Extracellular matrix binding compounds for topical loading of therapeutic or diagnostic agents

Abstract

In the field of biotherapy, a major disorder is the need for a platform technology that enables high bioavailability and long-term treatment exposure of biotherapy in the context of localized disease. Conventional methods have some troublesome tradeoff problems, such as toxic and off-target side effects. The present disclosure provides compounds comprising the follistatin domain 1 (FSD1) of a follistatin (FST) that are capable of binding to a biological structure, such as an extracellular matrix, by heparan sulfate without poorly neutralizing activin A, myostatin, and GDF11 activity, such as full-length follistatin. The present invention relates to fusion proteins or binding proteins and compositions thereof, constructs or vectors encoding the same, medical uses thereof and biotherapeutic methods.

Description

Technical Field

[0001] The present invention relates to compounds comprising the follistatin domain 1 (FSD1) of follistatin (FST) that binds to heparan sulfate present on the cell surface, extracellular matrix and basement membranes of mammalian tissues. Background Art

[0002] Half-life extension technology has accelerated the therapeutic application of proteins (biotherapy). These technologies include recombinant fusion with proteins with intrinsic long serum half-life (such as IgG Fc, transferrin or albumin), fusion with polypeptides to increase overall size and hydrodynamic radius, or change the glycosylation spectrum. Half-life extension technology is mainly to optimize the systemic delivery of biotherapies, that is, delivery to the entire organism (REF.1). When the disease is systemic, this systemic delivery is ideal. However, when the disease is confined to a specific tissue area, organ or physiological compartment, systemic treatment will affect tissues that are not intended to be targeted. In addition, due to the wide distribution between various physiological compartments in the body, the bioavailability of systemic treatment in the target tissue may be low (REF.2).

[0003] Attempts to direct biotherapeutics to specific tissues have included targeting cell surface proteins such as cluster of differentiation (CD) proteins. For example, an antibody can be bispecific for CD3 (a T cell coreceptor) and a tumor antigen, thereby bridging the interaction between T cells and tumor cells (REF. 2). Although selectivity for CD proteins can direct therapies to specific cell subsets, it does not eliminate the occurrence of toxicity and off-target side effects (REFs 3-5). This may be partly because CD proteins or similar marker proteins are not completely specific to the target cell type, and biotherapeutics are still distributed systemically (REFs 3-5).

[0004] There is a need for platform technologies that can achieve high bioavailability and long-term therapeutic exposure of biotherapeutics in the setting of localized diseases, such as solid tumors or ocular diseases. Local and limited fixation of biotherapeutics to tissues or organs may simultaneously minimize systemic toxicity and off-target side effects. Current platform strategies for systemic half-life extension and tagged protein targeting fail to meet this requirement. SUMMARY OF THE INVENTION

[0006] The present invention is defined by the appended claims.

[0007] The inventors of the present invention have developed compounds, such as fusion proteins or binding proteins comprising a polypeptide (P1) containing the follistatin domain 1 (FSD1) of follistatin (FST), which are capable of binding to biological structures such as the extracellular matrix via heparan sulfate without undesirably neutralizing the activin A, myostatin and GDF11 activities of, for example, full-length follistatin.

[0008] The disclosed polypeptides (P1) form a versatile platform that can be used to fuse and conjugate with therapeutic or diagnostic agents, such as the four types of compounds developed by the inventors. The compounds of the invention can be used to locally load therapeutic or diagnostic agents, bind therapeutic or diagnostic agents to biological structures, and improve the local half-life of the therapeutic or diagnostic agents they are loaded with. The inventors have also shown that the developed compounds can be taken up intracellularly, for example, into the cytosol or nucleus.

[0009] A first aspect of the invention relates to a fusion protein or binding protein comprising: (i) one or more polypeptides (P1) consisting of at least one FSD1 domain comprising or consisting of a polypeptide having at least 70%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 99% sequence identity to SEQ ID NO: 1, and (ii) a therapeutic agent or a diagnostic agent.

[0010] A second aspect of the present invention relates to one or more polynucleotides which, upon expression, encode a fusion protein or binding protein of the present invention.

[0011] The third aspect of the present invention relates to one or more constructs or vectors comprising one or more polynucleotides of the second aspect or encoding a fusion protein of the first aspect.

[0012] The fourth aspect of the present invention relates to a host cell comprising one or more polynucleotides of the second aspect or one or more constructs or vectors of the third aspect.

[0013] The fifth aspect of the present invention relates to a composition comprising the fusion protein or binding protein of the first aspect, one or more polynucleotides of the second aspect, one or more constructs or vectors of the third aspect, the host cell of the fourth aspect, or a mixture thereof.

[0014] The sixth aspect of the present invention relates to the use of the fusion protein or binding protein of the first aspect, the one or more polynucleotides of the second aspect, the one or more constructs or vectors of the third aspect, the host cell of the fourth aspect, or the composition of the fifth aspect in medicine.

[0015] The seventh aspect of the present invention relates to a method for treating a disease or condition, which comprises administering to a subject an effective amount of the fusion protein or binding protein of the first aspect, one or more polynucleotides of the second aspect, one or more constructs or vectors of the third aspect, the host cell of the fourth aspect, or the composition of the fifth aspect.

[0016] The eighth aspect of the present invention relates to the use of the fusion protein or binding protein of the first aspect, the one or more polynucleotides of the second aspect, the one or more constructs or vectors of the third aspect, the host cell of the fourth aspect or the composition of the fifth aspect in the manufacture of a medicament for treating a disease or condition such as an ophthalmic disease, a cancerous disease or an inflammatory disease.

[0017] The ninth aspect of the invention relates to a method for increasing the local half-life of a therapeutic or diagnostic agent at the site of administration, comprising obtaining the fusion protein or binding protein of the first aspect, the one or more polynucleotides of the second aspect, the one or more constructs or vectors of the third aspect, the host cell of the fourth aspect, or the composition of the fifth aspect, wherein the therapeutic or diagnostic agent (ii) of the invention, or the same or different therapeutic part or diagnostic part of the invention comprises or consists of the therapeutic or diagnostic agent.

[0018] A tenth aspect of the present invention relates to a method for increasing the local in vivo half-life of a therapeutic or diagnostic agent, the method comprising the steps of:

[0019] a) providing therapeutic or diagnostic agents;

[0020] b) obtaining the fusion protein or binding protein of the first aspect, wherein the therapeutic agent or diagnostic agent of step a) is a therapeutic agent or diagnostic agent (ii) as described herein,

[0021] The local in vivo half-life of the therapeutic or diagnostic agent is thereby increased.

[0022] The eleventh aspect of the invention relates to a method for increasing the binding of a therapeutic or diagnostic agent to an extracellular matrix, comprising administering to a subject the fusion protein or binding protein of the first aspect, the one or more polynucleotides of the second aspect, the one or more constructs or vectors of the third aspect, the host cell of the fourth aspect, or the composition of the fifth aspect, wherein the therapeutic or diagnostic agent (ii) of the invention, or the same or different therapeutic or diagnostic part of the invention comprises or consists of the therapeutic or diagnostic agent.

[0023] The twelfth aspect of the present invention relates to a method for increasing the binding of a therapeutic agent or diagnostic agent to a predetermined organ, which comprises administering to a subject the fusion protein or binding protein of the first aspect, one or more polynucleotides of the second aspect, one or more constructs or vectors of the third aspect, the host cell of the fourth aspect, or the composition of the fifth aspect, wherein the therapeutic agent or diagnostic agent (ii) of the present invention, or the same or different therapeutic part or diagnostic part of the present invention comprises or consists of the therapeutic agent or diagnostic agent.

[0024] The thirteenth aspect of the present invention relates to a method for increasing the intracellular uptake of a therapeutic agent or diagnostic agent, which comprises administering to a subject the fusion protein or binding protein of the first aspect, one or more polynucleotides of the second aspect, one or more constructs or vectors of the third aspect, the host cell of the fourth aspect, or the composition of the fifth aspect, wherein the therapeutic agent or diagnostic agent (ii) of the present invention, or the same or different therapeutic part or diagnostic part of the present invention comprises or consists of the therapeutic agent or diagnostic agent.

[0025] A fourteenth aspect of the present invention relates to a method for degrading intracellular proteins, comprising the step of coupling the fusion protein or binding protein described herein to a ligand of an E3 ubiquitin ligase, wherein the therapeutic agent or diagnostic agent (ii) of the fusion protein or binding protein binds to the intracellular protein.

[0026] A fifteenth aspect of the present invention relates to one or more constructs or vectors encoding one or more polypeptides (P1) (i) of the fusion protein or binding protein described herein.

[0027] The sixteenth aspect of the present invention relates to a composition comprising one or more polypeptides selected from the group consisting of FSD1 (Q124A) of SEQ ID NO: 47, FSD1 (E126A) of SEQ ID NO: 50, and FSD1 (Q124 E126A) of SEQ ID NO: 53, or variants thereof having at least 70%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 96%, such as at least 97%, such as 98%, such as at least 99% sequence identity thereto. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 :Compound Design

[0029] A conceptual diagram of four types of compounds is provided. For all types, FSD1 can be used in a multimerized or non-multimerized form and with or without a linker. A. FSD1 is fused as a module to any other protein (Type 1). B. Fc-FSD1, a homodimer (i) or heterodimer (ii), is fused as a module to any other protein (Type 2). C. FSD1 is covalently coupled to any conventional antibody or any other therapeutic or diagnostic agent (i), or an anti-Fc nanobody-FSD1 fusion is bound to any conventional antibody or any other therapeutic or diagnostic agent for which the nanobody has affinity (ii) (Type 3). D. Heterodimeric Fc is fused to FST or FSD1 on one entity, while any other protein is fused to another entity (Type 4).

[0030] Figure 2 : Heparin affinity chromatography

[0031] A. Chromatogram showing the elution profiles of FSD1, compounds linked to FSD1 or FST291, and therapeutic agents (A 280 ) (left axis). A linear gradient from 0% to 100% buffer B (2 M NaCl) is shown as a straight line (right axis). Each peak in the elution curve is labeled with a number (#1 or #2). B. Table showing the elution concentration ([NaCl]) of each peak in the sample. Nb (Nanobody).

[0032] Figure 3 :FSD1 is covalently coupled to the anti-TNFα antibody (adalimumab)

[0033] A and B. Chromatograms showing the elution profiles of adalimumab (A) or adalimumab covalently conjugated to FSD1 (B) (A 280 , left axis). A linear gradient from 0% to 100% buffer B (2 M NaCl) is shown as a straight line (right axis). C. SDS-PAGE electropherogram of fractions 7-12 from heparin affinity chromatography of adalimumab covalently coupled to FSD1.

[0034] Figure 4 : Bioassay-based validation of activin A neutralization

[0035] Neutralization of somatostatin A was assessed using a luciferase-based bioassay of pSmad2 / 3 activation. Neutralization of FSD1 (monomer or dimer) (A), type 2 and type 4 design compounds (B and C), and native full-length follistatin was performed. 315 (FST315, SEQ ID NO: 28) and FST315 with an altered heparin binding site fused to a murine Fc fragment (FST315dHBS-mFc, SEQ ID NO: 30, SEQ ID NO: 31) (A) were evaluated at concentrations up to 8.2 nM. The commercially available counterparts, VGFR(1 / 2)-hFc (Aflibercept) or TNFR2-hFc (Etanercept), were also evaluated at concentrations up to 8.2 nM (B and C). Positive control (Pos control) and negative control (Neg control). TNFR2 (tumor necrosis factor receptor 2), VGFR1 / 2 (ligand-binding domains of vascular endothelial growth factor receptors 1 and 2). All results are expressed as mean ± SEM.

[0036] Figure 5 : Bioassay-based validation of myostatin neutralization

[0037] Neutralization of the growth factor myostatin was assessed using a luciferase-based bioassay for pSmad2 / 3 activation. Neutralization of FSD1 (monomer or dimer) (A), type 2 and type 4 design compounds (B and C), and native full-length follistatin was performed. 315 (FST315, SEQ ID NO:28) and FST315 with an altered heparin-binding site fused to a murine Fc fragment (FST315dHBS-mFc, SEQ ID NO:30, SEQ ID NO:31) (A) were evaluated at concentrations up to 8.2 nM. The commercially available counterparts, VGFR(1 / 2)-hFc (aflibercept) or TNFR2-hFc (etanercept), were also evaluated at concentrations up to 8.2 nM (B and C). Positive controls (Pos control) and negative controls (Neg control) were used. All results are expressed as mean ± SEM.

[0038] Figure 6 : Bioassay-based validation of GDF11 neutralization

[0039] Neutralization of the growth factor GDF11 (growth differentiation factor 11) was assessed using a luciferase-based bioassay for pSmad2 / 3 activation. Neutralization of FSD1 (monomer or dimer) (A), type 2 and type 4 design compounds (B and C), and native full-length follistatin was performed. 315 (FST315, SEQ ID NO:28) and FST315 with an altered heparin-binding site fused to a murine Fc fragment (FST315dHBS-mFc, SEQ ID NO:30, SEQ ID NO:31) (A) were evaluated at concentrations up to 8.2 nM. The commercially available counterparts, VGFR(1 / 2)-hFc (aflibercept) or TNFR2-hFc (etanercept), were also evaluated at concentrations up to 8.2 nM (B and C). Positive controls (Pos control) and negative controls (Neg control) were used. All results are expressed as mean ± SEM.

[0040] Figure 7 : Ligand binding affinity determined by surface plasmon resonance

[0041] Left: Surface plasmon resonance sensorgrams of VEGF (vascular endothelial growth factor) binding to immobilized Type 2 or Type 4 design compounds containing VGFR(1 / 2), commercially available VGFR(1 / 2)-hFc (aflibercept), and a negative control (FST315dHBS-mFc, SEQ ID NO: 30, SEQ ID NO: 31). Right: Surface plasmon resonance sensorgrams of TNFα (tumor necrosis factor α) binding to immobilized Type 2 or Type 4 design compounds containing TNFR2, commercially available TNFR2-hFc (etanercept), and a negative control (FST315dHBS-mFc, SEQ ID NO: 30, SEQ ID NO: 31).

[0042] Figure 8 : Binding to the extracellular matrix

[0043] The binding properties of type 2 and type 4 design compounds to ECM were evaluated using culture plates pre-coated with extracellular matrix (ECM) extract (Matrigel). A. Isolated FSD1 (monomer or dimer), native full-length follistatin, and IL-67 detected by colorimetric assay. 288 A. ECM binding curves for FST288 and FST315dHBS-mFc (SEQ ID NO: 30, SEQ ID NO: 31). B. Controls for type 2 and type 4 designed compounds containing VGFR(1 / 2), a commercial VGFR(1 / 2)-hFc (aflibercept), and an isolated FSD1 format. C. Controls for type 2 and type 4 designed compounds containing TNFR2, a commercial TNFR2-hFc (etanercept), and an isolated FSD1 format. All results are presented as mean ± SEM.

[0044] Figure 9 In vivo assessment of compound half-life in mouse skeletal muscle using IVIS

[0045] Type 2 design or Type 4 design compounds with VGFR(1 / 2) as the therapeutic moiety were administered intramuscularly into the gastrocnemius muscle of naive mice and compared with commercially available VGFR(1 / 2)-hFc (aflibercept) or PBS (phosphate buffered saline). A. Longitudinal tracking of fluorescence intensity from the gastrocnemius muscle assessed using the IVIS Spectrum in vivo imaging system. B. IVIS scan of all mice on the last day (96 hours after treatment). C. Fluorescence intensity of the excised gastrocnemius muscle. D. IVIS scan of the excised gastrocnemius muscle. E. Fluorescence intensity of serum samples on the last day (96 hours after treatment). F. IVIS scan of serum samples in 1.5 mL microcentrifuge tubes. All results are expressed as mean ± SEM. Data were analyzed using a one-way ANOVA analysis including all groups. Multiple comparisons were corrected using the Bonferroni test. *P<0.05vs PBS, # P<0.05vs VGFR(1 / 2) / FST291-mFc, vs VGFR(1 / 2)-hFc(aflibercept).

[0046] Figure 10 In vivo evaluation of the half-life of four design compounds in mouse ocular tissue using IVIS

[0047] Type 4 design compounds with VGFR(1 / 2) as the therapeutic moiety were administered intravitreally into the eyes of naive mice and compared with commercially available VGFR(1 / 2)-hFc (aflibercept) or PBS. A. Longitudinal tracking of fluorescence intensity from the eyes assessed using the IVIS Spectrum in vivo imaging system. B. Fluorescence intensity of the enucleated eye on the last day (96 hours after treatment). C. IVIS scan of the enucleated eye. All results are expressed as mean ± SEM. Data were analyzed using a one-way ANOVA analysis including all groups. Multiple comparisons were corrected using the Bonferroni test. *P < 0.05 vs PBS, vs VGFR(1 / 2)-hFc(aflibercept).

[0048] Figure 11 Ex vivo assessment of the half-life of type 4 design compounds in porcine kidneys during normothermic machine perfusion using IVIS

[0049] Four design compounds with TNFR2 as the therapeutic moiety were administered via arterial line to perfused kidneys and compared with commercially available TNFR2-hFc (aflibercept) or PBS. A. Fluorescence intensity of a midline section of a pig kidney 6 hours after treatment. B. IVIS scan of a midline section of a pig kidney. C. Fluorescence intensity of a section of the outer cortex of a pig kidney 6 hours after treatment. D. IVIS scan of a section of the outer cortex of a pig kidney.

[0050] Figure 12 : Intracellular uptake of the platform after 18 hours of compound incubation

[0051] Confocal images of adherent HEK293 cells. Cells were incubated for 18 hours with ATTO 488-fluorescently labeled design compound type 1 (with an anti-vimentin nanobody as the therapeutic or diagnostic moiety, i.e., anti-vimentin nanobody-FSD1), anti-vimentin nanobody, or DMEM alone. Following compound incubation, cells were washed and incubated with Hoechst and Lysotracker, respectively, to visualize nuclei and lysosomes. A. Maximum intensity projection of the z-stack for each sample. B. Orthogonal projection showing x and y slices of the anti-vimentin nanobody-FSD1 sample alone.

[0052] Figure 13 : In vivo demonstration of compound ECM binding in skeletal muscle

[0053] NIR730 fluorescently labeled type 2 or type 4 design compounds (with VGFR(1 / 2) as the therapeutic moiety) were administered intramuscularly into the gastrocnemius muscle of naive mice and compared with VGFR(1 / 2)-hFc (aflibercept) or PBS. A. Serial sections of gastrocnemius muscle stained with DAPI and Sirius Red. DAPI staining visualizes cell nuclei. The fluorescent signal of NIR730 visualizes the administered compound. Sirius Red staining visualizes connective tissue morphology (connective tissue stroke) within the muscle tissue. B. High-magnification images of two areas of gastrocnemius muscle treated with VGFR(1 / 2)-mFc-FSD1. Area 1 is a magnified image of the parenchyma (P) and connective tissue (CT). Area 2 is a magnified image of the blood vessels.

[0054] Figure 14 In vivo evaluation of the half-lives of type 2 and type 4 design compounds in mouse ocular tissue using fluorescent fundus imaging

[0055] Type 2 and type 4 designed compounds with VGFR(1 / 2) as the therapeutic moiety were administered intravitreally into the eyes of naive mice and compared with commercially available VGFR(1 / 2)-hFc (aflibercept) or PBS. A+B. In vivo fluorescent fundus examination images at baseline (A) and 96 hours after treatment (B). C. Mean fluorescence intensity of fundus images 96 hours after treatment. All results are expressed as mean ± SEM. Data were analyzed using a one-way ANOVA analysis including all groups. Multiple comparisons were corrected using the Bonferroni test. * = P < 0.05 vs PBS, # = P < 0.05 vs VGFR(1 / 2) / FST291-mFc, vs VGFR(1 / 2)-hFc(aflibercept).

[0056] Figure 15 In vivo efficacy of type 2 designer compounds in a wet age-related macular degeneration model

[0057] Type 2 design compounds with VGFR(1 / 2) as the therapeutic moiety were administered intravitreally into the eyes of naive mice and compared with commercial VGFR(1 / 2)-hFc (aflibercept) or a mouse IgG2a isotype control. A. Representative images of CD31- and isolectin-positive CNV lesions in RPE / choroidal flat mounts. Images were adjusted for contrast and brightness, respectively, to obtain the best black and white visual representation of CNV lesions. Scale bar = 150 μM. B. Mean CNV lesion area per eye assessed by CD31 and isolectin immunohistochemical staining of RPE / choroidal flat mounts. One outlier was identified and excluded in the VGFR(1 / 2)-mFc-FSD1 group in the normally distributed QQ plot. One eye in the VGFR(1 / 2)-hFc (aflibercept) group was inadvertently damaged during sample preparation. Based on pre-defined CNV exclusion criteria, two eyes were excluded: one from the VGFR(1 / 2)-mFc-FSD1 group and one from the VGFR(1 / 2)-hFc (aflibercept) group. All results are presented as mean ± SEM. Data were analyzed using a one-way ANOVA including all groups. Multiple comparisons were corrected using the Bonferroni test. * = P < 0.05 vs. mouse IgG2a isotype control.

[0058] Figure 16 : Intracellular uptake of the platform after 3 hours of compound incubation

[0059] Maximum intensity projection of a Z-stack of confocal images of adherent HEK293 cells. Cells were incubated for 3 hours with either a design compound (with an anti-vimentin nanobody as the therapeutic or diagnostic moiety, i.e., anti-vimentin nanobody-FSD1) fluorescently labeled with ATTO 488 or an anti-vimentin nanobody. Following compound incubation, cells were washed and incubated with Hoechst and Lysotracker, respectively, to visualize nuclei and lysosomes.

[0060] Figure 17 Intracellular uptake of the platform after 18 h of compound incubation, imaged using Airyscan super-resolution confocal imaging.

[0061] Maximum intensity projection of a Z-stack of Airyscan super-resolution confocal images of HEK293 adherent cells. Cells were incubated for 18 hours with either a design compound (with an anti-vimentin nanobody as the therapeutic or diagnostic moiety, i.e., anti-vimentin nanobody-FSD1) or an anti-vimentin nanobody labeled with ATTO 488. Following compound incubation, cells were washed and incubated with Hoechst to visualize nuclei. Cells treated with the anti-vimentin nanobody did not provide sufficient signal to generate super-resolution images.

[0062] Figure 18 : Intracellular uptake of the platform in vivo

[0063] Maximum intensity projection of a Z-stack of confocal images of the gastrocnemius muscle. ATTO 488 fluorescently labeled type 1 design compound (anti-vimentin nanobody-FSD1) with an anti-vimentin nanobody as either a therapeutic or diagnostic moiety was administered intramuscularly to the gastrocnemius muscle of naive mice and compared to the anti-vimentin nanobody. Mice were sacrificed 18 hours after injection, and gastrocnemius muscles were fixed with formalin, sectioned, and mounted with DAPI-containing mounting medium. P, parenchyma. CT, connective tissue.

[0064] Figure 19 : Expanded bioassay-based validation of the neutralization effects of type 1 design compounds on activin A, myostatin, and GDF11

[0065] Neutralization of somatostatin A (A), myostatin (B), or GDF11 (C) was assessed using a luciferase-based bioassay for pSmad2 / 3 activation. FSD1 (monomer or dimer) and type 1 design compounds using anti-vimentin nanobodies as therapeutic or diagnostic moieties (anti-vimentin-nb-FSD1) were evaluated at concentrations up to 1 μM. Positive controls (Pos control) and negative controls (Neg control) were also used. All results are presented as mean ± SEM.

[0066] Figure 20 Bioassay-based validation of the neutralization of activin A and myostatin by FSD1 mutants

[0067] Neutralization of activin A (A) and myostatin (B) was assessed using a luciferase-based bioassay for pSmad2 / 3 activation. Native FSD1 (abbreviated as "FSD1") and FSD1 mutants FSD1(Q124A), FSD1(E126A), and FSD1(Q124A,E126A) were evaluated at concentrations up to 822 nM. Positive controls (Pos control) and negative controls (Neg control) were also used. All results are expressed as mean ± SEM.

[0068] Figure 21 Bioassay-based validation of activin A neutralization by type 2 designed compounds using FSD1 mutants

[0069] Neutralization of the growth factors activin A (A), myostatin (B), and GDF11 (C) was assessed using a luciferase-based bioassay for pSmad2 / 3 activation. Type 2 design compounds employing native FSD1, VGFR(1 / 2)-mFc-FSD1, and its commercial counterpart, VGFR(1 / 2)-hFc (aflibercept), were evaluated at concentrations up to 1 μM. Type 2 design compounds employing mutant FSD1, VGFR(1 / 2)-mFc-FSD1(Q124A), VGFR(1 / 2)-mFc-FSD1(E126A), and VGFR(1 / 2)-mFc-FSD1(Q124A, E126A), were evaluated at concentrations up to 750 nM (activin A) or 63 nM (myostatin and GDF11). Positive controls (Pos control) and negative controls (Neg control) were used. All results are expressed as mean ± SEM.

[0070] Figure 22 : Binding of multimeric FSD1, type 3 designed compounds, and type 2 designed compounds utilizing FSD1 mutants to the extracellular matrix

[0071] The ECM binding properties of multimeric FSD1, type 3 design compounds, and type 2 design compounds utilizing FSD1 mutants were evaluated using colorimetric assays using culture plates pre-coated with extracellular matrix (ECM) extract (Matrigel). A. ECM binding curves of individual multimeric FSD1 (FSD1, FSD1-FSD1, and FSD1-FSD1-FSD1). B. ECM binding curves of three different fractions of type 3 design compound, the anti-TNFα antibody-FSD1 (adalimumab-FSD1), with increasing heparin affinity (low, medium, and high). Commercially available adalimumab was included as a control. C. ECM binding curves of type 2 designed compounds VGFR(1 / 2)-mFc-FSD1 using native FSD1 or type 2 designed compounds VGFR(1 / 2)-mFc-FSD1(Q124A), VGFR(1 / 2)-mFc-FSD1(E126A), and VGFR(1 / 2)-mFc-FSD1(Q124A, E126A) using FSD1 mutants. All results are expressed as mean ± SEM.

[0072] Figure 23 : Bioassay-based validation of glucocorticoid neutralization

[0073] Neutralization of the endogenous glucocorticoid cortisol or the synthetic glucocorticoid prednisolone-21-hemisuccinate was assessed using a luciferase-based bioassay activated by a glucocorticoid response element (GRE). Design compounds of type 2 and type 4, using CBG as the therapeutic moiety, were tested at concentrations up to 2 μM. Positive (Pos) and negative (Neg) controls were also included. All results are presented as mean ± SEM.

[0074] Figure 24 : Using the platform to degrade intracellular proteins

[0075] Western blot of lysates from HEK293 cells treated for 24 hours with a type 1 design compound (anti-vimentin-nb-FSD1) containing an anti-vimentin nanobody as the therapeutic moiety, anti-vimentin-nb, or DMEM alone. Both compounds were conjugated to an E3 ligase ligand (VHL ligand) to mediate proteasomal degradation of the target protein. A. Western blot of vimentin and GAPDH (loading control). Lane 1: Size marker. Lane 2: DMEM lysate. Lane 3: Lysate of anti-vimentin-nb conjugated to VHL ligand. Lane 4: Lysate of anti-vimentin-nb-FSD1 conjugated to VHL ligand. B. Vimentin band intensity was normalized to GAPDH band intensity. Detailed Description of the Invention

[0077] definition

[0078] As used herein, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise.

[0079] It should also be understood that "one or more" can be interchangeably replaced with "a plurality" or "at least one".

[0080] As used herein, the term "compound" encompasses molecules comprising or consisting of follistatin domain 1 (FSD1) and a therapeutic or diagnostic agent. For example, the term "compound" encompasses fusion proteins and binding proteins of the present invention comprising or consisting of follistatin domain 1 (FSD1) and a therapeutic or diagnostic agent.

[0081] As used herein, "fusion protein" refers to a hybrid polypeptide comprising protein domains of at least two different polypeptides that would not normally or naturally be fused in a single amino acid sequence. A fusion protein can comprise a single amino acid sequence comprising two completely different amino acid sequences or two similar or identical polypeptide sequences, provided that these sequences would not normally be present together in the same configuration in a single amino acid sequence found in nature. Thus, the protein domains of a fusion protein can be located at the amino terminal portion or the carboxyl terminal portion of the fusion protein, thereby forming an "amino-terminal fusion protein" or a "carboxyl-terminal fusion protein," respectively. A fusion protein can also comprise a linker polypeptide between the constituent polypeptides of the fusion protein. The term "fusion construct" or "fusion protein construct" generally refers to a polynucleotide encoding a fusion protein.

[0082] As used herein, "conjugated protein" refers to a hybrid polypeptide comprising one or more protein domains that bind to non-polypeptide components (eg, small molecule compounds, nucleic acids, sugar chains, or nanoparticles).

[0083] As used herein, "follistatin" (FST) may refer to all forms of follistatin, such as the protein core and molecular weight forms that have been identified as being produced by alternatively spliced ​​mRNAs FS-315, FS-303, and FS-288. Therefore, it should also be understood to include any isoforms produced by alternative splicing of follistatin mRNA, or mutant or polymorphic forms of follistatin. It should also be understood to extend to any protein encoded by the follistatin (FST) gene, any subunit polypeptide (e.g., a precursor form that may be produced), and any follistatin protein, whether present as a monomer, multimer, or fusion protein.

[0084] As used herein, follistatin domain 1 (FSD1) refers to the first of the three follistatin domains FSD1 (SEQ ID NO: 1), FSD2 (SEQ ID NO: 41), and FSD3 (SEQ ID NO: 42).

[0085] As used herein, "module" is used interchangeably with "unit" and "part", such as a (poly)peptide module fused or combined with another polypeptide or non-polypeptide part as a fusion protein or a binding protein.

[0086] As used herein, the term "therapeutic agent" refers to a biological or chemical agent used to treat, cure, alleviate or prevent a harmful condition in a subject. The term "therapeutic agent" also includes substances and agents used to combat a disease, condition or illness in a subject, including drugs, diagnostic agents and instruments. "Therapeutic agent" also includes any substance used for medical diagnosis or to restore, correct or alter physiological function. Therapeutic agents include, for example, therapeutic polypeptides, therapeutic polynucleotides, therapeutic small molecules (such as, but not limited to, therapeutic antibodies and fragments thereof), and antibiotics.

[0087] As used herein, a "therapeutic moiety" refers to a portion of a therapeutic agent that exerts at least a portion of the therapeutic effect of the therapeutic agent. Thus, a therapeutic moiety can be a polypeptide having therapeutic activity, as well as constructs comprising such polypeptides. For example, a therapeutic moiety can be a ligand receptor that provides a therapeutic effect. A therapeutic moiety can be any moiety that can be used therapeutically, including but not limited to antibiotics, anti-inflammatory drugs, anti-tumor drugs, cytotoxins, antivirals, and radioactive moieties. "Therapeutic moieties" include prodrugs of biologically active moieties, constructs in which multiple therapeutic moieties are bound to a carrier, such as multivalent moieties.

[0088] As used herein, a "diagnostic agent" refers to an agent that can be used to detect, image, and / or monitor the presence and / or progression of a condition, pathological disorder, and / or disease.

[0089] As used herein, "diagnostic moiety" refers to a portion of a diagnostic agent that, attached or separate from the diagnostic agent, achieves at least a portion of the diagnostic effect of the diagnostic agent, such as an imaging moiety.

[0090] The term "click chemistry," as used herein, refers to the use of chemical building blocks to drive ligation reactions with suitable complementary sites in other building blocks. These chemical reactions (e.g., including but not limited to reactions between azide and alkyne groups) are specific and result in a covalent bond between the two molecules. Compounds of the invention, such as fusion proteins and binding proteins of the invention, can be obtained through production steps involving click chemistry.

[0091] IgG Fc as used herein refers to the C-terminal region of the IgG heavy chain.

[0092] As used herein, "full-length antibody" refers to an antibody comprising at least two heavy chains and two light chains. The term includes antibodies whose heavy chains contain an Fc region. Full-length antibodies can be native sequence antibodies or recombinant antibodies. Full-length antibodies can be, for example, human antibodies, humanized antibodies, mouse antibodies, murinized antibodies and / or affinity-matured antibodies. Antibodies can be of any immunoglobulin class, including IgG, IgM, IgE, IgA, IgD, and any subclass thereof. Full-length antibodies can contain other domains, such as scFv or scFab, which bind to one or more chains of the full-length antibody. These conjugates are also encompassed by the term "full-length antibody."

