JP5844971B2 - Aprotinin-like polypeptide for delivering conjugated substances to tissues - Google Patents

Description

  The present invention relates to improvements in the field of drug delivery. More specifically, the present invention relates to polypeptides, conjugates and pharmaceutical compositions comprising the polypeptides of the invention, and substances (agents) (e.g., therapeutic agents) up to certain cell types such as liver, lung, or kidney. ) Related to its use for transporting.

  Liver diseases such as hepatitis (eg viral hepatitis) and liver cancer (eg hepatocellular carcinoma), and lung diseases such as lung cancer (eg small cell and non-small cell lung cancer) are serious health problems. Many therapeutic agents for such diseases have undesired side effects (e.g. chemotherapeutic agents) or are sufficient in target tissues for reasons such as in vivo stability, transport, or other pharmacokinetic properties It is difficult to provide at a high concentration or for a period long enough to allow maximum therapeutic effect in the target tissue.

  Accordingly, there is a need for methods and compositions that increase the concentration of therapeutic and diagnostic agents in a target organ or tissue.

  We have efficiently transported the polypeptides described herein (e.g., Angiopep-7; SEQ ID NO: 112) to certain cell types (e.g., liver, lung, spleen, kidney, and muscle). However, it has been found that it is not efficiently transported across the blood-brain barrier (BBB). When the polypeptide is conjugated to a substance (drug), it can act as a vector and increase the concentration of the conjugated substance in cells. The inventors also have vectors such as Angiopep-3, Angiopep-4a, Angiopep-4b, Angiopep that can be efficiently transported across the BBB and can also be transported into specific cell types. -5 and Angiopep-6 (SEQ ID NOs: 107-111) were identified. Accordingly, the invention features polypeptides, conjugates comprising the polypeptides as vectors, and methods for diagnosing and treating diseases (eg, cancer) using such polypeptides and conjugates.

Thus, in a first aspect, the invention features a polypeptide comprising an amino acid sequence having the formula:
X1-X2-X3-X4-X5-X6-X7-X8-X9-X10-X11-X12-X13-X14-X15-X16-X17-X18-X19
Wherein each of X1-X19 (e.g., X1-X6, X8, X9, X11-X14, and X16-X19) is independently any amino acid (e.g., Ala, Arg, Asn, Asp, Cys , Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, and Val naturally occurring amino acids) or absent, and At least one of X1, X10, and X15 is arginine]. In some embodiments, X7 is Ser or Cys; or X10 and X15 are each independently Arg or Lys. In some embodiments, residues X1-X19, including both ends, include SEQ ID NOs: 1-105 and 107-112 (e.g., Angiopep-1, Angiopep-2, Angiopep-3, Angiopep-4a, Angiopep- 4b, Angiopep-5, Angiopep-6, and Angiopep-7) are substantially identical to any amino acid sequence. In some embodiments, at least one (e.g., 2, 3, 4, or 5) of amino acids X1-X19 is Arg (e.g., any one of X1, X10, and X15, 2 or 3).

  In other embodiments, the invention provides any one of SEQ ID NOs: 1-105 and 107-112 (e.g., Angiopep-3, Angiopep-4a, Angiopep-4b, Angiopep-5, Angiopep-6 and Angiopep-7). A polypeptide comprising an amino acid sequence having substantial identity to one. In certain embodiments, the polypeptide comprises or is Angiopep-3, Angiopep-4a, Angiopep-4b, Angiopep-5, Angiopep-6, or Angiopep-7 (SEQ ID NOs: 107-112). Often, it may comprise a sequence substantially identical to the Angiopep-7 sequence or a fragment thereof (eg a functional fragment). The polypeptide may have an amino acid sequence that is 10 to 50 amino acids long, such as 10 to 30 amino acids long. The invention also features functional derivatives (eg, chemical derivatives or variants) of the polypeptide.

  Exemplary polypeptides of the invention include lysine or arginine at position 10 (with respect to the amino acid sequence of SEQ ID NO: 1), or lysine or arginine at position 15 (with respect to the amino acid sequence of SEQ ID NO: 1), or positions 10 and Has lysine or arginine at both positions in position 15. The polypeptide of the present invention may have serine or cysteine at position 7 (relative to the amino acid sequence of SEQ ID NO: 1). If multimerization of the polypeptide is desired, the polypeptide may include a cysteine (eg, at position 7).

  In certain embodiments, a polypeptide of the invention (eg, any polypeptide described herein) is modified (eg, as described herein). Polypeptides may be amidated, acetylated, or both. For example, a polypeptide comprising Angiopep-6 or Angiopep-7 may be amidated or SEQ ID NO: 67 may be amidated (polypeptide number 67). In another example, the amino acid of any one of SEQ ID NOs: 107-112, or any other polypeptide of the invention, is amidated or acetylated. Such modifications to the polypeptides of the invention may be modifications at the amino terminus or carboxy terminus of the polypeptide. The invention also features peptidomimetics of any of the polypeptides described herein (eg, peptidomimetics described herein). The polypeptides of the present invention may be multimeric. For example, the polypeptide may be in a dimeric form (eg, a dimeric form formed by disulfide bonds with cysteine residues).

  The polypeptides of the invention can be efficiently transported into specific cells (e.g., liver, kidney, lung, muscle, or spleen cells) or can efficiently cross the BBB (e.g., Angiopep-3 Angiopep-4a, Angiopep-4b, Angiopep-5, and Angiopep-6). In some embodiments, the polypeptide is efficiently transported into specific cells (e.g., liver, kidney, lung, muscle, or spleen cells) and not efficiently transported across the BBB (e.g., Angiopep-7). The polypeptide is efficiently applied to at least one (eg, at least 2, 3, 4, or 5) cells or tissues selected from the group consisting of liver, kidney, lung, muscle, or spleen. Transported.

  For any polypeptide and conjugate described herein, the amino acid sequence is any of SEQ ID NOs: 1-105 and 107-112 (eg, SEQ ID NOs: 1-96, Angiopep-1, Angiopep-2, Angiopep -3, any of Angiopep-4a, Angiopep-4b, Angiopep-5, Angiopep-6, and Angiopep-7) may be specifically excluded. In some embodiments, the polypeptides and conjugates of the invention exclude the polypeptides of SEQ ID NOs: 102, 103, 104, and 105. In other embodiments, the polypeptides and conjugates of the invention comprise these polypeptides.

  In certain embodiments, the polypeptides of the invention have at least one amino acid substitution (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 substitutions). It has the amino acid sequence described herein. In certain embodiments, the polypeptide is any of SEQ ID NO: 1, Angiopep-1, Angiopep-2, Angiopep-3, Angiopep-4a, Angiopep-4b, Angiopep-5, Angiopep-6, and Angiopep-7. It has arginine at 1, 2, or 3 positions corresponding to position 1, 10 and 15 of the amino acid sequence of For example, the polypeptide may contain 1 to 12 amino acid substitutions (eg, SEQ ID NO: 91). For example, the amino acid sequence contains 1 to 10 (e.g., 9, 8, 7, 6, 5, 4, 3, 2) amino acid substitutions or 1 to 5 amino acid substitutions. It's okay. In the present invention, the amino acid substitution may be a conservative amino acid substitution or a non-conservative amino acid substitution.

  The polypeptides of the invention may be chemically synthesized (eg, solid phase synthesis) or produced by recombinant DNA technology, as is known in the art. Any polypeptide, composition, or conjugate described herein can be in isolated or substantially purified form.

  In another aspect, the invention provides a polypeptide of the invention (e.g., any polypeptide described herein, e.g., Angiopep-3, Angiopep-4a, Angiopep-4b, Angiopep-5, Angiopep-6, and Characterized by a polynucleotide sequence encoding Angiopep-7). More specifically, a nucleotide sequence (deoxyribonucleotide, ribonucleotide, or derivative thereof) encoding a polypeptide selected from the group consisting of SEQ ID NO: 1 to 97 and SEQ ID NO: 107 to 112 is encompassed by the present invention. Is done. The desired nucleotide sequence may be chemically synthesized by methods known in the art.

  In another aspect, the invention provides a polypeptide, analog, or derivative thereof described herein (e.g., Angiopep-3, Angiopep-4a, Angiopep-4b, Angiopep-5, Angiopep-6, and Angiopep- A vector selected from the group consisting of any one of 7) and a conjugate containing a substance to be conjugated to the vector.

  The agent consists of a therapeutic agent (e.g., a small molecule drug, e.g., an anticancer agent, antibiotic, or any of those described herein), a detectable label, a polypeptide (e.g., an enzyme), and a protein complex. Selected from the group. In some embodiments, the material has a maximum molecular weight of about 160,000 daltons. The substance may be a molecule that is active in the central nervous system. The substance may be any substance useful for the treatment or detection of nervous system, liver, lung, kidney, or spleen disease. Detectable labels are, for example, radioimaging agents (eg isotopes), fluorescent labels (eg rhodamine, FITC, cy5.5, alexa), reporter molecules (eg biotin). Other examples of detectable labels include green fluorescent protein, his tag protein, and β-galactosidase. The conjugate may be a fusion protein consisting essentially of a vector and a protein. Examples of protein-based compounds that can be conjugated to the vectors of the invention include antibodies (e.g., antibodies comprising heavy and / or light chains), antibody fragments (e.g., antibody-binding fragments, e.g., Fv fragments, F ( ab) 2, F (ab) 2 ', and Fab). Antibodies or fragments thereof that can be conjugated to a vector include monoclonal or polyclonal antibodies, which can be of any origin (eg, human, chimeric, and humanized antibodies). Other proteins or protein-based compounds include cytotoxins (e.g. monomethyl auristatin E (MMAE), bacterial endotoxins and exotoxins, diphtheria toxin, botulinum toxin, tetanus toxin, perussis toxin, staphylococcal enterotoxin, Toxin shock syndrome toxin TSST-1, adenylate cyclase toxin, Shiga toxin, and cholera enterotoxin) and anti-angiogenic compounds (endostatin, catechin, nutriceuticals, chemokine IP-10, matrix metalloproteinase inhibitor (MMPI) ), Anastellin, vironectin, antithrombin, tyrosine kinase inhibitor, VEGF inhibitor, receptor targeting antibody, herceptin, avastin, and panitumumab).

  Leptin exendin-4, GLP-1, PYY, or PYY (3-36) may be used, for example, in the treatment of obesity. Other polypeptides that can be included in the conjugates of the invention include adrenocorticotropic hormone (ACTH, corticotropin), growth hormone peptides (e.g., human placental lactogen (hPL), growth hormone, and prolactin (Prl)), Melanocyte stimulating hormone (MSH), oxytocin, vasopressin (ADH), corticotropin releasing factor (CRF), gonadotropin releasing hormone related peptide (GAP), growth hormone releasing factor (GRF), luteinizing hormone releasing hormone (LH-RH) ), Orexin, prolactin-releasing peptide, somatostatin, thyroid-stimulating hormone-releasing hormone (THR), calcitonin (CT), calcitonin precursor peptide, calcitonin gene-related peptide (CGRP), parathyroid hormone (PTH), parathyroid hormone-related Protein (PTHrP), amylin, glucagon, insulin and Insulin-like peptide, neuropeptide Y, pancreatic polypeptide (PP), peptide YY, somatostatin, cholecystokinin (CCK), gastrin releasing peptide (GRP), gastrin, gastrin inhibitory peptide, motilin, secretin, vasoactive intestinal peptide ( VIP), natriuretic peptides (e.g. atrial natriuretic peptide (ANP), B-type natriuretic peptide (BNP), brain natriuretic peptide, and C-type natriuretic peptide (CNP)), tachykinins (e.g. neurokinin A , Neurokinin B, and substance P), substance P, angiotensin (e.g., angiotensin I and angiotensin II), renin, endothelin (e.g., endothelin-1, endothelin-2, endothelin-3, sarafotoxin (snake venom) and scorpion) Poison), Sarafotoxinpe Tides, opioid peptides (e.g., casomorphin peptides, demorphins, endorphins, enkephalins, deltorphins, dynorphins), thymic peptides (e.g. thymopentin, thymopentin, thymosin, thymic factor) (THF)), adrenomedullin peptide (AM), allatostatin peptide, amyloid beta-protein fragment (Aβ fragment), antibacterial peptides (e.g., defensin, cecropin, buforin, and magainin), antioxidant peptides (e.g., natural Killer enhancing factor B (NKEF-B), bombesin, bone Gla protein peptide (eg, osteocalcin (bone Gla-protein or BGP), CART peptide, cell adhesion peptide, cortisatin peptide, fibronectin fragment and fibrin related Peptide, a FMRF peptide, galanin, guanylin and uroguanylin, and inhibin peptide.

  Small molecule drugs include anticancer agents (eg, any such substance described herein). Examples of anticancer agents include paclitaxel (Taxol), vinblastine, vincristine, etoposide, doxorubicin, cyclophosphamide, taxotere, melphalan, chlorambucil, and any anticancer agent described herein, or any of its anticancer agents. A combination is included. The anticancer agent may have a chemical moiety that allows conjugation to the vector of the invention.

In certain embodiments, the conjugates of the invention comprise the formula: V _x -L _y -A _z or a pharmaceutically acceptable salt thereof, wherein V is a polypeptide or A derivative thereof (eg, Angiopep-3, Angiopep-4a, Angiopep-4b, Angiopep-5, Angiopep-6, Angiopep-7, or any analog, derivative, or fragment described herein). V certain cell types (e.g., liver, lung, kidney, spleen or muscle cells) efficiently either transported into, or (for example, after attachment to L _y -A _z) efficiently across the BBB Be transported to. In certain embodiments, the vector or conjugate is not efficiently transported across the BBB, but is efficiently transported into a particular cell type (eg Angiopep-7). L is a linker or bond (eg, chemical bond or covalent bond). A is at the level of therapeutic agents (e.g., small molecule drugs), detectable labels, proteins, or protein-based compounds (e.g., antibodies, antibody fragments), antibiotics, anticancer agents, anti-angiogenic compounds, and the central nervous system. A substance such as a substance selected from the group consisting of an active polypeptide or any molecule.

X, Y, and Z are each independently any number greater than 0 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10), or 1 ( For example, it may be a series of values ranging from 2, 3, 4, 5, 6, 7, 8, 9) to 10 (eg, 9, 8, 7, 6, 5, 4, 3, 2). Thus, the formula: V _x -L _y -A _z is not limited to a specific order or ratio; for example, the conjugate is a 1-5 vector (1, 2, 3, 4, Or 5). In other embodiments, two or more substances are conjugated to a vector. A substance or conjugate is active when the substance is conjugated to a vector. In some embodiments, the compound can be released from the vector, for example after transport across the BBB or into a particular cell type. The compound becomes active after its release (eg as a prodrug). In some embodiments, the substance remains conjugated to the vector after transport. Here, the conjugate exhibits a lower excretion transport (eg by P-glycoprotein) compared to the unconjugated substance. In this case, it is desirable that the substance and the vector remain bound. The conjugate may be provided in a pharmaceutically acceptable composition.

  When a substance is conjugated to a vector, the modified (e.g., improved) bioavailability, improved efficacy (e.g., anti-cancer activity) compared to the substance when not conjugated to a vector, the substance Showing altered tissue distribution, reduced toxicity, or altered pharmacokinetics. Vectors (e.g., Angiopep-3, Angiopep-4a, Angiopep-4b, Angiopep-5, and Angiopep-6) are the brains of individuals (e.g., brains, lungs, breasts that contain tumor cells such as glioblastoma, Promotes the accumulation of substances in the colon, liver, pancreas, or spleen, tumor cells) or certain cells or tissues (eg, liver, lung, kidney, spleen, and muscle). Vectors such as Angiopep-7 can promote accumulation in cell types such as liver, lung, kidney, spleen or muscle, but do not promote accumulation in the brain. More specifically, the vector, when provided (eg, administered) to a subject, is a P-gp or P-gp-related protein (eg, a P-gp human or mammalian allelic variant, eg, a rodent The accumulation or efficacy of substances that are P-gp substrates or therapeutic agents released by mdr1a or mdr1b isoforms from) can be increased. The cell expresses or can express P-gp or a P-gp related protein. The cell to which the conjugate is transported expresses a vector receptor or transporter, such as a low density lipoprotein-related receptor (LRP) (e.g., co-expressing P-gp or P-gp related protein) cell). The cells can be, for example, normal cells, tumor cells, or metastatic cells (eg, multidrug resistant cancer cells). The cell or tumor may be a metastasis (eg, of any cancer described herein).

  The vector or conjugate can be efficiently transported into a specific cell type (e.g., liver, lung, kidney, spleen, or muscle cell) and can be efficiently transported across the BBB; Such conjugates include Angiopep-3, Angiopep-4a, Angiopep-4b, Angiopep-5, and Angiopep-6. In certain embodiments, the transport activity of the vector does not itself affect the integrity of the cells to which it is delivered or does not affect the integrity of the BBB. In other embodiments, vectors or conjugates such as Angiopep-7 are efficiently transported into certain cell types, but not efficiently across the BBB. Any vector or conjugate described herein is transported by receptor-mediated endocytosis or transcellular transport or adsorption-mediated endocytosis or transcellular transport.

