids 40-133. tensin(1-8), neurotensin(8-13), Asp(12)-neurotensin(8-13), In certain embodiments, the GDNF analog is a splice vari Asp(13)-neurotensin(8-13), Lys(8)-neurotensin(8-13), ant of GDNF. Such proteins are described in PCT Publication N-methyl-Arg(8)-Lys(9)-neo-Trp.(11)-neo-Leu(12)-neuro No. WO 2009/053536, and include the pre-(C)pro-GDNF, tensin(8-13), neurotensin(9-13), neurotensin 69 L, Arg(9)- pre-(B)pro-GDNF, and pre-(y)pro-GDNF splice variant, as neurotensin, azidobenzoyl-Lys(6)-Trp(11)-neurotensin, Gln 60 well as the variants lacking the pre-pro region: (Cl)pro-GDNF, (4)-neurotensin, iodo-Tyr(11)-neurotensin, iodo-Tyr3)- (B)pro-GDNF, and pre-(y)pro-GDNF. neurotensin, N-O-(fluoresceinylthiocarbamyl)glutamyl(1)- GDNF analogs are also described in U.S. Patent Applica neurotensin, Phe(11)-neurotensin, Ser(7)-neurotensin, Trp tion Publication No. 2009/0069230, which include a GDNF (11)-neurotensin, Tyr(11)-neurotensin, rat NTT7, PD analog having the sequence: 1491.63, proneurotensin, stearyl-Nle(17)-neurotensin(6-11) 65 VIP(7-28), ''"Tc-NT-XI, TJN950, and vasoactive intestinal Xaa-Pro-Xaa-Pro-Xaas-Xaa-Xaaz-Xaas (SEQID peptide-neurotensin hybrid. NO: 182) US 9, 161988 B2 49 50 where Xaa is Phe, Trp, or Tyr; Xaa is Leu, Ala, Ile, or Val; where Xaa, and Xaa, are, independently, Arg, Lys, or His Xaas is Ala, Leu, Ile, or Val: Xaa is Gly, is any amino acid or are absent; Xaa is Glu or Asp. Xaa, is Arg, Lys, or His: residue of the D configuration or is absent; Xaa, is Lys, Arg, Xaas is ASn, Gln, Ser, or Thr, Xaa, is Arg, Lys, or His; Xaa, or His or is absent; and Xaas is Arg, Lys, or His or is absent. is Gln, ASn, Ser, or Thr, Xaas, Xaao, Xaalo, and Xaa, are, Xaa represents an amino acid, which we may also refer to as independently, Ala, Gly, Ile, Leu, Met, or Val; Xaa is ASn, an amino acid residue. The Subscripts (here, the Subscripts Gln, Ser, or Thr; Xaa is Pro or Ser; Xaa, is Glu or Asp; 1-8) represent the positions of each amino acid in the peptide Xaas is ASn, Gln, Ser, or Thr, Xaa is Ser, Asn., Gln, or Thr, sequence. Thus, Xaa represents the first amino acid residue Xaa, is Lys, Arg, or His; Xaas is Gly, Ala, Ile, Leu, Met, or in a fragment of a GDNF precursor protein. Val; Xaa, is Lys, Arg, or His: Xaa is Gly, is any amino acid 10 residue of the D-configuration, or is not present; and Xaa In specific embodiments, the fragments of a GDNF precur and Xaa, are, independently, Arg, Lys, His, or are not sor protein can have a sequence represented by (1) Phe-Pro present. An exemplary peptide conforming to Formula III can Xaa-Pro-Xaas-Xaa-Xaa,-Xaas (SEQ ID NO: 183), (e.g., have the sequence Arg-Arg-Glu-Arg-Asn-Arg-Gln-Ala-Ala Phe-Pro-Leu-Pro-Ala-Gly-Lys-Arg (SEQ ID NO: 151); (2) Ala-Ala-Asn-Pro-Glu-Asn-Ser-Arg-Gly-Lys-Gly-Arg-Arg Xaa-Pro-Leu-Pro-Xaas-Xaa-Xaa-Xaas (SEQ ID NO: 15 (SEQ ID NO:192). 184); (3) Phe-Pro-Leu-Pro-Xaas-Xaa-Xaa,-Xaas (SEQ ID Other GDNF analogs are described in PCT Publication No. NO: 152); (4) Xaa-Pro-Xaa-Pro-Ala-Xaa-Xaa-Xaas WO 2008/069876. These analogs include ERNROAAAAN (SEQ ID NO: 185); (5) Phe-Pro-Xaa-Pro-Ala-Xaa-Xaa Xaas (SEQ ID NO: 153); (6) Phe-Pro-Leu-Pro-Ala-Xaa PENSRGK-amide (SEQID NO: 200): FPLPA-amide (SEQ Xaa-Xaas (SEQID NO: 154); (7) Xaa-Pro-Xaa-Pro-Xaas ID NO: 194); and PPEAPAEDRSL-amide (SEQ ID NO: Gly Xaa-Xaas SEQ ID NO : 186); (8) Phe-Pro-Xaa-Pro 195). Xaa-Gly-Xaa-Xaas (SEQID NO: 155); (9) Phe-Pro-Leu Still other GDNF analogs are described in PCT Publication Pro-Xaas-Gly-Xaa-Xaas (SEQID NO: 156); (10) Phe-Pro No. WO 2007/019860. The analogs include those having the Leu-Pro-Ala-Gly-Xaa-Xaas (SEQID NO: 157); (11) Xaa formula: Pro-Xaa-Pro-Xaas-Xaa-Lys-Xaas (SEQID NO: 187); (12) Phe-Pro-Xaa-Pro-Xaas-Xaa-Lys-Xaas (SEQID NO: 158); 25 (13) Phe-Pro-Leu-Pro-Xaas-Xaa-Lys-Xaas (SEQ ID NO: where X is D, E, A or G. (X) is a sequence of 2-3 amino acid 159); (14) Phe-Pro-Leu-Pro-Ala-Xaa-Lys-Xaas (SEQ ID residues or a single amino acid residue selected from the NO: 160); (15) Phe-Pro-Leu-Pro-Ala-Gly-Lys-Xaas (SEQ group consisting of amino acid residues A, D, E, G, I. K. L. P. ID NO:161); (16) Xaa-Pro-Xaa-Pro-Xaas-Xaa-Xaa,-Arg Q. S. T and V. X, is amino acid residue Y or H, or a hydro 30 phobic amino acid residue, and at least one of X, X or X, is (SEQ ID NO: 188); (17) Phe-Pro-Xaa-Pro-Xaas-Xaa a charged or hydrophobic amino acid residue. The analog Xaa-Arg (SEQID NO: 162): (18) Phe-Pro-Leu-Pro-Xaas Xaa-Xaa-Arg (SEQ ID NO: 163); (19) Phe-Pro-Leu-Pro may be 6-22 amino acids in length. Ala-Xaa-Xaa,-Arg (SEQ ID NO: 164); and (20) Phe-Pro Further GDNF analogs are described in U.S. Patent Appli Leu-Pro-Ala-Gly-Xaa-Arg (SEQ ID NO: 165). cation Publication No. 2006/0258576. These analogs include In another embodiment, the fragment of a GDNF precursor 35 FPLPA-amide, (SEQID NO: 194), PPEAPAEDRSL-amide protein can be a fragment or portion of a GDNF precursor (SEQ ID NO: 195), LLEAPAEDHSL-amide (SEQ ID NO: protein conforming to Formula I, where Xaa is Phe, Xaa is 196), SPDKQMAVLP (SEQ ID NO: 197), SPDKQAAALP Leu, Xaas is Ala, Xaa is Gly, Xaa, is Lysand Xaas is Arg (i.e., (SEQID NO:198), SPDKQTPIFS (SEQID NO:199), ERN Phe-Pro-Leu-Pro-Ala-Gly-Lys-Arg). At least one (e.g., one, RQAAAANPENSRGK-amide (SEQ ID NO: 200), ERN 40 RQAAAASPENSRGK-amide (SEQID NO: 201), and ERN two, or three) of the amino acid residues represented by RQSAATNVENSSKK-amide (SEQID NO: 202). Formula I can be absent. For example, Xaa, Xaa, and/or Additional GDNF analogs can include functional frag Xaas can be absent. ments (e.g., any of the fragments described herein), peptides In another embodiment, the fragment of a GDNF precursor having any of the modifications described herein, or peptido protein or the biologically active variants can have, or can 45 mimetics thereof. Activity of such analogs and fragments can include, a sequence of amino acid residues conforming to the be tested using any means known in the art. amino acid sequence: Brain-Derived Neurotrophic Factor (BDNF) and BDNF Pro-Pro-Xaa-Xaa-Pro-Xaa-Xaa-Xaas-Xaa-Xa-ao Derivatives Xaa-Xaa-Xaa-Xaa (SEQID NO: 189) where Xaa is The compounds of the invention may be or may include Glu or Asp;Xaa is Ala, Gly, Ile, Leu, Met, or Val: Xaa is Ala, 50 BDNF, BDNF analogs, or BDNF fragments. BDNF is glyco Gly, Ile, Leu, Met, or Val; Xaa, is Glu or Asp; Xaas is Asp or protein of the nerve growth factor family of proteins. The Glu, Xaa, is Arg, His, or Lys: Xaao is Ser, ASn, Gln, or Thr: protein is encoded as a 247 amino acid polypeptide (isoform Xaa is Leu, Ala, Gly, Ile, Leu, Met or Val; Xaa is Gly, is A), a 255 amino acid polypeptide (isoform B), a 262 amino any amino acid residue of the D-configuration, or is not acid polypeptide (isoform C), a 276 amino acid polypeptide present; Xaa is Arg, His, or Lys or is not present; Xaa, is 55 (isoform D), a 329 amino acid polylpeptide (isoform E). The Arg, His, or Lys or is not present. An exemplary peptide mature 119 amino acid glycoprotein is processed from the conforming to Formula II can have the sequence Pro-Pro larger precursor to yield a neutrophic factor that promotes the Glu-Ala-Pro-Ala-Glu-Asp-Arg-Ser-Leu-Gly-Arg-Arg (SEQ Survival of neuronal cell populations. The mature protein ID NO:190). includes amino acids 129-247 of the isoform A preprotein, In another embodiment, the fragments of a GDNF precur 60 amino acids 137-255 of the isoform B preprotein, amino acids Sorprotein or the biologically active variants can have, or can 144-162 of isoform C preprotein, amino acids 158-276 of the include, a sequence of amino acid residues conforming to the isoform D preprotein, or amino acids 211 (or 212)-329 of the amino acid sequence of Formula III: isoform E preprotein. BDNF acts at the TrkB receptor and at low affinity nerve growth factor receptor (LNGFR or p75). 65 BDNF is capable of supporting neuronal survival of existing neurons and can also promote growth and differentiation of NO: 191) (III). new neurons. The BDNF fragments or analogs of the inven US 9, 161988 B2 51 52 tion may have any of the aforementioned activities. Activity TABLE 3 - continued of such analogs and fragments can be tested using any means known in the art. Residue WT Residue Possible substitutions BDNF analogs are described in U.S. Patent Application Publication No. 2004/0072291, which include those having a 5 51 Q A C G P substitution of A, C, D, E, G, H, K, N P, QR, S, or T at one 52 Y M W W more positions selected from the group consisting of 10, 16. 20, 29, 31, 36,38, 39, 42,44, 49, 52,53,54, 61, 63,71, 76, 86, 53 M. W. Y 87, 90, 92, 98, 100, 102, 103, and 105. Additional substitu tions are described in Table 3 below. 10 55 E A C G H N P Q S T 56 T A C G P TABLE 3 st K A C G H P Q S T Residue WT Residue Possible substitutions 15 58 C D E G H K N P C R S T

