Nidogen-based scaffold proteins and therapeutic nanocomplexes

Abstract

The present invention relates to proteins suitable for use as scaffolds to which peptides of interest are attached, or proteins contained within complexes to which agents of interest are attached. The present invention also relates to complexes suitable for selectively delivering complexes of agents of interest to specific cell and tissue types, where the agents may be therapeutic or imaging agents. The present invention also relates to nanoparticles comprising such complexes and their therapeutic uses.

Description

[Technical Field]

[0001] The present invention relates to the field of nanostructured protein materials, and more particularly to therapeutic agent-carrying polypeptides that can be used in therapy. [Background technology]

[0002] Systemic administration of drugs in the form of nanocomplexes offers the advantage of improved drug stability compared to free molecules. By chemically incorporating functional groups into nanoscale media, researchers can benefit from the high surface area / volume ratio of nanomaterials and integrate additional beneficial properties, such as cell targeting, into existing hybrid composites. The resulting drug-loaded complexes, approximately 8–100 nm in size, avoid renal filtration when administered systemically unless there is aggregation in the lungs or other highly vascularized organs. This fact, combined with the appropriate physicochemical properties of the material, may result in extended circulation time and extended drug exposure to target organs, thus potentially enhancing therapeutic efficacy and benefit for patients.

[0003] Among the diverse materials under investigation as drug vehicles, including metals, ceramics, polymers, and carbon nanotubes, proteins offer unique properties with respect to biocompatibility and degradability, making them particularly desirable in the context of growing concerns about nanotoxicity. Rapid advances in engineering protein self-assembly into nanostructured materials allow for tighter control over the final shape and physicochemical properties, allowing protein materials to gain functional and structural diversity from chemically conjugated drugs as vehicles.

[0004] Indeed, the conjugation of a cytotoxic "payload" to an antibody to form an antibody-drug conjugate (ADC) has been shown to provide a mechanism for selective delivery of cytotoxic agents to cancer cells via specific antibody binding 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-targeting antibiotic used against acute myeloid leukemia. Furthermore, maytansinoids, highly potent microtubule-disrupting agents, were tested for maximum payload loading in ADCs, resulting in the formulation ado-trastuzumab emtansine for treating HER2-positive breast cancer.

[0005] Nevertheless, the structural complexity of antibodies can be a formidable obstacle in terms of cost and synthesis. We have previously explored the field of nanomedicine by applying nanostructural 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]. These terminal tags and the resulting charge balance across the fusion have been described in the art to promote self-assembly and oligomerization of monomeric proteins into robust toroidal nanoparticles, which are stable in plasma [Cespedes, MV et al. 2014. ACS Nano., 8:4166-4176] and, when cell-targeting peptides are added, enhance cell penetration [Xu, ZK et al. 2015. Materials Letters, 154:140-3]. These protein structural components may also include functional peptides such as cell targeting agents, endosomolytic agents or nuclear localization signals in the form of modular fusion stretches.

[0006] Since current treatments still show room for improvement, mainly due to the phenomenon of tumor resistance, which can be caused by intratumoral clonal selection of cells that are most resistant to chemotherapy, for example, there remains a need in the art to develop more specific therapeutic approaches that can target specific tumor cells responsible for treatment failure and tumor progression, while reducing side effects and off-target effects of therapeutic agents. Summary of the Invention

[0007] In a first aspect, the present invention provides a method for producing a medicament for the treatment of a pulmonary arthritis, comprising: (i) 11 beta-strand domains designated A, B, C, D, E, F, G, H, I, J, and K; and (ii) 10 loop regions connecting two consecutive β-strand 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 cognate loop region variant in SEQ ID NO:62, wherein the cognate 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 to 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 and wherein at least one of the beta strand domains is a variant of the cognate beta strand in SEQ ID NO: 62 and has at least 50% sequence identity with the cognate beta strand domain in SEQ ID NO: 62, wherein the cognate beta strand domain in SEQ ID NO: 62 is defined in SEQ ID NO: 9 (beta strand domain A), SEQ ID NO: 11 (beta strand domain B), SEQ ID NO: 12 (beta strand domain C), SEQ ID NO: 13 (beta strand domain D), SEQ ID NO: 14 (beta strand domain E), SEQ ID NO: 15 (beta strand domain F), SEQ ID NO: 16 (beta strand domain G), SEQ ID NO: 17 (beta strand domain H), SEQ ID NO: 18 (beta strand domain I), SEQ ID NO: 19 (beta strand domain J), and SEQ ID NO: 20 (beta strand domain K).

[0008] In a second aspect, the present invention relates to a polypeptide display library comprising a plurality of polypeptides according to the first aspect of the present invention, wherein the plurality of polypeptides is formed by polypeptides that differ in the sequence of one or more loop regions.

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

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

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

[0012] In a sixth aspect, the present invention provides a method for producing a composition comprising: (i) a first polypeptide region comprising the G2 domain of nidogen-1 or a functionally equivalent variant thereof; and (ii) the agent of interest The present invention relates to a complex comprising:

[0013] In a seventh aspect, the present invention provides a method for preparing a complex according to the sixth aspect of the invention, comprising the steps of: (i) providing a polypeptide of a complex according to the sixth aspect of the invention comprising the G2 domain of nidogen-1 or a functionally equivalent variant thereof, wherein the polypeptide is in an activated form; and (ii) contacting the polypeptide with a target agent capable of reacting with a reactive group in the polypeptide under conditions suitable for forming a bond between the reactive group in the polypeptide and a group in the target agent; The present invention relates to a method comprising:

[0014] In an eighth aspect, the present invention provides a method for producing a composition comprising: (i) a first region comprising the G2 domain of nidogen-1 or a functionally equivalent variant thereof; and (ii) a second region that contains an antagonistic CXCR4 ligand; The present invention relates to a polypeptide comprising:

[0015] In a ninth aspect, the present invention provides a method for preparing a complex according to the sixth aspect of the invention, comprising the steps of: (i) providing a polypeptide of a complex according to the sixth aspect of the invention comprising the G2 domain of nidogen-1 or a functionally equivalent variant thereof; and (ii) contacting said polypeptide with an activated form of an agent of interest of a conjugate according to the sixth aspect of the invention capable of reacting with at least one group in said polypeptide under conditions suitable to form a bond between a reactive group in said agent of interest and a group in said polypeptide. The present invention relates to a method comprising:

[0016] In a tenth aspect, the present invention provides a method for preparing a complex according to the sixth aspect of the invention, comprising the steps of: (i) providing a polypeptide of a complex according to the sixth aspect of the invention comprising the G2 domain of nidogen-1 or a functionally equivalent variant thereof, wherein the polypeptide is in an activated form; and (ii) contacting the polypeptide with a target agent capable of reacting with a reactive group in the polypeptide under conditions suitable for forming a bond between the reactive group in the polypeptide and a group in the target agent; The present invention relates to a method comprising:

[0017] In an eleventh aspect, the present invention provides a method for producing a composition comprising: (i) a first region comprising the G2 domain of nidogen-1 or a functionally equivalent variant thereof; and (ii) a second region that contains an antagonistic CXCR4 ligand; The present invention relates to a polypeptide comprising:

[0018] In a twelfth aspect, the present invention relates to a method for preparing nanoparticles comprising multiple copies of a polypeptide according to the eleventh aspect of the invention, the method comprising subjecting a preparation of said polypeptide to conditions suitable for assembling the multiple copies of said polypeptide into nanoparticles.

[0019] In a thirteenth aspect, the present invention provides a method for preparing nanoparticles comprising multiple copies of a complex according to the sixth aspect of the invention or multiple copies of a polypeptide according to the eleventh aspect of the invention, comprising the steps of: (i) subjecting a preparation of said complex or polypeptide to conditions suitable for assembling multiple copies of said complex or polypeptide into nanoparticles; or (ii) i. Each 1. A first polypeptide region which is the G2 domain of nidogen-1 or a functionally equivalent variant thereof; 2. a second polypeptide capable of specifically binding to a target of interest, the second polypeptide being a polycationic peptide; and 3. A third polypeptide region that is rich in positively charged amino acids under conditions suitable to form nanoparticles comprising multiple copies of said polypeptides, the polycationic peptide and the region rich in positively charged amino acids are located at the termini of a polypeptide, the polypeptide being provided in an activated form, the activated form of the polypeptide comprising a reactive group; and ii. contacting the nanoparticles obtained in step i with an activated form of an agent of interest that comprises a group capable of reacting with a reactive group in the polypeptide under conditions suitable for forming a bond between the reactive group in the polypeptide and a group in the agent of interest; A method including The present invention relates to a method selected from the group consisting of:

[0020] In a fourteenth aspect, the present invention provides a method for preparing biparatopic nanoparticles comprising multiple copies of a first type of complex and multiple copies of a second type of complex, said first and second type of complex being as defined in the sixth aspect, said first and second type of complex having different polycationic peptides; (i) contacting a preparation of said first type of complex with a preparation of said second type of complex under conditions suitable for assembling multiple copies of the two types of complex into nanoparticles; or (ii) i. contacting a preparation of a first polypeptide with a preparation of a second polypeptide, wherein said first and second types of polypeptides are: a. a first polypeptide region which is the G2 domain of nidogen-1 or a functionally equivalent variant thereof; b. a second polypeptide region capable of specifically binding to a target of interest, said second polypeptide being a polycationic peptide and / or comprising an additional positively charged peptide sequence and a polycationic sequence located at its N-terminus or C-terminus, wherein the polycationic peptide sequence of one polypeptide differs from the polycationic peptide sequence of the other polypeptide; c. a third polypeptide region that is a region rich in positively charged amino acids; d. optionally, a positively charged peptide sequence located at the N-terminus or C-terminus of the polycationic peptide; Including, the polycationic peptide and the region rich in positively charged amino acids are located at the termini of the polypeptide; the first and second polypeptides having different polycationic peptides; wherein said first and / or second polypeptides are present as a complex as defined in the sixth aspect of the invention, the contacting, wherein the first and / or second polypeptides are provided in an activated form, the activated form of the polypeptides comprising reactive groups, and the placing is carried out under conditions suitable to form nanoparticles comprising multiple copies of the polypeptides; ii. contacting the nanoparticles obtained in step i with an activated form of an agent of interest that contains a group capable of reacting with the reactive group in each polypeptide under conditions suitable for forming a bond between the reactive group in the polypeptide and the group in the agent of interest; A method including The present invention relates to a method selected from the group consisting of:

[0021] In a fifteenth aspect, the present invention provides a method for preparing biparatopic nanoparticles comprising multiple copies of at least one complex according to the sixth aspect of the invention and multiple copies of at least one polypeptide according to the eleventh aspect of the invention, wherein the sequences of the polycationic peptide of said first type of complex and the second region of said at least one polypeptide are different, (i) subjecting a preparation of multiple copies of said at least one complex and multiple copies of said at least one polypeptide under conditions suitable for assembling the multiple copies of said two complexes into nanoparticles; or (ii) i. contacting a preparation of a first polypeptide with a preparation of a second polypeptide, wherein said first and second types of polypeptides are: a. a first polypeptide region which is the G2 domain of nidogen-1 or a functionally equivalent variant thereof; b. a second polypeptide region capable of specifically binding to a target of interest, said second polypeptide being a polycationic peptide and / or comprising an additional positively charged peptide sequence and a polycationic sequence located at its N-terminus or C-terminus, wherein the sequence of the peptide in one polypeptide differs from the sequence of the polycationic peptide in the other polypeptide; c. a third polypeptide region that is rich in positively charged amino acids Including, the polycationic peptide and the region rich in positively charged amino acids are located at the termini of the polypeptide; wherein said first polypeptide is present as a complex as defined in the sixth aspect of the invention and said second polypeptide is as defined in the eleventh aspect of the invention; the polycationic peptide of the first polypeptide and the polycationic peptide of the second polypeptide are different; wherein the first and / or second polypeptides are provided in an activated form, the activated form of the polypeptide comprising a reactive group, and the placing is carried out under conditions suitable to form nanoparticles comprising multiple copies of the polypeptide; ii. contacting the nanoparticles obtained in step i with an activated form of an agent of interest that contains a group capable of reacting with the reactive group in each polypeptide under conditions suitable for forming a bond between the reactive group in the polypeptide and the group in the agent of interest; A method including The present invention relates to a method selected from the group consisting of:

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

[0023] In a seventeenth aspect, the present invention relates to biparatopic nanoparticles comprising multiple copies of a first and a second type of complex, wherein said first and second type of complex are both as defined in the sixth aspect of the invention or are present as polypeptides as defined in the eleventh aspect of the invention, and wherein said first and second type of complexes are different polycationic peptides or biparatopic nanoparticles obtainable by a method according to the fourteenth or fifteenth aspect of the invention.

[0024] In an eighteenth aspect, the present invention relates to a biparatopic nanoparticle comprising multiple copies of a complex according to the sixth aspect of the invention and multiple copies of a polypeptide according to the eleventh aspect of the invention, wherein the polycationic region of said complex and the first region of said polypeptide are different, or a biparatopic nanoparticle obtainable by a method according to the fourteenth or fifteenth aspect of the invention.

[0025] In a nineteenth aspect, the present invention relates to a conjugate according to the sixth aspect of the invention, a polypeptide according to the eleventh aspect of the invention, or a nanoparticle according to the sixteenth, seventeenth or eighteenth aspect of the invention for use in medicine.

[0026] In a twentieth aspect, the present invention provides a method for imaging a target cell comprising specific binding sites for one or more components of a complex according to the sixth aspect of the invention, one or more components of a polypeptide according to the eleventh aspect, or one or more components of a nanoparticle according to the sixteenth, seventeenth or eighteenth aspect of the invention, comprising: (i) contacting a sample comprising said cells with a complex according to the sixth aspect of the invention, a polypeptide according to the eleventh aspect of the invention or a nanoparticle according to the sixteenth, seventeenth or eighteenth aspect of the invention under conditions suitable for binding of said complex, polypeptide or nanoparticle to said cells, wherein said agent of interest is an imaging agent; and (ii) imaging the cells by detecting the signal provided by the imaging agent. The present invention relates to a method comprising:

[0027] In a twenty-first aspect, the present invention provides a method for identifying a polypeptide that binds to a target peptide, comprising the steps of: i) contacting a target peptide with a polypeptide display library according to the second aspect of the present invention under conditions that allow interaction between the polypeptide and the target peptide; ii) recovering library members that specifically interacted with said target peptide; iii) identifying the sequence of a polypeptide that interacts with the target peptide; The present invention relates to a method comprising:

[0028] In a twenty-second aspect, the present invention relates to the use of a polypeptide according to the first aspect of the invention for presenting a peptide, wherein said peptide is found in one of the loop regions.

[0029] In a twenty-third aspect, the present invention provides a method for determining the presence of a target peptide in a sample, comprising the steps of: i) contacting proteins present in a sample with a polypeptide according to the first aspect of the present invention, wherein the sequence of at least one loop region in said polypeptide is a sequence capable of specifically binding to said target peptide; ii) determining whether there is an interaction between the target peptide and the polypeptide, and determining that if there is an interaction between the polypeptide and the target peptide, the target peptide is present in the sample. The present invention relates to a method comprising: [Brief explanation of the drawings]

[0030] [Figure 1] Figure 1 shows the structure of the human nidogen-1 protein as shown in Takagi J. et al. (Nature 424, 969-974, 2003). G1, G2, and G3 represent the three major 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 characteristics of T22-STM-H6 (A), T22-NIDOmut2-H6 (B), and T22-GFP-H6 (C) nanoparticles. MALDI-TOF mass spectrometry spectra are shown at the top, Western blot immunodetection using an anti-His monoclonal antibody is shown as an inset, and the volumetric size distribution of each nanoparticle measured by DLS is shown at the bottom. [Figure 3]Figure 3 shows the characteristics of T22-NIDOmut2-H6 nanoparticles. A) Coomassie blue staining of purified T22-NIDOmut2-H6 peak 1 (Pico1) and peak 2 (Pico2) in carbonate (-) and carbonate + salt (+) buffers by SDS-PAGE electrophoresis. B) Western blot immunodetection of T22-NIDOmut2-H6 protein with an 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 an additional ATTO molecule. B) Volume size distribution of T22-NIDOmut2-H6-ATTO488 nanoparticles determined by DLS. [Figure 5] Figure 5 shows the CXCR4-specific internalization of T22-NIDOmut2-H6 protein in CXCR4+ cells. A) Internalization and competition (+AMD) of labeled T22-NIDOmut2-H6-ATTO488 nanoparticles on HeLa cells (CXCR4+) at different concentrations (1 nM and 10 nM) upon incubation for 24 hours. The percentage of cellular uptake inhibition in the presence of the CXCR4 receptor antagonist AMD3100 (+AMD) is shown. B) Confocal laser microscope images of HeLa cells incubated for 24 hours in the presence of 25 nM T22-NIDOmut2-H6-ATTO488. Cell nuclei are stained with Hoechst, and cell membranes are stained with CellMask. The punctuated patterns within the cells correspond to protein nanoparticles. [Figure 6]Figure 6 shows T22-NIDOmut2-H6 nanoparticles conjugated with oligo-FdU. A) Schematic of the two-step covalent attachment of thiol-linked oligo 5'-(FdU)5-hexaethyleneglycolthiol-3' (oligo-FdU-SH) via protein lysine amines using a 6-maleimidohexanoic acid N-hydroxysuccinimide ester (EMCS) bifunctional linker. B) MALDI-TOF mass spectrometry spectrum of the T22-NIDOmut2-H6-FdU nanocomplex. C) Volume size distribution of the T22-NIDOmut2-H6-FdU nanocomplex determined by DLS. [Figure 7] Figure 7 shows the cytotoxicity of oligo-FdU conjugated nanoparticles against CXCR4+ cells. The graph shows the percent viability of HeLa (CXCR4+) cells after 48 hours of 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 nanoconjugates, and 100 nM free oligo-FdU (FdU), as measured by an MTT viability assay. [Figure 8] Figure 8 shows that the T22-NIDOmut2-H6-FdU nanocomplex induces greater growth inhibition than T22-STM-H6-FdU or T22-GFP-H6-FdU in a CXCR4+ tumor model. The graph shows the time course of tumor volume for each nanocomplex-treated group (n=4) at 20 μg q3d x 5 doses, compared to buffer treatment (K, n=4) in a CXCR4+ M5 subcutaneous (SC) colorectal cancer (CRC) model. [Figure 9] Figure 9 shows that the T22-NIDOmut2-H6-FdU nanocomplex exhibits greater apoptosis induction than T22-STM-H6-FdU or T22-GFP-H6-FdU in CXCR4+M5 tumor tissue. A) The graph shows the number of apoptotic bodies observed in tumor sections from tumors treated with buffer or the nanocomplex at the end of the experiment. B) Representative photomicrographs of H&E-stained sections identifying apoptotic bodies (black arrows) observed in each group compared. [Figure 10]Figure 10 shows that there were no histological changes in the kidney or liver between the compared groups. Representative H&E stained sections of kidney and liver tissues show no structural or histological changes (no inflammatory or apoptotic features observed) in both organs at the end of treatment in control buffer-treated or nanocomplex-treated mice. [Figure 11] Figure 11 shows the correlation between intrinsic fluorescence and temperature for the isolated hexahistidine-tagged human nidogen G2 domain, the isolated hexahistidine-tagged human nidogen G2 domain containing the H459A, R468N, F639S, and R650A mutations, and stefin A. Arrows indicate sample heating. Unfolding curves for Nidomut2H6 (A), NidoWTH6 (C), and STMH6 (D) with CSM values ​​as a function of temperature. CSM values ​​were calculated from the experiment illustrated in Figure 1a and performed for each protein. Black and gray arrows indicate Tm and Tstart values, respectively. [Figure 12] Figure 12 shows the structure of the nidogen G2 domain. A) Schematic of the multidomain human nidogen 1 protein and its interaction with its natural ligand. The β-barrel structure within the G2 domain is shown. B) Secondary structure of the nidogen G2 β-barrel domain. Different β-sheets (A-K) are shown in gray and α-helices in blue. C) Schematic of the tertiary β-barrel structure 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 accession number 1QYO, version 1.2 from July 13, 2011) in the right panel. D) Superposition of the barrel structures of nidogen G2 and GFP β. [Figure 13]Figure 13 shows the design of the NidoMut2 peptide. A) Similarity of the human and mouse nidogen G2 domains: Amino acid alignment between the human nidogen 1 protein (P14543 in the Uniprot database, dated July 7, 2009) and the mouse nidogen 1 protein (P10493 in the Uniprot database, dated July 7, 2009) analyzed by Clustal Omega (EMBL-EMI). The black lines indicate the start and end of the G2 β barrel domain. "*" indicates amino acid matches, ":" indicates amino acids with high similarity, and "." indicates amino acids with low similarity. B) Amino acid sequences of the human nidogen 1 protein, HSNBT scaffold protein, and T22-HSNBT-H6 protein. In human nidogen, the G2 β barrel domain is highlighted in black, and candidate amino acids for mutation are shown in bold and underlined. In the sequence of NIDOmut2, the incorporated mutations are shown in bold black. In T22-NIDOmut2-H6, the N-terminal T22 ligand is underlined in black, the short linker is in bold black, the incorporated mutations are bold and underlined, and the C-terminal polyhistidine tail is highlighted in bold black italics. [Figure 14] FIG. 14 shows Western blots of the soluble fractions of cell lysates from expression studies of the proteins indicated in the figure. [Figure 15] Figure 15 shows MALDI-TOF mass spectrometry spectra of selected candidate proteins shown in each panel of the figure after expression testing. The theoretical size of all proteins is approximately 30.3 kDa. [Figure 16] FIG. 16 shows the volume size distribution of T22-NIDOmut2-H6 protein, T22-NIDOmut3-H6 protein, T22-NIDOmut4_T215V-H6 protein, and T22-NIDOmut5-H6 protein and 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 concentrations of ZnCl. Figure 17B shows the volumetric size distributions of T22-NIDOmut2-H6, T22-NIDOmut3-H6, T22-NIDOmut4_T215V-H6, and T22-NIDOmut5-H6 nanoparticles assembled at different ZnCl concentrations, as determined by DLS. [Figure 18] Figure 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. Graph showing key metrics (Tm, Tonset, and ΔT) for each buffer tested. [Figure 19] Figures 19A-B show MALDI-TOF mass spectra of T22-NIDOmut3-H6-FdU and T22-NIDOmut2-H6-FdU. Each peak, which represents an additional 2 kDa to the protein weight (30.3 kDa), belongs to the conjugated protein with an additional oligoFdU. C. Cytotoxicity assay after 48 hours of incubation of the T22-NIDOmut3-H6-FdU nanocomplex with the unconjugated negative control and the reference T22-NIDOmut2-H6-FdU positive control. D. Volumetric size distribution of T22-NIDOmut3-H6-FdU. [Figure 20]Figure 20 shows the structural and functional properties of EPI-X4-based NPs. A. Scheme of the modular proteins EPIX4-GFP-H6 (top) and EPIX4-(RK)-GFP-H6 (bottom) and the amino acid sequence of the latter. B. Mass spectrometry analysis of EPIX4-GFP-H6 (left) and EPIX4-(RK)-GFP-H6 (right). The molecular weight of the purified proteins was demonstrated by SDS-PAGE and Western blot (Anti-His). C. Hydrodynamic size and pdi (polydispersity index) determined by dynamic light scattering (DLS). Average peak size values ​​are shown (in nm). D. Size exclusion chromatography (SEC) of EPIX4-GFP-H6 (black) and EPIX4-(RK)-GFP-H6 (gray) using a Superdex 200 increase 10 / 300Gl column E. E. Representative FESEM (direct deposition) of EPIX4-GFP-H6 (top) and EPIX4-(RK)-GFP-H6 (bottom) protein NPs. Size bar represents 50 nm. F. Protein amount internalized into CXCR4+ HeLa cells after administration of 2 μM EPIX4-GFP-H6 and EPIX4-(RK)-GFP-H6 (dark green) at 4 h. Inhibition of uptake promoted by the natural CXCR4 ligand AMD3100 (light green). Intracellular fluorescence was corrected for their specific fluorescence to represent protein amount. Asterisks indicate significant differences in internalization of EPIX4-GFP-H6 and EPIX4-(RK)-GFP-H6 proteins, and hashes indicate significant differences in inhibition promoted by EPIX4-(RK)-GFP-H6 and AMD3100 (p≦0.001). G. Confocal images of HeLa cells exposed to EPIX4-GFP-H6 (left) and EPIX4-(RK)-GFP-H6 (right) for 24 hours. Blue: cell nucleus, red: cell membrane, green: internalized NPs. Size bar represents 10 μm. All data are expressed as mean ± SEM. [Figure 21]Figure 21 shows the formation and properties of biparatopic nanoparticles. A. Scheme of hybrid NP forming proteins EPIX4-(RK)-GFP-H6 (top) and T22-BFP-H6 (bottom). B. Controlled EPIX4-(RK)-GFP-H6 degraded with 0.2% SDS (black) and EPIX4-(RK)-GFP-H6 assembled by dialysis to remove SDS (gray) as determined by dynamic light scattering (DLS) (top). Hydrodynamic size comparison of T22-BFP-H6 (gray), EPIX4-(RK)-GFP-H6 (black), and biparatopic nanoNPs (gray dashed line) (bottom). Peak size values ​​(average) are shown in nm and Pdi (polydispersity index). C. Representative FESEM (direct deposition) of biparatopic NPs. Size bar represents 50 nm. D. FRET analysis of biparatopic NP formation. Samples of biparatopic NPs, a monomer mixture of T22-BFP-H6 and EPIX4-(RK)-GFP-H6, and a mixture of T22-BFP-H6 and EPIX4-(RK)-GFP-H6 NPs were excited with 405 nm light, and emission was collected between 350 and 650 nm. BFP was used as the donor fluorophore, and GFP was used as the acceptor. E. Time course kinetics of cellular internalization of EPIX4-(RK)-GFP-H6, T22-GFP-H6, and biparatopic NPs (1 μM) in CXCR4+ HeLa cells (left) and SW1417 cells (right). Intracellular fluorescence was corrected for specific fluorescence to represent protein abundance. Significant differences (p<0.05) between biparatopic NPs and both proteins forming them are indicated by *, and significant differences between biparatopic NPs and EPIX4-(RK)-GFP-H6 are indicated by #. E. Time course kinetics of cellular internalization of EPIX4-(RK)-GFP-H6, T22-GFP-H6, and biparatopic NPs (1 μM) in CXCR4+ HeLa cells (left) and SW1417 (right). Intracellular fluorescence was corrected for specific fluorescence to represent protein abundance. Significant differences (p<0.05) between biparatopic NPs and both proteins forming them are indicated by *, and significant differences between biparatopic NPs and EPIX4-(RK)-GFP-H6 are indicated by #.F. Uptake inhibition mediated by the CXCR4 antagonist AMD3100 (always at a 10:1 molar excess) in HeLa cells exposed to 1 μM for 1 h. & indicates significant difference between the inhibition promoted by NP and AMD3100 (p≦0.001). [Figure 22] Figure 22 shows in vivo biodistribution and toxicity assessment in a subcutaneous mouse model of CXCR4+ human colorectal cancer. A. Quantification of emitted fluorescence (measured as FLI ratio) in tumors at various time points. B. Number of apoptotic cell bodies after nanoparticle administration. Significant differences (p<0.05) between EPIX4-(RK)-GFP-H6 or biparatopic NPs versus control are indicated by *, and significant differences between biparatopic NPs and EPIX4-(RK)-GFP-H6 are indicated by #. C. Mitotic bodies after nanoparticle administration. Significant differences between EPIX4-(RK)-GFP-H6 or biparatopic NPs are indicated (*p<0.05). D. Lack of systemic toxicity in the kidney, liver, kidney, 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. DETAILED DESCRIPTION OF THE INVENTION