  Any vector or conjugate described herein may be present in a pharmaceutically acceptable carrier (eg, in pharmaceutical format). Accordingly, the present invention includes (a) a conjugate (e.g., any conjugate described herein); (b) a pharmaceutically acceptable carrier (e.g., any described herein). Features a pharmaceutical composition. The conjugate may further comprise (c) a solubilizing agent. The solubilizer may be, for example, a polyoxyethylene ester of a fatty acid (eg, Solutol® HS-15). The pharmaceutical composition may be used to alter the pharmacokinetics of a compound, reduce tumor cell growth, or detect tumor cells.

  In another aspect, the invention provides a vector of the invention for the diagnosis or treatment of liver disease, lung disease, kidney disease, spleen disease, or muscle disease (e.g., manufacture or use of a medicament in those treatments) or Characterized by the use of conjugates. In certain embodiments, a conjugate of the invention that is efficiently transported across the BBB is used to treat a nervous system disease or a central nervous system disease (e.g., any disease described herein). Good. For example, a vector or conjugate is used for in vivo detection of any of the above diseases. Treatment or diagnosis is performed in a mammal in need thereof (e.g. a human or non-human mammal), e.g. a mammal having or at risk of having a disease (e.g. cancer) (e.g. high risk). Good. Administration may be performed using any means known in the art, for example, intraarterial, intranasal, intraperitoneal, intravenous, intramuscular, subcutaneous, transdermal, or oral. Administration may be a therapeutically effective amount.

  By conjugating a substance to the vectors described herein, it is possible to reduce the toxicity of the substance and thus provide the substance at a higher dose than the recommended dose for the substance alone. . Accordingly, the present invention is a method of treating a subject having a disease or condition (eg, any disease or condition described herein, such as cancer), which provides the subject with a conjugate of the invention Wherein the conjugate has a higher dosage than the therapeutic amount of the substance alone (e.g., at least 5%, 10%, 25%, 50%, 75%, 100%, 250%, 500%, 1,000%, 5,000%, or 10,000% higher dose) substance.

  Since the vectors described herein can target a substance to a specific cell type (e.g., a cell type described herein), in certain embodiments, the substance is conjugated to a vector. When gated, it has high potency when administered to a subject. Accordingly, the present invention also provides a disease or condition (e.g., any disease or condition described herein, such as cancer or the like) by administering to a subject a conjugate of the invention or a composition comprising a conjugate of the invention. A subject having a symptom) wherein the conjugate is at a lower dose than the therapeutic amount of the substance alone (e.g., 5%, 10%, 15%, 20%, 30%, 50%, 70%, 80%, 90%, 95%, 98%, 99%, 99.9% lower dose) substance.

  The therapeutic method of the present invention comprises the step of assessing whether an individual's tumor contains multidrug resistant tumor cells (e.g., cells expressing P-gp (MDR1)) or the tumor has a multidrug resistant drug phenotype. It may further comprise the step of determining whether or not it has. The treatment method may include providing chemotherapy, radiation therapy, or both. Alternatively, the subject being treated (for example, within 1 year, 6 months, 3 months, 2 months, 1 month, 2 weeks, 1 week, 3 days, 2 days, or 1 day) May (or may) receive any treatment. The individual may have undergone surgery. The individual may have a brain tumor (eg, a primary brain tumor) or a tumor at a site other than the brain (eg, no brain tumor). An individual in need exhibits or is at risk of developing resistance to at least one drug (eg, multidrug resistance (MDR) phenotype). The tumor may be a lung tumor, an extracranial metastasis of a brain tumor (eg, from glioblastoma), a brain tumor of a metastatic origin (eg, from a lung tumor, breast tumor, melanoma, colorectal cancer, or urological tumor). Tumors may include cells expressing P-gp, LRP, or both, or cells capable of expressing P-gp, LRP, or both. P-gp and LRP are localized on the cell surface.

  In another aspect, the invention also features a method for increasing the concentration of a substance in a cell or transporting a substance into a cell, wherein the cell expresses a member of the LRP receptor family. . The method comprises contacting a cell with a conjugate comprising a substance conjugated to a vector (e.g., any polypeptide described herein), thereby increasing the concentration of the substance in the cell, or Transporting the substance into the cell. The invention also features a method for increasing the concentration of a substance in a cell, wherein the cell expresses a P-glycoprotein. The method comprises contacting a cell with a conjugate comprising a substance conjugated to a vector (e.g., any polypeptide described herein), thereby comparing the cell to the unconjugated substance. Increasing the concentration of the substance therein.

  In accordance with any aspect of the present invention, nervous system diseases include brain tumors, brain metastases, schizophrenia, epilepsy, Alzheimer's disease, Parkinson's disease, Huntington's disease, seizures, and BBB-related dysfunction (eg obesity). . The liver disease may be a cancer such as hepatocellular carcinoma. Other liver diseases include those described herein. The lung disease may be lung cancer (eg, as described herein).

  The conjugate can comprise a multimer, for example a dimeric form of the vector. Multimeric forms of vectors can increase the transport or accumulation of substances in some embodiments. The vector may be in a ratio of at least 1: 1 (substance: vector), 1: 2, 1: 3, 1: 4, 1: 5, 1: 6, or 1:10. As described herein, a high ratio of material to substance can cause an increase in transport. As such, the number of vectors per substance is not limited.

  The pharmaceutical composition may also be used in accordance with the present invention, for example to deliver a substance to an individual's CNS.

  Pharmaceutically acceptable salts of vectors (polypeptides) or conjugates are encompassed by the present invention, including pharmaceutically acceptable acid addition salts of substances.

  The vectors or conjugates of the invention may be used in combination with or separately from conventional treatment methods or therapies. Combination therapy with other substances includes sequential or simultaneous administration to an individual. A pharmaceutical composition of the invention comprises a vector-substance conjugate of the invention combined with a pharmaceutically acceptable excipient as described herein and another therapeutic agent known in the art or A combination of prophylactic agents may be included.

  By “vector” is meant a compound or molecule, eg, a polypeptide, that can be transported into a particular cell type (eg, liver, lung, kidney, spleen, or muscle) or across the BBB. A vector can be attached or conjugated to a substance (covalently or non-covalently), thereby transporting the substance within a particular cell type or across the BBB. In certain embodiments, the vector binds to a receptor present on the cancer cell or brain endothelial cell and is thereby transported into the cancer cell by transcellular transport or across the BBB. A vector may be a molecule that achieves a high level of transendothelial transport without affecting cell or BBB integrity. A vector can be a polypeptide or peptidomimetic and can be naturally occurring or produced by chemical synthesis or recombinant genetic technology.

  “Conjugate” means a vector linked to a substance. Conjugation is essentially chemical conjugation, such as conjugation via a linker, or conjugation in a fusion protein, eg, with a reporter molecule (eg, green fluorescent protein, β-galactosidase, His tag, etc.) It may be essentially gene conjugation, for example by recombinant gene technology.

  “Efficiently transported across the BBB” vector is at least as efficient as Angiopep-6 (ie, Angiopep-1 (250 nM) in the in situ brain perfusion assay described herein) Means a vector that can traverse the BBB (with 38.5% higher efficiency). Thus, a vector or conjugate that is “not efficiently transported across the BBB” is transported to the brain only at low levels (eg, transported less efficiently than Angiopep-6).

  A vector or conjugate that is “effectively transported to a particular cell type” is defined in that cell type (for example, due to increased transport into the cell, decreased efflux from the cell, or a combination thereof). At least 10% (e.g. 25%, 50%, 100%, 200%, 500%, 1,000%, 5,000%) compared to the control substance or, in the case of a conjugate, compared to the unconjugated substance. Or 10,000%) means a vector or conjugate that can accumulate to a high degree.

  By “substantially pure” or “isolated” is meant a compound (eg, a polypeptide or conjugate) that has been separated from other chemical components. Typically, a compound is substantially pure when it is at least 30% by weight separated from other components. In certain embodiments, the preparation is separated by at least 50%, 60%, 75%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% by weight from other components. Has been. A purified polypeptide can be obtained, for example, by expression of a recombinant polynucleotide encoding such a polypeptide, or by chemically synthesizing the polypeptide. Purity can be measured by any appropriate method, for example, column chromatography, polyacrylamide gel electrophoresis, or HPLC analysis.

  An “analog” is derived from the original sequence or portion of the original sequence and includes one or more modifications; for example, one or more modifications in an amino acid sequence (eg, amino acid addition, deletion, insertion, or substitution) Means a polypeptide comprising one or more modifications in the main chain or side chain of one or more amino acids, or the addition of a group or another molecule to one or more amino acids (side chain or main chain). An analog may have one or more amino acid insertions at one or both ends of the polypeptide or within the amino acid sequence of the polypeptide. Analogs may have sequence similarity and / or sequence identity (eg, may be substantially identical) to the original sequence or portions of the original sequence. An analog may comprise a modification of its structure, for example as described herein. The degree of similarity between two sequences is based on the identity (identical amino acids) and the percentage of conservative substitutions. Analogs are at least 35%, 50f%, 60%, 70%, 80% relative to the original sequence, with one or more modifications in the main chain or side chain of amino acids, or the addition of groups or another molecule There may be 90%, or 95% (eg, 96%, 97%, 98%, 99%, and 100%) sequence similarity. Typical amino acids (conserved amino acids) that are said to be similar to other amino acids are known in the art and include, for example, those listed in Table 3.

  `` Substantially identical '' means at least 35%, 40%, 50%, 55%, 60%, 65%, 70%, 75%, 85%, 90%, 95 relative to a reference amino acid or nucleic acid sequence A polypeptide or nucleic acid that shows as much as% or even 99% identity. For polypeptides, the length of the comparison sequence is generally at least 4 (e.g., at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 25, 50, or 100) amino acids. For nucleic acids, the length of comparison sequences is generally at least 60 nucleotides, preferably at least 90 nucleotides, and more preferably at least 120 nucleotides, or full length. It should be understood herein that gaps are found between analog amino acids that are identical or similar to the amino acids of the original polypeptide. A gap may contain one or more amino acids that are free of amino acids or that are not identical or similar to the original polypeptide. Biologically active analogs of the vectors (polypeptides) of the invention are encompassed therewith. The percent identity may be determined using default gap weights, for example, with GAP, BESTFIT, or FASTA algorithms of Wisconsin Genetics Software Package Release 7.0.

  “Functional derivative” means a “chemical derivative”, “fragment” or “variant” of the biologically active sequence or portion of the vectors or substances or conjugates and salts thereof of the present invention. A functional derivative of the vector can be attached to or conjugated to the substance and can enter a particular cell type and thereby transport the substance into the cell.

  “Chemical derivative” means a vector, substance, or conjugate of the invention that contains an additional chemical moiety that is not part of a vector, substance or vector-substance conjugate, including covalent modifications. Chemical derivatives may be made by direct chemical synthesis using methods known in the art. Such modifications can be introduced into a protein or peptide vector, substance, or vector-substance conjugate by reacting a target amino acid residue with an organic derivatizing agent capable of reacting with a selected side chain or terminal residue. Good. Chemical derivatives of the vector can cross the BBB or invade or accumulate in certain cell types (eg, those described herein). In a preferred embodiment, a very high level of transendothelial transport across the BBB is achieved without affecting the integrity of the BBB.

  "Fragment" means a polypeptide derived from the original sequence or a portion of the parent sequence or an analog of the parent sequence. Fragments include polypeptides having one or more amino acid truncations, which may be derived from the amino terminus (N terminus), carboxy terminus (C terminus) of the protein, or from within the protein. . A fragment may comprise a sequence identical to the corresponding portion of the original sequence. Functional fragments of the vectors (polypeptides) described herein are encompassed by the present invention. Fragments are at least 5 (e.g., at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 25, 28 , 30, 35, 40, 45, 50, 60, 75, 100, or 150) amino acids. The fragments of the invention may comprise, for example, a polypeptide of 7, 8, 9 or 10 amino acids to 18 amino acids. Fragments may contain any modification described herein (eg, acetylation, amidation, amino acid substitution).

  A “non-naturally occurring amino acid” is an amino acid that is not naturally produced or found in a mammal.

  By “substance (drug)” is meant any compound, such as an antibody or therapeutic agent, marker, tracer, or imaging compound.

  “Therapeutic agent” means a substance having biological activity. In some cases, therapeutic agents are used to treat disease signs, physical or mental conditions, injuries, or infections, including anticancer agents, antibiotics, anti-angiogenic agents, and the central nervous system. Active molecules at the level of

  By “small molecule drug” is meant a drug having a molecular weight of 1,000 g / mol or less (eg, less than 800, 600, 500, 400, or 200 g / mol).

  “Subject” means a human or non-human animal (eg, mammal).

  “Treatment”, “treating”, etc. refers to a desired pharmacological or physiological effect, such as inhibition of cancer cell growth, death of a cancer cell, disease or condition (eg, any of those described herein) Means to achieve reduction of at least one symptom associated with the disease or condition. The effect may be, for example, a prophylactic effect that completely or partially prevents the disease or its symptoms, or may be a therapeutic effect that relates to a partial or complete cure for the disease and / or the adverse effects resulting from the disease. Treatment includes (a) preventing (for example, preventing cancer) the occurrence of a disease or symptom in an individual (e.g., an individual who is susceptible to the disease but has not been diagnosed as having it); (b) a disease Inhibiting (eg, stopping its development); or (c) alleviating the disease (eg, reducing symptoms associated with the disease). Treatment includes any administration of a therapeutic agent to an individual to treat, cure, reduce, ameliorate, reduce or inhibit an individual's symptoms, including, but not limited to, a vector-substance conjugate. Is administered to an individual.

  By “cancer” is meant the growth of any cell whose characteristic trait is loss of normal control that can result in unregulated growth, lack of differentiation, or the ability to invade and metastasize into tissue. Cancer can develop in any tissue or any organ. Cancer is intended to include, but is not limited to, brain, liver, lung, kidney, or spleen cancer. Additional cancers are described herein.

  “Providing”, in the context of the vector or conjugate of the invention, means contacting the vector or conjugate with a target cell or tissue in vivo or in vitro. A vector or conjugate can be provided by administering the vector or conjugate to a subject.

  “Administering” and “administration” mean modes of delivery, including but not limited to intraarterial, intranasal, intraperitoneal, intravenous, intramuscular, subcutaneous, transdermal, or oral modes. Is included. The daily dose can be divided into 1, 2 or more doses in a manner suitable for administration once, twice or more over a period of time.

  A “therapeutically effective” or “effective amount” is a therapeutic agent sufficient to ameliorate, reduce, prevent, delay, suppress or stop any sign of the disease or condition being treated Means the amount. A therapeutically effective amount of the substance need not cure the disease or condition, but provides a treatment for the disease or condition to delay, prevent or prevent the onset of the disease or condition, or an indication of the disease or condition Or change the duration of the disease or condition, or, for example, be less severe, or expedite recovery in the individual.

  `` Symptoms '' means any situation that causes pain, discomfort, illness, disease or disability (mental or physical) to or in an individual, including nervous system disease, injury, Infections or include chronic or acute pain. Nervous system diseases include brain tumors, brain metastases, schizophrenia, epilepsy, Alzheimer's disease, Parkinson's disease, Huntington's disease, and seizures.

  “Pharmaceutical composition” means a therapeutically effective amount of a substance with a pharmaceutically acceptable diluent, preservative, solubilizer, emulsifier, or adjuvant, eg, any of those described herein. .

  “Therapeutic amount” means a dose of a substance, such as a drug (without a vector), that is acceptable for clinical use in terms of toxicity or efficacy. By conjugating a substance to the vector of the present invention, it is possible to administer a lower or higher dose of the substance than the therapeutic amount.

  Where a “range” or “group of substances” is described with respect to a particular characteristic (eg, temperature, concentration, time, etc.), the present invention shall include each and every particular member and sub-range or sub-part thereof. Regarding the combination of groups, they are explicitly incorporated herein. Thus, for example, with respect to the length of 9-18 amino acids, each and every individual length, for example, 18, 17, 15, 10, 9, and any number of lengths between them, are expressly set forth herein. It shall be understood that it is incorporated. Accordingly, unless stated otherwise, all ranges described herein are to be understood to include both ends. For example, the expression 5-19 amino acids in length shall include 5 and 19. This applies equally to other parameters such as sequence, length, concentration, element, etc.

  The sequences, regions, and portions defined herein are each and each and every individual sequence, region, and portion described thereby, and each and every possible subsequence, subregion, and subportion. Whether such subsequences, subregions, and subparts explicitly include a particular possibility, exclude a particular possibility, or are defined as combinations thereof. For example, an exclusive definition for a region can be read as follows: “provided that the polypeptide is not shorter than 4, 5, 6, 7, 8 or 9 amino acids”. Additional examples of passive limitations are the following; sequences comprising SEQ ID NO: X except for the polypeptide of SEQ ID NO: Y; etc. Additional examples of passive limitations are as follows; provided that the polypeptide is not SEQ ID NO: Z (does not contain or consist of SEQ ID NO: Z).