9 E A C F. G. I. L. M. P. W. W. Y 59 N A C G P T

O L M. F. W. W. Y 60 P T

1. S A C F. G. I. L. M. P. W. W. Y 2O 61 M I W W Y

3 C D E F H I K N P C R S T V Y 87 W I M W Y

4. D A C F. G. I. L. M. P. W. W. Y 88 R A C G P

5 S D F H I L N P O W, Y 89 A. D E H K N C R T 25 6 I W M Y 90 I M W W Y

7 S A C G P 91 T A C P G P

8 E T F H I P Q S 92 H I W Y 30 9 W A C D E G H K N P C R S T 93 D p T

2O W W Y 94 S A C G P

21 T D. F. H. I. L. P. W. Y 95 K H. P 35 22 A. D E H K N P C R S T 96 K d

23 A. HT 97 R A C G P

24 D H P T 98 I H. W.

28 A. HT 40 O1 R p T

31 M W Y O2 M W W Y

32 S A C G P O3 I M W Y

34 G T D E H K N P O RS 45 O4 R A C G P T

35 T A C G P OS I M. W.

36 W F I L. M. W. Y O6 D A C G H II M P T

38 W W Y F I M 50 O7 T A C D E G H K N P O S

39 L I M W W Y O8 S A C D G H P

41 K A C G H P S O9 C D E H K N P C R S T

42 W I 55 1O W T

44 W F. L. M. W. Y 11 C D E F H I K N P C R S T W W Y

45 S A C F P W Y 12 T A C F. G. I L H P W W. Y

46 K A C G P Q S T 60 13 L Any amino acid

47 G D E H N P C R S T BDNF analogs are also described in U.S. Pat. No. 6,800, 48 Q A C G P 607, which describes BDNF modified with 1-acyl-glycerol. 49 L I M W W Y These analogs include a modified BDNF, where is the com 65 pound of the formula: SO K I P T US 9, 161988 B2 53 54 where A is a residue of brain-derived neurotrophic factor, Bis 2 to 10 carbonatoms; (12) A modified BDNF according to the a residue of a 1-acyl-glycerol derivative having a hydroxyl above (10), where R' is trimethylene. group at the 2-position of the glycerol moiety, which is pre Other BDNF analogs include those described in PCT Pub pared by removing a hydrogenatom from the hydroxyl group, lication No. WO 96/15146, which described conjugates of X is a chemical cross-linkage, and m is an average number of 5 BDNF to water soluble polymers such as polyethylene gly the introduction and is not less than about 0.5; (3) A modified col. Additional BDNF analogs can include functional frag BDNF according to the above (2), wherein X is a group of the ments (e.g., any of the fragments described herein), peptides formula (2): having any of the modifications described herein, or peptido mimetics thereof. Activity of such analogs can be tested using -C-R-C- 10 any method known in the art. O O Modified Polypeptides The peptide vectors and peptide/polypeptide agents used in where R' is an alkylene group, or a group of the formula (3): the invention may have a modified amino acid sequence. In H certain embodiments, the modification does not destroy sig -C-R-C-N-R-C- 15 nificantly a desired biological activity (e.g., ability to cross the BBB or agonist activity). The modification may reduce O O O (e.g., by at least 5%, 10%, 20%, 25%, 35%, 50%, 60%, 70%, where R and Rare independently an alkylene group; (4) A 75%, 80%, 90%, or 95%), may have no effect, or may modified BDNF according to the above (2), wherein the increase (e.g., by at least 5%, 10%, 25%, 50%, 100%, 200%, 1-acyl-glycerol derivative is 1-acyl-glycero-3-phosphoryl 500%, or 1000%) the biological activity of the original choline, 1-acyl-glycero-3-phosphoryl serine, or 1-acyl-gryc polypeptide. The modified peptide or polypeptide may have ero-3-phosphoryl ethylamine; (5) A modified BDNF accord or may optimize a characteristic of a polypeptide. Such as in ing to the above (2), wherein B is a 1-acyl-glycero-3-phos Vivo Stability, bioavailability, toxicity, immunological activ phoryl choline residue of the formula (4): ity, immunological identity, and conjugation properties. 25 Modifications include those by natural processes, such as HC-O-R posttranslational processing, or by chemical modification techniques known in the art. Modifications may occur any -O-CH O where in a polypeptide including the polypeptide backbone, the amino acid side chains and the amino- or carboxy-termi HC-O-P-CHCHN (CH) nus. The same type of modification may be present in the 30 same or varying degrees at several sites in a given polypep O tide, and a polypeptide may contain more than one type of where R is an acyl group, a 1-acyl-glycero-3-phosphoryl modification. Polypeptides may be branched as a result of serine residue of the formula (5): ubiquitination, and they may be cyclic, with or without branching. Cyclic, branched, and branched cyclic polypep HC-O-R 35 tides may result from posttranslational natural processes or may be made synthetically. Other modifications include -O-CH pegylation, acetylation, acylation, addition of acetomidom ethyl (Acm) group, ADP-ribosylation, alkylation, amidation, HC-O-P-OCHCHCOOH biotinylation, carbamoylation, carboxyethylation, esterifica OH 40 tion, covalent attachment to flavin, covalent attachment to a heme moiety, covalent attachment of a nucleotide or nucle where R is an acyl group, or a 1-acyl-glycero-phosphoryl otide derivative, covalent attachment of drug, covalent attach ethylamine residue of the formula (6): ment of a marker (e.g., fluorescent or radioactive), covalent attachment of a lipid or lipid derivative, covalent attachment HC-O-R of phosphatidylinositol, cross-linking, cyclization, disulfide 45 -O-CH bond formation, demethylation, formation of covalent crosslinks, formation of cystine, formation of pyroglutamate, formylation, gamma-carboxylation, glycosylation, GPI anchor formation, hydroxylation, iodination, methylation, OH myristoylation, oxidation, proteolytic processing, phospho where R is an acyl group; (6) A modified BDNF according to 50 rylation, prenylation, racemization, selenoylation, Sulfation, the above (2) or (3), where B is a group of the formula (4): transfer-RNA mediated addition of amino acids to proteins Such as arginylation and ubiquitination. HC-O-R A modified polypeptide can also include an amino acid insertion, deletion, or Substitution, either conservative or non -O-CH 55 conservative (e.g., D-amino acids, desamino acids) in the HC-O-P-CHCHN (CH) polypeptide sequence (e.g., where such changes do not Sub stantially alter the biological activity of the polypeptide). In O particular, the addition of one or more cysteine residues to the where R is an acyl group; (7) A modified BDNF according to amino or carboxy terminus of any of the polypeptides of the any one of the above (2), (3), (4), (5) and (6), where the acyl 60 invention can facilitate conjugation of these polypeptides by, group is an alkanoyl group having 8 to 30 carbonatoms; (8)A e.g., disulfide bonding. For example, Angiopep-1 (SEQ ID modified BDNF according to any one of the above (2), (3), NO:67), Angiopep-2 (SEQID NO:97), or Angiopep-7 (SEQ (4), (5), (6) and (7), where the acyl group is palmitoyl group; ID NO:112) can be modified to include a single cysteine (9) A modified BDNF according to any one of the above (2), residue at the amino-terminus (SEQ ID NOS: 71, 113, and (3), (4), (5), (6), (7) and (8), where m is in the range of from 65 115, respectively) or a single cysteine residue at the carboxy about 1 to about 6; (11) A modified BDNF according to the terminus (SEQ ID NOS: 72, 114, and 116, respectively). above (10), where R' is a straight chain alkylenegroup having Amino acid Substitutions can be conservative (i.e., wherein a US 9, 161988 B2 55 56 residue is replaced by another of the same general type or TABLE 4 - continued group) or non-conservative (i.e., wherein a residue is replaced by an amino acidofanother type). In addition, a non-naturally Amino acid substitutions occurring amino acid can be substituted for a naturally occur Original Exemplary Conservative ring amino acid (i.e., non-naturally occurring conservative 5 residue substitution substitution amino acid substitution or a non-naturally occurring non Asp (D) Glu Glu conservative amino acid Substitution). Polypeptides made synthetically can include substitutions Cys (C) Ser Ser of amino acids not naturally encoded by DNA (e.g., non naturally occurring or unnatural amino acid). Examples of Glin (Q) Asn Asn non-naturally occurring amino acids include D-amino acids, Glu (E) Asp Asp an amino acid having an acetylaminomethyl group attached to a Sulfur atom of a cysteine, a pegylated amino acid, the Gly (G) Pro Pro omega amino acids of the formula NH2(CH2)COOH His (H) Asn., Gln, Lys, Arg Arg wherein n is 2-6, neutral nonpolar amino acids, such as Sar 15 cosine, t-butyl , t-butyl glycine, N-methyl , Ile (I) Lieu, Wal Met, Ala, Lell and norleucine. Phenylglycine may substitute for Trp, Tyr, or Phe, norleucine Phe, citrulline and methionine sulfoxide are neutral nonpolar, Leu (L) Norleucine, Ile, Wall, Ile cysteic acid is acidic, and ornithine is basic. Proline may be Met, Ala, Phe substituted with hydroxyproline and retain the conformation conferring properties. Llys (K) Arg, Gln, Asn Arg Analogs may be generated by Substitutional mutagenesis and retain the biological activity of the original polypeptide. Met (M) Lieu, Phe, Ile Lell Examples of substitutions identified as “conservative substi Phe (F) Lieu, Wall, Ile, Ala Lell tutions' are shown in Table 4. If such substitutions result in a 25 change not desired, then other type of Substitutions, denomi Pro (P) Gly Gly nated “exemplary substitutions” in Table 4, or as further Ser (S) Thir Thir described herein in reference to amino acid classes, are intro Thir (T) Ser Ser duced and the products screened. 30 Substantial modifications in function or immunological Trp (W) Tyr Tyr identity are accomplished by selecting Substitutions that dif fer significantly in their effect on maintaining (a) the structure Tyr (Y) Trp, Phe, Thr, Ser Phe of the polypeptide backbone in the area of the substitution, for example, as a sheet or helical conformation. (b) the charge or Wall (W) Ile, Lieu, Met, Phe, Lell hydrophobicity of the molecule at the target site, or (c) the 35 Ala, nor bulk of the side chain. Naturally occurring residues are divided into groups based on common side chain properties: Polypeptide Derivatives and Peptidomimetics (1) hydrophobic: norleucine, methionine (Met), Alanine In addition to polypeptides consisting of naturally occur (Ala), (Val), Leucine (Leu), Isoleucine (Ile). ringamino acids, peptidomimetics or polypeptide analogs are (His), (Trp), Tyrosine (Tyr), (Phe), 40 also encompassed by the present invention and can form the (2) neutral hydrophilic: Cysteine (Cys), Serine (Ser), peptide vectors or peptide/polypeptide agents used in the Threonine (Thr) compounds of the invention. Polypeptide analogs are com (3) acidic/negatively charged: Aspartic acid (Asp), monly used in the pharmaceutical industry as non-peptide Glutamic acid (Glu) drugs with properties analogous to those of the template (4) basic: (ASn), (Gln). Histidine 45 polypeptide. The non-peptide compounds are termed "pep (His), Lysine (LyS), Arginine (Arg) tide mimetics' or peptidomimetics (Fauchere et al., Infect. (5) residues that influence chain orientation: Glycine Immun. 54:283-287, 1986 and Evans et al., J. Med Chem. (Gly), Proline (Pro); (6) aromatic: Tryptophan (Trp), Tyrosine (Tyr), Phenyla 30:1229-1239, 1987). Peptide mimetics that are structurally 50 related to therapeutically useful peptides or polypeptides may lanine (Phe), Histidine (His), be used to produce an equivalent or enhanced therapeutic or (7) polar: Ser, Thr, Asn., Gln (8) basic positively charged: Arg, Lys, His, and; prophylactic effect. Generally, peptidomimetics are structur (9) charged: Asp, Glu, Arg, Lys, His ally similar to the paradigm polypeptide (i.e., a polypeptide Other amino acid substitutions are listed in Table 4. that has a biological or pharmacological activity) Such as 55 naturally-occurring receptor-binding polypeptides, but have one or more peptide linkages optionally replaced by linkages TABLE 4 Such as CH-NH CHS , CH2—CH2—, Amino acid substitutions —CH=CH-(cis and trans), —CHSO , —CH(OH) CH , —COCH2— etc., by methods well known in the art Original Exemplary Conservative 60 (Spatola, Peptide Backbone Modifications, Vega Data, 1:267, residue substitution substitution 1983: Spatola et al., Life Sci. 38:1243-1249, 1986; Hudsonet Ala (A) Wall, Lieu, Ile Wall al., Int. J. Pept. Res. 14:177-185, 1979; and Weinstein, 1983, Chemistry and Biochemistry, of Amino Acids, Peptides and Arg (R) Lys, Gln, Asn Lys Proteins, Weinstein eds., Marcel Dekker, New York). Such Asn (N) Gln, His, Lys, Arg Glin 65 polypeptide mimetics may have significant advantages over naturally occurring polypeptides including more economical production, greater chemical stability, enhanced pharmaco US 9, 161988 B2 57 58 logical properties (e.g., half-life, absorption, potency, effi purification procedure or (2) by removal of the polypeptide ciency), reduced antigenicity, and others. from the Support along with complete side chain de-protec While the peptide vectors described herein may efficiently tion, followed by oxidation of the free SH-functions in highly cross the BBB or target particular cell types (e.g., those dilute aqueous Solution. described herein), their effectiveness may be reduced by the The cyclic derivative containing an intramolecular amide presence of proteases. Likewise, the effectiveness of the pep bond may be prepared by conventional Solid phase synthesis tide/polypeptide agents used in the invention may be simi while incorporating Suitable amino and carboxyl side chain larly reduced. Serum proteases have specific substrate protected amino acid derivatives, at the position selected for requirements, including L-amino acids and peptide bonds for cyclization. The cyclic derivatives containing intramolecular cleavage. Furthermore, exopeptidases, which represent the 10 —S-alkyl bonds can be prepared by conventional Solid phase most prominent component of the protease activity in serum, chemistry while incorporating an amino acid residue with a usually act on the first peptide bond of the polypeptide and Suitable amino-protected side chain, and a suitable S-pro require a free N-terminus (Powell et al., Pharm. Res. tected cysteine or homocysteine residue at the position 10:1268-1273, 1993). In light of this, it is often advantageous selected for cyclization. to use modified versions of polypeptides. The modified 15 Another effective approach to confer resistance to pepti polypeptides retain the structural characteristics of the origi dases acting on the N-terminal or C-terminal residues of a nal L-amino acid polypeptides, but advantageously are not polypeptide is to add chemical groups at the polypeptide readily Susceptible to cleavage by protease and/or exopepti termini. Such that the modified polypeptide is no longer a dases. substrate for the peptidase. One such chemical modification is Systematic Substitution of one or more amino acids of a glycosylation of the polypeptides at either or both termini. consensus sequence with D-amino acid of the same type (e.g., Certain chemical modifications, in particular N-terminal gly an enantiomer, D-lysine in place of L-lysine) may be used to cosylation, have been shown to increase the stability of generate more stable polypeptides. Thus, a polypeptide polypeptides in human serum (Powell et al., Pharm. Res. derivative or peptidomimetic as described herein may be all 10:1268-1273, 1993). Other chemical modifications which L-, all D-, or mixed D. L. polypeptides. The presence of an 25 enhance serum stability include, but are not limited to, the N-terminal or C-terminal D-amino acid increases the in vivo addition of an N-terminal alkyl group, consisting of a lower stability of a polypeptide because peptidases cannot utilize a alkyl of from one to twenty carbons, such as an acetyl group, D-amino acid as a substrate (Powell et al., Pharm. Res. and/or the addition of a C-terminal amide or substituted 10:1268-1273, 1993). Reverse-D polypeptides are polypep amide group. In particular, the present invention includes tides containing D-amino acids, arranged in a reverse 30 modified polypeptides consisting of polypeptides bearing an sequence relative to a polypeptide containing L-amino acids. N-terminal acetyl group and/or a C-terminal amide group. Thus, the C-terminal residue of an L-amino acid polypeptide Also included by the present invention are other types of becomes N-terminal for the D-amino acid polypeptide, and so polypeptide derivatives containing additional chemical moi forth. Reverse D-polypeptides retain the same tertiary con eties not normally part of the polypeptide, provided that the formation and therefore the same activity, as the L-amino acid 35 derivative retains the desired functional activity of the polypeptides, but are more stable to enzymatic degradation in polypeptide. Examples of such derivatives include (1) N-acyl vitro and in Vivo, and thus have greater therapeutic efficacy derivatives of the amino terminal or of another free amino than the original polypeptide (Brady and Dodson, Nature group, wherein the acyl group may be an alkanoyl group (e.g., 368:692-693, 1994 and Jameson et al., Nature 368:744-746, acetyl, hexanoyl, octanoyl)anaroyl group (e.g., benzoyl) or a 1994). In addition to reverse-D-polypeptides, constrained 40 blocking group Such as F-moc (fluorenylmethyl-O-CO—); polypeptides including a consensus sequence or a Substan (2) esters of the carboxy terminal or of another free carboxy or tially identical consensus sequence variation may be gener hydroxyl group; (3) amide of the carboxy-terminal or of ated by methods well known in the art (Rizo et al., Ann. Rev. another free carboxyl group produced by reaction with Biochem. 61:387-418, 1992). For example, constrained ammonia or with a Suitable amine; (4) phosphorylated deriva polypeptides may be generated by adding cysteine residues 45 tives; (5) derivatives conjugated to an antibody or other bio capable of forming disulfide bridges and, thereby, resulting in logical ligand and other types of derivatives. a cyclic polypeptide. Cyclic polypeptides have no free N- or Longer polypeptide sequences which result from the addi C-termini. Accordingly, they are not susceptible to proteoly tion of additional amino acid residues to the polypeptides sis by exopeptidases, although they are, of course, Susceptible described herein are also encompassed in the present inven to endopeptidases, which do not cleave at polypeptide ter 50 tion. Such longer polypeptide sequences can be expected to mini. The amino acid sequences of the polypeptides with have the same biological activity and specificity (e.g., cell N-terminal or C-terminal D-amino acids and of the cyclic tropism) as the polypeptides described above. While polypeptides are usually identical to the sequences of the polypeptides having a Substantial number of additional amino polypeptides to which they correspond, except for the pres acids are not excluded, it is recognized that Some large ence of N-terminal or C-terminal D-amino acid residue, or 55 polypeptides may assume a configuration that masks the their circular structure, respectively. effective sequence, thereby preventing binding to a target A cyclic derivative containing an intramolecular disulfide (e.g., a member of the LRP receptor family such as LRP or bond may be prepared by conventional Solid phase synthesis LRP2). These derivatives could act as competitive antago while incorporating Suitable S-protected cysteine or nists. Thus, while the present invention encompasses homocysteine residues at the positions selected for cycliza 60 polypeptides or derivatives of the polypeptides described tion Such as the amino and carboxy termini (Sah et al., J. herein having an extension, desirably the extension does not Pharm. Pharmacol. 48:197, 1996). Following completion of destroy the cell targeting activity of the polypeptides or its the chain assembly, cyclization can be performed either (1) by derivatives. selective removal of the S-protecting group with a consequent Other derivatives included in the present invention are dual on-support oxidation of the corresponding two free SH-func 65 polypeptides consisting of two of the same, or two different tions, to form a S-S bonds, followed by conventional polypeptides, as described herein, covalently linked to one removal of the product from the Support and appropriate another either directly or through a spacer, Such as by a short US 9, 161988 B2 59 60 stretch of alanine residues or by a putative site for proteolysis platforms to display organic pharmocophores in libraries of (e.g., by cathepsin, see e.g., U.S. Pat. No. 5,126.249 and candidates designed to mimic the desired activity of the native European Patent No. 495.049): Multimers of the polypeptides polypeptide. In more detail the three phases are as follows. In described herein consist of a polymer of molecules formed phase 1, the lead candidate polypeptides are scanned and their from the same or different polypeptides orderivatives thereof. structure abridged to identify the requirements for their activ The present invention also encompasses polypeptide ity. A series of polypeptide analogs of the original are syn derivatives that are chimeric or fusion proteins containing a thesized. In phase 2, the best polypeptide analogs are inves polypeptide described herein, or fragment thereof, linked at tigated using the conformationally constrained dipeptide its amino- or carboxy-terminal end, or both, to an amino acid Surrogates. Indolizidin-2-one, indolizidin-9-one and quino sequence of a different protein. Such a chimeric or fusion 10 lizidinone amino acids (Iaa, I’aa and Qaa respectively) are protein may be produced by recombinant expression of a used as platforms for studying backbone geometry of the best nucleic acid encoding the protein. For example, a chimeric or peptide candidates. These and related platforms (reviewed in fusion protein may contain at least 6 amino acids shared with Halab et al., Biopolymers 55: 101-122, 2000 and Hanessian et one of the described polypeptides which desirably results in a al., Tetrahedron 53:12789-12854, 1997) may beintroduced at chimeric or fusion protein that has an equivalent or greater 15 specific regions of the polypeptide to orient the pharmacoph functional activity. ores in different directions. Biological evaluation of these Assays to Identify Peptidomimetics analogs identifies improved lead polypeptides that mimic the As described above, non-peptidyl compounds generated to geometric requirements for activity. In phase 3, the platforms replicate the backbone geometry and pharmacophore display from the most active lead polypeptides are used to display (peptidomimetics) of the polypeptides described herein often organic Surrogates of the pharmacophores responsible for possess attributes of greater metabolic stability, higher activity of the native peptide. The pharmacophores and scaf potency, longer duration of action, and better bioavailability. folds are combined in a parallel synthesis format. Derivation Peptidomimetics compounds can be obtained using any of of polypeptides and the above phases can be accomplished by the numerous approaches in combinatorial library methods other means using methods known in the art. known in the art, including biological libraries, spatially 25 Structure function relationships determined from the addressable parallel solid phase or solution phase libraries, polypeptides, polypeptide derivatives, peptidomimetics or synthetic library methods requiring deconvolution, the one other small molecules described herein may be used to refine bead one-compound library method, and synthetic library and prepare analogous molecular structures having similar or methods using affinity chromatography selection. The bio better properties. Accordingly, the compounds of the present logical library approach is limited to peptide libraries, while 30 invention also include molecules that share the structure, the other four approaches are applicable to peptide, non polarity, charge characteristics and side chain properties of peptide oligomer, or small molecule libraries of compounds the polypeptides described herein. (Lam, Anticancer Drug Des. 12:145, 1997). Examples of In Summary, based on the disclosure herein, those skilled in methods for the synthesis of molecular libraries can be found the art can develop peptides and peptidomimetics screening in the art, for example, in: DeWitt et al. (Proc. Natl. Acad. Sci. 35 assays which are useful for identifying compounds for target USA 90:6909, 1993); Erb et al. (Proc. Natl. Acad. Sci. USA ing an agent to particular cell types (e.g., those described 91:11422, 1994); Zuckermann et al. (J. Med. Chem. 37:2678, herein). The assays of this invention may be developed for 1994); Cho et al. (Science 261:1303, 1993); Carell et al. low-throughput, high-throughput, or ultra-high throughput (Angew. Chem, Int. Ed. Engl. 33:2059, 1994 and ibid 2061); screening formats. Assays of the present invention include and in Gallop et al. (Med. Chem. 37:1233, 1994). Libraries of 40 assays amenable to automation. compounds may be presented in Solution (e.g., Houghten, Diseases and Conditions Biotechniques 13:412-421, 1992) or on beads (Lam, Nature The compounds of the invention can be used to treat a 354:82-84, 1991), chips (Fodor, Nature 364:555-556, 1993), variety of diseases and conditions. Because the compounds of bacteria or spores (U.S. Pat. No. 5.223.409), plasmids (Cullet the invention are able to cross the BBB or enterparticular cell al., Proc. Natl. Acad. Sci. USA 89:1865-1869, 1992) or on 45 types, treatments of neurological disorders, including neuro phage (Scott and Smith, Science 249:386-390, 1990), or degenerative diseases and cancer, can be enhanced using the luciferase, and the enzymatic label detected by determination multimers of the invention. of conversion of an appropriate Substrate to product. Cancer Therapy Once a polypeptide as described herein is identified, it can Compounds of the invention including anticancer agents be isolated and purified by any number of standard methods 50 may be used to treat any brain or central nervous system including, but not limited to, differential Solubility (e.g., pre disease (e.g., a brain cancer Such as glioblastoma, astrocy cipitation), centrifugation, chromatography (e.g., affinity, ion toma, glioma, meduloblastoma, and oligodendroma, neuro exchange, and size exclusion), or by any other standard tech glioma, ependymoma, and meningioma). Compounds that niques used for the purification of peptides, peptidomimetics, are efficiently transported to the liver, lung, kidney, spleen or or proteins. The functional properties of an identified 55 muscle (e.g., AngioPep-1 through AngioPep-7) and therefore polypeptide of interest may be evaluated using any functional may also be used, in conjunction with an appropriate thera assay known in the art. Desirably, assays forevaluating down peutic agent, to treat a disease associated with these tissues stream receptor function in intracellular signaling are used (e.g., a cancer Such as hepatocellular carcinoma, liver cancer, (e.g., cell proliferation). Small cell carcinoma (e.g., oat cell cancer), mixed Small cell/ For example, the peptidomimetics compounds of the 60 large cell carcinoma, combined Small cell carcinoma, and present invention may be obtained using the following three metastatic tumors. Metastatic tumors can originate from can phase process: (1) Scanning the polypeptides described herein cer of any tissue, including breast cancer, colon cancer, pros to identify regions of secondary structure necessary for tar tate cancer, sarcoma, bladder cancer, neuroblastoma, Wilm's geting the particular cell types described herein; (2) using tumor, lymphoma, non-Hodgkin’s lymphoma, and certain conformationally constrained dipeptide Surrogates to refine 65 T-cell lymphomas). Additional exemplary cancers that may the backbone geometry and provide organic platforms corre be treated using a composition of the invention include hepa sponding to these Surrogates; and (3) using the best organic tocellular carcinoma, breast cancer, cancers of the head and US 9, 161988 B2 61 62 neck including various lymphomas Such as mantle cell lym satiety (U.S. Patent Application Publication No. 2003/ phoma, non-Hodgkin’s lymphoma, adenoma, squamous cell 0232754); treating irritable bowel syndrome (U.S. Pat. No. carcinoma, laryngeal carcinoma, cancers of the retina, can 6,348.447); reducing the morbidity and/or mortality associ cers of the esophagus, multiple myeloma, ovarian cancer, ated with myocardial infarction (U.S. Pat. No. 6,747.006)and uterine cancer, melanoma, colorectal cancer, bladder cancer, 5 stroke (PCT Publication No. WO 00/16797); treating acute prostate cancer, lung cancer (including non-Small cell lung coronary syndrome characterized by an absence of Q-wave carcinoma), pancreatic cancer, cervical cancer, head and neck myocardial infarction (U.S. Pat. No. 7,056,887); attenuating cancer, skin cancers, nasopharyngeal carcinoma, liposar post-surgical catabolic changes (U.S. Pat. No. 6,006,753); coma, epithelial carcinoma, renal cell carcinoma, gallbladder treating hibernating myocardium or diabetic cardiomyopathy adenocarcinoma, parotid adenocarcinoma, endometrial sar- 10 (U.S. Pat. No. 6,894,024); suppressing plasma blood levels of coma, multidrug resistant cancers; and proliferative diseases norepinepherine (U.S. Pat. No. 6,894,024); increasing uri and conditions, such as neovascularization associated with nary sodium excretion, decreasingurinary potassium concen tumor angiogenesis, macular degeneration (e.g., wet/dry tration (U.S. Pat. No. 6,703,359); treating conditions or dis AMD), corneal neovascularization, diabetic retinopathy, orders associated with toxic hypervolemia, e.g., renal failure, neovascular glaucoma, myopic degeneration and other pro- 15 congestive heart failure, nephrotic syndrome, cirrhosis, pull liferative diseases and conditions such as restenosis and poly monary edema, and hypertension (U.S. Pat. No. 6,703,359); cystic kidney disease. Brain cancers that may be treated with inducing an inotropic response and increasing cardiac con vector that is transported efficiently across the BBB include tractility (U.S. Pat. No. 6,703,359); treating polycystic ovary astrocytoma, pilocytic astrocytoma, dysembryoplastic neu syndrome (U.S. Pat. No. 7,105,489); treating respiratory dis roepithelial tumor, oligodendrogliomas, ependymoma, glio- 20 tress (U.S. Patent Application Publication No. 2004/ blastoma multiforme, mixed gliomas, oligoastrocytomas, 0235726); improving nutrition via a non-alimentary route, medulloblastoma, retinoblastoma, neuroblastoma, germi i.e., via intravenous, Subcutaneous, intramuscular, peritoneal, noma, and teratoma. or other injection or infusion (U.S. Pat. No. 6,852,690); treat GLP-1-Based Therapy ing nephropathy (U.S. Patent Application Publication No. The compounds of the invention including a GLP-1 agoinst 25 2004/0209803); treating left ventricular systolic dysfunction, can be used in any therapeutic application where a GLP-1 e.g., with abnormal left ventricular ejection fraction (U.S. Pat. agonist activity in the brain, or in a particular tissues, is No. 7,192,922); inhibiting antro-duodenal motility, e.g., for desired. GLP-1 agonist activity is associated with stimulation the treatment or prevention of gastrointestinal disorders such of insulin secretion (i.e., to act as an hormone) and as diarrhea, postoperative dumping syndrome and irritable inhibition glucagon Secretion, thereby contributing to limit 30 bowel syndrome, and as premedication in endoscopic proce postprandial glucose excursions. GLP-1 agonists can also dures (U.S. Pat. No. 6,579,851); treating critical illness poly inhibit gastrointestinal motility and secretion, thus acting as neuropathy (CIPN) and systemic inflammatory response syn an enterogastrone and part of the “ileal brake' mechanism. drome (SIRS) (U.S. Patent Application Publication No. 2003/ GLP-1 also appears to be a physiological regulator of appetite 0199445); modulating triglyceride levels and treating and food intake. Because of these actions, GLP-1 and GLP-1 35 dyslipidemia (U.S. Patent Application Publication Nos. receptor agonists can be used for therapy of metabolic disor 2003/0036504 and 2003/0143183); treating organ tissue ders, as reviewed in, e.g., Kinzig et al., J Neurosci 23:6163 injury caused by reperfusion of blood flow following 6170, 2003. Such disorders include obesity, hyperglycemia, ischemia (U.S. Pat. No. 6,284,725); treating coronary heart dyslipidemia, hypertriglyceridemia, syndrome X, insulin disease risk factor (CHDRF) syndrome (U.S. Pat. No. 6,528, resistance, IGT, diabetic dyslipidemia, hyperlipidemia, a car- 40 520); and others. diovascular disease, and hypertension. Leptin-Based Therapy GLP-1 is also has neurological effects including sedative Compounds of the invention that include leptin or a related oranti-anxiolytic effects, as described in U.S. Pat. No. 5,846, molecule can be used to treat metabolic disorders, neurologi 937. Thus, GLP-1 agonists can be used in the treatment of cal diseases, as well as other indications. anxiety, aggression, psychosis, seizures, panic attacks, hys- 45 In certain embodiments, the compound of the invention is teria, or sleep disorders. GLP-1 agonists can also be used to used to treat a metabolic disorder. Such disorders include treat Alzheimer's disease, as GLP-1 agonists have been diabetes (type I or type II), obesity, hyperglycemia, dyslipi shown to protect neurons against amyloid-fi peptide and demia, hypertriglyceridemia, syndrome X, insulin resistance, glutamate-induced apoptosis (Perry et al., Curr Alzheimer IGT, diabetic dyslipidemia, hyperlipidemia, a cardiovascular Res 2:377-85, 2005). 50 disease, and hypertension. Leptin decreases food intake and Other therapeutic uses for GLP-1 agonists include improv thus can be used to reduce weight and to treat diseases where ing learning, enhancing neuroprotection, and alleviating a reduced food intake or weight loss is beneficial. symptom of a disease or disorder of the central nervous sys Because peptide vectors described herein are capable of tem, e.g., through modulation of neurogenesis, and e.g., Par transporting an agent across the BBB, the compounds of the kinson's Disease, Alzheimer's Disease, Huntington's Dis- 55 invention are also useful for the treatment of neurological ease, ALS, stroke, ADD, and neuropsychiatric syndromes diseases such as neurodegenerative diseases or other condi (U.S. Pat. No. 6,969,702 and U.S. Patent Application No. tions of the central nervous system (CNS), the peripheral 2002/01 15605). Stimulation of neurogenesis using GLP-1 nervous system, or the autonomous nervous system (e.g., agonists has been described, for example, in Bertilsson et al., where neurons are lost or deteriorate). Many neurodegenera J Neurosci Res 86:326-338, 2008. 60 tive diseases are characterized by ataxia (i.e., uncoordinated Still other therapeutic uses include converting liver stem/ muscle movements) and/or memory loss. Neurodegenerative progenitor cells into functional pancreatic cells (U.S. Patent diseases include Alexander disease, Alper disease, Alzhe Application Publication No. 2005/0053588); preventing imer's disease, amyotrophic lateral Sclerosis (ALS, i.e., Lou beta-cell deterioration (U.S. Pat. Nos. 7.259,233 and 6,569, Gehrig's disease), ataxia telangiectasia, Batten disease 832) and stimulation of beta-cell proliferation (U.S. Patent 65 (Spielmeyer-Vogt-Sjogren-Batten disease), bovine spongi Application Publication No. 2003/0224983); treating obesity form encephalopathy (BSE), Canavan disease, Cockayne (U.S. Pat. No. 7,211,557); suppressing appetite and inducing syndrome, corticobasal degeneration, Creutzfeldt-Jakob dis US 9, 161988 B2 63 64 ease, Huntington's disease, HIV-associated dementia, Neurotensin is also known to have analgesic effects. Thus Kennedy's disease, Krabbé disease, Lewy body dementia, the compounds of the invention may be used to reduce pain in Machado-Joseph disease (Spinocerebellar ataxia type 3), a subject. The Subject may be suffering from an acute pain multiple Sclerosis, multiple system atrophy, narcolepsy, neu (e.g., selected from the group consisting of mechanical pain, roborreliosis, Parkinson's disease, Pelizaeus-Merzbacher heat pain, cold pain, ischemic pain, and chemical-induced disease, Pick's disease, primary lateral Sclerosis, prion dis pain). Other types of pain include peripheral or central neu eases, RefSum's disease, Schilder's disease (i.e., adrenoleu ropathic pain, inflammatory pain, migraine-related pain, kodystrophy), Schizophrenia, Spinocerebellar ataxia, spinal headache-related pain, irritable bowel syndrome-related muscularatrophy, Steele-Richardson, Olszewski disease, and pain, fibromyalgia-related pain, arthritic pain, skeletal pain, tabes dorsalis. 10 joint pain, gastrointestinal pain, muscle pain, angina pain, The compounds of the invention can also be used to treat facial pain, pelvic pain, claudication, postoperative pain, post diseases found in other organs or tissues. For example, Angio traumatic pain, tension-type headache, obstetric pain, gyne pep-7 (SEQ ID NO:112) is efficiently transported into liver, cological pain, or chemotherapy-induced pain. lung, kidney, spleen, and muscle cells, allowing for the pref There is evidence that neurotensin can be used to treat erential treatment of diseases associated with these tissues 15 metabolic disorders; see, e.g., U.S. Patent Application No. (e.g., hepatocellular carcinoma and lung cancer). The com 2001/0046956. Thus the compounds of the invention may be pounds of the presents invention may also be used to treat used to treat such disorders. The metabolic disorder may be genetic disorders, such as Down syndrome (i.e., trisomy 21), diabetes (e.g., Type I or Type II), obesity, diabetes as a con where down-regulation of particular gene transcripts may be sequence of obesity, hyperglycemia, dyslipidemia, hypertrig useful. lyceridemia, Syndrome X, insulin resistance, impaired glu Neurotensin-based Therapies cose tolerance (IGT), diabetic dyslipidemia, hyperlipidemia, The compounds of the invention can be used in any appro a cardiovascular disease, or hypertension. The Subject may be priate therapeutic application where the activity of neuro overweight, obese, or bulimic. tensin activity is beneficial. In brain, NT is associated with Neurotensin has also been suggested to be able to treat drug dopaminergic receptors and other neurotransmitter systems. 25 addiction or reduce drug abuse in Subjects, particularly with Peripheral NT acts as a paracrine and endocrine peptide on psychostimulant. Thus the compounds of the invention may both the digestive and cardiovascular systems. Various thera be useful in treating addiction to or abuse of drugs such as peutic applications have been suggested for neurotensin, amphetamine, methamphetamine, 3.4-methylene including psychiatric disorders, metabolic disorder, and pain. dioxymethamphetamine, nicotine, cocaine, methylpheni Because neurotensin has been shown to modulate neurotrans 30 date, and arecoline. mission in areas of the brain associated with Schizophrenia, GDNF/BDNF-based Therapy neurotensin and neurotensin receptoragonists have been pro GDNF and BDNF-based compounds may be used to treat posed as antipsychotic agents. any disease or condition where enhancing neuronal Survival Because polypeptides described herein are capable of (e.g., decreasing neuronal death rate) or increasing the rate of transporting an agent across the BBB, the compounds of the 35 neuronal formation is beneficial. Such conditions include invention are also useful for the treatment of neurological neurodegenerative disorders, e.g., a disorder selected from diseases such as neurodegenerative diseases or other condi the group consisting of a polyglutamine expansion disorder tions of the central nervous system (CNS), the peripheral (e.g., Huntington's disease (HD), dentatorubropallidoluysian nervous system, or the autonomous nervous system (e.g., atrophy, Kennedy's disease (also referred to as spinobulbar where neurons are lost or deteriorate). Neurotensin has been 40 muscular atrophy), and spinocerebellar ataxia (e.g., type 1, Suggested an antipsychotic therapy, and thus may be useful in type 2, type 3 (also referred to as Machado-Joseph disease), the treatment of diseases such as Schizophrenia and bipolar type 6, type 7, and type 17)), another trinucleotide repeat disorder. Many neurodegenerative diseases are characterized expansion disorder (e.g., fragile X syndrome, fragile XE by ataxia (i.e., uncoordinated muscle movements) and/or mental retardation, Friedreich's ataxia, myotonic dystrophy, memory loss. Neurodegenerative diseases include Alexander 45 spinocerebellar ataxia type 8, and spinocerebellar ataxia type disease, Alper disease, Alzheimer's disease, amyotrophic lat 12), Alexander disease, Alper's disease, Alzheimer's disease, eral Sclerosis (ALS, i.e., Lou Gehrig's disease), ataxiatelang amyotrophic lateral Sclerosis (ALS), ataxia telangiectasia, iectasia, Batten disease (Spielmeyer-Vogt-Sjogren-Batten Batten disease (also referred to as Spielmeyer-Vogt-Sjogren disease), bovine spongiform encephalopathy (BSE), Cana Batten disease), Canavan disease, Cockayne syndrome, cor Van disease, Cockayne syndrome, corticobasal degeneration, 50 ticobasal degeneration, Creutzfeldt-Jakob disease, ischemia Creutzfeldt-Jakob disease, Huntington's disease, HIV-asso stroke, Krabbe disease, Lewy body dementia, multiple scle ciated dementia, Kennedy’s disease, Krabbé disease, Lewy rosis, multiple system atrophy, Parkinson's disease, Peliza body dementia, Machado-Joseph disease (Spinocerebellar eus-Merzbacher disease, Pick's disease, primary lateral scle ataxia type 3), multiple Sclerosis, multiple system atrophy, rosis, Refsum’s disease, Sandhoff disease, Schilder's disease, narcolepsy, neuroborreliosis, Parkinson's disease, Pelizaeus 55 spinal cord injury, spinal muscular atrophy, Steele-Richard Merzbacher disease, Pick's disease, primary lateral sclerosis, son-Olszewski disease, and Tabes dorsalis. Other conditions prion diseases, Refsum’s disease, Schilder's disease (i.e., include injury (e.g., spinal cord injury), concussion, ischemic adrenoleukodystrophy), Schizophrenia, Spinocerebellar stroke, and hemorrhagic stroke. ataxia, spinal muscular atrophy, Steele-Richardson, Olsze Administration and Dosage wski disease, and tabes dorsalis. 60 The present invention also features pharmaceutical com The compounds of the invention may be used to reduce the positions that contain a therapeutically effective amount of a body temperature of a subject. Because reduction in body compound of the invention. The composition can be formu temperature has been shown to be beneficial in subjects who lated for use in a variety of drug delivery systems. One or may be suffering from, or may have recently Suffered from, a more physiologically acceptable excipients or carriers can stroke, cerebral ischemia, cardiac ischemia, or a nerve injury 65 also be included in the composition for proper formulation. Such as a spinal cord injury, Such a treatment would therefore Suitable formulations for use in the present invention are be useful in reducing complications of these conditions. found in Remington's Pharmaceutical Sciences, Mack Pub US 9, 161988 B2 65 66 lishing Company, Philadelphia, Pa., 17th ed., 1985. For a effective. A therapeutically effective amount of an agent or brief review of methods for drug delivery, see, e.g., Langer compound is not required to cure a disease or condition but (Science 249:1527-1533, 1990). will provide a treatment for a disease or condition such that The pharmaceutical compositions are intended for the onset of the disease or condition is delayed, hindered, or parenteral, intranasal, topical, oral, or local administration, 5 prevented, or the disease or condition symptoms are amelio Such as by a transdermal means, for prophylactic and/or rated, or the term of the disease or condition is changed or, for therapeutic treatment. The pharmaceutical compositions can example, is less severe or recovery is accelerated in an indi be administered parenterally (e.g., by intravenous, intramus vidual. cular, or Subcutaneous injection), or by oral ingestion, or by Amounts effective for this use may depend on the severity topical application or intraarticular injection at areas affected 10 of the disease or condition and the weight and general state of by the vascular or cancer condition. Additional routes of the Subject, but generally range from about 0.05ug to about administration include intravascular, intra-arterial, intratu 1000 ug (e.g., 0.5-100 lug) of an equivalent amount of the mor, intraperitoneal, intraventricular, intraepidural, as well as agent perdose per Subject. Suitable regimes for initial admin nasal, ophthalmic, intrascleral, intraorbital, rectal, topical, or istration and booster administrations are typified by an initial aerosol inhalation administration. Sustained release adminis 15 administration followed by repeated doses at one or more tration is also specifically included in the invention, by Such hourly, daily, weekly, or monthly intervals by a Subsequent means as depot injections or erodible implants or compo administration. The total effective amount of an agent present nents. Thus, the invention provides compositions for in the compositions of the invention can be administered to a parenteral administration that include the above mention mammal as a single dose, either as abolus or by infusion over agents dissolved or Suspended in an acceptable carrier, pref a relatively short period of time, or can be administered using erably an aqueous carrier, e.g., water, buffered water, Saline, a fractionated treatment protocol, in which multiple doses are PBS, and the like. The compositions may contain pharmaceu administered over a more prolonged period of time (e.g., a tically acceptable auxiliary Substances as required to approxi dose every 4-6, 8-12, 14-16, or 18-24 hours, or every 2-4 days, mate physiological conditions, such as pH adjusting and buff 1-2 weeks, once a month). Alternatively, continuous intrave ering agents, tonicity adjusting agents, wetting agents, 25 nous infusion sufficient to maintain therapeutically effective detergents and the like. The invention also provides compo concentrations in the blood are contemplated. sitions for oral delivery, which may contain inert ingredients The therapeutically effective amount of one or more agents such as binders or fillers for the formulation of a tablet, a present within the compositions of the invention and used in capsule, and the like. Furthermore, this invention provides the methods of this invention applied to mammals (e.g., compositions for local administration, which may contain 30 humans) can be determined by the ordinarily-skilled artisan inert ingredients such as solvents or emulsifiers for the for with consideration of individual differences in age, weight, mulation of a cream, an ointment, and the like. and the condition of the mammal. Because certain com These compositions may be sterilized by conventional ster pounds of the invention exhibit an enhanced ability to cross ilization techniques, or may be sterile filtered. The resulting the BBB, the dosage of the compounds of the invention can be aqueous Solutions may be packaged for use as is, or lyo 35 lower than (e.g., less than or equal to about 90%, 75%, 50%, philized, the lyophilized preparation being combined with a 40%, 30%, 20%, 15%, 12%, 10%, 8%, 7%, 6%. 5%, 4%, 3%, sterile aqueous carrier prior to administration. The pH of the 2%, 1%, 0.5%, or 0.1% of) the equivalent dose of required for preparations typically will be between 3 and 11, more pref atherapeutic effect of the unconjugatedagonist. The agents of erably between 5 and 9 or between 6 and 8, and most prefer the invention are administered to a Subject (e.g. a mammal, ably between 7 and 8, such as 7 to 7.5. The resulting compo 40 Such as a human) in an effective amount, which is an amount sitions in Solid form may be packaged in multiple single dose that produces a desirable result in a treated Subject (e.g., units, each containing a fixed amount of the above-mentioned preservation of neurons, new neuronal growth). Therapeuti agent or agents, such as in a sealed package of tablets or cally effective amounts can also be determined empirically by capsules. The composition in Solid form can also be packaged those of skill in the art. in a container for a flexible quantity, Such as in a squeezable 45 The Subject may also receive an agent in the range of about tube designed for a topically applicable cream or ointment. 0.05 to 1,000 ug equivalent dose as compared to unconju The compositions containing an effective amount can be gated agent per dose one or more times per week (e.g., 2, 3, 4, administered for prophylactic or therapeutic treatments. In 5, 6, or 7 or more times per week), 0.1 to 2,500 (e.g., 2,000, prophylactic applications, compositions can be administered 1,500, 1,000, 500, 100, 10, 1,0.5, or 0.1) ug dose per week. A to a subject with a clinically determined predisposition or 50 Subject may also receive an agent of the composition in the increased Susceptibility to a neurological or neurodegenera range of 0.1 to 3,000 ug per dose once every two or three tive disease. Compositions of the invention can be adminis weeks. tered to the Subject (e.g., a human) in an amount Sufficient to Single or multiple administrations of the compositions of delay, reduce, or preferably prevent the onset of clinical dis the invention including an effective amount can be carried out ease. In therapeutic applications, compositions are adminis 55 with dose levels and pattern being selected by the treating tered to a Subject (e.g., a human) already Suffering from physician. The dose and administration schedule can be disease (e.g., a neurological condition or neurodegenerative determined and adjusted based on the severity of the disease disease) in an amount Sufficient to cure or at least partially or condition in the Subject, which may be monitored through arrest the symptoms of the condition and its complications. out the course of treatment according to the methods com An amount adequate to accomplish this purpose is defined as 60 monly practiced by clinicians or those described herein. a “therapeutically effective amount, an amount of a com The compounds of the present invention may be used in pound Sufficient to Substantially improve some symptom combination with either conventional methods of treatment associated with a disease or a medical condition. For ortherapy or may be used separately from conventional meth example, in the treatment of a neurodegenerative disease ods of treatment or therapy. (e.g., those described herein), an agent or compound that 65 When the compounds of this invention are administered in decreases, prevents, delays, Suppresses, or arrests any symp combination therapies with other agents, they may be admin tom of the disease or condition would be therapeutically istered sequentially or concurrently to an individual. Alterna US 9, 161988 B2 67 tively, pharmaceutical compositions according to the present TABLE 5 invention may be comprised of a combination of a compound of the present invention in association with a pharmaceuti Purification cally acceptable excipient, as described herein, and another Column Flow Rate therapeutic or prophylactic agent known in the art. 5 Time (min) Volume (C.V.) (ml/min) % Solvent B O.OO O.OO 13.00 2O.O EXAMPLE1 S.12 1.52 13.00 2O.O 28.75 7.01 13.00 40.0 (over 23.63 min) Synthesis of Dimeric Angiopep-2. Using a TMEA 33.30 1.35 13.00 95.0 (over 4.6 min) Linker 10 38.00 1.39 13.00 95.0 1 CW = 43 ml The following scheme was used produce a dimeric form of Angiopep-2 joined by a TMEA linker. After coupling, ESI-TOF MS analysis showed the pres 15 ence of TMEA cross-linked monomer, dimer, and trimer in Solution. After evaporation of methanol and lyophilization, the dimer TMEA-(AN2) was obtained as a pure white solid O N (119 mg, 50%, purity).98%). The mass was checked by ESI O TOF MS (Bruker Daltonics). N In addition to the information in FIG. 1, the following N \ / / O AN2-Cys-NH22.4 equiv. apparatus was used in the analytic methods. A Waters Acquity N (2.4 equiv.) HPLC Column BEH phenyl, 1.7 m, 2.1 x50 mm was used. Urea 8M, pH 8.53 O O rt, 1 h Detection was performed at 229 nm. Solution A was 0.1% FA 50% 25 N in HO: Solution B was 0.1% FA in MeOH. A flow rate of 0.5 ml/min was used. The gradient settings are shown in Table 6 O 2 below.