[0093] As used herein, "activin A" refers to the active form of a homodimer of the polypeptide inhibin βA (βA) chain. "Activin A" refers to the activin protein with UniProt accession number P08476 (SEQ ID NO: 44), also known as inhibin subunit βA. Activins A, B, and AB are homodimers and heterodimers of the two polypeptide chains βA and βB, respectively.

[0094] As used herein, "myostatin" refers to the active form of the myostatin protein with UniProt Accession No. 014793 (SEQ ID NO: 45), also known as "GDF8" or "GDF-8," growth differentiation factor 8. Myostatin is a negative regulator of skeletal muscle mass.

[0095] As used herein, "GDF11" or "GDF-11" refers to the active form of growth differentiation factor 11, also known as bone morphogenetic protein 11 (BMP-11), UniProt Accession No. 095390 (SEQ ID NO: 46).

[0096] As used herein, "half-life" refers to the pharmacokinetic properties of a compound or agent, which measures the average survival time of a compound after its administration. Half-life can be expressed as the time required for 50% of a known amount of a compound or agent to be eliminated from a patient's body or a specific compartment thereof (e.g., measured in serum, i.e., circulating half-life, or other tissues). "Local half-life" refers to the half-life of a compound in a local area, such as at the site of administration of the compound or agent, such as in a specific organ, such as the local half-life of a compound or agent in muscle, or, for example, the local half-life of a compound or agent in the eye.

[0097] The "RCSB ID" used in this article refers to the Research Collaboration for Structural Bioinformatics (RCSB) Protein Data Bank (PDB) ID assigned to proteins. The PDB is a comprehensive repository of three-dimensional structural information of biological macromolecules, and each entry is assigned a unique identifier.

[0098] As used herein, "CNV" refers to choroidal neovascularization, such as, but not limited to, choroidal neovascularization in age-related macular degeneration (AMD).

[0099] As used herein, "RPE" refers to retinal pigment epithelium.

[0100] As used herein, a "variant" refers to a polypeptide or protein that differs in one or more amino acids from the sequence from which it is derived. Typically, a variant will have at least 70%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99% sequence identity with the sequence from which it is derived. As used herein, a "variant" includes mutant polypeptides obtained by mutagenesis, such as by making one or more point mutations in the sequence from which it is derived.

[0101] As used herein, "PROTAC" refers to a proteolysis-targeting chimera. A PROTAC consists of two covalently linked protein-binding molecules: one that is capable of engaging an E3 ubiquitin ligase and the other that binds to a target protein that is to be degraded, for example, by ubiquitination for intracellular degradation.

[0102] Fusion protein or binding protein construct

[0103] The present invention relates to fusion or binding protein structures comprising one or more polypeptides (P1) comprising or consisting of at least one FSD1 domain. Such polypeptides P1 and fusion or binding proteins comprising them are capable of binding to biological structures (e.g., extracellular matrix) via heparan sulfate without undesirably neutralizing the activities of, for example, full-length follistatin, activin A, myostatin, and GDF11.

[0104] Thus, the present invention provides a platform technology that is capable of, but not limited to, increasing the binding and therapeutic exposure of therapeutic or diagnostic agents while reducing off-target side effects.

[0105] Type 1

[0106] In one aspect, the present invention relates to a fusion protein or binding protein comprising:

[0107] (i) one or more polypeptides (P1) comprising or consisting of at least one FSD1 domain, said FSD1 domain comprising or consisting of a polypeptide having at least 70%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 99% sequence identity to SEQ ID NO: 1,

[0108] and

[0109] (ii) therapeutic or diagnostic agents.

[0110] In some embodiments, the fusion protein or binding protein comprises or consists of a polypeptide having at least 70% sequence identity, such as at least 80% sequence identity, such as at least 90% sequence identity, such as at least 95% sequence identity, such as 95% sequence identity, to the polypeptide shown in the group consisting of SEQ ID NO:2, SEQ ID NO:3 and SEQ ID NO:5.

[0111] In other embodiments, the fusion protein or binding protein comprises or consists of a polypeptide encoded by a polynucleotide having at least 70% sequence identity, such as at least 80% sequence identity, such as at least 90% sequence identity, such as at least 95% sequence identity, such as 95% sequence identity, to the polypeptide shown in the group consisting of SEQ ID NO:4 and SEQ ID NO:6.

[0112] Type 2

[0113] Those skilled in the art will appreciate that technological advances in the field of antibody engineering have enabled the development of novel therapeutic and diagnostic agents based on the engineering of the Fc domain of immunoglobulins (Ig, e.g., IgG). Such compounds include, for example, therapeutic or diagnostic Fc fusion proteins and derivatives thereof, which comprise one or more therapeutic or diagnostic moieties linked to an Ig Fc domain at the C-terminus.

[0114] In one embodiment, the present invention relates to a fusion protein or binding protein comprising:

[0115] (i) one or more polypeptides (P1) comprising or consisting of at least one FSD1 domain, said FSD1 domain comprising or consisting of a polypeptide having at least 70%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 99% sequence identity to SEQ ID NO: 1,

[0116] and

[0117] (ii) a therapeutic agent or a diagnostic agent, wherein the therapeutic agent or the diagnostic agent comprises or consists of an immunoglobulin Fc domain; and wherein the one or more polypeptides (P1) are linked to the immunoglobulin Fc domain at the C-terminus, preferably wherein the immunoglobulin is IgG, IgA, IgM, IgE or IgD.

[0118] The immunoglobulin Fc domain of the fusion protein or binding protein of the present invention may comprise two identical protein fragments, each fragment comprising a CH2 domain and a CH3 domain, such as a native IgG Fc region. In a preferred embodiment, the immunoglobulin Fc domain of the fusion protein or binding protein of the present invention may comprise different protein fragments, for example, protein fragments comprising one or more different mutations in the CH2 and / or CH3 domains of the Fc region.

[0119] Therefore, in one embodiment, the immunoglobulin Fc domain of the fusion protein or binding protein of the invention is an IgG Fc domain and comprises or consists of an IgG Fc-homodimer or an IgG Fc-heterodimer.

[0120] The Fc heterodimers of the present invention and / or fusion proteins or binding proteins comprising the Fc heterodimers can be prepared by protein engineering techniques known in the art, for example, using a scFV-Fc / Fc expression system, cloning into a plasmid, transfecting into cells for expression (e.g., HEK293 cells or CHO cells), and then collecting the supernatant and purifying the produced protein. Specifically, those skilled in the art are aware that techniques such as "knobs-into-holes" technology and other methods such as DD-KK variants with asymmetric electrostatic interactions, IgG / IgA chain exchange engineered domains, and HA-TF variants with asymmetric hydrophobic interactions can be used to promote the formation of heterodimers.

[0121] In a preferred embodiment, the fusion protein or binding protein of the invention comprises or consists of an IgG Fc-homodimer, wherein each monomer of the IgG Fc-homodimer is linked at the C-terminus to at least one polypeptide (P1), said polypeptide (P1) comprising or consisting of at least one FSD1 domain, said FSD1 domain comprising or consisting of a polypeptide having at least 70%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 99% sequence identity to SEQ ID NO: 1, and wherein each monomer of the IgG Fc-homodimer is linked at the N-terminus to the same or different therapeutic or diagnostic moiety.

[0122] Such fusion proteins or binding proteins can be represented herein by the general formula:

[0123] (Therapeutic moiety or diagnostic moiety)-Fc-(P1).

[0124] Human- and mouse-derived Fc regions may also be referred to herein as hFc and mFc, respectively.

[0125] In other preferred embodiments, the fusion protein or binding protein of the invention comprises or consists of an IgG Fc-heterodimer, wherein each monomer of the IgG Fc-heterodimer is linked at the C-terminus to at least one polypeptide (P1), said polypeptide (P1) comprising or consisting of at least one FSD1 domain, said FSD1 domain comprising or consisting of a polypeptide having a sequence identity to SEQ ID NO: 1 of at least 70%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 99%, or consisting of such a polypeptide, and wherein each monomer of the IgG Fc-heterodimer is linked at the N-terminus to the same or different therapeutic moiety or diagnostic moiety.

[0126] In some embodiments, the fusion protein or binding protein comprises or consists of a polypeptide having at least 70% sequence identity, such as at least 80% sequence identity, such as at least 90% sequence identity, such as at least 95% sequence identity, such as 95% sequence identity, to the polypeptide shown in the group consisting of SEQ ID NO:7 and SEQ ID NO:9.

[0127] In other embodiments, the fusion protein or binding protein comprises or consists of a polypeptide encoded by a polynucleotide having at least 70% sequence identity, such as at least 80% sequence identity, such as at least 90% sequence identity, such as at least 95% sequence identity, such as 95% sequence identity, to the polypeptide shown in the group consisting of SEQ ID NO: 8 and SEQ ID NO: 10.

[0128] Type 4

[0129] The at least one polypeptide (P1) comprising at least one FSD1 domain of the fusion protein or binding protein of the present invention may be linked at the N-terminus to an IgG Fc domain.

[0130] In one embodiment, the present invention relates to a fusion protein or binding protein of the present invention comprising:

[0131] (i) one or more polypeptides (P1) comprising or consisting of at least one FSD1 domain, said FSD1 domain comprising or consisting of a polypeptide having at least 70%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 99% sequence identity to SEQ ID NO: 1, and

[0132] (ii) a therapeutic or diagnostic agent; wherein the therapeutic or diagnostic agent comprises or consists of an IgG Fc-heterodimer, wherein at least one monomer of the IgG Fc-heterodimer is linked at the N-terminus to at least one polypeptide (P1), said polypeptide (P1) comprising or consisting of at least one FSD1 domain, said FSD1 domain comprising or consisting of a polypeptide having at least 70%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 99% sequence identity to SEQ ID NO: 1, and wherein at least one monomer of the IgG Fc-heterodimer is linked at the N-terminus to the same or different therapeutic or diagnostic moiety.

[0133] Such fusion proteins or binding proteins can be represented herein by the general formula:

[0134] (Therapeutic or diagnostic molecule) / (P1)-Fc.

[0135] Human- and mouse-derived Fc regions may also be referred to herein as hFc and mFc, respectively.

[0136] The fusion protein or binding protein of the present invention can be linked to one or more polypeptides (P1), for example, one or more polypeptides (P1), for example, a polymer of polypeptides (P1), for example, at least three polypeptides (P1), for example, at least four polypeptides, for example, at least five polypeptides (P1), for example, at least ten polypeptides (P1), for example, at least twenty-five polypeptides (P1), wherein the polypeptide (P1) comprises or consists of at least one FSD1 domain, wherein the FSD1 domain comprises or consists of a polypeptide having a sequence identity of at least 70%, for example, at least 80%, for example, at least 90%, for example, at least 95%, for example, at least 99% to SEQ ID NO: 1.

[0137] Therefore, in some preferred embodiments, at least one monomer of the IgG Fc-heterodimer of the fusion protein or binding protein of the present invention is linked at the N-terminus to at least one polypeptide (P1), for example, at least two polypeptides (P1), for example, at least three polypeptides (P1), for example, at least four polypeptides, for example, at least five polypeptides (P1), said polypeptide (P1) comprising at least one FSD1 domain, said FSD1 domain comprising or consisting of a polypeptide having a sequence identity to SEQ ID NO: 1 of at least 70%, for example, at least 80%, for example, at least 90%, for example, at least 95%, for example, at least 99%.

[0138] For certain applications, it may be beneficial for a therapeutic or diagnostic agent or therapeutic or diagnostic moiety to target (e.g., bind to) a specific protein (e.g., a specific protein associated with a disease or condition). For example, where a receptor is known to bind to the specific protein, the therapeutic or diagnostic moiety may, for example, be a receptor for the specific protein. The specific protein may be a protein associated with, for example, an immune response, an inflammatory response, or an angiogenic response. It will be appreciated by those skilled in the art that diseases such as, but not limited to, cancer, diabetes, arthritis, asthma, cardiovascular disease, and chronic inflammatory diseases may benefit from targeting the immune response, inflammatory response, or angiogenic response.

[0139] In other embodiments, the therapeutic or diagnostic agent, or at least one of the same or different therapeutic or diagnostic moieties, of the fusion or binding proteins of the invention is one or more cytokine receptors.

[0140] Those skilled in the art will appreciate that cytokines are immunomodulators that are widely used in cell communication. The term "cytokine" encompasses a variety of polypeptide modulators, such as interferons, interleukins, chemokines, or tumor necrosis factors.

[0141] In a further embodiment, the therapeutic or diagnostic agent of the fusion protein or binding protein of the invention, or at least one of the same or different therapeutic or diagnostic moieties, comprises or consists of the ligand binding domain of tumor necrosis factor receptor 2 (TNFR2) (SEQ ID NO: 21).

[0142] In other embodiments, the therapeutic or diagnostic agent of the fusion protein or binding protein of the invention, or at least one of the same or different therapeutic or diagnostic moieties, comprises or consists of a ligand binding domain of one or more growth factor receptors.

[0143] Examples of growth factor receptors include, but are not limited to, insulin growth factor receptors (IGF-1R, IR, and IRR); activin type IIA and type IIB receptors (ActRIIA, ActRIIB); epidermal growth factor family receptors (EGFR, ErbB2, and ErbB4); platelet-derived growth factor receptor (PDGFR), vascular endothelial growth factor receptor (VGFR), tyrosine kinase with immunoglobulin-like and epidermal growth factor homology domains (TIE-2), macrophage colony-stimulating factor (c-fms), c-kit, c-met, fibroblast growth factor receptor (FGFR), hepatocyte growth factor receptor (HGFR), Trk receptors (TrkA, TrkB, and TrkC), ephrin (Eph) receptors, and RET proto-oncogene.

[0144] In certain embodiments, the therapeutic or diagnostic agent of the fusion protein or binding protein of the invention, or at least one of the same or different therapeutic or diagnostic moieties, comprises or consists of the ligand binding domain of VGFR1 (SEQ ID NO: 19).

[0145] In other embodiments, the therapeutic or diagnostic agent of the fusion protein or binding protein of the invention, or at least one of the same or different therapeutic or diagnostic moieties, comprises or consists of the ligand binding domain of VGFR2 (SEQ ID NO: 20).

[0146] In a preferred embodiment, the one or more polypeptides (P1) (i) comprising or consisting of at least one FSD1 domain comprises or consists of a polypeptide having at least 70% sequence identity, such as at least 80% identity, such as at least 90% identity, such as at least 95% identity, such as 99% identity to SEQ.ID.NO:26.

[0147] In some embodiments, the IgG Fc-heterodimer of the fusion protein or binding protein of the present invention comprises or consists of a monomer having at least 70% sequence identity, such as at least 80% identity, such as at least 90% identity, such as at least 95% identity, such as at least 99% identity to SEQ ID NO: 11, and a monomer having at least 80% sequence identity, such as at least 85% identity, such as at least 90% identity, such as at least 95% identity, such as at least 99% identity to a sequence selected from SEQ ID NO: 13, SEQ ID NO: 15 and SEQ ID NO: 17.

[0148] In other embodiments, the IgG Fc-heterodimer of the fusion protein or binding protein of the present invention comprises or consists of: a monomer encoded by a sequence having at least 70% sequence identity, such as at least 80% identity, such as at least 90% identity, such as at least 95% identity, such as at least 99% identity to SEQ ID NO: 12, and a monomer encoded by a sequence having at least 80% sequence identity, such as at least 85% identity, such as at least 90% identity, such as at least 95% identity, such as at least 99% identity to a sequence selected from SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18.

[0149] Type 3

[0150] The at least one polypeptide (P1) comprising at least one FSD1 domain of the fusion protein or binding protein of the present invention may be linked to a full-length antibody.

[0151] Therefore, another embodiment of the present invention relates to a fusion protein or binding protein comprising:

[0152] (i) one or more polypeptides (P1) comprising or consisting of at least one FSD1 domain, said FSD1 domain comprising or consisting of a polypeptide having at least 70%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 99% sequence identity to SEQ ID NO: 1, and

[0153] (ii) therapeutic or diagnostic agents;

[0154] wherein the one or more polypeptides (P1)(i) are covalently linked to the therapeutic agent or diagnostic agent or are linked via non-covalent interactions.

[0155] In a further embodiment, one or more polypeptides (P1)(i) are covalently linked to the therapeutic or diagnostic agent using click chemistry.

[0156] In other embodiments, one or more polypeptides (P1)(i) are linked to the therapeutic or diagnostic agent via a non-covalent streptavidin-biotin interaction.

[0157] In other embodiments, one or more polypeptides (P1)(i) are linked to the therapeutic or diagnostic agent via a Nanobody (eg an anti-Fc Nanobody) having affinity for the therapeutic or diagnostic agent.

[0158] In some embodiments, the therapeutic or diagnostic agent comprises or consists of a full-length antibody.

[0159] The full-length antibody may be a multispecific antibody, such as a bispecific antibody.

[0160] In a preferred embodiment, the full-length antibody is a bispecific therapeutic antibody.

[0161] For certain applications of the fusion proteins or binding proteins described herein, it may be preferred that the FSD1 domain of the one or more polypeptides (P1) further comprises residues that allow chemical conjugation of the one or more polypeptides (P1) to the therapeutic or diagnostic agent (ii). These additional residues may be, for example, but not limited to, lysine-threonine-cysteine ​​(also referred to herein as lys-thr-cys or KTC) or other residues that provide a free C-terminal cysteine.

[0162] Thus, in some embodiments, one or more polypeptides (P1)(i) comprise or consist of a sequence having at least 70% similarity, such as at least 80% similarity, such as at least 90% similarity, such as at least 95% similarity, such as at least 99% similarity to a sequence selected from SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 64, SEQ ID NO: 65 and SEQ ID NO: 66.

[0163] In some embodiments, one or more polypeptides (P1)(i) comprise or consist of a sequence having at least 70% similarity, such as at least 80% similarity, such as at least 90% similarity, such as at least 95% similarity, such as at least 99% similarity to a sequence selected from SEQ ID NO: 23 and SEQ ID NO: 24.

[0164] In some embodiments, one or more polypeptides (P1)(i) are encoded by a sequence comprising or consisting of a sequence having at least 70% similarity, such as at least 80% similarity, such as at least 90% similarity, such as at least 95% similarity, such as at least 99% similarity to SEQ ID NO:25.

[0165] Variant FSD1 domain

[0166] In some embodiments of the fusion protein or binding protein of the present invention, at least one FSD1 domain comprising or consisting of a polypeptide having at least 70%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 99% sequence identity to SEQ ID NO: 1 is a variant of the FSD1 domain, which variant is defined by another polypeptide sequence as described herein having at least 70%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 99% sequence identity to SEQ ID NO: 1.

[0167] Therefore in a preferred embodiment, (i) at least one FSD1 domain of the one or more polypeptides (P1) comprises or consists of a variant FSD1 domain encoded by a sequence selected from the group consisting of SEQ ID NO: 47, SEQ ID NO: 50 and SEQ ID NO: 53, or a polypeptide having at least 70%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 96%, such as at least 97%, such as 98%, such as at least 99% sequence identity thereto.

[0168] Thus, in some embodiments, the fusion protein or binding protein of the present invention comprises:

[0169] (i) one or more polypeptides (P1) comprising or consisting of at least one FSD1 domain, said at least one FSD1 domain comprising or consisting of a variant FSD1 domain encoded by a sequence selected from the group consisting of SEQ ID NO: 47, SEQ ID NO: 50 and SEQ ID NO: 53, or a polypeptide having at least 70%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 96%, such as at least 97%, such as 98%, such as at least 99% sequence identity thereto;

[0170] and

[0171] (ii) therapeutic or diagnostic agents.

[0172] In some embodiments, the variant FSD1 domain is selected from the group consisting of: FSD1 (Q124A) of SEQ ID NO:47, FSD1 (E126A) of SEQ ID NO:50, and FSD1 (Q124 E126A) of SEQ ID NO:53.

[0173] Functional characteristics of fusion proteins or binding proteins

[0174] Lack of activin A, myostatin, and GDF-11 neutralization

[0175] The polypeptides (P1) of the present invention comprising at least one FSD1 domain do not neutralize the activity of growth factors such as activin A (SEQ ID NO: 44), myostatin (SEQ ID NO: 45), and GDF11 (SEQ ID NO: 46). This neutralization is in contrast to full-length follistatin, such as follistatin comprising the N-terminal domain (SEQ. ID NO: 43) and domains 1, 2, and 3 (FSD1 (SEQ ID NO: 1), FSD2 (SEQ ID NO: 41), and FSD3 (SEQ ID NO: 42). Importantly, the neutralization of these growth factors is associated with biological effects on muscle growth, such as skeletal muscle anabolism, which is detrimental to the versatile use of the (P1) polypeptide as a platform for developing fusion or binding proteins. The lack of neutralization of activin A, myostatin, and / or GDF11 can be measured by any assay known in the art, such as measuring the lack of activation of signaling downstream of these growth factors, such as the lack of activation of pSmad2 / 3 signaling in cells, for example, using a reporter gene bioassay of phosphorylated Smad2 / 3 signaling, as described in the Examples herein.

[0176] In a preferred embodiment of the present invention, the one or more polypeptides (P1) of the fusion protein or binding protein do not neutralize the activity of activin A (SEQ ID NO: 44), myostatin (SEQ ID NO: 45) and / or GDF11 (SEQ ID NO: 46).

[0177] In some other embodiments, the fusion protein or binding protein is expressed at a concentration range of up to at least 5 nM, such as at least 7.5 nM, such as at least 8 nM, such as at least 8.2 nM, such as at least 8.4 nM, such as at least 8.6 nM, such as at least 8.8 nM, such as at least 9 nM, such as at least 10 nM, such as at least 12 nM, such as at least 15 nM, such as at least 20 nM, such as at least 30 nM, such as at least 40 nM, such as at least 50 nM, such as at least 60 nM, such as at least 61 nM, such as at least 62 nM, such as at least 63 nM, such as at least 64 nM, such as at least 65 nM, such as at least For example, the present invention provides a method for treating a somatostatin-induced somatostatin-1 deficiency by at least 1 μM, for example, at least 2 μM, for example, at least 3 μM, for example, at least 4 μM, for example, at least 5 μM, for example, at least 6 μM, for example, at least 7 μM, for example, at least 8 μM, for example, at least 9 μM, for example, at least 10 μM, for example, at least 15 μM, for example, at least 20 μM, for example, at least 500 nM, for example, at least 600 nM, for example, at least 700 nM, for example, at least 750 nM, for example, at least 800 nM, for example, at least 820 nM, for example, at least 821 nM, for example, at least 822 nM, for example, at least 830 nM, for example, at least 1 μM, for example, at least 2 μM, for example, at least 3 μM, for example, at least 4 μM, for example, at least 5 μM, for example, at least 6 μM, for example, at least 7 μM, for example, at least 8 μM, for example, at least 9 μM, for example, at least 10 μM, for example, at least

[0178] In some embodiments, the fusion protein or binding protein does not neutralize the activity of myostatin, GDF11 and / or activin A at a concentration range of up to at least 5 nM, e.g., at least 7.5 nM, e.g., at least 8 nM, e.g., at least 8.2 nM, e.g., at least 8.4 nM, e.g., at least 8.6 nM, e.g., at least 8.8 nM, e.g., at least 9 nM, e.g., at least 10 nM, e.g., at least 12 nM, e.g., at least 15 nM, e.g., at least 20 nM, e.g., at least 30 nM, e.g., at least 40 nM, e.g., at least 50 nM, e.g., at least 80 nM, e.g., at least 100 nM, e.g., at least 150 nM, e.g., at least 200 nM.

[0179] In a further embodiment, the one or more polypeptides (P1)(i) are present in a concentration range of up to at least 5 nM, such as at least 7.5 nM, such as at least 8 nM, such as at least 8.2 nM, such as at least 8.4 nM, such as at least 8.6 nM, such as at least 8.8 nM, such as at least 9 nM, such as at least 10 nM, such as at least 12 nM, such as at least 15 nM, such as at least 20 nM, such as at least 30 nM, such as at least 40 nM, such as at least 50 nM, such as at least 60 nM, such as at least 61 nM, such as at least 62 nM, such as at least 63 nM, such as at least 64 nM, such as at least 65 nM, such as at least 66 nM, For example, the present invention does not neutralize the activity of myostatin (SEQ ID NO: 45), GDF11 (SEQ ID NO: 46) and / or activin A (SEQ ID NO: 44) at a concentration of at least 70 nM, such as at least 80 nM, such as at least 100 nM, such as at least 150 nM, such as at least 200 nM, such as at least 500 nM, such as at least 600 nM, such as at least 700 nM, such as at least 750 nM, such as at least 800 nM, such as at least 820 nM, such as at least 821 nM, such as at least 822 nM, such as at least 830 nM, such as at least 1 μM, such as at least 2 μM, such as at least 3 μM, such as at least 4 μM, such as at least 5 μM, such as at least 6 μM, such as at least 7 μM, such as at least 8 μM, such as at least 9 μM, such as at least 10 μM, wherein the neutralization or lack thereof is determined by a reporter gene bioassay of phosphorylated Smad2 / 3 signaling.

[0180] In a further embodiment, the one or more polypeptides (P1) do not neutralize the activity of myostatin, GDF11 and / or activin A at a concentration range of up to at least 5 nM, such as at least 7.5 nM, such as at least 8 nM, such as at least 8.2 nM, such as at least 8.4 nM, such as at least 8.6 nM, such as at least 8.8 nM, such as at least 9 nM, such as at least 10 nM, such as at least 12 nM, such as at least 15 nM, such as at least 20 nM, such as at least 30 nM, such as at least 40 nM, such as at least 50 nM, such as at least 80 nM, such as at least 100 nM, such as at least 150 nM, such as at least 200 nM.

[0181] In some embodiments, wherein the fusion protein or binding protein described herein is FSD1-FSD1, a Type 2 compound described herein, VGFR(1 / 2)-mFc-FSD1 and / or TNFR2-mFc-FSD1, the fusion protein or binding protein does not neutralize the activity of activin A, myostatin or GDF11 signaling at a concentration range of up to 8.2 nM.

[0182] In some embodiments, wherein the fusion protein or binding protein described herein is FSD1-FSD1, the fusion protein or binding protein does not neutralize the activity of activin A, myostatin, or GDF11 signaling at concentrations ranging up to 1 μM.

[0183] In other embodiments, the FSD1 domain variant selected from the group consisting of FSD1 (Q124A), FSD1 (E126A), and FSD1 (Q124A, E126A) does not neutralize activin A or myostatin at concentrations ranging up to 822 nM.

[0184] Heparan sulfate binding

[0185] One or more polypeptides (P1) comprising at least one FSD1 domain of the present invention bind to heparan sulfate (HS). Those skilled in the art understand that HS is a major component of the extracellular matrix (ECM) and can exist as free HS chains or as HS proteoglycans (e.g., cell surface proteoglycans). HS comprises a domain structure consisting of a repeating building block of a β-D-glucuronic acid-(1→4)-N-acetylated β-D-glucosamine disaccharide (NA domain) and a building block of a highly sulfated heparin-like α-L-iduronic acid-(1→4)-N-sulfated β-D-glucosamine disaccharide (NS domain), with a small proportion of mixed sequences of N-acetylated and N-sulfated disaccharides separating the two domains.

[0186] One or more polypeptides (P1)(i) conjugated to heparin sulfate are beneficial as modular protein platforms, for example for loading biologics into the ECM and / or specific organs, and / or increasing the half-life of therapeutic or diagnostic agents.

[0187] In some embodiments of the present invention, one or more polypeptides (P1)(i) of the fusion protein or binding protein of the present invention bind to heparan sulfate.

[0188] In a preferred embodiment, the one or more polypeptides (P1) (i) of the present invention comprising at least one FSD1 domain are encoded by a sequence comprising or consisting of a sequence that is at least 70% identical, such as at least 80% identical, such as at least 90% identical, such as at least 95% identical, such as at least 99% identical to SEQ.ID.NO:33.

[0189] In a preferred embodiment, the one or more polypeptides (P1 )(i) of the invention comprising at least one FSD1 domain comprise or consist of a sequence that is at least 100% identical to SEQ.ID.NO: 1 .

[0190] connector

[0191] In some embodiments, the fusion protein or binding protein of the present invention further comprises a linker between the one or more polypeptides (P1) comprising at least one FSD1 domain (i) and the therapeutic or diagnostic agent (ii).

[0192] In a further embodiment, the linker is a chemical linker.

[0193] Therapeutic or diagnostic agents

[0194] In a preferred embodiment, the therapeutic or diagnostic agent, or the same or different therapeutic or diagnostic moiety of the fusion protein or binding protein of the invention is selected from the group consisting of: a peptide, a protein such as an antibody or fragment thereof, a nanobody, streptavidin, a glycoprotein or an interleukin, a nucleic acid and a small molecule.

[0195] In other embodiments, the fusion protein or binding protein further comprises a detectable moiety. The detectable moiety can be selected from the group consisting of: fluorescent proteins, gold nanoparticles, radioisotopes, biotin or its derivatives, and enzymes.

[0196] In a preferred embodiment, the therapeutic or diagnostic agent (ii) of the fusion protein or binding protein of the invention, or the same or different therapeutic or diagnostic portion, binds to a target selected from the group consisting of: cluster of differentiation (CD) proteins, cytokines such as interleukins, growth factors such as colony stimulating factors, immune checkpoint proteins, angiogenic factors, hemostatic factors, chemokines, neurotrophic factors, inflammatory proteins, tumor antigens, bacterial proteins, and viral proteins.

[0197] In some embodiments, the therapeutic or diagnostic agent (ii) of the fusion protein or binding protein of the invention, or the same or different therapeutic or diagnostic portion, comprises or consists of a ligand binding domain of a protein selected from TNFR2, VGFR1, VGFR2, and CBG (corticosteroid binding globulin).

[0198] In a further embodiment, the therapeutic or diagnostic agent (ii) of the fusion protein or binding protein of the invention, or the same or different therapeutic or diagnostic portion, comprises or consists of a ligand binding domain of a protein having at least 70% similarity, such as at least 80% similarity, such as at least 90% similarity, such as at least 95% similarity, such as at least 99% similarity to a sequence selected from SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21 and SEQ ID NO: 22.

[0199] In another embodiment, the therapeutic or diagnostic agent or the same or different therapeutic or diagnostic moiety of the fusion protein or binding protein of the invention comprises or consists of an anti-vimentin Nanobody.

[0200] In some embodiments, the anti-vimentin Nanobody has at least 70% similarity to SEQ ID NO: 34, such as at least 80% similarity, such as at least 90% similarity, such as at least 95% similarity, such as at least 99% similarity.

[0201] In a further embodiment, the anti-vimentin Nanobody is encoded by a sequence that has at least 70% similarity, such as at least 80% similarity, such as at least 90% similarity, such as at least 95% similarity, such as at least 99% similarity to SEQ ID NO: 35.

[0202] In some embodiments, the fusion protein or binding protein is as described in the "Type 2" section herein, i.e., comprises:

[0203] (i) one or more polypeptides (P1) consisting of at least one FSD1 domain comprising or consisting of a polypeptide having at least 70%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 99% sequence identity to SEQ ID NO: 1,

[0204] and

[0205] (ii) a therapeutic or diagnostic agent, wherein the therapeutic or diagnostic agent comprises or consists of an immunoglobulin Fc domain; and wherein the one or more polypeptides (P1) are linked to the immunoglobulin Fc domain at the C-terminus, preferably wherein the immunoglobulin is IgG, IgA, IgM, IgE or IgD,

[0206] The therapeutic agent or diagnostic agent is selected from an anti-VEGF agent, an anti-TNFα agent, and a mutated CBG.

[0207] In a preferred embodiment, the fusion protein or binding protein comprises:

[0208] (i) two polypeptides (P1) consisting of one FSD1 domain comprising or consisting of a polypeptide having at least 70%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 99% sequence identity to SEQ ID NO: 1,

[0209] and

[0210] (ii) a therapeutic or diagnostic agent, wherein the therapeutic or diagnostic agent comprises or consists of an immunoglobulin Fc domain; and wherein the two polypeptides (P1) are linked to the immunoglobulin Fc domain at the C-terminus, preferably wherein the immunoglobulin is IgG, IgA, IgM, IgE or IgD,

[0211] The therapeutic agent or diagnostic agent is selected from an anti-VEGF agent, an anti-TNFα agent, and a mutated CBG.