FIG. 1 shows the structure of a typical polypeptide of the present invention. Figure 2 shows the protocol used aprotinin to IgG conjugated using crosslinkers BS ^3. FIG. 3 shows the protocol used to conjugate aprotinin to IgG using the crosslinker sulfo-EMCS. FIG. 4 shows the protocol used to conjugate Angiopep-2 with an antibody using the cross-linking agent ECMS. FIG. 5 shows the protocol used to conjugate Angiopep-2 with an antibody using the cross-linking agent SATA. FIG. 6 shows the protocol used to conjugate Angiopep-2 to an antibody using a carbohydrate target via hydrazide. FIG. 7 shows other linker candidates that can be used to make conjugates. FIG. 8 shows a method for attaching the vector of the invention to paclitaxel. FIG. 9 is a schematic diagram of a cell surface efflux pump, P-glycoprotein (P-gp or MDR1). The efflux pumps associated with multidrug resistance, P-gp or MDRl, are highly expressed on the cell surface of many cancer cells and various tissues including the blood brain barrier (BBB). FIGS. 10A and 10B are diagrams representing the tissue distribution of Taxol and TxlAnl conjugates. FIG. 11 is a diagram showing the lung distribution of Taxol and TxlAn1. FIG. 12 is a diagram representing the level of TxlAn1 conjugate in plasma and lung. FIG. 13 is an image set showing the in vivo accumulation of Angiopep-2 and Angiopep-7 in the rat brain 30 minutes after IV injection. Angiopep-2 accumulates in the brain to a greater extent than Angiopep-7. FIG. 14 is an image set showing fluorescence microscopy of brain fragments after 10 minutes of in situ perfusion of fluorescently labeled Angiopep-2 or Angiopep-7. These images indicate that Angiopep-2 is localized in the brain, while Angiopep-7 is localized in the capillary. FIG. 15A is an image set showing in vivo imaging of Angiopep-2 and Angiopep-7 in rat liver, lung, and kidney. Angiopep-7 is observed to accumulate in these tissues. FIG. 15B is a graph showing ex-vivo organ imaging 24 hours after injection of Angiopep-2 or Angiopep-7 in rats. Both polypeptides accumulate in the kidney, liver, and lung. Angiopep-2 accumulates in the brain to a greater extent than Angiopep-7. FIG. 16 shows the volume of distribution in the brain parenchyma of free and Angiopep conjugated IgG. FIG. 17 is a diagram of cell proliferation in the presence of the parent drug Taxol. Glioblastoma cells (U-87) were exposed to various concentrations of Taxol for 3 days. ³ H-thymidine incorporated into the cells was plotted as a function of Taxol concentration. FIG. 18A is a graph depicting the effect of TxlAn2 treatment on subcutaneous glioblastoma (U-87) tumor growth. FIG. 18B is a graph depicting the effect of TxlAn2 treatment on subcutaneous glioblastoma (U-87) tumor growth. FIG. 19 is a set of photomicrographs showing detection of β-tubulin in NCI-H460 cells by immunofluorescence or visible light in cancer cells exposed to Taxol or TxlAn2 conjugates. As a control, cells were exposed to 1% DMSO. FIG. 20 is a set of graphs showing the effect of Taxol and TxlAn2 conjugates on the NCI-H460 cell cycle as measured by FACS. Cells were exposed to vehicle (DMSO), Taxol (100 nM), or TxlAn2 conjugate (equivalent to 30 nM, 100 nM Taxol) for 24 hours. FIG. 21A is a graph showing the accumulation of various drugs in MDCK cells transfected with MDRl in the presence or absence (control) of 10 μM CsA, P-gp inhibitors. Experiments were performed in the presence of 1% DMSO. FIG. 21B is a graph showing the accumulation of conjugate in cells overexpressing P-gp. Figures 22A and 22B are photographs of Western blots showing immunodetection of LRP in human brain tumor biopsies. FIG. 23A is a schematic of drug conjugation to a vector of the invention. FIG. 23B is a schematic of drug conjugation to a vector of the invention. FIG. 24 is a chromatogram showing production of TxlAn2 (3: 1) conjugate. FIG. 25 is an HPLC analysis of the peak purified on a hydrophobic column using AKTA-Explorer. FIG. 26 shows the association of Angiopep-2 with IgG light and heavy chains. FIG. 27 shows the increase in brain distribution volume of IgG-Angiopep-2 conjugates cross-linked with sulfo-EMCS. FIG. 28 shows brain penetration of IgG-Angiopep-2 conjugate using in situ cerebral perfusion. FIG. 29 shows an autoradiogram of radiolabeled [ ¹²⁵ I] -IgG-Angiopep conjugate. FIG. 30 shows similar detection of EGFR on U87 cells with anti-EGFR and anti-EGFR-Angiopep-2 conjugates by FACS analysis. FIG. 31 shows the distribution volume in the brain parenchyma of free and Angiopep-2 conjugated EGFR antibody. FIG. 32 shows the distribution volume in the brain parenchyma of free and Angiopep-2 conjugated VEFG antibodies. FIG. 33 shows the incorporation of conjugates containing different ratios of vector to substance (antibody) in parenchyma.

  We have efficiently transported Angiopep-7 into certain cell types or organs, such as liver, lung, kidney, spleen, and muscle, but with polypeptides such as Angiopep-1 or Angiopep-2 In comparison, we found that they were not efficiently transported across the blood brain barrier (BBB). Based on this, we have transported the substance Angiopep-7 (eg, for the treatment of diseases associated with these tissues, such as cancer or any disease described herein). It was identified as a suitable vector. We have also identified other polypeptides, including Angiopep-3, Angiopep-4a, Angiopep-4b, Angiopep-5, and Angiopep-6, that can be used as vectors of the present invention as well. These polypeptides retain the ability to be efficiently transported across the BBB or transported into specific tissues or cell types (eg, liver, lung, kidney, spleen, and muscle). In these polypeptides, substances that cannot efficiently cross the BBB or are ineffective at that point can be conjugated to the polypeptide and transported across the BBB. The conjugate may be in the form of a composition, eg, a pharmaceutical composition, for the treatment or diagnosis of a condition or disease.

  The conjugates of the present invention (eg, any described herein) may have beneficial in vivo pharmacokinetic properties compared to a single substance. These properties include increased in vivo half-life or increased concentration in the desired cell type, increased accumulation rate, or decreased clearance from the desired cell type. Based on this, it is possible to administer the conjugates of the invention to a subject at lower doses, less frequently, or in shorter periods than unconjugated substances. Reduction in dosage, frequency, or duration of administration is particularly desirable for pharmacotherapy with known side effects. In other cases, conjugating substances to the polypeptides described herein results in high potency due to increased concentrations in target cells or tissues. Indeed, concentrating substances in the target cell type can reduce undesirable side effects (eg, with high potency). This reduces the required duration of treatment. In some cases, it is possible to administer the conjugate at a higher dose, frequency, or duration compared to the dose of unconjugated substance, and observe high potency, low side effects, or even both.

Polypeptides of the Invention The present invention relates to any of the polypeptides described herein, such as any of the polypeptides described in Table 1 (e.g., SEQ ID NO: 1-105 and 107-112, e.g., SEQ ID NO: 1-97). , 99, 100, 101, or 107-112), or any fragment, analog, derivative, or variant thereof. In certain embodiments, the polypeptide is at least 35%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, relative to the polypeptide described herein. Or even 100% identity. The polypeptide has one or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, compared to one of the sequences described herein. Or 15) substitutions. Other modifications are described in further detail below.

  The invention also features fragments (eg, functional fragments) of these polypeptides. In certain embodiments, the fragment can invade or accumulate in a particular cell type (e.g., liver, lung, kidney, spleen, or muscle) or BBB. Can be crossed. Polypeptide truncation can be from the N-terminus of the polypeptide, from the C-terminus of the polypeptide, or a combination of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or These amino acids may be used. Other fragments include sequences in which an internal portion of the polypeptide is deleted.

  Identify additional polypeptides by using one of the assays or methods described in US Patent Application Publication No. 2006/0189515 (incorporated herein by reference) or by any method known in the art. be able to. For example, vector candidates may be prepared by conventional polypeptide synthesis, conjugated with Taxol, and administered to experimental animals. Bioactive vectors, for example, comparing the survival of an animal injected with tumor cells and treated with the conjugate with a control not treated with the conjugate (e.g., treated with an unconjugated substance). Can be identified based on its increasing potency.

In another example, a bioactive polypeptide may be identified based on its location in the parenchyma in an in situ brain perfusion assay. Such vectors can be identified using in vitro BBB assays, such as models developed by CELLIAL ^™ technology.

Assays for determining accumulation in other tissues may be performed as well. See, for example, Example 1 herein. A labeled conjugate of the polypeptide can be administered to the animal to measure accumulation in different organs. For example, a polypeptide conjugated to a detectable label (eg, a near infrared fluorescence spectroscopic label, eg, Cy5.5) allows for live in vivo visualization. Such a polypeptide can be administered to an animal to determine the presence of the polypeptide in the organ, thus allowing the rate and amount of accumulation of the polypeptide in the desired organ to be determined. In other embodiments, the polypeptide can be labeled with a radioisotope (eg, ¹²⁵ I). The polypeptide is then administered to the animal. After a certain period, the animals are sacrificed and the organs of the animals are extracted. The amount of radioisotope in each organ can then be measured using any means known in the art. By comparing the amount of labeled candidate polypeptide in a particular organ without the amount of labeled control, the ability of the candidate polypeptide, the rate or amount of accumulation of candidate polypeptide in a particular tissue, I can confirm. Suitable negative controls include any polypeptide that is known not to be transported into a particular cell type.

  Peptide 5 contains the sequence of SEQ ID NO: 5 and is amidated at its C-terminus (see, eg, FIG. 1).

  Peptide 67 contains the sequence of SEQ ID NO: 67 and is amidated at its C-terminus (see, eg, FIG. 1).

  Peptide 76 contains the sequence of SEQ ID NO: 76 and is amidated at its C-terminus (see, eg, FIG. 1).

  Peptide 91 contains the sequence of SEQ ID NO: 91 and is amidated at its C-terminus (see, eg, FIG. 1).

  Peptide 107 contains the sequence of SEQ ID NO: 97 and is acetylated at its N-terminus.

  Peptide 109 contains the sequence of SEQ ID NO: 109 and is acetylated at its N-terminus.

  Peptide 110 contains the sequence of SEQ ID NO: 110 and is acetylated at its N-terminus.

The amine groups of Angiopep-1 (SEQ ID NO: 67) and Angiopep-2 (SEQ ID NO: 97) have been used as sites for substance conjugation. To study the role of the amine group in conjugation and its impact on the total transport capacity of these vectors, the Angiopep-1 and Angiopep-2 sequences with variable reactive amine groups and variable total charge A new vector based was designed. These polypeptides are shown in Table 2.

Modified polypeptides The invention also provides polypeptides having amino acid sequence modifications described herein (eg, SEQ ID NO: 1 such as Angiopep-3, -4a, -4b, -5, -6, or -7). A polypeptide having the sequence of any one of -105 and 107-112). In certain embodiments, the modification does not significantly destroy the desired biological activity. In some embodiments, the modification is of biological activity (e.g., at least 5%, 10%, 20%, 25%, 35%, 50%, 60%, 70%, 75%, 80%, 90% Or 95%). In other embodiments, the modification has no effect on biological activity, or the biological activity of the original polypeptide (e.g., at least 5%, 10%, 25%, 50%, 100 %, 200%, 500%, or 1,000%). Modified polypeptides have or optimize one or more properties of the polypeptides of the invention that are necessary or desirable in some cases. Such properties include in vivo stability, bioavailability, toxicity, immunological activity, or immunological identity.

  The polypeptides of the present invention may comprise amino acids or sequences that have been modified by natural processing, such as post-translational processing, or by chemical modification techniques known in the art. The modification may be anywhere in the polypeptide including the polypeptide backbone, the amino acid side chain, and the amino terminus or carboxy terminus. The same type of modification may be present in the same or varying degrees at several sites in a given polypeptide, and the polypeptide may contain more than one type of modification. A polypeptide may be branched as a result of ubiquitin binding, and it may be a cyclic polypeptide with or without branching. Cyclic, branched, and branched cyclic polypeptides may result from post-translation natural processing or may be made synthetically. Other modifications include pegylation, acetylation, acylation, addition of acetomidomethyl (Acm) group, ADP-ribosylation, alkylation, amidation, biotinylation, carbamoylation, carboxyethylation, ester , Covalent bond to fiavin, covalent bond to heme moiety, covalent bond of nucleotide or nucleotide derivative, covalent bond of drug, covalent bond of marker (e.g. fluorescent or radioactive marker) Bond, covalent bond of lipid or lipid derivative, covalent bond of phosphatidylinositol, crosslinking, cyclization, disulfide bond formation, demethylation, formation of covalent bridge, cystine formation, pyroglutamic acid formation, formyl , Gamma-carboxylation, glycosylation, GPI anchor formation, hydroxylation, iodination, methylation, milli Toyl, oxidation, proteolytic processing, phosphorylation, prenylation, racemization, selenoylation (selenoylation), sulfation, such as arginylation and ubiquitination, include transfer -RNA mediated addition of amino acids to proteins.

  The modified polypeptide may further comprise (for example, such conservative or non-conservative (e.g., D-amino acids, desamino acids) amino acid insertions, deletions, or substitutions) in the polypeptide sequence. Such changes do not substantially change the biological activity of the polypeptide).

  Substitutions are conservative (ie, a residue is replaced by another identical general type or group) or non-conservative (ie, a residue is replaced by another type of amino acid). Furthermore, non-naturally occurring amino acids may be used in place of naturally occurring amino acids (ie, non-naturally occurring conservative amino acid substitutions or non-naturally occurring non-conservative amino acid substitutions).

Polypeptides made synthetically may include substitutions of amino acids that are not naturally encoded by DNA (eg, non-naturally occurring or unnatural amino acids). Examples of non-naturally occurring amino acids include D-amino acids, amino acids having an acetylaminomethyl group bonded to the sulfur atom of cysteine, pegylated amino acids, formula: NH ₂ (CH ₂ ) _n COOH (where n Omega amino acids, neutral nonpolar amino acids such as sarcosine, t-butylalanine, t-butylglycine, N-methylisoleucine, and norleucine. Phenylglycine replaces Trp, Tyr, or Phe; citrulline and methionine sulfoxide are neutral apolar, cysteic acid is acidic, and ornithine is basic. Proline can be substituted with hydroxyproline to retain a conformation that confers properties.

  Analogs can be made by substitutional mutagenesis and retain the biological activity of the original polypeptide. Examples of substitutions identified as “conservative substitutions” are shown in Table 3. If such substitutions cause undesired changes, other types of substitutions referred to as “typical substitutions” in Table 3, or other types further described herein with respect to amino acid classes Substitution is introduced and the product is screened.

Substantial alterations in function or immunological identity include (a) the structure of the polypeptide backbone in the region of substitution, e.g., a sheet or helical (helical) conformation, (b) the charge of the molecule at the target site or It is achieved by choosing substitutions whose hydrophobicity, or (c) the side chain volume, whose influence on maintenance is significantly different. Naturally occurring residues are divided into groups based on common side chain properties:
(1) Hydrophobicity: norleucine, methionine (Met), alanine (Ala), valine (Val), leucine (Leu), isoleucine (Ile), histidine (His), tryptophan (Trp), tyrosine (Tyr), phenylalanine ( Phe),
(2) Neutral hydrophilicity: cysteine (Cys), serine (Ser), threonine (Thr)
(3) Acid / Negative charge: Aspartic acid (Asp), Glutamic acid (Glu)
(4) Basicity: Asparagine (Asn), Glutamine (Gln), Histidine (His), Lysine (Lys), Arginine (Arg)
(5) Residues that affect chain orientation: Glycine (Gly), Proline (Pro);
(6) Aromatic: Tryptophan (Trp), Tyrosine (Tyr), Phenylalanine (Phe), Histidine (His),
(7) Polarity: Ser, Thr, Asn, Gln
(8) Basic positive charge: Arg, Lys, His, and;
(9) Charge: Asp, Glu, Arg, Lys, His.

Other conservative amino acid substitutions are listed in Table 3.

Additional Analogs The polypeptides and conjugates of the present invention may comprise polypeptide analogs of aprotinin known in the art. For example, US Pat. No. 5,807,980 describes inhibitors derived from bovine pancreatic trypsin inhibitor (aprotinin), including the polypeptide of SEQ ID NO: 102, and methods for its production and therapeutic use. These polypeptides have been used to treat conditions characterized by abnormal appearance or amount of tissue factor and / or factor VIIIa, such as abnormal thrombosis. US Pat. No. 5,780,265 describes a serine protease inhibitor capable of inhibiting plasma kallikrein comprising SEQ ID NO: 103. US Pat. No. 5,118,668 describes a bovine pancreatic trypsin inhibitor variant comprising SEQ ID NO: 105. The amino acid sequence of aprotinin (SEQ ID NO: 98), Angiopep-1 amino acid sequence (SEQ ID NO: 67), and SEQ ID NO: 104, and several biologically active analog sequences can be found in International Application Publication No. WO 2004/060403.