[0031] I—Polypeptides of the Invention The present inventors have found that the G2 domain from nidogen protein, optionally modified to exhibit reduced affinity for its natural ligand, can act as a scaffold, sufficiently stable to present peptides inserted into one or more loop regions connecting the β-strands within the G2 domain. Furthermore, the present inventors have found that when complexed with a ligand that exhibits affinity for a receptor expressed by a cell of interest, the G2 domain of the nidogen protein can be used to deliver agents of interest, including therapeutic agents and diagnostic / imaging agents, to the cell of interest. The present inventors have found that a fusion protein comprising the nidogen G2 domain and the CXCR4-specific ligand T22, linked to an anticancer agent, can deliver the anticancer agent to CXCR4-expressing cells and inhibit cell proliferation to a degree significantly greater than that achieved by a comparable agent in which the T22 ligand is fused to a different protein, such as GFP or Stefin A.

[0032] Thus, in a first aspect, the present invention provides a method for producing a medicament for the treatment of a pulmonary arthritis, comprising: (i) 11 beta-strand domains designated A, B, C, D, E, F, G, H, I, J, and K; and (ii) 10 loop regions connecting two consecutive β-strand 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 cognate loop region variant in SEQ ID NO:62, wherein the cognate 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 to 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 beta strand domains is a variant of the cognate beta strand in SEQ ID NO: 62 and has at least 50% sequence identity with the cognate beta strand domain in SEQ ID NO: 62, wherein the cognate beta strand domain in SEQ ID NO: 62 is defined as SEQ ID NO: 9 (beta strand domain A), SEQ ID NO: 11 (beta strand domain B), SEQ ID NO: 12 (beta strand domain C), SEQ ID NO: 13 (beta strand domain D), SEQ ID NO: 14 (beta strand domain E), SEQ ID NO: 15 (beta strand domain F), SEQ ID NO: 16 (beta strand domain G), SEQ ID NO: 17 (beta strand domain H), SEQ ID NO: 18 (beta strand domain I), SEQ ID NO: 19 (beta strand domain J), and SEQ ID NO: 20 (beta strand domain K). Concerning polypeptides.

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

[0034] As used herein, the term "polypeptide" generally refers to a linear chain of amino acid residues of any length joined by peptide bonds. As used herein, the term "peptide" refers to a linear chain of amino acids, like a polypeptide, but shorter than a polypeptide. Peptides generally refer to amino acid chains of 2 to 50 amino acids. It will be understood that the terms "peptide bond," "peptide," "polypeptide," and "protein" are known to those skilled in the art.

[0035] The polypeptides of the present invention are variants 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) under accession number P14543-1 (SEQ ID NO: 72).

[0037] The term "G2 domain of nidogen-1" as used herein refers to the G2 domain of the nidogen-1 protein as defined above. The G2 domain of nidogen-1 is as set forth in SEQ ID NO: 62, which corresponds to amino acids 430 to 667 of the amino acid sequence of the nidogen-1 protein, with identification number 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 set forth in SEQ ID NO: 64, which lacks the first two amino acids of SEQ ID NO: 62 and thus corresponds to the region consisting of amino acids 432 to 667 of the amino acid sequence of the nidogen-1 protein precursor (SEQ ID NO: 72) 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 purposes of the present invention, the G2 domain of nidogen-1 lacks the N- or C-terminal EGF-like domains.

[0038] In one embodiment, the polypeptides of the invention comprise an N-terminal methionine residue, hi another embodiment, the polypeptides of the invention do not comprise a methionine at the N-terminal position.

[0039] As used herein, "amino acid residue" refers to 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 consecutive and do not contain non-amino acids interrupting 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-strand," "β-strand domain," or "β-strand sequence" refer to an extended polypeptide chain or sequence connected to another polypeptide chain or sequence through hydrogen bonds between the NH group of one chain and the CO group of the other chain. β-strands are typically about 3-10 amino acids in length, but can be longer, e.g., 13-15 amino acids in length. As a result of these inter-strand connections, β-strands form a sheet-like protein secondary structure. Such protein secondary structures are referred to herein as "β-sheets." Within a β-sheet, β-strands can be arranged in a parallel, antiparallel, or mixed (parallel and antiparallel) fashion. When arranged parallel, β-strands align in the same direction from one end (N or C) to the other. When arranged antiparallel, each β-strand aligns in the opposite direction to the strand to which it is connected.

[0041] As used herein, the term "β-barrel" refers to a protein secondary structure formed by β-sheets, with the first and last strands connected by hydrogen bonds to form a closed toroidal structure.

[0042] The term "α-helix" or "alpha-helix" as used herein refers to a protein secondary structure consisting of a right-handed helix in which the NH group of an amino acid bonds a hydrogen to the C=O group of the backbone of the amino acid located three or four residues earlier in the protein sequence.

[0043] The phrase "α-helical portion" or "alpha-helical portion" as used herein refers to a motif in the secondary structure of a protein that substantially comprises one or more α-helices.

[0044] As will be understood by one of skill in the art, a beta strand as used herein refers to a protein domain, and a beta sheet as used herein refers to a secondary structure of a protein.

[0045] In certain embodiments, the 11 β-strand domains of the polypeptide of the invention comprise or form a β-sheet secondary structure, hi a preferred embodiment, the 12 β-strand domains of the polypeptide of the invention comprise or form 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-repetitive protein structural motif consisting of a polypeptide chain of six or more amino acid residues with any amino acid sequence. The residues constituting the beginning and end of the loop are adjacent to each other via a gap, without any intervening conventional secondary structural motifs. They typically connect two protein domains contained within a secondary protein structure, such as a beta strand, or a direct secondary protein structure, such as an alpha helix. Such loops often allow a protein domain or protein secondary structure connected to one end of the loop to change its orientation (N- to C- or C- 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 proteins and are therefore typically involved in interactions between the protein to which they belong and other molecules.

[0047] In certain embodiments, the polypeptide of the first aspect of the invention is a nidogen G2 domain mutant comprising 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 amino acids found in the cognate 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 the loop region that partially or completely replaces the sequence of the loop region.

[0048] The length of the heterologous polypeptide is not particularly limited. Thus, the heterologous polypeptide can 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.

[0049] In another embodiment, the polypeptide of the first aspect of the invention comprises a heterologous polypeptide in one or more loop regions and comprises a mutation in one or more beta strands, the mutation being at position 9 of beta strand B as defined in SEQ ID NO: 11 (corresponding to amino acid position 30 of SEQ ID NO: 62, or to amino acid position 459 of the human nidogen 1 precursor as defined in SEQ ID NO: 72, or the sequence of accession number P14543-1 in the Uniprot database (version dated July 7, 2009) or SEQ ID NO: 72), or at position 1 of beta strand C as defined in SEQ ID NO: 12 (corresponding to amino acid position 39 of SEQ ID NO: 62, or to amino acid position 459 of the human nidogen 1 precursor as defined in SEQ ID NO: 72, or the sequence of accession number P14543-1 in the Uniprot database (version dated July 7, 2009) or SEQ ID NO: 72). the amino acid at position 10 of beta chain J as defined in SEQ ID NO: 19 (corresponding to amino acid at position 210 of SEQ ID NO: 62 or to amino acid at position 639 of the human nidogen-1 precursor as defined in the Uniprot database (version dated July 7, 2009) under accession number P14543-1 or SEQ ID NO: 72); or the amino acid at position 3 of beta chain K as defined in SEQ ID NO: 20 (corresponding to amino acid at position 221 of SEQ ID NO: 62 or to amino acid at position 650 of the human nidogen-1 precursor as defined in the Uniprot database (version dated July 7, 2009) under accession number P14543-1 or SEQ ID NO: 72).

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

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

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

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

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

[0055] In another embodiment, the polypeptide of the first aspect of the invention is a nidogen G2 domain mutant as defined in any of the above embodiments, in particular NIDOmut4, further comprising a mutation of the threonine at position 619 (corresponding to position 190 in SEQ ID NO: 62). Preferably, the threonine at position 619 is mutated to a valine. In another embodiment, the polypeptide of the first aspect of the invention is a nidogen G2 domain mutant as defined in any of the above embodiments, in which the amino acid at position 619 (corresponding to position 190 in SEQ ID NO: 62) is the same residue as found in the human nidogen G2 domain defined in UniProt database accession number P14534, i.e., a valine. Thus, in one embodiment, the polypeptide of the first aspect of the invention is a nidogen G2 domain mutant having the following mutations: H459A, R468N, F639S, R650A, H543K, H545N, V449T, V525Q, and F561E. In one embodiment, the polypeptide of the first aspect of the 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 invention is a nidogen G2 domain mutant as defined in any of the above embodiments, in particular NIDOMut4, further comprising a mutation of the cysteine ​​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 invention is a nidogen G2 domain mutant having the following mutations: H459A, R468N, F639S, R650A, H543K, H545N, V449T, V525Q, V619T, F561E, and C618S. In one embodiment, the polypeptide of the first aspect of the invention is a nidogen G2 domain mutant 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 invention is a nidogen G2 domain mutant as defined in any of the above embodiments, in particular a NIDOMut3 mutant further comprising a mutation selected from the group consisting of: A mutation of the 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 invention is a nidogen G2 domain mutant having the sequence of SEQ ID NO: 95 (hereinafter referred to as NIDOmut3-V176T). - a mutation of the 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 invention is a nidogen G2 domain mutant having the sequence of SEQ ID NO: 96 (hereinafter referred to as NIDOmut3-I200T). - A mutation of 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 invention is a nidogen G2 domain mutant having the sequence of SEQ ID NO: 97 (hereinafter referred to as NIDOmut3-V236Y). - A mutation of the leucine at position -641 (corresponding to position 212 in SEQ ID NO: 62). Preferably, the leucine at position 641 is mutated to threonine. In one embodiment, the polypeptide of the first aspect of the invention is a nidogen G2 domain mutant having the sequence of SEQ ID NO: 98 (hereinafter referred to as NIDOmut3-L237T). -A mutation of the serine at position 469 (corresponding to position 40 in SEQ ID NO: 62). Preferably, the serine at position 469 is mutated to Ile. In one embodiment, the polypeptide of the first aspect of the invention is a nidogen G2 domain mutant having the sequence of SEQ ID NO: 99 (hereinafter referred to as NIDOmut3-S65I). -A mutation of the arginine at position -518 (corresponding to position 89 in 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 invention is a nidogen G2 domain mutant having the sequence of SEQ ID NO: 100 (hereinafter referred to as NIDOmut3-R114I). - a mutation of the cysteine ​​at position -618 (corresponding to position 189 in 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 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 invention is a nidogen G2 domain mutant as defined in any of the above embodiments, in particular a NIDOmut3 mutant further comprising a mutation at position 469 (preferably an S469I mutation) and a mutation at position 518 (preferably an R518I mutation). Thus, in one embodiment, the polypeptide of the first aspect of the invention is a nidogen G2 domain mutant comprising H459A, R468N, F639S, R650A, H543K, H545N, S469I and R518I mutations, 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 invention is a nidogen G2 domain mutant as defined in any of the above embodiments, in particular a NIDOmut5 mutant further comprising a mutation at position 469 (preferably an S469I mutation) and a mutation at position 518 (preferably an R518I mutation). Thus, in one embodiment, the polypeptide of the first aspect of the invention is a nidogen G2 domain mutant as defined in SEQ ID NO: 103 comprising H459A, R468N, F639S, R650A, H543K, H545N, V449T, V525Q, V619T, F561E, S469I, and R518I mutations (hereinafter referred to as NIDOmut5-S65I_R114I).

[0060] In some embodiments, the polypeptide of the first aspect of the invention is a nidogen G2 domain mutant as defined in any of the above embodiments, in particular a NIDOmut5 mutant further comprising a mutation of serine 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 invention is a nidogen G2 domain mutant having the following mutations: H459A, R468N, F639S, R650A, H543K, H545N, V449T, V525Q, V619T, F561E, and S469I. In one embodiment, the polypeptide of the first aspect of the invention is a nidogen G2 domain mutant 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 invention is a nidogen G2 domain mutant as defined in any of the above embodiments, in particular a NIDOmut5 mutant further comprising a mutation of the 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 invention is a nidogen G2 domain mutant having the following mutations: H459A, R468N, F639S, R650A, H543K, H545N, V449T, V525Q, V619T, F561E, and R518I. In one embodiment, the polypeptide of the first aspect of the invention is a nidogen G2 domain mutant having the sequence of SEQ ID NO: 104 (hereinafter referred to as NIDOmut5-R114I).

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

[0063] A heterologous polypeptide can be inserted within the loop region, i.e., the loop region preserves all amino acids found in the cognate loop domain of SEQ ID NO:62 or SEQ ID NO:63, but the heterologous polypeptide is inserted between two consecutive amino acids. The length of the heterologous polypeptide is not particularly limited. Thus, the heterologous polypeptide can 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, the heterologous polypeptide can replace a portion of the loop region, i.e., the loop region contains a deletion relative to the sequence of the cognate loop domain of SEQ ID NO:62 or SEQ ID NO:63, and the deleted sequence is replaced with the heterologous polypeptide and inserted between two consecutive amino acids. It will be understood that the length of the deletion need not be the same as the length of the heterologous peptide. Thus, the loop region can 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. Thus, a heterologous polypeptide can comprise 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.

[0065] When the nidogen G2 domain mutant comprises multiple heterologous polypeptides, the heterologous polypeptides may be found within the same loop region, or preferably, may be found in different loop regions. Furthermore, when the nidogen G2 domain mutant comprises multiple heterologous polypeptides, the heterologous polypeptides may be the same or different.

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

[0067] As used herein, the terms "binding," "bond," and "binds" refer to the interaction of affinity binding molecules or specific binding pairs as a result of non-covalent bonds such as, but not limited to, hydrogen bonds, hydrophobic interactions, van der Waals bonds, ionic bonds, or combinations of the above.

[0068] When used in the present invention 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 due to complementarity between the three-dimensional structures of both the polypeptide and the target molecule, such that binding between the polypeptide and the target molecule occurs before binding of the polypeptide to other molecules present in the vicinity of the polypeptide, as in the case of a reaction mixture. The ability of a polypeptide to specifically bind to a target molecule in a reaction mixture can be tested, for example, by assessing the binding of the polypeptide to the target molecule of interest and several related molecules that are closer or more distant (structurally and / or functionally) under conventional conditions. Only if a polypeptide binds to a target molecule but does not or substantially does not bind to other related molecules close to the target molecule, is said binding considered to be specific for the target molecule. Binding between a polypeptide and a target molecule is considered to be specific when the binding affinity between them is 10 or higher. -6 Under M, 10 -7 Under M, 10 -8 Under M, 10-9 Under M, 10 -10 Under M, 10 -11 Under M, 10 -12 Under M, 10 -13 Under M, 10 -14 Less than M or 10 -15 If the dissociation constant (KD) is less than M, it can be considered specific. The method for determining the binding between polypeptide and target molecule and the KD of said binding includes methods well known by experts in the field.Non-limiting examples of such methods include gel shift assays such as electrophoretic mobility shift assay (EMSA), co-immunoprecipitation assays followed by mass spectrometry, gas chromatography associated with mass spectrometry, liquid chromatography associated with mass spectrometry, or Western blot analysis.Another method is the oil cushion method [see Hesselgesset et al., 1998, J.Immunol., 160:877-883].

