ES2488668T3 - Aprotinin-like polypeptides for administering agents conjugated therewith to tissues - Google Patents

Abstract

Polypeptide comprising an amino acid sequence having 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 are absent; at least one of X10 and X15 is Arg; and said amino acid sequence is at least 80% identical to Angiopep-7, which has the amino acid sequence of SEQ ID NO: 112; wherein said polypeptide is efficiently transported to at least one cell or tissue selected from the group consisting of liver, lung, and kidney, and wherein said polypeptide is transported across the blood-brain barrier at levels below Angiopep- 6, which has the amino acid sequence of SEQ ID NO: 111.

Description

Aprotinin-like polypeptides for administering agents conjugated therewith to tissues

5 Background of the invention

The invention relates to improvements in the field of drug administration. More particularly, the invention relates to polypeptides, conjugates and pharmaceutical compositions that include the polypeptides of the invention and their use to transport agents (eg, therapeutic agents) through particular cell types such as liver, lung or kidney.

Liver diseases, including hepatitis (for example, viral hepatitis) and liver cancers (for example, hepatocarcinoma), and lung diseases, such as lung cancer (for example, small and non-small cell lung cancer) are problems of serious health. Many therapeutic agents for such

15 diseases have unwanted side effects (for example, chemotherapeutic agents) or, for reasons such as in vivo stability, transport or other pharmacokinetic properties, are difficult to provide at a sufficiently high concentration in the target tissue or for a sufficiently long duration for allow a maximum therapeutic effect on the target tissue.

Therefore, there is a need for methods and compositions that increase concentrations of therapeutic and diagnostic agents in target tissues or organs.

WO 2007/009229 A1 discloses polypeptides that are captured by specific organs and, when conjugated with an agent, can deliver said agent to the organs.

WO 2006/086870 A1 discloses polypeptides that are captured by the brain and, when conjugated with an agent, can increase the transport of the agent through the blood brain barrier.

WO 2004/060403 A2 discloses a polypeptide that, when conjugated with an agent, enhances the transport of the agent through the blood brain barrier.

Summary of the invention

It has been found that the polypeptides described herein (eg, Angiopep-7; SEQ ID

35 NO: 112) are efficiently transported into particular cell types (for example, liver, lung, spleen, kidney and muscle), but are not transported effectively through the blood brain barrier (BHE). When conjugated with an agent, the polypeptides act as vectors and can increase the concentration of the conjugated agent in the cell. Angiopep-3, Angiopep-4a, Angiopep-4b, Angiopep-5 and Angiopep-6 (SEQ ID NOS: 107-111) have also been identified as vectors that can be efficiently transported through BHE and can be further transported to interior of particular cell types. Therefore, the invention features polypeptides, conjugates that include polypeptides as vectors, and methods for diagnosing and treating diseases (eg, cancer) with such polypeptides and conjugates.

In a first aspect, the present invention relates to a polypeptide comprising a 45 amino acid sequence having the following formula:

X1-X2-X3-X4-X5-X6-X7-X8-X9-X10-X11-X12-X13-X14-X15-X16-X17-X18-X19

in which X1-X19 are any amino acid or are absent;

at least one of X10 and X15 is Arg; Y

said amino acid sequence has at least 80% identity with Angiopep-7, which has the amino acid sequence of SEQ ID NO: 112, wherein said polypeptide is efficiently transported to at least one cell

55 or tissue selected from the group consisting of liver, lung and kidney, and in which said polypeptide is transported through the blood brain barrier to levels below Angiopep-6, which has the amino acid sequence of SEQ ID NO: 111 .

Accordingly, the invention features a polypeptide that includes 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 (for example, X1-X6, X8, X9, X11-X14 and X16-X19) is, independently, any

Amino acid (for example, a naturally occurring amino acid such as Ala, Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr and Val) or is absent and at least one of X1, X10 and X15

It is arginine. In some embodiments, X7 is Ser or Cys; or X10 and X15 are each independently Arg or Lys. In some embodiments, residues from X1 to X19, inclusive, are substantially identical to the amino acid sequence SEQ ID NO: 112 (for example, Angiopep-7). In additional examples disclosed herein, the residues from X1 to X19, inclusive, are substantially identical to any of the

5 amino acid sequences of any one of SEQ ID NOS: 1-105 and 107-111 (for example, Angiopep-1, Angiopep-2, Angiopep-3, Angiopep-4a, Angiopep-4b, Angiopep-5 and Angiopep-6 ). In some embodiments, at least one (for example, 2, 3, 4 or 5) of amino acids X1-X19 is Arg (for example, any one, two or three of X1, X10 and X15).

In other embodiments, the invention features a polypeptide that includes an amino acid sequence that has substantial identity with SEQ ID NO: 112 (eg, Angiopep-7). A polypeptide is also disclosed which includes an amino acid sequence that has substantial identity with any one of SEQ ID NOS: 1-105 and 107-111 (for example, Angiopep-3, Angiopep-4a, Angiopep-4b, Angiopep- 5 and Angiopep-6). In certain embodiments, the polypeptide may include or may be Angiopep-3, Angiopep-4a, Angiopep-4b, Angiopep-5,

Angiopep-6 or Angiopep-7 (SEQ ID NOS: 107-112), may include a sequence substantially identical to the Angiopep-7 sequence or a fragment thereof (for example, a functional fragment). The polypeptide may have an amino acid sequence of 10 to 50 amino acids, for example, 10 to 30 amino acids, in length. The invention also features functional derivatives (for example, chemical derivatives or variants) of these polypeptides.

Exemplary polypeptides of the invention have a lysine or arginine at position 10 (with respect to the amino acid sequence of SEQ ID NO: 1) or a lysine or arginine at position 15 (with respect to the amino acid sequence of SEQ ID NO: 1), or a lysine or arginine in both position 10 and position 15. The polypeptides of the invention may also have a serine or cysteine in position 7 (with respect to

25 amino acid sequence of SEQ ID NO: 1). When multimerization of polypeptides is desired, the polypeptide may include a cysteine (for example, at position 7).

In certain embodiments, the polypeptides of the invention (for example, any polypeptide described herein) are modified (for example, as described herein). The polypeptide can be amidated, acetylated, or both. For example, a polypeptide that includes Angiopep-6 or Angiopep-7 can be amidated, or SEQ ID NO: 67 can be amidated (polypeptide # 67). In another example, the amino acid of any one of SEQ ID NOS: 107-112, or any other polypeptide of the invention, is amide or acetylated. Such modifications in polypeptides of the invention can be at the amino or carboxyl-terminal end of said polypeptide. The invention also features peptidomimetics (for example, those described herein) of any of the

35 polypeptides described herein. The polypeptides of the invention may be in multimeric form. For example, the polypeptides may be in dimeric form (for example, formed by disulfide bond through cysteine residues).

The polypeptides of the invention can be efficiently transported into particular cells (for example, liver, kidney, lung, muscle or spleen cells) or can effectively cross the BHE (for example, Angiopep-3, Angiopep-4a , Angiopep-4b, Angiopep-5 and Angiopep-6). In some embodiments, the polypeptides are efficiently transported into particular cells (for example, liver, kidney, lung, muscle or spleen cells) and are not transported effectively through the BHE (e.g., Angiopep- 7). The polypeptide can be efficiently transported into at least one (for example, at least

45 two, three, four or five) of a cell or tissue selected from the group consisting of liver, kidney, lung, muscle or spleen.

For any of the polypeptides and conjugates described herein, the amino acid sequence may specifically exclude a polypeptide that includes or consists of any of SEQ ID NOS: 1-105 and 107-112 (for example, any of SEQ ID NOS: 1-96, Angiopep-1, Angiopep-2, Angiopep-3, Angiopep-4a, Angiopep-4b, Angiopep-5, Angiopep-6 and Angiopep-7). In some embodiments, the polypeptides and conjugates of the invention exclude the polypeptides of SEQ ID NOS: 102, 103, 104 and 105. In some embodiments, the polypeptides and conjugates of the invention include these polypeptides.

In certain embodiments, a polypeptide of the invention may have an amino acid sequence described herein with at least one amino acid substitution (eg, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 substitutions). The polypeptide may have an arginine in one, two or three of the positions corresponding to positions 1, 10 and 15 of the amino acid sequence of any of SEQ ID NO: 1, Angiopep-1, Angiopep-2, Angiopep-3, Angiopep-4a, Angiopep-4b, Angiopep-5, Angiopep-6 and Angiopep-7. For example, the polypeptide may contain 1 to 12 amino acid substitutions (for example, SEQ ID NO: 91). For example, the amino acid sequence may contain 1 to 10 (for example, 9, 8, 7, 6, 5, 4, 3, 2) amino acid substitutions or 1 to 5 amino acid substitutions. According to the invention, the amino acid substitution may be a conservative or non-conservative amino acid substitution.

The polypeptides of the invention can be chemically synthesized (eg, solid phase synthesis) or can be produced by recombinant DNA technology, as is known in the art. Any of the

Polypeptides, compositions or conjugates described herein may be in an isolated form or in a substantially purified form.

A polynucleotide sequence encoding a polypeptide of the invention is also disclosed (by

For example, any polypeptide described herein, such as Angiopep-3, Angiopep-4a, Angiopep-4b, Angiopep-5, Angiopep-6 and Angiopep-7). More particularly, nucleotide sequences (deoxyribonucleotides, ribonucleotides, or derivatives thereof) encoding a polypeptide selected from the group consisting of any one of SEQ ID NOS: 1-97 and SEQ ID NO: 107-112 are disclosed . A desired nucleotide sequence can be chemically synthesized by methods known in the art.

In another aspect, the invention features a conjugate that includes a vector selected from the group consisting of any one of the polypeptides, analogs or derivatives thereof described herein (eg, Angiopep-7), and an agent, when The agent is conjugated with the vector. Such conjugates are also disclosed which include a vector selected from the group consisting of any one of the polypeptides,

15 analogues or derivatives thereof described herein (for example, Angiopep-3, Angiopep-4a, Angiopep-4b, Angiopep-5 and Angiopep-6), and an agent, when the agent is conjugated to the vector.

The agent may be selected from the group consisting of a therapeutic agent (for example, a small molecule drug, such as an anticancer agent, antibiotic or any described herein), a detectable label, a polypeptide (for example, an enzyme ), and a protein complex. In some embodiments, the agent has a maximum molecular weight of approximately 160,000 Daltons. The agent can be an active molecule in the central nervous system. The agent can be any agent useful for treating or detecting a neurological, liver, lung, kidney or spleen disease. The detectable label can be, for example, a radioimage agent (for example, an isotope), a fluorescent label (for example, rhodamine, FITC, cy5.5, 25 alexa), an indicator molecule (for example, biotin). Other examples of detectable labels include green fluorescent protein, histag protein and β-galactosidase. The conjugate may be a fusion protein consisting essentially of the vector and a protein. Examples of protein-based compounds that can be conjugated to a vector of the invention include an antibody (for example, including heavy and / or light chains), an antibody fragment (for example, an antibody binding fragment such as Fv fragment , F (ab) 2, F (ab) 2 'and Fab). An antibody or fragment thereof that can be conjugated to the vector includes a monoclonal or polyclonal antibody and, in addition, can be of any origin (eg, human, chimeric and humanized antibodies). Other protein or protein-based compounds include cellular toxins (e.g., monomethyl auristatin E (MMAE), bacterial exotoxins and endotoxins, diphtheria toxins, botulinum toxin, tetanus toxin, Perussis toxins, staphylococcal enterotoxins, TSST toxic shock syndrome toxin -one,

35 toxin adenylate cyclase, shiga toxin and cholera enterotoxin) and antiangiogenic compounds (endostatin, catechins, nutritional, chemokine IP-10, matrix metalloproteinase inhibitors (MMPI), anasteline, vironectin, antithrombin, tyrosine kinase inhibitors, inhibitors of VEGF, antibodies against receptor, herceptin, avastine and panitumumab).

Leptin, exendin-4, GLP-1, PYY or PYY (3-36) can be used, for example, for the treatment of obesity. Other polypeptides that may be included in a conjugate of the invention are adrenocorticotropic hormones (ACTH, corticotropin), growth hormone peptides (eg, human placental lactogen (hPL), growth hormones and prolactin (Prl)), stimulating hormones of melanocytes (MSH), oxytocin, vasopressin (ADH), corticotropin releasing factor (CRF), peptides associated with gonadotropin releasing hormone (GAP), growth hormone releasing factor (GRF), luteinizing hormone releasing hormones (LH-RH), orexins, prolactin-releasing peptides, somatostatin, thyrotropin-releasing hormone (THR), calcitonin (CT), calcitonin precursor peptide, calcitonin gene-related peptide (CGRP), parathyroid hormones (PTH), parathyroid hormone-related proteins (PTHrP), amylin, glucagon, insulin and insulin-like peptides, neuropeptide Y, pancreatic polypeptide (PP), eg YY peptide, somatostatin, cholecystokinin (CCK), gastrin-releasing peptide (GRP), gastrin, gastrin-inhibiting peptide, motilin, secretin, vasoactive intestinal peptide (VIP), natriuretic peptides (e.g., atrial natriuretic peptide (ANP) , type B natriuretic peptide (BNP), brain natriuretic peptide and type C natriuretic peptide (CNP), tachykinins (for example, neurocinin A, neurocinin B and substance P), substance P, angiotensins (for example, angiotensin I and angiotensin II ), renin, endothelin (for example, endothelin-1, endothelin-2, endothelin-3, sarafotoxin (a poison

55 snake) and scorpion toxin), sarafotoxin peptides, opioid peptides (e.g., casomorphine peptides, demorphins, endorphins, enkephalins, deltorphins, dinorphins), thymic peptides (e.g., thimopoietin, thymulin, thymotine, thymosin, factor thymic humoral (THF)), adrenomedulin (AM) peptides, alatostatin peptides, beta-amyloid protein fragments (Aβ fragments), antimicrobial peptides (e.g., defensin, cecropin, buforin and magainin), antioxidant peptides (e.g., natural killer cell improvement factor B (NKEF-B), bombesin, bone Gla protein peptides (eg, osteocalcin (bone Gla protein or BGP), CART peptides, cell adhesion peptides, cortistatin peptides, fibronectin fragments and fibrin-related peptides, FMRF peptides, galanin, guaniline and uroguaniline and inhibin peptides.

65 Small molecule drugs include anti-cancer agents (for example, any such agent 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 combination thereof. An anticancer agent may have a chemical moiety that allows conjugation with the vector of the invention.

In certain embodiments, the conjugate of the invention may include the formula Vx-Ly-Az or a pharmaceutically acceptable salt thereof, wherein V is a polypeptide or derivative thereof described herein (for example, Angiopep- 7, or any of the analogs, derivatives or fragments described herein). Conjugates such that V is, for example, Angiopep-3, Angiopep-4a, Angiopep-4b, Angiopep-5, Angiopep-6), or any of the analogs, derivatives or fragments described herein are disclosed. document. V can be efficiently transported into a particular cell type (for example, a liver, lung, renal, spleen or muscle cell) or it can be transported effectively through the BHE (for example, after binding to Ly-Az). In certain embodiments, the vector or conjugate is not efficiently transported through the BHE, but is efficiently transported into a particular cell type (eg, Angiopep-7). L is a linker or a bond (for example, chemical or covalent bond). TO

15 is an agent such as those selected from the group consisting of a therapeutic agent (for example, a small molecule drug), a detectable label, a protein or protein-based compound (eg, antibody, an antibody fragment), an antibiotic, an anticancer agent, an antiangiogenic compound and a polypeptide or any active molecule at the level of the central nervous system.

X, Y and Z can each independently be any number greater than 0 (for example, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10) or they can represent a range of values between 1 (for example, 2, 3, 4, 5, 6, 7, 8, 9) and 10 (for example, 9, 8, 7, 6, 5, 4, 3, 2). Therefore, the formula Vx-Ly-Az is not limited to a specific order or specific reason; for example, the conjugate may include 1 to 5 vectors (1, 2, 3, 4 or 5) coupled to the agent. In other embodiments, more than one agent can be conjugated to the vector. The agent or conjugate can be active when the agent is

25 conjugates with the vector. In some embodiments, the compound can be released from the vector, for example, after transport through the BHE or into a particular cell type. The compound can become active upon release (for example, as a prodrug). In some embodiments, the agent remains conjugated to the vector after transport. Here, the conjugate may exhibit reduced efflux transport (eg, by P glycoprotein) compared to the unconjugated agent. In this case, it may be desirable that the agent and the vector remain attached. The conjugate can be provided in a pharmaceutically acceptable composition.