  A typical nucleotide sequence encoding an aprotinin analog is shown in SEQ ID NO: 106 (atgagaccag atttctgcct cgagccgccg tacactgggc cctgcaaagc tcgtatcatc cgttacttct acaatgcaaa ggcaggcggactcctgcactcgactact This sequence encodes lysine in place of valine found in SEQ ID NO: 98 at position 16. Mutations in the nucleotide sequence of SEQ ID NO: 106 may be introduced by methods known in the art to alter the polypeptide product of SEQ ID NO: 98 having a valine at position 16. Additional mutations or fragments can be obtained using any technique known in the art.

  Other examples of aprotinin analogs include the protein BLAST (Genebank: www.ncbi.nlm.nih.gov/BLAST/) using the synthetic aprotinin sequence (or portion thereof) disclosed in International Application No. PCT / CA2004 / 000011. ) Can be found. Typical aprotinin analogs can be found under accession numbers CAA37967 (GI: 58005) and 1405218C (GI: 3604747).

Production of Polypeptide Derivatives and Peptidomimetics In addition to polypeptides composed solely of naturally occurring amino acids, peptidomimetics or polypeptide analogs are also encompassed by the present invention. Polypeptide analogs are generally used in the pharmaceutical industry as non-polypeptide drugs that have properties similar to those of the template polypeptide. Non-polypeptide compounds are referred to as “polypeptide mimetics” or peptidomimetics (Fauchere et al., Infect. Immun. 54: 283-287, 1986; Evans et al., J. Med. Chem. 30: 1229-1239, 1987). Polypeptide mimetics that are structurally related to therapeutically useful polypeptides can be used to obtain an equivalent or improved therapeutic or prophylactic effect. In general, a peptidomimetic is structurally similar to an exemplary polypeptide (i.e., a polypeptide having biological or pharmacological activity), such as a naturally occurring receptor binding polypeptide, although the art by methods well known _{in, -CH 2 NH -, - CH} 2 S -, - CH 2 -CH 2 -, - CH = CH- ( cis and _{trans), - CH 2 SO -,} - CH (OH) CH 2 Has one or more peptide bonds optionally substituted by bonds such as-, -COCH _2-, etc. (Spatola, Peptide Backbone Modifications, Vega Data, 1 (3): 267, 1983); Spatola et al. (Life Sci. 38: 1243-1249, 1986); Hudson et al. (Int. J. Pept. Res. 14: 177-185, 1979); and Weinstein. B., 1983, Chemistry and Biochemistry, of Amino Acids, Peptides and Proteins, Weinstein eds, Marcel Dekker, New-York). Such polypeptide mimetics have significant advantages over naturally occurring polypeptides, including economic production, high chemical stability, enhanced pharmacological properties (e.g., half Phase, absorption, efficacy, efficiency), reduced antigenicity and other advantages.

  While the polypeptides of the present invention are effective for entry into certain cell types (eg, the cell types described herein), their effectiveness is reduced by the presence of proteases. Serum proteases have specific substrate requirements. The substrate must have both L-amino acid and peptide bonds for cleavage. Furthermore, exopeptidase, the largest component of protease activity in serum, usually acts on the first peptide bond of a polypeptide and requires a free N-terminus (Powell et al., Pharm. Res. 10 : 1268-1273, 1993). Given this, it is often beneficial to use a modified version of the polypeptide. The modified polypeptide retains the structural characteristics of the original L-amino acid polypeptide that confers biological activity on IGF-1, but is beneficially not readily susceptible to cleavage by proteases and / or exopeptidases .

  Using a systematic substitution of one or more amino acids of the consensus sequence with a D-amino acid of the same type (e.g., D-lysine instead of L-lysine) can produce a more stable polypeptide. it can. Thus, the polypeptide derivatives or peptidomimetics of the present invention may be all L, all D or mixed D, L polypeptides. The presence of the N-terminal or C-terminal D-amino acid increases the in vivo stability of the polypeptide. This is because peptidases cannot use D-amino acids as substrates (Powell et al., Pharm. Res. 10: 1268-1273, 1993). A reverse-D polypeptide is a polypeptide containing D-amino acids arranged in a reverse sequence compared to a polypeptide containing L-amino acids. Thus, the C-terminal residue of the L-amino acid polypeptide becomes the N-terminus of the D-amino acid polypeptide, and so forth. The reverse D-polypeptide retains the same tertiary conformation as the L-amino acid polypeptide and therefore retains the same activity, but is more stable to enzymatic degradation in vitro and in vivo than the original polypeptide. And therefore has high therapeutic efficacy (Brady and Dodson, Nature 368: 692-693, 1994; Jameson et al., Nature 368: 744-746, 1994). In addition to reverse-D-polypeptides, constrained polypeptides containing consensus sequences or substantially identical consensus sequence mutations may be made by methods well known in the art (Rizo and Gierasch, Ann. Rev. Biochem. 61: 387-418, 1992). For example, a constrained polypeptide may be made by adding a cysteine residue capable of forming a disulfide bond, thereby producing a cyclic polypeptide. Cyclic polypeptides do not have a free N-terminus or C-terminus. Thus, they are not sensitive to proteolysis by exopeptidases, but are naturally sensitive to endopeptidases that do not cleave the peptide ends. The amino acid sequence of a polypeptide having an N-terminal or C-terminal D-amino acid and the amino acid sequence of a cyclic polypeptide are usually their corresponding except for the presence of the N-terminal or C-terminal D-amino acid residue, respectively, or their cyclic structure Identical to the original polypeptide sequence.

  Cyclic derivatives containing intramolecular disulfide bonds are prepared by conventional solid phase synthesis and incorporate suitable S-protected cysteine or homocysteine residues at selected positions for cyclization, such as amino and carboxy termini (Sah et al., J. Pharm. Pharmacol. 48: 197, 1996). After chain assembly is complete, (1) the S-protecting group is selectively removed, resulting in oxidation of the corresponding two free SH functional groups on the support to form SS bonds, and then the product By conventional removal from the support and subject to appropriate purification procedures, or (2) removal of the polypeptide from the support and complete deprotection of the side chains, and then in a highly diluted aqueous solution. Cyclization can be performed by oxidizing the free SH functional group at

  Cyclic derivatives containing intramolecular amide linkages can be prepared by conventional solid phase synthesis, incorporating suitable amino and carboxyl side chain protected amino acid derivatives at selected positions for cyclization. Cyclic derivatives containing intramolecular -S-alkyl bonds are prepared by conventional solid-phase chemistry, and amino acid residues with suitable amino-protected side chains and suitable S-protection at selected positions for cyclization Cysteine or homocysteine residues can be incorporated.

  Another effective approach to confer resistance to peptidases acting on the N-terminal or C-terminal residues of a polypeptide is to add a chemical group to the end of the polypeptide so that the modified polypeptide is no longer a substrate for the peptidase It is not to be. One such chemical modification is glycosylation of the polypeptide at one or both ends. Certain chemical modifications, particularly N-terminal glycosylation, have been shown to increase the stability of polypeptides in human serum (Powell et al., Pharm. Res. 10: 1268-1273, 1993). Other chemical modifications that enhance serum stability include, but are not limited to, addition of N-terminal alkyl groups consisting of 1-20 carbon lower alkyls such as acetyl groups, and / or addition of C-terminal amide or substituted amide groups. Is included. In particular, the present invention includes modified polypeptides consisting of polypeptides that retain an N-terminal acetyl group and / or a C-terminal amide group.

  Other types of polypeptide derivatives that contain additional chemical moieties not normally part of the polypeptide are also included in the invention, as long as the derivative retains the desired functional activity of the polypeptide. Examples of such derivatives include (1) N-acyl derivatives at the amino terminus or another free amino group, where the acyl group is an alkanoyl group (e.g. acetyl, hexanoyl, octanoyl) F-moc (may be a protecting group such as fluorenylmethyl-O-CO-)); (2) carboxy-terminal or another free carboxy or hydroxyl ester; (3) ammonia or a suitable amine An amide at the carboxy terminus or another free carboxyl group resulting from the reaction with (4) a phosphorylated derivative; (5) a derivative conjugated to an antibody or other biological ligand, and other types of derivatives included.

  Long polypeptide sequences resulting from the addition of additional amino acid residues to the polypeptides of the present invention are also encompassed by the present invention. Such long polypeptide sequences are expected to have the same biological activity as the polypeptide (eg, entry into a particular cell type). Polypeptides with a considerable number of additional amino acids are not excluded, but some large polypeptides take a configuration that masks the effective sequence and thereby targets (e.g. LRP receptor families such as LRP or LRP2). It is recognized that the binding to the member is hindered. These derivatives can act as competitive antagonists. Thus, although the present invention includes a polypeptide or derivative of a polypeptide described herein having an extension, desirably the extension does not destroy the cell targeting activity of the polypeptide or derivative.

  Other derivatives included in the present invention may be directly or through a spacer, for example by short stretches of alanine residues or by putative sites for proteolysis (eg ligation by cathepsin. Eg US Pat. No. 5,126,249 and European patents) No. 495 049), a double polypeptide consisting of two identical or two different polypeptides of the invention covalently linked to each other. Multimers of polypeptides of the present invention are composed of polymers of molecules formed from the same or different polypeptides or derivatives thereof.

  The present invention also provides a polypeptide derivative that is a chimeric or fusion protein containing a polypeptide described herein, or a fragment thereof, linked to the amino acid sequence of a different protein at the amino terminus or carboxy terminus or both. Include. Such a chimeric or fusion protein may be produced by recombinant expression of a nucleic acid encoding the protein. For example, a chimeric or fusion protein contains at least 6 amino acids of the polypeptide of the present invention, and desirably has a functional activity equivalent to or higher than that of the polypeptide of the present invention.

  The polypeptide derivatives of the present invention may be modified by substitution, addition, or deletion or by changing the amino acid sequence by amino acid residues to produce functionally equivalent molecules, or molecules that are functionally enhanced or attenuated as desired. It can produce by obtaining. Derivatives of the invention include, but are not limited to, the amino acid sequences of the polypeptides described herein, including, but not limited to, modified sequences containing functionally equivalent amino acid residue substitutions (eg, SEQ ID NOs: 1-105 and 107). Those containing the whole or part of any one of -112) as a primary amino acid sequence. For example, one or more amino acid residues in a sequence can be replaced by another amino acid of similar polarity that functions as a functional equivalent to produce a silent change. Substitution for an amino acid in the sequence may be selected from other members of the class to which the amino acid belongs. For example, positively charged (basic) amino acids include arginine, lysine and histidine. Nonpolar (hydrophobic) amino acids include leucine, isoleucine, alanine, phenylalanine, valine, proline, tryptophan and methionine. Uncharged polar amino acids include serine, threonine, cysteine, tyrosine, asparagine and glutamine. Negatively charged (acidic) amino acids include glutamic acid and aspartic acid. The amino acid glycine is included in the nonpolar amino acid family or the uncharged (neutral) polar amino acid family. It is understood that substitutions made within a family of amino acids are generally conservative substitutions.

Assays for Identifying Peptidomimetics As noted above, non-peptidyl compounds (peptidomimetics) made to reproduce the polypeptide backbone and pharmacophore display identified by the methods of the present invention are: Often, it has attributes of high metabolic stability, high potency, long duration of action and good bioavailability.

  Peptidomimetic compounds of the present invention can be obtained from biological libraries; spatially addressable parallel solid or liquid phase libraries; synthetic library methods that require deconvolution integration; “one bead one compound” libraries And any of a number of approaches in combinatorial library methods known in the art, including synthetic library methods using affinity chromatography selection. Biological library approaches are limited to polypeptide libraries, while the other four approaches are applicable to compounds in polypeptides, non-peptide oligomers or small molecule libraries (Lam, Anticancer Drug Des. 12: 145 , 1997). Examples of methods for the synthesis of molecular libraries are known in the art, for example: DeWitt et al. (Proc. Natl. Acad. Sci. USA 90: 6909, 1993); Erb et al. (Proc. Natl. Acad. Sci. USA 91: 11422, 1994); Zuckermann et al., J. Med. Chem. 37: 2678, 1994); Cho et al. (Science 261: 1303, 1993); Carell et al. (Angew. Chem, Int. Ed. Engl. 33: 2059, 1994 and ibid 2061); and Gallop et al. (Med. Chem. 37: 1233, 1994). Compound libraries are available in solution (e.g. Houghten, Biotechniques 13: 412-421, 1992) or on beads (Lam, Nature 354: 82-84, 1991), chips (Fodor, Nature 364: 555-556, 1993) Bacteria or spores (US Pat.No. 5,223,409), plasmids (Cull et al., Proc. Natl. Acad. Sci. USA 89: 1865-1869, 1992) or on phage (Scott and Smith, Science 249: 386-390 1990) or luciferase and enzyme label may be detected by determining the conversion of the appropriate substrate to product.

  After identifying a polypeptide of the invention, or by any number of standard methods including, but not limited to, solubility differences (e.g. sedimentation), centrifugation, chromatography (e.g. affinity, ion exchange, size exclusion, etc.) or Can be isolated and purified by any other standard technique used for the purification of polypeptides, peptidomimetics or proteins. Any functional assay known in the art may be used to assess the functional properties of the identified polypeptide of interest. Desirably, an assay for assessing downstream receptor function in intracellular signaling is used (eg, cell proliferation).

For example, the following three-phase process: (1) Scanning a polypeptide of the invention to identify regions of secondary structure required for targeting of a particular cell type described herein; (2 ) Using conformationally constrained dipeptide surrogates to narrow down the backbone shape and providing organic platforms corresponding to these surrogates; and (3) using the best organic platforms to Displaying organic pharmocophores in a candidate library designed to mimic the desired activity of the peptide may be used to obtain peptidomimetic compounds of the invention. More specifically, the three phases are as follows. In phase 1, the lead candidate polypeptide is scanned and its structure is constrained to identify requirements for its activity. A series of polypeptide analogs of the original polypeptide are synthesized. In phase 2, the best polypeptide analog is investigated using a conformationally constrained dipeptide surrogate. To study the backbone shape of the best polypeptide candidates, indolizidin-2-one, indolizidin-9-one and quinolizidinone amino acids (I ² aa, I ⁹ aa and Qaa, respectively) are used as platforms. These and related platforms (reviewed in Halab et al., Biopolymers 55: 101-122, 2000; and Hanessian et al. Tetrahedron 53: 12789-12854, 1997) were introduced into specific regions of the polypeptide. The pharmacophore can be directed in different directions. Biological evaluation of these analogs identifies improved lead polypeptides that mimic morphological requirements for activity. In the three phase, the most active lead polypeptide-derived platform is used to display an organic substitute for the pharmacophore responsible for the activity of the native polypeptide. Combine pharmacophore and skeleton in parallel synthesis form. Derivation of the polypeptide and the above phases can be accomplished by other means using methods known in the art.

  Using structure-function relationships determined from the polypeptides, polypeptide derivatives, peptidomimetics, or other small molecules of the present invention to refine and produce similar molecular structures with similar or good properties it can. Accordingly, the compounds of the present invention also include molecules that share the structure, polarity, charge properties, and side chain properties of the polypeptides described herein.

  In summary, based on the disclosure herein, one of ordinary skill in the art will be able to identify compounds useful for identifying compounds for targeting a substance to a particular cell type (e.g., a cell type described herein). Peptide and peptidomimetic screening assays can be developed. The assays of the invention can be developed for low-throughput, high-throughput, or ultra-high-throughput screening formats. The assays of the present invention include assays that are amenable to automation.

Conjugates of the invention The polypeptides described herein or derivatives thereof can be linked to substances. For example, a polypeptide (eg, Angiopep-7) may be attached to a therapeutic agent, diagnostic agent, or label. In certain embodiments, the polypeptide is linked to or labeled with a detectable label, such as a radioimaging material, for diagnosis of a disease or condition. Examples of these substances include radioimaging substance-antibody-vector conjugates (eg for diagnosis or therapy) where the antibody binds to a disease or condition specific antigen. Other binding molecules are also envisioned by the present invention. In other cases, the polypeptide or derivative may be linked to a therapeutic agent for treating a disease or condition, or linked to a mixture or labeled with the mixture. The disease or condition can be treated by administering the vector-substance conjugate to an individual under conditions that allow for transport of the substance across the BBB or transport of the substance into a particular cell type. Each polypeptide may comprise at least 1, 2, 3, 4, 5, 6, or 7 substances. In other embodiments, each substance has at least 1, 2, 3, 4, 5, 67, 10, 15, 20, or more polypeptides attached thereto. The conjugates of the invention can promote the accumulation of substances (eg, due to increased uptake or decreased elimination) in a particular cell type or tissue of a subject, such as liver, lung, kidney, spleen or muscle. .

  The substance may be releasable from the vector after transport into a particular cell type or transport across the BBB. The substance can be released, for example, by enzymatic or other cleavage of the chemical bond between the vector and the substance. The released material will then function with respect to its intended ability in the absence of the vector.