[0212] In other embodiments, the fusion protein or binding protein is ubiquitinated.

[0213] In some embodiments, the therapeutic or diagnostic agent, or the same or different therapeutic or diagnostic moiety, of the fusion protein or binding protein of the invention binds to a target, and the fusion protein or binding protein is capable of binding heparan sulfate and the target simultaneously.

[0214] Platform Strategy

[0215] Increased local half-life

[0216] The functional properties of one or more (P1)(i) polypeptides described herein (e.g., heparin sulfate binding properties) enable the use of said polypeptides as modules as a platform technology to increase the half-life of the therapeutic or diagnostic agents described herein, or the same or different therapeutic or diagnostic moieties.

[0217] Thus, in some embodiments, the fusion protein or binding protein has a longer local half-life at the site of administration than the therapeutic or diagnostic agent alone, or the same or different therapeutic or diagnostic moiety.

[0218] ECM binding

[0219] The functional properties of the (P1) polypeptides described herein (e.g., heparan sulfate binding) enable the use of the polypeptides as modules as a platform technology to increase binding of therapeutic or diagnostic agents described herein, or the same or different therapeutic or diagnostic moieties, to ECM.

[0220] Binding to the ECM can be measured by any method known in the art, such as using an in vitro ECM binding assay, such as a colorimetric or fluorescence-based assay. For example, the half-maximal effective concentration (EC50) can be used to assess the concentration at which the test compound achieves 50% of the total binding capacity (e.g., 50% of the binding capacity to the ECM surface being tested).

[0221] Thus, in some embodiments, the fusion protein or binding protein increases binding of a therapeutic or diagnostic agent, or the same or different therapeutic or diagnostic moiety, to the extracellular matrix compartment compared to the therapeutic or diagnostic agent or the same or different therapeutic or diagnostic moiety alone.

[0222] In some embodiments, e.g., wherein the fusion protein or binding protein of the invention is linked to one or more polypeptides (P1), e.g., one or more polypeptides (P1), e.g., a polymer of polypeptides (P1), e.g., at least three polypeptides (P1), e.g., at least four polypeptides, e.g., at least five polypeptides (P1), e.g., at least ten polypeptides (P1), e.g., at least twenty-five polypeptides (P1), said polypeptides (P1) comprising or consisting of at least one FSD1 domain comprising or consisting of a polypeptide having at least 70%, e.g., at least 80%, e.g., at least 90%, e.g., at least 95%, e.g., at least 99% sequence identity to SEQ ID NO: 1, ECM binding increases with increasing number of polypeptides (P1).

[0223] For example, in some embodiments, the FSD1-FSD1-FSD1 compound has a higher ECM binding affinity, e.g., a higher ECM binding affinity than FSD1-FSD1, as measured by an ECM binding assay described herein. In other embodiments, the FSD1-FSD1 compound has a higher ECM binding affinity, e.g., a higher ECM binding affinity than FSD1, as measured by an ECM binding assay described herein.

[0224] Organ loading

[0225] In some applications, it may be beneficial for the increased binding of the fusion protein or binding protein of the invention to occur at a specific predetermined structure, such as a predetermined organ or tumor.

[0226] Those skilled in the art understand that, for example, binding of a therapeutic or diagnostic agent to a disease localized in a specific compartment (eg, an organ) and increased delivery to that disease may increase the efficacy of the therapeutic and / or diagnostic effect of the compound.

[0227] Thus, in some embodiments, the fusion protein or binding protein increases binding of the therapeutic or diagnostic agent (ii) or the same or different therapeutic or diagnostic moiety to a predetermined organ or tumor compared to the therapeutic or diagnostic agent or the same or different therapeutic or diagnostic moiety alone.

[0228] Increased binding can be measured by a decrease in EC50, which is the concentration of the test compound (e.g., therapeutic or diagnostic agent (ii) or the same or different therapeutic or diagnostic moiety) at which 50% of the total binding capacity is achieved (e.g., 50% of the binding capacity to the predetermined organ or tumor being tested).

[0229] Thus, in preferred embodiments, the EC50 of the therapeutic or diagnostic agent (ii) or the same or different therapeutic or diagnostic moiety for a predetermined organ or tumor is reduced by at least 2-fold, such as at least 5-fold, such as at least 10-fold, such as at least 20-fold, such as at least 50-fold, such as at least 80-fold, such as at least 100-fold, such as at least 250-fold, such as at least 500-fold, such as at least 1000-fold, such as at least 5000-fold, such as at least 10000-fold, such as at least 100000-fold, compared to the therapeutic or diagnostic agent (ii) or the same or different therapeutic or diagnostic moiety alone.

[0230] In a further embodiment, the predetermined organ is selected from the group consisting of organs of the musculoskeletal system, digestive system, respiratory system, urinary system, reproductive organ and endocrine system, circulatory system, nervous system, hematopoietic organ and integumentary system.

[0231] The organ may be selected from the group consisting of kidney, eye, liver, heart, lung, bladder, pancreas, gall bladder, intestine, prostate, brain, skin, muscle, bone, hematopoietic tissue and subcutaneous tissue, such as joint or synovial tissue.

[0232] Intracellular uptake

[0233] In some embodiments, the fusion protein or binding protein increases the intracellular uptake of the therapeutic or diagnostic agent (ii) or the same or different therapeutic or diagnostic moiety compared to the therapeutic or diagnostic agent (ii) or the same or different therapeutic or diagnostic moiety alone.

[0234] Multimers comprising FSD1 (P1) polypeptides

[0235] The fusion protein or binding protein of the present invention may be linked to one or more polypeptides (P1) as described herein.

[0236] In a preferred embodiment, the therapeutic or diagnostic agent, or the same or different therapeutic or diagnostic moiety of the fusion protein or binding protein of the present invention is linked to at least one polypeptide (P1) (i), such as at least two polypeptides (P1) (i), such as at least three polypeptides (P1) (i), such as at least four polypeptides, such as at least five polypeptides (P1) (i), wherein the polypeptide (P1) comprises at least one FSD1 domain or consists of at least one FSD1 domain, wherein the FSD1 domain comprises or consists of a polypeptide having a sequence identity of at least 70%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 99% to SEQ ID NO: 1.

[0237] In other embodiments, the therapeutic or diagnostic agent comprises or consists of an Fc dimer, each monomer of the Fc homodimer or Fc heterodimer is linked to at least one polypeptide (P1) (i), for example at least two polypeptides (P1) (i), for example at least three polypeptides (P1) (i), for example at least four polypeptides, for example at least five polypeptides (P1) (i), said polypeptide (P1) comprising or consisting of at least one FSD1 domain, said FSD1 domain comprising or consisting of a polypeptide having at least 70%, for example at least 80%, for example at least 90%, for example at least 95%, for example at least 99% sequence identity to SEQ ID NO: 1.

[0238] Multimerization and linkage of FSD1 domains

[0239] For example, to solve steric issues, it may be beneficial for at least one polypeptide (P1)(i) to comprise or consist of a plurality of FSD1 domains as described herein arranged in a multimer. For example, at least one of the at least one polypeptide (P1)(i) may comprise or consist of at least two FSD1 domains (dimer).

[0240] In a further embodiment, at least one of the at least one polypeptide (P1)(i) of the fusion protein or binding protein comprises or consists of a multimer of FSD1 domains, such as at least two FSD1 domains, such as at least three FSD1 domains, such as at least four FSD1 domains, such as at least five FSD1 domains, which FSD1 domains comprise or consist of a polypeptide having at least 70%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 99% sequence identity to SEQ ID NO: 1.

[0241] In yet other embodiments, at least two monomers in a multimer of FSD1 domains of a fusion protein or binding protein of the present invention are connected by a linker.

[0242] In some embodiments, the fusion protein or binding protein comprises or consists of a polypeptide having at least 70% sequence identity, such as at least 80% sequence identity, such as at least 90% sequence identity, such as at least 95% identity, such as at least 99% identity to a sequence selected from SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, and SEQ ID NO: 9.

[0243] In other embodiments, the fusion protein or binding protein comprises or consists of a polypeptide having at least 70% sequence identity, such as at least 80% sequence identity, such as at least 90% sequence identity, such as at least 95% identity, such as at least 99% identity to SEQ ID NO: 11, and a polypeptide having at least 70% sequence identity, such as at least 80% sequence identity, such as at least 90% sequence identity, such as at least 95% identity, such as at least 99% identity to a sequence selected from SEQ ID NO: 13, SEQ ID NO: 15 and SEQ ID NO: 17.

[0244] polynucleotides

[0245] Another aspect of the invention relates to one or more polynucleotides that, when expressed, encode a fusion protein or binding protein as described herein.

[0246] In some embodiments, the one or more polynucleotides have at least 70% sequence identity, such as at least 80% sequence identity, such as at least 90% sequence identity, such as at least 95% identity, such as at least 99% identity to a polynucleotide selected from SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8 and SEQ ID NO:10.

[0247] In other embodiments, the one or more polynucleotides have at least 70% sequence identity, such as at least 80% sequence identity, such as at least 90% sequence identity, such as at least 95% identity, such as at least 99% identity, to a combination of SEQ ID NO: 12 and at least one polynucleotide selected from SEQ ID NO: 14, SEQ ID NO: 16 and SEQ ID NO: 18.

[0248] Construct or vector

[0249] Another aspect of the present invention relates to one or more constructs or vectors comprising one or more polynucleotides described herein and / or encoding a fusion protein or binding protein described herein.

[0250] It will be appreciated by those skilled in the art that the polynucleotides and constructs or vectors used to express the compounds of the present invention may also contain additional sequences known in the art, such as sequences for facilitating compound production, expression, or purification. Such additional sequences may be, but are not limited to, purification tags known in the art, such as polyhistidine tags.

[0251] host cells

[0252] Those skilled in the art understand that expression of recombinant proteins (eg, fusion proteins or binding proteins described herein) requires an expression system, such as a mammalian, insect, yeast, bacterial, algal, or cell-free expression system.

[0253] One aspect of the present invention relates to a host cell comprising one or more polynucleotides described herein or one or more constructs or vectors described herein.

[0254] In preferred embodiments, the host cells described herein are mammalian cells.

[0255] In a more preferred embodiment, the host cell is a human cell, such as a human embryonic kidney 293 cell (HEK293).

[0256] In other embodiments, the host cell is a Chinese Hamster Ovary (CHO) cell.

[0257] Composition

[0258] Another aspect of the present invention relates to a composition comprising a fusion protein or binding protein as described herein, one or more polynucleotides as described herein, one or more constructs or vectors as described herein, a host cell as described herein, or a mixture thereof.

[0259] In preferred embodiments, the compositions described herein are pharmaceutical compositions.

[0260] Another aspect of the invention relates to a composition comprising one or more polypeptides selected from the group consisting of FSD1 (Q124A) of SEQ ID NO: 47, FSD1 (E126A) of SEQ ID NO: 50, and FSD1 (Q124E126A) of SEQ ID NO: 53, or variants thereof having at least 70%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 96%, such as at least 97%, such as 98%, such as at least 99% sequence identity thereto.

[0261] Medical uses and treatments

[0262] In a further embodiment, the fusion protein or binding protein as described herein, the one or more polynucleotides as described herein, the one or more constructs or vectors as described herein, the host cell as described herein, or the composition as described herein, is for use in medicine.

[0263] In a preferred embodiment, the fusion protein or binding protein as described herein, the one or more polynucleotides as described herein, the one or more constructs or vectors as described herein, the host cell as described herein, or the composition as described herein is used to treat ophthalmic diseases, cancerous diseases, inflammatory diseases, neurological diseases, cardiovascular diseases, metabolic diseases, respiratory diseases, musculoskeletal diseases, and aging-related diseases.

[0264] In a more preferred embodiment, the fusion protein or binding protein as described herein, the one or more polynucleotides as described herein, the one or more constructs or vectors as described herein, the host cell as described herein, or the composition as described herein is used to treat an ophthalmic disease, a cancerous disease, or an inflammatory disease.

[0265] In some embodiments, the ophthalmic disease is neovascular age-related macular degeneration (wet AMD). In further embodiments, the ophthalmic disease is neovascular age-related macular degeneration (wet AMD), and the therapeutic or diagnostic agent (ii) of the fusion protein or binding protein described herein is an anti-VEGF agent, preferably, wherein the fusion protein or binding protein is as described in the "Type 2" section herein.

[0266] In some embodiments of the fusion protein or binding protein as described herein, one or more polynucleotides as described herein, one or more constructs or vectors as described herein, a host cell as described herein, or a composition as described herein for treating an inflammatory disorder, the therapeutic or diagnostic agent (ii) of the fusion protein or binding protein as described herein is an anti-TNFα agent, preferably, wherein the fusion protein or binding protein is as described in the "Type 2" section herein.

[0267] In some embodiments of the fusion protein or binding protein as described herein, one or more polynucleotides as described herein, one or more constructs or vectors as described herein, a host cell as described herein, or a composition as described herein for treating a musculoskeletal condition, such as a steroid-induced myopathy, the therapeutic or diagnostic agent (ii) of the fusion protein or binding protein as described herein is a mutant CBG, preferably, wherein the fusion protein or binding protein is as described in the "Type 2" section herein.

[0268] In some embodiments of the fusion protein or binding protein as described herein, one or more polynucleotides as described herein, one or more constructs or vectors as described herein, a host cell as described herein, or a composition as described herein for treating a cancerous disease or an autoimmune disorder, the therapeutic or diagnostic agent (ii) of the fusion protein or binding protein as described herein is interleukin 2 (IL-2), preferably, wherein the fusion protein or binding protein is as described in the "Type 1" section herein.

[0269] In other embodiments, the inflammatory condition results from an organ transplant.

[0270] In yet other embodiments, the medical use is in a subject who donates or receives an organ, and the donated or transplanted organ is selected from the group consisting of a kidney, heart, lung, bone marrow, and liver.

[0271] Another aspect of the present invention relates to a method of treating a disease or condition, comprising administering to a subject an effective amount of a fusion protein or binding protein as described herein, one or more polynucleotides as described herein, one or more constructs or vectors as described herein, a host cell as described herein, or a composition as described herein.

[0272] Another aspect of the present invention relates to a method for treating an ophthalmic disease, an inflammatory disease, or a cancerous disease, comprising administering to a subject an effective amount of a fusion protein or binding protein as described herein, one or more polynucleotides as described herein, one or more constructs or vectors as described herein, a host cell as described herein, or a composition as described herein.

[0273] Another aspect of the present invention relates to the use of a fusion protein or binding protein described herein, one or more polynucleotides described herein, one or more constructs or vectors described herein, a host cell described herein, or a composition described herein in the manufacture of a medicament for treating a disease or condition (e.g., an ophthalmic disease, a cancerous disease, or an inflammatory disease).

[0274] In some embodiments of the uses of the fusion proteins or binding proteins described herein or the methods described herein, the fusion proteins or binding proteins, polynucleotide(s), construct(s) or vector(s), host cells, or compositions described herein are administered systemically.

[0275] In some embodiments of the uses of the fusion proteins or binding proteins described herein or the methods described herein, the fusion proteins or binding proteins, polynucleotide(s), construct(s) or vector(s), host cells, or compositions described herein are administered topically.

[0276] In some embodiments of the uses of the fusion proteins or binding proteins described herein or the methods described herein, the fusion proteins or binding proteins, polynucleotide(s), construct(s) or vector(s), host cells or compositions described herein are administered intraadiposally, intraarterially, intraarticularly, intracranially, intradermally, intralesionally, intramuscularly, intranasally, intraocularly, intraosseously, intrapericardially, intraperitoneally, intrapleurally, intraprostatically, intrarectally, intrathecally, intratracheally, intratumorally, intraumbilically, intravaginally, intravenously, intravesicularly, intravitreally, or liposomally.

[0277] Methods for increasing the local half-life of therapeutic or diagnostic agents

[0278] One aspect of the present invention relates to a method of increasing the local half-life of a therapeutic or diagnostic agent at the site of administration, comprising obtaining a fusion protein or binding protein as described herein, one or more polynucleotides as described herein, one or more constructs or vectors as described herein, a host cell as described herein, or a composition as described herein, wherein the therapeutic or diagnostic agent or the same or different therapeutic or diagnostic portion of the fusion protein or binding protein as described herein comprises or consists of the therapeutic or diagnostic agent.

[0279] Another aspect of the invention relates to a method of increasing the local half-life of a therapeutic or diagnostic agent in vivo at the site of administration in a subject, comprising administering to the subject a fusion protein or binding protein as described herein, one or more polynucleotides as described herein, one or more constructs or vectors as described herein, a host cell as described herein, or a composition as described herein, wherein the therapeutic or diagnostic agent or the same or different therapeutic or diagnostic portion of the fusion protein or binding protein as described herein comprises or consists of the therapeutic or diagnostic agent.

[0280] In some embodiments, the fusion proteins or binding proteins described herein, the one or more polynucleotides described herein, the one or more constructs or vectors described herein, the host cells described herein, or the compositions described herein are administered to a subject having an ophthalmic disease, a cancerous disease, an inflammatory disease, a neurological disease, a cardiovascular disease, a metabolic disease, a respiratory disease, a musculoskeletal disease, or an aging-related disease.

[0281] In preferred embodiments, the fusion protein or binding protein or composition is administered to a subject having an ophthalmic disease, and the fusion protein or binding protein or composition is administered intravitreally, subretinally, or suprachoroidally.

[0282] In a further embodiment, the ophthalmic disease is wet AMD.

[0283] In other embodiments, the fusion protein or binding protein or composition is administered to a subject donating or receiving an organ (preferably a kidney, heart, lung, bone marrow, or liver, even more preferably a kidney), or to the organ during ex vivo perfusion, and the therapeutic or diagnostic agent comprises or consists of a compound that reduces an inflammatory disease.

[0284] In preferred embodiments, the local half-life of the therapeutic or diagnostic agent at the site of administration is increased by the methods of the invention by at least 6 hours, such as at least 12 hours, such as at least 24 hours, such as at least 48 hours, such as at least 72 hours, such as at least 96 hours, such as at least 120 hours, such as at least one week, such as at least 2 weeks, such as at least 4 weeks, such as at least 8 weeks, such as at least 3 months, such as at least 6 months, such as at least 12 months.

[0285] Methods for increasing ECM binding of therapeutic or diagnostic agents

[0286] Another aspect of the invention relates to a method of increasing the binding of a therapeutic or diagnostic agent to an extracellular matrix, comprising administering to a subject a fusion protein or binding protein as described herein, one or more polynucleotides as described herein, one or more constructs or vectors as described herein, a host cell as described herein, or a composition as described herein, wherein the therapeutic or diagnostic agent or the same or different therapeutic or diagnostic portion of the fusion protein or binding protein as described herein comprises or consists of the therapeutic or diagnostic agent.

[0287] Method for increasing the binding of therapeutic or diagnostic agents to a predetermined organ

[0288] One aspect of the present invention relates to a method for increasing the binding of a therapeutic or diagnostic agent to a predetermined organ, comprising administering to a subject a fusion protein or binding protein as described herein, one or more polynucleotides as described herein, one or more constructs or vectors as described herein, a host cell as described herein, or a composition as described herein, wherein the therapeutic or diagnostic agent or the same or different therapeutic or diagnostic portion of the fusion protein or binding protein as described herein comprises or consists of the therapeutic or diagnostic agent.

[0289] Methods for increasing the intracellular uptake of therapeutic or diagnostic agents

[0290] One aspect of the invention relates to a method of increasing the intracellular uptake of a therapeutic or diagnostic agent, comprising administering to a subject a fusion protein or binding protein as described herein, one or more polynucleotides as described herein, one or more constructs or vectors as described herein, a host cell as described herein, or a composition as described herein, wherein the therapeutic or diagnostic agent or the same or different therapeutic or diagnostic portion of the fusion protein or binding protein as described herein comprises or consists of the therapeutic or diagnostic agent.

[0291] In further embodiments, the therapeutic or diagnostic agent is transported to the cytosol, eg, cytoskeletal filaments, or to the nucleus.

[0292] Methods for degrading intracellular proteins using proteolysis-targeting chimeras (PROTAC) strategies

[0293] The fusion proteins or binding proteins of the present invention can be used to degrade intracellular proteins using the proteolysis targeting chimera (PROTAC) strategy.

[0294] In some embodiments, the fusion protein or binding protein of the invention further comprises a PROTAC linker, preferably wherein the PROTAC linker is selected from the group consisting of a flexible aliphatic linker, a flexible PEGylated linker, a flexible heterochain, a rigid linker, a triazole-based linker, a bioorthogonal clickable linker, and a photoswitchable linker.

[0295] Therefore, one aspect of the present invention relates to a method for degrading an intracellular protein, comprising the step of coupling a fusion protein or binding protein according to any one of the preceding items to a ligand of an E3 ubiquitin ligase, wherein the therapeutic agent or diagnostic agent (ii) of the fusion protein or binding protein is bound to the intracellular protein.

[0296] In some embodiments, the ligand of the E3 ubiquitin ligase is (S,R,S)-AHPC. Preferably, the ligand of the E3 ubiquitin ligase is (S,R,S)-AHPC-PEG8-NHS.

[0297] In other embodiments, the E3 ubiquitin ligase is the Von-Hippel Lindau (VHL) tumor suppressor protein.

[0298] In some embodiments, the therapeutic or diagnostic agent (ii) is an anti-vimentin Nanobody.

[0299] In other embodiments, the fusion protein or binding protein is a Type 1 protein as described herein, preferably, the fusion protein or binding protein is anti-vimentin Nanobody-FSD1.

[0300] Subjects of medical uses and methods

[0301] In a preferred embodiment of the present invention, the subject of the use of the fusion protein or binding protein described herein or the subject of the method described herein is a human or non-human animal.

[0302] Routes of administration of the uses and methods of the present invention

[0303] In some embodiments of the uses and methods of the invention, a fusion protein or binding protein as described herein, one or more polynucleotides as described herein, one or more constructs or vectors as described herein, a host cell as described herein, or a composition as described herein is administered systemically.

[0304] In other embodiments of the uses and methods of the invention, a fusion protein or binding protein as described herein, one or more polynucleotides as described herein, one or more constructs or vectors as described herein, a host cell as described herein, or a composition as described herein is administered topically.

[0305] In further embodiments of the uses and methods of the present invention, the fusion protein or binding protein described herein, the one or more polynucleotides described herein, the one or more constructs or vectors described herein, the host cell described herein, or the composition described herein is administered intraadipose, intraarterially, intraarticularly, intracranially, intradermally, intralesionally, intramuscularly, intranasally, intraocularly, intraosseously, intrapericardially, intraperitoneally, intrapleurally, intraprostatically, intrarectally, intrathecally, intratracheally, intratumorally, intraumbilically, intravaginally, intravenously, intravesicularly, intravitreally, or via liposomes. Example

[0306] Example 1: Construct design and sequence optimization

[0307] Purpose:

[0308] The goal of this example was to develop four types of protein designs that combine therapeutic activity with strong affinity for heparan sulfate present in the cellular ECM and glycocalyx.

[0309] Materials and methods:

[0310] In constructs designed for mammalian expression systems, the serum albumin preproprotein signal peptide (MKWVTFISLLFLFSSAYS) is used as a leader sequence to promote extracellular expression (REF. 1). Vectors containing an IRES2 sequence are used for bicistronic expression of the target protein and an intracellular fluorescent marker. All sequences are optimized for mammalian cell expression. For bacterial expression, the gene encoding the target protein is optimized for bacterial expression and cloned into the pET22B(+) vector.

[0311] result:

[0312] Common to all four designs is the presence of FSD1 (monomer or multimer) or full-length FST variants as a platform, providing affinity for heparan sulfate. A linker can optionally be used in all designs between FSD1 and the fusion moiety.

[0313] In type 1 design, the gene encoding the therapeutic or diagnostic protein is fused to FSD1 (monomer or multimer) to express a single polypeptide chain containing the therapeutic or diagnostic moiety and FSD1 ( Figure 1 A). A llama anti-vimentin heavy chain variable domain (VHH or nanobody (nb)) fused to FSD1 (anti-vimentin-nb-FSD1) is an example molecule of Type 1 design.

[0314] Type 2 designs are based on immunoglobulin fragment crystallizable (Fc) platforms (homodimeric or heterodimeric) ( Figure 1 B). The therapeutic / diagnostic portion is fused to the N-terminus of Fc, and FSD1 (monomer or multimer) is fused to the C-terminus of Fc. The homodimeric Fc platform helps design dimeric therapeutic proteins or dimeric diagnostic proteins ( Figure 1 Bi), while the heterodimeric Fc platform makes it possible to design compounds containing two different therapeutic or diagnostic moieties ( Figure 1 Bii). An example of a Type 2 design is VGFR(1 / 2)-mFc-FSD1 or TNFR2-mFc-FSD1. VGRF(1 / 2)-mFc-FSD1 constructs in which FSD1 residues glutamine 124 or glutamate 126, or both, were mutated to alanine were also included to verify the loss of activin A, myostatin, and / or GDF11 neutralization.

[0315] The Type 3 design allows the use of FSD1 (monomer or multimer) as a platform to bind to any commercially obtained or expressed conventional antibody as a single entity ( Figure 1 C). Type 3 designs contain FSD1 covalently coupled (using click chemistry) to any conventional antibody ( Figure 1 Ci) or with anti-Fc single chain V H H nanobody expresses FSD1 together, thus can be mixed and used with any conventional antibody at the point of care (point-of-care) ( Figure 1Cii). Type 3 designs also include FSD1 fused to streptavidin for binding to biotinylated therapeutic or diagnostic proteins, or biotinylated FSD1 for binding to proteins fused to streptavidin. An example of a Type 3 design is an anti-tumor necrosis factor alpha (TNFα) antibody conjugated to FSD1 (anti-TNFα antibody-FSD1).

[0316] Type 4 designs are based on an Fc heterodimer platform, in which full-length FST or FSD1 (monomer or multimer) replaces one Fab region of the Fc heterodimer and the other Fab region is replaced by a therapeutic or diagnostic moiety ( Figure 1 D) Type 4 designs incorporate a C-terminal histidine tag on the Fc chain (does not contain full-length FST or FSD1) to aid in purification. Examples of Type 4 designs include VGFR(1 / 2) / FST291-mFc, TNFR2 / FST291-mFc, or CBG / FST291-mFc.

[0317] To chemically conjugate FSD1 to existing therapeutic antibodies, a version of FSD1 (FSD1(KTC)) was designed that contains three additional native C-terminal residues (lys-thr-cys). The free C-terminal cysteine ​​of FSD1(KTC) allows for its conjugation with different types of maleimide-containing click chemistry compounds.

[0318] The homodimeric Fc platform used in the Type 2 design is based on mouse IgG2A. The heterodimeric Fc platforms used in the Type 2 and Type 4 designs are both based on the “knobs-in-holes” approach (REF. 2). The “knobs-in-holes” approach uses two Fc chains (Fc A and Fc B ), and point mutations were introduced to promote the formation of heterodimers rather than homodimers.

[0319] Additional independent proteins, FSD1, FSD1-FSD1, and FSD1-FSD1-FSD1, were designed to verify affinity for heparan sulfate and lack of neutralization of activin A, myostatin, and / or GDF11.

[0320] in conclusion:

[0321] These four designs represent different strategies for constructing therapeutic or diagnostic moieties fused to FSD1 or FST as heparan sulfate affinity platforms.

[0322] Example 2: Protein expression and purification

[0323] Purpose:

[0324] The goal of this example was to generate cell lines that express the desired recombinant proteins and purify them using various chromatographic methods.

[0325] Materials and methods:

[0326] Mammalian expression systems:

[0327] The cloning vector was transformed into DH5alpha competent E. coli cells (ThermoFisher Scientific #18265017). High-yield plasmid purification was performed using the Maxiprep kit (Qiagen #12162). The plasmid was transfected into a mammalian expression platform based on Chinese hamster ovary (CHO) cells using lipofection. (REF.6) Monoclonal cell lines were inoculated based on fluorescence intensity using a cell sorter. Clones expressing the highest titer of target protein were then screened by dot blot and grown to a suspension. Protein was purified from filtered conditioned medium using independent protein A (Cytiva, #GE17-0403-01), IMAC (Roche, #6781535001, cOmplete His-Tag purification columns), or heparin chromatography (Cytiva, #GE17-0407-01), or a combination thereof. Protein purity was verified using SDS-PAGE.

[0328] Bacterial expression:

[0329] The vectors for bacterial expression were transformed into Escherichia coli BL21 (DE3) strain for expression. The cells were grown in LB medium (Sigma-Aldrich, #L3022) containing 100 μg / ml ampicillin (Sigma-Aldrich, #A9518) at 37°C in a shaking incubator. Expression was induced using 0.1 mM IPTG (Sigma-Aldrich, #I6758), and the temperature was then lowered to 20°C. After overnight expression, the cells were collected by centrifugation and stored at -20°C. The cells were lysed by ultrasonication and cell debris was removed by centrifugation. The recombinant protein was purified by cation exchange chromatography or heparin chromatography, and the purity was verified by SDS-PAGE.

[0330] result:

[0331] Type 2 compounds, VGFR(1 / 2)-mFc-FSD1 and TNFR2-mFc-FSD1, based on native FSD1, as well as type 4 compounds, VGFR(1 / 2) / FST291-mFc, TNFR2 / FST291-mFc, and CBG / FST291-mFc, have been successfully expressed in mammalian expression systems and purified by affinity chromatography. Type 2 compounds, VGFR(1 / 2)-mFc-FSD1(Q124A), VGFR(1 / 2)-mFc-FSD1(E126A), and VGFR(1 / 2)-mFc-FSD1(Q124A,E126A), based on mutant FSD1, have been successfully expressed in mammalian expression systems and purified by affinity chromatography. Type 1 compounds (anti-vimentin-nb-FSD1), FSD1(KTC) (used for type 3 compound design), and the independent proteins FSD1, FSD1-FSD1, and FSD1-FSD1-FSD1 have been successfully expressed in E. coli and purified by cation exchange or heparin chromatography. Independent mutant FSD1 proteins FSD1(Q124A), FSD1(E126A), and FSD1(Q124A,E126A) have been successfully expressed in E. coli and purified by cation exchange or heparin chromatography. Anti-vimentin-nb (His-tagged) has been successfully expressed in E. coli and purified by IMAC (this protein was used as a control in studies involving type 1 compounds (anti-vimentin-nb-FSD1)).

[0332] in conclusion:

[0333] Using recombinant fusions of native FSD1 or mutant FSD1 or full-length FST as platform technology, proteins were generated that could be correctly expressed, purified, and folded according to the described design.

[0334] Example 3: Heparin affinity chromatography

[0335] Purpose:

[0336] The purpose of this example was to evaluate the heparin affinity of the generated compounds using heparin affinity chromatography and compare it with commercial counterparts (if available) such as VGFR(1 / 2)-hFc (aflibercept) and TNFR2-hFc (etanercept).

[0337] Materials and methods:

[0338] Approximately 200 μg of sample was loaded onto a 1 ml HiTrap Heparin HP affinity column (Cytiva, #GE17-0407-01) equilibrated in buffer A (200 mM NaCl, Tris-HCl, pH 7.6) and eluted with a 15 ml gradient of 0-100% buffer B (2000 mM NaCl, Tris-HCl, pH 7.6). The eluted protein was detected by UV absorbance (A 280 )monitor.

[0339] result:

[0340] The independent compounds FSD1 and FSD1-FSD1 both showed high heparin affinity and were eluted from the column at 1133 mM and 1280 mM NaCl, respectively ( Figure 2 A+B). This indicates that the fusion of multiple FSD1 domains leads to an increase in heparin affinity.

[0341] Fusion of FSD1 to anti-vimentin nanobody (type 1) resulted in strong binding to heparin, eluted at 1277 mM NaCl, whereas the nanobody itself did not bind to the column ( Figure 2 AB).

[0342] Type 2 compounds VGFR(1 / 2)-mFc-FSD1 and TNFR2-mFc-FSD1 both showed high affinity for heparin, eluting at 1133 mM and 1080 mM NaCl, respectively ( Figure 2 AB).

