Nidogen-based scaffold proteins and therapeutic nanocomplexes

Abstract

We provide a therapeutic drug delivery polypeptide that can be used for treatment. [Solution] The present invention relates to a protein suitable for use as a scaffold to which a target peptide binds, or a protein contained within a complex to which a target agent binds. It also relates to a complex suitable for selectively delivering a complex of a target agent to specific types of cells and tissues. It also relates to nanoparticles containing such a complex. In one embodiment, a polypeptide is provided comprising (i) 11 β-chain domains designated A, B, C, D, E, F, G, H, I, J, and K, and (ii) 10 loop regions connecting two consecutive β-chain domains, designated AB, BC, CD, DE, EF, FG, GH, HI, IJ, and JK loops, wherein at least one of the loop regions is a homogeneous loop region variant, and at least one of the β-chain domains is a homogeneous β-chain variant.

Description

[Technical Field]

[0001] This invention relates to the field of nanostructured protein materials, and more specifically to therapeutic drug delivery polypeptides that can be used in treatment. [Background technology]

[0002] Systemic administration of drugs in the form of nanocomposites offers the advantage of improved drug stability compared to free molecules. By chemically incorporating functional groups into nanoscale media, the high surface area / volume ratio of nanomaterials can be leveraged, and beneficial additional properties such as cell targeting can be integrated into existing hybrid composite materials. The resulting drug-carrying complexes, approximately 8-100 nm in size, will avoid renal filtration when administered systemically, unless aggregation occurs in the lungs or other highly angiogenic organs. This fact, combined with appropriate physicochemical properties of the material, can lead to extended circulation time and prolonged drug exposure to target organs, thus potentially enhancing the therapeutic effect and benefits for patients.

[0003] Among the diverse materials being investigated as drug carriers, including metals, ceramics, polymers, and carbon nanotubes, proteins offer unique properties in terms of biocompatibility and degradability, making them particularly favorable given growing concerns about nanotoxicity. As the engineering of self-assembly of proteins into nanostructured materials rapidly advances and control over the final shape and physicochemical properties becomes more stringent, protein materials will gain functional and structural diversity as a carrier, moving beyond chemically bound drugs.

[0004] In fact, the formation of antibody-drug conjugates (ADCs) by binding a cytotoxic "payload" to an antibody has been shown to provide a mechanism for selective delivery of cytotoxic agents to cancer cells via the specific binding of antibodies to cancer-selective cell surface molecules. Several examples of this strategy have proven effective, such as gemtuzumab ozogamicin, which contains an anti-CD33 antibody conjugated to calicheamicin, a highly potent DNA-targeted antibiotic used for acute myeloid leukemia. Furthermore, mytansinoids, highly potent microtubule-disrupting agents, have been tested for the maximum payload of ADCs, resulting in adtrastuzumab emtansine, a formulation for treating HER2-positive breast cancer.

[0005] Nevertheless, the structural complexity of antibodies can still be a troublesome obstacle in terms of cost and synthesis. We have previously explored the field of nanomedicine by applying nanostructure principles based on the addition of cationic N-terminal domains and C-terminal polyhistidines to core proteins [Serna, N. et al. 2016. Nanomedicine, 12:1241-51]. The charge balance of these terminal tags and the resulting fusions has been described in the art as promoting the self-assembly and oligomerization of monomer proteins as robust toroidal nanoparticles, making them stable in plasma [Cespedes, MV et al. 2014. ACS Nano., 8:4166-4176], and increasing their permeability into cells when cell-targeted peptides are added [Xu, ZK et al. 2015. Materials Letters, 154:140-3]. These protein structural components may also include functional peptides such as cell-targeting agents, endosomal lysants, or nuclear localization signals, in the form of fusion stretches through modular structuring.

[0006] Current treatments still have room for improvement, mainly due to tumor resistance phenomena that can be caused by intratumoral clonal selection of cells that are most resistant to chemotherapy. For example, in this field, there is still a need to develop more specific therapeutic approaches that can target specific tumor cells involved in treatment failure and tumor progression while reducing the side effects and off-target effects of therapeutic drugs. [Overview of the project]

[0007] In the first embodiment, the present invention is (i) Eleven β-chain domains designated as A, B, C, D, E, F, G, H, I, J, and K, and (ii) Ten types of loop regions that connect two consecutive β-chain domains, designated as AB, BC, CD, DE, EF, FG, GH, HI, IJ, and JK loops. A polypeptide containing; At least one of the loop regions is a congeneral loop region variant in SEQ ID NO: 62, and the congeneral loop region in SEQ ID NO: 62 is defined by SEQ ID NO: 1 (loop region AB), SEQ ID NO: 2 (loop region BC), SEQ ID NO: 3 (loop region CD), SEQ ID NO: 4 (loop region DE), SEQ ID NO: 5 (loop region EF), SEQ ID NO: 6 (loop region FG), amino acids 149-150 of SEQ ID NO: 62 (loop region GH), SEQ ID NO: 7 (loop region HI), SEQ ID NO: 8 (loop region IJ), and SEQ ID NO: 9 (loop region JK), and The present invention relates to a polypeptide in which at least one of the aforementioned β-chain domains is a variant of the congeneral β-chain in SEQ ID NO: 62 and has at least 50% sequence identity with the congeneral β-chain domain, wherein the congeneral β-chain domain in SEQ ID NO: 62 is defined by SEQ ID NO: 9 (β-chain domain A), SEQ ID NO: 11 (β-chain domain B), SEQ ID NO: 12 (β-chain domain C), SEQ ID NO: 13 (β-chain domain D), SEQ ID NO: 14 (β-chain domain E), SEQ ID NO: 15 (β-chain domain F), SEQ ID NO: 16 (β-chain domain G), SEQ ID NO: 17 (β-chain domain H), SEQ ID NO: 18 (β-chain domain I), SEQ ID NO: 19 (β-chain domain J), and SEQ ID NO: 20 (β-chain domain K).

[0008] In a second embodiment, the present invention relates to a polypeptide display library comprising a plurality of polypeptides according to a first embodiment of the present invention, wherein the plurality of polypeptides are formed by polypeptides having one or more loop region sequences that differ.

[0009] In a third embodiment, the present invention relates to a polynucleotide encoding a polypeptide according to a first aspect of the present invention, or a polypeptide of a polypeptide display library according to a second aspect of the present invention.

[0010] In a fourth embodiment, the present invention relates to a vector comprising a polynucleotide according to a third aspect of the present invention.

[0011] In a fifth embodiment, the present invention relates to a host cell comprising a polynucleotide according to a third aspect of the present invention, or a vector according to a fourth aspect of the present invention.

[0012] In a sixth embodiment, the present invention is (i) A first polypeptide region comprising the G2 domain of nidogen-1 or a functionally equivalent variant thereof, and (ii) the agent of the target Regarding complexes that include this.

[0013] In a seventh embodiment, the present invention relates to a method for preparing a composite according to a sixth aspect of the present invention, (i) To provide a polypeptide of a complex according to a sixth aspect of the present invention comprising the G2 domain of nidogen-1 or a functionally equivalent variant, which is an activated polypeptide, and (ii) Contacting the polypeptide with a target agent that can react with the reactive group in the polypeptide under conditions suitable for forming a bond between the reactive group in the polypeptide and the group in the target agent. Regarding methods including

[0014] In the eighth embodiment, the present invention is (i) A first region comprising the G2 domain of nidogen-1 or a functionally equivalent variant thereof, and (ii) A second region containing an antagonistic CXCR4 ligand Regarding polypeptides including [specific polypeptides].

[0015] In the ninth embodiment, the present invention relates to a method for preparing a composite according to the sixth aspect of the present invention, (i) To provide a polypeptide of a complex according to a sixth aspect of the present invention, comprising the G2 domain of nidogen-1 or a functionally equivalent variant thereof, and (ii) Contacting the polypeptide with an activated form of the agent of interest of a complex according to a sixth aspect of the present invention, which can react with at least one group in the polypeptide, under conditions suitable for forming a bond between the reactive group in the agent of interest and the group in the polypeptide. Regarding methods including

[0016] In a tenth embodiment, the present invention relates to a method for preparing a composite according to a sixth aspect of the present invention, (i) To provide a polypeptide of a complex according to a sixth aspect of the present invention comprising the G2 domain of nidogen-1 or a functionally equivalent variant, which is an activated polypeptide, and (ii) Contacting the polypeptide with a target agent that can react with the reactive group in the polypeptide under conditions suitable for forming a bond between the reactive group in the polypeptide and the group in the target agent. Regarding methods including

[0017] In the eleventh embodiment, the present invention is (i) A first region comprising the G2 domain of nidogen-1 or a functionally equivalent variant thereof, and (ii) A second region containing an antagonistic CXCR4 ligand Regarding polypeptides including [specific polypeptides].

[0018] In a twelfth aspect, the present invention relates to a method for preparing nanoparticles comprising a plurality of copies of a polypeptide according to an eleventh aspect of the present invention, the method comprising placing the polypeptide preparation under conditions suitable for assembling the plurality of copies of the polypeptide to form nanoparticles.

[0019] In a thirteenth aspect, the present invention provides a method for preparing nanoparticles comprising multiple copies of a complex according to a sixth aspect of the present invention, or multiple copies of a polypeptide according to an eleventh aspect of the present invention, (i) A method comprising placing the preparation of the complex or polypeptide under conditions suitable for assembling multiple copies of the complex or polypeptide to form nanoparticles, or (ii) i. Each 1. The G2 domain of Nidogen-1 or a functionally equivalent variant of the first polypeptide region, 2. A second polypeptide that can specifically bind to a target of interest, the second polypeptide being a polycationic peptide, and 3. The third polypeptide region, which is rich in positively charged amino acids. Placing a plurality of polypeptides containing the polypeptide under conditions suitable for forming nanoparticles containing a plurality of copies of the polypeptide, The polycationic peptide and the region rich in positively charged amino acids are located at the terminus of the polypeptide, the polypeptide is provided in an activated form, and the polypeptide in the activated form contains a reactive group, and ii. The nanoparticles obtained in step i and the activated form of the target agent, which contains a group capable of reacting with the reactive group in the polypeptide, are brought into contact under conditions suitable for forming a bond between the reactive group in the polypeptide and the group in the target agent. Methods including Regarding the method of selection.

[0020] In a fourteenth embodiment, the present invention relates to a method for preparing biparatopic nanoparticles comprising a plurality of copies of a first type complex and a plurality of copies of a second type complex, wherein the first and second type complexes are defined in a sixth embodiment, and the first and second type complexes have different polycationic peptides. (i) A method comprising contacting a preparation of the first type of composite and a preparation of the second type of composite under conditions suitable for assembling multiple copies of the two types of composites to form nanoparticles, or (ii) i. Contacting a preparation of a first polypeptide with a preparation of a second polypeptide, wherein the first and second types of polypeptides are a. The G2 domain of Nidogen-1 or a functionally equivalent variant of the first polypeptide region, b. A second polypeptide region capable of specifically binding to a target of interest, wherein the second polypeptide is a polycationic peptide and / or comprises a positively charged additional peptide sequence and a polycationic sequence located at its N-terminus or C-terminus, wherein the polycationic peptide sequence of one polypeptide is different from the polycationic peptide sequence of the other polypeptide. c. The third polypeptide region, which is rich in positively charged amino acids. d. Optionally, a positively charged peptide sequence located at the N-terminus or C-terminus of a polycationic peptide. Includes, The region rich in the polycationic peptide and positively charged amino acids is located at the terminus of the polypeptide. The first and second polypeptides have different polycationic peptides, The first and / or second polypeptide exists as a complex as defined in the sixth aspect of the present invention, The first and / or second polypeptide is provided in an activated form, the polypeptide in the activated form comprises a reactive group, and the contact is carried out under conditions suitable for forming nanoparticles comprising multiple copies of the polypeptide. ii. The nanoparticles obtained in step i and the activated form of the target agent, which contains a group capable of reacting with the reactive group in each polypeptide, are brought into contact under conditions suitable for forming a bond between the reactive group in the polypeptide and the group in the target agent. Methods including Regarding the method of selection.

[0021] In a 15th aspect, the present invention relates to a method for preparing biparatopic nanoparticles comprising a plurality of copies of at least one complex according to a sixth aspect of the present invention and a plurality of copies of at least one polypeptide according to an 11th aspect of the present invention, wherein the sequence of the polycationic peptide of the first type of complex and the sequence of the second region of the at least one polypeptide are different. (i) A method comprising placing a preparation of multiple copies of the at least one complex and multiple copies of the at least one polypeptide under conditions suitable for assembling the multiple copies of the two complexes into nanoparticles, or (ii) i. Contacting a preparation of a first polypeptide with a preparation of a second polypeptide, wherein the first and second types of polypeptides are a. The G2 domain of Nidogen-1 or a functionally equivalent variant of the first polypeptide region, b. A second polypeptide region capable of specifically binding to a target of interest, wherein the second polypeptide is a polycationic peptide and / or comprises a positively charged additional peptide sequence and a polycationic sequence located at its N-terminus or C-terminus, and the peptide sequence of one polypeptide differs from the polycationic peptide sequence of the other polypeptide. c. The third polypeptide region, which is rich in positively charged amino acids. Includes, The region rich in the polycationic peptide and positively charged amino acids is located at the terminus of the polypeptide. The first polypeptide exists as a complex as defined in the sixth aspect of the present invention, and the second polypeptide is defined in the eleventh aspect of the present invention. The polycationic peptide of the first polypeptide and the polycationic peptide of the second polypeptide are different, The first and / or second polypeptide is provided in an activated form, the polypeptide in the activated form comprises a reactive group, and the process is carried out under conditions suitable for forming nanoparticles comprising multiple copies of the polypeptide. ii. The nanoparticles obtained in step i and the activated form of the target agent, which contains a group capable of reacting with the reactive group in each polypeptide, are brought into contact under conditions suitable for forming a bond between the reactive group in the polypeptide and the group in the target agent. Methods including Regarding the method of selection.

[0022] In a sixteenth aspect, the present invention relates to nanoparticles comprising multiple copies of a complex according to a sixth aspect of the present invention, multiple copies of a polypeptide according to an eleventh aspect of the present invention, or obtained by a method according to a twelfth or thirteenth aspect of the present invention.

[0023] In the 17th aspect, the present invention relates to biparatopic nanoparticles comprising multiple copies of first and second type complexes, wherein both of the first and second type complexes exist as polypeptides defined in the 6th aspect of the present invention or as polypeptides defined in the 11th aspect of the present invention, and the first and second type complexes are different biparatopic nanoparticles obtained by the method according to the 14th or 15th aspect of the present invention.

[0024] In the 18th aspect, the present invention relates to biparatopic nanoparticles comprising a plurality of copies of a complex according to the 6th aspect of the present invention and a plurality of copies of a polypeptide according to the 11th aspect of the present invention, wherein the polycation region of the complex and the first region of the polypeptide are different, or to biparatopic nanoparticles obtained by a method according to the 14th or 15th aspect of the present invention.

[0025] In a 19th embodiment, the present invention relates to a complex according to a sixth embodiment of the present invention, a polypeptide according to an eleventh embodiment of the present invention, or nanoparticles according to an 16th, 17th, or 18th embodiment of the present invention for use in pharmaceuticals.

[0026] In a 20th aspect, the present invention provides a method for imaging target cells comprising a specific binding site for one or more components of a complex according to the 6th aspect of the present invention, one or more components of a polypeptide according to the 11th aspect, or one or more components of nanoparticles according to the 16th, 17th, or 18th aspect of the present invention, (i) Contacting a sample containing the cells with a complex according to the sixth aspect of the present invention, a polypeptide according to the eleventh aspect of the present invention, or nanoparticles according to the sixteenth, seventeenth, or eighteenth aspect of the present invention under conditions suitable for binding of the complex, polypeptide, or nanoparticles to the cells, wherein the agent of interest is a contrast agent; and (ii) imaging the cells by detecting a signal provided by the contrast agent.

[0027] In a 21st embodiment, the present invention relates to a method for identifying polypeptides that bind to a target peptide, i) Contacting the target peptide with a polypeptide display library according to a second aspect of the present invention under conditions that allow the polypeptide and the target peptide to interact, ii) Recovering library members that have specifically interacted with the target peptide, iii) Identifying the sequence of the polypeptide that interacts with the target peptide. Regarding methods including

[0028] In a 22nd embodiment, the present invention relates to the use of a polypeptide according to a first embodiment of the present invention for presenting a peptide, wherein the peptide is found in one of the loop regions.

[0029] In a 23rd embodiment, the present invention relates to a method for determining the presence of a target peptide in a sample, i) Contacting a protein present in a sample with a polypeptide according to the first aspect of the present invention, wherein at least one sequence of the loop region in the polypeptide is a sequence that can specifically bind to the target peptide. ii) Determine whether there is an interaction between the target peptide and the polypeptide, and if there is an interaction between the polypeptide and the target peptide, determine that the target peptide is present in the sample. Regarding methods including [Brief explanation of the drawing]

[0030] [Figure 1] Figure 1 shows the structure of the human nidogen-1 protein as presented by Takagi J. et al. (Nature 424, 969-974, 2003). G1, G2, and G3 represent the three main globular domains. EG represents the EGF module, TY represents the thyroglobulin repeat, and LY represents the LDL receptor YWTD repeat. [Figure 2] Figure 2 shows the properties of T22-STM-H6 (A), T22-NIDOmut2-H6 (B), and T22-GFP-H6 (C) nanoparticles. The MALDI-TOF mass spectrometry spectra are shown above, the results of Western blot immunoassay using an anti-His monoclonal antibody are shown as an inset, and the volume size distribution of each nanoparticle measured by DLS is shown below. [Figure 3]Figure 3 shows the characteristics of T22-NIDOmut2-H6 nanoparticles. A) Coomassie blue staining of purified peaks 1 (Pico1) and 2 (Pico2) of T22-NIDOmut2-H6 in carbonate (-) and carbonate + salt (+) buffers by SDS-PAGE electrophoresis. B) Western blot immunoassay detection of T22-NIDOmut2-H6 protein with anti-His monoclonal antibody. C) Volume size distribution of T22-NIDOmut2-H6 nanoparticles determined by DLS. D) MALDI-TOF mass spectrometry spectrum of T22-NIDOmut2-H6 protein. [Figure 4] Figure 4 shows the labeling of T22-NIDOmut2-H6. A) MALDI-TOF mass spectrometry spectrum of the labeled T22-NIDOmut2-H6-ATTO488 protein. Each peak above 30.3 kDa corresponds to the incorporation of additional ATTO molecules. B) Volume size distribution of T22-NIDOmut2-H6-ATTO488 nanoparticles determined by DLS. [Figure 5] Figure 5 shows the CXCR4-specific internalization of the T22-NIDOmut2-H6 protein in CXCR4+ cells. A) Internalization and competition (+AMD) of labeled T22-NIDOmut2-H6-ATTO488 nanoparticles after 24 hours incubation on HeLa cells (CXCR4+) at different concentrations (1 nM and 10 nM). Percentage of inhibition of cellular uptake in the presence of the CXCR4 receptor antagonist AMD3100 (+AMD) is shown. B) Confocal laser microscopy images of HeLa cells after 24 hours incubation in the presence of 25 nM T22-NIDOmut2-H6-ATTO488. Cell nuclei are stained with Hoechst, cell membranes with CellMask, and intermittent patterns within the cell correspond to protein nanoparticles. [Figure 6]Figure 6 shows T22-NIDOmut2-H6 nanoparticles bound to oligoFdU. A) Schematic diagram of covalent bonding via a two-step reaction of thiol-linked oligo5'-(FdU)5-hexaethylene glycol thiol-3'(oligo-FdU-SH) via protein lysine amine using a 6-maleimidohexanoic acid N-hydroxysuccinimide (EMCS) bifunctional linker. B) MALDI-TOF mass spectrometry spectrum of the T22-NIDOmut2-H6-FdU nanocomposite. C) Volume size distribution of the T22-NIDOmut2-H6-FdU nanocomposite determined by DLS. [Figure 7] Figure 7 shows the cytotoxicity of oligo-FdU complex nanoparticles against CXCR4+ cells. The graph shows the survival rate (%) of HeLa cells (CXCR4+) after 48-hour incubation in the presence of 25 nM or 100 nM T22-STM-H6-FdU, T22-NIDOmut2-H6-FdU, or two different stocks of T22-GFP-H6-FdU nanocomplexes, and 100 nM free oligo-FdU (FdU), as measured by the MTT survival assay. [Figure 8] Figure 8 shows that the T22-NIDOmut2-H6-FdU nanocomplex induces higher growth inhibition than T22-STM-H6-FdU or T22-GFP-H6-FdU in the CXCR4+ tumor model. The graph shows the change in tumor volume over time for each nanocomplex treatment group (n=4) at a dose of 20 μg q3d x 5, and a comparison with buffer treatment (K, n=4) in the CXCR4+ M5 subcutaneous (SC) colorectal cancer (CRC) model. [Figure 9] Figure 9 shows that in CXCR4+M5 tumor tissue, the T22-NIDOmut2-H6-FdU nanocomplex induces apoptosis more effectively than T22-STM-H6-FdU or T22-GFP-H6-FdU. A) The graph shows the number of apoptotic bodies observed in tumor sections treated with buffer or the nanocomplex at the end of the experiment. B) Representative micrographs of H&E-stained sections identifying the apoptotic features (black arrows) observed in each group compared. [Figure 10]Figure 10 shows no histological changes in the kidney or liver between the compared groups. Representative H&E stained sections of kidney and liver tissue showing no structural or histological changes (no signs of inflammation or apoptosis) in both organs at the end of treatment in control buffer-treated mice or nanocomplex-treated mice. [Figure 11] Figure 11 shows the correlation between intrinsic fluorescence and temperature for isolated human nidogen G2 domains with hexahistidine tags, isolated human nidogen G2 containing hexahistidine-tagged mutations H459A, R468N, F639S, and R650A, and stefin A. Arrows indicate sample heating. Developed curves of Nidomut2H6(B), NidoWTH6(C), and STMH6(D) using CSM values ​​as a function of temperature. CSM values ​​were calculated for each protein from the experiments exemplified in Figure 1a. Black and gray arrows indicate Tm and T start values, respectively. [Figure 12] Figure 12 shows the structure of the Nidogen G2 domain. A) Schematic diagram of the interaction between the multi-domain human Nidogen 1 protein and its native ligand. Shows the β-barrel structure within the G2 domain. B) Secondary structure of the Nidogen G2 β-barrel domain. Different gray β-sheets (A-K) and a blue α-helix. C) Schematic diagrams of the tertiary β-barrel structures of Nidogen G2 (RCSB PDB database accession number 1GL4, version 1.2 from July 13, 2011) in the left panel and green fluorescent protein (RCSB PDB database chain A accession number 1QYO, version 1.2 from July 13, 2011) in the right panel. D) Superposition of the barrel structures of Nidogen G2 and GFPβ. [Figure 13]Figure 13 shows the design of the NidoMut2 peptide. A) Similarity of the human and mouse nidogen G2 domain: Amino acid alignment between human nidogen 1 protein (P14543 in the Uniprot database version dated July 7, 2009) and mouse nidogen 1 protein (P10493 in the Uniprot database version dated July 7, 2009) analyzed by Clustal Omega (EMBL-EMI). The black lines indicate the start and end points of the G2β barrel domain. "*" indicates amino acid agreement, ":" indicates amino acids with high similarity, and "." indicates amino acids with low similarity. B) Amino acid sequences of human nidogen 1 protein, HSNBT scaffold protein, and T22-HSNBT-H6 protein. In human nidogen, the G2β barrel domain is highlighted in black, and candidate amino acids for mutation are shown in bold black and underlined. In the NIDOmut2 sequence, incorporated mutations are shown in bold black. In T22-NIDOmut2-H6, the N-terminal T22 ligand is shown in underlined black, the short linker is shown in bold black, incorporated mutations are shown in bold and underlined, and the C-terminal polyhistidine tail is highlighted in bold black italics. [Figure 14] Figure 14 shows a Western blot of the soluble fraction of the cell lysate from the protein expression test shown in the figure. [Figure 15] Figure 15 shows the MALDI-TOF mass spectrometry spectra after expression testing of the selected candidate proteins shown in each panel of the figure. The theoretical size of all proteins is approximately 30.3 kDa. [Figure 16] Figure 16 shows the volume size distribution of the T22-NIDOmut2-H6 protein, T22-NIDOmut3-H6 protein, T22-NIDOmut4_T215V-H6 protein, and T22-NIDOmut5-H6 protein, as well as their nanoparticles, as determined by DLS. [Figure 17]Figure 17A shows the precipitation profiles of T22-NIDOmut2-H6, T22-NIDOmut3-H6, T22-NIDOmut4_T215V-H6, and T22-NIDOmut5-H6 after incubation with increasing ZnCl2 concentration. Figure 17B shows the volume size distribution of T22-NIDOmut2-H6, T22-NIDOmut3-H6, T22-NIDOmut4_T215V-H6, and T22-NIDOmut5-H6 nanoparticles assembled at different ZnCl2 concentrations, as determined by DLS. [Figure 18] Figures 18A-C. CSM profiles of T22-NIDOmut2-H6, T22-NIDOmut3-H6, and T22-NIDOmut5-H6 in carbonate buffer and three FDA-approved buffers (A9, B6, D1). D. Graphs showing the key indices (Tm, Tonset, and ΔT) for each buffer tested. [Figure 19] Figures 19A-B show the MALDI-TOF mass spectrometry spectra of T22-NIDOmut3-H6-FdU and T22-NIDOmut2-H6-FdU. Each peak with an additional 2kDa added to the protein weight (30.3kDa) belongs to the complex protein with the additional oligoFdU added. C. Cytotoxicity assay after incubation of T22-NIDOmut3-H6-FdU nanocomplexes for 48 hours with a non-complex negative control and a reference T22-NIDOmut2-H6-FdU positive control. D. Volume size distribution of T22-NIDOmut3-H6-FdU. [Figure 20]Figure 20 shows the structural and functional properties of EPI-X4-based NPs. A. Schemes of modular proteins EPIX4-GFP-H6 (top) and EPIX4-(RK)-GFP-H6 (bottom), and the amino acid sequence of the latter. B. Mass spectrometry of EPIX4-GFP-H6 (left) and EPIX4-(RK)-GFP-H6 (right). The molecular weight of the purified proteins is shown by SDS-PAGE and Western blot (Anti-His). C. Hydrodynamic size and pdi (polydispersion index) determined by dynamic light scattering (DLS). Peak size values ​​(average) are shown (in nm). D. Size exclusion chromatography (SEC) of EPIX4-GFP-H6 (black) and EPIX4-(RK)-GFP-H6 (gray) using Superdex200 increase 10 / 300Gl column E. Representative FESEM (direct deposition) images of E.EPIX4-GFP-H6 (top) and EPIX4-(RK)-GFP-H6 (bottom) protein NPs. Size bars represent 50 nm. F. Protein amount internalized in CXCR4+ HeLa cells after administration of 2 μM EPIX4-GFP-H6 and EPIX4-(RK)-GFP-H6 at 4 hours (dark green). Inhibition of uptake promoted by the native CXCR4 ligand AMD3100 (light green). Intracellular fluorescence was corrected for their specific fluorescence values ​​to represent protein amount. Asterisks indicate significant differences in the internalization of EPIX4-GFP-H6 protein and EPIX4-(RK)-GFP-H6 protein, and hashes indicate significant differences in the inhibition promoted by EPIX4-(RK)-GFP-H6 and AMD3100 (p ≤ 0.001). Confocal images of HeLa cells exposed to G.EPIX4-GFP-H6 (left) and EPIX4-(RK)-GFP-H6 (right) for 24 hours. Blue: cell nucleus, red: cell membrane, green: internalized NP. Size bars represent 10 μm. All data are expressed as mean ± SEM. [Figure 21]Figure 21 shows the formation and characterization of biparatopic nanoparticles. A. Scheme of hybrid NPs forming the proteins EPIX4-(RK)-GFP-H6 (top) and T22-BFP-H6 (bottom). B. Controlled EPIX4-(RK)-GFP-H6 (black) degraded with 0.2% SDS, and EPIX4-(RK)-GFP-H6 (gray) assembled after removing SDS by dialysis; determined by dynamic light scattering (DLS) (top). Hydrodynamic size comparison of T22-BFP-H6 (gray), EPIX4-(RK)-GFP-H6 (black), and biparatopic nano NPs (gray dashed line) (bottom). Peak size values ​​(mean) are shown in nm and Pdi (polydispersion index). C. Representative FESEM (direct deposition) of biparatopic NPs. Size bars represent 50 nm. D. FRET analysis of biparatopic NP formation. Samples of monomer mixtures of biparatopic NP, T22-BFP-H6, and EPIX4-(RK)-GFP-H6, as well as a mixture of T22-BFP-H6 and EPIX4-(RK)-GFP-H6 NP, were excited with 405 nm light, and emission from 350 to 650 nm was collected. BFP was used as the donor fluorescent dye, and GFP was used as the acceptor. Time-course dynamics of intracellular incorporation of EPIX4-(RK)-GFP-H6, T22-GFP-H6, and biparatopic NP (1 μM) in E.CXCR4+ HeLa cells (left) and SW1417 (right). Intracellular fluorescence was corrected for specific fluorescence values ​​to represent protein amounts. Significant differences (p<0.05) between biparatopic NP and both proteins that form them are indicated by *, and significant differences between biparatopic NP and EPIX4-(RK)-GFP-H6 are indicated by #. Time-course dynamics of intracellular incorporation of EPIX4-(RK)-GFP-H6, T22-GFP-H6, and biparatopic NP (1 μM) in E.CXCR4+ HeLa cells (left) and SW1417 cells (right). Intracellular fluorescence was corrected for specific fluorescence values ​​to represent protein levels. Significant differences (p<0.05) between biparatopic NP and both proteins that form them are indicated by *, and significant differences between biparatopic NP and EPIX4-(RK)-GFP-H6 are indicated by #.Inhibition of CXCR4 uptake mediated by the CXCR4 antagonist AMD3100 (always in an excess molar ratio of 10:1) in HeLa cells exposed to 1 μM F for 1 hour. The & indicates a significant difference between inhibition promoted by NP and inhibition promoted by AMD3100 (p ≤ 0.001). [Figure 22] Figure 22 shows the in vivo biodistribution and toxicity assessment in a subcutaneous mouse model of CXCR4+ human colorectal cancer. A. Quantification of fluorescence emitted in tumors at various time points (measured as FLI ratio). B. Number of apoptotic cell bodies after nanoparticle administration. Significant differences (p<0.05) between EPIX4-(RK)-GFP-H6 and biparatopic NP compared to control are indicated by *, and significant differences between biparatopic NP and EPIX4-(RK)-GFP-H6 are indicated by #. C. Mitocytes after nanoparticle administration. Significant differences between EPIX4-(RK)-GFP-H6 and biparatopic NP are shown (*p<0.05). D. Absence of systemic toxicity in the kidney, liver, nephroditic and spleen by histological analysis of tissue sections (H&E) 5 and 24 hours after treatment. All photographs were taken at 400x magnification. All data are expressed as mean ± SEM. [Modes for carrying out the invention]

[0031] I. Polypeptide of the present invention The inventors have found that a G2 domain derived from a nidogen protein, optionally modified to exhibit reduced affinity for its native ligand, acts as a scaffold and is sufficiently stable to present a peptide inserted into one or more loop regions connected to the β-chain within the G2 domain. Furthermore, the inventors have found that when complexed with a ligand exhibiting affinity for a receptor expressed by target cells, the G2 domain of the nidogen protein can be used to deliver the target agent, including therapeutic and diagnostic / contrast agents, to the target cells. The inventors have found that a fusion protein comprising the nidogen G2 domain and the CXCR4-specific ligand T22, bound to an anticancer agent, can deliver the anticancer agent to CXCR4-expressing cells and inhibit cell proliferation to a degree significantly better than the inhibition achieved by equivalent agents fused with the T22 ligand to different proteins such as GFP or StefinA.

[0032] Therefore, in the first embodiment, the present invention is (i) Eleven β-chain domains designated as A, B, C, D, E, F, G, H, I, J, and K, and (ii) Ten types of loop regions that connect two consecutive β-chain domains, designated as AB, BC, CD, DE, EF, FG, GH, HI, IJ, and JK loops. A polypeptide containing; At least one of the loop regions is a congeneral loop region variant in SEQ ID NO: 62, and the congeneral loop region in SEQ ID NO: 62 is defined by SEQ ID NO: 1 (loop region AB), SEQ ID NO: 2 (loop region BC), SEQ ID NO: 3 (loop region CD), SEQ ID NO: 4 (loop region DE), SEQ ID NO: 5 (loop region EF), SEQ ID NO: 6 (loop region FG), amino acids 149-150 of SEQ ID NO: 62 (loop region GH), SEQ ID NO: 7 (loop region HI), SEQ ID NO: 8 (loop region IJ), and SEQ ID NO: 9 (loop region JK), and At least one of the aforementioned β-chain domains is a variant of the congeneral β-chain in SEQ ID NO: 62, and has at least 50% sequence identity with the congeneral β-chain domain, and the congeneral β-chain domain in SEQ ID NO: 62 is defined by SEQ ID NO: 9 (β-chain domain A), SEQ ID NO: 11 (β-chain domain B), SEQ ID NO: 12 (β-chain domain C), SEQ ID NO: 13 (β-chain domain D), SEQ ID NO: 14 (β-chain domain E), SEQ ID NO: 15 (β-chain domain F), SEQ ID NO: 16 (β-chain domain G), SEQ ID NO: 17 (β-chain domain H), SEQ ID NO: 18 (β-chain domain I), SEQ ID NO: 19 (β-chain domain J), and SEQ ID NO: 20 (β-chain domain K). Regarding polypeptides.

[0033] The polypeptide is hereinafter referred to as "the polypeptide according to the first aspect of the present invention" or "the polypeptide of the present invention."

[0034] As used herein, the term “polypeptide” generally refers to a linear chain of amino acid residues of any length linked by peptide bonds. As used herein, the term “peptide” also refers to a linear chain of amino acids, like a polypeptide, but shorter than that of a polypeptide. A peptide generally refers to an amino acid chain of 2 to 50 amino acids. The terms “peptide bond,” “peptide,” “polypeptide,” and “protein” will be understood to be known to those skilled in the art.

[0035] The polypeptide of the present invention is a variant of the "nidogen G2 domain".

[0036] As used herein, the term "nidogen-1" refers to the glycoprotein previously known as entactin. Nidogen-1 is disclosed in the Uniprot database (version dated July 7, 2009) as accession number P14543-1 (sequence number 72).

[0037] As used herein, the term “G2 domain of Nidogen-1” refers to the G2 domain of the Nidogen-1 protein as defined above. The G2 domain of Nidogen-1 is as shown in SEQ ID NO: 62, which corresponds to amino acid numbers 430-667 of the Nidogen-1 protein amino acid sequence and is identified as P14543-1 (SEQ ID NO: 72) in the Uniprot database (version dated July 7, 2009). In another embodiment, the G2 domain of Nidogen-1 is as shown in SEQ ID NO: 64, which lacks the first two amino acids of SEQ ID NO: 62 and therefore corresponds to the region consisting of amino acid numbers 432-667 of the amino acid sequence (SEQ ID NO: 72) of the Nidogen-1 protein precursor with identification number P14543-1 in the Uniprot database (version dated July 7, 2009). In the wild-type Nidogen-1 sequence, the G2 domain is adjacent to a short EGF-like domain. However, for the purposes of the present invention, the G2 domain of nidogen-1 lacks an EGF-like domain at the N-terminus or C-terminus.

