Extracellular matrix binding compounds for localized loading of therapeutic or diagnostic agents - Patent Application 20070122999

Abstract

A major obstacle in the field of biotherapeutics is the need for platform technologies that enable high bioavailability and long-term therapeutic exposure of biotherapies in localized disease. Traditional approaches result in problematic trade-offs, such as toxicity and off-target side effects. The present disclosure provides compounds comprising follistatin domain 1 (FSD1) of follistatin (FST), which can bind to biological structures, such as the extracellular matrix, via heparan sulfate without the undesired neutralizing effects on the activity of activin A, myostatin, and GDF11, as full-length follistatin does. The present invention relates to fusion or conjugated proteins, compositions thereof, constructs or vectors encoding them, their medical uses, and methods for biotherapeutics.

Description

[Technical Field]

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

[0002] The therapeutic use of proteins (biologic therapies) has been accelerated by half-life extension technologies. These technologies include recombinant fusions with proteins with inherently long serum half-lives (e.g., IgG Fc, transferrin, or albumin), fusions with polypeptides to increase overall size and hydrodynamic radius, or altered glycosylation profiles. Prioritized, half-life extension technologies optimize the systemic delivery of biologic therapies, i.e., delivery throughout the entire organism (Ref. 1). This systemic delivery is desirable when disease is generalized. However, when disease is localized to a specific tissue region, organ, or physiological compartment, systemic therapies may affect tissues not intended to be targeted. Furthermore, systemic therapies may have low bioavailability in target tissues as a result of systemic distribution across physiological compartments within the body (Ref. 2).

[0003] Attempts to deliver biologic therapies to specific tissues include targeting cell surface proteins, such as cluster of differentiation (CD) proteins. For example, antibodies with dual specificity for CD3 (a T-cell co-receptor) and tumor antigens can bridge the interaction between T cells and tumor cells (Reference 2). While selectivity for CD proteins can be used to target therapy to specific cell populations, toxicity and off-target side effects remain (References 3-5). This is likely due, in part, to the lack of complete specificity of CD proteins or similar marker proteins for the desired cell type, and the biologic therapy still distributes throughout the body (References 3-5).

[0004] There is a need for platform technologies that enable high bioavailability and long-term therapeutic exposure of biotherapies in the setting of localized diseases such as solid tumors or ocular diseases. Localized and limited immobilization of biotherapies to tissues or organs is likely to simultaneously minimize systemic toxicity and off-target side effects. This requirement is not met by current platform strategies for systemic half-life extension and marker protein targeting. Summary of the Invention

[0005] The invention is defined in the claims.

[0006] The inventors of the present invention have developed compounds, such as fusion proteins or conjugated proteins, comprising a polypeptide (P1) containing follistatin domain 1 (FSD1) of follistatin (FST), which can bind to biological structures, such as the extracellular matrix, via heparan sulfate without the undesirable neutralizing effects of, for example, full-length follistatin on the activity of activin A, myostatin, and GDF11.

[0007] The disclosed polypeptide (P1) forms a versatile platform that can be used for fusion and conjugation to therapeutic or diagnostic agents, as exemplified by four compounds developed by the inventors. The compounds of the present invention can be used for localized loading and attachment of therapeutic or diagnostic agents to biological structures, improving the local half-life of the therapeutic or diagnostic agents they carry. The inventors have also shown that the developed compounds are internalized intracellularly, e.g., into the cytosol or nucleus.

[0008] A first aspect of the present invention relates to a fusion or conjugated protein comprising (i) one or more polypeptide(s) (P1) consisting of at least one FSD1 domain, said polypeptide(s) comprising or consisting of a polypeptide having at least 70%, such as at least 80%, for example at least 90%, such as at least 95%, for example at least 99% sequence identity to SEQ ID NO: 1, and (ii) a therapeutic or diagnostic agent.

[0009] A second aspect of the present invention relates to one or more polynucleotides which, upon expression, encode the fusion or conjugate protein of the present invention.

[0010] A third aspect of the invention relates to one or more constructs or vectors comprising one or more polynucleotides of the second aspect or encoding the fusion protein of the first aspect.

[0011] A fourth aspect of the 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.

[0012] A fifth aspect of the present invention relates to a composition comprising a fusion protein or conjugate protein of the first aspect, one or more polynucleotides of the second aspect, one or more constructs or vectors of the third aspect, a host cell of the fourth aspect, or a mixture thereof.

[0013] A sixth aspect of the present invention relates to the use of a fusion protein or conjugate protein of the first aspect, one or more polynucleotides of the second aspect, one or more constructs or vectors of the third aspect, a host cell of the fourth aspect, or a composition of the fifth aspect in a pharmaceutical application.

[0014] A seventh aspect of the invention relates to a method of treating a disease or condition, the method comprising administering to a subject an effective amount of a fusion protein or conjugated protein of the first aspect, one or more polynucleotides of the second aspect, one or more constructs or vectors of the third aspect, a host cell of the fourth aspect, or a composition of the fifth aspect.

[0015] An eighth aspect of the present invention relates to the use of a fusion protein or conjugate protein of the first aspect, one or more polynucleotides of the second aspect, one or more constructs or vectors of the third aspect, a host cell of the fourth aspect, or a composition of the fifth aspect in the manufacture of a medicament for the treatment of a disease or condition, such as an ophthalmic disease, a neoplastic disease, or an inflammatory disease.

[0016] A ninth aspect of the present invention relates to a method for increasing the local half-life of a therapeutic or diagnostic agent at a site of administration, comprising obtaining a fusion protein or conjugated protein of the first aspect, one or more polynucleotides of the second aspect, one or more constructs or vectors of the third aspect, a host cell of the fourth aspect, or a composition of the fifth aspect, wherein a therapeutic or diagnostic agent (ii) according to the invention, or a therapeutic or diagnostic moiety which is the same or different from that according to the invention, comprises or consists of said therapeutic or diagnostic agent.

[0017] A tenth aspect of the present invention is a method for 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 conjugate protein of the first aspect, wherein the therapeutic or diagnostic agent of step a) is a therapeutic or diagnostic agent (ii) as described herein; and increasing the local half-life in vivo of the therapeutic or diagnostic agent.

[0018] An eleventh aspect of the present invention relates to a method for enhancing binding of a therapeutic or diagnostic agent to an extracellular matrix, the method comprising administering to a subject a fusion protein or conjugated protein of the first aspect, one or more polynucleotides of the second aspect, one or more constructs or vectors of the third aspect, a host cell of the fourth aspect, or a composition of the fifth aspect, wherein the therapeutic or diagnostic agent (ii) according to the invention, or a therapeutic or diagnostic moiety which is the same as or different from that according to the invention, comprises or consists of the therapeutic or diagnostic agent.

[0019] A twelfth aspect of the present invention relates to a method for enhancing binding of a therapeutic or diagnostic agent to a predetermined organ, comprising administering to a subject a fusion protein or conjugated protein of the first aspect, one or more polynucleotides of the second aspect, one or more constructs or vectors of the third aspect, a host cell of the fourth aspect, or a composition of the fifth aspect, wherein the therapeutic or diagnostic agent (ii) according to the invention, or a therapeutic or diagnostic moiety which is the same as or different from that described in the invention, comprises or consists of the therapeutic or diagnostic agent.

[0020] A thirteenth aspect of the present invention relates to a method for increasing the cellular uptake of a therapeutic or diagnostic agent, the method comprising administering to a subject a fusion protein or conjugated protein of the first aspect, one or more polynucleotides of the second aspect, one or more constructs or vectors of the third aspect, a host cell of the fourth aspect, or a composition of the fifth aspect, wherein the therapeutic or diagnostic agent (ii) according to the invention, or a therapeutic or diagnostic moiety which is the same as or different from that according to the invention, comprises or consists of the therapeutic or diagnostic agent.

[0021] A fourteenth aspect of the present invention relates to a method for degrading an intracellular protein, the method comprising the step of binding a fusion protein or conjugated protein as described herein to a ligand of an E3 ubiquitin ligase, wherein (ii) a therapeutic or diagnostic agent of the fusion protein or conjugated protein binds to the intracellular protein.

[0022] A fifteenth aspect of the present invention relates to one or more constructs or vectors encoding one or more polypeptide(s) (P1)(i) of the fusion or conjugate proteins described herein.

[0023] A 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(Q124E126A) of SEQ ID NO: 53 or variants thereof having at least 70%, such as at least 80%, for example at least 90%, such as at least 95%, for example at least 96%, such as at least 97%, for example 98%, such as at least 99% sequence identity thereto. [Brief explanation of the drawings]

