ME02170B - Conjugates having a degradable linkage and polymeric reagents useful in preparing such conjugates - Google Patents

Description

INSULIN-LIKE GROWTH FACTOR RECEPTOR 1 BINDING PEPTIDES

[0001] This invention relates to insulin-like growth factor 1 receptor binding peptides, polynucleotides encoding them, and methods for their preparation and use.

Background of the invention

[0002] Recent advances in biomedical research and high-throughput drug screening have led to the emergence of numerous potential therapeutics for the treatment of CNS-related diseases. However, many therapeutics do not undergo in vivo testing due to inadequate transport through the blood-brain barrier (BBB).

[0003] Therapeutics can cross the BBB by several pathways, including saturable transporter systems, adsorptive transcytosis in which the transported therapeutic is internalized from the cell to the BBB and directed to the abluminal surface for deposition in the intracellular space of the cerebrospinal fluid, transmembrane diffusion in which the therapeutic dissolves in the lipid bilayer that forms cell membranes spanning the BBB and extracellular pathways in which the therapeutic utilizes the residual permeability of the BBB.

[0004] Several ways have been attempted to modify therapeutic agents in order to increase the permeability of the BBB, including conjugation with proteins that naturally pass through the BBB, for example insulin, insulin-growth factors 1 and 2 (IGF-1, IGF-2), leptin and transferrin (US Pat. Appl. No. US2007/ 0081992), linking polypeptides to cationized antibodies that bind to specific cellular receptors, such as the insulin receptor or the transferrin receptor (US Patent No. 6,329,508; Zhang and Pardridge, Brain Res 889 :49-56, 2001); by combining therapeutics with synthetic polymers such as poly(butylcyanoacrylate) or polyacrylamide coated with Polysorbate 80 (US Pat. Appl. No. 2002/0009491, US Pat. Appl No. 2002/0013266; US Pat. Appl. No. 2006/0051317) , and using liposomes or immunoliposomes.

[0005] Current approaches to improve transport of therapeutics across the BBB include inefficiency due to competition with endogenous ligand, lack of transport of therapeutics to the brain parenchyma, and degradation of therapeutics due to lysosomal targeting.

[0006] Thus, there is a need to develop methods for transporting therapeutic agents across the BBB.

Brief Description of the Drawing

[0007]

Sl. 1 shows the binding of selected phage lysates to IGF1R and IR.

Sl. 2 shows binding of peptide-AP fusions to IGF1R.

Summary of the invention

[0008] One aspect of the invention is an isolated polypeptide comprising a polypeptide having the sequence shown in SEQ ID NO: 1-13.

[0009] Another aspect of the invention is an isolated polynucleotide comprising a polynucleotide encoding a polypeptide comprising the amino acid sequence shown in SEQ ID NO: 1-13.

[0010] Another aspect of the invention is an isolated polynucleotide comprising a polynucleotide having the sequence shown in SEQ ID NOs: 14-26, or its complementary sequence.

[0011] Another aspect of the invention is an isolated vector comprising a polynucleotide having the sequence shown in SEQ ID NO: 14-26.

[0012] Another aspect of the invention is an isolated host cell containing a vector of the present invention.

[0013] Another aspect of the invention is an isolated fusion protein comprising a polypeptide having the sequence shown in SEQ ID NO: 1-13 fused to another polypeptide.

[0014] Another aspect of the invention is the method of expressing a polypeptide which contains the following steps:

a. providing a host cell of the present invention; and

b. culturing the host cell under conditions sufficient for the expression of the polypeptide having the sequence shown in SEQ ID NO: 1-13.

[0015] Another aspect of the invention is a method for delivering a therapeutic agent via endothelial cells, which comprises

a. conjugating a therapeutic agent to a polypeptide comprising a polypeptide having the sequence shown in SEQ ID NO: 1, 2, 4, 8, or 12 to form a conjugate;

b. contacting the conjugate with endothelial cells; and

c. measuring the amount of conjugate delivered via endothelial cells.

Detailed Description of the Invention

[0016] As used herein and in the claims, the singular forms include the plural unless the context clearly dictates otherwise. Thus, for example, reference to a "polypeptide" is a reference to one or more polypeptides and includes equivalents thereof known to one of ordinary skill in the art.

[0017] Unless otherwise defined, all technical and scientific terms used herein have the same meaning that is usually understood by one who is ordinarily versed in the state of the art to which the invention belongs. Although any compositions and methods similar or equivalent to those described herein may be used in the practice or testing of this invention, exemplary compositions and methods are described herein.

[0018] The term "polypeptide" means a molecule containing at least two amino acid residues joined by a peptide bond to form a polypeptide. Small polypeptides of less than 50 amino acids may be referred to as "peptides". Polypeptides may also be referred to as "proteins".

[0019] The term "polynucleotide" means a molecule containing a chain of nucleotides covalently linked by a backbone of sugar and phosphate groups or other equivalent covalent bonds. Single- and double-stranded DNA and RNA are typical examples of polynucleotides.

[0020] The term "complementary sequence" means a second isolated polynucleotide sequence that is antiparallel to the first isolated polynucleotide sequence, and contains nucleotides complementary to nucleotides in the first polynucleotide sequence. Typically, such "complementary sequences" can form a double-stranded polynucleotide molecule such as double-stranded DNA or double-stranded RNA when combined under appropriate conditions with the first isolated polynucleotide sequence.