[0343] Type 4 compounds VGFR(1 / 2) / FST291-mFc and TNFR2 / FST291-mFc both exhibited two peaks in the elution curve, indicating two different conformations with different heparin affinities ( Figure 2 AB) - The low-affinity conformation is represented by Peak 1 (VGFR(1 / 2) / FST291-mFc eluted at 507 mM NaCl, TNFR2 / FST291-mFc eluted at 440 mM NaCl), and the high-affinity conformation (VGFR(1 / 2) / FST291-mFc eluted at 933 mM NaCl, TNFR2 / FST291-mFc eluted at 907 mM NaCl). Commercially available VGFR(1 / 2)-hFc (aflibercept) and TNFR2-hFc (etanercept) have no affinity for heparin. In addition, FST315dHBS-mFc (SEQ ID NO: 30, SEQ ID NO: 31) exhibits very low heparin affinity, consistent with it containing an altered heparin binding site (REF. 3).

[0344] Type 4 compound CBG / FST291-mFc showed high affinity for heparin and eluted at 915 mM ( Figure 2 AB).

[0345] in conclusion:

[0346] This example demonstrates that the designed Type 1, Type 2 and Type 4 compounds have high heparin affinity.

[0347] Example 4: Protein binding using click chemistry

[0348] Purpose:

[0349] The purpose of this example is to conjugate FSD1 to an anti-TNFα antibody (adalimumab) using click chemistry.

[0350] Materials and methods:

[0351] The C-terminal cysteine ​​of FSD1 (KTC) was conjugated with DBCO-maleimide. Adalimumab was oxidized using sodium periodate and subsequently conjugated with aminooxy-PEG3-azide. The conjugated FSD1 and conjugated adalimumab were mixed in a 2:1 ratio to covalently link the two compounds.

[0352] result:

[0353] Sodium periodate oxidation of antibody N-linked glycosylation generates multiple aldehyde groups that bind to aminooxy groups. Similarly, FSD1(KTC) binds to maleimide groups. Using these groups and the click chemistry compounds azide and DBCO, FSD1(KTC) was covalently coupled to adalimumab. Although the binding was far from complete, we observed that several adalimumab-containing fractions had an enhanced affinity for heparin ( Figure 3 A+B). SDS-PAGE showed that the size of these fractions increased in direct proportion to their heparin affinity ( Figure 3 C), showing that multiple binding of FSD1(KTC) to adalimumab can increase heparin affinity.

[0354] in conclusion:

[0355] Using click chemistry, FSD1(KTC) can be conjugated to existing antibodies, thereby conferring heparin affinity to the antibodies. This example demonstrates that type 3(i) compounds with high heparin affinity can be constructed.

[0356] Example 5: Bioassay

[0357] Purpose:

[0358] The purpose of this example was to quantify the neutralization of activin A, myostatin, or GDF11 and to confirm that such neutralization is absent in the claimed compounds.

[0359] Materials and methods:

[0360] Using a reporter gene bioassay for phosphorylated Smad signaling, we quantitatively analyzed the neutralization of growth factors by activin A (R&D Systems, #338-AC), myostatin (R&D Systems, #788-G8-010), or GDF11 (R&D Systems, #1958-GD-010). Phosphorylated Smad2 or Smad3 forms a complex with Smad4, translocates to the nucleus, and recognizes a repetitive base sequence, 5'-CAGAC-3', termed the Smad binding element (SBE). HEK293 cells were transfected with a construct containing 12 repeats of the SBE motif (REF. 4), upstream of a minimal promoter controlling luciferase expression. The HEK293 cell line proliferated to a stable cell line with robust luciferase responsiveness to phosphorylated Smad2 or Smad3 signaling induced by growth factor stimulation. Briefly, cells were stimulated with activin A, myostatin, or GDF11 and co-treated with recombinant proteins at concentrations ranging from 8.2 nM, 63 nM, 750 nM, 822 nM, or 1 μM, as previously described (REF. 3). After 16 to 20 hours, cells were lysed (Promega, Glo Lysis Buffer, #E2661), luciferase substrate (Promega, Steady-Glo Luciferase Assay System, #E2520) was added, and luminescence signals were analyzed using a microplate reader (PerkinElmer, EnSpire 2300). Three to six positive controls (growth factor stimulation without inhibitors) and negative controls (without growth factors and inhibitors) were included. Data were analyzed and half-maximal inhibitory concentrations (IC50) were calculated using nonlinear regression with three parameters, no weighting, and no constraints (Graphpad, Prism version 9.4.1).

[0361] result:

[0362] FSD1, FSD1-FSD1, or type 2 design compounds VGFR(1 / 2)-mFc-FSD1 or TNFR2-mFc-FSD1 inhibited activin A ( Figure 4 ), myostatin ( Figure 5 ) or GDF11 signaling ( Figure 6) had no neutralizing effect. Similarly, the commercially available therapeutics TNFR2-hFc (etanercept) or VGFR(1 / 2)-hFc (aflibercept) did not neutralize growth factor signaling in the tested concentration range up to 8.2 nM ( Figure 4 BC, 5B-C, and 6B-C). In contrast, native FST315 (SEQ ID NO:28) and FST315dHBS-mFc (recombinant FST315 protein fused to a murine Fc fragment in which the heparin-binding sequence [HBS] of FSD1 has been replaced by the structurally related sequences SEQ ID NO:30 and SEQ ID NO:31) neutralized activin A, myostatin, and GDF11 with IC50s in the pM range (REF. 3)( Figure 4 A, 5A and 6A). Type 4 design compounds containing a single portion of full-length FST, VGFR(1 / 2) / FST291-mFc or TNFR2 / FST291-mFc, neutralized growth factors with IC50 in the mid-pM range to low nM range ( Figure 4 BC, 5B-C, and 6B-C). FSD1 and FSD1-FSD1 did not neutralize activin A, myostatin, or GDF11 signaling when tested at concentrations up to 1 μM ( Figure 19 The type 1 design compound, anti-vimentin nanobody-FSD1, significantly increased pSmad2 / 3 signaling when myostatin was used as a stimulus, and to a lesser extent when activin A or GDF11 were used as stimuli, in all cases over the tested concentration range starting from approximately 100 nM up to 1 μM ( Figure 19 The single entity FSD1 mutants FSD1(Q124A), FSD1(E126A), or FSD1(Q124A,E126A) had no neutralizing effect on activin A or myostatin when tested over a concentration range up to 822 nM ( Figure 20 When tested at concentrations up to 1 μM, type 2 design compounds neutralized activin A, myostatin, and GDF11 signaling with native FSD1 (VGFR(1 / 2)-mFc-FSD1) with IC50 values ​​in the low nM range, demonstrating that compound inhibition of growth factor signaling persisted even at concentrations exceeding 8.2 nM (compare Figure 4 B, 5B and 6B Figure 21 However, when FSD1 mutants (Q124A or E126A) were used instead of native FSD1 in the type 2 design, neutralization of activin A was reduced (to the low μM range when extrapolated) or abolished (myostatin and GDF11) across the concentration range tested ( Figure 21The commercial counterpart, VGFR(1 / 2)-hFc (aflibercept), did not neutralize activin A signaling over the tested concentration range up to 1 μM.

[0363] in conclusion:

[0364] FSD1 as a monomer or dimer (FSD1-FSD1) did not neutralize the signaling activity of activin A, myostatin, or GDF11 within a concentration range of at least 1 μM. Consistent with this, type 2 design compounds based on the native FSD1 platform did not show any neutralizing activity against activin A, myostatin, or GDF11 when tested at concentrations up to 8.2 nM. However, when concentrations exceeded 8.2 nM, type 2 design compounds based on native FSD1 neutralized activin A, myostatin, and GDF11 signaling with IC50s in the low nM range. This is likely due to the avidity of the Fc dimer for native FSD1. By replacing native FSD1 with the selected FSD1 mutants FSD1(Q124A), FSD1(E126A), or FSD1(Q124A,E126A) in a type 2 design, neutralization of activin A signaling was further reduced (IC50 values ​​in the low μM range), and neutralization of myostatin and GDF11 signaling was abolished across the concentration range tested. The mutations were selected based on structural analysis of the crystal structure of follistatin in complex with myostatin. Here, Gln124 and Glu126 from FSD1 were identified as the primary residues involved in the direct interaction with myostatin. The type 1 design compound, anti-vimentin nanobody-FSD1, increased pSmad2 / 3 signaling starting at approximately 100 nM. An interaction between vimentin and Smad2 / 3 has been reported, with phosphorylated vimentin enhancing pSmad2 / 3 signaling (REF 13). In cases where FSD1 confers the ability of its fusion partner (anti-vimentin nanobody) to enter the cytosol, this could affect the interaction between vimentin and Smad2 / 3 and lead to the observed enhancement of pSmad2 / 3 signaling. Type 4 designed compounds containing the full-length FST portion retained growth factor neutralization activity.

[0365] Example 6: Surface Plasmon Resonance

[0366] Purpose:

[0367] The purpose of this example was to quantify the affinity of the generated compounds for TNF-α and VEGF.

[0368] Materials and methods:

[0369] Surface Plasmon Resonance (SPR) experiments were performed on a Biacore 3000 instrument (Cytiva). A CM5 chip was prepared for compound capture according to the instructions of the Mouse Antibody Capture Kit (Cytiva, #BR100838). VGFR(1 / 2)-mFc-FSD1, TNFR2-mFc-FSD1, VGFR(1 / 2) / FST291-mFc, TNFR2 / FST291-mFc, VGFR(1 / 2)-hFc (aflibercept), TNFR2-hFc (etanercept), and FST315dHBS-mFc (SEQ ID NO:30, SEQ ID NO:31) were captured to approximately 1,000 RU (resonance units). TNFα (R&D Systems, #210-TA-005) or VEGF (R&D Systems, #293-VE-010) was then injected for 120 seconds, followed by a 300-second dissociation phase. At the end of each binding cycle, non-covalently bound molecules were removed from both surfaces by regeneration with 10 mM glycine (pH 1.7) for 180 seconds. All proteins were diluted in running buffer (10 mm HEPES, pH 7.5, 150 mm NaCl, 2 mm CaCl2, and 0.05% Tween 20). The flow rate for all steps of the experiment was 30 μl / min. Binding analysis was performed at 25°C, and data acquisition was performed at a rate of 1 Hz. Recorded signals were referenced using BIAevaluation 4.1.1 software (Cytiva); the signal from the parallel reference flow cell and the signal from the blank run (0 nM analyte) were subtracted.

[0370] result:

[0371] Both VGFR(1 / 2)-mFc-FSD1 and VGFR(1 / 2) / FST291-mFc showed very strong and almost irreversible binding to VEGF ( Figure 7 Similarly, TNFR2-mFc-FSD1 and TNFR2 / FST291-mFc also exhibited very strong and nearly irreversible binding to TNFα. These affinities are comparable to the VEGF / TNFα affinities of the commercial therapeutics VGFR(1 / 2)-hFc (aflibercept) and TNFR2-hFc (etanercept). Full-length FST315 (SEQ ID NO: 29), which has a mutated heparin-binding site, has no affinity for either VEGF or TNFα.

[0372] in conclusion:

[0373] VGFR(1 / 2)-mFc-FSD1, VGFR(1 / 2) / FST291-mFc, TNFR2-mFc-FSD1, and TNFR2 / FST291-mFc had similar ligand affinities to their commercial counterparts.

[0374] Example 7: ECM extract binding experiment

[0375] Purpose:

[0376] The purpose of this example was to evaluate the binding affinity of the claimed platform to extracellular matrix (ECM) extracts and to demonstrate the lack of binding of commercial therapeutics.

[0377] Materials and methods:

[0378] Binding to the ECM was quantified using a modified version of a previously described in vitro colorimetric assay (REF.5) based on ECM extract coated plates (Corning, BioCoat Matrigel #354607). The ECM contains various heparan sulfates that will immobilize the test compounds during the wash cycle depending on the strength of the compound and the interaction between them. Briefly, the plates were blocked overnight with 100 μL of 100% StartingBlock (Thermo Scientific, #37543) per well. The test compounds were serially diluted in blocking buffer at a concentration range starting from 60 nM or 120 nM and added to the plates in triplicate (50 μL). The plates were incubated at room temperature for two hours and then washed four times with 200 μL of tris-buffered saline containing 0.1% Tween 20 and 10% StartingBlock. A two-hour primary antibody incubation step was performed using a mixture of biotinylated anti-FST antibody (0.1 μg / mL, R&D Systems, #BAF669) and biotinylated anti-human IgG antibody (0.065 μg / mL, Invitrogen #31774) (50 μL / well). Where appropriate, single primary antibodies were used instead of a mixture for incubation. Binding of the biotinylated anti-human IgG antibody to the commercial therapeutics VGFR(1 / 2)-hFc (aflibercept) or TNFR2-hFc (etanercept) at the same concentrations was validated in a direct ELISA. Streptavidin conjugated to horseradish peroxidase (Streptavidin-HRP, 1:400 dilution, R&D Systems,

[0379] #DY998) (50 μL / well) was used for a one-hour secondary antibody incubation step. The primary antibody and streptavidin-HRP were diluted in 100% StartingBlock and washed four times with 200 μL of wash solution after each incubation step. TMB substrate solution (Thermo Scientific, #N301) (100 μL per well) was added, the color development was observed visually, and the color development was stopped with an equal volume of stop solution (ThermoScientific, #N600). The color reaction was evaluated at a wavelength of 450 nm using a microplate reader (PerkinElmer, EnSpire 2300). Background was determined by developing the color in blocking buffer. The data were analyzed and the half-maximal effective concentration (EC50) was calculated using nonlinear regression with three parameters, no weighting and no constraints (Graphpad, Prism version 9.4.1). The calculated EC50 is a relative measure of the binding strength of the compound to the ECM. The highest plateau of each curve does not represent affinity for the ECM, but is a function of the extent to which the primary antibody recognizes the test compound and how long the color reaction persists.

[0380] result:

[0381] The OD450 curves of FSD1, FSD1-FSD1, and FSD1-FSD1-FSD1 showed a left shift, indicating the increased ECM affinity of the various modules of FSD1 present, with EC50 values ​​ranging from 0.08 nM to 1 nM ( Figure 22 A). The maximum deviation of the curve occurs between one and two FSD1 modules. Correspondingly, the EC50 values ​​of type 2 design compounds VGFR(1 / 2)-mFc-FSD1 or TNFR2-mFc-FSD1 are lower than 0.5 nM ( Figure 8 BC). The EC50 of native FST288 (SEQ ID NO: 27) is in the range of 1 nM ( Figure 8 A), while the EC50 of type 4 design compounds VGFR(1 / 2) / FST291-mFc or TNFR2 / FST291-mFc (containing a full-length FST portion) was approximately 15 nM ( Figure 8 BC). FST315dHBS-mFc (lacking the heparin binding site, SEQ ID NO: 30, SEQ ID NO: 31) has a low but traceable ECM affinity with an EC50 of approximately 80 nM ( Figure 8 A). No commercially available therapeutic agent VGFR(1 / 2)-hFc (Aflibercept, Figure 8 B) or TNFR2-hFc (etanercept, Figure 8C) Binding to the ECM. To demonstrate the practicality of the Type 3 design, FSD1 (KTC) was covalently linked to the commercially available anti-TNFα antibody adalimumab (described in Example 4). During heparin affinity chromatography, fractions with different heparin affinities were collected, indicating that different numbers of FSD1 modules were bound to adalimumab. Fractions were labeled as adalimumab-FSD1 low, medium, or high depending on the increase in heparin affinity. The exact number of FSD1 (KTC) modules bound to adalimumab in each fraction is unknown and may appear as a continuous value. Therefore, the exact molecular weight cannot be determined, and the EC50 value cannot be determined. However, in the same manner as observed for multimeric FSD1, the OD450 curve of the adalimumab-FSD1 fraction showed a left shift from low to high, indicating an increased affinity for the ECM ( Figure 22 B). Adalimumab not employing this platform invention showed limited, if any, ECM binding ( Figure 22 B). Similar to the type 2 designer protein VGFR(1 / 2)-mFc-FSD1 using native FSD1, the type 2 designer proteins VGFR(1 / 2)-mFc-FSD1(Q124A), VGFR(1 / 2)-mFc-FSD1(E126A), and VGFR(1 / 2)-mFc-FSD1(Q124A,E126A) using FSD1 mutants also showed binding to the ECM ( Figure 22 C).

[0382] in conclusion:

[0383] When used as a platform, FSD1, whether as a standalone monomer, multimer, or as a portion of full-length FST, enabled compounds to bind to ECM extracts and remain resident for 12 wash cycles. ECM affinity increased with increasing amounts of the FSD1 moiety in the compound. Type 2 designer proteins utilizing FSD1 mutants also demonstrated binding to ECM extracts. FSD1-FSD1 or type 2 designer compounds exhibited EC50s 3- to 7-fold lower than those of FSD1 and native FST288, and 30- to 100-fold lower than those of type 4 designer compounds.

[0384] Example 8: In vivo evaluation of the half-life of type 2 and type 4 design compounds in mouse skeletal muscle using IVIS

[0385] Purpose:

[0386] The purpose of this example is to evaluate the half-life of type 2 and type 4 design compounds in mouse skeletal muscle using fluorescence.

[0387] Materials and methods:

[0388] Compound Labeling:

[0389] Compounds were fluorescently labeled using the near-infrared spectroscopy marker NIR730 (Sigma-Aldrich, #92315) according to the manufacturer's instructions. After labeling, compound concentrations were determined using the Pierce Coomassie (Bradford) Protein Assay Kit (ThermoScientific, #23200).

[0390] Animal experiments:

[0391] Twelve 16-week-old female C57BL / 6 mice were divided into four groups: PBS (vehicle), VGFR(1 / 2)-mFc-FSD1 (type 2 compound), VGFR(1 / 2) / FST291 (type 4 compound) and VGFR(1 / 2)-hFc (aflibercept). During anesthesia, the hair on the right hind limb of the mouse was shaved to expose the calf muscle. A single dose of 25 μg of fluorescent-labeled compound in a volume of 5 μL was administered intramuscularly to the gastrocnemius muscle. The mice were injected subcutaneously with analgesics (carprofen 5 mg / kg) to relieve pain caused by muscle tension. Immediately after injection and every 24 hours, the mice were fluorescently scanned using the IVIS Spectrum (PerkinElmer) in vivo imaging system. The exposure time for all scans (whole animals, cut muscles or serum) was set to 1 second, and the excitation / emission wavelengths were set to 675 / 760 nm. Four days later, the mice were sacrificed, the right gastrocnemius muscle was dissected, blood was collected and serum was separated by centrifugation. Gastrocnemius muscle and serum samples were immediately scanned to assess fluorescence intensity. Data were analyzed using Living Image version 4.3.1 (Caliper Life Sciences, Inc.).

[0392] result:

[0393] In skeletal muscle, the half-life of the type 2 design compound VGFR(1 / 2)-mFc-FSD1 was 63.5 hours ( Figure 9 AB), and the average radiation of the cut gastrocnemius muscle was 17.4 times that of the commercially available VGFR(1 / 2)-hFc (Aflibercept) ( Figure 9 CD), which was statistically significant. The half-life of type 4 design compound VGFR(1 / 2) / FST291-mFc was 8.3 hours ( Figure 9 AB), and the radiation of gastrocnemius muscle was significantly higher than that of PBS, but not higher than that of VGFR(1 / 2)-hFc(aflibercept)( Figure 9CD). The half-life of commercial VGFR(1 / 2)-hFc (aflibercept) is 7.5 hours, and the mean radiation of excised skeletal muscle was not significantly different from that of PBS, indicating that local presence was very low. Notably, the presence of type 2 compound was not detected in serum after 96 hours ( Figure 9 EF). Although some radioactivity was observed in the serum of mice treated with the type 4 designer compound, its level was not significantly different from that of PBS-treated mice. Finally, the serum of mice treated with the commercial VGFR(1 / 2)-hFc (aflibercept) showed abundant radioactivity, indicating high systemic presence.

[0394] in conclusion:

[0395] In skeletal muscle, the half-life of the type 2 design compound, VGFR(1 / 2)-mFc-FSD1, was nearly ten times that of its commercial counterpart, VGFR(1 / 2)-hFc (aflibercept). Consequently, the type 2 design compound still exhibited significant irradiation in gastrocnemius muscle 96 hours after injection. The type 4 design compound was also detectable in skeletal muscle after 96 hours, while the irradiation of VGFR(1 / 2)-hFc (aflibercept) was indistinguishable from that of PBS-treated samples. In serum, VGFR(1 / 2)-hFc (aflibercept) exhibited abundant irradiation, while the irradiation of the type 2 and type 4 design compounds was indistinguishable from that of PBS-treated mice.

[0396] Example 9: In vivo evaluation of the half-life of type 4 design compounds in mouse eye tissue using IVIS

[0397] Purpose:

[0398] The purpose of this example is to evaluate the half-life of type 4 design compounds in mouse eye tissue using fluorescence.

[0399] Materials and methods:

[0400] Compound Labeling:

[0401] Compounds were fluorescently labeled using the near-infrared spectroscopy marker NIR730 (Sigma-Aldrich, #92315) according to the manufacturer's instructions. After labeling, compound concentrations were determined using the Pierce Coomassie (Bradford) Protein Assay Kit (ThermoScientific, #23200).

[0402] Animal experiments:

[0403] Seven 12-week-old female BALB / c mice were divided into two groups: a PBS group (n=3, vehicle) or an active treatment group (n=4, treatment dependent on the eye). In the PBS group, mice received PBS in both the left and right eyes. In the active treatment group, mice received VGFR(1 / 2)-hFc (aflibercept) in the left eye and VGFR(1 / 2) / FST291 (type 4 compound) in the right eye. During anesthesia, Tropicamid eye drops were used to dilate the pupil, and a single dose of 10 μg of the fluorescently labeled compound was administered intravitreally in a volume of 1 μL. Post-injection, the eye was treated with chloramphenicol, and the mice received a subcutaneous injection of analgesics (carprofen 5 mg / kg) to relieve pain caused by ocular tension. Immediately after injection, and 48, 72, and 96 hours after injection, the mice were scanned for fluorescence using an IVIS Spectrum (PerkinElmer) in vivo imaging system. The exposure time for in vivo scans was set to 0.5 seconds, and the exposure time for ex vivo scans was set to 20 seconds. The excitation / emission wavelengths were set at 675 / 760 nm. Four days later, the mice were euthanized and their eyes were enucleated. Data were analyzed using Living Image version 4.3.1 (Caliper Life Sciences, Inc.).

[0404] result:

[0405] In ocular tissue, the half-life of type 4 design compound VGFR(1 / 2) / FST291-mFc was 26.4 hours ( Figure 10 A), the average radiation in the removed eyeball was 5.3 times that of the commercially available VGFR(1 / 2)-hFc (Aflibercept) ( Figure 10 The half-life of VGFR(1 / 2)-hFc (aflibercept) was 13.3 hours, and the radiation in the enucleated eye was not significantly different from that in PBS.

[0406] in conclusion:

[0407] The half-life of the Type 4 design compound, VGFR(1 / 2) / FST291-mFc, was twice that of its commercial counterpart, VGFR(1 / 2)-hFc (aflibercept), and the presence of the Type 4 design compound was still detectable in enucleated eyes 96 hours after injection.

[0408] Example 10: Ex vivo evaluation of the half-life of Type 4 design compounds in porcine kidney using IVIS during normothermic machine perfusion

[0409] Purpose:

[0410] The purpose of this example was to evaluate the half-life of Type 4 design compounds in isolated perfused porcine kidney using fluorescence.

[0411] Materials and methods:

[0412] Compound Labeling:

[0413] Compounds were fluorescently labeled using the near-infrared spectroscopy marker NIR730 (Sigma-Aldrich, #92315) according to the manufacturer's instructions. After labeling, compound concentrations were determined using the Pierce Coomassie (Bradford) Protein Assay Kit (ThermoScientific, #23200).

[0414] Ex vivo delivery to the kidney during normothermic machine perfusion (NMP):

[0415] Nephrectomy and red blood cell isolation were performed on female Danish Landrace pigs weighing approximately 60 kg as previously described (REF. 6). After nephrectomy, the kidneys were flushed with isotonic saline (Fresenius Kabi) and then with Belzer UW cold storage solution (Bridge to Life, UK) and stored in Belzer UW cold storage solution at 4°C for approximately 16 hours. Before perfusion, the kidneys were flushed with isotonic saline and connected to a basic perfusion device. A red blood cell-based perfusion solution (as previously described) was used. (6) Both kidneys from the same pig were perfused for 6 hours at 37°C under oxygenated conditions at a flow rate of approximately 400-500 mL / min. A total of 1 mg of labeled type 2 design compound TNFR2 / FST291-mFc or TNFR2-hFc (etanercept) was diluted in 10 mL of perfusate and administered via a slow infusion (60 mL / h) via the arterial line 5 minutes after the start of perfusion. At the end of perfusion, the kidneys were flushed with 1.5 L of isotonic saline to remove the perfusate. The kidneys were sectioned along the outer cortex and midline and analyzed using an IVIS Spectrum (PerkinElmer) in vitro imaging system. The exposure time was set to 8 seconds for midline sections and 6 seconds for outer cortical sections. The excitation / emission wavelengths were set to 675 / 760 nm. Data were analyzed using Living Image version 4.3.1 (Caliper Life Sciences, Inc.). To visualize background fluorescence, additional porcine kidneys from an independent experiment in which 10 mL of PBS was infused into the arterial line during NMP were scanned.

[0416] result:

[0417] In isolated perfused pig kidneys, the type 4 designer compound TNFR2 / FST291-mFc expressed in midline sections ( Figure 11 AB) and cortical sections ( Figure 11 The average radiation shown in CD) was twice that of commercial TNFR2-hFc (etanercept).

[0418] in conclusion:

[0419] When the therapeutic was delivered during ex vivo normothermic machine perfusion, the Type 4 design compound TNFR2 / FST291-mFc showed twice the radiance of its commercial counterpart TNFR2-hFc (etanercept), indicating high exposure of the Type 4 design compound.

[0420] Example 11: In vitro study of intracellular uptake

[0421] Purpose:

[0422] The purpose of this example was to evaluate the intracellular uptake of the claimed platform invention in vitro.

[0423] Materials and methods:

[0424] Compound Labeling:

[0425] Compounds were fluorescently labeled using the green fluorescent protein marker ATTO 488 (ATTO-TEC GmbH, #AD488) according to the manufacturer's instructions. After labeling, compound concentrations were determined using the Pierce Coomassie (Bradford) Protein Assay Kit (Thermo Scientific, #23200).

[0426] Intracellular uptake studies:

[0427] Briefly, HEK293 cells were seeded on coverslips (Thorlabs, #CG15NH) treated with poly-D-lysine (ThermoFisher, #A3890401) to promote adhesion. Cells were plated in DMEM (ThermoFisher, #11995073) supplemented with 10% fetal bovine serum (FBS). After 24 hours, cells were washed with PBS, and compound (1 μM) was added in 100% DMEM without FBS. A control sample containing 100% DMEM without compound was also included. After 3 and 18 hours of incubation, cells were washed three times with PBS. Then, the cells were incubated with a mixture of Hoechst33342 (3 μg / mL) (ThermoFisher, #H3570) and Lysotracker Red DND-99 (75 nM) (ThermoFisher, #L7528) for 30 minutes to visualize the nucleus and lysosomes, respectively. The cells were then washed three times with PBS and fixed with 4% formalin for 30 minutes. Coverslips were mounted using ProLong Glass Antifade Mountant (ThermoFisher, #P36980). Confocal imaging was performed using a Zeiss LSM800 laser scanning confocal microscope (Carl Zeiss Microscopy GmbH, Oberkochen, Germany) equipped with 2 GaAsP and an Airyscan detector and Zen Blue Edition software (CarlZeiss Microscopy GmbH, Version 2.5). Confocal images were taken using a PlanApo x63 Oil NA 1.4 objective lens. For conventional confocal imaging, ATTO 488 was excited with a 488 nm diode laser at 2.20% (3 hour samples) or 0.45% (18 hour samples) and detected at a wavelength of 510-575 nm. Hoechst 33342 was excited with a 405 nm diode laser at 0.50% (3 hour and 18 hour samples) and detected at a wavelength of 400-510 nm. Lysotracker Red DND-99 was excited with a 561 nm diode laser at 2.60% (3 hour and 18 hour samples) and detected at a wavelength of 575-700 nm. For each sample, Z-stacks were recorded at intervals of 0.270 μm (3 hour samples) or 0.170 μm (18 hour samples). For all scans, the pixel time was 1.84 μs, and the average value was set to 4x in per-line repetition mode and the average intensity method.For Airyscan super-resolution imaging, ATTO 488 was excited with a 488 nm diode laser at 0.03% and detected at wavelengths of 490–580 nm; Hoechst 33342 was excited with a 405 nm diode laser at 0.10% and detected at wavelengths of 400–469 nm. Images were processed using Fiji (v2.14.0 / 1.54f). All images were contrast- and brightness-adjusted equally for ATTO 488 and Lysotracker Red DND-99 signals at each time point. Hoechst 33342 signals were contrast- and brightness-adjusted separately for 3-hour samples and equally for 18-hour samples.

[0428] result:

[0429] In HEK293 cells, the strong fluorescence signal of ATTO 488 indicated that the type 1 designed compound anti-vimentin nanobody-FSD1 was taken up by the cells after 3 hours of incubation ( Figure 16 In contrast, very little ATTO 488 signal was found in cells treated with anti-vimentin nanobody after 3 hours of incubation ( Figure 16 At 3 hours, the ATTO 488 signal of anti-vimentin nanobody-FSD1 exhibited a punctate pattern characteristic of endocytosis. After 18 hours of incubation, the punctate ATTO 488 signal of anti-vimentin nanobody-FSD1-treated cells had been replaced by fluorescent signals resembling interwoven threads (or filaments) that bent and stretched throughout the cell ( Figure 12 A and Figure 17 In contrast, there was little or no ATTO 488 signal in cells treated with anti-vimentin nanobody or in cells treated with DMEM alone ( Figure 12 A). In cells treated with anti-vimentin nanobody-FSD1, there was no significant overlap between lysosomal (Lysotracker Red DND-99) and ATTO 488 signals, indicating that type 1 compounds are not localized within the lysosomal compartment ( Figure 12 A and Figure 16 ). The obvious filamentous structures in cells treated with anti-vimentin nanobody-FSD1 indicate that type 1 compounds escaped endosomal uptake and entered the cytosol, where they bound to vimentin (an intermediate filament protein that forms part of the cytoskeleton). In addition, the nuclei of cells treated with anti-vimentin nanobody-FSD1 showed very high ATTO 488 fluorescence intensity, indicating that type 1 compounds moved not only to the cytosol but also into the nucleus. In orthogonal projection, the x and y cutting planes can be visualized simultaneously ( Figure 12B). Here, it is apparent that in both the x and y cut planes, ATTO 488 fluorescence is localized not only to the membrane surface but also between the nucleus and the surface. This further confirms that the anti-vimentin Nanobody-FSD1 is present in the cytosol itself and not simply associated with the cell membrane. In cells treated with either the anti-vimentin Nanobody or DMEM, no nuclei or filamentous structures were visualized by ATTO 488 fluorescence. This suggests that without the Type 1 design of this platform, the anti-vimentin Nanobody is not taken up by cells to any significant extent.

[0430] in conclusion:

[0431] This platform invention, exemplified by the anti-vimentin nanobody FSD1, a type 1 design compound, was taken up by cells and enabled visualization of vimentin filaments in the cytosol. The type 1 design compound also appeared to enter the cell nucleus. Taken together, these data demonstrate that this platform invention can confer the ability of therapeutic or diagnostic agents to enter the cytosol and nucleus.

[0432] Example 12: Preparation of FSD1 variants

[0433] Purpose:

[0434] The purpose of this example was to apply structural analysis to generate FSD1 mutants that lack neutralizing activity against activin A, myostatin, and GDF11.