[0069] In certain embodiments, binding of the polypeptide to the target molecule is determined by measuring the binding between the polypeptide and the target molecule in increments of 10. -6 Under M, 10 -7 Under M, 10 -8 Under M, 10 -9 Under M, 10 -10 Under M, 10 -11 Under M, 10 -12 Under M, 10 -13 Under M, 10 -14 Less than M or 10 -15 A loop region is considered specific if it has a dissociation constant (KD) of less than 10 M. Similarly, binding between a loop region and a specific target molecule is considered specific if the binding between the loop region and a specific target molecule is less than 10 M. -6 Under M, 10 -7 Under M, 10 -8 Under M, 10 -9 Under M, 10 -10 Under M, 10 -11 Under M, 10 -12 Under M, 10 -13 Under M, 10 -14 Less than M or 10 -15If it has a dissociation constant (KD) less than M, it is considered specific.

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

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

[0072] As used herein, the phrase "cognate loop region in SEQ ID NO: 62" refers to the loop region that appears in SEQ ID NO: 62, the wild-type nidogen G2 domain of human origin. As will be understood by one of skill in the art, each loop region of the polypeptide of the first aspect of the invention has its cognate 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 show a certain degree of sequence identity and if they encode the same type of protein domain or secondary protein structure, i.e., if they both form a β-strand, a loop region, an α-helix, an α-helical portion, a β-sheet, or a β-barrel. In certain embodiments, amino acid sequences encoding two protein domains or secondary protein structures that are considered to be identical exhibit a degree of sequence identity 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%, or at least 99.99%. The degree of identity between two amino acid sequences can be determined by conventional methods, for example, by standard sequence alignment algorithms known in the art, such as 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 specific domain or protein secondary structure are well known to those skilled in the art, and include methods for determining the secondary structure of a protein using bioinformatics tools such as DSSPcont (Carter, Andersen & Rost, 2003) and STRIDE (Heinig & Frishman, 2004), after the atomic coordinates of the protein have been obtained according to methods well known to those skilled in the art, such as X-ray crystallography or protein NMR.

[0074] The cognate loop region in SEQ ID NO:62 in loop region AB comprises, substantially comprises, or consists of SEQ ID NO:1. In another specific embodiment, the cognate loop region in SEQ ID NO:62 in loop region BC comprises, substantially comprises, or consists of SEQ ID NO:2. In another specific embodiment, the cognate loop region in SEQ ID NO:62 in loop region CD comprises, substantially comprises, or consists of SEQ ID NO:3. In another specific embodiment, the cognate loop region in SEQ ID NO:62 in loop region DE comprises, substantially comprises, or consists of SEQ ID NO:4. In another specific embodiment, the cognate loop region in SEQ ID NO:62 in loop region EF comprises, substantially comprises, or consists of SEQ ID NO:5. In another specific embodiment, the cognate loop region in SEQ ID NO:62 in loop region FG comprises, substantially comprises, or consists of SEQ ID NO:6. In another specific embodiment, the cognate loop region in SEQ ID NO:62 in loop region GH comprises, substantially comprises, or consists of amino acids 149-150 of SEQ ID NO:62. In another specific embodiment, the cognate loop region in SEQ ID NO:62 of loop region HI comprises, substantially comprises, or consists of SEQ ID NO: 7. In another specific embodiment, the cognate loop region in SEQ ID NO:62 of loop region IJ comprises, substantially comprises, or consists of SEQ ID NO: 8. In another specific embodiment, the cognate loop region in SEQ ID NO:62 of loop region JK comprises, substantially comprises, or consists of SEQ ID NO:9.

[0075] In a particular embodiment, the loop region of the polypeptide of the first aspect of the invention is its cognate loop region variant in SEQ ID NO:62.

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

[0077] Thus, in certain embodiments, at least one loop region variant of the polypeptide of the first aspect of the invention is generated by mutation by deletion, substitution or addition of at least one amino acid in the sequence of its cognate loop region in SEQ ID NO:62.

[0078] In certain embodiments, loop region variants of the polypeptide of the first aspect have a degree of 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%, at least 99% to the sequence of the cognate loop region in SEQ ID NO: 62. Methods for determining the degree of sequence identity between two sequences are as described above.

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

[0080] In a particular embodiment, the sequence of the AB loop region of a nidogen G2 domain variant of the invention is a variant of SEQ ID NO: 1, while the sequences of 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 of the BC, CD, DE, EF, FG', GH, HI, IJ, and JK loop regions of the polypeptide of the first aspect of the 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 the sequences of 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 loop regions of the polypeptide of the first aspect of the invention are 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 the remaining sequence of the polypeptide of the first aspect of the invention is identical to the remaining sequence 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%, or at least 99% sequence identity to SEQ ID NO:1.

[0082] In another particular embodiment, the sequence of the loop region AB of the polypeptide of the first aspect of the invention is SEQ ID NO:1.

[0083] In a particular embodiment, 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 the sequences of 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 of the AB, CD, DE, EF, FG, GH, HI, IJ, and JK loop regions of the polypeptide of the first aspect of the invention are the sequences of their cognate loop regions in SEQ ID NO: 62, as set forth above. In another particular embodiment, the sequence of loop region BC is a variant of SEQ ID NO: 2, and the sequences of 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 of the loop regions of the polypeptide of the first aspect of the invention are the 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 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%, or at least 99% sequence identity to SEQ ID NO:2.

[0085] In another particular embodiment, the sequence of the loop region BC of the polypeptide of the first aspect of the invention is SEQ ID NO:2.

[0086] In a particular embodiment, 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 the sequences of 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 AB, BC, DE, EF, FG, GH, HI, IJ, and JK loop regions of the first aspect of the invention are the sequences of their cognate loop regions in SEQ ID NO: 62, as set forth 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 of the sequences of the loop regions of the polypeptide of the first aspect of the invention have the 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 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%, or at least 99% sequence identity to SEQ ID NO:3.

[0088] In another particular embodiment, the sequence of the loop region CD of the polypeptide of the first aspect of the invention is SEQ ID NO:3.

[0089] In a specific embodiment, the sequence of loop region CD is a cognate domain variant of SEQ ID NO: 62, in which the cognate region is modified in a loop region that does not exhibit α-helical structure. As shown by Hopf et al. (supra), the CD loop region of a wild-type G2 domain contains three regions with α-helical 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-strand 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-strand D (hereinafter referred to as the αD region), respectively. In one embodiment, the Cα region comprises, essentially comprises, or consists of SEQ ID NO: 24. In one embodiment, the αD region comprises, essentially comprises, or consists of the amino acid GG. In one embodiment, the loop region sequence is a variant of the cognate region of SEQ ID NO: 3, where the sequences of SEQ ID NOs: 21, 22, and 23 are conserved with respect to the cognate region. In another embodiment, the loop region sequence is a variant of the cognate region of SEQ ID NO: 3, and includes one or more mutations in the Cα region. In another embodiment, the loop region sequence is a variant of the cognate region of SEQ ID NO: 3, and includes one or more mutations in the αD region. In another embodiment, the loop region sequence is a variant of the cognate region of SEQ ID NO: 3, and includes one or more mutations in the Cα and αD regions.

[0090] In certain embodiments, the Cα region in a 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%, or at least 99% sequence identity to SEQ ID NO:24.

[0091] In another particular embodiment, the sequence of the loop region Cα of the polypeptide of the first aspect of the invention is SEQ ID NO: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%, or at least 99% sequence identity to a sequence consisting of the amino acids GG.

[0093] In a particular embodiment, 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 the sequences of 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 AB, BC, CD, EF, FG, GH, HI, IJ, and JK loop regions of the polypeptide of the first aspect of the invention comprise the sequences of their cognate loop regions in SEQ ID NO: 62, as set forth above. In another particular embodiment, the sequence of loop region DE is a variant of SEQ ID NO: 4, and the sequences of 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 loop regions of the polypeptide of the first aspect of the 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 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%, or at least 99% sequence identity to SEQ ID NO:4.

[0095] In another particular embodiment, the sequence of the loop region DE of the polypeptide of the first aspect of the invention is SEQ ID NO:4.

[0096] In a particular embodiment, 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 the sequences of 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 AB, BC, CD, DE, FG, GH, HI, IJ, and JK loop regions of the first aspect of the invention are the sequences of their cognate loop regions in SEQ ID NO: 62, as set forth above. In another particular embodiment, the sequence of loop region EF is a variant of SEQ ID NO: 5, and the sequences of 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 loop regions of the polypeptide of the first aspect of the invention are the 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 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%, or at least 99% sequence identity to SEQ ID NO:5.

[0098] In another particular embodiment, the sequence of the loop region EF of the polypeptide of the first aspect of the invention is SEQ ID NO: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 the sequences of their cognate loop regions in SEQ ID NO: 62, as set forth 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 regions of the polypeptide of the first aspect of the invention are the 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 invention is identical to the remaining sequence of SEQ ID NO: 62.

[0100] In certain embodiments, the loop region FG variant of SEQ ID NO: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%, or at least 99% sequence identity to SEQ ID NO:6.

[0101] In another particular embodiment, the sequence of the loop region FG of the polypeptide of the first aspect of the invention is SEQ ID NO:6.

[0102] In a specific embodiment, the sequence of loop region GH is a variant of 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 specific embodiment, the sequence of loop region GH is a variant of the sequence corresponding to amino acids 149-150 of SEQ ID NO: 62, wherein 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, FG, HI, IJ, and JK of the polypeptide of the first aspect of the invention are the sequences of their cognate loop regions in SEQ ID NO: 62 as shown above. In another specific embodiment, the sequence of loop region GH is a variant of the sequence corresponding to amino acids 149-150 of SEQ ID NO: 62, wherein 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 regions of the polypeptide of the first aspect of the invention are the sequences of their cognate loop regions in SEQ ID NO: 62 as shown above. In another specific embodiment, the sequence of the loop region GH is a variant of the sequence corresponding to amino acids 149 to 150 of SEQ ID NO: 62, and the remaining sequence of the polypeptide of the first aspect of the invention is the remaining sequence of SEQ ID NO: 62.

[0103] In certain embodiments, 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%, or at least 99% sequence identity to 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 of the first aspect of the present invention is a sequence corresponding to amino acids 149 to 150 of SEQ ID NO:62.

[0105] In a particular embodiment, 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 the sequences of 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 AB, BC, CD, DE, EF, FG, GH, IJ, and JK of the polypeptide of the first aspect of the invention are the sequences of their cognate loop regions in SEQ ID NO: 62, as set forth above. In another particular embodiment, the sequence of loop region HI is a variant of SEQ ID NO: 7, and the sequences of 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 loop regions of the polypeptide of the first aspect of the invention are the 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 sequence of the polypeptide of the first aspect of the invention is identical to the remaining sequence 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%, or at least 99% sequence identity to SEQ ID NO:7.

[0107] In another particular embodiment, the sequence of the loop region HI of the polypeptide of the first aspect of the invention is SEQ ID NO:7.

[0108] In a particular embodiment, 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 the sequences of 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 AB, BC, CD, DE, EF, FG, GH, HI, and JK loop regions of the polypeptide of the first aspect of the invention are the sequences of their cognate loop regions in SEQ ID NO: 62, as set forth above. In another particular embodiment, the sequence of loop region IJ is a variant of SEQ ID NO: 8, and the sequences of 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 loop regions of the polypeptide of the first aspect of the invention are the 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 invention are identical to the remaining sequences of SEQ ID NO: 62.

[0109] In certain embodiments, the loop region IJ variant of SEQ ID NO: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%, or at least 99% sequence identity to SEQ ID NO:8.

[0110] In another particular embodiment, the sequence of the loop region IJ of the polypeptide of the first aspect of the invention is SEQ ID NO:8.

[0111] In a particular embodiment, 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 the sequences of 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 AB, BC, CD, DE, EF, FG, GH, HI, and IJ of the polypeptide of the first aspect of the invention are the sequences of their cognate loop regions in SEQ ID NO: 62, as set forth above. In another particular embodiment, the sequence of loop region JK is a variant of SEQ ID NO: 9, and the sequences of 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 loop regions of the polypeptide of the first aspect of the invention are the 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 sequence of the polypeptide of the first aspect of the invention is identical to the remaining sequence of SEQ ID NO: 62.

[0112] In certain embodiments, the loop region JK variant of SEQ ID NO: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%, or at least 99% sequence identity to SEQ ID NO:10.

[0113] In another particular embodiment, the sequence of the loop region JK of the polypeptide of the first aspect of the invention is SEQ ID NO:9.

[0114] In certain embodiments, at least one of the sequences located upstream, near upstream, downstream or near downstream of the loop region variant of the polypeptide of the first aspect of the invention comprises 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 in the same position with respect to the cognate 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 of the first aspect of the invention as its reference sequence in the polypeptide having SEQ ID NO: 62. The methods for determining whether two amino acid sequences form the same protein domain or secondary structure, such as a loop region, an α-helical portion or an α-helix, are methods for determining whether two amino acid sequences form the same protein structure as described above.

[0115] In certain embodiments, at least one sequence comprising one or more amino acid modifications, insertions and / or deletions relative 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%, at least 99% sequence identity with the reference sequence in SEQ ID NO: 62. Methods for determining the degree of sequence identity between two amino acid sequences are as described above.

[0116] As used herein, the phrase "cognate β-strand domain in SEQ ID NO: 62" refers to a β-strand domain of SEQ ID NO: 62 located between amino acid sequences of a protein having SEQ ID NO: 62 between which a β-strand domain variant encodes the same loop region or protein secondary structure as the sequence in which the polypeptide of the first aspect of the invention is located. As will be understood by one of skill in the art, each β-strand domain of the first polypeptide of the invention has its cognate β-strand domain in SEQ ID NO: 62.

[0117] Each β-strand domain of the nidogen G2 domain variant may be identical to the cognate β-strand in SEQ ID NO: 62 or may differ by one or more amino acids such that the overall sequence identity between the β-strand of the nidogen G2 domain variant and the cognate β-strand domain in SEQ ID NO: 62 may be at least 50%. In preferred embodiments, the sequence identity between the β-strand of the nidogen G2 domain variant and the cognate β-strand 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, beta-strand domain A is a beta-strand domain variant of its cognate beta-strand domain in SEQ ID NO: 62. In certain embodiments, beta-strand domain B is a beta-strand domain variant of its cognate beta-strand domain in SEQ ID NO: 62. In certain embodiments, beta-strand domain C is a beta-strand domain variant of its cognate beta-strand domain in SEQ ID NO: 62. In certain embodiments, beta-strand domain D is a beta-strand domain variant of its cognate beta-strand domain in SEQ ID NO: 62. In certain embodiments, beta-strand domain E is a beta-strand domain variant of its cognate beta-strand domain in SEQ ID NO: 62. In certain embodiments, beta-strand domain F is a beta-strand domain variant of its cognate beta-strand domain in SEQ ID NO: 62. In certain embodiments, beta-strand domain G is a beta-strand domain variant of its cognate beta-strand domain in SEQ ID NO: 62. In certain embodiments, beta-strand domain H is a beta-strand domain variant of its cognate beta-strand domain in SEQ ID NO: 62. In certain embodiments, beta-strand domain I is a beta-strand domain variant of its cognate beta-strand domain in SEQ ID NO: 62. In certain embodiments, beta-strand domain J is a beta-strand domain variant of its cognate beta-strand domain in SEQ ID NO: 62. In certain embodiments, beta-strand domain K is a beta-strand domain variant of its cognate beta-strand domain in SEQ ID NO: 62.

[0119] In a particular embodiment, the beta strand domain of the polypeptide of the first aspect of the invention is a variant of its cognate beta strand in SEQ ID NO:62.

[0120] As used herein, the expression "beta strand domain variant" refers to a beta strand domain from the polypeptide of the first aspect of the invention which comprises in its sequence one or more amino acid modifications, insertions and / or deletions relative to the sequence of its cognate beta strand domain in SEQ ID NO: 62. In certain embodiments, the beta strand 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, a beta strand domain variant of the polypeptide of the first aspect has a degree of 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%, at least 99% to the sequence of its cognate beta strand domain in SEQ ID NO: 62. Methods for determining the degree of sequence identity are as described above.

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

[0123] Thus, in a particular embodiment, the sequence of beta-strand A is a variant of SEQ ID NO: 10. In another particular embodiment, the sequence of beta-strand 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 beta-strand domains B through K of the polypeptide of the first aspect of the invention have the sequence of their cognate beta-strand domain in SEQ ID NO: 62 shown above. In another particular embodiment, the sequence of beta-strand 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 beta-strand domains of the polypeptide of the first aspect of the invention have the sequence of their cognate beta-strand domain in SEQ ID NO: 62. In another particular embodiment, the sequence of beta-strand A is a variant of SEQ ID NO: 10, and the remaining sequence of the polypeptide of the first aspect of the invention is identical to the remaining sequence of SEQ ID NO: 62.

[0124] In certain embodiments, the beta strand 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%, or at least 99% sequence identity to SEQ ID NO: 11.

[0125] In another particular embodiment, the sequence of beta strand domain A of the polypeptide of the first aspect of the invention is SEQ ID NO:10.

[0126] In a particular embodiment, the sequence of beta strand B is a variant of SEQ ID NO: 11. In another particular embodiment, the sequence of beta strand 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 A, C-K beta strand domains of the polypeptide of the first aspect of the invention have the sequence of their cognate beta strand domains in SEQ ID NO: 62 shown above. In another particular embodiment, the sequence of beta strand 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 beta strand domains of the polypeptide of the first aspect of the invention have the sequence of their cognate beta strand domains in SEQ ID NO: 62. In another particular embodiment, the sequence of beta strand B is a variant of SEQ ID NO: 11, and the remaining sequence of the polypeptide of the first aspect of the invention is identical to the remaining sequence of SEQ ID NO: 62.

[0127] In certain embodiments, the beta strand 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%, or at least 99% sequence identity to SEQ ID NO: 12.

[0128] In another particular embodiment, the sequence of the beta strand domain B of the polypeptide of the first aspect of the invention is SEQ ID NO:11.

[0129] In a particular embodiment, the sequence of beta strand C is a variant of SEQ ID NO: 12. In another particular embodiment, the sequence of beta strand 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 A, B, D-K beta strand domains of the polypeptide of the first aspect of the invention have the sequence of their cognate beta strand domain in SEQ ID NO: 62 shown above. In another particular embodiment, the sequence of beta strand 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 beta strand domains of the polypeptide of the first aspect of the invention have the sequence of their cognate beta strand domain in SEQ ID NO: 62. In another particular embodiment, the sequence of beta strand C is a variant of SEQ ID NO: 12, and the remaining sequence of the polypeptide of the first aspect of the invention is identical to the remaining sequence of SEQ ID NO: 62.

[0130] In certain embodiments, the beta strand 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%, or at least 99% sequence identity to SEQ ID NO: 12.

[0131] In another particular embodiment, the sequence of beta strand domain C of the polypeptide of the first aspect of the invention is SEQ ID NO:12.

[0132] In a particular embodiment, the sequence of beta strand D is a variant of SEQ ID NO: 13. In another particular embodiment, the sequence of beta strand 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 beta strand domains A-C, E-K of the polypeptide of the first aspect of the invention have the sequence of their cognate beta strand domain in SEQ ID NO: 62 shown above. In another particular embodiment, the sequence of beta strand 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 beta strand domains of the polypeptide of the first aspect of the invention have the sequence of their cognate beta strand domain in SEQ ID NO: 62. In another particular embodiment, the sequence of beta strand D is a variant of SEQ ID NO: 13, and the remaining sequence of the polypeptide of the first aspect of the invention is identical to the remaining sequence of SEQ ID NO: 62.

[0133] In certain embodiments, the beta strand 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%, or at least 99% sequence identity to SEQ ID NO: 13.

[0134] In another particular embodiment, the sequence of beta strand domain D of the polypeptide of the first aspect of the invention is SEQ ID NO:13.

[0135] In a particular embodiment, the sequence of beta-strand E is a variant of SEQ ID NO: 14. In another particular embodiment, the sequence of beta-strand 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 beta-strand domains A-D, F-K of the polypeptide of the first aspect of the invention have the sequence of their cognate beta-strand domain in SEQ ID NO: 62 shown above. In another particular embodiment, the sequence of beta-strand 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 beta-strand domains of the polypeptide of the first aspect of the invention have the sequence of their cognate beta-strand domain in SEQ ID NO: 62. In another particular embodiment, the sequence of beta-strand E is a variant of SEQ ID NO: 14, and the remaining sequence of the polypeptide of the first aspect of the invention is identical to the remaining sequence of SEQ ID NO: 62.

[0136] In certain embodiments, the beta strand 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%, or at least 99% sequence identity to SEQ ID NO: 14.

[0137] In another particular embodiment, the sequence of the beta strand domain E of the polypeptide of the first aspect of the invention is SEQ ID NO:14.

[0138] In a particular embodiment, the sequence of beta-strand F is a variant of SEQ ID NO: 15. In another particular embodiment, the sequence of beta-strand 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 beta-strand domains A-E, G-K of the polypeptide of the first aspect of the invention have the sequence of their cognate beta-strand domain in SEQ ID NO: 62 shown above. In another particular embodiment, the sequence of beta-strand 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 beta-strand domains of the polypeptide of the first aspect of the invention have the sequence of their cognate beta-strand domain in SEQ ID NO: 62. In another particular embodiment, the sequence of beta-strand F is a variant of SEQ ID NO: 15, and the remaining sequence of the polypeptide of the first aspect of the invention is identical to the remaining sequence of SEQ ID NO: 62.