Therapeutic agent The therapeutic agent may be any biologically active substance. For example, a therapeutic agent is a drug, a medicament, a substance that releases radioactivity, a cytotoxin (eg, a chemotherapeutic agent), a biologically active fragment thereof, or a mixture thereof for treating a disease (eg, killing cancer cells) Or a substance for treating a disease or condition in an individual. The therapeutic agent may be a synthetic product or a product of fungal, bacterial or other microorganism (eg, mycoplasma or virus), animal, eg, reptile, or plant origin. The therapeutic agent and / or biologically active fragment thereof is an enzymatically active substance and / or fragment thereof, or is present in an important and / or essential naturally occurring by inhibiting or blocking an important and / or essential cellular pathway It works by competing with cellular components. Other therapeutic agents include antibodies and antibody fragments.

  Anticancer drugs. Any anticancer agent known in the art may be part of the conjugate of the invention. Brain cancer may be treated with conjugates containing vectors that are efficiently transported across the BBB (eg, Angiopep-3, Angiopep-4a, Angiopep-4b, Angiopep-5, or Angiopep-6). Cancer of the liver, lung, kidney, or spleen may be treated with an anticancer agent conjugated to a vector (eg, Angiopep-7) that is efficiently transported into the appropriate cell type. Typical substances include abarelix, aldesroykin, alemtuzumab, alitretinoin, allopurinol, altretamine, amifostine, anakinra, anastrozole, arsenic trioxide, asparaginase, azacitidine, raw BCG, bevacuzimab, bexarotene, bleomycin, Bleomycin, bortezombi, bortezomib, busulfan, busulfan, calusterone, capecitabine, carboplatin, carmustine, celecoxib, cetuximab, chlorambucil, cisplatin, cladribine, clofarabin, cyclophosphamide, cytaradine, dacarbino Mycin D, dalteparin (e.g. sodium salt), darbepoetin alfa, dasatinib, daunorubici , Daunomycin, decitabine, denileukine, denileukin diftitox, dexrazoxane, docetaxel, doxorubicin, drmostanolone propionate, eculizumab, epirubicin (e.g., HCl salt), epoetin alfa, erlotinib, estramustine, etoposide (e.g., cetomethane) Fentanyl (e.g. citrate), filgrastim, floxuridine, fludarabine, fluorouracil, 5-FU, fulvestrant, gefitinib, gemcitabine (e.g. HCl salt), gemtuzumab ozogamicin, goserelin (e.g. acetate), historelin (E.g. acetate), hydroxyurea, ibritumomab tiuxetan, idarubicin, ifosfamide, imatinib (e.g. mesylate), interferon alpha-2b, irinote Can, lapatinib ditosylate, lenalidomide, letrozole, leucovorin, leuprolide (e.g. acetate), levamisole, lomustine, CCNU, meclorethamine (nitrogen mustard), megestrol, melphalan (L-PAM), mercapto Purine (6-MP), mesna, methotrexate, methoxalene, mitomycin C, mitotane, mitoxantrone, nandrolone fenpropionate, nelarabine, nofetumomab, oprelbequin, oxaliplatin, paclitaxel, palifermin, pamidronumab, penitumumab , Pegaspargase, pegfilgrastim, peginterferon alfa-2b, pemetrexed (e.g. disodium salt), pentostatin, pipbloman, pricamycin (mitrama Isine), porfimers (e.g. sodium salt), procarbazine, quinacrine, rasburicase, rituximab, sargramostim, sorafenib, streptozocin, sunitinib (e.g. maleate), talc, tamoxifen, temozolomide, teniposide (VM-26), test lactone, thalidomide Thioguanine (6-TG), thiotepa, thiotepa, thiotepa, topotecan (e.g. hcl salt), toremifene, tositumomab / I-131 (tositumomab), trastuzumab, trastuzumab, tretinoin (ATRA), uracil mustard, valrubicin, vinblastine, vincristine, Includes vinorelbine, vorinostat, zoledronic acid, and zoledronic acid. Exemplary derivatives of paclitaxel are described in US Pat. No. 6,911,549, the entire contents of which are hereby incorporated by reference.

The conjugates of the invention may be labeled for detectable label detection or diagnostic purposes. The detectable label or marker may be a radioactive label, a fluorescent label, a nuclear magnetic resonance active label, a luminescent label, a chromophore label, a positron emitting isotope for a PET scanner, a chemiluminescent label, or an enzyme label. Typical radioimaging materials that release radioactivity (detectable radiolabels) include indium-111, technitium-99, or low doses of iodine-131. Gamma and beta emitting radionuclides include ⁶⁷ Cu, ⁶⁷ Ga, ⁹⁰ Y, ¹¹¹ In, ^99m Tc, and ²⁰¹ Tl). Positron emitting radionuclides include ¹⁸ F, ⁵⁵ Co, ⁶⁰ Cu, ⁶² Cu, ⁶⁴ Cu, ⁶⁶ Ga, ⁶⁸ Ga, ⁸² Rb, and ⁸⁶ Y. Fluorescent labels include Cy5.5, green fluorescent protein (GFP), fluorescein, and rhodamine. Chemiluminescent labels include luciferase and β-galactosidase. Enzymatic labels include peroxidase and phosphatase. The his tag is also a detectable label. For example, the conjugate may comprise a vector portion and an antibody portion (antibody or antibody fragment), which may further comprise a label. In this case, the label may be conjugated to a vector or antibody.

Antibodies Antibodies may be conjugated to the polypeptides of the invention by any means known in the art (eg, using the conjugation strategies described herein). Any diagnostic or therapeutic antibody may be conjugated to one or more (eg, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) vectors of the invention. Furthermore, an antibody fragment (eg, an antibody fragment capable of binding to an antigen) may be conjugated to the vector of the present invention. Antibody fragments include the Fab and Fc regions (eg, of any of the antibodies described herein), heavy chain, and light chain of the antibody. Typical antibodies for use in cancer diagnosis and therapy include ABX-EGF (Panitimumab), OvaRex (Oregovemab), Theragyn (pemtumomabytrrium-90), Therex, Bibatuzumab (Bivatuzumab), Panorex (Edrecolomab), ReoPro (Leopro) (Abciximab), Bexxar (Bexial) (Tositumomab), MAb, Idiotype 105AD7, Anti-EpCAM (Katsumaxomab), MAb Lung Cancer (Cytocloin Herp) (Trastuzumab), Rituxan (Rituxan) (Rituximab), Avastin (Avastin) (Bevacizumab), AMD Fab (Ranibizumab), E-26 (2 ^nd gen. IgE) (Omarizumab), Zevalin (Rituxan + Yttrium-90 ) (Ibritumomab tiuxetan), cetuximab, BEC2 (Mitumomab), IMC-1C11, nuC242-DM1, LymphoCide (Epratuzumab), LymphoCide Y-90, CEA-Cide (Labetuzumab), CEA-Cide Y-90, CEA-Scan (Tc-99m-labeled arsitomab), LeukoScan (Tc-99m-labeled sulesomab), LymphoScan (Tc-99m-labeled bectumomab), AFP -Scan (Tc-99m-labeled), HumaRAD-HN (+ yttrium-90), HumaSPECT (Votumumab), MDX-101 (CTLA-4), MDX-210 (her-2 overexpression), MDX- 210 / MAK, Vitaxin, MAb 425, IS-IL-2, Campath (campus) (alemtuzumab), CD20 streptavidin, Avidicin, (albumin + NRLU13), Oncolym (+ iodine-131) Cotara (+ iodine-131), C215 (+ staphylococcal enterotoxin, MAb lung / kidney cancer (Pharmacia Corp.), nacolomab tafenatox (C242 staphylococcal enterotoxin), Nuvion (visidizumab), SMART M195, SMART 1D10, CEAVac, Includes TriGem, TriAb, radiolabeled NovoMAb-G2, Monopharm C, GlioMAb-H (+ gelonin toxin), Rituxan (rituximab), and ING-1 That. Additional therapeutic antibodies include 5G1.1 (Ecluizumab), 5G1.1-SC (Pexelizumab), ABX-CBL (Gavilimomab), ABX-IL8, Antegren (natalizumab) ), Anti-CD11a (efarizumab), anti-CD18 (Genetech), anti-LFA1, Antova, BTI-322, CDP571, CDP850, Corsevin M, D2E7 (adalimumab), Humira (humira) (adalimumab), Hu23F2G (Robelizumab) (Rovelizumab)), IC14, IDEC-114, IDEC-131, IDEC-151, IDEC-152, Infliximab (Remicade), LDP-01, LDP-02, MAK-195F (Aferimomab), MDX-33, MDX-CD4, MEDI-507 (Ciplizumab), OKT4A, OKT3 (Muromonab-CD3), and ReoPro (Abciximab) are included.

Conjugation Linkers Any cross-linking (conjugation) reagent or protocol (many commercially available) known in the art can be used to obtain conjugates (eg, protein-protein conjugates). Such protocols and reagents include crosslinkers reactive with amino, carboxyl, sulfhydryl, carbonyl, carbohydrate and / or phenol groups. The amount, time, and conditions of such protocols can be varied to optimize the conjugation. Cross-linking reagents contain at least two reactive groups, generally homofunctional crosslinkers (containing identical reactive groups) and heterofunctional crosslinkers (containing non-identical reactive groups) It is divided into. The cross-linking agents of the present invention may be homobifunctional and / or heterobifunctional. Furthermore, the crosslinker may include a “spacer” between the reactive moieties, or the two reactive moieties in the crosslinker may be directly linked. The bond includes an ester bond.

Typical linkers include BS ³ [bis (sulfosuccinimidyl) suberate], NHS / EDC (N-hydroxysuccinimide and N-ethyl- (dimethylaminopropyl) carbodiimide), sulfo-EMCS ([Ne-maleimide) Caproic acid] hydrazide), SATA (N-succinimidyl-S-acetylthioacetate), and hydrazide. BS ³ is a homobifunctional N-hydroxysuccinimide ester that targets accessible primary amines. The conjugation scheme is illustrated in FIG. NHS / EDC allows the conjugation of primary amine groups and carboxyl groups. Sulfo-EMCS is a heterobifunctional reactive group (maleimide and NHS-ester) that is reactive towards sulfhydryl and amino groups. Amine coupling using sulfo-NHS / EDC activation can be used to crosslink the polypeptides of the invention and therapeutic antibodies, as illustrated in FIGS. This is a quick, simple and reproducible coupling technique. The resulting conjugate is stable and retains the biological activity of the antibody. Furthermore, it has a high conjugation capacity that can be easily controlled and has low non-specific interactions during the coupling procedure. SATA is reactive towards amines and adds protected sulfhydryl groups. NHS-esters react with primary amines to form stable amide bonds. Sulfhydryl groups can be deprotected using hydroxylamine. The conjugation method is illustrated in FIG. As shown in FIG. 6, hydrazide can be used to link a carboxyl group to a primary amine, and thus hydrazide is useful for glycoprotein linking. An additional exemplary linker is shown in FIG.

Small molecules, such as therapeutic agents, can be conjugated to the polypeptides of the present invention. A typical small molecule, paclitaxel, has two strategically important positions (C2 'and C7 positions) useful for conjugation. Conjugation of a vector or a vector of the invention with paclitaxel can be performed as follows (FIG. 8). Briefly, paclitaxel is reacted with anhydride succinic pyridine at room temperature for 3 hours to attach a succinyl group at the 2 ′ position. 2'-succinyl paclitaxel has a cleavable ester bond at the 2 'position, which can simply release succinic acid. This cleavable ester bond can be further used in various modifications with linkers if desired. The resulting 2'-O-succinyl-paclitaxel is then reacted with EDC / NHS in DMSO for 9 hours at room temperature, and then the vector or vector in Ringer / DMSO is added during an additional reaction time of 4 hours at room temperature . The conjugation reaction shown in FIG. 8 is monitored by HPLC. Each intermediate, such as paclitaxel, 2'-O-succinyl - paclitaxel and 2'-O-NHS-succinyl - purified paclitaxel, HPLC, thin layer (thin) liquid chromatography, NMR ⁽¹³ C or ¹ H exchange) Validate using different approaches such as melting, melting, or mass spectrometry. The final conjugate is analyzed by mass spectrometry and SDS-polyacrylamide gel electrophoresis. This makes it possible to determine the number of paclitaxel molecules conjugated to each vector.

Conjugation activity By conjugating the substance to the vectors described herein, desired properties such as altered pharmacokinetics, altered tissue distribution (e.g. specific tissues or cell types such as liver, brain, lungs). , Increased delivery to the spleen, or kidney). Briefly, the inventors have identified vectors (eg, Angiopep-3, Angiopep-4a, Angiopep-4b, Angiopep-5, and Angiopep-6) that efficiently transport material across the BBB. Like Angiopep-2, these vectors may also be able to target substances to other cell types or tissues (eg, liver, lung, kidney, spleen, or muscle). The inventors have also identified Angiopep-7, a vector that is not efficiently transported across the BBB, but is transported to specific tissues (eg, liver, lung, kidney, spleen, or muscle). Thus, vectors with this activity are useful when transport across the BBB is not desired.

  Because the conjugates of the present invention transport substances to specific tissues, the conjugated substances result from low toxicity (e.g., fewer side effects), high potency (e.g., increased uptake or decreased efflux from tissues or cells). The substance is concentrated in the target tissue, or has a high stability when conjugated), or a combination thereof. Such activities are described below and in WO 2007/009229, which is hereby incorporated by reference.

  In some cases, conjugating a substance to a vector allows the substance to avoid the action of P-glycoprotein (P-gp), an efflux pump that can excrete specific substances from cells. Become. By reducing the ability of P-gp to excrete a substance from the cell, the potency of the substance in the cell can be increased. Therefore, these conjugates actively inhibit cancer cell growth. Furthermore, the results obtained for in vivo tumor growth indicate that the vectors of the invention target the receptor LRP. Conjugation also alters the pharmacokinetics or biodistribution of unconjugated substances.

  Overall, conjugates can be used against primary tumors, including breast, lung, and skin cancers, and metastases from primary tumors.

P-glycoprotein bypass Since resistance to various chemotherapeutic agents such as vincristine, etoposide, and doxorubicin is mediated by P-gp (MDR1) overexpression (FIG. 9), bypassing this efflux pump results in various cancer types The action of these drugs on can be enhanced. By conjugating the vector to such a substance, it becomes possible to reduce the efflux of P-gp mediated substances. These conjugates are useful for increasing the potency of drugs associated with resistance mediated by P-gp. As illustrated below, the vectors described herein were tested for their ability to avoid elimination by P-gp.

LRP-mediated uptake Based on our previous work (see International Patent Application No.PCT / CA2004 / 00011, which is incorporated herein by reference), the receptor-related protein (RAP) is In vitro model of blood brain barrier inhibited the transcellular transport of aprotinin. LRP is expressed on a variety of cancer cells (see, eg, WO 2007/009229). We therefore proposed that the low density lipoprotein-related receptor (LRP) is involved in the penetration of aprotinin into the brain. Similar inhibition of Angiopep-1 and Angiopep-2 transport across an in vitro model of the blood brain barrier was also obtained (data not shown). This suggests that LRP is involved in the transcellular transport of these polypeptides across brain endothelial cells as well. Based on this, we generally believe that LRP is involved in the uptake of the aprotinin-related polypeptides described herein.

  LRP is a 600 kDa heterodimeric membrane receptor composed of two subunits; subunit-α (515 kDa) and subunit-β (85 kDa). Since LRP is involved in the transport of the vectors described herein, the conjugates of the invention target cells and tumors that express this receptor. Certain cancer cells, for example, express LRP members (eg, LRP or LRP2).

Therapeutic Methods The present invention also features therapeutic methods using the polypeptide conjugates described herein. Treat any brain or central nervous system disease with conjugates that are efficiently transported across the BBB (for example, Angiopep-3, Angiopep-4a, Angiopep-4b, Angiopep-5, and Angiopep-6) can do. These conjugates are also efficiently transported to the liver, lungs, kidneys, spleen or muscle, and therefore these conjugates are used in combination with appropriate therapeutic agents to treat diseases associated with these tissues ( For example, cancer) may be treated. Angiopep-7 is not efficiently transported to the brain, but it is efficiently transported to tissues and cells such as liver, lung, kidney, spleen and muscle, so Angiopep-7 is desirable for targeting substances to the brain If not, it is particularly well suited as a vector for the treatment of diseases associated with the tissue.

  Liver diseases include amoebic liver abscess, cirrhosis, disseminated coccidioidomycosis; drug-induced cholestasis, hemochromatosis, hepatitis A, hepatitis B, hepatitis C, hepatitis D, hepatocellular carcinoma, liver cancer, alcohol Liver disease, primary biliary cirrhosis, purulent hepatic abscess, Lewy syndrome, sclerosing cholangitis, and Wilson disease. Amebic liver abscess can be treated by administration of a vector conjugated to metronidazole. For example, hepatitis B can be treated by administration of a vector conjugated to interferon-alpha, lamivudine, adefovir dipivoxil, entecavir, or other antiviral drugs. For example, hepatitis C can be treated by administration of a vector conjugated to pegylated interferon or ribavirin, or a combination thereof.

  Lung diseases include lung cancer, such as small cell carcinoma (eg, oat cell carcinoma), mixed small / large cell carcinoma, complex small cell carcinoma, and metastatic tumor. Metastatic tumors can be derived from cancer of any tissue, including breast cancer, colon cancer, prostate cancer, sarcoma, bladder cancer, neuroblastoma, and Wilms tumor. Spleen diseases include cancers such as lymphoma, non-Hodgkin lymphoma, and certain T cell lymphomas.

