US 20140322132A1 (19) United States (12) Patent Application Publication (10) Pub. No.: US 2014/0322132 A1 Vitalis et al. (43) Pub. Date: Oct. 30, 2014

(54) FRAGMENTS OF P97 AND USES THEREOF CI2N 9/96 (2006.01) C07K 7/06 (2006.01) (71) Applicant: biOasis Technologies, Inc., Richmond C07K 16/00 (2006.01) (CA) (52) U.S. Cl. O O CPC. C07K 14/47 (2013.01); C07K 7/06 (2013.01); (72) Inventors: Timothy Z. Vitalis, Vancouver (CA); C07K 7/08 (2013.01); C07K 16/00 (2013.01); Reinhard Gabathuler, Montreal (CA) C07K 14/565 (2013.01); C12N 9/96 (2013.01); A61K 49/0002 (2013.01) (21) Appl. No.: 14/210,029 USPC ...... 424/1.69; 530/327: 530/326; 530/328; 530/329; 530/324; 530/395; 530/3917; (22) Filed: Mar 13, 2014 530/351; 435/188: 514/21.7: 514/21.6: O O 514/21.5; 514/214: 514/21.3; 514/21.2: Related U.S. Application Data 514/17.7: 514/17.8: 514/17.9; 514/19.3: (60) Provisional application No. 61/780,170, filed on Mar. 514/19.4: 514/19.5: 514/18.3: 514/2.3: 514/2.6; 13, 2013, provisional application No. 61/885,387, 514/2.8: 514/2.7: 514/3.7: 514/4.2: 514/3.8; filed on Oct. 1, 2013. 424/9.1; 424/9.6; 424/9.34 Publication Classification (57) ABSTRACT Provided are fragments of human p97 (melanotransferrin) (51) Int. Cl. polypeptides having blood-brain barrier (BBB) transport C07K I4/47 (2006.01) activity, including variants and combinations thereof, conju C07K 7/08 (2006.01) gates comprising said p97 fragments, and related methods of A6 IK 49/00 (2006.01) use thereof, for instance, to facilitate delivery of therapeutic C07K I4/565 (2006.01) or diagnostic agents across the BBB. Patent Application Publication Oct. 30, 2014 Sheet 1 of 23 US 2014/0322132 A1

Patent Application Publication Oct. 30, 2014 Sheet 2 of 23 US 2014/0322132 A1

Patent Application Publication Oct. 30, 2014 Sheet 3 of 23 US 2014/0322132 A1

azeunfi!--

Patent Application Publication Oct. 30, 2014 Sheet 4 of 23 US 2014/0322132 A1

&&&&&&&&&&&&&&&&&&&&&&& Patent Applica ion Publiicat ion Oct. 30, 2014 Sheet 5 of 23 US 2014/0322132 A1

qz3infi!-- Patent Application Publication US 2014/0322132 A1

vºeun61-I

Ipueg

Patent Application Publication Oct 30, 2014 Sheet 8 of 23 US 2014/0322132 A1

ºocaun61-I



£pueg §$$$$$$$$$$$ Patent Application Publication Oct. 30, 2014 Sheet 9 of 23 US 2014/0322132 A1

wytyaun61-I

Patent Application Publication Oct. 30, 2014 Sheet 10 of 23 US 2014/0322132 A1

ayaun61-I Patent Application Publication Oct. 30, 2014 Sheet 11 of 23 US 2014/0322132 A1

wygeun61-I

----

zptræg Patent Application Publication Oct. 30, 2014 Sheet 12 of 23 US 2014/0322132 A1

Patent Application Publication Oct. 30, 2014 Sheet 13 of 23 US 2014/0322132 A1

v9eun6!--

Patent Application Publication Oct. 30, 2014 Sheet 14 of 23 US 2014/0322132 A1

£19aun61-I Patent Application Publication Oct. 30, 2014 Sheet 15 of 23 US 2014/0322132 A1

i

Patent Application Publication Oct. 30, 2014 Sheet 16 of 23 US 2014/0322132 A1

8eun61-I ·SE?EH?efuldCz?LJe??na(Hº)

Patent Application Publication Oct. 30, 2014 Sheet 19 of 23 US 2014/0322132 A1

------,

Patent Application Publication Oct. 30, 2014 Sheet 21 of 23 US 2014/0322132 A1

*paedød Patent Application Publication Oct. 30, 2014 Sheet 22 of 23 US 2014/0322132 A1

i Patent Application Publication Oct. 30, 2014 Sheet 23 of 23 US 2014/0322132 A1

i US 2014/0322132 A1 Oct. 30, 2014

FRAGMENTS OF P97 AND USES THEREOF 0010. In certain embodiments, the p97 polypeptide com prises one or both of SEQ ID NO:13 and/or 14, optionally CROSS-REFERENCE TO RELATED including intervening sequences as defined by SEQID NO:1. APPLICATION In certain embodiments, the p97 polypeptide is about or up to 0001. This application claims the benefit under 35 U.S.C about 250, 240, 230, 220, 210, 200, 190, 180, 170, 160, 150, S119(e) of U.S. Provisional Patent Application No. 61/780, 140, 130, 120, 110, 100,90, 80, 70, 60, 50, 40, 30, 25, 20, 15, 170 filed Mar. 13, 2013 and U.S. Provisional Patent Applica or 10 amino acids in length. tion 61/885,387 filed Oct. 1, 2013, each of which is incorpo 0011. In certain embodiments, the p97 polypeptide is rated herein by reference in its entirety. fused to a heterologous protein. 0012. Also included are conjugates, comprising the p97 STATEMENT REGARDING THE SEQUENCE polypeptide described herein, where the p97 polypeptide is LISTING covalently or operatively linked to an agent, to form a p97 agent conjugate. In certain embodiments, the agent is a small 0002 The Sequence Listing associated with this applica molecule, a polypeptide, a peptide mimetic, a peptoid, an tion is provided in text format in lieu of a paper copy, and is aptamer, or a detectable entity. hereby incorporated by reference into the specification. The 0013. In certain embodiments, the small molecule is a name of the text file containing the Sequence Listing is cytotoxic or chemotherapeutic or anti-angiogenic agent BIOA 002 02 US ST25.txt. The text file is about 40 KB, selected from one or more of alkylating agents, anti-metabo was created on Mar. 13, 2014, and is being submitted elec lites, anthracyclines, anti-tumor antibiotics, platinums, type I tronically via EFS-Web. topoisomerase inhibitors, type II topoisomerase inhibitors, Vinca alkaloids, and taxanes. In certain embodiments, the BACKGROUND small molecule is selected from one or more of chlorambucil, 0003 1. Technical Field cyclophosphamide, cilengitide, lomustine (CCNU), mel 0004. The present invention relates generally to fragments phalan, procarbazine, thiotepa, carmustine (BCNU), enZas of human p97 (melanotransferrin) polypeptides having trans taurin, buSulfan, daunorubicin, doxorubicin, gefitinib, erlo port activity, including variants and combinations thereof, tinib idarubicin, temozolomide, epirubicin, mitoxantrone, conjugates comprising said p97 fragments, and related meth bleomycin, cisplatin, carboplatin, oxaliplatin, camptoth ods of use thereof, for instance, to facilitate delivery of thera ecins, irinotecan, topotecan, amsacrine, etoposide, etoposide peutic and/or diagnostic agents across the blood-brain barrier phosphate, teniposide, temsirolimus, everolimus, Vincristine, (BBB) and into the central nervous system. vinblastine, vinorelbine, vindesine, CT52923, paclitaxel, 0005 2. Description of the Related Art imatinib, dasatinib, Sorafenib, paZopanib, Sunitnib, Vatalanib, 0006 Overcoming the difficulties of delivering therapeu ge?tinib, erlotinib, AEE-788, dichoroacetate, tamoxifen, tic or diagnostic agents to specific regions of the brain repre fasudil, SB-681323, semaxanib, donepizil, galantamine, sents a major challenge to treatment or diagnosis of many memantine, rivastigmine, tacrine, rasigiline, naltrexone, lubi central nervous system (CNS) disorders, including those of prostone, Safinamide, istradefylline, pimavanserin, pitolisant, the brain. In its neuroprotective role, the blood-brain barrier isradipine, pridopidine (ACR16), tetrabenazine, bexarotene, (BBB) functions to hinder the delivery of many potentially glatirimer acetate, fingolimod, and mitoxantrone, including important diagnostic and therapeutic agents to the brain. pharmaceutically acceptable salts and acids thereof. 0007. Therapeutic molecules and genes that might other 0014. In certain embodiments, the polypeptide is an anti wise be effective in diagnosis and therapy do not cross the body or antigen-binding fragment thereof, or an immunoglo BBB in adequate amounts. It is reported that over 95% of all bulin-like molecule. therapeutic molecules do not cross the blood-brain barrier. 0015. In certain embodiments, the antibody or antigen 0008 Accordingly, there is a need for compositions and binding fragment thereof specifically binds to a cancer-asso methods that facilitate the delivery of therapeutic agents and ciated antigen. In certain embodiments, the cancer-associated other molecules across the blood-brain-barrier, for instance, antigen is one or more of human Her2/neu, Her1/EGF recep to effectively treat certain diseases of the central nervous tor (EGFR), Her3, A33 antigen, B7H3, CD5, CD19, CD20, system (CNS) such as cancers, particularly those that have CD22. CD23 (IgE Receptor), C242 antigen, 5T4, IL-6, IL-13, metastasized to the CNS. The present invention addresses vascular endothelial growth factor VEGF (e.g., VEGF-A) these needs and offers other related advantages. VEGFR-1, VEGFR-2, CD30, CD33, CD37, CD40, CD44, CD51, CD52, CD56, CD74, CD80, CD152, CD200, CD221, CCR4, HLA-DR, CTLA-4, NPC-1C, tenascin, vimentin, BRIEF SUMMARY OF THE INVENTION insulin-like growth factor 1 receptor (IGF-1R), alpha-feto 0009 Embodiments of the present invention include iso protein, insulin-like growth factor 1 (IGF-1), carbonic anhy lated p97 (melanotransferrin) polypeptides of up to about drase 9 (CA-IX), carcinoembryonic antigen (CEA), integrin 300, 400, 500, 600, or 700 amino acids in length, where the Of3, integrin Os3, folate receptor 1, transmembrane glyco polypeptide comprises an amino acid sequence at least 70% protein NMB, fibroblast activation protein alpha (FAP), gly identical to any one or more of SEQID NO:2-18, or an active coprotein 75, TAG-72, MUC1, MUC16 (or CA-125), phos fragment or variant thereof. In certain embodiments, the p97 phatidylserine, prostate-specific membrane antigen (PMSA), polypeptide comprises one of SEQ ID NO:2-18, optionally NR-LU-13 antigen, TRAIL-R1, recep including adjacent C-terminal and/or N-terminal sequences tor superfamily member 10b (TNFRSF10B or TRAIL-R2), as defined by SEQ ID NO:1. In certain embodiments, the SLAM family member 7 (SLAMF7), EGP40 pancarcinoma polypeptide comprises 2, 3, 4, or 5 of SEQ ID NOS:2-18, antigen, B-cell activating factor (BAFF), platelet-derived optionally including any intervening sequences as defined by growth factor receptor, glycoprotein EpCAM (17-1A). Pro SEQID NO:1. grammed Death-1, protein disulfide isomerase (PDI), Phos US 2014/0322132 A1 Oct. 30, 2014

phatase of Regenerating Liver 3 (PRL-3), prostatic acid phos lumab, , Volociximab, Votumumab, and Zalutu phatase, Lewis-Y antigen, GD2 (a disialoganglioside mumab, including antigen-binding fragments thereof. expressed on tumors of neuroectodermal origin), glypican-3 0021. In certain embodiments, the polypeptide is an inter (GPC3), or mesothelin. feron-B polypeptide, or an active fragment or variant thereof. 0016. In certain embodiments, antibody or antigen-bind 0022. In certain embodiments, the polypeptide associates ing fragment thereof specifically binds to a pain-associated with a lysosomal storage disease. In certain embodiments, the antigen. In certain embodiments, the pain associated-antigen polypeptide is selected from one or more of aspartylglu is one or more of nerve growth factor (NGF) or tropomyosin cosaminidase, acid lipase, cysteine transporter, Lamp-2, related kinase A (TrkA). O-galactosidase A, acid ceramidase, C-L-fucosidase, B-hex 0017. In certain embodiments, the antibody or antigen osaminidase A, GM2-ganglioside activator (GM2A), C-D- binding fragment thereof or immunoglobulin-like molecule mannosidase, B-D-mannosidase, arylsulfatase A, saposin B, specifically binds to a pro-inflammatory molecule, optionally neuraminidase, Cl-N-acetylglucosaminidase phosphotrans a pro-inflammatory cytokine or chemokine. ferase, phosphotransferase Y-Subunit, L-iduronidase, idur 0018. In certain embodiments, the pro-inflammatory mol onate-2-sulfatase, heparan-N-sulfatase, Cl-N-acetylglu ecule is one or more of TNF-ct, TNF-B, FasL, CD27L, cosaminidase, acetylCoA:N-acetyltransferase, CD3OL, CD40L, Ox40L, 4-1 BBL, TRAIL, TWEAK, and N-acetylglucosamine 6-sulfatase, galactose 6-sulfatase, Apo3L, IL-1C., IL-1B, IL-2, interferon-Y (IFN-Y), IFN-C. B-galactosidase, N-acetylgalactosamine 4-sulfatase, hyalu IFN-B, IL-6, IL-8, IL-12, IL-15, IL-17, IL-18, IL-21, LIF, ronoglucosaminidase, Sulfatases, palmitoyl protein CCL5, GROC, MCP-1, MIP-1C., MIP-1 B, macrophage thioesterase, tripeptidyl peptidase I, acid sphingomyelinase, colony stimulating factor (MCSF), or granulocyte macroph cathepsin A, cathepsin K. C.-galactosidase B, NPC1, NPC2, age colony stimulating factor (GM-CSF). In certain embodi sialin, and sialic acid transporter, including active fragments ments, the pro-inflammatory molecule is TNF-C., and the and variants thereof. antibody or immunoglobulin-like molecule is adalimumab, 0023. In certain embodiments, the detectable entity is certolizumab pegol, etanercept, golimumab, infliximab, selected from one or more of diatrizoic acid, a radioisotope, a D2E7, CDP571, or CDP870, oran antigen-binding fragment fluorophore/fluorescent dye, and a nanoparticle. or variant thereof. 0024. In certain embodiments, the agent is a cardiotoxic 0019. In certain embodiments, the antibody or antigen agent in its unconjugated form. In certain embodiments, the binding fragment thereof specifically binds to one or more of cardiotoxic agent is an anthracycline/anthraquinolone, cyclo human Her2/neu, Her1/EGFR, TNF-C., B7H3 antigen, CD20, phosphamide, antimetabolite, antimicrotubule agent, VEGF, CD52, CD33, CTLA-4, tenascin, alpha-4 (C4) inte tyrosine kinase inhibitor, , or . In grin, IL-23, amyloid-fi, Huntingtin, CD25, nerve growth fac certain embodiments, the cardiotoxic agent is cyclopentenyl tor (NGF), TrkA, or C-synuclein. cytosine, 5-fluorouracil, capecitabine, paclitaxel, docataxel, 0020. In certain embodiments, the antibody is selected adriamycin, doxorubucin, epirubicin, emetine, isotamide, from one or more of trastuzumab, , daclizumab, mitomycin C, erlotinib, gefitinib, imatinib, Sorafenib, Suni tanezumab, , 8H9, , , afutu tinib, cisplatin, thalidomide, busulfan, vinblastine, bleomy Zumab, , alacizumab (pegol), , cin, Vincristine, arsenic trioxide, methotrexate, rosiglitaZone, apolizumab, , , belimumab, bevaci or mitoxantrone. Zumab, bivatuZumab (mertansine), , 0025. Some embodiments include compositions (e.g., cantuzumab (mertansine), cantuzumab (ravitansine), capro pharmaceutical compositions, therapeutic compositions, mab (pendetide), , citatuZumab (bogatox), diagnostic compositions), comprising a p97 protein or con , clivatuZumab (tetraxetan), , jugate described herein, and a pharmaceutically acceptable or , , , , pharmaceutical grade carrier. , , , , 0026. Also included are methods of treating a subject in , epratuZumab, , , far need thereof, comprising administering to the Subject a p97 letuzumab, FBTA05, , , galiximab, conjugate or composition described herein. gemtuzumab, , gemtuzumab (oZogamicin), giren 0027 Certain embodiments include methods for treating a tuximab, glembatumumab (vedotin), , cancer of the central nervous system (CNS), optionally the , igovomab, , intetu brain. Certain embodiments include methods for treating pri mumab, , (MDX-101), mary cancer of the CNS, optionally the brain. Certain , , , , lorvo embodiments include methods for treating a metastatic can tuZumab (mertansine), , lumiliximab, mapatu cer of the CNS, optionally the brain. Certain embodiments mumab, , , , mogamuli include methods for treating a glioma, meningioma, pituitary Zumab, moxetumomab (pasudotox), nacolomab (tafenatox), adenoma, Vestibular Schwannoma, primary CNS lymphoma, naptumomab (estafenatox), , , neuroblastoma, or primitive neuroectodermal tumor , , Neuradiab(R) (with or without (medulloblastoma). In some embodiments, the glioma is an radioactive iodine), NR-LU-10. , , astrocytoma, oligodendroglioma, ependymoma, or a choroid , oportuZumab (monatox), , panitu plexus papilloma. Certain embodiments include methods for mumab, , , , pritu treating glioblastoma multiforme. In some embodiments, the mumab, , , , rilotu glioblastoma multiforme is a giant cell gliobastoma or a mumab, , , samalizumab, gliosarcoma. , , tabalumab, taplitumomab (pap 0028 Certain embodiments include methods for treating a tox), , , TGN1412, ticilimumab, lysosomal storage disease. In some embodiments, the lyso tremelimumab, tigatuzumab, TNX-650, to situmomab, Somal storage disease is selected from one or more of aspar TRBS07, tucotuzumab (celmoleukin), , ure tylglucosaminuria, cholesterolester storage disease, Wolman US 2014/0322132 A1 Oct. 30, 2014

disease, cystinosis, Danon disease, Fabry disease, Farber encephalitis, parasitic infection, an inflammatory demyele lipogranulomatosis, Farber disease, fucosidosis, galactosiali niating disorder, a CD8+ T Cell-mediated autoimmune dis dosis types I/II, Gaucher disease types I/II/III, Gaucher dis ease of the CNS, Parkinson's disease, myasthenia gravis, ease, globoid cell leucodystrophy, Krabbe disease, glycogen motor neuropathy, Guillain-Barre Syndrome, autoimmune storage disease II, Pompe disease, GM1-gangliosidosis types neuropathy, Lambert-Eaton myasthenic syndrome, paraneo I/II/III, GM2-gangliosidosis type I, Tay Sachs disease, GM2 plastic neurological disease, paraneoplastic cerebellar atro gangliosidosis type II, Sandhoff disease, GM2-gangliosido phy, non-paraneoplastic stiffman syndrome, progressive cer sis, C.-mannosidosis types I/II, B-mannosidosis, metachro ebellar atrophy, Rasmussen's encephalitis, amyotrophic matic leucodystrophy, mucolipidosis type I, Sialidosis types lateral sclerosis, Sydeham chorea, Gilles de la Tourette syn I/II mucolipidosis types II/III I-cell disease, mucolipidosis drome, autoimmune polyendocrinopathy, dysimmune neur type IIIC pseudo-Hurler polydystrophy, mucopolysacchari opathy, acquired neuromyotonia, arthrogryposis multiplex, dosis type I, mucopolysaccharidosis type II (Hunter Syn optic neuritis, stroke, traumatic brain injury (TBI), spinal drome), mucopolysaccharidosis type IIIA, Sanfilippo Syn Stenosis, acute spinal cord injury, and spinal cord compres drome, mucopolysaccharidosis type IIIB, S1O. mucopolysaccharidosis type IIIC, mucopolysaccharidosis 0035. In certain embodiments, the inflammatory condition type IIID, mucopolysaccharidosis type IVA, Morquio syn is associated with an infection of the central nervous system. drome, mucopolysaccharidosis type IVB. mucopolysaccha In certain embodiments, the infection is a bacterial infection ridosis type VI, mucopolysaccharidosis type VII, Sly Syn caused by one or more of group B streptococci (e.g., Subtypes drome, mucopolysaccharidosis type IX, multiple Sulfatase III), Streptococcus pneumoniae (e.g., serotypes 6, 9, 14, 18 deficiency, neuronal ceroid lipofuscinosis, CLN1 Batten dis and 23), Escherichia coli (e.g., carrying K1 antigen), Listeria ease, Niemann-Pick disease types NB, Niemann-Pick dis monocytogenes (e.g., serotype IVb), neisserial infection Such ease, Niemann-Pick disease type C1, Niemann-Pick disease as Neisseria meningitidis (meningococcus), staphylococcal type C2, pycnodysostosis, Schindler disease types I/II, Schin infection, heamophilus infection Such as Haemophilus influ dler disease, and sialic acid storage disease. enzae type B, Klebsiella, Mycobacterium tuberculosis, Tre 0029. Certain embodiments include methods for treating a ponema pallidum, or Borrelia burgdorferi. In certain embodi degenerative or autoimmune disorder of the central nervous ments, the infection is a viral infection caused by one or more system (CNS). In particular embodiments, the degenerative of an enterovirus, herpes simplex virus type 1 or 2, human or autoimmune disorder of the CNS is Alzheimer's disease, T-lymphotrophic virus, varicella Zoster virus, mumps virus, Huntington's disease, Parkinson’s disease, or multiple scle human immunodeficiency virus(HIV), or lymphocytic chori rosis (MS). omeningitis virus (LCMV). 0030. In some embodiments, the subject is undergoing 0036. In certain embodiments, the inflammatory condition therapy with an otherwise cardiotoxic agent. In certain is associated with a cancer of the CNS, optionally a malignant embodiments, the cardiotoxic agent is an anthracycline/an meningitis. thraquinolone, cyclophosphamide, antimetabolite, antimi 0037 Also included are methods for imaging an organ or crotubule agent, tyrosine kinase inhibitor, bevacizumab, or tissue component in a subject, comprising (a) administering trastuzumab. In particular embodiments, the cardiotoxic to the subject a human p97 polypeptide described herein, agent is cyclopentenyl cytosine, 5-fluorouracil, capecitabine, where the polypeptide is conjugated to a detectable entity, and paclitaxel, docataxel, adriamycin, doxorubucin, epirubicin, (b) visualizing the detectable entity in the subject. In certain emetine, isotamide, mitomycin C, erlotinib, gefitinib, ima embodiments, the organ or tissue compartment comprises the tinib, Sorafenib, Sunitinib, cisplatin, thalidomide, buSulfan, central nervous system. In certain embodiments, the organ or vinblastine, bleomycin, Vincristine, arsenic trioxide, methotr tissue compartment comprises the brain. In certain embodi exate, rosiglitaZone, or mitoxantrone. ments, visualizing the detectable entity comprises one or 0031. In certain embodiments, the subject has cancer. In more of fluoroscopy, projectional radiography, X-ray CT certain embodiments, the cancer is one or more of breast scanning, positron emission tomography (PET), single pho cancer, prostate cancer, gastrointestinal cancer, , ton emission computed tomography (SPECT), or magnetic , testicular cancer, head and neck cancer, stom resonance imaging (MRI). ach cancer, bladder cancer, , liver cancer, 0038. These and other aspects of the present invention will kidney cancer, squamous cell carcinoma, CNS or brain can become apparent upon reference to the following detailed cer, , non-melanoma cancer, thyroid cancer, description and attached drawings. All references disclosed endometrial cancer, an epithelial tumor, bone cancer, or a herein are hereby incorporated by reference in their entirety hematopoietic cancer. as if each was incorporated individually. 0032. In certain embodiments, administration of the con jugate reduces cardiotoxicity of the agent, relative to an BRIEF DESCRIPTION OF THE DRAWINGS unconjugated form of the agent. 0033 Certain embodiments include methods for treating 0039 FIG. 1 shows an SDS-PAGE analysis of CNBr-di pain. In some embodiments, the pain is acute pain, chronic gested human melanotransferrin (p97). pain, neuropathic pain, and/or central pain. 0040 FIGS. 2A-2D show a list ofp97 fragments identified 0034 Certain embodiments include methods for treating by MS/MS analysis of an in-solution trypsin digest of human an inflammatory condition. In some embodiments, the p97, and FIG. 2E shows the sequence coverage map of that inflammatory condition has a central nervous system compo analysis. nent. In certain embodiments, the inflammatory condition is 0041 FIG.3 shows the sequence coverage maps of the p97 one or more of meningitis, myelitis, encaphaloymyelitis, fragments identified by MS/MS analysis of a CNBr digest of arachnoiditis, sarcoidosis, granuloma, drug-induced inflam human p97. The 3 bands identified in the SDS-PAGE of FIG. mation, Alzheimer's disease, stroke, HIV-dementia, 1 were subject to trypsin digestion and LC-MS/MS analysis: US 2014/0322132 A1 Oct. 30, 2014

FIG. 3A shows the results for band 1, FIG. 3B shows the nostic arts, for instance, to improve transfer of agents across results for band 2, and FIG. 3C shows the results for band 3. the BBB. Also, by identifying the minimal fragments 0042 FIG. 4A shows the matching of the peptides required for BBB transport activity, certain aspects of the detected in band 1 to the amino acid sequence of human p97; present invention allow the use of smaller p97 polypeptides, the sequence coverage of the matched peptides is indicated in thereby reducing some of the difficulties associated with the bold. FIG. 4B lists the individual peptides along with certain synthesis/production, purification, and pharmaceutical for physical characteristics. mulation of larger polypeptides. 0043 FIG. 5A shows the matching of the peptides 0055. Other advantages and benefits will be apparent to detected in band 2 to the amino acid sequence of human p97; persons skilled in the art. the sequence coverage of the matched peptides is indicated in bold. FIG. 5B lists the individual peptides along with certain Definitions physical characteristics. 0056. Unless defined otherwise, all technical and scien 0044 FIG. 6A shows the matching of the peptides tific terms used herein have the same meaning as commonly detected in band 3 to the amino acid sequence of human p97; understood by those of ordinary skill in the art to which the the sequence coverage of the matched peptides is indicated in invention belongs. Although any methods and materials simi bold. FIG. 6B lists the individual peptides along with certain lar or equivalent to those described herein can be used in the physical characteristics. practice or testing of the present invention, preferred methods 004.5 FIG. 7 illustrates the in vitro model of the blood and materials are described. For the purposes of the present brain barrier (BBB), with endothelial cells on a filter (either a 3 or 4 um filter) in the luminal compartment to simulate the invention, the following terms are defined below. barrier from the blood to the central nervous system, and glial 0057 The articles “a” and “an are used herein to refer to cells in the abluminal compartment to simulate the central one or to more than one (i.e., to at least one) of the grammati nervous system. cal object of the article. By way of example, “an element' 0046 FIG. 8 shows a schematic of test protocols using the means one element or more than one element. in vitro model of the BBB. 0.058 By “about is meant a quantity, level, value, number, 0047 FIG.9 shows aYASPIN secondary structure predic frequency, percentage, dimension, size, amount, weight or tion (see Yin et al., Bioinformatics. 21:152-159, 2005) of length that varies by as much as 30, 25, 20, 15, 10,9,8,7,6, human soluble p97 (SEQID NO:91; residues 20-709 of SEQ 5, 4, 3, 2 or 1% to a reference quantity, level, value, number, ID NO: 1) along with some of the p97 peptide fragments frequency, percentage, dimension, size, amount, weight or identified as having significant transport activity in the in length. vitro model of the BBB. FIG.9A shows certain of the tryptic 0059. As used herein, the term “amino acid' is intended to digest peptide fragments that cross the BBB (underlined), and mean both naturally occurring and non-naturally occurring FIG. 9B shows three of the CNBr digest peptide fragments amino acids as well as amino acid analogs and mimetics. that cross the BBB (underlined). Naturally occurring amino acids include the 20 (L)-amino 0048 FIG. 10 shows the synthesis route for p97 (MTf)- acids utilized during protein biosynthesis as well as others antibody conjugates (see Example 3). Such as 4-hydroxyproline, hydroxylysine, desmosine, isodes mosine, homocysteine, citrulline and ornithine, for example. 0049 FIG. 11 shows the brain distribution of MTf-anti Non-naturally occurring amino acids include, for example, body conjugates and control proteins after administration to (D)-amino acids, norleucine, norvaline, p-fluorophenylala mice (see Example 3). nine, ethionine and the like, which are known to a person 0050 FIG. 12 shows the synthesis route for p97 (MTf)- skilled in the art. Amino acid analogs include modified forms HRP (12B) conjugates (see Example 4). of naturally and non-naturally occurring amino acids. Such 0051 FIGS. 13 A-13C show the results of three-dimen modifications can include, for example, Substitution or sional (3D) confocal microscopy that was performed to replacement of chemical groups and moieties on the amino evaluate brain biodistribution of test proteins. FIG. 13A acid or by derivatization of the amino acid. Amino acid shows the results for PBS, FIG. 13B shows the results for mimetics include, for example, organic structures which AF680-labeled HRP and FIG. 13C shows the results for exhibit functionally similar properties such as charge and AF680-labeled MTf-HRP conjugate. The arrows in FIG. charge spacing characteristic of the reference amino acid. For 13C highlight the AF680 fluorescence of the AF680-labeled example, an organic structure which mimics Arginine (Arg or MTT-HRP conjugate in brain tissues. R) would have a positive charge moiety located in similar molecular space and having the same degree of mobility as DETAILED DESCRIPTION OF THE INVENTION the e-amino group of the side chain of the naturally occurring 0052 Embodiments of the present invention are based Argamino acid. Mimetics also include constrained structures partly on the discovery of minimal fragments of human p97 So as to maintain optimal spacing and charge interactions of (melanotransferrin) having the ability to transport across the the amino acid or of the amino acid functional groups. Those blood-brain barrier (BBB). skilled in the art know or can determine what structures 0053 Hence, embodiments of the present invention relate constitute functionally equivalent amino acid analogs and to particular polypeptide fragments of human p97 and Vari amino acid mimetics. ants thereof, compositions that comprise the polypeptide 0060. Throughout this specification, unless the context fragments, conjugates or mixtures of p97 fragments having requires otherwise, the words “comprise.” “comprises.” and an attached or operatively linked agent of interest, and related “comprising will be understood to imply the inclusion of a methods of use, including methods of treatment, diagnosis, stated step or element or group of steps or elements but not the and testing. Such as medical imaging. exclusion of any other step or element or group of steps or 0054 The human p97 polypeptide fragments described elements. By "consisting of is meant including, and limited herein can find a variety of uses in the therapeutic and diag to, whatever follows the phrase “consisting of.” Thus, the US 2014/0322132 A1 Oct. 30, 2014 phrase “consisting of indicates that the listed elements are 1.6., 1.7. 1.8, etc.) the amount produced by no composition required or mandatory, and that no other elements may be (e.g., the absence of polypeptide of conjugate of the inven present. By "consisting essentially of is meant including any tion) or a control composition, sample or test Subject. A elements listed after the phrase, and limited to other elements “decreased' or “reduced amount is typically a “statistically that do not interfere with or contribute to the activity or action significant” amount, and may include a 1%, 2%. 3%, 4%. 5%, specified in the disclosure for the listed elements. Thus, the 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, phrase “consisting essentially of indicates that the listed 17%, 18%, 19%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, elements are required or mandatory, but that other elements 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% are optional and may or may not be present depending upon decrease in the amount produced by no composition or a whether or not they materially affect the activity or action of control composition, including all integers in between. As the listed elements. one non-limiting example, a control could compare the activ 0061 The term "conjugate' is intended to refer to the ity, Such as the amount or rate of transport/delivery across the entity formed as a result of covalent or non-covalent attach blood brain barrier, the rate and/or levels of distribution to ment or linkage of an agent or other molecule, e.g., a biologi central nervous system tissue, and/or the C for plasma, cally active molecule, to a p97 polypeptide. One example of central nervous system tissues, or any other systemic or a conjugate polypeptide is a “fusion protein' or “fusion peripheral non-central nervous system tissues, of a p97-agent polypeptide. that is, a polypeptide that is created through the conjugate relative to the agent alone. Other examples of com joining of two or more coding sequences, which originally parisons and “statistically significant amounts are described coded for separate polypeptides; translation of the joined herein. coding sequences results in a single, fusion polypeptide, typi 0067. In certain embodiments, the “purity” of any given cally with functional properties derived from each of the agent (e.g., a p97 conjugate Such as a fusion protein) in a separate polypeptides. composition may be specifically defined. For instance, cer 0062. As used herein, the terms “function' and “func tain compositions may comprise an agent that is at least 80%, tional and the like refer to a biological, enzymatic, or thera 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, peutic function. 99%, or 100% pure, including all decimals in between, as 0063 “Homology” refers to the percentage number of measured, for example and by no means limiting, by high amino acids that are identical or constitute conservative Sub pressure liquid chromatography (HPLC), a well-known form stitutions. Homology may be determined using sequence of column chromatography used frequently in biochemistry comparison programs such as GAP (Deveraux et al., Nucleic and analytical chemistry to separate, identify, and quantify Acids Research. 12, 387-395, 1984), which is incorporated compounds. herein by reference. In this way sequences of a similar or 0068. The terms “polypeptide' and “protein’ are used substantially different length to those cited herein could be interchangeably herein to refer to a polymer of amino acid compared by insertion of gaps into the alignment, such gaps residues and to variants and synthetic analogues of the same. being determined, for example, by the comparison algorithm Thus, these terms apply to amino acid polymers in which one used by GAP. or more amino acid residues are synthetic non-naturally 0064. By "isolated' is meant material that is substantially occurring amino acids, such as a chemical analogue of a or essentially free from components that normally accom corresponding naturally occurring amino acid, as well as to pany it in its native state. For example, an "isolated peptide' naturally-occurring amino acid polymers. The polypeptides or an "isolated polypeptide' and the like, as used herein, described herein are not limited to a specific length of the includes the in vitro isolation and/or purification of a peptide product; thus, peptides, oligopeptides, and proteins are or polypeptide molecule from its natural cellular environ included within the definition of polypeptide, and such terms ment, and from association with other components of the cell; may be used interchangeably herein unless specifically indi i.e., it is not significantly associated with in vivo Substances. cated otherwise. The polypeptides described herein may also 0065. The term “linkage.” “linker,” “linker moiety,” or “1” comprise post-expression modifications, such as glycosyla is used herein to refer to a linker that can be used to separate tions, acetylations, phosphorylations and the like, as well as a p97 polypeptide fragment from an agent of interest, or to other modifications known in the art, both naturally occurring separate a first agent from another agent, for instance where and non-naturally occurring. A polypeptide may be an entire two or more agents are linked to form a p97 conjugate. The protein, or a Subsequence, fragment, variant, or derivative linker may be physiologically stable or may include a releas thereof. able linker Such as an enzymatically degradable linker (e.g., 0069. A “physiologically cleavable' or “hydrolyzable” or proteolytically cleavable linkers). In certain aspects, the “degradable bond is a bond that reacts with water (i.e., is linker may be a peptide linker, for instance, as part of a p97 hydrolyzed) under physiological conditions. The tendency of fusion protein. In some aspects, the linker may be a non a bond to hydrolyze in water will depend not only on the peptide linker or non-proteinaceous linker. In some aspects, general type of linkage connecting two central atoms but also the linker may be particle. Such as a nanoparticle. on the Substituents attached to these central atoms. Appropri 0066. The terms “modulating and “altering include ate hydrolytically unstable or weak linkages include, but are “increasing. "enhancing or “stimulating as well as not limited to: carboxylate ester, phosphate ester, anhydride, “decreasing or “reducing, typically in a statistically signifi acetal, ketal, acyloxyalkyl ether, imine, orthoester, thioester, cantor a physiologically significant amount or degree relative thiol ester, carbonate, and hydrazone, peptides and oligo to a control. An "increased,” “stimulated' or "enhanced' nucleotides. amount is typically a 'statistically significant” amount, and 0070 A “releasable linker' includes, but is not limited to, may include an increase that is 1.1.1.2,2,3,4,5,6,7,8,9, 10, a physiologically cleavable linker and an enzymatically 15, 20, 30 or more times (e.g., 500, 1000 times) (including all degradable linker. Thus, a “releasable linker' is a linker that integers and decimal points in between and above 1, e.g., 1.5. may undergo either spontaneous hydrolysis, or cleavage by US 2014/0322132 A1 Oct. 30, 2014

Some other mechanism (e.g., enzyme-catalyzed, acid-cata parison window may comprise additions or deletions (i.e., lyzed, base-catalyzed, and so forth) under physiological con gaps) of about 20% or less as compared to the reference ditions. For example, a “releasable linker can involve an sequence (which does not comprise additions or deletions) elimination reaction that has a base abstraction of a proton, for optimal alignment of the two sequences. Optimal align (e.g., an ionizable hydrogen atom, Ha), as the driving force. ment of sequences for aligning a comparison window may be For purposes herein, a “releasable linker is synonymous conducted by computerized implementations of algorithms with a “degradable linker. An "enzymatically degradable (GAP, BESTFIT. FASTA, and TFASTA in the Wisconsin linkage' includes a linkage, e.g., amino acid sequence that is Genetics Software Package Release 7.0, Genetics Computer Subject to degradation by one or more enzymes, e.g., pepti Group, 575 Science Drive Madison, Wis., USA) or by inspec dases or proteases. In particular embodiments, a releasable tion and the best alignment (i.e., resulting in the highest linker has a halflife at pH 7.4, 25°C., e.g., a physiological pH, percentage homology over the comparison window) gener human body temperature (e.g., in vivo), of about 30 minutes, ated by any of the various methods selected. Reference also about 1 hour, about 2 hour, about 3 hours, about 4 hours, about may be made to the BLAST family of programs as for 5 hours, about 6 hours, about 12 hours, about 18 hours, about example disclosed by Altschul et al., Nucl. Acids Res. 24 hours, about 36 hours, about 48 hours, about 72 hours, or 25:3389, 1997. A detailed discussion of sequence analysis about 96 hours or less. can be found in Unit 19.3 of Ausubelet al., “Current Protocols 0071. The term “reference sequence” refers generally to a in Molecular Biology,” John Wiley & Sons Inc, 1994-1998, nucleic acid coding sequence, or amino acid sequence, to Chapter 15. which another sequence is being compared. All polypeptide 0074 By “statistically significant, it is meant that the and polynucleotide sequences described herein are included result was unlikely to have occurred by chance. Statistical as references sequences, including those described by name significance can be determined by any method known in the and those described in the Tables and the Sequence Listing. art. Commonly used measures of significance include the 0072 The terms “sequence identity” or, for example, com p-value, which is the frequency or probability with which the prising a “sequence 50% identical to.” as used herein, refer to observed event would occur, if the null hypothesis were true. the extent that sequences are identical on a nucleotide-by If the obtained p-value is smaller than the significance level, nucleotide basis or an amino acid-by-amino acid basis over a then the null hypothesis is rejected. In simple cases, the sig window of comparison. Thus, a “percentage of sequence nificance level is defined at a p-value of 0.05 or less. identity” may be calculated by comparing two optimally (0075. The term “solubility” refers to the property of a p97 aligned sequences over the window of comparison, determin polypeptide fragment or conjugate to dissolve in a liquid ing the number of positions at which the identical nucleic acid Solvent and form a homogeneous solution. Solubility is typi base (e.g., A, T, C, G, I) or the identical amino acid residue cally expressed as a concentration, either by mass of Solute (e.g., Ala, Pro, Ser, Thr, Gly, Val, Leu, Ile, Phe, Tyr, Trp, Lys, per unit volume of solvent (g of solute per kg of solvent, g per Arg, His, Asp, Glu, ASn, Gln, Cys and Met) occurs in both dL (100 mL), mg/ml, etc.), molarity, molality, mole fraction sequences to yield the number of matched positions, dividing or other similar descriptions of concentration. The maximum the number of matched positions by the total number of equilibrium amount of solute that can dissolve per amount of positions in the window of comparison (i.e., the window solvent is the solubility of that solute in that solvent under the size), and multiplying the result by 100 to yield the percent specified conditions, including temperature, pressure, pH, age of sequence identity. Included are nucleotides and and the nature of the solvent. In certain embodiments, solu polypeptides having at least about 50%, 55%, 60%. 65%, bility is measured at physiological pH, or other pH, for 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% example, at pH 5.0, pH 6.0, pH 7.0, or pH 7.4. In certain sequence identity to any of the reference sequences described embodiments, solubility is measured in water or a physiologi herein (see, e.g., Sequence Listing), typically where the cal buffer such as PBS or NaCl (with or without NaP). In polypeptide variant maintains at least one biological activity specific embodiments, solubility is measured at relatively of the reference polypeptide. lower pH (e.g., pH 6.0) and relatively higher salt (e.g., 500 0073 Terms used to describe sequence relationships mM NaCl and 10 mM NaP). In certain embodiments, solu between two or more polynucleotides or polypeptides include bility is measured in a biological fluid (solvent) such as blood “reference sequence.” “comparison window.' 'sequence or serum. In certain embodiments, the temperature can be identity.” “percentage of sequence identity, and “substantial about room temperature (e.g., about 20, 21, 22, 23, 24, 25°C.) identity.” A “reference sequence' is at least 12 but frequently or about body temperature (-37°C.). In certain embodiments, 15 to 18 and often at least 25 monomer units, inclusive of a p97 polypeptide or conjugate has a solubility of at least nucleotides and amino acid residues, in length. Because two about 0.1, 0.2,0.3, 0.4,0.5,0.6,0.7, 0.8, 0.9, 1,2,3,4, 5, 6, 7, polynucleotides may each comprise (1) a sequence (i.e., only 8,9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, or 30 mg/ml a portion of the complete polynucleotide sequence) that is at room temperature or at about 37°C. similar between the two polynucleotides, and (2) a sequence 0076 A“subject, as used herein, includes any animal that that is divergent between the two polynucleotides, sequence exhibits a symptom, or is at risk for exhibiting a symptom, comparisons between two (or more) polynucleotides are typi which can be treated or diagnosed with a p97 conjugate of the cally performed by comparing sequences of the two poly invention. Suitable subjects (patients) include laboratory ani nucleotides over a “comparison window” to identify and mals (such as mouse, rat, rabbit, or guinea pig), farm animals, compare local regions of sequence similarity. A "comparison and domestic animals or pets (such as a cat or dog). Non window' refers to a conceptual segment of at least 6 contigu human primates and, preferably, human patients, are ous positions, usually about 50 to about 100, more usually included. about 100 to about 150 in which a sequence is compared to a (0077 “Substantially” or “essentially” means nearly reference sequence of the same number of contiguous posi totally or completely, for instance, 95%, 96%, 97%, 98%, tions after the two sequences are optimally aligned. The com 99% or greater of some given quantity. US 2014/0322132 A1 Oct. 30, 2014