The agent, when conjugated with a vector, may exhibit modified bioavailability (e.g., improved), improved potency (e.g., anticancer activity), altered tissue distribution of the agent, decreased toxicity or altered pharmacokinetics compared to the agent when It does not conjugate with the vector. A vector (for example, Angiopep-3, Angiopep-4a, Angiopep-4b, Angiopep-5 and Angiopep-6) can promote the accumulation of the agent in an individual's brain (for example, a brain that contains such a tumor cell such as a glioblastoma, tumor cell of the lung, breast, colon, liver, pancreas, or spleen) or in a particular tissue or cell (for example, liver, lung, kidney, spleen and muscle). A vector such as Angiopep-7 can promote accumulation in cell types such as liver, lung, kidney, spleen or muscle, but does not promote accumulation in the brain. More particularly, the vector may increase the accumulation or potency of an agent that is a P-gp substrate or therapeutic agents that are expelled by P-gp or a P-gp-related protein (eg, a mammalian allelic variant or human P-gp, for example, mdr1a or mdr1b isoforms of a rodent), when provided (for example, administered) to a subject. The cell can express or it can be

45 capable of expressing a protein related to P-gp or P-gp. The cell within which the conjugate is transported can express a vector receptor or transporter, for example, a low density lipoprotein (LRP) related receptor (for example, a cell that jointly expresses P-gp or a related protein with P-gp). The cell can be, for example, a normal cell, a tumor cell or a metastatic cell (for example, a multi-drug resistant cancer cell). The cell or tumor may be a metastasis (for example, of any of the cancers described herein).

A vector or conjugate can be efficiently transported into a particular cell type (for example, liver, lung, kidney, spleen or muscle cells) and can be efficiently transported through the BHE; such conjugates include Angiopep-3, Angiopep-4a, Angiopep-4b, Angiopep-5 and Angiopep-6. In

In certain embodiments, the carrier activity of the vector, by itself, does not affect the integrity of the cell to which it is being administered or does not affect the integrity of the BHE. In other embodiments, the vector or conjugate, such as Angiopep-7, is efficiently transported into a particular cell type, but is not transported effectively through the BHE. Any vector or conjugate described herein may be transported by endocytosis or receptor-mediated transcytosis or by endocytosis or adsorption-mediated transcytosis.

Any vector or conjugate described herein may be present in a pharmaceutically acceptable carrier (for example, it may be in the form of a medicament). Therefore, the invention features a pharmaceutical composition that includes (a) a conjugate (for example, any conjugate described herein); (b) a pharmaceutically acceptable carrier (for example, any described herein). The conjugate may also include (c) a solubilizer. The solubilizer can be, by

for example, a fatty acid polyoxyethylene ester (for example, Solutol® HS-15). The pharmaceutical composition can be used to modify the pharmacokinetics of a compound, to reduce the growth of a tumor cell.

or for the detection of a tumor cell.

In another aspect, the invention presents the use of a vector or a conjugate of the invention for the diagnosis or treatment (for example, for the manufacture of a medicament or use in the treatment) of a liver disease, lung disease, kidney disease , spleen disease or muscle disease. In certain embodiments, a neurological disease or a disease of the central nervous system (for example, any disease described herein) can be treated using a conjugate of the invention that is

10 transports efficiently through the BHE. For example, the vector or conjugate can be used for in vivo detection of any of these diseases. The treatment or diagnosis can be made in a mammal (for example, a human being or a non-human mammal) that needs it, such as a mammal that has or is at risk (for example, increased risk) of having the disease (for example , a cancer). Administration can be performed using any means known in the art, for example orally, intraarterially, via

15 intranasally, intraperitoneally, intravenously, intramuscularly, subcutaneously, transdermally or by mouth. Administration can be in a therapeutically effective amount.

By conjugating an agent with a vector described herein, the toxicity of the agent can be reduced, thereby allowing the agent to be provided to the subject at a dose greater than the recommended dose for the agent.

20 agent alone. Accordingly, the invention presents a method of treating a subject having a disease or condition (for example, any disease or condition described herein such as cancer) that includes providing a conjugate of the invention to the subject, in the that said conjugate includes an agent, at a higher dose (for example, at least 5%, 10%, 25%, 50%, 75%, 100%, 250%, 500%, the 1,000%, 5,000% or 10,000% higher) at the therapeutic dose of the agent alone.

Because a vector described herein can direct an agent to a particular cell type (for example, those described herein), the agent when conjugated to a vector can, in certain embodiments, have an efficacy major when administered to the subject. Accordingly, the invention also presents a method of treating a subject who has a disease or condition (for example,

30 any disease or condition described herein as cancer) by administration to the subject of a conjugate or a composition that includes a conjugate of the invention, wherein the conjugate includes an agent, at a lower dose (e.g., 5%, 10%, 15%, 20%, 30%, 50%, 70%, 80%, 90%, 95%, 98%, 99%, 99 , 9% lower) at the therapeutic dose of the agent alone.

The methods of treatment of the invention may further comprise a step of assessing whether the individual's tumor includes a multi-drug resistant tumor cell (eg, a cell expressing P-gp (MDR1) or determining whether the tumor can have or have a multi-drug resistant phenotype). Treatment methods may include a stage of providing chemotherapy, radiotherapy, or both. Alternatively, the subject being treated may have received (or will receive) such therapies (for example, in the

40 term of 1 year, 6 months, 3 months, 2 months, 1 month, 2 weeks, 1 week, 3 days, 2 days or 1 day). The individual may have gone through surgery. The individual may have a brain tumor (for example, a primary brain tumor) or a tumor at a site other than the brain (for example, not having a brain tumor). An individual in need may be aware of or at risk of developing resistance to at least one drug (eg, a multi-drug resistant phenotype (MDR)). The tumor can be a lung tumor, a metastasis

45 extracranial of a brain tumor of (for example, from a glioblastoma), a brain tumor of metastatic origin (for example, from a lung tumor, a breast tumor, a melanoma, a colorectal cancer or a urinary organ tumor) . The tumor may include a cell that expresses P-gp, LRP, or both, or a cell that can express P-gp, LRP, or both. P-gp and LRP can be located on a cell surface.

In another aspect, the invention also presents the conjugate of the present invention for use in a method of increasing the concentration of an agent in a cell or transport of an agent to a cell, in which the cell expresses a member of the LRP receptor family. The method includes contacting the cell with a conjugate comprising the conjugate agent with a vector (for example, any polypeptide described herein), thereby increasing the concentration of the agent in the cell or transporting the agent.

55 to the cell. The invention also presents a method of increasing the concentration of agent in a cell, in which the cell expresses P-glycoprotein. The method includes contacting the cell with a conjugate that includes the conjugate agent with a vector (eg, any polypeptide described herein), thereby increasing the concentration of the agent in the cell, compared to the unconjugated agent.

According to any aspect of the invention, neurological diseases include a brain tumor, a brain metastasis, schizophrenia, epilepsy, Alzheimer's disease, Parkinson's disease, Huntington's disease, stroke and BHE-related malfunction (for example , obesity). Liver disease can be a cancer such as hepatocarcinoma. Other liver diseases include those described herein. Lung disease can be a lung cancer (for example, the

65 described herein).

Conjugates may include vectors in the form of multimers such as dimers. The multimeric forms of the vectors may, in some embodiments, increase the transport or accumulation of the agent. Vectors can also be in a ratio of at least 1: 1 (agent: vector), 1: 2, 1: 3, 1: 4, 1: 5, 1: 6 or 1:10. As indicated herein, the superior vector ratios with respect to agent may lead to transport.

5 increased. As such, it is not intended to limit the vector number per agent.

Also according to the invention, the pharmaceutical composition can be used, for example, for the administration of an agent to the CNS of an individual.

A pharmaceutically acceptable salt of a vector (polypeptide) or a conjugate is encompassed by the invention and may include pharmaceutically acceptable acid addition salts of the agent.

The vector or conjugate of the invention can be used in combination with or separately from conventional treatment or therapy methods. Combination therapy with other agents may include sequential administration or

15 simultaneous to an individual. The pharmaceutical compositions of the invention may include a combination of a vector-agent conjugate of the invention in association with a pharmaceutically acceptable excipient, as described herein, and another therapeutic or prophylactic agent known in the art.

By "vector" is meant a compound or molecule such as a polypeptide that can be transported into a particular cell type (eg, liver, lungs, kidney, spleen or muscle) or through the BHE. The vector can be linked to (covalently or not) or conjugated with an agent and thereby can transport the agent into a particular cell type or through the BHE. In certain embodiments, the vector can bind to receptors present in cancer cells or brain endothelial cells and thereby be transported into the cancer cell or through the BHE by transcytosis. The vector can be a molecule for

25 that high levels of transendothelial transport can be obtained, without affecting the integrity of the cell or BHE. The vector can be a polypeptide or a peptidomimetic and can be produced naturally or produced by chemical synthesis or recombinant genetic technology.

By "conjugate" is meant a vector bound to an agent. The conjugation can be of a chemical nature, such as through a linker, or of a genetic nature for example by recombinant genetic technology, such as in a fusion protein with for example an indicator molecule (for example, green fluorescent protein, β- galactosidase, Histag, etc.).

By a vector that "is efficiently transported through the BHE" means a vector that can cross the

35 BHE at least as effectively as Angiopep-6 (i.e. 38.5% higher than Angiopep-1 (250 nM) in the in situ cerebral perfusion test described herein). Accordingly, a vector or conjugate that "is not transported efficiently through BHE" is transported to the brain at lower levels (for example, transported less efficiently than Angiopep-6).

By a vector or conjugate that "is efficiently transported to a particular cell type" means a vector or conjugate that can accumulate (for example, or due to increased transport into the cell,

or decreased efflux from the cell, or a combination thereof) in that type of cell at least 10% (for example, 25%, 50%, 100%, 200%, 500%, 1,000%, 5,000% or 10,000%) to a greater degree than either a control substance, or, in the case of a conjugate, compared to the unconjugated agent.

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

55 "Analogous" means a polypeptide that originates from an original sequence or from a part of an original sequence and may include one or more modifications; for example, one or more modifications in the amino acid sequence (for example, an amino acid addition, deletion, insertion or substitution), one or more modifications in the main structure or side chain of one or more amino acids, or an addition of a group or other molecule to one or more amino acids (side chains or main structure). An analog may have one or more amino acid insertions, at either or both ends of the polypeptide or within the amino acid sequence of the polypeptide. An analog may have sequence similarity and / or sequence identity (for example, it may be substantially identical) with that of an original sequence or a part of an original sequence. The analogs may include a modification of their structure, for example, as described herein. The degree of similarity between two sequences is based on the percentage of identities (amino acids

65 identical) and conservative substitution. An analogue can have at least 35%, 50%, 60%, 70%, 80%, 90% or 95% (for example, 96%, 97%, 98%, the 99% and 100%) sequence similarity with a

original sequence with a combination of one or more modifications to a main structure or side chain of an amino acid, or an addition of a group or other molecule. Exemplary amino acids that are intended to be similar (a conservative amino acid) to others are known in the art and include, for example, those listed in Table 3.

5 By "substantially identical" means a polypeptide or nucleic acid having at least 35%, 40%, 50%, 55%, 60%, 65%, 70%, 75%, 85%, 90%, 95%, or even 99% identity with a reference amino acid or nucleic acid sequence. For polypeptides, the length of the comparison sequences will generally be at least 4 (for example, 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 the comparison sequences will generally be 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 can be found between amino acids of analogs that are identical or similar to amino acids of the original polypeptide. The gaps may not include amino acids, or include one or more amino acids that are not identical or similar to the polypeptide.

15 original. The biologically active analogs of the vectors (polypeptides) of the invention are encompassed with the same. The percent identity can be determined, for example, with a GAP, BESTFIT or FASTA algorithm in the Wisconsin Genetics Software Package Release 7.0, using default gap weights.

By "functional derivative" is meant a "chemical derivative", "fragment" or "variant" of biologically active sequence or part of a vector or agent or conjugate and a salt thereof of the invention. A functional derivative of the vector can bind to or conjugate with an agent and enter a particular type of cell, thereby transporting the agent into that cell.

By "chemical derivative" is meant a vector, an agent or a conjugate of the invention, which contains moieties.

Additional chemicals that are not part of the vector, agent or conjugate of vector-agent, including covalent modifications. A chemical derivative can be prepared by direct chemical synthesis using methods known in the art. Such modifications can be introduced into a protein or peptide vector, agent or conjugate of vector-agent by reacting selected amino acid residues as a target with an organic derivative agent that can react with selected side chains or terminal residues. A chemical vector derivative can cross the BHE or enter or accumulate in a particular cell type (for example, those described herein). In a preferred embodiment, very high levels of transendothelial transport are obtained through the BHE without affecting the integrity of the BHE.

By "fragment" is meant a polypeptide that originates from a part of an original sequence or

35 parental or from an analogue of said parental sequence. The fragments encompass polypeptides that have truncations of one or more amino acids, in which truncation can originate from the aminoterminal (N-terminal), carboxyl-terminal (C-terminal) end, or from within the protein. A fragment may include the same sequence as the corresponding part of the original sequence. The functional fragments of the vector (polypeptide) described herein are encompassed by the invention. The fragments can be at least 5 (for example, 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. Fragments of the invention may include, for example, a polypeptide of 7, 8, 9 or 10 amino acids at 18 amino acids. The fragments may contain any of the modifications described herein (eg, acetylation, amidation, amino acid substitutions).

A "naturally occurring amino acid" is an amino acid that does not occur naturally or is found in a mammal.

By "agent" is meant, any compound, for example, an antibody, or a therapeutic agent, a label, a tracer or an imaging compound.

By "therapeutic agent" is meant an agent that has a biological activity. In some cases, the therapeutic agent is used to treat the symptoms of a disease, a physical or mental state, an injury or an infection and includes anticancer agents, antibiotics, antiangiogenic agents, and active molecules at the system level

55 central nervous.

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

By "subject" is meant a human being or non-human animal (for example, a mammal).

By "treatment", "treating" and the like are meant to obtain a desired pharmacological or physiological effect, for example, inhibition of cancer cell growth, death of a cancer cell, improvement of a disease or condition (for example, any disease or condition described herein), or improvement of

At least one symptom associated with a disease or condition. The effect can be prophylactic, for example, to completely or partially prevent a disease or symptom thereof or it can be therapeutic in terms of a partial cure.

or complete for a disease and / or adverse effect attributable to the disease. Treatment includes: (a) preventing the occurrence of a disease or condition (for example, preventing cancer) in an individual (for example, someone who is predisposed to the disease but who has not been diagnosed to have it) ; (b) inhibit a disease (for example, stop its development); or (c) relieve a disease (for example, reduce symptoms

5 associated with a disease). Treatment includes any administration of a therapeutic agent to an individual to treat, cure, relieve, improve, decrease or inhibit a state in the individual, including, without limitation, administering a vector-agent conjugate to an individual.

By "cancer" means any cell proliferation whose only characteristic is the loss of controls

10 normal which can result in unregulated growth, lack of differentiation, or ability to invade tissues and metastases. Cancer can develop in any tissue or in any organ. The cancer is intended to include, without limitation, cancer of the brain, liver, lungs, kidney or spleen. Additional cancers are described herein.

By "providing" it is meant, in the context of a vector or conjugate of the invention, to bring the vector or conjugate into contact with a target tissue or cell either in vivo or in vitro. A vector or conjugate can be provided by administering the vector or conjugate to a subject.

By "administer" and "administration" means a way of administering including, without limitation, via

20 intraarterially, intranasally, intraperitoneally, intravenously, intramuscularly, subcutaneously, transdermally or by mouth. A daily dosage can be divided into one, two or more doses in a suitable form to be administered in one, two or more times over a period of time.

By "therapeutically effective" or "effective amount" is meant a sufficient amount of a therapeutic agent.