  Additional exemplary cancers that can be treated using the conjugates or compositions of the invention include hepatocellular carcinoma, breast cancer, head and neck cancer, such as various lymphomas such as mantle cell lymphoma, Non-Hodgkin lymphoma, adenoma, squamous cell carcinoma, laryngeal cancer, retinal cancer, esophageal cancer, multiple myeloma, ovarian cancer, uterine cancer, melanoma, colorectal cancer, bladder cancer, prostate cancer, lung cancer (non-small cell) (Including lung cancer), pancreatic cancer, cervical cancer, head and neck cancer, skin cancer, nasopharyngeal cancer, liposarcoma, epithelial cancer, renal cell cancer, gallbladder adenocarcinoma, parotid gland cancer, endometrial sarcoma, multiple Drug-resistant cancer; and proliferative diseases and symptoms, such as angiogenesis associated with tumor angiogenesis, macular degeneration (eg wet / non-wet AMD), comeal neovascularization, diabetic retinopathy, blood vessels Neonatal glaucoma, myopic degeneration and other proliferative diseases and symptoms, Examples include restenosis and polycystic kidneys. Brain cancers that can be treated with vectors that are efficiently transported across the BBB include astrocytoma, ciliary astrocytoma, germ dysplastic neuroepithelial tumor, oligodendroglioma, ependymoma , Pleomorphic glioblastoma, mixed glioma, oligoastrocytomas, medulloblastoma, retinoblastoma, neuroblastoma, germinomas, and teratomas.

  The conjugates or compositions of the invention can be administered to a subject by any means known in the art, for example, oral, intraarterial, intranasal, intraperitoneal, intravenous, intramuscular, subcutaneous, transdermal, or oral. Can be administered. The substance may be, for example, an anti-angiogenic compound.

Pharmaceutical Compositions A pharmaceutical composition of the invention may comprise a polypeptide or conjugate described herein together with a pharmaceutically acceptable carrier. Such compositions are liquid or lyophilized formulations or otherwise dried formulations, with various buffer contents (e.g., Tris-HCl, acetic acid, phosphate), pH and ionic strength diluents, to the surface Additives such as albumin or gelatin, surfactants (eg Tween 20, Tween 80, Pluronic F68, bile salts) are included. Solubilizers (e.g., glycerol, polyethylene glycerol), antioxidants (e.g., ascorbic acid, sodium metabisulfite), preservatives (e.g., thimerosal, benzyl alcohol, parabens), bulking agents or tonicity modifiers ) (E.g., lactose, mannitol), covalent addition of polymers such as polyethylene glycol to proteins, complexation with metal ions, or polymer compounds, e.g. in microparticle preparations such as polylactic acid, polyglycolic acid, hydrogel, or Incorporation of substances onto microparticle preparations or incorporation of substances onto liposomes, microemulsions, micelles, monolayer or multilamellar vesicles, erythrocyte ghosts, or spheroplasts. Such compositions affect the physical state, solubility, stability, rate of in vivo release, and rate of in vivo clearance. Controlled or sustained release compositions include the formulation in lipophilic depots (eg, fatty acids, waxes, oils). Also included in the present invention are particulate compositions coated with polymers (eg, poloxamers or poloxamines). Other embodiments of the compositions of the present invention include particulate form protective coatings, protease inhibitors or penetration enhancers for various administration routes such as parenteral, pulmonary, nasal, oral, vaginal, rectal routes. In one embodiment, the pharmaceutical composition is parenteral, paracancerally, transmucosally, transdermal, intramuscular, intravenous, intradermal, subcutaneous, intraperitoneal, intracerebroventricular, intracranial, and intratumoral. The thing is administered.

  The pharmaceutically acceptable carrier further comprises 0.01-0.1 M or 0.05 M phosphate buffer or 0.8% saline. In addition, such pharmaceutically acceptable carriers can be aqueous or non-aqueous solutions, suspensions, and emulsions. Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate. Aqueous carriers include water, alcoholic / aqueous solutions, emulsions or suspensions (including saline and buffered media). Parenteral vehicles include sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's or fixed oil. Intravenous vehicles include fluid and nutrient replenishers, electrolyte replenishers, such as those based on Ringer's dextrose, and the like. For example, antibacterial agents, antioxidants, collating agents, preservatives such as inert gases, and other additives may be added.

  Other formulations include poly-oxyethylene esters of fatty acids (eg 12-hydroxystearic acid), eg Solutol® HS15. Thus, in some embodiments, the pharmaceutical composition comprises a) a conjugate as described herein, b) Solutol® HS15 and c) an aqueous solution or buffer (e.g., Ringer / pH 5-7) Hepes solution). The concentration of Solutol® HS15 in the formulation is at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, or 60% (e.g. 30 %), Or within any range between any two of these numbers. The concentration of the conjugate can be determined based on the dose required to efficiently treat the subject or the amount of ester required for the solubility of the administered conjugate. The use of Solutol in formulations for administration of Taxol conjugates is described, for example, in International Publication No. WO 2007/009229, which is hereby incorporated by reference.

Solid dosage forms for oral use Oral formulations include tablets containing the active ingredient (s) in a mixture with non-toxic pharmaceutically acceptable excipients, and such formulations are well known to those skilled in the art. (Eg, USPN: 5,817,307, 5,824,300, 5,830,456, 5,846,526, 5,882,640, 5,910,304, 6,036,949, 6,036,949, 6,372,218, which is incorporated herein by reference). These excipients include, for example, inert diluents or fillers (e.g. sucrose, sorbitol, sugar, mannitol, microcrystalline cellulose, starch including potato starch, calcium carbonate, sodium chloride, lactose, calcium phosphate, sulfuric acid Calcium or sodium phosphate); granulating and disintegrating agents (e.g., cellulose derivatives including microcrystalline cellulose, starches including potato starch, croscarmellose sodium, alginate, or alginic acid); binders (e.g., sucrose) , Glucose, sorbitol, acacia, alginic acid, sodium alginate, gelatin, starch, pregelatinized starch, microcrystalline cellulose, magnesium aluminum silicate, sodium carboxymethylcellulose, methylcellulose, hydro And lubricants, glidants, and anti-adhesives (e.g., magnesium stearate, zinc stearate, stearic acid, silica, hydrogenated vegetable oil, or talc). It's okay. Other pharmaceutically acceptable excipients may be colorants, flavoring substances, plasticizers, wetting agents, buffering agents, and the like.

  The tablets may be uncoated or optionally coated by known techniques to delay disintegration and absorption in the gastrointestinal tract and provide a sustained action over a longer period. The coating is configured to release the substance in a predetermined pattern (e.g. to achieve a controlled release formulation) or not to release the substance (s) until after it has passed through the stomach (Enteric coating). The coating can be a sugar coating, a film coating (e.g., a coating based on hydroxypropylmethylcellulose, methylcellulose, methylhydroxyethylcellulose, hydroxypropylcellulose, carboxymethylcellulose, acrylate copolymer, polyethylene glycol, and / or polyvinylpyrrolidone), or an enteric coating (e.g., Methacrylic acid copolymer, cellulose acetate phthalate, hydroxypropylmethylcellulose phthalate, hydroxypropylmethylcellulose succinate acetate, polyvinyl acetate phthalate, shellac, and / or coatings based on ethylcellulose). In addition, a time delay material such as glyceryl monostearate or glyceryl distearate may be employed.

  The solid tablet composition may include a coating configured to protect the composition from unwanted chemical changes (eg, chemical degradation prior to release of the active agent). The coating may be applied onto the solid dosage form in a manner similar to that described in the Encyclopedia of Pharmaceutical Technology above.

  The compositions of the present invention may be mixed together in tablets or divided. In one example, the first substance is contained inside the tablet and the second substance is contained outside so that a significant portion of the second substance is released prior to the release of the first substance.

  Formulations for oral use are as chewable tablets or as hard gelatin capsules in which the active ingredient is mixed with an inert solid diluent (e.g. potato starch, lactose, microcrystalline cellulose, calcium carbonate, calcium phosphate or kaolin) Alternatively, it may be provided as a soft gelatin capsule in which the active ingredient is mixed with water or an oil medium such as peanut oil, liquid paraffin, or olive oil. Powders and granules may be produced using the ingredients described above under tablets and capsules in a conventional manner using, for example, a mixer, fluid bed apparatus, or spray drying apparatus.

Dosage The dose of any conjugate or composition described herein or identified using the methods described herein is the method of administration, the disease being treated (e.g., cancer), the disease It depends on a number of factors, including the severity, whether the cancer is being treated or prevented, and the age, weight, and health of the subject being treated.

  With respect to the therapeutic methods of the invention, administration of a vector, conjugate, or composition to a subject is not limited to a particular mode of administration, dose, or frequency of dosing; Is assumed. The conjugate, or composition may be administered to the subject in a single dose or in multiple doses. For example, the compounds, conjugates described herein or identified using the screening methods of the present invention may be, for example, 2, 3, 4, 5, 6, 7, 8, 10, 15, 20 Or once a week for more than one week. For any particular subject, the specific mode of administration is adjusted over time according to the individual needs and the professional judgment of the person administering or directing the administration of the vector, conjugate, or composition It should be understood that it should be. For example, if the low dose does not provide sufficient activity in the treatment of the diseases or conditions described herein (eg, cancer), the dose of the conjugate can be increased. Conversely, if the disease (eg, cancer) is reduced or eliminated, the dose of the compound can be reduced.

Ultimately the attending physician will decide on the appropriate amount and mode of administration, but therapeutically effective amounts of the vectors, conjugates, or compositions described herein may be, for example, 0.0035 μg-20 μg / kg body weight / day or 0.010. It may range from μg to 140 μg / kg body weight / week. Desirably, the therapeutically effective amount is in the range of 0.025 μg to 10 μg / kg administered daily, every other day, or twice a week, such as at least 0.025, 0.035, 0.05, 0.075, 0.1, 0.25, 0.5, 1.0, 1.5. 2.0, 2.5, 3.0, 3.5, 4.0, 5.0, 6.0, 7.0, 8.0, or 9.0 μg / kg body weight. Further, a therapeutically effective amount is in the range of 0.05 μg to 20 μg / kg administered weekly, every other week, or monthly, such as at least 0.05, 0.7, 0.15, 0.2, 1.0, 2.0, 3.0, 4.0, 5.0, It may be 6.0, 7.0, 8.0, 10.0, 12.0, 14.0, 16.0, or 18.0 μg / kg body weight. Furthermore, a therapeutically effective amount of a compound, for example, every other day, once a week, or a range of 100μg / m ² ~100,000μg / m ² administered every other week. In a preferred embodiment, the therapeutically effective amount is a daily, every other day, twice weekly, weekly, or 1,000μg / m ² ~20,000μg / m ² in the range to be administered every other week, for example, at least 1,000,1,500 , 4,000, or 14,000 μg / m ² of compound.

Hereinafter, embodiments of the present invention will be described.
(1) The following formula:
X1-X2-X3-X4-X5-X6-X7-X8-X9-X10-X11-X12-X13-X14-X15-X16-X17-X18-X19
[Where
X1-X19 are any amino acid or absent,
At least one of X1, X10, and X15 is Arg]
A polypeptide comprising an amino acid sequence having an amino acid sequence selected from the group consisting of Angiopep-4b, Angiopep-5, Angiopep-6, and Angiopep-7 (SEQ ID NOs: 109 to 112). Said polypeptide having at least 50% identity to it.
(2) The polypeptide according to (1), comprising an amino acid sequence having at least 50% identity to the sequence of Angiopep-7.
(3) The polypeptide according to (1), wherein the identity is at least 70%.
(4) The polypeptide according to (1), wherein the identity is at least 90%.
(5) The polypeptide according to (1), wherein X10 is Lys or Arg, X15 is Lys or Arg, or both.
(6) The polypeptide according to (1), wherein X1 is Arg.
(7) The polypeptide according to (1), wherein X10 is Arg.
(8) The polypeptide according to (1), wherein X15 is Arg.
(9) The polypeptide according to (1), wherein X10 and X15 are Arg.
(10) The polypeptide according to (1), wherein X7 is Ser or Cys.
(11) The polypeptide according to (1), wherein X7 is Ser.
(12) The polypeptide according to (1), comprising an amino acid sequence selected from the group consisting of Angiopep-4b, Angiopep-5, Angiopep-6, and Angiopep-7.
(13) The polypeptide according to (12), comprising the amino acid sequence of Angiopep-7.
(14) The polypeptide according to (12), comprising an amino acid sequence selected from the group consisting of Angiopep-4b, Angiopep-5, Angiopep-6, and Angiopep-7.
(15) The polypeptide according to (14), comprising the amino acid sequence of Angiopep-7.
(16) The polypeptide according to (1), which is transported to at least one cell or tissue selected from the group consisting of liver, lung, kidney, spleen, and muscle.
(17) The polypeptide according to (1), which is not efficiently transported across the blood brain barrier (BBB).
(18) The polypeptide according to (17), comprising a sequence having at least 80% identity to Angiopep-7.
(19) A composition comprising the polypeptide according to (1).
(20) The composition according to (19), comprising a pharmaceutically acceptable carrier.
(21) A conjugate comprising a vector comprising the polypeptide of (a) (1), and (b) a therapeutic agent, diagnostic agent or detectable label conjugated to the vector.
(22) The conjugate according to (21), wherein the vector comprises an amino acid sequence having at least 50% identity to the sequence of Angiopep-7.
(23) The conjugate according to (21), wherein the identity is at least 70%.
(24) The conjugate according to (21), wherein the identity is at least 90%.
(25) The conjugate according to (21), wherein X10 is Lys or Arg; X15 is Lys or Arg; or both.
(26) The conjugate according to (21), wherein X1 is Arg.
(27) The conjugate according to (21), wherein X10 is Arg.
(28) The conjugate according to (21), wherein X15 is Arg.
(29) The conjugate according to (21), wherein X10 and X15 are Arg.
(30) The conjugate according to (21), wherein X7 is Ser or Cys.
(31) The conjugate according to (21), wherein X7 is Ser.
(32) The conjugate according to (21), wherein the vector comprises an amino acid sequence selected from the group consisting of Angiopep-4b, Angiopep-5, Angiopep-6, and Angiopep-7.
(33) The conjugate according to (21), wherein the vector comprises the amino acid sequence of Angiopep-7.
(34) The conjugate according to (21), wherein the vector comprises an amino acid sequence selected from the group consisting of Angiopep-4b, Angiopep-5, Angiopep-6, and Angiopep-7.
(35) The conjugate according to (21), wherein the vector comprises the amino acid sequence of Angiopep-7.
(36) The conjugate according to (21), wherein the vector is conjugated to a therapeutic agent.
(37) The conjugate according to (21), wherein the therapeutic agent is an anticancer agent.
(38) The conjugate according to (21), wherein the vector is efficiently transported to at least one cell or tissue selected from the group consisting of liver, lung, kidney, spleen, and muscle.
(39) The conjugate according to (21), wherein the vector is not efficiently transported across the BBB.
40. Bortezombi, bortezomib, busulfan, calusterone, capecitabine, carboplatin, carmustine, celecoxib, cetuximab, chlorambucil, cisplatin, cladribine, clofarabine, cyclophosphamide, cytarabine, dacarbazine, dactinomycin, dactinomycin , Darbepoetin alfa, dasatinib, daunorubicin, daunomycin, decitabine, denyleukin, de Leukin diftitox, dexrazoxane, docetaxel, doxorubicin, drmostanolone propionate, eculizumab, epirubicin, epoetin alfa, erlotinib, estramustine, etoposide (e.g. phosphate), exemestane, fentanyl, filgrastim, floxuridine, fludavin Fluorouracil, 5-FU, fulvestrant, gefitinib, gemcitabine, gemtuzumab ozogamicin, goserelin, histrelin, hydroxyurea, ibritumomab tiuxetan, idarubicin, ifosfamide, imatinib, interferon alfa-2b, irinotecan, lapatinib , Letrozole, leucovorin, leuprolide, levamisole, lomustine, CCNU, mechloretamine (m (eclorethamine) (nitrogen mustard), megestrol, melphalan (L-PAM), mercaptopurine (6-MP), mesna, methotrexate, methoxalene, mitomycin C, mitotane, mitoxantrone, nandrolone fenpropionate, nelarabine , Nofetumomab, oprelbequin, oxaliplatin, paclitaxel, paliferum, pamidronate, panitumumab, pegademase, pegaspargase, pegfilgrastim, peginterferon alfa-2b, pemetrexed, pentostatin, pipobromantramycin , Porfimer, procarbazine, quinacrine, rasburicase, rituximab, sargramostim, sorafenib, streptozocin, sunitinib, sunitinib maleate, ta Luc, tamoxifen, temozolomide, teniposide (VM-26), test lactone, thalidomide, thioguanine (6-TG), thiotepa, topotecan, toremifene, tositumomab / I-131 (tositumomab), trastuzumab, tretinoin (ATRA), uracil mustard, The conjugate according to (37), selected from the group consisting of valrubicin, vinblastine, vincristine, vinorelbine, vorinostat, zoledronate, and zoledronic acid.
(41) The conjugate according to (40), wherein the anticancer agent is paclitaxel.
(42) The conjugate according to (37), wherein the drug is an antibody.
(43) The antibody is ABX-EGF (Panitimumab), OvaRex (Oregovemab), Theragyn (pemtumomabytrrium-90), Therex, Bivatuzumab, Ed ), ReoPro (Alepro) (Abciximab), Bexxar (Tositumomab), MAb, Idiotype 105AD7, Anti-EpCAM (Katumaxomab), MAb Lung Cancer (Cytoclonal), Herceptin (Herceptin) (Trastuzumab), Rituxan (Tituxanri) (Rituximab), Avastin (Bevacizumab), AMD Fab (Ranibizumab), E-26 (2 ^nd gen. IgE) (Omarizumab), Zevalin (Rituxan + Yttrium-90) (Ibritumomab Chiuxetane) , Cetuximab, BEC2 (Mitumomab), IMC-1C11, nuC242-DM1, LymphoCide (Epratuzumab), LymphoCide Y-90, CEA-Cide (Labetuzumab), CEA-Cide Y-90, HumaRAD-H (+ Yttrium-90), MDX-101 (CTLA-4), MDX-210, MDX-210 / MAK, Vitaxin, MAb 425, IS-IL-2, Campus (campus) (alemtuzumab), CD20 streptavidin, Avidicin , (Albumin + NRLU13), Oncolym (+ iodine-131) Cotara (+ iodine-131), C215 (+ staphylococcal enterotoxin), MAb lung / kidney cancer (Pharmacia Corp.), nacolomab tafenatox (nacolomab tafenatox) (C242 staphylococcal enterotoxin), Nuvion (Vigilizumab), SMART M195, SMART 1D10, CEAVac, TriGem, TriAb, radiolabeled NovoMAb-G2, Monopharm C, GlioMAb-H (+ gelonin toxin), Rituxan (rituximab), And the conjugate according to (42), selected from the group consisting of ING-1.
(44) P-gp-mediated accumulation in liver, lung, kidney, or spleen cells that accumulates rapidly, accumulates at a high concentration, and is reduced compared to the agent or label when not conjugated to the vector The conjugate according to (21), which exhibits sex efflux.
(45) A composition comprising the conjugate according to (21) and a pharmaceutically acceptable carrier.
(46) A method for treating a subject having cancer, the method comprising the step of providing the subject with a sufficient amount of the conjugate according to (37) to treat the subject.
(47) The method according to (46), wherein the cancer is lung, liver, kidney or spleen cancer.
(48) The cancer is hepatocellular carcinoma, breast cancer, head and neck cancer, lymphoma, mantle cell lymphoma, non-Hodgkin lymphoma, adenoma, squamous cell carcinoma, laryngeal cancer, retinal cancer, esophageal cancer, multiple Myeloma, ovarian cancer, uterine cancer, melanoma, colorectal cancer, bladder cancer, prostate cancer, lung cancer, non-small cell lung cancer, pancreatic cancer, cervical cancer, head and neck cancer, skin cancer, nasopharyngeal cancer, fat (46) The method according to (46), which is selected from the group consisting of sarcoma, epithelial cancer, renal cell carcinoma, gallbladder adenocarcinoma, parotid gland cancer, endometrial sarcoma and multidrug resistant cancer.
(49) The method according to (46), wherein the conjugate comprises a vector that is efficiently transported to at least one cell or tissue selected from the group consisting of liver, lung, kidney, spleen, or muscle.
(50) The method of (46), wherein the conjugate comprises a vector that is not efficiently transported across the BBB.
(51) The method according to (50), wherein the conjugate comprises Angiopep-7.
(52) The method according to (46), wherein the anticancer agent is paclitaxel.
(53) When the anticancer agent is conjugated to the polypeptide, the anticancer agent has increased efficacy or reduced side effects compared to the anticancer agent when not conjugated to the polypeptide. Method.
(54) The method according to (46), wherein the subject is a human.
The following examples are intended to illustrate the invention and are not intended to limit the invention.