0078 “Substantially free” refers to the nearly complete or comprises, consists essentially of or consists of 2, 3, 4, or 5 of complete absence of a given quantity for instance, less than the p97 sequences set forth in SEQID NOS:2-18, optionally about 10%, 5%, 4%, 3%, 2%, 1%, 0.5% or less of some given including any intervening p97 sequences (i.e. p97 sequences quantity. For example, certain compositions may be "sub from SEQ ID NO:1 that lie between SEQ ID NOS:2-18, if stantially free of cell proteins, membranes, nucleic acids, present) (see also FIGS. 9A and 9B for the relationships endotoxins, or other contaminants. between SEQID NOS:2-18 in the primary structure of human 0079. “Treatment” or “treating,” as used herein, includes p97). As one example, ap97 polypeptide could comprise SEQ any desirable effect on the symptoms or pathology of a dis ID NO:13 and 14, optionally including any intervening p97 ease or condition, and may include even minimal changes or improvements in one or more measurable markers of the sequences from SEQID NO:1, or variants thereof. disease or condition being treated. “Treatment' or “treating I0086. In certain embodiments, a p97 polypeptide frag does not necessarily indicate complete eradication or cure of ment is about, at least about, or up to about 5, 6,7,8,9, 10, 11, the disease or condition, or associated symptoms thereof. The 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, Subject receiving this treatment is any Subject in need thereof. 29, 30, 31, 32,33,34, 35,36, 37,38, 39, 40, 41,42, 43,44, 45, Exemplary markers of clinical improvement will be apparent 46,47, 48,49, 50, 51, 52,53,54, 55,56, 57,58, 59, 60, 61, 62, to persons skilled in the art. 63,64, 65,66, 67,68, 69,70, 71,72, 73,74, 75,76, 77,78,79, 0080. The term “wild-type” refers to a gene or gene prod 80, 81, 82, 83, 84,85, 86, 87, 88, 89,90,91, 92,93, 94, 95, 96, uct that has the characteristics of that gene or gene product 97, 98, 100,105, 110, 115, 120, 125, 130, 135, 140,145, 150, when isolated from a naturally-occurring source. A wild type 155, 160, 165, 170, 175, 180, 185, 190, 195, 200, 210, 220, gene or gene product (e.g., a polypeptide) is that which is 230, 240, 250, 260, 270, 280, 290, 300, 310,320, 330, 340, most frequently observed in a population and is thus arbi 350, 360, 370, 380,390, 400, 410, 420, 430, 440, 450, 460, trarily designed the “normal” or “wild-type' form of the gene. 470, 480,490, 500,510,520, 530, 540, 550,560,570,580, p97 Polypeptide Sequences and Conjugates Thereof 590, 600, 610, 620, 630, 640, 650, 660, 670, 680, 690, 700. 700, 710, 720, 730 or more amino acids in length, including 0081 Embodiments of the present invention relate gener all integers and ranges in between, and which may comprise ally to polypeptide fragments of human p97 (melanotransfer all or a portion of the sequence of a reference p97 sequence rin; MTf), compositions that comprise Such fragments, and (see, e.g., Sequence Listing, Tables 1-7, Table B, FIGS. 2-6 conjugates thereof. In certain instances, the p97 polypeptide fragments described herein have transport activity, that is, and 9), including any adjacent N-terminal and/or C-terminal they are ability to transport across the blood-brain barrier sequences of a reference p97 fragment, as defined by SEQID (BBB). In particular embodiments, the p97 fragments are NO:1. covalently, non-covalently, or operatively coupled to an agent I0087. In certain embodiments, a p97 polypeptide frag of interest, such as a therapeutic, diagnostic, or detectable ment is about 5-700, 5-600, 5-500, 5-400, 5-300, 5-200, agent, to form a p97-agent conjugate. Specific examples of 5-100, 5-50, 5-40, 5-30, 5-25, 5-20, 5-15, 5-10, 10-700, agents include Small molecules and polypeptides, such as 10-600, 10-500, 10-400, 10-300, 10-200, 10-100, 10-50, antibodies, among other agents described herein and known 10-40, 10-30, 10-25, 10-20, 10-15, 20-700, 20-600, 20-500, in the art. Exemplary p97 polypeptide sequences and agents 20-400, 20-300, 20-200, 20-100, 20-50, 20-40, 20-30, 20-25, are described below. Also described are exemplary methods 30-700, 30-600, 30-500, 30-400, 30-300, 30-200, 30-100, and components, such as linker groups, for coupling a p97 30-50, 30-40, 40-700, 40-600, 40-500, 40-400, 40-300, polypeptide to an agent of interest. 40-200, 40-100, 40-50, 50-700, 50-600, 50-500, 50-400, I0082 p97 Sequences. 50-300, 50-200, 50-100, 60-700, 60-600, 60-500, 60-400, 0083. In some embodiments, a p97 polypeptide com 60-300, 60-200, 60-100, 60-70, 70-700, 70-600, 70-500, prises, consists essentially of, or consists of at least one of the 70-400, 70-300, 70-200, 70-100, 70-80, 80-700, 80-600, human p97 fragments identified in Tables 1-7, or FIG. 2-6 or 80-500, 80-400, 80-300, 80-200, 80-100, 80-90, 90-700, 9. In specific embodiments, a p97 polypeptide comprises, 90-600, 90-500, 90-400, 90-300, 90-200, 90-100, 100-700, consists essentially of, or consists of at least one of the human 100-600, 100-500, 100-400, 100-300, 100-250, 100-200, p97 sequence set forth in SEQID NOS:2-18. 100-150, 200-700, 200-600, 200-500, 200-400, 200-300, or 0084. In other specific embodiments, described in greater 200-250 amino acids in length, and comprises all or a portion detail below, a p97 polypeptide sequence comprises a of a reference p97 sequence (see, e.g., Sequence Listing, sequence having at least 70%, 75%, 80%, 85%, 90%. 95%, Tables 1-7, Table B, FIGS. 2-6 and 9), including any adjacent 96%, 97%, 98%, or 99% identity or homology, along its N-terminal and/or C-terminal sequences of a reference p97 length, to at least one of the human p97 fragments identified fragment, as defined by SEQID NO:1. in Tables 1-7, or FIG. 2-6 or 9. In some embodiments, a variant of a p97 polypeptide sequence comprises a sequence I0088 Certain embodiments comprise one or more p97 having at least 70%, 75%, 80%, 85%, 90%. 95%, 96%, 97%, fragments, for example, 2, 3, 4, or 5 fragments, as illustrated 98%, or 99% identity or homology, along its length, to at least by the formula X, where X is a p97 fragment described one of the human p97 sequence set forth in SEQID NOS:2- herein and n is an integer from 1-5. In specific embodiments, 18. X is DSSHAFTLDELR (SEQID NO:13). 0085. In some embodiments, the p97 polypeptide com I0089. In particular embodiments, the p97 fragment or prises, consists essentially of, or consists of 2, 3, 4, or 5 of the variant thereofhas the ability to cross the BBB, and optionally p97 fragments identified in Tables 1-7, or FIG. 2-6 or 9. transport an agent of interest across the BBB and into the optionally including any intervening p97 sequences (i.e. p97 central nervous system. In certain embodiments, the p97 frag sequences from SEQID NO:1 that lie between the fragments, ment or variant thereof is capable of specifically binding to a if present). In particular embodiments, the p97 polypeptide p97 receptor, an LRP1 receptor, and/or an LRP1B receptor. US 2014/0322132 A1 Oct. 30, 2014

0090. In some embodiments, the p97 fragment has one or 0099 Exemplary small molecules include cytotoxic, che more terminal (e.g., N-terminal, C-terminal) cysteines and/or motherapeutic, and anti-angiogenic agents, for instance, tyrosines, which can be added for conjugation and iodination, those that have been considered useful in the treatment of respectively. various cancers, including cancers of the central nervous 0091 Variants and fragments of reference p97 polypep system and cancers that have metastasized to the central ner tides and other reference polypeptides are described in Vous system. Particular classes of Small molecules include, greater detail below. without limitation, alkylating agents, anti-metabolites, p97 Conjugates. anthracyclines, anti-tumor antibiotics, platinums, type I 0092 topoisomerase inhibitors, type II topoisomerase inhibitors, 0093. As noted above, certain embodiments comprise a p97 polypeptide that is linked to an agent of interest, for Vinca alkaloids, and taxanes. instance, a Small molecule, a polypeptide (e.g., peptide, anti 0100 Specific examples of small molecules include body), a peptide mimetic, a peptoid, an aptamer, a detectable chlorambucil, cyclophosphamide, cilengitide, lomustine entity, or any combination thereof. Also included are conju (CCNU), melphalan, procarbazine, thiotepa, carmustine (BCNU), enzastaurin, busulfan, daunorubicin, doxorubicin, gates that comprise more than one agent of interest, for gefitinib, erlotinib idarubicin, temozolomide, epirubicin, instance, a p97 fragment conjugated to an antibody and a mitoxantrone, bleomycin, cisplatin, carboplatin, oxaliplatin, Small molecule. camptothecins, irinotecan, topotecan, amsacrine, etoposide, 0094 Covalent linkages are preferred, however, non-co etoposide phosphate, teniposide, temsirolimus, everolimus, Valent linkages can also be employed, including those that Vincristine, vinblastine, vinorelbine, vindesine, CT52923, utilize relatively strong non-covalent protein-ligand interac and paclitaxel, and pharmaceutically acceptable salts, acids tions, such as the interaction between biotin and avidin. Operative linkages are also included, which do not necessar or derivatives of any of the above. ily require a directly covalent or non-covalent interaction 0101 Additional examples of small molecules include between the p97 fragment and the agent of interest; examples those that target protein kinases for the treatment of nervous of Such linkages include liposome mixtures that comprise a system (e.g., CNS) disorders, including imatinib, dasatinib, p97 polypeptide and an agent of interest. Exemplary methods Sorafenib, paZopanib, Sunitnib, Vatalanib, geftinib, erlotinib, of generating protein conjugates are described herein, and AEE-788, dichoroacetate, tamoxifen, fasudil, SB-681323, other methods are well-known in the art. and semaxanib (5U5416) (see Chico et al., Nat Rev Drug Discov. 8:829-909, 2009). Examples of small molecules also 0095 Small Molecules. include donepizil, galantamine, memantine, rivastigmine, 0096. In particular embodiments, the p97 fragment is con tacrine, rasigiline, naltrexone, lubiprostone, Safinamide, jugated to a small molecule. A 'small molecule' refers to an istradefylline, pimavanserin, pitolisant, isradipine, pridopi organic compound that is of synthetic or biological origin dine (ACR16), tetrabenazine, and bexarotene (e.g., for treat (biomolecule), but is typically not a polymer. Organic com ing Alzheimer's Disease, Parkinson's Disease, Huntington's pounds refer to a large class of chemical compounds whose Disease); and glatirimer acetate, fingolimod, mitoxantrone molecules contain carbon, typically excluding those that con (e.g., for treating MS). Also included are pharmaceutically tain only carbonates, simple oxides of carbon, or cyanides. A acceptable salts, acids or derivatives of any of the above. “biomolecule' refers generally to an organic molecule that is 0102. Further examples of small molecules include alky produced by a living organism, including large polymeric lating agents such as thiotepa, cyclophosphamide (CY molecules (biopolymers) Such as peptides, polysaccharides, TOXANTM); alkyl sulfonates such as busulfan, improsulfan and nucleic acids as well, and Small molecules Such as pri and piposulfan; aziridines such as benzodopa, carboquone, mary secondary metabolites, lipids, phospholipids, glycolip meturedopa, and uredopa; ethylenimines and methy ids, Sterols, glycerolipids, vitamins, and hormones. A "poly lamelamines including altretamine, triethylenemelamine, tri mer” refers generally to a large molecule or macromolecule etylenephosphoramide, triethylenethiophosphaoramide and composed of repeating structural units, which are typically trimethylolomelamine; nitrogen mustards such as chloram connected by covalent chemical bond. bucil, chlornaphazine, cholophosphamide, estramustine, 0097. In certain embodiments, a small molecule has a ifosfamide, mechlorethamine, mechlorethamine oxide molecular weight of less than about 1000-2000 Daltons, typi hydrochloride, melphalan, novembichin, phenesterine, pred cally between about 300 and 700 Daltons, and including nimustine, trofosfamide, uracil mustard; nitroSureas such as about 50, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, carmustine, chlorozotocin, fotemustine, lomustine, nimus 500, 650, 600, 750, 700, 850, 800,950, 1000 or 2000 Daltons. tine, ranimustine; antibiotics such as aclacinomysins, actino 0098 Certain small molecules can have the “specific bind mycin, authramycin, azaserine, bleomycins, cactinomycin, ing characteristics described for antibodies (infra). For calicheamicin, carabicin, carminomycin, carzinophilin, chro instance, a small molecule can specifically bind to a target momycins, dactinomycin, daunorubicin, detorubicin, described herein with a binding affinity (K) of at least about 6-diazo-5-oxo-L-norleucine, doxorubicin, epirubicin, esoru 0.01, 0.05, 0.1, 0.2,0.3, 0.4,0.5,0.6,0.7, 0.8, 0.9, 1, 2, 3, 4, bicin, idarubicin, marcellomycin, mitomycins, mycophe 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, nolic acid, nogalamycin, olivomycins, peplomycin, potfiro 23, 24, 25, 26, 27, 28, 29, 30, 40, or 50 nM. In certain mycin, puromycin, quelamycin, rodorubicin, Streptonigrin, embodiments a small specifically binds to a cell Surface streptozocin, tubercidin, ubenimex, Zinostatin, Zorubicin; receptor or other cell Surface protein. In some embodiments, anti-metabolites such as methotrexate and 5-fluorouracil the Small molecule specifically binds to at least one cancer (5-FU); folic acid analogues such as denopterin, methotrex associated antigen described herein. In particular embodi ate, pteropterin, trimetrexate; purine analogs such as fludara ments, the Small molecule specifically binds to at least one bine, 6-mercaptopurine, thiamiprine, thioguanine; pyrimi nervous system-associated, pain-associated, and/or autoim dine analogs such as ancitabine, azacitidine, 6-azauridine, mune-associated antigen described herein. carmofur, cytarabine, dideoxyuridine, doxifluridine, enocit US 2014/0322132 A1 Oct. 30, 2014

abine, floXuridine, 5-FU; androgens such as calusterone, dro Such polypeptides include aspartylglucosaminidase, acid mostanolone propionate, epitiostanol, mepitioStane, testolac lipase, cysteine transporter, Lamp-2, C-galactosidase A, acid tone; anti-adrenals such as aminoglutethimide, mitotane, ceramidase, C-L-fucosidase, 3-hexosaminidase A, GM2 triloStane; folic acid replenisher Such as frolinic acid; acegla ganglioside activator (GM2A), C-D-mannosidase, B-D-man tone; aldophosphamide glycoside; aminolevulinic acid; nosidase, arylsulfatase A, saposin B, neuraminidase, Cl-N- amsacrine; bestrabucil; bisantrene; ediatraxate, defofamine; acetylglucosaminidase phosphotransferase, demecolcine; diaziquone; elformithine; elliptinium acetate; phosphotransferase Y-subunit, L-iduronidase, iduronate-2- etoglucid, gallium nitrate; hydroxyurea; lentinan; Sulfatase, heparan-N-sulfatase, Cl-N-acetylglucosaminidase, lonidamine; mitoguaZone, mitoxantrone; mopidamol; nitra acetylCoA:N-acetyltransferase, N-acetylglucosamine 6-sul crine; pentostatin; phenamet; pirarubicin; podophyllinic fatase, galactose 6-sulfatase, B-galactosidase, N-acetylgalac acid; 2-ethylhydrazide; procarbazine; PSK; razoxane: sizofi tosamine 4-sulfatase, hyaluronoglucosaminidase, Sulfatases, ran; Spirogermanium; tenuaZonic acid; triaziquone; 2.2.2"- palmitoyl protein thioesterase, tripeptidyl peptidase I, acid trichlorotriethylamine, urethan; vindesine; dacarbazine; sphingomyelinase, cathepsin A, cathepsin K, C-galactosidase mannomustine, mitobronitol; mitolactol; pipobroman, gacy B, NPC1, NPC2, sialin, and sialic acid transporter, including tosine; arabinoside (Ara-C); cyclophosphamide; thiotepa; fragments, variants, and derivatives thereof. taxoids, e.g. paclitaxel (TAXOL(R), Bristol-Myers Squibb 0.108 Certain embodiments include polypeptides such as Oncology, Princeton, N.J.) and doxetaxel (TAXOTERER, interferon-B polypeptides, such as interferon-B1a (e.g., Rhine-Poulenc Rorer, Antony, France); chlorambucil; gemcit AVONEX, REBIF) and interferon-?31b (e.g., Betaseron), abine; 6-thioguanine; mercaptopurine; methotrexate; plati which are often used for the treatment of multiple sclerosis num analogs such as cisplatin and carboplatin: vinblastine; (MS). platinum: etoposide (VP-16); ifosfamide; mitomycin C: 0109. In some embodiments, as noted above, the polypep mitoxantrone; Vincristine; vinorelbine; navelbine; tide agent is an antibody or an antigen-binding fragment novantrone; teniposide; daunomycin; aminopterin: Xeloda; thereof. The antibody orantigen-binding fragment used in the ibandronate:CPT-11: topoisomerase inhibitor RFS 2000; dif conjugates or compositions of the present invention can be of luoromethylomithine (DMFO); retinoic acid derivatives such essentially any type. Particular examples include therapeutic as TargretinTM (bexarotene), PanretinTM (alitretinoin): and diagnostic antibodies. As is well known in the art, an ONTAKTM (denileukin diftitox); esperamicins; capecitabine; antibody is an immunoglobulin molecule capable of specific and pharmaceutically acceptable salts, acids or derivatives of binding to a target, Such as a carbohydrate, polynucleotide, any of the above. lipid, polypeptide, etc., through at least one epitope recogni 0103 Also included are anti-hormonal agents that act to tion site, located in the variable region of the immunoglobulin regulate or inhibit hormone action on tumors such as anti estrogens including for example tamoxifen, raloxifene, aro molecule. matase inhibiting 4(5)-imidazoles, 4-hydroxytamoxifen, tri 0110. As used herein, the term “antibody' encompasses oxifene, keoxifene, LY 1 17018, onapristone, and toremifene not only intact polyclonal or monoclonal antibodies, but also (Fareston); and anti-androgens such as flutamide, nilutamide, fragments thereof (such as dAb, Fab, Fab', F(ab'). Fv), single bicalutamide, leuprolide, and goserelin; and pharmaceuti chain (ScFV), synthetic variants thereof, naturally occurring cally acceptable salts, acids or derivatives of any of the above. variants, fusion proteins comprising an antibody portion with 0104. As noted above, in certain aspects the small mol an antigen-binding fragment of the required specificity, ecule is an otherwise cardiotoxic agent. Particular examples humanized antibodies, chimeric antibodies, and any other of cardiotoxic Small molecules include, without limitation, modified configuration of the immunoglobulin molecule that anthracyclines/anthraquinolones, cyclophosphamides, anti comprises an antigen-binding site or fragment (epitope rec metabolites, antimicrotubule agents, and tyrosine kinase ognition site) of the required specificity. inhibitors. Specific examples of cardiotoxic agents include 0111. The term “antigen-binding fragment as used herein cyclopentenyl cytosine, 5-fluorouracil, capecitabine, pacli refers to a polypeptide fragment that contains at least one taxel, docataxel, adriamycin, doxorubucin, epirubicin, emet CDR of an immunoglobulin heavy and/or light chains that ine, isotamide, mitomycin C, erlotinib, gefitinib, imatinib, binds to the antigen of interest. In this regard, an antigen Sorafenib, Sunitinib, cisplatin, thalidomide, buSulfan, Vin binding fragment of the herein described antibodies may blastine, bleomycin, Vincristine, arsenic trioxide, methotrex comprise 1,2,3,4, 5, or all 6 CDRs of a VHandVL sequence ate, rosiglitaZone, and mitoxantrone, among other Small mol from antibodies that bind to a therapeutic or diagnostic target. ecules described herein and known in the art. 0112 The term “antigen” refers to a molecule or a portion 0105 Polypeptide Agents. of a molecule capable of being bound by a selective binding 0106. In particular embodiments, the agent of interest is a agent, Such as an antibody, and additionally capable of being peptide or polypeptide. The terms “peptide' and “polypep used in an animal to produce antibodies capable of binding to tide' are used interchangeably herein, however, in certain an epitope of that antigen. An antigen may have one or more instances, the term "peptide' can refer to shorter polypep epitopes. tides, for example, polypeptides that consist of about 2, 3, 4, 0113. The term “epitope' includes any determinant, pref 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, erably a polypeptide determinant, capable of specific binding 35, 40, 45, or 50 amino acids, including all integers and to an immunoglobulin or T-cell receptor. An epitope is a ranges (e.g., 5-10, 8-12, 10-15) in between. Polypeptides and region of an antigen that is bound by an antibody. In certain peptides can be composed of naturally-occurring amino acids embodiments, epitope determinants include chemically and/or non-naturally occurring amino acids, as described active surface groupings of molecules Such as amino acids, herein. Antibodies are also included as polypeptides. Sugar side chains, phosphoryl or Sulfonyl, and may in certain 0107 Exemplary polypeptide agents include polypeptides embodiments have specific three-dimensional structural associated with lysosomal storage disorders. Examples of characteristics, and/or specific charge characteristics. US 2014/0322132 A1 Oct. 30, 2014

Epitopes can be contiguous or non-contiguous in relation to ments, the antibody or antigen-binding fragment or other the primary structure of the antigen. polypeptide specifically binds to an intracellular protein. 0114. A molecule such as an antibody is said to exhibit 0118. In certain embodiments, the antibody or antigen “specific binding or “preferential binding” if it reacts or binding fragment thereof or other polypeptide specifically associates more frequently, more rapidly, with greater dura binds to a cancer-associated antigen, or cancer antigen. tion and/or with greater affinity with a particular cell or sub Exemplary cancer antigens include cell Surface proteins such stance than it does with alternative cells or substances. An as cell Surface receptors. Also included as cancer-associated antibody “specifically binds” or “preferentially binds to a antigens are ligands that bind to Such cell Surface proteins or target if it binds with greater affinity, avidity, more readily, receptors. In specific embodiments, the antibody or antigen and/or with greater duration than it binds to other Substances. binding fragment specifically binds to a intracellular cancer For example, an antibody that specifically or preferentially antigen. In some embodiments, the cancer that associates binds to a specific epitope is an antibody that binds that with the cancer antigen is one or more of breast cancer, specific epitope with greater affinity, avidity, more readily, metastatic brain cancer, prostate cancer, gastrointestinal can and/or with greater duration than it binds to other epitopes. It cer, lung cancer, ovarian cancer, testicular cancer, head and is also understood by reading this definition that, for example, neck cancer, stomach cancer, bladder cancer, pancreatic can an antibody (or moiety or epitope) that specifically or prefer cer, liver cancer, kidney cancer, squamous cell carcinoma, entially binds to a first target may or may not specifically or CNS or brain cancer, melanoma, non-melanoma cancer, thy preferentially bind to a second target. As such, “specific bind roid cancer, endometrial cancer, epithelial tumor, bone can ing’ or "preferential binding does not necessarily require cer, or a hematopoietic cancer. (although it can include) exclusive binding. Generally, but not 0119. In particular embodiments, the antibody or antigen necessarily, reference to binding means preferential binding. binding fragment or other polypeptide specifically binds to at 0115 Immunological binding generally refers to the non least one cancer-associated antigen, or cancer antigen, Such as covalent interactions of the type which occur between an human Her2/neu, HerVEGF receptor (EGFR), Her3, A33 immunoglobulin molecule and an antigen for which the antigen, B7H3, CD5, CD19, CD20, CD22, CD23 (IgE immunoglobulin is specific, for example by way of illustra Receptor), C242 antigen, 5T4, IL-6, IL-13, vascular endot tion and not limitation, as a result of electrostatic, ionic, helial growth factor VEGF (e.g., VEGF-A) VEGFR-1, hydrophilic and/or hydrophobic attractions or repulsion, VEGFR-2, CD30, CD33, CD37, CD40, CD44, CD51, CD52, steric forces, hydrogen bonding, van der Waals forces, and CD56, CD74, CD80, CD152, CD200, CD221, CCR4, HLA other interactions. The strength, or affinity of immunological DR, CTLA-4, NPC-1C, tenascin, vimentin, insulin-like binding interactions can be expressed in terms of the disso growth factor 1 receptor (IGF-1R), alpha-fetoprotein, insu ciation constant (K) of the interaction, wherein a smaller K, lin-like growth factor 1 (IGF-1), carbonic anhydrase 9 (CA represents a greater affinity. Immunological binding proper IX), carcinoembryonic antigen (CEA), integrin C.B. integrin ties of selected polypeptides can be quantified using methods Os?, folate receptor 1, transmembrane glycoprotein NMB, well known in the art. One such method entails measuring the fibroblast activation protein alpha (FAP), glycoprotein 75, rates of antigen-binding site/antigen complex formation and TAG-72, MUC1, MUC16 (or CA-125), phosphatidylserine, dissociation, wherein those rates depend on the concentra prostate-specific membrane antigen (PMSA), NR-LU-13 tions of the complex partners, the affinity of the interaction, antigen, TRAIL-R1, tumor necrosis factor receptor Super and on geometric parameters that equally influence the rate in family member 10b (TNFRSF10B or TRAIL-R2), SLAM both directions. Thus, both the “on rate constant” (K) and family member 7 (SLAMF7), EGP40 pancarcinoma antigen, the “offrate constant” (K) can be determined by calculation B-cell activating factor (BAFF), platelet-derived growth fac of the concentrations and the actual rates of association and tor receptor, glycoprotein EpCAM (17-1A), Programmed dissociation. The ratio of K/K, enables cancellation of all Death-1, protein disulfide isomerase (PDI), Phosphatase of parameters not related to affinity, and is thus equal to the Regenerating Liver 3 (PRL-3), prostatic acid phosphatase, dissociation constant K. Lewis-Y antigen, GD2 (a disialoganglioside expressed on 0116. Immunological binding properties of selected anti tumors of neuroectodermal origin), glypican-3 (GPC3), and/ bodies and polypeptides can be quantified using methods well or mesothelin. known in the art (see Davies et al., Annual Rev. Biochem. I0120 In specific embodiments, the antibody or antigen 59:439-473, 1990). In some embodiments, an antibody or binding fragment thereof or other polypeptide specifically other polypeptide is said to specifically bind an antigen or binds to the human Her2/neu protein. Essentially any anti epitope thereof when the equilibrium dissociation constant is Her2/neu antibody, antigen-binding fragment or other Her2/ about s107 or 10 M. In some embodiments, the equilib neu-specific binding agent may be used in producing the rium dissociation constant of an antibody may be abouts10 p97-antibody conjugates of the present invention. Illustrative Mors10' M. In certain illustrative embodiments, an anti anti-Her2/neu antibodies are described, for example, in U.S. body or other polypeptide has an affinity (K) for an antigen Pat. Nos. 5,677,1715,720,937; 5,720,954; 5,725,856; 5,770, or target described herein (to which it specifically binds) of at 195; 5,772,997; 6,165,464; 6,387,371; and 6,399,063, the least about 0.01, 0.05, 0.1, 0.2,0.3, 0.4,0.5,0.6,0.7, 0.8, 0.9, contents of which are incorporated herein by reference in 1,2,3,4,5,6,7,8,9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, their entireties. 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 40, or 50 nM. I0121. In some embodiments, the antibody or antigen 0117. In some embodiments, the antibody or antigen binding fragment thereof or other polypeptide specifically binding fragment or other polypeptide specifically binds to a binds to the human Her1/EGFR (epidermal growth factor cell surface receptor or other cell surface protein. In some receptor). Essentially any anti-Her1/EGFR antibody, anti embodiments, the antibody or antigen-binding fragment or gen-binding fragment or other Her1-EGFR-specific binding other polypeptide specifically binds to a ligand of a cell Sur agent may be used in producing the p97-antibody conjugates face receptor or other cell surface protein. In some embodi of the present invention. Illustrative anti-Her1/EGFR anti US 2014/0322132 A1 Oct. 30, 2014

bodies are described, for example, in U.S. Pat. Nos. 5,844, composed of the Fv (variable; antigen-binding) regions of the 093; 7,132,511; 7,247,301; 7,595,378; 7,723,484; 7,939,072: 225 murine EGFR specific for the and 7,960,516, the contents of which are incorporated by N-terminal portion of human EGFR with human IgG1 heavy reference in their entireties. and kappa light chain constant (framework) regions. 0122. In certain embodiments, the antibody is a therapeu I0126. In some embodiments, the antibody or antigen tic antibody, such as an anti-cancer therapeutic antibody, binding fragment or other polypeptidespecifically binds to an including antibodies such as 3F8, 8H9, abagovomab, adeca antigen associated with (e.g., treatment of) at least one ner tumumab, afutuZumab, alemtuzumab, alacizumab (pegol), Vous system disorder, including disorders of the peripheral amatuximab, apolizumab, bavituximab, bectumomab, beli and/or central nervous system (CNS) disorder. In certain mumab, bevacizumab, bivatuZumab (mertansine), brentuX embodiments, the antibody or antigen-binding fragment or imab vedotin, cantuzumab (mertansine), cantuzumab other polypeptide specifically binds to an antigen associated (ravitansine), capromab (pendetide), catumaXomab, cetux with (e.g., treatment of) pain, including acute pain, chronic imab, citatuZumab (bogatox), cixutumumab, clivatuZumab pain, and neuropathic pain. In some embodiments, the anti (tetraxetan), conatumumab, dacetuZumab, dalotuzumab, body or antigen-binding fragment or other polypeptide spe detumomab, drozitumab, ecromeximab, edrecolomab, elotu cifically binds an antigen associated with (e.g., treatment of) Zumab, enavatuZumab, ensituximab, epratuZumab, ertumax an autoimmune disorder, including autoimmune disorders of omab, etaracizumab, , FBTA05, figitumumab, the nervous system or CNS. flanVotumab, galiximab, gemtuzumab, ganitumab, gemtu 0127 Examples of nervous system-, pain-, and/or autoim Zumab (oZogamicin), , glembatumumab (ve mune-associated antigens include, without limitation, dotin), ibritumomab tiuxetan, icrucumab, igovomab, alpha-4 (a4) integrin, CD20, CD52, IL-12, IL-23, the p40 indatuximab ravtansine, , inotuzumab ozogami subunit of IL-12 and IL-23, and the axonal regrowth and cin, ipilimumab (MDX-101), iratumumab, labetuzumab, remyelination inhibitors Nogo-A and LINGO, IL-23, amy lexatumumab, lintuZumab, lorVotuZumab (mertansine), luca loid-f(e.g., Afae), Huntingtin, CD25 (i.e., the alpha chain tumumab, lumiliximab, , matuZumab, milatu of the IL-2 receptor), nerve growth factor (NGF), neu Zumab, mitumomab, mogamuliZumab, moxetumomab (pa rotrophic tyrosine kinase receptor type 1 (TrkA; the high Sudotox), nacolomab (tafenatox), naptumomab affinity catalytic receptor for NGF), and C-synuclein. These (estafenatox), narnatumab, necitumumab, nimotuzumab, and other targets have been considered useful in the treatment nivolumab, Neuradiab(R) (with or without radioactive iodine), of a variety of nervous system, pain, and/or autoimmune NR-LU-10, ofatumumab, olaratumab, onartuzumab, oportu disorders, such as multiple sclerosis (C4 integrin, IL-23, Zumab (monatox), oregovomab, , patritumab, CD25, CD20, CD52, IL-12, IL-23, the p40 subunit of IL-12 pemtumomab, pertuZumab, , racotumomab, and IL-23, and the axonal regrowth and remyelination inhibi radretumab, ramucirumab, , rituximab, robatu tors Nogo-A and LINGO), Alzheimer's Disease (AB), Hun mumab, Samalizumab, sibrotuZumab, siltuximab, tabalumab, tington's Disease (Huntingtin), Parkinson's Disease (C-sy taplitumomab (paptoX), tenatumomab, teprotumumab, nuclein), and pain (NGF and TrkA). TGN1412, ticilimumab, tremelimumab, tigatuzumab, TNX I0128. In specific embodiments, the anti-CD25 antibody 650, to situmomab, TRBS07, trastuzumab, tucotuzumab (cel used in a p97 conjugate is daclizumab (i.e., ZenapaxTM), or a moleukin), ublituximab, urelumab, veltuzumab, Volocix fragment, variant or derivative thereof. Daclizumab a human imab, Votumumab, and . Also included are ized monoclonal antibody that specifically binds to CD25, the fragments, variants, and derivatives of these antibodies. alpha subunit of the IL-2 receptor. In other embodiments, the 0123. In particular embodiments, the antibody is a car antibody is rituximab, ocrelizumab. ofatumumab, or a variant diotoxic antibody, that is, an antibody that displays cardiotox or fragment thereof that specifically binds to CD20. In par icity when administered in an unconjugated form. Specific ticular embodiments, the antibody is alemtuzumab, or a vari examples of antibodies that display cardiotoxicity include ant or fragment thereof that specifically binds to CD52. In trastuzumab and bevacizumab. certain embodiments, the antibody is ustekinumab (CNTO 0.124. In specific embodiments, the anti-Her2/neu anti 1275), or a variant or fragment thereof that specifically binds body used in a p97 conjugate is trastuzumab (HerceptinR), or to the p40 subunit of IL-12 and IL-23. a fragment, variant or derivative thereof. Herceptin R) is a I0129. In specific embodiments, the anti-NGF antibody Her2/neu-specific monoclonal antibody approved for the used in a conjugate is taneZumab, or a fragment, variant or treatment of human breast cancer. In certain embodiments, a derivative thereof. Tanezumab specifically binds to NGF and Her2/neu-binding antigen-binding fragment comprises one prevents NGF from binding to its high affinity, membrane or more of the CDRs of a Her2/neu antibody. In this regard, it bound, catalytic receptor tropomyosin-related kinase A has been shown in some cases that the transfer of only the (TrkA), which is present on sympathetic and sensory neu VHCDR3 of an antibody can be performed while still retain rons; reduced stimulation of TrkA by NGF is believed to ing desired specific binding (Barbas et al., PNAS. 92: 2529 inhibit the pain-transmission activities of Such neurons. 2533, 1995). See also, McLane et al., PNAS USA. 92:5214 0.130. In some embodiments, the antibody or antigen 5218, 1995; and Barbas et al., J. Am. Chem. Soc. 116:2161 binding fragment thereof or other polypeptide (e.g., immu 2162, 1994. noglobulin-like molecule, soluble receptor, ligand) specifi (0.125. In other specific embodiments, the anti-Her1/EGFR cally binds to a pro-inflammatory molecule, for example, a antibody used in a conjugate of the invention is cetuximab pro-inflammatory cytokine or chemokine. In these and (ErbituxR), or a fragment or derivative thereof. In certain related embodiments, the p97 conjugate can be used to treat a embodiments, an anti-HerVEGFR binding fragment com variety of inflammatory conditions, as described herein. prises one or more of the CDRs of a HerVEGFR antibody Examples of pro-inflammatory molecules include tumor Such as cetuximab. Cetuximab is approved for the treatment necrosis factors (TNF) such as TNF-C. and TNF-B, TNF of head and neck cancer, and . Cetuximab is superfamily molecules such as FasL, CD27L, CD30L, US 2014/0322132 A1 Oct. 30, 2014