[0038] In one embodiment, the polypeptide of the present invention contains an N-terminal methionine residue. In another embodiment, the polypeptide of the present invention does not contain methionine at the N-terminus.

[0039] As used herein, “amino acid residue” means any naturally occurring amino acid, any amino acid derivative, or any amino acid mimic known in the art. In certain embodiments, the amino acid residue is an amino acid, i.e., a naturally occurring amino acid. In certain embodiments, the residues of a protein or peptide are continuous and do not contain any non-amino acids that interrupt the sequence of amino acid residues. In other embodiments, the sequence may contain one or more non-amino acid moieties. In certain embodiments, the sequence of residues of a protein or peptide may be interrupted by one or more non-amino acid moieties.

[0040] As used herein, the terms “β-chain,” “β-chain domain,” or “β-chain sequence” refer to an extended polypeptide chain or sequence connected to another polypeptide chain or sequence via hydrogen bonds between the NH group of one chain and the CO group of the other chain. Typically, β-chains are about 3 to 10 amino acids long, but can be longer, for example, 13 to 15 amino acids long. As a result of such inter-chain connections, β-chains form a sheet-like protein secondary structure, which is referred to herein as a “β-sheet.” Within a β-sheet, β-chains can be arranged in parallel, antiparallel, or mixed (parallel and antiparallel) configurations. When arranged parallel, β-chains are aligned in the same direction from one end (N or C) to the other. When arranged antiparallel, each β-chain is aligned in the opposite direction to the chain to which it is connected.

[0041] As used herein, the term "β-barrel" refers to a protein secondary structure formed by β-sheets, which is a closed, toroidal structure resulting from hydrogen bonding between the first and last chains.

[0042] As used herein, the term "α-helix" or "alpha-helix" refers to a protein secondary structure consisting of a right-hand helix in which the NH group of an amino acid is bonded to a hydrogen atom and to a C=O group of an amino acid backbone located three or four residues ahead in the protein sequence.

[0043] As used herein, the terms "alpha-helix moiety" or "alpha-helix moiety" refer to a motif in the secondary structure of a protein that substantially contains one or more alpha-helices.

[0044] As will be understood by those skilled in the art, as used herein, a β-chain refers to a protein domain, and as used herein, a β-sheet refers to a protein secondary structure.

[0045] In certain embodiments, 11 β-chain domains of the polypeptide of the present invention are included in or constitute a β-sheet secondary structure. In preferred embodiments, 12 β-chain domains of the polypeptide of the present invention are included in or constitute a β-barrel secondary structure.

[0046] As used herein, the terms “loop,” “loop region,” “loop sequence,” “omega-loop,” “omega-loop region,” or “omega-loop sequence” refer to an irregular, non-repeating protein structural motif consisting of a polypeptide chain of six or more amino acid residues having any amino acid sequence. The residues constituting the start and end of a loop are close to each other through a gap, without the interposition of a normal secondary structural motif between them. They generally connect two protein domains contained in a secondary protein structure, such as a β-chain, or a direct secondary protein structure, such as an α-helix. Such loops often allow a protein domain or protein secondary structure connected to one end of the loop to change its orientation (N-terminus to C-terminus, or C-terminus to N-terminus) relative to another protein domain or protein structure connected to the other end of the loop. Loops are most often located on the outer surface of a protein and are therefore generally involved in interactions between the protein to which the loop belongs and other molecules.

[0047] In certain embodiments, the polypeptide of the first aspect of the present invention is a nidogen G2 domain variant containing a heterologous polypeptide within one or more loop regions. In one embodiment, the heterologous polypeptide is inserted within a loop region, i.e., the loop region preserves all the amino acids found in the congeneral loop domain of SEQ ID NO: 62 or SEQ ID NO: 63, but the heterologous polypeptide is inserted between two consecutive amino acids. In another embodiment, the heterologous polypeptide within one or more loop regions is found as an insertion within a loop region that partially or completely replaces the sequence of the loop region.

[0048] The length of a heterologous polypeptide is not particularly limited. Therefore, a heterologous polypeptide may contain at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, at least ten, at least fifteen, at least twenty, at least twenty-five, at least thirty, at least thirty-five, at least forty, at least forty-five, at least fifty, at least fifty-five, at least sixty, at least sixty, at least seventy, at least seventy-five, at least eighty, at least eighty-five, at least ninety, at least ninety-five, at least one hundred or more amino acids.

[0049] In another embodiment, the polypeptide of the first aspect of the present invention comprises heterologous polypeptides within one or more loop regions, and comprises mutations in one or more β-chains, wherein the mutations are located at position 9 of β-chain B as defined by SEQ ID NO: 11 (corresponding to the amino acid at position 30 of SEQ ID NO: 62, or the sequence with accession number P14543-1 in the Uniprot database (version dated July 7, 2009), or the amino acid at position 459 of the human nidogen 1 precursor as defined by SEQ ID NO: 72), and at position 1 of β-chain C as defined by SEQ ID NO: 12 (corresponding to the amino acid at position 39 of SEQ ID NO: 62, or the sequence with accession number P14543-1 in the Uniprot database (version dated July 7, 2009), or the sequence defined by SEQ ID NO: 72). It is located at position 468 of the human nidogen 1 precursor as defined by SEQ ID NO: 19 (corresponding to the amino acid at position 210 of SEQ ID NO: 62, or the sequence with accession number P14543-1 in the Uniprot database (version dated July 7, 2009), or the amino acid at position 639 of the human nidogen 1 precursor as defined by SEQ ID NO: 72), or at position 3 of the β-chain K as defined by SEQ ID NO: 20 (corresponding to the amino acid at position 221 of SEQ ID NO: 62, or the sequence with accession number P14543-1 in the Uniprot database (version dated July 7, 2009), or the amino acid at position 650 of the human nidogen 1 precursor as defined by SEQ ID NO: 72).

[0050] In one embodiment, the mutation at position 9 of β-chain B, as defined by SEQ ID NO: 11, is an H459A mutation; the mutation at position 1 of β-chain C, as defined by SEQ ID NO: 12, is an R468N mutation; the mutation at position 10 of β-chain J, as defined by SEQ ID NO: 19, is an F639S mutation; and / or the mutation at position 3 of β-chain K, as defined by SEQ ID NO: 20, is an R650A mutation.

[0051] In one embodiment, the human nidogen G2 domain variant includes mutations H459A and R468N. In one embodiment, the human nidogen G2 domain variant includes mutations H459A and F639S. In one embodiment, the human nidogen G2 domain variant includes mutations H459A and R650A. In one embodiment, the human nidogen G2 domain variant includes mutations R468N and F639S. In one embodiment, the human nidogen G2 domain variant includes mutations R468N and R650A. In one embodiment, the human nidogen G2 domain variant includes mutations H459A, R468N, and F639S. In one embodiment, the human nidogen G2 domain variant includes mutations H459A, R468N, and R650A. In one embodiment, the human nidogen G2 domain variant includes the R468N, F639S, and R650A mutations. In another embodiment, the human nidogen G2 domain variant includes the H459A, R468N, F639S, and R650A mutations. In a preferred embodiment, the nidogen G2 domain variant has the sequence defined by SEQ ID NO: 64 or 65 (hereinafter referred to as NIDOmut2).

[0052] In another embodiment, the polypeptide of the first aspect of the present invention is a nidogen G2 domain variant as defined in any of the embodiments described above, and in particular, a nidogen G2 domain variant having mutations at positions 543 (corresponding to the histidine at position 114 of SEQ ID NO: 62) and 545 (corresponding to the histidine at position 116 of SEQ ID NO: 62), and further comprising a mutation at a position selected from the group consisting of these positions. In another embodiment, position H543 is mutated to Lys. In another embodiment, position H545 is mutated to Asn. In some embodiments, the polypeptide of the first aspect of the present invention is a nidogen G2 domain variant comprising mutations at positions H459A, R468N, F639S, R650A and H543K. In some embodiments, the polypeptide of the first aspect of the present invention is a nidogen G2 domain variant containing the H459A, R468N, F639S, R650A, and H545N mutations. In another embodiment, the nidogen G2 domain variant contains the H543K and H545N mutations. In one embodiment, the nidogen G2 domain variant contains or comprises SEQ ID NO: 87 (hereinafter referred to as NIDOmut3), characterized by containing the H459A, R468N, F639S, R650A, H543K, and H545N mutations.

[0053] In another embodiment, the polypeptide of the first aspect of the present invention is a nidogen G2 domain variant as defined in any of the above embodiments, and in particular, a NIDOmut3 variant further comprising a mutation selected from the group consisting of: A mutation in valine at position -449 (corresponding to position 20 of sequence number 62). Preferably, the valine at position 449 is mutated to Thr. In a preferred embodiment, the nidogen G2 domain mutant has the sequence defined in sequence number 88 (hereinafter referred to as NIDOmut3-V45T). A mutation in valine at position -525 (corresponding to position 96 of sequence number 62). Preferably, valine at position 449 is mutated to Gln. In a preferred embodiment, the nidogen G2 domain mutant has the sequence defined in sequence number 89 (hereinafter referred to as NIDOmut3-V212Q). A mutation in phenylalanine at position -561 (corresponding to position 142 of SEQ ID NO: 62). Preferably, the phenylalanine at position 561 is mutated to glutamic acid. In a preferred embodiment, the nidogen G2 domain mutant has the sequence defined in SEQ ID NO: 90 (hereinafter referred to as NIDOmut3-F157E). A mutation in valine at position -619 (corresponding to position 190 of sequence number 62). Preferably, the valine at position 619 is mutated to threonine. In a preferred embodiment, the nidogen G2 domain mutant has the sequence defined in sequence number 91 (hereinafter referred to as NIDOmut3-V215T).

[0054] In another embodiment, the polypeptide of the first aspect of the present invention is a nidogen G2 domain variant as defined in any of the above embodiments, further comprising the V449T, V525Q, F561E, and V619T mutations. In some embodiments, the polypeptide of the first aspect of the present invention is a nidogen G2 domain variant comprising the H459A, R468N, F639S, R650A, H543K, V449T, V525Q, F561E, and V619T mutations. In some embodiments, the polypeptide of the first aspect of the present invention is a nidogen G2 domain variant comprising the H459A, R468N, F639S, R650A, V449T, H545N, V525Q, F561E, and V619T mutations. In some embodiments, the polypeptide of the first aspect of the present invention is a nidogen G2 domain variant containing mutations in H459A, R468N, F639S, R650A, H543K, H545N, V449T, V525Q, F561E, and V619T. In another embodiment, the nidogen G2 domain variant contains or consists of the sequence defined by SEQ ID NO: 92 (hereinafter referred to as NIDOmut4).

[0055] In another embodiment, the polypeptide of the first aspect of the present invention is a nidogen G2 domain variant as defined in any of the above embodiments, and in particular, NIDOmut4 which further includes a threonine mutation at position 619 (corresponding to position 190 of SEQ ID NO: 62). Preferably, the threonine at position 619 is mutated to valine. In another embodiment, the polypeptide of the first aspect of the present invention is a nidogen G2 domain variant as defined in any of the above embodiments, wherein the amino acid at position 619 (corresponding to position 190 of SEQ ID NO: 62) is the same residue found in the human nidogen G2 domain as defined by accession number P14534 in the UniProt database, namely valine. Thus, in one embodiment, the polypeptide of the first aspect of the present invention is a nidogen G2 domain variant having mutations at H459A, R468N, F639S, R650A, H543K, H545N, V449T, V525Q and F561E. In one embodiment, the polypeptide of the first aspect of the present invention is a nidogen G2 domain mutant having the sequence of SEQ ID NO: 93 (hereinafter referred to as NIDOmut4_T215V).

[0056] In another embodiment, the polypeptide of the first aspect of the present invention is a nidogen G2 domain variant as defined in any of the above embodiments, and in particular, NIDOMut4 which further includes a cysteine ​​mutation at position 618 (corresponding to position 189 of SEQ ID NO: 62). Preferably, the cysteine ​​at position 618 is mutated to serine. Thus, in one embodiment, the polypeptide of the first aspect of the present invention is a nidogen G2 domain variant having the mutations H459A, R468N, F639S, R650A, H543K, H545N, V449T, V525Q, V619T, F561E and C618S. In one embodiment, the polypeptide of the first aspect of the present invention is a nidogen G2 domain variant having the sequence of SEQ ID NO: 94 (hereinafter referred to as NIDOmut5).

[0057] In another embodiment, the polypeptide of the first aspect of the present invention is a nidogen G2 domain variant as defined in any of the above embodiments, and in particular a NIDOMut3 variant further comprising a mutation selected from the group consisting of: A mutation in valine at position -580 (corresponding to position 151 of SEQ ID NO: 62). Preferably, the valine at position 580 is mutated to Thr. In one embodiment, the polypeptide of the first aspect of the present invention is a nidogen G2 domain mutant having the sequence of SEQ ID NO: 95 (hereinafter referred to as NIDOmut3-V176T). A mutation in isoleucine at position -604 (corresponding to position 175 of SEQ ID NO: 62). Preferably, the isoleucine at position 604 is mutated to Thr. In one embodiment, the polypeptide of the first aspect of the present invention is a nidogen G2 domain mutant having the sequence of SEQ ID NO: 96 (hereinafter referred to as NIDOmut3-I200T). A mutation in valine at position -638 (corresponding to position 209 of SEQ ID NO: 62). Preferably, the valine at position 638 is mutated to tyrosine. In one embodiment, the polypeptide of the first aspect of the present invention is a nidogen G2 domain mutant having the sequence of SEQ ID NO: 97 (hereinafter referred to as NIDOmut3-V236Y). A leucine mutation at position -641 (corresponding to position 212 of sequence number 62). Preferably, the leucine at position 641 is mutated to threonine. In one embodiment, the polypeptide of the first aspect of the present invention is a nidogen G2 domain mutant having the sequence of sequence number 98 (hereinafter referred to as NIDOmut3-L237T). A serine mutation at position -469 (corresponding to position 40 of sequence number 62). Preferably, the serine at position 469 is mutated to Ile. In one embodiment, the polypeptide of the first aspect of the present invention is a nidogen G2 domain mutant having the sequence of sequence number 99 (hereinafter referred to as NIDOmut3-S65I). - A mutation in the arginine at position 518 (corresponding to position 89 of SEQ ID NO: 62). Preferably, the arginine at position 518 is mutated to Ile. In one embodiment, the polypeptide of the first aspect of the present invention is a nidogen G2 domain mutant having the sequence of SEQ ID NO: 100 (hereinafter referred to as NIDOmut3-R114I). A mutation in cysteine ​​at position -618 (corresponding to position 189 of SEQ ID NO: 62). Preferably, the cysteine ​​at position 618 is mutated to serine. Therefore, in one embodiment, the polypeptide of the first aspect of the present invention is a nidogen G2 domain mutant having the sequence of SEQ ID NO: 101 (hereinafter referred to as NIDOmut3-C214S).

[0058] In some embodiments, the polypeptide of the first aspect of the present invention is a nidogen G2 domain variant as defined in any of the above embodiments, and in particular, a NIDOmut3 variant further comprising a mutation at position 469 (preferably a mutation at S469I) and a mutation at position 518 (preferably a mutation at R518I). Thus, in one embodiment, the polypeptide of the first aspect of the present invention is a nidogen G2 domain variant comprising mutations at H459A, R468N, F639S, R650A, H543K, H545N, S469I and R518I, corresponding to the sequence of SEQ ID NO: 102 (hereinafter referred to as NIDOmut3-S65I_R114I).

[0059] In some embodiments, the polypeptide of the first aspect of the present invention is a nidogen G2 domain variant as defined in any of the embodiments described above, and in particular, a NIDOmut5 variant further comprising a mutation at position 469 (preferably a mutation at S469I) and a mutation at position 518 (preferably a mutation at R518I). Thus, in one embodiment, the polypeptide of the first aspect of the present invention is a nidogen G2 domain variant comprising mutations at H459A, R468N, F639S, R650A, H543K, H545N, V449T, V525Q, V619T, F561E, S469I and R518I as defined in SEQ ID NO: 103 (hereinafter referred to as NIDOmut5-S65I_R114I).

[0060] In some embodiments, the polypeptide of the first aspect of the present invention is a nidogen G2 domain variant as defined in any of the above embodiments, and in particular, a NIDOmut5 variant further comprising a serine mutation at position 469 (corresponding to position 40 of SEQ ID NO: 62). Preferably, the serine at position 469 is mutated to Ile. Thus, in one embodiment, the polypeptide of the first aspect of the present invention is a nidogen G2 domain variant having mutations in H459A, R468N, F639S, R650A, H543K, H545N, V449T, V525Q, V619T, F561E and S469I. In one embodiment, the polypeptide of the first aspect of the present invention is a nidogen G2 domain variant having the sequence of SEQ ID NO: 104 (hereinafter referred to as NIDOmut5-S65I).

[0061] In some embodiments, the polypeptide of the first aspect of the present invention is a nidogen G2 domain variant as defined in any of the above embodiments, and in particular, a NIDOmut5 variant further comprising a mutation in arginine at position 518 (corresponding to position 89 of SEQ ID NO: 62). Preferably, the arginine at position 518 is mutated to Ile. Thus, in one embodiment, the polypeptide of the first aspect of the present invention is a nidogen G2 domain variant having mutations in H459A, R468N, F639S, R650A, H543K, H545N, V449T, V525Q, V619T, F561E and R518I. In one embodiment, the polypeptide of the first aspect of the present invention is a nidogen G2 domain variant having the sequence of SEQ ID NO: 104 (hereinafter referred to as NIDOmut5-R114I).

[0062] Nidogen G2 domain variants suitable for use in the present invention are summarized in the following table. [Table 1]

[0063] Heterogeneous polypeptides can be inserted within loop regions, i.e., the loop region preserves all amino acids found in the congeneral loop domain of SEQ ID NO: 62 or SEQ ID NO: 63, but heterogeneous polypeptides are inserted between two consecutive amino acids. The length of heterogeneous polypeptides is not particularly limited. Therefore, heterogeneous polypeptides may contain at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 15, at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, at least 100 or more amino acids.

[0064] In another embodiment, a heterologous polypeptide may substitute for a portion of the loop region, i.e., the loop region may contain a deletion with respect to the sequence of the congeneral loop domain of SEQ ID NO: 62 or SEQ ID NO: 63, and the deleted sequence may be replaced by the heterologous polypeptide, inserted between two consecutive amino acids. It will be understood that the length of the deletion does not have to be the same as the length of the heterologous peptide. Thus, the loop region may contain a deletion of at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% of the total length of the region. The length of the heterologous polypeptide is not particularly limited. Therefore, heterologous polypeptides may contain at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, at least ten, at least fifteen, at least twenty, at least twenty-five, at least thirty, at least thirty-five, at least forty, at least forty-five, at least fifty, at least fifty-five, at least sixty, at least sixty, at least seventy, at least seventy-five, at least eighty, at least eighty-five, at least ninety, at least ninety-five, at least one hundred or more amino acids.

[0065] If the nidogen G2 domain variant contains multiple heterologous polypeptides, these heterologous polypeptides may be found within the same loop region, or preferably in different loop regions. Furthermore, if the nidogen G2 domain variant contains multiple heterologous polypeptides, these heterologous polypeptides may be the same or different.

[0066] The heterologous polypeptide that forms part of the nidogen G2 domain variant of the present invention specifically binds to the target peptide. More preferably, the heterologous polypeptide specifically binds to the target peptide that does not show specific binding to other regions of the nidogen G2 domain variant of the present invention or to other regions of the first polypeptide of the present invention.

[0067] In the present invention, the terms “binding,” “bond,” and “binds” refer to interactions between affinity-bound molecules or specific bond pairs as a result of non-covalent bonds such as hydrogen bonds, hydrophobic interactions, van der Waals bonds, ionic bonds, or combinations thereof, but are not particularly limited.

[0068] In this invention, when used to refer to the binding of a polypeptide to a specific or target molecule, the expressions "specifically binds," "specifically binding," "specifically recognizes," or "specifically interacts" are understood to mean the ability of a polypeptide to specifically bind to a target molecule with substantially high affinity, preferably so that the binding between the polypeptide and the target molecule occurs before the polypeptide binds to other molecules present in close proximity to the polypeptide, as in the case of a reaction mixture, due to the complementarity between the three-dimensional structures of both the polypeptide and the target molecule. The ability of a polypeptide to specifically bind to a target molecule in a reaction mixture can be tested, for example, by evaluating the binding of the polypeptide to the target molecule of interest and to several related molecules that are (structurally and / or functionally) closer or further away, under conventional conditions. The binding is considered specific to the target molecule only if the polypeptide binds to the target molecule but does not bind to or substantially binds to other related molecules close to the target molecule. The binding between a polypeptide and a target molecule is considered specific to the target molecule if the binding affinity between them is 10 -6 Less than M, 10 -7 Less than M, 10 -8 Less than M, 10-9 Less than M, 10 -10 Less than M, 10 -11 Less than M, 10 -12 Less than M, 10 -13 Less than M, 10 -14 Less than or equal to M or 10 -15 If it has a dissociation constant (KD) less than M, it can be considered specific. Methods for determining the binding between a polypeptide and a target molecule and the KD of said binding include those well known to those skilled in the art. Non-limiting examples of such methods include gel shift assays such as electrophoretic mobility shift assay (EMSA), co-immunoprecipitation assay and subsequent mass spectrometry, gas chromatography related to mass spectrometry, liquid chromatography related to mass spectrometry, or Western blot analysis. A further method is the oil cushion method [see Hesselgesset et al, 1998, J. Immunol., 160:877-883].

[0069] In certain embodiments, the binding of the polypeptide to the target molecule is such that the binding between the polypeptide and the target molecule is 10 -6 Less than M, 10 -7 Less than M, 10 -8 Less than M, 10[[ID=!]] -9 Less than M, 10 -10 Less than M, 10 -11 Less than M, 10 -12 Less than M, 10 -13 Less than M, 10 -14 Less than or equal to M or 10 -15 If it has a dissociation constant (KD) less than M, it is considered specific. Similarly, the binding between the loop region and a specific target molecule is such that the binding between the loop region and the specific target molecule is 10 -6 Less than M, 10 -7 Less than M, 10 -8 Less than M, 10 -9 Less than M, 10 -10 Less than M, 10 -11 Less than M, 10 -12 Less than M, 10 -13 Less than M, 10 -14 Less than or equal to M or 10 -15A dissociation constant (KD) less than M is considered specific.

[0070] In the polypeptide of the present invention, loop region AB connects β chains A and B, loop region BC connects β chains B and C, loop region CD connects β chains C and D, loop region DE connects β chains D and E, loop region EF connects β chains E and F, loop region FG' connects β chains F and G, loop region GH connects β chains G and H, loop region HI connects β chains H and I, loop region IJ connects β chains I and J, and loop region JK connects β chains J and K.

[0071] In a particular embodiment, β-chain A is connected to β-chain B by loop region AB of the polypeptide according to the first embodiment of the present invention. In another particular embodiment, β-chain B is connected to β-chain C by loop region BC of the polypeptide according to the first embodiment of the present invention. In another particular embodiment, β-chain C is connected to β-chain D by loop region CD of the polypeptide according to the first embodiment of the present invention. In another particular embodiment, β-chain D is connected to β-chain E by loop region DE of the polypeptide according to the first embodiment of the present invention. In another particular embodiment, β-chain E is connected to β-chain F by loop region EF of the polypeptide according to the first embodiment of the present invention. In another particular embodiment, β-chain F is connected to β-chain G by loop region FG of the polypeptide according to the first embodiment of the present invention. In another particular embodiment, β-chain G is connected to β-chain H by loop region GH of the polypeptide according to the first embodiment of the present invention. In another particular embodiment, β-chain H is connected to β-chain I by loop region HI of the polypeptide according to the first embodiment of the present invention. In another particular embodiment, β-chain I is connected to β-chain J by loop region IJ of the polypeptide according to the first embodiment of the present invention. In another specific embodiment, the β-chain J is connected to the β-chain K by the loop region JK of the polypeptide according to the first aspect of the present invention.

[0072] As used herein, the expression “congeneral loop region in SEQ ID NO: 62” refers to the loop region appearing in SEQ ID NO: 62, which is a human-derived wild-type nidogen G2 domain. As will be understood by those skilled in the art, each loop region of the polypeptide in the first aspect of the present invention has its congeneral loop region in SEQ ID NO: 62.

[0073] As will be understood by those skilled in the art, two amino acid sequences are considered to encode the same protein domain or secondary protein structure if they exhibit a degree of sequence identity and they encode the same type of protein domain or secondary protein structure, i.e., if they both form a β-chain, loop region, α-helix, α-helix moiety, β-sheet, or β-barrel. In a particular embodiment, two amino acid sequences encoding protein domains or secondary protein structures that are considered identical exhibit sequence identity of a degree of at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, at least 99.5%, at least 99.75%, at least 99.8%, at least 99.9%, at least 99.95%, at least 99.975%, and at least 99.99%. The degree of identity between two amino acid sequences can be determined by conventional methods, such as standard sequence alignment algorithms known in the art, including BLAST [Altschul SF et al., J. Mol. Biol., 1990 Oct 5; 215(3):403-10]. Methods for determining whether an amino acid sequence within a protein forms or maintains a particular domain or protein secondary structure are well known to those skilled in the art. These methods include obtaining the atomic coordinates of the protein according to methods well known to those skilled in the art, such as X-ray crystallography or protein NMR, and then determining the protein's secondary structure using bioinformatics tools such as DSSPcont (Carter, Andersen & Rost, 2003) and STRIDE (Heinig & Frishman, 2004).

[0074] In another specific embodiment, the cognate loop region in sequence number 62 of loop region AB includes, substantially includes, or consists of sequence number 1. In another specific embodiment, the cognate loop region in sequence number 62 of loop region BC includes, substantially includes, or consists of sequence number 2. In another specific embodiment, the cognate loop region in sequence number 62 of loop region CD includes, substantially includes, or consists of sequence number 3. In another specific embodiment, the cognate loop region in sequence number 62 of loop region DE includes, substantially includes, or consists of sequence number 4. In another specific embodiment, the cognate loop region in sequence number 62 of loop region EF includes, substantially includes, or consists of sequence number 5. In another specific embodiment, the cognate loop region in sequence number 62 of loop region FG includes, substantially includes, or consists of sequence number 6. In another specific embodiment, the cognate loop region in sequence number 62 of loop region GH includes, substantially includes, or consists of amino acids 149-150 in sequence number 62. In another specific embodiment, the related loop region in sequence number 62 of loop region HI includes, substantially includes, or consists of sequence number 7. In another specific embodiment, the related loop region in sequence number 62 of loop region IJ includes, substantially includes, or consists of sequence number 8. In another specific embodiment, the related loop region in sequence number 62 of loop region JK includes, substantially includes, or consists of sequence number 9.

[0075] In certain embodiments, the loop region of the polypeptide according to the first aspect of the present invention is a homologous loop region variant in SEQ ID NO: 62.

[0076] As used herein, the term “loop region variant” refers to a loop region of a polypeptide of the first embodiment, which has one or more amino acid modifications, insertions, and / or deletions in its sequence relative to the cognate loop region in SEQ ID NO: 62. In certain embodiments, the loop region of the polypeptide of the first embodiment is selected from the group of loop regions consisting of AB, BC, CD, DE, EF, FG, GH, HI, IJ, and JK.

[0077] Therefore, in certain embodiments, at least one loop region variant of the polypeptide according to the first aspect of the present invention is caused by a mutation resulting from the deletion, substitution, or addition of at least one amino acid in the sequence of the cognate loop region in SEQ ID NO: 62.

[0078] In certain embodiments, loop region variants of the polypeptide of the first embodiment have sequence identity of at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, and at least 99% with respect to the sequence of the congeneral loop region in SEQ ID NO: 62. The method for determining the degree of sequence identity between the two sequences is as described above.

[0079] In a particular embodiment, loop region AB is a loop region variant of the congeneral loop region in SEQ ID NO: 62. In another particular embodiment, loop region BC is a loop region variant of the congeneral loop region in SEQ ID NO: 62. In another particular embodiment, loop region CD is a loop region variant of the congeneral loop region in SEQ ID NO: 62. In another particular embodiment, loop region DE is a loop region variant of the congeneral loop region in SEQ ID NO: 62. In another particular embodiment, loop region EF is a loop region variant of the congeneral loop region in SEQ ID NO: 62. In another particular embodiment, loop region FG is a loop region variant of the congeneral loop region in SEQ ID NO: 62. In another particular embodiment, loop region GH is a loop region variant of the congeneral loop region in SEQ ID NO: 62. In another particular embodiment, loop region HI is a loop region variant of the congeneral loop region in SEQ ID NO: 62. In another particular embodiment, loop region IJ is a loop region variant of the congeneral loop region in SEQ ID NO: 62. In another particular embodiment, loop region JK is a loop region variant of the congeneral loop region in SEQ ID NO: 62.

[0080] In certain embodiments, the sequence of the AB loop region of the nidogen G2 domain variant of the present invention is a variant of SEQ ID NO: 1, but at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, or all sequences of the BC, CD, DE, EF, FG', GH, HI, IJ, and JK loop regions of the polypeptide of the first aspect of the present invention are identical to the sequences of their cognate loop regions in SEQ ID NO: 62. In another particular embodiment, the sequence of loop region AB is a variant of SEQ ID NO: 1, and at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, or all sequences of the loop region of the polypeptide of the first aspect of the present invention are identical to the sequences of their cognate loop regions in SEQ ID NO: 62. In yet another particular embodiment, the sequence of loop region AB is a variant of SEQ ID NO: 1, and the remaining sequences of the polypeptide of the first aspect of the present invention are identical to the remaining sequences of SEQ ID NO: 62.

[0081] In certain embodiments, the loop region AB variant of SEQ ID NO: 1 has at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, and at least 99% sequence identity with SEQ ID NO: 1.

[0082] In another specific embodiment, the sequence of the loop region AB of the polypeptide according to the first aspect of the present invention is sequence number 1.

[0083] In certain embodiments, the sequence of loop region BC is a variant of SEQ ID NO: 2. In another particular embodiment, the sequence of loop region BC is a variant of SEQ ID NO: 2, and at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, or all sequences of the AB, CD, DE, EF, FG, GH, HI, IJ, and JK loop regions of the polypeptide of the first aspect of the present invention are sequences of their cognate loop regions in SEQ ID NO: 62 as shown above. In another particular embodiment, the sequence of loop region BC is a variant of SEQ ID NO: 2, and at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, or all sequences of the loop regions of the polypeptide of the first aspect of the present invention are sequences of their cognate loop regions in SEQ ID NO: 62. In another particular embodiment, the sequence of loop region BC is a variant of SEQ ID NO: 2, and the remaining sequences of the polypeptide of the first aspect of the present invention are identical to the remaining sequences of SEQ ID NO: 62.

[0084] In certain embodiments, the loop region BC variant of SEQ ID NO: 2 has at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, and at least 99% sequence identity with SEQ ID NO: 2.

[0085] In another specific embodiment, the sequence of the loop region BC of the polypeptide according to the first aspect of the present invention is sequence number 2.

[0086] In certain embodiments, the sequence of loop region CD is a variant of SEQ ID NO: 3. In another particular embodiment, the sequence of loop region CD is a variant of SEQ ID NO: 3, and at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, or all sequences of the loop regions AB, BC, DE, EF, FG, GH, HI, IJ, and JK of the first aspect of the present invention are sequences of their cognate loop regions in SEQ ID NO: 62 as shown above. In another particular embodiment, the sequence of loop region CD is a variant of SEQ ID NO: 3, and at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, or all sequences of the loop regions of the polypeptide of the first aspect of the present invention have sequences of their cognate loop regions in SEQ ID NO: 62. In another particular embodiment, the sequence of loop region CD is a variant of SEQ ID NO: 3, and the remaining sequences of the polypeptide of the first aspect of the present invention are identical to the remaining sequences of SEQ ID NO: 62.

[0087] In certain embodiments, the loop region CD variant of SEQ ID NO: 3 has at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, and at least 99% sequence identity with SEQ ID NO: 3.

[0088] In another specific embodiment, the sequence of the loop region CD of the polypeptide according to the first aspect of the present invention is sequence number 3.

[0089] In certain embodiments, the sequence of loop region CD is a congeneral domain variant in SEQ ID NO: 62 of a sequence in which the congeneral region is modified in a loop region that does not exhibit an α-helix structure. As shown by Hopf et al. (above), the CD loop region of the wild-type G2 domain contains three regions having an α-helix structure known as α1, α2, and α3. These regions are defined as SEQ ID NOs: 21, 22, and 23, respectively. These regions divide the CD loop region into four loop regions corresponding to the region between the end of β-chain C and α1 (hereinafter referred to as the Cα region), the region between α1 and α2, the region between α2 and α3, and the region between α3 and the beginning of β-chain D (hereinafter referred to as the αD region), respectively. In one embodiment, the Cα region contains, substantially contains, or consists of SEQ ID NO: 24. In one embodiment, the αD region contains, substantially contains, or consists of the amino acid GG. In one embodiment, the loop region sequence is a variant of the congenerate region of sequence number 3, where sequences 21, 22, and 23 are conserved with respect to the congenerate region. In another embodiment, the loop region sequence is a variant of the congenerate region of sequence number 3, containing one or more mutations in the Cα region. In yet another embodiment, the loop region sequence is a variant of the congenerate region of sequence number 3, containing one or more mutations in the αD region. In yet another embodiment, the loop region sequence is a variant of the congenerate region of sequence number 3, containing one or more mutations in the Cα and αD regions.

[0090] In certain embodiments, the Cα region of the polypeptide of the present invention has at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, and at least 99% sequence identity with SEQ ID NO: 24.

[0091] In another specific embodiment, the sequence of the loop region Cα of the polypeptide according to the first aspect of the present invention is sequence number 24.

[0092] In certain embodiments, the loop region αD variant has at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, and at least 99% sequence identity with the sequence consisting of amino acids GG.

[0093] In certain embodiments, the sequence of loop region DE is a variant of SEQ ID NO: 4. In another particular embodiment, the sequence of loop region DE is a variant of SEQ ID NO: 4, and at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, or all sequences of the AB, BC, CD, EF, FG, GH, HI, IJ, and JK loop regions of the polypeptide of the first aspect of the present invention contain the sequences of their cognate loop regions in SEQ ID NO: 62 as shown above. In another particular embodiment, the sequence of loop region DE is a variant of SEQ ID NO: 4, and at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, or all sequences of the loop regions of the polypeptide of the first aspect of the present invention are the sequences of their cognate loop regions in SEQ ID NO: 62. In another particular embodiment, the sequence of loop region DE is a variant of SEQ ID NO: 4, and the remaining sequences of the polypeptide of the first aspect of the present invention are identical to the remaining sequences of SEQ ID NO: 62.

