Neuropilin-1 Specific Binding Peptide, Fusion Protein Fused with Same, and Use Thereof

Abstract

A peptide that binds specifically to neuropilin-1 (NRP1) without binding to neuropilin-2 (NRP2) is provided. A fusion protein, a fusion antibody, small-molecule drug, a nanoparticle, or a liposome, which comprises the peptide, and a pharmaceutical composition for treating or preventing cancer or angiogenesis-related diseases, and a composition for diagnosing cancer or angiogenesis-related diseases are provided. A polynucleotide encoding the peptide that binds specifically to NRP1 and a method for screening the peptide that binds specifically to NRP1 are provided. An antibody heavy-chain constant region Fc-fused peptide binding specifically to NRP1 has the property of binding specifically to NRP1, and thus when it is administered in vivo, it accumulates selectively in tumor tissue, and widens the intercellular space between tumor-associated endothelial cells to promote its extravasation and increases its tumor tissue penetration.

1 . A peptide that binds specifically to neuropilin-1, without binding to neuropilin-2,
 wherein the peptide comprises 5 to 50 amino acids, and the C-terminus of the peptide is represented by X1-X2-X3-X4, wherein X1 is arginine, lysine, or any amino acid residue, X2 and X3 are each independently any amino acid residue, and X4 is arginine or lysine.   
     
     
         2 . The peptide of  claim 1 , wherein the amino acid residue constituting X3 from the N-terminus of the peptide is selected from the group consisting of histidine, glycine, asparagine,
 serine, glutamine, phenylalanine, valine, leucine, threonine, arginine, proline, isoleucine, alanine, and lysine.   
     
     
         3 . The peptide of  claim 1 , wherein the peptide has tumor tissue-penetrating activity and/or anti-angiogenesis activity. 
     
     
         4 . The peptide of  claim 1 , wherein the peptide comprises an amino acid sequence of any one of SEQ ID NO: 1 to SEQ ID NO: 3. 
     
     
         5 . The peptide of  claim 1 , wherein the peptide further comprises a linker peptide. 
     
     
         6 . The peptide of  claim 5 , wherein the linker peptide consists of 1 to 50 amino acids. 
     
     
         7 . The peptide of  claim 5 , wherein the linker peptide comprises an amino acid sequence of (GGGGS)n, wherein n is each independently an integer between 1 and 20. 
     
     
         8 . The peptide of  claim 7 , wherein the peptide comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 4 to SEQ ID NO: 6. 
     
     
         9 . A fusion protein comprising the peptide of any one of  claims 1  to  8 , which is fused thereto. 
     
     
         10 . The fusion protein of  claim 9 , wherein the protein is selected from the group consisting of antibodies, antibody fragments, immunoglobulin, peptides, enzymes, growth factors, cytokine, transcription factors, toxins, antigen peptides, hormones, carrier proteins, motor function proteins, receptors, signaling proteins, storage proteins, membrane proteins, transmembrane proteins, internal proteins, external proteins, secretory proteins, viral proteins, glycoproteins, cleaved proteins, protein complexes, and chemically modified proteins. 
     
     
         11 . The fusion protein of  claim 9 , wherein the peptide binds to neuropilin-1 bivalently ormultivalently. 
     
     
         12 . The fusion protein of  claim 9 , wherein the fusion is mediated by a linker peptide. 
     
     
         13 . The fusion protein of  claim 10 , wherein each of the antibody fragments is a heavy-chain constant region fragment (Fc), a heavy-chain constant region domain fragment (CH1, CH2, or CH3), an antigen binding fragment (Fab), a single-chain variable fragment (scFv), a heavy-chain variable region fragment (VH), a light-chain constant region fragment (CL), or a light-chain variable region fragment (VL). 
     
     
         14 . The fusion protein of  claim 10 , wherein a peptide is fused to the C-terminus of the heavy-chain constant region (Fc) of an antibody, in case where the protein is an antibody. 
     
     
         15 . The fusion protein of  claim 14 , wherein the fusion is mediated by a linker peptide. 
     
     
         16 . The fusion protein of  claim 14 , wherein the antibody is any one selected from the group consisting of IgG, IgM, IgA, IgD, and IgE. 
     
     
         17 . A nanoparticle comprising the peptide of any one of  claims 1  to  8 , which is fused thereto. 
     
     
         18 . A liposome comprising fused thereto the peptide of any one of  claims 1  to  8 , which is fused thereto. 
     
     
         19 . A small-molecule drug comprising fused thereto the peptide of any one of  claims 1  to  8 , which is fused thereto. 
     
     
         20 . A polynucleotide that encodes the peptide of any one of  claims 1  to  8 . 
     
     
         21 . A pharmaceutical composition for treating or preventing cancer or angiogenesis-related diseases, comprising the peptide of any one of  claims 1  to  8 , a fusion protein 20 comprising the peptide which is fused thereto, a nanoparticle comprising the peptide which is fused thereto, a liposome comprising the peptide which is fused thereto, or a small-molecule drug comprising the peptide which is fused thereto. 
     
     
         22 . A composition for diagnosing cancer or angiogenesis-related diseases, in which the composition comprises the peptide of any one of  claims 1  to  8 , a fusion protein comprising the peptide which is fused thereto, a nanoparticle 5 comprising the peptide which is fused thereto, a liposome comprising the peptide which is fused thereto, or a small-molecule drug comprising the peptide which is fused thereto. 
     
     
         23 . A method of screening the peptide according to  claim 1  comprises the steps of:
 (1) designing a peptide library capable of interacting with the VEGF-binding site (or arginine-binding pocket) of the b1 domain of NRP1; 
 (2) fusing the peptide library of step (1) to the C-terminus of an antibody heavy-chain constant region Fc; 
 (3) binding the Fc-fused library of step (2) to NRP1-b1b2 in the presence of high amount of NRP2-b1b2 as a competitor; and 
 (4) screening desirable Fc-fused peptides based on the binding affinity between the isolated Fc-fused peptide library and NRP1-b1b2 bound in step (3).