[0139] In certain embodiments, the beta strand 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%, or at least 99% sequence identity to SEQ ID NO: 15.

[0140] In another particular embodiment, the sequence of the beta strand domain F of the polypeptide of the first aspect of the invention is SEQ ID NO:15.

[0141] In a particular embodiment, the sequence of beta strand G is a variant of SEQ ID NO: 16. In another particular embodiment, the sequence of beta strand 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 beta strand domains A-F and H-K of the polypeptide of the first aspect of the invention have the sequence of their cognate beta strand domain in SEQ ID NO: 62 shown above. In another particular embodiment, the sequence of beta strand 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 beta strand domains of the polypeptide of the first aspect of the invention have the sequence of their cognate beta strand domain in SEQ ID NO: 62. In another particular embodiment, the sequence of beta strand G is a variant of SEQ ID NO: 16, and the remaining sequence of the polypeptide of the first aspect of the invention is identical to the remaining sequence of SEQ ID NO: 62.

[0142] In certain embodiments, the beta strand 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%, or at least 99% sequence identity to SEQ ID NO: 16.

[0143] In another particular embodiment, the sequence of the beta strand domain G of the polypeptide of the first aspect of the invention is SEQ ID NO:16.

[0144] In a particular embodiment, the sequence of beta-strand H is a variant of SEQ ID NO: 17. In another particular embodiment, the sequence of beta-strand 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 beta-strand domains A-G, I-K of the polypeptide of the first aspect of the invention have the sequence of their cognate beta-strand domain in SEQ ID NO: 62 shown above. In another particular embodiment, the sequence of beta-strand 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 beta-strand domains of the polypeptide of the first aspect of the invention have the sequence of their cognate beta-strand domain in SEQ ID NO: 62. In another particular embodiment, the sequence of beta-strand H is a variant of SEQ ID NO: 17, and the remaining sequence of the polypeptide of the first aspect of the invention is identical to the remaining sequence of SEQ ID NO: 62.

[0145] In certain embodiments, the beta strand 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%, or at least 99% sequence identity to SEQ ID NO: 17.

[0146] In another particular embodiment, the sequence of the beta strand domain H of the polypeptide of the first aspect of the invention is SEQ ID NO:17.

[0147] In a particular embodiment, the sequence of beta-strand I is a variant of SEQ ID NO: 18. In another particular embodiment, the sequence of beta-strand 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 beta-strand domains A-H, J, K of the polypeptide of the first aspect of the invention have the sequence of their cognate beta-strand domain in SEQ ID NO: 62 shown above. In another particular embodiment, the sequence of beta-strand 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 beta-strand domains of the polypeptide of the first aspect of the invention have the sequence of their cognate beta-strand domain in SEQ ID NO: 62. In another particular embodiment, the sequence of beta-strand I is a variant of SEQ ID NO: 18, and the remaining sequence of the polypeptide of the first aspect of the invention is identical to the remaining sequence of SEQ ID NO: 62.

[0148] In certain embodiments, the beta strand 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%, or at least 99% sequence identity to SEQ ID NO: 19.

[0149] In another particular embodiment, the sequence of beta strand domain I of the polypeptide of the first aspect of the invention is SEQ ID NO:18.

[0150] In a particular embodiment, the sequence of beta strand J is a variant of SEQ ID NO: 19. In another particular embodiment, the sequence of beta strand 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 beta strand domains A-I, K of the polypeptide of the first aspect of the invention have the sequence of their cognate beta strand domain in SEQ ID NO: 62 shown above. In another particular embodiment, the sequence of beta strand 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 beta strand domains of the polypeptide of the first aspect of the invention have the sequence of their cognate beta strand domain in SEQ ID NO: 62. In another particular embodiment, the sequence of beta strand J is a variant of SEQ ID NO: 19, and the remaining sequence of the polypeptide of the first aspect of the invention is identical to the remaining sequence of SEQ ID NO: 62.

[0151] In certain embodiments, the beta strand 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%, or at least 99% sequence identity to SEQ ID NO:19.

[0152] In another particular embodiment, the sequence of the beta strand domain J of the polypeptide of the first aspect of the invention is SEQ ID NO:19.

[0153] In a particular embodiment, the sequence of beta-strand K is a variant of SEQ ID NO: 20. In another particular embodiment, the sequence of beta-strand 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 beta-strand domains A to J of the polypeptide of the first aspect of the invention have the sequence of their cognate beta-strand domain in SEQ ID NO: 62 shown above. In another particular embodiment, the sequence of beta-strand 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 beta-strand domains of the polypeptide of the first aspect of the invention have the sequence of their cognate beta-strand domain in SEQ ID NO: 62. In another particular embodiment, the sequence of beta-strand K is a variant of SEQ ID NO: 20, and the remaining sequence of the polypeptide of the first aspect of the invention is identical to the remaining sequence of SEQ ID NO: 62.

[0154] In certain embodiments, the beta strand 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%, or at least 99% sequence identity to SEQ ID NO:20.

[0155] In another particular embodiment, the sequence of the beta strand domain K of the polypeptide of the first aspect of the invention is SEQ ID NO:20.

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

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

[0158] In preferred embodiments, the sequence identity between the α-helical portion CαD of the nidogen G2 domain variant and the cognate α-helical 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] Methods for determining the degree of sequence identity are as described above. In certain embodiments, the α-helical portion CαD, which differs by one or more amino acids as indicated above, is referred to as an α-helical portion variant of the cognate α-helical portion in SEQ ID NO:62.

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

[0161] The β-barrel domain of the G2 domain of nidogen-1 and the β-barrel structure of the sequence of SEQ ID NO: 62 are composed of an 11-stranded β-barrel. The β-strands of the β-barrel are designated herein as I, II, III, IV, V, VI, VII, VIII, IX, X, and XI. They correspond to the cognate β-strands of SEQ ID NO: 62 of β-strands A-K of the polypeptide of the first embodiment. The interior of the β-barrel is traversed by a hydrophobic, primarily α-helical segment connecting strands III and IV. The N-terminal half of the barrel is composed of two β-meanders (strands I-III and IV-VI) connected by a buried α-helical segment. The polypeptide chain then traverses the bottom of the barrel, forming a five-stranded Greek key motif in the C-terminal half of the domain.

[0162] As used herein, the term "β-meander" refers to two or more consecutive antiparallel β-strands connected by a hairpin loop. As used herein, the term "hairpin loop" refers to two antiparallel strands connected by a short loop of 2-5 residues, one of which is often glycine or proline, either of which can assume the dihedral-angle conformation required 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 which three antiparallel strands are connected by hairpins, and the fourth strand is adjacent to the first strand and connected to the third strand by a longer loop.

[0164] Thus, in certain embodiments, the β-strands A to C and the β-strands D to F of the G2 domain variant form a β-meander. In another specific embodiment, the β-meander is connected by the α-helical portion CαD of the G2 domain variant. In another specific embodiment, the β-strands G to K of the G2 domain variant form a five-stranded Greek key motif. In another specific embodiment, the β-strands of the G2 domain variant, except for the β-strands A and F, are arranged antiparallel.

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

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

[0167] As used herein, the phrase "polypeptide display library" refers to a library or pool of polypeptides comprising a plurality of polypeptides having different amino acid sequences, each polypeptide of the library being as defined in the first aspect of the invention and differing from at least another polypeptide of the library in the sequence of one or more loop regions.

[0168] As used herein, the phrase "polypeptides that differ in sequence in one or more loop regions" refers to the fact that each polypeptide in the library exhibits at least one difference in amino acid sequence relative to the amino acid sequence of at least one other polypeptide in the library, wherein said at least one difference is within the amino acid sequence of the loop region of the polypeptide. Thus, in certain embodiments, the polypeptides in the library are polypeptides of the first aspect that comprise at least one loop region variant defined in the first aspect that differs 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 definitions and embodiments of "loop region variant" of the first aspect of the invention.

[0169] As used herein, the phrase "exhibiting at least one difference in amino acid sequence relative to the amino acid sequence of at least one other polypeptide in the library, said sequence being comprised in the amino acid sequence of the loop region of the polypeptide" refers to the fact that the loop region variant as defined above of a first polypeptide in the library contains at least one insertion, deletion, or modification of at least one amino acid in its sequence relative to the amino acid sequence of the corresponding loop region in a 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, 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, 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, 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, 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, the corresponding loop region in the second polypeptide is also loop region EF in the second polypeptide. If the loop region variant in the first polypeptide is loop region FG, the corresponding loop region in the second polypeptide is also loop region FG in the second polypeptide. If the loop region variant in the first polypeptide is loop region GH, the corresponding loop region in the second polypeptide is also loop region GH in the second polypeptide. If the loop region variant in the first polypeptide is loop region HI, the corresponding loop region in the second polypeptide is also loop region HI in the second polypeptide. If the loop region variant in the first polypeptide is loop region IJ, the corresponding loop region in the second polypeptide is also loop region IJ in the second polypeptide.If the loop region variant in the first polypeptide is the loop region JK, then the corresponding loop region in the second polypeptide is also the loop region JK in the second polypeptide.

[0170] In certain embodiments, the loop region variants of a first polypeptide of the library exhibit a degree of sequence identity to the corresponding loop region of a second polypeptide of the library 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%, or at least 99%. In another specific embodiment, the entire sequence of the first polypeptide exhibits a degree of sequence identity with the entire sequence of the second polypeptide 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%, or at least 99.999%. Methods for determining the percentage of sequence identity are as described in the first aspect of the invention.

[0171] In certain embodiments, a first polypeptide of the library exhibits differences in amino acid sequence in at least two loop regions, at least three loop regions, at least four loop regions, at least five loop regions, at least six loop regions, at least seven loop regions, at least eight loop regions, at least nine loop regions, at least ten loop regions, or at least eleven loop regions relative to a corresponding loop region in at least one other polypeptide of the library, referred to as a 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 differences in amino acid sequence 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, also as defined above.

[0172] In certain embodiments, the polypeptides of the library can specifically bind to other molecules, preferably peptides or proteins, present in their vicinity, for example, in a sample, via at least one of their loop regions. In a preferred embodiment, the polypeptides of the library that exhibit differences in the amino acid sequence of one or more of their corresponding loop regions specifically bind to different molecules, preferably peptides or proteins, present in their vicinity, for example, in a sample. As will be understood by those skilled in the art, the difference in their binding ability may be that one polypeptide can specifically bind to one molecule, preferably a peptide or protein of the sample, while another polypeptide of the library with differences in one or more loop regions cannot specifically bind to the one molecule, preferably a peptide or protein of the sample. Alternatively, one polypeptide may specifically bind to one or more molecules, preferably peptides or proteins, while another polypeptide of the library with differences in one or more loop regions cannot specifically bind to the one or more molecules, but can bind to one or more other molecules, preferably peptides present in the sample. In certain embodiments, a first polypeptide of the library is capable of specifically binding to a target molecule of interest, preferably a peptide or protein of interest, by virtue of at least one loop region variant that exhibits a difference in its sequence relative to the corresponding loop region in a second polypeptide of the library, whereas the second polypeptide of the library is not capable of specifically binding to said target molecule.

[0173] In another particular embodiment, a loop region variant of a first polypeptide of the library specifically binds to a target peptide, whereas its cognate loop region in SEQ ID NO: 62 is unable to specifically bind to said target peptide.

[0174] In another specific embodiment, polypeptides in a polypeptide library having identical loop region variants 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 aspect of the invention. Methods for determining binding between a polypeptide and a target molecule, and the K of such binding, are also described in the above definitions.

[0176] In certain embodiments, binding of a polypeptide of the library to a target molecule is determined by measuring the binding between the polypeptide and the target molecule in a manner that is greater than or equal to 10. -6 Under M, 10 -7 Under M, 10 -8 Under M, 10 -9 Under M, 10 -10 Under M, 10 -11 Under M, 10 -12 Under M, 10 -13 Under M, 10 -14 Less than M or 10 -15 A dissociation constant (K d Similarly, binding between a loop region, preferably a loop region variant, and a particular target molecule is considered specific if the binding between the loop region and the target molecule is 10 -6 Under M, 10 -7 Under M, 10 -8 Under M, 10 -9 Under M, 10 -10 Under M, 10 -11 Under M, 10 -12 Under M, 10 -13 Under M, 10 -14Less than M or 10 -15 If it has a dissociation constant (KD) less than M, it is considered specific.

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

[0178] The term "genotype," as used herein, refers to a nucleic acid molecule that encodes one or several peptides, polypeptides, or proteins, or a nucleic acid molecule that contains a coding sequence. A group of peptides, polypeptides, or proteins corresponds to a 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 that encodes a single peptide, polypeptide, or protein. In this case, the phenotype corresponds to the peptide, polypeptide, or protein. The nucleic acid can be any of the nucleic acids specified in the definition of nucleic acid below.

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

[0180] The definitions of the terms peptide, polypeptide and protein are set out in the first aspect of the invention.

[0181] The terms "nucleic acid," "nucleotide sequence," or "polynucleotide" are used interchangeably herein and refer to the polymeric form of ribonucleoside phosphates (adenosine, guanosine, uridine, or cytidine; "RNA molecules") or deoxyribonucleosides (deoxyadenosine, deoxyguanosine, deoxythymidine, or deoxycytidine; "DNA molecules") or any of their phosphoester analogs, such as phosphorothioates and thioesters, in single- or double-stranded form. Thus, the term encompasses single-stranded DNA or RNA molecules. It also encompasses double-stranded molecules formed by DNA-DNA, DNA-RNA, 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 a particular type of tertiary structure. Thus, the 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, the nucleic acid is an RNA molecule.

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

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

[0184] The expression "a phenotype directly or indirectly linked to a nucleic acid as a genotype corresponding to the phenotype," as used herein, is understood as a polypeptide of a polypeptide display library (i.e., a phenotype as defined above) linked to a nucleic acid encoding it (i.e., a genotype as defined above). The linkage 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, a polypeptide display library is formed by a complex comprising a polypeptide of the first aspect of the present invention linked directly or indirectly to a nucleic acid encoding it. The polypeptide is considered to be the phenotype, and the nucleic acid is considered to be the genotype corresponding to the phenotype.

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

[0186] Each polypeptide in the library is referred to as a "library member," regardless of whether it is part of a complex as described above. Thus, as used herein, this term refers to any peptide in the library, where the polypeptide and the nucleic acid may be directly or indirectly linked, and the nucleic acid may encode or contain a sequence encoding the polypeptide, or may simply not be linked by any means to a nucleic acid containing a sequence encoding it. Thus, in certain embodiments, the library member is a polypeptide of the library as defined above. In another specific embodiment, the library member 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 of the library and the nucleic acid encoding it, resulting in a polypeptide-nucleic acid complex in which the polypeptide binds or covalently links to the nucleic acid encoding it, and the polypeptide is 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 include additional proteins and / or nucleic acids.

[0188] In certain embodiments, the association between the polypeptide and nucleic acid of the complex is direct, hi other specific embodiments, the association between the polypeptide and nucleic acid of the complex is indirect, where the polypeptide is bound or covalently linked 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," or "covalently coupled" refer to an interaction between two molecules that are directly covalently bonded to each other through a covalent chemical bond, or that are indirectly covalently bonded to each other through one or more intervening moieties, such as a linker, bridge, or spacer.

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

[0191] As used herein, the term "linker moiety" or "linker" refers to a molecule that links two molecules or compounds. It is also intended that the linking moiety is not limited in terms of its chemical nature and / or structure, and thus, the linking moiety may be, among others, a polysaccharide, a polypeptide, a fatty acid, a 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 the molecules, may be bonded to the linker via any chemical bond, such as a peptide bond, an isopeptide bond, an amide bond, an imine bond, etc.

[0192] In certain embodiments, the polypeptide display library is formed by a complex comprising a polypeptide of the first aspect of the invention directly linked to a nucleic acid encoding the polypeptide, the polypeptide-nucleic acid complex comprising: a complex comprising a polypeptide of the library bound to a nucleic acid encoding it, - a complex consisting of a polypeptide of the library and an additional protein, peptide and / or nucleic acid bound to a nucleic acid encoding it, -polypeptide-nucleic acid complexes, -ribosome or part of a ribosome is selected from the group consisting of:

[0193] As used herein, the term "complex" refers to any compound resulting from the covalent bonding of two or more individual compounds or molecules, a covalent bond as defined above. By definition, a complex is never found in nature.

[0194] The individual compounds covalently linked to the complex of the second aspect of the invention are a library polypeptide and its encoding nucleic acid. Thus, in certain embodiments, the complex of the second aspect of the invention comprises a library polypeptide attached directly to the encoding nucleic acid via a chemical covalent bond. In another specific embodiment, the complex of the second aspect of the invention comprises a library polypeptide attached to the encoding nucleic acid via an intervening moiety or moieties, such as a linker, bridge, spacer, moiety or moieties. In certain embodiments, they are attached via a linker.

[0195] As used herein, the term "ribosome" refers to the highly complex cellular machinery essential for protein synthesis. Ribosomes bind amino acids in the order specified by messenger RNA (mRNA) molecules. Ribosomes are composed of specialized RNA known as ribosomal RNA (rRNA) and dozens of different proteins (the exact number varies by species). Ribosomal proteins and rRNA are generally arranged into two distinct ribosomal fragments of different sizes known as the large and small ribosomal subunits.

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

[0197] In another specific embodiment, the polypeptide-nucleic acid complex of the second aspect of the invention comprises on its outer surface only one polypeptide of the library. In another specific embodiment, the polypeptide-nucleic acid complex of the second aspect of the invention comprises 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 x 10 3 In a particular embodiment, the complex contains copies of a polypeptide from the library. In a particular embodiment, the copies have the same amino acid sequence. In another particular embodiment, the complex does not contain other polypeptides from the library.

[0198] In another specific embodiment, the polypeptide-nucleic acid complex of the second aspect of the invention comprises only one nucleic acid encoding the amino acid sequence of a polypeptide of the library contained in the complex, said polypeptide and said polypeptide sequence being as specified in the above embodiments.

[0199] In certain embodiments, the complex of the second aspect of the invention comprises 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, or at least 100 copies of nucleic acids encoding the amino acid sequences of the polypeptides of the library comprised in the complex. The polypeptides and polypeptide sequences are as specified in the above embodiments. In certain embodiments, the nucleic acids have the same nucleotide sequence.

[0200] Indirect linkage consists of genetic fusion between the polypeptides and nucleic acids of the library, with the microorganism containing both the polypeptides and nucleic acids. The polypeptides of the library are contained on the outer surface of the microorganism. The nucleic acids are preferably contained internally of the microorganism.

[0201] In certain embodiments, the microorganism comprises only one polypeptide of the library on its outer surface. In certain embodiments, the microorganism comprises 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 x 10 3 In certain embodiments, the copies have the same amino acid sequence. In another particular embodiment, the microorganism is free of other polypeptides from the library.

[0202] In another specific embodiment, the microorganism comprises only one nucleic acid encoding the amino acid sequence of a polypeptide of the library contained in the microorganism, said polypeptide and said polypeptide sequence being as specified in the above embodiments.

[0203] In another specific embodiment, the microorganism comprises 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, or at least 100 copies of a nucleic acid encoding an amino acid sequence of a polypeptide from a library contained in the microorganism. The polypeptides and polypeptide sequences are as specified in the above embodiments. In a specific embodiment, the nucleic acids have the same nucleotide sequence.

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

[0205] In certain embodiments, the microorganism is selected from the group consisting of a phage, a bacteriophage, a virus, a bacterium, and a yeast. In a preferred embodiment, 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 Enterobacteriaceae phage M13, T4 bacteriophage, T7 bacteriophage, or Escherichia lambda virus.

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

[0208] III - Polynucleotides, Vectors and Host Cells In a third aspect, the present invention relates to a polynucleotide encoding a polypeptide according to the first aspect of the invention, or a polynucleotide encoding a polypeptide of a polypeptide display library according to the second aspect of the invention.

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

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

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

[0212] As used herein, the term "vector" refers to a vehicle by which a polynucleotide or DNA molecule can be manipulated or introduced into a cell. A vector may be a linear or circular polynucleotide, or any other type of construct, such as a larger polynucleotide, or DNA or RNA of a viral genome, a virion, or other biological construct that allows for the manipulation or introduction of DNA into a cell. It is understood that the terms "recombinant vector" and "recombinant system" are used interchangeably with the term vector. Those skilled in the art will understand that there is no limitation regarding the type of vector that can be used, as the vector can be a cloning vector suitable for propagation and obtaining a polynucleotide or an appropriate genetic construct or expression vector in a different heterologous organism suitable for producing the polynucleotide of the present invention.Therefore, suitable vectors according to the present invention include expression vectors in prokaryotes such as pET (such as pET14b), pUC18, pUC19, Bluescript and their derivatives, mp18, mp19, pBR322, pMB9, CoIE1, pCR1, RP4, phages, and shuttle vectors such as pSA3 and pAT28, expression vectors in yeast such as vectors of the 2 micron plasmid type, integrative plasmids, YEP vectors, centromeric plasmids, expression vectors in insect cells such as pAC series vectors and pVL series vectors, expression vectors in plants such as pIBI, pEarleyGate, pAVA, pCAMBIA, pGSA, pGWB, pMDC, pMY, pORE series vectors, and viral vectors (adenoviruses, viruses related to adenoviruses, viruses related to retroviruses, viruses related to lentiviruses) and pSilencer 4.1-CMV (Ambion), pcDNA3, pcDNA3.1 / hyg. Included are expression vectors in higher eukaryotic cells based on non-viral vectors such as pHCMV / Zeo, pCR3.1, pEF1 / His, pIND / GS, pRc / HCMV2, pSV40 / Zeo2, pTRACER-HCMV, pUB6 / V5-His, pVAX1, pZeoSV2, pCI, pSVL, and pKSV-10, pBPV-1, pML2d, and pTDT1.