[0024] [Figure 1]The design of compounds is shown. A conceptual diagram of four types of compounds is shown. For all types, FSD1 can be applied with or without multimerization and with or without a linker. A shows FSD1 modularly fused to any other protein (Type 1). B shows homodimeric (i) or heterodimeric (ii) Fc-FSD1 modularly fused to any other protein (Type 2). C shows FSD1 covalently linked to any conventional antibody or any other therapeutic or diagnostic agent (i), or an anti-Fc nanobody-FSD1 fusion (the nanobody has affinity) associated with any conventional antibody or any other therapeutic or diagnostic agent (Type 3). D shows a heterodimeric Fc (Type 4) in which either FST or FSD1 is fused to one entity and any other protein is fused to the other entity. [Figure 2] Heparin affinity chromatography. A shows a chromatogram (left axis) illustrating the elution profile (A280) of compounds and therapeutic agents linked to FSD1, FSD1, or FST291. A linear gradient of 0% to 100% buffer B (2 M NaCl) is shown as a line (right axis). Individual peaks in the elution profile are numbered (#1 or #2). B is a table showing the elution concentration ([NaCl]) of individual peaks in the sample. Nb (nanobody). [Figure 3] Covalent binding of FSD1 to an anti-TNFα antibody (adalimumab). A and B show chromatograms showing the elution profile (A280, left axis) of adalimumab (A) or adalimumab covalently bound to FSD1 (B). A linear gradient of 0% to 100% buffer B (2 M NaCl) is shown as a line (right axis). C shows SDS-PAGE of fractions 7 to 12 from heparin affinity chromatography of adalimumab covalently bound to FSD1. [Figure 4]Bioassay-based validation of activin A neutralization is shown. Neutralization of the growth factor activin A was evaluated using a luciferase-based bioassay of pSmad2 / 3 activation. FSD1 (monomer or dimer) (A), type II and type IV design compounds (B and C), native full-length follistatin 315 (FST315, SEQ ID NO: 28), and FST315 with a modified heparin-binding site fused to a murine Fc fragment (FST315dHBS-mFc, SEQ ID NO: 30, SEQ ID NO: 31) (A) were evaluated over a concentration range up to 8.2 nM. The commercially available counterparts, VGFR(1 / 2)-hFc (aflibercept) or TNFR2-hFc (etanercept), were also evaluated over a concentration range up to 8.2 nM (B and C). Pos control (positive control), Neg control (negative control). TNFR2 (tumor necrosis factor receptor 2), VGFR1 / 2 (ligand binding domain of vascular endothelial growth factor receptors 1 and 2). All results are presented as mean ± SEM. [Figure 5] Bioassay-based validation of myostatin neutralization is shown. Neutralization of the growth factor myostatin was evaluated using a luciferase-based bioassay of pSmad2 / 3 activation. FSD1 (monomer or dimer) (A), type II and type IV design compounds (B and C), native full-length follistatin 315 (FST315, SEQ ID NO: 28), and FST315 with a modified heparin-binding site fused to a murine Fc fragment (FST315dHBS-mFc, SEQ ID NO: 30, SEQ ID NO: 31) (A) were evaluated over a concentration range up to 8.2 nM. The commercially available counterparts, VGFR(1 / 2)-hFc (aflibercept) or TNFR2-hFc (etanercept), were also evaluated over a concentration range up to 8.2 nM (B and C). Pos control (positive control), Neg control (negative control). All results are presented as mean ± SEM. [Figure 6]Bioassay-based validation of GDF11 neutralization is shown. Neutralization of the growth factor GDF11 (growth differentiation factor 11) was evaluated using a luciferase-based bioassay of pSmad2 / 3 activation. FSD1 (monomer or dimer) (A), type 2 design, and type 4 design compounds (B and C), native full-length follistatin 315 (FST315, SEQ ID NO: 28), and FST315 with a modified heparin-binding site fused to a murine Fc fragment (FST315dHBS-mFc, SEQ ID NO: 30, SEQ ID NO: 31) (A) were evaluated over a concentration range up to 8.2 nM. The commercially available counterparts, VGFR(1 / 2)-hFc (aflibercept) or TNFR2-hFc (etanercept), were also evaluated over a concentration range up to 8.2 nM (B and C). Pos control (positive control), Neg control (negative control). All results are presented as mean ± SEM. [Figure 7] Ligand binding affinity measured by surface plasmon resonance (SPR) is shown. Left panel: SPR sensorgrams of VEGF (vascular endothelial growth factor) binding to immobilized type II or type IV design compounds, including VGFR(1 / 2), commercially available VGFR(1 / 2)-hFc (aflibercept), and negative control (FST315dHBS-mFc, SEQ ID NO: 30, SEQ ID NO: 31). Right panel: SPR sensorgrams of TNFα (tumor necrosis factor alpha) binding to immobilized type II or type IV design compounds, including TNFR2, commercially available TNFR2-hFc (etanercept), and negative control (FST315dHBS-mFc, SEQ ID NO: 30, SEQ ID NO: 31). [Figure 8]Figure 1 shows binding to extracellular matrix. The binding profiles of the Type 2 and Type 4 designed compounds to ECM were evaluated using plates precoated with extracellular matrix (ECM) extract (Matrigel). Figure 1A shows ECM binding curves of stand-alone FSD1 (monomer or dimer), native full-length follistatin 288 (FST288), and FST315dHBS-mFc (SEQ ID NO: 30, SEQ ID NO: 31), detected by colorimetric analysis. Figure 1B shows the Type 2 and Type 4 designed compounds, including VGFR(1 / 2), commercially available VGFR(1 / 2)-hFc (aflibercept), and a control in the form of stand-alone FSD1. Figure 1C shows the Type 2 and Type 4 designed compounds, including TNFR2, commercially available TNFR2-hFc (etanercept), and a control in the form of stand-alone FSD1. All results are shown as mean ± SEM. [Figure 9] Figure 1 shows in vivo estimation of compound half-life in mouse skeletal muscle using IVIS. Type II or Type IV design compounds containing VGFR(1 / 2) as the treatment site were administered intramuscularly to the gastrocnemius muscle of naive mice and compared with commercially available VGFR(1 / 2)-hFc (aflibercept) or PBS (phosphate-buffered saline). (A) Longitudinal follow-up of fluorescence intensity from the gastrocnemius muscle estimated using the IVIS Spectrum in vivo imaging system. (B) IVIS scans of all mice on the final day (96 hours after treatment). (C) Fluorescence intensity of excised gastrocnemius muscle. (D) IVIS scans of excised gastrocnemius muscle. (E) Fluorescence intensity of serum samples on the final day (96 hours after treatment). (F) IVIS scans of serum samples in 1.5 mL microcentrifuge tubes. All results are presented as mean ± SEM. Data were analyzed using one-way ANOVA 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, †P<0.05 vs. VGFR(1 / 2)-hFc(aflibercept). [Figure 10]Figure 1 shows in vivo estimation of the half-life of a type IV designed compound in mouse ocular tissues using IVIS. A type IV designed compound containing VGFR(1 / 2) as the treatment site was administered intravitreally to naive mouse eyes and compared with commercially available VGFR(1 / 2)-hFc (aflibercept) or PBS. (A) Longitudinal follow-up of fluorescence intensity from the eye estimated using the in vivo imaging system, IVIS Spectrum. (B) Fluorescence intensity of an enucleated eye on the final day (96 hours after treatment). (C) IVIS scan of the enucleated eye. All results are presented as mean ± SEM. Data were analyzed using one-way ANOVA including all groups. Multiple comparisons were corrected using Bonferroni's test. *P<0.05 vs. PBS; †P<0.05 vs. VGFR(1 / 2)-hFc (aflibercept). [Figure 11] Figure 1 shows ex vivo estimation of half-life of Type IV designed compounds using IVIS in normothermic machine perfused porcine kidneys. Type IV designed compounds containing TNFR2 as the treatment site were administered via an arterial line to the perfused kidney and compared with commercially available TNFR2-hFc (aflibercept) or PBS. A shows the fluorescence intensity of a porcine kidney midline section 6 hours after treatment. B shows an IVIS scan of a porcine kidney midline section. C shows the fluorescence intensity of a porcine kidney outer cortex section 6 hours after treatment. D shows an IVIS scan of a porcine kidney outer cortex section. [Figure 12] Figure 1 shows the cellular uptake of the platform after 18 hours of compound incubation. Confocal images of adherent HEK293 cells are shown. Cells were incubated for 18 hours with a Type 1 design compound (anti-vimentin-nanobody-FSD1) containing an anti-vimentin nanobody and fluorescently labeled with ATTO488, anti-vimentin nanobody, or DMEM alone as the therapeutic or diagnostic site. After compound incubation, cells were washed and incubated with Hoechst and Lysotracker to visualize nuclei and lysosomes, respectively. Figure 2 shows the maximum intensity estimates of the Z-stacks from each sample. Figure 3 shows orthogonal projections of the anti-vimentin-nanobody-FSD1 sample alone, showing the X- and Y-axis cross-sections. [Figure 13] This figure demonstrates compound ECM binding in vivo in skeletal muscle. NIR730 fluorescently labeled type 2 or type 4 design compounds containing VGFR(1 / 2) as the treatment site were intramuscularly administered to 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 are shown. DAPI staining visualized cell nuclei. The fluorescent signal from NIR730 visualized the administered compound. Sirius Red staining visualized linear connective tissue structures within the muscle tissue. (B) High-magnification images of two regions of gastrocnemius muscle treated with VGFR(1 / 2)-mFc-FSD1 are shown. Region 1 shows the parenchyma (P) and connective tissue (CT) at magnification. Region 2 shows the blood vessels at magnification. [Figure 14] Figure 1 shows in vivo evaluation of the half-life of Type 2 and Type 4 designed compounds in mouse ocular tissues using fluorescent fundus imaging. Type 2 and Type 4 designed compounds containing VGFR(1 / 2) as the treatment site were administered intravitreally to naive mouse eyes and compared with commercially available VGFR(1 / 2)-hFc (aflibercept) or PBS. A + B. Images of in vivo fluorescent fundus imaging are shown at baseline (A) and 96 hours after treatment (B). C shows the mean fluorescence intensity of fundus images 96 hours after treatment. All results are presented as mean ± SEM. Data were analyzed using one-way ANOVA including all groups. Multiple comparisons were corrected using Bonferroni's test. * = P < 0.05 vs. PBS, # = P < 0.05 vs. VGFR(1 / 2) / FST291-mFc, † = P < 0.05 vs. VGFR(1 / 2)-hFc (aflibercept). [Figure 15]The in vivo efficacy of type 2 designed compounds in a model of exudative age-related macular degeneration (AMD) was demonstrated. Type 2 designed compounds containing VGFR(1 / 2) as the treatment site were intravitreally administered to naive mouse eyes and compared with commercially available VGFR(1 / 2)-hFc (aflibercept) or a mouse IgG2a isotype control. (A) Representative images of CD31- and isolectin-positive CNV lesions in RPE / choroid flat mounts are shown. Image contrast and brightness were individually adjusted for optimal visualization of CNV lesions in black and white. Scale bar = 150 μm. (B) Average CNV lesion area per eye estimated from CD31 and isolectin immunohistochemical staining of RPE / choroid flat mounts. In a QQ plot of the normal distribution, one outlier was identified and removed in the VGFR(1 / 2)-mFc-FSD1 group. One eye in the VGFR(1 / 2)-hFc(aflibercept) group was inadvertently destroyed during sample preparation. Of the two eyes, one eye was excluded from the VGFR(1 / 2)-mFc-FSD1 group and one eye was excluded from the VGFR(1 / 2)-hFc(aflibercept) group according to the predetermined CNV lesion exclusion criteria. All results are presented as mean ± SEM. Data were analyzed using one-way ANOVA including all groups. Multiple comparisons were corrected using Bonferroni's test. * = P < 0.05 mouse vs. mouse IgG2a isotype control. [Figure 16] Figure 1 shows the cellular uptake of the platform after 3 hours of compound incubation. Figure 2 shows the maximum intensity projection of a Z-stack of confocal images of adherent HEK293 cells. Cells were incubated for 3 hours with either a Type 1 design compound (anti-vimentin nanobody-FSD1) or an anti-vimentin nanobody fluorescently labeled with ATTO488, containing an anti-vimentin nanobody, as a therapeutic or diagnostic site. After compound incubation, cells were washed and incubated with Hoechst and Lysotracker to visualize nuclei and lysosomes, respectively. [Figure 17]Figure 1 shows the cellular uptake of the platform after 18 hours of compound incubation, as imaged using Airyscan super-resolution confocal imaging. Figure 2 shows a maximum intensity projection of a Z-stack of Airyscan super-resolution confocal images of adherent HEK293 cells. Cells were incubated for 18 hours with a Type 1 design compound (anti-vimentin nanobody-FSD1) or an anti-vimentin nanobody fluorescently labeled with ATTO488, containing an anti-vimentin nanobody, as a treatment or diagnostic site. After compound incubation, cells were washed and incubated with Hoechst to visualize nuclei. Cells treated with anti-vimentin nanobody did not provide sufficient signal to generate super-resolution images. [Figure 18] Intracellular uptake of the platform in vivo is shown. This figure shows maximum intensity projections of Z-stacks of confocal images of the gastrocnemius muscle. A type 1 designed compound (anti-vimentin nanobody-FSD1) containing an anti-vimentin nanobody and fluorescently labeled with ATTO488 was intramuscularly administered to the gastrocnemius muscle of naive mice as a therapeutic or diagnostic site, and the results were compared with those of the anti-vimentin nanobody. Eighteen hours after injection, the mice were euthanized, and the gastrocnemius muscles were fixed in formalin, sectioned, and mounted with DAPI-containing mounting medium. P, parenchyma. CT, connective tissue. [Figure 19] Extended bioassay-based validation of type 1 designed compounds for neutralization of activin A, myostatin, and GDF11 is shown. Neutralization of the growth factors activin A (A), myostatin (B), or GDF11 (C) was evaluated using a luciferase-based bioassay of pSmad2 / 3 activation. Type 1 designed compounds, including FSD1 (monomer or dimer) and an anti-vimentin nanobody (anti-vimentin-nb-FSD1), were evaluated at concentrations up to 1 μM as therapeutic or diagnostic sites. Pos control (positive control), Neg control (negative control). All results are presented as mean ± SEM. [Figure 20]Bioassay-based validation of FSD1 mutations for neutralization of activin A and myostatin is shown. Neutralization of the growth factors activin A (A) and myostatin (B) was assessed using a luciferase-based bioassay of pSmad2 / 3 activation. Native FSD1 (simply referred to as "FSD1") and the FSD1 mutants FSD1(Q124A), FSD1(E126A), and FSD1(Q124A, E126A) were evaluated over a concentration range up to 822 nM. Pos control (positive control), Neg control (negative control). All results are presented as mean ± SEM. [Figure 21] Bioassay-based validation of activin A neutralization by type 2 designed compounds utilizing FSD1 mutations is shown. Neutralization of the growth factors activin A (A), myostatin (B), and GDF11 (C) was evaluated using a luciferase-based bioassay of pSmad2 / 3 activation. Type 2 designed compounds utilizing native FSD1, VGFR(1 / 2)-mFc-FSD1, and the commercially available counterpart, VGFR(1 / 2)-hFc (aflibercept), were evaluated over a concentration range up to 1 μM. The type II designed compounds utilizing 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 ranging up to 750 nM (activin A) or 63 nM (myostatin and GDF11). Pos control (positive control), Neg control (negative control). All results are shown as mean ± SEM. [Figure 22]Figure 1 shows the binding of FSD1 multimers utilizing FSD1 mutations, type 3 designed compounds, and type 2 designed compounds to the extracellular matrix. The ECM binding profiles of FSD1 multimers, type 3 designed compounds, and type 2 designed compounds were evaluated using plates precoated with extracellular matrix (ECM) extract (Matrigel) and FSD1 mutations detected by colorimetric analysis. Figure 1 shows the ECM binding curves of stand-alone FSD1 multimers (FSD1, FSD1-FSD1, and FSD1-FSD1-FSD1). Figure 2 shows the ECM binding curves of three different fractions of type 3 designed compounds with increased heparin affinity (low, medium, or high), anti-TNFα antibody-FSD1 (adalimumab-FSD1). Commercially available adalimumab was included as a control. C shows ECM binding curves of type 2 designed compounds utilizing either native FSD1, VGFR(1 / 2)-mFc-FSD1, or FSD1 mutants (VGFR(1 / 2)-mFc-FSD1(Q124A), VGFR(1 / 2)-mFc-FSD1(E126A), and VGFR(1 / 2)-mFc-FSD1(Q124A, E126A)). All results are shown as mean ± SEM. [Figure 23] Bioassay-based validation of glucocorticoid neutralization is shown. A luciferase-based bioassay of glucocorticoid response element (GRE) activation was used to evaluate neutralization of the endogenous glucocorticoid, cortisol, or the synthetic glucocorticoid, prednisolone 21-hemisuccinate. Type 2 and Type 4 designed compounds containing CBG as the therapeutic moiety were tested over a concentration range up to 2 μM. Pos control (positive control), Neg control (negative control). All results are presented as mean ± SEM. [Figure 24]This demonstrates the use of the platform to degrade intracellular proteins. Western blots of lysates from HEK293 cells treated for 24 hours with type 1 designed compounds using anti-vimentin nanobody (anti-vimentin-nb-FSD1), anti-vimentin-nb, or DMEM alone as the treatment moiety are shown. Both compounds were conjugated to an E3 ligase ligand (VHL ligand) to induce proteasomal degradation of the target protein. (A) Western blots of vimentin and GAPDH (loading control). Lane 1: size marker. Lane 2: DMEM lysate. Lane 3: anti-vimentin-nb bound to VHL ligand lysate. Lane 4: anti-vimentin-nb-FSD1 bound to VHL ligand lysate. (B) The intensity of the vimentin band normalized to the intensity of the GAPDH band. DETAILED DESCRIPTION OF THE INVENTION