[0021] The term "vector" means a polynucleotide capable of replicating within a biological system or capable of being transferred between such systems. Vector polynucleotides typically contain elements, such as replication initiation sites, a polyadenylation signal, or selectable markers, that function to facilitate the duplication or maintenance of these polynucleotides in a biological system. Examples of such biological systems may include cell, virus, animal, plant, and reconstituted biological systems using biological components capable of duplicating the vector. The vector-containing polynucleotides can be DNA or RNA molecules or their hybrids.

[0022] The term "expression vector" means a vector that can be used in a biological system or a reconstituted biological system to direct the translation of a polypeptide encoded by a polynucleotide sequence present in the expression vector.

[0023] The term "blood-brain barrier" or "BBB" as used herein refers to the barrier between the peripheral circulation and the brain and spinal cord formed by tight junctions between brain capillary endothelial plasma membranes, which form an extremely tight barrier that limits the transport of molecules. into the brain, even such small molecules as urea, which has a molecular weight of 60 Da. The blood-brain barrier in the brain, the blood-brain barrier in the spinal cord, and the blood-retinal barrier within the retina are the limiting capillary barriers in the central nervous system (CNS), collectively referred to as the blood-brain barrier.

[0024] The term "antibody" refers to a molecule that specifically binds to an antigen, and includes dimeric, trimeric, and multimeric antibodies, and chimeric, humanized, and fully human antibodies. Also, an antibody can be a whole antibody or a functional fragment of an antibody molecule, such as a fragment that has retained at least antigen-binding function, and includes Fab, F(ab'), F(ab')2, scFv, dsFv, and diabodies. For example, antibody fragments can be obtained using proteolytic enzymes (eg, the whole antibody is digested with papain to produce Fab fragments, and treatment with pepsin leads to the production of F(ab')2 fragments). Techniques for the preparation and use of various antibodies are well known in the art (Ausubel, et al., ed., Current Protocols in Molecular Biology, John Viley&Sons, Inc., NY 1987-2001, Sambrook, et al., Molecular cloning: A Laboratory Manual, 2nd Edition, Cold Spring Harbor, NY, 1989; Harlow and Lane, Antibodies, A Laboratory Manual, Cold Spring Harbor, NY, 1989; Colligan, et al., ed., Current Protocols in Immunology, John Wiley&Sons, Inc. , NY 1994-2001; Colligan et al. Current Protocols in Protein Science, John Wiley&Sons, NY, 1997-2001; Kohler et al., Nature 256:495-497, 1975; US 4,816,567, Queen et al., Proc Natl. Acad. Sci. 86:10029-10033, 1989). For example, fully human monoclonal antibodies lacking non-human sequences can be prepared from human immunoglobulin from transgenic mice or from phage libraries (Lonberg et al., Nature 368: 856-859, 1994; Fishwild et al., Nature Biotech 14:845 -851, 1996; Mendez et al., Nature Genetics 15:146-156, 1997; Knappik et al., J. Mol Biol. 296:57-86, 2000; Krebs et al., J. Immunol. Meth 265: 67-84, 2001).

[0025] An antibody molecule or preparation "specifically binds" a given antigen when it binds this antigen with greater affinity and in a specific, as opposed to non-specific, manner relative to another non-identical antigen. In other words, the "specific binding" of an antibody molecule or preparation can be used to distinguish between two different polypeptides.

[0026] The term "insulin-like growth factor 1 receptor" or "IGF1R" as used herein refers to human IGF1R (GenBank Acc. No. NP_000866) having the amino acid sequence shown in SEQ ID NO: 27. IGF1R pro-polypeptide is cleaved into alpha and beta chains to form the mature protein. The alpha chain has amino acid residues 31-740 of SEQ ID NO: 27 and the beta chain has amino acid residues 741-1367 of SEQ ID NO: 27. "Soluble IGF1R" or "sIGF1R" as used herein refers to the extracellular domain of IGF1R (amino acids 31-932 of SEQ ID NO: 27). Soluble IGF1R can be the extracellular domain of the unfolded pro-polypeptide or the extracellular domain of the mature IGF1R (amino acid residues 31-740 forming the alpha chain and amino acid residues 741-932 forming the extracellular part of the beta chain).

[0027] The term "conjugate" as used herein refers to a molecule comprising a chimeric peptide of the present invention having the amino acid sequence shown in SEQ ID NO: 1-13 and a therapeutic agent. The term "conjugated" or "conjugation" means that the therapeutic agent(s) and peptides of the present invention are physically linked, for example, by covalent chemical bonds, physical forces such as Van der Waals bonds or hydrophobic interactions, encapsulation, embedding, or combinations thereof. The therapeutic agent(s) and peptides of the present invention may be chemically linked via alcohol, acid, carbonyl, thiol or amino groups according to known methods of chemical synthesis (see, eg, US Pat. Appl. No. US2010/0028370). A therapeutic agent with the peptides of the present invention can be joined by a linker. Examples of linkers are glycine-rich linkers, such as Gly3SerGly3Ser (SEQ ID NO: 28) or Gly9SerGly4SerGly9Ser (SEQ ID NO: 29). When the therapeutic agent and the peptides according to the invention are conjugated by a covalent bond or the peptide and the therapeutic agent form a polypeptide, then the whole conjugate is a "fusion protein". Accordingly, the term "fusion protein" refers to a polypeptide consisting of two (or more) heterologous polypeptides that are not normally fused into a single amino acid sequence. Fusion proteins can generally be prepared using recombinant DNA technology, i.e. as a result of the transcription and translation of the fusion product of the recombinant genes, which includes the coding segment of the polypeptide of the present invention and the coding segment of the heterologous polypeptide.