[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 cell can be a prokaryotic or eukaryotic organism. For example, the vector into which the DNA sequence is introduced can be a plasmid or vector that, when introduced into a host cell, is integrated into the genome of the cell and replicates together with the chromosome (or chromosomes) into which it is integrated. 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. 1-3 a).

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

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

[0216] Suitable host cells for containing the polynucleotide of the third aspect of the invention or the vector of the fourth aspect of the invention include, but are not limited to, mammalian, plant, insect, fungal, and bacterial cells. Bacterial cells include, but are not limited to, Gram-positive bacterial cells such as Bacillus, Streptomyces, Listeria, and Staphylococcus species, and Gram-negative bacterial cells such as Escherichia, Salmonella, and Pseudomonas cells. Fungal cells preferably include yeast cells such as Saccharomyces cerevisiae, Pichia pastoris, and Hansenula polymorpha. Insect cells include, but are not limited to, Drosophila and Sf9 cells. Plant cells include cells of crop plants such as cereals, medicinal plants, ornamental plants or bulbous plants, among others. Suitable mammalian cells in the present invention include epithelial cell lines (human, ovine, porcine, etc.), osteosarcoma cell lines (human, etc.), neuroblastoma cell lines (human, etc.), epithelial carcinoma (human, etc.), glial cells (mouse, etc.), hepatic 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 line 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 invention are equally applicable to the third aspect of the invention. In another embodiment, all terms and embodiments described in the first, second and third aspects of the invention are equally applicable to the fourth aspect of the invention. In another specific embodiment, all terms and embodiments of the first, second, third and fourth aspects of the invention are equally applicable to the fifth aspect of the invention.

[0218] IV - Conjugates of the Invention In a further aspect, the present invention provides a method for producing a pharmaceutical composition comprising: (i) a polypeptide comprising the G2 domain of nidogen-1 or a functionally equivalent variant thereof; and (ii) the agent of interest The present invention relates to a complex comprising:

[0219] A polypeptide forming part of a complex and comprising the G2 domain of nidogen-1 or a functionally equivalent variant thereof (as identified in point (i) above) is also referred to as a "polypeptide of the complex of the sixth aspect of the invention", a "polypeptide of the complex of the invention" or a "polypeptide of the complex".

[0220] The G2 domain of nidogen-1 or a functionally equivalent variant thereof contained 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," "first polypeptide region of the complex," "first polypeptide region," or "first polypeptide region contained in the polypeptide of the complex."

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

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

[0223] As used herein, the term "agent of interest" refers to any compound, without limitation of chemical structure, provided that it can be covalently bound to the polypeptide of the complex. In certain embodiments, the agent is a therapeutic agent. In another specific embodiment, the agent is an imaging agent. The terms "therapeutic agent" and "imaging agent" are defined in Sections IV-E.1 and IV-E.2 below.

[0224] IV.A - First Polypeptide of the Complex The complexes of the present invention comprise a polypeptide comprising the G2 domain of nidogen-1 or a functionally equivalent variant thereof.

[0225] The term "nidogen-1" as used herein is defined in the above context with respect to variants of the nidogen G2 domain and applies equally to the complexes 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 adjacent to a short EGF-like domain. However, for purposes of the present invention, the G2 domain of nidogen-1 is formed by amino acids 430 to 667 of the amino acid sequence of the nidogen-1 protein identified as P14543-1 (SEQ ID NO: 62) in the Uniprot database (version dated July 7, 2009), and lacks an EGF-like domain at the N- 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 thus corresponds to the region consisting of amino acids 432 to 667 of the amino acid sequence of the nidogen-1 protein identified as P14543-1 (SEQ ID NO: 72) in the Uniprot database (version dated July 7, 2009).

[0227] The expression "functionally equivalent variant" as used herein refers to any peptide that shows a certain 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 that substantially maintains the function of the G2 domain of nidogen-1. The function of the G2 domain that is maintained in the complex of the sixth aspect of the present invention is preferably considered to be the tertiary structure of the domain when it is not part of the complex. Thus, functionally equivalent variants of the G2 domain preferably substantially maintain the tertiary structure of the G2 domain of nidogen-1 when it is not part of the complex. As will be understood by those skilled in the art, the tertiary structure of the G2 domain that is maintained is preferably the tertiary structure of the β-barrel domain of the G2 domain of nidogen-1. Said structure is as 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 beta-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 in the domain that maintain their relative position relative to the remaining amino acids of the domain within the tertiary structure of the domain.Methods for determining the tertiary structure of a protein, which allow determining the atomic coordinates of the protein, are well known to those skilled in the art, and include circular dichroism analysis, X-ray crystallography and protein NMR.

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

[0231] In certain embodiments, the degree of sequence identity between the G2 domain of nidogen1 having SEQ ID NO: 62 and a functionally equivalent variant 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 variant 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 the first aspect of the invention.

[0232] Once incorporated into the complex, the first polypeptide region of the complex need not maintain the cellular or physiological function of the nidogen G2 domain. Thus, in certain embodiments, the first polypeptide region is the G2 domain of nidogen-1 or a functionally equivalent variant thereof that has reduced physiological function upon incorporation into the fusion protein of the invention. In another embodiment, the first polypeptide region is the G2 domain of nidogen-1 or a functionally equivalent variant thereof that does not have the physiological function of G2 of nidogen-1 or the physiological function of the β-barrel domain of the G2 domain of nidogen-1 outside the complex. In a preferred embodiment, the first polypeptide region is a functionally equivalent variant of the nidogen G2 domain that has reduced physiological function compared to the wild-type G2 domain of nidogen-1 or compared to the wild-type β-barrel domain of the G2 domain of nidogen-1 prior to incorporation into the complex of the invention. More preferably, the first polypeptide region is a protein that does not have 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 prior to incorporation into the complex of the present invention due to the presence of an inactivating mutation.

[0233] As used herein, the phrase "physiological function" or "cellular function" refers to the function of a peptide within a cell or organism. Thus, 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 phrase is understood to refer to the function of the domain when it is part of the nidogen-1 protein. Thus, the phrase refers to the role of the domain in the biochemical pathway or molecular mechanism in which the nidogen-1 protein participates in the cell. Thus, the phrase directly relates to the ability of the domain to interact with a specific peptide or protein on the outer surface of the cell, the outside of the cell, or another cell. Thus, in certain embodiments, the phrase refers to the ability of the G2 domain or the β-barrel domain of the G2 domain to interact with a common protein binding partner. Non-limiting examples of such binding partners include collagen IV and perlecan. Thus, in certain embodiments, the first polypeptide of the complex is a functionally equivalent variant of the G2 domain of nidogen-1 that comprises 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 a normal binding partner of the G2 domain of nidogen-1 or the β-barrel domain of the G2 domain of nidogen-1, 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 mutant of the inactive G2 domain of nidogen-1. In a specific embodiment, the first polypeptide of the complex is inactive once incorporated into the complex. In another specific embodiment, the first polypeptide of the complex is already inactive before being incorporated into the complex.

[0235] As used herein, the term "inactive" when referring to a polypeptide, protein, protein fragment, or domain is understood to mean a polypeptide, protein, fragment, or domain that has no physiological or biological activity or the ability to specifically interact with other macromolecules for biological function, and a protein fragment or domain that lacks known therapeutic activity (e.g., antitumor activity). The inactive polypeptide portion of the complex is non-reactive and serves as a physical structure for binding to the agent of interest. An inactive polypeptide is intended to contain no motifs that have inherent enzymatic, physiological, or biological activity of their own, and to be non-immunoreactive, i.e., to stimulate neither the adaptive nor the innate immune response.

[0236] Generally, the intrinsic activity of the first polypeptide of the conjugate is irrelevant for purposes of the present invention and is not intended 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 any of the polypeptides described in the first aspect of the invention. Thus, in certain embodiments, the polypeptide of the complex is a functionally equivalent variant of the G2 domain of nidogen-1 as any of the polypeptides described in the first aspect of the invention.

[0238] In another specific embodiment, the first polypeptide region has a sequence corresponding to amino acids 430 to 667 of the sequence of human nidogen-1 as defined in the Uniprot database (version dated July 7, 2009) under accession number P14543-1, i.e., SEQ ID NO: 62. In another specific embodiment, the first polypeptide region has a sequence corresponding to amino acids 432 to 667 of the sequence of human nidogen-1 as defined in the Uniprot database (version dated July 7, 2009) under accession number P14543-1, i.e., SEQ ID NO: 63.

[0239] In another particular embodiment, the first polypeptide of the complex is a functionally equivalent variant of the G2 domain of nidogen-1 comprising a mutation 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 numbering of the sequence of human nidogen-1 as defined in the UniProt database (version dated July 7, 2009) under accession number P14543-1. Thus, in another particular embodiment, the polypeptide of the complex of the sixth aspect of the invention is a functionally equivalent variant of the G2 domain of nidogen-1 comprising a mutation 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 numbering of the sequence of human nidogen-1 as defined in the UniProt database (version dated July 7, 2009) under accession number P14543-1.

[0240] The term "mutation" as used herein 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 position of an amino acid modification or deletion is referred to by the position of the modified or deleted amino acid in the amino acid sequence before the mutation.In a specific embodiment, when the mutation is an insertion, the position of the mutation is defined by referring to the amino acid at the N-terminus of the inserted amino acid.In another specific embodiment, when the mutation is an insertion, the position of the mutation is defined by referring to the amino acid at the C-terminus of the inserted amino acid. Therefore, as will be understood by those skilled in the art, mutation of 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 set forth above refers to modification or deletion of the amino acid at said positions in the amino acid sequence of human nidogen-1 as defined in the UniProt database (version dated July 7, 2009) under accession number P14543-1. In a specific embodiment, it refers to insertion of at least one amino acid at the C-terminus of one or more of said amino acids. In another specific embodiment, it refers to insertion of at least one amino acid at the N-terminus of one or more of said amino acids.

[0241] In a preferred embodiment, the mutation is an amino acid modification. In a specific 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 specific embodiment, the mutation at position 650 shown above is an R650A mutation.

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

[0243] Suitable nidogen G2 domain mutants 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, NIDOmut3-I, NIDOmut4_I, NIDOmut5 ... and any of the nidogen G2 domain mutants defined above in the context of the first aspect of the invention, including mutants carrying NIDOmut3-S65I, 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 of the sixth aspect of the invention optionally comprises a second polypeptide region capable of specifically binding to a target of interest.

[0245] The second polypeptide region capable of specifically binding to a target of interest is also referred to as the "second polypeptide of the complex," the "second polypeptide region of the complex," the "second polypeptide region," or the "second polypeptide region comprised in the polypeptide of the complex."

[0246] The term "specifically binds" is defined in the second aspect of the invention. In a particular embodiment, the second polypeptide of the complex binds specifically to the first polypeptide of the complex. -6 Under M, 10 -7 Under M, 10 -8 Under M, 10 -9 Under M, 10 -10 Under M, 10 -11 Under M, 10 -12 Under M, 10 -13 Under M, 10 -14 Under M, 10 -15 A dissociation constant (K D ) is considered to specifically bind a target of interest. Methods for determining whether a polypeptide is capable of binding to a target molecule, and for determining the dissociation constant of said binding, are provided in the definition of "specific binding" of the second aspect of the invention.

[0247] In certain embodiments, the second polypeptide domain in the complex of the invention is a ligand for a cellular receptor. The term "cellular receptor" or "cell surface receptor" refers to a cell-associated protein that binds to a "ligand." Non-limiting examples of cellular receptors and agents of interest that are specific ligands for particular receptors, or the cell types to which they bind, are shown in Table 2 below. [Table 2-1] [Table 2-2]

[0248] In certain embodiments, 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 promote the internalization of the complex in the cell. When referring to the second polypeptide of the complex, the phrase "promoting the internalization of the complex in the cell" refers to a polypeptide that binds to a receptor on the cell surface that undergoes endocytosis in response to binding of the polypeptide. This binding specificity allows the second polypeptide of the complex, and the remainder of the complex containing it, to be delivered to cells, tissues, or organs that express the receptor. In this way, the complex containing the polypeptide region is specifically targeted to the cells when administered to an animal or contacted with a population of different types of cells in vitro.

[0250] As used herein, "internalization" refers to the process in 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, along with the molecule or construct, may then be localized to a specific cellular compartment. Preferably, the second polypeptide of the complex of the present invention promotes endosomal escape of the complex in addition to promoting internalization.

[0251] As used herein, the phrase "promotes endosomal escape" refers to the ability of a second polypeptide of the complex to induce release of the complex from an endosomal compartment following internalization by receptor-mediated endocytosis.

[0252] The ability of a complex of the present invention to be internalized by cells expressing a receptor to which the second polypeptide of the complex binds can be conveniently determined by fluorescence techniques if the polypeptide of the complex 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 polypeptide of the complex is fused in frame with the fluorescent protein and expressed in a suitable host cell or organism. The fusion protein can then be contacted with a culture of cells expressing the aforementioned receptor or with tissue expressing the receptor in vivo for an appropriate period of time, after which a fluorescence microscope can be used to determine whether the construct has penetrated 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 with a known cytoplasmic stain.

[0253] A wide variety of uptake receptors and carriers are known in the art, with many more receptor-specific ligands.

[0254] Non-limiting examples of receptors that can be targeted by the second polypeptide are provided above.

[0255] In certain embodiments, the second polypeptide of the complex is a polycationic peptide. As used herein, the term "polycationic peptide" or "polycationic region" refers to a polypeptide sequence containing multiple positively charged amino acids. The polycationic peptide may be formed solely by positively charged amino acids, or 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 they can be grouped according to their properties.Therefore, at physiological pH, five amino acids exhibit charge; arginine, histidine and lysine are positively charged, and aspartic acid and glutamic acid are negatively charged.Those skilled in the art will understand that the polycationic peptide of the present invention corresponds to a polypeptide that has a net charge of two or more positive charges under physiological pH conditions.Therefore, the polycationic peptide of the present invention is 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 produce two or more net positive charges.

[0257] Thus, in one embodiment of the present invention, the polycationic peptide of the present invention comprises: (i) a sequence capable of specifically interacting with a cell surface receptor to promote internalization of the complex into the cell; (ii) arginine-rich sequences; (iii) GW-H1 peptide, (iv) CD44 ligand, (v) peptides that can cross the blood-brain barrier; (vi) a cell-penetrating peptide, and (vii) nucleolin-binding peptide is selected from the group consisting of:

[0258] (i) a sequence capable of specifically binding to a cell surface receptor and promoting internalization of the complex into the cell; As used herein, the term "a sequence capable of specifically binding to a receptor on the surface of a cell to promote internalization of a complex into said cell" refers to any sequence encoding a polypeptide capable of specifically binding to a target of interest, said target being a receptor on the surface of a cell, as defined above, and wherein the polypeptide encoded by said sequence promotes internalization of a complex into said cell, as defined above.

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

[0260] Non-limiting examples of receptors that can be targeted by the polycationic peptide of the present invention, preferably by a sequence that can specifically bind to the above-mentioned cell surface receptors, 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 invention, a polycationic peptide comprising a sequence capable of specifically binding to a receptor on the cell surface and promoting 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 an important role in immune and inflammatory responses by mediating the directional migration and activation of leukocytes. CXCR4 is expressed or overexpressed in various cancer cells and tissues, including breast, prostate, ovarian, colon, colorectal, pancreatic, kidney, and brain, as well as in non-Hodgkin's 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 an important role in multiple stages of tumorigenesis, including tumor growth, invasion, angiogenesis, and metastasis.

[0263] The term "specifically binds" is defined in the second aspect of the present invention. As will be understood by those skilled in the art, the term "specifically binds to CXCR4" as used herein 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 can be measured, for example, by any of the methods provided in the definition of "specifically bind" in the second aspect of the present invention, preferably by the oil cushion method described by Tamamura et al. (See Hesselgesset et al., 1998, J. Immunol., 160:877-883). The method comprises contacting the peptide with a CXCR4-transfected cell line (e.g., CHO cells) and a labeled CXCR4 ligand (e.g., 125I-SDF-1α), and measuring the percentage inhibition of the target peptide against the binding of the labeled CXCR4 ligand.

[0265] Specific binding can occur, for example, when CXCR4 has multiple binding sites for ligands and ligands with low affinity can be useful for targeting, e.g., at least about 10 -4 Specific binding can be demonstrated by a low affinity targeting agent having a Kd of at least about 10 M. Specific binding can also be demonstrated by a high affinity ligand, e.g., a targeting agent having a Kd of at least about 10 M. -7 M, at least about 10 -8 M, at least about 10 -9 M, at least about 10 -10 M, or at least about 10 -11 M or 10 -12 It may have a Kd of equal to or greater than M. Both low and high affinity targeting ligands are useful for incorporation into the conjugates of the invention.

[0266] The ability of the complexes of the present invention to be internalized by cells expressing CXCR4 can be determined by fluorescence techniques in which the complex contains a fluorescent protein, such as GFP, as described above for any second polypeptide of the complex that binds to any cellular receptor. More specifically, complexes that are internalized by cells expressing CXCR4 can be obtained by preparing a recombinant nucleic acid in which a nucleic acid encoding a polycationic peptide is fused in frame with a nucleic acid encoding a fluorescent protein and expressed in a suitable host cell or organism. The fusion protein can then be contacted with a culture of cells expressing CXCR4 or with tissues expressing CXCR4 in vivo for an appropriate period of time, after which a fluorescence microscope can be used to determine whether the construct has penetrated 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 with known cytoplasmic stains.

[0267] In an even more preferred embodiment of the present invention, the CXCR4 ligand is selected from the group consisting of RRWCYRKCYKGYCYRKCR (SEQ ID NO: 25), the V1 peptide (SEQ ID NO: 26), the CXCL12 peptide (SEQ ID NO: 27), the vCCL2 peptide (SEQ ID NO: 28), the EPI-X4 sequence (SEQ ID NO: 29), or a functionally equivalent variant thereof, such as 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 polyphemusin II (extracted from blood cell debris from the American 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-terminus of vCCL2. CXCL12, a C-X-C motif chemokine 12, also known as stromal cell-derived factor 1 (SDF1), is a member of the chemokine family that functions as a proinflammatory 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 to bind to the CXCR4 receptor (Zirafi et al., 2015, Cell Reports, 11:1-11). In one embodiment, an optimized EPI-X4 tandem version (SEQ ID NO: 132) with higher receptor affinity and serum stability is used.

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

[0271] The terms "functional variant" and "functionally equivalent variant" are interchangeable and are herein understood as any peptide derived from the T22, V1, CXCL12, vCCL2 and / or EPI-X4 peptide 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 cationic polypeptides exhibit a degree of identity with human T22, V1, CXCL12, vCCL2, and / or EPI-X4 peptides, such as 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 to the respective SEQ ID NOs. Methods for determining the degree of identity between two amino acid sequences are provided in the first aspect of the invention. The cationic polypeptides of the invention may contain post-translational modifications such as glycosylation, acetylation, isoprenylation, myristoylation, and proteolytic processing.

[0273] Alternatively, suitable functional variants of cationic polypeptides include those containing, at one or more positions, amino acids that are conservative substitutions for the amino acids present in the T22, V1, CXCL12, vCCL2, and / or EPI-X4 peptides. A "conservative amino acid substitution" is a substitution of one amino acid with another amino acid that has similar structural and / or chemical properties. For example, the following six groups each contain amino acids that are conservative substitutions for 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 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 its functionally equivalent variant is, for example, the following: a putative T22, V1, CXCL12, vCCL2, or EPI-X4 peptide variant is fused in frame with a marker polypeptide (e.g., a fluorescent protein). Such a fusion protein can be obtained by preparing a recombinant nucleic acid in which a nucleic acid encoding the peptide is fused in frame with a nucleic acid encoding the fluorescent protein and expressed in a suitable host cell or organism. The fusion protein is then contacted with a culture of CXCR4 cells (e.g., HeLa cells) for an appropriate period of time, after which fluorescence microscopy can be used to determine whether the construct has penetrated 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 fluorescence microscopy images obtained from the fluorescent protein with images obtained with a known cytoplasmic stain (e.g., DAPI).

[0275] (ii) arginine-rich sequences As mentioned above, arginine amino acid and its residues exhibit a positive charge at physiological pH. It is understood that "arginine-rich sequence" refers to a polypeptide sequence containing multiple arginine residues. Thus, 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 in its complete sequence as arginine residues. It is understood that whenever an arginine-rich sequence contains less than 100% of the sequence as arginine residues, these residues do not all need to be adjacent or contiguous to each other.

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

[0277] In an embodiment of the invention, the polycationic peptide of the invention is 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]. Although 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. GW-H1 has been proposed to exert its cytolytic activity by folding into an amphipathic helix [Chen et al., supra]. Therefore, this peptide is thought to exert its cytolytic effect through two sequential events: binding to the cell membrane and subsequent permeation.

[0280] In a preferred embodiment of the present invention, the polycationic peptide of the present invention is the 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, 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, which are cell-specifically expressed and differentially glycosylated.

[0282] Thus, a "CD44 ligand" is a molecule capable of binding to CD44. CD44 has other ligands, including chondroitin sulfate, the major surface receptor for hyaluronic acid, a component of the extracellular matrix, the heparin-binding domain of fibronectin, osteopontin, serglycin, collagen, and laminin. Additionally, CD44 can interact with metalloproteinases and selectins.