25 to improve, decrease, prevent, delay, suppress or stop any symptoms of the disease or condition being treated. It is not necessary for a therapeutically effective amount of an agent to cure a disease or condition, but it will provide a treatment for a disease or condition so that the onset of the disease or condition is delayed, prevented or prevented, or the symptoms of the disease or condition, or the term of the disease or condition is changed or, for example, is less severe or recovery is accelerated in a

30 individual

By "state" means any situation that causes pain, discomfort, dizziness, illness or disability (mental or physical) to or in an individual, including neurological disease, injury, infection, or chronic or acute pain. Neurological diseases include brain tumors, brain metastases, schizophrenia, epilepsy,

35 Alzheimer's disease, Parkinson's disease, Huntington's disease and stroke.

By "pharmaceutical composition" is meant a therapeutically effective amount of an agent together with a pharmaceutically acceptable diluent, preservative, solubilizer, emulsifier or adjuvant, for example, any of those described herein.

40 By "therapeutic dose" is meant the dosage of an agent such as a drug (without the vector) acceptable for clinical use with respect to its toxicity or efficacy. By conjugating an agent with a vector of the invention, it may be possible to administer the agent at a dosage either lower or higher than the therapeutic dose.

If a "range" or a "group of substances" is mentioned with respect to a particular characteristic (for example, temperature, concentration, time and the like), the invention refers to and explicitly incorporates in each and every document specific members and combinations of subgames or subgroups in them. Therefore, for example, with respect to a length of from 9 to 18 amino acids, it should be understood that each and every individual length is specifically incorporated herein,

50 for example, a length of 18, 17, 15, 10, 9 and any number between them. Therefore, unless specifically mentioned, each interval mentioned in this document should be understood as being inclusive. For example, the expression from 5 to 19 amino acids in length includes 5 and 19. This similarity applies with respect to other parameters such as sequences, length, concentrations, elements and the like.

55 The sequences, regions, parts defined herein include each and every one of the sequences, regions and individual parts described in this manner as well as each and every possible sub-sequence, sub-region and sub-part if such sub-sequence, sub-region. and subparts are defined as positively including particular possibilities, such as excluding particular possibilities or a combination thereof. For example, an exclusive definition for a region can say the following: “provided that

60 polypeptide is not shorter than 4, 5, 6, 7, 8 or 9 amino acids. An additional example of a negative limitation is the following; a sequence that includes SEQ ID NO: X with the exclusion of a polypeptide of SEQ ID NO: Y; etc. An additional example of a negative limitation is the following; provided that said polypeptide is not (does not include or consists of) SEQ ID NO: Z.

65 Brief description of the drawings

Figure 1 illustrates the exemplary polypeptide structure of the invention.

Figure 2 illustrates the protocol used to conjugate aprotinin with IgG using the BS3 crosslinking agent.

5 Figure 3 illustrates the protocol used to conjugate aprotinin with IgG using the sulfo-EMCS crosslinking agent.

Figure 4 illustrates the protocol used to conjugate Angiopep-2 with antibodies using the ECMS crosslinking agent.

10 Figure 5 illustrates the protocol used to conjugate Angiopep-2 with antibodies using the SATA crosslinking agent.

Figure 6 illustrates the protocol used to conjugate Angiopep-2 with antibodies using carbohydrate targets through hydrazide. 15 Figure 7 illustrates other possible linkers that can be used in the preparation of a conjugate.

Figure 8 illustrates a method for attaching a vector of the invention to paclitaxel.

Figure 9 is a schematic representation of the efflux pump, P glycoprotein (P-gp or MDR1) to the cell surface. The efflux pump, P-gp or MDR1, associated with resistance to multiple drugs is highly expressed on the cell surface of many cancer cells and various tissues including the blood brain barrier (BHE).

Figure 10A and 10B are diagrams representing the tissue distribution of Taxol and TxlAn1 conjugate. 25 Figure 11 is a diagram representing the pulmonary distribution of Taxol and TxlAn1.

Figure 12 is a diagram representing the levels of TxlAn1 conjugate in plasma and lung.

30 Figure 13 is a set of images showing the in vivo accumulation of Angiopep-2 and Angiopep-7 in the brains of rats 30 minutes after IV injection. Angiopep-2 accumulates to a greater degree in the brain compared to Angiopep-7.

Figure 14 is a set of images showing fluorescence microscopy of brain sections after

35 an infusion in situ of 10 min. of either Angiopep-2 or Angiopep-7 fluorescently labeled. These images show that Angiopep-2 is located inside the brain, while Angiopep-7 is located inside the capillaries.

Figure 15A is a set of images showing the in vivo imaging of Angiopep-2 and 40 Angiopep-7 in the liver, lungs and kidneys of a rat. It is observed that Angiopep-7 accumulates in these tissues.

Figure 15B is a graph showing images of ex vivo organs 24 hours after the injection of either Angiopep2 or Angiopep-7 in a rat. Both polypeptides accumulate in the kidneys, liver and lungs. Angiopep-2 accumulates to a greater degree in the brain compared to Angiopep-7.

45 Figure 16 illustrates the volume of distribution in the cerebral parenchyma of IgG conjugated to Angiopeps and free.

Figure 17 is a diagram of cell proliferation in the presence of the parental drug Taxol. Glioblastoma cells (U-87) were exposed to various concentrations of Taxol for 3 days. The 3H-thymidine incorporated into 50 cells was plotted as a function of Taxol concentrations.

Figures 18A and 18B are graphs depicting the effect of TxlAn2 treatment on tumor growth of subcutaneous glioblastomas (U-87).

Figure 19 is a set of photomicrographs showing the detection of β-tubulin in NCI-H460 cells by immunofluorescence or visible light in cancer cells exposed to Taxol or TxlAn2 conjugate. As a control, the cells were exposed to 1% DMSO.

Figure 20 is a set of graphs showing the effect of Taxol and TxlAn2 conjugate in the NCI-H460 cell cycle measured by FACS. Cells were exposed for 24 h with the vehicle (DMSO), Taxol (100 nM) or TxlAn2 conjugate (30 nM, equivalent to 100 nM Taxol).

Figure 21A is a graph showing the accumulation of various drugs in MDCK cells transfected with MDR1 in the presence or absence (control) of 10 µM CsA, a P-gp inhibitor. The experiment was performed in the presence of 1% DMSO.

Figure 21B is a graph showing the accumulation of the conjugate in cells that overexpress P-gp.

Figures 22A and 22B are Western blot photographs showing immunodetection of LRP in human brain tumor biopsies.

Figures 23A and 23B are a schematic representation of the conjugation of a drug with the vector of the invention.

Figure 24 is a chromatogram illustrating the production of the TxlAn2 conjugate (3: 1).

Figure 25 is an HPLC analysis of the purified peak on a hydrophobic column using the AKTA scanner.

Figure 26 illustrates the association of Angiopep-2 with the IgG light and heavy chain.

Figure 27 illustrates the increase in brain distribution volume of IgG-Angiopep-2 conjugates crosslinked with sulfo-EMCS.

Figure 28 illustrates brain penetration for IgG-Angiopep-2 conjugates using in situ cerebral perfusion.

Figure 29 illustrates an autoradiogram of radiolabeled [125I] -IgG-Angiopep conjugates.

Figure 30 illustrates the similar detection of EGFR on U87 cells with anti-EGFR and anti-EGFRAngiopep-2 conjugate by FACS analysis.

Figure 31 illustrates the volume of distribution in the cerebral parenchyma of EGFR antibody conjugated to Angiopep-2 and free.

Figure 32 illustrates the volume of distribution in the cerebral parenchyma of VEFG antibody conjugated to Angiopep-2 and free.

Figure 33 illustrates the uptake of conjugates including different vector ratios with respect to the agent (antibody) in the parenchyma.

Detailed description of the invention

35 It has been discovered that Angiopep-7 can be efficiently transported into particular cell types or organs such as liver, lungs, kidneys, spleen and muscle, but it is not transported effectively through the blood brain barrier (BHE) , in relation to polypeptides such as Angiopep-1 or Angiopep-2. Based on this, Angiopep-7 has been identified as a suitable vector for transporting agents (for example, for the treatment of diseases associated with these tissues, such as cancer or any of the diseases described herein). Other polypeptides have also been identified, including Angiopep-3, Angiopep-4a, Angiopep-4b, Angiopep-5 and Angiopep-6, which can also be used as vectors in the invention. These polypeptides can retain the ability to be transported efficiently through the BHE, or transported into particular cell types or tissues (e.g., liver, lungs, kidneys, spleen and

45 muscle). For these polypeptides, agents that cannot or are ineffective in effectively crossing the BHE can be transported through the BHE when conjugated to the polypeptide. The conjugates may be in the form of a composition, such as a pharmaceutical composition, for the treatment or diagnosis of a condition or disease.

The conjugates of the invention (for example, any of those described herein) may also have advantageous in vivo pharmacokinetic properties compared to the agent alone. These properties may include increased half-life in vivo or increased concentration in, increased accumulation rate in, or decreased withdrawal of a desired cell type. Based on this, it may be possible to administer conjugates of the invention to a subject either at a lower dosage, or less frequently, or for a duration

55 shorter than the unconjugated agent. Lower amounts, frequency or duration of administration may be especially desirable for therapeutic agents with known side effects. In other cases, conjugation of the agent to a polypeptide described herein may result in superior efficacy, due to increased concentration in target tissues or cells. In fact, by concentrating the agent on a type of target cell, unwanted side effects can be reduced (for example, with increased efficacy). This may decrease the required duration of therapy. In some cases, it may even be possible to administer the conjugate at an increased dosage, frequency or duration, in relation to the dosage of the unconjugated agent and observe increased efficacy, reduced side effects, or both.

Polypeptides of the invention

The invention presents any of the polypeptides described herein, for example, any of

the polypeptides described in Table 1 (for example, a polypeptide defined in any of SEQ ID NOS: 1-105 and 107-112 such as SEQ ID NOS: 1-97, 99, 100, 101 or 107-112), or any fragment, analog, derivative or variant thereof. In certain embodiments, the polypeptide may have at least 35%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99% or even 100%. of identity with a polypeptide described herein. The polypeptide may have one or more (eg, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15) substitutions relative to one of the sequences described herein. document. Other modifications are described in greater detail below.

The invention also features fragments of these polypeptides (for example, a functional fragment). In certain embodiments, the fragments can enter or accumulate in a particular type of cell (e.g., liver, lung, kidney, spleen or muscle) or they can cross the EBR. The truncations of the polypeptide may have 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or more amino acids from either the N-terminal end of the polypeptide, the Cterminal end of the polypeptide or A combination of them. Other fragments include sequences in which internal parts of the polypeptide are deleted.

Additional polypeptides can be identified using one of the assays or methods described in U.S. Patent Application Publication No. 2006/0189515, or by any method known in the art. For example, a candidate vector can be produced by conventional polypeptide synthesis, conjugated with Taxol and administered to a laboratory animal. A biologically active vector can be identified, for example, based on its effectiveness in increasing the survival of an animal in which tumor cells have been injected and treated with the conjugate compared to a control that has not been treated with a conjugate (for example , treated with the unconjugated agent).

In another example, a biologically active polypeptide can be identified based on its location in the parenchyma in an in situ brain perfusion assay. In vitro BHE assays, such as the model developed by CELLIAL ™ Technologies, can be used to identify such vectors.

Tests may also be performed to determine the accumulation in other tissues. See, for example, example 1 in this document. Marked conjugates of a polypeptide can be administered to an animal, and accumulation in different organs can be measured. For example, a polypeptide conjugated to a detectable label (eg, a near IR fluorescence spectroscopy tag such as Cy5.5) allows live in vivo visualization. Such a polypeptide can be administered to an animal, and the presence of the polypeptide in an organ can be detected, thus allowing the determination of the rate and amount of accumulation of the polypeptide in the desired organ. In other embodiments, the polypeptide can be labeled with a radioactive isotope (for example, 125I). The polypeptide is then administered to an animal. After a period of time, the animal is sacrificed, and the animal's organs are removed. The amount of radioisotope in each organ can then be measured using any means known in the art. By comparing the amount of a labeled candidate polypeptide in a particular organ without the amount of labeled control, the capacity of the candidate polypeptide, the rate or amount of accumulation of a candidate polypeptide in a particular tissue can be determined. Appropriate negative controls include any polypeptide that is known not to be transported into a particular cell type.

Table 1

Peptide # 5 includes the sequence of SEQ ID NO: 5 and is amidated at its C-terminal end (see for example Figure 1)

5 Peptide # 67 includes the sequence of SEQ ID NO: 67 and is amidated at its C-terminal end (see for example Figure 1)

Peptide No. 76 includes the sequence of SEQ ID NO: 76 and is amidated at its C-terminal end (see for example Figure 1)

Peptide # 91 includes the sequence of SEQ ID NO: 91 and is amidated at its C-terminal end (see for example Figure 1)

15 Peptide No. 107 includes the sequence of SEQ ID NO: 97 and is acetylated at its N-terminal end

Peptide No. 109 includes the sequence of SEQ ID NO: 109 and is acetylated at its N-terminal end

Peptide # 110 includes the sequence of SEQ ID NO: 110 and is acetylated at its N-terminal end.

20 The amine groups of Angiopep-1 (SEQ ID NO: 67) and Angiopep-2 (SEQ ID NO: 97) have been used as sites for agent conjugation. To study the role of amine groups in conjugation and their impact on the overall transport capacity of these vectors, new vectors were designed, based on the sequence of Angiopep-1 and Angiopep-2, with variable reactive amine groups and variable global load . These polypeptides are shown in the table.

25 2.

Table 2: Vectors with variable amine group targets

Polypeptide name

Polypeptide sequences Reactive amines (positions) Load SEQ ID No.

Angiopep-3 *

Ac1-TFFYGGSRGKRNNFKTEEY 2 (10.15) +1 107

Angiopep-4b

RFFYGGSRGKRNNFKTEEY 3 (1,10,15) +3 108

Angiopep-4a

Ac1-RFFYGGSRGKRNNFKTEEY 2 (10.15) +2 109

Angiopep-5

Ac1-RFFYGGSRGKRNNFRTEEY 1 (10) +2 110

Angiopep-6

TFFYGGSRGKRNNFRTEEY 2 (1.10) +2 111

Angiopep-7

TFFYGGSRGRRNNFRTEEY eleven) +2 112

* Angiopep-3 is an acetylated form of Angiopep-2.1Ac represents acetylation.

Modified polypeptides

The invention also includes a polypeptide having a modification of a polypeptide having a sequence.

5 described in SEQ ID NO: 112 such as Angiopep-7. A polypeptide having a modification of an amino acid sequence described herein is also disclosed (eg, polypeptide having a sequence described in any one of SEQ ID NOS: 1-105 and 107-111 such as Angiopep- 3, -4a, -4b, -5 or -6). In certain embodiments, the modification does not significantly destroy a desired biological activity. In some embodiments, the modification may cause a reduction in biological activity (by

For example, in 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 may increase (for example, by at least 5%, 10%, 25%, 50%, 100%, 200%, 500% or 1,000%) the biological activity of the original polypeptide. The modified polypeptide may have or may optimize one or more of the characteristics of a polypeptide of the invention that, in some cases, may be necessary or desirable. Such features include

15 in vivo stability, bioavailability, toxicity, immunological activity or immunological identity.

The polypeptides of the invention can include amino acids or modified sequences either by natural methods, such as post-translational processing, or by chemical modification techniques known in the art. Modifications can occur anywhere in a polypeptide.

20 including the main structure of the polypeptide, the amino acid side chains and the amino-or carboxyl-terminal end. The same type of modification may be present in the same degree or varying degrees at several sites in a given polypeptide, and a polypeptide may contain more than one type of modification. The polypeptides may be branched as a result of ubiquitination, and they may be cyclic, with or without branching. Cyclic, branched and branched cyclic polypeptides can result from natural processes

25 post-translational or can be prepared synthetically. Other modifications include pegylation, acetylation, acylation, addition of the acetomidomethyl group (Acm), ADP-ribosylation, alkylation, amidation, biotinylation, carbamoylation, carboxylation, esterification, covalent binding to flavin, covalent binding to a heme moiety, covalent binding of a nucleotide or nucleotide derivative, covalent drug binding, covalent binding of a label (eg, fluorescent or radioactive), covalent binding of a lipid or lipid derivative, covalent binding of

Phosphatidylinositol, crosslinking, cyclization, disulfide bond formation, demethylation, covalent crosslinking formation, cystine formation, pyroglutamate formation, formylation, gamma-carboxylation, glycosylation, GPI anchor formation, hydroxylation, iodination, methylation, myrisylation, oxidation, proteolytic processing, phosphorylation, prepylation, racemization, selenoylation, sulfation, addition of amino acids mediated by transfer RNA to proteins such as arginylation and ubiquitination.