[0094] In certain embodiments, the loop region DE variant of SEQ ID NO: 4 has at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, and at least 99% sequence identity with SEQ ID NO: 4.

[0095] In another specific embodiment, the sequence of the loop region DE of the polypeptide according to the first aspect of the present invention is sequence number 4.

[0096] In certain embodiments, the sequence of loop region EF is a variant of SEQ ID NO: 5. In another particular embodiment, the sequence of loop region EF is a variant of SEQ ID NO: 5, and at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, or all sequences of the loop regions AB, BC, CD, DE, FG, GH, HI, IJ, and JK of the first aspect of the present invention are sequences of their cognate loop regions in SEQ ID NO: 62 as shown above. In another particular embodiment, the sequence of loop region EF is a variant of SEQ ID NO: 5, and at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, or all sequences of the loop regions of the polypeptide of the first aspect of the present invention are sequences of their cognate loop regions in SEQ ID NO: 62. In another particular embodiment, the sequence of loop region EF is a variant of SEQ ID NO: 5, and the remaining sequences of the polypeptide of the first aspect of the present invention are identical to the remaining sequences of SEQ ID NO: 62.

[0097] In certain embodiments, the loop region EF variant of SEQ ID NO: 5 has at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, and at least 99% sequence identity with SEQ ID NO: 5.

[0098] In another specific embodiment, the sequence of the loop region EF of the polypeptide according to the first aspect of the present invention is sequence number 5.

[0099] In a particular embodiment, the sequence of loop region FG is a variant of SEQ ID NO: 6. In another particular embodiment, the sequence of loop region FG is a variant of SEQ ID NO: 6, and at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, or all of the sequences AB, BC, CD, DE, EF, GH, HI, IJ, and JK are sequences of their cognate loop regions in SEQ ID NO: 62 as shown above. In another particular embodiment, the sequence of loop region FG is a variant of SEQ ID NO: 6, and at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, or all of the sequences of the loop region of the polypeptide of the first aspect of the present invention are sequences of their cognate loop regions in SEQ ID NO: 62. In another particular embodiment, the sequence of loop region FG is a variant of SEQ ID NO: 6, and the remaining sequence of the polypeptide of the first aspect of the present invention is identical to the remaining sequence of SEQ ID NO: 62.

[0100] In certain embodiments, the loop region FG variant of sequence number 6 has at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, and at least 99% sequence identity with sequence number 6.

[0101] In another specific embodiment, the sequence of the loop region FG of the polypeptide according to the first aspect of the present invention is sequence number 6.

[0102] In a particular embodiment, the sequence of the loop region GH is a variant of the sequence TS corresponding to amino acids 149-150 of SEQ ID NO: 62 (or amino acids 147-148 of SEQ ID NO: 63). In another particular embodiment, the sequence of the loop region GH is a variant of the sequence corresponding to amino acids 149-150 of SEQ ID NO: 62, and at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, or all of the sequences of AB, BC, CD, DE, EF, FG, HI, IJ, and JK of the polypeptide of the first aspect of the present invention are sequences of their cognate loop regions in SEQ ID NO: 62 as described above. In another particular embodiment, the sequence of the loop region GH is a variant of the sequence corresponding to amino acids 149-150 of SEQ ID NO: 62, and at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, or all of the sequences of the loop region of the polypeptide of the first aspect of the present invention are sequences of their cognate loop regions in SEQ ID NO: 62. In another specific embodiment, the sequence of the loop region GH is a variant of the sequence corresponding to amino acids 149-150 of SEQ ID NO: 62, and the rest of the polypeptide sequence of the first aspect of the present invention is the rest of the sequence of SEQ ID NO: 62.

[0103] In a particular embodiment, the loop region GH variant of the sequence corresponding to amino acids 149-150 of SEQ ID NO: 62 has at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, and at least 99% sequence identity with the sequence corresponding to amino acids 149-150 of SEQ ID NO: 62.

[0104] In another specific embodiment, the sequence of the loop region GH of the polypeptide according to the first aspect of the present invention is the sequence corresponding to amino acids 149-150 of SEQ ID NO: 62.

[0105] In certain embodiments, the sequence of loop region HI is a variant of SEQ ID NO: 7. In another particular embodiment, the sequence of loop region HI is a variant of SEQ ID NO: 7, and at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, or all sequences of AB, BC, CD, DE, EF, FG, GH, IJ, and JK of the polypeptide of the first aspect of the present invention are sequences of their cognate loop regions in SEQ ID NO: 62 as shown above. In another particular embodiment, the sequence of loop region HI is a variant of SEQ ID NO: 7, and at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, or all sequences of the loop region of the polypeptide of the first aspect of the present invention are sequences of their cognate loop regions in SEQ ID NO: 62. In another particular embodiment, the sequence of loop region HI is a variant of SEQ ID NO: 7, and the remaining sequences of the polypeptide of the first aspect of the present invention are identical to the remaining sequences of SEQ ID NO: 62.

[0106] In certain embodiments, the loop region HI variant of SEQ ID NO: 7 has at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, and at least 99% sequence identity with SEQ ID NO: 7.

[0107] In another specific embodiment, the sequence of the loop region HI of the polypeptide according to the first aspect of the present invention is sequence number 7.

[0108] In certain embodiments, the sequence of loop region IJ is a variant of SEQ ID NO: 8. In another particular embodiment, the sequence of loop region IJ is a variant of SEQ ID NO: 8, and at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, or all sequences of the AB, BC, CD, DE, EF, FG, GH, HI, and JK loop regions of the polypeptide of the first aspect of the present invention are sequences of their cognate loop regions in SEQ ID NO: 62 as shown above. In another particular embodiment, the sequence of loop region IJ is a variant of SEQ ID NO: 8, and at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, or all sequences of the loop regions of the polypeptide of the first aspect of the present invention are sequences of their cognate loop regions in SEQ ID NO: 62. In another particular embodiment, the sequence of loop region IJ is a variant of SEQ ID NO: 8, and the remaining sequences of the polypeptide of the first aspect of the present invention are identical to the remaining sequences of SEQ ID NO: 62.

[0109] In certain embodiments, the loop region IJ variant of sequence number 8 has at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, and at least 99% sequence identity with sequence number 8.

[0110] In another specific embodiment, the sequence of the loop region IJ of the polypeptide according to the first aspect of the present invention is sequence number 8.

[0111] In certain embodiments, the sequence of loop region JK is a variant of SEQ ID NO: 9. In another particular embodiment, the sequence of loop region JK is a variant of SEQ ID NO: 9, and at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, or all sequences of AB, BC, CD, DE, EF, FG, GH, HI, and IJ of the polypeptide of the first aspect of the present invention are sequences of their cognate loop regions in SEQ ID NO: 62 as shown above. In another particular embodiment, the sequence of loop region JK is a variant of SEQ ID NO: 9, and at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, or all sequences of the loop region of the polypeptide of the first aspect of the present invention are sequences of their cognate loop regions in SEQ ID NO: 62. In another particular embodiment, the sequence of loop region JK is a variant of SEQ ID NO: 9, and the remaining sequences of the polypeptide of the first aspect of the present invention are identical to the remaining sequences of SEQ ID NO: 62.

[0112] In certain embodiments, the loop region JK variant of sequence number 9 has at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, and at least 99% sequence identity with sequence number 10.

[0113] In another specific embodiment, the sequence of the loop region JK of the polypeptide according to the first aspect of the present invention is sequence number 9.

[0114] In certain embodiments, at least one sequence located upstream, near upstream, downstream, or near downstream of a loop region variant of a polypeptide according to the first aspect of the present invention includes one or more amino acid modifications, insertions, and / or deletions with respect to the sequence in SEQ ID NO: 62 (referred to as its reference sequence), which is located at the same position with respect to the congeneral loop region of the loop region variant. However, at least one sequence encodes the same protein domain or secondary structure as defined above in the polypeptide according to the first aspect of the present invention as its reference sequence in the polypeptide having SEQ ID NO: 62. A method for determining whether two amino acid sequences form the same protein domain or secondary structure, such as a loop region, an α-helix moiety, or an α-helix, is a method for determining whether the two amino acid sequences described above form the same protein structure.

[0115] In certain embodiments, at least one sequence comprising one or more amino acid modifications, insertions, and / or deletions with respect to a reference sequence has at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, and at least 99% sequence identity with that reference sequence in Sequence ID No. 62. The method for determining the degree of sequence identity between two amino acid sequences is as described above.

[0116] As used herein, the expression “congenerate β-chain domain in SEQ ID NO: 62” refers to the β-chain domain of SEQ ID NO: 62 located between the amino acid sequences of a protein having SEQ ID NO: 62, which encodes the same loop region or protein secondary structure as the sequence in which the polypeptide of the first aspect of the present invention is located. As will be understood by those skilled in the art, each β-chain domain of the first polypeptide of the present invention has its congenerate β-chain domain in SEQ ID NO: 62.

[0117] Each β-chain domain of the nidogen G2 domain mutant may be identical to the congenital β-chain in SEQ ID NO: 62, or it may differ by one or more amino acids such that the overall sequence identity between the β-chain of the nidogen G2 domain mutant and the congenital β-chain domain in SEQ ID NO: 62 is at least 50%. In a preferred embodiment, the sequence identity between the β-chain of the nidogen G2 domain mutant and the congenital β-chain domain in SEQ ID NO: 62 is at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%.

[0118] In certain embodiments, β-chain domain A is a β-chain domain variant of its homologous β-chain domain in SEQ ID NO: 62. In certain embodiments, β-chain domain B is a β-chain domain variant of its homologous β-chain domain in SEQ ID NO: 62. In certain embodiments, β-chain domain C is a β-chain domain variant of its homologous β-chain domain in SEQ ID NO: 62. In certain embodiments, β-chain domain D is a β-chain domain variant of its homologous β-chain domain in SEQ ID NO: 62. In certain embodiments, β-chain domain E is a β-chain domain variant of its homologous β-chain domain in SEQ ID NO: 62. In certain embodiments, β-chain domain F is a β-chain domain variant of its homologous β-chain domain in SEQ ID NO: 62. In certain embodiments, β-chain domain G is a β-chain domain variant of its homologous β-chain domain in SEQ ID NO: 62. In certain embodiments, β-chain domain H is a β-chain domain variant of its homologous β-chain domain in SEQ ID NO: 62. In certain embodiments, β-chain domain I is a β-chain domain variant of its homologous β-chain domain in SEQ ID NO: 62. In certain embodiments, β-chain domain J is a β-chain domain variant of its homologous β-chain domain in SEQ ID NO: 62. In certain embodiments, β-chain domain K is a β-chain domain variant of its homologous β-chain domain in SEQ ID NO: 62.

[0119] In certain embodiments, the β-chain domain of the polypeptide according to the first aspect of the present invention is a variant of its homologous β-chain in SEQ ID NO: 62.

[0120] As used herein, the term “β-chain domain variant” refers to a β-chain domain from a polypeptide of the first aspect of the present invention, which comprises one or more amino acid modifications, insertions, and / or deletions in its sequence relative to the sequence of its homologous β-chain domain in SEQ ID NO: 62. In a particular embodiment, the β-chain domain from the polypeptide of the first aspect is selected from the group consisting of A, B, C, D, E, F, G, H, I, J, or K.

[0121] In certain embodiments, the β-chain domain variant of the polypeptide of the first embodiment has sequence identity of at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, and at least 99% with the sequence of its homologous β-chain domain in SEQ ID NO: 62. The method for determining the degree of sequence identity is as described above.

[0122] In a particular embodiment, the cognate β-chain domain in SEQ ID NO: 62 for β-chain domain A has SEQ ID NO: 10. In another particular embodiment, the cognate β-chain domain in SEQ ID NO: 62 for β-chain domain B has SEQ ID NO: 11. In another particular embodiment, the cognate β-chain domain in SEQ ID NO: 62 for β-chain domain C has SEQ ID NO: 12. In another particular embodiment, the cognate β-chain domain in SEQ ID NO: 62 for β-chain domain D has SEQ ID NO: 13. In another particular embodiment, the cognate β-chain domain in SEQ ID NO: 62 for β-chain domain E has SEQ ID NO: 14. In another particular embodiment, the cognate β-chain domain in SEQ ID NO: 62 for β-chain domain F has SEQ ID NO: 15. In another particular embodiment, the cognate β-chain domain in SEQ ID NO: 62 for β-chain domain G has SEQ ID NO: 16. In another particular embodiment, the cognate β-chain domain in SEQ ID NO: 62 for β-chain domain H has SEQ ID NO: 17. In another particular embodiment, the cognate β-chain domain in SEQ ID NO: 62 for β-chain domain I has SEQ ID NO: 18. In another specific embodiment, the homologous β-chain domain in SEQ ID NO: 62 for β-chain domain J has SEQ ID NO: 19. In another specific embodiment, the homologous β-chain domain in SEQ ID NO: 62 for β-chain domain K has SEQ ID NO: 20.

[0123] Therefore, in certain embodiments, the sequence of β-chain A is a variant of sequence number 10. In another specific embodiment, the sequence of β-chain domain A is a variant of SEQ ID NO: 10, and at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, or all of the β-chain domains B-K of the polypeptide of the first aspect of the present invention have the sequences of their homogeneous β-chain domains in SEQ ID NO: 62 as shown above. In another specific embodiment, the sequence of β-chain A is a variant of SEQ ID NO: 10, and at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, or all of the β-chain domains of the polypeptide of the first aspect of the present invention have the sequences of their homogeneous β-chain domains in SEQ ID NO: 62. In another specific embodiment, the sequence of β-chain A is a variant of SEQ ID NO: 10, and the remaining sequences of the polypeptide of the first aspect of the present invention are identical to the remaining sequences of SEQ ID NO: 62.

[0124] In certain embodiments, the β-chain domain A variant of SEQ ID NO: 10 has at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, and at least 99% sequence identity with SEQ ID NO: 11.

[0125] In another specific embodiment, the sequence of the β-chain domain A of the polypeptide according to the first aspect of the present invention is SEQ ID NO: 10.

[0126] In a particular embodiment, the sequence of β-chain B is a variant of SEQ ID NO: 11. In another particular embodiment, the sequence of β-chain domain B is a variant of SEQ ID NO: 11, and at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine or all of the β-chain domains A, C-K of the polypeptide of the first aspect of the present invention have the sequences of their homogeneous β-chain domains in SEQ ID NO: 62 as shown above. In another particular embodiment, the sequence of β-chain B is a variant of SEQ ID NO: 11, and at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine or all of the β-chain domains of the polypeptide of the first aspect of the present invention have the sequences of their homogeneous β-chain domains in SEQ ID NO: 62. In another particular embodiment, the sequence of β-chain B is a variant of SEQ ID NO: 11, and the remaining sequences of the polypeptide of the first aspect of the present invention are identical to the remaining sequences of SEQ ID NO: 62.

[0127] In certain embodiments, the β-chain domain B variant of SEQ ID NO: 11 has at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, and at least 99% sequence identity with SEQ ID NO: 12.

[0128] In another specific embodiment, the sequence of the β-chain domain B of the polypeptide according to the first aspect of the present invention is SEQ ID NO: 11.

[0129] In certain embodiments, the sequence of β-chain C is a variant of SEQ ID NO: 12. In another particular embodiment, the sequence of β-chain domain C is a variant of SEQ ID NO: 12, and at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine or all of the β-chain domains A, B, D-K of the polypeptide of the first aspect of the present invention have the sequences of their homogeneous β-chain domains in SEQ ID NO: 62 as shown above. In another particular embodiment, the sequence of β-chain C is a variant of SEQ ID NO: 12, and at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine or all of the β-chain domains of the polypeptide of the first aspect of the present invention have the sequences of their homogeneous β-chain domains in SEQ ID NO: 62. In another particular embodiment, the sequence of β-chain C is a variant of SEQ ID NO: 12, and the remaining sequences of the polypeptide of the first aspect of the present invention are identical to the remaining sequences of SEQ ID NO: 62.

[0130] In certain embodiments, the β-chain domain C variant of SEQ ID NO: 12 has at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, and at least 99% sequence identity with SEQ ID NO: 12.

[0131] In another specific embodiment, the sequence of the β-chain domain C of the polypeptide according to the first aspect of the present invention is SEQ ID NO: 12.

[0132] In certain embodiments, the sequence of β-chain D is a variant of SEQ ID NO: 13. In another particular embodiment, the sequence of β-chain domain D is a variant of SEQ ID NO: 13, and at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine or all of the β-chain domains A-C, E-K of the polypeptide of the first aspect of the present invention have the sequences of their homogeneous β-chain domains in SEQ ID NO: 62 as shown above. In another particular embodiment, the sequence of β-chain D is a variant of SEQ ID NO: 13, and at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine or all of the β-chain domains of the polypeptide of the first aspect of the present invention have the sequences of their homogeneous β-chain domains in SEQ ID NO: 62. In another particular embodiment, the sequence of β-chain D is a variant of SEQ ID NO: 13, and the remaining sequences of the polypeptide of the first aspect of the present invention are identical to the remaining sequences of SEQ ID NO: 62.

[0133] In certain embodiments, the β-chain domain D variant of SEQ ID NO: 13 has at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, and at least 99% sequence identity with SEQ ID NO: 13.

[0134] In another specific embodiment, the sequence of the β-chain domain D of the polypeptide according to the first aspect of the present invention is SEQ ID NO: 13.

[0135] In certain embodiments, the sequence of β-chain E is a variant of SEQ ID NO: 14. In another particular embodiment, the sequence of β-chain domain E is a variant of SEQ ID NO: 14, and at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine or all of the β-chain domains A-D, F-K of the polypeptide of the first aspect of the present invention have the sequences of their homogeneous β-chain domains in SEQ ID NO: 62 as shown above. In another particular embodiment, the sequence of β-chain E is a variant of SEQ ID NO: 14, and at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine or all of the β-chain domains of the polypeptide of the first aspect of the present invention have the sequences of their homogeneous β-chain domains in SEQ ID NO: 62. In another particular embodiment, the sequence of β-chain E is a variant of SEQ ID NO: 14, and the remaining sequences of the polypeptide of the first aspect of the present invention are identical to the remaining sequences of SEQ ID NO: 62.

[0136] In certain embodiments, the β-chain domain E variant of SEQ ID NO: 14 has at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, and at least 99% sequence identity with SEQ ID NO: 14.

[0137] In another specific embodiment, the sequence of the β-chain domain E of the polypeptide according to the first aspect of the present invention is SEQ ID NO: 14.

[0138] In a particular embodiment, the sequence of β-chain F is a variant of SEQ ID NO: 15. In another particular embodiment, the sequence of β-chain domain F is a variant of SEQ ID NO: 15, and at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine or all of the β-chain domains A-E, G-K of the polypeptide of the first aspect of the present invention have the sequences of their homogeneous β-chain domains in SEQ ID NO: 62 as shown above. In another particular embodiment, the sequence of β-chain F is a variant of SEQ ID NO: 15, and at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine or all of the β-chain domains of the polypeptide of the first aspect of the present invention have the sequences of their homogeneous β-chain domains in SEQ ID NO: 62. In another particular embodiment, the sequence of β-chain F is a variant of SEQ ID NO: 15, and the remaining sequences of the polypeptide of the first aspect of the present invention are identical to the remaining sequences of SEQ ID NO: 62.

[0139] In certain embodiments, the β-chain domain F variant of SEQ ID NO: 15 has at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, and at least 99% sequence identity with SEQ ID NO: 15.

[0140] In another specific embodiment, the sequence of the β-chain domain F of the polypeptide according to the first aspect of the present invention is SEQ ID NO: 15.

[0141] In certain embodiments, the sequence of β-chain G is a variant of SEQ ID NO: 16. In another particular embodiment, the sequence of β-chain domain G is a variant of SEQ ID NO: 16, and at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine or all of the β-chain domains A-F and H-K of the polypeptide of the first aspect of the present invention have the sequences of their homogeneous β-chain domains in SEQ ID NO: 62 as shown above. In another particular embodiment, the sequence of β-chain G is a variant of SEQ ID NO: 16, and at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine or all of the β-chain domains of the polypeptide of the first aspect of the present invention have the sequences of their homogeneous β-chain domains in SEQ ID NO: 62. In another particular embodiment, the sequence of β-chain G is a variant of SEQ ID NO: 16, and the remaining sequences of the polypeptide of the first aspect of the present invention are identical to the remaining sequences of SEQ ID NO: 62.

[0142] In certain embodiments, the β-chain domain G variant of SEQ ID NO: 16 has at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, and at least 99% sequence identity with SEQ ID NO: 16.

[0143] In another specific embodiment, the sequence of the β-chain domain G of the polypeptide according to the first aspect of the present invention is SEQ ID NO: 16.

[0144] In certain embodiments, the sequence of β-chain H is a variant of SEQ ID NO: 17. In another particular embodiment, the sequence of β-chain domain H is a variant of SEQ ID NO: 18, and at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine or all of the β-chain domains A-G, I-K of the polypeptide of the first aspect of the present invention have the sequences of their homogeneous β-chain domains in SEQ ID NO: 62 as shown above. In another particular embodiment, the sequence of β-chain H is a variant of SEQ ID NO: 17, and at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine or all of the β-chain domains of the polypeptide of the first aspect of the present invention have the sequences of their homogeneous β-chain domains in SEQ ID NO: 62. In another particular embodiment, the sequence of β-chain H is a variant of SEQ ID NO: 17, and the remaining sequence of the polypeptide of the first aspect of the present invention is identical to the remaining sequence of SEQ ID NO: 62.

[0145] In certain embodiments, the β-chain domain H variant of SEQ ID NO: 17 has at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, and at least 99% sequence identity with SEQ ID NO: 17.

[0146] In another specific embodiment, the sequence of the β-chain domain H of the polypeptide according to the first aspect of the present invention is SEQ ID NO: 17.

[0147] In certain embodiments, the sequence of β-chain I is a variant of SEQ ID NO: 18. In another particular embodiment, the sequence of β-chain domain I is a variant of SEQ ID NO: 18, and at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine or all of the β-chain domains A-H, J, K of the polypeptide of the first aspect of the present invention have the sequences of their homogeneous β-chain domains in SEQ ID NO: 62 as shown above. In another particular embodiment, the sequence of β-chain I is a variant of SEQ ID NO: 18, and at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine or all of the β-chain domains of the polypeptide of the first aspect of the present invention have the sequences of their homogeneous β-chain domains in SEQ ID NO: 62. In another particular embodiment, the sequence of β-chain I is a variant of SEQ ID NO: 18, and the remaining sequence of the polypeptide of the first aspect of the present invention is identical to the remaining sequence of SEQ ID NO: 62.

[0148] In certain embodiments, the β-chain domain I variant of SEQ ID NO: 18 has at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, and at least 99% sequence identity with SEQ ID NO: 19.

[0149] In another specific embodiment, the sequence of the β-chain domain I of the polypeptide according to the first aspect of the present invention is SEQ ID NO: 18.

[0150] In certain embodiments, the sequence of β-chain J is a variant of SEQ ID NO: 19. In another particular embodiment, the sequence of β-chain domain J is a variant of SEQ ID NO: 19, and at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine or all of the β-chain domains A-I, K of the polypeptide of the first aspect of the present invention have the sequences of their homogeneous β-chain domains in SEQ ID NO: 62 as shown above. In another particular embodiment, the sequence of β-chain J is a variant of SEQ ID NO: 19, and at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine or all of the β-chain domains of the polypeptide of the first aspect of the present invention have the sequences of their homogeneous β-chain domains in SEQ ID NO: 62. In another particular embodiment, the sequence of β-chain J is a variant of SEQ ID NO: 19, and the remaining sequence of the polypeptide of the first aspect of the present invention is identical to the remaining sequence of SEQ ID NO: 62.

[0151] In certain embodiments, the β-chain domain J variant of SEQ ID NO: 19 has at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, and at least 99% sequence identity with SEQ ID NO: 19.

[0152] In another specific embodiment, the sequence of the β-chain domain J of the polypeptide according to the first aspect of the present invention is SEQ ID NO: 19.

[0153] In certain embodiments, the sequence of β-chain K is a variant of SEQ ID NO: 20. In another particular embodiment, the sequence of β-chain domain K is a variant of SEQ ID NO: 20, and at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine or all of the β-chain domains A-J of the polypeptide of the first aspect of the present invention have the sequences of their homogeneous β-chain domains in SEQ ID NO: 62 as shown above. In another particular embodiment, the sequence of β-chain K is a variant of SEQ ID NO: 20, and at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine or all of the β-chain domains of the polypeptide of the first aspect of the present invention have the sequences of their homogeneous β-chain domains in SEQ ID NO: 62. In another particular embodiment, the sequence of β-chain K is a variant of SEQ ID NO: 20, and the remaining sequences of the polypeptide of the first aspect of the present invention are identical to the remaining sequences of SEQ ID NO: 62.

[0154] In certain embodiments, the β-chain domain K variant of SEQ ID NO: 20 has at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, and at least 99% sequence identity with SEQ ID NO: 20.

[0155] In another specific embodiment, the sequence of the β-chain domain K of the polypeptide according to the first aspect of the present invention is SEQ ID NO: 20.

[0156] In one embodiment, the polypeptide according to the first aspect of the present invention includes at least one loop region variant with respect to the congenital loop region in SEQ ID NO: 62, which is caused by a mutation resulting from the deletion, substitution, or addition of at least one amino acid with respect to the sequence of the congenital loop region. In one embodiment, the nidogen G2 domain variant includes a mutation in loop region AB with respect to SEQ ID NO: 1, which is the congenital loop region AB. In one embodiment, the nidogen G2 domain variant includes a mutation in loop region BC with respect to SEQ ID NO: 2, which is the congenital loop region BC. In one embodiment, the nidogen G2 domain variant includes a mutation in loop region CD with respect to SEQ ID NO: 3, which is the congenital loop region CD. In one embodiment, the nidogen G2 domain variant includes a mutation in loop region DE with respect to SEQ ID NO: 4, which is the congenital loop region DE. In one embodiment, the nidogen G2 domain variant includes a mutation in loop region EF with respect to SEQ ID NO: 5, which is the congenital loop region EF. In one embodiment, the nidogen G2 domain variant includes a mutation in loop region FG with respect to SEQ ID NO: 6, which is the congenital loop region FG. In one embodiment, the nidogen G2 domain variant includes a mutation in the loop region GH with respect to the congeneral loop region GH corresponding to amino acids 149-150 of SEQ ID NO: 62. In one embodiment, the nidogen G2 domain variant includes a mutation in the loop region HI with respect to SEQ ID NO: 7, which is the congeneral loop region HI. In one embodiment, the nidogen G2 domain variant includes a mutation in the loop region IJ with respect to SEQ ID NO: 8, which is the congeneral loop region IJ. In one embodiment, the nidogen G2 domain variant includes a mutation in the loop region JK with respect to SEQ ID NO: 9, which is the congeneral loop region JK.

[0157] The α-helix portion CαD of the nidogen G2 domain mutant may be identical to the congenital α-helix portion in SEQ ID NO: 62, or it may differ by one or more amino acids such that there is at least 50% overall sequence identity between the α-helix portion CαD of the nidogen G2 domain mutant and the congenital α-helix portion of SEQ ID NO: 26 in SEQ ID NO: 62.

[0158] In a preferred embodiment, the sequence identity between the α-helix portion CαD of the nidogen G2 domain mutant and the congeneral α-helix portion of SEQ ID NO: 24 in SEQ ID NO: 62 is at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%.

[0159] The method for determining the degree of sequence identity is as described above. In certain embodiments, an α-helix moiety CαD that differs by one or more amino acids as shown above is called an α-helix moiety variant of the congeneral α-helix moiety in SEQ ID NO: 62.

[0160] In certain embodiments, the polypeptide of the first embodiment has a β-barrel structure. In preferred embodiments, the polypeptide of the first embodiment has a β-barrel structure of the β-barrel domain of the G2 domain of nidogen-1. In certain embodiments, the polypeptide of the first embodiment has a β-barrel structure of the sequence having SEQ ID NO: 62.

[0161] The β-barrel domain of the G2 domain of Nidogen-1 and the β-barrel structure of sequence SEQ ID NO: 62 consist of an 11-stranded β-barrel. Hereinafter, the β-chains of the β-barrel are referred to as I, II, III, IV, V, VI, VII, VIII, IX, X, and XI. They correspond to the homologous β-chains of sequence SEQ ID NO: 62, β-chains A-K of the polypeptide in the first embodiment. The interior of the β-barrel is traversed by a hydrophobic, primarily α-helix moiety connecting chains III and IV. The N-terminal half of the barrel consists of two beta-meanders (chains I-III and IV-VI) linked by an embedded α-helix moiety. The polypeptide chain then traverses the bottom of the barrel, forming a 5-stranded Greek key motif at the C-terminal half of the domain.

[0162] As used herein, the term "β-meaner" refers to two or more consecutive antiparallel β-chains linked by a hairpin loop. As used herein, the term "hairpin loop" refers to two antiparallel chains linked by a short loop of 2 to 5 residues, one of which is often glycine or proline, both of which can provide the dihedral-angle conformation necessary for a tight turn or β-bulge loop.

[0163] As used herein, the term "Greek key" refers to a secondary protein structure consisting of four adjacent antiparallel strands and their connecting loops. In this structure, three antiparallel strands are connected by hairpins, and a fourth strand is adjacent to the first strand and connected to the third by a longer loop.

[0164] Therefore, in certain embodiments, the β chains A-C and D-F of the G2 domain variant form a β-meaner. In another specific embodiment, the β-meaner is connected by the α-helix portion CαD of the G2 domain variant. In yet another specific embodiment, the β chains G-K of the G2 domain variant form a quintuple Greek key motif. In yet another specific embodiment, the β chains of the G2 domain variant are arranged antiparallel, except for the β chains A and F.

[0165] The method for determining the secondary structure of a polypeptide, or for determining whether two amino acid sequences encode the same domain or secondary protein structure, is as described above.

[0166] II-Polypeptide Display Library In a second embodiment, the present invention relates to a polypeptide display library comprising a plurality of polypeptides according to a first embodiment of the present invention, wherein the plurality of polypeptides are formed by polypeptides having one or more loop region sequences that differ.

[0167] As used herein, the term "polypeptide display library" refers to a library or pool of polypeptides comprising multiple polypeptides having different amino acid sequences. Each polypeptide in the library is as defined in the first embodiment of the present invention and differs from at least one other polypeptide in the library in the sequence of one or more loop regions.

[0168] As used herein, the expression “polypeptides with different loop region sequences” refers to the fact that each polypeptide in the library exhibits at least one difference in its amino acid sequence with respect to the amino acid sequence of at least one other polypeptide in the library, and that at least one difference is contained in the amino acid sequence of the polypeptide’s loop region. Thus, in certain embodiments, the polypeptides in the library are polypeptides of the first embodiment that include at least one loop region variant as defined in the first embodiment, which is different from the corresponding loop region of another polypeptide in the library. In certain embodiments, the loop region variant is selected from the group consisting of A, B, C, D, E, F, G, H, I, J, or K, as described in the definition and embodiments of “loop region variant” in the first embodiment of the present invention.

[0169] As used herein, the expression "shows at least one difference in amino acid sequence with respect to the amino acid sequence of at least one other polypeptide in the library, and the sequence is included in the amino acid sequence of the loop region of the polypeptide" means that the loop region variant of the first polypeptide in the library, as defined above, contains at least one insertion, deletion, or modification of at least one amino acid with respect to the amino acid sequence of the corresponding loop region in the second polypeptide in the library. As will be understood by those skilled in the art, if the loop region variant in the first polypeptide is loop region AB, then the corresponding loop region in the second polypeptide is also loop region AB in the second polypeptide. If the loop region variant in the first polypeptide is loop region BC, then the corresponding loop region in the second polypeptide is also loop region BC in the second polypeptide. If the loop region variant in the first polypeptide is loop region CD, then the corresponding loop region in the second polypeptide is also loop region CD in the second polypeptide. If the loop region variant in the first polypeptide is loop region DE, then the corresponding loop region in the second polypeptide is also loop region DE in the second polypeptide. If the loop region variant in the first polypeptide is loop region EF, then the corresponding loop region in the second polypeptide is also loop region EF. If the loop region variant in the first polypeptide is loop region FG, then the corresponding loop region in the second polypeptide is also loop region FG. If the loop region variant in the first polypeptide is loop region GH, then the corresponding loop region in the second polypeptide is also loop region GH. If the loop region variant in the first polypeptide is loop region HI, then the corresponding loop region in the second polypeptide is also loop region HI. If the loop region variant in the first polypeptide is loop region IJ, then the corresponding loop region in the second polypeptide is also loop region IJ.If the loop region variant in the first polypeptide is loop region JK, then the corresponding loop region in the second polypeptide is also loop region JK in the second polypeptide.

[0170] In certain embodiments, loop region variants of the first polypeptide in the library exhibit sequence identity of at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, and at least 99% with the corresponding loop region of the second polypeptide in the library. In another specific embodiment, the entire sequence of the first polypeptide exhibits sequence identity with the entire sequence of the second polypeptide to a degree of at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.75%, at least 99.9%, at least 99.95%, at least 99.975%, at least 99.98%, at least 99.99%, and at least 99.999%. The method for determining the percentage of sequence identity is as described in the first aspect of the present invention.

[0171] In certain embodiments, the first polypeptide of the library exhibits amino acid sequence differences of at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, at least ten, and at least eleven loop regions with respect to the corresponding loop region in at least one other polypeptide of the library, referred to as the second polypeptide of the library. The term "corresponding loop region in the second polypeptide" is as defined above for each loop region of the first polypeptide. The amino acid sequence differences are also as defined above. Furthermore, each amino acid sequence exhibits a degree of sequence identity with the sequence of the corresponding loop region in the second polypeptide, as similarly defined above.

[0172] In certain embodiments, polypeptides in a library can specifically bind to other molecules, preferably peptides or proteins, present in close proximity to them in a sample, for example, by at least one of their loop regions. In preferred embodiments, polypeptides in a library exhibiting differences in amino acid sequences in one or more of their corresponding loop regions can specifically bind to different molecules, preferably peptides or proteins, present in close proximity to them in a sample. As will be understood by those skilled in the art, the difference in their binding ability may be such that one polypeptide can specifically bind to one molecule, preferably a peptide or protein in the sample, while another polypeptide in the library having differences in one or more loop regions cannot specifically bind to one molecule, preferably a peptide or protein in the sample. Alternatively, one polypeptide may be able to specifically bind to one or more molecules, preferably peptides or proteins, while another polypeptide in the library having differences in one or more loop regions cannot specifically bind to the aforementioned one or more molecules, but can bind to one or more other molecules, preferably peptides present in the sample. In certain embodiments, the first polypeptide in the library can specifically bind to a target molecule, preferably a target peptide or protein, by at least one loop region variant exhibiting sequence differences with respect to the corresponding loop region in the second polypeptide in the library, whereas the second polypeptide in the library cannot specifically bind to the target molecule.

[0173] In another specific embodiment, a loop region variant of the first polypeptide in the library specifically binds to the target peptide, whereas its cognate loop region in SEQ ID NO: 62 cannot specifically bind to the target peptide.