CD40L, Ox40L, 4-1BBL, TRAIL, TWEAK, and Apo3L, sity of the human antibody repertoire is represented by seven interleukin-1 (IL-1) including IL-1C. and IL-1B, IL-2, inter heavy chain and seven light chain variable region genes. The feron-Y (IFN-Y), IFN-C/B, IL-6, IL-8, IL-12, IL-15, IL-17, combination of these genes gives rise to 49 frameworks in the IL-18, IL-21, LIF, CCL5, GROC, MCP-1, MIP-1C., MIP-1B, master library. By Superimposing highly variable genetic cas macrophage colony Stimulating factor (MCSF), granulocyte settes (CDRS complementarity determining regions) on macrophage colony stimulating factor (GM-CSF), CXCL2. these frameworks, the vast human antibody repertoire can be CCL2, among others. In some embodiments, the antibody or reproduced. Also included are human libraries designed with antigen-binding fragment thereof specifically binds to a human-donor-sourced fragments encoding a light-chain vari receptor of one or more of the foregoing pro-inflammatory able region, a heavy-chain CDR-3, synthetic DNA encoding molecules, such as TNF receptor (TNFR), an IL-1 receptor diversity in heavy-chain CDR-1, and synthetic DNA encod (IL-1R), or an IL-6 receptor (IL-6R), among others. ing diversity in heavy-chain CDR-2. Other libraries suitable 0131. In specific embodiments, as note above, the anti for use will be apparent to persons skilled in the art. The p97 body or antigen-binding fragment or other polypeptide spe polypeptides described herein and known in the art may be cifically binds to TNF-C. or TNF-B. In particular embodi used in the purification process in, for example, an affinity ments, the anti-TNF antibody or other TNF-binding chromatography step. polypeptide is adalimumab (HumiraR), certolizumab pegol I0135) In certain embodiments, antibodies and antigen (CimziaR), etanercept (EnbrelR), golimumab (CimziaR), or binding fragments thereofas described herein include a heavy infliximab (Remicade(R), D2E7, CDP 571, or CDP870, oran chain and a light chain CDR set, respectively interposed antigen-binding fragment or variant thereof. In some embodi between a heavy chain and a light chain framework region ments, the TNF-binding polypeptide is a soluble receptor or (FR) set which provide support to the CDRs and define the ligand, such as TNRFSF10B, TRAIL (i.e., CD253), spatial relationship of the CDRs relative to each other. As TNFSF10, TRADD (tumor necrosis factor receptor type used herein, the term “CDR set' refers to the three hypervari 1-associated DEATH domain protein), TRAFs (TNF receptor able regions of a heavy or light chain V region. Proceeding associated factors, including TRAFS 1-7), or RIP (ribosome from the N-terminus of a heavy or light chain, these regions inactivating proteins). Conjugates comprising an anti-TNF are denoted as “CDR1,” “CDR2, and “CDR3 respectively. antibody or TNF-binding polypeptide can be used, for An antigen-binding site, therefore, includes six CDRS, com instance, in the treatment of various inflammatory conditions, prising the CDR set from each of a heavy and a light chain V as described herein. Such p97 conjugates can also be used in region. A polypeptide comprising a single CDR, (e.g., a the treatment of various neurological conditions or disorders CDR1, CDR2 or CDR3) is referred to herein as a “molecular Such as Alzheimer's disease, stroke, traumatic brain injury recognition unit.” Crystallographic analysis of a number of (TBI), spinal Stenosis, acute spinal cord injury, and spinal antigen-antibody complexes has demonstrated that the amino cord compression (see U.S. Pat. Nos. 6,015,557; 6,177,077; acid residues of CDRs form extensive contact with bound 6,419,934; 6,419,944; 6,537,549; 6,982,089; and 7,214,658). antigen, wherein the most extensive antigen contact is with 0.132. In specific embodiments, as note above, the anti the heavy chain CDR3. Thus, the molecular recognition units body orantigen-binding fragment specifically binds to IL-1C. are primarily responsible for the specificity of an antigen or IL-1B. In particularembodiments, the anti-IL-1 antibody is binding site. canakinumab or gevokizumab, or a variant or fragment 0.136. As used herein, the term “FR set refers to the four thereofthat specifically binds to IL-1B. Among other inflam flanking amino acid sequences which frame the CDRS of a matory conditions described herein, p97 conjugates compris CDR set of a heavy or light chain V region. Some FR residues ing an anti-IL-1 antibody can be used to treat cryopyrin may contact bound antigen; however, FRS are primarily associated periodic syndromes (CAPS), including familial responsible for folding the V region into the antigen-binding cold autoinflammatory syndrome, Muckle-Wells syndrome, site, particularly the FR residues directly adjacent to the and neonatal-onset multisystem inflammatory disease. CDRs. Within FRs, certain amino residues and certain struc 0.133 Antibodies may be prepared by any of a variety of tural features are very highly conserved. In this regard, all V techniques known to those of ordinary skill in the art. See, region sequences contain an internal disulfide loop of around e.g., Harlow and Lane, Antibodies. A Laboratory Manual. 90 amino acid residues. When the V regions fold into a bind Cold Spring Harbor Laboratory, 1988. Monoclonal antibod ing-site, the CDRS are displayed as projecting loop motifs ies specific for a polypeptide of interest may be prepared, for which form an antigen-binding Surface. It is generally recog example, using the technique of Kohler and Milstein, Eur: J. nized that there are conserved structural regions of FRs which Immunol. 6:51 1-519, 1976, and improvements thereto. Also influence the folded shape of the CDR loops into certain included are methods that utilize transgenic animals such as “canonical structures—regardless of the precise CDR amino mice to express human antibodies. See, e.g., Neuberger et al., acid sequence. Further, certain FR residues are known to Nature Biotechnology 14:826, 1996: Lonberg et al., Hand participate in non-covalent interdomain contacts which sta book of Experimental Pharmacology 1 13:49-101, 1994; and bilize the interaction of the antibody heavy and light chains. Lonberg et al., Internal Review of Immunology 13:65-93, 0.137 The structures and locations of immunoglobulin 1995. Particular examples include the VELOCIMMUNE(R) variable domains may be determined by reference to Kabat, platform by REGENEREX(R) (see, e.g., U.S. Pat. No. 6,596, E. A. et al., Sequences of Proteins of Immunological Interest. 541). 4th Edition. US Department of Health and Human Services. 0134) Antibodies can also be generated or identified by the 1987, and updates thereof. use of phage display or yeast display libraries (see, e.g., U.S. 0.138 A "monoclonal antibody” refers to a homogeneous Pat. No. 7,244,592: Chao et al., Nature Protocols. 1:755-768, antibody population wherein the monoclonal antibody is 2006). Non-limiting examples of available libraries include comprised of amino acids (naturally occurring and non-natu cloned or synthetic libraries, such as the Human Combinato rally occurring) that are involved in the selective binding of an rial Antibody Library (HuCAL), in which the structural diver epitope. Monoclonal antibodies are highly specific, being US 2014/0322132 A1 Oct. 30, 2014

directed against a single epitope. The term "monoclonal anti tide within the multimer with the second domain of another body encompasses not only intact monoclonal antibodies polypeptide within the multimer (WO94/13804). A dAb frag and full-length monoclonal antibodies, but also fragments ment of an antibody consists of a VH domain (Ward et al., thereof (such as Fab, Fab'. F(abl. Fv), single chain (Sclv), Nature 341:544-546, 1989). Dia bodies and other multivalent variants thereof, fusion proteins comprising an antigen-bind or multispecific fragments can be constructed, for example, ing portion, humanized monoclonal antibodies, chimeric by gene fusion (see WO94/13804; and Holliger et al., PNAS monoclonal antibodies, and any other modified configuration USA. 90:6444-6448, 1993)). of the immunoglobulin molecule that comprises an antigen 0.143 Minibodies comprising a schv joined to a CH3 binding fragment (epitope recognition site) of the required domain are also included (see Hu et al., Cancer Res. 56:3055 specificity and the ability to bind to an epitope. It is not 3061, 1996). See also Wardet al., Nature. 341:544-546, 1989: intended to be limited as regards the source of the antibody or Bird et al., Science. 242:423-426, 1988; Huston et al., PNAS the manner in which it is made (e.g., by hybridoma, phage USA. 85:5879-5883, 1988); PCT/US92/09965; WO94/ selection, recombinant expression, transgenic animals). The 13804; and Reiter et al., Nature Biotech. 14:1239-1245, 1996. term includes whole immunoglobulins as well as the frag 0144. Where bispecific antibodies are to be used, these ments etc. described above under the definition of “antibody.” may be conventional bispecific antibodies, which can be 0.139. The proteolytic enzyme papain preferentially manufactured in a variety of ways (Holliger and Winter, Cur cleaves IgG molecules to yield several fragments, two of rent Opinion Biotechnol. 4:446-449, 1993), e.g. prepared which (the F(ab) fragments) each comprise a covalent het chemically or from hybrid hybridomas, or may be any of the erodimer that includes an intact antigen-binding site. The bispecific antibody fragments mentioned above. Dia bodies enzyme pepsin is able to cleave IgG molecules to provide and ScHv can be constructed without an Fc region, using only several fragments, including the F(ab') fragment which com variable domains, potentially reducing the effects of anti prises both antigen-binding sites. An Fv fragment for use idiotypic reaction. according to certain embodiments of the present invention 0145 Bispecific diabodies, as opposed to bispecific whole can be produced by preferential proteolytic cleavage of an antibodies, may also be particularly useful because they can IgM, and on rare occasions of an IgG or IgA immunoglobulin be readily constructed and expressed in E. coli. Diabodies molecule. Fv fragments are, however, more commonly (and many other polypeptides such as antibody fragments) of derived using recombinant techniques known in the art. The appropriate binding specificities can be readily selected using FV fragment includes a non-covalent V::V, heterodimer phage display (WO94/13804) from libraries. If one arm of the including an antigen-binding site which retains much of the diabody is to be kept constant, for instance, with a specificity antigen recognition and binding capabilities of the native directed againstantigen X, then a library can be made where antibody molecule. See Inbar et al., PNAS USA. 69:2659 the other arm is varied and an antibody of appropriate speci 2662, 1972; Hochman et al., Biochem. 15:2706-2710, 1976: ficity selected. Bispecific whole antibodies may be made by and Ehrlich et al., Biochem. 19:4091-4096, 1980. knobs-into-holes engineering (Ridgeway et al., Protein Eng., 0140. In certain embodiments, single chain Fv or scFV 9:616-621, 1996). antibodies are contemplated. For example, Kappa bodies (III 0146 In certain embodiments, the antibodies described et al., Prot. Eng. 10:949-57, 1997); minibodies (Martin et al., herein may be provided in the form of a UniBody(R). A Uni EMBOJ 13:5305-9, 1994); diabodies (Holliger et al., PNAS Body(R) is an IgG4 antibody with the hinge region removed 90:6444-8, 1993); or Janusins (Trauneckeret al., EMBOJ10: (see GenMab Utrecht, The Netherlands; see also, e.g., 3655-59, 1991; and Traunecker et al., Int. J. Cancer Suppl. US20090226,421). This antibody technology creates a stable, 7:51-52, 1992), may be prepared using standard molecular Smaller antibody format with an anticipated longer therapeu biology techniques following the teachings of the present tic window than current Small antibody formats. IgG4 anti application with regard to selecting antibodies having the bodies are considered inert and thus do not interact with the desired specificity. immune system. Fully human IgG4 antibodies may be modi 0141. A single chain Fv (sEv) polypeptide is a covalently fied by eliminating the hinge region of the antibody to obtain linked V::V, heterodimer which is expressed from a gene half-molecule fragments having distinct stability properties fusion including V- and V-encoding genes linked by a relative to the corresponding intact IgG4 (GenMab, Utrecht). peptide-encoding linker. Huston et al. (PNAS USA. 85(16): Halving the IgG4 molecule leaves only one area on the Uni 5879-5883, 1988). A number of methods have been described Body(R) that can bind to cognate antigens (e.g., disease tar to discern chemical structures for converting the naturally gets) and the UniBody(R) therefore binds univalently to only aggregated—but chemically separated—light and heavy one site on target cells. For certain cancer cell Surface anti polypeptide chains from an antibody V region into an SFV gens, this univalent binding may not stimulate the cancer cells molecule which will fold into a three dimensional structure to grow as may be seen using bivalent antibodies having the Substantially similar to the structure of an antigen-binding same antigen specificity, and hence UniBody(R) technology site. See, e.g., U.S. Pat. Nos. 5,091,513 and 5,132,405, to may afford treatment options for Some types of cancer that Huston et al.; and U.S. Pat. No. 4,946,778, to Ladner et al. may be refractory to treatment with conventional antibodies. 0142. In certain embodiments, an antibody as described The small size of the UniBody(R) can be a great benefit when herein is in the form of a “dia body.” Diabodies are multimers treating some forms of cancer, allowing for better distribution of polypeptides, each polypeptide comprising a first domain of the molecule over larger Solid tumors and potentially comprising a binding region of an immunoglobulin light increasing efficacy. chain and a second domain comprising a binding region of an 0.147. In certain embodiments, the antibodies provided immunoglobulin heavy chain, the two domains being linked herein may take the form of a nanobody. Minibodies are (e.g. by a peptide linker) but unable to associate with each encoded by single genes and are efficiently produced in other to forman antigenbinding site: antigenbinding sites are almost all prokaryotic and eukaryotic hosts, for example, E. formed by the association of the first domain of one polypep coli (see U.S. Pat. No. 6,765,087), moulds (for example US 2014/0322132 A1 Oct. 30, 2014

Aspergillus or Trichoderma) and yeast (for example Saccha embodiments, the heterologous Fc domain is of human ori romyces, Kluyvermyces, Hansenula or Pichia (see U.S. Pat. gin. In other embodiments, the heterologous Fc domain may No. 6,838,254). The production process is scalable and multi be from a different Ig class from the parent antibody, includ kilogram quantities of nanobodies have been produced. ing IgA (including Subclasses IgA1 and IgA2), Ig|D, IgE, IgG Nanobodies may be formulated as a ready-to-use Solution (including Subclasses IgG1, IgG2, IgG3, and IgG4), and IgM. having a long shelf life. The Nanoclone method (see WO In further embodiments, the heterologous Fc domain may be 06/079372) is a proprietary method for generating Nanobod comprised of CH2 and CH3 domains from one or more of the ies against a desired target, based on automated high-through different Ig classes. As noted above with regard to humanized put selection of B-cells. antibodies, the antigen-binding fragment of a chimeric anti 0148. In certain embodiments, the antibodies or antigen body may comprise only one or more of the CDRs of the binding fragments thereof are humanized. These embodi antibodies described herein (e.g., 1,2,3,4, 5, or 6 CDRs of the ments refer to a chimeric molecule, generally prepared using antibodies described herein), or may comprise an entire vari recombinant techniques, having an antigen-binding site able domain (VL, VH or both). derived from an immunoglobulin from a non-human species 0151 Peptide Mimetics. and the remaining immunoglobulin structure of the molecule 0152 Certain embodiments employ "peptide mimetics.” based upon the structure and/or sequence of a human immu Peptide analogs are commonly used in the pharmaceutical noglobulin. The antigen-binding site may comprise either industry as non-peptide drugs with properties analogous to complete variable domains fused onto constant domains or those of the template peptide. These types of non-peptide only the CDRS grafted onto appropriate framework regions in compound are termed "peptide mimetics' or "peptidomimet the variable domains. Epitope binding sites may be wild type ics’ (Luthman et al., A Textbook of Drug Design and Devel or modified by one or more amino acid substitutions. This opment, 14:386-406, 2nd Ed., Harwood Academic Publish eliminates the constant region as an immunogen in human ers, 1996; Joachim Grante, Angew. Chem. Int. Ed. Engl., individuals, but the possibility of an immune response to the 33:1699-1720, 1994: Fauchere, Adv. Drug Res., 15:29, 1986: foreign variable region remains (LoBuglio et al., PNAS USA Veber and FreidingerTINS, p. 392 (1985); and Evans et al., J. 86:4220-4224, 1989; Queen et al., PNAS USA. 86:1.0029 Med. Chem. 30:229, 1987). A peptidomimetic is a molecule 10033, 1988: Riechmann et al., Nature. 332:323-327, 1988). that mimics the biological activity of a peptide but is no longer Illustrative methods for humanization of antibodies include peptidic in chemical nature. Peptidomimetic compounds are the methods described in U.S. Pat. No. 7,462,697. known in the art and are described, for example, in U.S. Pat. 0149 Another approach focuses not only on providing No. 6,245,886. human-derived constant regions, but modifying the variable 0153. A peptide mimetic can have the “specific binding regions as well so as to reshape them as closely as possible to characteristics described for antibodies (supra). For example, human form. It is known that the variable regions of both a peptide mimetic can specifically bind to a target described heavy and light chains contain three complementarity-deter herein with a binding affinity (K) of at least about 0.01, 0.05, mining regions (CDRS) which vary in response to the 0.1, 0.2,0.3, 0.4,0.5,0.6,0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, epitopes in question and determine binding capability, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, flanked by four framework regions (FRs) which are relatively 27, 28, 29, 30, 40, or 50 nM. In some embodiments a peptide conserved in a given species and which putatively provide a mimetic specifically binds to a cell surface receptor or other scaffolding for the CDRs. When nonhuman antibodies are cell Surface protein. In some embodiments, the peptide prepared with respect to a particular epitope, the variable mimetic specifically binds to at least one cancer-associated regions can be “reshaped or “humanized by grafting CDRs antigen described herein. In particular embodiments, the pep derived from nonhuman antibody on the FRs present in the tide mimetic specifically binds to at least one nervous system human antibody to be modified. Application of this approach associated, pain-associated, and/or autoimmune-associated to various antibodies has been reported by Sato et al., Cancer antigen described herein. Res. 53:851-856, 1993; Riechmann et al., Nature 332:323 0154 Peptoids. 327, 1988; Verhoeyen et al., Science 239:1534-1536, 1988: 0155 The conjugates of the present invention also Kettleborough et al., Protein Engineering. 4:773-3783, 1991; includes "peptoids.” Peptoid derivatives of peptides represent Maeda et al., Human Antibodies Hybridoma 2:124-134, another form of modified peptides that retain the important 1991; Gorman et al., PNAS USA. 88:4181 -4185, 1991; Tem structural determinants for biological activity, yet eliminate pest et al., Bio/Technology 9:266-271, 1991; Co et al., PNAS the peptide bonds, thereby conferring resistance to proteoly USA. 88:2869-2873, 1991; Carteret al., PNAS USA. 89:4285 sis (Simon, et al., PNAS USA. 89:9367-9371, 1992). Peptoids 4289, 1992; and Co et al., J Immunol. 148:1149-1154, 1992. are oligomers of N-substituted glycines. A number of N-alkyl In some embodiments, humanized antibodies preserve all groups have been described, each corresponding to the side CDR sequences (for example, a humanized mouse antibody chain of a natural amino acid. The peptidomimetics of the which contains all six CDRs from the mouse antibodies). In present invention include compounds in which at least one other embodiments, humanized antibodies have one or more amino acid, a few amino acids or all amino acid residues are CDRs (one, two, three, four, five, six) which are altered with replaced by the corresponding N-Substituted glycines. Pep respect to the original antibody, which are also termed one or toid libraries are described, for example, in U.S. Pat. No. more CDRS “derived from one or more CDRs from the 5,811,387. original antibody. 0156 A peptoid can have the “specific binding charac 0150. In certain embodiments, the antibodies of the teristics described for antibodies (Supra). For instance, a pep present invention may be chimeric antibodies. In this regard, toid can specifically bind to a target described herein with a a chimericantibody is comprised of an antigen-binding frag binding affinity (K) of at least about 0.01, 0.05, 0.1, 0.2,0.3, ment of an antibody operably linked or otherwise fused to a 0.4,0.5,0.6,0.7, 0.8, 0.9, 1,2,3,4,5,6,7,8,9, 10, 11, 12, 13, heterologous Fc portion of a different antibody. In certain 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, US 2014/0322132 A1 Oct. 30, 2014

40, or 50 nM. In certain embodiments a peptoid specifically decay, and levels of energy which can be tailored to match the binds to a cell surface receptor or other cell surface protein. In needs of a particular protocol. Certain of these radioisotopes Some embodiments, the peptoid specifically binds to at least can be selectively targeted or better targeted to CNS tissues by one cancer-associated antigen described herein. In particular conjugation to p97 polypeptides, for instance, to improve the embodiments, the peptoid specifically binds to at least one medical imaging of Such tissues. nervous system-associated, pain-associated, and/or autoim 0.165 Examples offluorophores or fluorochromes that can mune-associated antigen described herein. be used as directly detectable entities include fluorescein, (O157 Aptamers. tetramethylrhodamine, Texas Red, Oregon Green(R), and a 0158. The p97 conjugates of the present invention also number of others (e.g., Haugland, Handbook of Fluorescent include aptamers (see, e.g., Ellington et al., Nature. 346, Probes-9th Ed., 2002, Molec. Probes, Inc., Eugene Oreg.: 818-22, 1990; and Tuerket al., Science. 249,505-10, 1990). Haugland, The Handbook: A Guide to Fluorescent Probes Examples of aptamers include nucleic acid aptamers (e.g., and Labeling Technologies-10th Ed., 2005, Invitrogen, Carls DNA aptamers, RNA aptamers) and peptide aptamers. bad, Calif.). Also included are light-emitting or otherwise Nucleic acid aptamers refer generally to nucleic acid species detectable dyes. The light emitted by the dyes can be visible that have been engineered through repeated rounds of in vitro light or invisible light, such as ultraviolet or infrared light. In selection or equivalent method, such as SELEX (systematic exemplary embodiments, the dye may be a fluorescence reso evolution of ligands by exponential enrichment), to bind to nance energy transfer (FRET) dye, a Xanthene dye, Such as various molecular targets such as Small molecules, proteins, fluorescein and rhodamine; a dye that has an amino group in nucleic acids, and even cells, tissues and organisms. See, e.g., the alpha or beta position (Such as a naphthylamine dye, U.S. Pat. Nos. 6,376, 190; and 6,387,620. 1-dimethylaminonaphthyl-5-Sulfonate, 1-anilino-8-naphtha 0159 Peptide aptamers typically include a variable pep lende Sulfonate and 2-p-touidinyl-6-naphthalene Sulfonate); tide loop attached at both ends to a protein scaffold, a double a dye that has 3-phenyl-7-isocyanatocoumarin; an acridine, structural constraint that typically increases the binding affin Such as 9-isothiocyanatoacridine and acridine orange; a ity of the peptide aptamer to levels comparable to that of an pyrene, a bensoxadiazole and a stilbene; a dye that has 3-(e- antibody’s (e.g., in the nanomolar range). In certain embodi carboxypentyl)-3'-ethyl-5,5-dimethyloxacarbocyanine ments, the variable loop length may be composed of about (CYA); 6-carboxy fluorescein (FAM); 5&6-carbox 10-20 amino acids (including all integers in between), and the yrhodamine-110 (R110); 6-carboxyrhodamine-6G (R6G); scaffold may include any protein that has good solubility and N.N.N',N'-tetramethyl-6-carboxyrhodamine (TAMRA); compacity properties. Certain exemplary embodiments may 6-carboxy-X-rhodamine (ROX); 6-carboxy-4,5'-dichloro-2', utilize the bacterial protein Thioredoxin-A as a scaffold pro 7'-dimethoxyfluorescein (JOE); ALEXA FLUORTM; Cyt: tein, the variable loop being inserted within the reducing Texas Red and Rhodamine Red: 6-carboxy-2',4,7,7-tetra active site (-Cys-Gly-Pro-Cys-loop in the wild protein), with chlorofluorescein (TET); 6-carboxy-2,4,4',5',7,7-hexachlo the two cysteines lateral chains being able to form a disulfide rofluorescein (HEX); 5-carboxy-2',4',5'7"-tetrachlorofluo bridge. Methods for identifying peptide aptamers are rescein (ZOE): NAN; NED: Cy3; Cy3.5; Cy5; Cy5.5; Cy7; described, for example, in U.S. Application No. 2003/ and Cy7.5; IR800CW, ICG, Alexa Fluor 350; Alexa Fluor O108532. 488; Alexa Fluor 532; Alexa Fluor 546; Alexa Fluor 568; 0160 Anaptamer can have the “specific binding charac Alexa Fluor 594; Alexa Fluor 647: Alexa Fluor 680, or Alexa teristics described for antibodies (Supra). For instance, an Fluor 750. Certain embodiments include conjugation to che aptamer can specifically bind to a target described herein with motherapeutic agents (e.g., paclitaxel, adriamycin) that are a binding affinity (K) of at least about 0.01, 0.05, 0.1, 0.2, labeled with a detectable entity, Such as a fluorophore (e.g., 0.3, 0.4,0.5,0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, Oregon Green R, Alexa Fluor 488). 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 0166 Nanoparticles usually range from about 1-1000 nm 29, 30, 40, or 50 nM. In particular embodiments, an aptamer in size and include diverse chemical structures such as gold specifically binds to a cell surface receptor or other cell sur and silver particles and quantum dots. When irradiated with face protein. In some embodiments, the aptamer specifically angled incident white light, silver or gold nanoparticles rang binds to at least one cancer-associated antigen described ing from about 40-120 nm will scatter monochromatic light herein. In particular embodiments, the aptamer specifically with high intensity. The wavelength of the scattered light is binds to at least one nervous system-associated, pain-associ dependent on the size of the particle. Four to five different ated, and/or autoimmune-associated antigen described particles in close proximity will each scatter monochromatic herein. light, which when Superimposed will give a specific, unique (0161. Detectable Entities. color. Derivatized nanoparticles Such as silver or gold par 0162. In some embodiments, the p97 fragment is conju ticles can be attached to abroad array of molecules including, gated to a “detectable entity.” Exemplary detectable entities proteins, antibodies, Small molecules, receptor ligands, and include, without limitation, iodine-based labels, radioiso nucleic acids. Specific examples of nanoparticles include topes, fluorophores/fluorescent dyes, and nanoparticles. metallic nanoparticles and metallic nanoshells such as gold 0163 Exemplary iodine-based labels include diatrizoic particles, silver particles, copper particles, platinum particles, acid (Hypaque(R), GE Healthcare) and its anionic form, dia cadmium particles, composite particles, gold hollow spheres, trizoate. Diatrizoic acid is a radio-contrast agent used in gold-coated silica nanoshells, and silica-coated gold shells. advanced X-ray techniques such as CT scanning. Also Also included are silica, latex, polystyrene, polycarbonate, included are iodine radioisotopes, described below. polyacrylate, PVDF nanoparticles, and colored particles of 0164. Exemplary radioisotopes that can be used as detect any of these materials. able entities include P, P, S, H, N, O, '''In, Yb, 0.167 Quantum dots are fluorescing crystals about 1-5 mm "TC, Fe, and isotopes of iodine such as I, I, I, and in diameter that are excitable by light over a large range of ''I. These radioisotopes have different half-lives, types of wavelengths. Upon excitation by light having an appropriate US 2014/0322132 A1 Oct. 30, 2014

wavelength, these crystals emit light, such as monochromatic TABLE A- continued light, with a wavelength dependent on their chemical compo sition and size. Quantum dots such as CdSe, ZnSe, InP, or Amino Acids Codons InAS possess unique optical properties; these and similar Asparagine ASn N AAC AAU quantum dots are available from a number of commercial sources (e.g., NN-Labs, Fayetteville, Ark., Ocean Nanotech, Proline Pro P CCA CCC CCG CCU Fayetteville, Ark.; Nanoco Technologies, Manchester, UK: Sigma-Aldrich, St. Louis, Mo.). Glutamine Glin Q CAA CAG 0168 Polypeptide Variants and Fragments. Arginine Arg R AGA AGG CGA CGC CGG CGU 0169 Certain embodiments include variants and/or frag ments of the reference polypeptides described herein, Serine Ser S AGC AGU UCA, UCC UCG UCU whether described by name or by reference to a sequence Threonine Thir T ACA ACC ACG ACU identifier, including p97 polypeptides and polypeptide-based agents such as antibodies. The wild-type or most prevalent Waline Wall W. GUA GUC GUG GUU sequences of these polypeptides are known in the art, and can Tryptophan Trp W UGG be used as a comparison for the variants and fragments described herein. Tyrosine Tyr Y UAC UAU 0170 A polypeptide “variant, as the term is used herein, is a polypeptide that typically differs from a polypeptide 0172 For example, certainamino acids may be substituted specifically disclosed herein by one or more Substitutions, for other amino acids in a protein structure without appre deletions, additions and/or insertions. Variant polypeptides ciable loss of interactive binding capacity with structures are biologically active, that is, they continue to possess the Such as, for example, antigen-binding regions of antibodies or enzymatic or binding activity of a reference polypeptide. binding sites on Substrate molecules. Since it is the interactive Such variants may result from, for example, genetic polymor capacity and nature of a protein that defines that protein’s phism and/or from human manipulation. biological functional activity, certain amino acid sequence 0171 In many instances, a biologically active variant will Substitutions can be made in a protein sequence, and, of contain one or more conservative substitutions. A "conserva course, its underlying DNA coding sequence, and neverthe tive substitution' is one in which an amino acid is substituted less obtain a protein with like properties. It is thus contem for another amino acid that has similar properties, such that plated that various changes may be made in the peptide one skilled in the art of peptide chemistry would expect the sequences of the disclosed compositions, or corresponding secondary structure and hydropathic nature of the polypep DNA sequences which encode said peptides without appre tide to be substantially unchanged. As described above, modi ciable loss of their utility. fications may be made in the structure of the polynucleotides 0173. In making such changes, the hydropathic index of and polypeptides of the present invention and still obtain a amino acids may be considered. The importance of the hydro functional molecule that encodes a variant or derivative pathic amino acid index in conferring interactive biologic polypeptide with desirable characteristics. When it is desired function on a protein is generally understood in the art (Kyte to alter the amino acid sequence of a polypeptide to create an & Doolittle, 1982, incorporated herein by reference). It is equivalent, or even an improved, variant or portion of a accepted that the relative hydropathic character of the amino polypeptide of the invention, one skilled in the art will typi acid contributes to the secondary structure of the resultant cally change one or more of the codons of the encoding DNA protein, which in turn defines the interaction of the protein sequence according to Table A below. with other molecules, for example, enzymes, Substrates, receptors, DNA, antibodies, antigens, and the like. Each TABLE A amino acid has been assigned a hydropathic index on the basis of its hydrophobicity and charge characteristics (Kyte & Amino Acids Codons Doolittle, 1982). These values are: isoleucine (+4.5); valine Alanine Ala A. GCA GCC GCG GCU (+4.2); leucine (+3.8); phenylalanine (+2.8); cysteine (+2.5); methionine (+1.9); alanine (+1.8); glycine (-0.4); threonine Cysteine Cys C UGC UGU (-0.7); serine (-0.8); tryptophan (-0.9); tyrosine (-1.3); pro line (-1.6); histidine (-3.2); glutamate (-3.5); glutamine (-3. Aspartic acid Asp D GAC GAU 5); aspartate (-3.5); asparagine (-3.5); lysine (-3.9); and Glutamic acid Glu E GAA GAG arginine (-4.5). It is known in the art that certain amino acids may be substituted by other amino acids having a similar Phenylalanine Phe F UUC UUU hydropathic index or score and still result in a protein with Glycine Gly G GGA. GGC GGG GGU similar biological activity, i.e., still obtain a biological func tionally equivalent protein. In making such changes, the Sub Histidine His H CAC CAU stitution of amino acids whose hydropathic indices are within +2 is preferred, those within +1 are particularly preferred, and Isoleucine Ile I AUA AUC AUU those within +0.5 are even more particularly preferred. Lysine Llys K AAA AAG 0.174. It is also understood in the art that the substitution of like amino acids can be made effectively on the basis of Leucine Leu L UUA UUG. CUA CUC CUG. CUU hydrophilicity. U.S. Pat. No. 4,554,101 (specifically incorpo Methionine Met M AUG rated herein by reference in its entirety), states that the great est local average hydrophilicity of a protein, as governed by the hydrophilicity of its adjacentamino acids, correlates with US 2014/0322132 A1 Oct. 30, 2014

a biological property of the protein. As detailed in U.S. Pat. 0176 Amino acid substitutions may further be made on No. 4,554,101, the following hydrophilicity values have been the basis of similarity in polarity, charge, solubility, hydro assigned to amino acid residues: arginine (+3.0); lysine (+3. phobicity, hydrophilicity and/or the amphipathic nature of the residues. For example, negatively charged amino acids O); aspartate (+3.0+1); glutamate (+3.0+1); serine (+0.3); include aspartic acid and glutamic acid; positively charged asparagine (+0.2); glutamine (+0.2); glycine (O); threonine amino acids include lysine and arginine; and amino acids with (-0.4); proline (-0.5+1); alanine (-0.5); histidine (-0.5); cys uncharged polar head groups having similar hydrophilicity teine (-1.0); methionine (-1.3); valine (-1.5); leucine (-1.8); values include leucine, isoleucine and valine; glycine and isoleucine (-1.8); tyrosine (-2.3); phenylalanine (-2.5); tryp alanine; asparagine and glutamine; and serine, threonine, tophan (-3.4). It is understood that an amino acid can be phenylalanine and tyrosine. Other groups of amino acids that substituted for another having a similar hydrophilicity value may represent conservative changes include: (1) ala, pro, gly, and still obtain a biologically equivalent, and in particular, an glu, asp, gln, asn, ser, thr; (2) cys, ser, tyr, thr; (3) Val, ile, leu, immunologically equivalent protein. In Such changes, the met, ala, phe; (4) lys, arg, his; and (5) phe, tyr, trp, his. substitution of amino acids whose hydrophilicity values are 0177. A variant may also, or alternatively, contain non conservative changes. In a preferred embodiment, variant within +2 is preferred, those within +1 are particularly pre polypeptides differ from a native sequence by Substitution, ferred, and those within +0.5 are even more particularly pre deletion or addition offewer than about 10,9,8,7,6, 5, 4, 3, ferred. 2 amino acids, or even 1 amino acid. Variants may also (or 0.175. As outlined above, amino acid substitutions are gen alternatively) be modified by, for example, the deletion or erally therefore based on the relative similarity of the amino addition of amino acids that have minimal influence on the acid side-chain Substituents, for example, their hydrophobic immunogenicity, secondary structure, enzymatic activity, ity, hydrophilicity, charge, size, and the like. Exemplary Sub and/or hydropathic nature of the polypeptide. stitutions that take various of the foregoing characteristics 0.178 In certain embodiments, variants of the into consideration are well known to those of skill in the art DSSHAFTLDELR (SEQ ID NO:13) can be based on the and include: arginine and lysine; glutamate and aspartate; sequence of p97 sequences from other organisms, as shown in serine and threonine; glutamine and asparagine; and valine, Table B below. Variant amino acids relative to the human leucine and isoleucine. sequence are underlined. TABLE B

Common 3. SEO ID Name Species Protein Name Identity Sequence NO :

Human Homo Sapien Melanotransferrin 1 OO& DSSHAFTLDELR 13

Black- Saimiri Melanotransferrin 1 OO& DSSHAFTLDELR 13 capped boliviensis squirrel boliviensis monkey

Bonobo Pan paniscus Melanotransferrin 1 OO& DSSHAFTLDELR 13

Chimpanzee Pan troglodytes Melanotransferrin 1 OO& DSSHAFTLDELR 13

Crab-eating Macaca hypothetical protein 1 OO& DSSHAFTLDELR 13 macaque fascicularis

Northern Nomascus Melanotransferrin 1 OO& DSSHAFTLDELR 13 white- leucogenys cheeked gibbon

Olive Papio anubis Melanotransferrin 1 OO& DSSHAFTLDELR 13 baboon

Rhesus Macaca mulatta hypothetical protein 1 OO& DSSHAFTLDELR 13 macaque

Rhesus Macaca mulatta hypothetical protein 1 OO& DSSHAFTLDELR 13 macaque

Western Gorilla gorilla Melanotransferrin 1 OO& DSSHAFTLDELR 13 lowland gorilla gorilla

White- Calli thrix liacchus Melanotransferrin 1 OO& DSSHAFTLDELR 13 tufted-ear marmoset

Lesser Jaculus jaculus Melanotransferrin 928. DSSDAFTLDELR 93 Egyptian jerboa US 2014/0322132 A1 Oct. 30, 2014 18

TABLE B- continued

Common 3. SEO ID Name Species Protein Name Identity Sequence NO :

Northern Otolemur Melanotransferrin DSSHSFTLDELR greater garnettii galago

Sumatran Pongo abelii Melanotransferrin 3. DSSDAFTLDELR orangutan

Thirteen- Ictidomys Melanotransferrin 3. DSSYAFTLDELR lined tridecemlineatus ground squirrel white Ceratotherium anotransferrin 3. NSSHAFTLDELR rhinoceros Simum simum alpaca Vicudna pacos anotransferrin 3. NSSYAFTLDELR

American Ochotona anotransferrin 3. DSSYAFPLDELR pika princeps black flying Pteropus alecto anotransferrin 3. NSSYAFTLDELR fox bottlenosed Tursiops anotransferrin 3. NSSYAFTLDELR dolphin tillcats

Chinese Tupaia chinensis anotransferrin 3. DSTHAFTWDELR tree shrew

Chiru Pantholops anotransferrin 3. NSSYAFTLDELR hodgsonii

Domestic Felis catus anotransferrin 3. NSSYAFTLDELR Cat

Domestic Bos taurus anotransferrin 3. NSSYAFTLDELR cattle

Domestic Mustela anotransferrin 3. NSSYAFTLDELR ferret putorius furo

Giant panda Ailuropoda anotransferrin 3. NSSYAFTLDELR Melanoleuca

Goat Capra hircus anotransferrin 3. NSSYAFTLDELR

House Mus musculus anotransferrin 3. DSSYSFTLDELR ColSee

Killer whale Orcinus orca anotransferrin 3. NSSNAFTLDELR

Long-tailed Chinchillia anotransferrin 3. DSSSAFTLNELR chinchilla lanigera

Nine- Dasypus anotransferrin 3. DSSYAFTLDELW banded novemcinctus armadillo

Norway rat Rattus anotransferrin 3. DSSYSFTLDELR norvegicus

Pacific Odobenius anotransferrin 3. NSSSAFTLDELR Walrus OSS divergens

Prairie vole Microtus anotransferrin 3. DSSYSFTLDELR ochrogaster

Sheep Ovis airies anotransferrin 3. NSSYAFTLDELR

Weddell Leptonychotes anotransferrin 3. NSSYAFTLDELR Seal Weddellii US 2014/0322132 A1 Oct. 30, 2014

TABLE B- continued

Common 3. SEO ID Name Species Protein Name Identity Sequence NO :

Wild Camelus ferus Melanotransferrin 838 NSSYAFTLDELR 18 Bactrian camel

Wild boar Sus scrofa Melanotransferrin 838 NSSYAFTLDELR 19

Yak Bos nutus Melanotransferrin 838 NSSYAFTLDELR 2O (Fungus) Cypheliophora hypothetical protein 75% ATSHAITLDELR 21 europaea

African Loxodonta elanotransferrin 75% NSSYAFTMDELR 22 Sawala africana elephant

Chinese Cricetulus elanotransferrin 75% DRSYSFTLDELR 23 hamster griseus

Common Oryctolagus elanotransferrin 75& DSAYAFTWDELR 24 rabbit cliniculus

Degu Octodon degus elanotransferrin 75% DSSSAFNLNELR 25 Domestic Canis lupus elanotransferrin 75% NSSDAFSLDELR 26 Dog familiaris

Domestic Cavia porcelius elanotransferrin 75% DSSSAFSLNELR 27 guinea pig

European Sorex arranels elanotransferrin 75% NSSDAFSLDELR 28 shrew

Florida Trichechus elanotransferrin 75% NSSYAFTMDELR 29 manatee naatlis latirostris

Golden Mesocricetus elanotransferrin 75& DRSYSFTLDELR 3 O hamster aliats

Gray short- Monodelphis elanotransferrin 75% NSSYSFTLDELR 31 tailed domestics opossum

Horse Equus cabalius elanotransferrin 75% NSSYAFTVDELR 32 Small Echinops telfairi elanotransferrin 75% NSSYAFTWDELR 33 Madagascar hedgehog

Star -nosed Condylura elanotransferrin 75% NSSYAFSLDELR 34 mole cristata

Human Homo sapien Transferrin 33% SASD LTWDNLK 35

Human Homo sapien Lactoferrin 173 SDTSLTWNSVK 36

0179 Hence, in certain embodiments, the p97 peptide 490, 500,510,520, 530, 540, 550,560,570,580,590, 600, comprises, consists, or consists essentially of a sequence in 610, 620, 630, 640, 650, 660, 670, 680, 690, 700. 700, 710, Table B. In specific aspects, the p97 peptide retains the short 720, 730, 740, 750, 760, 770, 780, 790, 800. 800, 810, 820, alpha-helix (LDEL) at the C-terminus of the 830, 840, 850, 860, 870, 880, 890, 900, 900, 910, 920, 930, DSSHAFTLDELR (SEQID NO:13) peptide. 940,950,960,970,980,990, 1000 or more contiguous amino 0180. In certain embodiments, a polypeptide sequence is acids in length, including all integers in between, and which about, at least about, or up to about 5, 6, 7, 8, 9, 10, 11, 12, 13, may comprise all or a portion of a reference sequence (see, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, e.g., Sequence Listing, Tables 1-7, Table B, FIGS. 2-6 and 9). 31, 32,33, 34,35,36, 37,38, 39, 40, 41, 42,43, 44, 45, 46,47, 0181. In other specific embodiments, a polypeptide 48, 49, 50, 55, 60, 65, 70, 75, 80, 85,90, 95, 100, 110, 120, sequence consists of about or no more than about 5, 6, 7, 8, 9. 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 250, 260,270, 280, 290, 300, 310,320, 330, 340,350, 360, 27, 28, 29, 30, 31, 32,33, 34,35, 36, 37,38, 39, 40, 41,42, 43, 370, 380,390, 400, 410, 420, 430, 440, 450, 460, 470, 480, 44, 45, 46, 47, 48, 49, 50, 55, 60, 65, 70, 75, 80, 85,90, 95, US 2014/0322132 A1 Oct. 30, 2014 20