TMEA 30 TABLE 6 Time Flow Method 1 Method 2

(min) (mL/min) Curve % A % B % A % B O.S 90 10 8O 2O ! 35 O.40 O.S 6 90 10 8O 2O O.70 O.S 6 70 30 50 50 O 2.20 O.S 6 30 70 15 85 O 2.40 O.S 6 10 90 5 95

2.70 O.S 6 10 90 5 95 2.80 O.S 6 90 10 8O 2O 40 2.81 O.S 6 90 10 8O 2O

Using ESI-TOF MS (Bruker Daltonics) the following m/z. values were calculated and identified: calculated 5193.62. TFA Salt 45 found 5193.68, m/z866.62 (+6), 1039.74 (+5), 1299.42 (+4), 1732.21 (+3). Purification was performed as follows, using a Waters Pre Dimer TMEA-(AN2) C232H328N66O68S2 pLC 4000 with a Phenyl OBD column (Waters X-Bridge) 5 MOI. Wt: 5193.62 50 um, 19x150 mm, 135A, Sample load: 282 mg, Urea 8M (27 ml), 20% MeOH in HO (2 ml), FA, Solution A was 0.1% FA in HO, Solution B was 0.1% FA in MeOHA flow rate of 13 ml/min was employed with a gradient: 20-40% B. Purifica C-terminally amidated Angiopep-2 with an additional tion of the crude was performed in 2 batches successively. C-terminal cysteine (Angiopep-2-Cys: SEQ ID NO:114) 55 (264.2 mg, 109.9 umol. 2.4 eq.) was dissolved in urea 8 M (18 Possible side reactions include hydrolysis of TMEA ml). This solution was added dropwise to a solution of TMEA (AN2) (55%, Mw =5211.63) might occur. Conjugate is then (tris-(2-maleimidoethyl)amine) (Pierce Biotechnology) stored under nitrogen atmosphere, below -20°C. (17.7 mg, 45.8 umol. 1 eq. in 9 ml of urea 8 M). Monitoring 60 of the reaction was done using the analytical methods 1 and 2 EXAMPLE 2 (which are described in chromatograms 1-2 of FIG. 1). The reaction (1.7 mM, pH 8.53) allowed to proceed at room tem Synthesis of Dimeric Angiopep-2 perature for 1 hour, and the mixture was purified by RP 65 HPLC chromatography (Waters PreplC4000; see chromato The following synthetic scheme was used to produce gram 3, Table 5). dimeric Angiopep-2 having an SATP linker. US 9, 161988 B2 69 70

1) HCTU/NMM Fmoc-Xaa He H-TFFYGGSRGKRNNFKTEEYC 2) 20% pip. SPPS

SEQID NO: 114)

O TFAH2O, EDTATES -e- Y S -noun 26% O O MPA-AN2 He DMSO, rt 30 min. S --nom 20% O O N SH SATP-AN2Cys-NH2 C12H5N3O33S2 Mol. Wit.: 2533.79 (H-TFFYGGSRGKRNNFKTEEYC-NH2: SEQID NO: 114 SATP

O

SATP-AN2Cys-AN2

(AN2: TFFYGGSRGKRNNFKTEEY: SEQID NO:97)