[0174] In another specific embodiment, polypeptides in a polypeptide library having the same loop region variant in one loop region selected from AB, BC, CD, DE, EF, FG, GH, HI, IJ, and JK specifically bind to a specific target peptide via the loop region variant, whereas polypeptides in a library not having the specific loop region variant do not specifically bind to a specific target peptide via the loop region variant.

[0175] The terms “bind,” “binding,” “specifically bind,” “specifically binding,” and “specifically interact” are defined in the first embodiment of the present invention. The binding between a polypeptide and a target molecule, and the method for determining K of such binding, are also described in the above definitions.

[0176] In a particular embodiment, the binding of the polypeptide from the library to the target molecule is such that the binding between the polypeptide and the target molecule is 10 -6 Less than M, 10 -7 Less than M, 10 -8 Less than M, 10 -9 Less than M, 10 -10 Less than M, 10 -11 Less than M, 10 -12 Less than M, 10 -13 Less than M, 10 -14 Less than M or 10 -15 Dissociation constant less than M (K d ) if it has a unique It is considered a target. Similarly, the binding between a loop region, preferably a loop region variant, and a specific target molecule is considered a binding between the loop region and the target molecule. -6 Less than M, 10 -7 Less than M, 10 -8 Less than M, 10 -9 Less than M, 10 -10 Less than M, 10 -11 Less than M, 10 -12 Less than M, 10 -13 Less than M, 10-14 Less than M or 10 -15 A dissociation constant (KD) less than M is considered specific.

[0177] In certain embodiments, each polypeptide of the library of the second aspect of the present invention, as a phenotype, is directly or indirectly linked to a nucleic acid, as a genotype, corresponding to the phenotype.

[0178] As used in this invention, the term "genotype" refers to a nucleic acid molecule that encodes one or more peptides, polypeptides, or proteins, or a nucleic acid molecule containing a sequence that encodes such a molecule. The group of peptides, polypeptides, or proteins corresponds to the phenotype corresponding to the genotype. As will be understood by those skilled in the art, a genotype can be formed by a single nucleic acid molecule encoding a single peptide, polypeptide, or protein. In this case, the phenotype corresponds to the peptide, polypeptide, or protein. A nucleic acid can be any of the nucleic acids specified in the following definition of nucleic acid.

[0179] In certain embodiments, the genotype is formed by a single nucleic acid molecule encoding a single polypeptide from the polypeptide display library. In another specific embodiment, the genotype is formed by several nucleic acid molecules encoding the same polypeptide from the polypeptide display library. In yet another specific embodiment, the nucleic acid molecules have the same nucleic acid sequence.

[0180] The definitions of the terms peptide, polypeptide, and protein are described in the first embodiment of the present invention.

[0181] The terms “nucleic acid,” “nucleotide sequence,” or “polynucleotide” are interchangeable in this invention and refer to single-stranded or double-stranded polymeric forms of ribonucleoside phosphate esters (adenosine, guanosine, uridine, or cytidine; “RNA molecule”) or deoxyribonucleosides (deoxyadenosine, deoxyguanosine, deoxythymidine, or deoxycytidine; “DNA molecule”) or any phosphoester analog thereof, such as phosphorothioates and thioesters. Therefore, this term includes single-stranded DNA or RNA molecules. It also includes double-stranded molecules formed by DNA-DNA strands, DNA-RNA strands, and RNA-RNA strands. The term “nucleic acid sequence,” particularly DNA or RNA molecules, refers only to the primary or secondary structure of the molecule and does not limit it to any particular type of tertiary structure. Therefore, this term encompasses linear or circular DNA molecules, supercoiled DNA plasmids, and double-stranded DNA contained in chromosomes. In certain embodiments, the nucleic acid is a DNA molecule. In another specific embodiment, nucleic acid is an RNA molecule.

[0182] As used in the present invention, the term "phenotype" refers to a peptide, polypeptide, or protein, or a group of peptides, polypeptides, or proteins. A nucleic acid that codes for a nucleic acid, or contains a sequence that codes for a nucleic acid, is the genotype corresponding to the phenotype. As will be understood by those skilled in the art, in the context of the present invention, the phenotype may consist of a single peptide, polypeptide, or protein. The genotype associated with a phenotype is the nucleic acid molecule or group of nucleic acid molecules that codes for it.

[0183] In certain embodiments, the phenotype is a polypeptide of a polypeptide library as defined above in the first embodiment of the present invention and in this embodiment of the present invention. In a particular embodiment, the phenotype is the first polypeptide of the library defined above.

[0184] The phrase "phenotype directly or indirectly linked to a nucleic acid as a genotype corresponding to the phenotype," as used in the present invention, is understood to mean a polypeptide of a polypeptide display library (i.e., the phenotype as defined above) linked to the nucleic acid encoding it (i.e., the genotype as defined above). Linking results in a complex formed by the polypeptide of the library and the nucleic acid encoding it. In certain embodiments, the polypeptide is exposed on the outer surface of the complex. Thus, in certain embodiments, the polypeptide display library is formed by a complex comprising a polypeptide of the first aspect of the present invention, directly or indirectly linked to the nucleic acid encoding it. The polypeptide is considered a phenotype, and the nucleic acid is considered a genotype corresponding to the phenotype.

[0185] In certain embodiments, the polypeptides in the library are not linked to any nucleic acids that encode them.

[0186] Each polypeptide in the library is referred to as a “member of the library,” whether or not it is part of a complex as described above. Therefore, as used herein, this term refers to any peptide in the library to which the polypeptide and nucleic acid may be directly or indirectly linked, and the nucleic acid either codes for or contains a sequence encoding the polypeptide, or is not linked by any means to a nucleic acid containing a sequence encoding it. Thus, in a particular embodiment, a member of the library is a polypeptide of the library as defined above. In another particular embodiment, a member of the library is a complex comprising a polypeptide of the first aspect of the present invention, directly or indirectly linked to a nucleic acid encoding it.

[0187] Direct linkage consists of a direct interaction between a polypeptide in the library and the nucleic acid encoding it, resulting in a polypeptide-nucleic acid complex in which the polypeptide binds or covalently to the nucleic acid encoding it, with the polypeptide contained on the outer surface of the polypeptide-nucleic acid complex. As will be understood by those skilled in the art, the complex may also contain additional proteins and / or nucleic acids.

[0188] In certain embodiments, the binding of the complex polypeptide to the nucleic acid is direct. In other specific embodiments, the binding of the complex polypeptide to the nucleic acid is indirect, and the polypeptide binds or covalently to the nucleic acid encoding it by another peptide, protein, protein complex, or molecule that binds to the nucleic acid.

[0189] As used herein, the terms “covalently attached,” “covalent attachment,” and “covalently coupled” refer to interactions between two molecules that are directly covalently bonded to each other via chemical covalent bonds, or that are indirectly covalently bonded to each other via one or more intervening parts such as linkers, bridges, or spacers.

[0190] In another specific embodiment, the covalent bond between the library polypeptide and the nucleic acid encoding it is direct, and the polypeptide is covalently bonded to the nucleic acid encoding it. In yet another specific embodiment, the covalent bond between the library polypeptide and the nucleic acid encoding it is indirect, and the polypeptide is bonded to the nucleic acid encoding it via one or more intervening parts such as a linker, bridge, or spacer. In a preferred embodiment, it is bonded via a linker.

[0191] As used herein, the terms “linker moiety” or “linker” refer to a molecule that links two molecules or compounds. The linker moiety is not intended to be limited in terms of its chemical properties and / or structure, and therefore, the linker moiety may, among other things, be a polysaccharide, polypeptide, fatty acid, phospholipid, or a chemical derivative thereof. Furthermore, it is intended that at least one of the molecules covalently bonded to another molecule via the linker, or both of such molecules, may be bonded to the linker via any chemical bond, such as a peptide bond, isopeptide bond, amide bond, or imine bond.

[0192] In certain embodiments, the polypeptide display library is formed by a complex comprising a polypeptide according to the first aspect of the present invention, which is directly linked to a nucleic acid encoding the polypeptide, and the polypeptide-nucleic acid complex is - A complex consisting of a library of polypeptides that bind to the nucleic acid that codes for it. -A complex consisting of a library polypeptide that binds to the nucleic acid encoding it, and additional proteins, peptides, and / or nucleic acids, -Polypeptide-nucleic acid complex, - Ribosome or part of a ribosome It is selected from the group consisting of the following.

[0193] As used herein, the term “complex” refers to any compound resulting from the covalent bonds of two or more individual compounds or molecules, as defined above. By definition, complexes are never found in nature.

[0194] The individual compounds covalently bonded to the complex of the second aspect of the present invention are a polypeptide from a library and the nucleic acid encoding it. Therefore, in certain embodiments, the complex of the second aspect of the present invention includes a polypeptide from a library directly attached to the encoding nucleic acid via chemical covalent bonds. In another particular embodiment, the complex of the second aspect of the present invention includes a polypeptide from a library attached to the encoding nucleic acid via one or more intervening parts, such as a linker, bridge, spacer, or one or more parts. In certain embodiments, they are attached via a linker.

[0195] As used herein, the term “ribosome” refers to a highly complex cellular mechanism essential for protein synthesis. Ribosomes link amino acids in an order specified by messenger RNA (mRNA) molecules. Ribosomes are composed of a specialized RNA known as ribosomal RNA (rRNA) and dozens (the exact number varies by species) different proteins. The ribosomal proteins and rRNA are arranged in two different ribosomal fragments of different sizes, commonly known as the large and small subunits of the ribosome.

[0196] As used herein, the term "ribosome portion" refers to an isolated portion of a ribosome, which may consist, for example, of an isolated large or small subunit of a ribosome. A ribosome portion may also refer to a ribosome containing only a portion of ribosomal protein or rRNA.

[0197] In another specific embodiment, the polypeptide-nucleic acid complex of the second aspect of the present invention contains only one polypeptide of the library on its outer surface. In another specific embodiment, the polypeptide-nucleic acid complex of the second aspect of the present invention contains at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 15, at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 150, at least 250, at least 500, at least 1×10 3 copies of the polypeptide of the library. In a specific embodiment, the copies have the same amino acid sequence. In another specific embodiment, the complex does not contain other polypeptides of the library.

[0198] In another specific embodiment, the polypeptide-nucleic acid complex of the second aspect of the present invention contains only one nucleic acid encoding the amino acid sequence of the polypeptide of the library contained in the complex. The polypeptide and the polypeptide sequence are those specified in the above embodiments.

[0199] In a specific embodiment, the complex of the second aspect of the present invention contains at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 15, at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100 copies of the nucleic acid encoding the amino acid sequence of the polypeptide of the library contained in the complex. The polypeptide and the polypeptide sequence are those specified in the above embodiments. In a specific embodiment, the nucleic acids have the same nucleotide sequence.

[0200] Indirect linkage consists of a genetic fusion between a library polypeptide and a nucleic acid by a microorganism containing both the polypeptide and the nucleic acid. The library polypeptide is contained on the outer surface of the microorganism. The nucleic acid is preferably contained inside the microorganism.

[0201] In certain embodiments, the microorganism contains only one polypeptide of the library on its outer surface. In certain embodiments, the microorganism contains at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 15, at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 150, at least 250, at least 500, at least 1×10 3 copies of the library polypeptide. In certain embodiments, the copies have the same amino acid sequence. In another certain embodiment, the microorganism does not contain other polypeptides of the library.

[0202] In another certain embodiment, the microorganism contains only one nucleic acid encoding the amino acid sequence of the library polypeptide contained in the microorganism. The polypeptide and the polypeptide sequence are those specified in the above embodiments.

[0203] In another specific embodiment, the microorganism contains nucleic acids encoding the amino acid sequence of a polypeptide in a library contained within the microorganism, with at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, at least ten, at least fifteen, at least twenty, at least twenty-five, at least thirty, at least thirty, at least thirty-five, at least forty, at least forty-five, at least fifty, at least sixty, at least seventy, at least eighty, at least ninety, and at least one hundred copies. The polypeptide and the polypeptide sequence are those identified in the embodiments described above. In a particular embodiment, the nucleic acids have the same nucleotide sequence.

[0204] In certain embodiments, a microorganism can be replicated. In certain embodiments, the replicated microorganism is an exact copy of the microorganism from which it originates. In another particular embodiment, the replication of the microorganism results in a microorganism having the same polypeptide and the same nucleic acid encoding it from a library contained in the microorganism from which it originates. Thus, as will be understood by those skilled in the art, the replication of a microorganism allows for the amplification of the polypeptide in the library and the amplification of the nucleic acid encoding it.

[0205] In certain embodiments, the microorganism is selected from the group consisting of phages, bacteriophages, viruses, bacteria, and yeasts. In preferred embodiments, the microorganism is a phage. In another preferred embodiment, the microorganism is a bacteriophage.

[0206] In certain embodiments, the bacteriophage is selected from the group consisting of enterobacteriaphage M13, T4 bacteriophage, T7 bacteriophage, or Escherichia λ virus.

[0207] In certain embodiments, all terms and embodiments described in the first aspect of the present invention are equally applicable to this aspect of the present invention.

[0208] III-polynucleotides, vectors, and host cells In a third embodiment, the present invention relates to a polynucleotide encoding a polypeptide according to a first aspect of the present invention, or a polynucleotide encoding a polypeptide of a polypeptide display library according to a second aspect of the present invention.

[0209] The term polynucleotide is defined in a second aspect of the present invention.

[0210] In certain embodiments, all terms and embodiments described in any of the above-described aspects of the present invention are equally applicable to a third aspect of the present invention.

[0211] In a fourth embodiment, the present invention relates to a vector comprising a polynucleotide according to a third aspect of the present invention.

[0212] As used herein, the term “vector” refers to a medium through which polynucleotides or DNA molecules can be manipulated or introduced into cells. A vector may be a linear or cyclic polynucleotide, or a larger polynucleotide, or any other type of construct such as viral genome DNA or RNA, virions, and other biological constructs that enable the manipulation or introduction of DNA into cells. The terms “recombinant vector” and “recombinant system” are understood to be interchangeable with the term “vector.” Since a vector may be a cloning vector suitable for growth and suitable for obtaining polynucleotides or suitable gene constructs or expression vectors in different heterologous organisms suitable for generating the polynucleotides of the present invention, those skilled in the art will understand that there are no limitations on the type of vector that can be used.Therefore, suitable vectors according to the present invention include prokaryotic expression vectors such as pET (pET14b, etc.), pUC18, pUC19, Bluescript and their derivatives, mp18, mp19, pBR322, pMB9, CoIE1, pCR1, RP4, phage, and shuttle vectors such as pSA3 and pAT28; 2-micron plasmid type vectors, embedded plasmids, yeast expression vectors such as YEP vectors and centromere plasmids; insect cell expression vectors such as pAC series vectors and pVL series vectors; plant expression vectors such as pIBI, pEarleyGate, pAVA, pCAMBIA, pGSA, pGWB, pMDC, pMY, and pORE series vectors; and viral vectors (adenovirus, adenovirus-related viruses, retrovirus-related viruses, lentivirus-related viruses) and pSilencer 4.1-CMV (Ambion), pcDNA3, pcDNA3.1 / hyg This includes nonviral vectors such as pHCMV / Zeo, pCR3.1, pEFl / His, pIND / GS, pRc / HCMV2, pSV40 / Zeo2, pTRACER-HCMV, pUB6 / V5-His, pVAX1, pZeoSV2, pCI, pSVL, and pKSV-10, pBPV-1, pML2d, and pTDT1, as well as expression vectors for higher eukaryotic cells.

[0213] The vector of the present invention can be used to transform, transfect, or infect cells that can be transformed, transfected, or infected by the vector. The cells may be prokaryotes or eukaryotes. For example, a vector into which a DNA sequence is introduced may be a plasmid or vector that, upon introduction into a host cell, integrates into the cell's genome and replicates together with the integrated chromosome (or multiple chromosomes). The vector can be obtained by conventional methods known to those skilled in the art (Sambrook et al., 2001, “Molecular cloning, to Laboratory Manual”, 2nd ed., Cold Spring Harbor Laboratory Press, NY Vol 1-3a).

[0214] Accordingly, in a fifth embodiment, the present invention relates to a host cell comprising a polynucleotide according to a third aspect of the present invention or a vector according to a fourth aspect of the present invention.

[0215] Transformed, transfected, or infected cells can be obtained by conventional methods known to those skilled in the art (Sambrook et al., 2001, above). In certain embodiments, the host cells are animal cells transfected or infected with a suitable vector.

[0216] Suitable host cells for containing the polynucleotide of the third aspect of the present invention or the vector of the fourth aspect of the present invention include, but are not limited to, mammalian, plant, insect, fungal, and bacterial cells. Suitable bacterial cells include, but are not limited to, Gram-positive bacterial cells such as species of the genera Bacillus, Streptomyces, Listeria, and Staphylococcus, as well as Gram-negative bacterial cells such as cells of the genera Escherichia, Salmonella, and Pseudomonas. Suitable fungal cells include yeast cells such as Saccharomyces cerevisiae, Pichia pastoris, and Hansenula polymorpha. Insect cells include, but are not limited to, those of Drosophila and Sf9. Plant cells include, in particular, those of crop plants such as cereals, medicinal plants, ornamental plants, or bulbous plants. Suitable mammalian cells in this invention include epithelial cell lines (human, sheep, pig, etc.), osteosarcoma cell lines (human, etc.), neuroblastoma cell lines (human, etc.), epithelial carcinoma (human, etc.), glial cells (mouse, etc.), hepatocyte cell lines (monkey, etc.), CHO (Chinese hamster ovary) cells, COS cells, BHK cells, HeLa cells, 911, AT1080, A549, 293 or PER.C6, NTERA-2 human ECC cells, mESC cells such as D3 cells, HS293 cells, BGV01 cells, SHEF1 cells, SHEF2 cells, HS181 cells, NIH3T3 cells, 293T cells, REH cells, MCF-7 cells, and human embryonic stem cells such as hMSC cells.

[0217] In certain embodiments, all terms and embodiments described in the first and second aspects of the present invention are equally applicable to the third aspect of the present invention. In another embodiment, all terms and embodiments described in the first, second and third aspects of the present invention are equally applicable to the fourth aspect of the present invention. In another specific embodiment, all terms and embodiments of the first, second, third and fourth aspects of the present invention are equally applicable to the fifth aspect of the present invention.

[0218] IV. The Invention Complex In a further aspect, the present invention relates to (i) a polypeptide comprising the G2 domain of nidogen-1 or a functionally equivalent variant thereof, and (ii) an agent of interest in a complex.

[0219] The polypeptide that forms part of the complex and comprises the G2 domain of nidogen-1 or a functionally equivalent variant thereof (specified in point (i) above) is also referred to as "the polypeptide of the complex of the sixth aspect of the present invention", "the polypeptide of the complex of the present invention" or "the polypeptide of the complex".

[0220] The G2 domain of nidogen-1 or a functionally equivalent variant thereof comprised in the polypeptide of the complex of the sixth aspect of the present invention is also referred to as "the first polypeptide of the complex", "the first polypeptide region of the complex", "the first polypeptide region" or "the first polypeptide region comprised in the polypeptide of the complex".

[0221] The term "complex" is defined in the second aspect of the present invention. The two components covalently bound to the complex of the sixth aspect of the present invention are the polypeptide of the complex and the agent of interest. Hereinafter, the complex of the sixth aspect of the present invention is also referred to as "the complex of the present invention".

[0222] The term "polypeptide" is defined in the first aspect of the present invention. The definition and embodiments of the term "amino acid residue" shown in the first aspect of the present invention also apply to this aspect of the present invention.

[0223] As used herein, the term “agent of interest” refers to any compound without restrictions on its chemical structure, provided that it can covalently bond with the polypeptide of the complex. In certain embodiments, the agent is a therapeutic agent. In other specific embodiments, the agent is a contrast agent. The terms “therapeutic agent” and “contrast agent” are defined in the following sections IV-E.1 and IV-E.2.

[0224] IV. The first polypeptide of the A-complex The complex of the present invention comprises a polypeptide containing the G2 domain of nidogen-1 or a functionally equivalent variant thereof.

[0225] As used herein, the term "nidogen-1" is defined in the above context relating to variants of the nidogen G2 domain and applies equally to the complex of the present invention.

[0226] As used herein, the term “G2 domain of Nidogen-1” refers to the G2 domain of the Nidogen-1 protein as defined above. In the wild-type Nidogen-1 sequence, the G2 domain is located alongside a short EGF-like domain. However, for the purposes of the present invention, the G2 domain of Nidogen-1 is formed by amino acids 430-667 of the amino acid sequence of the Nidogen-1 protein, identification number P14543-1 (SEQ ID NO: 62) in the Uniprot database (version dated July 7, 2009), and lacks an EGF-like domain at the N-terminus or C-terminus. In another embodiment, the G2 domain of Nidogen-1 lacks the first two amino acids of SEQ ID NO: 62 (SEQ ID NO: 63), and therefore corresponds to the region consisting of amino acids 432-667 of the amino acid sequence of the Nidogen-1 protein, identification number P14543-1 (SEQ ID NO: 72) in the Uniprot database (version dated July 7, 2009).

[0227] As used herein, the expression “functionally equivalent variant” refers to all peptides that exhibit a degree of sequence identity with the sequence of the G2 domain of nidogen-1, preferably the sequence of SEQ ID NO: 63, more preferably the sequence of SEQ ID NO: 62, and in which the function of the G2 domain of nidogen-1 is substantially maintained. The function of the G2 domain maintained in the complex of the sixth aspect of the present invention is preferably considered to be the tertiary structure of the domain, if it is not part of the complex. Thus, a functionally equivalent variant of the G2 domain preferably has the tertiary structure of the G2 domain of nidogen-1 substantially maintained, if it is not part of the complex. As will be understood by those skilled in the art, the tertiary structure of the maintained G2 domain is preferably the tertiary structure of the β-barrel domain of the G2 domain of nidogen-1, which is defined in the first aspect of the present invention.

[0228] As used herein, the expression “substantially maintained” is understood to mean that the structure of the G2 domain of nidogen-1, preferably the structure of the G2 β-barrel domain of nidogen-1, is maintained by at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, at least 99.9%, preferably 95%, more preferably 99%, and even more preferably 100%.

[0229] When expressed as a percentage, the maintenance of the tertiary structure of a protein domain is understood as the percentage of amino acids relative to the domain that maintain the relative position of the domain to the remaining amino acids within the domain's tertiary structure. Methods for determining the tertiary structure of a protein that enable the determination of the atomic coordinates of the protein are well known to those skilled in the art and include circular dichroism, X-ray crystallography, and protein NMR.

[0230] In certain embodiments, if a functionally equivalent variant of the G2 domain is incorporated into the complex of the sixth aspect of the present invention, a percentage of the structure of the nidogen-1 G2 domain is substantially maintained. In preferred embodiments, if the functionally equivalent variant of the G2 domain is not part of the complex, a percentage of the structure of the nidogen-1 G2 domain is substantially maintained. In preferred embodiments, the substantially maintained G2 domain structure is the structure of the β-barrel domain of the nidogen-1 G2 domain as described in the first aspect of the present invention. In certain embodiments, if the substantially maintained G2 domain is the first polypeptide of the complex, the substantially maintained G2 domain is the structure of the β-barrel domain of the nidogen-1 G2 domain.

[0231] In certain embodiments, the degree of sequence identity between the G2 domain of nidogen 1 having sequence number 62 and a functionally equivalent mutant is at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, or at least 99.9%. In another specific embodiment, the degree of sequence identity between the G2 domain of nidogen 1 having SEQ ID NO: 63 and a functionally equivalent mutant is at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, or at least 99.9%. A method for determining the degree of sequence identity between two amino acid sequences is provided in a first embodiment of the present invention.

[0232] When incorporated into the complex, the first polypeptide region of the complex does not need to maintain the cellular or physiological function of the nidogen G2 domain. Therefore, in certain embodiments, the first polypeptide region is the nidogen-1 G2 domain or a functionally equivalent variant that exhibits reduced physiological function when incorporated into the fusion protein of the present invention. In another embodiment, the first polypeptide region is the nidogen-1 G2 domain or a functionally equivalent variant that lacks the physiological function of nidogen-1 G2 or the physiological function of the β-barrel domain of the nidogen-1 G2 domain outside the complex. In a preferred embodiment, the first polypeptide region is a functionally equivalent variant of the nidogen G2 domain that exhibits reduced physiological function before being incorporated into the complex of the present invention, compared to the wild-type nidogen-1 G2 domain or compared to the wild-type β-barrel domain of the nidogen-1 G2 domain. More preferably, the first polypeptide region is a protein that, due to the presence of an inactivating mutation, does not possess the physiological function of either the G2 domain of nidogen-1 or the wild-type β-barrel domain of the G2 domain of nidogen-1 before being incorporated into the complex of the present invention.

[0233] As used herein, the terms “physiological function” or “cellular function” refer to the function of a peptide within a cell or organism. Therefore, when referring to the physiological function of the G2 domain of Nidogen-1, or the physiological function of the β-barrel domain of the G2 domain of Nidogen-1, the above expression is understood to mean the function of the domain when it is part of the Nidogen-1 protein. Thus, the above expression refers to the role of the domain in the biochemical pathways or molecular mechanisms in which the Nidogen-1 protein is involved in cells. Therefore, the above expression directly relates to the ability of the domain to interact with specific peptides or proteins on cells, outside cells, or on the outer surface of another cell. Thus, in certain embodiments, the above expression refers to the ability of the G2 domain, or the β-barrel domain of the G2 domain, to interact with its usual protein-binding partners. Non-limiting examples of such binding partners include collagen IV and perlecan. Therefore, in certain embodiments, the first polypeptide of the complex is a functionally equivalent variant of the G2 domain of nidogen-1, comprising a mutation that inhibits the interaction of the G2 domain of nidogen-1 or the β-barrel domain of the G2 domain of nidogen-1 with its usual binding partner, preferably with collagen IV and / or perlecan.

[0234] In another embodiment of the present invention, the first polypeptide of the complex is a functionally equivalent variant of the G2 domain of nidogen-1, which is inactive. In a particular embodiment, the first polypeptide of the complex is inactive once incorporated into the complex. In another particular embodiment, the first polypeptide of the complex is already inactive before being incorporated into the complex.

[0235] As used herein, the term “inactive” refers to polypeptides, proteins, protein fragments, or domains that lack physiological or biological activity, or the ability to specifically interact with other macromolecules for biological function, and protein fragments or domains that lack known therapeutic activity (e.g., antitumor activity). The inactive polypeptide portion of a complex is nonreactive and functions as a physical structure for binding with the agent of interest. Inactive polypeptides are intended to not contain motifs that themselves have intrinsic enzymatic, physiological, or biological activity, and to be immunoreactive, i.e., not to stimulate either adaptive or innate immune responses.

[0236] In general, the intrinsic activity of the first polypeptide of the complex is irrelevant to the purpose of this invention and is intended not to contribute to or interfere with the biological activity of the agent of interest.

[0237] In certain embodiments, the first polypeptide of the complex is a functionally equivalent variant of the G2 domain of nidogen-1, as is the polypeptide described in the first aspect of the present invention. Therefore, in certain embodiments, the polypeptide of the complex is a functionally equivalent variant of the G2 domain of nidogen-1, as is the polypeptide described in the first aspect of the present invention.

[0238] In another specific embodiment, the first polypeptide region has a sequence corresponding to amino acids 430-667 of the sequence of human nidogen-1 as defined by accession number P14543-1 in the Uniprot database (version dated July 7, 2009), i.e., sequence number 62. In another specific embodiment, the first polypeptide region has a sequence corresponding to amino acids 432-667 of the sequence of human nidogen-1 as defined by accession number P14543-1 in the Uniprot database (version dated July 7, 2009), i.e., sequence number 63.

[0239] In another specific embodiment, the first polypeptide of the complex is a functionally equivalent variant of the G2 domain of nidogen-1, comprising mutations in one or more amino acid residues at positions 459, 468, 639, 650, 543, 545, 449, 525, 561, 618, 619, 151, 604, 638, 641, 469, and 518 in the sequence numbering of human nidogen-1 as defined by accession number P14543-1 in the UniProt database (version dated July 7, 2009). Accordingly, in another specific embodiment, the polypeptide of the complex according to the sixth aspect of the present invention is a functionally equivalent variant of the G2 domain of nidogen-1, comprising mutations in one or more amino acid residues at positions 459, 468, 639, 650, 543, 545, 449, 525, 561, 618, 619, 151, 604, 638, 641, 469, and 518 in the sequence numbering of human nidogen-1 as defined by accession number P14543-1 in the UniProt database (version dated July 7, 2009).

[0240] As used herein, the term “mutation” refers to any modification or deletion of an amino acid in an amino acid sequence, or the insertion of at least one amino acid before or after an amino acid in an amino acid sequence. As will be understood by those skilled in the art, the location of an amino acid modification or deletion is referred to by the location of the modified or deleted amino acid in the amino acid sequence prior to the mutation. In a particular embodiment, if the mutation is an insertion, the location of the mutation is defined by referring to the N-terminal amino acid of the inserted amino acid. In another particular embodiment, if the mutation is an insertion, the location of the mutation is defined by referring to the C-terminal amino acid of the inserted amino acid. Therefore, as will be understood by those skilled in the art, mutations in one or more amino acid residues at positions 459, 468, 639, 650, 543, 545, 449, 525, 561, 618, 619, 151, 604, 638, 641, 469, and 518 of the protein sequence shown above refer to alterations or deletions of amino acids at those positions in the amino acid sequence of human nidogen-1 as defined by accession number P14543-1 in the UniProt database (version dated July 7, 2009). In a particular embodiment, it refers to the insertion of at least one amino acid at the C-terminus of one or more of the aforementioned amino acids. In another particular embodiment, it refers to the insertion of at least one amino acid at the N-terminus of one or more of the aforementioned amino acids.

[0241] In a preferred embodiment, the mutation is an amino acid modification. In a particular embodiment, the mutation at position 459 shown above is an H459A mutation. In another preferred embodiment, the mutation at position 468 shown above is an R468N mutation. In another preferred embodiment, the mutation at position 639 shown above is an F639S mutation. In another particular embodiment, the mutation at position 650 shown above is an R650A mutation.

[0242] Therefore, in a particular embodiment, one or more mutations at the 459, 468, 639, or 650 positions in the first polypeptide of the complex, as indicated above, are H459A mutations, R468N mutations, F639S mutations, or R650A mutations. In another particular embodiment, one or more mutations at the 459, 468, 639, or 650 positions in the polypeptide of the complex, as indicated above, are H459A mutations, R468N mutations, F639S mutations, or R650A mutations.

[0243] Suitable variants of the nidogen G2 domain that can be included in the first polypeptide region of the complex include, but are not limited to, NIDOmut2, NIDOmut3, NIDOmut3-V45T, NIDOmut3_V121Q, NIDOmut3-F157E, NIDOmut3-V215T, NIDOmut4, NIDOmut4_T215V, NIDOmut5, NIDOmut3-V176T, and NIDOmut3-I, defined as SEQ ID NOs. 64, 65, and 87-104, respectively. Examples of nidogen G2 domain mutants defined above in the context of the first aspect of the present invention include any of the nidogen G2 domain mutants defined above, including mutants carrying 200T, NIDOmut3-V236Y, NIDOmut3-L237T, NIDOmut3-S65I, NIDOmut3-R114I, NIDOmut3-C214S, NIDOmut3-S65I_R114I, NIDOmut5-S65I_R114I, NIDOmut3-S65I_R114I, and NIDOmut5-S65I_R114I.

[0244] IV-B. Second polypeptide region of the complex The polypeptide of the complex according to the sixth aspect of the present invention optionally includes a second polypeptide region that can specifically bind to a target of interest.

[0245] A second polypeptide region capable of specifically binding to a target of interest is also called the "second polypeptide of the complex," the "second polypeptide region of the complex," the "second polypeptide region," or the "second polypeptide region contained within the complex polypeptide."

[0246] The expression "specifically binds" is defined in a second embodiment of the present invention. In a particular embodiment, the second polypeptide of the complex is 10 -6 Less than M, 10 -7 Less than M, 10 -8 Less than M, 10 -9 Less than M, 10 -10 Less than M, 10 -11 Less than M, 10 -12 Less than M, 10 -13 Less than M, 10 -14 Less than M, 10 -15 Dissociation constant less than M (K D When a polypeptide binds to a target, it is considered to bind specifically to the target of interest. Methods for determining whether a polypeptide can bind to a target molecule, and for determining the dissociation constant of such binding, are provided in the definition of “specific binding” in a second aspect of the present invention.

[0247] In certain embodiments, the second polypeptide region in the complex of the present invention is a ligand for a cell receptor. The terms “cell receptor” or “cell surface receptor” refer to a cell-associated protein that binds to a “ligand.” Non-limiting examples of the agent of interest that is a cell receptor and a specific ligand for a particular receptor, or the cell types to which they bind, are shown in Table 2 below. [Table 2-1] [Table 2-2]

[0248] In a particular embodiment, the second polypeptide of the complex is selected from the group of ligands shown in Table 2.

[0249] In certain embodiments, the target of interest is a receptor on the surface of a cell, and the second polypeptide of the complex can facilitate the internalization of the complex in the cell. When referring to the second polypeptide of the complex, the expression "facilitates the internalization of the complex in the cell" refers to the polypeptide that binds to the cell surface receptor that undergoes endocytosis in response to polypeptide binding. This binding specificity allows the second polypeptide of the complex, and the rest of the complex containing it, to be delivered to cells, tissues, or organs expressing the receptor. In this way, the complex containing the polypeptide region is directed particularly to such cells when administered to an animal or when in contact with different types of cell populations in vitro.

[0250] As used herein, “internalization” refers to the process by which a molecule or a construct containing a molecule binds to a target element on the outer surface of the cell membrane, and the resulting complex is internalized by the cell. The resulting complex may dissociate in the cytoplasm after internalization. The target element, together with the molecule or construct, can then be localized to a specific cellular compartment. Preferably, the second polypeptide of the complex of the present invention promotes endosomal extrusion of the complex in addition to promoting internalization.

[0251] As used herein, the expression “promotes endosomal escape” refers to the ability of the complex’s second polypeptide to induce the release of the complex from the endosomal compartment after internalization by receptor-mediated endocytosis.

[0252] The ability of the complex of the present invention to be internalized by cells expressing the receptor to which the complex's second polypeptide binds can be conveniently determined by fluorescence if the complex polypeptide contains a fluorescent protein such as GFP. Such a fusion protein can be obtained by preparing a recombinant nucleic acid in which the nucleic acid encoding the complex polypeptide and the fluorescent protein are fused in frame and expressed in a suitable host cell or organism. The fusion protein can then be brought into contact with a culture of cells expressing the aforementioned receptor, or into contact with tissue expressing the receptor in vivo for an appropriate time, and then a fluorescence microscope can be used to determine whether the construct has permeated the cells. The presence of fluorescence in the cytoplasm can be further investigated by comparing fluorescence microscopy images obtained from the fluorescent protein with those obtained from known cytoplasmic stains.

[0253] A wide variety of uptake receptors and carriers with a greater number of receptor-specific ligands are known in this field.