Conjugate distribution and the effect of conjugating a pharmacokinetic agent (drug) to a vector on the distribution of the substance, administration of a labeled polypeptide or conjugate to an animal and distribution of the polypeptide or conjugate to an organ It can be evaluated by measuring. In one example, mice are administered ³ H-taxol (Taxol) (5 mg / kg) or ¹²⁵ I-Taxol-Angiopep-1 (TxlAn-1) (equivalent to 10 mg / kg, 5 mg Taxol / kg). Similar experiments can be performed using any of Angiopep-3, Angiopep-4a, Angiopep-4b, Angiopep-5, Angiopep-6, and Angiopep-7. Here, unconjugated anticancer agents and conjugates are injected intravenously into mice as a bolus. Tissues are collected and homogenized at various time points (0.25, 0.5, 1, and 4 hours). To quantify the amount of ³ H-Taxol, the tissue homogenate was digested with a tissue solubilizer and 10 ml of liquid scintillator was added to the sample. The amount of ¹²⁵ I labeled conjugate in the various tissues is measured after TCA precipitation. Quantify the radioactivity associated with the tissue. The area under the curve (AUC 0-4) is estimated using Prism software and plotted for various tissues. With the Angiopep-1 conjugate, the AUC 0-4 values obtained for the conjugate are higher than Taxol in various tissues including brain, kidney, liver, and eye (FIG. 10A). This indicates a high conjugate accumulation in these tissues compared to unconjugated material. In the lung, conjugate accumulation is much higher than unconjugated material (FIG. 10B).

The results of similar experiments performed with Taxol-Angiopep-2 conjugate are summarized in Table 4 below. Although different from the results obtained for the TxlAn-1 conjugate, the conjugates in Table 4 also accumulate more efficiently in the lungs, brain, and liver than the unconjugated Taxol.

  The kinetics of Taxol and Taxol-Angiopep-1 accumulation in the lung were studied (FIG. 11). The amount of conjugate measured in the lung at various time points is much higher than the unconjugated material. Conjugate accumulation in the lung is also much higher than its concentration in serum (plasma) at various time points (FIG. 12). These results indicate that the biodistribution or pharmacokinetics of anticancer agents such as Taxol can be altered by conjugation to the vectors of the present invention (eg Angiopep-1 or 2).

Angiopep-7 is not efficiently transported to the brain
Angiopep-7 is not efficiently transported across the blood brain barrier. As measured by the accumulation of Angiopep-2 and Angiopep-7 in the rat brain 30 minutes after IV injection, Angiopep-7 was only present in the brain at a lower level than Angiopep-1 or Angiopep-2 (FIG. 13). ).

  We also investigated the accumulation of Angiopep-2 and Angiopep-7 in the brain using fluorescence microscopy after 10 minutes of in situ cerebral perfusion. Here, 2 mM Angiopep-2 or Angiopep-7 conjugated to Cy5.5 fluorescent marker, respectively, was injected into the carotid artery. The animal brain was then evaluated by fluorescence microcopy. In each case, capillaries were stained with Vessel Green (FITC-lectin), brain cell nuclei were stained with DAPI (blue), and Angiopep was visualized using Cy5.5 labeling (FIG. 14). From these sections, it is observed that Angiopep-2 is localized in the brain and Angiopep-7 is localized in the capillary. Low brain accumulation of Angiopep-7 compared to Angiopep-2 is also observed in vivo. Each of Angiopep-2 and Angiopep-7 was conjugated to Cy5.5 and administered to rats. After 30 minutes, accumulation in the brain was measured using fluorescence of Cy5.5 indicator. Based on this, the inventors conclude that Angiopep-7 is not efficiently transported across the BBB, while Angiopep-2 is efficiently transported across the BBB.

Angiopep-7 is efficiently transported to the liver, kidneys, lungs, spleen, and muscles Use rats administered with Angiopep-2 or Angiopep-7 conjugated to Cy5.5 as described in Example 2 Then, levels of Angiopep-7 and Angiopep-2 were measured in organs such as liver, kidney, and lung. In these organs, similar levels of polypeptide were observed when using Angiopep-7 compared to Angiopep-2. In view of these results, we believe that Angiopep-7 is particularly useful for delivering substances to the liver, kidneys, and lungs in applications where delivery to the brain is undesirable.

  In order to determine the concentration of Angiopep-7 in animals, an imaging study of Angiopep-7 conjugated to Cy5.5 was performed. Based on an in vivo imaging study 30 minutes after injection, accumulation of Cy5.5 signal was observed in the kidney, liver, and lung (FIG. 15A). Ex vivo organ analysis was performed 24 hours after Angiopep-7 injection and compared to injection using Angiopep-2. These studies also showed significant accumulation of both Angiopep-2 and Angiopep-7 in the liver, lung, and kidney. However, Angiopep-7 showed significantly less accumulation in the brain compared to Angiopep-2 (FIG. 15B). Based on these results, we have found that Angiopep-7 is useful as a vector of material into peripheral organs such as liver, kidney, lung, spleen, or muscle in applications where delivery to the brain is undesirable I believe that.

Transport of IgG conjugates
In situ brain perfusion experimental model was used to measure the uptake of the vector in the brain. The results (shown in Table 5 below) showed that the 10th and 15th lysines are important for the ability of the vector to cross the BBB.

  Angiopep polypeptide was conjugated to IgG using SATA crosslinker and its transport ability was studied using in situ brain perfusion. As shown in FIG. 16, Angiopep-2 is a conjugate that is most distributed in the brain parenchyma. Polypeptide dimers also appear to be efficiently transported. Angiopep-7 was not efficiently transported into the brain.

Effect of conjugates on in vitro cell growth Testing of the ability of conjugates, including vectors and anticancer agents, to kill cancer cells can be performed in vitro. In one example, Taxol (unconjugated) is shown to block the growth of gliobiastoma cells (U-87) with an IC50 value of about 10 nM (FIG. 17). The effect of conjugated Taxol when conjugated to the vectors described herein is evaluated and compared to unconjugated Taxol. As shown in Table 6A, the IC50 values obtained for the Taxol-Angiopep-2 (TxlAn2) conjugate were very similar to unconjugated Taxol in many cancer cells. Rat brain-derived endothelial cells (RBE4) were less sensitive than the cancer cell lines tested. For comparison, the obtained results were expressed in terms of Taxol concentration.

Most said cells (U-87, U-118, NC1-H460, A549) express LRP. However, this data is not available for RBE4 cells. Assess the anti-proliferative activity of the conjugate against cancer cells in vitro. In this assay, cancer cells (U87 and U118) are exposed to anticancer agents (eg Taxol) and conjugates (eg TxlAn2 (3: 1) conjugate) for 48 hours. [ ³ H] -thymidine incorporation in U87 and U118 cells decreases with substance concentration. The value (1C50) required to inhibit cell growth by 50% is indicated. The results obtained from the proliferation assay demonstrate that the IC50 value required for inhibition of cancer cell growth is expressed in nM units and that the TxlAn2 (3: 1) conjugate is 3 times more potent than paclitaxel, It is shown to be within the same range when reported for paclitaxel equivalent (Table 6B). Similar experiments can be performed using any of the vectors or conjugates described herein.

We also estimate the ability of conjugates (eg TxlAn2 (3: 1)) to block the growth of other cancer cell types. Such assays include lung cancer cells (NC1-H460) and breast cancer cell lines (MDA-MB231, MDA-MB-468, HCC-1954, BT-474) hepatocellular carcinoma (SK-Hepl) and glioblastoma ( U-87MG). We have shown that such cells are also very sensitive to TxlAn2 (3: 1) conjugates (Table 6B).

In vivo tumor growth inhibition (U-87)
The ability of the conjugate to inhibit tumor growth can be assessed in an in vivo model (see, eg, FIG. 18A). U-87 cells are implanted subcutaneously into the right flank of mice and 3 days after transplantation, vehicle (DMSO / Ringer: 80/20; control), unconjugated substance (e.g. Taxol (5 mg / kg)) or conjugate Mice are injected with the substance as part of the gate (eg Taxol-Angiopep-2 (10 mg / kg) equivalent to 5 mg Taxol / kg). Inhibition of tumor growth is more pronounced in mice treated with the conjugate than in mice treated with unconjugated anticancer agents.

In one example, TxlAn2 was used to inhibit tumor growth by more than 75% 17 days after transplantation, whereas using unconjugated material only inhibited tumor growth by 34% (Table 7). Therefore, conjugates are more efficient than unconjugated materials with respect to inhibiting tumor growth in vivo. Ultimately, 2.2 times the tumor growth inhibition level was measured for the conjugate compared to the unconjugated material.

In vivo tumor growth inhibition (hepatocellular carcinoma)
An in vivo study is performed to determine if the conjugate can inhibit the growth of subcutaneously transplanted hepatocellular carcinoma cells (SK-Hep 1). A subcutaneous injection of 2.5 × 10 ⁶ human SK-Hep 1 cells is given to the right flank of nude mice. Treatment begins when the size of the transplanted tumor reaches approximately 200 mm ³ . Animals are treated with conjugate or vehicle by intraperitoneal injection.

  Results obtained from experiments performed with TxlAn2 (3: 1) administered at 80 mg / kg by intraperitoneal injection are shown in FIG. 18B; treatments are indicated by black arrows. In this experiment, treatments were given twice weekly for 5 treatments at a designated dose of up to 80 mg / kg. The i.p. administered TxlAn2 (3: 1) conjugate shows high potency in inhibiting the growth of hepatocellular carcinoma, a type of cancer that is usually not sensitive to Taxol® (FIG. 18B).

Method for Assessing Conjugate Activity In the examples described herein, the following method was used.

Cell proliferation assay
For in vitro cell proliferation assays, U87 or A549 cells ranging from 2.5 to 5 x 10 ⁴ are seeded in a final volume of 1 ml medium containing 10% serum in a 24-well tissue culture microplate, and 5% CO at 37 ° C. Incubate at ₂ for 24 hours. The medium is then replaced with serum-free medium and incubated overnight. The next morning, the material is freshly dissolved in dimethyl sulfoxide (DMSO) and the medium is changed to a complete medium containing various concentrations of the substance in duplicate. The final concentration of DMSO is 0.1%. The control used is a microplate well containing cells but no material. Incubate cells for 48-72 hours at 37 ° C. with 5% CO ₂ . After incubation, the medium is changed and replaced with 1 ml complete medium containing [ ³ H] -thymidine (1 pCi / assay). Incubate the plate at 37 ° C. with 5% CO ₂ for 4 hours. Remove the medium and wash the cells with PBS at 37 ° C. Cells are fixed with a mixture of ethanol: acetic acid (3: 1), washed with water and precipitated three times with 10% ice-cold TCA (trichloroacetic acid). Finally, 500 μl of PCA (perchloric acid) is added to the wells and the microplate is heated at 65 ° C. for 30 minutes and 75 ° C. for 30 minutes. Then, the contents are transferred to each well into a scintillation vial containing 10 ml of the scintillation reaction mixture, and the activity is measured in units of CPM (count / min) with a Packard liquid scintillation counter Tri-Carb.

Polypeptide iodination
Polypeptides are iodinated using standard procedures using iodo-beads from Sigma. Briefly, the polypeptide is diluted in 0.1 M phosphate buffer (pH 6.5) (PB). Use two iodo-beads for each protein. These beads are washed twice with 3 ml PB on Whatman filters and resuspended in 60 μl PB. ¹²⁵ I (1 mCi) from Amersham-Pharmacia biotech was added to the bead suspension for 5 minutes at room temperature. Each iodination was initiated by the addition of polypeptide (100 μg). After 10 minutes incubation at room temperature, free iodine was removed by HPLC.

In order to estimate the efficacy of the conjugates and formulations against subcutaneous transplant tumor growth, we developed a subcutaneous model of glioblastoma. In this model, 2.5 × 10 ⁶ cells in 100 μl cell medium without serum containing 1% methylcellulose are injected subcutaneously into the flank of mice. The tumor is clearly visible and can be measured using a vernier caliper. The estimated tumor volume was then plotted as a function of time.

In situ mouse brain perfusion [ ¹²⁵ I] -poly to the luminal side of mouse brain capillaries using the in situ brain perfusion method constructed in our laboratory to study substance uptake in the mouse brain Peptide uptake is measured. Briefly, the right common carotid artery of anesthetized mice with ketamine / xylazine (140/8 mg / kg ip) is exposed and ligated at the level of the common carotid branch on the rostral side of the occipital artery. A catheter is then inserted into the rostral side of the common carotid artery using a polyethylene tube filled with heparin (25 U / ml) and mounted on a 26 gauge needle. Inject a syringe containing perfusate ([ ¹²⁵ I] -polypeptide or [ ¹⁴ C] -inulin in pH 7.4 Krebs / bicarbonate buffer gasified with 95% O ₂ and 5% CO ₂ ) Place in pump (Harvard pump PHD 2000; Harvard Apparatus) and connect to catheter. Prior to perfusion, the contralateral blood flow contribution is eliminated by disrupting the ventricles. Perfuse the brain for a specified time at a flow rate of 1.15 ml / min. After 14.5 minutes of perfusion, the brain is further perfused with Krebs buffer for 60 seconds to wash excess [ ¹²⁵ I] -protein. The head of the mouse is then decapitated to terminate perfusion and the right hemisphere is isolated on ice before subjecting to capillary depletion. Aliquots of homogenate, supernatant, pellet and perfusate are collected and their [ ¹²⁵ I] -conjugate content measured by TCA precipitation to assess the apparent volume of distribution.