100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, acid sequences, the sequences are aligned for optimal com 220, 230, 240, 250, 260, 270, 280, 290, 300, 310,320, 330, parison purposes (e.g., gaps can be introduced in one or both 340, 350, 360, 370, 380,390, 400, 410, 420, 430, 440, 450, of a first and a second amino acid or nucleic acid sequence for 460, 470,480,490, 500,510,520, 530, 540, 550,560, 570, optimal alignment and non-homologous sequences can be 580, 590, 600, 610, 620, 630, 640, 650, 660, 670, 680, 690, disregarded for comparison purposes). In certain embodi 700, 710, 720, 730, 740, 750, 760, 770, 780, 790, 800. 800, ments, the length of a reference sequence aligned for com 810, 820, 830, 840, 850, 860, 870, 880, 890, 900, 900, 910, parison purposes is at least 30%, preferably at least 40%, 920, 930, 940, 950, 960, 970, 980, 990, 1000 or more con more preferably at least 50%. 60%, and even more preferably tiguous amino acids, including all integers in between, and at least 70%, 80%, 90%, 100% of the length of the reference which may comprise all or a portion of a reference sequence sequence. The amino acid residues or nucleotides at corre (see, e.g., Sequence Listing, Tables 1-7, Table B, FIGS. 2-6 sponding amino acid positions or nucleotide positions are and 9). then compared. When a position in the first sequence is occu 0182. In still other specific embodiments, a polypeptide pied by the same amino acid residue or nucleotide as the sequence is about 10-1000, 10-900, 10-800, 10-700, 10-600, corresponding position in the second sequence, then the mol 10-500, 10-400, 10-300, 10-200, 10-100, 10-50, 10-40, ecules are identical at that position. 10-30, 10-20, 20-1000, 20-900, 20-800, 20-700, 20-600, 0186 The percent identity between the two sequences is a 20-500, 20-400, 20-300, 20-200, 20-100, 20-50, 20-40, function of the number of identical positions shared by the 20-30, 50-1000, 50-900, 50-800, 50-700, 50-600, 50-500, sequences, taking into account the number of gaps, and the 50-400, 50-300, 50-200, 50-100, 100-1000, 100-900, 100 length of each gap, which need to be introduced for optimal 800, 100-700, 100-600, 100-500, 100-400, 100-300, 100 alignment of the two sequences. 200, 200-1000, 200-900, 200-800, 200-700, 200-600, 200 0187. The comparison of sequences and determination of 500, 200-400, or 200-300 contiguous amino acids, including percent identity between two sequences can be accomplished all ranges in between, and comprises all or a portion of a using a mathematical algorithm. In a preferred embodiment, reference sequence. In certain embodiments, the C-terminal the percent identity between two amino acid sequences is or N-terminal region of any reference polypeptide may be determined using the Needleman and Wunsch, (J. Mol. Biol. truncated by about 1,2,3,4,5,6,7,8,9, 10, 15, 20, 25.30,35, 48: 444-453, 1970) algorithm which has been incorporated 40, 45, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, into the GAP program in the GCG Software package, using 170, 180, 190, 200, 250, 300, 350, 400, 450, 500, 550, 600, either a Blossum 62 matrix or a PAM250 matrix, and a gap 650, 700, 750, or 800 or more amino acids, or by about 10-50, weight of 16, 14, 12, 10, 8, 6, or 4 and a length weight of 1, 2, 20-50, 50-100, 100-150, 150-200, 200-250, 250-300, 300 3, 4, 5, or 6. In yet another preferred embodiment, the percent 350, 350-400, 400-450, 450-500, 500-550, 550-600, 600 identity between two nucleotide sequences is determined 650, 650-700,700-750, 750-800 or more amino acids, includ using the GAP program in the GCG Software package, using ing all integers and ranges in between (e.g., 101, 102, 103. a NWSgapdna. CMP matrix and a gap weight of 40, 50, 60. 104, 105), so long as the truncated polypeptide retains the 70, or 80 and a length weight of 1, 2, 3, 4, 5, or 6. A particu binding properties and/or activity of the reference polypep larly preferred set of parameters (and the one that should be tide. Typically, the biologically-active fragment has no less used unless otherwise specified) are a Blossum 62 scoring than about 1%, about 5%, about 10%, about 25%, or about matrix with a gap penalty of 12, a gap extend penalty of 4, and 50% of an activity of the biologically-active reference a frameshift gap penalty of 5. polypeptide from which it is derived. 0188 The percent identity between two amino acid or 0183 In general, variants will display at least about 30%, nucleotide sequences can be determined using the algorithm 40%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, of E. Meyers and W. Miller (Cabios. 4:11-17, 1989) which 91%, 92%.93%, 94%, 95%, 96%,97%.98%,99% similarity has been incorporated into the ALIGN program (version 2.0), or sequence identity or sequence homology to a reference using a PAM120 weight residue table, a gap length penalty of polypeptide sequence. Moreover, sequences differing from 12 and a gap penalty of 4. the native or parent sequences by the addition (e.g., C-termi 0189 The nucleic acid and protein sequences described nal addition, N-terminal addition, both), deletion, truncation, herein can be used as a "query sequence' to perform a search insertion, or substitution of about 1,2,3,4,5,6,7,8,9, 10, 11, against public databases to, for example, identify other family 12, 13, 14, 15, 16, 17, 18, 19, 20, 30, 40, 50, 60, 70, 80,90, 100 members or related sequences. Such searches can be per or more amino acids but which retain the properties or activi formed using the NBLAST and XBLAST programs (version ties of a parent or reference polypeptide sequence are con 2.0) of Altschul, et al., (1990, J. Mol. Biol, 215: 403-10). templated. BLAST nucleotide searches can be performed with the 0184. In some embodiments, variant polypeptides differ NBLAST program, score=100, wordlength=12 to obtain from reference sequence by at least one but by less than 50, nucleotide sequences homologous to nucleic acid molecules 40, 30, 20, 15, 10, 8, 6, 5, 4, 3 or 2 amino acid residue(s). In of the invention. BLAST protein searches can be performed other embodiments, variant polypeptides differ from a refer with the XBLAST program, score=50, wordlength=3 to ence sequence by at least 1% but less than 20%, 15%, 10% or obtain amino acid sequences homologous to protein mol 5% of the residues. (If this comparison requires alignment, ecules of the invention. To obtain gapped alignments for the sequences should be aligned for maximum similarity. comparison purposes, Gapped BLAST can be utilized as “Looped out sequences from deletions or insertions, or mis described in Altschul et al., (Nucleic Acids Res. 25: 3389 matches, are considered differences.) 3402, 1997). When utilizing BLAST and Gapped BLAST 0185. Calculations of sequence similarity or sequence programs, the default parameters of the respective programs identity between sequences (the terms are used interchange (e.g., XBLAST and NBLAST) can be used. ably herein) are performed as follows. To determine the per 0190. In one embodiment, as noted above, polynucle cent identity of two amino acid sequences, or of two nucleic otides and/or polypeptides can be evaluated using a BLAST US 2014/0322132 A1 Oct. 30, 2014 alignment tool. A local alignment consists simply of a pair of aligned with a reference polypeptide sequence (see, e.g., sequence segments, one from each of the sequences being Sequence Listing) to generate a BLAST bit scores or compared. A modification of Smith-Waterman or Sellers sequence similarity scores of at least about 50, 60, 70, 80.90, algorithms will find all segment pairs whose scores cannot be 100, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, improved by extension or trimming, called high-scoring seg 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310,320, ment pairs (HSPs). The results of the BLAST alignments 330, 340, 350, 360, 370, 380,390, 400, 410, 420, 430, 440, include statistical measures to indicate the likelihood that the 450, 460, 470, 480,490, 500,510,520, 530, 540, 550,560, BLAST score can be expected from chance alone. 570, 580, 590, 600, 610, 620, 630, 640, 650, 660, 670, 680, 0191 The raw score, S, is calculated from the number of 690, 700, 710, 720, 730, 740, 750, 760, 770, 780, 790, 800, gaps and Substitutions associated with each aligned sequence 810, 820, 830, 840, 850, 860, 870, 880, 890, 900, 910, 920, wherein higher similarity Scores indicate a more significant 930, 940, 950, 960, 970, 980, 990, 1000, or more, including alignment. Substitution scores are given by a look-up table all integers and ranges in between, wherein the BLAST align (see PAM, BLOSUM). ment used the BLOSUM62 matrix, a gap existence penalty of 0.192 Gap scores are typically calculated as the sum of G, 11, and a gap extension penalty of 1. the gap opening penalty and L, the gap extension penalty. For 0198 As noted above, a reference polypeptide may be a gap of length n, the gap cost would be G+Ln. The choice of altered in various ways including amino acid Substitutions, gap costs, G and L is empirical, but it is customary to choose deletions, truncations, additions, and insertions. Methods for a high value for G (10-15), e.g., 11, and a low value for L (1-2) Such manipulations are generally known in the art. For e.g., 1. example, amino acid sequence variants of a reference 0193 The bit score, S', is derived from the raw alignment polypeptide can be prepared by mutations in the DNA. Meth score S in which the statistical properties of the scoring sys ods for mutagenesis and nucleotide sequence alterations are tem used have been taken into account. Bit scores are normal well known in the art. See, for example, Kunkel (PNAS USA. ized with respect to the scoring system, therefore they can be 82: 488-492, 1985); Kunkelet al., (Methods in Enzymol. 154: used to compare alignment scores from different searches. 367-382, 1987), U.S. Pat. No. 4,873,192, Watson, J. D. et al., The terms “bit score” and “similarity score are used inter (“Molecular Biology of the Gene. Fourth Edition, Benjamin/ changeably. The bit score gives an indication of how good the Cummings, Menlo Park, Calif., 1987) and the references alignment is; the higher the score, the better the alignment. cited therein. Guidance as to appropriate amino acid Substi 0194 The E-Value, or expected value, describes the like tutions that do not affect biological activity of the protein of lihood that a sequence with a similar score will occur in the interest may be found in the model of Dayhoffet al., (1978) database by chance. It is a prediction of the number of differ Atlas of Protein Sequence and Structure (Natl. Biomed. Res. ent alignments with scores equivalent to or better than S that Found. Washington, D.C.). are expected to occur in a database search by chance. The 0199 Methods for screening gene products of combina smaller the E-Value, the more significant the alignment. For torial libraries made by Such modifications, and for screening example, an alignment having an Evalue of e' means that cDNA libraries for gene products having a selected property a sequence with a similar score is very unlikely to occur are known in the art. Such methods are adaptable for rapid simply by chance. Additionally, the expected score for align screening of the gene libraries generated by combinatorial ing a random pair of amino acids is required to be negative, mutagenesis of reference polypeptides. As one example, otherwise long alignments would tend to have high score recursive ensemble mutagenesis (REM), a technique which independently of whether the segments aligned were related. enhances the frequency of functional mutants in the libraries, Additionally, the BLAST algorithm uses an appropriate sub can be used in combination with the screening assays to stitution matrix, nucleotide or amino acid and for gapped identify polypeptide variants (Arkin and Yourvan, PNAS alignments uses gap creation and extension penalties. For USA89: 7811-7815, 1992: Delgrave et al., Protein Engineer example, BLAST alignment and comparison of polypeptide ing. 6: 327-331, 1993). sequences are typically done using the BLOSUM62 matrix, a 0200 Exemplary Methods for Conjugation. gap existence penalty of 11 and a gap extension penalty of 1. 0201 Conjugation or coupling of a p97 polypeptide 0.195. In one embodiment, sequence similarity scores are sequence to an agent of interest can be carried out using reported from BLAST analyses done using the BLOSUM62 standard chemical, biochemical and/or molecular techniques. matrix, a gap existence penalty of 11 and a gap extension Indeed, it will be apparent how to make a p97 conjugate in penalty of 1. light of the present disclosure using available art-recognized 0196. In a particular embodiment, sequence identity/simi methodologies. Of course, it will generally be preferred when larity scores provided herein refer to the value obtained using coupling the primary components of a p97 conjugate of the GAPVersion 10 (GCG, Accelrys, San Diego, Calif.) using the present invention that the techniques employed and the result following parameters:% identity and% similarity for a nucle ing linking chemistries do not substantially disturb the otide sequence using GAPWeight of 50 and Length Weight of desired functionality or activity of the individual components 3, and the nwsgapdna.cmp scoring matrix; % identity and % of the conjugate. similarity for an amino acid sequence using GAP Weight of 8 0202 The particular coupling chemistry employed will and Length Weight of 2, and the BLOSUM62 scoring matrix depend upon the structure of the biologically active agent (Henikoff and Henikoff, PNAS USA. 89:10915-10919, 1992). (e.g., Small molecule, polypeptide), the potential presence of GAP uses the algorithm of Needleman and Wunsch (J Mol multiple functional groups within the biologically active Biol. 48:443-453, 1970) to find the alignment of two com agent, the need for protection/deprotection steps, chemical plete sequences that maximizes the number of matches and stability of the agent, and the like, and will be readily deter minimizes the number of gaps. mined by one skilled in the art. Illustrative coupling chemistry 0197) In one particular embodiment, the variant polypep useful for preparing the p97 conjugates of the invention can tide comprises an amino acid sequence that can be optimally be found, for example, in Wong (1991), “Chemistry of Pro US 2014/0322132 A1 Oct. 30, 2014 22 tein Conjugation and Crosslinking, CRC Press, Boca Raton, reaction between an agent comprising a trichlorophenyl car Fla.; and Brinkley “A Brief Survey of Methods for Preparing bonate functional group and a P97 polypeptide comprising an Protein Conjugates with Dyes, Haptens, and Crosslinking amino group forms an carbamate linkage; a reaction between Reagents.” in Bioconjug. Chem., 3:2013, 1992. Preferably, an agent comprising a thioester functional group and a P97 the binding ability and/or activity of the conjugate is not polypeptide comprising an n-terminal amino group forms an Substantially reduced as a result of the conjugation technique amide linkage; a reaction between an agent comprising a employed, for example, relative to the unconjugated agent or proprionaldehyde functional group and a P97 polypeptide the unconjugated p97 polypeptide. comprisinganamino group forms a secondary amine linkage. 0203. In certain embodiments, a p97 polypeptide 0207. In some exemplary embodiments, a reaction sequence may be coupled to an agent of interest either directly between an agent comprising a butyraldehyde functional or indirectly. A direct reaction between a p97 polypeptide group and a P97 polypeptide comprising an amino group sequence and an agent of interest is possible when each pos forms a secondary amine linkage; a reaction between an agent sesses a substituent capable of reacting with the other. For comprising an acetal functional group and a P97 polypeptide example, a nucleophilic group, Such as an amino or Sulfhydryl comprisinganamino group forms a secondary amine linkage; group, on one may be capable of reacting with a carbonyl a reaction between an agent comprising a piperidone func containing group, such as an anhydride or an acid halide, or tional group and a P97 polypeptide comprising an amino with an alkyl group containing a good leaving group (e.g., a group forms a secondary amine linkage; a reaction between halide) on the other. an agent comprising a methylketone functional group and a 0204 Alternatively, it may be desirable to indirectly P97 polypeptide comprising an amino group forms a second couple a p97 polypeptide sequence and an agent of interest ary amine linkage; a reaction between an agent comprising a via a linker group, including non-peptide linkers and peptide tresylate functional group and a P97 polypeptide comprising linkers. A linker group can also function as a spacer to dis an amino group forms a secondary amine linkage; a reaction tance an agent of interest from the p97 polypeptide sequence between an agent comprising a maleimide functional group in order to avoid interference with binding capabilities, tar and a P97 polypeptide comprising an amino group forms a geting capabilities or other functionalities. A linker group can secondary amine linkage; a reaction between an agent com also serve to increase the chemical reactivity of a substituent prising a aldehyde functional group and a P97 polypeptide on an agent, and thus increase the coupling efficiency. An comprisinganamino group forms a secondary amine linkage; increase in chemical reactivity may also facilitate the use of and a reaction between an agent comprising a hydrazine agents, or functional groups on agents, which otherwise functional group and a P97 polypeptide comprising an car would not be possible. The selection of releasable or stable boxylic acid group forms a secondary amine linkage. linkers can also be employed to alter the pharmacokinetics of 0208. In particular exemplary embodiments, a reaction a p97 conjugate and attached agent of interest. Illustrative between an agent comprising a maleimide functional group linking groups include, for example, disulfide groups, thioet and a P97 polypeptide comprising a thiol group forms a thio her groups, acid labile groups, photolabile groups, peptidase ether linkage; a reaction between an agent comprising a vinyl labile groups and esterase labile groups. In other illustrative Sulfone functional group and a P97 polypeptide comprising a embodiments, the conjugates include linking groups such as thiol group forms a thioether linkage; a reaction between an those disclosed in U.S. Pat. No. 5,208,020 or EP Patent 0.425 agent comprising a thiol functional group and a P97 polypep 235 B1, and Charietal., Cancer Research. 52: 127-131, 1992. tide comprising a thiol group forms a di-sulfide linkage; a Additional exemplary linkers are described below. reaction between an agent comprising a orthopyridyl disul 0205. In some embodiments, it may be desirable to couple fide functional group and a P97 polypeptide comprising a more than one p97 polypeptide sequence to an agent, or vice thiol group forms a di-sulfide linkage; and a reaction between versa. For example, in certain embodiments, multiple p97 an agent comprising an iodoacetamidefunctional group and a polypeptide sequences are coupled to one agent, or alterna P97 polypeptide comprising a thiol group forms a thioether tively, one or more p97 polypeptides are conjugated to mul linkage. tiple agents. The p97 polypeptide sequences can be the same 0209. In a specific embodiment, an amine-to-sulfhydryl or different. Regardless of the particular embodiment, conju crosslinker is used for preparing a conjugate. In one preferred gates containing multiple p97 polypeptide sequences may be embodiment, for example, the crosslinker is succinimidyl-4- prepared in a variety of ways. For example, more than one (N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC) polypeptide may be coupled directly to an agent, or linkers (Thermo Scientific), which is a sulfhydryl crosslinker con that provide multiple sites for attachment can be used. Any of taining NHS-ester and maleimide reactive groups at opposite a variety of known heterobifunctional crosslinking strategies ends of a medium-length cyclohexane-stabilized spacer arm can be employed for making conjugates of the invention. It (8.3 angstroms). SMCC is a non-cleavable and membrane will be understood that many of these embodiments can be permeable crosslinker that can be used to create sulfhydryl achieved by controlling the stoichiometries of the materials reactive, maleimide-activated agents (e.g., polypeptides, used during the conjugation/crosslinking procedure. antibodies) for subsequent reaction with p97 polypeptide 0206. In certain exemplary embodiments, a reaction sequences. NHS esters react with primary amines at pH 7-9 to between an agent comprising a succinimidyl ester functional form stable amide bonds. Maleimides react with sulfhydryl group and a p97 polypeptide comprising an amino group groups at pH 6.5-7.5 to form stable thioether bonds. Thus, the forms an amide linkage; a reaction between an agent com amine reactive NHS ester of SMCC crosslinks rapidly with prising a oxycarbonylimidizaole functional group and a P97 primary amines of an agent and the resulting sulfhydryl polypeptide comprising an amino group forms an carbamate reactive maleimide group is then available to react with cys linkage; a reaction between an agent comprising a p-nitro teine residues of p97 to yield specific conjugates of interest. phenyl carbonate functional group and a P97 polypeptide 0210. In certain specific embodiments, the p97 polypep comprising an amino group forms an carbamate linkage; a tide sequence is modified to contain exposed sulfhydryl US 2014/0322132 A1 Oct. 30, 2014 groups to facilitate crosslinking, e.g., to facilitate crosslink 0216 where Z is cysteine or serine; Z is a proline or ing to a maleimide-activated agent. In a more specific alanine residue, X is present or absent and, when present, is embodiment, the p97 polypeptide sequence is modified with any amino acid, where X is preferably present when the a reagent which modifies primary amines to add protected heterologous sulfatase motif is at an N-terminus of the alde thiol Sulfhydryl groups. In an even more specific embodi hyde tagged polypeptide; and X and X are each indepen ment, the reagent N-Succinimidyl-S-acetylthioacetate dently any amino acid. (SATA) (Thermo Scientific) is used to produce thiolated p97 0217 Polypeptides with the above-described motif can be polypeptides. modified by an FGE enzyme to generate a motif having a 0211. In other specific embodiments, a maleimide-acti FGly residue, which, as noted above, can then be used for vated agent is reacted under suitable conditions with thiolated site-specific attachment of an agent, such as a second p97 polypeptides to produce a conjugate of the present inven polypeptide, for instance, via a linker moiety. Such modifi tion. It will be understood that by manipulating the ratios of cations can be performed, for example, by expressing the SMCC, SATA, agent, and p97 polypeptide in these reactions Sulfatase motif-containing polypeptide (e.g. p97, antibody) it is possible to produce conjugates having differing Stoichi in a mammalian, yeast, or bacterial cell that expresses an FGE ometries, molecular weights and properties. enzyme or by in vitro modification of isolated polypeptide 0212. In still other illustrative embodiments, conjugates with an isolated FGE enzyme (see Wu et al., PNAS. 106:3000 are made using bifunctional protein coupling agents such as 3005, 2009: Rush and Bertozzi, J. Am ChemSoc. 130:12240 N-succinimidyl-3-(2-pyridyldithio)propionate (SPDP), suc 1, 2008; and Carlson et al., J Biol Chem. 283:2011 7-25, cinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxy 2008). late, iminothiolane (IT), bifunctional derivatives of imi 0218. Hence, some embodiments include a p97 polypep doesters (such as dimethyl adipimidate HCL), active esters tide or polypeptide agent (e.g., antibody) that comprises 1, 2, (such as disuccinimidyl Suberate), aldehydes (such as glu 3, 4, 5, 6, 7, 8, 9, 10 or more heterologous sulfatase motifs tareldehyde), bis-azido compounds (such as bis (p-azidoben having a formylglycine residue, where the motif comprises Zoyl)hexanediamine), bis-diazonium derivatives (such as bis the following structure: (p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (such as toluene 2,6-diisocyanate), and bis-active fluorine compounds (such as 1,5-difluoro-2,4-dinitrobenzene). Par (SEQ ID NO: 2O) ticular coupling agents include N-Succinimidyl-3-(2-py X1 (FGly) XZ2X ridyldithio)propionate (SPDP) (Carlsson et al., Biochem. J. 0219 where FGly is a formylglycine residue; Z is a pro 173:723-737 (1978) and N-succinimidyl-4-(2-pyridylthio) line or alanine residue: X is present or absent and, when pentanoate (SPP) to provide for a disulfide linkage. present, is any amino acid, where X is preferably present 0213. The specific crosslinking strategies discussed when the heterologous sulfatase motif is at an N-terminus of herein are but a few of many examples of suitable conjugation the aldehyde tagged polypeptide; and X and X are each strategies that may be employed in producing conjugates of independently any amino acid. the invention. It will be evident to those skilled in the art that 0220. In particular embodiments, X, X, and X are each a variety of other bifunctional or polyfunctional reagents, independently an aliphatic amino acid, a Sulfur-containing both homo- and hetero-functional (such as those described in amino acid or a polar, uncharged amino acid. For instance, X the catalog of the Pierce Chemical Co., Rockford, Ill.), may can be L.M.V. SorT; andX, and/or X can be independently be employed as the linker group. Coupling may be effected, S, T, A, V, G or C. for example, through amino groups, carboxyl groups, sulfhy 0221. In some embodiments, the heterologous sulfatase dryl groups or oxidized carbohydrate residues. There are motifs) can be (a) less than 16 amino acid residues in length, numerous references describing Such methodology, e.g., U.S. including about 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 Pat. No. 4,671,958, to Rodwell et al. residues in length, (b) positioned at the N-terminus of the 0214 Particular embodiments may employ one or more polypeptide, (c) positioned at the C-terminus of the polypep aldehyde tags to facilitate conjugation between a p97 tide, (d) positioned at an internal site of an amino acid polypeptide and an agent (see U.S. Pat. Nos. 8,097,701 and sequence native to the polypeptide, (e) positioned in a termi 7.985,783, incorporated by reference). Here, enzymatic nal loop of the polypeptide, (f) positioned at a site of post modification at a Sulfatase motif of the aldehyde tag through translational modification of the polypeptide (e.g., glycosy action of a formylglycine generating enzyme (FGE) gener lation site), or any combination thereof. ates a formylglycine (FGly) residue. The aldehyde moiety of 0222 Some embodiments relate to conjugates of (i) a sul the FGly residue can then be exploited as a chemical handle fatase motif (or aldehyde tag)-containing p97 polypeptide, for site-specific attachment of a moiety of interest to the and (ii) an agent (A) Such as Small molecule that is function polypeptide. In some aspects, the moiety of interest is a small alized with an aldehyde reactive group, where (i) and (ii) are molecule, peptoid, aptamer, or peptide mimetic. In some covalently linked via the FGly residue of the sulfatase motif aspects, the moiety of interest is another polypeptide, such as and the aldehyde reactive group. Such conjugates can have an antibody. one of the following general structures: 0215 Particularembodiments thus include ap97 polypep 0223 p97(FGly)-R-A tide or polypeptide agent that comprises 1, 2, 3, 4, 5, 6, 7, 8, 0224 where R is at least one aldehyde reactive linkage; 9, 10 or more heterologous sulfatase motifs, where the motif and FGly is a formylglycine residue within a heterologous comprises the following structure: sulfatase motif. 0225. Some embodiments relate to conjugates of (i) a sul (SEQ ID NO: 19) fatase motif (or aldehyde tag)-containing p97 polypeptide, XZX222X3 and (ii) a polypeptide agent (pA) that is functionalized with an aldehyde reactive group, or vice versa, where (i) and (ii) are US 2014/0322132 A1 Oct. 30, 2014 24 covalently linked via the FGly residue of the sulfatase motif reactive groups include aminooxy, hydrazide, and thiosemi and the aldehyde reactive group. Such conjugates can have carbazide groups, which will form Schiff-base containing one of the following general structures: linkages with a FGly residue, including oxime linkages, 0226 p97(FGly)-R-pA or p97-R-(FGly)p A hydrazine linkages, and hydrazine carbothiamide linkages, 0227 where R is at least one aldehyde reactive linkage; respectively. Hence, RandR can be independently a linkage and FGly is a formylglycine residue within a heterologous that comprises a Schiff base, such as an oxime linkage, a sulfatase motif. hydrazine linkage, or a hydrazine carbothiamide linkage. 0228. The agent or non-aldehyde tag-containing polypep 0234. In some embodiments, the aldehyde tag-containing tide (e.g., antibody, p97 polypeptide) can be functionalized p97 polypeptide and the aldehyde tag-containing agent are with one or more aldehyde reactive groups such as aminooxy, linked (e.g., covalently linked) via a multi-functionalized hydrazide, and thiosemicarbazide, and then covalently linked linker (e.g., bi-functionalized linker), the latter being func to the aldehyde tag-containing polypeptide via the at least one tionalized with the same or different aldehyde reactive group FGly residue, to form an aldehyde reactive linkage. The (s). In these and related embodiments, the aldehyde reactive attachment of an aminooxy functionalized agent (or non groups allow the linker to form a covalent bridge between the aldehyde tag-containing polypeptide) creates an oxime link p97 polypeptide and the agent via their respective FGly resi age between the FGly residue and the functionalized agent (or dues. Linker moieties include any moiety or chemical that can non-aldehyde tag-containing polypeptide); attachment of a be functionalized and preferably bi- or multi-functionalized hydrazide-functionalized agent (or non-aldehyde tag-con with one or more aldehyde reactive groups. Particular taining polypeptide) creates a hydrazine linkage between the examples include peptides, water-soluble polymers, detect FGly residue and the functionalized agent (or non-aldehyde able entities, other therapeutic compounds (e.g., cytotoxic tag-containing polypeptide); and attachment of a thiosemi compounds), biotin/streptavidin moieties, and glycans (see carbazide-functionalized agent (or non-aldehyde tag-con Hudak et al., JAm ChemSoc. 133:16127-35, 2011). Specific taining polypeptide) creates a hydrazine carbothiamide link examples of glycans (or glycosides) include aminooxy gly age between the FGly residue and the functionalized agent (or cans, such as higher-order glycans composed of glycosyl non-aldehyde tag-containing polypeptide). Hence, in these N-pentenoyl hydroxamates intermediates (Supra). Exem and related embodiments, R can be a linkage that comprises plary linkers are described herein, and can be functionalized a Schiffbase. Such as an oxime linkage, a hydrazine linkage, with aldehyde reactive groups according to routine tech or a hydrazine carbothiamide linkage. niques in the art (see, e.g., Carrico et al., Nat Chem Biol. 0229 Certain embodiments include conjugates of (i) a 3:321-322, 2007; and U.S. Pat. Nos. 8,097,701 and 7,985, Sulfatase motif (or aldehyde tag)-containing p97 polypeptide 783). and (ii) a sulfatase motif (or aldehyde tag)-containing 0235 p97 conjugates can also be prepared by a various polypeptide agent (A), where (i) and (ii) are covalently linked "click chemistry’ techniques, including reactions that are via their respective FGly residues, optionally via a bi-func modular, wide in Scope, give very high yields, generate tionalized linker moiety or group. For instance, certain p97 mainly inoffensive byproducts that can be removed by non conjugates may comprise the following structure: chromatographic methods, and can be stereospecific but not 0230 p97(FGly)-R-L-R-(FGly)A necessarily enantioselective (see Kolb et al., Angew Chem Int 0231 where R and R are the same or different aldehyde Ed Engl. 40:2004-2021, 2001). Particular examples include reactive linkage; L is a linker moiety, p97(FGly) is a alde conjugation techniques that employ the Huisgen 1,3-dipolar hyde-tag containingp97 polypeptide, and (FGly)A is an alde cycloaddition of azides and alkynes, also referred to as hyde tag-containing agent, Such as an antibody or other “azide-alkyne cycloaddition reactions (see Hein et al., polypeptide-based agent. Pharm Res. 25:2216-2230, 2008). Non-limiting examples of 0232 Merely by way of illustration, in some embodi azide-alkyne cycloaddition reactions include copper-cata ments, the at least one heterologous Sulfatase motif can be at lyzed azide-alkyne cycloaddition (CuAAC) reactions and the C-terminus of the p97 polypeptide and the N-terminus of ruthenium-catalyzed azide-alkyne cycloaddition (RuAAC) the polypeptide-based agent. In other embodiments, the at reactions. least one heterologous sulfatase motif can be at the N-termi 0236 CuAAC works over a broad temperature range, is nus of the p97 polypeptide and the C-terminus of the polypep insensitive to aqueous conditions and a pH range over 4 to 12, tide-based agent. In still other embodiments, the at least one and tolerates abroad range of functional groups (see Himo et heterologous sulfatase motif can be at the N-terminus of the al, J Am Chem Soc. 127:210-216, 2005). The active Cu(I) p97 polypeptide and the N-terminus of the polypeptide-based catalyst can be generated, for example, from Cu(I) salts or agent. In further embodiments, the at least one heterologous Cu(II) salts using sodium ascorbate as the reducing agent. sulfatase motif can be at the C-terminus of the p97 polypep This reaction forms 1,4-substituted products, making it tide an the C-terminus of the polypeptide-based agent. As region-specific (see Hein et al., Supra). noted above, the at least one heterologous motif can be at an 0237 RuAAC utilizes pentamethylcyclopentadienyl internal position in the p97 polypeptide and/or the polypep ruthenium chloride Cp*RuCl complexes that are able to tide-based agent. Persons skilled in the art will recognize that catalyze the cycloaddition of azides to terminal alkynes, regi other combinations are possible. oselectively leading to 1,5-disubstituted 1,2,3-triazoles (see 0233. The aldehyde reactive linkages of R and R can be Rasmussenet al., Org. Lett. 9:5337-5339, 2007). Further, and independently formed by any aldehyde reactive group that in contrast to CuAAC, RuAAC can also be used with internal will form a covalent bond between (i) the formylglycine alkynes to provide fully substituted 1,2,3-triazoles. (FGly) residue of the aldehyde tag and (ii) a linker moiety that 0238 Certain embodiments thus include p97 polypeptides is functionalized with said aldehyde reactive group (e.g., a that comprise at least one unnatural amino acid with an azide bi-functionalized linker with two aldehyde reactive groups, side-chain or an alkyne side-chain, including internal and which can be the same or different). Examples of aldehyde terminal unnatural amino acids (e.g., N-terminal, C-termi US 2014/0322132 A1 Oct. 30, 2014 nal). Certain of these p97 polypeptides can be formed by in ments include a p97 polypeptide that comprises at least one Vivo or in vitro (e.g., cell-free systems) incorporation of internal unnatural amino acid with an azide side-chain con unnatural amino acids that contain azide side-chains or jugated to an agent that comprises an alkyne group. Addi alkyne side-chains. Exemplary in vivo techniques include cell tional embodiments include a p97 polypeptide that comprises culture techniques, for instance, using modified E. coli (see at least one internal unnatural amino acid with an alkyne Travis and Schultz, The Journal of Biological Chemistry. side-chain conjugated to an agent that comprises an azide 285:11039-44, 2010; and Deiters and Schultz, Bioorganic & side-group Medicinal Chemistry Letters. 15:1521-1524, 2005), and 0244 Particular embodiments include a p97 polypeptide exemplary in vitro techniques include cell-free systems (see that comprises an N-terminal unnatural amino acid with an Bundy, Bioconjug Chem. 21:255-63, 2010). azide side-chain conjugated to a polypeptide agent that com 0239. In some embodiments, a p97 polypeptide that com prises an N-terminal unnatural amino acid with an alkyne prises at least one unnatural amino acid with an azide side side-chain. Other embodiments include a p97 polypeptide chain is conjugated by azide-alkyne cycloaddition to an agent that comprises a C-terminal unnatural amino acid with an (or linker) that comprises at least one alkyne group, such as a azide side-chain conjugated to a polypeptide agent that com polypeptide agent that comprises at least one unnatural amino prises a C-terminal unnatural amino acid with an alkyne acid with an alkyne side-chain. In other embodiments, a p97 side-chain. Still other embodiments include a p97 polypep polypeptide that comprises at least one unnatural amino acid tide that comprises an N-terminal unnatural amino acid with with an alkyne side-chain is conjugated by azide-alkyne an azide side-chain conjugated to a polypeptide agent that cycloaddition to an agent (or linker) that comprises at least comprises a C-terminal unnatural amino acid with an alkyne one azide group. Such as a polypeptide agent that comprises at side-chain. Further embodiments include a p97 polypeptide least one unnatural amino acid with an azide side-chain. that comprises a C-terminal unnatural amino acid with an Hence, certain embodiments include conjugates that com azide side-chain conjugated to a polypeptide agent that com prise a p97 polypeptide covalently linked to an agent via a prises an N-terminal unnatural amino acid with an alkyne 1,2,3-triazole linkage. side-chain. 0240 Specific p97 conjugates can be formed by the fol 0245. Other embodiments include a p97 polypeptide that lowing CuAAC-based or RuAAC-based reactions, to com comprises an N-terminal unnatural amino acid with an alkyne prise the following respective structures (I) or (II). side-chain conjugated to a polypeptide agent that comprises an N-terminal unnatural amino acid with an azide side-chain. Still further embodiments include a p97 polypeptide that (I) comprises a C-terminal unnatural amino acid with an alkyne side-chain conjugated to a polypeptide agent that comprises a n/ S. C-terminal unnatural amino acid with an azide side-chain. Cu(I) (cat) N NN Additional embodiments include a p97 polypeptide that com prises an N-terminal unnatural amino acid with an alkyne R side-chain conjugated to a polypeptide agent that comprises a (II) C-terminal unnatural amino acid with an azide side-chain. Still further embodiments include a p97 polypeptide that Cp*RuCl(PPh3) (cat) RN N comprises a C-terminal unnatural amino acid with an alkyne R-N, + = -R - dioxane, A - NN NN side-chain conjugated to a polypeptide agent that comprises an N-terminal unnatural amino acid with an azide side-chain. 0246. Also included are methods of producing a p97 con R jugate, comprising: (a) performing an azide-alkyne cycload dition reaction between (i) a p97 polypeptide that comprises 0241 where R is a p97 polypeptide and R is an agent of at least one unnatural amino acid with an azide side-chain and interest (or linker); or where R is an agent of interest (or an agent that comprises at least one alkyne group (for linker) and R is a p97 polypeptide. instance, an unnatural amino acid with an alkyne side chain); 0242. In certain embodiments, the unnatural amino acid or (ii) a p97 polypeptide that comprises at least one unnatural with the azide side-chain and/or the unnatural amino acid amino acid with an alkyne side-chain and an agent that com with alkyne side-chain are terminal amino acids (N-terminal, prises at least one azide group (for instance, an unnatural C-terminal). In certain embodiments, one or more of the amino acid with an azide side-chain); and (b) isolating a p97 unnatural amino acids are internal. conjugate from the reaction, thereby producing a p97 conju 0243 For instance, certain embodiments include a p97 gate. polypeptide that comprises an N-terminal unnatural amino 0247. In the case where the p97 conjugate is a fusion acid with an azide side-chain conjugated to an agent that polypeptide, the fusion polypeptide may generally be pre comprises an alkyne group. Some embodiments, include a pared using standard techniques. Preferably, however, a p97 polypeptide that comprises a C-terminal unnatural amino fusion polypeptide is expressed as a recombinant polypeptide acid with an azide side-chain conjugated to an agent that in an expression system, described herein and known in the comprises an alkyne group. Particular embodiments include a art. Fusion polypeptides of the invention can contain one or p97 polypeptide that comprises an N-terminal unnatural multiple copies of a p97 polypeptide sequence and may con amino acid with an alkyne side-chain conjugated to an agent tain one or multiple copies of a polypeptide-based agent of that comprises an azide side-group. Further embodiments interest (e.g., antibody or antigen-binding fragment thereof), include a p97 polypeptide that comprises an C-terminal present in any desired arrangement. unnatural amino acid with an alkyne side-chain conjugated to 0248 For fusion proteins, DNA sequences encoding the an agent that comprises an azide side-group. Some embodi p97 polypeptide, the polypeptide agent (e.g., antibody), and US 2014/0322132 A1 Oct. 30, 2014 26 optionally peptide linker components may be assembled sequences, enhancer sequences, marker genes and other separately, and then ligated into an appropriate expression sequences as appropriate. Vectors may be plasmids, viral e.g. vector. The 3' end of the DNA sequence encoding one phage, or phagemid, as appropriate. For further details see, polypeptide component is ligated, with or without a peptide for example, Molecular Cloning: a Laboratory Manual: 2nd linker, to the 5' end of a DNA sequence encoding the other edition, Sambrook et al., 1989, Cold Spring Harbor Labora polypeptide component(s) so that the reading frames of the tory Press. Many known techniques and protocols for sequences are in phase. The ligated DNA sequences are oper manipulation of nucleic acid, for example in preparation of ably linked to Suitable transcriptional or translational regula nucleic acid constructs, mutagenesis, sequencing, introduc tory elements. The regulatory elements responsible for tion of DNA into cells and gene expression, and analysis of expression of DNA are located only 5' to the DNA sequence proteins, are described in detail in Current Protocols in encoding the first polypeptides. Similarly, stop codons Molecular Biology, Second Edition, Ausubel et al. eds., John required to end translation and transcription termination sig Wiley & Sons, 1992, or subsequent updates thereto. nals are only present 3' to the DNA sequence encoding the 0255. The term “host cell is used to refer to a cell into most C-terminal polypeptide. This permits translation into a which has been introduced, or which is capable of having single fusion polypeptide that retains the biological activity of introduced into it, a nucleic acid sequence encoding one or both component polypeptides. more of the polypeptides described herein, and which further 0249 Similar techniques, mainly the arrangement of regu expresses or is capable of expressing a selected gene of inter latory elements such as promoters, stop codons, and tran est, such as a gene encoding any herein described polypep Scription termination signals, can be applied to the recombi tide. The term includes the progeny of the parent cell, whether nant production of non-fusion proteins, for instance, p97 or not the progeny are identical in morphology or in genetic polypeptides and polypeptide agents (e.g., antibody agents) make-up to the original parent, so long as the selected gene is for the production of non-fusion conjugates. present. Host cells may be chosen for certain characteristics, 0250 Polynucleotides and fusion polynucleotides of the for instance, the expression of a formylglycine generating invention can contain one or multiple copies of a nucleic acid enzyme (FGE) to convert a cysteine or serine residue within encoding a p97 polypeptide sequence, and/or may contain a sulfatase motif into a formylglycine (FGly) residue, or the one or multiple copies of a nucleic acid encoding a polypep expression of aminoacyl tRNA synthetase(s) that can incor tide agent. porate unnatural amino acids into the polypeptide, including 0251. In some embodiments, a nucleic acids encoding a unnatural amino acids with an azide side-chain, alkyne side subject p97 polypeptide, polypeptide agent, and/or p97 chain, or other desired side-chain, to facilitate conjugation. polypeptide fusion are introduced directly into a host cell, and 0256 Accordingly there is also contemplated a method the cell incubated under conditions sufficient to induce comprising introducing such nucleic acid(s) into a host cell. expression of the encoded polypeptide(s). The polypeptide The introduction of nucleic acids may employ any available sequences of this disclosure may be prepared using standard technique. For eukaryotic cells, Suitable techniques may techniques well known to those of skill in the art in combi include calcium phosphate transfection, DEAE-Dextran, nation with the polypeptide and nucleic acid sequences pro electroporation, liposome-mediated transfection and trans vided herein. duction using retrovirus or other virus, e.g. Vaccinia or, for 0252. Therefore, according to certain related embodi insect cells, baculovirus. For bacterial cells, suitable tech ments, there is provided a recombinant host cell which com niques may include calcium chloride transformation, elec prises a polynucleotide or a fusion polynucleotide that troporation and transfection using bacteriophage. The intro encodes a polypeptide described herein. Expression of a p97 duction may be followed by causing or allowing expression polypeptide, polypeptide agent, or p97-polypeptide agent from the nucleic acid, e.g., by culturing host cells under fusion in the host cell may conveniently be achieved by cul conditions for expression of the gene. In one embodiment, the turing under appropriate conditions recombinant host cells nucleic acid is integrated into the genome (e.g. chromosome) containing the polynucleotide. Following production by of the host cell. Integration may be promoted by inclusion of expression, the polypeptide(s) may be isolated and/or purified sequences which promote recombination with the genome, in using any suitable technique, and then used as desired. accordance-with standard techniques. 0253 Systems for cloning and expression of a polypeptide 0257 The present invention also provides, in certain in a variety of different host cells are well known. Suitable embodiments, a method which comprises using a nucleic acid host cells include bacteria, mammalian cells, yeast and bacu construct described herein in an expression system in order to lovirus systems. Mammalian cell lines available in the art for express a particular polypeptide, Such as a p97 polypeptide, expression of a heterologous polypeptide include Chinese polypeptide agent, orp97-polypeptide agent fusion protein as hamster ovary (CHO) cells, HeLa cells, baby hamster kidney described herein. cells, HEK-293 cells, NSO mouse melanoma cells and many 0258 As noted above, certain p97 conjugates, such as others. A common, preferred bacterial host is E. coli. The fusion proteins, may employ one or more linker groups, expression of polypeptides in prokaryotic cells Such as E. coli including non-peptide linkers (e.g., non-proteinaceous link is well established in the art. For a review, see for example ers) and peptide linkers. Such linkers can be stable linkers or Pluckthun, A. Bio/Technology. 9:545-551 (1991). Expres releasable linkers. sion in eukaryotic cells in culture is also available to those 0259 Exemplary non-peptide stable linkages include suc skilled in the art as an option for recombinant production of cinimide, propionic acid, carboxymethylate linkages, ethers, polypeptides (see Ref, Curr: Opinion Biotech. 4:573-576, carbamates, amides, amines, carbamides, imides, aliphatic 1993; and Trillet al., Curr. Opinion Biotech. 6:553-560, 1995. C-C bonds, thioether linkages, thiocarbamates, thiocarba 0254 Suitable vectors can be chosen or constructed, con mides, and the like. Generally, a hydrolytically stable linkage taining appropriate regulatory sequences, including promoter is one that exhibits a rate of hydrolysis of less than about 1-2% sequences, terminator sequences, polyadenylation to 5% per day under physiological conditions. US 2014/0322132 A1 Oct. 30, 2014 27

