Human and mouse targeting peptides identified by phage display

Abstract

The present invention concerns methods and compositions for in vivo and in vitro targeting. A large number of targeting peptides directed towards human organs, tissues or cell types are disclosed. The peptides are of use for targeted delivery of therapeutic agents, including but not limited to gene therapy vectors. A novel class of gene therapy vectors is disclosed. Certain of the disclosed peptides have therapeutic use for inhibiting angiogenesis, inhibiting tumor growth, inducing apoptosis, inhibiting pregnancy or inducing weight loss. Methods of identifying novel targeting peptides in humans, as well as identifying endogenous receptor-ligand pairs are disclosed. Methods of identifying novel infectious agents that are causal for human disease states are also disclosed. A novel mechanism for inducing apoptosis is further disclosed.

What is claimed is:  
     
         1 . A method comprising 
 a) injecting a subject with a phage display library;    b) obtaining samples of one or more organs or tissues;    c) producing thin sections of the samples; and    d) recovering phage from the thin sections.    
     
     
         2 . The method of  claim 1 , further comprising selecting one or more portions of a thin section by PALM (Positioning and Ablation with Laser Microbeams).  
     
     
         3 . The method of  claim 2 , wherein the selected portion contains a specific cell type.  
     
     
         4 . The method of  claim 2 , wherein the selected portion contains a homogenous population of cells.  
     
     
         5 . The method of  claim 3 , wherein the cells are cancer cells.  
     
     
         6 . The method of  claim 1 , wherein the phage are recovered by infecting bacteria with the phage.  
     
     
         7 . The method of  claim 1 , wherein the phage are recovered by amplifying phage inserts and ligating the amplified inserts to phage DNA to produce new phage.  
     
     
         8 . A method of preparing a phage display library comprising: 
 a) immunizing a host animal with a target organ, tissue or cell type;    b) obtaining mRNAs encoding antibodies from the host animal;    c) preparing cDNAs from the mRNAs encoding antibodies; and    d) preparing a phage display library from the cDNAs.    
     
     
         9 . The method of  claim 8 , further comprising using antibody specific primers to amplify cDNAs that encode antibodies.  
     
     
         10 . The method of  claim 8 , wherein the target organ, tissue or cell is diseased.  
     
     
         11 . The method of  claim 10 , wherein the target comprises cancer cells.  
     
     
         12 . The method of  claim 8 , further comprising: (i) injecting the phage display library into a subject; and (ii) recovering phage from one or more organs, tissues or cell types.  
     
     
         13 . The method of  claim 8 , further comprising screening said library against a target protein or peptide.  
     
     
         14 . A phage display library prepared by the method of  claim 8 .  
     
     
         15 . A method of interfering with pregnancy comprising; 
 a) obtaining a peptide comprising at least three contiguous amino acids of a sequence selected from SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44 or SEQ ID NO:45; and    b) administering the peptide to a female subject.    
     
     
         16 . The method of  claim 15 , wherein the subject is pregnant.  
     
     
         17 . The method of  claim 15 , further comprising attaching an agent to the peptide.  
     
     
         18 . A method of delivering an agent to a fetus comprising: 
 a) obtaining a peptide comprising at least three contiguous amino acids of a sequence selected from SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44 or SEQ ID NO:45;    b) attaching the peptide to an agent; and    b) administering the peptide to a pregnant subject.    
     
     
         19 . The method of claims  17  or  18 , wherein the agent is a drug, a pro-apoptotic agent, an anti-angiogenic agent, an enzyme, a hormone, a cytokine, a growth factor, a cytotoxic agent, a peptide, a protein, an antibiotic, an antibody, a Fab fragment of an antibody, an imaging agent, an antigen, a survival factor, an anti-apoptotic agent, a hormone antagonist, a virus, a bacteriophage, a bacterium, a liposome, a microparticle, a microdevice, a cell or an expression vector.  
     
     
         20 . A method of targeting delivery to adipose tissue comprising: 
 a) obtaining a targeting peptide comprising an amino acid sequence of at least three contiguous amino acids selected from SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:49, SEQ ID NO:50, SEQ ID NO:51, SEQ ID NO:52, SEQ ID NO:53, SEQ ID NO:54 or SEQ ID NO:55;    b) attaching the peptide to an agent to form a complex; and    c) administering the complex to a subject.    
     
     
         21 . The method of  claim 20 , further comprising inducing weight loss in said subject.  
     
