Peptides whose uptake by cells is controllable

Abstract

A generic structure for the peptides of the present invention includes A-X-B-C, where C is a cargo moiety, the B portion includes basic amino acids, X is a cleavable linker sequence, and the A portion includes acidic amino acids. The intact structure is not significantly taken up by cells; however, upon extracellular cleavage of X, the B-C portion is taken up, delivering the cargo to targeted cells. Cargo may be, for example, a contrast agent for diagnostic imaging, a chemotherapeutic drug, or a radiation-sensitizer for therapy. X may be cleaved extracellularly or intracellularly. The molecules of the present invention may be linear, cyclic, branched, or have a mixed structure.

1 . A cyclic molecule of the structure A-X1-B-X2, wherein 
 B is a peptide portion of about 5 to about 20 basic amino acid residues, which is suitable for cellular uptake,    A is a peptide portion of about 2 to about 20 acidic amino acid residues, which when linked with portion B is effective to inhibit or prevent cellular uptake of portion B; and    A and B are linked together and linked via linkers X1 and X2 to form a cyclic compound; where    X1 and X2 are each a cleavable linker of about 2 to about 100 atoms joining A with B to form a cyclic compound where each of X1 and X2 link to both A and B.    
     
     
         2 . The molecule of  claim 1 , wherein at least one of cleavable linkers X1 or X2 is configured for cleavage exterior to a cell.  
     
     
         3 . The molecule of  claim 1 , wherein at least one of cleavable linkers X1 or X2 is configured for cleavage interior to a cell.  
     
     
         4 . The molecule of  claim 2 , wherein at least one of cleavable linkers X1 or X2 is configured for cleavage by an enzyme.  
     
     
         5 . The molecule of  claim 4 , wherein said enzyme is a matrix metalloprotease.  
     
     
         6 . The molecule of  claim 5  wherein at least one of cleavable linkers X1 or X2 comprises the amino acid sequence PLGLAG (SEQ ID NO:1).  
     
     
         7 . The molecule of  claim 5  wherein at least one of cleavable linkers X1 or X2 comprises the amino acid sequence EDDDDKA (SEQ ID NO:2).  
     
     
         8 . The molecule of  claim 1  wherein at least one of cleavable linkers X1 or X2 comprises a S-S linkage.  
     
     
         9 . The molecule of  claim 1 , further comprising a cargo moiety C linked to peptide portion B.  
     
     
         10 . The molecule of  claim 9 , wherein said cargo moiety C comprises a label.  
     
     
         11 . The molecule of  claim 10 , wherein said label comprises a label selected from the group consisting of fluorescent labels, luminescent labels, radioactive labels, contrast agents, radiation sensitizers, antibodies, and antigens.  
     
     
         12 . The molecule of  claim 9 , wherein said cargo moiety C comprises a phage.  
     
     
         13 . The molecule of  claim 11 , wherein said cargo moiety C comprises a radioactive technetium label.  
     
     
         14 . The molecule of  claim 9 , wherein said cargo moiety C comprises a contrast agent.  
     
     
         15 . The molecule of  claim 14 , wherein said cargo moiety C comprises a gadolinium contrast agent.  
     
     
         16 . A pharmaceutical composition comprising: 
 A cyclic molecule of the structure A-X1-B-X2, wherein    B is a peptide portion of about 5 to about 20 basic amino acid residues, which is suitable for cellular uptake,    A is a peptide portion of about 2 to about 20 acidic amino acid residues, which when linked with portion B is effective to inhibit or prevent cellular uptake of portion B; and    A and B are linked together and linked via linkers X1 and X2 to form a cyclic compound; where    X1 and X2 are each a cleavable linker of about 2 to about 100 atoms joining A with B to form a cyclic compound where each of X1 and X2 link to both A and B,    and a pharmaceutically acceptable carrier.    
     
     
         17 . The pharmaceutical composition of  claim 16 , further comprising a cargo moiety C linked to peptide portion B.  
     
     
         18 . The pharmaceutical composition of  claim 17 , wherein said cargo moiety C comprises a therapeutic agent.  
     