[0028] The term "therapeutic agent" as used herein refers to a molecule that is administered to induce a desired therapeutic effect in a subject. The subject is a human or a non-human animal, including a mammal or primate. Examples of therapeutic agents are proteins, peptides, antibodies, small molecules, or polynucleotides. Therapeutic agents can also be toxins or radioisotopes, where the intended therapeutic effect is, for example, killing cancer cells.

[0029] The present invention provides isolated polypeptides that bind to IGF1R, polynucleotides that encode polypeptides, vectors containing these polynucleotides, isolated host cells, polypeptides that can be obtained by expressing these polynucleotides, methods for expressing polypeptides according to the present invention and methods for using polynucleotides and polypeptides of the present invention. Polypeptides of the invention bind to IGF1R, and are transported by transcytosis across endothelial cells. Since the IFG1R is expressed on endothelial cells in the blood-brain barrier (BBB), the polypeptides of the present invention may provide a means of delivering therapeutic agents across the BBB.

[0030] One aspect of the invention is an isolated polypeptide comprising a polypeptide having the sequence shown in SEQ ID NO: 1-13.

[0031] The polypeptides of the present invention can be produced by chemical synthesis, such as solid phase peptide synthesis, on an automated peptide synthesizer. Alternatively, polypeptides of the present invention can be obtained from polynucleotides encoding these polypeptides using cell-free expression systems such as expression systems based on reticulocyte lysate, wheat germ extract, and Escherichia coli extract. Polypeptides of the present invention can also be obtained by expression and isolation from cells having a single nucleic acid sequence of the present invention, by techniques well known in the art, such as recombinant expression of affinity-tagged polypeptides that are readily isolated. Those skilled in the art will recognize other techniques for preparing polypeptides of the invention.

[0032] Another aspect of the invention is an isolated fusion protein comprising a polypeptide having the sequence shown in SEQ ID NO: 1-13 fused to another polypeptide. This second polypeptide can be a leader or secretory signal sequence. Such a second polypeptide may be a therapeutic agent coupled to the peptides of the present invention. The therapeutic agent and peptide of the present invention can be combined in various ways. The C-terminus or N-terminus of a peptide of the present invention may be directly linked to the N-terminus or C-terminus of a suitable therapeutic agent via an amide bond or a peptide linker. Therapeutic agents can be linked to the peptide of the present invention by chemical crosslinking well known in the art.

[0033] A further aspect of the invention is an isolated polynucleotide comprising a polynucleotide encoding the polypeptides of the present invention.

[0034] Polynucleotides of the present invention can be produced by chemical synthesis such as solid phase polynucleotide synthesis on an automated polynucleotide synthesizer. Alternatively, the polynucleotides of the present invention can be produced by other techniques such as PCR-based duplication, vector-based duplication, or restriction enzyme DNA manipulation techniques. Techniques for producing or obtaining polynucleotides with a particular sequence are well known in the art.

[0035] Polynucleotides according to the present invention may also contain at least one non-coding sequence, such as transcribed but not translated sequences, termination signals, ribosome binding sites, mRNA stabilization sequences, introns and polyadenylation signals. Polynucleotide sequences may also contain additional sequences encoding additional amino acids. These additional polynucleotide sequences may, for example, encode a marker or tag sequence such as a hexahistidine peptide (Gentz et al., Proc. Natl. Acad. Sci. (USA) 86:821-284, 1989) or an HA peptide tag ( Wilson et al., Cell 37:767-778, 1984) which facilitates purification of the fused polypeptides. Exemplary polynucleotides are polynucleotides having the sequence shown in SEQ ID NO: 14-26.

[0036] Another variant of the invention is a vector containing an isolated polynucleotide having the sequence shown in SEQ ID NO: 14-26. Vectors of the present invention are useful for maintaining polynucleotides, duplicating polynucleotides, or directing the expression of a polypeptide encoded by a vector of the present invention in biological systems including reconstituted biological systems. Vectors can be chromosomal-, episomal- and viral-derived such as vectors derived from bacterial plasmids, bacteriophages, transposons, yeast episomes, insertion elements, yeast chromosomal elements, bacilloviruses, papoviruses such as SV40, vaccinia viruses, adenoviruses, fowl pox viruses, pseudorabies viruses, picornaviruses and retroviruses and vectors derived from their combinations, such as cosmids and phagemids.

[0037] The vectors of the present invention may be formulated in the form of microparticles, with adjuvants, lipid, buffer or other excipients as required for a particular application.

[0038] In one variant of the invention, the vector is an expression vector. Expression vectors typically contain nucleic acid sequence elements capable of controlling, regulating, causing, or permitting expression of the polypeptide encoded by such vector. Such elements may contain transcription enhancer binding sites, RNA polymerase initiation sites, ribosome binding sites, and other sites that facilitate expression of the encoded polypeptides in a particular expression system. Such expression systems can be cell-based, or "cell-free" systems that are well known in the art. The elements of the nucleic acid sequence, that is, the sequence of the parent vector, suitable for use in the expression of coded polypeptides are also well known. An exemplary plasmid-derived expression vector useful for expressing a polypeptide of the present invention contains an E. coli origin of replication, a chloramphenicol acetyltransferase (CAT) gene, a T7 bacteriophage promoter, a pelB signal sequence, and a T7 terminator sequence.