[0435] Materials and methods:

[0436] The crystal structure of the follistatin-myostatin complex (RCSB ID: 3HH2) was used to structurally analyze the interaction between FSD1 and myostatin. The inventors discovered that Glu126 (glutamic acid at amino acid residue position 126) of FSD1 forms a salt bridge with Lys39 (lysine at amino acid residue position 39) of myostatin. Furthermore, Gln124 (glutamine at amino acid residue position 124) of FSD1 was found to form a hydrogen bond with Asn83 (asparagine at amino acid residue position 83) of myostatin. Aside from these specific interactions, no salt bridges or hydrogen bonds were observed between FSD1 and myostatin. The inventors mutated FSD1 Glu126 and / or Gln124 to alanine at these sites, thereby obtaining the FSD1 domain variants E126A, Q124A, and Q124A E126A used herein, respectively, to disrupt the interaction and prevent FSD1 from binding to and neutralizing myostatin. These mutations were introduced by site-directed mutagenesis using the InFusion HD Cloning Kit (Clontech).

[0437] The GDF11 residues corresponding to myostatin Lys39 and Asn83 are conserved, suggesting a similar interaction between GDF11 and FSD1. In activin A, the residues corresponding to myostatin Lys39 and Asn83 are His36 and Ser90, respectively. Therefore, the interaction between FSD1 and activin A is expected to be weaker.

[0438] in conclusion

[0439] In summary, by mutating FSD1 Glu126 and Gln124 to alanine, the inventors tested their hypothesis that the interaction between FSD1 and the growth factors activin A, myostatin, and GDF11 would be disrupted. The FSD1 domain variants E126A, Q124A, and Q124A E126A used herein were obtained.

[0440] Example 13: Demonstration of ECM binding of Type 2 and Type 4 design compounds in vivo

[0441] Purpose:

[0442] The purpose of this example was to demonstrate in vivo ECM binding and to visualize the increased localization of Type 2 or Type 4 designer compounds in tissues compared to non-FSD1 fusion compounds.

[0443] Materials and methods:

[0444] This example is based on material harvested from animals treated as in Example 8. Briefly, PBS (vehicle), VGFR(1 / 2)-mFc-FSD1 (Type 2 compound), VGFR(1 / 2) / FST291 (Type 4 compound), or VGFR(1 / 2)-hFc (aflibercept, Bayer AG, Leverkusen, Germany) were injected intramuscularly into the right gastrocnemius muscle of C57BL / 6 mice (3 per group). Compounds were fluorescently labeled with NIR730. Four days after injection, gastrocnemius muscles were excised and fixed in 4% formaldehyde for 48 hours. Gastrocnemius muscles were dehydrated with ethanol, embedded in paraffin, and then cut into 2 μm-thick serial sections using a microtome. Serial sections were stained with Sirius Red (a common stain for viewing connective tissue) or left unstained and mounted on microscope slides with DAPI-containing mounting medium (#P36962, ProLong Diamond Antifade Mountant, containing DAPI). Slides were imaged using an Olympus VS120 slide scanner equipped with a Spectra X optical engine and a Semrock quintuple filter set (DAPI / FITC / Cy3 / Cy5 / Cy7 quintuple LED HC filter set, #F68-050) using a Hamamatsu ORCA-FLASH4.0 V2 (QE82%) camera. Images were acquired using a x20 Air NA 0.75 objective. DAPI excitation used a 395 / 25 bandpass filter, emission bandpass 425 / 50, with an exposure of 5 ms. NIR730 excitation used a 730 / 40 bandpass filter, emission bandpass 800 / 100, with an exposure of 150 ms. Images were processed using Fiji (v2.14.0 / 1.54f). All images were adjusted equally for DAPI contrast and brightness, with the exception of VGFR(1 / 2)-mFc-FSD1, which received a smaller adjustment due to its stronger signal. All images were adjusted equally for NIR730 signal.

[0445] result:

[0446] In skeletal muscle, the presence of the type 2 designer compound VGFR(1 / 2)-mFc-FSD1 was readily visualized by its NIR730 fluorescence signal ( Figure 13A). In contrast, no NIR730 signal was observed in PBS-treated muscle. Weak NIR730 signals were detected in both VGFR(1 / 2) / FST291-mFc (Type 4 design compound) and VGFR(1 / 2)-hFc (aflibercept)-treated muscle. Strong signals from VGFR(1 / 2)-mFc-FSD1 were present in two distinct compartments. The first compartment showed the strongest signal, consistent with the distribution of connective tissue morphology within the muscle, as visualized by Sirius Red staining. The second compartment was present throughout the muscle parenchyma ( Figure 13 B, Enlarged area 1) and vascular tissue penetrating the muscle ( Figure 13 B, magnified area 2) forms a fluorescent signal network. NIR730 signals from the substance are emitted from the interior of the subsarcolemmal muscle fibers (subsarcolemmal region) or from the outer surface of the cells close to the sarcolemma. NIR730 signals from vascular tissue visualize all three layers of the vascular system (intima, media, and adventitia). Similar to the substance, VGFR (1 / 2) -mFc-FSD1 signals are emitted from cells inside the vascular system, or from the extracellular compartment, or from both.

[0447] in conclusion:

[0448] The ECM is a major component of connective tissue and may serve as a reservoir for the platform invention being protected. In this way, the local presence of the type 2 design compound VGFR(1 / 2)-mFc-FSD1 in skeletal muscle was significantly increased, as reflected by the compound's strong NIR730 fluorescence signal. In contrast, both VGFR(1 / 2) / FST291-mFc (type 4 design compound) and VGFR(1 / 2)-hFc (aflibercept) showed limited NIR730 signals, indicating that the presence of these compounds was minimal or absent four days after administration.

[0449] Example 14: In vivo evaluation of the half-life of type 2 and type 4 design compounds in mouse ocular tissue using fluorescent fundus imaging

[0450] Purpose:

[0451] The purpose of this example is to evaluate the half-lives of type 2 and type 4 design compounds in mouse ocular tissue using fluorescent ophthalmoscopy.

[0452] Materials and methods:

[0453] Compound Labeling:

[0454] Compounds were fluorescently labeled using the green fluorescent protein spectral marker ATTO 488 (ATTO-TEC GmbH, #AD488) according to the manufacturer's instructions. After labeling, compound concentrations were determined using the Pierce Coomassie (Bradford) Protein Assay Kit (Thermo Scientific, #23200).

[0455] Animal experiments:

[0456] Eight 12-week-old female BALB / c mice were divided into four groups of two mice each: PBS (vehicle), VGFR(1 / 2)-mFc-FSD1 (type 2 compound), VGFR(1 / 2) / FST291 (type 4 compound) and VGFR(1 / 2)-hFc (Aflibercept, Bayer AG, Leverkusen, Germany). During anesthesia, tropicamide (Mydriacyl 0.5%) eye drops were used to dilate the pupil. Experienced operators administered a single dose of 10 μg of fluorescently labeled compound in a volume of 1 μL through the vitreous. The injected eyes were treated with chloramphenicol gel (Kloramfenikol, 1%) to prevent infection, and mice received a subcutaneous injection of analgesics (carprofen 5 mg / kg) after surgery. Immediately after intravitreal administration, baseline fluorescent fundus imaging (Micron IV, Phoenix Research Laboratories, OR, USA) was performed with an exposure of 200 ms to verify the correct delivery and fluorescent signal of the compound. Four days after treatment, the mice were anesthetized and fluorescent fundus imaging was performed again with an exposure time of 500 ms. The fluorescence intensity of the images was quantified using Fiji (v 2.14.0 / 1.54f).

[0457] result:

[0458] Fluorescence ophthalmoscopy at baseline showed no statistically significant differences in mean fluorescence intensity between the groups ( Figure 14 However, four days after treatment, the mean fluorescence intensity of eyes treated with VGFR(1 / 2)-mFc-FSD1 (type 2 compound) was significantly higher than that of eyes treated with VGFR(1 / 2) / FST291-mFc (type 4 compound) or VGFR(1 / 2)-hFc (aflibercept) ( Figure 14 Four days after treatment, the fluorescence signals in eyes treated with VGFR(1 / 2) / FST291-mFc or VGFR(1 / 2)-hFc (aflibercept) were not different from those in eyes treated with PBS or other eyes ( Figure 14 C).

[0459] in conclusion:

[0460] In ocular tissue, the Type 2 design compound, VGFR(1 / 2)-mFc-FSD1, exhibited superior intraocular half-life characteristics compared to the Type 4 design compounds, VGFR(1 / 2) / FST291-mFc and VGFR(1 / 2)-hFc (aflibercept). This was demonstrated by significantly higher fluorescence signals in eyes treated with VGFR(1 / 2)-mFc-FSD1 compared to the other compounds four days after treatment.

[0461] Example 15: In vivo efficacy of type 2 design compounds in a wet age-related macular degeneration model

[0462] Purpose:

[0463] The purpose of this example is to investigate the efficacy of type 2 design compounds in a mouse choroidal neovascularization (CNV) disease model of wet age-related macular degeneration (wet AMD).

[0464] Materials and methods:

[0465] Animal experiments:

[0466] Twenty-three 8-week-old male C57BL6 / J mice were divided into three groups: mouse IgG2a isotype control (Invitrogen, #02-6200; n=7), VGFR(1 / 2)-mFc-FSD1 (type 2 compound, n=8), and VGFR(1 / 2)-hFc (aflibercept, Bayer AG, Leverkusen, Germany; n=8). During anesthesia, tropicamide (Mydriacyl 0.5%) eye drops were used to dilate the pupil. CNV was laser-induced using an image-guided laser system (Micron IV, Phoenix Research Laboratories, OR, USA) according to the method described by Gong et al. (REF. 7). Laser settings were: wavelength 532 nm and 240 mW; duration: 70 ms; size: 50 μm. Four laser ablations were performed in the eye in a clockwise rotation direction (12, 3, 6, and 9 o'clock). The distance between the two laser burns and the distance between the laser burn and the optic nerve was approximately two optic disc diameters of the optic nerve. After CNV induction, a single dose of 10 μg of the compound was administered intravitreally in a volume of 1 μL by an experienced operator who was blinded to the compound. The compound vehicle used was PBS. The injected eyes were treated with chloramphenicol gel (Kloramfenikol, 1%) to prevent infection, and mice were given subcutaneous analgesics (carprofen 5 mg / kg) after surgery. One week after CNV induction and compound injection, mice were euthanized, their eyeballs were removed, fixed with 4% paraformaldehyde for 2 hours at room temperature, and then washed with PBS.

[0467] Retinal Pigment Epithelium / Choroid Flat Mounting and Immunohistochemistry:

[0468] Retinal pigment epithelium (RPE) / choroid flat sealing is carried out according to Askou et al. (REF.8) description. In 96-well plates, flat sealing material is permeabilized and blocked in PBB buffer (1x PBS, 4% BSA [Millipore, #81-068-3] and 0.5% Triton X-100 [Millipore, #1.08603]) at 4 ° C for 2 hours. In order to visualize vascular structure, samples are incubated overnight at 4 ° C with a dilution of 1:100 in PBB with rat anti-mouse CD31 primary antibody (BD Pharmingen, #557355) and with biotin-conjugated isolectin GS-IB4 (Invitrogen, #I21414) in PBB with a dilution of 1:100. The samples were washed four times in PBS-X wash solution (1× PBS and 0.5% Triton X-100) and incubated with Alexa Fluor 568-conjugated goat anti-rat IgG secondary antibody (Invitrogen, #A-11077) at a dilution of 1:500 in PBB and Alexa Fluor 405 conjugated to streptavidin (Invitrogen, #S32351) at a dilution of 1:100 in PBB for 2 hours at room temperature, followed by six washes in PBS-X. RPE / choroid flat mounts were mounted on microscope slides and imaged using an Olympus VS120 slide scanner equipped with a Spectra X optical engine and Semrock five-pass filters (DAPI / FITC / Cy3 / Cy5 / Cy7 five-pass LED HC filter set, #F68-050) using a Hamamatsu ORCA-FLASH4.0 V2 (QE82%) camera. Images were taken using a x20 Air NA 0.75 objective. Alexa Fluor 405 excitation used a 395 / 25 bandpass filter, an emission bandpass of 425 / 50, and an exposure of 10 ms. Alexa Fluor 568 excitation used a 575 / 25 bandpass filter, an emission bandpass of 600 / 60, and an exposure of 10 ms. Images were processed using Fiji (v2.14.0 / 1.54f). To quantify the mean CNV lesion area per eye, samples were adjusted equally for contrast and brightness and filtered using a global threshold. Thresholds were visually assessed across five samples to determine optimal low and high cutoff values. During quantification, investigators were blinded to the samples. Exclusion of CNV lesions was based on the recommendations of Gong et al. (REF. 7) and was performed prior to unblinding.

[0469] result:

[0470] In the mouse CNV disease model of wet AMD, CD31 and isolectin immunohistochemical staining of RPE / choroid flat mounts provides two complementary methods for visualizing CNV lesions ( Figure 15 A). Based on CD31 and isolectin staining, eyes treated with VGFR(1 / 2)-mFc-FSD1 (Type 2 compound) or VGFR(1 / 2)-hFc (aflibercept) showed statistically significant reductions in CNV lesion area compared to eyes treated with mouse IgG2a isotype control ( Figure 15 B). No significant differences were observed in CNV lesion area assessed by CD31 and isolectin staining between eyes treated with VGFR(1 / 2)-mFc-FSD1 and VGFR(1 / 2)-hFc (aflibercept).

[0471] in conclusion:

[0472] In the CNV disease model of wet AMD in mice, the type 2 design compound VGFR(1 / 2)-mFc-FSD1 was as effective as its commercially available counterpart, VGFR(1 / 2)-hFc (aflibercept), in reducing CNV lesion area. The results demonstrate that this platform invention (here exemplified by the type 2 design compound) does not interfere with the ligand neutralization of the therapeutic moiety (i.e., VGFR(1 / 2)). In this experimental setting, the therapeutic effect of VGFR(1 / 2)-mFc-FSD1 is not expected to exceed that of VGFR(1 / 2)-hFc (aflibercept) because both compounds were administered on the same day of CNV induction.

[0473] Example 16: In vivo study of intracellular uptake

[0474] Purpose:

[0475] The purpose of this example is to evaluate the intracellular uptake of the claimed platform invention in vivo.

[0476] Materials and methods:

[0477] Compound Labeling:

[0478] Compounds were fluorescently labeled using the green fluorescent protein marker ATTO 488 (ATTO-TEC GmbH, #AD488) according to the manufacturer's instructions. After labeling, compound concentrations were determined using the Pierce Coomassie (Bradford) Protein Assay Kit (Thermo Scientific, #23200).

[0479] Animal experiments:

[0480] Two 12-week-old female BALB / c mice were assigned to be injected with anti-vimentin nanobody-FSD1 (type 1 compound, n=1) or anti-vimentin nanobody (n=1). During anesthesia, the hair of the right hind limb was shaved to expose the calf muscle. A single dose of 8 μg of fluorescently labeled compound was administered intramuscularly in the gastrocnemius muscle in a volume of 5 μl. The mice received a subcutaneous injection of analgesics (carprofen 5 mg / kg) to relieve any pain caused by muscle tension. 18 hours after injection, the gastrocnemius muscle was cut and immersion fixed in 4% formaldehyde. The gastrocnemius muscle was dehydrated with ethanol, embedded in paraffin, and sliced ​​into 4 μm thick sections using a microtome. The sections remained unstained and mounted on microscope slides with a mounting medium containing DAPI (#P36962, ProLong Diamond Antifade Mountant, containing DAPI). Confocal imaging was performed using a Zeiss LSM800 laser scanning confocal microscope (Carl Zeiss Microscopy GmbH, Oberkochen, Germany) equipped with two GaAsP and one Airyscan detector and Zen Blue Edition software (Carl Zeiss Microscopy GmbH, version 2.5). Confocal images were taken using a PlanApo x63 Oil NA 1.4 objective. ATTO 488 was excited at 1.30% using a 488nm diode laser and detected at a wavelength of 510-575nm. DAPI was excited at 1.00% using a 405nm diode laser and detected at a wavelength of 400-510nm. For each sample, Z-stacks were recorded at intervals of 0.190μm. For all scans, the pixel time was 1.10μs, and the average value was set to 4x in per-line repetition mode and the average intensity method. Image processing was performed using Fiji (v 2.14.0 / 1.54f). For ATTO 488 and DAPI, all images were adjusted equally for contrast and brightness.

[0481] result:

[0482] In skeletal muscle, the presence of the type 1 designer compound anti-vimentin nanobody-FSD1 was easily visualized by its ATTO 488 fluorescence signal ( Figure 18 In contrast, no ATTO488 signal was detected in muscles injected with the anti-vimentin nanobody. The anti-vimentin nanobody-FSD1 signal was most pronounced in cells resident in connective tissue, some of which exhibited the spindle-shaped morphology characteristic of fibroblasts. The ATTO 488 fluorescent signal resembled interwoven threads (or filaments) that curved and stretched throughout the cell and relative to the nucleus (stained with DAPI). No clear fluorescent signature was detected in the muscle parenchyma.

[0483] in conclusion:

[0484] This platform invention, taking the type 1 designed compound anti-vimentin nanobody-FSD1 as an example, is taken up by cells and realizes the visualization of vimentin filaments. This is only possible if the compound enters the cytosol after cell uptake. In contrast, no ATTO 488 signal was detected from the sample injected with the anti-vimentin nanobody, indicating that the nanobody itself cannot enter the cytosol without this platform invention. The intermediate filament protein vimentin is not expressed in all cell types, but it is a marker of mesenchymal-derived cells (including fibroblasts) (REF.9, 10). No obvious ATTO 488 signal was detected in the parenchyma of the muscle injected with the anti-vimentin nanobody-FSD1, which is consistent with the fact that vimentin is not expressed in mature and intact skeletal muscle fibers (REF.11). Therefore, the anti-vimentin nanobody-FSD1 taken up by the parenchyma is likely to be dispersed in the cytosol of skeletal muscle fibers, and will not be enriched at the target protein vimentin. Skeletal muscle fibers are much larger than most other cell types, and any ATTO 488 fluorescence signal from the parenchyma is likely diluted beyond the detection point of this study. Taken together, these data suggest that this platform invention can confer the ability for therapeutic or diagnostic agents to enter the cytosol and potentially the nucleus.

[0485] Example 17: Neutralization of Glucocorticoid Signaling

[0486] Purpose:

[0487] The purpose of this example is to quantify the neutralizing effect of compounds designed based on the Type 2 or Type 4 design of this platform invention on glucocorticoid signaling.

[0488] Materials and methods:

[0489] The glucocorticoid neutralization of cortisol (Supelco, #C-106-1ML) or prednisolone 21-hemisuccinate (a water-soluble form of prednisolone, Sigma-Aldrich, #P4153-1G) was quantified using a glucocorticoid response element (GRE)-activated reporter gene bioassay (REF. 12). HEK293 cells were transfected with a construct containing three repeats of the GRE motif upstream of a minimal promoter controlling luciferase expression. The HEK293 cell line was propagated as a stable cell line with robust luciferase responsiveness to glucocorticoid signaling induced by endogenous or synthetic glucocorticoids. Briefly, cells were stimulated with cortisol or prednisolone 21-hemisuccinate and co-treated with recombinant protein at concentrations starting at 2 μM. After 16-20 hours, cells were lysed (Promega, Glo Lysis Buffer, #E2661), luciferase substrate (Promega, Steady-Glo Luciferase Assay System, #E2520) was added, and the luminescent signal was analyzed using a microplate reader (PerkinElmer, EnSpire 2300). Six or more positive controls (containing glucocorticoids and not containing inhibitors) and negative controls (containing no glucocorticoids and inhibitors) were included. Data were analyzed and half-maximal inhibitory concentrations (IC50) were calculated using a three-parameter, unweighted, and unconstrained nonlinear regression (Graphpad, Prism 9.4.1).

[0490] result:

[0491] Compounds containing either type 2 (CBG-mFc-FSD1) or type 4 (CBG / FST291-mFc) designs fused to CBG in this platform neutralized endogenous glucocorticoid signaling (cortisol) or synthetic glucocorticoid signaling (prednisolone 21-hemisuccinate) at concentrations up to 2 μM with IC50 values ​​in the low to mid-nM range. Type 2 design compounds have two CBG moieties, resulting in ligand binding that is approximately twice as strong as type 4 design compounds with a single CBG moiety.

[0492] in conclusion:

[0493] CBG typically acts as a serum glucocorticoid transporter or buffer protein and can be used to neutralize glucocorticoids. This article uses the Type 2 and Type 4 designs of this platform invention as examples for illustration. By integrating CBG with this platform invention, a local glucocorticoid neutralizing compound was developed. Because this platform invention is relatively limited to the injected tissue (depending on the number of FSD1 modules, as shown in Examples 7 and 8), the glucocorticoid neutralizing compound does not produce systemic effects.

[0494] Example 18: Degrading intracellular proteins using this platform invention

[0495] Purpose:

[0496] The purpose of this example is to demonstrate the utility of this platform invention for degrading intracellular proteins using a proteolysis targeting chimera (PROTAC) strategy.

[0497] Materials and methods:

[0498] Generation of PROTAC Nanobodies:

[0499] Anti-vimentin Nanobody and anti-vimentin Nanobody-FSD1 (Type 1 compound) were conjugated to (S,R,S)-AHPC-PEG8-NHS, a ligand of the Von Hippel-Lindau tumor suppressor protein (VHL) linked to an NHS ester via a PEG8 linker (Broadpharm, #BP-25703). Briefly, each compound was incubated with the VHL ligand in a buffer of 500 mM KCl, 20 mM Hepes, pH 8.3 for 1 hour. The buffer was then exchanged for PBS, and protein concentration was determined using a NanoDrop 2000c (Thermo Scientific, #ND-2000C).

[0500] In vitro degradation of vimentin:

[0501] HEK293 cells were seeded in DMEM (ThermoFisher, #11995073) supplemented with 2% FBS in 6-well plates treated with Nunclon Delta (ThermoFisher, #140685) at a density of 600,000 cells per well. At the time of seeding, PROTAC nanobody compounds (500 nM) were added, and a control condition without compound (DMEM control) was included. After 24 hours, cells were trypsinized, transferred to a microcentrifuge tube, and centrifuged at 200 x g for 5 minutes at room temperature. The medium was discarded, and the pellet was washed with PBS and centrifuged again at 200 x g for 5 minutes. The PBS was removed, and the pellet was resuspended in 100 μL RIPA buffer (ThermoFisher, #89901) containing 1 mM phenylmethylsulfonyl fluoride (PMSF, Sigma-Aldrich, #93482). The resulting homogenate was incubated at 4°C, shaken at 750 RPM for 30 minutes, and then centrifuged at 16,000 x g for 20 minutes at 4°C. The supernatant (lysate) was stored at -20°C, and the protein concentration was determined using a Pierce Coomassie (Bradford) protein assay kit (Thermo Scientific, #23200). The lysate was diluted 1 / 10 with PBS.

[0502] Western blotting:

[0503] For Western blotting, 16 μg of total protein was subjected to stain-free SDS-PAGE and total protein analysis was performed using the Gel Doc EZ system (Bio-Rad, #1708270). The gel was transferred to a PVDF membrane and blocked with 5% skim milk for 1 hour. A 1-hour incubation step was performed using a rabbit polyclonal anti-vimentin primary antibody (Invitrogen, #PA5-27231) diluted 1:10,000 in 5% skim milk. After washing five times with Tris-buffered saline containing 0.1% Tween 20 (TBST), a 1-hour incubation step was performed using a goat anti-rabbit IgG-peroxidase secondary antibody (Sigma-Aldrich, #A0545) diluted 1:5,000 in 5% skim milk. After the blot was washed 5 times in TBST, it was imaged using the Invitrogen iBright FL1500 imaging system (Invitrogen, #A44241) with an exposure time of 14 minutes. The background-corrected vimentin band intensity was quantified using iBright analysis software (version 1.8.1). Next, a protein blot was performed on the same membrane for glyceraldehyde 3-phosphate dehydrogenase (GAPDH) as a loading control. The same procedure as above was performed using a 1:1,000 dilution of mouse monoclonal anti-GAPDH primary antibody (Invitrogen, #MA5-15738) and a 1:5,000 dilution of goat anti-mouse IgG-peroxidase secondary antibody (Sigma-Aldrich, #A2554). The blot was imaged for 35 seconds, and the background-adjusted GAPDH band intensity was quantitatively analyzed.

[0504] result:

[0505] In HEK293 cells, treatment with the anti-vimentin Nanobody-FSD1 conjugated to the VHL ligand (Type 1 compound) for 24 h significantly reduced vimentin levels (normalized to GAPDH) compared to lysates from cells treated with DMEM control or anti-vimentin Nanobody conjugated to VHL ( Figure 24 AB). Anti-vimentin nanobody (without FSD1) did not reduce vimentin levels compared to DMEM controls. To ensure data consistency, vimentin band intensity was also quantified using Fiji and normalized to unstained total protein estimates (rather than GAPDH intensity). Similar results were obtained in this analysis (data not shown).

[0506] in conclusion:

[0507] This platform invention (here taking type 1 design compounds as an example) when used in the PROTAC strategy can promote the degradation of vimentin (a constitutively expressed intracellular protein). In contrast, anti-vimentin nanobodies coupled to VHL ligands that are not used in this platform invention do not degrade vimentin. The results show that this platform invention can give therapeutic agents or diagnostic agents the ability to enter the cytosol. In this way, the platform invention is recombinantly fused or chemically coupled with, for example, nanobodies against intracellular target proteins, and then the compound (nanobody-FSD1) is fused or coupled with an E3 ligase ligand to degrade intracellular proteins.

[0508] References

[0509] 1.Kober,L.et al.(2013)Optimized signal peptides for the development of high expressing CHO cell lines.Biotechnol.Bioeng.110,1164–1173

[0510] 2.Choi,H.-J.et al.(2013)A Heterodimeric Fc-Based Bispecific AntibodySimultaneously Targeting VEGFR-2and Met Exhibits Potent Antitumor Activity.

[0511] 3. Dennler, S. et al. (1998) Direct binding of Smad3 and Smad4 to criticalTGF beta-inducible elements in the promoter of human plasminogen activatorinhibitor-type 1gene. EMBO J.17,3091–3100

[0512] 4.Lodberg,A.et al.(2019)A follistatin-based molecule increases muscleand bone mass without affecting the red blood cell count in mice.FASEB J.33,6001–6010

[0513] 5.Pearsall,R.S.et al.(2019)Follistatin-based ligand trap ACE-083induces localized hypertrophy of skeletal muscle with functionalimprovement in models of neuromuscular disease.Sci.Rep.9,11392

[0514] 6.Lohmann,S.et al.(2021)Mesenchymal stromal cell treatment of donorkidneys during ex vivo normothermic machine perfusion:A porcine renalautotransplantation study.Am.J.Transplant 21,2348–2359

[0515] 7.Gong,Y.et al.(2015)Optimization of an Image-Guided Laser-InducedChoroidal Neovascularization Model in Mice.PLoS One 10

[0516] 8.Askou,A.L.et al.(2017)Suppression of Choroidal Neovascularizationin Mice by Subretinal Delivery of Multigenic Lentiviral Vectors EncodingAnti-Angiogenic MicroRNAs.Hum.Gene Ther.Methods 28,222–233

[0517] 9.Ostrowska-Podhorodecka,Z.et al.(2022)Impact of Vimentin onRegulation of Cell Signaling and Matrix Remodeling.Front Cell Dev Biol 10,869069.

[0518] 10. Kidd, M.E., et al. (2014) The role of Vimentin intermediate filaments in the progression of lung cancer. Am J Respir Cell Mol Biol 50, 1–6 (2014).

[0519] 11. Vater, R., et al. (1994) The expression of vimentin in satellite cells of regenerating skeletal muscle in vivo. The Histochemical Journal 26:12 26, 916–928

[0520] 12. Novotna, A., Pavek, P. & Dvorak, Z. (2012) Construction and characterization of a reporter gene cell line for assessment of human glucocorticoid receptor activation. European Journal of Pharmaceutical Sciences 47, 842–847

[0521] 13. Jang HR, Shin SB, Kim CH, et al. (2021) PLK1 / vimentin signaling facilitates immune escape by recruiting Smad2 / 3 to PD-L1 promoter in metastatic lung adenocarcinoma [published correction appears in Cell Death Differ. 2021 Aug 17;:]. Cell Death Differ. 2021;28(9):2745-2764. doi:10.1038 / s41418-021-00781-4

[0522] Sequence Listing

[0523] SEQ ID NO:1: Human FSD1

[0524] ETCENVDCGPGKKCRMNKKNKPRCVCAPDCSNITWKGPVCGLDGKTYRNECALLKARCKEQPELEVQYQGRCK

[0525] SEQ ID NO: 2: Human FSD1-FSD1

[0526] ETCENVDCGPGKKCRMNKKNKPRCVCAPDCSNITWKGPVCGLDGKTYRNECALLKARCKEQPELEVQYQGRCKETCENVDCGPGKKCRMNKKNKPRCVCAPDCSNITWKGPVCGLDGKTYRNECALLKARCKEQPELEVQYQGRCK

[0527] SEQ ID NO: 3: Human FSD1 - FSD1 produced in E. coli as a single entity, containing a starting methionine (M) amino acid.

[0528] METCENVDCGPGKKCRMNKKNKPRCVCAPDCSNITWKGPVCGLDGKTYRNECALLKARCKEQPELEVQYQGRCKETCENVDCGPGKKCRMNKKNKPRCVCAPDCSNITWKGPVCGLDGKTYRNECALLKARCKEQPELEVQYQGRCK

[0529] SEQ ID NO: 4: Polynucleotide sequence encoding human FSD1 - FSD1 produced in E. coli as a single entity containing an initial methionine (M) amino acid.

[0530] ATGGAGACATGCGAGAACGTGGATTGTGGACCAGGCAAGAAGTGCCGGATGAACAAGAAGAACAAGCCCAGATGCGTGTGCGCTCCTGACTGCAGCAACATCACCTGGAAGGGACCCGTGTGCGGCCTGGATGGCAAGACATACCGGAATGAGTGCGCCCTGCTGAAGGCTAGGTGTAAGGAGCAGCCTGAGCTGGAGGTGCAGTATCAGGGCCGGTGCA AGGAGACATGCGAGAACGTGGATTGTGGACCAGGCAAGAAGTGCCGGATGAACAAGAAGAACAAGCCCAGATGCGTGTGCGCTCCTGACTGCAGCAACATCACCTGGAAGGGACCCGTGTGCGGCCTGGATGGCAAGACATACCGGAATGAGTGCGCCCTGCTGAAGGCTAGGTGTAAGGAGCAGCCTGAGCTGGAGGTGCAGTATCAGGGCCGGTGCAAG

[0531] SEQ ID NO: 5: Anti-vimentin Nanobody-FSD1. Expressed in E. coli, therefore contains a starting methionine (M) amino acid.

[0532] MQVQLVESGGGLVQSGGSLTLTCAASGFTFSAASMRWVRQVPGKGLEWVATIDGTGANSYYSESAKGRFTISRDNARNTLYLQMNNLKPDDTAVYYCANFGRNYWGKGTQVTVSSETCENVDCGPGKKCRMNKKNKPRCVCAPDCSNITWKGPVCGLDGKTYRNECALLKARCKEQPELEVQYQGRCK

[0533] SEQ ID NO: 6: Polynucleotide encoding the anti-vimentin nanobody FSD1. Expressed in E. coli, it contains an initial methionine (M) amino acid.

[0534] ATGCAAGTACAGCTAGTTGAATCAGGTGGAGGGTTGGTGCAAAGCGGTGGTTCTCTGACCTTGACGTGCGCGGCGTCTGGCTTCACCTTCAGCGCTGCCTCCATGCGTTGGGTACGTCAGGTGCCGGGTAAGGGCCTTGAG TGGGTCGCAACCATCGACGGTACTGGTGCAAACAGCTATTACAGCGAGTCGGCGAAAGGCCGTTTTACGATCAGCCGTGATAATGCGCGTAACACCTTATATCTGCAGATGAATAACCTGAAACCGGATGACACCGCAGTT TACTACTGCGCGAACTTTGGTCGCAACTATTGGGGTAAGGGCACGCAAGTGACCGTTTCCTCCGAAACCTGTGAAAATGTCGATTGCGGTCCGGGCAAAAAGTGCCGTATGAACAAGAAGAACAAGCCGCGTTGTGTTTGC GCTCCGGACTGTAGCAATATTACCTGGAAAGGCCCAGTGTGCGGCCTGGACGGCAAAACCTACCGCAACGAATGTGCGCTGCTGAAAGCCAGATGCAAAGAGCAGCCGGAACTGGAGGTTCAATATCAGGGTCGCTGCAAG

[0535] SEQ ID NO: 7: VGFR(1 / 2)-mFc-FSD1. The serum albumin preproprotein signal peptide is shown in bold, mouse IgG2A is underlined, and FSD1 is double underlined.