     
         19 . The pharmaceutical composition of  claim 17 , wherein said cargo moiety C comprises a radioactive agent.  
     
     
         20 . A method of modulating cellular uptake of a cargo moiety C, comprising: 
 covalently attaching a cargo moiety C to a peptide B of about 5 to about 20 basic amino acid residues to form a molecule BC;    linking said molecule BC to a peptide A of about 2 to about 20 acidic amino acid residues with cleavable linkers X1 and X2 of about 3 to about 30 atoms to form a cyclic molecule of the structure A-X1-BC-X2 where each of X1 and X2 link to both A and BC, and    cleaving said cleavable linkers X1 and X2 effective to separate BC from said peptide A.    
     
     
         21 . A molecule for transporting a cargo moiety across a cell membrane of the structure A-X-B-C, wherein 
 C is a portion comprising a cargo moiety,    B is a peptide portion of about 5 to about 20 basic amino acid residues, which is suitable for cellular uptake, is covalently linked to portion C, and is effective to enhance transport of cargo portion C across a cell membrane,    A is a peptide portion of about 2 to about 20 acidic amino acid residues, which when linked with portion B is effective to inhibit or prevent cellular uptake of B-C, and    X is a cleavable linker of about 2 to about 100 atoms joining A with B-C, which can be cleaved under conditions in or near a target cell.    
     
     
         22 . The molecule for transporting a cargo moiety across a cell membrane of  claim 21 , wherein said portion comprising a cargo moiety C comprises a phage.  
     
     
         23 . The molecule of  claim 21 , wherein said portion comprising a cargo moiety C comprises a label.  
     
     
         24 . The molecule of  claim 23 , wherein said label comprises a label selected from the group consisting of fluorescent labels, luminescent labels, radioactive labels, contrast agents, radiation sensitizers, antibodies, and antigens.  
     
     
         25 . The molecule of  claim 21 , wherein said portion comprising a cargo moiety C comprises a radioactive technetium label.  
     
     
         26 . The molecule of  claim 21 , wherein said portion comprising a cargo moiety C comprises a contrast agent.  
     
     
         27 . The molecule of  claim 21 , wherein said portion comprising a cargo moiety C comprises a gadolinium contrast agent.  
     
     
         28 . A method of guiding surgical resection of a cancer, the method comprising the steps of administering to the subject a molecule of  claim 21 , wherein the cargo is a fluorescent molecule.  
     
     
         29 . The method of  claim 28 , wherein the cancer has metastasized to lymph nodes which are resected during the surgery.  
     
     
         30 . A method of diagnosing metastatic cancer in lymph nodes by in vivo imaging, the method comprising the steps of administering to a subject a molecule of  claim 21 , wherein the cargo is a gamma emitting radionuclide, and imaging the lymph nodes using SPECT or planar gamma camera imaging.  
     
     
         31 . The method of  claim 30 , wherein the gamma emitting radionuclide is selected from the group consisting of  64 Cu,  99m Tc,  111 In,  123 I,  124 I or  131 I.  
     
     
         32 . A method of diagnosing metastatic cancer in lymph nodes by in vivo imaging, the method comprising the steps of administering to a subject a molecule of  claim 21 , wherein the cargo is a contrast agent, and imaging the lymph nodes using MRI.  
     
     
         33 . The method of  claim 32 , wherein the contrast agent is Gd 3+ .  
     
     
         34 . A method of in vivo selection of protease cleavage sites in linker X, the method comprising the steps of: 
 (a) providing a molecule of  claim 21 , wherein the cargo is a phage, the molecule is attached to the phage via a coat protein attached to at portion B, and X is a test cleavable linker from a random amino acid library;    (b) administering the phage to a non-human subject;    (c) removing tumor tissue comprising the phage;    (d) isolating the phage from the tumor tissue;    (e) amplifying the phage by re-administering the phage to a non-human subject; and    (f) isolating and sequencing the phage to identify protease cleavage sites.    
     
     
         35 . The method of  claim 34 , wherein the phage is administered to the non-human subject for a total of seven cycles.  
     