[0039] Another variant of the present invention is an isolated host cell containing a vector of the present invention. Representative representatives of the host cell include Archaea cells; cells of bacteria such as Streptococci, Staphylococci, Enterococci, E. coli, Streptomyces, cyanobacteria, B. subtilis and S. aureus; fungal cells such as Kluveromyces, Saccharomyces, Basidomycetes, Candida albicans or Aspergillus; insect cells, such as Drosophila S2 and Spodoptera Sf9; animal cells such as CHO, COS, HeLa, C127, 3T3, BHK, 293, CV-1, Bowes melanoma and myeloma; and plant cells, such as gymnosperm or angiosperm cells. Host cells in the methods of the present invention may be provided as single cells or as cell populations. Cell populations may comprise isolated or cultured cell populations or cells present in a matrix such as tissue.

[0040] Introduction of a polynucleotide, such as a vector, into a host cell can be performed according to methods known to those skilled in the art (Davis et al., Basic Methods in Molecular Biology, 2nd ed., Appleton & Lange, Norwalk, CT, 1994; Sambrook et al. Molecular Cloning: A Laboratory Manual, 3rd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 2001). These procedures include calcium phosphate transfection, DEAE-dextran-mediated transfection, microinjection, cationic lipid-mediated transfection, electroporation, transduction, scrape loading, ballistic entry, and infection.

[0041] It is possible to modify the structure of polypeptides or fragments of the invention for such purposes as increasing substrate specificity, stability, solubility and the like. For example, a modified polypeptide can be produced in which the amino acid sequence has been changed, such as an amino acid substitution, deletion, or addition. It is predicted that the isolated replacement of leucine with isoleucine or valine, aspartate with glutamate, threonine with serine, or a similar replacement of an amino acid with a structurally related amino acid (ie conservative mutations), in some cases but not in all, will not have a major impact on the biological activity of the resulting molecule . Conservative substitutions are those that occur in families of amino acids that bind to their side chains. Genetically coded amino acids can be divided into four families: (1) acidic (aspartate, glutamate); (2) basic (lysine, arginine, histidine); (3) non-polar (alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan); and (4) uncharged polar (glycine, asparagine, glutamine, cysteine, serine, threonine, tyrosine). Phenylalanine, tryptophan, and tyrosine are sometimes classified together as aromatic amino acids. Alternatively, the repertoire of amino acids can be grouped as (1) acidic (aspartate, glutamate); (2) basic (lysine, arginine histidine), (3) aliphatic (glycine, alanine, valine, leucine, isoleucine, serine, threonine), and serine and threonine can optionally be grouped separately as aliphatic-hydroxyl; (4) aromatic (phenylalanine, tyrosine, tryptophan); (5) amides (asparagine, glutamine); and (6) those containing sulfur (cysteine and methionine) (Stryer (ed.), Biochemistry, 2nd. ed., WH Freeman and Co., 1981). A change in the amino acid sequence of a polypeptide or a change in a fragment thereof results in a functional homologue being readily determined by assessing the ability of the modified polypeptide or fragment to produce a response in a manner similar to the unmodified polypeptide or fragment using the assays described herein. Peptides, polypeptides or proteins in which more than one substitution has been made can easily be tested in the same way.

[0042] The polypeptides of the present invention may also be formulated in a pharmaceutically acceptable carrier or diluent. Different water carriers can be used, e.g. 0.4% saline solution, 0.3% glycine and the like. These solutions are sterile and generally free of particulates. These solutions can be sterilized by conventional, well-known sterilization techniques (eg, filtration). The compositions may contain pharmaceutically acceptable excipients required for approximately physiological conditions, such as pH adjusting agents and buffers. The concentration of the polypeptide of the invention in such a pharmaceutical formulation can vary widely, from below about 0.5%, usually up to or at least about 1% to as much as 15 or 20% by weight and will be selected primarily based on liquid volume, viscosity and other factors, in accordance with the particular by the chosen administration method. An appropriate therapeutically effective dose can be readily determined by one skilled in the art. A given dose may, if necessary, be repeated at appropriate time intervals selected as necessary by a physician or other expert in the relevant state of the art (eg nurse, veterinarian or veterinary technician) during the treatment period.

[0043] The polypeptides of the present invention may be lyophilized for storage and reconstituted in a suitable vehicle prior to use. This technique has been shown to be effective with conventional protein preparations. Lyophilization and reconstitution techniques are well known in the art.

[0044] A further embodiment of the present invention is a method for expressing a polypeptide comprising the steps of providing a host cell of the present invention; culturing the host cell under conditions sufficient for the expression of at least one polypeptide of the invention.

[0045] Host cells can be cultured under any conditions suitable for maintaining or propagating a particular type of host cell and sufficient for polypeptide expression. Culture conditions, media, and similar procedures sufficient for polypeptide expression are well known in the art. For example, many mammalian cell types can be aerobically cultured at 37°C using appropriately buffered DMEM medium, while bacterial, yeast, and other cell types can be cultured at 37°C under appropriate atmospheric conditions in LB medium.