[0536]

[0537] SEQ ID NO: 8: Polynucleotide sequence encoding VGFR(1 / 2)-mFc-FSD1.

[0538]

[0539] SEQ ID NO: 9: TNFR2-mFc-FSD1. The serum albumin preproprotein signal peptide is shown in bold, mouse IgG2A is underlined, and FSD1 is double underlined.

[0540]

[0541] SEQ ID NO: 10: Polynucleotide encoding TNFR2-mFc-FSD1

[0542]

[0543] SEQ ID NO: 11: FST291-mFc Knob-In-Hole A Chain (for use with VGFR(1 / 2) B Chain, TNFR2 B Chain, or CBG B Chain). Serum albumin preproprotein signal peptide is shown in bold, and the mouse IgG2A A chain is underlined.

[0544]

[0545]

[0546] SEQ ID NO: 12: Polynucleotide encoding FST291-mFc Knob-In-Hole A chain (for use with VGFR(1 / 2) B chain, TNFR2 B chain, or CBG B chain).

[0547]

[0548] SEQ ID NO: 13: VGFR(1 / 2)-mFc knob-in-hole B chain. The serum albumin preproprotein signal peptide is shown in bold, and the mouse IgG2A B chain is underlined. The C-terminal thrombin site and his tag are double underlined.

[0549]

[0550] SEQ ID NO: 14: Polynucleotide encoding VGFR(1 / 2)-mFc knob-in-hole B chain.

[0551]

[0552] SEQ ID NO: 15: TNFR2-mFc knob-in-hole B chain. The serum albumin preproprotein signal peptide is shown in bold, and the mouse IgG2A B chain is underlined. The C-terminal thrombin site and his-tag are double-underlined.

[0553]

[0554]

[0555] SEQ ID NO: 16: Polynucleotide encoding TNFR2-mFc knob-in-hole B chain.

[0556]

[0557] SEQ ID NO: 17: CBG-mFc knob-in-hole B chain. The serum albumin preproprotein signal peptide is shown in bold, and the mouse IgG2A B chain is underlined. The C-terminal thrombin site and his tag are double underlined.

[0558]

[0559] SEQ ID NO: 18: Polynucleotide encoding CBG-mFc knob-in-hole B chain.

[0560]

[0561] SEQ ID NO: 19: Portion of the native human VGFR1 extracellular domain that binds to VEGF (immunoglobulin-like type 2)

[0562] SDTGPFVEMYSEIPEIIHMTEGRELVIPCRVTSPNITVTLKKFPLDTLIPDGKRIIWDSRKGFIISNATYKEIGLLTCEATVNGHLYKTNYLTHRQTNTII

[0563] SEQ ID NO: 20: Portion of the native human VGFR2 extracellular domain that binds to VEGF (Immunoglobulin type 3-like protein 3)

[0564] DVVLSPSHGIELSVGEKLVLNCTARTELNVGIDFNWEYPSSKHQHKKLVNRDLKTQSGSEMKKFLSTLTIDGVTRSDQGLYTCAASSGLMTKKNSTFVRVHEK SEQ ID NO: 21: Native human TNFR2 extracellular domain (TNFα binding domain)

[0565] LPAQVAFTPYAPEPGSTCRLREYYDQTAQMCCSKCSPGQHAKVFCTKTSDTVCDSCEDSTYTQLWNWVPECLSCGSRCSSDQVETQACTREQNRICTCRPGWYCALSKQEGCRLCAP LRKCRPGFGVARPGTETSDVVCKPCAPGTFSNTTSSTDICRPHQICNVVAIPGNASMDAVCTSTSPTRSMAPGAVHLPQPVSTRSQHTQPTPEPSTAPSTSFLLPMGPSPPAEGSTGD

[0566] SEQ ID NO: 22: Native human CBG. The native signal peptide is shown in bold.

[0567]

[0568] SEQ ID NO: 23: Human FSD1 (KTC)

[0569] ETCENVDCGPGKKCRMNKKNKPRCVCAPDCSNITWKGPVCGLDGKTYRNECALLKARCKEQPELEVQYQGRCKKTC

[0570] SEQ ID NO:24: Human FSD1 (KTC), produced as a single entity in E. coli, containing a starting methionine (M) amino acid.

[0571] METCENVDCGPGKKCRMNKKNKPRCVCAPDCSNITWKGPVCGLDGKTYRNECALLKARCKEQPELEVQYQGRCKKTC

[0572] SEQ ID NO:25: Polynucleotide encoding human FSD1 (KTC), produced as a single entity in E. coli, containing an initial methionine (M) amino acid:

[0573] ATGGAGACATGCGAGAACGTGGATTGTGGACCAGGCAAGAAGTGCCGGATGAACAAGAAGAACAAGCCCAGATGCGTGTGCGCTCCTGACTGCAGCAACATCACCTGGAAGGGACCCGTGTGCGGCCTGGATGGCAAGACATACCGGAATGAGTGCGCCCTGCTGAAGGCTAGGTGTAAGGAGCAGCCTGAGCTGGAGGTGCAGTATCAGGGCCGGTGCAAGAAAACATGT

[0574] SEQ ID NO: 26: Native human follistatin 291. The native signal peptide is shown in bold.

[0575]

[0576] SEQ ID NO: 27: Native human follistatin 288. The native signal peptide is shown in bold.

[0577]

[0578] SEQ ID NO: 28: Native human follistatin 315. The native signal peptide is shown in bold.

[0579]

[0580] SEQ ID NO: 29: Human follistatin 315, wherein the heparin binding sequence (HBS) of FSD1 has been replaced by a structurally related sequence. The native signal peptide is shown in bold.

[0581]

[0582] SEQ ID NO: 30: Human follistatin 315 fused to a mouse IgG2A Fc fragment (underlined), wherein the heparin binding sequence (HBS) of FSD1 has been replaced by a structurally related sequence. The protein encoded by this sequence is designated FST315dHBS-mFc. The native signal peptide is in bold.

[0583]

[0584]

[0585] SEQ ID NO:31: Polynucleotide sequence encoding human follistatin 315 fused to a murine IgG2A Fc fragment, wherein the heparin binding sequence (HBS) of FSD1 has been replaced by a structurally related sequence.

[0586]

[0587] SEQ ID NO:32: Human FSD1 produced as a single entity in E. coli with an initial methionine (M) amino acid.

[0588] METCENVDCGPGKKCRMNKKNKPRCVCAPDCSNITWKGPVCGLDGKTYRNECALLKARCKEQPELEVQYQGRCK

[0589] SEQ ID NO:33: Polynucleotide encoding human FSD1, produced as a single entity in E. coli, containing an initial methionine (M) amino acid

[0590] ATGGAGACATGCGAGAACGTGGATTGTGGACCAGGCAAGAAGTGCCGGATGAACAAGAAGAACAAGCCCAGATGCGTGTGCGCTCCTGACTGCAGCAACATCACCTGGAAGGGACCCGTGTGCGGCCTGGATGGCAAGACATACCGGAATGAGTGCGCCCTGCTGAAGGCTAGGTGTAAGGAGCAGCCTGAGCTGGAGGTGCAGTATCAGGGCCGGTGCAAG

[0591] SEQ ID NO: 34: Anti-vimentin Nanobody. Expressed in E. coli, therefore contains an initial methionine (M) amino acid. The C-terminal thrombin site and his-tag are double underlined.

[0592]

[0593] SEQ ID NO: 35: Polynucleotide encoding anti-vimentin nanobody. Expressed in E. coli, thus containing an initial methionine (M) amino acid

[0594] ATGCAAGTACAGCTAGTTGAATCAGGTGGAGGGTTGGTGCAAAGCGGTGGTTCTCTGACCTTGACGTGCGCGGCGTCTGGCTTCACCTTCAGCGCTGCCTCCATGCGTTGGGTACGTCAGGTGCCGGGTAAGGGCCTTGAGTGGGTCGCAACCATCGACGGTACTGGTGCAAACAGCTATTACAGCGAGTCGGCGAAAGGCCGTTTTACGATCAGCCGTGATAATGCGCGTAACACCTTATATCTGCAGATGAATAACCTGAAACCGGATGACACCGCAGTTTACTACTGCGCGAACTTTGGTCGCAACTATTGGGGTAAGGGCACGCAAGTGACCGTTTCCTCCCTAGTACCCAGGGGTAGCCACCACCACCACCACCACSEQ ID NO:36: Heparin-binding domain of human FSD1

[0595] KKCRMNKKNKPR

[0596] SEQ ID NO:37: Murine IgG2A Fc

[0597] PRGPTIKPCPPCKCPAPNLLGGPSVFIFPPKIKDVLMISLSPIVTCVVVDVSEDDPDVQISWFVNNVEVHTAQTQTHREDYNSTLRVVSALPIQHQDWMSGKEFKCKVNNKDLPAPIERTISKPKGSVRAPQVYVLPPPEEEMTKKQVTLTCMVTDFMPEDIYVEWTNNGKTELNYKNTEPVLDSDGSYFMYSKLRVEKKNWVERNSYSCSVVHEGLHNHHTTKSFSRTPGK

[0598] SEQ ID NO:38: Murine IgG2A Fc knob-into-hole A chain

[0599] PRGPTIKPCPPCKCPAPNLLGGPSVFIFPPKIKDVLMISLSPIVTCVVVDVSEDDPDVQISWFVNNVEVHTAQTQTHREDYNSTLRVVSALPIQHQDWMSGKEFKCKVNNKDLPAPIERTISKPKGSVRAPQVYVLPPPEEEMTEKQVTLTCMVTDFMPEDIYVEWTNNGKTELNYKNTEPVLDSDGSYFMYSWLRVEKKNWVERNSYSCSVVHEGLHNHHTTKSFSRTPGK

[0600] SEQ ID NO:39: Mouse IgG2A Fc Knob-into-Hole B chain

[0601] PRGPTIKPCPPCKCPAPNLLGGPSVFIFPPKIKDVLMISLSPIVTCVVVDVSEDDPDVQISWFVNNVEVHTAQTQTHREDYNSTLRVVSALPIQHQDWMSGKEFKCKVNNKDLPAPIERTISKPKGSVRAPRVYVLPPPEEEMTKKQVTLTCMVTDFMPEDIYVEWTNNGKTELNYKNTEPVLVSDGSYTMYSKLRVEKKNWVERNSYSCSVVHEGLHNHHTTKSFSRTPGK

[0602] SEQ ID NO:40: Human VGFR(1 / 2)

[0603] SDTGRPFVEMYSEIPEIIHMTEGRELVIPCRVTSPNITVTLKKFPLDTLIPDGKRIIWDSRKGFIISNATYKEIGLLTCEATVNGHLYKTNYLTHRQTNTIIDVVLSPSHGIELSVGEKLVLNCTARTELNVGIDFNWEYPSSKHQHKKLVNRDLKTQSGSEMKKFLSTLTIDGVTRSDQGLYTCAASSGLMTKKNSTFVRVHEK

[0604] SEQ ID NO:41: Human FSD2

[0605] KTCRDVFCPGSSTCVVDQTNNAYCVTCNRICPEPASSEQYLCGNDGVTYSSACHLRKATCLLGRSIGLAYEGKCI

[0606] SEQ ID NO:42: Human FSD3

[0607] KAKSCEDIQCTGGKKCLWDFKVGRGRCSLCDELCPDSKSDEPVCASDNATYASECAMKEAACSSGVLLEVKHSGSCN

[0608] SEQ ID NO:43: Human follistatin N-terminal domain

[0609] GNCWLRQAKNGRCQVLYKTELSKEECCSTGRLSTSWTEEDVNDNTLFKWMIFNGGAPNCIPCK

[0610] SEQ ID NO:44: Human activin A (inhibin βA chain) (signal peptide shown in bold)

[0611]

[0612] SEQ ID NO:45: Myostatin (also known as growth differentiation factor 8) (signal peptide shown in bold)

[0613]

[0614] SEQ ID NO:46: Growth differentiation factor 11 (GDF11)

[0615]

[0616] SEQ ID NO: 47: Human FSD1 (Q124A)

[0617] ETCENVDCGPGKKCRMNKKNKPRCVCAPDCSNITWKGPVCGLDGKTYRNECALLKARCKEAPELEVQYQGRCK

[0618] SEQ ID NO:48: Human FSD1 (Q124A), produced as a single entity in E. coli, containing an initial methionine (M) amino acid.

[0619] METCENVDCGPGKKCRMNKKNKPRCVCAPDCSNITWKGPVCGLDGKTYRNECALLKARCKEAPELEVQYQGRCK

[0620] SEQ ID NO:49: Polynucleotide sequence encoding human FSD1 (Q124A), produced as a single entity in E. coli, containing an initial methionine (M) amino acid.

[0621] ATGGAGACATGCGAGAACGTGGATTGTGGACCAGGCAAGAAGTGCCGGATGAACAAGAAGAACAAGCCCAGATGCGTGTGCGCTCCTGACTGCAGCAACATCACCTGGAAGGGACCCGTGTGCGGCCTGGATGGCAAGACATACCGGAATGAGTGCGCCCTGCTGAAGGCTAGGTGTAAGGAGGCGCCTGAGCTGGAGGTGCAGTATCAGGGCCGGTGCAAG

[0622] SEQ ID NO: 50: Human FSD1 (E126A)

[0623] ETCENVDCGPGKKCRMNKKNKPRCVCAPDCSNITWKGPVCGLDGKTYRNECALLKARCKEQPALEVQYQGRCK

[0624] SEQ ID NO:51: Human FSD1 (E126A), produced as a single entity in E. coli, containing an initial methionine (M) amino acid.

[0625] METCENVDCGPGKKCRMNKKNKPRCVCAPDCSNITWKGPVCGLDGKTYRNECALLKARCKEQPALEVQYQGRCK

[0626] SEQ ID NO:52: Polynucleotide sequence encoding human FSD1 (E126A), produced as a single entity in E. coli, containing an initial methionine (M) amino acid.

[0627] ATGGAGACATGCGAGAACGTGGATTGTGGACCAGGCAAGAAGTGCCGGATGAACAAGAAGAACAAGCCCAGATGCGTGTGCGCTCCTGACTGCAGCAACATCACCTGGAAGGGACCCGTGTGCGGCCTGGATGGCAAGACATACCGGAATGAGTGCGCCCTGCTGAAGGCTAGGTGTAAGGAGCAGCCTGCGCTGGAGGTGCAGTATCAGGGCCGGTGCAAG

[0628] SEQ ID NO: 53: Human FSD1 (Q124A, E126A)

[0629] ETCENVDCGPGKKCRMNKKNKPRCVCAPDCSNITWKGPVCGLDGKTYRNECALLKARCKEAPALEVQYQGRCK

[0630] SEQ ID NO:54: Human FSD1 (Q124A, E126A), produced as a single entity in E. coli, containing a starting methionine (M) amino acid.

[0631] METCENVDCGPGKKCRMNKKNKPRCVCAPDCSNITWKGPVCGLDGKTYRNECALLKARCKEAPALEVQYQGRCK

[0632] SEQ ID NO:55: Polynucleotide sequence encoding human FSD1 (Q124A, E126A), produced as a single entity in E. coli, containing an initial methionine (M) amino acid.

[0633] ATGGAGACATGCGAGAACGTGGATTGTGGACCAGGCAAGAAGTGCCGGATGAACAAGAAGAACAAGCCCAGATGCGTGTGCGCTCCTGACTGCAGCAACATCACCTGGAAGGGACCCGTGTGCGGCCTGGATGGCAAGACATACCGGAATGAGTGCGCCCTGCTGAAGGCTAGGTGTAAGGAGGCGCCTGCGCTGGAGGTGCAGTATCAGGGCCGGTGCAAG

[0634] SEQ ID NO: 56: VGFR(1 / 2)-mFc-FSD1(Q124A). The serum albumin preproprotein signal peptide is shown in bold, mouse IgG2A is underlined, and FSD1(Q124A) is double underlined.

[0635]

[0636]

[0637] SEQ ID NO: 57: Polynucleotide sequence encoding VGFR(1 / 2)-mFc-FSD1(Q124A).

[0638]

[0639] SEQ ID NO: 58: VGFR(1 / 2)-mFc-FSD1(E126A). The serum albumin preproprotein signal peptide is shown in bold, mouse IgG2A is underlined, and FSD1(E126A) is double underlined.

[0640]

[0641] SEQ ID NO: 59: Polynucleotide sequence encoding VGFR(1 / 2)-mFc-FSD1(E126A).

[0642]

[0643] SEQ ID NO: 60: VGFR(1 / 2)-mFc-FSD1(Q124A, E126A). The serum albumin preproprotein signal peptide is shown in bold, mouse IgG2A is underlined, and FSD1(Q124A, E126A) is double underlined.

[0644]

[0645]

[0646] SEQ ID NO: 61: Polynucleotide sequence encoding VGFR(1 / 2)-mFc-FSD1(Q124A, E126A).

[0647]

[0648] SEQ ID NO: 62: CBG-mFc-FSD1. The serum albumin preproprotein signal peptide is shown in bold, murine IgG2A is underlined, and FSD1 is double underlined.

[0649]

[0650] SEQ ID NO: 63: Polynucleotide sequence encoding CBG-mFc-FSD1.

[0651]

[0652] SEQ.ID NO:64:FSD1(KTC)(Q124A)

[0653] ETCENVDCGPGKKCRMNKKNKPRCVCAPDCSNITWKGPVCGLDGKTYRNECALLKARCKEAPELEVQYQGRCKKTC

[0654] SEQ.ID NO:65:FSD1(KTC)(E126A)

[0655] ETCENVDCGPGKKCRMNKKNKPRCVCAPDCSNITWKGPVCGLDGKTYRNECALLKARCKEQPALEVQYQGRCKKTC

[0656] SEQ.ID NO:66:FSD1(KTC)(Q124A,E126A)

[0657] ETCENVDCGPGKKCRMNKKNKPRCVCAPDCSNITWKGPVCGLDGKTYRNECALLKARCKEAPALEVQYQGRCKKTC

[0658] SEQ.IDNO:67: Serum albumin preproprotein signal peptide

[0659] MKWVTFISLLFLFSSAYS

[0660] item

[0661] 1. A fusion protein or binding protein comprising:

[0662] (i) one or more polypeptides (P1) comprising or consisting of at least one FSD1 domain, said FSD1 domain comprising or consisting of a polypeptide having at least 70%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 99% sequence identity to SEQ ID NO: 1,

[0663] and

[0664] (ii) therapeutic or diagnostic agents.

[0665] 2. The fusion protein or binding protein according to item 1, comprising:

[0666] (i) one or more polypeptides (P1) consisting of at least one FSD1 domain comprising or consisting of a polypeptide having at least 70%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 99% sequence identity to SEQ ID NO: 1,

[0667] and

[0668] (ii) therapeutic or diagnostic agents.

[0669] 3. The fusion protein or binding protein according to item 1, comprising:

[0670] (i) one or more polypeptides (P1) comprising or consisting of at least one FSD1 domain, said FSD1 domain comprising or consisting of a polypeptide having at least 70%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 99% sequence identity to SEQ ID NO: 1,

[0671] and

[0672] (ii) a therapeutic or diagnostic agent comprising or consisting of an immunoglobulin domain;

[0673] And wherein the one or more polypeptides (P1)(i) are linked to the immunoglobulin Fc domain at the C-terminus.

[0674] 4. The fusion protein or binding protein according to item 3, wherein the immunoglobulin is selected from the group consisting of IgG, IgA, IgM, IgE and IgD.

[0675] 5. A fusion protein or binding protein according to any one of the preceding items, comprising:

[0676] (i) one or more polypeptides (P1) consisting of at least one FSD1 domain comprising or consisting of a polypeptide having at least 70%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 99% sequence identity to SEQ ID NO: 1,

[0677] and

[0678] (ii) a therapeutic or diagnostic agent comprising or consisting of an immunoglobulin domain;

[0679] And wherein said one or more polypeptides (P1)(i) are linked at the C-terminus to said immunoglobulin Fc domain, preferably wherein said immunoglobulin is IgG.

[0680] 6. The fusion protein or binding protein according to any one of items 3 to 5, wherein the immunoglobulin is IgG, and wherein the IgG Fc domain comprises or consists of an IgG Fc-homodimer or an IgG Fc-heterodimer.

[0681] 7. The fusion protein or binding protein according to item 6, wherein the fusion protein or binding protein comprises or consists of an IgG Fc-homodimer, wherein each monomer of the IgG Fc-homodimer is linked at the C-terminus to at least one polypeptide (P1), said polypeptide (P1) comprising or consisting of at least one FSD1 domain, said FSD1 domain comprising or consisting of a polypeptide having at least 70%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 99% sequence identity to SEQ ID NO: 1, and wherein each monomer of the IgG Fc-homodimer is linked at the N-terminus to the same or different therapeutic or diagnostic moiety.

[0682] 8. The fusion or binding protein according to any of the preceding items, wherein the fusion or binding protein comprises or consists of an IgG Fc-homodimer, wherein each monomer of the IgG Fc-homodimer is linked at the C-terminus to at least one polypeptide (P1)(i), said polypeptide (P1)(i) consisting of at least one FSD1 domain comprising or consisting of a polypeptide having a sequence identity to SEQ ID NO: 1 of at least 70%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 99%,

[0683] And wherein each monomer of said IgG Fc-homodimer is linked at the N-terminus to the same or different therapeutic or diagnostic moiety.

[0684] 9. The fusion protein or binding protein according to item 6, wherein the fusion protein or binding protein comprises or consists of an IgG Fc-heterodimer, wherein each monomer of the IgG Fc-heterodimer is linked at the C-terminus to at least one polypeptide (P1), said polypeptide (P1) comprising or consisting of at least one FSD1 domain, said FSD1 domain comprising or consisting of a polypeptide having at least 70%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 99% sequence identity to SEQ ID NO: 1, and wherein each monomer of the IgG Fc-heterodimer is linked at the N-terminus to the same or different therapeutic or diagnostic moiety.

[0685] 10. The fusion or binding protein according to any of the preceding items, wherein the fusion or binding protein comprises or consists of an IgG Fc-heterodimer, wherein each monomer of the IgG Fc-heterodimer is linked at the C-terminus to at least one polypeptide (P1)(i), said polypeptide (P1)(i) consisting of at least one FSD1 domain comprising or consisting of a polypeptide having a sequence identity to SEQ ID NO: 1 of at least 70%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 99%,

[0686] And wherein each monomer of said IgG Fc-heterodimer is linked at the N-terminus to the same or different therapeutic or diagnostic moiety.

[0687] 11. The fusion protein or binding protein according to item 1, comprising:

[0688] (i) one or more polypeptides (P1)(i) comprising or consisting of at least one FSD1 domain, said FSD1 domain comprising or consisting of a polypeptide having at least 70%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 99% sequence identity to SEQ ID NO: 1,

[0689] and

[0690] (ii) a therapeutic or diagnostic agent; wherein the therapeutic or diagnostic agent comprises or consists of an IgG Fc-heterodimer, wherein at least one monomer of the IgG Fc-heterodimer is linked at the N-terminus to at least one of the one or more polypeptides (P1) (i), and wherein at least one monomer of the IgG Fc-heterodimer is linked at the N-terminus to the same or different therapeutic or diagnostic moiety.

[0691] 12. The fusion protein or binding protein according to any of the preceding items, wherein at least one monomer of the IgG Fc-heterodimer is linked at the N-terminus to at least one of the one or more polypeptides (P1) (i), such as at least two polypeptides (P1), such as at least three polypeptides (P1), such as at least four polypeptides, such as at least five polypeptides (P1), said polypeptides (P1) comprising or consisting of at least one FSD1 domain, said FSD1 domain comprising or consisting of a polypeptide having a sequence identity of at least 70%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 99% to SEQ ID NO: 1.

[0692] 13. A fusion protein or binding protein according to any of the preceding items, wherein the therapeutic or diagnostic agent of any of items 1 to 3, or at least one identical or different therapeutic or diagnostic part of any of the preceding items, is one or more cytokine receptors.

[0693] 14. The fusion protein or binding protein according to any of the preceding items, wherein the therapeutic or diagnostic agent of any of the preceding items, or at least one identical or different therapeutic or diagnostic moiety of any of the preceding items, comprises or consists of the ligand binding domain of TNFR2.

[0694] 15. A fusion protein or binding protein according to any of the preceding items, wherein the therapeutic agent or diagnostic agent of any of the preceding items, or at least one identical or different therapeutic part or diagnostic part of any of the preceding items, comprises the ligand binding domain of one or more growth factor receptors, or consists of the ligand binding domain of one or more growth factor receptors.

[0695] 16. A fusion protein or binding protein according to any of the preceding items, wherein the therapeutic agent or diagnostic agent of any of the preceding items, or at least one identical or different therapeutic part or diagnostic part of any of the preceding items, comprises or consists of the ligand binding domain of VGFR1.

[0696] 17. A fusion protein or binding protein according to any of the preceding items, wherein the therapeutic agent or diagnostic agent of any of the preceding items, or at least one identical or different therapeutic part or diagnostic part of any of the preceding items, comprises or consists of the ligand binding domain of VGFR2.

[0697] 18. A fusion protein or binding protein according to any of the preceding items, wherein the one or more polypeptides (P1) comprise or consist of at least one FSD1 domain, said FSD1 domain comprising or consisting of a polypeptide having at least 70% sequence identity, such as at least 80% identity, such as at least 90% identity, such as at least 95% identity, such as 99% identity to SEQ.ID.NO:26.

[0698] 19. The fusion protein or binding protein according to item 1, comprising:

[0699] (i) one or more polypeptides (P1) comprising or consisting of at least one FSD1 domain, said FSD1 domain comprising or consisting of a polypeptide having at least 70%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 99% sequence identity to SEQ ID NO: 1,

[0700] and

[0701] (ii) therapeutic or diagnostic agents;

[0702] wherein the one or more polypeptides (P1) (i) are covalently linked to the therapeutic or diagnostic agent (ii) or linked via non-covalent interactions.

[0703] 20. The fusion protein or binding protein according to item 19, wherein the one or more polypeptides (P1) (i) and the therapeutic agent or diagnostic agent (ii) are covalently linked via click chemistry.

[0704] 21. The fusion protein or binding protein according to item 19, wherein the one or more polypeptides (P1) (i) and the therapeutic or diagnostic agent (ii) are linked via a non-covalent streptavidin-biotin interaction.

[0705] 22. A fusion protein or binding protein according to item 19, wherein said one or more polypeptides (P1) (i) and said therapeutic or diagnostic agent (ii) are linked via a Nanobody having affinity for the therapeutic or diagnostic agent, such as an anti-Fc Nanobody.

[0706] 23. A fusion protein or binding protein according to any one of the preceding items, comprising:

[0707] (i) one or more polypeptides (P1) consisting of at least one FSD1 domain comprising or consisting of a polypeptide having at least 70%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 99% sequence identity to SEQ ID NO: 1,

[0708] and

[0709] (ii) therapeutic or diagnostic agents;

[0710] wherein the one or more polypeptides (P1) (i) are covalently linked to the therapeutic or diagnostic agent (ii), for example via click chemistry or via non-covalent interactions, such as streptavidin-biotin interactions, or via Nanobodies having affinity for the therapeutic or diagnostic agent (ii).

[0711] 24. The fusion protein or binding protein according to any one of items 19 to 23, wherein the therapeutic or diagnostic agent (ii) comprises or consists of a full-length antibody.

[0712] 25. The fusion protein or binding protein according to item 24, wherein the full-length antibody is a bispecific therapeutic antibody.

[0713] 26. A fusion protein or binding protein according to any of the preceding items, wherein the one or more polypeptides (P1) (i) comprise or consist of a sequence having at least 70% similarity, such as at least 80% similarity, such as at least 90% similarity, such as at least 95% similarity, such as at least 99% similarity to a sequence selected from SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 64, SEQ ID NO: 65 and SEQ ID NO: 66.

[0714] 27. A fusion protein or binding protein according to any of the preceding items, wherein the one or more polypeptides (P1) (i) comprise or consist of a sequence having at least 70% similarity, such as at least 80% similarity, such as at least 90% similarity, such as at least 95% similarity, such as at least 99% similarity to a sequence selected from SEQ ID NO: 23 and SEQ ID NO: 24.

[0715] 28. A fusion protein or binding protein according to any of the preceding items, wherein the one or more polypeptides (P1) are encoded by a sequence comprising or consisting of a sequence having at least 70% similarity, such as at least 80% similarity, such as at least 90% similarity, such as at least 95% similarity, such as at least 99% similarity to SEQ ID NO: 25.

[0716] 29. The fusion protein or binding protein according to any one of the preceding items, wherein the one or more polypeptides (P1)(i) do not neutralize the activity of myostatin (SEQ ID NO: 45), GDF11 (SEQ ID NO: 46) and / or activin A (SEQ ID NO: 44).

[0717] 30. The fusion protein or binding protein according to any one of the preceding items, wherein the fusion protein or binding protein is expressed at a concentration of up to at least 5 nM, such as at least 7.5 nM, such as at least 8 nM, such as at least 8.2 nM, such as at least 8.4 nM, such as at least 8.6 nM, such as at least 8.8 nM, such as at least 9 nM, such as at least 10 nM, such as at least 12 nM, such as at least 15 nM, such as at least 20 nM, such as at least 30 nM, such as at least 40 nM, such as at least 50 nM, such as at least 60 nM, such as at least 61 nM, such as at least 62 nM, such as at least 63 nM, such as at least 64 nM, such as at least 65 nM, For example, the present invention does not neutralize the activity of myostatin (SEQ ID NO: 45), GDF11 (SEQ ID NO: 46) and / or activin A (SEQ ID NO: 44) within a concentration range of at least 70 nM, for example, at least 80 nM, for example, at least 100 nM, for example, at least 150 nM, for example, at least 200 nM, for example, at least 500 nM, for example, at least 600 nM, for example, at least 700 nM, for example, at least 750 nM, for example, at least 800 nM, for example, at least 820 nM, for example, at least 821 nM, for example, at least 822 nM, for example, at least 830 nM, for example, at least 1 μM, for example, at least 2 μM, for example, at least 3 μM, for example, at least 4 μM, for example, at least 5 μM, for example, at least 6 μM, for example, at least 7 μM, for example, at least 8 μM, for example, at least 9 μM, for example, at least 10 μM, wherein the neutralization or lack thereof is determined by a reporter gene bioassay of phosphorylated Smad2 / 3 signaling.

[0718] 31. The fusion protein or binding protein according to any of the preceding items, wherein the fusion protein or binding protein does not neutralize the activity of myostatin, GDF11 and / or activin A within a concentration range of up to at least 5 nM, such as at least 7.5 nM, such as at least 8 nM, such as at least 8.2 nM, such as at least 8.4 nM, such as at least 8.6 nM, such as at least 8.8 nM, such as at least 9 nM, such as at least 10 nM, such as at least 12 nM, such as at least 15 nM, such as at least 20 nM, such as at least 30 nM, such as at least 40 nM, such as at least 50 nM, such as at least 80 nM, such as at least 100 nM, such as at least 150 nM, such as at least 200 nM.

[0719] 32. The fusion protein or binding protein according to any one of the preceding items, wherein the one or more polypeptides (P1) (i) is expressed in an amount up to at least 5 nM, such as at least 7.5 nM, such as at least 8 nM, such as at least 8.2 nM, such as at least 8.4 nM, such as at least 8.6 nM, such as at least 8.8 nM, such as at least 9 nM, such as at least 10 nM, such as at least 12 nM, such as at least 15 nM, such as at least 20 nM, such as at least 30 nM, such as at least 40 nM, such as at least 50 nM, such as at least 60 nM, such as at least 61 nM, such as at least 62 nM, such as at least 63 nM, such as at least 64 nM, such as at least 65 nM, The invention also provides a method for treating a somatostatin-induced somatostatin-1 deficiency by inhibiting the expression of a novel phospho-Smad2 / 3 inhibitor. The method further provides a method for treating a somatostatin-induced somatostatin-1 deficiency by inhibiting the expression of a novel phospho-Smad2 / 3 inhibitor. The method further provides a method for treating a somatostatin-induced somatostatin-1 deficiency by inhibiting the expression of a novel phospho-Smad2 / 3 inhibitor.