0260 Exemplary non-peptide releasable linkages include 2,000 to about 20,000 Dabeing of particular interest. Linear, carboxylate ester, phosphate ester, anhydride, acetal, ketal, branched, and terminally charged water Soluble polymers are acyloxyalkyl ether, imine, orthoester, thioester, thiol ester, also included. carbonate, and hydrazone linkages. Additional illustrative 0265 Polymers useful as linkers between aldehyde tagged embodiments of hydrolytically unstable or weak linkages polypeptides can have a wide range of molecular weights, and include, but are not limited to:—OC-(CH), O—, where polymer Subunits. These Subunits may include a biological b is from 1 to 5, —O—(CH) CO (CH) , where b is polymer, a synthetic polymer, or a combination thereof. from 1 to 5 and c is from 2-5. —O—(CH), CO (CH) Examples of such water-soluble polymers include: dextran O—, where b is from 1 to 5 and c is from 2-5. —(CH) and dextran derivatives, including dextran Sulfate, P-amino OPO (CH), where b is 1-5 and b' is 1-5, —C(O)— cross linked dextrin, and carboxymethyl dextrin, cellulose (NH CHR-CO), NH CHR-, wherea is 2 to 20 and R and cellulose derivatives, including methylcellulose and car is a Substituent found on an O-amino acid, —O-(CH2) - boxymethyl cellulose, starch and dextrines, and derivatives CO. CHCH-CH , whereb is from 1-5, —O CH and hydroylactes of starch, polyalklyene glycol and deriva CH=N-(CH), O—, where b is from 1-5, and —O— tives thereof, including polyethylene glycol (PEG), methoxy (CH) CH-CH=N (CH), O—, where each b is polyethylene glycol, polyethylene glycol homopolymers, independently from 1-5. polypropylene glycol homopolymers, copolymers of ethyl 0261. Other illustrative examples of releasable linkers can ene glycol with propylene glycol, wherein said homopoly be benzyl elimination-based linkers, trialkyl lock-based link mers and copolymers are unsubstituted or Substituted at one ers (or trialkyl lock lactonization based), bicine-based link end with an alkyl group, heparin and fragments of heparin, ers, and acid labile linkers. Among the acid labile linkers can polyvinyl alcohol and polyvinyl ethyl ethers, polyvinylpyr be disulfide bond, hydrazone-containing linkers and thiopro rolidone, aspartamide, and polyoxyethylated polyols, with pionate-containing linkers. the dextran and dextran derivatives, dextrine and dextrine 0262 Also included are linkers that are releasable or derivatives. It will be appreciated that various derivatives of cleavable during or upon internalization into a cell. The the specifically described water-soluble polymers are also mechanisms for the intracellular release of an agent from included. these linker groups include cleavage by reduction of a disul fide bond (e.g., U.S. Pat. No. 4,489,710, to Spitler), by irra 0266 Water-soluble polymers are known in the art, par diation of a photolabile bond (e.g., U.S. Pat. No. 4,625,014, to ticularly the polyalkylene oxide-based polymers such as Senter et al.), by hydrolysis of derivatized amino acid side polyethylene glycol “PEG” (see Poly(ethylene glycol) chains (e.g., U.S. Pat. No. 4,638.045, to Kohnet al.), by serum Chemistry: Biotechnical and Biomedical Applications, J. M. complement-mediated hydrolysis (e.g., U.S. Pat. No. 4,671, Harris, Ed., Plenum Press, New York, N.Y. (1992); and Poly 958, to Rodwell et al.), and acid-catalyzed hydrolysis (e.g., (ethylene glycol) Chemistry and Biological Applications, J. U.S. Pat. No. 4,569,789, to Blattler et al.). In one embodi M. Harris and S. Zalipsky, Eds. ACS (1997); and Interna ment, an acid-labile linker may be used (Cancer Research tional Patent Applications: WO 90/13540, WO 92/00748, 52:127-131, 1992; and U.S. Pat. No. 5,208,020). WO92/16555, WO 94/04193, WO 94/14758, WO 94/17039, 0263. In certain embodiments, “water soluble polymers' WO94/18247, WO 94/28937, WO95/11924, WO 96/00080, are used in a linker for coupling a p97 polypeptide sequence WO 96/23794, WO 98/07713, WO 98/41562, WO 98/48837, to an agent of interest. A “water-soluble polymer refers to a WO 99/30727, WO 99/32134, WO 99/33483, WO 99/53951, polymer that is soluble in water and is usually substantially WO 01/26692, WO95/13312, WO 96/21469, WO 97/03106, non-immunogenic, and usually has an atomic molecular WO 99/45964, and U.S. Pat. Nos. 4,179,337; 5,075,046; weight greater than about 1,000 Daltons. Attachment of two 5,089,261; 5,100,992; 5,134,192: 5,166,309; 5,171,264; polypeptides via a water-soluble polymer can be desirable as 5,213,891; 5,219,564: 5,275,838; 5,281,698; 5,298,643; Such modification(s) can increase the therapeutic index by 5,312,808: 5,321,095; 5,324,844; 5,349,001: 5,352,756; increasing serum half-life, for instance, by increasing pro 5,405,877; 5,455,027: 5,446,090; 5,470,829; 5,478,805; teolytic stability and/or decreasing renal clearance. Addition 5,567,422; 5,605,976; 5,612,460; 5,614,549; 5,618,528: ally, attachment via of one or more polymers can reduce the 5,672,662; 5,637,749; 5,643,575; 5,650,388: 5,681,567; immunogenicity of protein pharmaceuticals. Particular 5,686,110; 5,730,990; 5,739,208; 5,756,593; 5,808.096; examples of water soluble polymers include polyethylene 5,824,778; 5,824,784; 5,840,900; 5,874,500; 5,880,131; glycol, polypropylene glycol, polyoxyalkylenes, or copoly 5,900,461; 5,902,588; 5,919,442; 5,919,455; 5,932,462: mers of polyethylene glycol, polypropylene glycol, and the 5,965,119; 5,965,566; 5,985,263; 5,990.237; 6,011,042: like. 6,013,283; 6,077,939; 6,113,906; 6,127,355; 6,177,087; 0264. In some embodiments, the water-soluble polymer 6,180,095; 6,194,580; 6,214,966, incorporated by reference). has an effective hydrodynamic molecular weight of greater 0267 Exemplary polymers of interest include those con than about 10,000 Da, greater than about 20,000 to 500,000 taining a polyalkylene oxide, polyamide alkylene oxide, or Da, greater than about 40,000 Da to 300,000 Da, greater than derivatives thereof, including polyalkylene oxide and polya about 50,000 Da to 70,000 Da, usually greater than about mide alkylene oxide comprising an ethylene oxide repeat unit 60,000 Da. The “effective hydrodynamic molecular weight' of the formula —(CH, CH O)—. Further exemplary refers to the effective water-solvated size of a polymer chain polymers of interest include a polyamide having a molecular as determined by aqueous-based size exclusion chromatog weight greater than about 1,000 Daltons of the formula—C raphy (SEC). When the water-soluble polymer contains poly (O) X-C(O) NH Y NHL or -NH Y NH merchains having polyalkylene oxide repeat units, such as C(O)—X—C(O), , where X and Y are divalent radicals ethylene oxide repeat units, each chain can have an atomic that may be the same or different and may be branched or molecular weight of between about 200 Da and about 80,000 linear, and n is a discrete integer from 2-100, usually from 2 Da, or between about 1,500 Da and about 42,000 Da, with to 50, and where either or both of X and Y comprises a US 2014/0322132 A1 Oct. 30, 2014 28 biocompatible, Substantially non-antigenic water-soluble residues. Other near neutral amino acids, such as Thrand Ala repeat unit that may be linear or branched. may also be employed in the peptide linker sequence, if desired. 0268. Further exemplary water-soluble repeat units com 0272. Certain exemplary linkers include Gly, Ser and/or prise an ethylene oxide of the formula—(CH, CH, O)— ASn-containing linkers, as follows: G, IS, N, GS, or —(CH2—CH2—O)—. The number of such water-soluble GGS), GSS, GSGS) (SEQID NO:21), GGSGI, (SEQ repeat units can vary significantly, with the usual number of ID NO:22), GGGS), (SEQID NO:23), GGGGS),(SEQID such units being from 2 to 500, 2 to 400, 2 to 300, 2 to 200, 2 NO:24), IGN, GGN, GNN, IGNGNI, (SEQ ID NO: to 100, and most usually 2 to 50. An exemplary embodiment 25), GGNG), (SEQID NO:26), GGGNI, (SEQID NO: 27), is one in which one or both of X and Y is selected from: GGGGN (SEQ ID NO: 28) linkers, where is 1, 2, 3, 4, 5, ((CH)—(CH-CH O), (CH) O ((CH) 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 or more. —(O—CH2—CH2), (CH)—), where n1 is 1 to 6, 1 to Other combinations of these and related amino acids will be 5, 1 to 4 and most usually 1 to 3, and where n2 is 2 to 50, 2 to apparent to persons skilled in the art. 25, 2 to 15, 2 to 10, 2 to 8, and most usually 2 to 5. A further 0273. In specific embodiments, the linker sequence com exemplary embodiment is one in which X is —(CH2— prises a Gly3 linker sequence, which includes three glycine CH)—, and where Y is —(CH (CH-CH O)— residues. In particular embodiments, flexible linkers can be CH, CH, CH) O (CH, CH, CH2—(O rationally designed using a computer program capable of CH-CH-)-CH2)—, among other variations. modeling both DNA-binding sites and the peptides them 0269. In certain embodiments, a peptide linker sequence selves (Desjarlais & Berg, PNAS. 90:2256-2260, 1993; and may be employed to separate or couple the components of a PNAS. 91:11099-11103, 1994) or by phage display methods. p97 conjugate. For instance, for polypeptide-polypeptide 0274 The peptide linkers may be physiologically stable or conjugates, peptide linkers can separate the components by a may include a releasable linker Such as a physiologically distance sufficient to ensure that each polypeptide folds into degradable or enzymatically degradable linker (e.g., pro its secondary and tertiary structures. Such a peptide linker teolytically cleavable linker). In certain embodiments, one or sequence may be incorporated into the conjugate (e.g., fusion more releasable linkers can result in a shorter half-life and protein) using standard techniques described herein and well more rapid clearance of the conjugate. These and related known in the art. Suitable peptide linker sequences may be embodiments can be used, for example, to enhance the solu chosen based on the following factors: (1) their ability to bility and blood circulation lifetime of p97 conjugates in the adopt a flexible extended conformation; (2) their inability to bloodstream, while also delivering an agent into the blood adopt a secondary structure that could interact with functional stream (or across the BBB) that, subsequent to linker degra epitopes on the first and second polypeptides; and (3) the lack dation, is substantially free of the p97 sequence. These of hydrophobic or charged residues that might react with the aspects are especially useful in those cases where polypep polypeptide functional epitopes. Amino acid sequences tides or other agents, when permanently conjugated to a p97 which may be usefully employed as linkers include those sequence, demonstrate reduced activity. By using the linkers disclosed in Maratea et al., Gene 40:39-46, 1985; Murphy et as provided herein, Such antibodies can maintain their thera al., Proc. Natl. Acad. Sci. USA 83:8258-8262, 1986; U.S. Pat. peutic activity when in conjugated form. In these and other No. 4,935,233 and U.S. Pat. No. 4,751,180. ways, the properties of the p97 conjugates can be more effec tively tailored to balance the bioactivity and circulating half 0270. In certain illustrative embodiments, a peptide linker life of the antibodies over time. is between about 1 to 5 amino acids, between 5 to 10 amino 0275. Enzymatically degradable linkages suitable for use acids, between 5 to 25 amino acids, between 5 to 50 amino in particular embodiments of the present invention include, acids, between 10 to 25 amino acids, between 10 to 50 amino but are not limited to: an amino acid sequence cleaved by a acids, between 10 to 100 amino acids, or any intervening serine protease such as thrombin, chymotrypsin, trypsin, range of amino acids. In other illustrative embodiments, a elastase, kallikrein, or substilisin. Illustrative examples of peptide linker comprises about 1, 5, 10, 15, 20, 25, 30, 35, 40, thrombin-cleavable amino acid sequences include, but are not 45, 50 or more amino acids in length. Particular linkers can limited to: -Gly-Arg-Gly-Asp-(SEQ ID NO:29), -Gly-Gly have an overall amino acid length of about 1-200amino acids, Arg-, -Gly-Arg-Gly-Asp-Asn-Pro-(SEQ ID NO: 30), -Gly 1-150 amino acids, 1-100 amino acids, 1-90 amino acids, Arg-Gly-Asp-Ser-(SEQID NO:31), -Gly-Arg-Gly-Asp-Ser 1-80 amino acids, 1-70 amino acids, 1-60 amino acids, 1-50 Pro-Lys-(SEQ ID NO: 32), -Gly-Pro-Arg-, -Val-Pro-Arg amino acids, 1-40 amino acids, 1-30 amino acids, 1-20 amino and -Phe-Val-Arg-. Illustrative examples of elastase-cleav acids, 1-10 amino acids, 1-5 amino acids, 1-4 amino acids, able amino acid sequences include, but are not limited to: 1-3 amino acids, or about 1, 2,3,4,5,6,7,8,9, 10, 11, 12, 13, -Ala-Ala-Ala- -Ala-Ala-Pro-Val-(SEQ ID NO:33). --Ala 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, Ala-Pro-Leu-(SEQ ID NO:34), -Ala-Ala-Pro-Phe-(SEQ ID 31, 32,33, 34,35,36, 37,38, 39, 40, 41, 42,43, 44, 45, 46,47, NO:35), -Ala-Ala-Pro-Ala-(SEQ ID NO:36), and -Ala-Tyr 48, 49, 50, 60, 70, 80, 90, 100 or more amino acids. Leu-Val-(SEQID NO:37). 0271 A peptide linker may employ any one or more natu 0276 Enzymatically degradable linkages suitable for use rally-occurring amino acids, non-naturally occurring amino in particular embodiments of the present invention also acid(s), amino acid analogs, and/or amino acid mimetics as include amino acid sequences that can be cleaved by a matrix described elsewhere herein and known in the art. Certain metalloproteinase such as collagenase, stromelysin, and amino acid sequences which may be usefully employed as gelatinase. Illustrative examples of matrix metalloproteinase linkers include those disclosed in Maratea et al., Gene 40:39 cleavable amino acid sequences include, but are not limited 46, 1985; Murphy et al., PNAS USA. 83:8258-8262, 1986: to:-Gly-Pro-Y-Gly-Pro-Z-(SEQID NO:38), -Gly-Pro-, Leu U.S. Pat. No. 4,935,233 and U.S. Pat. No. 4,751,180. Particu Gly-Pro-Z-(SEQID NO:39), -Gly-Pro-Ile-Gly-Pro-Z-(SEQ lar peptide linker sequences contain Gly, Ser, and/or ASn ID NO: 40), and -Ala-Pro-Gly-Leu-Z-(SEQ ID NO:41), US 2014/0322132 A1 Oct. 30, 2014 29 where Y and Z are amino acids. Illustrative examples of ing a composition that comprises a p97 conjugate described collagenase-cleavable amino acid sequences include, but are herein. Also included are methods of delivering an agent to not limited to: -Pro-Leu-Gly-Pro-D-Arg-Z-(SEQ ID NO: the nervous system (e.g., central nervous system tissues) of a 42), -Pro-Leu-Gly-Leu-Leu-Gly-Z-(SEQ ID NO: 43), -Pro Subject, comprising administering a composition that com Gln-Gly-Ile-Ala-Gly-Trp-(SEQ ID NO. 44), -Pro-Leu-Gly prises a p97 conjugate described herein. In certain of these Cys(Me)-His-(SEQID NO:45), -Pro-Leu-Gly-Leu-Tyr-Ala and related embodiments, the methods increase the rate of (SEQID NO: 46), -Pro-Leu-Ala-Leu-Trp-Ala-Arg-(SEQID delivery of the agent to the central nervous system tissues, NO:47), and -Pro-Leu-Ala-Tyr-Trp-Ala-Arg-(SEQ ID NO: relative, for example, to delivery by a composition that com 48), where Z is an amino acid. An illustrative example of a prises the agent alone. stromelysin-cleavable amino acid sequence is -Pro-Tyr-Ala 0283. In some instances, a subject has a disease, disorder, Tyr-Tyr-Met-Arg-(SEQ ID NO: 49); and an example of a or condition of the CNS, where increased delivery of a thera gelatinase-cleavable amino acid sequence is -Pro-Leu-Gly peutic agent across the blood brain barrier to CNS tissues Met-Tyr-Ser-Arg-(SEQID NO: 50). relative to peripheral tissues can improve treatment, for 0277 Enzymatically degradable linkages suitable for use instance, by reducing side-effects associated with exposure of in particular embodiments of the present invention also an agent to peripheral tissues. Exemplary diseases, disorders, include amino acid sequences that can be cleaved by an and conditions of the CNS include various cancers, including angiotensin converting enzyme. Such as, for example, -Asp primary and metastatic CNS cancers, lysosomal storage dis Lys-Pro-, -Gly-Asp-Lys-Pro-(SEQ ID NO. 51), and -Gly eases, neurodegenerative diseases such as Alzheimer's dis Ser-Asp-Lys-Pro-(SEQID NO: 52). ease, and auto-immune diseases such as multiple Sclerosis. 0278 Enzymatically degradable linkages suitable for use 0284 Certain embodiments thus relate to methods for in particular embodiments of the present invention also treating a cancer of the central nervous system (CNS), option include amino acid sequences that can be degraded by cathe ally the brain, where the subject in need thereof has such a psin B. Such as, for example, -Val-Cit-, -Ala-Leu-Ala-Leu cancer or is at risk for developing such a condition. In some (SEQ ID NO:53), -Gly-Phe-Leu-Gly- (SEQID NO: 54) and embodiments, the cancer is a primary cancer of the CNS, such -Phe-Lys-. as a primary cancer of the brain. For instance, the methods can 0279. In certain embodiments, however, any one or more be for treating a glioma, meningioma, pituitary adenoma, of the non-peptide or peptide linkers are optional. For vestibular Schwannoma, primary CNS lymphoma, or primi instance, linker sequences may not required in a fusion pro tive neuroectodermal tumor (medulloblastoma). In some tein where the first and second polypeptides have non-essen embodiments, the glioma is an astrocytoma, oligodendro tial N-terminal and/or C-terminal amino acid regions that can glioma, ependymoma, or a choroid plexus papilloma. In cer be used to separate the functional domains and prevent steric tain embodiments, the primary CNS or brain cancer is glio interference. blastoma multiforme, such as a giant cell gliobastoma or a 0280. The functional properties of the p97 polypeptides gliosarcoma. and p97 polypeptide conjugates described herein may be 0285. In particular embodiments, the cancer is a meta assessed using a variety of methods known to the skilled static cancer of the CNS, for instance, a cancer that has person, including, e.g., affinity/binding assays (for example, metastasized to the brain. Examples of such cancers include, Surface plasmon resonance, competitive inhibition assays); without limitation, breast cancers, lung cancers, genitouri cytotoxicity assays, cell viability assays, cell proliferation or nary tract cancers, gastrointestinal tract cancers (e.g., col differentiation assays, cancer cell and/or tumor growth inhi orectal cancers, pancreatic carcinomas), osteosarcomas, bition using in vitro or in Vivo models. For instance, the , head and neck cancers, prostate cancers (e.g., conjugates described herein may be tested for effects on prostatic adenocarcinomas), and lymphomas. Certain receptor internalization, in vitro and in vivo efficacy, etc., embodiments thus include methods for treating, inhibiting or including the rate of transport across the blood brain barrier. preventing metastasis of a cancer by administering to a Such assays may be performed using well-established proto patient a therapeutically effective amount of a herein dis cols known to the skilled person (see e.g., Current Protocols closed conjugate (e.g., in an amount that, following adminis in Molecular Biology (Greene Publ. Assoc. Inc. & John Wiley tration, inhibits, prevents or delays metastasis of a cancer in a & Sons, Inc., NY, NY); Current Protocols in Immunology statistically significant manner, i.e., relative to an appropriate (Edited by: John E. Coligan, Ada M. Kruisbeek, David H. control as will be known to those skilled in the art). In par Margulies, Ethan M. Shevach, Warren Strober 2001 John ticular embodiments, the Subject has a cancer that has not yet Wiley & Sons, NY, NY); or commercially available kits. metastasized to the central nervous system, including one or more of the above-described cancers, among others known in Methods of Use and Pharmaceutical Compositions the art. 0281 Certain embodiments of the present invention relate 0286. In particular embodiments, the cancer (cell) to methods of using the compositions of p97 polypeptides and expresses or overexpresses one or more of Her2/neu, B7H3. p97 conjugates described herein. Examples of such methods CD20, Her1/EGF receptor(s), VEGF receptor(s), PDGF include methods of treatment and methods of diagnosis, receptor(s), CD30, CD52, CD33, CTLA-4, or tenascin. including for instance, the use of p97 conjugates for medical 0287. Also included is the treatment of other cancers, imaging of certain organs/tissues, such as those of the nervous including breast cancer, prostate cancer, gastrointestinal can system. Specific embodiments include methods of diagnos cer, lung cancer, ovarian cancer, testicular cancer, head and ing and/or treating disorders or conditions of the central ner neck cancer, stomach cancer, bladder cancer, pancreatic can vous system (CNS), or disorders or conditions having a CNS cer, liver cancer, kidney cancer, squamous cell carcinoma, component. melanoma, non-melanoma cancer, thyroid cancer, endome 0282. Accordingly, certain embodiments include methods trial cancer, epithelial tumor, bone cancer, or a hematopoietic of treating a subject in need thereof, comprising administer cancer. Hence, in certain embodiments, the cancer cell being US 2014/0322132 A1 Oct. 30, 2014 30 treated by a p97 conjugate overexpresses or is associated with storage disease II, Pompe disease, GM1-gangliosidosis types a cancer antigen, Such as human Her2/neu, Her1/EGF recep I/II/III, GM2-gangliosidosis type I, Tay Sachs disease, GM2 tor (EGFR), Her3, A33 antigen, B7H3, CD5, CD19, CD20, gangliosidosis type II, Sandhoff disease, GM2-gangliosido CD22. CD23 (IgE Receptor), C242 antigen, 5T4, IL-6, IL-13, sis, C.-mannosidosis types I/II, B-mannosidosis, metachro vascular endothelial growth factor VEGF (e.g., VEGF-A) matic leucodystrophy, mucolipidosis type I, Sialidosis types VEGFR-1, VEGFR-2, CD30, CD33, CD37, CD40, CD44, I/II mucolipidosis types II/III I-cell disease, mucolipidosis CD51, CD52, CD56, CD74, CD80, CD152, CD200, CD221, type IIIC pseudo-Hurler polydystrophy, mucopolysacchari CCR4, HLA-DR, CTLA-4, NPC-1C, tenascin, vimentin, dosis type I, mucopolysaccharidosis type II, Hunter Syn insulin-like growth factor 1 receptor (IGF-1R), alpha-feto drome, mucopolysaccharidosis type IIIA, Sanfilippo Syn protein, insulin-like growth factor 1 (IGF-1), carbonic anhy drome, mucopolysaccharidosis type IIIB, drase 9 (CA-IX), carcinoembryonic antigen (CEA), integrin mucopolysaccharidosis type IIIC, mucopolysaccharidosis O?3, integrin Os3, folate receptor 1, transmembrane glyco type IIID, mucopolysaccharidosis type IVA, Morquio syn protein NMB, fibroblast activation protein alpha (FAP), gly drome, mucopolysaccharidosis type IVB Morquio syn coprotein 75, TAG-72, MUC1, MUC16 (or CA-125), phos drome, mucopolysaccharidosis type VI, mucopolysacchari phatidylserine, prostate-specific membrane antigen (PMSA). dosis type VII, Sly Syndrome, mucopolysaccharidosis type NR-LU-13 antigen, TRAIL-R1, tumor necrosis factor recep IX, multiple Sulfatase deficiency, neuronal ceroid lipofusci tor superfamily member 10b (TNFRSF10B or TRAIL-R2), nosis, CLN1 Batten disease, Niemann-Pick disease types SLAM family member 7 (SLAMF7), EGP40 pancarcinoma NB, Niemann-Pick disease, Niemann-Pick disease type C1, antigen, B-cell activating factor (BAFF), platelet-derived Niemann-Pick disease type C2, pycnodysostosis, Schindler growth factor receptor, glycoprotein EpCAM (17-1A). Pro disease types I/II, Schindler disease, and Sialic acid storage grammed Death-1, protein disulfide isomerase (PDI), Phos disease. In these and related embodiments, the p97 polypep phatase of Regenerating Liver 3 (PRL-3), prostatic acid phos tide can be conjugated to one or more polypeptides associated phatase, Lewis-Y antigen, GD2 (a disialoganglioside with a lysosomal storage disease, as described herein. expressed on tumors of neuroectodermal origin), glypican-3 0291. In certain instances, the subject has or is at risk for (GPC3), and/or mesothelin. having an auto-immune disorder and/or a neurodegenerative 0288 The use of p97 conjugates for treating cancers disorder, optionally of the CNS. Hence, also included are including cancers of the CNS can be combined with other methods of treating a degenerative or autoimmune disorder of therapeutic modalities. For example, a composition compris the central nervous system (CNS) in a subject in need thereof. ing a p97 conjugate can be administered to a subject before, For instance, in specific embodiments, the degenerative or during, or after other therapeutic interventions, including autoimmune disorder of the CNS is Alzheimer's disease, symptomatic care, radiotherapy, Surgery, transplantation, Huntington's disease, Parkinson's disease, or multiple scle , hormone therapy, photodynamic therapy, rosis (MS). Hence, certain embodiments include administer antibiotic therapy, or any combination thereof. Symptomatic ing a p97 conjugate to a subject having Alzheimer's disease, care includes administration of corticosteroids, to reduce Huntington's disease, Parkinson's disease, or MS. In particu cerebral edema, headaches, cognitive dysfunction, and eme lar embodiments, the p97 polypeptide is conjugated to an sis, and administration of anti-convulsants, to reduce Sei antibody or other agent that specifically binds to amyloid-f Zures. Radiotherapy includes whole-brain irradiation, frac (e.g., AR, 2) for Alzheimer's Disease, Huntingtin for Hun tionated radiotherapy, and radioSurgery, such as stereotactic tington's Disease, C-synuclein for Parkinson's Disease, or C4 radiosurgery, which can be further combined with traditional integrin, CD25, or IL-23 for MS. In some embodiments, the Surgery. p97 polypeptide is conjugated to an interferon-B polypeptide, 0289. In specific combination therapies, the antibody por for the treatment of MS. In specific embodiments, the p97 tion of an p97-antibody conjugate comprises cetuximab, and polypeptide is conjugated to daclizumab for the treatment of the p97-cetuximab conjugate is used for treating a subject MS. with locally or regionally advanced squamous cell carcinoma 0292 Also included are methods of treating pain in a of the head and neck in combination with radiation therapy. In Subject in need thereof. Examples include acute pain, chronic other aspects, the p97-cetuximab conjugate is used for treat pain, and neuropathic pain, including combinations thereof. inga Subject with recurrent locoregional disease or metastatic In some aspects, the pain has a centrally-acting component, squamous cell carcinoma of the head and neck in combina Such as central pain syndrome (CPS), where the pain is asso tion with platinum-based therapy with 5-fluorouracil (5-FU). ciated with damage to or dysfunction of the CNS, including In some aspects, the p97-cetuximab conjugate is used in the brain, brainstem, and/or spinal cord. In particular embodi combination with irinotecan for treating a Subject with ments, the p97 polypeptide is conjugated to an antibody or EGFR-expressing colorectal cancer and that is refractory to other agent that specifically binds to NGF or TrkA. In specific irinotecan-based . embodiments, the p97 polypeptide is conjugated to tan 0290. In some instances, the subject has or is at risk for eZumab for the treatment of pain, optionally for the treatment having a lysosomal storage disease. Certain methods thus of osteoarthritis of the knee or hip, chronic low back pain, relate to the treatment of lysosomal storage diseases in a bone cancer pain, or interstitial cystitis. Subject in need thereof, optionally those lysosomal storage 0293 Also included are methods of treating inflammation diseases associated with the central nervous system. Exem or an inflammatory condition in a subject in need thereof. plary lysosomal storage diseases include aspartylglu “Inflammation” refers generally to the biological response of cosaminuria, cholesterol ester storage disease, Wolman dis tissues to harmful stimuli. Such as pathogens, damaged cells ease, cystinosis, Danon disease, Fabry disease, Farber (e.g., wounds), and irritants. The term “inflammatory lipogranulomatosis, Farber disease, fucosidosis, galactosiali response' refers to the specific mechanisms by which inflam dosis types I/II, Gaucher disease types I/II/III, Gaucher dis mation is achieved and regulated, including, merely by way of ease, globoid cell leucodystrophy, Krabbe disease, glycogen illustration, immune cell activation or migration, cytokine US 2014/0322132 A1 Oct. 30, 2014 production, vasodilation, including kinin release, fibrinoly lymphocytes, plasma cells, and fibroblasts, though in contrast sis, and coagulation, among others described herein and to acute inflammation, which is mediated mainly by granu known in the art. Ideally, inflammation is a protective attempt locytes, chronic inflammation is mainly mediated by mono by the body to both remove the injurious stimuli and initiate nuclear cells Such as monocytes and lymphocytes. Chronic the healing process for the affected tissue or tissues. In the inflammation also involves a variety of inflammatory media absence of inflammation, wounds and infections would never tors, such as IFN-Y and other cytokines, growth factors, reac heal, creating a situation in which progressive destruction of tive oxygen species, and hydrolytic enzymes. Chronic the tissue would threaten survival. On the other hand, exces inflammation may last for many months or years, and may sive or chronic inflammation may associate with a variety of result in undesired tissue destruction and fibrosis. diseases, such as hay fever, atherosclerosis, and rheumatoid 0299 Clinical signs of chronic inflammation are depen arthritis, among others described herein and known in the art. dent upon duration of the illness, inflammatory lesions, cause 0294 p97 conjugates of the invention may modulate acute and anatomical area affected. (see, e.g., Kumaret al., Robbins inflammation, chronic inflammation, or both. Depending on Basic Pathology-8" Ed., 2009 Elsevier, London; Miller, LM, the needs of the subject, certain embodiments relate to reduc Pathology Lecture Notes, Atlantic Veterinary College, Char ing acute inflammation or inflammatory responses, and cer lottetown, PEI, Canada). Chronic inflammation is associated tain embodiments relate to reducing chronic inflammation or with a variety of pathological conditions or diseases, includ chronic inflammatory responses. ing, for example, allergies, Alzheimer's disease, anemia, aor 0295 Acute inflammation relates to the initial response of tic valve Stenosis, arthritis Such as rheumatoid arthritis and the body to presumably harmful stimuli and involves osteoarthritis, cancer, congestive heart failure, fibromyalgia, increased movement of plasma and leukocytes from the blood fibrosis, heart attack, kidney failure, lupus, pancreatitis, into the injured tissues. It is a short-term process, typically stroke, Surgical complications, inflammatory lung disease, beginning within minutes or hours and ending upon the inflammatory bowel disease, atherosclerosis, and psoriasis, removal of the injurious stimulus. Acute inflammation may among others described herein and known in the art. Hence, be characterized by any one or more of redness, increased p97 conjugates may be used to treat or manage chronic heat, Swelling, pain, and loss of function. Redness and heat inflammation, modulate any of one or more of the individual are due mainly to increased blood flow at body core tempera chronic inflammatory responses, or treat any one or more ture to the inflamed site, Swelling is caused by accumulation diseases or conditions associated with chronic inflammation. of fluid, pain is typically due to release of chemicals that 0300. In certain embodiments, p97 conjugates may modu stimulate nerve endings, and loss of function has multiple late inflammatory responses at the cellular level, such as by CalSCS. modulating the activation, inflammatory molecule secretion 0296 Acute inflammatory responses are initiated mainly (e.g., cytokine or kinin secretion), proliferation, activity, by local immune cells, such as resident macrophages, den migration, or adhesion of various cells involved in inflamma dritic cells, histiocytes, Kuppfer cells and mastocytes. At the tion. Examples of Such cells include immune cells and vas onset of an infection, burn, or other injuries, these cells cular cells. Immune cells include, for example, granulocytes undergo activation and release inflammatory mediators Such as neutrophils, eosinophils and basophils, macrophages/ responsible for the clinical signs of inflammation, Such as monocytes, lymphocytes such as B-cells, killer T-cells (i.e., vasoactive amines and eicosanoids. Vasodilation and its CD8+ T-cells), helper T-cells (i.e., CD4+ T-cells, including resulting increased blood flow cause the redness and T1 and T2 cells), natural killer cells, Yö T-cells, dendritic increased heat. Increased permeability of the blood vessels cells, and mast cells. Examples of vascular cells include results in an exudation or leakage of plasma proteins and fluid smooth muscle cells, endothelial cells, and fibroblasts. Also into the tissue, which creates Swelling. Certain released included are methods of modulating an inflammatory condi mediators such as bradykinin increase sensitivity to pain, and tion associated with one or more immune cells or vascular alter the blood vessels to permit the migration or extravasa cells, including neutrophil-mediated, macrophage-mediated, tion of leukocytes, such as neutrophils, which typically and lymphocyte-mediated inflammatory conditions. migrate along a chemotactic gradient created by the local 0301 In certain embodiments, p97 conjugates may modu immune cells. late the levels or activity of inflammatory molecules, includ 0297 Acute inflammatory responses also includes one or ing plasma-derived inflammatory molecules and cell-derived more acellular biochemical cascade systems, consisting of inflammatory molecules. Included are pro-inflammatory preformed plasma proteins modulate, which act in parallel to molecules and anti-inflammatory molecules. Examples of initiate and propagate the inflammatory response. These sys plasma-derived inflammatory molecules include, without tems include the complement system, which is mainly acti limitation, proteins or molecules of any one or more of the vated by bacteria, and the coagulation and fibrinolysis sys complement system, kinin system, coagulation system, and tems, which are mainly activated by necrosis, such as the type the fibrinolysis system. Examples of members of the comple of tissue damage that is caused by certain infections, burns, or ment system include C1, which exists in blood serum as a other trauma. Hence, p97 conjugates may be used to modu molecular complex containing about 6 molecules of C1q, 2 late acute inflammation, or any of one or more of the indi molecules of C1r, and 2 molecules of C1s, C2 (a and b), C3 (a vidual acute inflammatory responses. and B), C4 (a and b), C5, and the membrane attack complex 0298 Chronic inflammation, a prolonged and delayed of C5a, C5b, C6, C7, C8, and C9. Examples of the kinin inflammatory response, is characterized by a progressive shift system include bradykinin, kallidin, kallidreins, carboxypep in the type of cells that are present at the site of inflammation, tidases, angiotensin-converting enzyme, and neutral and often leads to simultaneous or near simultaneous destruc endopeptidase. tion and healing of the tissue from the inflammatory process. 0302 Examples of cell-derived inflammatory molecules At the cellular level, chronic inflammatory responses involve include, without limitation, enzymes contained within lyso a variety of immune cells such as monocytes, macrophages, Some granules, vasoactive amines, eicosanoids, cytokines, US 2014/0322132 A1 Oct. 30, 2014 32 acute-phase proteins, and soluble gases such as nitric oxide. tiple Sclerosis, autoimmune encephalomyelitis), arachnoidi Vasoactive amines contain at least one amino group, and tis (i.e., inflammation of the arachnoid, one of the membranes target blood vessels to alter their permeability or cause that surround and protect the nerves of the central nervous vasodilation. Examples of vasoactive amines include hista system), granuloma, drug-induced inflammation or meningi mine and serotonin. Eicosanoids refer to signaling molecules tis, neurodegenerative diseases such as Alzheimer's disease, made by oxidation of twenty-carbon essential fatty acids, and stroke, HIV-dementia, encephalitis such viral encephalitis include prostaglandins, prostacyclins, thromboxanes, and and bacterial encephalitis, parasitic infections, inflammatory leukotrienes. demyelinating disorders, and auto-immune disorders such as 0303 p97 conjugates may also modulate levels or activity CD8+ T Cell-mediated autoimmune diseases of the CNS. of acute-phase proteins. Examples of acute-phase proteins Additional examples include Parkinson's disease, myasthe include C-reactive protein, serum amyloid A, serum amyloid nia gravis, motor neuropathy, Guillain-Barre syndrome, P. and vasopressin. In certain instances, expression of acute autoimmune neuropathy, Lambert-Eaton myasthenic Syn phase proteins can cause a range of undesired systemic effects drome, paraneoplastic neurological disease, paraneoplastic including amyloidosis, fever, increased blood pressure, cerebellar atrophy, non-paraneoplastic stiff man syndrome, decreased Sweating, malaise, loss of appetite, and somno progressive cerebellar atrophy, Rasmussen's encephalitis, lence. Accordingly, p97 conjugates may modulate the levels amyotrophic lateral sclerosis, Sydeham chorea, Gilles de la or activity of acute-phase proteins, their systemic effects, or Tourette syndrome, autoimmune polyendocrinopathy, dys both. immune neuropathy, acquired neuromyotonia, arthrogrypo 0304. In certain embodiments, p97 conjugates reduce sis multiplex, optic neuritis, stiff-man syndrome, stroke, trau local inflammation, Systemic inflammation, or both. In cer matic brain injury (TBI), spinal Stenosis, acute spinal cord tain embodiments, p97 conjugates may reduce or maintain injury, and spinal cord compression. (i.e., prevent further increases) local inflammation or local 0307 As noted above, also included is inflammation asso inflammatory responses. In certain embodiments, p97 conju ciated with infections of the nervous system or CNS. Specific gates may reduce or maintain (i.e., prevent further increases) examples of bacterial infections associated with inflamma systemic inflammation or systemic inflammatory responses. tion of the nervous system include, without limitation, Strep 0305. In certain embodiments, the modulation of inflam tococcal infection Such as group B streptococci (e.g., Sub mation or inflammatory responses can be associated with one types III) and Streptococcus pneumoniae (e.g., serotypes 6,9. or more tissues or organs. Non-limiting examples of Such 14, 18 and 23), Escherichia coli (e.g., carrying K1 antigen), tissues or organs include skin (e.g., dermis, epidermis, Sub Listeria monocytogenes (e.g., serotype IVb), neisserial infec cutaneous layer), hair follicles, nervous system (e.g., brain, tion Such as Neisseria meningitidis (meningococcus), Staphy spinal cord, peripheral nerves, meninges including the dura lococcal infection, heamophilus infection such as Haemophi mater, arachnoid mater, and pia mater), auditory system or lus influenzae type B. Klebsiella, and Mycobacterium balance organs (e.g., inner ear, middle ear, outer ear), respi tuberculosis. Also included are infections by staphylococci ratory system (e.g., nose, trachea, lungs), gastroesophogeal and pseudomonas and other Gram-negative bacilli, mainly tissues, the gastrointestinal system (e.g., mouth, esophagus, with respect to trauma to the skull, which gives bacteria in the stomach, Small intestines, large intestines, rectum), vascular nasal cavity the potential to enter the meningeal space, or in system (e.g., heart, blood vessels and arteries), liver, gallblad persons with cerebral shunt or related device (e.g., extraven der, lymphatic/immune system (e.g., lymph nodes, lymphoid tricular drain, Ommaya reservoir). Specific examples of viral follicles, spleen, thymus, bone marrow), uro-genital system infections associated with inflammation of the nervous sys (e.g., kidneys, ureter, bladder, urethra, cervix, Fallopian tem include, without limitation, enteroviruses, herpes sim tubes, ovaries, uterus, Vulva, prostate, bulbourethral glands, plex virus type 1 and 2, human T-lymphotrophic virus, vari epidlymis, prostate, seminal vesicles, testicles), musculosk cella Zoster virus (chickenpox and shingles), mumps virus, eletal system (e.g., skeletal muscles, Smooth muscles, bone, human immunodeficiency virus (HIV), and lymphocytic cartilage, tendons, ligaments), adipose tissue, mammaries, choriomeningitis virus (LCMV). Meningitis may also result and the endocrine system (e.g., hypothalamus, pituitary, thy from infection by Spirochetes such as Treponema pallidum roid, pancreas, adrenal glands). Accordingly, p97 conjugates (syphilis) and Borrelia burgdorferi (Lyme disease), parasites may be used to modulate inflammation associated with any of Such as malaria (e.g., cerebral malaria), fungi such as Cryp these tissues or organs, such as to treat conditions or diseases tococcus neoformans, and ameoba Such as Naegleria fowleri. that are associated with the inflammation of these tissues or 0308 Meningitis or otherforms of nervous system inflam Organs. mation may also associate with the spread of cancer to the 0306 In particular embodiments, the inflammatory con meninges (malignant meningitis), certain drugs such as non dition has a nervous system or central nervous system com steroidal anti-inflammatory drugs, antibiotics and intrave ponent, including inflammation of the brain, spinal cord, nous immunoglobulins, sarcoidosis (or neurosarcoidosis), and/or the meninges. In particular embodiments, the inflam connective tissue disorders such as Systemic lupus erythema matory condition of the CNS in meningitis (e.g., bacteria, tosus, and certain forms of vasculitis (inflammatory condi viral), encephalitis (e.g., caused by infection or autoimmune tions of the blood vessel wall) such as Behcet’s disease. inflammation Such as Acute Disseminated Enchephalomyeli Epidermoid cysts and dermoid cysts may cause meningitis by tis), sarcoidosis, non-metastatic diseases associated with neo releasing irritant matter into the Subarachnoid space. Accord plasia. Particular examples of nervous system or CNS asso ingly, p97 conjugates may be used to treat or manage any one ciated inflammatory conditions include, without limitation, or more of these conditions. meningitis (i.e., inflammation of the protective membranes 0309 As noted above, certain subjects are about to covering the brain and spinal cord), myelitis, encaphaloymy undergo, are undergoing, or have undergone therapy with an elitis (e.g., myalgic encephalomyelitis, acute disseminated otherwise cardiotoxic agent, that is, an agent that displays encephalomyelitis, encephalomyelitis disseminata or mul cardiotoxicity in its unconjugated form (an agent that is not US 2014/0322132 A1 Oct. 30, 2014 conjugated to p97). Such subjects can benefit from adminis to the Subject a composition comprising a human p97 (mel tration of a p97-agent conjugate, relative to administration of anotransferrin) polypeptide, or a variant thereof, where the the agent alone, partly because p97 can exert a cardioprotec p97 polypeptide is conjugated to a detectable entity, and (b) tive effect on otherwise cardiotoxic agents by a mechanism visualizing the detectable entity in the Subject, organ, or tis that is believed to differ from its BBB transport properties. SU Hence, such subjects can be treated with a p97-cardiotoxic 0316. In particular embodiments, the organ or tissue com agent conjugate for a variety of disease conditions, including partment comprises the central nervous system (e.g., brain, diseases of the CNS described herein, and diseases relating to brainstem, spinal cord). In specific embodiments, the organ or peripheral, non-CNS tissues. tissue compartment comprises the brain or a portion thereof, 0310 Exemplary cardiotoxic agents are described else for instance, the parenchyma of the brain. where herein, and can be identified according to well-known 0317. A variety of methods can be employed to visualize in Vivo diagnostic and in vitro screening techniques. See the detectable entity in the Subject, organ, or tissue. Exem Bovelli et al., 2010, supra; Inoue et al., AATEX 14, Special plary non-invasive methods include radiography, such as Issue, 457-462, 2007; and Dorr et al., Cancer Research. fluoroscopy and projectional radiographs, CT-Scanning or 48:5222-5227, 1988. CAT-Scanning (computed tomography (CT) or computed 0311 For instance, subjects undergoing therapy with a axial tomography (CAT)), whether employing X-ray Suspected cardiotoxic agent can be monitored by imaging CT-Scanning, positron emission tomography (PET), or single techniques to asses LV systolic and diastolic dysfunction, photon emission computed tomography (SPECT), and cer heart valve disease, pericarditis and pericardial effusion, and tain types of magnetic resonance imaging (MRI), especially carotidartery lesions. LV fractional shortening and LVEF are those that utilize contrast agents, including combinations the most common indexes of LV systolic function for cardiac thereof. function assessment, for instance, during chemotherapy. 0318 Merely by way of example, PET can be performed Also, Doppler-derived diastolic indexes represent an early with positron-emitting contrast agents or radioisotopes such sign of LV dysfunction in patients undergoing therapy, so that as F. SPECT can be performed with gamma-emitting con evaluation of mitral diastolic flow pattern, early peak flow trast agents or radioisotopes such as 'Tl, ''"TC, 'I, and velocity to atrial peak flow velocity (E/A) ratio, deceleration 'Ga, and MRI can be performed with contrast agents or time of E wave and isoVolumic relaxation time can be useful radioisotopes such as H, C, F, ''O, 'Na, 'P, and Xe, to detect diastolic changes of LV function before systolic and Gd (gadolidinium; chelated organic Gd (III) complexes). dysfunction occurs. Pulsed tissue Doppler may be performed Any one or more of these exemplary contrast agents or radio during a standard Doppler echocardiographic examination; it isotopes can be conjugated to or otherwise incorporated into can be reliable in providing quantitative information on myo a p97 polypeptide and administered to a subject for imaging cardial diastolic relaxation and systolic performance (E" purposes. For instance, p97 polypeptides can be directly wave. A wave and S wave velocity). Tissue Doppler of LV labeled with one or more of these radioisotopes, or conju lateral mitral annulus has a recognized prognostic role and, in gated to molecules (e.g., Small molecules) that comprise one combination with PW Doppler of mitral inflow, provides or more of these radioisotopic contrast agents, or any others accurate information about the degree of LV filling pressure. described herein. Early changes in LV myocardial function have been identified 0319 For in vivo use, for instance, for the treatment of by pulsed tissue Doppler of multiple LV sites, and can be human disease, medical imaging, or testing, the conjugates relevant determinants of cardiotoxicity. described herein are generally incorporated into a pharma 0312. In particular embodiments, the cardiotoxic agent is ceutical composition prior to administration. A pharmaceuti a chemotherapeutic, and the Subject has cancer. Specific cal composition comprises one or more of the p97 polypep examples of cancers include, without limitation, breast can tides or conjugates described herein in combination with a cers, prostate cancers, gastrointestinal cancers, lung cancers, physiologically acceptable carrier or excipient. ovarian cancers, testicular cancers, head and neck cancers, 0320 To prepare a pharmaceutical composition, an effec stomach cancers, bladder cancers, pancreatic cancers, liver tive or desired amount of one or more of the p97 polypeptides cancers, kidney cancers, squamous cell carcinomas, CNS or or conjugates is mixed with any pharmaceutical carrier(s) or brain cancers (described herein), melanomas, non-melanoma excipient known to those skilled in the art to be suitable for the cancers, thyroid cancers, endometrial cancers, epithelial particular mode of administration. A pharmaceutical carrier tumors, bone cancers, and hematopoietic cancers. may be liquid, semi-liquid or Solid. Solutions or Suspensions 0313. In specific embodiments, the subject has a Her2/ used for parenteral, intradermal, Subcutaneous or topical neu-expressing cancer, Such as a breast cancer, ovarian can application may include, for example, a sterile diluent (Such cer, stomach cancer, aggressive uterine cancer, or metastatic as water), Saline Solution (e.g., phosphate buffered saline; cancer. Such as a metastatic CNS cancer, and the p97 polypep PBS), fixed oil, polyethylene glycol, glycerin, propylene gly tide is conjugated to trastuzumab. Such patients can benefit color other synthetic solvent; antimicrobial agents (such as not only from the therapeutic synergism resulting from the benzyl alcohol and methyl parabens); antioxidants (such as combination of p97 and trastuzumab, especially for CNS ascorbic acid and sodium bisulfite) and chelating agents (such cancers, but also from the reduced cardiotoxicity of trastu as ethylenediaminetetraacetic acid (EDTA)); buffers (such as Zumab, resulting from the potential cardioprotective effects acetates, citrates and phosphates). If administered intrave of p97. nously, Suitable carriers include physiological saline orphos 0314 Methods for identifying subjects with one or more phate buffered saline (PBS), and solutions containing thick of the diseases or conditions described herein are known in ening and solubilizing agents, such as glucose, polyethylene the art. glycol, polypropylene glycol and mixtures thereof. 0315 Also included are methods for imaging an organ or 0321) Administration of the polypeptides and conjugates tissue component in a subject, comprising (a) administering described herein, in pure form or in an appropriate pharma US 2014/0322132 A1 Oct. 30, 2014 34 ceutical composition, can be carried out via any of the non-covalently conjugated to each other, as described herein, accepted modes of administration of agents for serving simi and then mixed with lipids to form a liposome. The p97 lar utilities. The pharmaceutical compositions can be pre polypeptide, the agent, or the p97-agent conjugate may be pared by combining a polypeptide or conjugate or conjugate entrapped in microcapsules prepared, for example, by coac containing composition with an appropriate physiologically ervation techniques or by interfacial polymerization (for acceptable carrier, diluent or excipient, and may be formu example, hydroxymethylcellulose or gelatin-microcapsules lated into preparations in Solid, semi-solid, liquid or gaseous and poly-(methylmethacylate)microcapsules, respectively), forms, such as tablets, capsules, powders, granules, oint in colloidal drug delivery systems (for example, liposomes, ments, Solutions, Suppositories, injections, inhalants, gels, albumin microspheres, microemulsions, nano-particles and microspheres, and aerosols. In addition, other pharmaceuti nanocapsules), or in macroemulsions. Such techniques are cally active ingredients (including other anti-cancer agents as disclosed in Remington's Pharmaceutical Sciences, 16th edi described elsewhere herein) and/or suitable excipients such tion, Oslo, A., Ed., (1980). The particle(s) or liposomes may as salts, buffers and stabilizers may, but need not, be present further comprise other therapeutic or diagnostic agents. Such within the composition. as cytotoxic agents. 0322 Administration may be achieved by a variety of 0325 The precise dosage and duration of treatment is a different routes, including oral, parenteral, nasal, intrave function of the disease being treated and may be determined nous, intradermal, Subcutaneous or topical. Preferred modes empirically using known testing protocols or by testing the of administration depend upon the nature of the condition to compositions in model systems known in the art and extrapo be treated or prevented. lating therefrom. Controlled clinical trials may also be per 0323 Carriers can include, for example, pharmaceutically formed. Dosages may also vary with the severity of the con acceptable carriers, excipients, or stabilizers that are nontoxic dition to be alleviated. A pharmaceutical composition is to the cell or mammal being exposed thereto at the dosages generally formulated and administered to exert a therapeuti and concentrations employed. Often the physiologically cally useful effect while minimizing undesirable side effects. acceptable carrier is an aqueous pH buffered solution. The composition may be administered one time, or may be Examples of physiologically acceptable carriers include buff divided into a number of smaller doses to be administered at ers such as phosphate, citrate, and other organic acids; anti intervals of time. For any particular subject, specific dosage oxidants including ascorbic acid; low molecular weight (less regimens may be adjusted over time according to the indi than about 10 residues) polypeptide; proteins. Such as serum vidual need. albumin, gelatin, or immunoglobulins; hydrophilic polymers 0326 Typical routes of administering these and related Such as polyvinylpyrrolidone; amino acids Such as glycine, pharmaceutical compositions thus include, without limita glutamine, asparagine, arginine or lysine; monosaccharides, tion, oral, topical, transdermal, inhalation, parenteral, Sublin disaccharides, and other carbohydrates including glucose, gual, buccal, rectal, vaginal, and intranasal. The term mannose, or dextrins; chelating agents such as EDTA: Sugar parenteral as used herein includes Subcutaneous injections, alcohols such as mannitol or Sorbitol; salt-forming counteri intravenous, intramuscular, intrasternal injection or infusion ons such as sodium; and/or nonionic Surfactants such as techniques. Pharmaceutical compositions according to cer polysorbate 20 (TWEENTM) polyethylene glycol (PEG), and tain embodiments of the present invention are formulated so poloxamers (PLURONICSTM), and the like. as to allow the active ingredients contained therein to be 0324. In certain aspects, the p97 polypeptide sequence and bioavailable upon administration of the composition to a the agent are each, individually or as a pre-existing conjugate, patient. Compositions that will be administered to a subjector bound to or encapsulated within a particle, e.g., a nanopar patient may take the form of one or more dosage units, where ticle, bead, lipid formulation, lipid particle, or liposome, e.g., for example, a tablet may be a single dosage unit, and a immunoliposome. For instance, in particular embodiments, container of a herein described conjugate in aerosol form may the p97 polypeptide sequence is bound to the surface of a hold a plurality of dosage units. Actual methods of preparing particle, and the agent of interest is bound to the surface of the Such dosage forms are known, or will be apparent, to those particle and/or encapsulated within the particle. In some of skilled in this art; for example, see Remington. The Science these and related embodiments, the p97 polypeptide and the and Practice of Pharmacy, 20th Edition (Philadelphia Col agent are covalently or operatively linked to each other only lege of Pharmacy and Science, 2000). The composition to be via the particle itself (e.g., nanoparticle, liposome), and are administered will, in any event, contain a therapeutically not covalently linked to each other in any other way; that is, effective amount of a p97 polypeptide, agent, or conjugate they are bound individually to the same particle. In other described herein, for treatment of a disease or condition of embodiments, the p97 polypeptide and the agent are first interest. covalently or non-covalently conjugated to each other, as 0327. A pharmaceutical composition may be in the form described herein (e.g., via a linker molecule), and are then of a solid or liquid. In one embodiment, the carrier(s) are bound to or encapsulated within a particle (e.g., immunoli particulate, so that the compositions are, for example, in posome, nanoparticle). In specific embodiments, the particle tablet or powder form. The carrier(s) may be liquid, with the is a liposome, and the composition comprises one or more compositions being, for example, an oral oil, injectable liquid p97 polypeptides, one or more agents of interest, and a mix or an aerosol, which is useful in, for example, inhalatory ture of lipids to form a liposome (e.g., phospholipids, mixed administration. When intended for oral administration, the lipid chains with Surfactant properties). In some aspects, the pharmaceutical composition is preferably in either Solid or p97 polypeptide and the agent are individually mixed with the liquid form, where semi-solid, semi-liquid, Suspension and lipid/liposome mixture, such that the formation of liposome gel forms are included within the forms considered herein as structures operatively links the p97 polypeptide and the agent either solid or liquid. without the need for covalent conjugation. In other aspects, 0328. As a solid composition for oral administration, the the p97 polypeptide and the agent are first covalently or pharmaceutical composition may be formulated into a pow US 2014/0322132 A1 Oct. 30, 2014 der, granule, compressed tablet, pill, capsule, chewing gum, in a pharmaceutical composition for topical administration. If wafer or the like. Such a solid composition will typically intended for transdermal administration, the composition contain one or more inert diluents or edible carriers. In addi may include a transdermal patch or iontophoresis device. tion, one or more of the following may be present: binders 0333. The pharmaceutical composition may be intended Such as carboxymethylcellulose, ethyl cellulose, microcrys for rectal administration, in the form, for example, of a Sup talline cellulose, gum tragacanthor gelatin; excipients such as pository, which will melt in the rectum and release the drug. starch, lactose or dextrins, disintegrating agents such as alg The composition for rectal administration may contain an inic acid, Sodium alginate, Primogel, corn starch and the like; oleaginous base as a suitable nonirritating excipient. Such lubricants such as magnesium Stearate or Sterotex, glidants bases include, without limitation, lanolin, cocoa butter, and Such as colloidal silicon dioxide; Sweetening agents such as polyethylene glycol. Sucrose or saccharin; a flavoring agent Such as peppermint, 0334. The pharmaceutical composition may include vari methyl salicylate or orange flavoring; and a coloring agent. ous materials, which modify the physical form of a solid or When the pharmaceutical composition is in the form of a liquid dosage unit. For example, the composition may include capsule, for example, a gelatin capsule, it may contain, in materials that form a coating shell around the active ingredi addition to materials of the above type, a liquid carrier Such as ents. The materials that form the coating shell are typically polyethylene glycolor oil. inert, and may be selected from, for example, Sugar, shellac, 0329. The pharmaceutical composition may be in the form and other enteric coating agents. Alternatively, the active of a liquid, for example, an elixir, syrup, Solution, emulsion or ingredients may be encased in a gelatin capsule. The pharma Suspension. The liquid may be for oral administration or for ceutical composition in Solid or liquid form may include an delivery by injection, as two examples. When intended for agent that binds to the conjugate or agent and thereby assists oral administration, preferred composition contain, in addi in the delivery of the compound. Suitable agents that may act tion to the present compounds, one or more of a Sweetening in this capacity include monoclonal or polyclonal antibodies, agent, preservatives, dye? colorant and flavor enhancer. In a one or more proteins or a liposome. composition intended to be administered by injection, one or 0335 The pharmaceutical composition may consist more of a Surfactant, preservative, wetting agent, dispersing essentially of dosage units that can be administered as an agent, Suspending agent, buffer, stabilizer and isotonic agent aerosol. The term aerosol is used to denote a variety of sys may be included. tems ranging from those of colloidal nature to systems con 0330. The liquid pharmaceutical compositions, whether sisting of pressurized packages. Delivery may be by a lique they be solutions, suspensions or other like form, may include fied or compressed gas or by a suitable pump system that one or more of the following adjuvants: sterile diluents such dispenses the active ingredients. Aerosols may be delivered in as water for injection, Saline solution, preferably physiologi single phase, bi-phasic, or tri-phasic systems in order to cal saline, Ringer's Solution, isotonic sodium chloride, fixed deliver the active ingredient(s). Delivery of the aerosol oils such as synthetic mono or diglycerides which may serve includes the necessary container, activators, valves, Subcon as the solvent or Suspending medium, polyethylene glycols, tainers, and the like, which together may form a kit. One of glycerin, propylene glycol or other solvents; antibacterial ordinary skill in the art, without undue experimentation may agents such as benzyl alcohol or methyl paraben; antioxidants determine preferred aerosols. Such as ascorbic acid or sodium bisulfite; chelating agents 0336. The compositions comprising conjugates as Such as ethylenediaminetetraacetic acid; buffers such as described herein may be prepared with carriers that protect acetates, citrates orphosphates and agents for the adjustment the conjugates against rapid elimination from the body, Such of tonicity such as sodium chloride or dextrose. The as time release formulations or coatings. Such carriers parenteral preparation can be enclosed in ampoules, dispos include controlled release formulations, such as, but not lim able syringes or multiple dose vials made of glass or plastic. ited to, implants and microencapsulated delivery systems, Physiological saline is a preferred adjuvant. An injectable and biodegradable, biocompatible polymers, such as ethylene pharmaceutical composition is preferably sterile. vinyl acetate, polyanhydrides, polyglycolic acid, polyorthoe 0331. A liquid pharmaceutical composition intended for sters, polylactic acid and others known to those of ordinary either parenteral or oral administration should contain an skill in the art. amount of a p97 polypeptide or conjugate as herein disclosed 0337 The pharmaceutical compositions may be prepared such that a suitable dosage will be obtained. Typically, this by methodology well known in the pharmaceutical art. For amount is at least 0.01% of the agent of interest in the com example, a pharmaceutical composition intended to be position. When intended for oral administration, this amount administered by injection can be prepared by combining a may be varied to be between 0.1 and about 70% of the weight composition that comprises a conjugate as described herein of the composition. Certain oral pharmaceutical composi and optionally, one or more of salts, buffers and/or stabilizers, tions contain between about 4% and about 75% of the agent of with sterile, distilled water so as to form a solution. A surfac interest. In certain embodiments, pharmaceutical composi tant may be added to facilitate the formation of a homoge tions and preparations according to the present invention are neous solution or Suspension. Surfactants are compounds that prepared so that a parenteral dosage unit contains between non-covalently interact with the conjugate so as to facilitate 0.01 to 10% by weight of the agent of interest prior to dilution. dissolution or homogeneous Suspension of the conjugate in 0332 The pharmaceutical composition may be intended the aqueous delivery system. for topical administration, in which case the carrier may 0338. The compositions may be administered in a thera Suitably comprise a solution, emulsion, ointment or gel base. peutically effective amount, which will vary depending upon The base, for example, may comprise one or more of the a variety of factors including the activity of the specific com following: petrolatum, lanolin, polyethylene glycols, bee pound (e.g., conjugate) employed; the metabolic stability and wax, mineral oil, diluents such as water and alcohol, and length of action of the compound; the age, body weight, emulsifiers and stabilizers. Thickening agents may be present general health, sex, and diet of the patient; the mode and time US 2014/0322132 A1 Oct. 30, 2014 36 of administration; the rate of excretion; the drug combination; temperature in a chemical fume hood. To quench the reaction, the severity of the particular disorder or condition; and the 10 volumes of MS Grade Water was added. The digestion Subject undergoing therapy. Generally, a therapeutically material was frozen at -80° C. and lyophilized overnight. The effective daily dose is (for a 70kg mammal) from about 0.001 sample was stored at -20° C. until purification. Digestion mg/kg (i.e., 0.07 mg) to about 100 mg/kg (i.e., ~7.0 g); pref material was re-solubilized in 5 mL 0.1% formic acid and erably a therapeutically effective dose is (for a 70 kg mam purified using Sep-Pack C8 12cc cartridges from Waters. The mal) from about 0.01 mg/kg (i.e., -0.7 mg) to about 50 mg/kg purified digestion material was frozen at -80° C. and lyo (i.e., -3.5 g); more preferably a therapeutically effective dose philized overnight. The lyophilized product was then stored at is (for a 70kg mammal) from about 1 mg/kg (i.e., ~70 mg) to -20°C. Table 1 shows an example of predicted p97 fragments about 25 mg/kg (i.e., ~1.75 g). from the CNBr digest. 0339 Compositions comprising the conjugates described herein may also be administered simultaneously with, prior TABLE 1 to, or after administration of one or more other therapeutic agents, as described herein. For instance, in one embodiment, CNBr Predicted Digest the conjugate is administered with an anti-inflammatory Position Peptide Peptide Predicted p97 fragment- SEQ agent. Anti-inflammatory agents or drugs include, but are not Cleavage Length Mass Residues of Full-Length ID limited to, Steroids and glucocorticoids (including Site (AA) (Da) Human p97 (SEQID NO: 1) NO: betamethasone, budesonide, dexamethasone, hydrocortisone 2 2 2O6.3 1-2 NA acetate, hydrocortisone, hydrocortisone, methylpredniso 2O 18 2091.3 3-2O 19 lone, prednisolone, prednisone, triamcinolone), nonsteroidal 137 117 12432.1 21-137 2O anti-inflammatory drugs (NSAIDS) including aspirin, ibu 293 1S6 16894.7 138-293 21 333 40 4578.1 294-333 22 profen, naproxen, methotrexate, Sulfasalazine, leflunomide, 363 30 3447.1 334-363 23 anti-TNF , cyclophosphamide and mycopheno 388 25 2884.4 364-388 24 late. 609 221 24044.7 389-609 25 641 32 3670.1 610-641 26 0340 Such combination therapy may include administra 685 44 4892.5 642-685 27 tion of a single pharmaceutical dosage formulation which 692 7 695.7 686-692 28 contains a compound of the invention and one or more addi tional active agents, as well as administration of compositions comprising conjugates of the invention and each active agent (0345 SDS-PAGE analysis was performed on the digested in its own separate pharmaceutical dosage formulation. For and purified product. Native and digested protein Samples example, a conjugate as described herein and the other active were loaded onto a 4-12% Bis-Tris gel, and the gel was run agent can be administered to the patient together in a single using a constant voltage of 200V for 35 minutes with a start oral dosage composition Such as a tablet or capsule, or each ing current of 114 mA and an ending current of 65 mA. After agent administered in separate oral dosage formulations. electrophoresis, the gel was rinsed 3x for five minutes each Similarly, a conjugate as described herein and the other active with 200 mL of Milli-Q water. The gel was then stained with agent can be administered to the patient together in a single 20 mL of GelCode Blue Stain Reagent overnight, and subse parenteral dosage composition Such as in a saline solution or quently de-stained with 200 mL of Milli-Q water for one other physiologically acceptable solution, or each agent hour. The SDS-PAGE analysis is shown in FIG. 1 (Lane 1, administered in separate parenteral dosage formulations. empty; Lane 2, SeeBlue Latter; Lanes 2-5, empty; Lane 6, 50 Where separate dosage formulations are used, the composi ug undigested p97; lanes 7-9, empty; Lane 10, 50 ug CNBr tions comprising conjugates and one or more additional digested p97; lanes 11-12, empty). Lane 6, the undigested active agents can be administeredatessentially the same time, protein sample, had many bands indicating that the p97 pro i.e., concurrently, or at separately staggered times, i.e., tein had impurities. Lane 10, the CNBr digest, and at least sequentially and in any order; combination therapy is under three bands visible as large digest fragments. stood to include all these regimens. 0346. These three bands were excised, in-gel digested 0341 The following Examples are offered by way of illus with trypsin, and extracted and analyzed by LC-MS/MS tration and not by way of limitation. analysis. The results are shown in FIGS. 3-6. FIG.3 shows the sequence coverage maps of the p97 fragments identified by EXAMPLES MS/MS analysis of a CNBr digest of human p97: FIG. 3A shows the results for band 1, FIG. 3B shows the results for Example 1 band 2, and FIG. 3B shows the results for band 3. Generation Fragments of Human Melanotransferrin 0347 FIG. 4A shows the matching of the peptides (p97) detected in band 1 to the amino acid sequence of human p97; the sequence coverage of the matched peptides is indicated in 0342 Scaled chemical and enzymatic digestions of bold. FIG. 4B lists the individual peptides along with certain human melanotransferrin (p97) were performed using cyano physical characteristics. FIG. 5A shows the matching of the gen bromide (CNBr) and trypsin, to generate p97 fragments peptides detected in band 2 to the amino acid sequence of for testing in an in vitro model of blood-brain barrier (BBB) human p97; the sequence coverage of the matched peptides is transport. indicated in bold. FIG. 5B lists the individual peptides along (0343 CNBr Digestion: with certain physical characteristics. FIG. 6A shows the 0344) To a 500 uL protein sample of human p97 (10 matching of the peptides detected in band 3 to the amino acid mg/ml), 2.664 ml of 88% formic acid and 166.5 LL of 5 M sequence of human p97; the sequence coverage of the CNBr in acetonitrile was added. The sample was vortexed, matched peptides is indicated in bold. FIG. 6B lists the indi covered in aluminum foil, and incubated for 24 hours at room vidual peptides along with certain physical characteristics. US 2014/0322132 A1 Oct. 30, 2014 37