40 Angiopep-2-Cys-NH2 (H-TFFYGGSRGKRNNFK ing the free N-terminal amino peptide bound to the resin with TEEYC-NH; SEQID NO:114) was synthesized using solid a solution of SATP (N-succinimidyl S-acetylthiopropionate) phase (SPPS). G'S' is coupled using (Pierce Biotechnology) (24.5 mg, 100 umol. 1 eq. in 4 ml of pseudoproline dipeptide GS to optimize the synthesis. SPPS DMF, 25 mM) for one hour at room temperature. The modi was carried out on a Protein Technologies, Inc. Symphony(R) 45 fication with SATP solution was repeated once for 1 h 30. peptide synthesizer using Fmoc (9-fluorenylmethyloxycar Cleavage of the resin-bound product was carried out using bonyl)amino-terminus protection. Angiopep-2-Cys-NH TFA/water/EDT/TES (94/2.5/2.5/1) for two hours at room temperature. The crude modified peptide was precipitated (H-TFFYGGSRGKRNNFKTEECY-NH; SEQID NO:114) using ice-cold ether and purified by RP-HPLC chromatogra was synthesized on a 100-umol scale using a 5-fold excess of phy (Waters PreplC4000, See chromatograms 1-3 in FIGS. Fmoc-amino acids (200 mM) relative to the resin. Coupling 50 1 and 2, Table 9). was performed from a Rink amide MBHA resin (with Nle) Methanol was evaporated from the collected fractions and (0.40 mmol/g) for carboxyl-terminus amides using 1:1:2 lyophilized to give SATP-AN2Cys-NH2 as a pure white solid amino acid/activator/NMM in DMF with HCTU (2-(1H-6- (736 mg, 26%, purity-95%). The mass was confirmed by chlorobenzotriazol-1-yl)-1,1,3,3-tetramethyluronium ESI-TOF MS (Bruker Daltonics): calculated 2533.79. found hexafluorophosphate) and NMM (N-methylmorpholine). 55 2533.18, m/z 1267.59 (+2), 845.41 (+3). Deprotection was carried out using 20% piperidine/DMF. Dimeric SATP-AN2-AN2 was produced as follows. MPA Rink amide MBHA resin (with Nle) (0.40 mmol/g), Fmoc AN2, AN2 vector activated by BMPS(N-B-Maleimidopro amino acids and HCTU were purchased from ChemImpex, pyloxysuccinimide ester), (120 mg, 39.4 umol. 1 eq., and the pseudoproline dipeptide GS was purchased from 80.25% peptide content) was dissolved in DMSO (2 ml). This Novabiochem. Side protecting groups for amino acids were 60 solution was added to a solution of SATP-AN2-CysNH2 (100 trityl (Trt) for cysteine and aspargine, (tRu) for glutamic acid, mg, 39.4 umol. 1 eq. in 2.5 ml of DMSO). Monitoring of the tyrosine, serine, and threonine, pentamethyldihydrobenzofu reaction was done with the analytical methods 1 and 2 (See ran-5-sulfonyl (Pbf) for arginine, and tButyloxycarbonyl chromatograms 4-5 in FIG. 3). The reaction (8.8 mM) (tBoc) for lysine. allowed to proceed at room temperature for 30 minutes and The SATP-AN2Cys-NH was generated as follows. After 65 filtered. The mixture was purified by RP-HPLC chromatog deprotection of the last threonine residue, N-terminal raphy (Waters PreplC 4000, See chromatograms 4-6 in S-acetylthiopropionic acyl groups were introduced by treat FIGS. 3 and 4 and Table 9). US 9, 161988 B2 71 72 After evaporation of methanol and lyophilization, the TABLE 10 dimer SATP-AN2-AN2 was obtained as a pure white solid (39 mg, 20%, purity).98%). The mass was confirmed by Purification of SATP-AN2-AN2 ESI-TOF MS (Bruker Daltonics): calculated 4986.39. found Column Flow Rate 4986.42, m/z 1247.60 (+4),998.08 (+5), 832.07 (+6), 713.35 5 (+7). Time (min) Volume (C.V.) (ml/min) % Solvent B Analytical methods 1 and 2 were performed as follows. O.OO O.OO 13.00 3S.O Both methods used a Waters Acquity HPLC system with a S.12 1.51 13.00 3S.O 28.75 6.98 13.00 50.0 (over 23.63 min) Waters Acquity HPLC BEH phenyl column (1.7 um, 2.1x50 33.30 1.34 13.00 95.0 (over 4.55 min) mm). Detection was performed at 229 nm. Solution A was 40.00 1.98 13.00 95.0 0.1% FA in HO: Solution B was 0.1% FA in MeOH, with a 10 flow rate of 0.5 ml/min. Flow gradients are shown in the 1. C.W. = 44 ml Tables 7 and 8 below for each method. The conjugate was stored under nitrogen atmosphere, TABLE 7 below -20° C. 15 Method 1 EXAMPLE 3

Time Flow (min) (mL/min) % A % B Curve Synthesis of an Angiopep-1 Dimer Using a Disulfide Bond O.S 90 10 O4O O.S 90 10 6 O.70 O.S 70 30 6 An Angiopep-1 dimer was prepared by incubating the 2.2O O.S 30 70 6 Angiopep-1 peptide (SEQID NO:67) at 37° C. for 2 hours in 240 O.S 10 90 6 2.70 O.S 10 90 6 phosphate buffered saline (PBS) at pH 8.5. This reaction 2.8O O.S 90 10 6 25 resulted in formation of Angiopep-1 dimers joined by a dis 2.81 O.S 90 10 6 ulfide bond through the cysteine amino acid on each protein (FIG. 5). Volume of brain parenchymal distribution was measured TABLE 8 using the in situ brain perfusion assay (FIG. 6). Great uptake 30 Volumes using the Angiopep-1 dimer were observed, espe Method 2: cially at lower concentrations, as compared to monomeric Time Flow Angiopep-2. Parenchymal uptake of the Angiopep-1 dimer (min) (mL/min) % A % B Curve and Angiopep-2 was also measured at various concentrations in situ (FIG. 7). Here, the Angiopep-1 dimer, especially at O.S 8O 2O 35 O4O O.S 8O 2O 6 lower concentrations (<500 nmol), exhibited higher transport 3.00 O.S 60 40 6 than the Angiopep-2 monomer. 3.30 O.S 5 95 6 3.80 O.S 5 95 6 EXAMPLE 4 4.OO O.S 8O 2O 6 4.20 O.S 8O 2O 6 4.21 O.S 8O 2O 6 40 Transport of Angiopep-2 Dimers and Trimers Purification of SATP-AN2Cys-NH, was performed as fol Transport of Angiopep-2 (synthetic) as well as Angiopep-2 lows using a Waters PrepDC 4000 with a Kromasil column (recombinant) was compared to transport of Angiopep-2 (C18, 10 m, 50x250 mm, 100 A). Solution A was 0.1% FA dimers and Angiopep-2 trimers (e.g., prepared as described in HO: Solution B was 0.1% FA in MeOH, with a flow rate 45 above) at 50 nM. Transport of recombinant Angiopep-2 into of 48 ml/min and gradient of 20-45% B. Purification results diet-induced obese mice (DIO) was also tested (FIG. 8). are shown in Table 9. Transport of the dimers and trimers into brain was observed.

TABLE 9 EXAMPLE 5 50 Purification of SATP-AN2-CYS-NH2 Generation of an Exendin-4-Angioep-2 Dimer Conjugate Column Flow Rate Time (min) Volume (C.V.) (ml/min) % Solvent B Using the conjugation chemistry described herein or simi O.OO O.OO 48.19 2O.O 55 lar chemistry, an Exendin-4-Angiopep-2 dimer was gener 18.08 1980 48.19 2O.O 26.91 9.67 48.19 35.0 (over 5.46 min) ated having the structure shown in FIG.9A. Briefly, the amine 109.26 90.19 48.19 45.0 (over 23.61 min) group in the C-terminal lysine of Lys Exendin-4 was con 130.00 22.72 48.19 95.0 (over 5.00 min) jugated to an Angiopep-2 dimer throughan MHA linker at the 154.OO 26.29 48.19 95.0 N-terminal threonine of the first Angiopep-2 peptide. A 60 N-succinimidyl-5-acetylthiopropionate (SATP) linker was 1 CW = 44 mil attached to an Angiopep-2-CyS peptide at its N-terminus. Purification of SATP-AN2-AN2 was performed as follows Through this cysteine, the Angiopep-2-CyS peptide was con using a Waters PrepLC 4000 with a BEH phenyl column (5 jugated to a second Angiopep-2 peptide, which had been um, 19x150 mm, 135A). Solution A was 0.1% FA in HO: modified to containan MPA linker. The dimer was then linked Solution B was 0.1% FA in MeOH with a flow rate of 13 65 to the Lys Exendin-4 through an MHA linker. A control ml/min and a gradient of 35-50% B. Purification results are molecule (EXen-S4) was also generated using a scrambed shown in Table 10. form of Angiopep-2 conjugated at its N-terminal to the cys US 9, 161988 B2 73 74 teine of Cys'Exendin-4 through an MHA linker (FIG.9B). BBB as compared to either the unconjugated exendin-4 or to These conjugates were prepared as trifluoroacetate (TFA) the exendin-4 conjugated to a single Angiopep-2 (FIG. 10). salts. We also tested the ability of the exendin-4-Angiopep-2 dimer conjugate to reduce glycemia in DIO mice. Mice were EXAMPLE 6 5 injected with a bolus containing a control, exendin-4, or the exendin-4-Angiopep-2 dimer conjugate. Mice receiving Characterization of an Exendin-4-Angiopep-2 Dimer either exendin-4 or the conjugate exhibited reduced glycemia Conjugate as compared to mice receiving the control (FIG. 11). Brain uptake of the exemplary GLP-1 agonist, exendin-4, 10 Other Embodiments was measured in situ when unconjugated, conjugated to a single Angiopep-2 using variable linker lengths, conjugated All patents, patent applications including U.S. Provisional to a scrambled Angiopep-2 (S4), or conjugated to a dimeric Application Nos. 61/222,785, filed Jul. 2, 2009, and 61/252, form of Angiopep-2. The experiments were performed as 024, filed Oct. 15, 2009, and publications mentioned in this described in Example 2 above. 15 specification are herein incorporated by reference to the same From these results, we observed that conjugation of the extent as if each independent patent, patent application, or exendin-4 analog to the dimeric form of Angiopep-2 results in publication was specifically and individually indicated to be a conjugate with a Surprisingly greater ability to cross the incorporated by reference.

SEQUENCE LISTING

<16 Os NUMBER OF SEO ID NOS: 2O3

SEO ID NO 1 LENGTH 19 TYPE PRT ORGANISM: Artificial Sequence FEATURE; OTHER INFORMATION: Description of Artificial Sequence: Synthetic peptide

<4 OOs SEQUENCE: 1 Thr Phe Val Tyr Gly Gly Cys Arg Ala Lys Arg Asn. Asn. Phe Llys Ser 1. 5 1O 15 Ala Glu Asp

SEO ID NO 2 LENGTH 19 TYPE PRT ORGANISM: Artificial Sequence FEATURE; OTHER INFORMATION: Description of Artificial Sequence: Synthetic peptide

<4 OOs SEQUENCE: 2 Thr Phe Glin Tyr Gly Gly Cys Met Gly Asn Gly Asn Asn Phe Val Thr 1. 5 1O 15 Glu Lys Glu

SEO ID NO 3 LENGTH 19 TYPE PRT ORGANISM: Artificial Sequence FEATURE; OTHER INFORMATION: Description of Artificial Sequence: Synthetic peptide

<4 OOs SEQUENCE: 3 Pro Phe Phe Tyr Gly Gly Cys Gly Gly Asn Arg Asn Asn Phe Asp Thr 1. 5 1O 15 Glu Glu Tyr

SEO ID NO 4 LENGTH 19 TYPE PRT ORGANISM: Artificial Sequence FEATURE; US 9, 161988 B2 75 76 - Continued <223> OTHER INFORMATION: Description of Artificial Sequence: Synthetic peptide

<4 OOs, SEQUENCE: 4 Ser Phe Tyr Tyr Gly Gly Cys Lieu. Gly Asn Lys Asn. Asn Tyr Lieu. Arg 1. 5 1O 15

Glu Glu Glu

<210s, SEQ ID NO 5 &211s LENGTH: 19 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Description of Artificial Sequence: Synthetic peptide

<4 OOs, SEQUENCE: 5 Thir Phe Phe Tyr Gly Gly Cys Arg Ala Lys Arg Asn. Asn. Phe Lys Arg 1. 5 1O 15 Ala Lys Tyr

<210s, SEQ ID NO 6 &211s LENGTH: 19 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Description of Artificial Sequence: Synthetic peptide

<4 OOs, SEQUENCE: 6 Thir Phe Phe Tyr Gly Gly Cys Arg Gly Lys Arg Asn. Asn. Phe Lys Arg 1. 5 1O 15 Ala Lys Tyr

<210s, SEQ ID NO 7 &211s LENGTH: 19 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Description of Artificial Sequence: Synthetic peptide

<4 OO > SEQUENCE: 7 Thir Phe Phe Tyr Gly Gly Cys Arg Ala Lys Lys Asn. Asn Tyr Lys Arg 1. 5 1O 15 Ala Lys Tyr

<210s, SEQ ID NO 8 &211s LENGTH: 19 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Description of Artificial Sequence: Synthetic peptide

<4 OOs, SEQUENCE: 8 Thir Phe Phe Tyr Gly Gly Cys Arg Gly Lys Lys Asn. Asn. Phe Lys Arg 1. 5 1O 15

Ala Lys Tyr

<210s, SEQ ID NO 9 &211s LENGTH: 19 212. TYPE: PRT <213> ORGANISM: Artificial Sequence US 9, 161988 B2 77 78 - Continued

22 Os. FEATURE: <223> OTHER INFORMATION: Description of Artificial Sequence: Synthetic peptide

<4 OOs, SEQUENCE: 9 Thir Phe Glin Tyr Gly Gly Cys Arg Ala Lys Arg Asn. Asn. Phe Lys Arg 1. 5 1O 15 Ala Lys Tyr

<210s, SEQ ID NO 10 &211s LENGTH: 19 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Description of Artificial Sequence: Synthetic peptide

<4 OOs, SEQUENCE: 10 Thir Phe Glin Tyr Gly Gly Cys Arg Gly Lys Lys Asn. Asn. Phe Lys Arg 1. 5 1O 15 Ala Lys Tyr

<210s, SEQ ID NO 11 &211s LENGTH: 19 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Description of Artificial Sequence: Synthetic peptide

< 4 OO SEQUENCE: 11 Thir Phe Phe Tyr Gly Gly Cys Lieu. Gly Lys Arg Asn. Asn. Phe Lys Arg 1. 5 1O 15 Ala Lys Tyr

<210s, SEQ ID NO 12 &211s LENGTH: 19 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Description of Artificial Sequence: Synthetic peptide

<4 OOs, SEQUENCE: 12 Thir Phe Phe Tyr Gly Gly Ser Lieu. Gly Lys Arg Asn. Asn. Phe Lys Arg 1. 5 1O 15 Ala Lys Tyr

<210s, SEQ ID NO 13 &211s LENGTH: 19 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Description of Artificial Sequence: Synthetic peptide

<4 OOs, SEQUENCE: 13 Pro Phe Phe Tyr Gly Gly Cys Gly Gly Lys Lys Asn. Asn. Phe Lys Arg 1. 5 1O 15 Ala Lys Tyr

<210s, SEQ ID NO 14 &211s LENGTH: 19 212. TYPE: PRT US 9, 161988 B2 79 80 - Continued <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Description of Artificial Sequence: Synthetic peptide

<4 OOs, SEQUENCE: 14 Thir Phe Phe Tyr Gly Gly Cys Arg Gly Lys Gly Asn. Asn Tyr Lys Arg 1. 5 1O 15 Ala Lys Tyr

<210s, SEQ ID NO 15 &211s LENGTH: 19 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Description of Artificial Sequence: Synthetic peptide

<4 OOs, SEQUENCE: 15 Pro Phe Phe Tyr Gly Gly Cys Arg Gly Lys Arg Asn. Asn. Phe Lieu. Arg 1. 5 1O 15 Ala Lys Tyr

<210s, SEQ ID NO 16 &211s LENGTH: 19 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Description of Artificial Sequence: Synthetic peptide

<4 OOs, SEQUENCE: 16 Thir Phe Phe Tyr Gly Gly Cys Arg Gly Lys Arg Asn. Asn. Phe Lys Arg 1. 5 1O 15 Glu Lys Tyr

<210s, SEQ ID NO 17 &211s LENGTH: 19 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Description of Artificial Sequence: Synthetic peptide

<4 OOs, SEQUENCE: 17 Pro Phe Phe Tyr Gly Gly Cys Arg Ala Lys Lys Asn. Asn. Phe Lys Arg 1. 5 1O 15 Ala Lys Glu

<210s, SEQ ID NO 18 &211s LENGTH: 19 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Description of Artificial Sequence: Synthetic peptide

<4 OOs, SEQUENCE: 18 Thir Phe Phe Tyr Gly Gly Cys Arg Gly Lys Arg Asn. Asn. Phe Lys Arg 1. 5 1O 15

Ala Lys Asp

<210s, SEQ ID NO 19 &211s LENGTH: 19 US 9, 161988 B2 81 82 - Continued

212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Description of Artificial Sequence: Synthetic peptide

<4 OOs, SEQUENCE: 19 Thir Phe Phe Tyr Gly Gly Cys Arg Ala Lys Arg Asn. Asn. Phe Asp Arg 1. 5 1O 15 Ala Lys Tyr

<210s, SEQ ID NO 2 O &211s LENGTH: 19 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Description of Artificial Sequence: Synthetic peptide

<4 OOs, SEQUENCE: 2O Thir Phe Phe Tyr Gly Gly Cys Arg Gly Lys Lys Asn. Asn. Phe Lys Arg 1. 5 1O 15 Ala Glu Tyr

<210s, SEQ ID NO 21 &211s LENGTH: 19 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Description of Artificial Sequence: Synthetic peptide

<4 OOs, SEQUENCE: 21 Pro Phe Phe Tyr Gly Gly Cys Gly Ala Asn Arg Asn. Asn. Phe Lys Arg 1. 5 1O 15 Ala Lys Tyr

<210s, SEQ ID NO 22 &211s LENGTH: 19 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Description of Artificial Sequence: Synthetic peptide

<4 OOs, SEQUENCE: 22 Thr Phe Phe Tyr Gly Gly Cys Gly Gly Lys Lys Asn Asn Phe Llys Thr 1. 5 1O 15 Ala Lys Tyr

<210s, SEQ ID NO 23 &211s LENGTH: 19 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Description of Artificial Sequence: Synthetic peptide

<4 OOs, SEQUENCE: 23 Thir Phe Phe Tyr Gly Gly Cys Arg Gly Asn Arg Asn. Asn. Phe Lieu. Arg 1. 5 1O 15

Ala Lys Tyr

<210s, SEQ ID NO 24 US 9, 161988 B2 83 84 - Continued

&211s LENGTH: 19 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Description of Artificial Sequence: Synthetic peptide

<4 OOs, SEQUENCE: 24 Thr Phe Phe Tyr Gly Gly Cys Arg Gly Asn Arg Asn Asn Phe Llys Thr 1. 5 1O 15 Ala Lys Tyr

<210s, SEQ ID NO 25 &211s LENGTH: 19 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Description of Artificial Sequence: Synthetic peptide

<4 OOs, SEQUENCE: 25 Thr Phe Phe Tyr Gly Gly Ser Arg Gly Asn Arg Asn Asn Phe Llys Thr 1. 5 1O 15 Ala Lys Tyr

<210s, SEQ ID NO 26 &211s LENGTH: 19 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Description of Artificial Sequence: Synthetic peptide

<4 OOs, SEQUENCE: 26 Thr Phe Phe Tyr Gly Gly Cys Lieu. Gly Asin Gly Asn Asn Phe Lys Arg 1. 5 1O 15 Ala Lys Tyr

<210s, SEQ ID NO 27 &211s LENGTH: 19 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Description of Artificial Sequence: Synthetic peptide

<4 OOs, SEQUENCE: 27 Thir Phe Phe Tyr Gly Gly Cys Lieu. Gly Asn Arg Asn. Asn. Phe Lieu. Arg 1. 5 1O 15 Ala Lys Tyr

<210s, SEQ ID NO 28 &211s LENGTH: 19 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Description of Artificial Sequence: Synthetic peptide

<4 OOs, SEQUENCE: 28 Thr Phe Phe Tyr Gly Gly Cys Lieu. Gly Asn Arg Asn Asn Phe Llys Thr 1. 5 1O 15 Ala Lys Tyr US 9, 161988 B2 85 86 - Continued

<210s, SEQ ID NO 29 &211s LENGTH: 19 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Description of Artificial Sequence: Synthetic peptide

<4 OOs, SEQUENCE: 29 Thr Phe Phe Tyr Gly Gly Cys Arg Gly Asin Gly Asn Asn Phe Llys Ser 1. 5 1O 15 Ala Lys Tyr

<210s, SEQ ID NO 3 O &211s LENGTH: 19 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Description of Artificial Sequence: Synthetic peptide

<4 OOs, SEQUENCE: 30 Thir Phe Phe Tyr Gly Gly Cys Arg Gly Lys Lys Asn. Asn. Phe Asp Arg 1. 5 1O 15 Glu Lys Tyr

<210s, SEQ ID NO 31 &211s LENGTH: 19 212. TYPE: PRT <213> ORGANISM: Artificial Sequence & 22 O FEATURE; <223> OTHER INFORMATION: Description of Artificial Sequence: Synthetic peptide

<4 OOs, SEQUENCE: 31 Thir Phe Phe Tyr Gly Gly Cys Arg Gly Lys Arg Asn. Asn. Phe Lieu. Arg 1. 5 1O 15 Glu Lys Glu

<210s, SEQ ID NO 32 &211s LENGTH: 19 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Description of Artificial Sequence: Synthetic peptide