     
         22 . An isolated peptide of 100 amino acids or less in size, comprising at least 3 contiguous amino acids of a sequence selected from any of SEQ ID NO:5 through SEQ ID NO:45, SEQ ID NO:47 through SEQ ID NO:121, SEQ ID NO:123 and SEQ ID NO:125 through SEQ ID NO:250.  
     
     
         23 . The isolated peptide of  claim 22 , wherein said peptide is 50 amino acids or less in size.  
     
     
         24 . The isolated peptide of  claim 22 , wherein said peptide is 25 amino acids or less in size.  
     
     
         25 . The isolated peptide of  claim 22 , wherein said peptide is 10 amino acids or less in size.  
     
     
         26 . The isolated peptide of  claim 22 , wherein said peptide is 7 amino acids or less in size.  
     
     
         27 . The isolated peptide of  claim 22 , wherein said peptide is 5 amino acids or less in size.  
     
     
         28 . The isolated peptide of  claim 22 , wherein said peptide comprises at least 5 contiguous amino acids of a sequence selected from any of SEQ ID NO:5 through SEQ ID NO:45, SEQ ID NO:47 through SEQ ID NO:121, SEQ ID NO:123 and SEQ ID NO:125 through SEQ ID NO:250.  
     
     
         29 . The isolated peptide of  claim 22 , wherein said peptide is attached to a molecule.  
     
     
         30 . The isolated peptide of  claim 29 , wherein said molecule is a drug, a chemotherapeutic agent, a radioisotope, a pro-apoptosis agent, an anti-angiogenic agent, a hormone, a cytokine, a growth factor, a cytotoxic agent, a peptide, a protein, an antibiotic, an antibody, a Fab fragment of an antibody, an imaging agent, survival factor, an anti-apoptotic agent, a hormone antagonist or an antigen.  
     
     
         31 . The isolated peptide of  claim 30 , wherein said pro-aptoptosis agent is selected from the group consisting of gramicidin, magainin, mellitin, defensin, cecropin, (KLAKLAK) 2  (SEQ ID NO: 1), (KLAKKLA) 2  (SEQ ID NO:2), (KAAKKAA) 2  (SEQ ID NO:3) and (KLGKKLG)3 (SEQ ID NO:4).  
     
     
         32 . The isolated peptide of  claim 30 , wherein said anti-angiogenic agent is selected from the group consisting of thrombospondin, angiostatin5, pigment epithelium-drived factor, angiotensin, laminin peptides, fibronectin peptides, plasminogen activator inhibitors, tissue metalloproteinase inhibitors, interferons, interleukin 12, platelet factor 4, IP-10, Gro-β, thrombospondin, 2-methoxyoestradiol, proliferin-related protein, carboxiamidotriazole, CM101, Marimastat, pentosan polysulphate, angiopoietin 2 (Regeneron), interferon-alpha, herbimycin A, PNU145156E, 16K prolactin fragment, Linomide, thalidomide, pentoxifylline, genistein, TNP470, endostatin, paclitaxel, Docetaxel, polyamines, a proteasome inhibitor, a kinase inhibitor, a signaling peptide, accutin, cidofovir, vincristine, bleomycin, AGM-1470, platelet factor 4 and minocycline.  
     
     
         33 . The isolated peptide of  claim 30 , wherein said cytokine is selected from the group consisting of interleukin 1 (IL-l), IL-2, IL-5, IL-10, IL-11, IL-12, IL-18, interferon-γ (IF-γ), IF-α, IF-β, tumor necrosis factor-α (INF-α), or GM-CSF (granulocyte macrophage colony stimulating factor).  
     
     
         34 . The isolated peptide of  claim 22 , wherein said peptide is attached to a macromolecular complex.  
     
     
         35 . The isolated peptide of  claim 34 , wherein said complex is a virus, a bacteriophage, a bacterium, a liposome, a microparticle, a magnetic bead, a yeast cell, a mammalian cell or a cell.  
     
     
         36 . The isolated peptide of  claim 34 , wherein said peptide is attached to a eukaryotic expression vector.  
     
     
         37 . The isolated peptide of  claim 36 , wherein said vector is a gene therapy vector.  
     
     
         38 . The isolated peptide of  claim 22 , wherein said peptide is attached to a solid support.  
     
     
         39 . A composition comprising the isolated peptide of  claim 22  in a pharmaceutically acceptable carrier.  
     
     
         40 . The composition of  claim 39 , wherein the isolated peptide is attached to a molecule or a macromolecular complex.  
     