0348 Trypsin Digestion: were stabilized and composed of astrocytes (-60%), oligo (0349. To a 500 uL protein sample of human p97 (10 dendrocytes, and microglial cells (Descamps et al., Glia. mg/ml), 0.5 ml of 25 mMammonium bicarbonate was added. 42:46-58, 2003). Fifty microliters of 200 mM DTT (in 25 mM Ambic) was 0357 Preparation of Filter Inserts. added and reduced for 30 minutes at 37° C. Two hundred microliters of 200 mMiodoacetamide (in 25 mMAmbic) was 0358 Culture plate inserts (Transwell PE3 um pore size; added and free cysteines were alkylated for 30 minutes at 37° 24-mm diameter, COSTAR, 3452/Transwell PC3 um pore C. Next, 250 ug of porcine trypsin (Promega) was added to the size; 24-mm diameter, COSTAR, 3414) were coated on the sample and digestion was performed overnight at 37°C. The upper side with rat-tail collagen. digestion material was purified using Oasis HLB 6cc car 0359 Co-Culture of Brain Capillary Endothelial Cells tridges from Waters. The purified digestion material was fro with Glial Cells. Zen at 80 Cand lyophilized overnight. The lyophilized prod 0360. The glial cells were plated at a concentration of uct was stored at -20°C. about 1.25x10 cells/ml in plastic six-well plates and incu 0350 For MS analysis, the lyophilized p97 tryptic digests bated at 37°C. with 5% CO. The medium was changed twice were rehydrated in 1 mL 0.1% formic acid and 3% acetoni a week. Three weeks after seeding, cultures of glial cells trile. One microgram was loaded onto a C18 column and became stabilised. Then, sub-clones of endothelial cells fro injected into an LTQ Orbitrap Velos mass spectrometer Zen at passage 3 were cultured on a 60-mm-diameter gelatin (Thermo). MS/MS analysis showed that the sample contained coated Petri dish. Confluent endothelial cells were a number of protein contaminants, but also confirmed that the trypsinized and plated on the upper side of the filters at a p97 trypsin digest was successful. density of 4x10 cells/ml. The medium used for the co-culture 0351. The results are shown in FIG. 2. FIGS. 2A-2D show was DMEM supplemented with 10% (v/v) calf serum (CS) a list of p97 fragments identified by MS/MS analysis of an and 10% (v/v) horse serum (HS), 2 mM glutamine, and 50 in-solution trypsin digest of human p97, and FIG. 2E shows ug/ml of gentamycin, and 1 ng/ml of basic fibroblast growth the sequence coverage map of that analysis. factor was added every other day. Under these conditions, endothelial cells formed a confluent monolayer after about 12 Example 2 days. 0361 Lucifer Yellow was used as a paracellular marker Testing P97 Fragments in an In Vitro Model of the during evaluation of the test peptides to confirm the integrity Blood Brain Barrier of the BBB model. This small hydrophilic molecule presents 0352 Experiments were performed to evaluate the pas a low cerebral penetration and its endothelial permeability sage of mixtures of p97 peptide fragments across the blood coefficient reveals the endothelial cell monolayer integrity, brain barrier (BBB) using a relevant and predictive BBB in thereby serving as a useful control. On the day of the experi vitro model (see Cecchelli et al., Adv. Drug Deliv: Rev. ments, Ringer-HEPES (NaCl, 150 mM, KC1, 5.2 mM: CaCl, 36:165-178, 1999). The model utilizes brain capillary endot 2.2 mM: MgCl, 6 HO, 0.2 mM; NaHCOs, 6 mM; HEPES, 5 helial cells co-cultured with glial cells, to closely mimic the in mM; glucose, 2.8 mM) was added to the lower compartment vivo BBB (see Lundquist et al., Pharm. Res. 16:976-981, (abluminal side) of a six-well plate (3 mL per well). Filters 2002). with or without endothelial cells were washed with the 0353 Cell-Based Model of the BBB: Ringer-HEPES solution for 10 minutes at 37° C. to minimize 0354) To provide an in vitro system for studying brain traces of serum, and were then transferred to each well of the capillary functions, a process of co-culture that closely mim six-well plate. A volume of 1 mL Ringer-HEPES solution ics the in vivo BBB was established by culturing brain capil containing the peptide fragments incombination with Lucifer lary endothelial cells on one side of a filter and supportive Yellow (20 uM) was placed in the upper compartment (lumi glial cells on the other side. Specifically, endothelial cells nal side) of the well. were cultured in the upper compartment on the filter and glial 0362 Experiments were performed in triplicate with fil cells in the lower compartment on the plastic of a six-wells ters containing a confluent monolayer of endothelial cells (for plate (see FIGS. 7 and 8). Under these conditions, endothelial BBB integrity testing or evaluation of peptide fragment pas cells retain the appropriate endothelial markers (e.g., factor sage), or in triplicate with empty filters coated only with VIII—related antigen, non-thrombogenic Surface, produc collagen (filter test). Incubations were performed on a rock tion of prostacyclin, angiotensin-converting enzyme activ ing platform for 120 minutes at 37° C. At the end of the ity), and also retain the relevant characteristics of the BBB incubation period, aliquots of the luminal and abluminal liq (e.g., presence of tight junctions, paucity of pinocytotic uids were collected for fluorescence counting to evaluate vesicles, monoamine oxidase activity, Y-glutamyltranspepti membrane integrity (Lucifer Yellow), and LC/MS analysis to dase activity, P-glycoprotein activity, specific receptors for evaluate passage of the p97 peptide fragments across the low density lipoproteins, and transferrin). empty filter or the endothelial monolayer, as detailed below. 0355 Glial Cell Culture. 0363 Fluorescence Analysis. 0356. Primary cultures of glial cells were isolated from 0364 Lucifer Yellow (20 uM) was used as a paracellular newborn rat cerebral cortex (Booher & Sensenbrenner, Neu marker for monitor the permeability of the BBB, and was robiology. 2:97-105, 1972). After removing the meninges, the analyzed by a fluorescence counter (Flouroskan Ascent, brain tissue was forced gently through a nylon sieve. DMEM Thermolabs Systems). Fluorescence was determined in rep (Dulbecco's modified Eagle medium) supplemented with resentative samples from each lower compartment of the 10% (v/v) fetal calf serum (FCS, same as Fetal Bovine triplicate and from the initial Solution (containing test pep Serum: FBS), 2 mM glutamine, and 50 ugml of gentamy tides and Luciferyellow). For the abluminal side (lower com cin was used for the dissociation of cerebral tissue and devel partment), aliquots of 200 uL were added to 96-well plates opment of glial cells. Three weeks after seeding, glial cultures and measured by fluorescence counting, and for the luminal US 2014/0322132 A1 Oct. 30, 2014

side (upper compartment), aliquots of 204 from TO and T120 spectrometer (Thermo), and the data (Raw files) were ana minutes were added to 96-well plates and measured by fluo lyzed with the Proteome Discoverer 1.3.0.339 software suite rescence counting. (Thermo Scientific). The peak lists were submitted to a Mas 0365 LC/MS Analysis of Trypsin Digests. cot 2.3 server against the Uniprot-Swissprot database. The 0366 Three hundred microliters was removed from each peak areas were calculated for the top three peptides for each well and pooled into a single tube for each timepoint/fraction/ protein detected with high confidence. pore size. Five hundred microliters of 0.1% formic acid was 0367 Tryptic peptides were detected in the luminal and added to each sample for acidification. The peptides were abluminal compartments for both the 0.3 and 0.4 um pore purified using Oasis HLB 10cc cartridges from Waters, and sizes after 120 minutes. Based on the peak area of the top the purified peptides were frozen at -80° C. and lyophilized three p97 peptides, the ratio of peptides in the luminal side to overnight. The samples were rehydrated in 30 ul (20% aceto the abluminal side was about 2:1. The results for specific p97 nitrile, 0.1% FA). Fifteen microliters of each sample was peptides are shown in Table 2 (3 micron pore size) and Table analyzed by LC-MS/MS on an LTQ Orbitrap Velos mass 3 (4 micron pore size) below. TABLE 2 Tryptic Peptides at 3 Micron Pore Size Exp Exp Tryptic Peptide Ablum Lulum Ablum IonScore Walue Lull IonScore Walue Sequence 12O: 12O: 12O Ablum Ablum 12 O Lium Lulum (SEQ ID NO: ) Area Area conf 2O 12 O conf 2O 12O

LFSHEGSSFOMFS 1.28E-O9 4.55E-09 High 15 2.5OE-11 High 3 O 8.4 OE-13 SEAYGOK (SEO ID NO : 55)

HTTWFDNTNGHNS 1.28E-O9 1. O5E-10 High O6 280E-10 High O3 5.8 OE-10 EPWAAELR (SEO ID NO. 56)

HTTWFDNTNGHNS 7. O4E-O9 192E-10 High O1 9.5OE-10 High O9 14 OE-10 EPWAAELR (SEO ID NO. 56)

AWSDYFGGSCWPG 5. 49E-08 s 51E-O9 High O1 280E-10 High 25 1.2 OE-12 AGETSYSESLCR (SEO ID NO : 57)

NYPSSLCALCWGD 7.34E-Of 6.15E-08 High OO 6. 6 OE-10 High 11 6.4 OE-11 EOGR (SEO ID NO. 58)

TLPSWGOALLSOD 2.25E-O 6 194E-O9 High 94 5.1 OE-09 High 33 6.8 OE-13 FELLCR (SEO ID NO. 59)

AODLFGDDHNKNGFK 9. O9E-08 5.4 OE-08 High 87 2. 4 OE-08 High 72 7.1 OE-Of (SEQ ID NO: 15)

CLAEGAGDWAFWK 22 OE-09 438E-O9 High 87 3.1OE-08 High 92 7.9 OE-09 (SEQ ID NO: 6O)

MFDSSNYHGODLLFK 9. 62E-O8 2. O6E-O9 High 86 2.5OE-08 High 81 72 OE-08 (SEQ ID NO : 61)

ADTDGGLIFR 1.59E-10 111E-10 High 82 8.5OE-08 High 82 9. 1 OE-08 (SEQ ID NO: 10)

LFSHEGSSFOMFS 194E-08 1. O6E-O9 High 81 5.5OE-08 High 104 3.2 OE-10 SEAYGOK (SEO ID NO : 55)

MFDSSNYHGODLLFK 5. 67E-O9 173E-10 High 79 14 OE-07 High 79 1.5 OE-Of (SEQ ID NO : 61)

MFDSSNYHGODLLFK 3.22E-Of 101E-O8 High 79 1. 10E-O7 High 77 1. 6 OE-07 (SEQ ID NO : 61)

CGDMAWAFR 3.58E-O9 7.79E-09 High 76 1. SOE-Of High 79 7.3 OE-08 (SEQ ID NO: 11)

GDSSGEGWCDKSPLER 193E-O9 5. O8E-08 High 74 3.1OE-07 High 82 42 OE-08 (SEQ ID NO : 6)

US 2014/0322132 A1 Oct. 30, 2014 44

0368 LC/MS Analysis of CNBr Digests. 30 ul (20% acetonitrile, 0.1% FA). Fifteen microliters of each sample was analyzed by LC-MS/MS on an LTQ Orbitrap 0369 Three hundred microliters was removed from each Velos mass spectrometer (Thermo), and the data (Raw files) well and pooled into a single tube for each timepoint/fraction/ were analyzed with the Proteome Discoverer 1.3.0.339 soft pore size. Five hundred microliters of 0.1% formic acid was ware suite (Thermo Scientific). The peak lists were submitted added to each sample for acidification. The CNBr protein to a Mascot 2.3 server against the Uniprot-Swissprot data fragments were purified using Sep-Pak Vac 12cc C8 car base. The peak areas were calculated for the top three peptides tridges. Purified fragments were frozen at -80° C. and lyo for each protein detected with high confidence. philized overnight. The CNBr fragments were rehydrated 0370 Tryptic peptides from CNBr p97 fragments were with 25 mMammonium bicarbonate, reduced with DTT, and detected in the luminal and abluminal compartments for both alkylated with iodoacetamide. The alkylation was quenched the 0.3 and 0.4 um pore sizes after 120 minutes. Based on the with a section addition of DTT. Six micrograms of purified peak area of the top three p97 peptides, the ratio of peptides in porcine trypsin was then added to each well and the samples the luminal side to the abluminal side was about 200:1. The were placed overnight in a 37°C. incubator. The following results for specific p97 peptides are shown in Table 4 (3 morning, the peptides were purified using Oasis HLB 10cc micron pore size) and Table 5 (4 micron pore size) below. cartridges from Waters. Purified peptides were frozen at -80° Tryptic peptides from three distinct p97 CNBr fragments C. and lyophilized overnight. The samples were rehydrated in were detected (see FIG.9B). TABLE 4 CNBr dicests at 3 Micron Pore Size Exp Ablum Lulum Ablumn IonScore Walue Lull CNBr Peptide 12 O : 12O: 12 O Ablum Ablum 12 O IonScore Exp Value Sequence Area Area conf 12O 12O conf Lulum 12O Lulum 12 O

SEDYELLCPNGAR 3.92E- O6 9, 18E-08 High 65 1.5OE-O 6 High st 8. 6 OE-O 6 (SEQ ID NO: 14)

FDSSNYHGODLLFK 1. 46E- 08 2.16E-1. O High f 8.2OE-O 6 High 68 6, 4 OE-07 (SEO ID NO : 87)

WRPDTNIFTWYGLLDK 171E-- Of 173E-10 High 56 3.7OE-O 6 High 79 21 OE-08 (SEQ ID NO: 88)

FSSEAYGOK 17OE-06 2.39E-08 High 42 3. 4 OE-O4 High 42 3.2OE-O4 (SEO ID NO: 89)

DSSHAFTLDELR 1.79E-06 1.18E+07 Medium 32 41 OE-03 High 55 2. 4 OE-Os (SEQ ID NO: 13)

HTTWFDNTNGHNSEPW O. OOE- OO 3.96E-O8 High 11O 3.2OE-11 AAELR (SEO ID NO. 56)

AWSDYFGGSCWPGAGE O. OOE- OO 1 O2E-O7 High 1. Of 18OE-11 TSYSESLCR (SEO ID NO: 57)

NYPSSLCALCVGDEOGR O. OOE- OO 2.34E-O7 High 87 5. OOE-09 (SEO ID NO: 58)

ADTDGGLIFR O. OOE- OO 3.25E-Of Hilgn 85 2.8OE-08 (SEQ ID NO: 10)

SEDYELLCPNGAR O. OOE- OO 5. O4E-O7 High 77 7.9 OE-08 (SEQ ID NO: 14)

CLVENAGDWAFVR O. OOE- OO 2. 69E-07 Hilgn 7s 2. OOE-07 (SEO ID NO: 73)

TWGWNWPWGYLWESGR O. OOE- OO 4.96E-O7 Hilgn 67 6.9 OE-07 (SEO ID NO: 74)

FDSSNYHGODLLFK O. OOE- OO 9.22E-O7 High 66 7.9 OE-Of (SEQ ID NO: 86)

WCVLSTPEIOK O. OOE- OO 1. O4E-08 High 66 18OE-O 6 (SEO ID NO: 67)

EAGIOPSLLCVR O. OOE- OO 626E-08 High 64 1.5OE-O 6 (SEQ ID NO: 66)

US 2014/0322132 A1 Oct. 30, 2014 47

0371 Using the abluminal 120/luminal 120 peak area ratios as one possible criteria, the p97 peptides having the highest BBB transport activity are shown in Tables 6 (tryptic digests) and 7 below (CNBr digests). However, any of the p97 fragments in Tables 2-5 showing a value in the abluminal 120 area could be of potential interest for having BBB transport activity. TABLE 6 Tryptic peptides that cross the BBB based on abluminal/luminal peak area ratios Ábl 12 O/Lum12 0

Peptide O. 4 lum 3 O um AA SEQ ID Sequence CONF insert insert position Structure NO :

WCATSDPEOHK High 892 7. 41 25-35 S-H 2

RSSHWTIDTLK High 2. O6 1.23 115-125 C-H 3 SSHWTIDTLKGWK High 2. 42 2.38 116-128 4.