[0720] 33. The fusion protein or binding protein according to any of the preceding items, wherein the one or more polypeptides (P1)(i) do not neutralize the activity of myostatin, GDF11 and / or activin A within a concentration range of up to at least 5 nM, such as at least 7.5 nM, such as at least 8 nM, such as at least 8.2 nM, such as at least 8.4 nM, such as at least 8.6 nM, such as at least 8.8 nM, such as at least 9 nM, such as at least 10 nM, such as at least 12 nM, such as at least 15 nM, such as at least 20 nM, such as at least 30 nM, such as at least 40 nM, such as at least 50 nM, such as at least 80 nM, such as at least 100 nM, such as at least 150 nM, such as at least 200 nM.

[0721] 34. The fusion protein or binding protein according to any of the preceding items, wherein the one or more polypeptides (P1 )(i) bind to heparan sulfate.

[0722] 35. A fusion protein or binding protein according to any of the preceding items, wherein the one or more polypeptides (P1) (i) comprising at least one FSD1 domain are encoded by a sequence comprising or consisting of a sequence that is at least 70% identical, such as at least 80% identical, such as at least 90% identical, such as at least 95% identical, such as at least 99% identical to SEQ.ID.NO:33.

[0723] 36. The fusion protein or binding protein according to any of the preceding items, wherein said one or more polypeptides (P1) (i) comprising at least one FSD1 domain comprises or consists of a sequence having 100% identity to SEQ.ID.NO: 1.

[0724] 37. The fusion protein or binding protein according to any of the preceding items, further comprising a linker between the one or more polypeptides (P1 ) comprising or consisting of at least one FSD1 domain (i) and the therapeutic or diagnostic agent (ii).

[0725] 38. The fusion protein or binding protein according to item 37, wherein the linker is a chemical linker.

[0726] 39. A fusion protein or binding protein according to any of the preceding items, wherein the therapeutic or diagnostic agent (ii) according to any of the preceding items, or the same or different therapeutic or diagnostic part according to any of the preceding items, is selected from the group consisting of: peptides, proteins such as antibodies or fragments thereof, nanobodies, glycoproteins, streptavidin or interleukins, nucleic acids and small molecules.

[0727] 40. The fusion protein or binding protein according to any one of the preceding items, wherein the fusion protein or binding protein further comprises a detectable moiety.

[0728] 41. The fusion protein or binding protein according to item 40, wherein the detectable moiety is selected from the group consisting of: a fluorescent protein, a gold nanoparticle, a radioisotope, biotin or a derivative thereof, and an enzyme.

[0729] 42. A fusion protein or binding protein according to any of the preceding items, wherein the therapeutic agent or diagnostic agent (ii) of any of the preceding items, or the same or different therapeutic part or diagnostic part of any of the preceding items, binds to a target selected from the group consisting of: cluster of differentiation (CD) proteins, cytokines such as interleukins, growth factors such as colony stimulating factors, immune checkpoint proteins, angiogenic factors, hemostatic factors, chemokines, neurotrophic factors, inflammatory proteins, tumor antigens, bacterial proteins and viral proteins.

[0730] 43. A fusion protein or binding protein according to item 42, wherein the therapeutic agent or diagnostic agent (ii) of any of the preceding items, or the same or different therapeutic part or diagnostic part of any of the preceding items, comprises or consists of a ligand binding domain of a protein selected from the group consisting of TNFR2, VGFR1, VGFR2 and CBG.

[0731] 44. A fusion protein or binding protein according to any of the preceding items, wherein the therapeutic agent or diagnostic agent (ii) of any of the preceding items, or the same or different therapeutic part or diagnostic part of any of the preceding items, comprises or consists of: a ligand binding domain of a protein having at least 70% similarity, such as at least 80% similarity, such as at least 90% similarity, such as at least 95% similarity, such as at least 99% similarity to a sequence selected from SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21 and SEQ ID NO: 22.

[0732] 45. A fusion protein or binding protein according to any of the preceding items, wherein the therapeutic or diagnostic agent (ii) of any of the preceding items or the same or different therapeutic or diagnostic part of any of the preceding items comprises or consists of an anti-vimentin Nanobody.

[0733] 46. ​​The fusion protein according to item 45, wherein the anti-vimentin Nanobody has at least 70% similarity to SEQ ID NO: 34, such as at least 80% similarity, such as at least 90% similarity, such as at least 95% similarity, such as at least 99% similarity.

[0734] 47. The fusion protein according to any one of items 45 to 46, wherein the anti-vimentin Nanobody is encoded by a sequence having at least 70% similarity, such as at least 80% similarity, such as at least 90% similarity, such as at least 95% similarity, such as at least 99% similarity to SEQ ID NO: 35.

[0735] 48. The fusion protein or binding protein according to any of the preceding items, wherein the therapeutic agent or diagnostic agent (ii) of any of the preceding items, or the same or different therapeutic part or diagnostic part of any of the preceding items, comprises or consists of:

[0736] (a) a ligand binding domain of a protein selected from the group consisting of TNFR2 (SEQ ID NO: 21), VGFR1 (SEQ ID NO: 19), VGFR2 (SEQ ID NO: 20), and CBG (SEQ ID NO: 22); and / or

[0737] (b) VGFR (1 / 2) (SEQ ID NO: 40)

[0738] (b) an anti-vimentin Nanobody that has at least 70% similarity, such as at least 80% similarity, such as at least 90% similarity, such as at least 95% similarity, such as at least 99% similarity to SEQ ID NO: 34; and / or

[0739] (c) Full-length antibodies, such as full-length anti-TNFα antibodies.

[0740] 49. The fusion protein or binding protein according to any one of the preceding items, wherein the fusion protein or binding protein is ubiquitinated.

[0741] 50. A fusion protein or binding protein according to any of the preceding items, wherein the therapeutic agent or diagnostic agent (ii) of any of the preceding items, or the same or different therapeutic part or diagnostic part of any of the preceding items binds to a target, and wherein the fusion protein or binding protein according to any of the preceding items is capable of binding to heparan sulfate and the target simultaneously.

[0742] 51. A fusion protein or binding protein according to any of the preceding items, wherein the local half-life of the fusion protein or binding protein at the site of administration is longer than that of the therapeutic agent or diagnostic agent (ii) of any of the preceding items alone or the same or different therapeutic part or diagnostic part of any of the preceding items.

[0743] 52. A fusion protein or binding protein according to any of the preceding items, wherein the fusion protein or binding protein increases the binding of the therapeutic or diagnostic agent (ii) of any of the preceding items or the same or different therapeutic or diagnostic portion of any of the preceding items to the extracellular matrix compartment compared to the therapeutic or diagnostic agent (ii) of any of the preceding items alone or the same or different therapeutic or diagnostic portion of any of the preceding items alone.

[0744] 53. A fusion protein or binding protein according to any of the preceding items, wherein the fusion protein or binding protein increases the binding of the therapeutic or diagnostic agent (ii) of any of the preceding items or the same or different therapeutic or diagnostic portion of any of the preceding items to a predetermined organ or tumor compared to the therapeutic or diagnostic agent (ii) of any of the preceding items alone or the same or different therapeutic or diagnostic portion of any of the preceding items alone.

[0745] 54. The fusion protein or binding protein of any of the preceding items, wherein the EC50 of therapeutic or diagnostic agent (ii) or the same or different therapeutic or diagnostic moiety for a predetermined organ or tumor is reduced by at least 2-fold, such as at least 5-fold, such as at least 10-fold, such as at least 20-fold, such as at least 50-fold, such as at least 80-fold, such as at least 100-fold, such as at least 250-fold, such as at least 500-fold, such as at least 1000-fold, such as at least 5000-fold, such as at least 1000-fold, such as at least 5000-fold, such as at least 10000-fold, such as at least 100000-fold, compared to the therapeutic or diagnostic agent (ii) or the same or different therapeutic or diagnostic moiety alone.

[0746] 55. The fusion protein or binding protein according to item 54, wherein the predetermined organ is selected from the group consisting of: organs of the musculoskeletal system, organs of the digestive system, organs of the respiratory system, organs of the urinary system, organs of the reproductive system and organs of the endocrine system, organs of the circulatory system, organs of the nervous system, organs of the hematopoietic system and organs of the integumentary system.

[0747] 56. The fusion protein or binding protein according to item 55, wherein the organ is selected from the group consisting of kidney, eye, liver, heart, lung, bladder, pancreas, gall bladder, intestine, prostate, brain, skin, muscle, bone, hematopoietic tissue and subcutaneous tissue, such as joint or synovial tissue.

[0748] 57. A fusion protein or binding protein according to any of the preceding items, wherein the fusion protein or binding protein increases the intracellular uptake of the therapeutic or diagnostic agent (ii) of any of the preceding items or the same or different therapeutic or diagnostic portion of any of the preceding items compared to the therapeutic or diagnostic agent (ii) of any of the preceding items alone or the same or different therapeutic or diagnostic portion of any of the preceding items alone.

[0749] 58. A fusion protein or binding protein according to any of the preceding items, wherein the therapeutic agent or diagnostic agent (ii) of any of the preceding items or the same or different therapeutic moiety or diagnostic moiety of any of the preceding items is linked to at least one polypeptide (P1) (i), such as at least two polypeptides (P1) (i), such as at least three polypeptides (P1) (i), such as at least four polypeptides, such as at least five polypeptides (P1) (i), said polypeptide (P1) (i) comprising or consisting of at least one FSD1 domain, said FSD1 domain comprising or consisting of a polypeptide having a sequence identity to SEQ ID NO: 1 of at least 70%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 99%.

[0750] 59. A fusion protein or binding protein according to any of the preceding items, wherein each monomer of the Fc-homodimer or Fc-heterodimer is linked to at least one polypeptide (P1)(i), such as at least two polypeptides (P1)(i), such as at least three polypeptides (P1)(i), such as at least four polypeptides, such as at least five polypeptides (P1)(i), said polypeptides (P1)(i) comprising or consisting of at least one FSD1 domain, said FSD1 domain comprising or consisting of a polypeptide having a sequence identity to SEQ ID NO: 1 of at least 70%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 99%.

[0751] 60. A fusion protein or binding protein according to any of the preceding items, wherein at least one of the at least one polypeptide (P1) (i) comprises or consists of a multimer of FSD1 domains, such as at least two FSD1 domains, such as at least three FSD1 domains, such as at least four FSD1 domains, such as at least five FSD1 domains, wherein the FSD1 domain comprises or consists of a polypeptide having at least 70%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 99% sequence identity to SEQ ID NO: 1.

[0752] 61. The fusion protein or binding protein according to item 60, wherein at least two monomers of the multimer of FSD1 domains are connected via a linker.

[0753] 62. A fusion protein or binding protein according to any of the preceding items, wherein the fusion protein or binding protein comprises a polypeptide having at least 70% sequence identity, such as at least 80% sequence identity, such as at least 90% sequence identity, such as at least 95% identity, such as at least 99% identity to a sequence selected from SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7 and SEQ ID NO: 9, or a composition thereof.

[0754] 63. A fusion protein or binding protein according to any of the preceding items, wherein the fusion protein or binding protein comprises or consists of a polypeptide having at least 70% sequence identity, such as at least 80% sequence identity, such as at least 90% sequence identity, such as at least 95% identity, such as at least 99% identity to SEQ ID NO: 11, and a polypeptide having at least 70% sequence identity, such as at least 80% sequence identity, such as at least 90% sequence identity, such as at least 95% identity, such as at least 99% identity to a sequence selected from SEQ ID NO: 13, SEQ ID NO: 15 and SEQ ID NO: 17.

[0755] 64. The fusion protein or binding protein according to any of the preceding items, wherein the fusion protein or binding protein comprises or consists of a polypeptide having at least 70% sequence identity, such as at least 80% sequence identity, such as at least 90% sequence identity, such as at least 95% identity, such as at least 99% identity to the following sequence:

[0756] (a) a sequence selected from the group consisting of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 56, SEQ ID NO: 58, SEQ ID NO: 60, and SEQ ID NO: 62; and / or

[0757] (b) SEQ ID NO: 11 and polypeptides having at least 70% sequence identity, such as at least 80% sequence identity, such as at least 90% sequence identity, such as at least 95% identity, such as at least 99% identity to a sequence selected from SEQ ID NO: 13, SEQ ID NO: 15 and SEQ ID NO: 17.

[0758] 65. A fusion protein or binding protein according to any of the preceding items, wherein the fusion protein or binding protein does not comprise any other follistatin domains FSD2, FSD3 and / or N-terminal follistatin domains, preferably wherein the fusion protein or binding protein does not comprise any of the human FSD2 of SEQ ID NO: 41, the human FSD3 of SEQ ID NO: 42 and the human follistatin N-terminal domain of SEQ ID NO: 43.

[0759] 66. A fusion protein or binding protein according to any of the preceding items, wherein the one or more polypeptides (P1) (i) do not contain any other follistatin domains FSD2, FSD3 and / or N-terminal follistatin domains, preferably wherein the fusion protein or binding protein does not contain any of the human FSD2 of SEQ ID NO: 41, the human FSD3 of SEQ ID NO: 42 and the human follistatin N-terminal domain of SEQ ID NO: 43.

[0760] 67. A fusion protein or binding protein according to any of the preceding items, wherein at least one FSD1 domain of said (i) one or more polypeptides (P1) comprises or consists of a variant FSD1 domain encoded by a sequence selected from the group consisting of: SEQ ID NO: 47, SEQ ID NO: 50 and SEQ ID NO: 53, or a polypeptide having at least 70%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 96%, such as at least 97%, such as 98%, such as at least 99% sequence identity thereto.

[0761] 68. The fusion protein or binding protein according to any one of the preceding items, wherein the fusion protein or binding protein further comprises a PROTAC linker, preferably wherein the PROTAC linker is selected from the group consisting of a flexible aliphatic linker, a flexible PEGylated linker, a flexible heterotether, a rigid linker, a triazole-based linker, a bioorthogonal clickable linker and a photoswitchable linker.

[0762] 69. One or more polynucleotides which, upon expression, encode a fusion protein or binding protein according to any one of the preceding items.

[0763] 70. The one or more polynucleotides according to item 69, wherein the one or more polynucleotides have at least 70% sequence identity, such as at least 80% sequence identity, such as at least 90% sequence identity, such as at least 95% identity, such as at least 99% identity with a polynucleotide selected from the group consisting of SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8 and SEQ ID NO: 10.

[0764] 71. The one or more polynucleotides according to item 69, wherein the one or more polynucleotides have at least 70% sequence identity, such as at least 80% sequence identity, such as at least 90% sequence identity, such as at least 95% identity, such as at least 99% identity, to a combination of SEQ ID NO: 12 and at least one polynucleotide selected from SEQ ID NO: 14, SEQ ID NO: 16 and SEQ ID NO: 18.

[0765] 72. One or more constructs or vectors comprising one or more polynucleotides according to any one of items 69 to 71 or encoding a fusion protein or binding protein according to any one of items 1 to 68.

[0766] 73. One or more constructs or vectors encoding a fusion protein according to any of the preceding items.

[0767] 74. A host cell comprising one or more polynucleotides of any one of items 69 to 71 or one or more constructs or vectors of any one of items 72 to 73.

[0768] 75. The host cell according to item 74, wherein the host cell is a mammalian cell.

[0769] 76. The host cell according to item 75, wherein the host cell is a human cell.

[0770] 77. The host cell according to item 76, wherein the host cell is a human embryonic kidney 293 (HEK293) cell.

[0771] 78. The host cell according to item 75, wherein the host cell is a Chinese Hamster Ovary (CHO) cell.

[0772] 79. A composition comprising the fusion protein or binding protein according to any one of items 1 to 68, one or more polynucleotides according to any one of items 69 to 71, one or more constructs or vectors according to any one of items 72 to 73, a host cell according to any one of items 74 to 78, or a mixture thereof.

[0773] 80. The composition according to item 79, wherein the composition is a pharmaceutical composition.

[0774] 81. The fusion protein or binding protein according to any one of items 1 to 68, the one or more polynucleotides according to any one of items 69 to 71, the one or more constructs or vectors according to any one of items 72 to 73, the host cell according to any one of items 74 to 78, or the composition according to any one of items 79 to 80, for use in medicine.

[0775] 82. The fusion protein or binding protein according to any one of items 1 to 68, the one or more polynucleotides according to any one of items 69 to 71, the one or more constructs or vectors according to any one of items 72 to 73, the host cell according to any one of items 74 to 78, or the composition according to any one of items 79 to 80, for use in treating an ophthalmic disease, a cancerous disease or an inflammatory disease, such as a rheumatic disease.

[0776] 83. The use according to item 82, wherein the ophthalmic disease is neovascular age-related macular degeneration (AMD).

[0777] 84. The use according to item 82, wherein the inflammatory disease is caused by organ transplantation.

[0778] 85. The use according to any one of items 81 to 82 and 84, for a subject donating or receiving an organ, wherein the donated or transplanted organ is selected from the group consisting of kidney, heart, lung, bone marrow and liver.

[0779] 86. A method of treating a disease or condition comprising administering to a subject an effective amount of a fusion protein or binding protein according to any one of items 1 to 68, one or more polynucleotides according to any one of items 69 to 71, one or more constructs or vectors according to any one of items 72 to 73, a host cell according to any one of items 74 to 78, or a composition according to any one of items 79 to 80.

[0780] 87. A method for treating an ophthalmic disease, an inflammatory disease, or a cancerous disease, comprising administering to a subject an effective amount of a fusion protein or binding protein according to any one of items 1 to 68, one or more polynucleotides according to any one of items 69 to 71, one or more constructs or vectors according to any one of items 72 to 73, a host cell according to any one of items 74 to 78, or a composition according to any one of items 79 to 80.

[0781] 88. Use of a fusion protein or binding protein according to any one of items 1 to 68, one or more polynucleotides according to any one of items 69 to 71, one or more constructs or vectors according to any one of items 72 to 73, a host cell according to any one of items 74 to 78, or a composition according to any one of items 79 to 80 in the manufacture of a medicament for treating a disease or disorder, such as an ophthalmic disease, a cancerous disease, or an inflammatory disease.

[0782] 89. The use according to any one of items 81 to 85, the method according to any one of items 86 to 87 or the use according to item 88, wherein the fusion protein or binding protein according to any one of items 1 to 68, the one or more polynucleotides according to any one of items 69 to 71, the one or more constructs or vectors according to any one of items 72 to 73, the host cell according to any one of items 74 to 78 or the composition according to any one of items 79 to 80 is administered systemically.

[0783] 90. The use according to any one of items 81 to 85, the method according to any one of items 86 to 87 or the use according to item 88, wherein the fusion protein or binding protein according to any one of items 1 to 68, the one or more polynucleotides according to any one of items 69 to 71, the one or more constructs or vectors according to any one of items 72 to 73, the host cell according to any one of items 74 to 78 or the composition according to any one of items 79 to 80 is applied topically.

[0784] 91. The use according to any one of items 81 to 85, the method according to any one of items 86 to 87, or the use according to item 88, wherein the fusion protein or binding protein according to any one of items 1 to 68, the one or more polynucleotides according to any one of items 69 to 71, the one or more constructs or vectors according to any one of items 72 to 73, the host cell according to any one of items 74 to 78, or the composition according to any one of items 79 to 80 is administered intraadipose, intraarterially, intraarticularly, intracranially, intradermally, intralesionally, intramuscularly, intranasally, intraocularly, intraosseously, intrapericardially, intraperitoneally, intrapleurally, intraprostatically, intrarectally, intrathecally, intratracheally, intratumorally, intraumbilically, intravaginally, intravenously, intravesicularly, intravitreally, or via liposomes.

[0785] 92. A method of increasing the local half-life of a therapeutic or diagnostic agent at the site of administration, comprising obtaining a fusion protein or binding protein according to any one of items 1 to 68, one or more polynucleotides according to any one of items 69 to 71, one or more constructs or vectors according to any one of items 72 to 73, a host cell according to any one of items 74 to 78, or a composition according to any one of items 79 to 80, wherein the therapeutic or diagnostic agent (ii) of any one of items 1 to 68, or the same or different therapeutic or diagnostic moiety of items 1 to 68, comprises or consists of said therapeutic or diagnostic agent.

[0786] 93. A method of increasing the in vivo half-life of a therapeutic or diagnostic agent at a site of administration in a subject, comprising administering to the subject a fusion protein or binding protein according to any one of items 1 to 68, one or more polynucleotides according to any one of items 69 to 71, one or more constructs or vectors according to any one of items 72 to 73, a host cell according to any one of items 74 to 78, or a composition according to any one of items 79 to 80, wherein the therapeutic or diagnostic agent (ii) of any one of items 1 to 68 or the same or different therapeutic or diagnostic moiety of items 1 to 68 comprises or consists of the therapeutic or diagnostic agent.

[0787] 94. A method of increasing the local in vivo half-life of a therapeutic or diagnostic agent comprising the steps of:

[0788] a) providing therapeutic or diagnostic agents;

[0789] b) obtaining a fusion protein or binding protein according to any one of items 1 to 68, wherein the therapeutic agent or diagnostic agent of step a) is the therapeutic agent or diagnostic agent (ii) according to any one of items 1 to 68,

[0790] The local in vivo half-life of the therapeutic or diagnostic agent is thereby increased.

[0791] 95. The method of any one of items 93 to 94, wherein the fusion protein or binding protein or the composition is administered to a subject suffering from an ophthalmic disease, and wherein the fusion protein or binding protein or the composition is administered intravitreally, subretinally or suprachoroidally.

[0792] 96. The method according to item 95, wherein the ophthalmic disease is wet AMD.

[0793] 97. The method according to item 93, wherein the fusion protein or binding protein or the composition is administered to a subject donating or receiving an organ, preferably a kidney, heart, lung, bone marrow or liver, even more preferably a kidney, or is administered to the organ by ex vivo perfusion, and wherein the therapeutic or diagnostic agent comprises or consists of a compound that reduces an inflammatory disease.

[0794] 98. The method of any one of items 92 to 97, wherein the local half-life of the therapeutic or diagnostic agent is increased at the site of administration by at least 6 hours, such as at least 12 hours, such as at least 24 hours, such as at least 48 hours, such as at least 72 hours, such as at least 96 hours, such as at least 120 hours, such as at least one week, such as at least 2 weeks, such as at least 4 weeks, such as at least 8 weeks, such as at least 3 months, such as at least 6 months, such as at least 12 months.

[0795] 99. A method of increasing binding of a therapeutic or diagnostic agent to an extracellular matrix, comprising administering to a subject a fusion protein or binding protein according to any one of items 1 to 68, one or more polynucleotides according to any one of items 69 to 71, one or more constructs or vectors according to any one of items 72 to 73, a host cell according to any one of items 74 to 78, or a composition according to any one of items 79 to 80, wherein the therapeutic or diagnostic agent (ii) of any one of items 1 to 68 or the same or different therapeutic or diagnostic part of any one of items 1 to 68 comprises or consists of said therapeutic or diagnostic agent.

[0796] 100. A method of increasing the binding of a therapeutic or diagnostic agent to a predetermined organ, comprising administering to a subject a fusion protein or binding protein according to any one of items 1 to 68, one or more polynucleotides according to any one of items 69 to 71, one or more constructs or vectors according to any one of items 72 to 73, a host cell according to any one of items 74 to 78, or a composition according to any one of items 79 to 80, wherein the therapeutic or diagnostic agent (ii) of any one of items 1 to 68 or the same or different therapeutic or diagnostic part of any one of items 1 to 68 comprises or consists of said therapeutic or diagnostic agent.

[0797] 101. A method of increasing the intracellular uptake of a therapeutic or diagnostic agent, comprising administering to a subject a fusion protein or binding protein according to any one of items 1 to 68, one or more polynucleotides according to any one of items 69 to 71, one or more constructs or vectors according to any one of items 72 to 73, a host cell according to any one of items 74 to 78, or a composition according to any one of items 79 to 80, wherein the therapeutic or diagnostic agent (ii) of any one of items 1 to 68 or the same or different therapeutic or diagnostic part of any one of items 1 to 68 comprises or consists of said therapeutic or diagnostic agent.

[0798] 102. A method for degrading an intracellular protein, the method comprising the step of coupling the fusion protein or binding protein according to any one of the preceding items to a ligand of an E3 ubiquitin ligase, wherein the therapeutic agent or diagnostic agent (ii) of the fusion protein or binding protein binds to the intracellular protein.

[0799] 103. The method according to item 102, wherein the ligand of the E3 ubiquitin ligase is (S,R,S)-AHPC-PEG8-NHS.

[0800] 104. The method according to any one of items 102 to 103, wherein the E3 ubiquitin ligase is Von-Hippel Lindau (VHL) tumor suppressor protein.

[0801] 105. The method according to any one of items 102 to 104, wherein the therapeutic or diagnostic agent (ii) is an anti-vimentin Nanobody.

[0802] 106. The method according to any one of items 102 to 105, wherein the fusion protein or binding protein is a Type 1 protein as described herein, preferably wherein the fusion protein or binding protein is anti-vimentin Nanobody-FSD1.

[0803] 107. The method according to any one of items 102 to 106, wherein the method is in vitro, in vivo or ex vivo.

[0804] 108. The use according to any one of items 81 to 85, 88 to 91 or the method according to any one of items 92 to 107, wherein the subject is a human or a non-human animal.

[0805] 109. The use according to any one of items 81 to 85, 88 to 91 or the method according to any one of items 92 to 107, wherein the fusion protein or binding protein according to any one of items 1 to 68, the one or more polynucleotides according to any one of items 69 to 71, the one or more constructs or vectors according to any one of items 72 to 73, the host cell according to any one of items 74 to 78, or the composition according to any one of items 79 to 80 is administered systemically.

[0806] 110. The use according to any one of items 81 to 85, 88 to 91 or the method according to any one of items 92 to 107, wherein the fusion protein or binding protein according to any one of items 1 to 68, the one or more polynucleotides according to any one of items 69 to 71, the one or more constructs or vectors according to any one of items 72 to 73, the host cell according to any one of items 74 to 78 or the composition according to any one of items 79 to 80 is applied topically.

[0807] 111. The use according to any one of items 81 to 85, 88 to 91 or the method according to any one of items 92 to 107, wherein the fusion protein or binding protein according to any one of items 1 to 68, the one or more polynucleotides according to any one of items 69 to 71, the one or more constructs or vectors according to any one of items 72 to 73, the host cell according to any one of items 74 to 78 or the composition according to any one of items 79 to 80 is administered intraadipose, intraarterially, intraarticularly, intracranially, intradermally, intralesionally, intramuscularly, intranasally, intraocularly, intraosseously, intrapericardially, intraperitoneally, intrapleurally, intraprostatically, intrarectally, intrathecally, intratracheally, intratumorally, intraumbilically, intravaginally, intravenously, intravesicularly, intravitreally or via liposomes.

[0808] 112. The method according to any one of items 101 to 111, wherein the therapeutic or diagnostic agent is transported to the cytosol, such as the cytoskeleton or the nucleus.

[0809] 113. One or more constructs or vectors encoding one or more polypeptides (P1 ) of a fusion protein or binding protein according to any one of the preceding items (i).

[0810] 114. A composition comprising one or more polypeptides selected from the group consisting of FSD1 (Q124A) of SEQ ID NO: 47, FSD1 (E126A) of SEQ ID NO: 50, and FSD1 (Q124 E126A) of SEQ ID NO: 53, or variants thereof having at least 70%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99% sequence identity thereto.

Claims

1. A fusion protein or binding protein comprising: (i) one or more polypeptides (P1) consisting of at least one FSD1 domain comprising or consisting of a polypeptide having at least 70%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 99% sequence identity to SEQ ID NO: 1, and (ii) therapeutic or diagnostic agents.

2. The fusion protein or binding protein according to claim 1, comprising: (i) one or more polypeptides (P1) consisting of at least one FSD1 domain comprising or consisting of a polypeptide having at least 70%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 99% sequence identity to SEQ ID NO: 1, and (ii) a therapeutic or diagnostic agent comprising or consisting of an immunoglobulin domain; And wherein the one or more polypeptides (P1)(i) are linked to the immunoglobulin Fc domain at the C-terminus.

3. The fusion protein or binding protein according to claim 2, wherein the immunoglobulin is selected from the group consisting of IgG, IgA, IgM, IgE and IgD.

4. The fusion protein or binding protein according to any one of the preceding claims, comprising: (i) one or more polypeptides (P1) consisting of at least one FSD1 domain comprising or consisting of a polypeptide having at least 70%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 99% sequence identity to SEQ ID NO: 1, and (ii) a therapeutic or diagnostic agent comprising or consisting of an immunoglobulin domain; And wherein said one or more polypeptides (P1)(i) are linked at the C-terminus to said immunoglobulin Fc domain, preferably wherein said immunoglobulin is IgG.

5. The fusion protein or binding protein according to any one of claims 2 to 4, wherein the immunoglobulin is IgG, and wherein the IgG Fc domain comprises or consists of an IgG Fc homodimer or an IgG Fc heterodimer.

6. The fusion protein or binding protein according to any one of the preceding claims, wherein the fusion protein or binding protein comprises or consists of an IgG Fc-homodimer, wherein each monomer of the IgG Fc-homodimer is linked at the C-terminus to at least one polypeptide (P1)(i), said polypeptide (P1)(i) consisting of at least one FSD1 domain comprising or consisting of a polypeptide having a sequence identity of at least 70%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 99% to SEQ ID NO: 1, And wherein each monomer of said IgG Fc-homodimer is linked at the N-terminus to the same or different therapeutic or diagnostic moiety.

7. The fusion protein or binding protein according to any one of the preceding claims, wherein the fusion protein or binding protein comprises or consists of an IgG Fc-heterodimer, wherein each monomer of the IgG Fc-heterodimer is linked at the C-terminus to at least one polypeptide (P1)(i), said polypeptide (P1)(i) consisting of at least one FSD1 domain comprising or consisting of a polypeptide having a sequence identity of at least 70%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 99% to SEQ ID NO: 1, And wherein each monomer of said IgG Fc-heterodimer is linked at the N-terminus to the same or different therapeutic or diagnostic moiety.

8. The fusion protein or binding protein according to any one of the preceding claims, comprising: (i) one or more polypeptides (P1)(i) consisting of at least one FSD1 domain comprising or consisting of a polypeptide having at least 70%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 99% sequence identity to SEQ ID NO: 1, and (ii) a therapeutic or diagnostic agent; wherein the therapeutic or diagnostic agent comprises or consists of an IgG Fc-heterodimer, wherein at least one monomer of the IgG Fc-heterodimer is linked at the N-terminus to at least one of the one or more polypeptides (P1)(i), and wherein at least one monomer of the IgG Fc-heterodimer is linked at the N-terminus to the same or different therapeutic or diagnostic moiety.

9. The fusion protein or binding protein according to any one of the preceding claims, wherein at least one monomer of the IgG Fc-heterodimer is linked at the N-terminus to at least one of the one or more polypeptides (P1) (i), such as at least two polypeptides (P1), such as at least three polypeptides (P1), such as at least four polypeptides, such as at least five polypeptides (P1), said polypeptides (P1) consisting of at least one FSD1 domain comprising or consisting of a polypeptide having a sequence identity to SEQ ID NO: 1 of at least 70%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 99%.

10. The fusion protein or binding protein of any of the preceding claims, wherein the therapeutic or diagnostic agent of any of the preceding claims, or at least one of the same or different therapeutic or diagnostic moieties of any of the preceding claims, is one or more cytokine receptors.

11. The fusion protein or binding protein of any of the preceding claims, wherein the therapeutic or diagnostic agent of any of the preceding claims, or at least one identical or different therapeutic moiety or diagnostic moiety of any of the preceding claims, comprises or consists of the ligand binding domain of TNFR2.

12. The fusion protein or binding protein of any one of claims 1 to 11, wherein the therapeutic or diagnostic agent of any of the preceding claims, or at least one of the same or different therapeutic or diagnostic moieties, comprises or consists of the ligand binding domains of one or more growth factor receptors.

13. The fusion protein or binding protein of any one of claims 1 to 12, wherein the therapeutic or diagnostic agent of any one of the preceding claims, or at least one identical or different therapeutic or diagnostic moiety, comprises or consists of the ligand binding domain of VGFR1.