[0046] In the methods of the present invention, the expression of the polypeptide can be confirmed using various well-known methods. For example, polypeptide expression can be confirmed using detection reagents, such as antibodies using, for example, FACS or immunofluorescence techniques, or by SDS-PAGE or HPLC.

[0047] A further aspect of the invention is a method for delivering a therapeutic agent via endothelial cells, comprising:

a. conjugating a therapeutic agent to a polypeptide comprising a polypeptide having the sequence shown in SEQ ID NO: 1, 2, 4, 8, or 12 to form a conjugate;

b. contacting the conjugate with endothelial cells; and

c. measurement of the amount of conjugate delivered via endothelial cells.

[0048] The polypeptides of the present invention facilitate the delivery of a therapeutic agent across endothelial cells through the binding of the polypeptides of the invention to the IGF1R. The peptides are selected so that the conjugated therapeutic agent does not interfere with the binding of the polypeptide of the invention to the IGF1R. The present invention describes the conjugation of proteins having a molecular weight of approximately 100 kD (921 amino acids) to the polypeptides of the present invention without loss of transcytotic activity. Other polypeptides of comparable size should also likely be successfully conjugated to the polypeptides of the present invention and delivered to the brain via endothelial cells.

[0049] Conjugate delivery across endothelial cells can be measured using well-known in vitro or in vivo methods. Examples of in vitro measurements can be performed using polarized monolayers of endothelial cells and measuring conjugate transcytosis using, for example, conjugated antibodies. In vivo measurements can be performed on a subject using e.g. radioactive conjugates and by measuring their distribution in the brain after their administration. A good correlation was observed between in vitro and in vivo methods. For example, Perrier et al., showed a good correlation (R=0.94) between the permeability coefficients of a series of compounds and their corresponding in vivo blood-brain transfer coefficients in rodents using co-cultures of rat brain astrocytes and endothelial cells (Perrier et al, Brain Res. 1150: 1-13, 2007).

[0050] The present invention will now be described with reference to the following specific, non-limiting examples.

First 1

Identification of IGF1R binding peptides

Selection of phage "Phage panning"

[0051] pIX phage libraries displaying random peptides were constructed according to the methods described in US Pat. Appl. No. US2010/0021477, and are used as a source of human IGF1R binding peptides. This library was panned in solution with a biotinylated form of purified soluble IGF1R (sIGF1R) bearing a carboxy-terminal hexahistidine tag (R&D Systems, Minneapolis, MN) in three rounds. Biotinylation of sIGF1R was performed using EZ-Link No-Weigh Sulfo-NHS-LC-Biotin Microtubes (Pierce, Rockford, IL). Due to the size of sIGF1R (~330 kDa), Tetralink Avidin beads were used for the first round of selection due to their ~10-fold higher binding capacity than Dynal magnetic beads.

[0052] A total of 384 selected individual phage lysates were tested for binding specificity to sIGF1R in a solid phase phage ELISA. Briefly, 100 µl/well of 5 µg/ml sIGF1R (R&D Systems, Minneapolis, MN) was bound to Black Maxisorp Plates (Nunc, Rochester, NY), 25 µl of phage lysate was added, and the reaction was detected using anti-M13-HRP antibodies (EMD Biosciences, Gibbstown, NJ) and POD substrate (Roche, Indianapolis, IN), and the signal was detected using a TEKAN plate reader. Unbound protein that has been shown not to cross the BBB was used as a negative control. Positive lysates were defined as clones with a sIGF1R specific signal that was three times stronger than the background of the negative control. A total of 13 clones with unique peptide sequences confirmed to bind to sIGF1R were obtained (Table 1). Of those 13 clones, 3 (clones 5, 13 and 16) also cross-react with the insulin receptor.

[0053] Peptides from clones 5-14, 16 and 17 were cloned "in-frame" as peptide-Alkaline phosphatase-His6 (peptide-AP) fusion proteins in a modified pET20b+ vector having genetically cloned chloramphenicol acetyltransferase (CAT). The resulting peptide-AP fusions were expressed in bacteria and purified using Ni-NTA (EMD Biosciences, Gibbstown, NJ) according to the manufacturer's instructions. The amino acid sequence of the alkaline phosphatase used is shown in SEQ ID NO: 30.

Table la. Polypeptide sequences of identified IGF1R binding peptides

Peptide Sequence

Clone br:

SEQ ID NO peptide:

TGCDFPELCRGCHP

5

1

AECEWPWLTLELCQS

6

2

PFCYSGGPLPYPCTY

7

3

PVCPSFCYDQYVCPT

8

4

FTCAVYSLSELDCRD

9

5

LSCYDPTLRTLYCHV

10

6

HTCFYPTLMPPELCFD

11

7

SNCPPLDMRLTELCVM

12

8

WHCTPLTQIADPGSIIHILECTV

13

9

VECDTPSITFSPGLEALFWNTCSP

14

10

VECDTPSITFSPGLEALFWNTCSP

15

11

AGCPSPMPPVDPGFYSAIVQLCRE

16

12

DIDEFLHQLHNLVNNVH

17

13

[0054] Binding of purified peptide-AP fusion proteins to sIGF1R was determined by ELISA with immobilized sIGF1R. Briefly, bacteria transformed with each peptide-AP fusion expression vector were grown overnight and the following day, and cultures were cleared by centrifugation at 4500 rpm, 4 °C. Supernatants were collected and 75 ml of each supernatant was analyzed for binding to 2 µg/ml sIGF1R immobilized on ELISA plates. Bound peptides were detected using Attophos Substrate (Roche, Indianapolis, IN) following the manufacturer's recommendations and read using a Molecular Devices M5 plate reader. Fusions with peptide clones 7 and 10 are not well expressed. All other peptide-AP fusions showed binding activity to sIGF1R (Figure 2).