     
         41 . The isolated peptide of  claim 22 , wherein said sequence is selected from any of SEQ ID NO:5 through SEQ ID NO:19.  
     
     
         42 . The isolated peptide of  claim 22 , wherein said sequence is selected from any of SEQ ID NO:20 through SEQ ID NO:38.  
     
     
         43 . The isolated peptide of  claim 22 , wherein said sequence is selected from any of SEQ ID NO:210 through SEQ ID NO:234.  
     
     
         44 . The isolated peptide of  claim 22 , wherein said sequence is selected from any of SEQ ID NO:56 through SEQ ID NO:68.  
     
     
         45 . The isolated peptide of  claim 22 , wherein said sequence is selected from any of SEQ ID NO:69 through SEQ ID NO:88.  
     
     
         46 . The isolated peptide of  claim 22 , wherein said sequence is selected from any of SEQ ID NO:235 through SEQ ID NO:250.  
     
     
         47 . The isolated peptide of  claim 22 , wherein said sequence is selected from SEQ ID NO:89, SEQ ID NO:90, SEQ ID NO:91 or SEQ ID NO:92.  
     
     
         48 . A kit comprising the isolated peptide of  claim 22  and a control peptide, each in a container.  
     
     
         49 . An antibody that selectively binds to an isolated peptide, the peptide comprising at least three contiguous amino acids selected from any of SEQ ID NO:5 through SEQ ID NO:45, SEQ ID NO:47 through SEQ ID NO:121, SEQ ID NO:123 and SEQ ID NO:125 through SEQ ID NO:250.  
     
     
         50 . A method comprising; 
 a) injecting a subject with a phage display library;    b) recovering at least one sample of at least one organ, tissue or cell type;    c) separating the sample into isolated cells or clumps of cells;    d) centrifuging the cells through an organic phase to form a pellet; and    e) recovering phage from the pellet.    
     
     
         51 . The method of  claim 50 , further comprising preselecting the phage display library against a different organ, tissue or cell type.  
     
     
         52 . A gene therapy vector, wherein the vector expresses a targeting peptide sequence as part of a surface protein, the targeting peptide comprising at least three contiguous amino acids selected from any of SEQ ID NO:5 through SEQ ID NO:45, SEQ ID NO:47 through SEQ ID NO:121, SEQ ID NO: 123 and SEQ ID NO: 125 through SEQ ID NO:250.  
     
     
         53 . A method of targeting delivery to an organ or tissue, comprising: 
 a) obtaining a peptide according to  claim 22;     b) attaching the peptide to an agent; and    c) administering the agent to a subject.    
     
     
         54 . The method of  claim 53 , wherein the subject is a human or a mouse.  
     
     
         55 . The method of  claim 53 , wherein the agent is a drug, a chemotherapeutic agent, a radioisotope, a pro-apoptosis agent, an anti-angiogenic agent, an enzyme, a hormone, a cytokine, a growth factor, a cytotoxic agent, a peptide, a protein, an antibiotic, an antibody, a Fab fragment of an antibody, an imaging agent, an antigen, a survival factor, an anti-apoptotic agent, a hormone antagonist, a virus, a bacteriophage, a bacterium, a liposome, a microparticle, a magnetic bead, a microdevice, a yeast cell, a mammalian cell, a cell or an expression vector.  
     
     
         56 . The method of  claim 53 , wherein the agent is an imaging agent.  
     
     
         57 . The method of  claim 56 , further comprising obtaining an image of the subject.  
     
     
         58 . The method of  claim 57 , wherein the image is diagnostic for a disease.  
     
     
         59 . The method of  claim 58 , wherein the disease is cancer, arthritis, diabetes, inflammatory disease, atherosclerosis, autoimmune disease, bacterial infection, viral infection, cardiovascular disease or degenerative disease.  
     
     
         60 . The method of  claim 53 , wherein the organ or tissue is bone marrow, prostate, prostate cancer, ovary, ureter, placenta, adipose, spleen, angiogenic tissue or ascites.  
     
     
         61 . A method of targeting delivery to prostate cancer comprising: 
 a) obtaining a targeting peptide comprising at least three contiguous amino acids selected from any of SEQ ID NO:20 through SEQ ID NO:38;    b) attaching the peptide to a therapeutic agent to form a complex; and    c) administering the complex to a subject with prostate cancer.    
     
     
         62 . A method of diagnosing prostate cancer comprising: 
 a) obtaining a targeting peptide comprising at least three contiguous amino acids selected from any of SEQ ID NO:20 through SEQ ID NO:38;    b) administering the peptide to a subject suspected of having prostate cancer; and    c) detecting the peptide bound to prostate cancer cells.    
     