A modified polypeptide may further include an insertion, deletion or substitution of amino acids either conservative or non-conservative (eg, D-amino acids, deamino acids) in the polypeptide sequence (for example, in which such changes do not substantially alter the biological activity of the polypeptide).

40 Substitutions may be conservative (that is, in which one residue is replaced by another of the same group

or general type) or non-conservative (ie, in which a residue is replaced by an amino acid of another type). In addition, an amino acid that does not occur naturally can be substituted for a naturally occurring amino acid (i.e., conservative amino acid substitution that does not occur naturally or a non-conservative amino acid substitution that does not occur naturally natural).

45 Synthetically prepared polypeptides may include amino acid substitutions not naturally encoded by DNA (eg, unnatural or non-naturally occurring amino acid). Examples of naturally occurring amino acids include D-amino acids, an amino acid that has an acetylaminomethyl group attached to a sulfur atom of a cysteine, a pegylated amino acid, the omega amino acids of

50 formula NH2 (CH2) nCOOH in which n is 2-6, neutral non-polar amino acids, such as sarcosine, t-butyl alanine, t-butyl glycine, N-methyl isoleucine and norleucine. Trp, Tyr or Phe can be substituted with phenylglycine; Citrulline and methionine sulfoxide are non-polar neutral, cysteic acid is acidic and ornithine is basic. Proline can be substituted with hydroxyproline and retain the properties that confer conformation.

55 Analogues can be generated by substitution mutagenesis and preserve the biological activity of the original polypeptide. Examples of substitutions identified as "conservative substitutions" are shown in Table 3. If such substitutions result in an unwanted change, then other types of substitutions, called "example substitutions" are introduced in Table 3, or such as further described herein with reference to classes of amino acids, and the products are examined.

60 Substantial modifications in immunological function or identity are achieved by selecting substitutions that differ significantly in their effect on the maintenance of (a) the structure of the main structure of the polypeptide in the area of substitution, for example, as a sheet conformation or helical, (b) the charge or hydrophobicity of the molecule at the target site or (c) the volume of the side chain. The waste that is produced from

65 naturally divided into groups based on common side chain properties:

(1) hydrophobic: norleucine, methionine (Met), alanine (Ala), valine (Val), leucine (Leu), isoleucine (Ile), histidine (His), tryptophan (Trp), tyrosine (Tyr), phenylalanine (Phe ),

(2)

neutral hydrophilic: cysteine (Cys), serine (Ser), threonine (Thr) 5

(3) negatively charged / acids: aspartic acid (Asp), glutamic acid (Glu)

(4)

basic: asparagine (Asn), glutamine (Gln), histidine (His), lysine (Lys), arginine (Arg) 10 (5) residues that influence chain orientation: glycine (Gly), proline (Pro);

(6) aromatic: tryptophan (Trp), tyrosine (Tyr), phenylalanine (Phe), histidine (His),

(7)

Polar: Ser, Thr, Asn, Gln 15

(8)

positively charged basic: Arg, Lys, His, and;

(9)

Loaded: Asp, Glu, Arg, Lys, His

20 Other conservative amino acid substitutions are listed in Table 3. Table 3: Amino acid substitution

Original waste

Example substitution Conservative substitution

Wing (A)

Val, Leu, Ile Val

Arg (R)

Lys, Gln, Asn Lys

Asn (N)

Gln, His, Lys, Arg Gln

Asp (D)

Glu Glu

Cys (C)

Be Be

Gln (Q)

Asn Asn

Glu (E)

Asp Asp

Gly (G)

Pro Pro

His (H)

Asn, Gln, Lys, Arg Arg

Ile (I)

Leu, Val, Met, Ala, Phe, Norleucine Leu

Leu (L)

Norleucine, Ile, Val, Met, Ala, Phe Ile

Lys (K)

Arg, Gln, Asn Arg

Met (M)

Leu, Phe, Ile Leu

Phe (F)

Leu, Val, Ile, Ala Leu

Pro (P)

Gly Gly

To be

Thr Thr

Thr (T)

Be Be

Trp (W)

Tyr Tyr

Tyr (Y)

Trp, Phe, Thr, Being Phe

Val (V)

Ile, Leu, Met, Phe, Ala, Norleucine Leu

Additional analogues

The polypeptides and conjugates of the invention may include aprotinin polypeptide analogs known in the art. For example, U.S. Patent No. 5,807,980 describes inhibitors derived from the bovine pancreatic trypsin inhibitor (aprotinin) as well as a method for its preparation and therapeutic use, including the polypeptide of SEQ ID NO: 102. These polypeptides have been used for the treatment of a condition characterized by a

Anomalous amount or appearance of tissue factor and / or factor VIIIa such as abnormal thrombosis. U.S. Patent No. 5,780,265 describes serine protease inhibitors that can inhibit plasma kallikrein, including SEQ ID NO: 103. U.S. Patent No. 5,118,668 describes trypsin inhibitor variants

bovine pancreatic, including SEQ ID NO: 105. The aprotinin amino acid sequence (SEQ ID NO: 98), the Angiopep-1 amino acid sequence (SEQ ID NO: 67) and SEQ ID NO: 104, as well as some biologically active analog sequences can be found in the publication of International application No. WO 2004/060403.

5 A nucleotide sequence exemplary encoding aprotinin analogue is illustrated in SEQ ID NO: 106 (atgagaccag atttctgcct cgagccgccg tacactgggc cctgcaaagc tcgtatcatc cgttacttct acaatgcaaa ggcaggcctg tgtcagacct tcgtatacgg cggctgcaga gctaagcgta acaacttcaa atccgcggaa gactgcatgc gtacttgcgg tggtgcttag; No registration of from Genbank X04666). This sequence encodes a lysine at position 16 instead of a valine, as found in SEQ ID NO: 98. A mutation can be introduced into the nucleotide sequence of SEQ ID NO: 106 by methods known in the art to change the production of the polypeptide 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.

15 Other examples of aprotinin analogs can be found using the BLAST protein database (Genebank: www.nc-bi.nlm.nih.gov/BLAST/) using the synthetic aprotinin sequence (or part thereof) given to know in international application no. PCT / CA2004 / 000011. By way of example, aprotinin analogues are found with registration numbers CAA37967 (GI: 58005) and 1405218C (GI: 3604747).

Preparation of polypeptide and peptidomimetic derivatives

In addition to polypeptides consisting only of naturally occurring amino acids, the present invention also encompasses peptidomimetics or polypeptide analogs. Polypeptide analogs are commonly used in the pharmaceutical industry as non-polypeptide drugs with properties analogous to those of the template polypeptide. Non-polypeptide compounds are called "polypeptidomimetics" or peptidomimetics (Fauchere et al., Infect. Immun. 54: 283-287, 1986; Evans et al., J. Med. Chem. 30: 1229-1239, 1987). Polypeptidomimetics that are structurally related to therapeutically useful polypeptides can be used to produce an equivalent or enhanced therapeutic or prophylactic effect. In general, peptidomimetics are structurally similar to the paradigm polypeptide (i.e., a polypeptide that has a biological or pharmacological activity) such as receptor binding polypeptides that occur naturally, but have one or more peptide junctions optionally replaced by junctions. such as -CH2NH-, -CH2S-, -CH2-CH2-, -CH = CH- (cis and trans), -CH2SO-, -CH (OH) CH2-, -COCH2-etc., by methods well known in the technique (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

35 Proteins, Weinstein ed, Marcel Dekker, New York). Such polypeptidomimetics may have significant advantages over naturally occurring polypeptides including more economical production, greater chemical stability, enhanced pharmacological properties (e.g., half-life, absorption, potency, efficacy), reduced antigenicity and others.

While the polypeptides of the invention may be effective to be introduced into particular cell types (for example, those described herein), their efficacy can be reduced by the presence of proteases. Serum proteases have specific substrate requirements. The substrate must have both L-amino acids and peptide bonds for cleavage. In addition, exopeptidases, which represent the most prominent component of serum protease activity, usually act on the first peptide bond of the polypeptide and require a

45 N-terminal free end (Powell et al., Pharm. Res. 10: 1268-1273, 1993). In view of this, it is often advantageous to use modified versions of polypeptides. The modified polypeptides retain the structural characteristics of the original L-amino acid polypeptides that confer biological activity with respect to IGF-1, but are advantageously not easily susceptible to cleavage by protease and / or exopeptidases.

Systematic substitution of one or more amino acids of a consensus sequence with D-amino acids of the same type (eg, D-lysine instead of L-lysine) can be used to generate more stable polypeptides. Thus, a polypeptide or peptidomimetic derivative of the present invention can be a whole L, all D or D, L mixed polypeptide. The presence of a D-amino acid at the N-terminal or C-terminal end increases the in vivo stability of a polypeptide because peptidases cannot use a D-amino acid as a substrate (Powell et al., Pharm. 55 Res. 10: 1268-1273, 1993). Inverse D-polypeptides are polypeptides containing D-amino acids, arranged in an inverse sequence in relation to a polypeptide containing L-amino acids. Therefore, the Cterminal residue of a polypeptide with L-amino acids becomes N-terminal for the polypeptide with D-amino acids, etc. Inverse D-polypeptides retain the same tertiary conformation and therefore the same activity, as L-amino acid polypeptides, but are more stable to enzymatic degradation in vitro and in vivo, and therefore have greater therapeutic efficacy than the original polypeptide. (Brady and Dodson, Nature 368: 692-693, 1994; Jameson et al., Nature 368: 744-746, 1994). In addition to the reverse D-polypeptides, restricted polypeptides comprising a consensus sequence or a substantially identical consensus sequence variation, can be generated by methods well known in the art (Rizo and Gierasch, Ann. Rev. Biochem. 61: 387-418, 1992). For example, restricted polypeptides can be generated by adding cysteine residues that can form disulphide bridges and thereby result in a cyclic polypeptide. Cyclic polypeptides have no N or C-terminal free end. Therefore, they are not susceptible to proteolysis by exopeptidases, although they are, by

of course, susceptible to endopeptidases, which do not cleave at the terminal ends of the peptide. The amino acid sequences of the polypeptides with D-amino acids at the N-terminal or C-terminal end and of the cyclic polypeptides are usually identical to the sequences of the polypeptides to which they correspond, except for the presence of the D-amino acid residue. at the N-terminal or C-terminal end, or its circular structure,

5 respectively.

A cyclic derivative containing an intramolecular disulfide bond can be prepared by conventional solid phase synthesis while incorporating suitable S-protected homocysteine or cysteine residues at the positions selected for cyclization such as the terminal amino and carboxyl ends (Sah et al., J Pharm. Pharmacol. 48: 197, 1996). After completing the chain assembly, cyclization can be performed either (1) by selective removal of the protective group of S with a consequent oxidation on support of the two corresponding free SH functions, to form SS bonds, followed by conventional removal of the product from the support and appropriate purification procedure or (2) by removal of the polypeptide from the support together with complete side chain deprotection, followed by oxidation of free SH functions in aqueous solution

15 highly diluted.

The cyclic derivative containing an intramolecular amide bond can be prepared by conventional solid phase synthesis while suitable amino acid derivatives are incorporated into the carboxyl and amino side chain, in the position selected for cyclisation. Cyclic derivatives containing intramolecular -S-alkyl bonds can be prepared by conventional solid phase chemistry while incorporating an amino acid residue with a suitable amino protected side chain, and a suitable S-protected cysteine or homocysteine residue at the position selected for cyclization

Another effective approach to confer resistance to peptidases that act on residues at the N-terminal or

C-terminal of a polypeptide is to add chemical groups to the terminal ends of the polypeptide, so that the modified polypeptide is no longer a substrate for peptidase. A chemical modification of this type is the glycosylation of the polypeptides at either or both terminal ends. It has been shown that certain chemical modifications, in particular N-terminal glycosylation, increase the stability of human serum polypeptides (Powell et al., Pharm. Res. 10: 1268-1273, 1993). Other chemical modifications that enhance serum stability include, but are not limited to, the addition of an N-terminal alkyl group, which consists of a lower alkyl of from one to twenty carbons, such as an acetyl group, and / or the addition of a C-terminal amide or substituted amide group. In particular, the present invention includes modified polypeptides consisting of polypeptides bearing an N-terminal acetyl group and / or a C-terminal amide group.

Other types of polypeptide derivatives containing additional chemical moieties that are not normally part of the polypeptide are also included by the present invention, provided that the derivative retains the desired functional activity of the polypeptide. Examples of such derivatives include (1) N-acyl derivatives of the amino terminal or other free amino group, in which the acyl group may be an alkanoyl group (eg, acetyl, hexanoyl, octanoyl) an aroyl group ( for example, benzoyl) or a blocking group such as F-moc (fluorenylmethyl-O-CO-); (2) esters of the carboxyl terminal or other carboxyl or free hydroxyl group; (3) amide of the carboxyl terminal group or other free carboxyl group produced by reaction with ammonia or with a suitable amine; (4) phosphorylated derivatives; (5) derivatives conjugated with an antibody or other biological ligand and other types of derivatives.

The present invention also encompasses longer polypeptide sequences that result from the addition of additional amino acid residues to the polypeptides of the invention. It would be expected that such longer polypeptide sequences would have the same biological activity (eg, introduction into particular cell types) as the polypeptides described above. Although polypeptides having a substantial number of additional amino acids are not excluded, it is recognized that some large polypeptides may adopt a configuration that masks the effective sequence, thereby preventing binding to a target (e.g., a member of the receptor family LRP such as LRP or LRP2). These derivatives could act as competitive antagonists. Therefore, although the present invention encompasses polypeptides or derivatives of the polypeptides described herein that have an extension, desirably the extension does not destroy the targeting activity of a cell of the polypeptide or derivative.

Other derivatives included in the present invention are double polypeptides consisting of two equal polypeptides.

or two different polypeptides of the present invention covalently linked to each other either directly or through a spacer, such as by a short extension of alanine residues or by a supposed site for proteolysis (for example, by cathepsin, see for example , U.S. Patent No. 5,126,249 and European Patent No. 495,049). The multimers of the polypeptides of the present invention consist of a polymer of molecules formed from the same or different polypeptides or derivatives thereof.

The present invention also encompasses polypeptide derivatives that are chimeric or fusion proteins containing a polypeptide described herein, or fragment thereof, attached at an amino or carboxy-terminal end, or both, to an amino acid sequence of a different protein Such a chimeric or fusion protein can be produced by recombinant expression of a nucleic acid encoding the

protein. For example, a chimeric or fusion protein may contain at least 6 amino acids of a polypeptide of the present invention and desirably has a functional activity equivalent to or greater than a polypeptide of the invention.

The polypeptide derivatives of the present invention can be prepared by altering the amino acid sequences by substitution, addition or deletion or an amino acid residue to provide a functionally equivalent molecule, or functionally enhanced or decreased molecule, as desired. Derivatives of the present invention include, but are not limited to, those which contain, as a primary amino acid sequence, all or part of the amino acid sequence of the polypeptides described herein (for example, any one of SEQ ID NOS: 1-105 and 107-112) including altered sequences containing functionally equivalent amino acid residue substitutions. For example, one or more amino acid residues within the sequence may be substituted by another amino acid of a similar polarity that acts as a functional equivalent, resulting in a silent alteration. The substitution of an amino acid within the sequence can be selected from other members of the class to which the amino acid belongs. For example, amino acids

15 positively charged (basic) include, arginine, lysine and histidine. Non-polar (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 amino acids (acids) include glutamic acid and aspartic acid. The amino acid glycine can be included in either the non-polar family of amino acids or in the family of uncharged (neutral) polar amino acids. In general, it is understood that substitutions made within a family of amino acids are conservative substitutions.

Tests to identify peptidomimetics

As described above, the non-peptidyl compounds generated to replicate the geometry of

The main structure and presentation to pharmacophore (peptidomimetics) of the polypeptides identified by the methods of the present invention often have attributes of greater metabolic stability, greater potency, longer duration of action and better bioavailability.