<4 OOs, SEQUENCE: 32 Thir Phe Phe Tyr Gly Gly Cys Arg Gly Lys Gly Asn. Asn. Phe Asp Arg 1. 5 1O 15 Ala Lys Tyr

<210s, SEQ ID NO 33 &211s LENGTH: 19 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Description of Artificial Sequence: Synthetic peptide

<4 OOs, SEQUENCE: 33 Thir Phe Phe Tyr Gly Gly Ser Arg Gly Lys Gly Asn. Asn. Phe Asp Arg 1. 5 1O 15

Ala Lys Tyr US 9, 161988 B2 87 88 - Continued

<210s, SEQ ID NO 34 &211s LENGTH: 19 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Description of Artificial Sequence: Synthetic peptide

<4 OOs, SEQUENCE: 34 Thr Phe Phe Tyr Gly Gly Cys Arg Gly Asin Gly Asn Asn Phe Val Thr 1. 5 1O 15 Ala Lys Tyr

<210s, SEQ ID NO 35 &211s LENGTH: 19 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Description of Artificial Sequence: Synthetic peptide

<4 OOs, SEQUENCE: 35 Pro Phe Phe Tyr Gly Gly Cys Gly Gly Lys Gly Asn Asn Tyr Val Thr 1. 5 1O 15 Ala Lys Tyr

<210s, SEQ ID NO 36 &211s LENGTH: 19 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Description of Artificial Sequence: Synthetic peptide

<4 OOs, SEQUENCE: 36 Thr Phe Phe Tyr Gly Gly Cys Lieu. Gly Lys Gly Asn Asn Phe Lieu. Thr 1. 5 1O 15 Ala Lys Tyr

<210s, SEQ ID NO 37 &211s LENGTH: 19 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Description of Artificial Sequence: Synthetic peptide

<4 OO > SEQUENCE: 37 Ser Phe Phe Tyr Gly Gly Cys Lieu. Gly Asn Lys Asn Asn Phe Lieu. Thr 1. 5 1O 15 Ala Lys Tyr

<210s, SEQ ID NO 38 &211s LENGTH: 19 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Description of Artificial Sequence: Synthetic peptide

<4 OOs, SEQUENCE: 38 Thr Phe Phe Tyr Gly Gly Cys Gly Gly Asn Lys Asn Asn Phe Val Arg 1. 5 1O 15

Glu Lys Tyr US 9, 161988 B2 89 90 - Continued

<210s, SEQ ID NO 39 &211s LENGTH: 19 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Description of Artificial Sequence: Synthetic peptide

<4 OOs, SEQUENCE: 39 Thr Phe Phe Tyr Gly Gly Cys Met Gly Asn Lys Asn Asn Phe Val Arg 1. 5 1O 15 Glu Lys Tyr

<210s, SEQ ID NO 4 O &211s LENGTH: 19 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Description of Artificial Sequence: Synthetic peptide

<4 OOs, SEQUENCE: 4 O Thr Phe Phe Tyr Gly Gly Ser Met Gly Asn Lys Asn Asn Phe Val Arg 1. 5 1O 15 Glu Lys Tyr

<210s, SEQ ID NO 41 &211s LENGTH: 19 212. TYPE PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Description of Artificial Sequence: Synthetic peptide

<4 OOs, SEQUENCE: 41 Pro Phe Phe Tyr Gly Gly Cys Lieu. Gly Asn Arg Asn. Asn Tyr Val Arg 1. 5 1O 15 Glu Lys Tyr

<210s, SEQ ID NO 42 &211s LENGTH: 19 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Description of Artificial Sequence: Synthetic peptide

<4 OOs, SEQUENCE: 42 Thr Phe Phe Tyr Gly Gly Cys Lieu. Gly Asn Arg Asn Asn Phe Val Arg 1. 5 1O 15 Glu Lys Tyr

<210s, SEQ ID NO 43 &211s LENGTH: 19 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Description of Artificial Sequence: Synthetic peptide

<4 OOs, SEQUENCE: 43 Thr Phe Phe Tyr Gly Gly Cys Lieu. Gly Asn Lys Asn Asn Tyr Val Arg 1. 5 1O 15 US 9, 161988 B2 91 92 - Continued Glu Lys Tyr

<210s, SEQ ID NO 44 &211s LENGTH: 19 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Description of Artificial Sequence: Synthetic peptide

<4 OOs, SEQUENCE: 44 Thr Phe Phe Tyr Gly Gly Cys Gly Gly Asin Gly Asn Asn Phe Lieu. Thr 1. 5 1O 15 Ala Lys Tyr

<210s, SEQ ID NO 45 &211s LENGTH: 19 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Description of Artificial Sequence: Synthetic peptide

<4 OOs, SEQUENCE: 45 Thr Phe Phe Tyr Gly Gly Cys Arg Gly Asn Arg Asn Asn Phe Lieu. Thr 1. 5 1O 15 Ala Glu Tyr

<210s, SEQ ID NO 46 & 211 LENGTH 19 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Description of Artificial Sequence: Synthetic peptide

<4 OOs, SEQUENCE: 46 Thr Phe Phe Tyr Gly Gly Cys Arg Gly Asin Gly Asn Asn Phe Llys Ser 1. 5 1O 15 Ala Glu Tyr

<210s, SEQ ID NO 47 &211s LENGTH: 19 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Description of Artificial Sequence: Synthetic peptide

<4 OOs, SEQUENCE: 47 Pro Phe Phe Tyr Gly Gly Cys Lieu. Gly Asn Lys Asn Asn Phe Llys Thr 1. 5 1O 15 Ala Glu Tyr

<210s, SEQ ID NO 48 &211s LENGTH: 19 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Description of Artificial Sequence: Synthetic peptide

<4 OOs, SEQUENCE: 48 Thr Phe Phe Tyr Gly Gly Cys Arg Gly Asn Arg Asn Asn Phe Llys Thr 1. 5 1O 15 US 9, 161988 B2 93 94 - Continued

Glu Glu Tyr

<210s, SEQ ID NO 49 &211s LENGTH: 19 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Description of Artificial Sequence: Synthetic peptide

<4 OOs, SEQUENCE: 49 Thr Phe Phe Tyr Gly Gly Cys Arg Gly Lys Arg Asn Asn Phe Llys Thr 1. 5 1O 15 Glu Glu Asp

<210s, SEQ ID NO 50 &211s LENGTH: 19 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Description of Artificial Sequence: Synthetic peptide

<4 OOs, SEQUENCE: 50 Pro Phe Phe Tyr Gly Gly Cys Gly Gly Asin Gly Asn Asn Phe Val Arg 1. 5 1O 15 Glu Lys Tyr

<210 SEQ ID NO 51 &211s LENGTH: 19 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Description of Artificial Sequence: Synthetic peptide

<4 OOs, SEQUENCE: 51 Ser Phe Phe Tyr Gly Gly Cys Met Gly Asin Gly Asn Asn Phe Val Arg 1. 5 1O 15 Glu Lys Tyr

<210s, SEQ ID NO 52 &211s LENGTH: 19 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Description of Artificial Sequence: Synthetic peptide

<4 OOs, SEQUENCE: 52 Pro Phe Phe Tyr Gly Gly Cys Gly Gly Asin Gly Asn Asn Phe Leu Arg 1. 5 1O 15 Glu Lys Tyr

<210s, SEQ ID NO 53 &211s LENGTH: 19 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Description of Artificial Sequence: Synthetic peptide

<4 OOs, SEQUENCE: 53 Thr Phe Phe Tyr Gly Gly Cys Lieu. Gly Asin Gly Asn Asn Phe Val Arg US 9, 161988 B2 95 96 - Continued

1. 5 1O 15 Glu Lys Tyr

<210s, SEQ ID NO 54 &211s LENGTH: 19 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Description of Artificial Sequence: Synthetic peptide

<4 OOs, SEQUENCE: 54 Ser Phe Phe Tyr Gly Gly Cys Lieu. Gly Asn Gly Asn. Asn Tyr Lieu. Arg 1. 5 1O 15 Glu Lys Tyr

<210s, SEQ ID NO 55 &211s LENGTH: 19 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Description of Artificial Sequence: Synthetic peptide

<4 OO > SEQUENCE: 55 Thr Phe Phe Tyr Gly Gly Ser Leu Gly Asin Gly Asn Asn Phe Val Arg 1. 5 1O 15 Glu Lys Tyr

<210s, SEQ ID NO 56 &211s LENGTH: 19 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Description of Artificial Sequence: Synthetic peptide

<4 OOs, SEQUENCE: 56 Thr Phe Phe Tyr Gly Gly Cys Arg Gly Asin Gly Asn Asn Phe Val Thr 1. 5 1O 15 Ala Glu Tyr

<210s, SEQ ID NO 57 &211s LENGTH: 19 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Description of Artificial Sequence: Synthetic peptide

<4 OO > SEQUENCE: 57 Thr Phe Phe Tyr Gly Gly Cys Lieu. Gly Lys Gly Asn Asn Phe Val Ser 1. 5 1O 15

Ala Glu Tyr

<210s, SEQ ID NO 58 &211s LENGTH: 19 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Description of Artificial Sequence: Synthetic peptide

<4 OOs, SEQUENCE: 58 US 9, 161988 B2 97 98 - Continued Thir Phe Phe Tyr Gly Gly Cys Lieu. Gly Asn Arg Asn. Asn. Phe Asp Arg 1. 5 1O 15 Ala Glu Tyr

<210s, SEQ ID NO 59 &211s LENGTH: 19 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Description of Artificial Sequence: Synthetic peptide

<4 OO > SEQUENCE: 59 Thir Phe Phe Tyr Gly Gly Cys Lieu. Gly Asn Arg Asn. Asn. Phe Lieu. Arg 1. 5 1O 15 Glu Glu Tyr

<210s, SEQ ID NO 60 &211s LENGTH: 19 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Description of Artificial Sequence: Synthetic peptide

<4 OOs, SEQUENCE: 60 Thir Phe Phe Tyr Gly Gly Cys Lieu. Gly Asn Lys Asn. Asn Tyr Lieu. Arg 1. 5 1O 15 Glu Glu Tyr

<210s, SEQ ID NO 61 &211s LENGTH: 19 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Description of Artificial Sequence: Synthetic peptide

<4 OOs, SEQUENCE: 61 Pro Phe Phe Tyr Gly Gly Cys Gly Gly Asn Arg Asn. Asn Tyr Lieu. Arg 1. 5 1O 15 Glu Glu Tyr

<210s, SEQ ID NO 62 &211s LENGTH: 19 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Description of Artificial Sequence: Synthetic peptide

<4 OOs, SEQUENCE: 62 Pro Phe Phe Tyr Gly Gly Ser Gly Gly Asn Arg Asn Asn Tyr Lieu. Arg 1. 5 1O 15

Glu Glu Tyr

<210s, SEQ ID NO 63 &211s LENGTH: 19 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Description of Artificial Sequence: Synthetic peptide

<4 OOs, SEQUENCE: 63 US 9, 161988 B2 99 100 - Continued

Met Arg Pro Asp Phe Cys Lieu. Glu Pro Pro Tyr Thr Gly Pro Cys Val 1. 5 1O 15 Ala Arg Ile

<210s, SEQ ID NO 64 &211s LENGTH: 21 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Description of Artificial Sequence: Synthetic peptide

<4 OOs, SEQUENCE: 64 Ala Arg Ile Ile Arg Tyr Phe Tyr Asn Ala Lys Ala Gly Lieu. Cys Glin 1. 5 1O 15 Thr Phe Val Tyr Gly 2O

<210s, SEQ ID NO 65 &211s LENGTH: 22 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Description of Artificial Sequence: Synthetic peptide

<4 OOs, SEQUENCE: 65 Tyr Gly Gly Cys Arg Ala Lys Arg Asn. Asn Tyr Lys Ser Ala Glu Asp 1. 5 1O 15 Cys Met Arg Thr Cys Gly 2O

<210s, SEQ ID NO 66 &211s LENGTH: 22 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Description of Artificial Sequence: Synthetic peptide

<4 OOs, SEQUENCE: 66 Pro Asp Phe Cys Lieu. Glu Pro Pro Tyr Thr Gly Pro Cys Val Ala Arg 1. 5 1O 15 Ile Ile Arg Tyr Phe Tyr 2O

<210s, SEQ ID NO 67 &211s LENGTH: 19 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Description of Artificial Sequence: Synthetic peptide

<4 OO > SEQUENCE: 67 Thr Phe Phe Tyr Gly Gly Cys Arg Gly Lys Arg Asn Asn Phe Llys Thr 1. 5 1O 15

Glu Glu Tyr

<210s, SEQ ID NO 68 &211s LENGTH: 19 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: US 9, 161988 B2 101 102 - Continued <223> OTHER INFORMATION: Description of Artificial Sequence: Synthetic peptide

<4 OOs, SEQUENCE: 68 Llys Phe Phe Tyr Gly Gly Cys Arg Gly Lys Arg Asn. Asn. Phe Llys Thr 1. 5 1O 15 Glu Glu Tyr

<210s, SEQ ID NO 69 &211s LENGTH: 19 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Description of Artificial Sequence: Synthetic peptide

<4 OOs, SEQUENCE: 69 Thr Phe Tyr Tyr Gly Gly Cys Arg Gly Lys Arg Asn Asn Tyr Lys Thr 1. 5 1O 15 Glu Glu Tyr

<210s, SEQ ID NO 70 &211s LENGTH: 19 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Description of Artificial Sequence: Synthetic peptide

<4 OO > SEQUENCE: 7 O Thr Phe Phe Tyr Gly Gly Ser Arg Gly Lys Arg Asn Asn Phe Llys Thr 1. 5 1O 15 Glu Glu Tyr

<210s, SEQ ID NO 71 &211s LENGTH: 2O 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Description of Artificial Sequence: Synthetic peptide

<4 OOs, SEQUENCE: 71 Cys Thr Phe Phe Tyr Gly Cys Cys Arg Gly Lys Arg Asn Asin Phe Lys 1. 5 1O 15 Thr Glu Glu Tyr 2O

<210s, SEQ ID NO 72 &211s LENGTH: 2O 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Description of Artificial Sequence: Synthetic peptide

<4 OOs, SEQUENCE: 72 Thr Phe Phe Tyr Gly Gly Cys Arg Gly Lys Arg Asn Asn Phe Llys Thr 1. 5 1O 15

Glu Glu Tyr Cys 2O

<210s, SEQ ID NO 73 &211s LENGTH: 2O US 9, 161988 B2 103 104 - Continued

212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Description of Artificial Sequence: Synthetic peptide

<4 OO > SEQUENCE: 73 Cys Thr Phe Phe Tyr Gly Ser Cys Arg Gly Lys Arg Asn Asin Phe Lys 1. 5 1O 15 Thr Glu Glu Tyr 2O

<210s, SEQ ID NO 74 &211s LENGTH: 2O 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Description of Artificial Sequence: Synthetic peptide

<4 OOs, SEQUENCE: 74 Thr Phe Phe Tyr Gly Gly Ser Arg Gly Lys Arg Asn Asn Phe Llys Thr 1. 5 1O 15 Glu Glu Tyr Cys 2O

<210s, SEQ ID NO 75 &211s LENGTH: 19 212. TYPE: PRT <213> ORGANISM: Artificial Sequence & 22 O FEATURE; <223> OTHER INFORMATION: Description of Artificial Sequence: Synthetic peptide

<4 OO > SEQUENCE: 75 Pro Phe Phe Tyr Gly Gly Cys Arg Gly Lys Arg Asn Asn Phe Llys Thr 1. 5 1O 15 Glu Glu Tyr

<210s, SEQ ID NO 76 &211s LENGTH: 19 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Description of Artificial Sequence: Synthetic peptide

<4 OO > SEQUENCE: 76 Thr Phe Phe Tyr Gly Gly Cys Arg Gly Lys Arg Asn Asn Phe Llys Thr 1. 5 1O 15 Lys Glu Tyr

<210s, SEQ ID NO 77 &211s LENGTH: 19 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Description of Artificial Sequence: Synthetic peptide

<4 OO > SEQUENCE: 77 Thr Phe Phe Tyr Gly Gly Lys Arg Gly Lys Arg Asn Asn Phe Llys Thr 1. 5 1O 15

Glu Glu Tyr US 9, 161988 B2 105 106 - Continued

<210s, SEQ ID NO 78 &211s LENGTH: 19 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Description of Artificial Sequence: Synthetic peptide

<4 OO > SEQUENCE: 78 Thr Phe Phe Tyr Gly Gly Cys Arg Gly Lys Arg Asn Asn Phe Llys Thr 1. 5 1O 15 Lys Arg Tyr

<210s, SEQ ID NO 79 &211s LENGTH: 19 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Description of Artificial Sequence: Synthetic peptide

<4 OO > SEQUENCE: 79 Thr Phe Phe Tyr Gly Gly Lys Arg Gly Lys Arg Asn Asn Phe Llys Thr 1. 5 1O 15 Ala Glu Tyr

<210s, SEQ ID NO 8O &211s LENGTH: 19 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Description of Artificial Sequence: Synthetic peptide

<4 OOs, SEQUENCE: 80 Thr Phe Phe Tyr Gly Gly Lys Arg Gly Lys Arg Asn Asn Phe Llys Thr 1. 5 1O 15 Ala Gly Tyr

<210s, SEQ ID NO 81 &211s LENGTH: 19 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Description of Artificial Sequence: Synthetic peptide

<4 OOs, SEQUENCE: 81 Thir Phe Phe Tyr Gly Gly Lys Arg Gly Lys Arg Asn. Asn. Phe Lys Arg 1. 5 1O 15 Glu Lys Tyr

<210s, SEQ ID NO 82 &211s LENGTH: 19 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Description of Artificial Sequence: Synthetic peptide

<4 OOs, SEQUENCE: 82 Thir Phe Phe Tyr Gly Gly Lys Arg Gly Lys Arg Asn. Asn. Phe Lys Arg 1. 5 1O 15

Ala Lys Tyr US 9, 161988 B2 107 108 - Continued

<210s, SEQ ID NO 83 &211s LENGTH: 19 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Description of Artificial Sequence: Synthetic peptide

<4 OOs, SEQUENCE: 83 Thr Phe Phe Tyr Gly Gly Cys Lieu. Gly Asn Arg Asn Asn Phe Llys Thr 1. 5 1O 15 Glu Glu Tyr

<210s, SEQ ID NO 84 &211s LENGTH: 19 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Description of Artificial Sequence: Synthetic peptide

<4 OOs, SEQUENCE: 84 Thr Phe Phe Tyr Gly Cys Gly Arg Gly Lys Arg Asn Asn Phe Llys Thr 1. 5 1O 15 Glu Glu Tyr

<210s, SEQ ID NO 85 &211s LENGTH: 19 212. TYPE PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Description of Artificial Sequence: Synthetic peptide

<4 OOs, SEQUENCE: 85 Thr Phe Phe Tyr Gly Gly Arg Cys Gly Lys Arg Asn Asn Phe Llys Thr 1. 5 1O 15 Glu Glu Tyr

<210s, SEQ ID NO 86 &211s LENGTH: 19 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Description of Artificial Sequence: Synthetic peptide

<4 OOs, SEQUENCE: 86 Thr Phe Phe Tyr Gly Gly Cys Lieu. Gly Asin Gly Asn Asn Phe Asp Thr 1. 5 1O 15

Glu Glu Glu

<210s, SEQ ID NO 87 &211s LENGTH: 19 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Description of Artificial Sequence: Synthetic peptide

<4 OO > SEQUENCE: 87 Thr Phe Glin Tyr Gly Gly Cys Arg Gly Lys Arg Asn Asn Phe Llys Thr 1. 5 1O 15 US 9, 161988 B2 109 110 - Continued Glu Glu Tyr

<210s, SEQ ID NO 88 &211s LENGTH: 19 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Description of Artificial Sequence: Synthetic peptide

<4 OOs, SEQUENCE: 88 Tyr Asn Lys Glu Phe Gly Thr Phe Asn Thr Lys Gly Cys Glu Arg Gly 1. 5 1O 15 Tyr Arg Phe

<210s, SEQ ID NO 89 &211s LENGTH: 19 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Description of Artificial Sequence: Synthetic peptide

<4 OOs, SEQUENCE: 89 Arg Phe Llys Tyr Gly Gly Cys Lieu. Gly Asn Met Asn Asn Phe Glu Thr 1. 5 1O 15

Lieu. Glu Glu

<210s, SEQ ID NO 90 & 211 LENGTH 19 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Description of Artificial Sequence: Synthetic peptide

<4 OOs, SEQUENCE: 90 Arg Phe Llys Tyr Gly Gly Cys Lieu. Gly Asn Lys Asn. Asn. Phe Lieu. Arg 1. 5 1O 15 Lieu Lys Tyr

<210s, SEQ ID NO 91 &211s LENGTH: 19 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Description of Artificial Sequence: Synthetic peptide

<4 OOs, SEQUENCE: 91 Arg Phe Llys Tyr Gly Gly Cys Lieu. Gly Asn Lys Asn. Asn Tyr Lieu. Arg 1. 5 1O 15 Lieu Lys Tyr

<210s, SEQ ID NO 92 &211s LENGTH: 22 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Description of Artificial Sequence: Synthetic peptide

<4 OOs, SEQUENCE: 92 Llys Thir Lys Arg Lys Arg Llys Lys Glin Arg Val Lys Ile Ala Tyr Glu 1. 5 1O 15 US 9, 161988 B2 111 112 - Continued

Glu Ile Phe Lys Asn Tyr 2O

<210s, SEQ ID NO 93 &211s LENGTH: 15 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Description of Artificial Sequence: Synthetic peptide