LCRGDSSGEGWCDK High 2.45 1.21 188-201 C-H-C 5 GDSSGEGVCDKSPLER High 4.93 3. 8O 191-2O6 6 YYOYSGAFR High 7.4 O NA 2O7-215 7

ADWEWR High 1276 11.26 263-269 C 8

WPAHAWWR High 3.32 3.56 276-284 C-S-H 9 ADTDGGEFR High 1.95 1. 44 285-294 1O

CGDMAWAFR High 2.26 2. O6 379-387 H 11

LKPEIOCVSAK High 4.81 4.37 391 - 4 O1 C-S-CE 12

DSSHAFTDER High O.98 NA 460 - 471 C-H-C 13

SEDYELLCPNGAR High 13. O5 8.38 596 - 608 C-S-C 14

AODLFGDDHNKNGFK High 5.45 1.68 645 - 659 H-C 15

TABLE F Example 3 p97 Fragment in an In Vivo Model of the Blood CNBr pe7 Fragments that Cross the BBB based Brain Barrier on abluminal /lumina peak area ratios 0372 Ap97 (Mtf) fragment (DSSHAFTLDELR; SEQID SEO ID NO:13) was conjugated to a monoclonal antibody (mAb), Peptide Sequence NO: administered peripherally to mice along with control pro teins, and tested relative to the control proteins for distribu FSSEAYGOKDLLFKDSTSELVPIATOTYEAWLGHEYLHAM 16 tion into brain tissues. For quantitative detection, all test proteins were labeled with Alexa Fluor 647 (AF647) accord ERIOAEOVDAVTLSGEDIYTAGKTYGLVPAAGEHYAPEDS 17 ing to routine techniques. SNSYYWWAWWRRDSSHAFTLDELRGKRSCHAGFGSPAGWD 0373 The following test proteins were prepared: AF647 WPVGALIORGFIRPKDCDVLTAVSEFFNASCVPVNNPKNY labeled monoclonal antibody (mAb), AF647-labeled MTf PSSLCALCVGDEOGRNKCVGNSOERYYGYRGAFRCLVENA mAb conjugate (MTf-mAb; MTf is soluble human p97), GDWAFWRHTTWEDNTNGHNSEPWAAELRSEDYELLCPNGA AF647-labeled MTf-mAb conjugate (MTf-mAb; RAEWSOFAACNLAQIPPHAVM MTF is the DSSHAFTLDELRYC (SEQID NO:92) frag ment of human p97); and AF647-labeled MTffragment with out antibody (MTf). The synthesis route of the MTf-mAb VRPDTNIFTWYGLLDKAODLFGDDHNKNGFKM 18 and MTf-mAb conjugates is illustrated in FIG. 10. 0374. The AF647-labeled test articles were administered to mice according to the study design in Table 8 below. TABLE 8 Study Design for Testing Brain Biodistribution in Mice Time Dose Level Dose Level Vascular Number Test Proteins Route? Point (h) (mg/kg) (nanomoles/kg) Perfusion of Mice" mAb IV 2 10 66.7 yes 3 MTf-mAb IV 2 15 65.2 yes 3 US 2014/0322132 A1 Oct. 30, 2014 48

TABLE 8-continued Study Design for Testing Brain Biodistribution in Mice Time Dose Level Dose Level Vascular Number Test Proteins Route? Point (h) (mg/kg) (nanomoles/kg) Perfusion of Mice MT?e, IV 2 5 1690.9 yes 3 MT-mAb IV 2 102 63.0 yes 3 Injection Wolume = 0.10 mLimouse *Injection Route = IV (tail vein) Vascular Perfusion = 5 min (a 4 ml/min with PBS pH 7.4 with 2.7% BSA, 100 UmL heparin 'Mouse Strain = BALB/c female 6-8 weeks old (17.4 + 1.1 grams (mean, S.D.)

0375. At 2 hours post-administration of test proteins, parenchyma), and V (Volume fraction of test proteins in Texas Red was administered, animals were sacrificed, and brain capillaries and brain parenchyma) were calculated. As brain tissues were removed. Five to six random fields were shown in FIG. 11, the unconjugated mAb did not effectively cryosected from the mid-coronal sections and the cerebral cross the BBB as illustrated by its low distribution in the brain cortex of brain tissues. Confocal microscopy was then per parenchyma. In contrast, conjugation of the mab to either formed to evaluate brain biodistribution of test proteins. MTfor MT?, increased distribution of the mab to the brain 0376 For confocal microscopy, confocal images of fluo parenchyma by about 5-fold. Also, the unconjugated MTf rescently labeled cells were acquired with an A Leica AOBS effectively crossed the BBB and distributed to brain paren SP8 laser Scanning confocal microscope (Leica, Heidelberg, chyma. These results illustrate that conjugation to fragments Germany). The excitation wavelengths were at 405 (DAPI), of p97 can be used to significantly improve the delivery of 595 nm (Texas Red), and 653 nm (AF647), and an 80 MHz polypeptides such as antibodies across the BBB and into CNS white light laser was used to collect the respective emission tissues such as the brain. signals. All images and spectral data (except DAPI) were generated using highly sensitive HyD detectors. The back Example 4 scattered emission signals from the sample were delivered through the tunable filter (AOBS). p97 Peptide Conjugates 0377 For three-dimensional (3D) image/volume fraction 0379 A p97 fragment (DSSHAFTLDELR; SEQ ID analysis, a series of two-dimensional (2D) Images (1024x NO:13) was conjugated to the 44 kd test protein horseradish 1024 pixels) for a 3D stack volume were acquired. The 3D peroxidase (HRP). This conjugate was administered periph stack images with optical section thickness (Z-axis) of erally (by IV injection) to mice along with control proteins, approximately 0.3 microns were captured from 20 micron and tested relative to the control proteins for distribution into brain tissue sections. For each tissue Volume, Z-section brain tissues. For quantitative detection, all test proteins were images were compiled and the 3-dimensional image restora labeled with Alexa Fluor 680 (AF680) according to routine tion was performed with Imaris (BITPLANE Scientific Soft techniques. ware). The Volume estimation was made on the 3D image data 0380. The following test proteins were prepared: AF680 sets recorded from five or more different areas of the cerebral labeled HRP (HRP): AF680-labeled MTf-HRP conjugate cortex. Gaussian noise removal filter was applied to define the (MTF-HRP; MTF is the DSSHAFTLDELRYC (SEQ boundary between foreground and background, and the lower ID NO:92) fragment of human p97). C-terminal cysteine and threshold level in the histogram was set to exclude all possible tyrosine residues were added to the MTf peptide for conju background voxel values. The sum of all the voxels above this gation and iodination, respectively. The synthesis route of the threshold level was determined to be the volume. HRP conjugates is illustrated in FIG. 12. 0378. The V (volume fraction of test proteins in brain 0381. The AF680-labeled test articles were administered capillaries), V. (Volume fraction of test proteins in brain to mice according to the study design in Table 9 below. TABLE

Study Design for Testing Brain Biodistribution in Mice

Time Dose Level Dose Level Vascular Number Test Proteins Route? Point (h) (mg/kg) (nanomoles/kg) Perfusion of Mice"

PBS IV 2 NA NA Yes 1 HRP IV 2 1O.O 227 yes 3 MTF-HRP IV 2 10.3 227 yes 3

'Injection Wolume = 0.10 mLimouse *Injection Route = IV (tail vein) Vascular Perfusion = 10 min (a 1 ml/min with PBS pH 7.4 with 2.7% BSA, 100 UmL heparin 'Mouse Strain = BALB/c female 6-8 weeks old (16-20 grams) US 2014/0322132 A1 Oct. 30, 2014 49

0382. At 2 hours post-administration of test proteins, 0384 The various embodiments described herein can be tomato Lectin-FITC was administered (80 ug for 10 minutes) combined to provide further embodiments. All of the U.S. to stain the brain vasculature followed by intracardiac perfu patents, U.S. patent application publications, U.S. patent sion of 10 ml heparinized saline, and brain tissues were application, foreign patents, foreign patent application and removed and processed for microscopy analysis. Three ran non-patent publications referred to in this specification and/or dom areas were cryosected from the mid-coronal sections brain tissues, fixed in cold acetone/methanol, and mounted listed in the Application Data Sheet are incorporated herein for microscopy. Three-dimensional (3D) confocal micros by reference, in their entirety. Aspects of the embodiments copy was then performed to evaluate brain biodistribution of can be modified, if necessary to employ concepts of the test proteins. various patents, application and publications to provide yet 0383. The results are shown in FIGS. 13 A-C. FIG. 13A further embodiments. shows the results for PBS, FIG. 13B shows the results for AF680-labeled HRP and FIG. 13C shows the results for 0385. These and other changes can be made to the embodi AF680-labeled MTf-HRP conjugate. FIGS. 13A and 13B ments in light of the above-detailed description. In general, in show no detectable AF680-labeling in brain tissues. In con the following claims, the terms used should not be construed trast, FIG. 13C shows detectable AF680-labeling, as illus to limit the claims to the specific embodiments disclosed in trated by the arrows. These results show that conjugation to the specification and the claims, but should be construed to the DSSHAFTLDELR peptide can significantly enhance the include all possible embodiments along with the full scope of delivery of a protein of interest across the BBB and into equivalents to which Such claims are entitled. Accordingly, tissues of the brain. the claims are not limited by the disclosure.

SEQUENCE LISTING

<16 Os NUMBER OF SEO ID NOS : 136

<21 Os SEQ ID NO 1 &211s LENGTH: 738 212s. TYPE: PRT <213> ORGANISM: Homo sapiens <4 OOs, SEQUENCE: 1

Met Arg Gly Pro Ser Gly Ala Lell Trp Lieu. Luell Luell Ala Lieu Arg Thr 1. 5 15

Wall Lell Gly Gly Met Glu Wall Arg Trp Ala Thir Ser Asp Pro Glu 25 3 O

Glin His Lys Cys Gly As yet Ser Glu Ala Phe Arg Glu Ala Gly Ile 35 4 O 45

Glin Pro Ser Luell Luell Wall Arg Gly Thir Ser Ala Asp His Wall SO 55 60

Glin Lell Ile Ala Ala Glin Glu Ala Asp Ala Ile Thir Luell Asp Gly Gly 65 70

Ala Ile Glu Ala Gly Lys Glu His Gly Luell Pro Wall Wall Gly 85 90 95

Glu Wall Asp Glin Glu Wall Gly Thir Ser Ala Wall Ala Wall 105 110

Wall Arg Arg Ser Ser His Wall Thir Ile Asp Thir Luell Lys Gly Wall 115 12O 125

Ser Cys His Thir Gly Ile ASn Arg Thir Wall Gly Trp Asn Wall Pro Wall 13 O 135 14 O

Gly Tyr Lell Wall Glu Ser Gly Arg Lell Ser Wall Met Gly Asp Wall 145 15 O 155 16 O

Luell Ala Wall Ser Asp Tyr Phe Gly Gly Ser Wall Pro Gly Ala 1.65 17 O 17s

Gly Glu Thir Ser Ser Glu Ser Lell Arg Luell Arg Gly Asp 18O 185 190

Ser Ser Gly Glu Gly Val Cys Asp Ser Pro Luell Glu Arg 195 2 OO 2O5

Asp Tyr Ser Gly Ala Phe Arg Lell Ala Glu Gly Ala Gly Asp Wall 210 215 22 O US 2014/0322132 A1 Oct. 30, 2014 50

- Continued

Ala Phe Val Llys His Ser Thr Val Lieu. Glu Asn Thr Asp Gly Lys Thr 225 23 O 235 24 O Lieu Pro Ser Trp Gly Glin Ala Lieu. Lieu. Ser Glin Asp Phe Glu Lieu. Lieu. 245 250 255 Cys Arg Asp Gly Ser Arg Ala Asp Val Thr Glu Trp Arg Glin Cys His 26 O 265 27 O Lieu Ala Arg Val Pro Ala His Ala Val Val Val Arg Ala Asp Thr Asp 27s 28O 285 Gly Gly Lieu. Ile Phe Arg Lieu. Lieu. Asn. Glu Gly Glin Arg Lieu. Phe Ser 29 O 295 3 OO His Glu Gly Ser Ser Phe Gln Met Phe Ser Ser Glu Ala Tyr Gly Glin 3. OS 310 315 32O Lys Asp Lieu. Lieu. Phe Lys Asp Ser Thir Ser Glu Lieu Val Pro Ile Ala 3.25 330 335 Thr Glin Thr Tyr Glu Ala Trp Leu Gly His Glu Tyr Lieu. His Ala Met 34 O 345 35. O Lys Gly Lieu. Lieu. Cys Asp Pro Asn Arg Lieu Pro Pro Tyr Lieu. Arg Trp 355 360 365 Cys Val Lieu Ser Thr Pro Glu Ile Gln Lys Cys Gly Asp Met Ala Val 37 O 375 38O Ala Phe Arg Arg Glin Arg Lieu Lys Pro Glu Ile Glin Cys Val Ser Ala 385 390 395 4 OO Llys Ser Pro Gln His Cys Met Glu Arg Ile Glin Ala Glu Glin Val Asp 4 OS 41O 415 Ala Val Thr Lieu Ser Gly Glu Asp Ile Tyr Thr Ala Gly Llys Thr Tyr 42O 425 43 O Gly Lieu Val Pro Ala Ala Gly Glu. His Tyr Ala Pro Glu Asp Ser Ser 435 44 O 445 Asn Ser Tyr Tyr Val Val Ala Val Val Arg Arg Asp Ser Ser His Ala 450 45.5 460 Phe Thr Lieu. Asp Glu Lieu. Arg Gly Lys Arg Ser Cys His Ala Gly Phe 465 470 47s 48O Gly Ser Pro Ala Gly Trp Asp Val Pro Val Gly Ala Lieu. Ile Glin Arg 485 490 495 Gly Phe Ile Arg Pro Lys Asp Cys Asp Val Lieu. Thir Ala Val Ser Glu SOO 505 51O Phe Phe Asn Ala Ser Cys Val Pro Val Asn Asn Pro Lys Asn Tyr Pro 515 52O 525 Ser Ser Lieu. Cys Ala Lieu. Cys Val Gly Asp Glu Glin Gly Arg Asn Lys 53 O 535 54 O Cys Val Gly Asn Ser Glin Glu Arg Tyr Tyr Gly Tyr Arg Gly Ala Phe 5.45 550 555 560 Arg Cys Lieu Val Glu Asn Ala Gly Asp Wall Ala Phe Val Arg His Thr 565 st O sts Thr Val Phe Asp Asn Thr Asn Gly His Asn Ser Glu Pro Trp Ala Ala 58O 585 59 O

Glu Lieu. Arg Ser Glu Asp Tyr Glu Lieu. Lieu. Cys Pro Asn Gly Ala Arg 595 6OO 605

Ala Glu Val Ser Glin Phe Ala Ala Cys Asn Lieu Ala Glin Ile Pro Pro 610 615 62O US 2014/0322132 A1 Oct. 30, 2014 51

- Continued His Ala Val Met Val Arg Pro Asp Thr Asn Ile Phe Thr Val Tyr Gly 625 630 635 64 O Lieu. Lieu. Asp Lys Ala Glin Asp Lieu. Phe Gly Asp Asp His Asn Lys Asn 645 650 655 Gly Phe Llys Met Phe Asp Ser Ser Asn Tyr His Gly Glin Asp Lieu. Lieu. 660 665 67 O Phe Lys Asp Ala Thr Val Arg Ala Val Pro Val Gly Glu Lys Thir Thr 675 68O 685 Tyr Arg Gly Trp Lieu. Gly Lieu. Asp Tyr Val Ala Ala Lieu. Glu Gly Met 69 O. 695 7 OO Ser Ser Glin Glin Cys Ser Gly Ala Ala Ala Pro Ala Pro Gly Ala Pro 7 Os 71O 71s 72O Lieu. Lieu Pro Lieu Lleu Lleu Pro Ala Lieu Ala Ala Arg Lieu. Lieu Pro Pro 72 73 O 73

Ala Lieu

<210s, SEQ ID NO 2 &211s LENGTH: 11 212. TYPE: PRT <213> ORGANISM: Homo sapiens <4 OOs, SEQUENCE: 2 Trp. Cys Ala Thr Ser Asp Pro Glu Gln His Lys 1. 5 1O

<210s, SEQ ID NO 3 &211s LENGTH: 11 212. TYPE: PRT <213> ORGANISM: Homo sapiens

<4 OOs, SEQUENCE: 3 Arg Ser Ser His Val Thir Ile Asp Thir Lieu Lys 1. 5 1O

<210s, SEQ ID NO 4 &211s LENGTH: 13 212. TYPE: PRT <213> ORGANISM: Homo sapiens

<4 OOs, SEQUENCE: 4 Ser Ser His Val Thr Ile Asp Thr Lieu Lys Gly Val Lys 1. 5 1O

<210s, SEQ ID NO 5 &211s LENGTH: 14 212. TYPE: PRT <213> ORGANISM: Homo sapiens

<4 OOs, SEQUENCE: 5 Lieu. Cys Arg Gly Asp Ser Ser Gly Glu Gly Val Cys Asp Llys 1. 5 1O

<210s, SEQ ID NO 6 &211s LENGTH: 16 212. TYPE: PRT <213> ORGANISM: Homo sapiens

<4 OOs, SEQUENCE: 6 Gly Asp Ser Ser Gly Glu Gly Val Cys Asp Llys Ser Pro Lieu. Glu Arg US 2014/0322132 A1 Oct. 30, 2014 52

- Continued

1. 5 1O 15

<210s, SEQ ID NO 7 &211s LENGTH: 9 212. TYPE: PRT <213> ORGANISM: Homo sapiens

<4 OO > SEQUENCE: 7 Tyr Tyr Asp Tyr Ser Gly Ala Phe Arg 1. 5

<210s, SEQ ID NO 8 &211s LENGTH: 7 212. TYPE: PRT <213> ORGANISM: Homo sapiens <4 OOs, SEQUENCE: 8 Ala Asp Val Thr Glu Trp Arg 1. 5

<210s, SEQ ID NO 9 &211s LENGTH: 9 212. TYPE: PRT <213> ORGANISM: Homo sapiens <4 OOs, SEQUENCE: 9 Val Pro Ala His Ala Val Val Val Arg 1. 5

<210s, SEQ ID NO 10 &211s LENGTH: 10 212. TYPE: PRT <213> ORGANISM: Homo sapiens <4 OOs, SEQUENCE: 10 Ala Asp Thir Asp Gly Gly Lieu. Ile Phe Arg 1. 5 1O

<210s, SEQ ID NO 11 &211s LENGTH: 9 212. TYPE: PRT <213> ORGANISM: Homo sapiens <4 OOs, SEQUENCE: 11 Cys Gly Asp Met Ala Val Ala Phe Arg 1. 5

<210s, SEQ ID NO 12 &211s LENGTH: 11 212. TYPE: PRT <213> ORGANISM: Homo sapiens

<4 OOs, SEQUENCE: 12 Lieu Lys Pro Glu Ile Glin Cys Val Ser Ala Lys 1. 5 1O

<210s, SEQ ID NO 13 &211s LENGTH: 12 212. TYPE: PRT <213> ORGANISM: Homo sapiens

<4 OOs, SEQUENCE: 13 US 2014/0322132 A1 Oct. 30, 2014 53

- Continued Asp Ser Ser His Ala Phe Thr Lieu. Asp Glu Lieu. Arg 1. 5 1O

<210s, SEQ ID NO 14 &211s LENGTH: 13 212. TYPE: PRT <213> ORGANISM: Homo sapiens

<4 OOs, SEQUENCE: 14 Ser Glu Asp Tyr Glu Lieu. Lieu. Cys Pro Asn Gly Ala Arg 1. 5 1O

<210s, SEQ ID NO 15 &211s LENGTH: 15 212. TYPE: PRT <213> ORGANISM: Homo sapiens

<4 OOs, SEQUENCE: 15 Ala Glin Asp Lieu. Phe Gly Asp Asp His Asn Lys Asn Gly Phe Lys 1. 5 1O 15

<210s, SEQ ID NO 16 &211s LENGTH: 4 O 212. TYPE: PRT <213> ORGANISM: Homo sapiens

<4 OOs, SEQUENCE: 16 Phe Ser Ser Glu Ala Tyr Gly Gln Lys Asp Lieu Lleu Phe Lys Asp Ser 1. 5 1O 15 Thir Ser Glu Lieu Val Pro Ile Ala Thr Glin Thr Tyr Glu Ala Trp Leu 2O 25 3O Gly His Glu Tyr Lieu. His Ala Met 35 4 O

<210s, SEQ ID NO 17 &211s LENGTH: 221 212. TYPE: PRT <213> ORGANISM: Homo sapiens

<4 OOs, SEQUENCE: 17 Glu Arg Ile Glin Ala Glu Glin Val Asp Ala Val Thir Lieu. Ser Gly Glu 1. 5 1O 15 Asp Ile Tyr Thr Ala Gly Llys Thr Tyr Gly Lieu Val Pro Ala Ala Gly 2O 25 3O Glu. His Tyr Ala Pro Glu Asp Ser Ser Asn Ser Tyr Tyr Val Val Ala 35 4 O 45 Val Val Arg Arg Asp Ser Ser His Ala Phe Thr Lieu. Asp Glu Lieu. Arg SO 55 6 O Gly Lys Arg Ser Cys His Ala Gly Phe Gly Ser Pro Ala Gly Trp Asp 65 70 7s 8O

Val Pro Val Gly Ala Lieu. Ile Glin Arg Gly Phe Ile Arg Pro Lys Asp 85 90 95

Cys Asp Val Lieu. Thir Ala Val Ser Glu Phe Phe Asn Ala Ser Cys Val 1OO 105 11 O

Pro Val Asn. Asn. Pro Lys Asn Tyr Pro Ser Ser Lieu. Cys Ala Lieu. Cys 115 12 O 125

Val Gly Asp Glu Glin Gly Arg Asn Lys Cys Val Gly Asn. Ser Glin Glu 13 O 135 14 O US 2014/0322132 A1 Oct. 30, 2014 54

- Continued

Arg Tyr Tyr Gly Tyr Arg Gly Ala Phe Arg Cys Lieu Val Glu Asn Ala 145 150 155 160 Gly Asp Val Ala Phe Val Arg His Thr Thr Val Phe Asp Asn Thr Asn 1.65 17O 17s Gly His Asn. Ser Glu Pro Trp Ala Ala Glu Lieu. Arg Ser Glu Asp Tyr 18O 185 19 O Glu Lieu. Lieu. Cys Pro Asn Gly Ala Arg Ala Glu Val Ser Glin Phe Ala 195 2OO 2O5 Ala Cys Asn Lieu Ala Glin Ile Pro Pro His Ala Wal Met 21 O 215 22O

<210s, SEQ ID NO 18 &211s LENGTH: 32 212. TYPE: PRT <213> ORGANISM: Homo sapiens <4 OOs, SEQUENCE: 18 Val Arg Pro Asp Thr Asn. Ile Phe Thr Val Tyr Gly Lieu. Lieu. Asp Llys 1. 5 1O 15 Ala Glin Asp Lieu. Phe Gly Asp Asp His Asn Lys Asn Gly Phe Llys Met 2O 25 3O

<210s, SEQ ID NO 19 &211s LENGTH: 5 212. TYPE PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Heterologous sulfatase motif 22 Os. FEATURE: <221 > NAMEAKEY: SC FEATURE <222s. LOCATION: (1) . . (1) <223> OTHER INFORMATION: Xaa = any amino acid or may be absent 22 Os. FEATURE: <221 > NAMEAKEY: SC FEATURE <222s. LOCATION: (2) ... (2) <223> OTHER INFORMATION: Xaa = Cys or Ser 22 Os. FEATURE: <221 > NAMEAKEY: SC FEATURE <222s. LOCATION: (3) ... (3) <223> OTHER INFORMATION: Xaa = any amino acid 22 Os. FEATURE: <221 > NAMEAKEY: SC FEATURE <222s. LOCATION: (4) ... (4) <223 is OTHER INFORMATION: Xaa = Pro or Ala 22 Os. FEATURE: <221 > NAMEAKEY: SC FEATURE <222s. LOCATION: (5) . . (5) <223> OTHER INFORMATION: Xaa = Any amino acid <4 OOs, SEQUENCE: 19

Xaa Xala Xala Xala Xala 1. 5

<210s, SEQ ID NO 2 O &211s LENGTH: 5 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Heterologous sulfatase motif 22 Os. FEATURE: <221s NAME/KEY: MISC FEATURE <222s. LOCATION: (1) . . (1) <223> OTHER INFORMATION: X = Any Amino Acid or may be absent 22 Os. FEATURE: <221s NAME/KEY: MOD RES US 2014/0322132 A1 Oct. 30, 2014 55

- Continued

<222s. LOCATION: (2) ... (2) <223> OTHER INFORMATION: C-alpha-formylglycine (FGly) 22 Os. FEATURE: <221s NAME/KEY: MISC FEATURE <222s. LOCATION: (3) ... (3) 223 OTHER INFORMATION: Xaa Any Amino Acid 22 Os. FEATURE: <221s NAME/KEY: MISC FEATURE <222s. LOCATION: (4) ... (4) 223 OTHER INFORMATION: Xaa Ala or Pro 22 Os. FEATURE: <221s NAME/KEY: MISC FEATURE <222s. LOCATION: (5) . . (5) 223 OTHER INFORMATION: Xaa Any amino acid <4 OOs, SEQUENCE: 2O Xaa Gly Xaa Xala Xaa 1. 5

<210s, SEQ ID NO 21 &211s LENGTH: 4 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Peptide linker <4 OOs, SEQUENCE: 21 Gly Ser Gly Ser 1.

<210s, SEQ ID NO 22 &211s LENGTH: 4 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Peptide linker <4 OOs, SEQUENCE: 22 Gly Gly Ser Gly 1.

<210s, SEQ ID NO 23 &211s LENGTH: 4 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Peptide linker <4 OOs, SEQUENCE: 23 Gly Gly Gly Ser 1.

<210s, SEQ ID NO 24 &211s LENGTH: 5 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Peptide Linker

<4 OOs, SEQUENCE: 24 Gly Gly Gly Gly Ser 1. 5

<210s, SEQ ID NO 25 &211s LENGTH: 4 212. TYPE: PRT US 2014/0322132 A1 Oct. 30, 2014 56

- Continued <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Peptide linker <4 OOs, SEQUENCE: 25 Gly Asn Gly Asn 1.

<210s, SEQ ID NO 26 &211s LENGTH: 4 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Peptide linker <4 OOs, SEQUENCE: 26 Gly Gly Asn Gly 1.

<210s, SEQ ID NO 27 &211s LENGTH: 4 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Peptide linker <4 OOs, SEQUENCE: 27 Gly Gly Gly Asn 1.

<210s, SEQ ID NO 28 &211s LENGTH: 5 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Peptide linker <4 OOs, SEQUENCE: 28 Gly Gly Gly Gly Asn 1. 5

<210s, SEQ ID NO 29 &211s LENGTH: 4 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Thrombin cleavable linker

<4 OOs, SEQUENCE: 29 Gly Arg Gly Asp 1.

<210s, SEQ ID NO 3 O &211s LENGTH: 6 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Thrombin cleavable linker

<4 OOs, SEQUENCE: 30 Gly Arg Gly Asp Asn Pro 1. 5

<210s, SEQ ID NO 31 US 2014/0322132 A1 Oct. 30, 2014 57

- Continued

&211s LENGTH: 5 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Thrombin cleavable linker

<4 OOs, SEQUENCE: 31 Gly Arg Gly Asp Ser 1. 5

<210s, SEQ ID NO 32 &211s LENGTH: 7 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Thrombin cleavable linker

<4 OOs, SEQUENCE: 32 Gly Arg Gly Asp Ser Pro Llys 1. 5

<210s, SEQ ID NO 33 &211s LENGTH: 4 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Elastase cleavable linker

<4 OOs, SEQUENCE: 33

Ala Ala Pro Wall 1.

<210s, SEQ ID NO 34 &211s LENGTH: 4 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Elastase cleavable linker

<4 OOs, SEQUENCE: 34

Ala Ala Pro Lieu. 1.

<210s, SEQ ID NO 35 &211s LENGTH: 4 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Elastase cleavable linker

<4 OOs, SEQUENCE: 35

Ala Ala Pro Phe 1.

<210s, SEQ ID NO 36 &211s LENGTH: 4 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Elastase cleavable linker

<4 OOs, SEQUENCE: 36

Ala Ala Pro Ala 1. US 2014/0322132 A1 Oct. 30, 2014 58

- Continued

<210s, SEQ ID NO 37 &211s LENGTH: 4 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Elastase cleavable linker

<4 OO > SEQUENCE: 37 Ala Tyr Lieu Val 1.

<210s, SEQ ID NO 38 &211s LENGTH: 6 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Matrix metalloproteinase cleavable linker 22 Os. FEATURE: <221s NAME/KEY: VARIANT <222s. LOCATION: (3) ... (3) 223 OTHER INFORMATION: Xaa Any amino acid 22 Os. FEATURE: <221s NAME/KEY: VARIANT <222s. LOCATION: (6) . . (6) 223 OTHER INFORMATION: Xaa Any amino acid <4 OOs, SEQUENCE: 38 Gly Pro Xaa Gly Pro Xaa 1. 5

<210s, SEQ ID NO 39 &211s LENGTH: 4 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Matrix metalloproteinase cleavable linker 22 Os. FEATURE: <221s NAME/KEY: VARIANT <222s. LOCATION: (4) ... (4) <223> OTHER INFORMATION: Xaa = Any amino acid <4 OOs, SEQUENCE: 39 Lieu. Gly Pro Xaa 1.

<210s, SEQ ID NO 4 O &211s LENGTH: 6 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Matrix metalloproteinase cleavable linker 22 Os. FEATURE: <221s NAME/KEY: VARIANT <222s. LOCATION: (6) . . (6) <223> OTHER INFORMATION: Xaa = Any amino acid

<4 OOs, SEQUENCE: 4 O Gly Pro Ile Gly Pro Xaa 1. 5

<210s, SEQ ID NO 41 &211s LENGTH: 5 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Matrix metalloproteinase cleavable linker 22 Os. FEATURE: US 2014/0322132 A1 Oct. 30, 2014 59

- Continued <221s NAME/KEY: VARIANT <222s. LOCATION: (5) . . (5) <223> OTHER INFORMATION: Xaa = Any amino acid <4 OOs, SEQUENCE: 41 Ala Pro Gly Lieu. Xaa 1. 5

<210s, SEQ ID NO 42 &211s LENGTH: 7 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Collagenase cleavable linker 22 Os. FEATURE: <221s NAME/KEY: VARIANT <222s. LOCATION: (7) . . (7) <223> OTHER INFORMATION: Xaa = Any amino acid <4 OOs, SEQUENCE: 42 Pro Lieu. Gly Pro Asp Arg Xaa 1. 5

<210s, SEQ ID NO 43 &211s LENGTH: 7 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Collagenase cleavable linker & 22 O FEATURE; <221s NAME/KEY: VARIANT <222s. LOCATION: (7) . . (7) <223> OTHER INFORMATION: Xaa = Any amino acid <4 OOs, SEQUENCE: 43 Pro Lieu. Gly Lieu. Lieu. Gly Xaa 1. 5

<210s, SEQ ID NO 44 &211s LENGTH: 7 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Collagenase cleavable linker <4 OOs, SEQUENCE: 44 Pro Glin Gly Ile Ala Gly Trp 1. 5

<210s, SEQ ID NO 45 &211s LENGTH: 5 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Collagenase cleavable linker

<4 OOs, SEQUENCE: 45 Pro Leu Gly Cys His 1. 5

<210s, SEQ ID NO 46 &211s LENGTH: 6 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Collagenase cleavable linker US 2014/0322132 A1 Oct. 30, 2014 60

- Continued

<4 OOs, SEQUENCE: 46 Pro Lieu. Gly Lieu. Tyr Ala 1. 5

<210s, SEQ ID NO 47 &211s LENGTH: 7 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Collagenase cleavable linker <4 OOs, SEQUENCE: 47 Pro Lieu Ala Lieu. Trp Ala Arg 1. 5

<210s, SEQ ID NO 48 &211s LENGTH: 7 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Collagenase cleavable linker <4 OOs, SEQUENCE: 48 Pro Lieu Ala Tyr Trp Ala Arg 1. 5

<210 SEQ ID NO 49 &211s LENGTH: 7 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Stromelysin cleavable linker <4 OOs, SEQUENCE: 49 Pro Tyr Ala Tyr Tyr Met Arg 1. 5

<210s, SEQ ID NO 50 &211s LENGTH: 7 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Gelatinase cleavable linker

<4 OOs, SEQUENCE: 50 Pro Leu Gly Met Tyr Ser Arg 1. 5

<210s, SEQ ID NO 51 &211s LENGTH: 4 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Angiotensin converting enzyme cleavable linker

<4 OOs, SEQUENCE: 51 Gly Asp Llys Pro 1.

<210s, SEQ ID NO 52 &211s LENGTH: 5 212. TYPE: PRT <213> ORGANISM: Artificial Sequence US 2014/0322132 A1 Oct. 30, 2014 61

- Continued

22 Os. FEATURE: <223> OTHER INFORMATION: Angiotensin converting enzyme cleavable linker <4 OOs, SEQUENCE: 52 Gly Ser Asp Llys Pro 1. 5

<210s, SEQ ID NO 53 &211s LENGTH: 4 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Cathepsin B cleavable linker <4 OOs, SEQUENCE: 53

Ala Lieu Ala Lieu. 1.

<210s, SEQ ID NO 54 &211s LENGTH: 4 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Cathepsin B cleavable linker <4 OOs, SEQUENCE: 54 Gly Phe Leu Gly 1.