The peptidomimetic compounds of the present invention can be obtained using any of the numerous approaches in combinatorial library methods known in the art, including: biological libraries; spatially addressable parallel or solid phase parallel libraries; synthetic library methods that require convolution; the library method "a pearl one compound"; and synthetic library methods using affinity chromatography selection. The biological library approach is limited to polypeptide library, while the other four approaches are applicable libraries of polypeptide compounds,

35 non-peptide oligomers or small molecules (Lam, Anticancer Drug Des. 12: 145, 1997). Examples of methods for the synthesis of molecular libraries can be found in the art, for example, in: 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 in Gallop et al. (Med. Chem. 37: 1233, 1994). Compound libraries can be presented in solution (for example, Houghten, Biotechniques 13: 412-421, 1992) or in beads (Lam, Nature 354: 82-84, 1991), lacquers (Fodor, Nature 364: 555-556, 1993), bacteria or spores (U.S. Patent No. 5,223,409), plasmids (Cull et al., Proc. Natl. Acad. Sci. USA 89: 1865-1869, 1992) or phage (Scott and Smith, Science 249: 386-390, 1990), or luciferase, and the enzymatic tag detected by determining conversion of an appropriate substrate into product.

Once a polypeptide of the present invention is identified, it can be isolated and purified by any of several conventional methods including, but not limited to, differential solubility (eg, precipitation), centrifugation, chromatography (eg, affinity, ion exchange , molecular exclusion, and the like) or by any other conventional technique used for the purification of polypeptides, peptidomimetics or proteins. The functional properties of an identified polypeptide of interest can be evaluated using any functional assay known in the art. Desirably, assays are used to evaluate the function of the downstream receptor in intracellular signaling (eg, cell proliferation).

For example, the peptidomimetic compounds of the present invention can be obtained using the following

Three-phase procedure: (1) examine the polypeptides of the present invention to identify regions of secondary structure necessary to select as target types of particular cells described herein; (2) use conformationally restricted dipeptide substitutes to refine the geometry of the main structure and provide organic platforms corresponding to these substitutes; and (3) use the best organic platforms to present organic pharmacophore in candidate libraries designed to mimic the desired activity of the native polypeptide. In more detail the three phases are as follows. In phase 1, the main candidate polypeptides and their limited structure are examined to identify the requirements for their activity. A series of polypeptide analogs of the original is synthesized. In phase 2, the best polypeptide analogs are investigated using conformationally restricted dipeptide substitutes. Amino acids of indolizidin-2-one, indolizidin-9-one and quinolizidinone (I2aa, I9aa and Qaa respectively) are used as platforms for

65 study the geometry of the main structure of the best polypeptide candidates. These platforms and related platforms (reviewed in Halab et al., Biopolymers 55: 101-122, 2000; and Hanessian et al. Tetrahedron

53: 12789-12854, 1997) can be introduced into specific regions of the polypeptide to orient the pharmacophors in different directions. The biological evaluation of these analogs identifies improved main polypeptides that mimic the geometric requirements for the activity. In phase 3, the most active main polypeptide platforms are used to present organic substitutes for the pharmacophore responsible for the activity of the

5 native polypeptide. Pharmacophore and support structures are combined in a parallel synthesis format. The derivation of the polypeptides and the above phases can be performed by other means using methods known in the art.

Function-structure relationships determined from polypeptides, polypeptide derivatives, peptidomimetics or other small molecules of the present invention can be used to refine and prepare analogous molecular structures that have better or similar properties. Accordingly, the compounds of the present invention also include molecules that share the structure, polarity, charge characteristics and side chain properties of the polypeptides described herein.

In summary, based on the description herein, those skilled in the art can develop test assays for polypeptides and peptidomimetics that are useful for identifying compounds for directing an agent to particular cell types (eg, those described in the present document). The assays of this invention can be developed for low performance, high performance or ultra-high performance test formats. The tests of the present invention include tests that are susceptible to automation.

Conjugates of the invention

The polypeptides described herein or derivatives thereof can be linked to an agent. For example, the polypeptide (for example, Angiopep-7) can be linked to a therapeutic agent, a diagnostic agent or a label. In certain embodiments, the polypeptide binds to or is labeled with a detectable label, such as a radioimage agent, for the diagnosis of a disease or condition. Examples of these agents include a radioimage-antibody-vector agent conjugate, in which the antibody binds to a disease or condition specific antigen (for example, for diagnosis or therapy). Other binding molecules are also contemplated by the invention. In other cases, the polypeptide or derivative binds to a therapeutic agent, to treat a disease or a condition, or it can be attached to or labeled with mixtures thereof. The disease or condition can be treated by administering a vector-agent conjugate to an individual under conditions that allow transport of the agent through the BHE or to a particular cell type. Each polypeptide can include at least 1, 2, 3, 4, 5, 6 or 7 agents. In other embodiments, each agent has at least 1, 2, 3, 4, 5, 6, 7, 10, 15, 20, or more polypeptides bound thereto. The conjugates of the invention may promote accumulation (for example, due to

Increased uptake or reduced elimination) of the agent in a particular type of cell or tissue such as liver, lung, kidney, spleen or muscle of a subject.

The agent may be releasable from the vector after transport to a particular cell type or through the BHE. The agent can be released, for example, by enzymatic cleavage or other breakage of a chemical bond between the vector and the agent. Then the released agent can function in its intended capacity in the absence of the vector.

Therapeutic agents

A therapeutic agent can be any biologically active agent. For example, a therapeutic agent can

45 being a drug, a drug, a radiation emitting agent, a cellular toxin (for example, a chemotherapeutic agent), a biologically active fragment thereof, or a mixture thereof to treat a disease (for example, to destroy cancer cells) or it can be an agent to treat a disease or condition in an individual. A therapeutic agent can be a synthetic product or a product of fungal, bacterial or other microorganism (for example, mycoplasma or virus), animal, such as reptile or plant. A therapeutic agent and / or biologically active fragment thereof can be an enzymatically active agent and / or fragment thereof, or it can act by inhibiting or blocking an essential and / or important cellular pathway or by competing with a naturally occurring cellular component. Essential and / or important. Other therapeutic agents include antibodies and antibody fragments.

55 Anti-cancer agents. Any anticancer agent known in the art can be part of a conjugate of the invention. Brain cancers can be treated with a conjugate that contains a vector that is efficiently transported through BHE (for example, Angiopep-3, Angiopep-4a, Angiopep-4b, Angiopep-5 or Angiopep6). Liver, lung, kidney or spleen cancers can be treated with an anticancer agent conjugated to a vector that is effectively transported in the appropriate cell type (eg, Angiopep-7). Exemplary agents include abarelix, aldesleucine, alemtuzumab, alitretinoin, allopurinol, altretamine, amifostine, anakinra, anastrozole, arsenic trioxide, asparaginase, azacitidine, live BCG, bevacuzimab, bexarotamine, bibarotamine, calbibonaceous, calxinone, caprine brometone, blexatone, caprine brometenes, blexate carboplatin, carmustine, celecoxib, cetuximab, chlorambucil, cisplatin, cladribine, clofarabine, cyclophosphamide, cytarabine, dacarbacin, dactinomycin, actinomycin D, dalteparin (for example, sodium), darbepoetin alfa, dasatinina, daoromycin, daatinibine, daatinibine, daatinibine, daatinibine, daatinibine, daatomycin, daatomycin, daatomycin, daatomycin

65 denileucine, denileucine diftitox, dexrazoxane, docetaxel, doxorubicin, dromostanolone propionate, eculizumab, epirubicin (for example, HCl), epoetin alfa, erlotinib, estramustine, etoposide (for example, phosphate), exemestane,

fentanyl (e.g. citrate), filgrastim, floxuridine, fludarabine, fluorouracil, 5-FU, fulvestrant, gefitinib, gemcitabine (e.g., HCl), gemtuzumab ozogamycin, goserelin (e.g., acetate), histrelin (e.g., acetate) , hydroxyurea, ibritumomab thiuxethan, idarubicin, ifosfamide, imatinib (for example, mesylate), interferon alfa2b, irinotecan, lapatinib ditosylate, lenalidomide, letrozole, leucovorin, leuprolide (for example, acetate), levamisole,

5 lomustine, CCNU, mechlorethamine (nitrogen mustard), megestrol, melphalan (L-PAM), mercaptopurine (6-MP), mesna, methotrexate, methoxsalene, mitomycin C, mitothane, mitoxantrone, nandrolone phenopromine, opherominebine, nofrabinebine , oxaliplatin, paclitaxel, palifermin, pamidronate, panitumumab, pegademasa, pegaspargasa, pegfilgrastim, peginterferon alfa-2b, pemetrexed (for example, disodium), pentostatin, pipobroman, plicamycin (mitramycin), porphyrinum, for example, chimeric, for example , rasburicase, rituximab, sargramostim, sorafenib, streptozocin, sunitinib (for example, maleate), talc, tamoxifen, temozolomide, teniposide (VM-26), testolactone, thalidomide, thioguanine (6-TG), thiotepa, topotecan (for example, hcl), toremifene, Tositumomab / I-131 (tositumomab), trastuzumab, tretinoin (ATRA), uracil mustard, valrubicin, vinblastine, vincristine, vinorelbine, vorinostat, zoledronate and zoledronic acid. Exemplary paclitaxel derivatives are described in U.S. Patent No. 6,911,549.

15 Detectable tags

For the purpose of detection or diagnosis, the conjugate of the invention can be labeled. The detectable tags, or markers, can be a radiolabel, a fluorescent tag, an active nuclear magnetic resonance tag, a luminescent tag, a chromophore tag, an isotope that emits positrons for a PET scanner, chemiluminescence tag or an enzymatic tag . Radioimage agents that emit radiation (detectable radiolabels) by way of example include indium-111, technetium-99 or iodine-131 at a low dose. Radionuclides that emit gamma and beta include 67Cu, 67Ga, 90Y, 111In, 99mTc and 201Tl). Positron emitting radionuclides include 18F, 55Co, 60Cu, 62Cu, 64Cu, 66Ga, 68Ga, 82Rb and 86Y. Fluorescent labels include Cy5.5,

25 green fluorescent protein (GFP), fluorescein and rhodamine. Chemiluminescence labels include luciferase and β-galactosidase. Enzymatic labels include peroxidase and phosphatase. A histag can also be a detectable label. For example, the conjugates may include a vector moiety and an antibody moiety (antibody or antibody fragment), which may further include a tag. In this case, the tag can be linked to either the vector or the antibody.

Antibodies

The antibodies can also be conjugated with the polypeptides of the invention by any means known in the art (for example, using the conjugation strategies described herein). Any diagnostic or therapeutic antibody can be conjugated with one or more (eg, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) vectors of the invention. In addition, antibody fragments (for example, which can bind to an antigen) can also be conjugated to the vectors of the invention. Antibody fragments include the Fab and Fc, heavy chain and light chain regions of an antibody (for example, of any antibody described herein). Exemplary antibodies for use in cancer diagnosis and therapy include ABX-EGF (Panitimumab), OvaRex (Oregovemab), Theragyn (pemtumomab-yttrium-90), Therex, bivatuzumab, Panorex (edrecolomab), ReoPro (abciximab ), Bexxar (tositumomab), AcM, 105AD7 idiotypic, Anti-EpCAM (catumaxomab), AcM against lung cancer (Cytoclonal), Herceptin (trastuzumab), Rituxan (rituximab), Avastin (bevacizumab), AMD Fab (ranibizumab) , E-26 (2nd gen. IgE) (omalizumab), Zevalin (Rituxan + yttrium-90) (ibritumomab tiuxethan), cetuximab, BEC2 (mitumomab), IMC-1C11, nuC242-DM1, LymphoCide (epratuzumab), 45 LymphoCide Y -90, CEA-Cide (labetuzumab), CEA-Cide Y-90, CEA-Scan (arcitumomab marked with Tc-99m), LeukoScan (sulesomab marked with Tc-99m), LymphoScan (bectumomab marked with Tc-99m), AFP -Scan (marked with Tc-99m), HumaRAD-HN (+ yttrium-90), HumaSPECT (votumumab), MDX-101 (CTLA-4), MDX-210 (over-expression of her-2), MDX-210 / MAK , Vitaxin, MAb 425, IS-IL-2, Campath (alemtuzumab), Streptavidin from CD20, Avidicin, (albumin + NRLU13), Oncolym (+ iodine-131) Cotara (+ iodine-131), C215 (+ staphylococcal enterotoxin, ACM against lung / kidney cancer (from Pharmacia Corp.), nacolomab tafenatox (staphylococcal enterotoxin C242), Nuvion (visilizumab), SMART M195, SMART 1D10, CEAVac, TriGem, TriAb, NovoMAb-G2 radiolabelled, Monopharm C, GlioMAb-H (+ gelonin toxin), Rituxan (rituxima-IN) . Additional therapeutic antibodies include 5G1,1 (ecluizumab), 5G1.1-SC (pexelizumab), ABX-CBL (gavilimomab), ABX-IL8, Antegren (natalizumab), Anti-CD11a (efalizumab), Anti-CD18 (from Genetech) , Anti-LFA1, Antova, BTI-322, CDP571, CDP850,

55 Corsevin M, D2E7 (adalimumab), Humira (adalimumab), Hu23F2G (rovelizumab), IC14, IDEC-114, IDEC-131, IDEC151, IDEC-152, infliximab (Remicade), LDP-01, LDP-02, MAK- 195F (afelimomab), MDX-33, MDX-CD4, MEDI-507 (siplizumab), OKT4A, OKT3 (muromonab-CD3) and ReoPro (abciximab).

Conjugation Linkers

The conjugate (for example, a protein-protein conjugate) can be obtained using any cross-linking reagent (conjugation) or protocol known in the art, many of which are commercially available. Such protocols and reagents include crosslinking reagent with amino, carboxyl, sulfhydryl, carbonyl, carbohydrate and / or phenol groups. The amounts, times and conditions of such protocols can be varied to optimize conjugation. Crosslinking reagents contain at least two reactive groups and are generally divided into homofunctional crosslinking agents (containing identical reactive groups) and agents

heterofunctional crosslinking (which do not contain identical reactive groups). The crosslinking agents of the invention can be either homobifunctional and / or heterobifunctional. In addition, the crosslinking agent can incorporate a "spacer" between the reactive moieties, or the two reactive moieties in the crosslinking agent can be attached directly. The links may include ester links.

5 Exemplary linkers include BS3 [Bis (sulfosuccinimidyl) suberate], NHS / EDC (N-hydroxysuccinimide and Netyl- (dimethylaminopropyl) carbodimide, sulfo-EMCS ([Ne-maleimidocaproic acid] hydrazide), SATA (N-succinimidyl -Sacetyl thioacetate) and hydrazide BS3 is a homobifunctional N-hydroxysuccinimide ester that selects accessible primary amines as a target A conjugation scheme is shown as an example in Figure 2. NHS / EDC allows the conjugation of primary amine groups with carboxyl groups. Sulfo-EMCS are heterobifunctional reactive groups (maleimide ester and NHS) that are reactive for sulfhydryl and amino groups.The amine coupling using sulfo-NHS / EDC activation can be used to cross-link the therapeutic antibodies with the polypeptides of the invention, as shown as an example in figures 3 and 4. This is a quick, simple and reproducible coupling technique.The resulting conjugate is stable and retains the biological activity ica of

15 antibody. In addition, it has a high conjugation capacity that can be reliably controlled and a low non-specific interaction during the coupling procedures. SATA is reactive for amines and adds protected sulfhydryl groups. The NHS ester reacts with primary amines to form stable amide bonds. The sulfhydryl groups can be deprotected using hydroxylamine. This method of conjugation is shown as an example in Figure 5. Hydrazide can be used to bind carboxyl groups to primary amines, as shown in Figure 6, and therefore may be useful for binding glycoproteins. Additional exemplary linkers are illustrated in Figure 7.

Small molecules such as therapeutic agents can be conjugated with the polypeptides of the invention. The small example molecule, paclitaxel, has two strategic positions (position C2 ’and C7) useful for conjugation. The conjugation of a vector or vector of the invention with paclitaxel can be performed as follows (Figure 8). In summary, paclitaxel is reacted with anhydrous succinic pyridine for three hours at room temperature to bind a succinyl group at position 2 ’. 2’-succinyl-paclitaxel has an ester bond that can be cleaved at the 2 ’position, it can easily release succinic acid. This ester linkage that can be cleaved can be used additionally for various modifications with linkers, if desired. The resulting 2'-O-succinyl-paclitaxel is then reacted with EDC / NHS in DMSO for nine hours at room temperature, followed by the addition of the vector or vector in Ringer / DMSO for an additional reaction time of four hours at temperature ambient. The conjugation reaction depicted in Figure 8 is monitored by HPLC. Each intermediate product, such as paclitaxel, 2’-O-succinyl-paclitaxel and 2’-O-NHS-succinylpaclitaxel, is purified and validated using different approaches such as HPLC, layer liquid chromatography

35 fine, NMR (13C or 1H exchange), melting point or mass spectrometry. The final conjugate is analyzed by mass spectrometry and SDS-polyacrylamide gel electrophoresis. This allows to determine the number of paclitaxel molecules conjugated with each vector.