[0283] In an embodiment of the present invention, the polycationic peptide of the present invention is a CD44 ligand. In a preferred embodiment 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 fibronectin HBFN fragment V. Peptide A5G27 corresponds to a peptide of the α5 chain 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 the art that one major obstacle to developing therapeutic approaches for brain pathologies 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 thought to be the primary route of serum ligand uptake, as its surface area is approximately 5,000 times that of the BCSFB. The brain endothelium, which constitutes the BBB, presents a major obstacle to the use of potential drugs for many CNS disorders. In principle, only small lipophilic molecules can cross the BBB, i.e., move from circulating systemic blood to the brain. Many drugs with larger sizes or higher hydrophobicity have shown promising results in animal studies for treating CNS disorders.

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

[0287] The peptide β-casomorphin-5 was reported to be able to overcome the BBB in 1983 [Ermisch, A. et al. 1983. J. of Neurochemistry. 41:1229-1233]. Since then, many other peptides with BBB penetration 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 conjugates of the present invention.

[0288] In an embodiment of the present invention, the polycationic peptide of the present invention is a peptide that can cross the blood-brain barrier. In a preferred embodiment 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-penetrating peptides (CPPs) The term "cell-penetrating peptide" (CPP) refers to peptides, typically about 5-60 amino acid residues in length, that can facilitate cellular uptake of molecular cargo, particularly proteins, in which they are contained. Proteins may exhibit one or more CPPs. CPPs can also be characterized as being capable of facilitating the movement or traversal of molecular cargo across / through one or more of a lipid bilayer, a cell membrane, an organelle membrane, a vesicle membrane, or a cell wall. As used herein, CPPs are polycationic.

[0290] Examples of CPPs useful herein, as well as further description of CPPs in general, 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 are transporter- and receptor-independent and facilitate the transport of proteins, including CPPs, directly across lipid bilayers without the involvement of other cellular components.

[0291] (vii) nucleolin-binding peptide Thus, a "nucleolin-binding peptide" is a peptide that can bind to intracellular nucleolin protein, preferably the cell surface-expressed fraction of nucleolin.

[0292] In an embodiment of the invention, the polycationic peptide of the invention is a nucleolin-binding peptide.

[0293] International Patent Application Publication No. WO2011 / 031477A2 provides numerous examples of nucleolin-binding peptides suitable for use in the conjugates of the invention.

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

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

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

[0297] As used herein, the terms "positively charged amino acid," "positively charged amino acid-rich region," or "third polypeptide region that is a positively charged amino acid-rich region" refer to a polypeptide sequence of a third polypeptide of a complex that is distinct from a second polypeptide region of the complex and characterized by containing multiple positively charged amino acids. The positively charged amino acid-rich region may be formed solely by positively charged amino acids or may contain other amino acids, provided that the net charge of the entire region is positive at pH 7. Thus, a positively charged amino acid-rich region 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 in 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 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 contains lysine and arginine residues. In one embodiment, the positively charged amino acid-rich region contains lysine and histidine residues. In one embodiment, the positively charged amino acid-rich region contains arginine and histidine residues. In one embodiment, the positively charged amino acid-rich region contains lysine, arginine, and histidine residues.

[0299] In some embodiments, the positively charged amino acid rich region comprises 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 11, at least 12, at least 13, at least 14, or at least 15 positively charged amino acid residues, where 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 comprises fewer than 100, fewer than 90, fewer than 80, fewer than 70, fewer than 60, fewer than 50, fewer than 40, fewer than 30, fewer than 29, fewer than 28, fewer than 27, fewer than 26, fewer than 25, fewer than 24, fewer than 23, fewer than 22, fewer than 21, fewer than 20, fewer than 19, fewer than 18, fewer than 17, fewer than 16, fewer than 15, fewer than 14, fewer than 13, fewer than 12, fewer than 11, fewer than 10 or even fewer positively charged amino acid residues, which may be histidine, lysine, arginine, or a combination thereof.

[0301] In some embodiments, the positively charged amino acid-rich region comprises 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 comprises 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 comprises 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 comprises 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 an embodiment of the present invention, the positively charged amino acid-rich region of the complex of the present invention is a polyhistidine region. In a preferred embodiment of the present invention, the polyhistidine region contains 2 to 10, preferably 6, adjacent histidine residues.

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

[0305] In an embodiment of the present invention, the region rich in positively charged amino acids of the fusion protein of the present invention is a polylysine region, which in a preferred embodiment of the present invention comprises 2 to 10, preferably 6, adjacent polylysine residues.

[0306] In certain embodiments, the positively charged peptide sequence is 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 of the complex The different components of the polypeptides of the complex of the present invention (the first, second and third polypeptides of the complex) can be positioned in any relative positions, as long as the second polypeptide, which is preferably a polycationic peptide, and the third polypeptide (or region rich in positively charged amino acids) function at any position in the complex and the first polypeptide remains fully or partially functional (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 refer to the free amino acid residues at the end of a polypeptide chain formed by a protein, and are in common usage among those skilled in the art.

[0309] Thus, in an embodiment 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 another embodiment of the present invention, the first polypeptide region can be located at either the C-terminus or the N-terminus of the polypeptide of the complex, the second polypeptide can be located in the middle of the polypeptide, and the positively charged amino acid-rich region can be located at the end of the polypeptide opposite to the first polypeptide region, or the positively charged amino acid-rich region can be located in the middle of the polypeptide, and the second polypeptide can be located at the end of the polypeptide opposite to the first polypeptide region.

[0310] Thus, the relative order of the polypeptide elements of the complex of the invention is: · N - second polypeptide region - first polypeptide region - region rich in positively charged amino acids - C; · N - region rich in positively charged amino acids - first polypeptide region - second polypeptide region - C; · N - second polypeptide region - region rich in positively charged amino acids; · first polypeptide region - C; · N - region rich in positively charged amino acids - second polypeptide region - first polypeptide region - C; N-first polypeptide region-second polypeptide region-positively charged amino acid-rich region-C; or N - first polypeptide region - region rich in positively charged amino acids - second polypeptide region - C It could be.

[0311] In certain embodiments, the order of the elements in the polypeptide of the complex of the sixth aspect of the invention is any of those set out above.

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

[0313] The terms "N-terminus" and "C-terminus" do not imply that the components must be directly attached end-to-end, but rather that the components maintain their relative positions regardless of the presence of additional elements, such as linkers / spacers, inserted at the end of either component or between the components.

[0314] Thus, the polypeptides of the complexes of the present invention comprise the above-described elements ((1) the second polypeptide region, (2) the first polypeptide region, and (3) the region rich in positively charged amino acids), which may be joined end-to-end, and may also include a "linker" or "spacer," which is one or more arbitrary peptides or polypeptides inserted therebetween and preferably linked by peptide bonds.

[0315] According to the present invention, the spacer or linker amino acid sequence 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 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 flexibility that allows 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, it is ensured that the secondary and tertiary structures of components (1), (2), or (3) are not affected by the presence of any of the others. The spacer is of polypeptide nature. The linker peptide preferably comprises 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 connect two adjacent polypeptide components of the complex of the invention by a covalent bond, preferably a peptide bond; and preferably the spacer is essentially 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 that can be used to link proteins without substantially impairing the function of the linked peptide, or at least one function of the linked peptide. More preferably, the spacer or linker used to link the peptides comprises a coiled-coil structure.

[0318] A preferred example of a linker peptide contains two or more amino acids selected from the group consisting of glycine, serine, alanine, and threonine. A preferred example of a flexible linker is a polyglycine linker. Possible examples of linker / spacer sequences include GGSSRSS (SEQ ID NO: 39), GGSSRSSS (SEQ ID NO: 76), SGGTSGSTSGTGST (SEQ ID NO: 49), AGSSTGSSTGPGSTT (SEQ ID NO: 50), or GGSGGAP (SEQ ID NO: 51). These sequences are used to connect designed coiled-coils to 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 residue sequence of the upper hinge region of mouse IgG3 used in the generation of coiled-coil dimerized antibodies (PKPSTPPGSS, SEQ ID NO: 53) [Pack, P. and Pluckthun, A., 1992, Biochemistry 31:1579-1584], the peptide of the sequence APAETKAEPMT (SEQ ID NO: 54), the peptide of the sequence GAP, the peptide of the sequence AAA, and the peptide of the sequence AAALE (SEQ ID NO: 55). In another preferred embodiment, the linker is GGSSRSS (SEQ ID NO: 39).

[0319] Alternatively, polypeptide components of the complexes of the invention can be connected by peptides whose sequences include protease cleavage targets, allowing for the separation of any of the components. Suitable protease cleavage sites for incorporation into polypeptides of the complexes of the 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), PreScission protease (cleavage site LEVLFQGP, SEQ ID NO: 60), intein, and the like.

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

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

[0322] As will be understood by one of skill in the art, the linker connecting the second polypeptide and the first polypeptide region and the linker connecting the first polypeptide region and the region rich in positively charged amino acids may comprise the same or different sequences, within the above-mentioned limitations that the presence and / or sequence of the linker does not result in a functional alteration of the second polypeptide, the first polypeptide region and / or the region rich in positively charged amino acids (e.g., but not limited to, due to alteration of the secondary or tertiary structure of the polypeptides of the complex, or the formation of disulfide bonds).

[0323] The above considerations regarding the relative positions, from N- to C-terminus, of the polypeptide elements of the complex apply even when there is a linker between them, regardless of their number or which elements are interposed between them. Thus, possible combinations and relative ordering of elements are as follows (the above numbering of 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 invention, the linker of the polypeptide of the complex of the invention comprises the sequence GGSSRSS (SEQ ID NO: 39) or the sequence GGGNS (SEQ ID NO: 40).

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

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

[0327] As will be understood by one of skill in the art, the protease cleavage site connecting the second polypeptide and the first polypeptide region and the protease cleavage site connecting the first polypeptide region and the positively charged amino acid-rich region can comprise the same or different sequences, within the above-mentioned limitation that the presence and / or sequence of the protease cleavage site does not result in a functional alteration of the second polypeptide, the first polypeptide region and / or the positively charged amino acid-rich region (e.g., but not limited to, due to alteration of the secondary or tertiary structure of the polypeptides of the complex or the formation of disulfide bonds).

[0328] The above considerations regarding the relative N- to C-terminal positions of the polypeptide elements of the complex apply regardless of their number or which elements are interposed, even if there is a protease cleavage site between them. Thus, possible combinations and relative ordering of elements are as follows (the above numbering of 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 comprises a linker connecting two polypeptides of the complex and a protease cleavage site connecting the other two polypeptides of the complex. In this case, the above-mentioned considerations regarding the relative positions of the elements of the complex from N-terminus to C-terminus apply, even in the presence of the linker and the protease cleavage site between them, regardless of their number or which elements are interposed. Thus, possible combinations and relative orders of elements are as follows (the above-mentioned numbering of 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 the elements in the polypeptide of the complex is N-(1)-linker-(2)-protease cleavage site-(3)-C. Thus, in a preferred embodiment, a second polypeptide is connected to a first polypeptide region via a linker, and the first polypeptide is connected to a third polypeptide region via a protease cleavage site.

[0331] In another preferred embodiment, the linker comprises the sequence GGSSRSS (SEQ ID NO: 39), GGSSRSSS (SEQ ID NO: 76) or the sequence GGGNS (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 invention comprises a polypeptide comprising the following elements: [Table 3]

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

[0334] In another preferred embodiment, a polypeptide forming part of a complex of the invention comprises, consists essentially of or consists of the amino acid sequence of SEQ ID NO: 61, optionally including an amino-terminal methionine.

[0335] In some embodiments, a polypeptide forming part of a complex of the invention comprises, essentially comprises, or consists of the amino acid sequence of any of SEQ ID NOs: 61 or 106-124, optionally including an amino-terminal methionine.

[0336] In certain embodiments, the agent of interest of the conjugate of the sixth aspect of the invention is a therapeutic agent or an imaging agent.

[0337] IV-E. Target Agent In certain embodiments, the agent of interest of the conjugate of the sixth aspect of the invention is a therapeutic agent or an imaging agent.

[0338] IV-E.1 Therapeutic drugs The term "therapeutic agent" as used herein refers to any compound suitable for the therapy and / or treatment of a condition, disorder, or disease, without limitation of chemical structure.

[0339] The nature of the therapeutic agent is not particularly limiting of the present invention, so long as it remains active in the complex or can be activated when delivered to the interior of a cell. Thus, any therapeutic agent can be used in the complex, provided that the therapeutic agent is active when delivered to the interior of a cell or exhibits at least 100%, at least 90%, at least 80%, at least 70%, at least 60%, at least 50%, or less of the activity of the unconjugated therapeutic agent. Alternatively, because the objective of the present invention is to enhance the action of a therapeutic agent by increasing the selectivity of the therapeutic agent and reducing its off-target effects, it is believed that the effect of a therapeutic agent conjugated to a polypeptide in a complex can be synergistic and exceed previously known parameterized values ​​for a particular therapeutic agent. Thus, some embodiments of a therapeutic agent attached to a polypeptide of a complex of the invention are also contemplated 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 functionality of the therapeutic agent alone.

[0340] In an embodiment of the invention, the therapeutic agent attached to the polypeptide of the complex of the invention is (i) a chemotherapeutic agent, (ii) a cytotoxic polypeptide; (iii) anti-angiogenic polypeptides; (iv) a polypeptide encoded by a tumor suppressor gene; (v) pro-apoptotic polypeptides; (vi) a polypeptide having anti-metastatic activity; (vii) a polypeptide encoded by the polynucleotide capable of activating an immune response against a tumor; (viii) anti-angiogenic molecules, and (ix) toxin is selected from the group consisting of:

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

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

[0343] It will be understood that the term "chemotherapeutic agent" refers to an anti-cancer agent.

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

[0345] Some anticancer drugs can be classified as DNA damaging agents, including topoisomerase inhibitors (e.g., etoposide, lamprothecin, topotecan, teniposide, mitoxantrone), DNA alkylating agents (e.g., cisplatin, mechlorethamine, cyclophosphamide, ifosfamide, melphalan, colambucil, busulfan, thiotepa, carmustine, lomustine, carboplatin, dacarbazine, procarbazine), and DNA strand break inducers (e.g., cyclophosphamide, methylprednisolone ... For example, 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 epipodophyllotoxins.

[0346] Further examples of anti-cancer drugs include, but are not limited to, acivicin; aclarubicin; acodazole hydrochloride; acronine; adzelesin; aldesleukin; altretamine; ambomycin; amethanthrone acetate; aminoglutethimide; amsacrine; anastrozole; anthramycin; asparaginase; asperlin; azacitidine; azetepa; azotomycin; batimastat; benzodepa; bicalutamide; byzanthrene hydrochloride; visnafide dimesylate; bizelesin; bleomycin sulfate; bortezomib (VELCADE); brequinar Sodium; Bropirimine; Busulfan; Cactinomycin; Calsterone; Caracemide; Carbetimer; Carboplatin (platinum-containing regimens); Carmustine; Carubicin hydrochloride; Carzelesin; Cedefingol; Chlorambucil; Cilolemycin; Cisplatin (platinum-containing regimens); Cladribine; Crisnatol mesylate; Cyclophosphamide; Cytarabine; Dacarbazine; Dactinomycin; Daunorubicin; Decitabine; Dexormaplatin; Dezaguanine; Diaziquone; Docetaxel (TAXOTERE); Doxorubicin cin; droloxifene; dromostanolone; duazomycin; edatrexate; eflornithine; elsamitrucin; enloplatin; enpromate; epipropidine; epirubicin; elbrozole; erlotinib (TARCEVA), esorubicin; estramustine; etanidazole; etoposide; etopurine; fadrozole; fazarabine; fenretinide; floxuridine; fludarabine; 5-fluorouracil; flurocitabine; fosquidone; fostriecin; gefitinib (IRESSA), gemcitabine; hydroxyurea Antibiotics; Idarubicin; Ifosfamide; Ilmofosine; Imatinib mesylate (GLEEVAC); Interferon alpha-2a; Interferon alpha-2b; Interferon alpha-nl; Interferon alpha-n3; Interferon beta-Ia; Interferon gamma-Ib; Iproplatin; Irinotecan; Lanreotide; Lenalidomide (REVLLM1D, REVIMID); Letrozole; Leuprolide; Liarozole; Lometrexol; Lomustine; Losoxantrone; Masoprocol; Maytansine; Mechlorethamine; Megestrol;Melengestrol; Melphalan; Menogaril; Mercaptopurine; Methotrexate; Metoprine; Meturedepa; Mitindomide; Mitocalcine; Mitochromin; Mitogillin; Mitomarcin; Mitomycin; Mitospar; Mitotane; Mitoxantrone; Mycophenolic acid; Nocodazole; Nogalamycin; Ormaplatin; Oxisuran; Paclitaxel; Pemetrexed (ALIMTA), Pegaspargase; Pe Riomycin; Pentamustine; Pentomone; Peplomycin; Perfosfamide; Pipobroman; Piposulfan; Piritrexim; Isethionate; Piroxantrone; Plicamycin; Promestane; Porfimer; Porfiromycin; Prednimustine; Procarbazine; Puromycin; Pirazofurin; Ribopurin; Rogletimide; Safingol; Semustine; Simtrazene; Sitoglucide; Sparfosate; Sparso Mycin; spirogermanium; spiromustine; spiroplatin; streptonigrin; streptozocin; sulofenur; tallysomycin; tamsulosin; taxol; taxotere; tecogalan; tegafur; teloxantrone; temoporfin; temozolomide (TEMODAR); teniposide; teroxylon; testolactone; thalidomide (THALOMID) and its derivatives; thiamiprine; thioguanine; thiotepa; These include tiazofurin, tirapazamine, topotecan, toremifene, trestron, triciribine, trimetrexate, triptorelin, tubrozole, uracil, mustard, uredepa, vapreotide, verteporfin, vinblastine, vincristine, vindesine, vinepidine, vinglisinate, vinleurosine, vinorelbine, vinrocidine, vinzolidine, vorozole, zeniplatin, zinostatin, and zorubicin.

[0347] In one embodiment, the anticancer agent is provided as an oligomer containing several units of the anticancer molecule. 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 five floxuridine molecules.

[0348] The anti-cancer 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 is not limited to, genistein (4',5,7-trihydroxyisoflavone), tyrphostin 25 (3,4,5-trihydroxyphenyl), methylene]-propanedinitrile, herbimycin A, daidzein (4',7-dihydroxyisoflavone), 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 is not limited to, p21, p27, p57, p5 , pl6, pl8, or pl9. The MAP kinase inhibitor can be KY12420 (C23H24O8), CNI-1493, PD98059, or 4-(4-fluorophenyl)-2-(4-methylsulfinylphenyl)-5-(4-pyridyl)1H-imidazole. The EGFR inhibitor can be, but is 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 anti-cancer agent can be a VEGF inhibitor, including, but not limited to, bevacizumab (AVASTIN), ranibizumab (LUCENTIS), pegaptanib (MACUGEN), sorafenib, sunitinib (SUTENT), vatalanib, ZD-6474 (ZACTIMA), anecortave (RETAANE), squalamine lactate, and semaphorins. Anticancer drugs include, but 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 (anti-HER-2 / neu oncogene protein product and immunoglobulin G (IgG)). The antibody or antibody fragment may include: a bispecific antibody that simultaneously binds to type I Fc receptors (FcγRI), oregovomab (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's lymphoma), cetuximab (ERBITUX), MDX-447, MDX-22, MDX-220 (anti-TAG-72), IOR-C5, IOR-T6 (anti-CD1), IOR EGF / R3, seroglobulin (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 above-mentioned anti-angiogenic polypeptides, marimastat; AG3340; COL-3, BMS-275291, thalidomide, endostatin, SU5416, SU6668, EMD121974, 2-methoxyestradiol, carboxyamidotriazole, CMIOL, pentosan polysulfate, angiopoietin 2 (Regeneron), herbimycin A, PNU145156E, 16K prolactin fragment, linomide, thalidomide, pentoxifylline, genistein, TNP470, endostatin, paclitaxel, accutane, angiostatin, cidofovir, vincristine, bleomycin, AGM-1470, platelet factor 4, or minocycline.

[0351] Other suitable active agents are DNA cleavage agents.Examples of DNA cleavage agents suitable for inclusion as cytotoxins in the complex used in carrying out the method include but are not limited to anthraquinone-oligopyrrole-carboxamide, benzimidazole, leinamycin; dynemicin A; enediyne; and their biologically active analogues or derivatives (i.e., those with substantially the same 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 U.S. Pat. No. 5,622,958 (1998). Other examples include, but are not limited to, enediyne quinone imine (U.S. Pat. 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 under a common category: antimetabolites. As used herein, "antimetabolites" refers to compounds that inhibit the utilization of metabolites that are part of normal metabolism. Antimetabolites are often structurally similar to the metabolites they interfere with, such as antifolates, which interfere with the utilization of folic acid. Non-limiting 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, ifosfamide, 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 another specific embodiment, the pyrimidine analog is floxuridine or a pentameric form thereof.

[0354] As used herein, the term "pyrimidine analog" refers to a nucleoside analog antimetabolite that mimics the structure of pyrimidine. Pyrimidine analogs inhibit nucleic acid synthesis. Their antiproliferative effect is achieved by incorporation into DNA, causing chain termination and inhibition of DNA synthesis. They can 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, floxuridine, fluorouracil, capecitabine, and tegafur-uracil.