[0025] definition As used herein, the singular forms "a," "an," and "the" include the plural forms unless the context clearly indicates otherwise.

[0026] It will also be understood that "one or more" can be interchangeably substituted with "plurality" or "at least one."

[0027] As used herein, the term "compound" encompasses molecules comprising or consisting of follistatin domain 1 (FSD1) and a therapeutic or diagnostic agent.

[0028] For example, the term compound includes fusion proteins and conjugate proteins of the invention comprising or consisting of follistatin domain 1 (FSD1) and a therapeutic or diagnostic agent.

[0029] As used herein, a "fusion protein" refers to a hybrid polypeptide containing protein domains derived from at least two distinct polypeptides that are not normally or naturally fused together in a single amino acid sequence. A fusion protein can contain a single amino acid sequence containing two completely different amino acid sequences, or two similar or identical polypeptide sequences, provided that these sequences are not normally found together in the same configuration in a single amino acid sequence that occurs in nature. Thus, the protein domain of the fusion protein can be located at the amino-terminal portion or the carboxy-terminal portion of the fusion protein, thereby forming an "amino-terminal fusion protein" or a "carboxy-terminal fusion protein," respectively. A fusion protein can also contain a linker polypeptide between the polypeptides that make up the fusion protein. The term "fusion construct" or "fusion protein construct" is generally meant to refer to a polynucleotide encoding a fusion protein.

[0030] As used herein, a "conjugated protein" refers to a hybrid polypeptide that includes one or more protein domains attached to a non-polypeptide component, e.g., a small molecule compound, a nucleic acid, a sugar chain, or a nanoparticle.

[0031] As used herein, "follistatin" (FST) may refer to all forms of follistatin, including, by way of example, the protein core and molecular weight forms identified as arising from alternatively spliced ​​mRNAs FS-315, FS-303, and FS-288. It should therefore be understood to include isoforms, or mutant or polymorphic forms of follistatin, that may arise from alternative splicing of the follistatin mRNA. It should also be understood to extend to any protein encoded by the follistatin (FST) gene, any subunit polypeptides, e.g., precursor forms that may be produced, and any follistatin protein, whether present as a monomer, multimer, or fusion protein.

[0032] Follistatin domain 1 (FSD1), as used herein, refers to the first of three follistatin domains: FSD1 (SEQ ID NO: 1), FSD2 (SEQ ID NO: 41), and FSD3 (SEQ ID NO: 42).

[0033] As used herein, the term "module" may be used interchangeably with "building block" and "part", for example, a (poly)peptide module fused or bound to another polypeptide or non-polypeptide moiety as a fusion protein or conjugated protein.

[0034] As used herein, the term "therapeutic agent" refers to a biological or chemical agent used to treat, cure, alleviate, or prevent an adverse condition in a subject. The term "therapeutic agent" also includes substances and agents for combating a disease, condition, or disorder in a subject, including drugs, diagnostic agents, and devices. "Therapeutic agents" also include those used in medical diagnosis or to restore, correct, or alter physiological function. Therapeutic agents include, but are not limited to, therapeutic polypeptides, therapeutic polynucleotides, therapeutic small molecules, such as therapeutic antibodies and fragments thereof, and antibiotics.

[0035] As used herein, a "therapeutic moiety" refers to a portion of a therapeutic agent that provides at least part of the therapeutic effect of a drug. Thus, a therapeutic moiety can be a polypeptide having therapeutic activity, and a construct comprising such a polypeptide; for example, a therapeutic moiety can be a ligand receptor that provides a therapeutic effect. A therapeutic moiety can be any moiety useful in therapy, including, but not limited to, antibiotics, anti-inflammatory agents, anti-tumor agents, cytotoxins, antivirals, and radioactive moieties. A "therapeutic moiety" includes prodrugs of biologically active moieties, constructs in which multiple therapeutic moieties are attached to a carrier, e.g., a multivalent moiety.

[0036] 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 pathological condition(s), pathological disorder(s), and / or disease(s).

[0037] As used herein, a "diagnostic moiety" refers to a portion of a diagnostic agent that, upon binding to or dissociating from the diagnostic agent, provides at least part of the diagnostic effect of the agent, e.g., an imaging moiety.

[0038] As used herein, the term "click chemistry" refers to the use of chemical moieties that facilitate a conjugation reaction with an appropriate complementary site in another block. These specific chemical reactions (e.g., including but not limited to, between an azide group and an alkyne group) result in a covalent bond between the two molecules. Compounds of the present invention, such as the fusion proteins and conjugated proteins of the present invention, can be obtained by manufacturing steps that involve click chemistry.

[0039] As used herein, IgG Fc refers to the C-terminal region of the IgG heavy chain.

[0040] As used herein, a "full-length antibody" refers to an antibody comprising at least two heavy chains and two light chains. This term includes antibodies having a heavy chain comprising an Fc region. A full-length antibody can be a native sequence antibody or a recombinant antibody. A full-length antibody can be, for example, human, humanized, murine, murine, and / or affinity matured. The antibody can be of any immunoglobulin class, including IgG, IgM, IgE, IgA, IgD, and subclasses thereof. A full-length antibody may also comprise additional domains, such as an scFv or scFab, conjugated to one or more of the chains of the full-length antibody. These conjugates are also encompassed by the term "full-length antibody."

[0041] As used herein, "activin A" refers to the active form of the homodimer of the polypeptide inhibitory beta A (βA) chain. "Activin A" refers to the activin protein corresponding to UniProt accession number P08476 (SEQ ID NO: 44), also known as the inhibin βA subunit. Activins A, B, and AB are homodimers or heterodimers composed of two polypeptide chains, βA and βB, respectively.

[0042] As used herein, "myostatin" refers to the active form of the myostatin protein, also known as "GDF8" or "GDF-8," growth differentiation factor 8, and corresponding to UniProt accession number O14793 (SEQ ID NO: 45). Myostatin is a negative regulator of skeletal muscle mass.

[0043] 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), which corresponds to UniProt accession number O95390 (SEQ ID NO: 46).

[0044] As used herein, "half-life" refers to the pharmacokinetic properties of a compound or drug, and is a measure of the average survival time of the compound after its administration.Half-life can be expressed as the time required for 50% of a known amount of compound or drug to be removed from a patient's body or a specific compartment thereof, for example, as measured in serum, i.e., circulating half-life, or in other tissues. "Local half-life" refers to the local half-life of a compound, for example, at the administration site of a compound or drug in a specific organ, such as the local half-life of a compound or drug in muscle, or the local half-life of a compound or drug in the eye.

[0045] As used herein, "rcsb id" refers to the protein's assigned ID in the Research Consortium for Structural Bioinformatics (RCSB) Protein Data Bank (PDB). Each entry in the PDB, a comprehensive resource providing information on the three-dimensional structure of biological macromolecules, is assigned a unique identifier.

[0046] As used herein, "CNV" refers to, but is not limited to, choroidal neovascularization, such as choroidal neovascularization in age-related macular degeneration (AMD).

[0047] As used herein, "RPE" refers to the retinal pigment epithelium.

[0048] As used herein, "variant" refers to a polypeptide or protein that differs from the sequence from which it is derived by one or more amino acids. Typically, a variant has at least 70%, such as at least 80%, for example at least 90%, for example at least 95%, for example at least 96%, for example at least 97%, for example at least 98%, for example at least 99% sequence identity to the sequence from which it is derived. As used herein, "variant" includes mutant polypeptides obtained by mutagenesis, for example, mutant polypeptides obtained by one or more point mutation(s) in the sequence from which it is derived.

[0049] As used herein, "PROTAC" refers to a proteolytic chimera. A PROTAC consists of two covalently linked protein-binding molecules, one of which can bind to an E3 ubiquitin ligase, and the other of which binds to a target protein for degradation, e.g., intracellular degradation by ubiquitination.

[0050] Fusion or conjugated protein structures The present invention relates to fusion or conjugated protein structures comprising one or more polypeptide(s) (P1) comprising or consisting of at least one FSD1 domain. Such polypeptides P1, and fusion or conjugated proteins comprising them, are capable of binding to biological structures, such as extracellular matrices, via heparan sulfate without the undesirable neutralizing effect of, for example, full-length follistatin on the activin A, myostatin, and GDF11 activities.

[0051] Thus, the present invention provides a platform technology that allows for increased binding and therapeutic exposure of therapeutic or diagnostic agents with reduced off-target side effects, but is not limited to this.

[0052] Type 1 In one aspect, the present invention provides a fusion protein or conjugated protein comprising: (i) one or more polypeptide(s) (P1) comprising or consisting of at least one FSD1 domain comprising or consisting of a polypeptide having at least 70%, such as 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; (ii) The fusion protein or conjugate protein includes a therapeutic or diagnostic agent.

[0053] In some embodiments, the fusion protein or conjugated protein comprises or consists of a polypeptide having at least 70% sequence identity, such as at least 80% sequence identity, for example at least 90% sequence identity, for example at least 95% sequence identity, to a polypeptide set forth in the group consisting of SEQ ID NO:2, SEQ ID NO:3, and SEQ ID NO:5.