<4 OOs, SEQUENCE: 93 Llys Thir Lys Arg Lys Arg Llys Lys Glin Arg Val Lys Ile Ala Tyr 1. 5 1O 15

<210s, SEQ ID NO 94 &211s LENGTH: 17 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Description of Artificial Sequence: Synthetic peptide

<4 OOs, SEQUENCE: 94 Arg Gly Gly Arg Lieu Ser Tyr Ser Arg Arg Phe Ser Thr Ser Thr Gly 1. 5 1O 15 Arg

<210s, SEQ ID NO 95 & 211 LENGTH: 10 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Description of Artificial Sequence: Synthetic peptide

<4 OO > SEQUENCE: 95 Arg Arg Lieu. Ser Tyr Ser Arg Arg Arg Phe 1. 5 1O

<210s, SEQ ID NO 96 &211s LENGTH: 16 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Description of Artificial Sequence: Synthetic peptide

<4 OOs, SEQUENCE: 96 Arg Glin Ile Lys Ile Trp Phe Glin Asn Arg Arg Met Lys Trp Llys Llys 1. 5 1O 15

<210s, SEQ ID NO 97 &211s LENGTH: 19 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Description of Artificial Sequence: Synthetic peptide

<4 OO > SEQUENCE: 97 Thr Phe Phe Tyr Gly Gly Ser Arg Gly Lys Arg Asn Asn Phe Llys Thr 1. 5 1O 15

Glu Glu Tyr US 9, 161988 B2 113 114 - Continued

<210s, SEQ ID NO 98 &211s LENGTH: 59 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Description of Artificial Sequence: Synthetic polypeptide

<4 OOs, SEQUENCE: 98 Met Arg Pro Asp Phe Cys Lieu. Glu Pro Pro Tyr Thr Gly Pro Cys Val 1. 5 1O 15 Ala Arg Ile Ile Arg Tyr Phe Tyr Asn Ala Lys Ala Gly Lieu. Cys Glin 2O 25 3O Thir Phe Val Tyr Gly Gly Cys Arg Ala Lys Arg Asn. Asn. Phe Llys Ser 35 4 O 45 Ala Glu Asp Cys Met Arg Thr Cys Gly Gly Ala SO 55

<210s, SEQ ID NO 99 &211s LENGTH: 19 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Description of Artificial Sequence: Synthetic peptide

<4 OOs, SEQUENCE: 99 Thr Phe Phe Tyr Gly Gly Cys Arg Gly Lys Arg Asn Asn Phe Llys Thr 1. 5 1O 15 Lys Glu Tyr

<210s, SEQ ID NO 100 &211s LENGTH: 19 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Description of Artificial Sequence: Synthetic peptide

<4 OOs, SEQUENCE: 1.OO Arg Phe Llys Tyr Gly Gly Cys Lieu. Gly Asn Lys Asn. Asn Tyr Lieu. Arg 1. 5 1O 15 Lieu Lys Tyr

<210s, SEQ ID NO 101 &211s LENGTH: 19 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Description of Artificial Sequence: Synthetic peptide

<4 OOs, SEQUENCE: 101 Thir Phe Phe Tyr Gly Gly Cys Arg Ala Lys Arg Asn. Asn. Phe Lys Arg 1. 5 1O 15

Ala Lys Tyr

<210s, SEQ ID NO 102 &211s LENGTH: 35 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Description of Artificial Sequence: Synthetic polypeptide US 9, 161988 B2 115 116 - Continued

<4 OOs, SEQUENCE: 102 Asn Ala Lys Ala Gly Lieu. Cys Glin Thr Phe Val Tyr Gly Gly Cys Lieu 1. 5 1O 15 Ala Lys Arg Asn. Asn. Phe Glu Ser Ala Glu Asp Cys Met Arg Thr Cys 2O 25 3O Gly Gly Ala 35

<210s, SEQ ID NO 103 &211s LENGTH: 24 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Description of Artificial Sequence: Synthetic peptide

<4 OOs, SEQUENCE: 103 Tyr Gly Gly Cys Arg Ala Lys Arg Asn. Asn. Phe Llys Ser Ala Glu Asp 1. 5 1O 15 Cys Met Arg Thr Cys Gly Gly Ala 2O

<210s, SEQ ID NO 104 &211s LENGTH: 22 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Description of Artificial Sequence: Synthetic peptide

<4 OOs, SEQUENCE: 104 Gly Lieu. Cys Glin Thr Phe Val Tyr Gly Gly Cys Arg Ala Lys Arg Asn 1. 5 1O 15 Asn Phe Llys Ser Ala Glu 2O

<210s, SEQ ID NO 105 &211s LENGTH: 2O 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Description of Artificial Sequence: Synthetic peptide

<4 OOs, SEQUENCE: 105 Lieu. Cys Glin Thir Phe Val Tyr Gly Gly Cys Glu Ala Lys Arg Asn. Asn 1. 5 1O 15 Phe Llys Ser Ala 2O

<210s, SEQ ID NO 106 &211s LENGTH: 18O &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Description of Artificial Sequence: Synthetic Construct

<4 OOs, SEQUENCE: 106 atgagaccag atttctgcct cago.cgc.cg tacactgggc cctgcaaagc ticgitat catc 6 O cgtt acttct acaatgcaaa ggcaggcctg. itt Cagacct tcgtatacgg C9gctgcaga 12 O gctaag.cgta acaact tcaa atc.cgcggala gactgcatgc gtacttgcgg toggtgct tag 18O US 9, 161988 B2 117 118 - Continued

<210s, SEQ ID NO 107 &211s LENGTH: 19 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Description of Artificial Sequence: Synthetic peptide

<4 OOs, SEQUENCE: 107 Thr Phe Phe Tyr Gly Gly Ser Arg Gly Lys Arg Asn Asn Phe Llys Thr 1. 5 1O 15 Glu Glu Tyr

<210s, SEQ ID NO 108 &211s LENGTH: 19 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Description of Artificial Sequence: Synthetic peptide

<4 OOs, SEQUENCE: 108 Arg Phe Phe Tyr Gly Gly Ser Arg Gly Lys Arg Asn. Asn. Phe Llys Thr 1. 5 1O 15 Glu Glu Tyr

<210s, SEQ ID NO 109 &211s LENGTH: 19 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Description of Artificial Sequence: Synthetic peptide

<4 OOs, SEQUENCE: 109 Arg Phe Phe Tyr Gly Gly Ser Arg Gly Lys Arg Asn. Asn. Phe Llys Thr 1. 5 1O 15 Glu Glu Tyr

<210s, SEQ ID NO 110 &211s LENGTH: 19 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Description of Artificial Sequence: Synthetic peptide

<4 OOs, SEQUENCE: 110 Arg Phe Phe Tyr Gly Gly Ser Arg Gly Lys Arg Asn. Asn. Phe Arg Thr 1. 5 1O 15 Glu Glu Tyr

<210s, SEQ ID NO 111 &211s LENGTH: 19 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Description of Artificial Sequence: Synthetic peptide

<4 OOs, SEQUENCE: 111 Thr Phe Phe Tyr Gly Gly Ser Arg Gly Lys Arg Asn Asn Phe Arg Thr 1. 5 1O 15

Glu Glu Tyr US 9, 161988 B2 119 120 - Continued

<210s, SEQ ID NO 112 &211s LENGTH: 19 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Description of Artificial Sequence: Synthetic peptide

<4 OOs, SEQUENCE: 112 Thr Phe Phe Tyr Gly Gly Ser Arg Gly Arg Arg Asn Asn Phe Arg Thr 1. 5 1O 15 Glu Glu Tyr

<210s, SEQ ID NO 113 &211s LENGTH: 2O 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Description of Artificial Sequence: Synthetic peptide

<4 OOs, SEQUENCE: 113 Cys Thr Phe Phe Tyr Gly Gly Ser Arg Gly Lys Arg Asn Asin Phe Lys 1. 5 1O 15 Thr Glu Glu Tyr 2O

<210s, SEQ ID NO 114 & 211 LENGTH: 2O 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Description of Artificial Sequence: Synthetic peptide

<4 OOs, SEQUENCE: 114 Thr Phe Phe Tyr Gly Gly Ser Arg Gly Lys Arg Asn Asn Phe Llys Thr 1. 5 1O 15 Glu Glu Tyr Cys 2O

<210s, SEQ ID NO 115 &211s LENGTH: 2O 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Description of Artificial Sequence: Synthetic peptide

<4 OOs, SEQUENCE: 115 Cys Thr Phe Phe Tyr Gly Gly Ser Arg Gly Arg Arg Asn Asin Phe Arg 1. 5 1O 15

Thr Glu Glu Tyr 2O

<210s, SEQ ID NO 116 &211s LENGTH: 2O 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Description of Artificial Sequence: Synthetic peptide

<4 OOs, SEQUENCE: 116 US 9, 161988 B2 121 122 - Continued Thr Phe Phe Tyr Gly Gly Ser Arg Gly Arg Arg Asn Asn Phe Arg Thr 1. 5 1O 15 Glu Glu Tyr Cys 2O

<210s, SEQ ID NO 117 &211s LENGTH: 19 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Description of Artificial Sequence: Synthetic peptide

<4 OOs, SEQUENCE: 117 Tyr Glu Glu Thir Llys Phe Asn. Asn Arg Lys Gly Arg Ser Gly Gly Tyr 1. 5 1O 15

Phe Phe Thr

<210s, SEQ ID NO 118 &211s LENGTH: 19 212. TYPE : RNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Description of Artificial Sequence: Synthetic Construct

<4 OOs, SEQUENCE: 118 ggagculgc cc alugaga aalu 19

<210 SEQ ID NO 119 &211s LENGTH: 19 212. TYPE : RNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Description of Artificial Sequence: Synthetic Construct

<4 OOs, SEQUENCE: 119 aluulu culcalug ggcagcucc 19

<210s, SEQ ID NO 120 &211s LENGTH: 19 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Description of Artificial Sequence: Synthetic Construct 22 Os. FEATURE: <221 > NAMEAKEY: misc feature <222s. LOCATION: (1) . . (19 <223> OTHER INFORMATION: synthetic construct for RNAi <4 OOs, SEQUENCE: 120 ggagtaccct gatgagat C 19

<210s, SEQ ID NO 121 &211s LENGTH: 13 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Description of Artificial Sequence: Synthetic peptide 22 Os. FEATURE: <221s NAME/KEY: MOD RES <222s. LOCATION: (1) . . (1) <223> OTHER INFORMATION: L-pyroglutamic acid 22 Os. FEATURE: <221s NAME/KEY: MOD RES US 9, 161988 B2 123 124 - Continued

<222s. LOCATION: (13) . . (13) <223> OTHER INFORMATION: C-terminus hydroxylation

<4 OOs, SEQUENCE: 121 Xaa Lieu. Tyr Glu Asn Llys Pro Arg Arg Pro Tyr Ile Lieu. 1. 5 1O

<210s, SEQ ID NO 122 &211s LENGTH: 78 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Description of Artificial Sequence: Synthetic peptide 22 Os. FEATURE: <221s NAME/KEY: MISC FEATURE <222s. LOCATION: (39) . . (40) <223> OTHER INFORMATION: The linkage between Lys at position 39 and Thr at position 4 O is maleimido hexanoic acid (MHA) 22 Os. FEATURE: <221s NAME/KEY: MISC FEATURE <222s. LOCATION: (59) . . (60) <223> OTHER INFORMATION: The linkage between Cys at position 59 and Thr at position 6 O is maleimido propionic acid (MHA) 22 Os. FEATURE: <221s NAME/KEY: MOD RES <222s. LOCATION: (78) . . (78) <223> OTHER INFORMATION: C-terminus hydroxylation

<4 OOs, SEQUENCE: 122 His Gly Glu Gly Thr Phe Thir Ser Asp Leu Ser Lys Gln Met Glu Glu 1. 5 1O 15 Glu Ala Val Arg Lieu Phe Ile Glu Trp Lieu Lys Asn Gly Gly Pro Ser 2O 25 3O Ser Gly Ala Pro Pro Pro Llys Thr Phe Phe Tyr Gly Gly Ser Arg Gly 35 4 O 45 Lys Arg Asn Asn Phe Lys Thr Glu Glu Tyr Cys Thr Phe Phe Tyr Gly SO 55 6 O Gly Ser Arg Gly Lys Arg Asn. Asn. Phe Llys Thr Glu Glu Tyr 65 70 7s

<210s, SEQ ID NO 123 &211s LENGTH: 58 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Description of Artificial Sequence: Synthetic peptide 22 Os. FEATURE: <221s NAME/KEY: MISC FEATURE <222s. LOCATION: (39) . . (40) <223> OTHER INFORMATION: The linkage between Ser at position 39 and Gly at position 4 O is maleimido hexanoic acid (MHA) 22 Os. FEATURE: <221s NAME/KEY: MOD RES <222s. LOCATION: (58) ... (58) <223> OTHER INFORMATION: C-terminus hydroxylation

<4 OOs, SEQUENCE: 123 His Gly Glu Gly Thr Phe Thir Ser Asp Leu Ser Lys Gln Met Glu Glu 1. 5 1O 15

Glu Ala Val Arg Lieu. Phe Ile Glu Trp Lieu Lys Asn Gly Gly Pro Cys 2O 25 3O

Ser Gly Ala Pro Pro Pro Ser Gly Tyr Lys Gly Glu Arg Tyr Arg Gly 35 4 O 45

Phe Lys Glu Thir Asn Phe Asn Thr Phe Ser SO 55 US 9, 161988 B2 125 126 - Continued

<210s, SEQ ID NO 124 &211s LENGTH: 30 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Description of Artificial Sequence: Synthetic peptide 22 Os. FEATURE: <221s NAME/KEY: MISC FEATURE <222s. LOCATION: (1) . . (5) <223> OTHER INFORMATION: This sequence may encompass 1-6 'Gly-Gly-Gly Gly-Ser' repeating units

<4 OOs, SEQUENCE: 124 Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly 1. 5 1O 15 Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser 2O 25 3O

<210s, SEQ ID NO 125 &211s LENGTH: 25 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Description of Artificial Sequence: Synthetic peptide 22 Os. FEATURE: <221s NAME/KEY: MISC FEATURE <222s. LOCATION: (1) . . (5) <223> OTHER INFORMATION: This sequence may encompass 1-5 'Xaa-Xaa-Xaa Xaa-Gly" repeating units. See specification as filed for detailed description of Substitutions and preferred embodiments 22 Os. FEATURE: <221s NAME/KEY: MOD RES <222s. LOCATION: (1) ... (4) 223 OTHER INFORMATION: Thir or Ser 22 Os. FEATURE: <221 > NAMEAKEY OD RES <222s. LOCATION: (6) ... (9) 223 OTHER INFORMATION: Thir or Ser 22 Os. FEATURE: <221s NAME/KEY: MOD RES <222s. LOCATION: (11) . . (14) 223 OTHER INFORMATION: Thir or Ser 22 Os. FEATURE: <221s NAME/KEY: MOD RES <222s. LOCATION: (16) ... (19) 223 OTHER INFORMATION: Thir or Ser 22 Os. FEATURE: <221s NAME/KEY: MOD RES <222s. LOCATION: (21) ... (24) 223 OTHER INFORMATION: Thir or Ser

<4 OOs, SEQUENCE: 125 Xaa Xala Xala Xala Gly Xaa Xaa Xala Xala Gly Xaa Xaa Xaa Xala Gly Xaa 1. 5 1O 15 Xaa Xala Xala Gly Xaa Xaa Xaa Xala Gly 2O 25

<210s, SEQ ID NO 126 &211s LENGTH: 5 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Description of Artificial Sequence: Synthetic peptide 22 Os. FEATURE: <221s NAME/KEY: MISC FEATURE <222s. LOCATION: (1) . . (5) <223> OTHER INFORMATION: This sequence may encompass more than one 'Ser Ser-Ser-Ser-Gly" repeating units US 9, 161988 B2 127 128 - Continued

<4 OOs, SEQUENCE: 126 Ser Ser Ser Ser Gly 1. 5

<210s, SEQ ID NO 127 &211s LENGTH: 5 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Description of Artificial Sequence: Synthetic peptide

<4 OOs, SEQUENCE: 127

Pro Ala Pro Ala Pro 1. 5

<210s, SEQ ID NO 128 &211s LENGTH: 15 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Description of Artificial Sequence: Synthetic peptide 22 Os. FEATURE: <221s NAME/KEY: MISC FEATURE <222s. LOCATION: (1) ... (2) <223> OTHER INFORMATION: This sequence may encompass 1-7 'Pro-Thr' repeating units

<4 OOs, SEQUENCE: 128

Pro Thr Pro Thr Pro Thr Pro Thr Pro Thr Pro Thr Pro Thr Pro 1. 5 1O 15

<210s, SEQ ID NO 129 &211s LENGTH: 27 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Description of Artificial Sequence: Synthetic peptide 22 Os. FEATURE: <221s NAME/KEY: MISC FEATURE <222s. LOCATION: (2) . . (6) <223> OTHER INFORMATION: This sequence may encompass 1-5 'Glu-Ala-Ala Ala-Lys' repeating units

<4 OOs, SEQUENCE: 129 Ala Glu Ala Ala Ala Lys Glu Ala Ala Ala Lys Glu Ala Ala Ala Lys 1. 5 1O 15 Glu Ala Ala Ala Lys Glu Ala Ala Ala Lys Ala 2O 25

<210s, SEQ ID NO 130 &211s LENGTH: 4 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Description of Artificial Sequence: Synthetic peptide

<4 OOs, SEQUENCE: 130

Ala Lieu Ala Lieu. 1.

<210s, SEQ ID NO 131 &211s LENGTH: 4 212. TYPE: PRT US 9, 161988 B2 129 130 - Continued <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Description of Artificial Sequence: Synthetic peptide

<4 OOs, SEQUENCE: 131 Gly Phe Leu Gly 1.

<210s, SEQ ID NO 132 &211s LENGTH: 39 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Description of Artificial Sequence: Synthetic peptide 22 Os. FEATURE: <221s NAME/KEY: MOD RES <222s. LOCATION: (39) . . (39) 223 OTHER INFORMATION: AMIDATION

<4 OOs, SEQUENCE: 132 His Gly Glu Gly Thr Phe Thir Ser Asp Leu Ser Lys Gln Met Glu Glu 1. 5 1O 15 Glu Ala Val Arg Lieu. Phe Ile Glu Trp Leu Lys Asn Gly Gly Pro Ser 2O 25 3O

Ser Gly Ala Pro Pro Pro Ser 35

<210s, SEQ ID NO 133 & 211 LENGTH 39 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Description of Artificial Sequence: Synthetic peptide

<4 OOs, SEQUENCE: 133 His Gly Glu Gly Thr Phe Thir Ser Asp Leu Ser Lys Gln Met Glu Glu 1. 5 1O 15 Glu Ala Val Arg Lieu. Phe Ile Glu Trp Lieu Lys Asn Gly Gly Pro Cys 2O 25 3O

Ser Gly Ala Pro Pro Pro Ser 35

<210s, SEQ ID NO 134 &211s LENGTH: 39 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Description of Artificial Sequence: Synthetic peptide

<4 OOs, SEQUENCE: 134 His Gly Glu Gly Thr Phe Thir Ser Asp Leu Ser Lys Gln Met Glu Glu 1. 5 1O 15

Glu Ala Val Arg Lieu. Phe Ile Glu Trp Leu Lys Asn Gly Gly Pro Ser 2O 25 3O

Ser Gly Ala Pro Pro Pro Llys 35

<210s, SEQ ID NO 135 &211s LENGTH: 39 212. TYPE: PRT <213> ORGANISM: Artificial Sequence US 9, 161988 B2 131 132 - Continued

FEATU RE: OTHER INFORMATION: Description of Artificial Sequence: Synthetic pepti de FEATU RE: NAME/ KEY: MOD RES LOCAT ION: (1) . . (1) INFORMATION: His, Arg or Tyr RE: KEY OD RES ION: (2) ... (2) INFORMATION: Ser, Gly, Ala or Thr RE: KEY OD RES ION: (3) ... (3) INFORMATION: Asp or Glu RE: KEY OD RES ION: (6) (6) INFORMATION: Phe, Tyr or naphthylalanine (Nal)