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

[0356] As used herein, the term "oligomeric form thereof" refers to a molecule formed by several repeating units, each unit being 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 ranges from 5 to 100. Thus, as used herein, an oligomeric form of a pyrimidine analog refers to a molecule formed by a sequence of several pyrimidine analogs. In certain embodiments, an oligomeric or polymeric form of a pyrimidine analog refers to 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 pyrimidine analogs. In another specific embodiment, it refers to a molecule consisting of 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.

[0357] In certain embodiments, oligomeric or polymeric forms of pyrimidine analogs are molecules 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 floxuridine units. In another specific embodiment, oligomeric forms of pyrimidine analogs are molecules consisting of a sequence of at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, or more floxuridine units.

[0358] The expression "pentameric form thereof", when referring to a floxuridine analogue, is understood as a molecule comprising or consisting of a sequence of five floxuridines.

[0359] (ii) a cytotoxic polypeptide As used herein, the term "cytotoxic polypeptide" refers to a drug capable of inhibiting cellular function. The drug may inhibit proliferation or may be toxic to the cell. Polypeptides that, when internalized by a cell, inhibit or detrimentally alter cellular metabolism or in any way inhibit cell growth or proliferation are included within the scope of this term, including, but not limited to, drugs whose toxic effects are mediated when delivered intracellularly and drugs whose toxic effects are mediated at the cell surface. Useful cytotoxic polypeptides include proteinaceous toxins, such as bacterial toxins.

[0360] Examples of proteinaceous cytotoxins useful for incorporation into conjugates according to the present invention include, but are not limited to, type 1 and type 2 ribosome-inactivating proteins (RIPs). Useful type 1 plant RIPs include, but are not limited to, dianthin 30, dianthin 32, likunin, saporins 1-9, pokeweed activator protein (PAP), PAP II, PAP-R, PAP-S, PAP-C, mapalmin, dodecandrin, bryodin-L, bryodin, colicin 1 and 2, rufin-A, rufin-B, rufin-S, 19K-protein synthesis inhibitory protein (PSI), 15K-PSI, 9K-PSI, α-kirilowin, β-kirilowin, gelonin, momordin, momordin-II, momordin-Ic, MAP-30, α-momorcarin, β-momorcarin, trichosanthin, TAP-29, trichochirin; barley RIP; flax RIP, tritin, maize RIP, and asparin 1 and 2 [Stirpe et al., 1992]. Bio / Technology 10:405-12]. Useful type 2 RIPs include, but are not limited to, volkensin, ricin, nigrin-b, CIP-29, abrin, modeccin, ebrutin-α, ebrutin-β, ebrutin-γ, vircumin, porectin, and their biologically active enzymatic 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]. Truncated and mutant toxin enzyme subunits can also be used as cytotoxin moieties (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. Pat. No. 5,082,927).Other targeted agents include, but are not limited to, the colicin family of over 34 described RNase toxins, including colicins A, B, D, E1-9, cloacin DF13, and the fungal RNase, 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 Tumor cell growth is highly dependent on extensive tumor angiogenesis, which accompanies cancer progression. Therefore, inhibition of new blood vessel formation and targeted destruction of existing blood vessels by antiangiogenic agents has been introduced as an effective and relatively non-toxic approach to tumor therapy.

[0363] As used herein, the term "anti-angiogenic polypeptide" refers to a polypeptide that can inhibit angiogenesis. Suitable anti-angiogenic polypeptides include, but are not limited to, angiostatin, endostatin, 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) a polypeptide encoded by a tumor suppressor gene As used herein, a "tumor suppressor" refers to a gene or gene product whose normal biological role is to suppress uncontrolled cell proliferation. 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 an apoptotic outcome. Suitable pro-apoptotic polypeptides include, but are not limited to, pro-apoptotic members of the BCL-2 family of proteins, such as 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, the adenoviral E4orf4 gene, p53 pathway genes, pro-apoptotic ligands such as TNF, FasL, and TRAIL, and / or their receptors such as TNFR, Fas, TRAIL-R1, and TRAIL-R2.

[0366] (vi) a polypeptide having anti-metastatic activity As used herein, the term "metastasis inhibitor" refers to a protein that acts to slow or prevent metastasis (secondary tumors) from spreading within a cancer-bearing organism. 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) a polypeptide encoded by the polynucleotide that is capable of activating an immune response against a tumor. As used herein, an immunostimulatory polypeptide agent is a polypeptide encoded by a polynucleotide that can activate or stimulate an immune response (including enhancing an existing immune response) in a subject to which it is administered, alone or in combination with other agents. Suitable non-limiting examples of immunostimulatory peptides include flagellin, muramyl dipeptide, 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.), immunostimulatory antibodies (e.g., anti-CTLA-4, anti-CD28, anti-CD3, or single chain / antibody fragments of these molecules), etc.

[0368] (viii) anti-angiogenic molecules; In certain embodiments, the intervening region of the fusion protein of the present invention is intended to correspond to a protein that acts as a tumor-targeting angiogenesis inhibitor. These agents include, in addition to the anti-angiogenic polypeptides described above, marimastat; AG3340; COL-3, BMS-275291, thalidomide, endostatin, SU5416, SU6668, EMD121974, 2-methoxyestradiol, carboxyamidotriazole, CMIOL, pentosan polysulfate, angiopoietin 2 (Regeneron), herbimycin A, PNU145156E, 16K prolactin fragment, linomide, thalidomide, pentoxifylline, genistein, TNP470, endostatin, paclitaxel, accutane, angiostatin, cidofovir, vincristine, bleomycin, AGM-1470, platelet factor 4, and minocycline. Also included are VEGF inhibitors, including, but not limited to, bevacizumab (AVASTIN), ranibizumab (LUCENTIS), pegaptanib (MACUGEN), sorafenib, sunitinib (SUTENT), vatalanib, ZD-6474 (ZACTIMA), anecortave (RETAANE), squalamine lactate, and semaphorins.

[0369] (ix) toxin As used herein, the term "toxin" refers to non-proteinaceous / non-polypeptide cytotoxic compounds obtained from different organisms, as well as chemically modified derivatives of the same compounds and compounds obtained by chemical synthesis. Compounds of this category, which have biological origins, can be obtained from microorganisms (bacteria, archaea, protozoa, 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; therefore, one or more amino acids may be part of their structure, whether as part of their base composition or as a result of chemical derivatization, 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 are calicheamicin γ1, dolastatin 10, maytansinoids (DM1) and pyrrolobenzodiazepine dimers (PBD).

[0371] (x) Additional therapeutic agent In certain embodiments of the conjugates of the invention, the therapeutic agent is selected from those shown in column 3 of Table 4 below. [Table 4-1] [Table 4-2] [Table 4-3]

[0372] In certain embodiments of the complexes of the invention, the second polypeptide region is a ligand selected from those shown in Table 4, and the agent of interest is selected from those therapeutic agents shown in the same row as the ligand in Table 4. Another particular embodiment refers to a complex of the invention as defined in an earlier embodiment in this section for use in treating a disease or disorder, wherein the disease or disorder is selected from those shown in Table 4. Another particular embodiment refers to a complex of the invention as defined in an earlier embodiment in this section for use in treating a disease or disorder selected from the corresponding row in Table 4 in which the second polypeptide region of the complex appears.

[0373] IV-E.2 Contrast agents The term "imaging 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. Thus, the term "imaging agent" encompasses agents used to enhance the quality of images that can be produced without such agents (e.g., as in MRI), and agents for preprocessing to produce an image (e.g., as in nuclear imaging). Suitable contrast agents include, but are not limited to, contrast agents for radionuclide imaging, computed tomography, Raman spectroscopy, magnetic resonance imaging (MRI), and optical imaging.

[0374] 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 fluorogenic agents include gadolinium and renografin. Examples of paramagnetic ions include chromium(III), manganese(II), iron(III), iron(II), cobalt(II), nickel(H), copper(II), neodymium(III), samarium(III), ytterbium(III), gadolinium(III), vanadium(II), terbium(III), dysprosium(III), holmium(III), and erbium(III) ions.

[0375] Contrast agents for optical imaging include, for example, 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, pyridyloxazole derivatives, cascade yellow dyes, and dapoxyl dyes. Contrast agents for optical imaging also include fluorescent proteins, which as used herein refer to proteins with atomic structures that allow them to exhibit fluorescence, a phenomenon well known in the art. Non-limiting examples of commonly used fluorescent proteins suitable for the conjugates of the present invention include green fluorescent protein (GFP, first discovered in 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 conjugates of the invention are enhanced green fluorescent protein (eGFP), enhanced cyan fluorescent protein CFP (ECFP), enhanced YFP (EYFP), GFPS65T, emerald, topaz (TYFP), Venus, citrine, mCitrine, 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, dimer2, t-dimer(12), mRFP1, pocilloporin, renilla GFP, monster GFP, paGFP, Kaede proteins and kindling proteins, phycobiliproteins and phycobiliprotein complexes, including B-phycoerythrin, R-phycoerythrin, and allophycocyanin. In another embodiment, the imaging agent is a fluorescent protein selected from the group consisting of mHoneydew, mBanana, mOrange, dTomato, tdTomato, mTangerine, mStrawberry, mCherry, mGrapel, mRaspbery, mGrape2, mPlum, etc. [Shaner et al. (2005) Nat. Methods 2:905-909].

[0377] Contrast agents for magnetic resonance imaging devices are gadolinium chelates, manganese chelates, chromium chelates, 19F and iron particles.

[0378] 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 Agent of Interest to the Polypeptide of the Complex The polypeptide of the complex can be conjugated with a single agent of interest or multiple agents of interest. When multiple agents are conjugated to the polypeptide of the complex, the agents can be the same or different. In certain embodiments, the multiple agents of interest are therapeutic agents, and as defined above, they are the same or different therapeutic agents. In another specific embodiment, the multiple agents of interest are imaging agents, and as defined above, they are the same or different imaging agents.

[0380] The following applies to any agent of interest, i.e., a therapeutic agent, an imaging agent, multiple therapeutic agents, and multiple imaging agents, as defined above. Thus, in certain embodiments, any of the following embodiments relating to an agent applies to a therapeutic agent by substituting the term "agent" with "therapeutic agent." In another specific embodiment, any of the following embodiments relating to an agent applies to an imaging agent by substituting the term "agent" with "imaging agent." In another specific embodiment, any of the following embodiments applies to multiple therapeutic agents by substituting the term "multiple agents" with "multiple therapeutic agents," wherein the multiple therapeutic agents are as defined above. In another specific embodiment, any of the following embodiments applies to multiple imaging agents by substituting the term "multiple agents" with "multiple imaging agents," wherein the multiple imaging agents are as defined above.

[0381] One or more agents of interest can be conjugated to any sequence of the polypeptide of the conjugate.

[0382] Thus, in certain embodiments, the agent of interest is conjugated to the first polypeptide region. In certain embodiments, the agent of interest is conjugated to the second polypeptide region of the complex. In another specific embodiment, the agent of interest is conjugated to the third polypeptide region of the complex. In another specific embodiment, the agent of interest is conjugated to any of the linking regions between the first and second polypeptides. In another specific embodiment, the agent of interest is conjugated to the linking region between the second and third polypeptide regions. In another specific embodiment, the agent of interest is conjugated to the linking region between the second and third polypeptide regions. In another specific embodiment, the agent of interest is conjugated to the protease cleavage site between the first and second polypeptide regions. In another specific embodiment, the agent of interest is conjugated to the protease cleavage site between the first and third polypeptide regions. In another specific embodiment, the agent of interest is conjugated to the protease cleavage site between the second and third polypeptide regions.

[0383] In certain embodiments, multiple agents of interest are conjugated to the same polypeptide region of the complex. In preferred embodiments, multiple agents of interest are conjugated to the first polypeptide region. In certain embodiments, multiple agents of interest are conjugated to the second polypeptide region of the complex. In another specific embodiment, multiple agents of interest are conjugated to the third polypeptide region of the complex. In another specific embodiment, multiple agents of interest are conjugated to all three polypeptide regions of the polypeptide of the complex. In another specific embodiment, multiple agents of interest are conjugated to the first and second polypeptide regions of the polypeptide. In another specific embodiment, multiple agents of interest are conjugated to the first and third polypeptide regions of the complex. In another specific embodiment, multiple agents of interest are conjugated to the second and third polypeptide regions of the complex.

[0384] In another specific embodiment, the plurality of agents of interest are conjugated to a junction region of the polypeptide of the complex. In another specific embodiment, the plurality of agents of interest are conjugated to a junction region between the second polypeptide region and the first polypeptide region. In another specific embodiment, the plurality of agents of interest are conjugated to a junction region between the first polypeptide region and the third polypeptide region. In another specific embodiment, the plurality of agents of interest are conjugated to a junction region between the second polypeptide region and the third polypeptide region. In another specific embodiment, the plurality of agents of interest are conjugated to a protease cleavage site of the polypeptide of the complex. In another specific embodiment, the plurality of agents of interest are conjugated to a protease cleavage site between the second polypeptide region and the first polypeptide region. In another specific embodiment, the plurality of agents of interest are conjugated to a protease cleavage site between the first polypeptide region and the third polypeptide region. In another specific embodiment, the plurality of agents of interest are conjugated to a protease cleavage site between the second polypeptide region and the third polypeptide region. In another specific embodiment, multiple agents of interest are conjugated to all of the protease cleavage sites of the polypeptide.

[0385] In another specific embodiment, multiple agents of interest are conjugated to any of the above polypeptide regions and to the linking region of the polypeptide of the complex. In another specific embodiment, multiple agents of interest are conjugated to any of the above polypeptide regions and to the linking region between the second polypeptide region and the first polypeptide region. In another specific embodiment, multiple agents of interest are conjugated to any of the above polypeptide regions and to the linking region between the first polypeptide region and the third polypeptide region. In another specific embodiment, multiple agents of interest are conjugated to any of the above polypeptide regions and to the linking region between the second polypeptide region and the third polypeptide region. In another specific embodiment, multiple agents of interest are conjugated to any of the above polypeptide regions of the polypeptide and to all of the linking regions of the polypeptide.

[0386] In another specific embodiment, multiple agents of interest are complexed to any of the above polypeptide regions and to the protease cleavage site of the polypeptide of the complex. In another specific embodiment, multiple agents of interest are complexed to any of the above polypeptide regions and to the protease cleavage site between the first polypeptide region and the second polypeptide region. In another specific embodiment, multiple agents of interest are complexed to any of the polypeptide regions of the polypeptide and to the protease cleavage site between the first polypeptide region and the third polypeptide region. In another specific embodiment, multiple agents of interest are complexed to any of the polypeptide regions of the polypeptide and to the protease cleavage site between the second polypeptide region and the third polypeptide region. In another specific embodiment, multiple agents of interest are complexed to the above polypeptide regions and to all of the cleavage sites of the polypeptide.

[0387] In another specific embodiment, multiple agents of interest are conjugated to the linking region of the polypeptide and the protease cleavage site of the polypeptide. In another specific embodiment, multiple agents of interest are conjugated to any of the above polypeptide regions, linking regions, and protease cleavage sites of the polypeptide of the conjugate.

[0388] As mentioned above, the agent of interest is conjugated to a polypeptide of the complex, and the position of conjugation within the polypeptide with respect to its N-terminus and C-terminus is not intended to be limited. Thus, the agent of interest may be conjugated to a position equidistant from the N-terminus and C-terminus of the polypeptide of the complex, or may be close to either of them. Thus, an agent of interest may be conjugated to a 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 amino acid residue at the N-terminus or C-terminus of the polypeptide, or may be conjugated to a polypeptide region at the same residue at the N-terminus or C-terminus of the polypeptide. This paragraph applies to each agent conjugated to a polypeptide; multiple agents may be conjugated to a polypeptide in a complex.

[0389] The only limitations intended on the attachment location of the agent of interest are that the agent and polypeptide elements are functional and that attachment of the agent does not interfere with the activity of either agent, polypeptide, or complex.

[0390] Thus, the functionalities of the target agent, second polypeptide, first polypeptide region, and positively charged amino acid-rich region are preserved 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% relative to their unconjugated forms. This applies regardless of the position of attachment in the polypeptide of the complex. This paragraph also applies to each agent conjugated to a polypeptide in which multiple agents are conjugated to the polypeptide of the complex. It is contemplated that the agent can be directly attached to a residue of the polypeptide of the complex or indirectly attached via a linking moiety.

[0391] Thus, in certain embodiments, the agent of interest is directly attached to the polypeptide of the complex, hi another particular embodiment, the agent of interest is attached to the polypeptide of the complex via a linking moiety.

[0392] In another specific embodiment, where multiple agents are conjugated to a polypeptide of a complex, all of them are directly bound to residues of the polypeptide. In another specific embodiment, where multiple agents are bound to a polypeptide of a complex, some of them are directly bound to residues of the polypeptide and the rest are indirectly bound via linking moieties. In another specific embodiment, all of the agents are bound via linking moieties.

[0393] The expression "linking moiety" or "linker" has already been defined in the second aspect of the invention.

[0394] Those skilled in the art will understand that when a linking moiety mediates the bond between an agent of interest and a polypeptide of a complex, the provisions made above regarding the functionality of the components of the polypeptide of the complex and the agent apply. Thus, when an agent of interest is bound to a polypeptide of a complex via a linking moiety, regardless of the position of the bond in the polypeptide of the complex, the chemical composition or structure of the linking moiety, and the chemical nature of the bond between the linking moiety and the agent and between the linking moiety and the polypeptide of the complex, the agent of interest, the second polypeptide region, the first polypeptide region, and the region rich in positively charged amino acids will retain 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 relative to their unconjugated forms. This applies to all agents of interest when multiple agents are conjugated to the polypeptide of the complex.

[0395] In a preferred embodiment of the invention, the linking moiety mediating the bond between the agent and the complex polypeptide is 6-maleimidohexanoic acid N-hydroxysuccinimide ester or 4-maleimidohexanoic acid N-hydroxysuccinimide ester. In another specific embodiment in which multiple agents are conjugated to the complex polypeptide, all of the linking moieties mediating the bond between the agent and the complex polypeptide are 6-maleimidohexanoic acid N-hydroxysuccinimide ester or 4-maleimidohexanoic acid N-hydroxysuccinimide ester. In another specific embodiment in which multiple agents are conjugated to the complex polypeptide, some of the linking moieties mediating the bond between the agent and the complex polypeptide are 6-maleimidohexanoic acid N-hydroxysuccinimide ester or 4-maleimidohexanoic acid N-hydroxysuccinimide ester.

[0396] In another preferred embodiment, the linking moiety that mediates the bond between the agent and the polypeptide of the conjugate is a moiety that can react with sulfhydryl groups present in the side chain of the polypeptide and with the active group of the agent of interest. Suitable linking groups that can react with sulfhydryl groups 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 bind to sulfhydryls either by alkylation (usually forming a thioether bond) or disulfide exchange (forming a disulfide bond).

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

[0398] As used herein, the term "spacer" refers to a moiety that connects at least two other moieties to one another. In some embodiments, the spacer is a polymer.

[0399] As used herein, the term "polymer" refers to a molecule comprising repeating structural units, i.e., monomers, connected by chemical bonds in a linear, cyclic, branched, cross-linked, or dendritic form, or a combination thereof, and may be of synthetic or biological origin, or a combination of both. The monomers may be the same, 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 referred to as "copolymers," and include, for example, 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 joined by covalent bonds; and gradient copolymers, in which the composition of different monomers gradually changes along the polymer chain. In some embodiments, the polymer comprises one or more other moieties, and in certain embodiments, the other moieties are C 1-50 Alkyl, C 2-50 Alkenyl, C 2-50 Alkynyl, C 3-10 In some embodiments, 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 comprises PEG. Such PEG-based moieties or reagents include 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 percent of the PEG-based moiety or reagent is: C 1-50 Alkyl, C 2-50 Alkenyl, C 2-50 Alkynyl, C 3-10 Cycloalkyl, 3- to 10-membered heterocyclyl, 8- to 11-membered heterobicyclyl, phenyl, naphthyl, indenyl, indanyl, and tetralinyl; branch points such as -CR<, >C<, or -N<; and dashed lines indicate attachment to the remainder of the moiety or reagent, and -R and -Ra are each independently -H and Ci -6 and a bond selected from the group consisting of alkyl; and the moiety and bond may be optionally further substituted.

[0401] In some embodiments of the invention, the linking moiety joining the agent and the polypeptide of the conjugate is susceptible to processing by enzymes present in the cytoplasm, such that the therapeutic agent conjugated to the fusion protein is released from the fusion protein upon internalization of the cell.

[0402] Furthermore, some agents can be polymerized to form polymers in which multiple copies of the same molecule are linked together, with each monomer of the polymer being one of the molecules. A non-limiting example of such a polymer is 5-fluoro-2'-deoxyuridine (FdU), resulting in oligo-FdU. It is contemplated that some embodiments of the present invention may include such polymers. It is also contemplated that some other embodiments of the present invention may 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 those embodiments of the invention featuring polymers of an agent of interest may feature 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 15, 30, 40, 50 or more polymerized molecules of one or more different agents of interest in ratios of 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10 or more.

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

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

[0405] Reporter proteins useful in the present invention include luciferase-4-monooxygenase from Photinus pyralis, β-galactosidase, thymidine kinase, etc. 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 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 is subject to the above-mentioned considerations regarding the relative position of the elements of the fusion protein.Therefore, regardless of the position of the positively charged amino acid-rich region in the fusion protein, the fluorescent protein is always adjacent to it, either directly or separated by a linker.