[0054] In another embodiment, the fusion protein or conjugated protein comprises or consists of a polypeptide encoded by a polynucleotide having at least 70% sequence identity, such as at least 80% sequence identity, for example at least 90% sequence identity, for example at least 95% sequence identity, to a polypeptide set forth in the group consisting of SEQ ID NO:4 and SEQ ID NO:6.

[0055] Type 2 Those skilled in the art will appreciate that technological advances in the field of antibody engineering have enabled the development of a new class of therapeutic and diagnostic agents based on engineering the Fc domain of immunoglobulins (Igs), such as IgG. Such compounds include, for example, therapeutic or diagnostic Fc fusion proteins and derivatives thereof, which consist of one or more therapeutic or diagnostic moieties attached to the C-terminus of an Ig Fc domain.

[0056] In one embodiment, the present invention provides a fusion protein or conjugated protein comprising: (i) one or more polypeptide(s) (P1) comprising or consisting of at least one FSD1 domain comprising or consisting of a polypeptide having at least 70%, such as 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; (ii) a therapeutic or diagnostic agent, said therapeutic or diagnostic agent comprising or consisting of an immunoglobulin Fc domain, wherein said one or more polypeptide(s) (P1) are C-terminally linked to said immunoglobulin Fc domain, preferably said immunoglobulin being IgG, IgA, IgM, IgE, or IgD.

[0057] The immunoglobulin Fc domain of the fusion or conjugated protein of the invention may be composed of two identical protein fragments, each comprising a CH2 and CH3 domain, e.g., a native IgG Fc region. In a preferred embodiment, the immunoglobulin Fc domain of the fusion or conjugated protein of the invention may be composed of different protein fragments, e.g., protein fragments comprising one or more different mutation(s) in the CH2 and / or CH3 domain of the Fc region.

[0058] Thus, in one embodiment, the immunoglobulin Fc domain of the fusion or conjugate protein of the invention is an IgG Fc domain, comprising or consisting of an IgG Fc homodimer or an IgG Fc heterodimer.

[0059] The Fc heterodimers and / or fusion or conjugated proteins of the present invention, including Fc heterodimers, may be produced by protein engineering techniques known in the art, for example, by using an scFV-Fc / Fc expression system, cloning into a plasmid, transfecting into cells (such as HEK293 cells or CHO cells) for expression, followed by collecting the supernatant and purifying the produced protein. In particular, those skilled in the art will understand that techniques such as the "knobs-into-holes" method, as well as DD-KK mutants with asymmetric electrostatic interactions, IgG / IgA strand-swapped engineered domains, and HA-TF mutants with asymmetric hydrophobic interactions can be used to promote heterodimer formation.

[0060] In a preferred embodiment, the fusion or conjugate protein of the invention comprises or consists of an IgG Fc homodimer, each monomer of which is linked to the C-terminus of at least one polypeptide (P1) comprising or consisting of at least one FSD1 domain, said polypeptide having at least 70%, such as at least 80%, for example at least 90%, such as at least 95%, for example at least 99% sequence identity to SEQ ID NO: 1, and each monomer of the IgG Fc homodimer is linked to the N-terminus of the same or different therapeutic or diagnostic moieties.

[0061] Such fusion or conjugated proteins may be represented herein by the following general formula:

[0062] (therapeutic or diagnostic site)-Fc-(P1) The Fc regions of human and mouse origin may be further designated herein as hFc and mFc, respectively.

[0063] In another preferred embodiment, the fusion protein or conjugate protein of the invention comprises or consists of an IgG Fc heterodimer, each monomer of which is linked to the C-terminus of at least one polypeptide (P1) comprising or consisting of at least one FSD1 domain, said polypeptide having at least 70%, such as at least 80%, for example at least 90%, such as at least 95%, for example at least 99% sequence identity to SEQ ID NO: 1, and each monomer of the IgG Fc heterodimer is linked to the N-terminus of the same or a different therapeutic or diagnostic moiety.

[0064] In some embodiments, the fusion protein or conjugated protein comprises or consists of a polypeptide having at least 70% sequence identity, such as at least 80% sequence identity, for example at least 90% sequence identity, for example at least 95% sequence identity, to a polypeptide set forth in the group consisting of SEQ ID NO:7 and SEQ ID NO:9.

[0065] In another embodiment, the fusion protein or conjugated protein comprises or consists of a polypeptide encoded by a polynucleotide having at least 70% sequence identity, such as at least 80% sequence identity, for example at least 90% sequence identity, such as at least 95% sequence identity, for example 95% sequence identity, to a polypeptide set forth in the group consisting of SEQ ID NO:8 and SEQ ID NO:10.

[0066] Type 4 At least one polypeptide (P1) comprising at least one FSD1 domain of the fusion or conjugated protein of the present invention may be linked at its N-terminus to an IgG Fc domain.

[0067] In one embodiment, the present invention provides a fusion protein or conjugate protein of the present invention, comprising: (i) one or more polypeptide(s) (P1) comprising or consisting of at least one FSD1 domain comprising or consisting of a polypeptide having at least 70%, such as 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; (ii) a therapeutic or diagnostic agent, said therapeutic or diagnostic agent comprising or consisting of an IgG Fc heterodimer, wherein at least one monomer of said IgG Fc heterodimer is linked to the N-terminus of at least one polypeptide (P1) comprising or consisting of at least one FSD1 domain, said polypeptide having at least 70%, such as at least 80%, for example at least 90%, such as at least 95%, for example at least 99% sequence identity to SEQ ID NO: 1, and wherein at least one monomer of the IgG Fc heterodimer is linked to the N-terminus of the same or a different therapeutic or diagnostic moiety.

[0068] Such fusion or conjugated proteins may be represented herein by the following general formula:

[0069] (treatment or diagnostic site) / (P1)-Fc The Fc regions of human and mouse origin may be further designated herein as hFc and mFc, respectively.

[0070] The fusion protein or conjugate protein of the present invention may bind to one or more polypeptides (P1), such as one or more polypeptides (P1), such as a polypeptide (P1) polymer, for example at least three polypeptides (P1), such as at least four polypeptides, for example at least five polypeptides, such as at least 10 polypeptides (P1), for example at least 25 polypeptides (P1), comprising or consisting of at least one FSD1 domain having at least 70%, such as 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.

[0071] Thus, in some preferred embodiments, at least one monomer of the IgG Fc heterodimer of the fusion protein or conjugate protein of the invention may be N-terminally linked to at least one polypeptide (P1), 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, comprising or consisting of at least one FSD1 domain comprising or consisting of a polypeptide having at least 70%, such as at least 80%, for example at least 90%, such as at least 95%, for example at least 99% sequence identity to SEQ ID NO: 1.

[0072] In some applications, it may be beneficial for a therapeutic or diagnostic agent or target of a therapeutic or diagnostic moiety to bind to a specific protein, such as, for example, a specific protein involved in a disease or condition. For example, if the receptor is known to bind to the specific protein, the therapeutic or diagnostic moiety may be, for example, a receptor for the specific protein. The specific protein may be, for example, a protein involved in immune, inflammatory, or angiogenic responses. Those skilled in the art will appreciate that conditions such as, but not limited to, cancer, diabetes, arthritis, asthma, cardiovascular disease, and chronic inflammatory diseases may benefit from targeting immune, inflammatory, or angiogenic responses.

[0073] In other embodiments, the therapeutic or diagnostic agent of the fusion or conjugated protein of the invention, or at least one therapeutic or diagnostic moiety that is the same as or different from the fusion or conjugated protein of the invention, is one or more cytokine receptors.

[0074] Those skilled in the art will appreciate that cytokines are immunomodulators and are widely used in cell-cell communication. The term cytokine encompasses a wide range of polypeptide regulatory factors, such as interferons, interleukins, chemokines, or tumor necrosis factors.

[0075] In a further embodiment, the therapeutic or diagnostic agent of the fusion or conjugated protein of the invention, or at least one therapeutic or diagnostic moiety the same as or different from the fusion or conjugated protein of the invention, comprises or consists of the ligand binding domain of tumor necrosis factor receptor 2 (TNFR2) (SEQ ID NO: 21).

[0076] In yet other embodiments, the therapeutic or diagnostic agent of the fusion or conjugated protein of the invention, or at least one therapeutic or diagnostic moiety that is the same as or different from the fusion or conjugated protein of the invention, comprises or consists of the ligand binding domain of one or more growth factor receptors.

[0077] 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-oncogen.

[0078] In certain embodiments, the therapeutic or diagnostic agent of the fusion protein or conjugate protein of the invention, or at least one therapeutic or diagnostic moiety that is the same as or different from the fusion protein or conjugate protein of the invention, comprises or consists of the ligand binding domain of VGFR1 (SEQ ID NO: 19).

[0079] In other embodiments, the therapeutic or diagnostic agent of the fusion protein or conjugate protein of the invention, or at least one therapeutic or diagnostic moiety that is the same as or different from the fusion protein or conjugate protein of the invention, comprises or consists of the ligand binding domain of VGFR2 (SEQ ID NO: 20).

[0080] In preferred embodiments, the one or more polypeptide(s) (P1)(i) comprising or consisting of at least one FSD1 domain comprise or consist 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 at least 99% identity to SEQ ID NO: 26. In some embodiments, the IgG Fc heterodimer of the fusion protein or conjugate protein of the 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 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 the group consisting of SEQ ID NO: 13, SEQ ID NO: 15, and SEQ ID NO: 17.

[0081] In another embodiment the IgG Fc heterodimer of the fusion or conjugate protein of the invention comprises or consists of a monomer encoded by a sequence having at least 70% sequence identity, such as at least 80% identity, for example at least 90% identity, such as at least 95% identity, for example at least 99% identity to SEQ ID NO: 12 and a monomer encoded by a sequence having at least 80% sequence identity, such as 85% identity, for example at least 90% identity, such as at least 95% identity, for example at least 99% identity to a sequence selected from the group consisting of SEQ ID NO: 14, SEQ ID NO: 16 and SEQ ID NO: 18.

[0082] Type 3 At least one polypeptide (P1) comprising at least one FSD1 domain of the fusion or conjugated protein of the present invention may be bound to a full-length antibody.

[0083] Therefore, another embodiment of the present invention is a fusion protein or conjugated protein comprising: (i) one or more polypeptide(s) (P1) comprising or consisting of at least one FSD1 domain comprising or consisting of a polypeptide having at least 70%, such as 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; (ii) a therapeutic or diagnostic agent, In the context of such a fusion protein or conjugated protein, the one or more polypeptide(s) (P1)(i) and the therapeutic or diagnostic agent are linked by covalent or non-covalent interactions.

[0084] In a further embodiment, the one or more polypeptide(s) (P1)(i) and the therapeutic or diagnostic agent are covalently linked using click chemistry.

[0085] In other embodiments, the one or more polypeptide(s) (P1)(i) and the therapeutic or diagnostic agent are linked by a streptavidin-biotin non-covalent interaction.

[0086] In yet other embodiments, the one or more polypeptide(s) (P1)(i) and the therapeutic or diagnostic agent are bound by a Nanobody, e.g., an anti-Fc Nanobody, that has affinity for the therapeutic or diagnostic agent.

[0087] In some embodiments, the therapeutic or diagnostic agent comprises or consists of a full-length antibody.

[0088] The full-length antibody may be a multispecific antibody, for example a bispecific antibody.

[0089] In a preferred embodiment, the full-length antibody is a bispecific therapeutic antibody.

[0090] In some applications of the fusion or conjugated proteins described herein, it may be preferable that the FSD1 domain of one or more polypeptides (P1) further comprises residues that allow chemical conjugation of one or more polypeptides (P1) to a therapeutic or diagnostic agent (ii). The additional residues may be, for example, but are 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.

[0091] Thus, in some embodiments, one or more polypeptide(s) (P1)(i) comprises or consists of a sequence having at least 70% similarity, such as at least 80% similarity, such as at least 90% similarity, for example at least 95% similarity, such as at least 99% similarity to a sequence selected from the group consisting of SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:64, SEQ ID NO:65, and SEQ ID NO:66.