Table 1b. Polynucleotide sequences of IGF1R binding peptides

Clone Number:

SEQ ID NO:

Sequence

5

14

ACGGGTTGTGATTTTCCGGAGTTGTGTCGTGGTTGTCATCCG

6

15

GCTGAGTGTGAGTGGCCGTGGCTTACGCTGGAGCTTTGTCAGTCT

7

16

CCTTTTTGTTATTCTGGTGGGCCGCTGCCGTATCCTTGTACGTAT

8

17

CCTGTGTGTCCGTCGTTTTGTTATGATCAGTATGTGTGTCCGACT

9

18

TTTACGTGTGCTGTTTATTCGTTGTCTGAGCTGGATTGTAGGGAT

10

19

TTGAGTTGTTATGATCCGACGCTGCGTACGTTGTATTGTCATGTT

11

20

12

21

13

22

14

23

15

24

16

25

17

26

First 2

Characterization of IGF1R binding peptides

[0055] The identified IFG1R binding peptides were cloned "in-frame" into the C-terminus of the protein G IgG domain (PG) in a modified pET17b vector (EMD Chemicals, Gibbstown, NJ) that has a ligation-free cloning site (LIC) to generate PG-peptide fusions. The IgG binding domain of protein G is stable, allowing easy purification of the fusion protein from bacterial lysates. The amino acid sequence of the IgG domain of protein G used is shown in SEQ ID NO: 31. PG-peptide fusions were expressed in bacteria after 1 mM IPTG induction and purified using IgG Sepharose beads (GE Healthcare Life Sciences, Piscataway, NJ) from bacterial lysates cleared by centrifugation. at 16,000 g, 4 °C for 20 min.

[0056] The relative binding affinities of the PG-peptide fusions to the sIGF1R were measured using an ELISA assay. Kds were determined using GraphPad Prism 4 software with a single binding site equation. Black Maxisorp 96-well plates (Nunc, Rochester, NI) were coated with 100 µl of 2 µg/ml sIGF1R (R&D Systems, Minneapolis, MN) in Dulbecco's phosphate buffered saline (DPBS (-/-)) overnight at 4°C. . Plates were washed with TBST and wells were blocked with 200 µl/well Starting Block T20 (TBS) (Pierce, Rockford, IL) for 1 h at room temperature with shaking at 300 rpm. 75 µl/well of purified, serially diluted PG-peptide fusions, with an initial concentration of 40 µM, was added to the wells and incubated for 1 h, at room temperature with shaking at 300 rpm; the volume was brought up to 100 µl with Starting Block T20. Plates were washed 3x with TBST and then probed with 100 µl/well Peroxidase-labeled-rabbit antibody (1:5000) (Rockland, Gilbertsville, PA), washed, and signal detected using 100 µl/well POD substrate (Roche, Indianapolis, IN). Chemiluminescence was detected using a Molecular Devices M5 plate reader. For competition experiments, insulin-like growth factor 1 (IGF-1) (R&D Systems, Minneapolis, MN) was added to the T20 Starting Block used to bring the volume to 100 µl at a final concentration of 400 nM.

[0057] Characterization of PG-peptide fusions is summarized in Table 2. Most peptides bound with relative affinities in the low µM range (0.75-8 µM). The fusion protein with peptide clone 17 is unique in its binding profile in that it has a bell-shaped binding curve. This is similar to insulin binding to the insulin receptor and IGF-1 binding to the IFG1R, ligands that induce conformational shifts in the receptors upon binding and have negative cooperativity profiles at higher ligand concentrations. The results of competition studies with IGF-1 are shown in Table 2.

Table 2.

Clone Number:

Peptid SEQ ID NO:

EC50 (µM)

Competition with IGF-1

Transcitoza

5

1

0.75

-

and

6

2

1

-

and

7

3

1.8

-

not done

8

4

1.6

-

and

9

5

3.5

-

it is

10

6

4.3

-

not done

11

7

1.2

and

it is

12

8

1.8

-

and

13

9

>50

and

it is

14

10

0.85

and

it is

15

11

2.6

-

not done

16

12

8.2

and

and

17

13

not done

and

it is

First 3.

In vitro BBB model

[0058] Selected sIGF1R binding peptides were further characterized in an in vitro model of the blood-brain barrier, in a rat brain microvascular endothelial cell model.