[0407] Thus, in embodiments of the invention that include a fluorescent protein, possible relative positions for the polypeptide elements of the complexes of the invention fit the following scheme (where RP refers to the reporter protein and the above numbering for 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 Conjugates of the Invention A preferred embodiment of the present invention is a conjugate whose components are as defined above in Table 3 and wherein the agent of interest is one or more copies of floxuridine or floxuridine pentanucleotide. In a more preferred embodiment, the conjugate as defined above results from a linkage between an amino or thiol group in a side chain of a first region of the conjugated polypeptide and a thiol or hydroxy or phosphate or amino or carboxy group in a therapeutic agent, which may or may not be attached by a linking moiety of 2 to 35 atoms.

[0409] IV-H. Stoichiometry of the Complexes of the Invention The number of target agents conjugated to the fusion proteins of the present invention depends on, but is not limited to, the number of residues available in the polypeptides of the present invention that are available for chemical conjugation with target agents. Since most conjugation occurs via amino or sulfhydryl groups present in the side chains of amino acids forming part of the conjugated polypeptide, the number of target agents conjugated to the conjugated polypeptide depends on the number of lysine and arginine residues (in the case of conjugation via side chain amino groups) or the number of cysteine ​​residues (in the case of conjugation via side chain sulfhydryl groups), and on the yield of the conjugation reaction. Thus, in certain embodiments of the present invention, the conjugated polypeptides of the present invention are conjugated to at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 15, 17, 20, 25, or 30 target agents.

[0410] In certain cases where agents are provided as polymers, it will be understood that the number of agents will also depend on the number of monomers in the polymer. In the specific case of FdU oligomers, the number of agents of interest in a given conjugate is the result of multiplying the number of oligomers attached to the polypeptide of the conjugate by the number of monomers. In the preferred case of FdU pentamers, preferred embodiments include conjugates comprising 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 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, nanoparticles according to the present invention result from the assembly of multiple copies of the complex of the present invention. In a preferred embodiment, the nanoparticles comprise 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] Thus, the total number of agents of interest 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 nanoparticle. In preferred embodiments, 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, or 300 agents of interest. In even more preferred embodiments, the nanoparticles are bound to at least 30, 35, 40, 45, 50, 60, 65, 70, 57, 80, 85, 90, 59, 100, or 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 aspects of the invention are equally applicable to the sixth aspect of the invention.

[0414] V—Methods for preparing the conjugates of the invention In a seventh aspect, the present invention provides a method for preparing a conjugate of the sixth aspect of the invention, comprising: (i) providing a polypeptide of the complex of the sixth aspect of the invention comprising the G2 domain of nidogen-1 or a functionally equivalent variant thereof; and (ii) contacting the polypeptide with an activated form of an agent of interest of the conjugate of the sixth aspect of the invention, which is capable of reacting with at least one group in the polypeptide, under conditions suitable to form a bond between a reactive group in the agent of interest and a group in the polypeptide. The present invention relates to a method comprising:

[0415] In another embodiment, the present invention provides a method for preparing a conjugate of the sixth aspect of the invention, comprising: (i) providing a polypeptide of the complex of the sixth aspect of the invention comprising the G2 domain of nidogen-1 or a functionally equivalent variant thereof, wherein the polypeptide is in an activated form; and (ii) contacting the polypeptide with an agent of interest capable of reacting with a reactive group in the polypeptide under conditions suitable for forming a bond between the reactive group in the polypeptide and a group in the agent of interest; The present invention relates to a method comprising:

[0416] Those skilled in the art will understand that "reactive group," as used herein, refers to any portion of a molecule that can chemically bond with another portion of another molecule in such a way that the two molecules are joined 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] "Activated," as used herein when referring to a molecule, refers to a modified form of the molecule, including a chemical modification, that allows the molecule to chemically react in a way not previously present on the molecule (e.g., activation adds a reactive group not previously present, allowing a bond not previously possible), or to become more reactive (i.e., requires a lower activation energy for the molecule to react with other molecules compared to the inactivated state). The present invention contemplates the possibility of activating an agent of interest and then contacting the activating agent with a polypeptide of the complex, or activating a polypeptide of the complex and then contacting the activated polypeptide with the agent of interest. In either case, activation of the polypeptide or agent of interest is typically performed by reacting the molecule to be activated with a reagent that introduces a reactive group onto the molecule to be activated. Examples of reactive groups that allow activation of an agent of interest or a polypeptide of the complex include, but are not limited to, carboxyl moieties, amine moieties, imine moieties, thiol moieties, sulfone moieties, hydroxyl moieties, sulfate moieties, and phosphate moieties, among many others commonly known to those of skill in the art. The activated form of an agent of interest is also referred to herein as an "activated agent of interest." The activated form of a polypeptide of a complex is also referred to herein as an "activated polypeptide." As described in the sixth aspect of the present invention, one or more reactive groups in an activated polypeptide are located in the region of the polypeptide to which the agent of interest is complexed. Thus, in certain embodiments, the reactive group is located in the first polypeptide region, the second polypeptide region, the third polypeptide region, the linker between any of the polypeptide regions, or the protease cleavage site between the polypeptide regions of the complex. In another specific embodiment, the reactive group is located in the first polypeptide region, the second polypeptide region, the third polypeptide region, the linker between any of the polypeptide regions, and / or the protease cleavage site between the polypeptide regions of the complex.

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

[0419] In embodiments of the present invention in which a linking moiety mediates the bond between a conjugated polypeptide and a target agent, the linking moiety is a bifunctional crosslinker that reacts with groups in the target agent and groups in the conjugated polypeptide, more preferably a heterobifunctional crosslinker that reacts sequentially (either with an activated agent followed by a polypeptide, or with a polypeptide followed by an activated agent) or simultaneously using, among others, a thioether, an amino bond, a carbon-nitrogen double bond, or a bond formed by cycloaddition as disclosed in Kalia J et al. (Advances in bioconjugation. Curr Org Chem 2010 January, 14(2):138-147). For example, typical thiol-reactive functional groups include iodoacetamide, maleimide, and disulfide. Additionally, proteins can be treated with small molecules or surfaces that present activated esters (e.g., N-hydroxysuccinimidyl esters) to form amide bonds with lysine side chains and N-terminal amino groups. In another embodiment, the linking moiety is a heterobifunctional crosslinker that contains a reactive group capable of reacting with a thiol group and a reactive group capable of reacting with an amino group. In one embodiment, the heterobifunctional crosslinker is 6-maleimidohexanoic acid N-hydroxysuccinimide ester.

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

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

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

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

[0424] In an even 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 moiety is 6-maleimidohexanoic acid N-hydroxysuccinimide ester, which mediates the bond between the activated agent and a side chain on the polypeptide of the conjugate shown in the embodiments described above in this section. In an even more preferred embodiment, the linking moiety 6-maleimidohexanoic acid N-hydroxysuccinimide ester is attached in a first step to the agent of interest, preferably activated FdU, even more preferably sulfhydryl-functionalized FdU, and in a second step to a side chain on the polypeptide of the conjugate, more preferably to a lysine on the exterior of the polypeptide of the conjugate, even more preferably to a lysine on the exterior of the first polypeptide region of the conjugate of the 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 moiety is 6-maleimidohexanoic acid N-hydroxysuccinimide ester, which mediates the bond between the activated agent and a side chain in the polypeptide of the conjugate shown in the embodiments described above in this section. In an even more preferred embodiment, the linking moiety 6-maleimidohexanoic acid N-hydroxysuccinimide ester is attached in a first step to the agent of interest, preferably activated FdU, even more preferably amino-functionalized FdU, and in a second step to a side chain of the polypeptide of the conjugate, more preferably to a cysteine ​​outside the polypeptide of the conjugate, even more preferably to a cysteine ​​outside the first polypeptide region of the conjugate of the 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, even more preferably FdU functionalized with a carboxylic acid in an activated form, reacts with a carboxyl group in the agent of interest with a reactive group in the polypeptide (e.g., an amino group capable of forming an amide bond with a carboxyl group in the agent of interest), a side chain of the polypeptide of the complex, more preferably a lysine on the outside of the polypeptide of the complex, even more preferably a lysine on the outside of the first polypeptide region of the complex.

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

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

[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, such as those disclosed 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), the contents of which are incorporated herein by reference.

[0433] Thus, in one embodiment of the invention, the activated form of the polypeptide of the conjugate comprises a group that reacts with at least one moiety of the agent of interest. In a further preferred embodiment of the invention, the group of the agent of interest, preferably a chemotherapeutic agent, that reacts with the activated polypeptide of the conjugate is a thiol group.

[0434] In a further preferred embodiment of the invention, the activated polypeptide of the conjugate 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 contains a second activated carboxyl group that can react with an amino group, a thiol group, or a hydroxyl group in the agent, as appropriate.

[0435] In a further preferred embodiment, the linking moiety is 6-maleimidohexanoic acid N-hydroxysuccinimide ester, which mediates the bond between an amino group in the conjugate polypeptide and a thiol group in the agent of interest. In an even more preferred embodiment, the linking moiety 6-maleimidohexanoic acid N-hydroxysuccinimide ester is attached to the conjugate polypeptide, more preferably to a lysine on the exterior of the conjugate polypeptide, in a first step, and attached to a side chain in the agent, preferably a thiol group of the activated agent, in a second step.

[0436] In a further preferred embodiment, the linking moiety is 6-maleimidohexanoic acid N-hydroxysuccinimide ester, which mediates a bond between a thiol group in the conjugate polypeptide and an amino group in the agent of interest. In an even more preferred embodiment, the linking moiety 6-maleimidohexanoic acid N-hydroxysuccinimide ester is attached to the conjugate polypeptide, more preferably to a cysteine ​​on the exterior of the conjugate polypeptide, in a first step, and attached to the agent of interest, preferably to an amino group in the agent of interest, in a second step.

[0437] In a further preferred embodiment, the linking moiety is 6-maleimidohexanoic acid N-hydroxysuccinimide ester, which mediates the bond between a thiol group in the conjugate polypeptide and an amino group in the agent of interest. In an even more preferred embodiment, the linking moiety 6-maleimidohexanoic acid N-hydroxysuccinimide ester is coupled in a first step to the agent of interest, preferably to an amino group in the agent of interest, and in a second step to the conjugate polypeptide, more preferably to a thiol group of a cysteine ​​on the exterior of the conjugate polypeptide.

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

[0439] VI—Polypeptides of the Invention Containing Antagonistic CXCR4 Ligands We have observed that a fusion protein containing a competitive CXCR4 ligand and the G2 domain of nidogen-1 or its mutants can target and penetrate CXCR4-expressing cells. Furthermore, when this fusion protein is further modified by the presence of a polycationic region, the fusion protein spontaneously assembles into nanoparticles, which can target, penetrate, and induce apoptosis in CXCR4-expressing cells.

[0440] Thus, in another aspect, the present invention provides a method for producing a pharmaceutical composition comprising: (i) a first region comprising the G2 domain of nidogen-1 or a functionally equivalent variant thereof; and (ii) a second region that contains an antagonistic CXCR4 ligand; (also known as the second polypeptide of the invention, the fusion protein of the invention, or the CXCR4 antagonistic polypeptide of the invention) comprising:

[0441] The first region is defined above in the context of the first polypeptide of the invention and in the context of the first region of a polypeptide forming part of a complex of the invention, and applies equally to the second polypeptide of the invention. In some embodiments, the first region is a functionally equivalent variant of the G2 domain of nidogen-1, which functionally equivalent variant of the G2 domain of nidogen-1 is any of the variants defined above in the context of the first polypeptide of the invention. In some embodiments, the polypeptide of claim 53 or 54, wherein the first region comprises amino acids 430 to 667 of the sequence of human nidogen-1 as defined in UniProt database accession number P14543-1. In some embodiments, functionally equivalent variants of the G2 domain of nidogen-1 forming part of the first region comprise mutations at 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 numbering of the sequence of human nidogen-1 as defined in the UniProt database accession number P14543-1 in the version dated July 7, 2009. Thus, in another particular embodiment, the polypeptide of the complex of the sixth aspect of the invention is a functionally equivalent variant of the G2 domain of nidogen-1 comprising a mutation 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 numbering of the sequence of human nidogen-1 as defined in the UniProt database (version dated 7 July 2009) under accession number P14543-1.In some embodiments, nidogen G2 domain mutants 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, NIDOmut3-I, NIDOmut4_I, NIDOmut5_V215T, NIDOmut5_V176T, NIDOmut3-I, NIDOmut4_V215T, NIDOmut5 ... and any of the nidogen G2 domain mutants defined above in the context of the first aspect of the invention, including mutants having NIDOmut3-S65I, NIDOmut3-R114I, NIDOmut3-C214S, NIDOmut3-S65I_R114I, NIDOmut5-S65I_R114I, NIDOmut3-S65I_R114I and NIDOmut5-S65I_R114I.

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

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

[0444] Antagonists suitable for use according to the invention are characterised by competing for binding to CXCR4 by the natural ligand CXCL12 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 or less, 5 μM or less, 6 μM or less, 7 μM or less, 8 μM or less, 9 μM or less, 10 μM or less, 15 μM or less, 20 μM, 30 μM or less, 40 μM or less, 50 μM or less, 60 μM or less, 70 μM or less, 80 μM or less, 90 μM or less or 100 μM or less.

[0445] Suitable methods for determining whether a molecule is a competitive CXCR4 ligand are, for example, those described in Zirafi et al. (Cell Rep. 2015, 11, 737), the contents of which are incorporated herein by reference. These methods include assays based on detecting the ability of an antagonist to inhibit the binding of CXCL12 to CXCR4 (to determine whether the molecule is a ligand), and methods based on detecting the ability of a molecule to inhibit Ca2+ release from CXCR4-expressing cells such as HEX293 cells, CXCL12-directed transwell migration of Jurkat T cells, and / or CXCL12-induced migration of human CD34+ stem cells (to determine whether the molecule acts as an antagonist).

[0446] In some embodiments, the second region of a polypeptide having an antagonistic CXCR4 ligand according to the present invention further comprises a positively charged amino acid region.

[0447] The positively charged peptide sequence may contain only one type of positively charged amino acid, or may contain multiple types of positively charged amino acids. In one embodiment, the positively charged peptide sequence is a polyarginine region. In one embodiment, the positively charged peptide sequence is a polylysine region. In one embodiment, the positively charged peptide sequence is a polyhistidine region. In one embodiment, the positively charged peptide sequence contains a lysine residue and an arginine residue. In one embodiment, the positively charged peptide sequence contains a lysine residue and a histidine residue. In one embodiment, the positively charged peptide sequence contains an arginine res...

Claims

1. (i) comprising amino acids corresponding to positions 430 to 667 of human nidogen-1 of SEQ ID NO: 72; Mutations H459A, R468N, F639S and R650A, Mutations H459A, R468N, F639S, R650A, H543K and H545N, Mutations H459A, R468N, F639S, R650A, H543K, H545N and V449T, Mutations H459A, R468N, F639S, R650A, H543K, H545N and V525Q, Mutations H459A, R468N, F639S, R650A, H543K, H545N and F561E, Mutations H459A, R468N, F639S, R650A, H543K, H545N and V619T, Mutations H459A, R468N, F639S, R650A, H543K, H545N, V449T, V525Q, F561E and V619T, Mutations H459A, R468N, F639S, R650A, H543K, H545N, V449T, V525Q and F561E, and Mutations H459A, R468N, F639S, R650A, H543K, H545N, V449T, V525Q, V619T, F561E and C618S and having a mutation selected from the group consisting of: a first polypeptide region which is a variant of the G2 domain of nidogen-1, wherein said mutation is as defined with respect to nidogen-1 of SEQ ID NO: 72; (ii) a second polypeptide region that is a polycationic peptide; (iii) a third polypeptide region that is a region rich in positively charged amino acids, and (iv) the agent of interest A complex comprising:

2. 2. The complex of claim 1, wherein the first polypeptide region has a sequence selected from the group consisting of SEQ ID NOs: 64, 65, 87, 88, 89, 90, 91, 92, 93 and 94.

3. the mutation of the first polypeptide region is (i) mutations H459A, R468N, F639S and R650A; (ii) mutations H459A, R468N, F639S, R650A, H543K and H545N; (iii) mutations H459A, R468N, F639S, R650A, H543K, H545N, V449T, V525Q, F561E, and V619T, and (iv) mutations H459A, R468N, F639S, R650A, H543K, H545N, V449T, V525Q, V619T, F561E, and C618S 2. The complex of claim 1, selected from the group consisting of:

4. 4. The complex of claim 3, wherein the first polypeptide region has a sequence selected from the group consisting of SEQ ID NOs: 64, 65, 87, 92 and 94.

5. The polycationic peptide is (i) a peptide that is a CXCR4 ligand and can specifically bind to a receptor on the cell surface to promote internalization of the complex into the cell; (ii) arginine-rich peptides; (iii) GWH1 peptide, (iv) CD44 ligand, (v) peptides that can cross the blood-brain barrier; (vi) a cell-penetrating peptide, and (vii) nucleolin-binding peptide The complex according to any one of claims 1 to 4, selected from the group consisting of:

6. The complex of claim 5, wherein the polycationic peptide is a peptide that can specifically interact with a cell surface receptor to promote internalization of the complex into the cell, and the peptide is a CXCR4 ligand.

7. 7. The complex of claim 6, wherein the CXCR4 ligand is selected from the group consisting of a peptide comprising 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).

8. 8. The conjugate of claim 6 or 7, further comprising a positively charged peptide sequence RKRKRK (SEQ ID NO: 77) located at the N-terminus or C-terminus of the CXCR4 ligand.

9. The polycationic peptide is - an arginine-rich peptide comprising a sequence selected from the group consisting of RRRRRRRRR (SEQ ID NO: 30), RRRGRGRRR (SEQ ID NO: 31), RARGRGRRR (SEQ ID NO: 32) and RARGRGGGA (SEQ ID NO: 33); CD44 ligand A5G27 (SEQ ID NO: 34) or FNI / II / V (SEQ ID NO: 35); or A peptide capable of crossing the blood-brain barrier 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). The complex of claim 5, wherein

10. 10. The complex of claim 9, wherein the region rich in positively charged amino acids is a polyhistidine region, the polyhistidine region comprising 2 to 10 adjacent histidine residues.

11. 11. The complex of claim 10, wherein the polycationic peptide is located at the N-terminus of the first polypeptide and the region rich in positively charged amino acids is located at the C-terminus of the first polypeptide, or the region rich in positively charged amino acids is located at the N-terminus of the first polypeptide and the polycationic peptide is located at the C-terminus of the first polypeptide.

12. The complex of any one of claims 1 to 11, further comprising a lysine between the first polypeptide region and the third polypeptide region of the complex.

13. The complex of any one of claims 1 to 12, wherein the region rich in positively charged amino acids comprises the sequence RKRKRK (SEQ ID NO: 77).

14. 14. The complex of any one of claims 1 to 13, wherein the second polypeptide region is connected to the first polypeptide region via a first peptide linker and / or the first polypeptide region is connected to the third polypeptide region via a second peptide linker.

15. 15. The conjugate of claim 14, wherein the first peptide linker comprises a GGSSRSS sequence (SEQ ID NO: 39), a GGSSRSS sequence (SEQ ID NO: 76), or a GGGNS sequence (SEQ ID NO: 40).

16. 2. The complex of claim 1, having a sequence selected from the group consisting of SEQ ID NOs: 61, 106, 107, 108, 109, 110, 111, 112 and 113 bound to the agent of interest.

17. 2. The complex of claim 1, having a sequence selected from the group consisting of SEQ ID NOs: 61, 106, 111 and 113 bound to the agent of interest.

18. The conjugate of claim 16 or 17, further comprising a methionine at the N-terminus of the sequence selected from the group consisting of SEQ ID NOs: 61, 106, 107, 108, 109, 110, 111, 112 and 113.

19. the agent of interest is a therapeutic agent or an imaging agent, and the therapeutic agent is (i) a chemotherapeutic agent, (ii) a cytotoxic polypeptide; (iii) an anti-angiogenic polypeptide; (iv) a polypeptide encoded by a tumor suppressor gene; (v) a pro-apoptotic polypeptide; (vi) a polypeptide having anti-metastatic activity; (vii) a polypeptide encoded by the polynucleotide capable of activating an immune response against a tumor; (viii) anti-angiogenic molecules, and (ix) Toxin The complex of any one of claims 1 to 18, selected from the group consisting of:

20. 20. The conjugate of claim 19, wherein the conjugate is conjugated to multiple therapeutic agents, and the multiple therapeutic agents are the same or different.

21. 21. The conjugate of claim 19 or 20, wherein the therapeutic agent is connected to the first polypeptide via a linker.

22. The conjugate of any one of claims 19 to 21, wherein the therapeutic agent is a chemotherapeutic agent, and the chemotherapeutic agent is an antimetabolite or an anti-microtubule agent.

23. 23. The conjugate of claim 22, wherein the antimetabolite is a pyrimidine analog or an oligomeric form thereof, and the pyrimidine analog is floxuridine or a pentameric form thereof.

24. Nanoparticles comprising multiple copies of the complex of any one of claims 1 to 23 and having a diameter of 1 to 100 nm.

25. A pharmaceutical composition comprising the complex of any one of claims 1 to 23 or the nanoparticle of claim 24.

26. 26. The pharmaceutical composition of claim 25 for the treatment of cancer.

27. 27. The pharmaceutical composition of claim 26, wherein the cancer comprises cancer cells that express or overexpress CXCR4.

28. The pharmaceutical composition of claim 27, wherein the cancer cells are metastatic stem cells.

29. 27. The pharmaceutical composition of claim 26, wherein the cancer is pancreatic cancer or colorectal cancer.

30. 27. The pharmaceutical composition of claim 26, wherein the cancer is a primary tumor or a metastasis.

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