Conjugate activities

By conjugating the agent with a vector described herein, desirable properties can be achieved, such as altered pharmacokinetics, altered tissue distribution (eg, increased supply to particular tissues or cell types such as liver, brain, lung, spleen or kidney). ). In summary, vectors that efficiently transport agents through BHE have been identified (for example, Angiopep-3, Angiopep-4a,

45 Angiopep-4b, Angiopep-5 and Angiopep-6). Like Angiopep-2, these vectors can also select agents for other types of cells or tissues (for example, liver, lung, kidney, spleen or muscle). A vector, Angiopep-7, has also been identified, which is not transported efficiently through BHE, but is transported to particular tissues (eg, liver, lung, kidney, spleen or muscle). Therefore, vectors with this activity can be useful when transport through the BHE is not desired.

Because the conjugates of the invention transport agents to specific tissues, the conjugated agents may result in lower toxicity (e.g., minor side effects), superior efficacy (e.g., because the agent is concentrated in a target tissue due to to increased uptake or decreased efflux of tissue or cells or because the agent has greater stability when conjugated), or a combination of the

55 same. Such activities are described below and in International Publication No. WO 2007/009229.

In some cases, the conjugation of an agent with a vector allows the agent to escape the action of glycoprotein P (P-gp), an efflux pump that can expel certain agents from a cell. By decreasing the ability of P-gp to expel an agent from a cell, the potency of this agent in a cell can be increased. Therefore these conjugates can actively inhibit the proliferation of cancer cells. In addition, the results obtained for tumor growth in vivo indicate that the vectors of the invention can target the LRP receptor. In addition, conjugation may modify the pharmacokinetics or biodistribution of the unconjugated agent.

65 Taken together, the conjugates can be used against primary tumors including breast, lung and skin cancers as well as metastases originating from primary tumors.

Avoid P glycoprotein

Because resistance to various chemotherapeutic agents such as vincristine, etoposide and doxorubicin

5 is mediated through the overexpression of P-gp (MDR1) (figure 9), avoiding this efflux pump can enhance the action of these drugs in various types of cancer. By conjugating a vector with such an agent, it is now possible to decrease the efflux of the agent mediated by P-gp. These conjugates may be useful for increasing the potency of drugs associated with resistance mediated by P-gp. As shown as an example below, the vectors described herein were tested to determine their ability to prevent expulsion by P-gp.

LRP mediated acquisition

Based on previous work (see International Patent Application No. PCT / CA2004 / 00011), associated protein

15 with receptor (RAP) inhibited aprotinin transcytosis in an in vitro model of the blood brain barrier. LRP is expressed in a variety of cancer cells (see, for example, International Publication No. WO 2007/009229). Therefore it has been proposed that the low density lipoprotein-related receptor (LRP) is involved in the penetration of aprotinin in the brain. Similar inhibition of the transport of Angiopep-1 and Angiopep-2 was also obtained through an in vitro model of the blood-brain barrier (data not shown) suggesting that transcytosis of this polypeptide through the cerebral endothelial cell also involved LRP Based on this, it is believed that LRP may be involved, in general, in the uptake of the aprotinin-related polypeptides described herein.

LRP is a 600 kDa heterodimeric membrane receptor composed of two subunits; the subunit-�

25 (515 kDa) and the β-subunit (85 kDa). Because LRP may be involved in the transport of a vector as described herein, the conjugates of the invention can select as target cells and tumors expressing this receptor. Certain cancer cells, for example, express a member of the LRP (for example, LRP or LRP2).

Treatments

The invention also presents the polypeptide conjugates described herein for use in a method for treatment. Conjugates that are efficiently transported through BHE (for example, Angiopep-3, Angiopep-4a, Angiopep-4b, Angiopep-5 and Angiopep-6) can be used in a method to treat

35 any disease of the brain or central nervous system. These conjugates are also efficiently transported to the liver, lung, kidney, spleen or muscle and therefore can also be used, in conjunction with an appropriate therapeutic agent, to treat a disease associated with these tissues (eg, cancer). Because Angiopep-7 is not transported effectively to the brain, but it is transported effectively to tissues and cells such as liver, lung, kidney, spleen and muscle, Angiopep-7 can be especially well suited as a vector treatment. diseases associated with these tissues when targeting the agent to the brain is not desired.

Liver diseases include amoebic liver abscess, cirrhosis, disseminated coccidioidomycosis; drug-induced cholestasis, hemochromatosis, hepatitis A, hepatitis B, hepatitis C, hepatitis D, carcinoma

45 hepatocellular, liver cancer, liver disease due to alcohol, primary biliary cirrhosis, pyogenic liver abscess, Reye's syndrome, sclerosing cholangitis and Wilson's disease. Amebic liver abscess can be treated by administering a vector conjugated with metronidazole. Hepatitis B can be treated, for example, by administering a conjugate vector with interferon-alpha, lamivudine, adefovir dipivoxil, entecavir or another antiviral agent. Hepatitis C can be treated, for example, by administering a vector conjugated to ribavirin or pegylated interferon, or a combination thereof.

Lung diseases include lung cancers such as small cell carcinoma (eg, oat cell cancer), mixed large / small cell carcinoma, combined small cell carcinoma and metastatic tumors. Metastatic tumors can originate from cancer of

55 any tissue, including breast cancer, colon cancer, prostate cancer, sarcoma, bladder cancer, neuroblastoma and Wilm's tumor. Spleen diseases include cancers such as lymphoma, non-Hodgkin lymphoma and certain T-cell lymphomas.

Additional exemplary cancers that can be treated using a conjugate or a composition of the invention include hepatocellular carcinoma, breast cancer, head and neck cancers including various lymphomas such as mantle cell lymphoma, non-Hodgkin lymphoma, adenoma, carcinoma squamous cell carcinoma, laryngeal carcinoma, retinal cancers, esophageal cancers, multiple myeloma, ovarian cancer, uterine cancer, melanoma, colorectal cancer, bladder cancer, prostate cancer, lung cancer (including non-cell lung carcinoma small), pancreatic cancer, cervical cancer, head and neck cancer, 65 skin cancers, nasopharyngeal carcinoma, liposarcoma, epithelial carcinoma, renal cell carcinoma, gallbladder adenocarcinoma, parotid adenocarcinoma, endometrial sarcoma, cancers resistant to

multiple drugs; and proliferative diseases and conditions, such as neovascularization associated with tumor angiogenesis, macular degeneration (e.g., wet / dry AMD), corneal neovascularization, diabetic retinopathy, neovascular glaucoma, myopic degeneration and other proliferative diseases and conditions such as restenosis and disease polycystic kidney. Brain cancers that can be treated with a vector that is effectively transported through BHE include astrocytoma, pilocytic astrocytoma, disembrioplastic neuroepithelial tumor, oligodendrogliomas, ependymoma, glioblastoma multiforme, mixed gliomas, oligoastrocytomas, medulloblastoma, neuroblastoma, retinoblastoma, neuroblastoma, retinoblastoma, neuroblastoma, retinoblastoma, neuroblastoma .

A conjugate or composition of the invention can be administered by any means known in the art; for example, orally, intraarterially, intranasally, intraperitoneally, intravenously, intramuscularly, subcutaneously, transdermally, or by mouth to the subject. The agent can be, for example, an antiangiogenic compound.

Pharmaceutical compositions

The pharmaceutical compositions of the invention may include a polypeptide or conjugate described herein, in association with a pharmaceutically acceptable carrier. Such compositions are liquid or lyophilized or otherwise dry formulations and include diluents of various buffer contents (eg, Tris-HCl, acetate, phosphate), pH and ionic strength, additives such as albumin or gelatin to avoid surface absorption, detergents (e.g., Tween 20, Tween 80, Pluronic F68, bile acid salts). Solubilizing agents (for example, glycerol, polyethylene glycerol), antioxidants (for example, ascorbic acid, sodium metabisulfite), preservatives (for example, thimerosal, benzyl alcohol, parabens), fillers or tonicity modifiers (for example, lactose, mannitol), covalent binding of polymers such as polyethylene glycol to the protein, complexation with metal ions, or incorporation of the material into or on particulate preparations of polymeric compounds such as poly (lactic acid), poly (glycolic acid), hydrogels, etc., or on liposomes, microemulsions, micelles, unilamellar or multilamellar vesicles, erythrocyte ghosts, or spheroplasts. Such compositions will influence the physical state, solubility, stability, in vivo release rate, and in vivo clearance rate. Controlled or sustained release compositions include formulation in lipophilic deposits (eg, fatty acids, waxes, oils). Also included by the invention are particulate compositions coated with polymers (for example, poloxamers or poloxamers). Other embodiments of the compositions of the invention incorporate protective coatings of particulate forms, protease inhibitors or permeation enhancers for various routes of administration, including parenteral, pulmonary, nasal, oral, vaginal, rectal routes. In one embodiment the pharmaceutical composition is administered parenterally, paracancerally, transmucosally, transdermally, intramuscularly, intravenously, intradermally, subcutaneously, intraperitoneally, intraventricularly, intraventricularly. intracranial and intratumoral.

Pharmaceutically acceptable carriers further include 0.01-0.1 M or 0.05 M phosphate buffer or 0.8% saline. Additionally, such pharmaceutically acceptable carriers may 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 dextrose, dextrose and sodium chloride, lactated fixed or Ringer oils. Intravenous vehicles include fluid and nutrient replenishers, electrolyte replenishers such as those based on Ringer's dextrose and the like. Preservatives and other additives may also be present, such as, for example, antimicrobial agents, antioxidants, intercalating agents, inert gases and the like.

Other formulations include polyoxyethylene esters of a fatty acid (eg, 12-hydroxystearic acid) such as Solutol® HS 15. Thus, in some embodiments, a pharmaceutical composition may comprise a) a conjugate described herein, b) Solutol® HS15 and c) an aqueous solution or buffer (for example, Ringer / Hepes solution at a pH of 5 to 7). The concentration of Solutol® HS 15 in the formulation can be at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45% , 50% or 60% (for example, 30%) or within any interval between any two of these numbers. The concentration of conjugate can be determined based on the dose required to effectively treat a subject, or the amount of the ester required for the solubility of the conjugate being administered. The use of Solutol in a formulation for the administration of a taxol conjugate is described, for example, in International Publication No. WO 2007/009229.

Solid pharmaceutical forms for oral use

Formulations for oral use include tablets containing the active ingredient (s) in a mixture with non-toxic pharmaceutically acceptable excipients, and those skilled in the art are aware of such formulations (e.g., U.S. Pat. Nos. 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). These excipients may be, for example, inert diluents or fillers (for example, sucrose, sorbitol, sugar, mannitol, microcrystalline cellulose, starches including potato starch, calcium carbonate, sodium chloride, lactose, calcium phosphate, calcium sulfate or sodium phosphate); agents of

granulation and disintegration (eg, cellulose derivatives including microcrystalline cellulose, starches including potato starch, croscarmellose sodium, alginates or alginic acid); binding agents (eg, sucrose, glucose, sorbitol, gum arabic, alginic acid, sodium alginate, gelatin, starch, pregelatinized starch, microcrystalline cellulose, magnesium aluminum silicate, sodium carboxymethylcellulose, methylcellulose,

Hydroxypropyl methylcellulose, ethyl cellulose, polyvinyl pyrrolidone or polyethylene glycol); and lubricating, sliding, and non-stick agents (for example, magnesium stearate, zinc stearate, stearic acid, silica, hydrogenated vegetable oils or talc). Other pharmaceutically acceptable excipients may be colorants, flavoring agents, plasticizers, humectants, buffering agents, and the like.

The tablets may be uncoated or they may be coated by known techniques, optionally to delay disintegration and absorption in the digestive tract and thereby provide sustained action over a longer period. The coating can be adapted to release the agent in a predetermined pattern (for example, in order to achieve a controlled release formulation) or it can be adapted to not release the agent (s) until after passing through the stomach (enteric coating ). The coating can be a

Sugar coating, a film coating (for example, based on hydroxypropylmethylcellulose, methylcellulose, methylhydroxyethylcellulose, hydroxypropylcellulose, carboxymethylcellulose, acrylate copolymers, polyethylene glycols and / or polyvinylpyrrolidone), or an enteric coating (for example, methacrylic acid , cellulose phthalate-acetate, hydroxypropylmethylcellulose phthalate, hydroxypropylmethylcellulose succinate-acetate, polyvinyl phthalate-acetate, shellac and / or ethyl cellulose). In addition, a temporary delay material such as, for example, glyceryl monostearate or glyceryl distearate can be employed.

Solid tablet compositions may include a coating adapted to protect the composition from unwanted chemical changes, (eg, chemical degradation before the release of the active substances). The coating may be applied on the solid pharmaceutical form in a manner similar to that described in the

25 Encyclopedia of Pharmaceutical Technology, cited above.

The compositions of the invention can be mixed together in the tablet, or they can be distributed. In one example, a first agent is contained inside the tablet, and a second agent is outside, so that a substantial part of the second agent is released before the release of the first agent.

Formulations for oral use may also be presented as chewable tablets, or as hard gelatin capsules in which the active ingredient is mixed with an inert solid diluent (for example, potato starch, lactose, microcrystalline cellulose, calcium carbonate, phosphate of calcium or kaolin), or as soft gelatin capsules in which the active substance is mixed with water or an oily medium, for example, peanut oil,

35 liquid paraffin or olive oil. Powders and granules can be prepared using the above-mentioned components in tablets and capsules in a conventional manner using, for example, a mixer, a fluid bed apparatus, or spray drying equipment.

Dosages

The dosage of any conjugate or composition described herein or identified using the methods described herein depends on several factors, including: the method of administration, the disease (eg, cancer) to be treated, the severity of the disease, if the cancer is to be treated or prevented, and the age, weight and health of the subject to be treated.

With respect to the methods of treatment of the invention, it is not intended that the administration of a vector, conjugate or composition to a subject be limited to a particular mode of administration, dosage or dosage frequency; The invention contemplates all modes of administration. The conjugate or composition can be administered to the subject in a single dose or in multiple doses. For example, a compound described herein or that is identified using conjugate selection methods of the invention can be administered once a week for, for example, 2, 3, 4, 5, 6, 7, 8, 10, 15, 20, or more weeks. It should be understood that, for any particular subject, the specific dosage regimens must be adjusted over time according to the individual need and professional judgment of the person administering or supervising the administration of the vector, conjugate or composition. For example, the dosage of a conjugate can be increased if the lower dose does not

55 provides sufficient activity in the treatment of a disease or condition described herein (eg, cancer). Conversely, the dosage of the compound can be decreased if the disease (for example, cancer) is reduced or eliminated.

Although the attending physician will ultimately decide the appropriate amount and dosage regimen, a therapeutically effective amount of a vector, conjugate or composition described herein, may be, for example, in the range of 0.0035 µg to 20 µg / kg body weight / day or 0.010 µg to 140 µg / kg body weight / week. Desirably a therapeutically effective amount is in the range of 0.025 µg to 10 µg / kg, for example, 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 administered daily, every other day, or twice a week. In addition, a therapeutically effective amount may be in the range of 0.05 µg to 20 µg / kg, for example, at least 0.05, 0.7,

0.15, 0.2, 1.0, 2.0, 3.0, 4.0, 5.0, 6.0, 7.0, 8.0, 10.0, 12.0, 14, 0, 16.0 or 18.0 µg / kg of body weight administered weekly, every two weeks or once a month. In addition, a therapeutically effective amount of a compound may be, for example, in the range of 100 µg / m2 to 100,000 µg / m2 administered every two days, once a week, or every two weeks. In a desirable embodiment, the therapeutically effective amount is in the

5 range of 1,000 µg / m2 to 20,000 µg / m2, for example, at least 1,000, 1,500, 4,000 or 14,000 µg / m2 of the compound administered daily, every other day, twice a week, weekly, or every two weeks.

The following examples are intended to illustrate, rather than limit the invention.