[0254] A non-limiting example of receptors that may be targeted by the second polypeptide is provided above.

[0255] In certain embodiments, the second polypeptide of the complex is a polycationic peptide. As used herein, the terms “polycationic peptide” or “polycationic region” correspond to a polypeptide sequence comprising a plurality of positively charged amino acids. The polycationic peptide may be formed solely from positively charged amino acids, or it may contain other amino acids, provided that the net charge of the entire region is positive at pH 7.

[0256] It is well known in the art that amino acids and their corresponding amino acid residues have different properties depending on their side chains, and that they can be grouped according to these properties. Therefore, at physiological pH, five amino acids exhibit charge; arginine, histidine, and lysine are positively charged, while aspartic acid and glutamic acid are negatively charged. Those skilled in the art will understand that the polycationic peptides of the present invention correspond to polypeptides having a net positive charge of two or more under physiological pH conditions. Thus, the polycationic peptides of the present invention are not limited to the presence of one or more negatively charged amino acid residues, as long as there are always enough positively charged amino acid residues to result in a net positive charge of two or more.

[0257] Therefore, in one embodiment of the present invention, the polycationic peptide of the present invention has a sequence that can specifically interact with a cell surface receptor and promote the internalization of the complex into the cell, (ii) Arginine-rich sequence, (iii) GW-H1 peptide, (iv) CD44 ligand, (v) Peptides that can cross the blood-brain barrier, (vi) Cell permeable peptides, and (vii) Nucleoline-bound peptide It is selected from the group consisting of the following.

[0258] (i) A sequence that can specifically bind to a cell surface receptor and promote the internalization of the complex into the cell. As used herein, the term “sequence capable of specifically binding to a cell surface receptor to promote the internalization of the complex into the cell” means any sequence encoding a polypeptide capable of specifically binding to a target of interest, wherein the target is a cell surface receptor as defined above, and the polypeptide encoded by the sequence promotes the internalization of the complex into the cell as defined above.

[0259] The embodiments provided above for the second polypeptide of the complex also apply to the polycationic peptide.

[0260] Non-limiting examples of receptors that can be targeted by the polycationic peptide of the present invention, preferably a sequence that can specifically bind to the cell surface receptor described above, include any of the cell receptors provided above. In certain embodiments, the receptor is selected from the group consisting of CXCR4 receptor, angiotensin receptor, bombesin receptor, bradykinin receptor, calcitonin receptor, chemokine receptor, cholecystokinin receptor, corticotropin-releasing factor receptor, endothelin receptor, ephrin receptor, formyl peptide receptor, frizzled receptor, galanin receptor, growth hormone secretagogue receptor (ghrelin) receptor, kisspeptin receptor, melanocortin receptor, neuropeptide FF / neuropeptide AF receptor, neuropeptide S receptor, neuropeptide W / neuropeptide B receptor, neuropeptide Y receptor, neurotensin receptor, orexin receptor, peptide P518 receptor, somatostatin receptor, tachykinin receptor, Toll-like receptor, vasopressin and oxytocin receptor, and VEGF receptor.

[0261] In a preferred embodiment of the present invention, the polycationic peptide comprising a sequence that can specifically bind to a cell surface receptor and promote the internalization of the complex into the cell is a CXCR4 ligand.

[0262] As used herein, the term “CXCR4” refers to a G protein-coupled, seven-transmembrane chemokine receptor. Like other chemokine receptors, CXCR4 plays a crucial role in immune and inflammatory responses by mediating the directional migration and activation of leukocytes. CXCR4 is expressed or overexpressed in a variety of cancer cells and tissues, including those of the chest, prostate, ovaries, colon, colorectal, pancreas, kidney, and brain, as well as in non-Hodgkin lymphoma and chronic lymphocytic leukemia. The only known ligand for CXCR4 is stromal cell-derived factor-1 (SDF-1 or CXCL12). The interaction between CXCR4 and SDF-1 plays a crucial role in multiple stages of tumorigenesis, including tumor growth, invasion, angiogenesis, and metastasis.

[0263] The expression “specifically binds” is defined in a second embodiment of the present invention. As will be understood by those skilled in the art, as used herein, the expression “specifically binds to CXCR4” refers to the ability of the complex of the present invention to bind to CXCR4 or cells expressing CXCR4 more frequently, more rapidly, for a longer duration, and / or with higher affinity than other receptors or cells, without substantially binding to other molecules.

[0264] The binding affinity is measured by one of the methods provided in the definition of “specifically binding” in a second aspect of the present invention, preferably by the method described by Tamamura et al., the oil cushion method [see Hesselgesset et al, 1998, J.Immunol., 160:877-883]. The method involves contacting a cell line transfected with the peptide and CXCR4 (e.g., CHO cells) with a labeled CXCR4 ligand (e.g., 125I-SDF-1α) and measuring the percentage of inhibition of the target peptide against the binding of the labeled CXCR4 ligand.

[0265] Specific binding occurs, for example, when CXCR4 has multiple binding sites for ligands and low-affinity ligands may be useful for targeting, for example, at least about 10 -4 Specific binding can also be demonstrated by low-affinity targeting agents having M's Kd. Specific binding can also be demonstrated by high-affinity ligands, e.g., at least about 10 -7 M, at least about 10 -8 M, at least about 10 -9 M, at least about 10 -10 M may be represented by a ligand having Kd, or at least about 10 -11 M or 10 -12 It may have a Kd of M or higher. Both low-affinity and high-affinity targeted ligands are useful for incorporation into the complex of the present invention.

[0266] The ability of the present invention to be internalized by cells expressing CXCR4 can be determined by fluorescence methods, including the use of a fluorescent protein such as GFP, as shown above for any second polypeptide of the complex that binds to any cell receptor. More specifically, the complex internalized by cells expressing CXCR4 can be obtained by preparing recombinant nucleic acids that are expressed in a suitable host cell or organism, by in-frame fusion of a nucleic acid encoding a polycationic peptide and a nucleic acid encoding a fluorescent protein. The fusion protein can then be brought into contact with a culture of cells expressing CXCR4 or with tissue expressing CXCR4 in vivo for an appropriate amount of time, and then a fluorescence microscope can be used to determine whether the construct has permeated the cells. The presence of fluorescence in the cytoplasm can be further investigated by comparing fluorescence microscopy images obtained from the fluorescent protein with those obtained from known cytoplasmic stains.

[0267] In a more preferred embodiment of the present invention, the CXCR4 ligand is selected from the group consisting of functionally equivalent variants thereof, such as RRWCYRKCYKGYCYRKCR (SEQ ID NO: 25), V1 peptide (SEQ ID NO: 26), CXCL12 peptide (SEQ ID NO: 27), vCCL2 peptide (SEQ ID NO: 28), EPI-X4 sequence (SEQ ID NO: 29), or the peptide of SEQ ID NO: 132.

[0268] The sequence RRWCYRKCYKGYCYRKCR (SEQ ID NO: 25) is the amino acid sequence of the T22 peptide. This peptide corresponds to a peptide derived from the protein polyfemusin II (extracted from blood cell fragments from the horseshoe crab (Limulus polyphemus)). vCCL2 corresponds to viral macrophage inflammatory protein-II, a homolog of the human chemokine CCL2 encoded by human herpesvirus 8. The V1 peptide corresponds to residues 1-21 of the N-terminal portion of vCCL2. CXCL12, also known as stromal cell-derived factor 1 (SDF1), CXC motif chemokine 12, is a member of the chemokine family that functions as a pro-inflammatory mediator. As shown in Liang, X. 2008. Chem. Biol. Drug. Des. 72:91-110, all four peptides are known to interact with the CXCR4 receptor.

[0269] EPI-X4 corresponds to residues 408-423 of human serum albumin (HSA). It has also been described as binding to the CXCR4 receptor (Zirafi et al., 2015, Cell reports, 11:1-11). In one embodiment, an optimized tandem version of EPI-X4 (SEQ ID NO: 132) with higher receptor affinity and serum stability is used.

[0270] In one embodiment, the polycationic peptide is -The T140 peptide has the sequence RRX1CYRKX2PYRX3CR (SEQ ID NO: 41), where X1 is L-3-(2-naphthyl)alanine, X2 is D-Lys, and X3 is L-citrulline. -TN14003 peptide having the sequence RRX1CYX2KX3PYRX4CR (SEQ ID NO: 42), where X1 is L-3-(2-naphthyl)alanine, X2 is L-citrulline, X3 is dLys and X4 is L-citrulline. -The TC14012 peptide has the sequence RRX1CYEKX2PYRX3CR (SEQ ID NO: 43), where X1 is L-3-(2-naphthyl)alanine, X2 is D-citrulline, and X3 is L-citrulline. -TE14011 peptide having the sequence RRX1CYX2KX3PYRX4CR (SEQ ID NO: 44), where X1 is L-3-(2-naphthyl)alanine, X2 is L-citrulline, X3 is D-Glu and X4 is L-citrulline, and - The TZ14011 peptide (known as Ac-TZ14011) has the sequence RRX1CYX2KX3PYRX4CR (SEQ ID NO: 45), where X1 is L-3-(2-naphthyl)alanine, X2 is L-citrulline, X3 is D-Lys, and X4 is L-citrulline or a variant thereof, and the N-terminal arginine residue is acetylated. It is selected from the group consisting of the following.

[0271] The terms “functional variant” and “functionally equivalent variant” are interchangeable and, as used herein, are understood as all peptides derived from T22, V1, CXCL12, vCCL2, and / or EPI-X4 peptides by modification, insertion, and / or deletion of one or more amino acids, provided that the function of binding to CXCR4 and internalizing the complex is substantially maintained.

[0272] In one embodiment, functionally equivalent variants of a cationic polypeptide exhibit some degree of identity with respect to human T22, V1, CXCL12, vCCL2, and / or EPI-X4 peptides, exhibiting at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with respect to their respective SEQ ID NOs. A method for determining the degree of identity between two amino acid sequences is provided in a first embodiment of the present invention. The cationic polypeptides of the present invention may include post-translational modifications such as glycosylation, acetylation, isoprenylation, myristoylation, and proteolytic processing.

[0273] Alternatively, a suitable functional variant of a cationic polypeptide contains, at one or more positions, an amino acid that is a conserved substitution of an amino acid present in the T22, V1, CXCL12, vCCL2, and / or EPI-X4 peptides. A "conservative amino acid substitution" is the substitution of one amino acid with another amino acid having similar structural and / or chemical properties. For example, the following six groups each contain amino acids that are conserved substitutions of each other: 1) alanine (A), serine (S), threonine (T); 2) aspartic acid (D), glutamic acid (E); 3) asparagine (N), glutamine (Q); 4) arginine (R), lysine (K); 5) isoleucine (I), leucine (L), methionine (M), valine (V); and 6) phenylalanine (F), tyrosine (Y), tryptophan (W). The selection of such conservative amino acid substitutions is within the scope of the skill of those skilled in the art and is described, for example, in Dordo et al. et al. [J. Mol. Biol, 1999, 217;721-739] and Taylor et al. [J. Theor. Biol., 1986, 119:205-218].

[0274] A suitable assay for determining whether a given peptide can be considered a functionally equivalent variant is, for example, the following assay: a variant of the putative T22, V1, CXCL12, vCCL2, or EPI-X4 peptide is fused in-frame with a marker polypeptide (e.g., a fluorescent protein). Such a fusion protein can be obtained by preparing recombinant nucleic acids in which the nucleic acid encoding the peptide and the nucleic acid encoding the fluorescent protein are fused in-frame and expressed in a suitable host cell or organism. The fusion protein can then be exposed to a culture of cells CXCR4 (e.g., HeLa cells) for an appropriate time, and then a fluorescence microscope can be used to determine whether the construct has permeated the cells. If the peptide is a functionally equivalent variant of the corresponding peptide, the marker protein will be internalized, and the presence of fluorescence in the cell cytoplasm will be visible. Furthermore, the performance of the functionally equivalent variant can be analyzed by comparing the fluorescence microscope image obtained from the fluorescent protein with the image obtained with a known cytoplasmic stain (e.g., DAPI).

[0275] (ii) Arginine-rich sequences As mentioned above, arginine amino acids and their residues exhibit a positive charge at physiological pH. It will be understood that an "arginine-rich sequence" refers to a polypeptide sequence containing multiple arginine residues. Therefore, a polypeptide sequence may contain 33%, preferably 40%, preferably 45%, preferably 50%, preferably 55%, preferably 60%, preferably 65%, preferably 70%, preferably 75%, preferably 80%, preferably 85%, more preferably 90%, more preferably 95%, even more preferably 99%, and even more preferably 100% of the amino acid residues of its complete sequence as arginine residues. It will be understood that whenever an arginine-rich sequence contains less than 100% of its sequence as arginine residues, these residues do not all need to be adjacent or contiguous to one another.

[0276] Those skilled in the art will recognize that a polypeptide having one or more arginine residues is a polycationic peptide insofar as the total positive charge of the polypeptide at physiological pH is 2 or greater, not only from the positive charge of the arginine residues but also from the positive charges of other positively charged amino acids.

[0277] In embodiments of the present invention, the polycationic peptide of the present invention has an arginine-rich sequence.

[0278] In a preferred embodiment of the present invention, the arginine-rich sequence of the polycationic peptide of the present invention is selected from the group consisting of SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, and SEQ ID NO: 33.

[0279] (iii) GW-H1 peptide The GW-H1 peptide has been previously described by Chen et al. [Chen, YL.S. et al. 2012. Peptides, 36:257-265]. While initially selected as an antimicrobial peptide, the GW-H1 peptide is also characterized by its ability to bind to the cell membrane, internalize into the cytoplasm, and translocate to the nucleus of eukaryotic cells. Once inside the cell, GW-H1 can induce apoptosis. It has been proposed that GW-H1 exerts its cytolytic activity by folding into an amphipathic helix [Chen et al., as mentioned above]. Therefore, it is thought that this peptide exerts its cytolytic effect through two consecutive events: binding to the cell membrane and subsequent translocation.

[0280] In a preferred embodiment of the present invention, the polycationic peptide of the present invention is a GW-H1 peptide having SEQ ID NO: 46.

[0281] (iv) CD44 ligand CD44 is a cell surface transmembrane glycoprotein involved in cell-cell and cell-matrix interactions, as well as cell adhesion and migration. CD44 is involved in inflammation and diseases such as cancer [Bajorath, J. 2000. Proteins. 39:103-111]. Many isoforms are known, and they are expressed and glycosylated in a cell-specific manner.

[0282] Therefore, a "CD44 ligand" is a molecule that can bind to CD44. CD44 is the primary surface receptor for hyaluronic acid, a component of the extracellular matrix, and also possesses other ligands such as chondroitin sulfate, the heparin-binding domain of fibronectin, osteopontin, cerglycine, collagen, and laminin. Furthermore, CD44 can also interact with metalloproteinases and selectins.

[0283] In embodiments of the present invention, the polycationic peptide of the present invention is a CD44 ligand. In preferred embodiments of the present invention, the CD44 ligand is selected from the group consisting of A5G27 (SEQ ID NO: 34) and FNI / II / V (SEQ ID NO: 35).

[0284] Peptide FNI / II / V corresponds to the HBFN fragment V of fibronectin. Peptide A5G27 corresponds to the α5 chain peptide of laminin [Pesarrodona, M. et al. 2014. Int. J. of Pharmaceutics. 473:286-295].

[0285] (v) Peptides that can cross the blood-brain barrier It is well known in this field that one major obstacle to the development of therapeutic approaches for brain disorders is the blood-brain barrier (BBB). The brain is protected from potentially toxic substances by the presence of two barrier systems: the blood-brain barrier (BBB) ​​and the blood-cerebrospinal fluid barrier (BCSFB). The BBB is considered to be the primary pathway for serum ligand uptake, given that its surface area is approximately 5000 times that of the BCSFB. The brain endothelium that constitutes the BBB presents a major obstacle to the use of potential drugs for many CNS disorders. In principle, only small lipophilic molecules can pass through the BBB, i.e., move from the circulating systemic blood to the brain. Many drugs that are larger in size or more hydrophobic have shown promising results in animal studies for treating CNS disorders.

[0286] Therefore, a "peptide capable of crossing the blood-brain barrier" is a peptide that can transport itself, and any molecule, preferably a protein, to which it binds, from the bloodstream into the central nervous system.

[0287] The peptide β-casomorphin-5 was reported to be able to overcome the blood-brain barrier (BBB) ​​in 1983 [Ermisch, A. et al. 1983. J. of Neurochemistry. 41:1229-1233]. Since then, many other peptides with BBB-permeable properties have been identified, characterized, and cataloged, and a comprehensive database was established in 2012, as reported by Van Dorpe et al. [Van Dorpe, S. et al. 2012. Brain Struct. Funct. 217:687-718]. Most of the peptides listed in the aforementioned database are suitable for the complex of the present invention.

[0288] In embodiments of the present invention, the polycationic peptide of the present invention is a peptide that can cross the blood-brain barrier. In preferred embodiments of the present invention, the peptide that can cross the blood-brain barrier is selected from the group consisting of Seq-1-7 (SEQ ID NO: 36), Seq-1-8 (SEQ ID NO: 37), and Angiopep-2-7 (SEQ ID NO: 38).

[0289] (vi) Cell-permeable peptides (CPPs) The term “cell-permeable peptide” (CPP) refers to peptides, typically about 5–60 amino acid residues in length, that can facilitate the cellular uptake of molecular cargo, particularly the proteins they contain. A protein may exhibit one or more CPPs. CPPs can also be characterized as being able to facilitate the movement or transposition of molecular cargo across / through one or more lipid bilayers, cell membranes, organelle membranes, vesicle membranes, or cell walls. In this specification, CPPs are polycationic.

[0290] Examples of CPPs useful in this specification, and a further general description of CPPs, are described in Schmidt et al. [2010. FEBS Lett. 584:1806-1813], Holm et al. [2006. Nature Protocols 1:1001-1005], Yandek et al. [2007. Biophys. J. 92:2434-2444], Morris et al. [2001. Nat. Biotechnol. 19:1173-1176] and U.S. Patent Application Publication No. 2014 / 0068797. CPPs facilitate the direct transport of CPP-containing proteins across the lipid bilayer, independent of transporters and receptors and without the involvement of other cellular components.

[0291] (vii) Nucleoline-bound peptide Therefore, a "nucleoline-binding peptide" is a peptide that can bind to intracellular nucleoline proteins, preferably to the cell surface expression fraction of nucleolins.

[0292] In embodiments of the present invention, the polycationic peptide of the present invention is a nucleoline-bound peptide.

[0293] The international patent application publication WO2011 / 031477A2 provides numerous examples of nucleoline-conjugated peptides suitable for use in the complex of the present invention.

[0294] In a preferred embodiment of the present invention, the nucleolin-bound peptide of the present invention is a peptide having the sequence of SEQ ID NO: 47 or a peptide having the sequence of SEQ ID NO: 48.

[0295] Third polypeptide region of the IV-C complex In certain embodiments, the polypeptide of the complex according to the sixth aspect of the present invention further comprises a third polypeptide region which is a region rich in positively charged amino acids.

[0296] The region rich in positively charged amino acids, and the third polypeptide region included in the complex polypeptide, is also called the "third polypeptide of the complex," the "third polypeptide region of the complex," the "third polypeptide region," or the "third polypeptide region included in the complex polypeptide." As will be understood by those skilled in the art, the expressions "third polypeptide of the complex," the "third polypeptide region of the complex," or the "third polypeptide region included in the complex polypeptide" are interchangeable with "region rich in positively charged amino acids."

[0297] As used herein, the terms “positively charged amino acids,” “regions rich in positively charged amino acids,” or “third polypeptide region which is a region rich in positively charged amino acids” refer to the polypeptide sequence of a third polypeptide of a complex, distinct from the second polypeptide region of the complex, characterized by containing multiple positively charged amino acids. Furthermore, the region rich in positively charged amino acids may be formed solely by positively charged amino acids, or it may contain other amino acids under the condition that the net charge of the entire region is positive at pH 7. Accordingly, the sequence of a region rich in positively charged amino acids may contain 33%, preferably 40%, preferably 45%, preferably 50%, preferably 55%, preferably 60%, preferably 65%, preferably 70%, preferably 75%, preferably 80%, preferably 85%, more preferably 90%, more preferably 95%, even more preferably 99%, and even more preferably 100% of the amino acid residues of its complete sequence as positively charged amino acid residues.

[0298] The positively charged amino acid-rich region may contain only one type of positively charged amino acid, or it may contain multiple types of positively charged amino acids. In one embodiment, the positively charged amino acid-rich region is a polyhistidine region. In one embodiment, the positively charged amino acid-rich region is a polyarginine region. In one embodiment, the positively charged amino acid-rich region is a polyhistidine region. In one embodiment, the positively charged amino acid-rich region includes lysine and arginine residues. In one embodiment, the positively charged amino acid-rich region includes lysine and histidine residues. In one embodiment, the positively charged amino acid-rich region includes arginine and histidine residues. In one embodiment, the positively charged amino acid-rich region includes lysine, arginine, and histidine residues.

[0299] In some embodiments, the positively charged amino acid-rich region comprises at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, at least ten, at least eleven, at least twelve, at least thirteen, at least fourteen, or at least fifteen positively charged amino acid residues, the positively charged amino acids may be histidine, lysine, arginine, or a combination thereof.

[0300] In some embodiments, the positively charged amino acid-rich region contains fewer than 100, 90, 80, 70, 60, 50, 40, 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, or even fewer positively charged amino acid residues, where the positively charged amino acids may be histidine, lysine, arginine, or a combination thereof.

[0301] In some embodiments, the positively charged amino acid-rich region contains 2 to 50 amino acids, 2 to 40 amino acids, 2 to 30 amino acids, 2 to 25 amino acids, 2 to 20 amino acids, 2 to 10 amino acids, or 2 to 8 amino acids.

[0302] In some embodiments, the positively charged amino acid-rich region contains 3 to 50 amino acids, 3 to 40 amino acids, 3 to 30 amino acids, 3 to 25 amino acids, 3 to 20 amino acids, 3 to 10 amino acids, or 3 to 8 amino acids. In some embodiments, the positively charged amino acid-rich region contains 4 to 50 amino acids, 4 to 40 amino acids, 4 to 30 amino acids, 4 to 25 amino acids, 4 to 20 amino acids, 4 to 10 amino acids, or 4 to 8 amino acids. In some embodiments, the positively charged amino acid-rich region contains 5 to 50 amino acids, 5 to 40 amino acids, 5 to 30 amino acids, 5 to 25 amino acids, 5 to 20 amino acids, 5 to 5 amino acids, 5 to 10 amino acids, or 5 to 8 amino acids.

[0303] In embodiments of the present invention, the positively charged amino acid-rich region of the complex is a polyhistidine region. In preferred embodiments of the present invention, the polyhistidine region contains 2 to 10, preferably 6, adjacent histidine residues.

[0304] In embodiments of the present invention, the positively charged amino acid-rich region of the complex is a polyarginine region. In preferred embodiments of the present invention, the polyarginine region contains 2 to 10, preferably 6, adjacent arginine residues.

[0305] In embodiments of the present invention, the positively charged amino acid-rich region of the fusion protein of the present invention is a polylysine region. In preferred embodiments of the present invention, the polylysine region contains 2 to 10, preferably 6, adjacent polylysine residues.

[0306] In certain embodiments, the positively charged peptide sequences are RKRKRK (SEQ ID NO: 77), RRRRRR (SEQ ID NO: 78), KKKKKK (SEQ ID NO: 79), HHHHHH (SEQ ID NO: 80), RHRHRH (SEQ ID NO: 81), RKRKRKRK (SEQ ID NO: 82), RKRHRK (SEQ ID NO: 83), RKRHRH (SEQ ID NO: 84), RHRHRH (SEQ ID NO: 85), or RKRKRKR (SEQ ID NO: 86).

[0307] IV-D. Relative positions of polypeptide elements and linking elements in the complex. The different elements of the polypeptide of the complex of the present invention (the first, second, and third polypeptides of the complex) can be positioned at any relative position, as long as the second polypeptide, preferably a polycationic peptide, and the third polypeptide (or a region rich in positively charged amino acids) function at any position in the complex, and the first polypeptide continues to function completely or partially (i.e., the structure of the G2 domain of nidogen-1 is substantially maintained).

[0308] As used herein, the terms “N-terminal end,” “N-terminus,” and “amino-terminus” of a polypeptide are not distinguished. Similarly, the terms “C-terminal end,” “C-terminus,” and “carboxy-terminus” are considered equivalent. These terms are common usage to those skilled in the art with respect to the free amino acid portions at the ends of polypeptide chains composed of proteins.

[0309] Therefore, in embodiments of the present invention, the second polypeptide of the complex is located at the N-terminus of the polypeptide of the complex, and the positively charged amino acid-rich region of the polypeptide (i.e., the third polypeptide of the complex) is located at the C-terminus of the polypeptide. In another embodiment of the present invention, the positively charged amino acid-rich region of the polypeptide of the complex is located at the N-terminus of the polypeptide, and the second polypeptide region is located at the C-terminus of the polypeptide. In yet another embodiment of the present invention, the first polypeptide region may be located at either the C-terminus or N-terminus of the polypeptide of the complex, the second polypeptide may be located in the middle of the polypeptide with the positively charged amino acid-rich region at the opposite end of the polypeptide from the first polypeptide region, or the positively charged amino acid-rich region may be located in the center of the polypeptide, and the second polypeptide may be located at the opposite end of the polypeptide from the first polypeptide region.

[0310] Therefore, the relative order of the polypeptide elements of the complex of the present invention is: • N-second polypeptide region - first polypeptide region - positively charged amino acid-rich region - C; • N-positively charged amino acid-rich region - first polypeptide region - second polypeptide region - C; • N-second polypeptide region - region rich in positively charged amino acids - first polypeptide region - C; • N-positively charged amino acid-rich region - second polypeptide region - first polypeptide region - C; • N-1 polypeptide region - 2 polypeptide region - positively charged amino acid-rich region - C; or • N-1 polypeptide region - region rich in positively charged amino acids - 2 polypeptide region - C It is possible.

[0311] In a particular embodiment, the order of elements in the polypeptide of the composite of the sixth aspect of the present invention is one of those shown above.

[0312] In a preferred embodiment, the order of elements in the polypeptide of the complex of the sixth aspect of the present invention is N-second polypeptide region-first polypeptide region-positively charged amino acid-rich region-C.

[0313] The terms "N-terminus" and "C-terminus" do not mean that the components must be directly joined from end to end, but rather that the components maintain their relative positions without the presence of additional elements such as linkers / spacers inserted at the ends of any of the components or between the components.

[0314] Accordingly, the polypeptide of the complex of the present invention comprises the elements described above ((1) a second polypeptide region, (2) a first polypeptide region, and (3) a region rich in positively charged amino acids), which may be linked terminally, and may also include a "linker" or "spacer" which is one or more arbitrary peptides or polypeptides inserted between them and preferably linked by peptide bonds.

[0315] According to the present invention, the amino acid sequence of a spacer or linker acts as a hinge region between components (1) and (2), and between components (2) and (3), and the presence of the peptide spacer or linker does not alter the function of any of the components (1), (2), and (3). In this sense, a preferred intermediate amino acid sequence of the present invention is a hinge region characterized by structural ductility that enables this movement. In certain embodiments, the intermediate amino acid sequence is a flexible linker. The effect of the linker region is to provide space between components (1) and (2), and between (2) and (3). Thus, the secondary and tertiary structures of components (1), (2), or (3) are ensured not to be affected by the presence of any of the others. The spacer is of polypeptide nature. The linker peptide preferably contains at least 2 amino acids, at least 3 amino acids, at least 5 amino acids, at least 10 amino acids, at least 15 amino acids, at least 20 amino acids, at least 30 amino acids, at least 40 amino acids, at least 50 amino acids, at least 60 amino acids, at least 70 amino acids, at least 80 amino acids, at least 90 amino acids, or about 100 amino acids.

[0316] The spacer or linker can be bonded by covalent bonds, preferably by peptide bonds, to two adjacent components of the polypeptide complex of the present invention; also preferably the spacer is intrinsically functional and / or not prone to proteolytic cleavage and / or does not contain cysteine ​​residues. Similarly, the three-dimensional structure of the spacer is preferably linear or substantially linear.

[0317] Preferred examples of spacer or linker peptides include those used to bind proteins without substantially reducing the function of the binding peptide, or substantially reducing the function of at least one of the binding peptides. More preferably, the spacer or linker used to bind the peptides includes a coiled-coil structure.

[0318] Preferred linker peptides include two or more amino acids selected from the group consisting of glycine, serine, alanine, and threonine. A preferred example of a flexible linker is the polyglycine linker. Possible examples of linker / spacer sequences include GGSSRSS (SEQ ID NO: 39), GGSRSSS (SEQ ID NO: 76), SGGTSGSTSGTGST (SEQ ID NO: 49), AGSSTGSSTGPGSTT (SEQ ID NO: 50), or GGSGAP (SEQ ID NO: 51). These sequences are used to link designed coiled coils with other protein domains [Muller, KM, Arndt, KM and Alber, T., Meth. Enzymology, 2000, 328: 261-281]. Further non-limiting examples of suitable linkers include the amino acid sequence GGGVEGGG (SEQ ID NO: 52), the 10-amino acid sequence of the upper hinge region of mouse IgG3 used in the production of dimerized antibodies by coiled coils (PKPSTPPGSS, SEQ ID NO: 53) [Pack, P. and Pluckthun, A., 1992, Biochemistry 31:1579-1584], the peptide of sequence APAETKAEPMT (SEQ ID NO: 54), the peptide of sequence GAP, the peptide of sequence AAA, and the peptide of sequence AAALE (SEQ ID NO: 55). In another preferred embodiment, the linker is GGSSRSS (SEQ ID NO: 39).

[0319] Alternatively, the polypeptide components of the complex of the present invention may be linked by peptides whose sequences contain protease cleavage targets and enable the separation of any component. Suitable protease cleavage sites for incorporation into the polypeptide of the complex of the present invention include enterokinase (cleavage site DDDDK, SEQ ID NO: 56), factor Xa (cleavage site IEDGR, SEQ ID NO: 57), thrombin (cleavage site LVPRGS, SEQ ID NO: 58), TEV protease (cleavage site ENLYFQG, SEQ ID NO: 59), prescision protease (cleavage site LEVLFQGP, SEQ ID NO: 60), intein, and the like.

[0320] In a preferred embodiment, the N-terminal polypeptide is linked to the intermediate polypeptide of the complex polypeptide by a linker, preferably selected from any of the above examples of linkers. In another preferred embodiment, the intermediate polypeptide is linked to the C-terminal polypeptide of the complex polypeptide by a linker, preferably selected from any of the above examples of linkers. Thus, in one embodiment of the present invention, the second polypeptide is linked to the first polypeptide region via a linker. In another embodiment of the present invention, the first polypeptide region is linked to a positively charged amino acid-rich region via a linker. In yet another embodiment of the present invention, the second polypeptide is linked to the first polypeptide region via a linker, and the first polypeptide region is also linked to a positively charged amino acid-rich region via a linker.

[0321] Therefore, in certain embodiments, the second polypeptide region is linked to the first polypeptide region via the first peptide linker, and / or the first polypeptide region is linked to the third polypeptide region via the second peptide linker. In certain embodiments, the first peptide linker includes the GGSSRSS sequence (SEQ ID NO: 39), GGSSRSS (SEQ ID NO: 76), or GGGNS sequence (SEQ ID NO: 40). In preferred embodiments, the first peptide linker includes the GGSSRSS sequence (SEQ ID NO: 39). In another preferred embodiment, the first peptide linker includes GGSSRSS (SEQ ID NO: 39).

[0322] As will be understood by those skilled in the art, the linker connecting the second polypeptide and the first polypeptide region, and the linker connecting the first polypeptide region and the positively charged amino acid-rich region, may include the same or different sequences, within the given limitation that the presence and / or sequence of the linkers does not result in any functional change (but not limited to, for example, alteration of the secondary or tertiary structure of the polypeptide of the complex, or the formation of disulfide bonds) of the second polypeptide, the first polypeptide region, and / or the positively charged amino acid-rich region.

[0323] The above considerations regarding the relative positions of the elements of the complex polypeptide from the N-terminus to the C-terminus also apply when a linker is present between them, regardless of the number of elements or which elements are positioned in between. Therefore, the possible combinations and relative orders of the elements are as follows (the above numbering of the elements is retained: (1) second polypeptide, (2) first polypeptide, (3) positively charged amino acid-rich region): ·N-(1)-(2)-(3)-C N-(1)-Linker-(2)-(3)-C N-(1)-(2)-Linker-(3)-C · N-(1)-linker-(2)-linker-(3)-C ·N-(3)-(2)-(1)-C N-(3)-Linker-(2)-(1)-C N-(3)-(2)-Linker-(1)-C · N-(3)-linker-(2)-linker-(1)-C N-(2)-(1)-(3)-C N-(2)-Linker-(1)-(3)-C N-(2)-(1)-Linker-(3)-C · N-(2)-linker-(1)-linker-(3)-C N-(2)-(3)-(1)-C N-(2)-Linker-(3)-(1)-C N-(2)-(3)-Linker-(1)-C · N-(2)-linker-(3)-linker-(1)-C N-(1)-(3)-(2)-C N-(1)-(3)-Linker-(2)-C N-(1)-Linker-(3)-(2)-C N-(1)-linker-(3)-linker-(2)-C ·N-(3)-(1)-(2)-C N-(3)-Linker-(1)-(2)-C N-(3)-(1)-Linker-(2)-C N-(3)-linker-(1)-linker-(2)-C.

[0324] In a preferred embodiment of the present invention, the polypeptide linker of the complex of the present invention comprises the sequence GGSSRSS (SEQ ID NO: 39) or the sequence GGGNS (SEQ ID NO: 40).

[0325] In a preferred embodiment, the N-terminal polypeptide is linked to the intermediate polypeptide of the complex via a protease cleavage site, preferably a protease cleavage site selected from any of the examples of protease cleavage sites provided above. In another preferred embodiment, the intermediate polypeptide is linked to the C-terminal polypeptide of the complex via a protease cleavage site, preferably a protease cleavage site from any of the examples of cleavage sites provided above.

[0326] In another embodiment, the second polypeptide is linked to the first polypeptide region via a protease cleavage site. In yet another embodiment of the present invention, the first polypeptide region is linked to a positively charged amino acid-rich region via a protease cleavage site. In yet another embodiment of the present invention, the second polypeptide is linked to the first polypeptide region via a protease cleavage site, and the first polypeptide region is also linked to a positively charged amino acid-rich region via a protease cleavage site.

[0327] As will be understood by those skilled in the art, the protease cleavage sites connecting the second polypeptide and the first polypeptide region, and the protease cleavage sites connecting the first polypeptide region and the positively charged amino acid-rich region, may include the same or different sequences, within the above-mentioned limitation that the presence and / or sequence of the protease cleavage sites do not result in any functional changes to the second polypeptide, the first polypeptide region, and / or the positively charged amino acid-rich region (for example, alteration of the secondary or tertiary structure of the polypeptide of the complex, or the formation of disulfide bonds).