     
         36 . The method of  claim 34 , wherein the molecule has a structure A-X-B-C, wherein B is linked to C at the N-terminus of the pIII coat protein of M13 phage, or C-B-X-A, wherein B is linked to C at the C-terminus of the 10B capsid protein of T7 phage.  
     
     
         37 . A method of in vitro selection of protease cleavage sites in linker X, the method comprising the steps of: 
 (a) provide a molecule of  claim 21 , wherein the cargo is a phage, the molecule is attached to the phage via a coat protein attached to at portion B, and X is a test cleavable linker from a random amino acid library;    (b) incubating the phage with protease from a non-cancerous cell;    (c) isolating phage that has not entered the cell;    (d) incubating the phage with protease from a cancerous cell;    (e) isolating phage cleaved by protease from the cancerous cell; and    (f) sequencing the phage to identify protease cleavage sites.    
     
     
         38 . The method of  claim 37 , wherein the phage is amplified for a total of six cycles.  
     
     
         39 . The method of  claim 37 , wherein the protease is purified.  
     
     
         40 . The method of  claim 37 , wherein the protease is from a tissue extract.  
     
     
         41 . A method of imaging a tumor in vivo, the method comprising the steps of 
 administering to a subject a molecule of  claim 21 , wherein the cargo is a contrast agent, and imaging the tumor using MRI.    
     
     
         42 . The method of  claim 41 , wherein the contrast agent is Gd 3+ .  
     
     
         43 . A method of imaging a tumor in vivo, the method comprising the steps of administering to a subject a molecule of  claim 21 , wherein the cargo is a gamma emitting radionuclide, and imaging the tumor using SPECT or planar gamma camera imaging.  
     
     
         44 . The method of  claim 43 , wherein the gamma emitting radionuclide is selected from the group consisting of  64 Cu,  99m Tc,  111 In,  123 I,  124 I or  131 I.  
     
     
         45 . A method of treating cancer in a subject, the method comprising the steps of administering to the subject a molecule of  claim 21 , wherein the cargo is a therapeutic radionuclide.  
     
     
         46 . The method of  claim 45 , wherein the therapeutic radionuclide is selected from the group consisting of  90 Y or  131 I,  32 P,  64 Cu,  67 Cu,  89 Sr,  111 In,  117m Sn,  153 Sm,  177 Lu,  186 Re, and  188 Re.  
     
     
         47 . The molecule of  claim 21 , wherein peptide portion A is further linked to a macromolecule having a size of about 50-70 kD.  
     
     
         48 . The molecule of  claim 21 , wherein peptide portion A is further linked to a macromolecule having a size of about 35-70 kD.  
     
     
         49 . The molecule of  claim 48 , wherein the macromolecule is selected from the group consisting of dextran, serum albumin, PSA, PEG, and PAMAM dendrimer.  
     
     
         50 . The molecule of  claim 21 , wherein the cargo is linked to a nuclear localization signal.  
     
     
         51 . The molecule of  claim 21 , wherein the cargo is linked to an endosomal release signal.  
     
     
         52 . The molecule of  claim 21 , wherein the cargo is selected from the group consisting of  64 Cu,  99m Tc,  111 In,  123 I,  124 I or  131 I.  
     
     
         53 . The molecule of  claim 21 , wherein the cargo is selected from the group consisting of  90 Y  32 P,  64 Cu,  67 Cu,  89 Sr,  111 In,  117m Sn,  153 Sm,  177 Lu,  186 Re, and  188 Re.  
     
     
         54 . The molecule of  claim 21 , wherein the cargo is selected from the group consisting of  32 P,  99m Tc,  47 Sc,  64 Cu,  67 Cu,  89 Sr,  86 Y,  87 Y,  90 Y,  105 Rh,  111 Ag,  111 In,  117m Sn,  149 Pm,  153 Sm,  166 Ho,  177 Lu,  186 Re,  188 Re,  211 At,  123 I,  124 I,  131 I and  212 Bi.  
     
     
         55 . The molecule of  claim 21 , wherein the cleavable linker is cleaved by MMP-2, MMP-9, MMP11m MMP-13, MMP-14, uPA, or PSA.  
     
     
         56 . A pharmaceutical composition comprising the molecule of  claim 21.