14. The fusion protein or binding protein of any one of claims 1 to 13, wherein the therapeutic or diagnostic agent of any one of the preceding claims, or at least one identical or different therapeutic or diagnostic moiety, comprises or consists of the ligand binding domain of VGFR2.

15. A fusion protein or binding protein according to any one of the preceding claims, wherein the one or more polypeptides (P1) consist of at least one FSD1 domain, which comprises or consists of a polypeptide having at least 70% sequence identity, such as at least 80% identity, such as at least 90% identity, such as at least 95% identity, such as 99% identity to SEQ.ID.NO:

26.

16. A fusion protein or binding protein according to any one of the preceding claims, comprising: (i) one or more polypeptides (P1) consisting of at least one FSD1 domain comprising or consisting of a polypeptide having at least 70%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 99% sequence identity to SEQ ID NO: 1, and (ii) therapeutic or diagnostic agents; wherein the one or more polypeptides (P1) (i) are covalently linked to the therapeutic or diagnostic agent (ii) or linked via non-covalent interactions.

17. The fusion protein or binding protein of claim 16, wherein the one or more polypeptides (P1) (i) and the therapeutic or diagnostic agent (ii) are covalently linked using click chemistry.

18. The fusion protein or binding protein of claim 16, wherein the one or more polypeptides (P1) (i) and the therapeutic or diagnostic agent (ii) are linked via a non-covalent streptavidin-biotin interaction.

19. The fusion protein or binding protein according to claim 16, wherein the one or more polypeptides (P1) (i) and the therapeutic or diagnostic agent (ii) are linked via a nanobody having affinity for the therapeutic or diagnostic agent, such as an anti-Fc nanobody.

20. The fusion protein or binding protein according to any one of the preceding claims, comprising: (i) one or more polypeptides (P1) consisting of at least one FSD1 domain consisting of a polypeptide having at least 70%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 99% sequence identity to SEQ ID NO: 1, and (ii) therapeutic or diagnostic agents; wherein the one or more polypeptides (P1) (i) are covalently linked to the therapeutic or diagnostic agent (ii), for example via click chemistry or via non-covalent interactions, such as streptavidin-biotin interactions, or via Nanobodies having affinity for the therapeutic or diagnostic agent (ii).

21. The fusion protein or binding protein of any one of claims 16 to 20, wherein the therapeutic or diagnostic agent (ii) comprises or consists of a full-length antibody.

22. The fusion protein or binding protein of claim 21, wherein the full-length antibody is a bispecific therapeutic antibody.

23. A fusion protein or binding protein according to any one of the preceding claims, wherein the one or more polypeptides (P1) (i) consists of a sequence having at least 70% similarity, such as at least 80% similarity, such as at least 90% similarity, such as at least 95% similarity, such as at least 99% similarity to a sequence selected from SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 64, SEQ ID NO: 65 and SEQ ID NO:

66.

24. A fusion protein or binding protein according to any one of the preceding claims, wherein the one or more polypeptides (P1) (i) consists of a sequence having at least 70% similarity, such as at least 80% similarity, such as at least 90% similarity, such as at least 95% similarity, such as at least 99% similarity to a sequence selected from SEQ ID NO: 23 and SEQ ID NO:

24.

25. A fusion protein or binding protein according to any one of the preceding claims, wherein the one or more polypeptides (P1) are encoded by a sequence comprising or consisting of a sequence having at least 70% similarity, such as at least 80% similarity, such as at least 90% similarity, such as at least 95% similarity, such as at least 99% similarity to SEQ ID NO:

25.

26. The fusion protein or binding protein of any one of the preceding claims, wherein the one or more polypeptides (P1)(i) do not neutralize the activity of myostatin (SEQ ID NO: 45), GDF11 (SEQ ID NO: 46) and / or activin A (SEQ ID NO: 44).

27. according to the fusion protein or the associated protein in any one of the preceding claims, wherein said fusion protein or the associated protein is up to at least 5nM, for example, at least 7.5nM, for example, at least 8nM, for example, at least 8.2nM, for example, at least 8.4nM, for example, at least 8.6nM, for example, at least 8.8nM, for example, at least 9nM, for example, at least 10nM, for example, at least 12nM, for example, at least 15nM, for example, at least 20nM, for example, at least 30nM, for example, at least 40nM, for example, at least 50nM, for example, at least 60nM, for example, at least 61nM, for example, at least 62nM, for example For example, the present invention does not neutralize the activity of myostatin (SEQ ID NO: 45), GDF11 (SEQ ID NO: 46) and / or activin A (SEQ ID NO: 44) within a concentration range of at least 63 nM, such as at least 64 nM, such as at least 64 nM, such as at least 65 nM, such as at least 70 nM, such as at least 80 nM, such as at least 100 nM, such as at least 150 nM, such as at least 200 nM, such as at least 500 nM, such as at least 600 nM, such as at least 700 nM, such as at least 750 nM, such as at least 800 nM, such as at least 820 nM, such as at least 821 nM, such as at least 822 nM, such as at least 830 nM, such as at least 1 μM, such as at least 2 μM, wherein the neutralization or lack thereof is determined by a reporter gene bioassay of phosphorylated Smad2 / 3 signaling.

28. The fusion protein or binding protein of any one of the preceding claims, wherein the fusion protein or binding protein does not neutralize the activity of myostatin, GDF11 and / or activin A over a concentration range of up to at least 5 nM, such as at least 7.5 nM, such as at least 8 nM, such as at least 8.2 nM, such as at least 8.4 nM, such as at least 8.6 nM, such as at least 8.8 nM, such as at least 9 nM, such as at least 10 nM, such as at least 12 nM, such as at least 15 nM, such as at least 20 nM, such as at least 30 nM, such as at least 40 nM, such as at least 50 nM, such as at least 80 nM, such as at least 100 nM, such as at least 150 nM, such as at least 200 nM.

29. according to any one of the preceding claims fusion protein or binding protein, wherein the one or more polypeptides (P1) (i) is up to at least 5nM, such as at least 7.5nM, such as at least 8nM, such as at least 8.2nM, such as at least 8.4nM, such as at least 8.6nM, such as at least 8.8nM, such as at least 9nM, such as at least 10nM, such as at least 12nM, such as at least 15nM, such as at least 20nM, such as at least 30nM, such as at least 40nM, such as at least 50nM, such as at least 60nM, such as at least 61nM, such as at least 62nM , for example, at least 63 nM, for example, at least 64 nM, for example, at least 64 nM, for example, at least 65 nM, for example, at least 70 nM, for example, at least 80 nM, for example, at least 100 nM, for example, at least 150 nM, for example, at least 200 nM, for example, at least 500 nM, for example, at least 600 nM, for example, at least 700 nM, for example, at least 750 nM, for example, at least 800 nM, for example, at least 820 nM, for example, at least 821 nM, for example, at least 822 nM, for example, at least at least 830 nM, for example, at least 1 μM, for example, at least 2 μM, wherein the neutralization or lack thereof is determined by a reporter gene bioassay of phosphorylated Smad2 / 3 signaling.

30. The fusion protein or binding protein of any one of the preceding claims, wherein the one or more polypeptides (P1)(i) do not neutralize the activity of myostatin, GDF11 and / or activin A within a concentration range of up to at least 5 nM, such as at least 7.5 nM, such as at least 8 nM, such as at least 8.2 nM, such as at least 8.4 nM, such as at least 8.6 nM, such as at least 8.8 nM, such as at least 9 nM, such as at least 10 nM, such as at least 12 nM, such as at least 15 nM, such as at least 20 nM, such as at least 30 nM, such as at least 40 nM, such as at least 50 nM, such as at least 80 nM, such as at least 100 nM, such as at least 150 nM, such as at least 200 nM.

31. The fusion protein or binding protein of any one of the preceding claims, wherein the one or more polypeptides (P1 )(i) binds to heparan sulfate.

32. A fusion protein or binding protein according to any one of the preceding claims, wherein the one or more polypeptides (P1) (i) comprising at least one FSD1 domain are encoded by a sequence comprising or consisting of a sequence that is at least 70% identical, such as at least 80% identical, such as at least 90% identical, such as at least 95% identical, such as at least 99% identical to SEQ.ID.NO:

33.

33. The fusion protein or binding protein according to any one of the preceding claims, wherein the one or more polypeptides (P1) (i) comprising at least one FSD1 domain comprise or consist of a sequence having 100% identity to SEQ.ID.NO:

1.

34. The fusion protein or binding protein of any preceding claim, further comprising a linker between the one or more polypeptides (P1 ) consisting of at least one FSD1 domain (i) and the therapeutic or diagnostic agent (ii).

35. The fusion protein or binding protein of claim 34, wherein the linker is a chemical linker.

36. A fusion protein or binding protein according to any of the preceding claims, wherein the therapeutic or diagnostic agent (ii) or the same or different therapeutic or diagnostic moiety according to any of the preceding claims is selected from the group consisting of: peptides, proteins such as antibodies or fragments thereof, nanobodies, glycoproteins, streptavidin or interleukins, nucleic acids and small molecules.

37. The fusion protein or binding protein of any one of the preceding claims, wherein the fusion protein or binding protein further comprises a detectable portion.

38. The fusion protein or binding protein of claim 37, wherein the detectable moiety is selected from the group consisting of a fluorescent protein, a gold nanoparticle, a radioisotope, biotin or a derivative thereof, and an enzyme.

39. A fusion protein or binding protein according to any of the preceding claims, wherein the therapeutic agent or diagnostic agent (ii) or the same or different therapeutic or diagnostic portion of any of the preceding claims binds to a target selected from the group consisting of: cluster of differentiation (CD) proteins, cytokines such as interleukins, growth factors such as colony stimulating factors, immune checkpoint proteins, angiogenic factors, hemostatic factors, chemokines, neurotrophic factors, inflammatory proteins, tumor antigens, bacterial proteins, and viral proteins.

40. The fusion protein or binding protein of claim 39, wherein the therapeutic or diagnostic agent (ii) or the same or different therapeutic or diagnostic moiety of any of the preceding claims comprises or consists of a ligand binding domain of a protein selected from TNFR2, VGFR1, VGFR2, and CBG.

41. A fusion protein or binding protein according to any of the preceding claims, wherein the therapeutic agent or diagnostic agent (ii) or the same or different therapeutic part or diagnostic part of any of the preceding claims comprises or consists of a ligand binding domain of a protein having at least 70% similarity, such as at least 80% similarity, such as at least 90% similarity, such as at least 95% similarity, such as at least 99% similarity to a sequence selected from SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21 and SEQ ID NO:

22.

42. The fusion or binding protein of claim 36, wherein the therapeutic or diagnostic agent (ii) of any preceding claim or the same or different therapeutic or diagnostic moiety of any one of claims 5 to 32 comprises or consists of an anti-vimentin Nanobody.

43. The fusion protein of claim 42, wherein the anti-vimentin Nanobody has at least 70% similarity to SEQ ID NO: 34, such as at least 80% similarity, such as at least 90% similarity, such as at least 95% similarity, such as at least 99% similarity.

44. The fusion protein according to any one of claims 42 to 43, wherein the anti-vimentin Nanobody is encoded by a sequence having at least 70% similarity, such as at least 80% similarity, such as at least 90% similarity, such as at least 95% similarity, such as at least 99% similarity to SEQ ID NO:

35.

45. The fusion protein or binding protein of any of the preceding claims, wherein the therapeutic or diagnostic agent (ii) of any of the preceding claims or the same or different therapeutic or diagnostic moiety of any of the preceding claims comprises or consists of: (a) a ligand binding domain of a protein selected from the group consisting of TNFR2 (SEQ ID NO: 21), VGFR1 (SEQ ID NO: 19), VGFR2 (SEQ ID NO: 20), and CBG (SEQ ID NO: 22); and / or (b) VGFR(1 / 2) (SEQ ID NO:40); and / or (c) an anti-vimentin Nanobody that has at least 70% similarity, such as at least 80% similarity, such as at least 90% similarity, such as at least 95% similarity, such as at least 99% similarity to SEQ ID NO: 34; and / or (d) Full-length antibodies, such as full-length anti-TNFα antibodies.

46. ​​The fusion protein or binding protein of any one of the preceding claims, wherein the fusion protein or binding protein is ubiquitinated.

47. The fusion protein or binding protein of any of the preceding claims, wherein the therapeutic or diagnostic agent (ii) or the same or different therapeutic or diagnostic moiety of any of the preceding claims binds to a target, and wherein the fusion protein or binding protein of any of the preceding claims is capable of binding heparan sulfate and the target simultaneously.

48. The fusion protein or binding protein of any of the preceding claims, wherein the fusion protein or binding protein has a longer local half-life at the site of administration than any of the preceding therapeutic or diagnostic agents (ii) or the same or different therapeutic or diagnostic moiety alone.

49. The fusion protein or binding protein of any of the preceding claims, wherein the fusion protein or binding protein increases binding of the therapeutic or diagnostic agent (ii) or the same or different therapeutic or diagnostic moiety of any of the preceding claims to an extracellular matrix compartment compared to the therapeutic or diagnostic agent (ii) or the same or different therapeutic or diagnostic moiety of any of the preceding claims alone.

50. The fusion protein or binding protein of any of the preceding claims, wherein the fusion protein or binding protein increases binding of the therapeutic or diagnostic agent (ii) or the same or different therapeutic or diagnostic moiety of any of the preceding claims to a predetermined organ or tumor compared to the therapeutic or diagnostic agent (ii) or the same or different therapeutic or diagnostic moiety of any of the preceding claims alone.

51. The fusion protein or binding protein of any of the preceding claims, wherein the EC50 of therapeutic or diagnostic agent (ii) or the same or different therapeutic or diagnostic moiety against a predetermined organ or tumor is reduced by at least 2-fold, e.g., at least 5-fold, e.g., at least 10-fold, e.g., at least 20-fold, e.g., at least 50-fold, e.g., at least 80-fold, e.g., at least 100-fold, e.g., at least 250-fold, e.g., at least 500-fold, e.g., at least 1000-fold, e.g., at least 5000-fold, e.g., at least 10000-fold, e.g., at least 100000-fold, compared to the therapeutic or diagnostic agent (ii) alone or the same or different therapeutic or diagnostic moiety.

52. The fusion protein or binding protein of claim 51, wherein the predetermined organ is selected from the group consisting of organs of the musculoskeletal system, digestive system, respiratory system, urinary system, reproductive organ, and endocrine system, circulatory system, nervous system, hematopoietic organ, and integumentary system.

53. The fusion protein or binding protein of claim 52, wherein the organ is selected from the group consisting of kidney, eye, liver, heart, lung, bladder, pancreas, gallbladder, intestine, prostate, brain, skin, muscle, bone, hematopoietic tissue, and subcutaneous tissue, such as joint or synovial tissue.

54. The fusion protein or binding protein of any of the preceding claims, wherein the fusion protein or binding protein increases the intracellular uptake of the therapeutic or diagnostic agent (ii) or the same or different therapeutic or diagnostic moiety as described in any of the preceding claims alone, compared to the therapeutic or diagnostic agent (ii) or the same or different therapeutic or diagnostic moiety described in any of the preceding claims alone.

55. A fusion protein or binding protein according to any of the preceding claims, wherein the therapeutic agent or diagnostic agent (ii) of any of the preceding claims or the same or different therapeutic moiety or diagnostic moiety of any of the preceding claims is linked to at least one polypeptide (P1) (i), such as at least two polypeptides (P1) (i), such as at least three polypeptides (P1) (i), such as at least four polypeptides, such as at least five polypeptides (P1) (i), said polypeptides (P1) (i) consisting of at least one FSD1 domain, said FSD1 domain comprising or consisting of a polypeptide having a sequence identity to SEQ ID NO: 1 of at least 70%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 99%.

56. The fusion protein or binding protein of any one of claims 5 to 55, wherein each monomer of the Fc-homodimer or Fc-heterodimer is linked to at least one polypeptide (P1) (i), such as at least two polypeptides (P1) (i), such as at least three polypeptides (P1) (i), such as at least four polypeptides, such as at least five polypeptides (P1) (i), said polypeptides (P1) (i) consisting of at least one FSD1 domain comprising or consisting of a polypeptide having a sequence identity to SEQ ID NO: 1 of at least 70%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 99%.

57. A fusion protein or binding protein according to any of the preceding claims, wherein at least one of the at least one polypeptide (P1) (i) comprises or consists of a multimer of FSD1 domains, such as at least two FSD1 domains, such as at least three FSD1 domains, such as at least four FSD1 domains, such as at least five FSD1 domains, and the FSD1 domains consist of a polypeptide having at least 70%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 99% sequence identity to SEQ ID NO:

1.

58. The fusion protein or binding protein of claim 57, wherein at least two monomers of the multimer of FSD1 domains are connected by a linker.

59. A fusion protein or binding protein according to any of the preceding claims, wherein the fusion protein or binding protein comprises or consists of a polypeptide having at least 70% sequence identity, such as at least 80% sequence identity, such as at least 90% sequence identity, such as at least 95% identity, such as at least 99% identity to a sequence selected from SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7 and SEQ ID NO:

9.

60. The fusion protein or binding protein of any one of the preceding claims, wherein the fusion protein or binding protein comprises or consists of: A polypeptide having at least 70% sequence identity, such as at least 80% sequence identity, such as at least 90% sequence identity, such as at least 95% identity, such as at least 99% identity to SEQ ID NO: 11, and a polypeptide having at least 70% sequence identity, such as at least 80% sequence identity, such as at least 90% sequence identity, such as at least 95% identity, such as at least 99% identity to a sequence selected from SEQ ID NO: 13, SEQ ID NO: 15 and SEQ ID NO:

17.

61. The fusion protein or binding protein of any one of the preceding claims, wherein the fusion protein or binding protein comprises or consists of a polypeptide having at least 70% sequence identity, such as at least 80% sequence identity, such as at least 90% sequence identity, such as at least 95% identity, such as at least 99% identity to the following sequence: (a) a sequence selected from the group consisting of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 56, SEQ ID NO: 58, SEQ ID NO: 60, and SEQ ID NO: 62; and / or (b) SEQ ID NO: 11 and polypeptides having at least 70% sequence identity, such as at least 80% sequence identity, such as at least 90% sequence identity, such as at least 95% identity, such as at least 99% identity to a sequence selected from SEQ ID NO: 13, SEQ ID NO: 15 and SEQ ID NO:

17.

62. A fusion protein or binding protein according to any of the preceding claims, wherein the fusion protein or binding protein does not comprise any other follistatin domains FSD2, FSD3 and / or N-terminal follistatin domains, preferably wherein the fusion protein or binding protein does not comprise any of the human FSD2 of SEQ ID NO: 41, the human FSD3 of SEQ ID NO: 42 and the human follistatin N-terminal domain of SEQ ID NO:

43.

63. A fusion protein or binding protein according to any of the preceding claims, wherein the one or more polypeptides (P1) (i) do not contain any other follistatin domains FSD2, FSD3 and / or N-terminal follistatin domains, preferably wherein the fusion protein or binding protein does not contain any of the human FSD2 of SEQ ID NO: 41, the human FSD3 of SEQ ID NO: 42 and the human follistatin N-terminal domain of SEQ ID NO:

43.

64. The fusion protein or binding protein of any one of the preceding claims, wherein at least one FSD1 domain of said (i) one or more polypeptides (P1) comprises or consists of a variant FSD1 domain encoded by a sequence selected from the group consisting of: SEQ ID NO:47, SEQ ID NO:50 and SEQ ID NO:53, or a polypeptide having at least 70%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 96%, such as at least 97%, such as 98%, such as at least 99% sequence identity thereto.

65. The fusion protein or binding protein of any one of the preceding claims, wherein the fusion protein or binding protein further comprises a PROTAC linker, preferably wherein the PROTAC linker is selected from the group consisting of a flexible aliphatic linker, a flexible PEGylated linker, a flexible heterochain, a rigid linker, a triazole-based linker, a bioorthogonal clickable linker, and a photoswitchable linker.

66. One or more polynucleotides which, when expressed, encode the fusion protein or binding protein of any one of the preceding claims.

67. One or more polynucleotides according to claim 66, wherein the one or more polynucleotides have at least 70% sequence identity, such as at least 80% sequence identity, such as at least 90% sequence identity, such as at least 95% identity, such as at least 99% identity with a polynucleotide selected from SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8 and SEQ ID NO:

10.

68. One or more polynucleotides according to claim 66, wherein the one or more polynucleotides have at least 70% sequence identity, such as at least 80% sequence identity, such as at least 90% sequence identity, such as at least 95% identity, such as at least 99% identity with the combination of SEQ ID NO: 12 and at least one polynucleotide selected from SEQ ID NO: 14, SEQ ID NO: 16 and SEQ ID NO:

18.

69. One or more constructs or vectors comprising one or more polynucleotides according to any one of claims 66 to 68 or encoding a fusion protein or binding protein according to any one of claims 1 to 65.

70. One or more constructs or vectors encoding a fusion protein according to any one of the preceding claims.

71. A host cell comprising one or more polynucleotides of any one of claims 66 to 68 or one or more constructs or vectors of any one of claims 69 to 70.

72. The host cell of claim 71, wherein the host cell is a mammalian cell.

73. The host cell of claim 72, wherein the host cell is a human cell.

74. The host cell of claim 73, wherein the host cell is a human embryonic kidney 293 cell (HEK293) cell.

75. The host cell of claim 72, wherein the host cell is a Chinese Hamster Ovary (CHO) cell.

76. A composition comprising a fusion protein or binding protein according to any one of claims 1 to 65, one or more polynucleotides according to any one of claims 66 to 68, one or more constructs or vectors according to any one of claims 69 to 70, a host cell according to any one of claims 71 to 75, or a mixture thereof.

77. The composition of claim 76, wherein the composition is a pharmaceutical composition.

78. The fusion protein or conjugated protein of any one of claims 1 to 65, the one or more polynucleotides of any one of claims 66 to 68, the one or more constructs or vectors of any one of claims 69 to 70, the host cell of any one of claims 71 to 75, or the compositions of any one of claims 76 to 77, for use in medicine.

79. The fusion protein or binding protein of any one of claims 1 to 65, the one or more polynucleotides of any one of claims 66 to 68, the one or more constructs or vectors of any one of claims 69 to 70, the host cell of any one of claims 71 to 75, or the composition of any one of claims 76 to 77 for the treatment of ophthalmic diseases, cancerous diseases or inflammatory diseases, such as rheumatism.

80. The use according to claim 79, wherein the ophthalmic disease is neovascular age-related macular degeneration (AMD).

81. The use according to claim 79, wherein the inflammatory disease is caused by organ transplantation.

82. The use according to any one of claims 78 to 79, 81, for a subject donating or receiving an organ, wherein the donated or transplanted organ is selected from the group consisting of kidney, heart, lung, bone marrow and liver.

83. A method of treating a disease or condition comprising administering to a subject an effective amount of a fusion protein or binding protein according to any one of claims 1 to 65, one or more polynucleotides according to any one of claims 66 to 68, one or more constructs or vectors according to any one of claims 69 to 70, a host cell according to any one of claims 71 to 75, or a composition according to any one of claims 76 to 77.

84. A method of treating an ophthalmic disease, an inflammatory disease, or a cancerous disease, comprising administering to a subject an effective amount of a fusion protein or binding protein according to any one of claims 1 to 65, one or more polynucleotides according to any one of claims 66 to 68, one or more constructs or vectors according to any one of claims 69 to 70, a host cell according to any one of claims 71 to 75, or a composition according to any one of claims 76 to 77.

85. according to the fusion protein or the associated protein described in any one of claims 1 to 65, according to the one or more polynucleotides described in any one of claims 66 to 68, according to the one or more constructs or the carrier described in any one of claims 69 to 70, according to the host cell described in any one of claims 71 to 75, or according to the purposes of the compositions described in any one of claims 76 to 77 in the manufacture of a medicament for the treatment of a disease or condition such as an ophthalmic disease, a cancerous disease or an inflammatory disease.

86. The use of any one of claims 78 to 82, the method of any one of claims 83 to 84, or the use of claim 85, wherein the fusion protein or binding protein of any one of claims 1 to 65, the one or more polynucleotides of any one of claims 66 to 68, the one or more constructs or vectors of any one of claims 69 to 70, the host cell of any one of claims 71 to 75, or the composition of any one of claims 76 to 77 is administered systemically.

87. The use according to any one of claims 78 to 82, the method according to any one of claims 83 to 84, or the use according to claim 85, wherein the fusion protein or binding protein according to any one of claims 1 to 65, the one or more polynucleotides according to any one of claims 66 to 68, the one or more constructs or vectors according to any one of claims 69 to 70, the host cell according to any one of claims 71 to 75, or the composition according to any one of claims 76 to 77 is administered topically.

88. The use of any one of claims 78 to 82, the method of any one of claims 83 to 84, or the use of claim 85, wherein the fusion protein or binding protein of any one of claims 1 to 65, the one or more polynucleotides of any one of claims 66 to 68, the one or more constructs or vectors of any one of claims 69 to 70, the host cell of any one of claims 71 to 75, or the composition of any one of claims 76 to 77 is administered intraadipose, intraarterially, intraarticularly, intracranially, intradermally, intralesionally, intramuscularly, intranasally, intraocularly, intraosseously, intrapericardially, intraperitoneally, intrapleurally, intraprostatically, intrarectally, intrathecally, intratracheally, intratumorally, intraumbilically, intravaginally, intravenously, intravesicularly, intravitreally, or liposomes.

89. A method of increasing the local half-life of a therapeutic or diagnostic agent at the site of administration, comprising obtaining the fusion protein or binding protein of any one of claims 1 to 65, the one or more polynucleotides of any one of claims 66 to 68, the one or more constructs or vectors of any one of claims 69 to 70, the host cell of any one of claims 71 to 75, or the composition of any one of claims 76 to 77, wherein the therapeutic or diagnostic agent (ii) of any one of claims 1 to 65 or the same or different therapeutic or diagnostic moiety of claims 6 to 65 comprises or consists of the therapeutic or diagnostic agent.

90. A method of increasing the local half-life of a therapeutic or diagnostic agent in vivo comprising the steps of: a) providing therapeutic or diagnostic agents; b) obtaining a fusion protein or binding protein according to any one of claims 1 to 65, wherein the therapeutic agent or diagnostic agent of step a) is a therapeutic agent or diagnostic agent (ii) according to any one of claims 1 to 65, The local in vivo half-life of the therapeutic or diagnostic agent is thereby increased.

91. according to the method described in any one of the preceding claims, also comprise the step of applying described fusion protein or binding protein to the experimenter with ophthalmologic disease, and wherein described fusion protein or binding protein or described composition is applied intravitreally, subretinally or suprachoroidally.

92. The method of claim 91, wherein the ophthalmic disease is wet AMD.

93. The method of any one of claims 89 to 90, wherein the fusion protein or binding protein or the composition is administered to a subject donating or receiving an organ, preferably a kidney, heart, lung, bone marrow or liver, even more preferably a kidney, or is administered to the organ by ex vivo perfusion, and wherein the therapeutic or diagnostic agent comprises or consists of a compound that reduces inflammation.

94. The method of any one of claims 89 to 93, wherein the local half-life of the therapeutic or diagnostic agent is increased at the site of administration by at least 6 hours, such as at least 12 hours, such as at least 24 hours, such as at least 48 hours, such as at least 72 hours, such as at least 96 hours, such as at least 120 hours, such as at least one week, such as at least 2 weeks, such as at least 4 weeks, such as at least 8 weeks, such as at least 3 months, such as at least 6 months, such as at least 12 months.

95. A method of increasing the binding of a therapeutic or diagnostic agent to an extracellular matrix, comprising administering to a subject a fusion protein or conjugated protein according to any one of claims 1 to 65, one or more polynucleotides according to any one of claims 66 to 68, one or more constructs or carriers according to any one of claims 69 to 70, a host cell according to any one of claims 71 to 75, or a composition according to any one of claims 76 to 77, wherein the therapeutic or diagnostic agent (ii) according to any one of claims 1 to 65, or the same or different therapeutic or diagnostic moiety according to any one of claims 6 to 65 comprises or consists of the therapeutic or diagnostic agent.

96. A method of increasing the binding of a therapeutic or diagnostic agent to a predetermined organ, comprising administering to a subject a fusion protein or binding protein according to any one of claims 1 to 65, one or more polynucleotides according to any one of claims 66 to 68, one or more constructs or vectors according to any one of claims 69 to 70, a host cell according to any one of claims 71 to 75, or a composition according to any one of claims 76 to 77, wherein the therapeutic or diagnostic agent (ii) according to any one of claims 1 to 65 or the same or different therapeutic moiety or diagnostic moiety according to any one of claims 6 to 65 comprises or consists of the therapeutic or diagnostic agent.

97. A method of increasing the intracellular uptake of a therapeutic or diagnostic agent, comprising administering to a subject a fusion protein or binding protein according to any one of claims 1 to 65, one or more polynucleotides according to any one of claims 66 to 68, one or more constructs or vectors according to any one of claims 69 to 70, a host cell according to any one of claims 71 to 75, or a composition according to any one of claims 76 to 77, wherein the therapeutic or diagnostic agent (ii) according to any one of claims 1 to 65 or the same or different therapeutic moiety or diagnostic moiety according to any one of claims 6 to 65 comprises or consists of the therapeutic or diagnostic agent.

98. A method for degrading an intracellular protein, the method comprising the step of coupling the fusion protein or binding protein according to any one of the preceding claims to a ligand of an E3 ubiquitin ligase, wherein the therapeutic or diagnostic agent (ii) of the fusion protein or binding protein binds to the intracellular protein.

99. The method of claim 98, wherein the ligand for the E3 ubiquitin ligase is (S,R,S)-AHPC-PEG8-NHS.

100. The method of any one of claims 98 to 99, wherein the E3 ubiquitin ligase is the Von-Hippel Lindau (VHL) tumor suppressor protein.

101. The method according to any one of claims 98 to 100, wherein the therapeutic or diagnostic agent (ii) is an anti-vimentin Nanobody.

102. The method according to any one of claims 98 to 101, wherein the fusion protein or binding protein is a Type 1 protein as described herein, preferably wherein the fusion protein or binding protein is anti-vimentin Nanobody-FSD1.

103. The method of any one of claims 98 to 102, wherein the method is in vitro, in vivo or ex vivo.

104. The use of any one of claims 78 to 82, 85 to 88 or the method of any one of claims 83 to 103, wherein the subject is a human or non-human animal.

105. The method of any one of claims 78 to 82, 85 to 88, or 83 to 103, wherein the fusion protein or binding protein of any one of claims 1 to 65, the one or more polynucleotides of any one of claims 66 to 68, the one or more constructs or vectors of any one of claims 69 to 70, the host cell of any one of claims 71 to 75, or the composition of any one of claims 76 to 77 is administered systemically.

106. The method of any one of claims 78 to 82, 85 to 88, or 83 to 103, wherein the fusion protein or binding protein of any one of claims 1 to 65, the one or more polynucleotides of any one of claims 66 to 68, the one or more constructs or vectors of any one of claims 69 to 70, the host cell of any one of claims 71 to 75, or the composition of any one of claims 76 to 77 is administered topically.

107. The method of any one of claims 78 to 82, 85 to 88, or 83 to 103, wherein the fusion protein or binding protein of any one of claims 1 to 65, the one or more polynucleotides of any one of claims 66 to 68, the one or more constructs or vectors of any one of claims 69 to 70, the host cell of any one of claims 71 to 75, or the composition of any one of claims 76 to 77 is administered intraadipose, intraarterially, intraarticularly, intracranially, intradermally, intralesionally, intramuscularly, intranasally, intraocularly, intraosseously, intrapericardially, intraperitoneally, intrapleurally, intraprostatically, intrarectally, intrathecally, intratracheally, intratumorally, intraumbilically, intravaginally, intravenously, intravesicularly, intravitreally, or liposomeically.

108. The method of any one of claims 97 to 107, wherein the therapeutic or diagnostic agent is transported to the cytosol, eg, the cytoskeleton or nucleus.

109. One or more constructs or vectors encoding one or more polypeptides (P1 ) (i) of a fusion protein or binding protein according to any one of the preceding claims.

110. A composition comprising one or more polypeptides selected from the group consisting of FSD1 (Q124A) of SEQ ID NO: 47, FSD1 (E126A) of SEQ ID NO: 50, and FSD1 (Q124 E126A) of SEQ ID NO: 53, or variants thereof having at least 70%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99% sequence identity thereto.

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