[0328] The above considerations regarding the relative positions of the elements of the complex polypeptide from the N-terminus to the C-terminus also apply when protease cleavage sites are present between them, regardless of their number or which elements are positioned in between. Therefore, the possible combinations and relative orders of the elements are as follows (the above numbering of the elements is retained: (1) second polypeptide, (2) first polypeptide, (3) positively charged amino acid-rich region): N-(1)-(2)-(3)-C N-(1)-Protease cleavage site-(2)-(3)-C N-(1)-(2)-Protease cleavage site-(3)-C N-(1)-Protease cleavage site-(2)-Protease cleavage site-(3)-C ·N-(3)-(2)-(1)-C N-(3)-Protease cleavage site-(2)-(1)-C N-(3)-(2)-Protease cleavage site-(1)-C ·N-(3)-Protease cleavage site-(2)-Protease cleavage site-(1)-C N-(2)-(1)-(3)-C N-(2)-Protease cleavage site-(1)-(3)-C N-(2)-(1)-Protease cleavage site-(3)-C N-(2)-Protease cleavage site-(1)-Protease cleavage site-(3)-C N-(2)-(3)-(1)-C N-(2)-Protease cleavage site-(3)-(1)-C N-(2)-(3)-Protease cleavage site-(1)-C ·N-(2)-Protease cleavage site-(3)-Protease cleavage site-(1)-C N-(1)-(3)-(2)-C N-(1)-(3)-Protease cleavage site-(2)-C N-(1)-Protease cleavage site-(3)-(2)-C N-(1)-Protease cleavage site-(3)-Protease cleavage site-(2)-C ·N-(3)-(1)-(2)-C N-(3)-Protease cleavage site-(1)-(2)-C N-(3)-(1)-Protease cleavage site-(2)-C N-(3)-Protease cleavage site-(1)-Protease cleavage site-(2)-C.

[0329] In certain embodiments, the complex includes a linker connecting two polypeptides of the complex and a protease cleavage site connecting two other polypeptides of the complex. In this case, the above considerations regarding the relative positions of the elements of the complex from the N-terminus to the C-terminus apply regardless of the number of linkers or which elements are positioned between them, even in the presence of the linker and the protease cleavage sites between them. Thus, the possible combinations and relative orders of the elements are as follows (the above numbering of the elements is retained: (1) second polypeptide, (2) first polypeptide, (3) positively charged amino acid-rich region): N-(1)-linker-(2)-protease cleavage site-(3)-C N-(1)-Protease cleavage site-(2)-Linker-(3)-C N-(1)-linker-(2)-protease cleavage site-(3)-C N-(1)-Protease cleavage site-(2)-Linker-(3)-C N-(2)-linker-(1)-protease cleavage site-(3)-C N-(2)-Protease cleavage site-(1)-Linker-(3)-C N-(2)-linker-(3)-protease cleavage site-(1)-C N-(2)-Protease cleavage site-(3)-Linker-(1)-C N-(1)-linker-(3)-protease cleavage site-(2)-C N-(1)-Protease cleavage site-(3)-Linker-(2)-C N-(3)-linker-(1)-protease cleavage site-(2)-C N-(3)-Protease cleavage site-(1)-Linker-(2)-C.

[0330] In a preferred embodiment, the combination and relative order of elements in the complex polypeptide is N-(1)-linker-(2)-protease cleavage site-(3)-C. Thus, in a preferred embodiment, the second polypeptide is linked to the first polypeptide region via the linker, and the first polypeptide is linked to the third polypeptide region via the protease cleavage site.

[0331] In another preferred embodiment, the linker includes the sequence GGSSRSS (SEQ ID NO: 39), GGSSRSSS (SEQ ID NO: 76), or GGGGNS (SEQ ID NO: 40), preferably the sequence GGSSRSS (SEQ ID NO: 39).

[0332] In a preferred embodiment, the complex of the sixth aspect of the present invention comprises a polypeptide comprising the following elements: [Table 3]

[0333] In another specific embodiment, a positively charged amino acid, preferably arginine or lysine, more preferably lysine, is included between the first polypeptide region and the third polypeptide region of the complex of the sixth embodiment.

[0334] In another preferred embodiment, the polypeptide forming part of the complex of the present invention comprises, substantially comprises, or consists of the amino acid sequence of SEQ ID NO: 61, and optionally comprises methionine at the amino terminus.

[0335] In some embodiments, the polypeptides forming part of the complex of the present invention contain, substantially contain, or consist of any of the amino acid sequences of SEQ ID NOs. 61 or 106-124, and optionally contain methionine at the amino terminus.

[0336] In certain embodiments, the target agent of the complex according to the sixth aspect of the present invention is a therapeutic agent or a contrast agent.

[0337] IV-E. Target agent In certain embodiments, the target agent of the complex according to the sixth aspect of the present invention is a therapeutic agent or a contrast agent.

[0338] IV-E.1 Therapeutic drugs As used herein, the term “therapeutic agent” refers to any compound suitable for the therapy and / or treatment of a condition, disorder, or disease, without any limitations on its chemical structure.

[0339] The properties of the therapeutic agent are not particularly limited to the present invention, as long as it can maintain its activity within the complex or be activated when delivered into the cell. Therefore, any therapeutic agent can be used in the complex, provided that it exhibits activity when delivered into the cell or exhibits at least 100%, at least 90%, at least 80%, at least 70%, at least 60%, at least 50%, or less than the activity of the uncomplexed therapeutic agent. Alternatively, since the object of the present invention is to enhance the action of a therapeutic agent by increasing its selectivity and reducing its off-target effects, the effects of the therapeutic agent bound to the polypeptide of the complex may be synergistic and may exceed parameterized values ​​already known for a particular therapeutic agent. Accordingly, some embodiments of the therapeutic agent conjugated to the polypeptide of the complex of the present invention are also intended to exhibit at least 101%, at least 105%, at least 110%, at least 115%, at least 120%, at least 125%, at least 130%, at least 135%, at least 140%, at least 145%, at least 150%, at least 175%, at least 200%, at least 300%, at least 400%, 500%, at least 1000% or more of the function of the therapeutic agent alone.

[0340] In embodiments of the present invention, the therapeutic agent conjugated to the polypeptide of the complex of the present invention is (i) Chemotherapy agents, (ii) Cytotoxic polypeptides, (iii) Anti-angiogenic polypeptides, (iv) polypeptides encoded by tumor suppressor genes, (v) Apoptosis-promoting polypeptides, (vi) Polypeptides having antitransfer activity, (vii) Polynucleotide-encoded polypeptides that can activate an immune response against tumors, (viii) Anti-angiogenic molecules, and (ix) toxin It is selected from the group consisting of the following.

[0341] In certain embodiments, the polypeptide of the complex is bound to multiple therapeutic agents, which may be the same or different.

[0342] (i) Chemotherapy agents In certain embodiments, the therapeutic agent is a chemotherapeutic agent.

[0343] It should be understood that the term "chemotherapeutic agent" refers to anticancer drugs.

[0344] As used herein, an anticancer agent is a drug that inhibits, at least partially, the development or progression of cancer, including inhibiting all or some of the symptoms associated with cancer, even if only for a short period.

[0345] Some anticancer drugs can be classified as DNA damaging agents, and these include topoisomerase inhibitors (e.g., etoposide, amptothecin, topotecan, teniposide, mitoxantrone), DNA alkylating agents (e.g., cisplatin, mechloretamine, cyclophosphamide, ifosphamide, melphalan, colambucil, busulfan, thiotepa, carmustine, lomustine, carboplatin, dacarbazine, procarbazine), and DNA strand break inducers (e.g., Examples include bleomycin, doxorubicin, daunorubicin, idarubicin, mitomycin C), antimicrotubule agents (e.g., vincristine, vinblastine), antimetabolites (e.g., cytarabine, methotrexate, hydroxyurea, 5-fluorouracil, floxuridine, 6-thioguanine, 6-mercaptopurine, fludarabine, pentostatin, chlorodeoxyadenosine), anthracyclines, vinca alkaloids, and epipodophyllotoxin.

[0346] Further examples of anticancer drugs include, but are not limited to, asibicin; acralubicin; acodazole hydrochloride; acronin; adzeresin; aldesleukin; altretamine; ambomycin; amethantrone acetate; aminoglutethimide; amsacrin; anastrozole; anthramycin; asparaginase; asperlin; azacitidine; azetepa; azotomycin; batimatsut; benzodepa; bicalutamide; bizanantren hydrochloride; bisnafido dimesylate; bizeresin; bleomycin sulfate; bortezomib (VELCADE); and Brekinal. Sodium; Bropyrimine; Busulfan; Cactinomycin; Carsterone; Calasemide; Carbetimer; Carboplatin (platinum-containing regimen); Carmustine; Carbicin hydrochloride; Carzeresin; Sedefingol; Chlorambucil; Ciloremycin; Cisplatin (platinum-containing regimen); Cladribine; Cristonatol mesylate; Cyclophosphamide; Cytarabine; Dacarbazine; Dactinomycin; Daunorubicin; Decitabine; Dexormaplatin; Dezaguanine; Diaziquan; Docetaxel (TAXOTERE); Doxorubicin Syn; droloxifen; dromostanolone; duazomycin; edatrexate; eflornithine; erusamitrusin; enloplatin; empromate; epipropidine; epirubicin; erbrozol; erlotinib (TARCEVA), esorubicin; estramustine; etanidazole; etoposide; etopurine; fadrozol; fazarabine; fenretinide; phloxuridine; fludarabine; 5-fluorouracil; flurocitabine; fosquidone; fostriesin; gefitinib (IRESSA), gemcitabine; hydroxyuria Idarubicin; Ifosfamide; Irmofosin; Imatinib mesylate (GLEEVAC); Interferon α-2a; Interferon α-2b; Interferon α-nl; Interferon α-n3; Interferon β-Ia; Interferon γ-Ib; Iproplatin; Irinotecan; Lanreotide; Lenalidomide (REVLLM1D, REVIMID); Letrozole; Leuprolide; Rialozol; Lometrexol; Lomustine; Loxoxantrone; Masopropyl; Mytansine; Mechloretamine; Megestrol;Merengestrol; Melphalan; Menogalil; Mercaptopurine; Methotrexate; Metoprine; Meturedepa; Mitindomide; Mitocalcin; Mitochromin; Mitogyrine; Mitomarcin; Mitomycin; Mitospar; Mitotan; Mitoxantrone; Mycophenolic acid; Nocodazole; Nogaramycin; Ormaplatin; Oxythran; Paclitaxel; Pemetrexed (ALIMTA), Pegaspar gauze; Pe Liomycin; Pentamustine; Pentomone; Peplomycin; Perphosphamide; Pipobroman; Piposulfan; Pyritrexime; Isethionate; Pyroxantrone; Plicamycin; Promestan; Porfimer; Porphyromycin; Prednimustine; Procarbazine; Puromycin; Pyrazofrine; Ribopurine; Logretimide; Safingol; Semustine; Simtrazene; Citogluside; Sparfosate; Sparso Mycin; Spirogermanium; Spiromustin; Spiroplatin; Streptonigrin; Streptozocin; Slophenur; Talisomycin; Tamsulosin; Taxol; Taxotere; Tecogalan; Tegafur; Teroxantrone; Temoporfin; Temozolomide; Teniposide; Teroxylone; Testotractone; Thalidomide and its derivatives; Thiamiprine; Thioguanine; Thiotepa; Thiazofrine; tirapazamine; topotecan; toremifene; trestron; trisilibine; trimethrexate; triptrelin; tuburosol; uracil; mustard; uredepa; bapreotide; verteporfin; vinblastine; vincristine; vindesine; vinepidine; vingricinate; vinleulosine; vinorelbine; vinrosidine; vinzolidine; borozol; zeniplatin; dinostatin; and zolubicin are examples.

[0347] In one embodiment, the anticancer agent is provided as an oligomer containing several units of anticancer molecules. In one embodiment, the anticancer agent is a floxuridine polynucleotide or oligonucleotide containing several floxuridine molecules. The floxuridine polynucleotide or oligonucleotide contains at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50 or more floxuridine molecules. In a preferred embodiment, the floxuridine polynucleotide is a floxuridine pentanucleotide, i.e., an oligonucleotide containing 5 floxuridine molecules.

[0348] The anticancer agent may be an enzyme inhibitor, including but not limited to a tyrosine kinase inhibitor, a CDK inhibitor, a MAP kinase inhibitor, or an EGFR inhibitor. The tyrosine kinase inhibitor may be, but not limited to, genistein (4',5,7-trihydroxyisoflavon), tilphostine 25 (3,4,5-trihydroxyphenyl), methylene]-propanedinitrile, helvimycin A, daidzein (4',7-dihydroxyisoflavon), AG-126, trans-1-(3'-carboxy-4'-hydroxyphenyl)-2-(2'',5''-dihydroxyphenyl)ethane, or HDBA (2-hydroxy5-(2,5-dihydroxybenzylamino)-2-hydroxybenzoic acid). The CDK inhibitor may be, but not limited to, p21, p27, p57, or pl5 , pl6, pl8 or pl9. MAP kinase inhibitors may be KY12420 (C23H24O8), CNI-1493, PD98059 or 4-(4-fluorophenyl)-2-(4-methylsulfinylphenyl)-5-(4-pyridyl)1H-imidazole. EGFR inhibitors may be, but are not limited to, erlotinib (TARCEVA), gefitinib (IRESSA), WHI-P97 (quinazoline derivative), LFM-A12 (leflunomide metabolite analog), ABX-EGF, lapatinib, canertinib, ZD-6474 (ZACTIMA), AEE788 and AG1458.

[0349] The anticancer drugs may include, but are not limited to, bevacizumab (AVASTIN), ranibizumab (LUCENTIS), pegaptanib (MACUGEN), sorafenib, sunitinib (SUTENT), vataranib, ZD-6474 (ZACTIMA), anecoltab (RETAANE), squalamine lactate, and VEGF inhibitors including semaphorin. Anticancer drugs are not limited to bevacizumab (AVASTIN), trastuzumab (HERCEPTIN), alemtuzumab (CAMPATH, indicated for B-cell chronic lymphocytic leukemia), gemtuzumab (MYLOTARG, hP67.6, anti-CD33, indicated for leukemias such as acute myeloid leukemia), rituximab (RITUXAN), tositumomab (BEXXAR, anti-CD20, indicated for B-cell malignancies), and MDX-210 (HER-2 / neu tumor gene protein product and immunoglobulin G (IgG)). This may include antibodies or antibody fragments that co-bind bispecific antibodies to type I Fc receptors (FcγRI), olegobomab (OVAREX, indicated for ovarian cancer), edrecolomab (PANOREX), daclizumab (ZENAPAX), palivizumab (SYNAGIS, indicated for respiratory diseases such as RSV infection), ibritumomab tiuxetan (Zevalin, indicated for non-Hodgkin lymphoma), cetuximab (ERBITUX), MDX-447, MDX-22, MDX-220 (anti-TAG-72), I0R-C5, 10R-T6 (anti-CD1), IOR EGF / R3, celogobab (ONCOSCINT OV 103), epratuzumab (LYMPHOCIDE), pemtumomab (THERAGYN), and gliomab-H (indicated for brain tumors and melanoma).

[0350] In certain embodiments of the present invention, proteins acting as angiogenesis inhibitors are intended to target tumors. These drugs include, in addition to the anti-angiogenic polypeptides listed above, marimast;AG3340;COL-3, BMS-275291, thalidomide, endostatin, SU5416, SU6668, EMD121974, 2-methoxyestradiol, carboxamide triazole, CMIol, pentosan polysulfate, angiopoietin 2 (Regeneron), herbimycin A, PNU145156E, 16K prolactin fragment, linamide, thalidomide, pentoxifylline, genistein, TNP470, endostatin, paclitaxel, acutine, angiostatin, cidofovir, vincristine, bleomycin, AGM-1470, platelet factor 4, or minocycline.

[0351] Other suitable activators are DNA cleavage agents. Examples of DNA cleavage agents suitable for inclusion as cytotoxins in the complex used to carry out the method include, but are not limited to, anthraquinone-oligopyrrole-carboxamide, benzimidazole, raynamycin; dynemycin A; enediyne; and biologically active analogues or derivatives thereof (i.e., those having substantially equivalent biological activity). For example, Islam et al., J. Med. Chem. 34 2954-61, 1991;Skibo et al., J. Med. Chem. 37:78-92, 1994;Behroozi et al., Biochemistry 35:1568-74, 1996;Helissey et al., Anticancer Drug Res. 11:527-51, 1996;Unno et al., Chem. Pharm. Bull. 45:125-33, 1997;Unno et al., Bioorg. Med. Chem., 5:903-19, 1997;Unno et al., Bioorg. Med. Chem., 5: 883-901, 1997; and Xu et al. al., Biochemistry 37:1890-7, Known analogs and derivatives are disclosed in 1998. Other examples include, but are not limited to, enediinquinoneimine (U.S. Patent No. 5,622,958); 2,2r-bis(2-aminoethyl)-4-4'-bithiazole [Lee et al., Biochem. Mol. Biol. Int. 40:151-7, 1996]; and epirithicin-salen-copper complex [Routier et al., Bioconjug. Chem., 8: 789-92, 1997].

[0352] Some of the chemotherapeutic agents mentioned above can be grouped together into a common category: antimetabolites. As used herein, “antimetabolites” refer to compounds that inhibit the utilization of metabolites that are part of normal metabolism. Antimetabolites often have a similar structure to the metabolites they interfere with, such as folate antagonists, which interfere with the utilization of folate. Non-exclusive examples of antimetabolites include the following compounds: bleomycin, busulfan, capecitabine, carmustine, carboplatin, chlorodeoxyadenosine, cisplatin, cyclophosphamide, cytarabine, dacarbazine, daunorubicin, docetaxel, doxorubicin, etoposide, fludarabine, fluorouracil, gemcitabine, hydroxyurea, idarubicin, ifosphamide, irinotecan, lomustine, melphalan, mercaptopurine, methotrexate, mitomycin, mitoxantrone, oxaliplatin, paclitaxel, procarbazine, SN-38, thioguanine, thiotepa, teniposide, vinblastine, vincristine, and vinorelbine.

[0353] In certain embodiments, the anticancer agent is an antimetabolite. In another specific embodiment, the antimetabolite is a pyrimidine analog or an oligomeric form thereof. In yet another specific embodiment, the pyrimidine analog is phloxuridine or a pentameric form thereof.

[0354] As used herein, the term “pyrimidine analog” refers to nucleoside analog antimetabolites that mimic the structure of pyrimidines. Pyrimidine analogs inhibit nucleic acid synthesis. Their antiproliferative effects are achieved by incorporation into DNA, leading to strand termination and inhibition of DNA synthesis. They may also inhibit enzymes involved in nucleic acid synthesis, such as DNA polymerase and ribonucleotide reductase. Non-limiting examples of pyrimidine analogs include azacitidine, 6-azauracil, cytarabine, decitabine, gemcitabine, troxacitabine, phloxuridine, fluorouracil, capecitabine, and tegafururacil.

[0355] As used herein, the term "phloxuridine" refers to an anticancer agent classified as an antimetabolite, which is a pyrimidine analog classified as deoxyuridine. The IUPAC name of this anticancer agent is 5-fluoro-1-[4-hydroxy-5-(hydroxymethyl)tetrahydrofuran-2-yl]-1H-pyrimidine-2,4-dione.

[0356] As used herein, the expression “its oligomeric form” refers to a molecule formed by several repeating units, each unit called a monomer, as opposed to a polymer which is not limited to a specific number of units. Generally, the number of monomers in an oligomer is between 5 and 100. Therefore, as used herein, the oligomeric form of a pyrimidine analog refers to a molecule formed by a sequence of several pyrimidine analogs. In certain embodiments, the oligomeric or polymeric form of a pyrimidine analog refers to a molecule containing a sequence of at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50 or more pyrimidine analogs. In another specific embodiment, it refers to a molecule consisting of a sequence of at least two, three, four, five, six, seven, eight, nine, ten, fifteen, twenty, twenty-five, thirty, thirty, thirty-five, forty, forty, fifty, or more pyrimidine analogs.

[0357] In certain embodiments, the oligomeric or polymeric form of the pyrimidine analog is a molecule comprising a sequence of at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50 or more phloxuridine molecules. In another particular embodiment, the oligomeric form of the pyrimidine analog is a molecule comprising a sequence of at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50 or more phloxuridine molecules.

[0358] The expression "its pentameric form," when referring to a phloxuridine analog, is understood to mean a molecule containing or consisting of a sequence of five phloxuridine molecules.

[0359] (ii) Cytotoxic polypeptides As used herein, the term “cytotoxic polypeptide” refers to a drug that can inhibit cellular function. A drug may inhibit proliferation or exhibit cellular toxicity. Polypeptides that, when internalized by cells, inhibit or adversely alter cellular metabolism, or inhibit cell growth or proliferation in any way, are included in this term, but are not limited to drugs whose toxicity is mediated when transported into cells, and drugs whose toxicity is mediated on the cell surface. Examples of useful cytotoxic polypeptides include protein toxins such as bacterial toxins.

[0360] Examples of proteinaceous cytotoxins useful for incorporation into the complex according to the present invention include, but are not limited to, type 1 and type 2 ribosome-inactivating proteins (RIPs). Useful plant RIPs of type 1 include, but are not limited to, dianthin 30, dianthin 32, lichen, saporin 1-9, pokeweed activating protein (PAP), PAP II, PAP-R, PAP-S, PAP-C, maparmin, dodecandrin, briodin-L, briodin, cholin 1 and 2, rufin-A, rufin-B, rufin-S, 19K-protein synthesis inhibitory protein (PSI), 15K-PSI, 9K-PSI, α-quilllowine, β-quilllowine, geronin, momordin, momordin-II, momordin-Ic, MAP-30, α-momorukarin, β-momorukarin, tricosanthin, TAP-29, trichoquilin; barley RIP; flax RIP; tritin, maize RIP, asparin 1 and 2 [Stirpe et al., 1992]. [Bio / Technology 10:405-12]. Useful type 2 RIPs include, but are not limited to, volkensin, lysine, nigrin-β, CIP-29, abrin, modesin, ebrintin-α, ebrintin-β, ebrintin-γ, bilcumin, polectin, and their biologically active enzyme subunits [Stirpe et al., 1992. Bio / Technology 10:405-12; Pastan et al., 1992. Annu. Rev. Biochem. 61:331-54; Brinkmann and Pastan, 1994. Biochim. et Biophys. Acta 1198:27-45,; and Sandvig and Van Deurs, 1996. Physiol. Rev. 76:949-66].

[0361] Examples of bacterial toxins useful as cytotoxins include, but are not limited to, Shiga toxins and Shiga-like toxins (i.e., toxins with the same activity or structure), as well as their catalytic subunits and biologically functional fragments. These bacterial toxins are also type 2 RIPs [Sandvig and Van Deurs, 1996. Physiol. Rev. 76:949-66; Armstrong, 1995. J. Infect. Dis., 171:1042-5; Kim et al., 1997. Microbiol. Immunol. 41:805-8; and Skinner et al., 1998. Microb. Pathog. 24:117-22]. Further examples of useful bacterial toxins include, but are not limited to, Pseudomonas exotoxin and diphtheria toxin [Pastan et al., 1992. Annu. Rev. Biochem. 61:331-54; and Brinkmann and Pastan, 1994. Biochim. et Biophys. Acta 1198:27-45]. Shortened and mutant forms of the toxin enzyme subunit can also be used as the cytotoxin portion (Pastan et al., Annu. Rev. Biochem. 61:331-54; Brinkmann and Pastan, Biochim. et Biophys. Acta 1198:27-45, 1994; Mesri et al., J. Biol. Chem. 268:4852-62, 1993; Skinner et al., Microb. Pathog. 24:117-22, 1998; and U.S. Patent No. 5,082,927).Other target agents include, but are not limited to, the colicin family of over 34 described RNase toxins, including colicin A, B, D, E1-9, croacin DF13, and fungal RNases, α-sarcin [Ogawa et al. 1999. Science 283: 2097-100; Smarda et al., 1998. Folia Microbiol (Praha) 43:563-82; Wool et al., 1992. Trends Biochem. Sci., 17: 266-69].

[0362] (iii) Anti-angiogenic polypeptide The proliferation of tumor cells is largely dependent on extensive tumor angiogenesis, which is associated with cancer progression. Therefore, inhibiting new angiogenesis and targeting and destroying existing blood vessels with anti-angiogenic agents has been introduced as an effective and relatively non-toxic approach to tumor treatment.

[0363] As used herein, the term “anti-angiogenic polypeptide” means a polypeptide capable of inhibiting angiogenesis. Suitable anti-angiogenic polypeptides include, but are not limited to, angiostatins, endostatins, anti-angiogenic antithrombin III, sFRP-4 as described in WO2007115376, and anti-VEGF antibodies such as anivizumab, bevacizumab (Avastin), Fab IMC1121, and F200Fab.

[0364] (iv) polypeptides encoded by tumor suppressor genes As used herein, “tumor suppressor” refers to a gene or gene product that has the normal biological role of suppressing the uncontrolled proliferation of cells. The functional counterpart of a tumor suppressor is an oncogene, and genes that promote normal cell proliferation are known as “proto-oncogenes.” Mutations that activate such genes or gene products further convert them into “oncogenes,” in which cell proliferation activity continues but is uncontrolled. Examples of tumor suppressor genes and gene products are well known in the literature and include PTC, BRCA1, BRCA2, p16, APC, RB, WT1, EXT1, p53, NF1, TSC2, NF2, VHL, ST7, ST14, PTEN, APC, CD95, and SPARC.

[0365] (v) Pro-apoptotic polypeptides As used herein, the term “pro-apoptotic polypeptide” refers to a protein capable of inducing cell death in a cell or cell population. Overexpression of these proteins involved in apoptosis shifts the delicate balance between anti-apoptotic and pro-apoptotic factors toward apoptotic outcomes. Suitable pro-apoptotic polypeptides include, but are not limited to, BAX, BAK, BOK / MTD, BID, BAD, BIK / NBK, BLK, HRK, BIM / BOD, BNIP3, NIX, NOXA, PUMA, BMF, EGL-I and viral homologs, caspases such as caspase-8, adenovirus E4orf4 gene, p53 pathway gene, pro-apoptotic ligands such as TNF, FasL, TRAIL, and / or pro-apoptotic members of the BCL-2 family of proteins such as TNFR, Fas, TRAIL-R1, and TRAIL-R2.

[0366] (vi) Polypeptides having antitransfer activity As used herein, the term “metastasis inhibitor” refers to a protein that acts to slow or prevent the spread of metastases (secondary tumors) within an organism with cancer. Suitable metastasis inhibitors include, but are not limited to, proteins such as BRMS1, CRSP3, DRG1, KAI1, KISS-1, NM23, TIMP family proteins, and uteroglobin.

[0367] (vii) Polynucleotide-encoded polypeptides that can activate an immune response against tumors. As used herein, an immunostimulatory polypeptide is a polypeptide encoded by a polynucleotide that, alone or in combination with other agents, can activate or stimulate an immune response (including enhancing an existing immune response) in a target to which it is administered. Suitable non-limiting examples of immunostimulatory peptides include flagellins, muramyl dipeptides, cytokines including interleukins (e.g., IL-2, IL-7, IL-15 (or superagonists / mutants of these cytokines), IL-12, IFN-γ, IFN-α, GM-CSF, FLT3-ligand, etc.), and immunostimulatory antibodies (e.g., anti-CTLA-4, anti-CD28, anti-CD3, or single-stranded / antibody fragments of these molecules).

[0368] (viii) anti-angiogenic molecules; In certain embodiments, the intervening region of the fusion protein of the present invention is also intended to correspond to proteins that act as tumor-targeting angiogenesis inhibitors. These agents include, in addition to the anti-angiogenic polypeptides described above, marimast;AG3340;COL-3, BMS-275291, thalidomide, endostatin, SU5416, SU6668, EMD121974, 2-methoxyestradiol, carboxamide triazole, CMIol, pentosan polysulfate, angiopoietin 2 (Regeneron), herbimycin A, PNU145156E, 16K prolactin fragment, linamide, thalidomide, pentoxifylline, genistein, TNP470, endostatin, paclitaxel, acutine, angiostatin, cidofovir, vincristine, bleomycin, AGM-1470, platelet factor 4, and minocycline. Other examples, though not limited to them, include VEGF inhibitors such as bevacizumab (AVASTIN), ranibizumab (LUCENTIS), pegaptanib (MACUGEN), sorafenib, sunitinib (SUTENT), vataranib, ZD-6474 (ZACTIMA), anecortab (RETAANE), squalamine lactate, and semaphorin.

[0369] (ix) toxin As used herein, the term “toxin” refers to non-proteinogenic / non-polypeptide cytotoxic compounds obtained from different organisms, as well as chemically modified derivatives and compounds obtained by chemical synthesis of the same compounds. Compounds in this category of biological origin can be obtained from microorganisms (bacteria, archaea, protists, or unicellular fungi) or multicellular organisms (multicellular fungi, plants, or animals such as mollusks). The chemical composition and structure of these toxins are not intended to be limited beyond their non-polypeptide nature, and therefore, one or more amino acids may be part of their structure, whether as part of their basic composition or as a result of chemical induction, as long as the amino acids involved in the structure are not linked by peptide bonds.

[0370] Examples of toxins suitable for the present invention include calicheamycin γ1, drastatin 10, meitansinoid (DM1), and pyrrolobenzodiazepine dimer (PBD).

[0371] (x) Additional medications In a particular embodiment of the complex of the present invention, the therapeutic agent is selected from the therapeutic agents shown in the third column of Table 4 below. [Table 4-1] [Table 4-2] [Table 4-3]

[0372] In certain embodiments of the complex of the present invention, the second polypeptide region is a ligand selected from the ligands shown in Table 4, and the agent of interest is selected from the therapeutic agents shown in the same column as the ligand in Table 4. Another particular embodiment refers to the complex of the present invention as defined in the embodiments described above in this section for use in the treatment of a disease or disorder, the disease or disorder being selected from those shown in Table 4. Another particular embodiment refers to the complex of the present invention as defined in the embodiments described above in this section for use in the treatment of a disease or disorder being selected in Table 4 from those corresponding to the column showing the second polypeptide region of the complex.

[0373] IV-E.2 Contrast agent The term “contrast agent” is used herein to refer to a biocompatible compound whose use facilitates the distinction between different parts of an image by increasing the contrast between different regions of the image. Therefore, the term “contrast agent” encompasses agents used to enhance the quality of images that could otherwise be produced without such an agent (e.g., in MRI), and agents used for image pretreatment (e.g., in nuclear imaging). Suitable contrast agents include, but are not limited to, those for radionuclide imaging, computed tomography, Raman spectroscopy, magnetic resonance imaging (MRI), and optical imaging.

[0374] Examples of contrast agents for radionuclide imaging include ions of iodine-123, technetium-99, indium-111, rhenium-188, rhenium-186, copper-67, iodine-131, yttrium-90, iodine-125, astatine-211, gallium-67, iridium-192, cobalt-60, radium-226, gold-198, cesium-137, and phosphorus-32. Examples of fluorescent agents include gadolinium and lenographin. Examples of paramagnetic ions include chromium(III), manganese(II), iron(III), iron(II), cobalt(II), nickel(H)₃, copper(II), neodymium(III), samarium(III), ytterbium(III), gadolinium(III), vanadium(II), terbium(III), dysprosium(III), holmium(III), and erbium(III).

[0375] Examples of contrast agents for optical imaging include fluorescein, fluorescein derivatives, indocyanine green, Oregon green, Oregon green derivatives, rhodamine green, rhodamine green derivatives, eosin, erythrosine, Texas red, Texas red derivatives, malachite green, nanogold sulfosuccinimidyl ester, Cascade blue, coumarin derivatives, naphthalene, pyridyl oxazole derivatives, Cascade yellow dye, and dapoxyl dye. Also, as used herein, contrast agents for optical imaging include fluorescent proteins, which refer to proteins having an atomic structure that enables them to exhibit fluorescence, a phenomenon well known in the art. Non-limiting examples of commonly used fluorescent proteins suitable for the complex of the present invention include green fluorescent protein (GFP, first discovered by Aequorea victoria), red fluorescent protein (RFP), yellow fluorescent protein (YFP), blue fluorescent protein (BFP), cyan fluorescent protein, or other variants, examples of which can be found in Kremers et al. [Kremers, GJ- et al. 2011. J.Cell Sci. 124:157-160].

[0376] Further non-limiting examples of fluorescent proteins suitable for the complexes of the present invention include phycobiliproteins and phycobiliprotein complexes comprising enhanced green fluorescent protein (eGFP), enhanced cyan fluorescent protein CFP (ECFP), enhanced YFP (EYFP), GFPS65T, emerald, topaz (TYFP), Venus, citrine, m-citrine, GFPuv, destabilized EGFP (dEGFP), destabilized ECFP (dECFP), destabilized EYFP (dEYFP), mCFPm, cerulean, T-sapphire, CyPet, YPet, mKO, HcRed, t-HcRed, DsRed, DsRed2, DsRed-monomer, J-red, dimer 2, t-dimer (12), mRFP1, posyloporin, reniraGFP, monsterGFP, paGFP, maple protein and kindling protein, β-phycoerythrin, R-phycoerythrin and allophycocyanin. In another embodiment, the contrast agent is a fluorescent protein selected from the group consisting of mHoneydew, mBanana, mOrange, dTomato, tdTomato, mTangerine, mStrawberry, mCherry, mGrapel, mRaspberry, mGrape2, and mPlum [Shaner et al. (2005) Nat. Methods 2:905-909].

[0377] Contrast agents for magnetic resonance imaging systems include gadolinium chelate, manganese chelate, chromium chelate, 19F, and iron particles.

[0378] Examples of MRI contrast agents include chromium(III), manganese(II), iron(III), iron(II), cobalt(II), nickel(II), copper(II), neodymium(III), samarium(III), ytterbium(III), gadolinium(III), vanadium(II), terbium(III), dysprosium(III), holmium(III), and erbium(III).

[0379] IV-E.3 Binding of the target agent to the polypeptide of the complex. The polypeptide of the complex can be complexed with a single or more agents of interest. When multiple agents are complexed with the polypeptide of the complex, the agents may be the same or different. In certain embodiments, the multiple agents of interest are therapeutic agents, and as defined above, they may be the same therapeutic agent or different therapeutic agents. In other particular embodiments, the multiple agents of interest are contrast agents, and as defined above, they may be the same contrast agent or different contrast agents.

[0380] The following applies to agents for any purpose, i.e., therapeutic agents, contrast agents, multiple therapeutic agents, and multiple contrast agents as defined above. Therefore, in a particular embodiment, any of the following embodiments relating to an agent applies to a therapeutic agent by substituting the term "agent" with "therapeutic agent." In another particular embodiment, any of the following embodiments relating to an agent applies to a contrast agent by substituting the term "agent" with "contrast agent." In another particular embodiment, any of the following embodiments applies to multiple therapeutic agents by substituting the expression "multiple agents" with "multiple therapeutic agents," wherein the multiple therapeutic agents are as defined above. In another particular embodiment, any of the following embodiments applies to multiple contrast agents by substituting the expression "multiple agents" with "multiple contrast agents," wherein the multiple contrast agents are as defined above.