Mechanisms of action of conjugates Studies of the mechanism of action of conjugated therapeutic agents can also be performed. In one example, lung cancer cells (NCI-H460) are incubated with free Taxol (30 nM) or a Taxol conjugate (eg, TxlAn2 10 nM; equivalent to 30 nM taxol) for 24 hours (FIG. 19). After labeling the cell β-tubulin using a secondary antibody linked to FITC, photographs are taken in a visible and fluorescent manner. In this example, both Taxol and Taxol conjugates have a similar effect on β-tubulin leading to polymerization. As illustrated in FIG. 20, addition of Taxol and Taxol conjugate induces blocking of NCI-H460 cells at G2 / M phase. These results suggest that the TxlAn conjugate has a mechanism of action on cancer cells similar to Taxol.

Conjugate Preparation Conjugation of substances to the vectors described herein is illustrated in FIGS. 23A-23B. In this example, Taxol is first activated to an N-succinimide (2′-NHS-Txl) derivative. For example, amines found in vectors (eg, amino terminal amine or lysine residues) are reacted with activated Taxol to form peptide bonds (amide bonds). If multiple amines are available, this reaction will give rise to multiple combinations of conjugates in which 1, 2 or 3 Taxols have been added to the polypeptide, for example depending on the molar ratio used. The conjugation product is analyzed by HPLC and the conjugation is confirmed by mass spectrometry (Maldi-Tof). Taxol is found to be releasable from the vector by cleavage of the ester bond with esterase.

  The method can be used to conjugate any of the vectors described herein to a substance. Preparation of TxlAn2 (3: 1) conjugate was performed by adding 1 molar equivalent of Angiopep-2 directly to a solution of 2.5 molar equivalents of 2′-NHS-Taxol. The reaction was performed in 68% DMSO in Ringer's solution (pH 7.3) for 1 hour at 12 ° C. After removing the cold bath, the reaction was allowed to continue for approximately 22 hours at room temperature (FIG. 24). Angiopep-2, 2'-NHS-Taxol and TxlAn2 (3: 1) conjugates are indicated by arrows on the chromatogram. Aliquots of the reaction were taken and analyzed by HPLC after 25 minutes, 2 hours 15 minutes, 5 hours and 23 hours as shown in FIG. The peaks of Angiopep-2, 2'-NHS-Taxol and TxlAn2 (3: 1) conjugate are indicated by arrows on the chromatogram. The results in FIG. 24 show the disappearance of Angiopep-2 and 2′-NHS-Taxol during the reaction, mainly versus the benefit of the TxlAn2 (3: 1) conjugate.

  This product mixture was separated by hydrophobic chromatography on an RPC 300 mm column using an AKTA-explorer at a flow rate of 4 ml / min (FIG. 25). Fractions were pooled and analyzed by HPLC and MS for the peak corresponding to the TxlAn2 (3: 1) conjugate. In FIG. 25, the upper chromatogram corresponds to the effluent reactant at t = 23 hours, and the lower chromatogram is the TxlAn2 (3: 1) conjugate confirmed by mass spectrometry after AKTA purification (MW 5107). Corresponding to

Antibody Conjugation and Uptake Antibodies can be conjugated to aprotinin as described in US Patent Publication No. 2006/0189515. Similar results can be obtained using any of the vectors described herein.

  We therefore conjugated Angiopep-2 to IgG using SATA. After conjugation, approximately 3-5 molecules of Angiopep-2 bind to both the light and heavy chains of IgG (FIG. 26). Thus, using this approach, 2-6 molecules of vector (eg, Angiopep-1, Angiopep-2) are expected to be conjugated to each molecule (heavy and light chain) of the antibody.

Also, as shown in FIG. 4, conjugation of Angiopep-2 using sulfo-EMCS was performed. The brain distribution of IgG-Angiopep-2 conjugates coupled via sulfhydryl groups using EMCS-Angiopep-2 was examined in various brain tissues (whole brain, capillaries, parenchyma). Uptake in the brain of [ ¹²⁵ I] -IgG-Angiopep-2 conjugate is observed higher than unconjugated [ ¹²⁵ I] -IgG (approximately 3 fold difference) (see FIG. 27). Thus, conjugation of IgG and Angiopep-2 increases IgG accumulation in the brain in vivo.

  In addition, transport of Angiopep-2-IgG conjugates coupled using the SATA method was examined using in situ brain perfusion experiments. IgG-An2 conjugate is transported into brain tissue (Figure 28). The IgG-Angiopep-2 conjugate accumulated in the parenchyma approximately 50 times more than IgG.

After IgG and vector conjugation, SDS-polyacrylamide gel electrophoresis, immunodetection, and autoradiography were performed to ensure proper conjugation; for example, only [ ¹²⁵ I] -IgG-Angiopep conjugate was detected See FIG. There was no evidence of free Angiopep-2 conjugate. This indicates the efficiency of the conjugation approach.

Coupling of anti-EGFR antibodies to vectors via epidermal growth factor receptor induces cell proliferation signals and is associated with transformation from normal cells to malignant cells. Several mutations in EGFR are detected in tumor cells. One of the most common mutations in EGFR is an EGFRvIII mutation that lacks amino acids 6-273.

  To demonstrate that the vectors of the present invention can be coupled to antibodies other than IgG and that the coupling maintains antibody function, the cross-linking agent SATA is used as a typical therapeutic polypeptide. EGFR antibody (monoclonal 528 available from ATCC) was cross-linked to the vector. The biological activity of the coupled EGFR antibody was examined by staining EGFR positive U87 cells. FACS analysis demonstrated similar detection of EGFR in U87 using both uncoupled and coupled EGFR antibodies (Figure 30). This indicates that the antibody coupling of the vector of the present invention did not change its activity.

In Table 8, we show that the antibodies are not inactivated by conjugation and show the same binding affinity in in vitro assays.

The labeled conjugate was then used to measure the transport of EGFR antibody conjugates (eg anti-EGFR-Angiopep-2) across the BBB. As shown in FIG. 31, uptake of anti- [ ¹²⁵ I] -EGFR-Angiopep-2 into the luminal side of mouse brain capillaries was measured using in situ brain perfusion. In all test tissues, higher uptake of [ ¹²⁵ I] -EGFR-Angiopep-2 conjugate in the brain than unconjugated [ ¹²⁵ I] -EGFR Ab was observed. Thus, conjugation of EGFR Ab and Angiopep-2 increases its accumulation in the brain parenchyma in vivo. These conjugates are an interesting tool because several different tumor types have been shown to express EGFR at various levels (see Table 9).

Coupling and transporting anti-VEGF antibodies to vectors To further demonstrate the versatility and applicability of coupling antibodies to the vectors of the invention, another exemplary therapeutic antibody, Avastin, is used. used. Avastin is an anti-VEGF recombinant humanized monoclonal IgG1 kappa isotype antibody available from Roche Biochemical, which binds to and inhibits a bioactive form of vascular endothelial growth factor (VEGF).

Following conjugation, transport of Avastin conjugates across the BBB was measured using in situ brain perfusion. Unconjugated ^[125 I] above ^{-Avastin [125 I] -Avastin-Angiopep} -2 uptake in the brain of the conjugates is observed (Figure 32).

  Thus, conjugation of therapeutic antibodies (e.g. MAb 528 available from Avastin or ATCC) to vectors (e.g. Angiopep-1, -2, -3, -4a, -4b, 5, and 6) Is a useful strategy for transporting blood into the brain.

Dimer transport vector dimers can also be transported. In one example, Angiopep-1 was incubated at 4 ° C. for at least 12 hours, thereby causing polypeptide multimerization / dimerization. The ability of Angiopep-1 dimer to transcellularly was evaluated in vitro. Angiopep-1 dimers are better transcellularly transported than IgG. Furthermore, as shown in Table 5, the dimeric form of Angiopep-1 exhibits a higher distribution volume than Angiopep-1. Hence, vector dimers can be transported across the BBB.

Conjugation of antibodies to two or more vectors Typically, the cross-linking reaction yields 1-6 molecules of vector per antibody, heavy chain and light chain.

  Angiopep-2 to IgG after deprotection of SATA on the amine of IgG, using an assay that allows titration of the amount of sulfhydryl groups before and after reaction with the maleimide group on the N-terminus of Angiopep-2 The level of conjugation of was estimated. By varying the relative concentrations of the various reactants used in the conjugation protocol, the amount of Angiopep-2 conjugated to IgG can be optimized.

  Using in vivo brain perfusion experiments, the higher the level of conjugation, the higher the conjugation uptake by the conjugate (Figure 33). It is therefore desirable to have a large number of vectors per molecule. As such, seven or more vectors can be used to transport a compound (substance) across the BBB.

Other Embodiments The contents of each publication, patent, and patent application described in this application are incorporated by reference. The publications, patents, and patent applications include US Application No. 11 / 807,597, filed May 29, 2007, and 11 / 807,917, filed May 30, 2007, and December 20, 2007. Includes filed US Provisional Application No. 61 / 008,825.

  While the invention has been described in detail herein and illustrated in the accompanying drawings, the invention is not limited to the embodiments described herein and departs from the scope or spirit of the invention. It should be understood that various changes and modifications can be made.

  Although the invention has been described with reference to specific embodiments thereof, it will be understood that it is capable of additional modifications, and the application will generally follow the principles of the invention, but the invention is of relevance. Any of the inventions, including deviations from the present disclosure, that fall within the scope of known or customary practice within the technical field and can be applied to essential features as described above and in the claims as follows: Variations, uses, or adaptations.

Claims (20)

A polypeptide comprising the amino acid sequence TFFYGGSRGRRNNFRTEEY,
The polypeptide is transported to at least one cell or tissue selected from the group consisting of liver, lung, kidney, spleen and muscle, but more efficiently than the blood brain barrier than Angiopep-6 (SEQ ID NO: 111). Not transported across (BBB),
Said polypeptide. The polypeptide according to claim 1, which consists of the amino acid sequence TFFYGGSRGRRNNFRTEEY .   A composition comprising the polypeptide of claim 1. 4. The composition of claim 3 , comprising a pharmaceutically acceptable carrier. (a) a vector comprising the amino acid sequence TFFYGGSRGRRNNFRTEEY , said vector being transported to at least one cell or tissue selected from the group consisting of liver, lung, kidney, spleen and muscle, wherein Angiopep-6 (sequence The vector, which is not transported across the blood brain barrier (BBB) more efficiently than the number 111), and
(b) a therapeutic agent, diagnostic agent or detectable label conjugated to the vector,
Conjugates comprising 6. A conjugate according to claim 5 , wherein the vector consists of the amino acid sequence TFFYGGSRGRRNNFRTEEY . 6. The conjugate of claim 5 , wherein the vector is conjugated to a therapeutic agent. 6. The conjugate according to claim 5 , wherein the therapeutic agent is an anticancer agent. The anticancer drug is abarelix, aldesleukin, alemtuzumab, alitretinoin, allopurinol, altretamine, amifostine, anakinra, anastrozole, arsenic trioxide, asparaginase, azacitidine, raw BCG, bevacuzimab, bexarotene, bortezo ombi ), Bortezomib, busulfan, calusterone, capecitabine, carboplatin, carmustine, celecoxib, cetuximab, chlorambucil, cisplatin, cladribine, clofarabine, cyclophosphamide, cytarabine, dacarbazine, dactinomycin, actinomycin D, darteparin Poetin alfa, dasatinib, daunorubicin, daunomycin, decitabine, denileukine, denililoy Diftitox, dexrazoxane, docetaxel, doxorubicin, drmostanolone propionate, eculizumab, epirubicin, epoetin alfa, erlotinib, estramustine, etoposide (e.g. phosphate), exemestane, fentanyl, filgrastim, floxuridine, fludrasil, fludavir -FU, fulvestrant, gefitinib, gemcitabine, gemtuzumab ozogamicin, goserelin, histrelin, hydroxyurea, ibritumomab tiuxetane, idarubicin, ifosfamide, imatinib, interferon alfa-2b, irinotecan, lapatinib ditosylate, lenalidomizole , Leucovorin, leuprolide, levamisole, lomustine, CCNU, meclorethamine (mecloreth amine) (nitrogen mustard), megestrol, melphalan (L-PAM), mercaptopurine (6-MP), mesna, methotrexate, methoxalene, mitomycin C, mitotane, mitoxantrone, nandrolone phenpropionate, nelarabine , Nofetumomab, oprelbequin, oxaliplatin, paclitaxel, paliferum, pamidronate, panitumumab, pegademase, pegaspargase, pegfilgrastim, peginterferon alfa-2b, pemetrexed, pentostatin, pipobromantramycin , Porfimer, procarbazine, quinacrine, rasburicase, rituximab, sargramostim, sorafenib, streptozocin, sunitinib, sunitinib maleate, talc, Tamoxifen, temozolomide, teniposide (VM-26), test lactone, thalidomide, thioguanine (6-TG), thiotepa, topotecan, toremifene, tositumomab / I-131 (tositumomab), trastuzumab, tretinoin (ATRA), uracil mustard, valrubicin 9. The conjugate of claim 8 , wherein the conjugate is selected from the group consisting of vinblastine, vincristine, vinorelbine, vorinostat, zoledronic acid, and zoledronic acid. 10. The conjugate according to claim 9 , wherein the anticancer agent is paclitaxel. 9. The conjugate according to claim 8 , wherein the anticancer agent is an antibody. Said antibody is ABX-EGF (Panitimumab), OvaRex (Oregovemab), Theragyn (pemtumomabytrrium-90), Therex, Bivatuzumab, Pronorma, (Leopro) (Abciximab), Bexxar (Tositumomab), MAb, Idiotype 105AD7, Anti-EpCAM (Katsumakisomab), MAb lung cancer (Cytoclonal), Herceptin (Herceptin) (Trastuzumab), Rituxan (Rituxan) (Rituxan) (Rituxan) , Avastin (bevacizumab), AMD Fab (ranibizumab), E-26 (2 ^nd gen. IgE) (Omarizumab), Zevalin (Rituxan + Yttrium-90) (ibritumomab tiuxetane), cetuximab, BEC2 (Mitumomab), IMC-1C11, nuC242-DM1, LymphoCide (Epratuzumab), LymphoCide Y-90, CEA-Cide, CEA-Cide Y-90, HumaRAD-HN (+ Yttrium- 90), MDX-10 1 (CTLA-4), MDX-210, MDX-210 / MAK, Vitaxin, MAb 425, IS-IL-2, Campath (campus) (alemtuzumab), CD20 streptavidin, Avidicin, (albumin + NRLU13), Oncolym ( + Iodine-131) Cotara (+ iodine-131), C215 (+ staphylococcal enterotoxin), MAb lung / kidney cancer (Pharmacia Corp.), nacolomab tafenatox (C242 staphylococcal enterotoxin), Nuvion ( Selected from the group consisting of (Nuvion) (Vigilizumab), SMART M195, SMART 1D10, CEAVac, TriGem, TriAb, radiolabeled NovoMAb-G2, Monopharm C, GlioMAb-H (+ gelonin toxin), Rituxan (Rituximab), and ING-1 12. The conjugate according to claim 11, wherein P-gp-mediated accumulation in liver, lung, kidney or spleen cells, which accumulates rapidly, accumulates at a high concentration, and is reduced compared to the therapeutic or diagnostic agent or label when not conjugated to the vector 6. A conjugate according to claim 5 exhibiting sexual efflux. A composition comprising the conjugate of claim 5 and a pharmaceutically acceptable carrier. 9. A conjugate according to claim 8 for use in a method of treating a subject having cancer. 16. The conjugate according to claim 15 , wherein the cancer is lung, liver, kidney or spleen cancer. The cancer is hepatocellular carcinoma, breast cancer, head and neck cancer, lymphoma, mantle cell lymphoma, non-Hodgkin lymphoma, adenoma, squamous cell carcinoma, laryngeal cancer, retinal cancer, esophageal cancer, multiple myeloma, Ovarian cancer, uterine cancer, melanoma, colorectal cancer, bladder cancer, prostate cancer, lung cancer, non-small cell lung cancer, pancreatic cancer, cervical cancer, head and neck cancer, skin cancer, nasopharyngeal cancer, liposarcoma, epithelium 16. The conjugate of claim 15 , selected from the group consisting of cancer, renal cell carcinoma, gallbladder adenocarcinoma, parotid gland cancer, endometrial sarcoma and multidrug resistant cancer. 16. The conjugate according to claim 15 , wherein the conjugate comprises a vector that is efficiently transported into at least one cell or tissue selected from the group consisting of liver, lung, kidney, spleen or muscle. When said anticancer agent is conjugated to said vector, as compared to the anticancer agent when the said vector is not conjugated, having increased potency or reduced side effects, a conjugate of claim 15, wherein. 16. A conjugate according to claim 15 , wherein the subject is a human.

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