[0092] In some embodiments, one or more polypeptide(s) (P1)(i) comprises or 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, for example at least 99% similarity to a sequence selected from the group consisting of SEQ ID NO:23 and SEQ ID NO:24.

[0093] In some embodiments, the one or more polypeptide(s) (P1)(i) are encoded by a sequence that comprises or consists of a sequence having at least 70% similarity to SEQ ID NO: 25, such as at least 80% similarity, such as at least 90% similarity, such as at least 95% similarity, for example at least 99% similarity.

[0094] Variant FSD1 domain In some embodiments of the fusion protein or conjugate protein of the invention, at least one FSD1 domain comprising or consisting of a polypeptide having at least 70%, such as 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 is a variant of said FSD1 domain defined by a further polypeptide sequence described herein 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.

[0095] Thus, in a preferred embodiment, (i) at least one FSD1 domain of the one or more polypeptide(s) (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%, for example at least 90%, for example at least 95%, for example at least 96%, for example at least 97%, for example 98%, for example at least 99% sequence identity to those SEQ ID NOs.

[0096] Thus, in some embodiments, the fusion or conjugate protein of the invention comprises: (i) one or more polypeptide(s) (P1) comprising or consisting of at least one FSD1 domain, wherein the variant FSD1 domain is 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%, for example at least 90%, for example at least 95%, such as at least 96%, for example at least 97%, for example 98%, for example at least 99% sequence identity to said SEQ ID NOs; (ii) Includes therapeutic or diagnostic agents.

[0097] 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.

[0098] Functional characteristics of fusion or conjugated proteins Lack of neutralization of activin A, myostatin, and GDF-11 One or more polypeptide(s) (P1) comprising at least one FSD1 domain of the present invention 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). In contrast, neutralization is observed with the N-terminal domain of follistatin (SEQ ID NO: 43) and full-length follistatin, including domains 1, 2, and 3 (FSD1 (SEQ ID NO: 1), FSD2 (SEQ ID NO: 41), and FSD3 (SEQ ID NO: 42)). Importantly, neutralization of these growth factors is associated with biological effects on muscle growth (e.g., skeletal muscle anabolism), which is undesirable for the versatile use of the (P1) polypeptide as a platform for developing fusion or conjugated proteins. The lack of neutralization of activin A, myostatin, and / or GDF11 can be measured using any assay known in the art, such as measuring the lack of activation of downstream signaling by these growth factors, e.g., the lack of activation of intracellular pSmad2 / 3 signaling, using a reporter gene bioassay of phosphorylated Smad2 / 3 signaling, as shown in the Examples herein.

[0099] In a preferred embodiment of the present invention, one or more polypeptide(s) (P1) of the fusion protein or composite protein does not neutralize the activity of activin A (SEQ ID NO: 44), myostatin (SEQ ID NO: 45), and / or GDF11 (SEQ ID NO: 46).

[0100] In some other embodiments the fusion protein or conjugated protein increases the activity of myostatin (SEQ ID NO: 45), GDF11 (SEQ ID NO: 46) and / or activin A (SEQ ID NO: 44) by up to at least 5 nM, such as at least 7.5 nM, for example at least 8 nM, such as at least 8.2 nM, for example at least 8.4 nM, such as at least 8.6 nM, for example at least 8.8 nM, such as at least 9 nM, for example at least 10 nM, such as at least 12 nM, for example at least 15 nM, such as at least 20 nM, for example at least 30 nM, such as at least 40 nM, for example at least 50 nM, such as at least 60 nM, for example at least 61 nM, such as at least 62 nM, for example at least 63 nM, such as at least 64 nM, for example at least 64 nM, For example, it does not neutralize within a concentration range of at least 70 nM, such as at least 80 nM, for example at least 100 nM, such as at least 150 nM, for example at least 200 nM, such as at least 500 nM, for example at least 600 nM, such as at least 700 nM, for example at least 750 nM, such as at least 800 nM, for example at least 820 nM, such as at least 821 nM, for example at least 822 nM, such as at least 830 nM, for example at least 1 μM, such as at least 2 μM, for example at least 3 μM, such as at least 4 μM, for example at least 5 μM, such as at least 6 μM, for example at least 7 μM, such as at least 8 μM, for example at least 9 μM, such as at least 10 μM, and the presence or absence of said neutralization is measured by a reporter gene bioassay of Smad2 / 3 phosphorylation signaling.

[0101] In some embodiments, the fusion protein or conjugated protein does not neutralize the activity of myostatin, GDF11, and / or activin A in a concentration range of up to at least 5 nM, such as at least 7.5 nM, for example at least 8 nM, at least 8.2 nM, for example at least 8.4 nM, such as at least 8.6 nM, for example at least 8.8 nM, for example at least 9 nM, for example at least 10 nM, for example at least 12 nM, at least 15 nM, for example at least 20 nM, such as at least 30 nM, for example at least 40 nM, for example at least 50 nM, for example at least 80 nM, for example at least 100 nM, such as at least 150 nM, for example at least 200 nM.

[0102] In a further embodiment, the one or more polypeptide(s) (P1)(i) increases the activity of myostatin (SEQ ID NO: 45), GDF11 (SEQ ID NO: 46) and / or activin A (SEQ ID NO: 44) by up to at least 5 nM, such as at least 7.5 nM, for example at least 8 nM, such as at least 8.2 nM, for example at least 8.4 nM, such as at least 8.6 nM, for example at least 8.8 nM, such as at least 9 nM, for example at least 10 nM, such as at least 12 nM, for example at least 15 nM, such as at least 20 nM, for example at least 30 nM, such as at least 40 nM, for example at least 50 nM, such as at least 60 nM, for example at least 61 nM, such as at least 62 nM, for example at least 63 nM, such as at least 64 nM, for example at least 64 nM, , such as at least 70 nM, for example at least 80 nM, such as at least 100 nM, for example at least 150 nM, such as at least 200 nM, for example at least 500 nM, such as at least 600 nM, for example at least 700 nM, such as at least 750 nM, for example at least 800 nM, such as at least 820 nM, for example at least 821 nM, such as at least 822 nM, for example at least 830 nM, such as at least 1 μM, for example at least 2 μM, such as at least 3 μM, for example at least 4 μM, such as at least 5 μM, for example at least 6 μM, such as at least 7 μM, for example at least 8 μM, such as at least 9 μM, for example at least 10 μM, and said neutralization or non-neutralization is measured by a reporter gene bioassay of Smad2 / 3 phosphorylation signaling.

[0103] In a further embodiment, the one or more polypeptide(s) (P1)(i) does not neutralize the activity of myostatin, GDF11, and / or activin A in a concentration range of up to at least 5 nM, such as at least 7.5 nM, for example at least 8 nM, at least 8.2 nM, such as at least 8.4 nM, for example at least 8.6 nM, such as at least 8.8 nM, for example at least 9 nM, such as at least 10 nM, for example at least 12 nM, at least 15 nM, such as at least 20 nM, for example at least 30 nM, such as at least 40 nM, for example at least 50 nM, such as at least 80 nM, for example at least 100 nM, such as at least 150 nM, for example at least 200 nM.

[0104] In some embodiments, the fusion protein or conjugate protein described herein is FSD1-FSD1, a type 2 compound described herein, VGFR(1 / 2)-mFc-FSD1, and / or TNFR2-mFc-FSD1, and the fusion protein or conjugate protein does not neutralize the activity of activin A, myostatin, or GDF11 signaling in a concentration range of up to 8.2 nM.

[0105] In some embodiments, the fusion protein or conjugated protein described herein is FSD1-FSD1, and the fusion protein or conjugated protein does not neutralize the activity of activin A, myostatin, or GDF11 signaling in a concentration range up to 1 μM.

[0106] 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 in a concentration range up to 822 nM.

[0107] Heparan sulfate binding 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 will understand that HS is a major component of the extracellular matrix (ECM) and can be found both as free HS chains and as HS chains on HS proteoglycans, such as cell surface proteoglycans. HS has a compartmentalized domain structure consisting of a repeating block of β-D-glucuronic acid-(1→4)-N-acetylated β-D-glucosamine disaccharides (NA domain) and a highly sulfated, heparin-like block of α-L-iduronic acid-(1→4)-N-sulfated β-D-glucosamine disaccharides (NS domain), with a small amount of mixed N-acetylated and N-sulfated disaccharide sequences separating these two domains.

[0108] Thus, it would be beneficial for one or more polypeptides to be conjugated to (P1)(i) heparin sulfate as a modular protein platform for, for example, loading biological materials into the ECM and / or specific organs, and / or extending the half-life of therapeutic or diagnostic agents.

[0109] In some embodiments of the invention, one or more polypeptide(s) (P1)(i) of the fusion or conjugated protein of the invention binds heparan sulfate.

[0110] In a preferred embodiment, one or more polypeptide(s) (P1)(i) comprising at least one FSD1 domain of the present invention comprises or consists of a sequence having at least 70% identity, such as at least 80% identity, for example at least 90% identity, such as at least 95% identity, for example at least 99% identity to SEQ ID NO: 33. In a preferred embodiment, one or more polypeptide(s) (P1)(i) comprising at least one FSD1 domain of the present invention comprises or consists of a sequence having at least 100% identity to SEQ ID NO: 1. Linker In some embodiments, the fusion or conjugated proteins of the invention further comprise a linker between the one or more polypeptide(s) comprising at least one FSD1 domain (P1)(i) and the therapeutic or diagnostic agent (ii).

[0111] In a further embodiment, the linker is a chemical linker.

[0112] Therapeutic or diagnostic agents In a preferred embodiment, the therapeutic or diagnostic agent of the fusion or conjugated protein of the invention, or the same or different therapeutic or diagnostic moiety of the fusion or conjugated 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.

[0113] In other embodiments, the fusion protein or conjugated protein further comprises a detectable moiety, which may be selected from the group consisting of a fluorescent protein, a gold nanoparticle, a radioisotope, biotin or a derivative thereof, and an enzyme.

[0114] In a preferred embodiment, the therapeutic or diagnostic agent (ii) of the fusion or conjugated protein of the invention, or a therapeutic or diagnostic moiety that is the same as or different from the fusion or conjugated protein of the invention, binds to a target selected from the group consisting of differentiation antigen (CD) proteins, cytokines such as interleukins, growth factors such as colony stimulating factors, immune checkpoint proteins, angiogenic factors, hemostatic factors, chemotactic factors, neurotrophic factors, inflammatory proteins, tumor antigens, bacterial proteins, and viral proteins.

[0115] In some embodiments, the therapeutic or diagnostic agent (ii) of the fusion protein or conjugate protein of the invention, or a therapeutic or diagnostic moiety that is the same or different from the fusion protein or conjugate protein of the invention, comprises or consists of a ligand binding domain of a protein selected from the group consisting of TNFR2, VGFR1, VGFR2, and CBG (corticosteroid binding globulin).

[0116] In a further embodiment, the therapeutic or diagnostic agent (ii) of the fusion protein or conjugate protein of the invention, or a therapeutic or diagnostic moiety which is the same or different from the fusion protein or conjugate protein of the invention, comprises or consists of a ligand binding domain of a protein having at least 70% similarity, such as at least 80% similarity, for example at least 90% similarity, such as at least 95% similarity, for example at least 99% similarity, to a sequence selected from the group consisting of SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21 and SEQ ID NO: 22.

[0117] In another embodiment, the therapeutic or diagnostic agent of the fusion or conjugated protein of the invention, or a therapeutic or diagnostic moiety that is the same or different from the fusion or conjugated protein of the invention, comprises or consists of an anti-vimentin-nanobody.

[0118] In some embodiments, the anti-vimentin nanobody has at least 70% similarity to SEQ ID NO: 34, such as at least 80% similarity, for example at least 90% similarity, such as at least 95% similarity, for example at least 99% similarity.