[0381] One or more target agents can be complexed with any sequence of the polypeptide of the complex.

[0382] Therefore, in a particular embodiment, the agent of interest is complexed with the first polypeptide region. In a particular embodiment, the agent of interest is complexed with the second polypeptide region of the complex. In another particular embodiment, the agent of interest is complexed with the third polypeptide region of the complex. In another particular embodiment, the agent of interest is complexed with either of the linking regions between the first polypeptide and the second polypeptide. In another particular embodiment, the agent of interest is complexed with the linking region between the second polypeptide region and the third polypeptide region. In another particular embodiment, the agent of interest is complexed with the linking region between the second polypeptide region and the third polypeptide region. In another particular embodiment, the agent of interest is complexed with the protease cleavage site between the first polypeptide region and the second polypeptide region. In another particular embodiment, the agent of interest is complexed with the protease cleavage site between the first polypeptide region and the third polypeptide region. In another particular embodiment, the agent of interest is complexed with the protease cleavage site between the second polypeptide region and the third polypeptide region.

[0383] In certain embodiments, multiple target agents are complexed into the same polypeptide region of the complex. In preferred embodiments, multiple target agents are complexed into the first polypeptide region. In certain embodiments, multiple target agents are complexed into the second polypeptide region of the complex. In other specific embodiments, multiple target agents are complexed into the third polypeptide region of the complex. In other specific embodiments, multiple target agents are complexed into all three polypeptide regions of the polypeptide of the complex. In other specific embodiments, multiple target agents are complexed into the first and second polypeptide regions of the polypeptide. In other specific embodiments, multiple target agents are complexed into the first and third polypeptide regions of the complex. In other specific embodiments, multiple target agents are complexed into the second and third polypeptide regions of the complex.

[0384] In another specific embodiment, the multiple-purpose agent is complexed with the linking region of the polypeptide of the complex. In another specific embodiment, the multiple-purpose agent is complexed with the linking region between the second polypeptide region and the first polypeptide region. In another specific embodiment, the multiple-purpose agent is complexed with the linking region between the first polypeptide region and the third polypeptide region. In another specific embodiment, the multiple-purpose agent is complexed with the linking region between the second polypeptide region and the third polypeptide region. In another specific embodiment, the multiple-purpose agent is complexed with the protease cleavage site of the polypeptide of the complex. In another specific embodiment, the multiple-purpose agent is complexed with the protease cleavage site between the second polypeptide region and the first polypeptide region. In another specific embodiment, the multiple-purpose agent is complexed with the protease cleavage site between the first polypeptide region and the third polypeptide region. In another specific embodiment, the multiple-purpose agent is complexed with the protease cleavage site between the second polypeptide region and the third polypeptide region. In another specific embodiment, multiple agents of interest are complexed with all protease cleavage sites of the polypeptide.

[0385] In another specific embodiment, the multiple-purpose agent is complexed with any of the polypeptide regions described above and the linking region of the polypeptide of the complex. In another specific embodiment, the multiple-purpose agent is complexed with any of the polypeptide regions described above and the linking region between the second polypeptide region and the first polypeptide region. In another specific embodiment, the multiple-purpose agent is complexed with any of the polypeptide regions described above and the linking region between the first polypeptide region and the third polypeptide region. In another specific embodiment, the multiple-purpose agent is complexed with any of the polypeptide regions described above and the linking region between the second polypeptide region and the third polypeptide region. In another specific embodiment, the multiple-purpose agent is complexed with any of the polypeptide regions of the polypeptide described above and all of the linking regions of the polypeptide.

[0386] In another specific embodiment, the multiple-purpose agent is complexed with any of the polypeptide regions described above and with the protease cleavage site of the polypeptide of the complex. In another specific embodiment, the multiple-purpose agent is complexed with any of the polypeptide regions described above and with the protease cleavage site between the first polypeptide region and the second polypeptide region. In another specific embodiment, the multiple-purpose agent is complexed with any of the polypeptide regions of the polypeptide and with the protease cleavage site between the first polypeptide region and the third polypeptide region. In another specific embodiment, the multiple-purpose agent is complexed with any of the polypeptide regions of the polypeptide and with the protease cleavage site between the second polypeptide region and the third polypeptide region. In another specific embodiment, the multiple-purpose agent is complexed with the polypeptide regions described above and with all of the cleavage sites of the polypeptide.

[0387] In another specific embodiment, multiple agents of interest are complexed with the linking region of the polypeptide and the protease cleavage site of the polypeptide. In another specific embodiment, multiple agents of interest are complexed with any of the polypeptide regions, the linking region, and the protease cleavage site of the polypeptide of the complex.

[0388] As described above, the agent of interest is intended to be complexed with the polypeptide of the complex, and the position of complexation within the polypeptide with respect to its N-terminus and C-terminus is not restricted. Therefore, the agent of interest may be complexed with respect to the polypeptide of the complex at equidistant positions with respect to the N-terminus and C-terminus, or close to either of them. Therefore, the agent of interest may be complexed with the polypeptide region at a distance of 500, 450, 400, 350, 325, 300, 275, 250, 236, 230, 220, 210, 200, 190, 180, 170, 160, 100, 90, 80, 75, 70, 65, 60, 55, 50, 45, 40, 35, 30, 25, 30, 25, 20, 15, 20, 10, 5 or fewer amino acid residues from the N-terminus or C-terminus of the polypeptide, or it may be complexed with the polypeptide region at the same residues at the N-terminus or C-terminus of the polypeptide. This paragraph applies to each agent complexed with a polypeptide, and multiple agents may be complexed with the polypeptide of the complex.

[0389] The only intended restriction on the binding site of the target agent is that the elements of the agent and polypeptide remain functional, and that the binding of the agent does not interfere with the activity of any of the agents, polypeptides, or complexes.

[0390] Therefore, for the agent of interest, the second polypeptide, the first polypeptide region, and the positively charged amino acid-rich region, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, preferably 95%, more preferably 99%, and even more preferably 100% of their functionality is preserved compared to their uncomplexed forms. This applies regardless of the binding position in the polypeptide of the complex. This paragraph also applies to each agent complexed into a polypeptide in which multiple agents are complexed into the polypeptide of the complex. The agents are intended to be able to bind directly to residues of the polypeptide of the complex or indirectly via binding sites.

[0391] Therefore, in certain embodiments, the agent of interest binds directly to the polypeptide of the complex. In other specific embodiments, the agent of interest binds to the polypeptide of the complex via a linking portion.

[0392] In another specific embodiment, where multiple agents are complexed into a polypeptide of the complex, all of them bind directly to the residues of the polypeptide. In another specific embodiment, where multiple agents are bound to the polypeptide of the complex, some of them bind directly to the residues of the polypeptide, while the rest bind indirectly via linking portions. In yet another specific embodiment, all agents bind via linking portions.

[0393] The terms "connecting portion" or "linker" have already been defined in a second aspect of the present invention.

[0394] Those skilled in the art will understand that when a linking portion mediates the binding between the agent of interest and the polypeptide of the complex, the above-mentioned provisions apply to the elements of the polypeptide of the complex and the functionality of the agent. Therefore, when the agent of interest is bound to the polypeptide of the complex via a linking portion, regardless of the position of the binding in the polypeptide of the complex, the chemical composition or structure of the linking portion, and the chemical properties of the binding between the linking portion and the agent and the binding between the linking portion and the polypeptide of the complex, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, preferably 95%, more preferably 99%, and even more preferably 100% of the functionality of the agent of interest, the second polypeptide region, the first polypeptide region, and the positively charged amino acid-rich region is preserved compared to their uncomplexed forms. This applies to all agents of interest when multiple agents are complexed into the polypeptide of the complex.

[0395] In a preferred embodiment of the present invention, the linking portion mediating the binding between the agent and the polypeptide of the complex is 6-maleimidohexanoate N-hydroxysuccinimide or 4-maleimidohexanoate N-hydroxysuccinimide. In another specific embodiment in which multiple agents are complexed into the polypeptide of the complex, all linking portions mediating the binding between the agent and the polypeptide of the complex are 6-maleimidohexanoate N-hydroxysuccinimide or 4-maleimidohexanoate N-hydroxysuccinimide. In another specific embodiment in which multiple agents are complexed into the polypeptide of the complex, some of the linking portions mediating the binding between the agent and the polypeptide of the complex are 6-maleimidohexanoate N-hydroxysuccinimide or 4-maleimidohexanoate N-hydroxysuccinimide.

[0396] In another preferred embodiment, the linking portion mediating the binding between the agent and the complex polypeptide is a portion that can react with a sulfhydryl group present in the side chain of the polypeptide and the active group of the agent of interest. Suitable linking groups that can react with a sulfhydryl group present in the side chain of the polypeptide include, but are not limited to, maleimide reagents, haloacetyl, aziridine, acryloyl, arylating agents, vinyl sulfone, pyridyl disulfide, TNB-thiol, and disulfide reducing agents. Most of these groups are linked to sulfhydryls by either alkylation (usually the formation of a thioether bond) or disulfide exchange (formation of a disulfide bond).

[0397] In some embodiments, the linking portion includes a spacer region connecting a portion of the linking portion that is connected to a polypeptide forming part of the complex, and a portion of the linking portion that is connected to the agent of interest. In some embodiments, the linking portion is connected to the agent of interest by the spacer, and the linking portion connects the spacer agent of interest to the polypeptide. In one embodiment, the linking portion is connected to the polypeptide, and the linking portion connects the spacer polypeptide to the agent of interest.

[0398] As used herein, the term “spacer” refers to a portion that connects at least two other portions to one another. In some embodiments, the spacer is a polymer.

[0399] As used herein, the term “polymer” means a molecule containing repeating structural units, i.e., monomers, that are chemically bonded together in a linear, cyclic, branched, crosslinked, or dendritic, or a combination thereof, and which may be of synthetic or biological origin, or a combination of both. The monomers may be identical, in which case the polymer is a homopolymer; or the monomers may be different, in which case the polymer is a heteropolymer. Heteropolymers are sometimes called “copolymers,” and examples include alternating copolymers in which different types of monomers are arranged alternately; periodic copolymers in which different types of monomers are arranged in a repeating sequence; statistical copolymers in which different types of monomers are arranged randomly; block copolymers in which blocks of different homopolymers consisting of only one type of monomer are covalently bonded together; and gradient copolymers in which the composition of different monomers gradually changes along the polymer chain. In some embodiments, the polymer includes one or more other parts, and in certain embodiments, the other parts are C 1-50 Alkyl, C 2-50 Alkenil, C 2-50 Alkinyl, C 3-10 The spacer is selected from the group consisting of cycloalkyl, 3-10 membered heterocyclyl, 8-11 membered heterobicyclyl, phenyl, naphthyl, indenyl, indanyl, and tetralinyl. In some embodiments, the spacer is a PEG-based spacer.

[0400] As used herein, the term “PEG-based” with respect to a spacer means that the spacer contains PEG. Such a PEG-based portion or reagent contains at least 10% (w / w) PEG, such as at least 20% (w / w) PEG, at least 30% (w / w) PEG, at least 40% (w / w) PEG, at least 50% (w / w), at least 60% (w / w) PEG, at least 70% (w / w) PEG, at least 80% (w / w) PEG, at least 90% (w / w) PEG, or at least 95% (w / w) PEG. The remaining weight % of the PEG-based portion or reagent is: 1-50 Alkyl, C 2-50 Alkenil, C 2-50 Alkinyl, C 3-10 Cycloalkyl, 3-10 membered heterocyclyl, 8-11 membered heterobicyclyl, phenyl, naphthyl, indenyl, indanyl, and tetralinyl; branching points such as -CR<, >C<, or -N<; and dashed lines indicating binding to the portion or the rest of the reagent, where -R and -Ra are independently -H and Ci -6 A bond selected from the group consisting of alkyl groups; the part and the bond may be further substituted as desired; or other parts selected from the group consisting of alkyl groups.

[0401] In some embodiments of the present invention, the linking portion that connects the agent to the polypeptide of the complex is readily processed by enzymes present in the cytoplasm, and when the therapeutic agent complexed with the fusion protein is internalized in the cell, the therapeutic agent is released from the fusion protein.

[0402] Furthermore, some agents can be polymerized such that multiple copies of the same molecule are bonded together to form a polymer in which each monomer of the polymer is one of the aforementioned molecules. A non-limiting example of such a polymer is 5-fluoro-2'-deoxyuridine (FdU), which yields oligo-FdU. Some embodiments of the present invention are intended to include such polymers. Also, some other embodiments of the present invention are intended to include polymers of two or more different molecular agents, provided that the agents do not interfere with each other's physiological or biological effects. Those skilled in the art will understand that the embodiments of the present invention, which feature polymers of the agent of interest, may feature two, three, four, five, six, seven, eight, nine, ten, fifteen, twenty, fifteen, thirty, forty, fifty or more polymerized molecules of one or more different agents of interest in a ratio of 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10 or higher.

[0403] IV-F reporter protein In another embodiment of the present invention, the polypeptide of the complex of the present invention further comprises a reporter protein.

[0404] Those skilled in the art will understand the term “reporter protein” to refer to a protein resulting from the expression of a “reporter gene.” Reporter proteins are well known and commonly used in the art as markers suitable for multiple purposes, such as the location of tissue, cell or organelle, protein-protein interactions, transport across plasma membranes or intracellular membranes, vesicular transport, and ligand-receptor interactions.

[0405] Useful reporter proteins in this invention include luciferase-4-monooxygenase, β-galactosidase, and thymidine kinase derived from Photinus pyralis. Reporter proteins also include the fluorescent proteins already described.

[0406] The reporter protein contained in the polypeptide of the complex of the present invention is either directly adjacent to the positively charged amino acid-rich region or separated by a linker. However, the relative position of the positively charged amino acid-rich region follows the considerations described above regarding the relative positions of the elements of the fusion protein. Therefore, regardless of the position of the positively charged amino acid-rich region within the fusion protein, the fluorescent protein is always adjacent to it, either directly or separated by a linker.

[0407] Therefore, in embodiments of the present invention comprising a fluorescent protein, the possible relative positions of the polypeptide elements of the complex of the present invention conform to the following scheme (where RP refers to the reporter protein, and the above numbering of the elements is retained: (1) second polypeptide region, (2) first polypeptide region, (3) positively charged amino acid region): ·N-(1)-(2)-RP-(3)-C ·N-(1)-Linker-(2)-RP-(3)-C N-(1)-Protease cleavage site-(2)-RP-(3)-C N-(1)-(2)-Linker-RP-(3)-C N-(1)-(2)-Protease cleavage site-RP-(3)-C · N-(1)-Linker-(2)-Linker-RP-(3)-C N-(1)-Protease cleavage site-(2)-Protease cleavage site-RP-(3)-C N-(1)-linker-(2)-protease cleavage site-RP-(3)-C N-(1)-Protease cleavage site-(2)-Linker-RP-(3)-C ·N-(3)-RP-(2)-(1)-C N-(3)-RP-Linker-(2)-(1)-C • N-(3)-RP-Protease cleavage site-(2)-(1)-C N-(3)-RP-(2)-Linker-(1)-C N-(3)-RP-(2)-Linker-(1)-C ·N-(3)-RP-Linker-(2)-Linker-(3)-C ·N-(3)-RP-Protease cleavage site-(2)-Protease cleavage site-(3)-C N-(3)-RP-linker-(2)-protease cleavage site-(3)-C N-(3)-RP-Protease cleavage site-(2)-Linker-(3)-C N-(1)-(2)-RP-Linker-(3)-C N-(1)-(2)-RP-Protease cleavage site-(3)-C ·N-(1)-Linker-(2)-RP-Linker-(3)-C N-(1)-Protease cleavage site-(2)-RP-Protease cleavage site-(3)-C N-(1)-linker-(2)-RP-protease cleavage site-(3)-C N-(1)-Protease cleavage site-(2)-RP-Linker-(3)-C N-(1)-(2)-Linker-RP-Linker-(3)-C N-(1)-(2)-Protease cleavage site-RP-Protease cleavage site-(3)-C N-(1)-(2)-Linker-RP-Protease cleavage site-(3)-C N-(1)-(2)-Protease cleavage site-RP-Linker-(3)-C · N-(1)-linker-(2)-linker-RP-linker-(3)-C N-(1)-Protease cleavage site-(2)-Protease cleavage site-RP-Protease cleavage site-(3)-C • N-(1)-Protease cleavage site-(2)-Linker-RP-Protease cleavage site-(3)-C N-(1)-Protease cleavage site-(2)-Protease cleavage site-RP-Linker-(3)-C N-(1)-linker-(2)-protease cleavage site-RP-protease cleavage site-(3)-C N-(1)-linker-(2)-linker-RP-protease cleavage site-(3)-C N-(1)-linker-(2)-protease cleavage site-RP-linker-(3)-C N-(1)-Protease cleavage site-(2)-Linker-RP-Linker-(3)-C N-(3)-Linker-RP-(2)-(1)-C N-(3)-Protease cleavage site-RP-(2)-(1)-C N-(3)-Linker-RP-Linker-(2)-(1)-C N-(3)-Protease cleavage site-RP-Protease cleavage site-(2)-(1)-C N-(3)-Protease cleavage site-RP-Linker-(2)-(1)-C N-(3)-linker-RP-protease cleavage site-(2)-(1)-C ·N-(3)-Linker-RP-(2)-Linker-(1)-C N-(3)-Protease cleavage site-RP-(2)-Protease cleavage site-(1)-C ·N-(3)-linker-RP-(2)-protease cleavage site-(1)-C N-(3)-Protease cleavage site-RP-(2)-Linker-(1)-C ·N-(3)-Linker-RP-Linker-(2)-Linker-(3)-C • N-(3)-Protease cleavage site-RP-Protease cleavage site-(2)-Protease cleavage site-(3)-C ·N-(3)-linker-RP-protease cleavage site-(2)-protease cleavage site-(3)-C ·N-(3)-Protease cleavage site-RP-Linker-(2)-Protease cleavage site-(3)-C N-(3)-Protease cleavage site-RP-Protease cleavage site-(2)-Linker-(3)-C N-(3)-linker-RP-linker-(2)-protease cleavage site-(3)-C N-(3)-linker-RP-protease cleavage site-(2)-linker-(3)-C N-(3)-Protease cleavage site-RP-Linker-(2)-Linker-(3)-C ·N-(2)-(1)-RP-(3)-C N-(2)-Linker-(1)-RP-(3)-C N-(2)-Protease cleavage site-(1)-RP-(3)-C N-(2)-(1)-Linker-RP-(3)-C N-(2)-(1)-Protease cleavage site-RP-(3)-C ·N-(2)-Linker-(1)-Linker-RP-(3)-C N-(2)-Protease cleavage site-(1)-Protease cleavage site-RP-(3)-C N-(2)-linker-(1)-protease cleavage site-RP-(3)-C N-(2)-Protease cleavage site-(1)-Linker-RP-(3)-C ·N-(2)-RP-(3)-(1)-C ·N-(2)-(3)-RP-(1)-C N-(2)-Linker-RP-(3)-(1)-C N-(2)-Protease cleavage site-RP-(3)-(1)-C ·N-(2)-Linker-(3)-RP-(1)-C N-(2)-Protease cleavage site-(3)-RP-(1)-C N-(2)-RP-(3)-Linker-(1)-C N-(2)-RP-(3)-Protease cleavage site-(1)-C N-(2)-(3)-RP-Linker-(1)-C N-(2)-(3)-RP-Protease cleavage site-(1)-C ·N-(2)-Linker-RP-(3)-Linker-(1)-C N-(2)-Protease cleavage site-RP-(3)-Protease cleavage site-(1)-C N-(2)-linker-RP-(3)-protease cleavage site-(1)-C N-(2)-Protease cleavage site-RP-(3)-Linker-(1)-C ·N-(2)-Linker-(3)-RP-Linker-(1)-C N-(2)-Protease cleavage site-(3)-RP-Protease cleavage site-(1)-C N-(2)-linker-(3)-RP-protease cleavage site-(1)-C ·N-(2)-Protease cleavage site-(3)-RP-Linker-(1)-C N-(1)-RP-(3)-(2)-C N-(1)-(3)-RP-(2)-C ·N-(1)-RP-(3)-Linker-(2)-C N-(1)-RP-(3)-Protease cleavage site-(2)-C N-(1)-(3)-RP-Linker-(2)-C N-(1)-(3)-RP-Protease cleavage site-(2)-C N-(1)-Linker-RP-(3)-(2)-C N-(1)-Protease cleavage site-RP-(3)-(2)-C ·N-(1)-Linker-(3)-RP-(2)-C N-(1)-Protease cleavage site-(3)-RP-(2)-C ·N-(1)-Linker-RP-(3)-Linker-(2)-C N-(1)-Protease cleavage site-RP-(3)-Protease cleavage site-(2)-C N-(1)-linker-RP-(3)-protease cleavage site-(2)-C N-(1)-Protease cleavage site-RP-(3)-Linker-(2)-C ·N-(1)-Linker-(3)-RP-Linker-(2)-C N-(1)-Protease cleavage site-(3)-RP-Protease cleavage site-(2)-C N-(1)-linker-(3)-RP-protease cleavage site-(2)-C N-(1)-Protease cleavage site-(3)-RP-Linker-(2)-C ·N-RP-(3)-(1)-(2)-C ·N-(3)-RP-(1)-(2)-C N-RP-(3)-Linker-(1)-(2)-C • N-RP-(3)-Protease cleavage site-(1)-(2)-C N-(3)-RP-Linker-(1)-(2)-C • N-(3)-RP-Protease cleavage site-(1)-(2)-C N-RP-(3)-(1)-Linker-(2)-C N-RP-(3)-(1)-Protease cleavage site-(2)-C N-(3)-RP-(1)-Linker-(2)-C N-(3)-RP-(1)-Protease cleavage site-(2)-C ·N-RP-(3)-Linker-(1)-Linker-(2)-C ·N-RP-(3)-Protease cleavage site-(1)-Protease cleavage site-(2)-C N-RP-(3)-Linker-(1)-Protease cleavage site-(2)-C N-RP-(3)-Protease cleavage site-(1)-Linker-(2)-C ·N-(3)-RP-Linker-(1)-Linker-(2)-C ·N-(3)-RP-Protease cleavage site-(1)-Protease cleavage site-(2)-C N-(3)-RP-linker-(1)-protease cleavage site-(2)-C N-(3)-RP-protease cleavage site-(1)-linker-(2)-C.

[0408] IV-G. Preferred composite of the present invention A preferred embodiment of the present invention is a complex in which the components are as defined in Table 3 above, and the agent of interest is one or more copies of phloxuridine or phloxuridine pentanucleotide. In a more preferred embodiment, the complex defined above arises from a linkage between an amino group or thiol group in the side chain of a first region of the polypeptide forming the complex and a thiol group or hydroxyl group or phosphate group or amino group or carboxyl group in the therapeutic agent, which is linked or unlinked by a linkage of 2 to 35 atoms.

[0409] IV-H. Stoichiometry of the Complex of the Invention The number of target agents to be complexed with the fusion protein of the present invention is not particularly limited, but depends on the number of available residues in the polypeptide of the present invention that can be used for chemical complexation with the target agents. Since most binding occurs via amino or sulfhydryl groups present in the side chains of amino acids that form part of the polypeptide of the complex, the number of agents that bind to the polypeptide of the complex depends on the number of lysine and arginine residues (in the case of binding via amino groups in the side chains) or the number of cysteine ​​residues (in the case of binding via sulfhydryl groups in the side chains), and on the yield of the binding reaction. Accordingly, in certain embodiments of the present invention, the polypeptide of the complex of the present invention binds with at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 15, 17, 20, 25, or 30 target agents.

[0410] In the particular case where the agent is provided as a polymer, it will be understood that the number of agents also depends on the number of monomers in the polymer. In the particular case of FdU oligomers, the number of desired agents in a given complex is the result of multiplying the number of oligomers bound to the polypeptide of the complex by the number of monomers. In the preferred case of FdU pentamers, preferred embodiments include complexes containing at least 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 75, 85, 100, 125, 150 or more therapeutic agents per polypeptide of the present invention, each corresponding to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 15, 17, 20, 25, or 30 FdU pentamers per molecule.

[0411] Furthermore, the nanoparticles according to the present invention are derived from an aggregate of multiple copies of the complex of the present invention. In a preferred embodiment, the nanoparticles contain at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 17, 20, 25, and more preferably at least 15 monomers of the complex of the present invention.

[0412] Therefore, the total number of target agents bound to each nanoparticle depends on (i) the number of agents bound to each polypeptide in the complex, (ii) the oligomerization state of the agents, and (iii) the number of complexes forming the nanoparticles. In a preferred embodiment, the nanoparticles are bound to at least 30, 35, 40, 45, 50, 60, 65, 70, 57, 80, 85, 90, 59, 100, 125, 150, 175, 200, 225, 250, 275, and 300 target agents. In a more preferred embodiment, the nanoparticles are bound to at least 30, 35, 40, 45, 50, 60, 65, 70, 57, 80, 85, 90, 59, and 100, more preferably at least 60 FdU pentamer molecules.

[0413] In certain embodiments, all terms and embodiments described in the first, second, third, fourth, and fifth embodiments of the present invention are equally applicable to the sixth embodiment of the present invention.

[0414] V. Method for preparing the composite of the present invention In a seventh embodiment, the present invention relates to a method for preparing a composite according to a sixth embodiment of the present invention: (i) A step of providing a polypeptide of a complex according to a sixth aspect of the present invention comprising the G2 domain of nidogen-1 or a functionally equivalent variant, and (ii) The step of bringing the polypeptide and an activated form of the agent of interest of a sixth aspect of the present invention, which can react with at least one group in the polypeptide, into contact under conditions suitable for forming a bond between the reactive group in the agent of interest and the group in the polypeptide. Regarding methods including

[0415] In another embodiment, the present invention relates to a method for preparing a composite according to a sixth aspect of the present invention: (i) A step of providing a polypeptide of a complex according to a sixth aspect of the present invention comprising the G2 domain of nidogen-1 or a functionally equivalent variant, which is an activated polypeptide, and (ii) A step of bringing the polypeptide and a target agent that can react with the reactive group in the polypeptide into contact under conditions suitable for forming a bond between the reactive group in the polypeptide and the group in the target agent. Regarding methods including

[0416] Those skilled in the art will understand that as used herein, “reactive group” refers to any part of a molecule that can be chemically bonded to another part of another molecule in such a way that the two molecules are bonded together, usually with the release of one or more additional molecules. Many such reactions are known in the art, such as the formation of a peptide bond between a carboxyl group and an amine group, which is one non-limiting example.

[0417] In this specification, "activation" as used to refer to a molecule refers to a modified molecule, including chemical modifications, which enable the molecule to react chemically in ways not previously present in the molecule (for example, activation adds a previously absent reactive group, enabling previously unattainable bonding) or increase its reactivity (i.e., requires lower activation energy for reactions between the molecule and other molecules compared to the inactivated state). The present invention envisions the possibility of activating a target agent and then contacting the activator with a complex polypeptide, or activating a complex polypeptide and then contacting the activated polypeptide with the target agent. In either case, activation of the polypeptide or the target agent is usually carried out by reacting the molecule to be activated with a reagent that introduces a reactive group into the molecule to be activated. Examples of reactive groups that enable activation of the target agent or the complex polypeptide include, but are not limited to, carboxyl, amine, imine, thiol, sulfone, hydroxyl, sulfate, and phosphate moieties, among many others commonly known to those skilled in the art. The activated form of the agent of interest is also referred to herein as the “activated agent of interest.” The activated form of the polypeptide of the complex is also referred to herein as the “activated polypeptide.” As described in a sixth aspect of the present invention, one or more reactive groups in the activated polypeptide are located in the region of the polypeptide to which the agent of interest is complexed. Thus, in a particular embodiment, the reactive group is located in the first polypeptide region, the second polypeptide region, the third polypeptide region, a linker between any of the polypeptide regions, or a protease cleavage site between the polypeptide regions of the complex. In another particular embodiment, the reactive group is located in the first polypeptide region, and / or the second polypeptide region, and / or the third polypeptide region, and / or a linker between any of the polypeptide regions, and / or a protease cleavage site between the polypeptide regions of the complex.

[0418] In a preferred embodiment, the reactive group is located in a first polypeptide region of the complex polypeptide. In a particular embodiment, the reactive group is located in the first polypeptide region of the complex and also in other regions of the complex polypeptide of the sixth embodiment described above.

[0419] In embodiments of the present invention in which the linking portion mediates the binding between the polypeptide of the complex and the agent of interest, the linking portion is a bifunctional crosslinking agent, more preferably a heterobifunctional crosslinking agent that reacts sequentially (either reacting with the activated agent first and then the polypeptide, or reacting with the polypeptide first and then the activated agent) or simultaneously with the group in the polypeptide of interest, using a thioether, amino bond, carbon-nitrogen double bond, or a bond resulting from cyclization as disclosed in Kalia J et al. (Advances in bioconjugation. Curr Org Chem 2010 January, 14(2):138-147). Typical thiol-reactive functional groups include iodoacetamide, maleimide, and disulfide. Furthermore, the protein can be treated with a small molecule or surface presenting an activated ester (e.g., N-hydroxysuccinimidyl ester) to form an amide bond with the lysine side chain and the N-terminal amino group. In another embodiment, the crosslinking portion is a heterobifunctional crosslinking agent comprising a reactive group that can react with a thiol group and a reactive group that can react with an amino group. In one embodiment, the heterobifunctional crosslinking agent is 6-maleimidohexanoic acid N-hydroxysuccinimide ester.

[0420] In a preferred embodiment, the linking portion reacts with the target agent activated in the first step and reacts with the polypeptide of the complex in the second step. In another embodiment, the linking portion reacts with the polypeptide of the complex in the first step and reacts with the target agent in the second step.

[0421] The step of contacting the polypeptide of the complex with the activated form of the agent of interest according to the sixth aspect of the present invention is intended to be carried out in a medium favorable to the reaction that establishes the bond between them. Suitable media for the reaction are generally known to those skilled in the art, including aqueous buffers and non-aqueous buffers. A solid support is also intended to be used in combination with the medium for either the synthesis of the activated agent, or the reaction step resulting in the complexation of the polypeptide of the complex, the agent of interest, and the linking moiety in one embodiment. Furthermore, the method for preparing a complex between a polypeptide and a therapeutic agent is not limited to including the polypeptide, the activated agent of interest, and the linking moiety, and some embodiments are intended to also include the use of one or more catalysts and cofactors in the reaction.

[0422] Accordingly, in one embodiment of the present invention, the activated form of the agent of interest comprises a group that reacts with the polypeptide of the complex, preferably with at least one of the side chains of a first polypeptide region contained in the polypeptide of the complex. As those skilled in the art will understand, “polypeptide side chain” refers to the side chain of an amino acid residue of the polypeptide sequence.

[0423] In another preferred embodiment, the residue is an outer lysine. In a further preferred embodiment of the present invention, the activated target agent, preferably a chemotherapeutic agent, that reacts with at least one of the side chains of the polypeptide of the complex is a thiol group.

[0424] In a more preferred embodiment of the present invention, the activated therapeutic agent is an activated chemotherapeutic agent, more preferably a thiol-functionalized oligofloxuridine.

[0425] In a preferred embodiment, the linking portion is 6-maleimidohexanoate N-hydroxysuccinimide, which mediates the binding between the activated agent and the side chains of the complex polypeptide as described in the embodiments above in this section. In a more preferred embodiment, the linking portion 6-maleimidohexanoate N-hydroxysuccinimide is bound in a first step to the agent of interest, preferably activated FdU, more preferably sulfhydryl-functionalized FdU, and in a second step to the side chains of the complex polypeptide, more preferably the lysine outside the complex polypeptide, and even more preferably the lysine outside the first polypeptide region of the complex of the present invention.

[0426] In another preferred embodiment of the present invention, the activated therapeutic agent is an activated chemotherapeutic agent, more preferably an amino-functionalized oligofloxuridine.

[0427] In a preferred embodiment, the linking portion is 6-maleimidohexanoate N-hydroxysuccinimide, which mediates the binding between the activated agent and the side chains of the complex polypeptide as described in the embodiments above in this section. In a more preferred embodiment, the linking portion 6-maleimidohexanoate N-hydroxysuccinimide is bound in a first step to the agent of interest, preferably activated FdU, more preferably amino-functionalized FdU, and in a second step to the side chains of the complex polypeptide, more preferably to the cysteine ​​outside the complex polypeptide, and even more preferably to the cysteine ​​outside the first polypeptide region of the complex of the present invention.

[0428] In another preferred embodiment of the present invention, the activated therapeutic agent is an activated chemotherapeutic agent, more preferably a carboxy-functionalized oligofloxuridine.

[0429] In a preferred embodiment, activated FdU, more preferably FdU functionalized with an activated carboxylic acid, reacts the carboxyl group in the agent of interest with a reactive group in the polypeptide (e.g., an amino group that can form an amide bond with the carboxyl group in the agent of interest), the side chain of the polypeptide of the complex, more preferably the lysine outside the polypeptide of the complex, and even more preferably the lysine outside the first polypeptide region of the complex.

[0430] In a more preferred embodiment, the agent of interest, more preferably FdU or its pentameric form, is functionalized with an amino group, and the linking portion is a bifunctional reagent that reacts with an amino group in the agent of interest and a reactive group in the polypeptide. In some embodiments, the bifunctional reagent includes a portion that reacts with an amino group (e.g., a carboxylate group that can form an amide group with an amino group in the agent of interest) and a portion that reacts with a sulfhydryl group in the protein side chain (e.g., a maleimide group that can form a thioether with a sulfhydryl group in the polypeptide side chain).

[0431] In a more preferred embodiment, the agent of interest, more preferably FdU or its pentameric form, is functionalized with carboxyl groups, and the linking portion is a bifunctional reagent that reacts with carboxyl groups in the agent of interest and reactive groups in the polypeptide. In some embodiments, the bifunctional reagent includes a portion that reacts with carboxyl groups (e.g., an amino group that can form an amide group with the carboxyl groups in the agent of interest) and a portion that reacts with sulfhydryl groups in the side chains of proteins (e.g., a maleidimide group that can form a thioether with the sulfhydryl groups in the side chains of the polypeptide) or a portion that reacts with amino groups in proteins.

[0432] Additional linkers that may be used between the agent of interest and the polypeptide in the context of the present invention include those commonly used in the preparation of antibody-drug conjugates, as disclosed by reference in Leung et al. (Antibodies 2020, 9, 2; doi:10.3390 / antib9010002) (see Figure 6) and Bargh et al. (Chem. Soc. Rev., 2019, DOI: 10.1039 / c8cs00676h).

[0433] Therefore, in one embodiment of the present invention, the activated form of the complex polypeptide includes a group that reacts with at least one portion of the agent of interest. In a more preferred embodiment of the present invention, the group of the agent of interest, preferably the chemotherapeutic agent, that reacts with the activated complex polypeptide is a thiol group.

[0434] In a more preferred embodiment of the present invention, the activated polypeptide of the complex is obtained by reacting one or more amino groups in the side chain of the polypeptide with a bifunctional reagent containing an activated carboxyl group such as an N-hydroxysuccinimide group. In one embodiment, the bifunctional reagent optionally contains a second activated carboxyl group that can react with an amino group, thiol group, or hydroxyl group in the reagent.