O A.CA.CA.CCf ION: (7) . . (7) INFORMATION: Thir or Ser RE: KEY OD RES ION: (8) ... (8) INFORMATION: Seir or Thr RE: KEY OD RES ION: (9) ... (9) INFORMATION: Asp or Glu RE: KEY: MOD RES O C f ION: (10) ... (10) INFORMATION: Leu, Ile, Val, pentylglycine (pgly) or Met RE: KEY: MOD RES ION: (14) . . (14) NFORMATION: Leu, Ile, pentylglycine (p.Gly), Val or Met RE: KEY: MOD RES ION: (22) ... (22) NFORMATION: Phe, Tyr, or naphthylalanine (Nal) RE: EOEOEA.CA.CA. KEY: MOD RES O C ION: (23) . . (23) NFORMATION: Ile, Val, Leu, pentylglycine (p.Gly), t glycine (t-BuG) or Met RE: KEY: MOD RES ION: (24) . . (24) OTH E NFORMATION: Glu or Asp FEAT RE: NA KEY: MOD RES LOCAT ION: (25) . . (25) OTHER NFORMATION: Trp, Phe, Tyr, or naphthylalanine (Nal) FEATU RE: NAME/ KEY: MOD RES LOCAT ION: (31) ... (31) OTHER NFORMATION: Pro, homoproline (HPro), 3 Hyp, 4Hyp, thiop roline (TPro), N-alkylglycine, N-alkyl-pentylglycine alky -pGly) or N-alkylalanine FEATU RE: NAME/ KEY: MOD RES LOCAT ION: (36) ... (36) OTHER INFORMATION: Pro, homoproline (HPro), 3 Hyp, 4Hyp, thiop roline (TPro), N-alkylglycine, N-alkyl-pentylglycine (N-a kyl-pCly) or N-alkylalanine FEATU RE: NAME/ KEY: MOD RES LOCAT ION: (37) . . (37) OTHER INFORMATION: Pro, homoproline (HPro), 3 Hyp, 4Hyp, thiop roline (TPro), N-alkylglycine, N-alkyl-pentylglycine (N-a kyl-pCly) or N-alkylalanine FEATU RE: NAME/ KEY: MOD RES LOCAT ION: (38) ... (38) OTHER INFORMATION: Pro, homoproline (HPro), 3 Hyp, 4Hyp, thiop roline (TPro), N-alkylglycine, N-alkyl-pentylglycine US 9, 161988 B2 133 134 - Continued (N-alkyl-pCly) or N-alkylalanine 22 Os. FEATURE: <221s NAME/KEY: MOD RES <222s. LOCATION: (39) . . (39) <223> OTHER INFORMATION: Ser, Thr, or Tyr 22 Os. FEATURE: <221s NAME/KEY: MOD RES <222s. LOCATION: (39) . . (39) <223> OTHER INFORMATION: C-terminus hydroxylation or amidation <4 OOs, SEQUENCE: 135 Xaa Xala Xala Gly Thr Xaa Xaa Xala Xala Xala Ser Lys Glin Xala Glu Glu 1. 5 1O 15 Glu Ala Val Arg Lieu. Xaa Xaa Xala Xala Lieu Lys Asn Gly Gly Xaa Ser 2O 25 3O Ser Gly Ala Xala Xaa Xaa Xaa 35

<210s, SEQ ID NO 136 &211s LENGTH: 4 O 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Description of Artificial Sequence: Synthetic peptide 22 Os. FEATURE: <221s NAME/KEY: MOD RES <222s. LOCATION: (14) . . (14) <223> OTHER INFORMATION: Arg, Leu, Ile, or Met 22 Os. FEATURE: <221s NAME/KEY: MOD RES <222s. LOCATION: (2O) . . (2O) <223> OTHER INFORMATION: His, Arg, or Lys 22 Os. FEATURE: <221s NAME/KEY: MOD RES <222s. LOCATION: (40) ... (40) <223> OTHER INFORMATION: Arg, Lys, or absent 22 Os. FEATURE: <221s NAME/KEY: MOD RES <222s. LOCATION: (40) ... (40) <223> OTHER INFORMATION: C-terminus hydroxylation or amidation, where -NH2 is present when Arg or Lys is absent

<4 OOs, SEQUENCE: 136 His Gly Glu Gly Thr Phe Thir Ser Asp Leu Ser Lys Glin Xaa Glu Glu 1. 5 1O 15 Glu Ala Val Xala Lieu. Phe Ile Glu Trp Lieu Lys Asn Gly Gly Pro Ser 2O 25 3O Ser Gly Ala Pro Pro Pro Ser Xaa 35 4 O

<210s, SEQ ID NO 137 &211s LENGTH: 32 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Description of Artificial Sequence: Synthetic peptide 22 Os. FEATURE: <221s NAME/KEY: MOD RES <222s. LOCATION: (6) . . (6) <223> OTHER INFORMATION: Phe or Tyr 22 Os. FEATURE: <221s NAME/KEY: MOD RES <222s. LOCATION: (14) . . (14) <223> OTHER INFORMATION: Met, Ile or Lieu. 22 Os. FEATURE: <221s NAME/KEY: MOD RES <222s. LOCATION: (2O) . . (2O) <223> OTHER INFORMATION: Lys 22 Os. FEATURE: US 9, 161988 B2 135 136 - Continued <221s NAME/KEY: MOD RES <222s. LOCATION: (30) ... (30) <223> OTHER INFORMATION: Gly or absent 22 Os. FEATURE: <221s NAME/KEY: MOD RES <222s. LOCATION: (32) ... (32) <223> OTHER INFORMATION: Arg or absent <4 OOs, SEQUENCE: 137 His Gly Glu Gly Thr Xaa Thir Ser Asp Lieu. Ser Lys Glin Xala Glu Glu 1. 5 1O 15 Glu Ala Val Xala Lieu. Phe Ile Glu Trp Lieu Lys Asn Gly Xaa Pro Xaa 2O 25 3O

<210s, SEQ ID NO 138 &211s LENGTH: 31 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Description of Artificial Sequence: Synthetic peptide 22 Os. FEATURE: <221s NAME/KEY: MOD RES <222s. LOCATION: (29).. (29) <223> OTHER INFORMATION: Gly or absent, depending on indicated chain length 22 Os. FEATURE: <221s NAME/KEY: MOD RES <222s. LOCATION: (30) ... (30) <223> OTHER INFORMATION: Arg or absent, depending on indicated chain length 22 Os. FEATURE: <221s NAME/KEY: MOD RES <222 LOCATION: (31) . . (31) <223> OTHER INFORMATION: Gly or absent, depending on indicated chain length

<4 OOs, SEQUENCE: 138 His Ala Glu Gly Thr Phe Thir Ser Asp Val Ser Ser Tyr Lieu. Glu Gly 1. 5 1O 15 Asn Ala Ala Lys Glu Phe Ile Ala Trp Lieu Val Lys Xaa Xala Xala 2O 25 3O

<210s, SEQ ID NO 139 &211s LENGTH: 30 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Description of Artificial Sequence: Synthetic peptide 22 Os. FEATURE: <221s NAME/KEY: MOD RES <222s. LOCATION: (1) . . (11 <223> OTHER INFORMATION: Amino acids from positions 1 to 10 (Ala-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser) may be absent, and Ser at position 11 may be amidated 22 Os. FEATURE: <221s NAME/KEY: MOD RES <222s. LOCATION: (1) . . (10 <223> OTHER INFORMATION: Amino acids from positions 1 to 9 (Ala-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val) may be absent, and Ser at position 10 may be amidated 22 Os. FEATURE: <221s NAME/KEY: MOD RES <222s. LOCATION: (1) ... (9) <223> OTHER INFORMATION: Amino acids from positions 1 to 8 (Ala-Glu-Gly-Thr-Phe-Thr-Ser-Asp) may be absent, and Val at position 9 may be amidated 22 Os. FEATURE: <221s NAME/KEY: MOD RES <222s. LOCATION: (1) ... (8) <223> OTHER INFORMATION: Amino acids from positions 1 to 7 (Ala-Glu-Gly Thir-Phe-Thr-Ser) may be absent, and Asp at position 8 may be US 9, 161988 B2 137 138 - Continued

amidated 22 Os. FEATURE: <221s NAME/KEY: MOD RES <222s. LOCATION: (1) . . (7) <223> OTHER INFORMATION: Amino acids from positions 1 to 6 (Ala-Glu-Gly Thr-Phe-Thr) may be absent, and Ser at position 7 may be amidated 22 Os. FEATURE: <221s NAME/KEY: MOD RES <222s. LOCATION: (1) . . (6) <223> OTHER INFORMATION: Amino acids from positions 1 to 5 (Ala-Glu-Gly Thr-Phe) may be absent, and Thr at position 6 may be amidated 22 Os. FEATURE: <221s NAME/KEY: MOD RES <222s. LOCATION: (1) . . (5) <223> OTHER INFORMATION: Amino acids from positions 1 to 4 (Ala-Glu-Gly Thr) may be absent, and Phe at position 5 may be amidated 22 Os. FEATURE: <221s NAME/KEY: MOD RES <222s. LOCATION: (1) ... (4) <223> OTHER INFORMATION: Amino acids from positions 1 to 3 (Ala-Glu-Gly) may be absent, and Thr at position 4 may be amidated 22 Os. FEATURE: <221s NAME/KEY: MOD RES <222s. LOCATION: (1) ... (3) <223> OTHER INFORMATION: Amino acids from positions 1 to 2 (Ala-Glu) may be absent, and Gly at position 3 may be amidated 22 Os. FEATURE: <221s NAME/KEY: MOD RES <222s. LOCATION: (1) ... (2) <223> OTHER INFORMATION: Amino acid at position 1 (Ala) may be absent, and Glu at position 2 may be amidated 22 Os. FEATURE: <221s NAME/KEY: MOD RES <222s. LOCATION: (1) . . (1) <223> OTHER INFORMATION: Ala at position 1 may be amidated 22 Os. FEATURE: <221. NAME/KEY: MOD RES <222s. LOCATION: (27) . . (27) <223> OTHER INFORMATION: Lys or Arg 22 Os. FEATURE: <221s NAME/KEY: MOD RES <222s. LOCATION: (29).. (29) <223> OTHER INFORMATION: C-terminus hydroxylation or amidation when Gly at position 3 O is absent 22 Os. FEATURE: <221s NAME/KEY: MOD RES <222s. LOCATION: (30) ... (30) <223> OTHER INFORMATION: Gly or absent 22 Os. FEATURE: <221s NAME/KEY: MOD RES <222s. LOCATION: (30) ... (30) <223> OTHER INFORMATION: C-terminus hydroxylation or amidation when Gly at position 3 O is present

<4 OOs, SEQUENCE: 139 Ala Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Lieu. Glu Gly Glin 1. 5 1O 15 Ala Ala Lys Glu Phe Ile Ala Trp Lieu Val Xaa Gly Arg Xaa 2O 25 3O

<210s, SEQ ID NO 140 &211s LENGTH: 30 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Description of Artificial Sequence: Synthetic peptide 22 Os. FEATURE: <221s NAME/KEY: MOD RES <222s. LOCATION: (28) ... (30) <223> OTHER INFORMATION: The sequence Lys-Gly-Arg at positions 28 to 30 may be absent 22 Os. FEATURE: <221s NAME/KEY: MOD RES <222s. LOCATION: (29).. (30) <223> OTHER INFORMATION: The sequence Gly-Arg at positions 29 to 3O may US 9, 161988 B2 139 140 - Continued

be absent 22 Os. FEATURE: <221s NAME/KEY: MOD RES <222s. LOCATION: (30) ... (30) <223> OTHER INFORMATION: The amino acid Arg at position 30 may be absent <4 OOs, SEQUENCE: 140 His Ala Glu Gly Thr Phe Thir Ser Asp Val Ser Ser Tyr Lieu. Glu Gly 1. 5 1O 15 Glin Ala Ala Lys Glu Phe Ile Ala Trp Lieu Val Lys Gly Arg 2O 25 3O

<210s, SEQ ID NO 141 &211s LENGTH: 30 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Description of Artificial Sequence: Synthetic

22 Os. FEATURE: <221s NAME/KEY: MOD RES <222s. LOCATION: (1) . . (1) <223> OTHER INFORMATION: N-terminus amidation 22 Os. FEATURE: <221s NAME/KEY: MOD RES <222s. LOCATION: (30) ... (30) <223> OTHER INFORMATION: C-terminus modification with -OH, - OM, or -NR2R3, where M is a pharmaceutically acceptable cation or a lower branched or unbranched alkyl group; and R2 and 3 are independently hydrogen or a lower branched or unbranched alkyl group

<4 OOs, SEQUENCE: 141 His Ala Glu Gly Thr Phe Thir Ser Asp Val Ser Ser Tyr Lieu. Glu Gly 1. 5 1O 15 Glin Ala Ala Lys Glu Phe Ile Ala Trp Lieu Val Lys Gly Arg 2O 25 3O

<210s, SEQ ID NO 142 &211s LENGTH: 31 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Description of Artificial Sequence: Synthetic peptide 22 Os. FEATURE: <221s NAME/KEY: MOD RES <222s. LOCATION: (1) . . (1) <223> OTHER INFORMATION: His D-His desamino-His 2-amino - His bis hydroxy-His, homohistidine, alpha-fluoromethyl-Hills, or alpha methyl-His 22 Os. FEATURE: <221s NAME/KEY: MOD RES <222s. LOCATION: (2) ... (2) <223> OTHER INFORMATION: Met, Asp, Lys, Thr, Leu, Asn., Gln, Phe, Val, or Tyr 22 Os. FEATURE: <221s NAME/KEY: MOD RES <222s. LOCATION: (15) . . (15) <223> OTHER INFORMATION: Glu, Gln, Ala, Thr, Ser, and Gly 22 Os. FEATURE: <221s NAME/KEY: MOD RES <222s. LOCATION: (21) ... (21) <223> OTHER INFORMATION: Glu, Gln, Ala, Thr, Ser, and Gly 22 Os. FEATURE: <221s NAME/KEY: MOD RES <222s. LOCATION: (31) ... (31) <223> OTHER INFORMATION: Gly or absent 22 Os. FEATURE: <221s NAME/KEY: MOD RES <222s. LOCATION: (31) ... (31) <223> OTHER INFORMATION: C-terminus hydroxylation, when Gly at position 31 is present, or amidation, when Gly at position 31 is absent US 9, 161988 B2 141 142 - Continued

<4 OOs, SEQUENCE: 142 Xaa Xaa Glu Gly Thr Phe Thir Ser Asp Val Ser Ser Tyr Lieu. Xaa Lieu. 1. 5 1O 15 Glin Ala Ala Lys Xaa Phe Ile Ala Trp Lieu Val Lys Gly Arg Xaa 2O 25 3O

<210s, SEQ ID NO 143 &211s LENGTH: 31 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Description of Artificial Sequence: Synthetic peptide 22 Os. FEATURE: <221s NAME/KEY: MOD RES <222s. LOCATION: (1) . . (1) <223> OTHER INFORMATION: 4-imidazopropionyl (des-amino-histidyl), 4 imidazoacetyl, or 4-imidazo-alpha, alpha-dimethyl-acetyl 22 Os. FEATURE: <221s NAME/KEY: MOD RES <222s. LOCATION: (2O) . . (2O) <223> OTHER INFORMATION: Lys or Arg 22 Os. FEATURE: <221s NAME/KEY: MOD RES <222s. LOCATION: (28) ... (28) <223> OTHER INFORMATION: R2, which is bound to the side chain of the Lys (e.g., through the sigma amino group), is C6-10 unbranched acyl or is absent 22 Os. FEATURE: <221s NAME/KEY: MOD RES <222s. LOCATION: (31) ... (31) <223> OTHER INFORMATION: Gly or absent 22 Os. FEATURE: <221. NAME/KEY: MOD RES <222s. LOCATION: (31) ... (31) <223> OTHER INFORMATION: C-terminus hydroxylation, when Gly at position 31 is present, or amidation, when Gly at position 31 is absent <4 OOs, SEQUENCE: 143 Xaa Ala Glu Gly Thr Phe Thir Ser Asp Val Ser Ser Tyr Lieu. Glu Gly 1. 5 1O 15 Glin Ala Ala Xala Glu Phe Ile Ala Trp Lieu Val Lys Gly Arg Xaa 2O 25 3O

<210s, SEQ ID NO 144 &211s LENGTH: 31 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Description of Artificial Sequence: Synthetic peptide 22 Os. FEATURE: <221s NAME/KEY: MOD RES <222s. LOCATION: (2) ... (2) <223> OTHER INFORMATION: Gly, Ala, Val, Leu, Ile, Ser, or Thr 22 Os. FEATURE: <221s NAME/KEY: MOD RES <222s. LOCATION: (5) . . (5) <223> OTHER INFORMATION: Asp, Glu, Arg, Thr, Ala, Lys, or His 22 Os. FEATURE: <221s NAME/KEY: MOD RES <222s. LOCATION: (6) . . (6) <223> OTHER INFORMATION: His, Trp, Phe, or Tyr 22 Os. FEATURE: <221s NAME/KEY: MOD RES <222s. LOCATION: (10) ... (10) <223> OTHER INFORMATION: Leu, Ser, Thr, Trp, His, Phe, Asp, Val, Tyr, Glu, or Ala 22 Os. FEATURE: <221s NAME/KEY: MOD RES <222s. LOCATION: (16) ... (16) <223> OTHER INFORMATION: Gly, Asp, Glu, Gln, Asn., Lys, Arg, Cys, or cysteic acid (Cya) US 9, 161988 B2 143 144 - Continued

22 Os. FEATURE: <221s NAME/KEY: MOD RES <222s. LOCATION: (17) . . (17) <223> OTHER INFORMATION: His, Asp, Lys, Glu, or Glin 22 Os. FEATURE: <221s NAME/KEY: MOD RES <222s. LOCATION: (18) ... (18) <223> OTHER INFORMATION: Glu, His, Ala, or Lys 22 Os. FEATURE: <221s NAME/KEY: MOD RES <222s. LOCATION: (2O) . . (2O) <223> OTHER INFORMATION: Asp, Lys, Glu, or His 22 Os. FEATURE: <221s NAME/KEY: MOD RES <222s. LOCATION: (21) ... (21) <223> OTHER INFORMATION: Ala, Glu, His, Phe, Tyr, Trp, Arg, or Lys 22 Os. FEATURE: <221s NAME/KEY: MOD RES <222s. LOCATION: (25) ... (25) <223> OTHER INFORMATION: Ala, Glu, Asp, Ser, or His 22 Os. FEATURE: <221s NAME/KEY: MOD RES <222s. LOCATION: (27) . . (27) <223> OTHER INFORMATION: Asp, Arg, Val, Lys, Ala, Gly, or Glu 22 Os. FEATURE: <221s NAME/KEY: MOD RES <222s. LOCATION: (28) ... (28) <223> OTHER INFORMATION: Glu, Lys, or Asp 22 Os. FEATURE: <221s NAME/KEY: MOD RES <222s. LOCATION: (29).. (29) <223> OTHER INFORMATION: Thr, Ser, Lys, Arg, Trp, Tyr, Phe, Asp, Gly, Pro His or Glu 22 Os. FEATURE: <221s NAME/KEY: MOD RES <222s. LOCATION: (30) ... (30) <223> OTHER INFORMATION: Arg, Glu, or His 22 Os. FEATURE: <221s NAME/KEY: MOD RES <222s. LOCATION: (31) ... (31) <223> OTHER INFORMATION: Lys, Arg, Thr, Ser, Glu, Asp, Trp, Tyr, Phe, His, -NH2, Gly, Gly-Pro, Gly-Pro-NH2, or absent

<4 OOs, SEQUENCE: 144 His Xaa Glu Gly Xaa Xaa Thir Ser Asp Xaa Ser Ser Tyr Lieu. Glu Xaa 1. 5 1O 15

Xaa Xaa Ala Xala Xaa Phe Ile Ala Xala Lieu. Xaa Xaa Xala Xala Xala 2O 25 3O

<210s, SEQ ID NO 145 &211s LENGTH: 31 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Description of Artificial Sequence: Synthetic peptide 22 Os. FEATURE: <221s NAME/KEY: MOD RES <222s. LOCATION: (2) ... (2) <223> OTHER INFORMATION: Gly, Ala, Val, Leu, Ile, Ser, or Thr 22 Os. FEATURE: <221s NAME/KEY: MOD RES <222s. LOCATION: (6) . . (6) <223> OTHER INFORMATION: His, Trp, Phe, or Tyr 22 Os. FEATURE: <221s NAME/KEY: MOD RES <222s. LOCATION: (10) ... (10) <223> OTHER INFORMATION: Leu, Ser, Thr, Trp, His, Phe, Asp, Val, Glu, or Ala 22 Os. FEATURE: <221s NAME/KEY: MOD RES <222s. LOCATION: (16) ... (16) <223> OTHER INFORMATION: Gly, Asp, Glu, Gln, Asn., Lys, Arg, Cys, o cysteic acid (Cya) 22 Os. FEATURE: <221s NAME/KEY: MOD RES US 9, 161988 B2 145 146 - Continued

<222s. LOCATION: (17) . . (17) <223> OTHER INFORMATION: His, Asp, Lys, Glu, or Glin 22 Os. FEATURE: <221s NAME/KEY: MOD RES <222s. LOCATION: (2O) . . (2O) <223> OTHER INFORMATION: Asp, Lys, Glu, or His 22 Os. FEATURE: <221s NAME/KEY: MOD RES <222s. LOCATION: (24) . . (24) <223> OTHER INFORMATION: Ala, Glu, Asp, Ser, or His 22 Os. FEATURE: <221s NAME/KEY: MOD RES <222s. LOCATION: (29).. (29) <223> OTHER INFORMATION: Thr, Ser, Lys, Arg, Trp, Tyr, Phe, Asp, Gly, Pro His or Glu 22 Os. FEATURE: <221s NAME/KEY: MOD RES <222s. LOCATION: (31) ... (31) <223> OTHER INFORMATION: Lys, Arg, Thr, Ser, Glu, Asp, Trp, Tyr, Phe, His, -NH2, Gly, Gly-Pro, Gly-Pro-NH2, or absent

<4 OOs, SEQUENCE: 145 His Xaa Glu Gly Thr Xaa Thir Ser Asp Xaa Ser Ser Tyr Lieu. Glu Xaa 1. 5 1O 15 Xaa Ala Ala Xala Glu Phe Ile Xaa Trp Lieu Val Lys Xaa Arg Xaa 2O 25 3O