[0119] In some embodiments, the anti-vimentin nanobody is encoded by a sequence having at least 70% similarity to SEQ ID NO: 35, such as at least 80% similarity, for example at least 90% similarity, such as at least 95% similarity, for example at least 99% similarity.

[0120] In some embodiments, the fusion protein or conjugated protein is as described in the "Type 2" section herein, i.e., (i) one or more polypeptide(s) (P1) comprising or consisting of at least one FSD1 domain having at least 70%, such as 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; (ii) a therapeutic or diagnostic agent comprising or consisting of an immunoglobulin Fc domain, wherein the one or more polypeptide(s) (P1) are C-terminally linked to the immunoglobulin Fc domain, preferably the immunoglobulin is IgG, IgA, IgM, IgE, or IgD; The therapeutic or diagnostic agent is selected from the list consisting of an anti-VEGF agent, an anti-TNFα agent, and a mutant CBG.

[0121] In a preferred embodiment, the fusion protein or conjugated protein comprises: (i) two polypeptide(s) (P1) consisting of one FSD1 domain comprising or consisting of a polypeptide having at least 70%, such as 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; (ii) a therapeutic or diagnostic agent, the therapeutic or diagnostic agent comprising or consisting of an immunoglobulin Fc domain, wherein the two polypeptide(s) (P1) are C-terminally linked to the immunoglobulin Fc domain, preferably the immunoglobulin is IgG, IgA, IgM, IgE, or IgD.

[0122] The therapeutic or diagnostic agent is selected from the list consisting of an anti-VEGF agent, an anti-TNFα agent, and a mutant CBG.

[0123] In other embodiments, the fusion protein or conjugated protein is ubiquitinated.

[0124] In some embodiments, the therapeutic or diagnostic agent of a fusion protein or conjugated protein of the invention, or a therapeutic or diagnostic moiety that is the same or different from a fusion protein or conjugated protein of the invention, binds to a target, and the fusion protein or conjugated protein can simultaneously bind to heparan sulfate and the target.

[0125] Platform Strategy Prolonged local half-life The functional properties (e.g., heparin sulfate binding properties) of one or more (P1)(i) polypeptides described herein enable the use of such polypeptides as modules as platform technologies to extend the half-life of therapeutic or diagnostic agents described herein, or the same or different therapeutic or diagnostic moieties described herein.

[0126] Thus, in some embodiments, the fusion protein or conjugated protein has a longer local half-life at the site of administration than the therapeutic or diagnostic agent, or the same or different therapeutic or diagnostic site alone.

[0127] ECM binding The functional properties of the (P1) polypeptides described herein (e.g., heparin sulfate binding) enable their use as modules as platform technologies to enhance ECM binding of therapeutic or diagnostic agents described herein, or therapeutic or diagnostic moieties the same or different as those described herein.

[0128] Binding to ECM can be measured by any method known in the art, for example, using an in vitro ECM binding assay, for example, a colorimetric or fluorescence-based assay. For example, the half maximal effective concentration (EC50) can be used to assess the concentration at which a test compound achieves 50% of its total binding capacity, for example, 50% of its binding capacity to the ECM surface being evaluated.

[0129] Thus, in some embodiments, the fusion protein or conjugated protein enhances binding of a therapeutic or diagnostic agent, or the same or different therapeutic or diagnostic moiety, to an extracellular matrix compartment compared to the therapeutic or diagnostic agent, or the same or different therapeutic or diagnostic moiety alone.

[0130] In some embodiments, the fusion protein or conjugate protein of the present invention binds to one or more polypeptides (P1), such as one or more polypeptides (P1), such as a polypeptide (P1) polymer, for example at least three polypeptides (P1), for example at least four polypeptides, for example at least five polypeptides, for example at least 10 polypeptides (P1), for example at least 25 polypeptides (P1), comprising or consisting of at least one FSD1 domain 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, and ECM binding is enhanced with increasing number of polypeptides (P1).

[0131] 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.

[0132] Organ loading In some applications, it may be beneficial for enhanced binding of the fusion or conjugated proteins of the invention to occur at specific, predetermined structures, such as predetermined organs or tumors.

[0133] Those skilled in the art will appreciate that, for example, enhancing the binding and delivery of a therapeutic or diagnostic agent to a disease-located compartment, such as an organ, is likely to improve the efficacy of the therapeutic and / or diagnostic effects of the compound.

[0134] Thus, in some embodiments, the fusion protein or conjugated protein enhances binding of the therapeutic or diagnostic agent (ii), or the same or different therapeutic or diagnostic moiety, to a given organ or tumor compared to the therapeutic or diagnostic agent (ii), or the same or different therapeutic or diagnostic moiety alone.

[0135] The enhancement of binding may be measured by the EC50, i.e., the reduction in the concentration of the compound being tested, such as a therapeutic or diagnostic agent (ii), or the same or different therapeutic or diagnostic site, that achieves 50% of the total binding capacity, e.g., 50% of the binding capacity of a given organ or tumor tested.

[0136] Thus, the EC50 of the therapeutic or diagnostic agent (ii) or the same or different therapeutic or diagnostic site for a given organ or tumor is reduced by at least 2 fold, such as at least 5 fold, for example at least 10 fold, such as at least 20 fold, for example at least 50 fold, such as at least 80 fold, for example at least 100 fold, such as at least 250 fold, for example at least 500 fold, for example at least 1000 fold, such as at least 5000 fold, for example 100000 fold, such as 100000 fold, for example 100000 fold, compared to the therapeutic or diagnostic agent (ii) or the same or different therapeutic or diagnostic site alone.

[0137] In further embodiments, the organ of interest is selected from the group consisting of a musculoskeletal organ, a digestive organ, a respiratory organ, a urinary organ, a reproductive organ, an endocrine organ, a circulatory organ, a nervous system organ, a hematopoietic organ, and an integumentary organ.

[0138] The organ may be selected from the group consisting of kidney, eye, liver, heart, lung, bladder, pancreas, gallbladder, intestine, prostate, brain, skin, muscle, bone, hematopoietic tissue, joint or subcutaneous tissue such as synovial tissue.

[0139] Intracellular uptake In some embodiments, the fusion protein or conjugated protein increases the cellular 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.

[0140] Multimers of FSD1-containing (P1) polypeptides The fusion or conjugated proteins of the present invention may be linked to one or more polypeptides (P1) described herein.

[0141] In a preferred embodiment, the therapeutic or diagnostic agent of the fusion protein or conjugate protein of the invention, or a therapeutic or diagnostic moiety which is the same as or different from the fusion protein or conjugate protein of the invention, binds 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, for example at least five polypeptides (P1)(i), comprising or consisting of at least one FSD1 domain which comprises or consists of a polypeptide having at least 70%, such as at least 80%, for example at least 90%, such as at least 95%, for example at least 99% sequence identity to SEQ ID NO: 1.

[0142] In another embodiment, the therapeutic or diagnostic agent comprises or consists of an Fc dimer, wherein each monomer of the Fc homodimer or Fc heterodimer binds 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, for example at least five polypeptides (P1)(i), comprising or consisting of at least one FSD1 domain having at least 70%, such as at least 80%, for example at least 90%, such as at least 95%, for example at least 99% sequence identity to SEQ ID NO: 1.

[0143] Multimerization and binding of FSD1 domains For example, to solve steric problems, it may be beneficial for at least one polypeptide (P1)(i) to comprise or consist of several FSD1 domains described herein arranged as 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 (dimers).

[0144] In a further embodiment, the fusion protein or conjugate protein comprises or consists of a multimer of FSD1 domains, such as at least two FSD1 domains, for example at least three FSD1 domains, for example at least four FSD1 domains, for example at least five FSD1 domains, wherein at least one of the at least one polypeptides (P1)(i) comprises or consists of a polypeptide having at least 70%, such as 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.

[0145] In yet another embodiment, at least two monomers of the multimer of the FSD1 domain of the fusion protein or conjugate protein of the invention are joined by a linker.

[0146] In some embodiments, the fusion protein or conjugated protein comprises or consists of a polypeptide having at least 70% sequence identity, such as at least 80% sequence identity, for example at least 90% sequence identity, such as at least 95% sequence identity, for example at least 99% sequence identity, to a sequence selected from the group consisting of SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, and SEQ ID NO:9.

[0147] In another embodiment, the fusion protein or conjugated protein comprises or consists of a polypeptide having at least 70% sequence identity, such as at least 80% identity, for example at least 90% identity, such as at least 95% identity, for example at least 99% identity to SEQ ID NO: 11 and a polypeptide having at least 70% sequence identity, at least 80% sequence identity, such as at least 90% identity, for example at least 95% identity, for example at least 99% identity to a sequence selected from the group consisting of SEQ ID NO: 13, SEQ ID NO: 15 and SEQ ID NO: 17.

[0148] Polynucleotides Another aspect of the present invention pertains to one or more polynucleotides that, upon expression, encode the fusion or conjugate proteins described herein.

[0149] In some embodiments, the one or more polynucleotides have at least 70% sequence identity, such as at least 80% sequence identity, for example at least 90% sequence identity, such as at least 95% identity, for example at least 99% identity, to 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.

[0150] In other embodiments, the one or more polynucleotides have at least 70% sequence identity, such as at least 80% sequence identity, for example at least 90% sequence identity, such as at least 95% identity, for example at least 99% identity, to a combination of SEQ ID NO:12 and at least one polynucleotide selected from the group consisting of SEQ ID NO:14, SEQ ID NO:16, and SEQ ID NO:18.

[0151] Constructs or Vectors A further aspect of the present invention relates to one or more constructs or vectors comprising one or more of the polynucleotides described herein and / or encoding the fusion or conjugate proteins described herein.

[0152] Those skilled in the art will appreciate that the polynucleotides and constructs or vectors for expression of the compounds of the present invention may further comprise additional sequences known in the art, such as sequences used to facilitate the manufacture, expression, or purification of the compounds, including, but not limited to, purification tags known in the art, such as polyhistidine tag(s).

[0153] host cell Those skilled in the art will understand that expression of recombinant proteins, such as the fusion or conjugated proteins described herein, requires an expression system, such as a mammalian, insect, yeast, bacterial, algal, or cell-free expression system.

[0154] One aspect of the present invention pertains to a host cell comprising one or more of the polynucleotides described herein, or one or more of the constructs or vectors described herein.

[0155] In a preferred embodiment, the host cell is a mammalian cell.

[0156] In a more preferred embodiment, the host cell is a human cell, such as a human embryonic kidney 293 cell (HEK293).

[0157] In other embodiments, the host cell is a Chinese hamster ovary (CHO) cell.

[0158] composition Another aspect of the present invention relates to a composition comprising a fusion protein or conjugated protein described herein, one or more nucleotides described herein, a construct or vector described herein, a host cell described herein, or a mixture thereof.

[0159] In a preferred embodiment, the compositions described herein are pharmaceutical compositions.

[0160] Another 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(Q124E126A) of SEQ ID NO: 53, or variants having at least 70%, such as at least 80%, for example at least 90%, such as at least 95%, for example at least 96%, such as at least 97%, for example 98%, such as at least 99% sequence identity thereto.

[0161] Treatment method In a further embodiment, the fusion protein or conjugated protein described herein, one or more polynucleotides described herein, one or more constructs or vectors described herein, host cells described herein, or compositions described herein are for use in medicine.

[0162] In a preferred embodiment, the fusion protein or conjugate protein described herein, one or more polynucleotides described herein, one or more constructs or vectors described herein, host cells described herein, or compositions described herein are for use in the treatment of an ophthalmic disease, a neoplastic disease, an inflammatory disease, a neurological disease, a cardiovascular disease, a metabolic disease, a respiratory disease, a musculoskeletal disease, or an age-related disease.

[0163] In a more preferred embodiment, the fusion protein or conjugated protein described herein, one or more polynucleotides described herein, one or more constructs or vectors described herein, host cells described herein, or compositions described herein are for use in the treatment of an ophthalmic disease, a neoplastic disease, or an inflammatory disease.