10 Example 1

Conjugate distribution and pharmacokinetics

The effect of conjugation of an agent with a vector on agent distribution can be assessed by administering

15 a conjugate or polypeptide labeled to an animal and measuring the distribution of the polypeptide or conjugate to organs. In one example, either 3H-Taxol (5 mg / kg) or 125I-Taxol-Angiopep-1 (TxlAn-1) (10 mg / kg, equivalent to 5 mg Taxol / kg) to mice. Similar experiments can be performed with any of Angiopep-3, Angiopep-4a, Angiopep-4b, Angiopep-5, Angiopep-6 and Angiopep-7. In this case, the unconjugated anticancer agent and the conjugates are injected intravenously into mice as a bolus. Tissues are collected at different times (0.25,

20 0.5, 1 and 4 h) and homogenize. To quantify the amount of 3H-Taxol, tissue homogenates are digested with tissue solubilizer, and 10 ml of liquid scintillator was added to samples. The amount of the conjugate labeled with 125I in the different tissues is measured after TCA precipitation. The radioactivity associated with tissues is quantified. The area under the curve (AUC 0-4) used by the Prism software is estimated and plotted for the different tissues. Using an Angiopep-1 conjugate, the AUC 0-4 values obtained for the conjugate are

25 higher than those of Taxol in various tissues including the brain, kidney, liver and eyes (Figure 10A) indicating a superior accumulation of the conjugate in these tissues compared to the unconjugated agent. The accumulation of the conjugate is much higher than the unconjugated agent in the lung (Figure 10B).

The results of a similar experiment performed with the Taxol-Angiopep-2 conjugate are summarized in Table 4.

30 below. Although there is a difference with respect to the results obtained for the TxlAn-1 conjugate, the conjugate in Table 4 also accumulates in the lungs, brain and liver more effectively than unconjugated Taxol.

Table 4

AUC 0-4 ( g / g of tissue)

TxlAn-2 Taxol Reason (TxlAn-2 / Taxol)

Plasma 170 2.2 77.3

Brain 0.32 0.07 4.6

Lung 3.4 1.1 3.0

Kidney 11.2 8.0 1.4

Heart 5.0 2.5 2.0

Liver 513 22 23

Eye 0.99 0.57 1.7

Urine 35.7 88 0.4

35 Treatments equivalent to 5 mg / kg of Taxol

The kinetics of the accumulation of Taxol and Taxol-Angiopep-1 in the lung were studied (Figure 11). The amount of the conjugate measured in the lungs at different times is much higher than that of the unconjugated agent. The accumulation of the conjugate in the lung is also much higher than its concentration in the serum (plasma) to

40 different times (figure 12). These results indicate that the biodistribution or pharmacokinetics of an anticancer agent such as Taxol can be altered by conjugation with a vector of the invention (for example, Angiopep-1 or 2).

Example 2 Angiopep-7 is not transported effectively to the brain

Angiopep-7 is not transported effectively through the blood brain barrier. Measuring the accumulation of Angiopep-2 and Angiopep-7 in the rat brain 30 minutes after IV injection, Angiopep-7 was present in

the brain at levels below either Angiopep-1 or Angiopep-2 (Figure 13).

The accumulation of Angiopep-2 and Angiopep-7 in the brain has also been examined using fluorescence microscopy after a 10-minute cerebral perfusion in situ. In this case, 2 mM of Angiopep-2 or 5 Angiopep-7, each conjugated with the fluorescent marker Cy5.5, was injected into the carotid artery. The brains of the animals were then evaluated by fluorescence microscopy. In each case, the capillaries were stained with Vessel Green (FITC-lectin), the brain nuclei were stained with DAPI (blue), and the Angiopeps were visualized using the Cy5.5 label (Figure 14). From these sections, it is observed that Angiopep-2 is going to be located inside the brain, while it is observed that Angiopep-7 is going to be located inside the capillaries. The

10 inferior cerebral accumulation of Angiopep-7 compared to Angiopep-2 in vivo. Each of Angiopep-2 and Angiopep-7 were conjugated with Cy5.5 and administered to rats. After 30 minutes, the accumulation in the brain was measured using the fluorescence of the Cy5.5 indicator. Based on this, it is concluded that Angiopep-7 is not transported effectively through the BHE, while Angiopep-2 is transported through the BHE effectively.

Example 3

Angiopep-7 is effectively transported to the liver, kidney, lungs, spleen and muscle

20 Using rats administered Angiopep-2 or Angiopep-7 conjugated with Cy5.5 as described in example 2, the 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 with Angiopep-7 compared to Angiopep-2. In view of these results, it is believed that Angiopep-7 may be especially useful for the administration of agent to the liver, kidney and lungs in applications where administration to the brain is not desired.

25 To measure Angiopep-7 concentrations in animals, imaging studies of Angiopep-7 conjugated with Cy5.5 have been performed. Based on imaging studies in vivo 30 minutes after injection, the accumulation of the Cy5.5 signal was observed in the kidneys, liver and lungs (Figure 15A). Ex vivo organ analysis was performed 24 hours after Angiopep-7 injection, and compared with injection using

30 Angiopep-2. These studies also revealed significant accumulation of both Angiopep-2 and Angiopep-7 in the liver, lungs and kidneys. However, Angiopep-7 showed significantly lower brain accumulation compared to Angiopep-2 (Figure 15B). Based on these results, Angiopep-7 is believed to be useful as a vector for agents in peripheral organs such as the liver, kidney, lungs, spleen or muscle in applications where administration to the brain is not desired.

Example 4

IgG conjugate transport

40 The brain uptake of these vectors was measured using the experimental brain perfusion model in situ. The results (shown in table 5 below) revealed that lysines at position 10 and 15 are important in the ability of these vectors to cross the EBR.

Table 5: Angiopeps brain uptake measured by cerebral perfusion in situ

Vector

Parenchyma (ml / 100 g) Transport with respect to Angiopep-1 (250 nM), expressed as a percentage

Angiopep-1 (50 nM)

34.9 155.46

Angiopep-1 (250 nM)

22.45 100.00

Angiopep-1 (dimer) (250 nM)

27.03 120.40

Angiopep-2 (250 nM)

19.62 87.39

Angiopep-3 (250 nM)

17.08 76.08

Angiopep-4a (250 nM)

17.57 78.26

Angiopep-4b (250 nM)

12.05 53.67

Angiopep-5 (250 nM)

11.82 52.65

Angiopep-6 (250 nM)

8.64 38.49

Angiopep-7 (250 nM)

2.99 13.32

The Angiopep polypeptides were conjugated with IgG using the SATA crosslinking agent and their transport capacity was studied using brain perfusion in situ. As shown in Figure 16, Angiopep-2 is the

most highly distributed conjugate in the brain parenchyma. It also seems that polypeptide dimers are transported efficiently. Angiopep-7 was not transported effectively in the brain.

Example 5

Effect of conjugates on cell growth in vitro

To test the ability of conjugates that include a vector and an anticancer agent to kill cancer cells, an in vitro test can be performed. In one example, Taxol (unconjugated) 10 is shown to block the proliferation of glioblastoma cells (U-87) with an IC50 value of about 10 nM (Figure 17). The effect of conjugated Taxol is evaluated when conjugated with a vector described herein and compared to unconjugated Taxol. As shown in Table 6A, the IC50 values obtained for the Taxol-Angiopep-2 conjugate (TxlAn2) were very similar to those for unconjugated Taxol in many cancer cells. Rat brain endothelial cells (RBE4) were less sensitive than cell lines

15 cancerous women tested. For comparison purposes, the results obtained regarding the concentration of Taxol were expressed.

Most of these cells (U-87, U-118, NC1-H460, A549) express LRP. However, these data are not available for RBE4 cells. The antiproliferation activity of the conjugate against cancer cells in vitro is evaluated. In this assay, cancer cells (U87 and U118) are exposed for 48 hours to an anticancer agent (for example, Taxol) and conjugate (for example, TxlAn2 conjugate (3: 1)). The incorporation of [3 H] -thymidine into U87 and U 118 cells decreases as a function of the concentration of the agent. The values required to inhibit cell proliferation in 50% (IC50) are expressed. The results obtained from the proliferation assays indicate that the IC50 values required for the inhibition of cancer cell proliferation are expressed in nM and

25 demonstrate that the TxlAn2 conjugate (3: 1) is 3 times more potent than paclitaxel, and they are in the same range when they are reported as equivalent to paclitaxel (Table 6B). Similar experiments can be performed using any of the vectors or conjugates described herein.

The ability of a conjugate (for example, TxlAn2 (3: 1)) to block the proliferation of others is also estimated

30 types of cancer cells. Such tests can be performed in lung cancer cells (NC1-H460) as well as the breast cancer cell line (MDAMB231, MDA-MB-468, HCC-1954, BT-474) in hepatocarcinomas (SK-Hepl) and glioblastomas (U-87MG). It has been shown that such cells are also very sensitive to the TxlAn2 conjugate (3: 1) (table 6B).

35 Table 6A: Effect of the conjugate on cell proliferation.

IC50 (nM)

Taxol cell lines

Taxol-Angiopep-2 (3: 1)

Glioblastomas 5 U-87 9.5 9.7 U-118 7.2 8.1

Lung carcinoma NCl * -H460 9.3 12.5 A549 3.6 6.0 10 Calu-3 17.2 25.0

Endothelial cells RBE4 137 139

Table 6B: Taxol in vitro cytotoxicity and TxlAn2 conjugate (3: 1)

Paclitaxel

TxlAn2 conjugate (3: 1)

Line

IC50 % of survival IC50 % of survival

mobile

(nM) residual (nM) residual

Half

FROM  Half FROM  Half FROM  Half FROM

BT-474

62.86 - 52.24 3.87 40.22 - 51.83 1.70

HCC1954

6.12 - 48.82 11.17 8.26 3.37 44.08 8.63

MDA-MB-231

17.61 - 45.62 13.89 28.16 - 46.95 7.50

MDA-MB-468

13.52 - 54.26 10.34 1.41 - 55.34 5.55

30

NCl-H460 6.61 3.35 24.98 10.80 12, 68 10.08 30.44 9.36

SK-HEP-1 8.84 -55.18 13.17 7.83 -54.09 11.03

U-87 MG 12.94 2.49 40.35 2.40 17.75 10.82 44.13 1.85

Example 6

In vivo tumor growth inhibition (U-87)

5 The ability of a conjugate to inhibit tumor growth in an in vivo model can be evaluated (see, for example, Figure 18A). U-87 cells are implanted subcutaneously on the right side of mice and, on day 3 after implantation, the vehicle is injected into the mice (DMSO / Ringer: 80/20; control), an unconjugated agent (by for example, Taxol (5 mg / kg)) or the agent as part of a conjugate (for example, Taxol-Angiopep-2 (10 mg / kg)

10 equivalent to 5 mg of Taxol / kg). Tumor growth inhibition is more pronounced in mice treated with the conjugate than in mice treated with the unconjugated anticancer agent.

In one example, on day 17 after implantation, tumor growth was inhibited in more than 75% using TxlAn2, while tumor growth was inhibited in 34% using the unconjugated agent (Table 7). Therefore, the

15 conjugates are more effective than unconjugated agents in inhibiting tumor growth in vivo. In general, a tumor growth inhibition level of 2.2 times for the conjugate was measured compared to the unconjugated agent.

Table 7. Inhibition of tumor growth with conjugates.

Growth Inhibition T / C

Groups Tumor volume (mm3)

tumor (%) (%) (mean eem) Days after injection Δ (mm3) Day 0 * Day 14 **

Control 79 ± 7 289 ± 50 203 ± 47 100

Taxol

74 ± 5 219 ± 52 134 ± 55 34 66

(5 mg / kg)

TxlAn2 (3: 1)

88 ± 9 144 ± 27 56 ± 32 73 27

(10 mg / kg)

20 Treatment equivalent to 5 mg / kg of Taxol * corresponds to 3 days after implantation (first treatment) ** corresponds to 17 days after implantation (after 4 treatments)

Example 7

In vivo tumor growth inhibition (hepatocarcinoma)

In vivo studies are performed to determine if a conjugate can inhibit the growth of hepatocarcinoma cells (SK-Hep 1) implanted subcutaneously. Naked mice receive an injection

30 subcutaneous 2.5 x 106 human SK-Hep 1 cells on its right side. Treatments are initiated when the size of the implanted tumor reached approximately 200 mm 3. Animals are treated with a conjugate or vehicle by intraperitoneal injections.

The results of an experiment performed with a TxlAn2 (3: 1) administered to

35 80 mg / kg by intraperitoneal injection; treatments are indicated by black arrows. In this experiment, treatments were provided twice a week for five maximum treatments at the indicated dose of 80 mg / kg. The conjugate of TxlAn2 (3: 1) administered i.p. It shows high efficacy in the inhibition of the growth of hepatocarcinomas, a type of cancer usually not sensitive to Taxol® (Figure 18B).

40 Example 8

Methods to evaluate conjugate activity

The following methods were used in the examples described herein. 45 Cell Proliferation Assay

For the in vitro cell proliferation assay, between 2.5 and 5 x 104 U87 or A549 cells are seeded in a 24-well tissue culture microplate in a final volume of 1 ml of medium with 10% serum and incubated for 24 hours at 37 ° C and 5% CO2. The medium is then replaced with serum-free medium and incubated overnight. The next morning the agent was dissolved in dimethylsulfoxide (DMSO) recently and the

5 medium completely containing the agent at different concentrations in triplicate. The final concentration of DMSO is 0.1%. The control used is a microplate well with cells and without agent. The cells are incubated for 48 to 72 h at 37 ° C and 5% CO2. After incubation, the medium is changed and replaced with 1 ml of complete medium containing [3 H] -thymidine (1 pCi / assay). The plate is incubated at 37 ° C and 5% CO2 for 4 h. The medium is removed, and the cells are washed with PBS at 37 ° C. The cells are fixed with a mixture of ethanol: acetic acid (3: 1), washed with water, and precipitated 3 times with 10% TCA (trichloroacetic acid) ice cream. Finally, 500 µl of PCA (perchloric acid) is added to the wells and the microplates are heated for 30 min at 65 ° C and 30 min at 75 ° C. The contents of each well are then transferred in a scintillation vessel with 10 ml scintillation cocktail and the activity in CPM (counts per minute) is measured in a Packard Tri-Carb liquid scintillation counter.

15 Polypeptide iodination

Polypeptides are iodinated with conventional procedures using Sigma iodine beads. In summary, the polypeptides are diluted in 0.1 M phosphate buffer, pH 6.5 (PB). Two pearls of iodine are used for each protein. These beads are washed twice with 3 ml of PB in a Whatman filter and resuspended in 60 µl of PB. 125I (1 mCi) of Amersham-Pharmacia biotech is added to the pearl suspension for 5 min at room temperature. Each iodination was initiated by the addition of the polypeptide (100 µg). After a 10 min incubation at room temperature, free iodine was removed by HPLC.

Subcutaneous implantation

25 In order to estimate the efficacy of conjugates and formulations on tumor growth, a subcutaneous model of glioblastomas has been developed. In this model, 2.5 x 106 cells are injected subcutaneously in 100 µl of serum-free cell medium containing 1% methylcellulose on the side of mice. The tumor is clearly visible and can be measured using a Vernier caliber. Then the estimated tumor volume was plotted as a function of time.

Mouse brain perfusion in situ

The uptake of [125I] -polypeptides on the luminal side of mouse cerebral capillaries is measured using the method of

35 cerebral perfusion in situ adapted in the laboratory for the study of agent uptake in the mouse brain. In summary, the right common carotid of mice anesthetized with ketamine / xylazine (140/8 mg / kg i.p.) is exposed and linked to the level of the common carotid bifurcation, rostral to the occipital artery. The common carotid is then catheterized in a rostral manner with polyethylene tube loaded with heparin (25 U / ml) and mounted on a 26 gauge needle. The syringe containing the perfusion fluid ([125I] -polypeptides or [14C] is placed ] -inulin in Krebs / bicarbonate buffer at pH 7.4 gasified with 95% O2 and 5% CO2) in an infusion pump (Harvard pump PHD 2000; Harvard apparatus) and connects to the catheter. Before perfusion, the contribution of contralateral blood flow is eliminated by rupturing ventricles of the heart. The brain is perfused during the indicated times at a flow rate of 1.15 ml / min. After 14.5 min of perfusion, the brain is further perfused for 60 s with Krebs buffer to wash the excess of [125I] -proteins. Then the mice are decapitated

45 to end the perfusion and the right hemisphere is isolated on ice before being subjected to capillary depletion. Aliquots of homogenates, supernatants, granules and perfusions are taken to measure their content in [125I] -conjugates by precipitation with TCA and evaluate the apparent volume of distribution.