<210s, SEQ ID NO 55 &211s LENGTH: 2O 212. TYPE: PRT <213> ORGANISM: Homo sapiens

<4 OO > SEQUENCE: 55 Lieu. Phe Ser His Glu Gly Ser Ser Phe Glin Met Phe Ser Ser Glu Ala 1. 5 1O 15 Tyr Gly Glin Lys 2O

<210s, SEQ ID NO 56 &211s LENGTH: 21 212. TYPE: PRT <213> ORGANISM: Homo sapiens

<4 OOs, SEQUENCE: 56 His Thr Thr Val Phe Asp Asn Thr Asn Gly His Asn Ser Glu Pro Trp 1. 5 1O 15 Ala Ala Glu Lieu. Arg 2O

<210s, SEQ ID NO 57 &211s LENGTH: 25 212. TYPE: PRT <213> ORGANISM: Homo sapiens

<4 OO > SEQUENCE: 57 Ala Val Ser Asp Tyr Phe Gly Gly Ser Cys Val Pro Gly Ala Gly Glu 1. 5 1O 15

Thir Ser Tyr Ser Glu Ser Lieu. Cys Arg 2O 25 US 2014/0322132 A1 Oct. 30, 2014 62

- Continued

<210s, SEQ ID NO 58 &211s LENGTH: 17 212. TYPE: PRT <213> ORGANISM: Homo sapiens <4 OOs, SEQUENCE: 58 Asn Tyr Pro Ser Ser Lieu. Cys Ala Lieu. CyS Val Gly Asp Glu Glin Gly 1. 5 1O 15 Arg

<210s, SEQ ID NO 59 &211s LENGTH: 19 212. TYPE: PRT <213> ORGANISM: Homo sapiens <4 OO > SEQUENCE: 59 Thir Lieu Pro Ser Trp Gly Glin Ala Lieu. Lieu. Ser Glin Asp Phe Glu Lieu. 1. 5 1O 15 Lieu. Cys Arg

<210s, SEQ ID NO 60 &211s LENGTH: 13 212. TYPE: PRT <213> ORGANISM: Homo sapiens

<4 OOs, SEQUENCE: 60 Cys Lieu Ala Glu Gly Ala Gly Asp Val Ala Phe Val Lys 1. 5 1O

<210s, SEQ ID NO 61 &211s LENGTH: 15 212. TYPE: PRT <213> ORGANISM: Homo sapiens

<4 OOs, SEQUENCE: 61 Met Phe Asp Ser Ser Asn Tyr His Gly Glin Asp Lieu. Lieu. Phe Lys 1. 5 1O 15

<210s, SEQ ID NO 62 &211s LENGTH: 25 212. TYPE: PRT <213> ORGANISM: Homo sapiens

<4 OOs, SEQUENCE: 62 Lieu. Phe Ser His Glu Gly Ser Ser Phe Glin Met Phe Ser Ser Glu Ala 1. 5 1O 15 Tyr Gly Glin Lys Asp Lieu. Lieu. Phe Lys 2O 25

<210s, SEQ ID NO 63 &211s LENGTH: 13 212. TYPE: PRT <213> ORGANISM: Homo sapiens

<4 OOs, SEQUENCE: 63 Arg Asp Ser Ser His Ala Phe Thr Lieu. Asp Glu Lieu. Arg 1. 5 1O

<210s, SEQ ID NO 64 &211s LENGTH: 11 US 2014/0322132 A1 Oct. 30, 2014 63

- Continued

212. TYPE: PRT <213> ORGANISM: Homo sapiens <4 OOs, SEQUENCE: 64 Ala Glin Asp Lieu. Phe Gly Asp Asp His Asn Lys 1. 5 1O

<210s, SEQ ID NO 65 &211s LENGTH: 10 212. TYPE: PRT <213> ORGANISM: Homo sapiens <4 OOs, SEQUENCE: 65 Lieu. Ser Wal Met Gly Cys Asp Val Lieu Lys 1. 5 1O

<210s, SEQ ID NO 66 &211s LENGTH: 12 212. TYPE: PRT <213> ORGANISM: Homo sapiens <4 OOs, SEQUENCE: 66 Glu Ala Gly Ile Glin Pro Ser Lieu. Lieu. Cys Val Arg 1. 5 1O

<210s, SEQ ID NO 67 &211s LENGTH: 10 212. TYPE PRT <213> ORGANISM: Homo sapiens

<4 OO > SEQUENCE: 67 Ser Ser His Val Thr Ile Asp Thr Lieu Lys 1. 5 1O

<210s, SEQ ID NO 68 &211s LENGTH: 11 212. TYPE: PRT <213> ORGANISM: Homo sapiens

<4 OOs, SEQUENCE: 68 Trp. Cys Val Lieu Ser Thr Pro Glu Ile Glin Lys 1. 5 1O

<210s, SEQ ID NO 69 &211s LENGTH: 8 212. TYPE: PRT <213> ORGANISM: Homo sapiens

<4 OOs, SEQUENCE: 69 Gly Lieu. Lieu. Cys Asp Pro Asn Arg 1. 5

<210s, SEQ ID NO 70 &211s LENGTH: 14 212. TYPE: PRT <213> ORGANISM: Homo sapiens

<4 OO > SEQUENCE: 7 O Asp Ser Ser His Ala Phe Thr Lieu. Asp Glu Lieu. Arg Gly Lys 1. 5 1O

<210s, SEQ ID NO 71 US 2014/0322132 A1 Oct. 30, 2014 64

- Continued

&211s LENGTH: 14 212. TYPE: PRT <213> ORGANISM: Homo sapiens

<4 OOs, SEQUENCE: 71 Gly Lieu. Lieu. Cys Asp Pro Asn Arg Lieu Pro Pro Tyr Lieu. Arg 1. 5 1O

<210s, SEQ ID NO 72 &211s LENGTH: 28 212. TYPE: PRT <213> ORGANISM: Homo sapiens <4 OOs, SEQUENCE: 72 Glu. His Gly Lieu Lys Pro Val Val Gly Glu Val Tyr Asp Glin Glu Val 1. 5 1O 15 Gly. Thir Ser Tyr Tyr Ala Wall Ala Val Val Arg Arg 2O 25

<210s, SEQ ID NO 73 &211s LENGTH: 13 212. TYPE: PRT <213> ORGANISM: Homo sapiens <4 OO > SEQUENCE: 73 Cys Val Gly Asn Ser Glin Glu Arg Tyr Tyr Gly Tyr Arg 1. 5 1O

<210s, SEQ ID NO 74 &211s LENGTH: 13 212. TYPE: PRT <213> ORGANISM: Homo sapiens

<4 OOs, SEQUENCE: 74 Cys Lieu Val Glu Asn Ala Gly Asp Val Ala Phe Val Arg 1. 5 1O

<210s, SEQ ID NO 75 &211s LENGTH: 27 212. TYPE: PRT <213> ORGANISM: Homo sapiens

<4 OO > SEQUENCE: 75 Asp Ser Thr Ser Glu Lieu Val Pro Ile Ala Thr Glin Thr Tyr Glu Ala 1. 5 1O 15 Trp Lieu. Gly. His Glu Tyr Lieu. His Ala Met Lys 2O 25

<210s, SEQ ID NO 76 &211s LENGTH: 21 212. TYPE: PRT <213> ORGANISM: Homo sapiens

<4 OO > SEQUENCE: 76 Ile Glin Ala Glu Glin Val Asp Ala Val Thir Lieu. Ser Gly Glu Asp Ile 1. 5 1O 15 Tyr Thr Ala Gly Lys 2O

<210s, SEQ ID NO 77 &211s LENGTH: 11 US 2014/0322132 A1 Oct. 30, 2014 65

- Continued

212. TYPE: PRT <213> ORGANISM: Homo sapiens <4 OO > SEQUENCE: 77 His Ser Thr Val Lieu. Glu Asn. Thir Asp Gly Lys 1. 5 1O

<210s, SEQ ID NO 78 &211s LENGTH: 16 212. TYPE: PRT <213> ORGANISM: Homo sapiens <4 OO > SEQUENCE: 78 Thr Val Gly Trp Asn Val Pro Val Gly Tyr Lieu Val Glu Ser Gly Arg 1. 5 1O 15

<210s, SEQ ID NO 79 &211s LENGTH: 7 212. TYPE: PRT <213> ORGANISM: Homo sapiens <4 OO > SEQUENCE: 79 Lieu. Lieu. Asn. Glu Gly Glin Arg 1. 5

<210s, SEQ ID NO 8O &211s LENGTH: 25 212. TYPE PRT <213> ORGANISM: Homo sapiens

<4 OOs, SEQUENCE: 80 Lieu. Phe Ser His Glu Gly Ser Ser Phe Glin Met Phe Ser Ser Glu Ala 1. 5 1O 15 Tyr Gly Glin Lys Asp Lieu. Lieu. Phe Lys 2O 25

<210s, SEQ ID NO 81 &211s LENGTH: 17 212. TYPE: PRT <213> ORGANISM: Homo sapiens

<4 OOs, SEQUENCE: 81 Ala Asp Thir Asp Gly Gly Lieu. Ile Phe Arg Lieu. Lieu. Asn. Glu Gly Glin 1. 5 1O 15 Arg

<210s, SEQ ID NO 82 &211s LENGTH: 9 212. TYPE: PRT <213> ORGANISM: Homo sapiens

<4 OOs, SEQUENCE: 82 Cys Gly Asn Met Ser Glu Ala Phe Arg 1. 5

<210s, SEQ ID NO 83 &211s LENGTH: 13 212. TYPE: PRT <213> ORGANISM: Homo sapiens

<4 OOs, SEQUENCE: 83 US 2014/0322132 A1 Oct. 30, 2014 66

- Continued Ala Asp Val Thr Glu Trp Arg Glin Cys His Lieu Ala Arg 1. 5 1O

<210s, SEQ ID NO 84 &211s LENGTH: 32 212. TYPE: PRT <213> ORGANISM: Homo sapiens

<4 OOs, SEQUENCE: 84 Asp Lieu. Lieu. Phe Lys Asp Ser Thir Ser Glu Lieu Val Pro Ile Ala Thr 1. 5 1O 15 Gln Thr Tyr Glu Ala Trp Lieu. Gly His Glu Tyr Lieu. His Ala Met Lys 2O 25 3O

<210s, SEQ ID NO 85 &211s LENGTH: 2O 212. TYPE: PRT <213> ORGANISM: Homo sapiens

<4 OOs, SEQUENCE: 85 Met Phe Asp Ser Ser Asn Tyr His Gly Glin Asp Lieu. Lieu. Phe Lys Asp 1. 5 1O 15 Ala Thr Val Arg 2O

<210s, SEQ ID NO 86 & 211 LENGTH; 11 212. TYPE: PRT <213> ORGANISM: Homo sapiens <4 OOs, SEQUENCE: 86 Ala Val Pro Val Gly Glu Lys Thr Thr Tyr Arg 1. 5 1O

<210s, SEQ ID NO 87 &211s LENGTH: 14 212. TYPE: PRT <213> ORGANISM: Homo sapiens

<4 OO > SEQUENCE: 87 Phe Asp Ser Ser Asn Tyr His Gly Glin Asp Lieu Lleu Phe Lys 1. 5 1O

<210s, SEQ ID NO 88 &211s LENGTH: 16 212. TYPE: PRT <213> ORGANISM: Homo sapiens

<4 OOs, SEQUENCE: 88 Val Arg Pro Asp Thr Asn. Ile Phe Thr Val Tyr Gly Lieu. Lieu. Asp Llys 1. 5 1O 15

<210s, SEQ ID NO 89 &211s LENGTH: 9 212. TYPE: PRT <213> ORGANISM: Homo sapiens

<4 OOs, SEQUENCE: 89 Phe Ser Ser Glu Ala Tyr Gly Gln Lys 1. 5 US 2014/0322132 A1 Oct. 30, 2014 67

- Continued

<210s, SEQ ID NO 90 &211s LENGTH: 31 212. TYPE: PRT <213> ORGANISM: Homo sapiens

<4 OOs, SEQUENCE: 90 Gly. Thir Ser Ala Asp His Cys Val Glin Lieu. Ile Ala Ala Glin Glu Ala 1. 5 1O 15 Asp Ala Ile Thr Lieu. Asp Gly Gly Ala Ile Tyr Glu Ala Gly Lys 2O 25 3O

<210s, SEQ ID NO 91 &211s LENGTH: 690 212. TYPE: PRT <213> ORGANISM: Homo sapiens <4 OOs, SEQUENCE: 91 Gly Met Glu Val Arg Trp Cys Ala Thr Ser Asp Pro Glu Gln His Lys 1. 5 1O 15 Cys Gly Asn Met Ser Glu Ala Phe Arg Glu Ala Gly Ile Glin Pro Ser 2O 25 3O Lieu. Lieu. Cys Val Arg Gly. Thir Ser Ala Asp His Cys Val Glin Lieu. Ile 35 4 O 45 Ala Ala Glin Glu Ala Asp Ala Ile Thir Lieu. Asp Gly Gly Ala Ile Tyr SO 55 6 O Glu Ala Gly Lys Glu. His Gly Lieu Lys Pro Val Val Gly Glu Val Tyr 65 70 7s 8O Asp Glin Glu Val Gly. Thir Ser Tyr Tyr Ala Val Ala Val Val Arg Arg 85 90 95 Ser Ser His Val Thr Ile Asp Thr Lieu Lys Gly Val Lys Ser Cys His 1OO 105 11 O Thr Gly Ile Asn Arg Thr Val Gly Trp Asin Val Pro Val Gly Tyr Lieu. 115 12 O 125 Val Glu Ser Gly Arg Lieu. Ser Val Met Gly Cys Asp Val Lieu Lys Ala 13 O 135 14 O Val Ser Asp Tyr Phe Gly Gly Ser Cys Val Pro Gly Ala Gly Glu Thr 145 150 155 160 Ser Tyr Ser Glu Ser Lieu. Cys Arg Lieu. Cys Arg Gly Asp Ser Ser Gly 1.65 17O 17s Glu Gly Val Cys Asp Llys Ser Pro Lieu. Glu Arg Tyr Tyr Asp Tyr Ser 18O 185 19 O Gly Ala Phe Arg Cys Lieu Ala Glu Gly Ala Gly Asp Wall Ala Phe Val 195 2OO 2O5 Lys His Ser Thr Val Lieu. Glu Asn Thr Asp Gly Lys Thr Lieu. Pro Ser 21 O 215 22O

Trp Gly Glin Ala Lieu Lleu Ser Glin Asp Phe Glu Lieu. Lieu. Cys Arg Asp 225 23 O 235 24 O Gly Ser Arg Ala Asp Val Thr Glu Trp Arg Glin Cys His Lieu Ala Arg 245 250 255 Val Pro Ala His Ala Val Val Val Arg Ala Asp Thr Asp Gly Gly Lieu 26 O 265 27 O

Ile Phe Arg Lieu. Lieu. Asn. Glu Gly Glin Arg Lieu. Phe Ser His Glu Gly 27s 28O 285

Ser Ser Phe Gln Met Phe Ser Ser Glu Ala Tyr Gly Gln Lys Asp Leu US 2014/0322132 A1 Oct. 30, 2014 68

- Continued

29 O 295 3 OO Lieu. Phe Lys Asp Ser Thr Ser Glu Lieu Val Pro Ile Ala Thr Glin Thr 3. OS 310 315 32O Tyr Glu Ala Trp Lieu. Gly His Glu Tyr Lieu. His Ala Met Lys Gly Lieu. 3.25 330 335 Lieu. Cys Asp Pro Asn Arg Lieu Pro Pro Tyr Lieu. Arg Trp Cys Val Lieu. 34 O 345 35. O Ser Thr Pro Glu Ile Glin Lys Cys Gly Asp Met Ala Val Ala Phe Arg 355 360 365 Arg Glin Arg Lieu Lys Pro Glu Ile Glin Cys Val Ser Ala Lys Ser Pro 37 O 375 38O Glin His Cys Met Glu Arg Ile Glin Ala Glu Glin Val Asp Ala Val Thr 385 390 395 4 OO Lieu. Ser Gly Glu Asp Ile Tyr Thr Ala Gly Lys Thr Tyr Gly Lieu Val 4 OS 41O 415 Pro Ala Ala Gly Glu. His Tyr Ala Pro Glu Asp Ser Ser Asn Ser Tyr 42O 425 43 O Tyr Val Val Ala Val Val Arg Arg Asp Ser Ser His Ala Phe Thr Lieu. 435 44 O 445 Asp Glu Lieu. Arg Gly Lys Arg Ser Cys His Ala Gly Phe Gly Ser Pro 450 45.5 460 Ala Gly Trp Asp Val Pro Val Gly Ala Lieu. Ile Glin Arg Gly Phe Ile 465 470 475 48O Arg Pro Lys Asp Cys Asp Val Lieu. Thir Ala Val Ser Glu Phe Phe Asn 485 490 495 Ala Ser Cys Val Pro Val Asn Asn Pro Lys Asn Tyr Pro Ser Ser Leu SOO 505 51O Cys Ala Lieu. Cys Val Gly Asp Glu Glin Gly Arg Asn Lys Cys Val Gly 515 52O 525 Asn Ser Glin Glu Arg Tyr Tyr Gly Tyr Arg Gly Ala Phe Arg Cys Lieu 53 O 535 54 O Val Glu Asn Ala Gly Asp Val Ala Phe Val Arg His Thr Thr Val Phe 5.45 550 555 560 Asp Asn. Thir Asn Gly. His Asn. Ser Glu Pro Trp Ala Ala Glu Lieu. Arg 565 st O sts Ser Glu Asp Tyr Glu Lieu. Lieu. Cys Pro Asn Gly Ala Arg Ala Glu Val 58O 585 59 O Ser Glin Phe Ala Ala Cys Asn Lieu Ala Glin Ile Pro Pro His Ala Val 595 6OO 605 Met Val Arg Pro Asp Thr Asn Ile Phe Thr Val Tyr Gly Lieu. Leu Asp 610 615 62O Lys Ala Glin Asp Lieu. Phe Gly Asp Asp His Asn Lys Asn Gly Phe Lys 625 630 635 64 O Met Phe Asp Ser Ser Asn Tyr His Gly Glin Asp Lieu. Lieu. Phe Lys Asp 645 650 655

Ala Thr Val Arg Ala Val Pro Val Gly Glu Lys Thr Thr Tyr Arg Gly 660 665 67 O

Trp Lieu. Gly Lieu. Asp Tyr Val Ala Ala Lieu. Glu Gly Met Ser Ser Glin 675 68O 685

Gln Cys 69 O. US 2014/0322132 A1 Oct. 30, 2014 69

- Continued

<210s, SEQ ID NO 92 &211s LENGTH: 14 212. TYPE: PRT <213> ORGANISM: Homo sapiens <4 OOs, SEQUENCE: 92 Asp Ser Ser His Ala Phe Thr Lieu. Asp Glu Lieu. Arg Tyr Cys 1. 5 1O

<210s, SEQ ID NO 93 &211s LENGTH: 12 212. TYPE: PRT <213> ORGANISM: Jaculus jaculus <4 OOs, SEQUENCE: 93 Asp Ser Ser Asp Ala Phe Thr Lieu. Asp Glu Lieu. Arg 1. 5 1O

<210s, SEQ ID NO 94 &211s LENGTH: 12 212. TYPE: PRT <213> ORGANISM: Otolemur garnettii <4 OOs, SEQUENCE: 94 Asp Ser Ser His Ser Phe Thr Lieu. Asp Glu Lieu. Arg 1. 5 1O

<210s, SEQ ID NO 95 &211s LENGTH: 12 212. TYPE: PRT <213> ORGANISM: Pongo abelii <4 OO > SEQUENCE: 95 Asp Ser Ser Asp Ala Phe Thr Lieu. Asp Glu Lieu. Arg 1. 5 1O

<210s, SEQ ID NO 96 &211s LENGTH: 12 212. TYPE: PRT <213> ORGANISM: Ictidomys tridecemlineatus <4 OOs, SEQUENCE: 96 Asp Ser Ser Tyr Ala Phe Thr Lieu. Asp Glu Lieu. Arg 1. 5 1O

<210s, SEQ ID NO 97 &211s LENGTH: 12 212. TYPE: PRT <213> ORGANISM: Ceratotherium Simum simum

<4 OO > SEQUENCE: 97 Asn Ser Ser His Ala Phe Thr Lieu. Asp Glu Lieu. Arg 1. 5 1O

<210s, SEQ ID NO 98 &211s LENGTH: 12 212. TYPE: PRT <213> ORGANISM: Wicugna pacos

<4 OOs, SEQUENCE: 98 Asn Ser Ser Tyr Ala Phe Thr Lieu. Asp Glu Lieu. Arg US 2014/0322132 A1 Oct. 30, 2014 70

- Continued

<210s, SEQ ID NO 99 &211s LENGTH: 12 212. TYPE: PRT <213> ORGANISM: Ochotona princeps

<4 OOs, SEQUENCE: 99 Asp Ser Ser Tyr Ala Phe Pro Lieu. Asp Glu Lieu. Arg 1. 5 1O

<210s, SEQ ID NO 100 &211s LENGTH: 12 212. TYPE: PRT <213> ORGANISM: Pteropus alecto <4 OOs, SEQUENCE: 1.OO Asn Ser Ser Tyr Ala Phe Thr Lieu. Asp Glu Lieu. Arg 1. 5 1O

<210s, SEQ ID NO 101 &211s LENGTH: 12 212. TYPE: PRT <213> ORGANISM: Tursiops truncatus <4 OOs, SEQUENCE: 101 Asn Ser Ser Tyr Ala Phe Thr Lieu. Asp Glu Lieu. Arg 1. 5 1O

<210s, SEQ ID NO 102 &211s LENGTH: 12 212. TYPE: PRT <213> ORGANISM: Tupaia chinensis <4 OOs, SEQUENCE: 102 Asp Ser Thr His Ala Phe Thr Val Asp Glu Lieu. Arg 1. 5 1O

<210s, SEQ ID NO 103 &211s LENGTH: 12 212. TYPE: PRT <213> ORGANISM: Pantholops hodgsonii

<4 OOs, SEQUENCE: 103 Asn Ser Ser Tyr Ala Phe Thr Lieu. Asp Glu Lieu. Arg 1. 5 1O

<210s, SEQ ID NO 104 &211s LENGTH: 12 212. TYPE: PRT <213> ORGANISM: Felis catus

<4 OOs, SEQUENCE: 104 Asn Ser Ser Tyr Ala Phe Thr Lieu. Asp Glu Lieu. Arg 1. 5 1O

<210s, SEQ ID NO 105 &211s LENGTH: 12 212. TYPE: PRT &213s ORGANISM: Bos taurus

<4 OOs, SEQUENCE: 105 US 2014/0322132 A1 Oct. 30, 2014 71

- Continued Asn Ser Ser Tyr Ala Phe Thr Lieu. Asp Glu Lieu. Arg 1. 5 1O

<210s, SEQ ID NO 106 &211s LENGTH: 12 212. TYPE: PRT <213> ORGANISM: Mustela putorius furo <4 OOs, SEQUENCE: 106 Asn Ser Ser Tyr Ala Phe Thr Lieu. Asp Glu Lieu. Arg 1. 5 1O

<210s, SEQ ID NO 107 &211s LENGTH: 12 212. TYPE: PRT <213> ORGANISM: Ailuropoda Melanoleuca

<4 OOs, SEQUENCE: 107 Asn Ser Ser Tyr Ala Phe Thr Lieu. Asp Glu Lieu. Arg 1. 5 1O

<210s, SEQ ID NO 108 &211s LENGTH: 12 212. TYPE: PRT <213> ORGANISM: Capra hircus <4 OOs, SEQUENCE: 108 ASn Ser Ser Tyr Ala Phe Thr Lieu. Asp Glu Lieu. Arg 1. 5 1O

<210s, SEQ ID NO 109 &211s LENGTH: 12 212. TYPE: PRT <213s ORGANISM: Mus musculus

<4 OOs, SEQUENCE: 109 Asp Ser Ser Tyr Ser Phe Thr Lieu. Asp Glu Lieu. Arg 1. 5 1O

<210s, SEQ ID NO 110 &211s LENGTH: 12 212. TYPE: PRT <213> ORGANISM: Orcinus orca

<4 OOs, SEQUENCE: 110 Asn Ser Ser Asn Ala Phe Thr Lieu. Asp Glu Lieu. Arg 1. 5 1O

<210s, SEQ ID NO 111 &211s LENGTH: 12 212. TYPE: PRT <213> ORGANISM: Chinchilla lanigera

<4 OOs, SEQUENCE: 111 Asp Ser Ser Ser Ala Phe Thr Lieu. Asn. Glu Lieu. Arg 1. 5 1O

<210s, SEQ ID NO 112 &211s LENGTH: 12 212. TYPE: PRT <213> ORGANISM: Dasypus novemcinctus

<4 OOs, SEQUENCE: 112 US 2014/0322132 A1 Oct. 30, 2014 72

- Continued

Asp Ser Ser Tyr Ala Phe Thr Lieu. Asp Glu Lieu. Trp 1. 5 1O

<210s, SEQ ID NO 113 &211s LENGTH: 12 212. TYPE: PRT <213> ORGANISM: Rattus norvegicus <4 OOs, SEQUENCE: 113 Asp Ser Ser Tyr Ser Phe Thr Lieu. Asp Glu Lieu. Arg 1. 5 1O

<210s, SEQ ID NO 114 &211s LENGTH: 12 212. TYPE: PRT <213> ORGANISM: Odobenus rosmarus divergens <4 OOs, SEQUENCE: 114 Asn Ser Ser Ser Ala Phe Thr Lieu. Asp Glu Lieu. Arg 1. 5 1O

<210s, SEQ ID NO 115 &211s LENGTH: 12 212. TYPE: PRT <213> ORGANISM: Microtus ochrogaster

<4 OOs, SEQUENCE: 115 Asp Ser Ser Tyr Ser Phe Thr Lieu. Asp Glu Lieu. Arg 1. 5 1O

<210s, SEQ ID NO 116 &211s LENGTH: 12 212. TYPE: PRT <213> ORGANISM: Ovis airies

<4 OOs, SEQUENCE: 116 Asn Ser Ser Tyr Ala Phe Thr Lieu. Asp Glu Lieu. Arg 1. 5 1O

<210s, SEQ ID NO 117 &211s LENGTH: 12 212. TYPE: PRT <213> ORGANISM: Leptonychotes weddellii

<4 OOs, SEQUENCE: 117 Asn Ser Ser Tyr Ala Phe Thr Lieu. Asp Glu Lieu. Arg 1. 5 1O

<210s, SEQ ID NO 118 &211s LENGTH: 12 212. TYPE: PRT <213s ORGANISM: Camelus ferus

<4 OOs, SEQUENCE: 118 Asn Ser Ser Tyr Ala Phe Thr Lieu. Asp Glu Lieu. Arg 1. 5 1O

<210s, SEQ ID NO 119 &211s LENGTH: 12 212. TYPE: PRT <213s ORGANISM: Sus scrofa US 2014/0322132 A1 Oct. 30, 2014 73

- Continued

<4 OOs, SEQUENCE: 119 Asn Ser Ser Tyr Ala Phe Thr Lieu. Asp Glu Lieu. Arg 1. 5 1O

<210s, SEQ ID NO 120 &211s LENGTH: 12 212. TYPE: PRT &213s ORGANISM: Bos mutus

<4 OOs, SEQUENCE: 120 Asn Ser Ser Tyr Ala Phe Thr Lieu. Asp Glu Lieu. Arg 1. 5 1O

<210s, SEQ ID NO 121 &211s LENGTH: 12 212. TYPE: PRT <213> ORGANISM: Cyphellophora europaea

<4 OOs, SEQUENCE: 121 Ala Thir Ser His Ala Ile Thr Lieu. Asp Glu Lieu. Arg 1. 5 1O

<210s, SEQ ID NO 122 &211s LENGTH: 12 212. TYPE: PRT <213> ORGANISM: Loxodonta africana

< 4 OO SEQUENCE: 122 Asn Ser Ser Tyr Ala Phe Thr Met Asp Glu Lieu. Arg 1. 5 1O

<210s, SEQ ID NO 123 &211s LENGTH: 12 212. TYPE: PRT <213> ORGANISM: Cricetulus griseus <4 OOs, SEQUENCE: 123 Asp Arg Ser Tyr Ser Phe Thr Lieu. Asp Glu Lieu. Arg 1. 5 1O

<210s, SEQ ID NO 124 &211s LENGTH: 12 212. TYPE: PRT <213> ORGANISM: Oryctolagus cuniculus

<4 OOs, SEQUENCE: 124 Asp Ser Ala Tyr Ala Phe Thr Val Asp Glu Lieu. Arg 1. 5 1O

<210s, SEQ ID NO 125 &211s LENGTH: 12 212. TYPE: PRT <213> ORGANISM: Octodon degus

<4 OOs, SEQUENCE: 125 Asp Ser Ser Ser Ala Phe Asn Lieu. Asn. Glu Lieu. Arg 1. 5 1O

<210s, SEQ ID NO 126 &211s LENGTH: 12 212. TYPE: PRT <213> ORGANISM: Canis lupus familiaris US 2014/0322132 A1 Oct. 30, 2014 74

- Continued

<4 OOs, SEQUENCE: 126 Asn Ser Ser Asp Ala Phe Ser Lieu. Asp Glu Lieu. Arg 1. 5 1O

<210s, SEQ ID NO 127 &211s LENGTH: 12 212. TYPE: PRT <213> ORGANISM: Cavia porcellus

<4 OOs, SEQUENCE: 127 Asp Ser Ser Ser Ala Phe Ser Lieu. Asn. Glu Lieu. Arg 1. 5 1O

<210s, SEQ ID NO 128 &211s LENGTH: 12 212. TYPE: PRT &213s ORGANISM: Sorex alranels

<4 OOs, SEQUENCE: 128 Asn Ser Ser Asp Ala Phe Ser Lieu. Asp Glu Lieu. Arg 1. 5 1O

<210s, SEQ ID NO 129 &211s LENGTH: 12 212. TYPE: PRT <213> ORGANISM: Trichechus manatus latirostris

<4 OOs, SEQUENCE: 129 Asn Ser Ser Tyr Ala Phe Thr Met Asp Glu Lieu. Arg 1. 5 1O

<210s, SEQ ID NO 130 &211s LENGTH: 12 212. TYPE: PRT <213> ORGANISM: Mesocricetus auratus

<4 OOs, SEQUENCE: 130 Asp Arg Ser Tyr Ser Phe Thr Lieu. Asp Glu Lieu. Arg 1. 5 1O

<210s, SEQ ID NO 131 &211s LENGTH: 12 212. TYPE: PRT <213> ORGANISM: Monodelphis domestica <4 OOs, SEQUENCE: 131 Asn Ser Ser Tyr Ser Phe Thr Lieu. Asp Glu Lieu. Arg 1. 5 1O

<210s, SEQ ID NO 132 &211s LENGTH: 12 212. TYPE: PRT <213> ORGANISM: Equus caballus

<4 OOs, SEQUENCE: 132 Asn Ser Ser Tyr Ala Phe Thr Val Asp Glu Lieu. Arg 1. 5 1O

<210s, SEQ ID NO 133 &211s LENGTH: 12 212. TYPE: PRT US 2014/0322132 A1 Oct. 30, 2014 75

- Continued <213> ORGANISM: Echinops telfairi <4 OOs, SEQUENCE: 133 Asn Ser Ser Tyr Ala Phe Thr Val Asp Glu Lieu. Arg 1. 5 1O

<210s, SEQ ID NO 134 &211s LENGTH: 12 212. TYPE: PRT <213> ORGANISM: Condylura cristata

<4 OOs, SEQUENCE: 134 Asn Ser Ser Tyr Ala Phe Ser Lieu. Asp Glu Lieu. Arg 1. 5 1O

<210s, SEQ ID NO 135 &211s LENGTH: 11 212. TYPE: PRT <213> ORGANISM: Homo sapien <4 OOs, SEQUENCE: 135 Ser Ala Ser Asp Lieu. Thir Trp Asp Asn Lieu Lys 1. 5

SEQ ID NO 136 LENGTH: 11 TYPE PRT ORGANISM; Homo sapien

< 4 OOs SEQUENCE: 136 Ser Asp Thr Ser Lieu. Thir Trp Asn Ser Val Lys 1. 5 1O

1. An isolated p97 polypeptide of up to about 300 amino lites, anthracyclines, anti-tumor antibiotics, platinums, type I acids in length, where the polypeptide comprises an amino topoisomerase inhibitors, type II topoisomerase inhibitors, acid sequence at least 70% identical to DSSHAFTLDELR Vinca alkaloids, and taxanes. (SEQ ID NO:13) or any one or more of SEQID NO:2-18. 10. The conjugate of claim 8, where the small molecule is 2. The isolated p97 polypeptide of claim 1, where the selected from one or more of chlorambucil, cyclophospha polypeptide comprises DSSHAFTLDELR (SEQID NO:13) mide, cilengitide, lomustine (CCNU), melphalan, procarba or one or more of SEQID NO:2-18. Zine, thiotepa, carmustine (BCNU), enZastaurin, buSulfan, daunorubicin, doxorubicin, gefitinib, erlotinib idarubicin, 3. The isolated p97 polypeptide of claim 1, where the temozolomide, epirubicin, mitoxantrone, bleomycin, cispl polypeptide comprises 2, 3, 4, or 5 of DSSHAFTLDELR atin, carboplatin, oxaliplatin, camptothecins, irinotecan, (SEQ ID NO:13) or SEQID NOS:2-18. topotecan, amsacrine, etoposide, etoposide phosphate, teni 4. The isolated p97 polypeptide of claim 1, where the poside, temsirolimus, everolimus, Vincristine, vinblastine, polypeptide comprises one or both of SEQID NO:13 and/or vinorelbine, vindesine, CT52923, paclitaxel, imatinib, dasa 14. tinib, Sorafenib, paZopanib, Sunitnib, Vatalanib, geftinib, erlo 5. The isolated p97 polypeptide of claim 1, where the tinib, AEE-788, dichoroacetate, tamoxifen, fasudil, polypeptide is up to about 250, 200, 150, 100, 50, 20, or 10 SB-681323, Semaxanib, donepizil, galantamine, memantine, amino acids in length. rivastigmine, tacrine, rasigiline, naltrexone, lubiprostone, 6. The isolated p97 polypeptide of claim 1, where the Safinamide, istradefylline, pimavanserin, pitolisant, israd polypeptide is fused to a heterologous protein. ipine, pridopidine (ACR16), tetrabenazine, bexarotene, glatirimer acetate, fingolimod, and mitoxantrone, including 7. A conjugate, comprising the p97 polypeptide of claim 1, pharmaceutically acceptable salts and acids thereof. where the p97 polypeptide is covalently or operatively linked 11. The conjugate of claim 8, where the polypeptide is an to an agent, to form a p97-agent conjugate. antibody or antigen-binding fragment thereof. 8. The conjugate of claim 7, where the agent is a small 12. The conjugate of claim 11, where the antibody or molecule, a polypeptide, a peptide mimetic, a peptoid, an antigen-binding fragment thereof specifically binds to a can aptamer, or a detectable entity. cer-associated antigen. 9. The conjugate of claim 8, where the small molecule is a 13. The method of claim 12, where the cancer-associated cytotoxic or chemotherapeutic or anti-angiogenic agent antigen is one or more of human Her2/neu, Her1/EGF recep selected from one or more of alkylating agents, anti-metabo tor (EGFR), Her3, A33 antigen, B7H3, CD5, CD19, CD20, US 2014/0322132 A1 Oct. 30, 2014 76

CD22. CD23 (IgE Receptor), C242 antigen, 5T4, IL-6, IL-13, tumab, galiximab, gemtuzumab, ganitumab, gemtuzumab vascular endothelial growth factor VEGF (e.g., VEGF-A) (oZogamicin), girentuximab, glembatumumab (vedotin), VEGFR-1, VEGFR-2, CD30, CD33, CD37, CD40, CD44, ibritumomab tiuxetan, icrucumab, igovomab, indatuximab CD51, CD52, CD56, CD74, CD80, CD152, CD200, CD221, ravtansine, intetumumab, inotuZumab ozogamicin, ipili CCR4, HLA-DR, CTLA-4, NPC-1C, tenascin, vimentin, mumab (MDX-101), iratumumab, labetuzumab, lexatu insulin-like growth factor 1 receptor (IGF-1R), alpha-feto mumab, lintuZumab, lorVotuZumab (mertansine), lucatu protein, insulin-like growth factor 1 (IGF-1), carbonic anhy mumab, lumiliximab, mapatumumab, matuZumab, drase 9 (CA-IX), carcinoembryonic antigen (CEA), integrin milatuZumab, mitumomab, mogamulizumab, moxetumomab CVB3, integrin C.531, folate receptor 1, transmembrane gly (pasudotox), nacolomab (tafenatox), naptumomab (estafena coprotein NMB, fibroblast activation protein alpha (FAP), tox), narnatumab, necitumumab, nimotuZumab, nivolumab, glycoprotein 75, TAG-72, MUC1, MUC16 (or CA-125), Neuradiab(R) (with or without radioactive iodine), NR-LU-10, phosphatidylserine, prostate-specific membrane antigen ofatumumab, olaratumab, onartuzumab, oportuZumab (PMSA), NR-LU-13 antigen, TRAIL-R1, tumor necrosis fac (monatox), oregovomab, panitumumab, patritumab, pemtu tor receptor superfamily member 10b (TNFRSF10B or momab, pertuzumab, pritumumab, racotumomab, TRAIL-R2), SLAM family member 7 (SLAMF7), EGP40 radretumab, ramucirumab, rillotumumab, rituximab, robatu pancarcinoma antigen, B-cell activating factor (BAFF), mumab, Samalizumab, sibrotuZumab, siltuximab, tabalumab, platelet-derived growth factor receptor, glycoprotein taplitumomab (paptoX), tenatumomab, teprotumumab, EpCAM (17-1A), Programmed Death-1, protein disulfide TGN1412, ticilimumab, tremelimumab, tigatuzumab, TNX isomerase (PDI), Phosphatase of Regenerating Liver 3 (PRL 650, , TRBS07, tucotuzumab (celmoleukin), 3), prostatic acid phosphatase, Lewis-Y antigen, GD2 (a ublituximab, urelumab, veltuzumab, Volociximab, votu disialoganglioside expressed on tumors of neuroectodermal mumab, and Zalutumumab, including antigen-binding frag origin), glypican-3 (GPC3), or mesothelin. ments thereof. 14. The conjugate of claim 11, where the antibody or 21. The conjugate of claim 8, where the polypeptide is an antigen-binding fragment thereof specifically binds to a pain interferon-B polypeptide, or an active fragment or variant associated antigen. thereof. 15. The method of claim 14, where the pain associated 22. The conjugate of claim 8, where the polypeptide asso antigen is one or more of nerve growth factor (NGF) or ciates with a lysosomal storage disease. tropomyosin-related kinase A (TrkA). 23. The conjugate of claim 22, where the polypeptide is 16. The conjugate of claim 11, where the antibody or selected from one or more of aspartylglucosaminidase, acid antigen-binding fragment thereof specifically binds to a pro lipase, cysteine transporter, Lamp-2, C-galactosidase A, acid inflammatory molecule, optionally a pro-inflammatory ceramidase, C-L-fucosidase, 3-hexosaminidase A, GM2 cytokine or chemokine. ganglioside activator (GM2A), C-D-mannosidase, B-D-man 17. The conjugate of claim 16, where the pro-inflammatory nosidase, arylsulfatase A, saposin B, neuraminidase, Cl-N- molecule is one or more of TNF-ct, TNF-B, FasL, CD27L, acetylglucosaminidase phosphotransferase, CD3OL, CD40L, Ox40L, 4-1 BBL, TRAIL, TWEAK, and phosphotransferase Y-subunit, L-iduronidase, iduronate-2- Apo3L, IL-1C., IL-1B, IL-2, interferon-Y (IFN-Y), IFN-C. Sulfatase, heparan-N-sulfatase, Cl-N-acetylglucosaminidase, IFN-B, IL-6, IL-8, IL-12, IL-15, IL-17, IL-18, IL-21, LIF, acetylCoA:N-acetyltransferase, N-acetylglucosamine 6-sul CCL5, GROC, MCP-1, MIP-1C., MIP-1 B, macrophage fatase, galactose 6-sulfatase, B-galactosidase, N-acetylgalac colony stimulating factor (MCSF), or granulocyte macroph tosamine 4-sulfatase, hyaluronoglucosaminidase, Sulfatases, age colony stimulating factor (GM-CSF). palmitoyl protein thioesterase, tripeptidyl peptidase I, acid 18. The conjugate of claim 17, where the pro-inflammatory sphingomyelinase, cathepsin A, cathepsin K, C-galactosidase molecule is TNF-C., and where the antibody is adalimumab, B, NPC1, NPC2, sialin, and sialic acid transporter, including certolizumab pegol, etanercept, golimumab, infliximab, active fragments and variants thereof. D2E7, CDP571, or CDP870, oran antigen-binding fragment 24. The conjugate of claim 8, where the detectable entity is or variant thereof. selected from one or more of diatrizoic acid, a radioisotope, a 19. The conjugate of claim 11, wherein the antibody or fluorophore/fluorescent dye, and a nanoparticle. antigen-binding fragment thereof specifically binds to one or 25. The conjugate of claim 8, where the agent is a car more of human Her2/neu, Her1/EGFR, TNF-C., B7H3 anti diotoxic agent in its unconjugated form. gen, CD20, VEGF, CD52, CD33, CTLA-4, tenascin, alpha-4 26. The conjugate of claim 25, where the cardiotoxic agent (C4) integrin, IL-23, amyloid-fi, Huntingtin, CD25, nerve is an anthracycline/anthraquinolone, cyclophosphamide, growth factor (NGF), TrkA, or C-synuclein. antimetabolite, antimicrotubule agent, tyrosine kinase inhibi 20. The conjugate of any of claim 11, where the antibody is tor, bevacizumab, or trastuzumab. selected from one or more of trastuzumab, cetuximab, dacli 27. The conjugate of claim 25, where the cardiotoxic agent Zumab, tanezumab, 3F8, 8H9, abagovomab, adecatumumab, is cyclopentenyl cytosine, 5-fluorouracil, capecitabine, pacli afutuZumab, alemtuzumab, alacizumab (pegol), amatuX taxel, docataxel, adriamycin, doxorubucin, epirubicin, emet imab, apolizumab, bavituximab, bectumomab, belimumab, ine, isotamide, mitomycin C, erlotinib, gefitinib, imatinib, bevacizumab, bivatuZumab (mertansine), brentuximab Sorafenib, Sunitinib, cisplatin, thalidomide, buSulfan, Vin Vedotin, cantuzumab (mertansine), cantuzumab (ravitansine), blastine, bleomycin, Vincristine, arsenic trioxide, methotrex capiromab (pendetide), catumaXomab, citatuZumab (boga ate, rosiglitaZone, or mitoxantrone. tox), cixutumumab, clivatuZumab (tetraxetan), conatu 28. A composition, comprising a conjugate of claim 7, and mumab, dacetuZumab, dalotuZumab, detumomab, drozitu a pharmaceutically acceptable carrier. mab, ecromeximab, edrecolomab, elotuZumab, 29. A method of treating a subject in need thereof, com enavatuZumab, ensituximab, epratuZumab, ertumaxomab, prising administering to the Subject a composition of claim etaracizumab, farletuzumab, FBTA05, figitumumab, flanvo 28.