[0164] 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 conjugated protein described herein is an anti-VEGF agent, preferably the fusion protein or conjugated protein is as described in Section 2 herein.

[0165] In some embodiments, the fusion protein or conjugated protein described herein, one or more polynucleotides described herein, one or more constructs or vectors described herein, host cells described herein, or compositions described herein are for use in treating an inflammatory disease, and the therapeutic or diagnostic agent (ii) of the fusion protein or conjugated protein described herein is an anti-TNFα agent, preferably the fusion protein or conjugated protein is as described in section 2 herein.

[0166] In some embodiments, the fusion protein or conjugated protein described herein, one or more polynucleotides described herein, one or more constructs or vectors described herein, host cell described herein, or composition described herein is for use in treating a musculoskeletal disorder, such as steroid-induced myopathy, and the therapeutic or diagnostic agent (ii) of the fusion protein or conjugated protein described herein is a mutant CBG, preferably the fusion protein or conjugated protein is as described in section 2 herein.

[0167] In some embodiments, the fusion protein or conjugated protein described herein, one or more polynucleotides described herein, one or more constructs or vectors described herein, host cells described herein, or compositions described herein are for use in the treatment of a neoplastic disease or an autoimmune disease, and the therapeutic or diagnostic agent (ii) of the fusion protein or conjugated protein described herein is interleukin 2 (IL-2), preferably the fusion protein or conjugated protein is as described in section 1 herein.

[0168] In another embodiment, the inflammatory disease is caused by organ transplantation.

[0169] In yet another embodiment, the medical use is in a subject donating an organ or receiving an organ, wherein the donated or transplanted organ is selected from the group consisting of kidney, heart, lung, bone marrow, and liver.

[0170] 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 conjugated 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.

[0171] A further aspect of the present invention relates to a method for treating an ophthalmic disease, an inflammatory disease, or a neoplastic disease, comprising administering to a subject an effective amount of a fusion protein or conjugated 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.

[0172] Yet another aspect of the present invention relates to the use of a fusion protein or conjugate 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 the treatment of a disease or condition, such as an ophthalmic disease, a neoplastic disease, or an inflammatory disease.

[0173] In some embodiments of the use of the fusion or conjugated proteins described herein or the methods described herein, the fusion or conjugated proteins, one or more polynucleotides, one or more constructs or vectors, host cells, or compositions described herein are administered systemically.

[0174] In some embodiments of the use of the fusion or conjugated proteins described herein or the methods described herein, the fusion or conjugated proteins, one or more polynucleotides, one or more constructs or vectors, host cells, or compositions described herein are administered locally.

[0175] In some embodiments of the use of the fusion proteins or conjugated proteins described herein, or the methods described herein, the fusion proteins or conjugated proteins, one or more polynucleotides, one or more constructs or vectors, host cells, or compositions described herein are administered intraadiposely, intra-arterially, intra-articularly, intracranially, intradermally, intralesionally, intramuscularly, intranasally, intraocularly, intraosseously, intrapericardially, intraperitoneally, intrapleurally, intraprostatically, intrarectally, intrathecally, intratracheally, intratumorally, intraumbilically, intravaginally, intravenously, intravesically, intravitreally, or via liposomes.

[0176] Methods for extending the local half-life of therapeutic or diagnostic agents Aspects of the invention relate to methods for increasing the local half-life of a therapeutic or diagnostic agent at a site of administration, comprising obtaining a fusion protein or conjugated 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, wherein the therapeutic or diagnostic agent of the fusion protein or conjugated protein described herein, or a therapeutic or diagnostic moiety that is the same or different from the fusion protein or conjugated protein described herein, comprises or consists of the therapeutic or diagnostic agent.

[0177] A further aspect of the present invention relates to a method for increasing the local half-life in vivo of a therapeutic or diagnostic agent at a site of administration to a subject, comprising administering to said subject a fusion protein or conjugated 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 of the fusion protein or conjugated protein as described herein, or a therapeutic or diagnostic moiety that is the same or different from the fusion protein or conjugated protein as described herein, comprises or consists of the therapeutic or diagnostic agent.

[0178] In some embodiments, a fusion protein or conjugated 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 is administered to a subject suffering from an ophthalmic disease, a neoplastic disease, an inflammatory disease, a neurological disease, a cardiovascular disease, a metabolic disease, a respiratory disease, a musculoskeletal disease, or an aging-related disease.

[0179] In a preferred embodiment, the fusion or conjugated protein, or composition is administered to a subject suffering from an ophthalmic disease, and the fusion or conjugated protein, or composition is administered intravitreally, subretinal, or suprachoroidally.

[0180] In a further embodiment, the ophthalmic disease is wet AMD.

[0181] In other embodiments, the fusion or conjugated protein or composition is administered to an organ, preferably a kidney, heart, lung, bone marrow, or liver, even more preferably to a subject donating or receiving a kidney, or to the organ during extracorporeal perfusion, and the therapeutic or diagnostic agent comprises or consists of a compound that reduces inflammation.

[0182] In a preferred embodiment, the local half-life of the therapeutic or diagnostic agent is extended at the site of administration by the method of the invention by at least 6 hours, such as at least 12 hours, for example at least 24 hours, such as at least 48 hours, for example at least 72 hours, such as at least 96 hours, at least 120 hours, such as at least 1 week, for example at least 2 weeks, such as at least 4 weeks, for example at least 8 weeks, such as at least 3 months, for example at least 6 months, such as at least 12 months.

[0183] Methods for increasing ECM binding of therapeutic or diagnostic agents Another aspect of the invention relates to a method of enhancing binding of a therapeutic or diagnostic agent to an extracellular matrix, comprising administering to a subject a fusion protein or conjugated 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, wherein the therapeutic or diagnostic agent of the fusion protein or conjugated protein described herein, or a therapeutic or diagnostic moiety that is the same as or different from the fusion protein or conjugated protein described herein, comprises or consists of the therapeutic or diagnostic agent.

[0184] A method for enhancing the binding of a therapeutic or diagnostic agent to a selected organ.

[0185] Aspects of the invention relate to methods for enhancing binding of a therapeutic or diagnostic agent to a selected organ, comprising administering to a subject a fusion protein or conjugated 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, wherein the therapeutic or diagnostic agent of the fusion protein or conjugated protein described herein, or a therapeutic or diagnostic moiety that is the same or different from the fusion protein or conjugated protein described herein, comprises or consists of the therapeutic or diagnostic agent.

[0186] A method for increasing the cellular uptake of a therapeutic or diagnostic agent.

[0187] Aspects of the invention relate to methods for increasing cellular uptake of a therapeutic or diagnostic agent, comprising administering to a subject a fusion protein or composite 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, wherein the therapeutic or diagnostic agent of the fusion protein or composite protein described herein, or a therapeutic or diagnostic moiety that is the same as or different from the fusion protein or composite protein described herein, comprises or consists of the therapeutic or diagnostic agent.

[0188] In further embodiments, the therapeutic or diagnostic agent is transported to the cytosol, eg, cytoskeletal filaments, or the nucleus.

[0189] A method for degrading intracellular proteins using a proteolysis-directed chimeric molecule (PROTAC) strategy.

[0190] The fusion or conjugated proteins of the present invention can be used to degrade intracellular proteins using a proteolysis-directed chimeric molecule (PROTAC) strategy.

[0191] In some embodiments, the fusion protein or conjugate protein of the invention further comprises a PROTAC linker, preferably 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.

[0192] An aspect of the present invention therefore relates to a method for degrading an intracellular protein, comprising binding a fusion or conjugated protein according to any one of the preceding paragraphs to a ligand of an E3 ubiquitin ligase, wherein the therapeutic or diagnostic agent of the fusion or conjugated protein (ii) binds to the intracellular protein.

[0193] In some embodiments, the ligand of the E3 ubiquitin ligase is (S,R,S)-AHPC, and preferably, the ligand of the E3 ubiquitin ligase is (S,R,S)-AHPC-PEG8-NHS.

[0194] In another embodiment, the E3 ubiquitin ligase is the von Hippel-Lindau (VHL) tumor suppressor protein.

[0195] In some embodiments, the therapeutic or diagnostic agent (ii) is an anti-vimentin-nanobody.

[0196] In another embodiment, the fusion protein or conjugated protein is a Type 1 protein as described herein, preferably the fusion protein or conjugated protein is anti-vimentin-nanobody-FSD1.

[0197] Subject matter in medical uses and methods.

[0198] In a preferred embodiment of the present invention, the subject in which the fusion or conjugated proteins described herein or in the methods described herein are used is a human or non-human animal.

[0199] Routes of administration in the uses and methods of the present invention.

[0200] In some embodiments of the uses and methods of the present invention, the fusion protein or conjugated protein described herein, one or more polynucleotides described herein, one or more constructs or vectors described herein, host cells described herein, or compositions described herein are administered systemically.

[0201] In other embodiments of the uses and methods of the present invention, the fusion protein or conjugate protein described herein, one or more polynucleotides described herein, one or more constructs or vectors described herein, host cells described herein or compositions described herein are administered locally.

[0202] In further embodiments of the uses and methods of the present invention, the fusion protein or conjugated protein described herein, one or more polynucleotides described herein, one or more constructs or vectors described herein, host cell described herein, or composition described herein is administered intraadiposely, intra-arterially, intra-articularly, intracranially, intradermally, intralesionally, intramuscularly, intranasally, intraocularly, intraosseously, intrapericardially, intraperitoneally, intrapleurally, intraprostatically, intrarectally, intrathecally, intratracheally, intratumorally, intraumbilically, intravaginally, intravenously, intravesically, intravitreally, or via liposomes. [Example]

[0203] Example 1: Construct design and sequence optimization the purpose: 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 both the ECM and cellular glycocalyx.

[0204] Materials and Methods: In constructs designed for mammalian expression systems, the serum albumin preproprotein signal peptide (MKWVTFISLLFLFSSAYS) was used as a leader sequence to facilitate extracellular expression (Reference 1). A vector containing an IRES2 sequence was used for bicistronic expression of the target protein and an intracellular fluorescent marker. All sequences were optimized for expression in mammalian cells. For bacterial expression, the gene encoding the target protein was optimized for bacterial expression and cloned into the pET22B(+) vector.

[0205] result: Common to all four designs was the presence of FSD1 (either monomeric or multimeric) or full-length FST variants as a platform to provide affinity for heparan sulfate. In all designs, a linker may optionally be introduced between FSD1 and the fusion moiety.

[0206] In type 1 designs, genes encoding therapeutic or diagnostic proteins were fused to FSD1 (monomer or multimer) for expression of a single polypeptide chain containing both the therapeutic or diagnostic moiety and FSD1 (Figure 1A). A llama-derived anti-vimentin heavy chain variable domain (VHH or nanobody (nb)) fused to FSD1 (anti-vimentin-nb-FSD1) was an exemplary molecule of type 1 design.

[0207] Type II designs were based on immunoglobulin fragment crystallizable (Fc) platforms (homodimers or heterodimers) (Figure 1B). Here, a therapeutic / diagnostic moiety was fused to the N-terminus of Fc, and FSD1 (monomer or multimer) was fused to the C-terminus of Fc. The homodimeric Fc platform facilitates the design of dimeric therapeutic or dimeric diagnostic proteins (Figure 1B). The heterodimeric Fc platform allows the design of compounds containing two different therapeutic or diagnostic moieties (Figure 1B). Examples of type II designs include VGFR(1 / 2)-mFc-FSD1 or TNFR2-mFc-FSD1. To verify the lack of neutralization of activin A, myostatin, and / or GDF11, a VGRF(1 / 2)-mFc-FSD1 construct in which either the glutamine 124 residue or the glutamic acid 126 residue, or both, of FSD1 were mutated to alanine was also included.