[0059] Rat brain capillary endothelial cells were prepared as described (Perriere et al., J. Neurochem, 93:279-289, 2005). Briefly, the brains of 6-8 week old male Sprague Dawley rats were rolled on 3MM chromatography paper to remove the meninges, cut sagittally and the white matter dissected to leave the cortices, which were then finely ground. Ground cortices were transferred to a 50 ml polypropylene conical tube with 20 ml DMEM supplemented with 39 units/ml DNase I (Worthington, Lakewood, NJ) and 0.7 mg/ml Collagenase type 2 (Worthington, Lakewood, NJ) at a final concentration of incubated at 37 °C with gentle agitation for 1.25 hours. After a brief centrifugation, the pellet obtained was resuspended in 20 ml of 20% BSA (Sigma, St. Louis, MO) in DMEM, centrifuged and the microvascular enriched pellet, isolated and digested a second time with 20 ml of DMEM supplemented with 39 units/ml of DNase I and 1 mg/ml Collagenase/Dispase (Roche, Indianapolis, IN) at 37 °C for 1 hour. The digestion product was briefly centrifuged and the resulting cell pellet resuspended in 2 ml DMEM and then separated at the top of a 33% continuous Percoll gradient, centrifuged, and the enriched microvascular fraction was removed and briefly centrifuged again. The cell pellet was resuspended in 10 ml of complete brain microvascular endothelial cell medium (DMEM, 20% Plasma Derived Serum (PDS), 100 mg/ml Heparin, 2 mM L-Glutamine, 100 U/ml penicillin, 100 µg/ml streptomycin, 0.25 µg/mL amphotericin) and plated on a 10 cm tissue culture dish for 4 hours at 37 °C, 5% CO2. After 4 hours, cells that did not bind were pipetted off and counted in a hemacytometer using Trypan Blue. Cells were plated in the upper chamber of a Transwell device (pore size 0.4 µm, diameter 1.12 cm, Corning, Acton, MA) treated with 400 µg/ml Collagen type IV (Sigma, St. Louis, MO) and 100 µg/ml fibronectin ( Sigma, St. Louis, MO), at a density of 6 x 105 cells/ml, 500 µl per well. 1ml of medium was placed in the lower chamber. Both chambers were supplemented with 4 µg/ml pyromycin (Clontech, Mountain View, CA) and the plates were incubated at 37 °C, 5% CO2 overnight. The following day, media were replaced with fresh complete culture media with 4 µg/ml pyromycin and cells were returned to the incubator overnight. The next day the media were replaced with complete culture media and then again two days later. Cultures were monitored by eye until they reached 100% confluency, ~6-7 days after seeding. The developed in vitro BBB model had a high transendothelial electrical resistance (> 100 ohm-cm2), as measured by Millicell-ERS (Millipore, Billercia, MA) and a very low Na-Fluorescein permeability (about 1-5 x 10-6 cm/s ).

[0060] 25 µg of purified peptide-AP fusions were added to the upper chamber of an in vitro BBB model and transcytically transported peptide-AP fusions were detected at 15- and 30-minute time intervals in the lower chamber by ELISA. Briefly, 75 µl of each sample was transferred to plates coated with 5 mg/ml mouse monoclonal anti-bacterial AP antibody (Sigma, St. Louis, MO). The plate was incubated for 1 hour, washed, the signal was developed with Attophos Substrate (Roche, Indianapolis, IN) according to the manufacturer's recommendation and read using a Molecular Devices M5 plate reader with 440 nm excitation and 550 nm emission. The results are shown in Table 2.

[0061] For the present invention which has now been fully described, it will be apparent to one of ordinary skill in the art that many changes and modifications may be made thereto without departing from the scope of the appended claims.

SEQUENCE LIST

[0062]

<110> Centocor Ortho Biotech Inc.