Example 9

Mechanism 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 for 24 h with either free Taxol (30 nM) or a conjugate

55 of Taxol (for example, 10 nM TxlAn2; equivalent to 30 nM of Taxol; Figure 19). After the cells are labeled for β-tubulin using a secondary antibody bound to FITC, photographs are taken in visible and fluorescence modes. In this example, both Taxol and the Taxol conjugate have similar effects on β-tubulin that lead to its polymerization. As shown by way of example in Figure 20, the addition of Taxol and the Taxol conjugate induces the blocking of NCI-H460 cells in the G2 / M phase. These results suggest that the TxlAn conjugate has a similar mechanism of action on cancer cells as Taxol.

Example 10

Conjugate Preparation

Figures 23A-23B show by way of example the conjugation of an agent with a vector described herein. In this example, Taxol is first activated in an N-succinimide derivative (2’-NHS-Txl). The amines found are reacted, for example, in a vector (for example, aminoterminal amine or lysine residues) in the activated Taxol to form a peptide bond (amide bond). If they are available

5 multiple amines, this reaction produces multiple combinations of conjugates, for example, with the addition of 1, 2 or 3 Taxols to the polypeptide, depending on the molar ratio used. The conjugation products are analyzed by HPLC, and the conjugation is confirmed by mass spectroscopy (Maldi-Tof). It is found that Taxol can be released from the vector by cleavage of the ester linkage with an esterase.

Any vector described herein can be conjugated with an agent using this method. Production of the TxlAn2 conjugate (3: 1) was carried out by directly adding 1 molar equivalent of Angiopep-2 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 h at 12 ° C. After removing the cooling bath, the reaction was allowed to continue for approximately 22 h at room temperature (Figure 24). Angiopep-2, 2’-NHSTaxol and conjugate of

15 TxlAn2 (3: 1) on the chromatogram by arrows. Aliquots of the reaction were sampled and analyzed by HPLC after 25 min, 2 h 15 min, 5 h and 23 h as indicated in Figure 24. The peaks of Angiopep-2, 2'-NHS-Taxol and conjugate are shown of TxlAn2 (3: 1) by arrows on the chromatogram. The results in Figure 24 illustrate the disappearance of Angiopep-2 and 2’-NHS-Taxol during the reaction mainly for the benefit of the TxlAn2 conjugate (3: 1).

This product mixture was separated by hydrophobic chromatography on a 300 mm RPC column with a flow rate at 4 ml / min using AKTA-explorer (Figure 25). For the peak corresponding to the TxlAn2 conjugate (3: 1), fractions were collected, analyzed by HPLC and MS. In Figure 25, the upper chromatogram corresponds to the reaction in execution at t = 23 h while the lower one corresponds to the TxlAn2 conjugate (3: 1)

25 which has been confirmed by mass spectrometry (PM 5107) after purification with AKTA.

Example 11

Antibody uptake and conjugation

Aprotinin antibodies can be conjugated as described in US Patent Application Publication No. 2006/0189515. Similar results can be obtained with any vector described herein.

35 Thus, Angiopep-2 has been conjugated with IgG using SATA. After conjugation, approximately 3 to 5 molecules of Angiopep-2 are associated with both light and heavy IgG chains (Figure 26). Therefore, using this approach, it is expected that 2 to 6 vector molecules (for example, Angiopep-1, Angiopep-2) are conjugated for each antibody molecule (heavy and light chains).

Angiopep-2 conjugation was also performed using sulfo-EMCS, as illustrated in Figure 4. Brain distribution of IgG-Angiopep-2 conjugates coupled through sulfhydryl groups using EMCS-Angiopep- was tested. 2 in various brain tissues (total brain, capillaries, parenchyma). There is superior brain uptake (about three times the difference) of conjugate of [125I] -IgG-Angiopep-2 than that of unconjugated [125I] -IgG (see Figure 27). Therefore the conjugation of IgG with Angiopep-2 increases the accumulation of

45 IgG in the brain in vivo.

The transport of coupled Angiopep-2-IgG conjugates was also tested using the SATA method using in situ brain perfusion experiments. IgG-An2 conjugates are transported in brain tissues (Figure 28). IgG-Angiopep-2 conjugates accumulated in the parenchyma approximately 50 times more than IgG.

After conjugation of IgG with vectors, polyacrylamide-SDS gel electrophoresis, immunodetection and autoradiography were performed to ensure appropriate conjugation; see, for example, Figure 29, which shows that only conjugates of [125I] -IgG-Angiopep were detected. There was no evidence of free Angiopep-2 conjugate, which

55 shows the effectiveness of the conjugation approach.

Example 12

Coupling of an anti-EGFR antibody with a vector

Signaling through the epidermal growth factor receptor induces cell proliferative signals and is associated with the transformation of normal to malignant cells. Several mutations in EGFR can be detected in tumor cells. One of the most common EGFR mutations is the EGFRvIII mutation in which amino acids 6-273 are eliminated.

To show that the coupling of vectors of the invention to antibodies other than IgG was possible, and that the

coupling maintained the antibody function, an antibody to EGFR (monoclonal 528 available from ATCC) was cross-linked with the vector using the SATA cross-linking agent as an example therapeutic polypeptide. The biological activity of the antibody against coupled EGFR was tested by staining EG87 positive U87 cells. A similar detection of EGFR in U87 was demonstrated using both coupled antibodies against EGFR

5 as not coupled by FACS analysis (Figure 30) showing that the coupling of the antibodies of the vectors of the invention did not alter their activity.

Table 8 shows that antibodies are not inactivated by conjugation and have the same affinity for binding in an in vitro assay. 10 Table 8: Analysis of the kinetics of the conjugate that binds to the EGFR receptor by FACS Anti-EGFR antibody Anti-EGFR-Angiopep-2 antibody

Bmax (RFU) 23.6 22.0

Medium saturation (nM) 1.7 1.8

The transport of antibody conjugates against EGFR (eg, anti-EGFRAngiopep-2 antibody) was then measured through the BHE using a labeled conjugate. The uptake of anti-[125I] EGFR-Angiopep-2 antibody to the luminal side of mouse brain capillaries was measured using the in situ cerebral perfusion method as shown in Figure 31. There was greater brain uptake for the conjugate [125I] -EGFR-Angiopep-2 than that of Ac versus [125I] -EGFR not conjugated in all tissues under test. Therefore, the conjugation of Ac versus EGFR with Angiopep-2 increases its accumulation in the cerebral parenchyma in vivo. These conjugates represent an interesting route since it was shown that several different types of tumors expressed EGFR at

20 different levels (see table 9).

Table 9

Type of tumor Tumors with expressed EGFR (%)

Head and neck 90-95 Breast 82-90 Renal carcinoma 76-89

Cervix / uterus 90

Esophagus 43-89 Pancreatic 30-89 Non-small cell lung 40-80 Prostate 40-80 Colon 25-77 Ovary 35-70

Glioma 40-63 Bladder 31-48 Gastric 4-33

Example 13

Coupling and transport of an anti-VEGF antibody with a vector

To further demonstrate the versatility and applicability of antibody coupling to vectors of the invention, another exemplary therapeutic antibody, Avastin, was used. Avastin is a recombinant humanized monoclonal monoclonal IgG1 kappa isotype anti-VEGF antibody available from Roche Biochemical that binds to, and inhibits, biologically active forms of vascular endothelial growth factor (VEGF).

After conjugation, then the transport of Avastin conjugates was measured through the BHE using the brain perfusion method in situ. There is superior brain uptake for conjugate of [125I] -Avastin-Angiopep-2 than 35 for [125I] -Avastin unconjugated (Figure 32).

Thus the conjugation of therapeutic antibodies (such as Avastin or MAb 528 available from ATCC) with vectors (for example, Angiopep-1, -2, -3, -4a, -4b, 5 and 6) is a useful strategy for their transport in the brain.

Example 14

Dimer transport

5 Vector dimers can also be transported. In one example, Angiopep-1 was incubated at 4 ° C for at least 12 hours, thereby causing multimerization / dimerization of the polypeptide. The transcytosis capacity of the Angiopep-1 dimer was evaluated in vitro. Angiopep-1 dimer translates better than IgG. In addition, as shown in Table 5, the dimeric form of Angiopep-1 shows a volume of distribution greater than that of Angiopep-1. Therefore, vector dimers can be transported through the BHE.

Example 15

Conjugation of an antibody with more than one vector

15 Normally, crosslinking reactions produce between 1 and 6 vector molecules per one antibody molecule, heavy and light chains.

The level of conjugation of Angiopep-2 with IgG was estimated using an assay that allows the assessment of the amount of sulfhydryl groups after deprotection of SATA in the IgG amine before and after the reaction with the maleimide group in N Angiopep-2 terminal. By changing the relative concentration of the different reagents used in the conjugation protocol, the amount of Angiopep-2 conjugated to the IgGs can be optimized.

When using brain perfusion experiments in vivo, the higher the level of conjugation, the greater the uptake in the parenchyma of conjugates (Figure 33). Therefore, it may be desirable to have a larger number of vectors per molecule. As such, more than 6 vectors can be used to transport a compound (agent) through the BHE.

Claims (20)

1. Polypeptide comprising an amino acid sequence having the following formula: 5 X1-X2-X3-X4-X5-X6-X7-X8-X9-X10-X11-X12-X13-X14-X15-X16-X17-X18-X19 in which X1-X19 are any amino acid or are absent; at least one of X10 and X15 is Arg; Y said amino acid sequence has at least 80% identity with Angiopep-7, which has the sequence of amino acids of SEQ ID NO: 112; wherein said polypeptide is efficiently transported to at least one cell or tissue selected from group 15 consisting of liver, lung and kidney, and wherein said polypeptide is transported through the blood brain barrier to levels below Angiopep -6, which has the amino acid sequence of SEQ ID NO: 111.

2.

Polypeptide according to claim 1, wherein said polypeptide, when conjugated with an agent, can increase the accumulation of this agent by at least 50% in said at least one cell or tissue compared to the unconjugated agent.

3.

Polypeptide according to claim 2, wherein said agent is paclitaxel.

4. Pharmaceutical composition comprising the polypeptide according to any one of claims 1 to 3 and a pharmaceutically acceptable carrier. 5. Conjugate comprising:

(to)

a vector comprising the polypeptide according to any one of claims 1 to 3; Y

(b)

a therapeutic agent, diagnostic agent or detectable label, wherein said agent or label is conjugated with said vector.

6. Polypeptide according to any one of claims 1 to 3 or conjugate according to claim 5, wherein said identity is at least 90%. 7. Polypeptide according to any one of claims 1 to 3 or conjugate according to claim 5, wherein: a.) X10 is Lys or Arg; X15 is Lys or Arg; or both b.) in which X1 is Arg, c.) in which X10 is Arg, 45 d.) In which X15 is Arg, e.) In which X10 and X15 are Arg, f.) In which X7 is Ser or Cys, or g.) In which X7 is Ser. 8. Polypeptide according to any one of claims 1 to 3 or conjugate according to claim 5, wherein said polypeptide or said vector comprises the amino acid sequence of SEQ ID NO: 112. 55

9.

Polypeptide according to any one of claims 1 to 3 or conjugate according to claim 5, wherein said polypeptide or said vector consists of the amino acid sequence of SEQ ID NO: 112.

10.

Conjugate according to claim 5, wherein said vector is conjugated with a therapeutic agent.

eleven.

Conjugate according to claim 5, wherein said therapeutic agent is an anticancer agent.

12.

Conjugate according to claim 11, wherein said anticancer agent is selected from the group consisting of abarelix, aldesleucine, alemtuzumab, alitretinoin, allopurinol, altretamine, amifostine, anakinra,

65 anastrozole, arsenic trioxide, asparaginase, azacitidine, live BCG, bevacuzimab, bexarotene, bleomycin, bleomycin, bortezomib, busulfan, calusterone, capecitabine, carboplatin, carmustine, celecoxib, cetuximab, chlorambucil, cisplatin, cladribine, clofarabine, cyclophosphamide, cytarabine, dacarbazine, dactinomycin, actinomycin D, dalteparin, darbepoetin alfa, dasatinib, daunorubicin, daunomycin, decitabine, denileukin, Denileukin diftitox, dexrazoxane, docetaxel, doxorubicin, dromostanolone propionate, eculizumab, epirubicin , epoetin alfa, erlotinib, estramustine, etoposide, exemestane, fentanyl, filgrastim, floxuridine, fludarabine, fluorouracil, 5-FU, 5 fulvestrant, gefitinib, gemcitabine, gemtuzumab ozogamycin, goserelin, histrelin, ibumibetimidamide imumomabidamide , interferon alfa-2b, irinotecan, lapatinib ditosylate, lenalidomide, letrozole, leucovorin, leuprolide, levamisole, lomustine, CCNU, mechlorethamine, megestrol, melphalan, mercaptopurine, mesna, methotrexate, methoxsalene, mitomycinthioproane, mitomycinthioproxone, mitochondrone, phenyleproxone , nelarabine, nofetumomab, oprelvecina, oxaliplatin, paclitaxel, palifermina, pamidron ato, panitumumab, pegademasa, 10 pegaspargasa, pegfilgrastim, peginterferon alfa-2b, pemetrexed, pentostatin, pipobroman, plicamycin, porphimer, procarbazine, quinacrine, rasburicase, rituximab, sargramostim, sorafenib, streptozocine, sunitinib, tamphoctazole, tenottostenoid, tetanotamide, tetanothozole Thalidomide, thioguanine, thiotepa, topotecan, toremifene, Tositumomab / I-131, trastuzumab, tretinoin, uracil mustard, valrubicin, vinblastine, vincristine, vinorelbine, vorinostat, zoledronate and zoledronic acid.

13.

Conjugate according to claim 12, wherein said agent is paclitaxel.

14.

Conjugate according to claim 11, wherein said agent is an antibody.

15. Conjugate according to claim 14, wherein said antibody is selected from the group consisting of ABX-EGF, OvaRex, Theragyn, Therex, Bivatuzumab, Panorex, ReoPro, Bexxar, AcM, 105AD7 idiotypic, Anti-EpCAM, AcM against Lung cancer, Herceptin, Rituxan, Avastin, AMD Fab, E-26, Zevalin, Cetuximab, BEC2, IMC1C11, nuC242-DM1, LymphoCide, LymphoCide Y-90, CEA-Cide, CEA-Cide Y-90, HumaRAD- HN, MDX-101, MDX210, MDX-210 / MAK, Vitaxin, MAb 425, IS-IL-2, Campath, CD20 streptavidin, Avidicin, Oncolym, Cotara, C215, 25 nacolomab tafenatox, Nuvion, SMART M195, SMART 1D10, CEA Vac, TriGem, TriAb, NovoMAb-G2 radiolabelled, Monopharm C, GlioMAb-H, Rituxan and ING-1. 16. Conjugate according to claim 5, wherein said conjugate accumulates more rapidly, accumulates at a higher concentration or has reduced P-gp mediated efflux in a liver, lung, renal or spleen cell, compared to said agent or label when not conjugated to said vector.

17.

Composition comprising the conjugate according to claim 5 and a pharmaceutically acceptable carrier.

18.

Conjugated according to any of claims 11 to 15 for use in a method of treating a

A subject having a cancer, said method comprising providing said subject said conjugate in an amount sufficient to treat said subject. 19. Conjugated for use according to claim 18, wherein said cancer is a lung, liver, kidney cancer or spleen 40 20. Conjugated for use according to claim 19, wherein said cancer is selected from the group consisting of hepatocellular carcinoma, breast cancer, head and neck cancers, lymphoma, mantle cell lymphoma, non-Hodgkin lymphoma, adenoma , squamous cell carcinoma, laryngeal carcinoma, retinal cancers, esophageal cancers, multiple myeloma, ovarian cancer, uterine cancer, melanoma, colorectal cancer, cancer 45 bladder, prostate cancer, lung cancer, non-small cell lung carcinoma, pancreatic cancer, cervical cancer, head and neck cancer, skin cancers, nasopharyngeal carcinoma, liposarcoma, epithelial carcinoma, renal cell carcinoma, adenocarcinoma of Gallbladder, parotid adenocarcinoma, endometrial sarcoma and multi-drug resistant cancers. 21. Conjugated for use according to claim 18, wherein said anticancer agent has increased efficacy or reduced side effects when conjugated to said polypeptide, compared to said agent when not conjugated to said polypeptide. 22. Conjugated for use according to claim 18, wherein said subject is a human being.