<210s, SEQ ID NO 146 &211s LENGTH: 31 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Description of Artificial Sequence: Synthetic peptide 22 Os. FEATURE: <221s NAME/KEY: MOD RES <222s. LOCATION: (2) ... (2) <223> OTHER INFORMATION: Gly, Ala, Val, Leu, Ile, Ser, or Thr 22 Os. FEATURE: <221s NAME/KEY: MOD RES <222s. LOCATION: (16) ... (16) <223> OTHER INFORMATION: Gly, Asp, Glu, Gln, Asn., Lys, Arg, Cys, or cysteic acid (Cya) 22 Os. FEATURE: <221s NAME/KEY: MOD RES <222s. LOCATION: (17) . . (17) <223> OTHER INFORMATION: His, Asp, Lys, Glu, or Glin 22 Os. FEATURE: <221s NAME/KEY: MOD RES <222s. LOCATION: (21) ... (21) <223> OTHER INFORMATION: Ala, Glu, His, Phe, Tyr, Trp, Arg, or Lys 22 Os. FEATURE: <221s NAME/KEY: MOD RES <222s. LOCATION: (24) . . (24) <223> OTHER INFORMATION: Ala, Glu, Asp, Ser, or His 22 Os. FEATURE: <221s NAME/KEY: MOD RES <222s. LOCATION: (31) ... (31) <223> OTHER INFORMATION: Lys, Arg, Thr, Ser, Glu, Asp, Trp, Tyr, Phe, His, -NH2, Gly, Gly-Pro, Gly-Pro-NH2, or absent

<4 OOs, SEQUENCE: 146 His Xaa Glu Gly Thr Phe Thir Ser Asp Val Ser Ser Tyr Lieu. Glu Xaa 1. 5 1O 15

Xaa Ala Ala Lys Xaa Phe Ile Xaa Trp Lieu Val Lys Gly Arg Xaa 2O 25 3O

<210s, SEQ ID NO 147 &211s LENGTH: 31 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Description of Artificial Sequence: Synthetic US 9, 161988 B2 147 148 - Continued peptide 22 Os. FEATURE: <221s NAME/KEY: MOD RES <222s. LOCATION: (1) . . (1) <223> OTHER INFORMATION: L-His D-His desamino-His 2amino - His beta hydroxy-His, homo-His, alpha-fluoromethyl-His or alpha-methyl-His 22 Os. FEATURE: <221s NAME/KEY: MOD RES <222s. LOCATION: (2) ... (2) <223> OTHER INFORMATION: Gly, Ala, Val, Leu, Ile, Ser or Thr 22 Os. FEATURE: <221s NAME/KEY: MOD RES <222s. LOCATION: (16) ... (16) <223> OTHER INFORMATION: Asp, Glu, Gln, Asn., Lys, Arg, Cys, or cysteic acid (Cya) 22 Os. FEATURE: <221s NAME/KEY: MOD RES <222s. LOCATION: (16) ... (16) <223> OTHER INFORMATION: Asp, Glu, Gln, Asn., Lys, Arg, Cys, or cysteic acid (Cya) 22 Os. FEATURE: <221s NAME/KEY: MOD RES <222s. LOCATION: (31) ... (31) <223> OTHER INFORMATION: Gly or absent 22 Os. FEATURE: <221s NAME/KEY: MOD RES <222s. LOCATION: (31) ... (31) <223> OTHER INFORMATION: C-termimus hydroxylation, when Gly at position 31 is present, or amidation, when Gly at position 31 is absent

<4 OOs, SEQUENCE: 147 Xaa Xala Glu Gly Thr Phe Thir Ser Asp Wall Asp Asp Tyr Lieu. Glu Xaa 1. 5 1O 15 Glin Ala Ala Lys Glu Phe Ile Ala Trp Lieu Val Lys Gly Arg Xaa 2O 25 30

<210s, SEQ ID NO 148 &211s LENGTH: 31 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Description of Artificial Sequence: Synthetic peptide 22 Os. FEATURE: <221s NAME/KEY: MOD RES <222s. LOCATION: (1) . . (1) <223> OTHER INFORMATION: L-His D-His desamino-His 2-amino - His beta hydroxy-His, homohistidine, alpha-fluoromethyl-Hills, or alpha methyl-His 22 Os. FEATURE: <221s NAME/KEY: MOD RES <222s. LOCATION: (2) ... (2) <223> OTHER INFORMATION: Ala, Gly, Val, Leu, Ile, Ser, or Thr 22 Os. FEATURE: <221 > NAMEAKEY OD RES <222s. LOCATION: (6) . . (6) <223> OTHER INFORMATION: Phe, Trp, or Tyr 22 Os. FEATURE: <221s NAME/KEY: MOD RES <222s. LOCATION: (10) ... (10) <223> OTHER INFORMATION: Val, Trp, Ile, Leu, Phe, or Tyr 22 Os. FEATURE: <221s NAME/KEY: MOD RES <222s. LOCATION: (12) ... (12) <223> OTHER INFORMATION: Ser, Trp, Tyr, Phe, Lys, Ile, Leu, or Val 22 Os. FEATURE: <221s NAME/KEY: MOD RES <222s. LOCATION: (13) . . (13) <223> OTHER INFORMATION: Tyr, Trp, or Phe 22 Os. FEATURE: <221s NAME/KEY: MOD RES <222s. LOCATION: (14) . . (14) <223> OTHER INFORMATION: Leu, Phe, Tyr, or Trp 22 Os. FEATURE: <221s NAME/KEY: MOD RES <222s. LOCATION: (16) ... (16) US 9, 161988 B2 149 150 - Continued OTHER INFORMATION: Gly, Glu, Asp, or Lys FEATURE: NAME/KEY: MOD RES LOCATION: (19) . . (19) OTHER INFORMATION: Ala Wall, Ile, or Lieu. FEATURE: NAME/KEY: MOD RES LOCATION: (21) . . (21) OTHER INFORMATION: Glu, Ile, or Ala FEATURE: NAME/KEY: MOD RES LOCATION: (24) . . (24) OTHER INFORMATION: Ala or Gul FEATURE: NAME/KEY: MOD RES LOCATION: (27) . . (27) OTHER INFORMATION: Val, or Ile FEATURE: NAME/KEY: MOD RES LOCATION: (31) . . (31) OTHER INFORMATION: Gly, His, or absent FEATURE: NAME/KEY: MOD RES LOCATION: (31) . . (31) OTHER INFORMATION: C-terminus amidation, when Gly at position 31 is absent

<4 OOs, SEQUENCE: 148 Xaa Xala Glu Gly Thr Xaa Thir Ser Asp Xaa Ser Xaa Xaa Xala Glu Xaa 1. 5 1O 15 Glin Ala Xala Lys Xaa Phe Ile Xaa Trp Lieu. Xaa Lys Gly Arg Xaa

<210 SEQ ID NO 149 LENGTH: 31 TYPE PRT ORGANISM: Artificial Sequence FEATURE: OTHER INFORMATION: Description of Artificial Sequence: Synthetic peptide FEATURE: NAME/KEY: MOD RES LOCATION: (1) ... (1) OTHER INFORMATION: L-His D-His desamino - His 2-amino-His beta hydroxy-His, homohistidine, alpha-fluoromethyl-Hills, or alpha methyl-His FEATURE: NAME/KEY: MOD RES LOCATION: (2) ... (2) OTHER INFORMATION: Gly, Ala, Val, Leu, Ile, Ser, or Thr FEATURE: NAME/KEY: MOD RES LOCATION: (10) . . (10) OTHER INFORMATION: Val, Phe, Tyr, or Trp FEATURE: NAME/KEY: MOD RES LOCATION: (12) ... (12) OTHER INFORMATION: Ser, Tyr, Trp, Phe, Lys, Ile, Leu, or Val FEATURE: NAME/KEY: MOD RES LOCATION: (16) . . (16) 2 2 3 OTHER INFORMATION: Gly, Glu, Asp, or Lys FEATURE: NAME/KEY: MOD RES LOCATION: (19) . . (19) OTHER INFORMATION: Ala Wall, Ile, or Lieu. FEATURE: NAME/KEY: MOD RES LOCATION: (27) . . (27) OTHER INFORMATION: Wall or Ile FEATURE: NAME/KEY: MOD RES LOCATION: (31) . . (31) OTHER INFORMATION: Gly or absent FEATURE: NAME/KEY: MOD RES US 9, 161988 B2 151 152 - Continued

<222s. LOCATION: (31) ... (31) <223> OTHER INFORMATION: C-terminus amidation, when Gly at position 31 is absent

<4 OOs, SEQUENCE: 149 Xaa Xala Glu Gly Thr Phe Thir Ser Asp Xaa Ser Xaa Tyr Lieu. Glu Xaa 1. 5 1O 15 Glin Ala Xala Lys Glu Phe Ile Ala Trp Lieu. Xaa Lys Gly Arg Xaa 2O 25 3O

<210s, SEQ ID NO 150 &211s LENGTH: 13 212. TYPE: PRT <213> ORGANISM: Homo sapiens <4 OOs, SEQUENCE: 150 Glin Lieu. Tyr Glu Asn Llys Pro Arg Arg Pro Tyr Ile Lieu. 1. 5 1O

<210s, SEQ ID NO 151 &211s LENGTH: 8 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Description of Artificial Sequence: Synthetic peptide

<4 OOs, SEQUENCE: 151 Phe Pro Lieu Pro Ala Gly Lys Arg 1. 5

<210s, SEQ ID NO 152 &211s LENGTH: 8 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Description of Artificial Sequence: Synthetic peptide 22 Os. FEATURE: <221s NAME/KEY: MOD RES <222s. LOCATION: (5) . . (5) <223> OTHER INFORMATION: Ala, Leu, Ile, or Val 22 Os. FEATURE: <221s NAME/KEY: MOD RES <222s. LOCATION: (6) . . (6) <223> OTHER INFORMATION: Gly, any amino acid residue of the D configuration, or absent 22 Os. FEATURE: <221s NAME/KEY: MOD RES <222s. LOCATION: (7) . . (7) <223> OTHER INFORMATION: Lys, Arg, His, or absent 22 Os. FEATURE: <221s NAME/KEY: MOD RES <222s. LOCATION: (8) ... (8) <223> OTHER INFORMATION: Arg, Lys, His, or absent <4 OOs, SEQUENCE: 152

Phe Pro Lieu Pro Xaa Xaa Xaa Xala 1. 5

<210s, SEQ ID NO 153 &211s LENGTH: 8 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Description of Artificial Sequence: Synthetic peptide 22 Os. FEATURE: <221s NAME/KEY: MOD RES <222s. LOCATION: (3) ... (3) US 9, 161988 B2 153 154 - Continued

<223> OTHER INFORMATION: Leu, Ala, Ile, or Val 22 Os. FEATURE: <221s NAME/KEY: MOD RES <222s. LOCATION: (6) . . (6) <223> OTHER INFORMATION: Gly, any amino acid residue of the D configuration, or absent 22 Os. FEATURE: <221s NAME/KEY: MOD RES <222s. LOCATION: (7) . . (7) <223> OTHER INFORMATION: Lys, Arg, His, or absent 22 Os. FEATURE: <221s NAME/KEY: MOD RES <222s. LOCATION: (8) ... (8) <223> OTHER INFORMATION: Arg, Lys, His, or absent <4 OOs, SEQUENCE: 153

Phe Pro Xala Pro Ala Xaa Xaa Xala 1. 5

<210s, SEQ ID NO 154 &211s LENGTH: 8 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Description of Artificial Sequence: Synthetic peptide 22 Os. FEATURE: <221s NAME/KEY: MOD RES <222s. LOCATION: (6) . . (6) <223> OTHER INFORMATION: Gly, any amino acid residue of the D configuration, or absent 22 Os. FEATURE: <221s NAME/KEY: MOD RES <222s. LOCATION: (7) . . (7) <223> OTHER INFORMATION: Lys, Arg, His, or absent 22 Os. FEATURE: <221s NAME/KEY: MOD RES <222s. LOCATION: (8) ... (8) <223> OTHER INFORMATION: Arg, Lys, His, or absent <4 OOs, SEQUENCE: 154

Phe Pro Lieu Pro Ala Xaa Xaa Xala 1. 5

<210s, SEQ ID NO 155 &211s LENGTH: 8 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Description of Artificial Sequence: Synthetic peptide 22 Os. FEATURE: <221s NAME/KEY: MOD RES <222s. LOCATION: (3) ... (3) <223> OTHER INFORMATION: Leu, Ala, Ile, or Val 22 Os. FEATURE: <221s NAME/KEY: MOD RES <222s. LOCATION: (5) . . (5) <223> OTHER INFORMATION: Ala, Leu, Ile, or Val 22 Os. FEATURE: <221s NAME/KEY: MOD RES <222s. LOCATION: (7) . . (7) <223> OTHER INFORMATION: Lys, Arg, His, or absent 22 Os. FEATURE: <221s NAME/KEY: MOD RES <222s. LOCATION: (8) ... (8) <223> OTHER INFORMATION: Arg, Lys, His, or absent

<4 OO > SEQUENCE: 155 Phe Pro Xaa Pro Xaa Gly Xaa Xala 1. 5

<210s, SEQ ID NO 156 US 9, 161988 B2 155 156 - Continued

&211s LENGTH: 8 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Description of Artificial Sequence: Synthetic peptide 22 Os. FEATURE: <221s NAME/KEY: MOD RES <222s. LOCATION: (5) . . (5) <223> OTHER INFORMATION: Ala, Leu, Ile, or Val 22 Os. FEATURE: <221s NAME/KEY: MOD RES <222s. LOCATION: (7) . . (7) <223> OTHER INFORMATION: Lys, Arg, His, or absent 22 Os. FEATURE: <221s NAME/KEY: MOD RES <222s. LOCATION: (8) ... (8) <223> OTHER INFORMATION: Arg, Lys, His, or absent <4 OOs, SEQUENCE: 156 Phe Pro Lieu Pro Xaa Gly Xaa Xala 1. 5

<210s, SEQ ID NO 157 &211s LENGTH: 8 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Description of Artificial Sequence: Synthetic peptide 22 Os. FEATURE: <221s NAME/KEY: MOD RES <222s. LOCATION: (7) . . (7) <223> OTHER INFORMATION: Lys, Arg, His, or absent & 22 O FEATURE; <221s NAME/KEY: MOD RES <222s. LOCATION: (8) ... (8) <223> OTHER INFORMATION: Arg, Lys, His, or absent <4 OO > SEQUENCE: 157 Phe Pro Phe Pro Ala Gly Xaa Xaa 1. 5

<210s, SEQ ID NO 158 &211s LENGTH: 8 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Description of Artificial Sequence: Synthetic peptide 22 Os. FEATURE: <221s NAME/KEY: MOD RES <222s. LOCATION: (3) ... (3) <223> OTHER INFORMATION: Leu, Ala, Ile, or Val 22 Os. FEATURE: <221s NAME/KEY: MOD RES <222s. LOCATION: (5) . . (5) <223> OTHER INFORMATION: Ala, Leu, Ile, or Val 22 Os. FEATURE: <221s NAME/KEY: MOD RES <222s. LOCATION: (6) . . (6) <223> OTHER INFORMATION: Gly, any amino acid residue of the D configuration, or absent 22 Os. FEATURE: <221s NAME/KEY: MOD RES <222s. LOCATION: (8) ... (8) <223> OTHER INFORMATION: Arg, Lys, His, or absent

<4 OOs, SEQUENCE: 158 Phe Pro Xaa Pro Xaa Xaa Lys Xaa 1. 5

<210s, SEQ ID NO 159 US 9, 161988 B2 157 158 - Continued

&211s LENGTH: 8 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Description of Artificial Sequence: Synthetic

22 Os. FEATURE: <221s NAME/KEY: MOD RES <222s. LOCATION: (5) . . (5) <223> OTHER INFORMATION: Ala, Leu, Ile, or Val 22 Os. FEATURE: <221s NAME/KEY: MOD RES <222s. LOCATION: (6) . . (6) <223> OTHER INFORMATION: Gly, any amino acid residue of the D configuration, or absent 22 Os. FEATURE: <221s NAME/KEY: MOD RES <222s. LOCATION: (8) ... (8) <223> OTHER INFORMATION: Arg, Lys, His, or absent <4 OOs, SEQUENCE: 159 Phe Pro Lieu Pro Xaa Xaa Lys Xaa 1. 5

<210s, SEQ ID NO 160 &211s LENGTH: 8 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Description of Artificial Sequence: Synthetic peptide 22 Os. FEATURE: <221s NAME/KEY: MOD RES <222s. LOCATION: (6) . . (6) <223> OTHER INFORMATION: Gly, any amino acid residue of the D configuration, or absent 22 Os. FEATURE: <221s NAME/KEY: MOD RES <222s. LOCATION: (8) ... (8) <223> OTHER INFORMATION: Arg, Lys, His, or absent <4 OOs, SEQUENCE: 160 Phe Pro Lieu Pro Ala Xaa Lys Xaa 1. 5

<210s, SEQ ID NO 161 &211s LENGTH: 8 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Description of Artificial Sequence: Synthetic peptide 22 Os. FEATURE: <221s NAME/KEY: MOD RES <222s. LOCATION: (8) ... (8) <223> OTHER INFORMATION: Arg, Lys, His, or absent <4 OOs, SEQUENCE: 161 Phe Pro Lieu Pro Ala Gly Lys Xaa 1. 5

<210s, SEQ ID NO 162 &211s LENGTH: 8 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Description of Artificial Sequence: Synthetic peptide 22 Os. FEATURE: <221s NAME/KEY: MOD RES <222s. LOCATION: (3) ... (3) <223> OTHER INFORMATION: Leu, Ala, Ile, or Val 22 Os. FEATURE: US 9, 161988 B2 159 160 - Continued <221s NAME/KEY: MOD RES <222s. LOCATION: (5) . . (5) <223> OTHER INFORMATION: Ala, Leu, Ile, or Val 22 Os. FEATURE: <221s NAME/KEY: MOD RES <222s. LOCATION: (6) . . (6) <223> OTHER INFORMATION: Gly, any amino acid residue of the D configuration, or absent 22 Os. FEATURE: <221s NAME/KEY: MOD RES <222s. LOCATION: (7) . . (7) <223> OTHER INFORMATION: Lys, Arg, His, or absent <4 OOs, SEQUENCE: 162 Phe Pro Xaa Pro Xaa Xaa Xaa Arg 1. 5

<210s, SEQ ID NO 163 &211s LENGTH: 8 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Description of Artificial Sequence: Synthetic peptide 22 Os. FEATURE: <221s NAME/KEY: MOD RES <222s. LOCATION: (5) . . (5) <223> OTHER INFORMATION: Ala, Leu, Ile, or Val 22 Os. FEATURE: <221s NAME/KEY: MOD RES <222s. LOCATION: (6) . . (6) <223> OTHER INFORMATION: Gly, any amino acid residue of the D configuration, or absent 22 Os. FEATURE: <221. NAME/KEY: MOD RES <222s. LOCATION: (7) . . (7) <223> OTHER INFORMATION: Lys, Arg, His, or absent <4 OOs, SEQUENCE: 163 Phe Pro Lieu Pro Xaa Xaa Xaa Arg 1. 5

<210s, SEQ ID NO 164 &211s LENGTH: 8 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Description of Artificial Sequence: Synthetic peptide 22 Os. FEATURE: <221s NAME/KEY: MOD RES <222s. LOCATION: (6) . . (6) <223> OTHER INFORMATION: Gly, any amino acid residue of the D configuration, or absent 22 Os. FEATURE: <221s NAME/KEY: MOD RES <222s. LOCATION: (7) . . (7) <223> OTHER INFORMATION: Lys, Arg, His, or absent <4 OOs, SEQUENCE: 164 Phe Pro Lieu Pro Ala Xaa Xaa Arg 1. 5

<210s, SEQ ID NO 165 &211s LENGTH: 8 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Description of Artificial Sequence: Synthetic peptide 22 Os. FEATURE: <221s NAME/KEY: MOD RES <222s. LOCATION: (7) . . (7) US 9, 161988 B2 161 162 - Continued <223> OTHER INFORMATION: Lys, Arg, His, or absent <4 OOs, SEQUENCE: 165 Phe Pro Lieu Pro Ala Gly Xaa Arg 1. 5

<210s, SEQ ID NO 166 &211s LENGTH: 14 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Description of Artificial Sequence: Synthetic peptide

<4 OOs, SEQUENCE: 166 Pro Pro Glu Ala Pro Ala Glu Asp Arg Ser Lieu. Gly Arg Arg 1. 5 1O

<210s, SEQ ID NO 167 &211s LENGTH: 22 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Description of Artificial Sequence: Synthetic peptide

<4 OOs, SEQUENCE: 167 Arg Arg Glu Arg Asn Arg Glin Ala Ala Ala Ala Asn Pro Glu Asn. Ser 1. 5 1O 15 Arg Gly Lys Gly Arg Arg 2O

<210s, SEQ ID NO 168 &211s LENGTH: 17 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Description of Artificial Sequence: Synthetic peptide 22 Os. FEATURE: <221s NAME/KEY: MOD RES <222s. LOCATION: (17) . . (17) <223> OTHER INFORMATION: C-terminus amidation

<4 OOs, SEQUENCE: 168 Glu Arg Asn Arg Glin Ala Ala Ala Ala Asn. Pro Glu Asn. Ser Arg Gly 1. 5 1O 15 Lys

<210s, SEQ ID NO 169 &211s LENGTH: 5 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Description of Artificial Sequence: Synthetic peptide 22 Os. FEATURE: <221s NAME/KEY: MOD RES <222s. LOCATION: (5) . . (5) <223> OTHER INFORMATION: C-terminus amidation

<4 OOs, SEQUENCE: 169

Phe Pro Leu Pro Ala 1. 5

<210s, SEQ ID NO 170 &211s LENGTH: 11