     
         63 . A method of identifying targeting peptides to angiogenic tissue comprising: 
 a) inducing hypoxia in a neonatal subject;    b) administering a phage display library to the subject; and    c) recovering phage from the retina of the subject.    
     
     
         64 . A method of inducing apoptosis in a cell comprising: 
 a) obtaining a targeting peptide comprising at least three contiguous amino acids selected from any of SEQ ID NO:93 through SEQ ID NO:121;    b) attaching the peptide to a permeabilizing agent to form a complex; and    c) administering the complex to the cell.    
     
     
         65 . The method of  claim 64 , wherein the permeabilizing agent is selected from a peptide with an amino acid sequence of SEQ ID NO:122 or HIV Tat protein.  
     
     
         66 . The method of  claim 64 , wherein the targeting peptide has the amino acid sequence of SEQ ID NO:112.  
     
     
         67 . A method of inducing apoptosis in a cell comprising: 
 a) attaching Annexin V to a permeabilizing agent to form a complex; and    b) administering the complex to the cell.    
     
     
         68 . The method of  claim 67 , wherein the permeabilizing agent is selected from a peptide with an amino acid sequence of SEQ ID NO: 122 or HIV Tat protein  
     
     
         69 . A method of modulating angiogenesis comprising: 
 a) obtaining a peptide comprising at least three contiguous amino acids selected from SEQ ID NO:93 through SEQ ID NO:11; and    b) administering the peptide to a subject.    
     
     
         70 . The method of  claim 69  wherein the subject has a tumor and the peptide inhibits tumor growth or survival.  
     
     
         71 . The method of  claim 69 , wherein the peptide is attached to an agent.  
     
     
         72 . The method of  claim 71 , wherein the agent is thrombospondin, angiostatin5, pigment epithelium-drived factor, angiotensin, laminin peptides, fibronectin peptides, plasminogen activator inhibitors, tissue metalloproteinase inhibitors, interferons, interleukin 12, platelet factor 4, IP-10, Gro-β, thrombospondin, 2-methoxyoestradiol, proliferin-related protein, carboxiamidotriazole, C M101, Marimastat, pentosan polysulphate, angiopoietin 2 (Regeneron), interferon-alpha, herbimycin A, PNU145156E, 16K prolactin fragment, Linomide, thalidomide, pentoxifylline, genistein, TNP-470, endostatin, paclitaxel, accutin, cidofovir, vincristine, bleomycin, AGM-1470, platelet factor 4 or minocycline.  
     
     
         73 . The method of  claim 69 , wherein the peptide has anti-angiogenic activity.  
     
     
         74 . The method of  claim 69 , wherein the peptide has pro-angiogenic activity.  
     
     
         75 . The method of  claim 73 , further comprising administering the peptide to a subject with ischemnia.  
     
     
         76 . The method of  claim 73 , further comprising administering the peptide to a subject with cardiovascular disease.  
     
     
         77 . The method of  claim 69 , further comprising administering the peptide to a subject with cancer, arthritis, diabetes, cardiovascular disease, inflammation or macular degeneration.  
     
     
         78 . A method of targeting delivery to an angiogenic tissue comprising: 
 a) obtaining a peptide comprising at least three contiguous amino acids selected from SEQ ID NO:123, SEQ ID NO:125, SEQ ID NO: 126, SEQ ID NO:127, SEQ ID NO:128, SEQ ID NO:129, SEQ ID NO:130 or SEQ ID NO:131;    b) attaching the peptide to a therapeutic agent to form a complex; and    b) administering the complex to a subject.    
     
     
         79 . The method of  claim 78 , wherein the peptide has an amino acid sequence of SEQ ID NO:123.  
     
     
         80 . The method of claim,  78 , wherein the angiogenic tissue is from a subject with cancer, arthritis, diabetes, cardiovascular disease, inflammation or macular degeneration.  
     
     
         81 . A method of detecting receptors for endostatin or angiostatin comprising 
 a) obtaining a sample from a tissue or organ;    b) incubating the sample with endostatin or angiostatin; and    c) detecting the presence of endostatin or angiostatin bound to the sample.    
     
     
         82 . The method of  claim 81 , wherein the sample is a thin section of a tissue or organ.  
     
     
         83 . The method of  claim 82 , further comprising assessing specificity by inhibiting binding with a targeting peptide selective for endostatin or angiostatin.