[0435] In a more preferred embodiment, the linking portion is 6-maleimidohexanoic acid N-hydroxysuccinimide, which mediates the bonding between the amino group in the complex polypeptide and the thiol group in the agent of interest. In an even more preferred embodiment, the linking portion of 6-maleimidohexanoic acid N-hydroxysuccinimide is bonded to the complex polypeptide, more preferably to the lysine on the outside of the complex polypeptide, in a first step, and to the side chain in the agent, preferably to the thiol group of the activated agent, in a second step.

[0436] In a more preferred embodiment, the linking portion is 6-maleimidohexanoic acid N-hydroxysuccinimide ester, which mediates the bonding between the thiol group in the complex polypeptide and the amino group in the agent of interest. In an even more preferred embodiment, the linking portion 6-maleimidohexanoic acid N-hydroxysuccinimide ester is bonded to the complex polypeptide, more preferably to the cysteine ​​on the outside of the complex polypeptide, in a first step, and then bonded to the agent of interest, more preferably to the amino group in the agent of interest, in a second step.

[0437] In a more preferred embodiment, the linking portion is 6-maleimidohexanoic acid N-hydroxysuccinimide ester, which mediates the bonding between the thiol group in the complex polypeptide and the amino group in the agent of interest. In an even more preferred embodiment, the linking portion 6-maleimidohexanoic acid N-hydroxysuccinimide ester is bonded in the first step to the agent of interest, preferably to the amino group in the agent of interest, and in the second step to the thiol group of the cysteine ​​on the outside of the complex polypeptide, more preferably to the cysteine ​​on the outside of the complex polypeptide.

[0438] In certain embodiments, all terms and embodiments described in the above-described aspects of the present invention are equally applicable to seventh aspects of the present invention.

[0439] VI-Polypeptide of the present invention containing an antagonistic CXCR4 ligand The inventors have observed that a fusion protein containing an antagonistic CXCR4 ligand and the G2 domain or a variant of nidogen-1 can target and permeate CXCR4-expressing cells. Furthermore, when this fusion protein is further modified by the presence of a polycationic domain, the fusion protein can spontaneously assemble into nanoparticles, target and permeate CXCR4-expressing cells, and induce apoptosis in those cells.

[0440] Therefore, in another embodiment, the present invention is (i) A first region comprising the G2 domain of nidogen-1 or a functionally equivalent variant thereof, and (ii) A second region containing an antagonistic CXCR4 ligand This invention relates to polypeptides containing (also known as the second polypeptide of the present invention, the fusion protein of the present invention, and the CXCR4 antagonist polypeptide of the present invention).

[0441] The first region is defined above in the context of the first polypeptide of the present invention and in the context of the first region of a polypeptide that forms part of a complex of the present invention, and applies equally to the second polypeptide of the present invention. In some embodiments, the first region is a functionally equivalent variant of the G2 domain of nidogen-1, and the functionally equivalent variant of the G2 domain of nidogen-1 is any of the variants defined above in the first polypeptide of the present invention. In some embodiments, the polypeptide according to claim 53 or 54, wherein the first region comprises amino acids 430-667 of the sequence of human nidogen-1 as defined by accession number P14543-1 in the UniProt database. In some embodiments, functionally equivalent variants of the G2 domain of nidogen-1 that form part of the first region include mutations in one or more amino acid residues at positions 459, 468, 639, 650, 543, 545, 449, 525, 561, 618, 619, 151, 604, 638, 641, 469, and 518, with respect to the sequence numbering of human nidogen-1 as defined in UniProt database accession number P14543-1 as of July 7, 2009. Accordingly, in another specific embodiment, the polypeptide of the complex according to the sixth aspect of the present invention is a functionally equivalent variant of the G2 domain of nidogen-1, with respect to the sequence numbering of human nidogen-1 as defined by accession number P14543-1 in the UniProt database (version dated July 7, 2009), comprising mutations in one or more amino acid residues at positions 459, 468, 639, 650, 543, 545, 449, 525, 561, 618, 619, 151, 604, 638, 641, 469, and 518.In some embodiments, nidogen G2 domain variants that may be included in the first polypeptide region include, but are not limited to, NIDOmut2, NIDOmut3, NIDOmut3-V45T, NIDOmut3_V121Q, NIDOmut3-F157E, NIDOmut3-V215T, NIDOmut4, NIDOmut4_T215V, NIDOmut5, NIDOmut3-V176T, and NIDOmut3-I, as defined by SEQ ID NOs. 64, 65, and 87-104, respectively. This includes any of the nidogen G2 domain mutants defined above, in the context of a first aspect of the present invention, including mutants having 200T, NIDOmut3-V236Y, NIDOmut3-L237T, NIDOmut3_S65I, NIDOmut3-R114I, NIDOmut3-C214S, NIDOmut3-S65I_R114I, NIDOmut5-S65I_R114I, NIDOmut3-S65I_R114I, and NIDOmut5-S65I_R114I.

[0442] A second region of the second polypeptide of the present invention comprises an antagonistic CXCR4 ligand. As used herein, the term “antagonistic CXCR4 ligand” refers to any polypeptide, peptide, or peptide mime that can specifically bind to CXCR4 and, in response to interaction with an agonist, reduces, inhibits, or prevents the biological activity of the molecule. In one embodiment, the antagonistic CXCR4 ligand is a competitive antagonist, i.e., an antagonist that reversibly binds to CXCR4 at the same binding site (active site) as an endogenous ligand or agonist without necessarily activating the receptor.

[0443] A suitable method for determining whether a given peptide can bind to CXCR4 is defined above in the context of the complex of the present invention and is equally applicable to the polypeptide of the present invention. In some embodiments, the second region is 10 -6 Less than M, 10 -7 Less than M, 10 -8 Less than M, 10 -9 Less than M, 10 -10 Less than M, 10-11 less than M, 10 -12 less than M, 10 -13 less than M, 10 -14 less than or 10 -15 dissociation constant (K D ) that can specifically bind to CXCR4. The method for determining whether a polypeptide can bind to a target molecule and the method for determining the dissociation constant of said binding are provided in the definition of "specific binding" of the second aspect of the present invention.

[0444] Antagonists suitable for use according to the present invention are characterized by competing with a binding of natural ligand CXCL12 to CXCR4 with an IC50 of 0.1 μM or less, 0.2 μM or less, 0.3 μM or less, 0.4 μM or less, 0.5 μM or less, 0.6 μM or less, 0.7 μM or less, 0.8 μM or less, 0.9 μM or less, 1 μM or less, 2 μM or less, 3 μM or less, 4 μM...

Claims

1. (i) Eleven β-chain domains designated as A, B, C, D, E, F, G, H, I, J and K, and (ii) Ten types of loop regions that connect two consecutive β-chain domains, designated as AB, BC, CD, DE, EF, FG, GH, HI, IJ, and JK loops. A polypeptide comprising; At least one of the loop regions is a congeneral loop region variant in SEQ ID NO: 62, and the congeneral loop region in SEQ ID NO: 62 is defined by SEQ ID NO: 1 (loop region AB), SEQ ID NO: 2 (loop region BC), SEQ ID NO: 3 (loop region CD), SEQ ID NO: 4 (loop region DE), SEQ ID NO: 5 (loop region EF), SEQ ID NO: 6 (loop region FG), amino acids 149-150 of SEQ ID NO: 62 (loop region GH), SEQ ID NO: 7 (loop region HI), SEQ ID NO: 8 (loop region IJ), and SEQ ID NO: 9 (loop region JK), and At least one of the β-chain domains is a variant of the congeneral β-chain in SEQ ID NO: 62, and has at least 50% sequence identity with the congeneral β-chain domain, and the congeneral β-chain domain in SEQ ID NO: 62 is defined by SEQ ID NO: 9 (β-chain domain A), SEQ ID NO: 11 (β-chain domain B), SEQ ID NO: 12 (β-chain domain C), SEQ ID NO: 13 (β-chain domain D), SEQ ID NO: 14 (β-chain domain E), SEQ ID NO: 15 (β-chain domain F), SEQ ID NO: 16 (β-chain domain G), SEQ ID NO: 17 (β-chain domain H), SEQ ID NO: 18 (β-chain domain I), SEQ ID NO: 19 (β-chain domain J), and SEQ ID NO: 20 (β-chain domain K). Polypeptide.

2. The polypeptide according to claim 1, wherein at least one variant of the loop region arises from a mutation resulting from the deletion, substitution, or addition of at least one amino acid in the sequence of the congeneral loop region.

3. The polypeptide according to claim 1 or 2, wherein one or more β-chains contain a mutation, the mutation being located at position 9 of β-chain B as defined by SEQ ID NO: 11, position 1 of β-chain C as defined by SEQ ID NO: 11, position 1 of β-chain C as defined by SEQ ID NO: 12, position 10 of β-chain J as defined by SEQ ID NO: 19, or position 3 of β-chain K as defined by SEQ ID NO:

20.

4. A polypeptide according to any one of claims 1 to 3, comprising one or more mutations, wherein the mutations are located at positions 459, 468, 639, 650, 543, 545, 449, 525, 561, 618, 619, 580, 604, 638, 640, 641, 469 and / or 518, and the numbers are defined in the protein sequence of accession number P14543-1 in the Uniprot database as of July 7, 2009.

5. The polypeptide according to claim 4, wherein one or more mutations at positions 459, 468, 639, 650, 543, 545, 449, 525, 561, 618, 619, 580, 604, 638, 641, 469 and / or 518 of Sequence ID No. 62 are H459A, R468N, F639S, R650A, H543K, H545N, V449T, V525Q, F561E, V619T, V619T, C618S, V580T, I604T, V640Y, L641T, S469I and R518I.

6. The polypeptide according to claim 4 or 5, wherein the one or more mutations are defined in Table 1.

7. A polypeptide display library comprising a plurality of polypeptides according to any one of claims 1 to 6, wherein the plurality of polypeptides are formed by polypeptides in which one or more loop region sequences differ.

8. The polypeptide display library according to claim 7, wherein each polypeptide as a phenotype and a nucleic acid as a genotype corresponding to the phenotype are directly or indirectly linked in the library.

9. A polynucleotide encoding a polypeptide according to any one of claims 1 to 6, or a polynucleotide assembly encoding a plurality of polypeptides of a display library according to claim 7 or 8.

10. A vector comprising the polynucleotide described in claim 9.

11. A host cell comprising the polynucleotide described in claim 9 or the vector described in claim 10.

12. (i) A first polypeptide region comprising the G2 domain of nidogen-1 or a functionally equivalent variant thereof, and (ii) Target agent A complex that includes this.

13. The complex according to claim 12, wherein the polypeptide is a functionally equivalent variant of the G2 domain of nidogen-1 as defined in any one of claims 1 to 6.

14. The complex according to claim 12 or 13, wherein the polypeptide region comprises amino acids 430 to 667 of the sequence of human nidogen-1 as defined by accession number P14543-1 in the UniPro database version dated July 7, 2009.

15. The complex according to any one of claims 12 to 14, wherein the polypeptide is a functionally equivalent variant of the G2 domain of nidogen-1 containing one or more mutations, the mutations being located at positions 459, 468, 639, 650, 543, 545, 449, 525, 561, 618, 619, 580, 604, 638, 640, 641, 469 and / or 518, and the numbers are defined by the protein sequence with accession number P14543-1 in the Uniprot database as of July 7, 2009.

16. The complex according to claim 15, wherein one or more mutations at positions 459, 468, 639, 650, 543, 545, 449, 525, 561, 618, 619, 580, 604, 638, 641, 469 and / or 518 of Sequence ID No. 62 are H459A, R468N, F639S, R650A, H543K, H545N, V449T, V525Q, F561E, V619T, V619T, C618S, V580T, I604T, V640Y, L641T, S469I and R518I.

17. The complex according to claim 15 or 16, wherein the one or more mutations are defined in Table 1.

18. The complex according to any one of claims 12 to 17, wherein the polypeptide further comprises a second polypeptide region capable of specifically binding to a target of interest.

19. The complex according to claim 18, wherein the second polypeptide region capable of specifically binding to a target of interest is a polycationic peptide.

20. The polycationic peptide is (i) A sequence that can specifically bind to a cell surface receptor and promote the internalization of the complex into the cell, (ii) Arginine-rich sequence, (iii) GWH1 peptide, (iv) CD44 ligand, (v) Peptides that can cross the blood-brain barrier, (vi) Cell permeable peptides, and (vii) Nucleoline-bound peptide A composite according to claim 19, selected from the group consisting of the following.

21. The polycationic peptide specifically interacts with receptors on the cell surface, and then reaches the cell. The complex according to claim 20, comprising a sequence that can promote the internalization of the complex, wherein the sequence is a CXCR4 ligand.

22. The complex according to claim 21, wherein the CXCR4 ligand is a peptide selected from the group consisting of a peptide containing the sequence RRWCYRKCYKGYCYRKCR (SEQ ID NO: 25), V1 peptide (SEQ ID NO: 26), CXCL12 peptide (SEQ ID NO: 27), vCCL2 (SEQ ID NO: 28), EPI-X4 sequence (SEQ ID NO: 29), or functionally equivalent variants thereof.

23. The complex according to claim 22, comprising a positively charged peptide sequence, preferably RKRKRK (SEQ ID NO: 77), located at the N-terminus or C-terminus of the CXCR4 ligand, preferably at the C-terminus of the CXCR4 ligand.

24. The complex according to claim 23, wherein the CXCR4 ligand is the EPI-X4 sequence (SEQ ID NO: 29) or a functionally equivalent variant thereof.

25. The complex according to claim 20, wherein the polycationic peptide is an arginine-rich sequence comprising a sequence selected from the group consisting of RRRRRRRRRR (SEQ ID NO: 30), RRRGRGRRRR (SEQ ID NO: 31), RARGRGRRRR (SEQ ID NO: 32), and RARGRGGGA (SEQ ID NO: 33).

26. The complex according to claim 20, wherein the polycationic peptide is CD44 ligand A5G27 (SEQ ID NO: 34) or FNI / II / V (SEQ ID NO: 35).

27. The complex according to claim 20, wherein the polycationic peptide is a peptide that can pass through the blood-brain barrier, selected from the group consisting of Sep-1-7 (SEQ ID NO: 36), Sep-1-8 (SEQ ID NO: 37), and Angiopep-2-7 (SEQ ID NO: 38).

28. The complex according to any one of claims 12 to 27, wherein the polypeptide further comprises a third polypeptide region which is a region rich in positively charged amino acids.

29. The complex according to claim 28, wherein the positively charged amino acid-rich region is a polyhistidine region.

30. The complex according to claim 29, wherein the polyhistidine region comprises 2 to 10 adjacent histidine residues.

31. The complex according to any one of claims 12 to 30, wherein the polycationic peptide is located at the N-terminus of the polypeptide and the positively charged amino acid-rich region is located at the C-terminus of the polypeptide, or the positively charged amino acid-rich region is located at the N-terminus of the polypeptide and the polycationic peptide is located at the C-terminus of the polypeptide.

32. The complex according to any one of claims 12 to 31, wherein the agent for the purpose is a therapeutic agent or a contrast agent.

33. The aforementioned drug (i) Chemotherapy agents, (ii) Cytotoxic polypeptides, (iii) Anti-angiogenic polypeptide, (iv) polypeptides encoded by tumor suppressor genes, (v) Apoptosis-promoting polypeptides, (vi) polypeptides having antitransfer activity, (vii) Polynucleotide-encoded polypeptides that can activate an immune response against tumors. (viiii) Anti-angiogenic molecules, and (ix) Toxin A composite according to claim 32, selected from the group consisting of the following.

34. The complex according to claim 32 or 33, wherein the polypeptide is bound to a plurality of therapeutic agents, and the plurality of therapeutic agents are the same or different.

35. The complex according to claim 33 or 34, wherein the therapeutic agent is a chemotherapeutic agent.

36. The complex according to claim 35, wherein the chemotherapeutic agent is an antimetabolite.

37. The complex according to claim 36, wherein the antimetabolite is a pyrimidine analog or an oligomeric form thereof.

38. The complex according to claim 37, wherein the pyrimidine analog is phloxuridine or its pentameric form.

39. The complex according to any one of claims 28 to 38, wherein the second polypeptide region and the first polypeptide region are linked via a first peptide linker, and / or the first polypeptide region and the third polypeptide region are linked via a second peptide linker.

40. A method for preparing a complex according to any one of claims 12 to 39, (i) To provide a polypeptide of the complex according to any one of claims 12 to 39, comprising the G2 domain of nidogen-1 or a functionally equivalent variant thereof, and (ii) Contacting the polypeptide with an activated form of the agent of interest of any one of claims 12 to 39, which can react with at least one group in the polypeptide, under conditions suitable for forming a bond between the reactive group in the agent of interest and the group in the polypeptide. Methods that include...

41. The method according to claim 40, wherein the activated form of the target agent includes a group that reacts with at least one of the side chains of the polypeptide.

42. The method according to claim 41, wherein the group that reacts with at least one of the side chains of the polypeptide region is a thiol group, an amino group, or a carboxylic acid group.

43. A method for preparing a complex according to any one of claims 12 to 39, (i) To provide a polypeptide of the complex according to any one of claims 12 to 39, comprising the G2 domain of nidogen-1 or a functionally equivalent variant, which is an activated polypeptide, and (ii) Contacting the polypeptide with a target agent capable of reacting with the reactive group in the polypeptide under conditions suitable for forming a bond between the reactive group in the polypeptide and the group in the target agent. Methods that include...

44. (i) A first region comprising the G2 domain of nidogen-1 or a functionally equivalent variant thereof, and (ii) A second region containing an antagonistic CXCR4 ligand Polypeptides containing this material.

45. The polypeptide according to claim 44, wherein the first region is a functionally equivalent variant of the G2 domain of nidogen-1 as defined in any one of claims 1 to 6.

46. The polypeptide according to claim 44 or 45, wherein the first region comprises amino acids 430 to 667 of the sequence of human nidogen-1 as defined by accession number P14543-1 in the UniPro database version dated July 7, 2009.

47. The polypeptide according to claim 44 or 45, wherein the polypeptide is a functionally equivalent variant of the G2 domain of nidogen-1 containing one or more mutations, the mutations being located at positions 459, 468, 639, 650, 543, 545, 449, 525, 561, 618, 619, 580, 604, 638, 640, 641, 469 and / or 518, and the numbers are defined in the protein sequence of accession number P14543-1 in the Uniprot database as of July 7, 2009.

48. The polypeptide according to claim 47, wherein one or more mutations at positions 459, 468, 639, 650, 543, 545, 449, 525, 561, 618, 619, 580, 604, 638, 641, 469 and / or 518 of Sequence ID No. 62 are H459A, R468N, F639S, R650A, H543K, H545N, V449T, V525Q, F561E, V619T, V619T, C618S, V580T, I604T, V640Y, L641T, S469I and R518I.

49. The polypeptide according to claim 47 or 48, wherein the one or more mutations are defined in Table 1.

50. The polypeptide according to any one of claims 44 to 49, wherein the second region comprises the EPI-X4 sequence (SEQ ID NO: 29) or a functionally equivalent variant thereof.

51. The polypeptide according to claim 50, further comprising a positively charged amino acid region in the second region.

52. The polypeptide according to claim 51, wherein at least 50% of the amino acids in the second region are positively charged amino acids.

53. The polypeptide according to claim 51 or 52, wherein the positively charged amino acid region comprises an RKRKRK sequence.

54. The polypeptide according to any one of claims 44 to 53, wherein the first region is located at the N-terminus of the polypeptide and the positively charged amino acid-rich region is located at the C-terminus of the polypeptide.

55. The polypeptide according to any one of claims 44 to 54, wherein the polypeptide further comprises a third polypeptide region which is a region rich in positively charged amino acids.

56. The polypeptide according to claim 55, wherein the positively charged amino acid-rich region is a polyhistidine region.

57. The polypeptide according to claim 56, wherein the polyhistidine region comprises 2 to 10 adjacent histidine residues.

58. The polypeptide according to any one of claims 55 to 57, wherein the first region is located at the N-terminus of the polypeptide and the third region is located at the C-terminus of the polypeptide.

59. A method for preparing nanoparticles comprising a plurality of copies of a polypeptide according to any one of claims 44 to 58, comprising placing the polypeptide preparation under conditions suitable for assembling the plurality of copies of the polypeptide to form nanoparticles.

60. A method for preparing nanoparticles comprising a plurality of copies of the complex described in any one of claims 28 to 39 or a plurality of copies of the polypeptide described in any one of claims 55 to 58, (i) A method comprising placing the preparation of the complex or polypeptide under conditions suitable for assembling multiple copies of the complex or polypeptide to form nanoparticles, or (ii) i. Each 1. The G2 domain of Nidogen-1 or a first polypeptide region which is a functionally equivalent variant, 2. A second polypeptide that can specifically bind to a target of interest, the second polypeptide being a polycationic peptide, and 3. The third polypeptide region, which is rich in positively charged amino acids. Placing a plurality of polypeptides containing the polypeptide under conditions suitable for forming nanoparticles containing a plurality of copies of the polypeptide, The polycationic peptide and the region rich in positively charged amino acids are located at the terminus of the polypeptide, the polypeptide is provided in an activated form, and the polypeptide in the activated form contains a reactive group, and ii. The nanoparticles obtained in step i and the activated form of the target agent, which contains a group capable of reacting with the reactive group in the polypeptide, are brought into contact under conditions suitable for forming a bond between the reactive group in the polypeptide and the group in the target agent. Methods including A method to be selected from.

61. The method according to claim 60, wherein the first, second, and third polypeptide regions are defined in the complex according to any one of claims 13 to 31 and 45 to 58.

62. A method for preparing biparatopic nanoparticles comprising multiple copies of a first type complex and multiple copies of a second type complex, wherein the first and second type complexes are defined in any one of claims 28 to 39, and the first and second type complexes have different polycationic peptide sequences. (i) A method comprising contacting a preparation of the first type of composite and a preparation of the second type of composite under conditions suitable for assembling multiple copies of the two types of composites to form nanoparticles, or (ii) i. Contacting a preparation of a first polypeptide with a preparation of a second polypeptide, wherein the first and second types of polypeptides are a. The G2 domain of Nidogen-1 or a first polypeptide region which is a functionally equivalent variant, b. A second polypeptide region capable of specifically binding to a target of interest, wherein the second polypeptide is a polycationic peptide and / or comprises an additional positively charged peptide sequence and a polycationic sequence located at its N-terminus or C-terminus, wherein the sequence of the polycationic peptide of one polypeptide is different from the sequence of the polycationic peptide of the other polypeptide. c. A third polypeptide region, which is rich in positively charged amino acids. d. Optionally, a positively charged peptide sequence located at the N-terminus or C-terminus of a polycationic peptide. Includes, The region rich in the polycationic peptide and positively charged amino acids is located at the terminus of the polypeptide. The first and second polypeptides have different polycationic peptides, The first and / or second polypeptide is defined in the complex according to any one of claims 28 to 39, The first and / or second polypeptide is provided in an activated form, the polypeptide in the activated form comprises a reactive group, and the contact is carried out under conditions suitable for forming nanoparticles comprising multiple copies of the polypeptide. ii. The nanoparticles obtained in step i and the activated form of the target agent, which contains a group capable of reacting with the reactive group in each polypeptide, are brought into contact under conditions suitable for forming a bond between the reactive group in the polypeptide and the group in the target agent. Methods including A method to be selected from.

63. The method according to claim 62, wherein the first, second, and third polypeptide regions are defined in the complex of any one of claims 13 to 31.

64. A method for preparing biparatopic nanoparticles comprising a plurality of copies of at least one complex according to any one of claims 28 to 39 and a plurality of copies of at least one polypeptide according to any one of claims 55 to 58, wherein the sequence of the polycationic peptide of the first type of complex and the sequence of the second region of the at least one polypeptide are different. (i) A method comprising placing a preparation of multiple copies of the at least one complex and multiple copies of the at least one polypeptide under conditions suitable for assembling the multiple copies of the two complexes into nanoparticles, or (ii) i. Contacting a preparation of a first polypeptide with a preparation of a second polypeptide, wherein the first and second types of polypeptides are a. The G2 domain of Nidogen-1 or a first polypeptide region which is a functionally equivalent variant, b. A second polypeptide region capable of specifically binding to a target of interest, wherein the second polypeptide is a polycationic peptide and / or comprises a positively charged additional peptide sequence and a polycationic sequence located at its N-terminus or C-terminus, and the peptide sequence of one polypeptide is different from the polycationic peptide sequence of the other polypeptide. c. A third polypeptide region rich in positively charged amino acids. Includes, The region rich in the polycationic peptide and positively charged amino acids is located at the terminus of the polypeptide. The first polypeptide exists as a complex defined in any one of claims 28 to 39, and the second polypeptide is defined in any one of claims 55 to 58. The polycationic peptide of the first polypeptide and the polycationic peptide of the second polypeptide are different, The first and / or second polypeptide is provided in an activated form, the polypeptide in the activated form comprises a reactive group, and the process is carried out under conditions suitable for forming nanoparticles comprising multiple copies of the polypeptide. ii. The nanoparticles obtained in step i and the activated form of the target agent, which contains a group capable of reacting with the reactive group in each polypeptide, are brought into contact under conditions suitable for forming a bond between the reactive group in the polypeptide and the group in the target agent. Methods including A method to be selected from.

65. The method according to claim 64, wherein the first, second, and third polypeptide regions of the alternative (i) complex and polypeptide, or the first, second, and third polypeptide regions of the alternative (ii) first and second polypeptides, are defined in any one of the complexes of claims 13 to 31 and 44 to 58.

66. The method according to claim 64 or 65, wherein the polycationic peptide of the alternative (i) complex, or the polycationic peptide of the first polypeptide of alternative (ii), is selected from the group consisting of sequence RRWCYRKCYKGYCYRKCR (SEQ ID NO: 25), V1 peptide (SEQ ID NO: 26), CXCL12 (SEQ ID NO: 27) peptide, vCCL2 (SEQ ID NO: 28), and functionally equivalent variants thereof.

67. The method according to claim 66, wherein the polycationic peptide of the alternative (i) complex or the polycationic peptide of the first polypeptide of alternative (ii) is selected from the sequence RRWCYRKCYKGYCYRKCR (SEQ ID NO: 25), V1 peptide (SEQ ID NO: 26), CXCL12 peptide (SEQ ID NO: 27), vCCL2 (SEQ ID NO: 28) and functionally equivalent variants thereof, preferably RRWCYRKCYKGYCYRKCR (SEQ ID NO: 25), and the polycationic peptide of the alternative (i) polypeptide or the polycationic peptide of the second polypeptide of alternative (ii) is the EPI-X4 sequence (SEQ ID NO: 29) or a functionally equivalent variant thereof.

68. The method according to claim 67, wherein the EPI-X4 sequence (sequence number 29) and the RKRKRK sequence (sequence number 77) are conjugated.

69. The method according to any one of claims 59 to 68, wherein suitable conditions for assembling multiple copies of the polypeptide to form nanoparticles include incubation in a low-salt buffer.

70. The method according to claim 69, wherein the low-salt buffer is selected from the group consisting of carbonate buffer, citrate buffer, acetate buffer, Tris buffer, and phosphate buffer.

71. The method according to claim 70, wherein the pH of the buffer solution is pH 4 to pH 8, preferably pH 5 to pH 7.5, and more preferably about pH 5.3, pH 6.5, or pH 7.

2.

72. The method according to claim 70 or 71, wherein the citrate buffer, acetate buffer and / or phosphate buffer further comprises polysorbate 80 and / or sucrose.

73. The method according to claim 72, wherein the sucrose is found at a concentration of 20 mg / ml to 100 mg / ml.

74. The method according to claim 73, wherein the sucrose is found at a concentration of 50 mg / ml to 90 mg / ml, preferably 70 mg / ml.

75. The method according to any one of claims 70 to 74, wherein the citrate buffer comprises polysorbate 80 (0.4 mg / ml), sucrose (80 mg / ml), sodium citrate dihydrate (2.7 mg / ml), and anhydrous citric acid (0.146 mg / ml), and has a pH of about 6.

5.

76. The method according to any one of claims 70 to 74, wherein the acetate buffer comprises sucrose (70 mg / ml), glacial acetic acid (0.12 mg / ml), sodium acetate trihydrate (2.45 mg / ml), and has a pH of about 5.

3.

77. The method according to any one of claims 70 to 74, wherein the phosphate buffer comprises polysorbate 80 (0.05 mg / ml), sucrose (50 mg / ml), sodium dihydrogen phosphate monohydrate (0.22 mg / ml), and sodium dihydrogen phosphate anhydrous (0.49 mg / ml), and has a pH of about 7.

2.

78. Nanoparticles comprising a plurality of copies of the complex according to any one of claims 28 to 39, a plurality of copies of the polypeptide according to any one of claims 55 to 58, or obtained by the method according to any one of claims 59 to 77.

79. Biparatopic nanoparticles comprising multiple copies of first and second type complexes, wherein both of the first and second type complexes exist as polypeptides defined in any one of claims 28 to 39 or in any one of claims 55 to 58, and the first and second type complexes differ in polycationic peptides or biparatopic nanoparticles obtained by the method of any one of claims 62 to 77.

80. The biparatopic nanoparticles according to claim 79, wherein the polycationic peptide of the first type complex and the second type complex is a CXCR4 ligand, preferably selected from the group consisting of peptides containing the sequence RRWCYRKCYKGYCYRKCR (SEQ ID NO: 25), V1 peptide (SEQ ID NO: 26), CXCL12 peptide (SEQ ID NO: 27), vCCL2 peptide (SEQ ID NO: 28), EPI-X4 sequence (SEQ ID NO: 29), and functionally equivalent variants thereof.

81. The biparatopic nanoparticle according to claim 80, wherein the polycationic peptide of the first type of complex comprises a peptide containing the sequence RRWCYRKCYKGYCYRKCR, and the polycationic peptide of the second type of complex is the EPI-X4 sequence (SEQ ID NO: 29).

82. A biparatopic nanoparticle comprising a plurality of copies of the complex described in any one of claims 28 to 39 and a plurality of copies of the polypeptide described in any one of claims 55 to 58, wherein the polycationic region of the complex and the first region of the polypeptide are different, or a biparatopic nanoparticle obtained by the method described in any one of claims 62 to 77.

83. The biparatopic nanoparticle according to claim 82, wherein the polycationic peptide of the complex is a CXCR4 ligand, preferably selected from the group consisting of a peptide containing the sequence RRWCYRKCYKGYCYRKCR (SEQ ID NO: 25), a V1 peptide (SEQ ID NO: 26), a CXCL12 peptide (SEQ ID NO: 27), and a vCCL2 peptide (SEQ ID NO: 28).

84. The biparatopic nanoparticle according to claim 83, wherein the antagonistic CXCR4 ligand is the EPI-X4 sequence (SEQ ID NO: 29).

85. Nanoparticles according to any one of claims 78 to 84, having a diameter of 1 to 100 nm.

86. A complex according to any one of claims 12 to 39, a polypeptide according to any one of claims 44 to 58, or a nanoparticle according to any one of claims 78 to 85, for use in pharmaceuticals.

87. A complex according to any one of claims 12 to 39, a polypeptide according to any one of claims 44 to 58, or a nanoparticle according to any one of claims 78 to 85 for use in the treatment of cancer, wherein the complex or polypeptide comprises a sequence that can specifically interact with a cell surface receptor to promote the internalization of the complex or nanoparticle into a cell, and the cell is a tumor cell present in cancer.

88. The complex, polypeptide, or nanoparticle according to claim 87, wherein the polycationic peptide of the complex or polypeptide forming the nanoparticles is a CXCR4 ligand, and the cancer comprises cancer cells expressing or overexpressing CXCR4.

89. The complex or nanoparticles for use according to claim 88, wherein the CXCR4 ligand is selected from the group consisting of a peptide containing the sequence RRWCYRKCYKGYCYRKCR (SEQ ID NO: 25), a V1 peptide (SEQ ID NO: 26), a CXCL12 peptide (SEQ ID NO: 27), a vCCL2 peptide (SEQ ID NO: 28), and an EPI-X4 sequence (SEQ ID NO: 29).

90. The complex or nanoparticles for use according to claim 89, wherein the CXCR4 ligand is a peptide having the sequence RRWCYRKCYKGYCYRKCR (SEQ ID NO: 25).

91. The complex or nanoparticles for use according to claim 90, wherein the CXCR4 ligand is EPI-X4 peptide (SEQ ID NO: 29).

92. The nanoparticles for use according to any one of claims 86 to 91, wherein the nanoparticles are biparatopic nanoparticles as defined in any one of claims 79 to 85.

93. The complex, polypeptide, or nanoparticle for use according to any one of claims 88 to 92, wherein the cancer cells expressing or overexpressing CXCR4 are metastatic stem cells.

94. The complex, polypeptide, or nanoparticle for use according to any one of claims 87 to 93, wherein the cancer is pancreatic cancer or colorectal cancer.

95. A complex, polypeptide, or nanoparticle for use according to any one of claims 87 to 94, wherein the cancer is a primary tumor or metastasis.

96. A method for imaging target cells comprising a specific binding site for one or more components of a complex according to any one of claims 12 to 39, one or more components of a polypeptide according to any one of claims 44 to 58, or one or more components of a nanoparticle according to any one of claims 78 to 85, (i) Contacting a sample containing the cells with a complex according to any one of claims 12 to 39, a polypeptide according to any one of claims 44 to 58, or nanoparticles according to any one of claims 78 to 85, under conditions suitable for the binding of the complex, polypeptide, or nanoparticles to the cells, wherein the agent of interest is a contrast agent, and (ii) A method comprising imaging cells by detecting a signal provided by the contrast agent.

97. The method according to claim 96, wherein the cells express or overexpress CXCR4, and one or more components of the complex or nanoparticles are polycationic peptides, and the polycationic peptide is a CXCR4 ligand.

98. A method for identifying polypeptides that bind to a target peptide, i) Contacting the target peptide with the polypeptide display library according to claim 7 or 8 under conditions that allow the polypeptide and the target peptide to interact, ii) Recovering library members that have specifically interacted with the target peptide, and iii) Identifying the sequence of the polypeptide that interacts with the target peptide. Methods that include...

99. The method according to claim 98, wherein steps i) to ii) are repeated at least once, and in each iteration the polypeptide library used in step i) is formed from members of the library recovered in step (ii).

100. The method according to claim 98 or 99, wherein the target peptide is immobilized on a solid support.

101. Use of a polypeptide according to any one of claims 1 to 6 for presenting a peptide, wherein the peptide is found in one of the loop regions.

102. A method for determining the presence of a target peptide in a sample, i) Contacting a protein present in a sample with a polypeptide according to any one of claims 1 to 6, wherein at least one sequence of a loop region in the polypeptide is a sequence that can specifically bind to the target peptide. ii) Determine whether there is an interaction between the target peptide and the polypeptide, and if there is an interaction between the polypeptide and the target peptide, determine whether the target peptide is present in the sample. Methods that include...

103. The method according to claim 102, wherein the polypeptide is immobilized on a solid support.

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