<120> Insulin-like growth factor receptor 1 binding peptides

<130>CEN5297PCT

<140> to be assigned

<141> 2010-05-25

<160> 31

<170> Fast SEC. for Windows Version 4.0

<210> 1

<211> 14

<212> PRT

<213> Artificial sequence

<220>

<223> IGF1R binding peptide from the pIX library

<400> 1

<210> 2

<211> 15

<212> PRT

<213> Artificial sequence

<220>

<223> IGF1R binding peptide from the pIX library

<400> 2

<210> 3

<211> 15

<212> PRT

<213> Artificial sequence

<220>

<223> IGF1R binding peptide from the pIX library

<400> 3

<210> 4

<211> 15

<212> PRT

<213> Artificial sequence

<220>

<223> IGF1R binding peptide from the pIX library

<400> 4

<210> 5

<211> 15

<212> PRT

<213> Artificial sequence

<220>

<223> IGF1R binding peptide from the pIX library

<400> 5

<210> 6

<211> 15

<212> PRT

<213> Artificial sequence

<220>

<223> IGF1R binding peptide from the pIX library

<400> 6

<210> 7

<211> 16

<212> PRT

<213> Artificial sequence

<220>

<223> IGF1R binding peptide from the pIX library

<400> 7

<210> 8

<211> 16

<212> PRT

<213> Artificial sequence

<220>

<223> IGF1R binding peptide from the pIX library

<400> 8

<210> 9

<211> 23

<212> PRT

<213> Artificial sequence

<220>

<223> IGF1R binding peptide from the pIX library

<400> 9

<210> 10

<211> 24

<212> PRT

<213> Artificial sequence

<220>

<223> IGF1R binding peptide from the pIX library

<400> 10

<210> 11

<211> 24

<212> PRT

<213> Artificial sequence

<220>

<223> IGF1R binding peptide from the pIX library

<400> 11

<210> 12

<211> 24

<212> PRT

<213> Artificial sequence

<220>

<223> IGF1R binding peptide from the pIX library

<400> 12

<210> 13

<211> 18

<212> PRT

<213> Artificial sequence

<220>

<223> IGF1R binding peptide from the pIX library

<400> 13

<210> 14

<211> 42

<212> DNA

<213> Artificial sequence

<220>

<223> DNA encoding IGF1R binding peptide from pIX library

<400> 14

acgggttgtg attttccgga gttgtgtcgt ggttgtcatc cg 42

<210> 15

<211> 45

<212> DNA

<213> Artificial sequence

<220>

<223> DNA encoding IGF1R binding peptide from pIX library

<400> 15

gctgagtgtg agtggccgtg gcttacgctg gagctttgtc agtct 45

<210> 16

<211> 45

<212> DNA

<213> Artificial sequence

<220>

<223> DNA encoding IGF1R binding peptide from pIX library

<400> 16

cctttttgtt attctggtgg gccgctgccg tatccttgta cgtat 45

<210> 17

<211> 45

<212> DNA

<213> Artificial sequence

<220>

<223> DNA encoding IGF1R binding peptide from pIX library

<400> 17

cctgtgtgtc cgtcgttttg ttatgatcag tatgtgtgtc cgact 45

<210> 18

<211> 45

<212> DNA

<213> Artificial sequence

<220>

<223> DNA encoding IGF1R binding peptide from pIX library

<400> 18

tttacgtgtg ctgtttattc gttgtctgag ctggattgta gggat 45

<210> 19

<211> 45

<212> DNA

<213> Artificial sequence

<220>

<223> DNA encoding IGF1R binding peptide from pIX library

<400> 19

ttgagttgtt atgatccgac gctgcgtacg ttgtattgtc atgtt 45

<210> 20

<211> 48

<212> DNA

<213> Artificial sequence

<220>

<223> DNA encoding IGF1R binding peptide from pIX library

<400> 20

catacgtgtt tttatcctac gctgatgcct cctgagctgt gtttcgat 48

<210> 21

<211> 48

<212> DNA

<213> Artificial sequence

<220>

<223> DNA encoding IGF1R binding peptide from pIX library

<400> 21

agtaattgtc ctccgttgga tatgcggctg actgagcttt gtgttatg 48

<210> 22

<211> 72

<212> DNA

<213> Artificial sequence

<220>

<223> DNA encoding IGF1R binding peptide from pIX library

<400> 22

<210> 23

<211> 72

<212> DNA

<213> Artificial sequence

<220>

<223> DNA encoding IGF1R binding peptide from pIX library

<400> 23

<210> 24

<211> 72

<212> DNA

<213> Artificial sequence

<220>

<223> DNA encoding IGF1R binding peptide from pIX library

<400> 24

<210> 25

<211> 72

<212> DNA

<213> Artificial sequence

<220>

<223> DNA encoding IGF1R binding peptide from pIX library

<400> 25

<210> 26

<211> 54

<212> DNA

<213> Artificial sequence

<220>

<223> DNA encoding IGF1R binding peptide from pIX library

<400> 26

gatgacatag acgaatttct tcatcaactc cacaacctag taaacaatgt tcac 54

<210> 27

<211> 1367

<212> PRT

<213> A wise man

<400> 27

<210> 28

<211> 8

<212> PRT

<213> Artificial sequence

<220>

<223> Linker

<400> 28

<210> 29

<211> 15

<212> PRT

<213> Artificial sequence

<220>

<223> Linker

<400> 29

<210> 30

<211> 921

<212> PRT

<213> Artificial sequence

<220>

<223> variant of alkaline phosphate

<400> 30

<210> 31

<211> 54

<212> PRT

<213> Unknown

<220>

<223> Protein G phys of unknown bacteria

<400> 31

Claims (12)

1. An isolated polypeptide comprising a polypeptide having the sequence shown in SEQ ID NO: 1-13, wherein said polypeptide is capable of: (a) binding to IGF1R; and (b) be transported by transcytosis across endothelial cells. 2. An isolated polynucleotide containing a polynucleotide encoding a polypeptide according to claim 1. 3. The isolated polynucleotide according to claim 2, which contains the polynucleotide having the sequence shown in SEQ ID NO: 14-26, or its complementary sequence. 4. An isolated vector comprising a polynucleotide having the sequence shown in SEQ ID NO: 14-26, wherein said polynucleotide encodes a polypeptide according to claim 1. 5. The vector according to claim 4, wherein the vector is an expression vector. 6. An isolated host cell containing the vector according to claim 4. 7. An isolated fusion protein containing the polypeptide of claim 1 joined to another polypeptide. 8. The fusion protein according to claim 7, wherein the second polypeptide encodes an immunoglobulin or a fragment thereof. 9. A method for expressing a polypeptide comprising the following steps: a. providing a host cell according to claim 6; and b. culturing the host cell under conditions sufficient for the expression of the polypeptide according to claim 1. 10. An in vitro method for delivering a therapeutic agent via endothelial cells, comprising: a. conjugating a therapeutic agent to a polypeptide comprising a polypeptide having the sequence shown in SEQ ID NO: 1, 2, 4, 8, or 12 to form a conjugate; b. contacting the conjugate with endothelial cells; go. measurement of the amount of conjugate delivered via endothelial cells. 11. A polypeptide having the sequence shown in SEQ ID NO: 1, 2, 4, 8 or 12 for use in a method for delivering a therapeutic agent via endothelial cells, said use comprising: a. conjugating a therapeutic agent to a polypeptide comprising a polypeptide having the sequence shown in SEQ ID NO: 1, 2, 4, 8, or 12 to form a conjugate; b. contacting the conjugate with endothelial cells; go. measuring the amount of conjugate delivered via endothelial cells. 12. The method according to claim 11, in which the endothelial cells form a blood-brain barrier.