[0208] The type 3 design allows the use of FSD1 (monomer or multimer) as a platform with any conventional antibody that is commercially available or expressed as a single entity (Figure 1C). The type 3 design includes FSD1 covalently conjugated to any conventional antibody (using click chemistry) (Figure 1C), or to an anti-Fc single-chain V H These include FSD1 (Figure 1Cii) that can be expressed with H nanobodies and "mix and use" with any conventional antibody in a therapeutic setting. Type 3 designs also include FSD1 fused to streptavidin for conjugation with biotinylated therapeutic or diagnostic proteins, or biotinylated FSD1 for conjugation with streptavidin fusion proteins. One example of a type 3 design was an anti-tumor necrosis factor alpha (TNFα) antibody conjugated to FSD1 (anti-TNFα antibody-FSD1).

[0209] In the type IV designs, based on the Fc heterodimer platform, either full-length FST or FSD1 (monomer or multimer) replaced one of the Fab regions of the Fc heterodimer, and the other Fab region was replaced by a therapeutic or diagnostic moiety (Figure 1D). The type IV designs included a histidine tag at the C-terminus of the Fc chain without full-length FST or FSD1 to facilitate purification procedures. Examples of type IV designs included VGFR(1 / 2) / FST291-mFc, TNFR2 / FST291-mFc, or CBG / FST291-mFc.

[0210] For chemical conjugation of FSD1 to existing therapeutic antibodies, we designed an FSD1 version (FSD1(KTC)) containing three additional natural C-terminal residues (lys-thr-cys). The free C-terminal cysteine ​​in FSD1(KTC) enabled conjugation with different types of click chemistry compounds containing maleimides.

[0211] The homodimeric Fc platform used in the Type 2 design was based on murine IgG2A. The heterodimeric Fc platform used in both Type 2 and Type 4 designs was based on the "knobs-in-holes" approach (Reference 2). The "knobs-in-holes" approach involves the separation of two Fc chains (Fc) into which point mutations have been introduced to promote heterodimerization rather than homodimerization. A and Fc B ) was used.

[0212] Additional standalone 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.

[0213] Conclusion: Each of the four designs represented a different strategy for constructing therapeutic or diagnostic moieties fused to FSD1 or FST as a platform for heparan sulfate affinity.

[0214] Example 2: Protein expression and purification the purpose: The goal of this example was to generate cell lines expressing desired recombinant proteins and purify them using various chromatographic methods.

[0215] Materials and Methods: Mammalian expression systems: The cloning vector was transformed into DH5 alpha-competent E. coli cells (ThermoFisher Scientific, 18265017). High-yield plasmid purification was performed using the Maxiprep kit (Qiagen, 12162). Plasmids were transfected into a mammalian expression platform based on Chinese hamster ovary cells (CHO) using lipofection (see reference 6). Monoclonal cell lines were seeded based on fluorescence intensity using a cell sorter. Clones with the highest titers of the target protein were selected based on dot blot analysis and expanded into suspension culture. Proteins were purified from filtered conditioned medium using either Protein A (Cytiva, GE17-0403-01), IMAC (Roche, 6781535001, cOmpleteHis-Tag purification column), or heparin chromatography (Cytiva, GE17-0407-01) alone or in combination. Protein purity was verified using SDS-PAGE.

[0216] Bacterial expression: The vector for bacterial expression was transformed into the expression strain E. coli BL21(DE3). 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, cells were harvested by centrifugation and stored at -20°C. Cells were lysed by sonication and centrifuged to remove cellular debris. Recombinant proteins were purified using cation exchange or heparin chromatography, and purity was verified using SDS-PAGE.

[0217] result: Type 2 compounds using native FSD1, VGFR(1 / 2)-mFc-FSD1, and TNFR2-mFc-FSD1, as well as type 4 compounds using VGFR(1 / 2) / FST291-mFc, TNFR2 / FST291-mFc, and CBG / FST291-mFc, were successfully expressed in a mammalian expression system and purified using affinity chromatography. Type 2 compounds using mutant FSD1, including VGFR(1 / 2)-mFc-FSD1(Q124A), VGFR(1 / 2)-mFc-FSD1(E126A), and VGFR(1 / 2)-mFc-FSD1(Q124A, E126A), were expressed in a mammalian expression system and purified using affinity chromatography. The type 1 compound (anti-vimentin-nb-FSD1), FSD1(KTC) (used in the design of type 3 compounds), and the stand-alone proteins FSD1, FSD1-FSD1, and FSD1-FSD1-FSD1 were successfully expressed in E. coli and purified using cation exchange or heparin chromatography. Stand-alone mutant FSD1 proteins, including FSD1(Q124A), FSD1(E126A), and FSD1(Q124A,E126A), were successfully expressed in E. coli and purified using cation exchange or heparin chromatography. Anti-vimentin-nb (His-tagged) was successfully expressed in E. coli and purified using IMAC (this protein was used as a control in studies involving the type 1 compound (anti-vimentin-nb-FSD1)).

[0218] Conclusion: Recombinant fusions with native or mutated FSD1 or full-length FST as a platform technology yielded proteins that could be expressed, purified, and correctly folded according to the described design.

[0219] Example 3: Heparin affinity chromatography the purpose: The purpose of this example was to estimate the heparin affinity of compounds prepared using heparin affinity chromatography and compare them to their commercially available counterparts, such as VGFR(1 / 2)-hFc (aflibercept) and TNFR2-hFc (etanercept), if available.

[0220] Materials and Methods: 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% to 100% buffer B (2000 mM NaCl, Tris-HCl, pH 7.6). Eluted proteins were analyzed by UV absorbance (A 280 ) was monitored.

[0221] result: Both the standalone compounds FSD1 and FSD1-FSD1 showed high heparin affinity and were eluted from the column at 1133 mM and 1280 mM NaCl, respectively (Fig. 2A+B), indicating that fusion of multiple FSD1 domains leads to increased heparin affinity.

[0222] Fusion of FSD1 with anti-vimentin nanobody (type 1) resulted in strong binding to heparin, eluting at 1277 mM NaCl, whereas the nanobody itself did not bind to the column (Figure 2A-B).

[0223] Both type 2 compounds, VGFR(1 / 2)-mFc-FSD1 and TNFR2-mFc-FSD1, showed high affinity for heparin and were eluted at 1133 mM and 1080 mM NaCl, respectively (FIG. 2A to B).

[0224] Both the fourth-type compounds, VGFR(1 / 2) / FST291-mFc and TNFR2 / FST291-mFc, showed elution profiles with two peaks representing two distinct conformations with different heparin affinities (Figure 2A-B). Of these, the low-affinity conformation was represented by peak 1 (eluted at 507 mM NaCl for VGFR(1 / 2) / FST291-mFc and 440 mM NaCl for TNFR2 / FST291-mFc), and the high-affinity conformation (eluted at 933 mM NaCl for VGFR(1 / 2) / FST291-mFc and 907 mM NaCl for TNFR2 / FST291-mFc). Commercially available VGFR(1 / 2)-hFc (aflibercept) and TNFR2-hFc (etanercept) showed no affinity for heparin, and FST315dHBS-mFc (SEQ ID NO: 30 and SEQ ID NO: 31) showed very low heparin affinity, consistent with containing an altered heparin-binding site (Reference 3).

[0225] The fourth type of compound, CBG / FST291-mFc, showed high affinity for heparin and was eluted at 915 mM (Figure 2, A to B).

[0226] Conclusion: This example demonstrated that the designed Type 1, Type 2, and Type 4 compounds have high heparin affinity.

[0227] Example 4: Protein conjugation using click chemistry the purpose: The aim of this example was to conjugate FSD1 to an anti-TNFα antibody (adalimumab) using click chemistry.

[0228] Materials and Methods: 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 conjugate the two compounds.

[0229] result: Oxidation of N-linked glycosylation of antibodies using sodium periodate resulted in the generation of multiple aldehyde groups, which were conjugated with aminooxy groups. Similarly, FSD1(KTC) was conjugated with maleimide groups. These groups, along with azide and DBCO, compounds used in click chemistry, were used to covalently attach FSD1(KTC) to adalimumab. Although conjugation was not complete, multiple fractions containing adalimumab with increased affinity for heparin were observed (Figure 3A+B). SDS-PAGE showed that these fractions increased in size in proportion to their heparin affinity (Figure 3C), indicating that multiple conjugations of FSD1(KTC) to adalimumab lead to increased heparin affinity.

[0230] Conclusion: Using click chemistry, it became possible to conjugate FSD1 (KTC) to an existing antibody to impart heparin affinity to the antibody. This example confirms that it is possible to create Type 3 (i) compounds with high heparin affinity.

[0231] Example 5: Bioassay the purpose: The purpose of this example was to quantify the neutralization of activin A, myostatin, or GDF11 and to confirm the lack of such neutralization in the compounds claimed in this invention.

[0232] Materials and Methods: Growth factor neutralization of activin A (R&D Systems, 338-AC), myostatin (R&D Systems, 788-G8-010), or GDF11 (R&D Systems, 1958-GD-010) was quantified using a reporter gene bioassay of phosphorylated Smad signaling. Phosphorylated Smad2 or Smad3 form a complex with Smad4, which translocates to the nucleus and recognizes the repeat sequence 5'-CAGAC-3', known as the Smad-binding element (SBE). HEK293 cells were transfected with a construct containing 12 repeats of the SBE motif upstream of a minimal promoter controlling luciferase expression (Ref. 4). HEK293 cell lines were passaged into stable lines that showed robust luciferase response to growth factor-induced phosphorylated Smad2 or Smad3 signaling. 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 16–20 h, cells were lysed (Promega, Glo Cell Lysis Buffer, E2661), luciferase substrate was added (Promega, Steady-Glo Luciferase Assay System, E2520), and luminescence signals were analyzed using a plate reader (PerkinElmer, EnSpire 2300). Three to six positive controls (growth factor stimulation without inhibitors) and negative controls (no growth factors and no inhibitors) were included. Data were analyzed and the half maximal inhibitory concentrations (IC50s) were calculated using three-parameter, weighted, and unconstrained nonlinear regression (GraphPad, Prism version 9.4.1).

[0233] result: FSD1, FSD1-FSD1, or the type 2 designed compounds VGFR(1 / 2)-mFc-FSD1 or TNFR2-mFc-FSD1 had no neutralizing effect on activin A (Figure 4), myostatin (Figure 5), or GDF11 (Figure 6) signaling at concentrations up to 8.2 nM. Similarly, the commercially available therapeutic agents TNFR2-hFc (etanercept) or VGFR(1 / 2)-hFc (aflibercept) did not neutralize growth factor signaling at concentrations up to 8.2 nM tested (Figures 4B-C, 5B-C, and 6B-C). In contrast, activin A, myostatin, and GDF11 were neutralized by native FST315 (SEQ ID NO: 28) and FST315dHBS-mFc (a recombinant FST315 protein fused to a murine Fc fragment, in which the heparin-binding sequence [HBS] of FSD1 was replaced by the structurally related sequences SEQ ID NO: 30 and SEQ ID NO: 31) with IC50s in the pM range (Reference 3) (Figures 4A, 5A, and 6A). Type 4 designed compounds containing full-length FST, VGFR(1 / 2) / FST291-mFc, or a single site of TNFR2 / FST291-mFc neutralized growth factors with IC50s in the mid-pM to low-nM range (Figures 4B-C, 5B-C, and 6B-C). When tested over a concentration range of up to 1 μM, FSD1 and FSD1-FSD1 had no neutralizing effect on activin A, myostatin, or GDF11 signaling (Figure 19). The type 1 designed compound, anti-vimentin-nanobody-FSD1, significantly increased pSmad2 / 3 signaling when myostatin was used as the stimulator, and to a lesser extent when activin A or GDF11 was used as the stimulator, but in both cases, the effect was observed over a tested concentration range of approximately 100 nM up to 1 μM (Figure 19). Single-entity FSD1 mutants, including FSD1(Q124A), FSD1(E126A), or FSD1(Q124A, E126A), had no neutralizing effect on activin A or myostatin when tested over a concentration range of up to 822 nM (Figure 20).The type 2 design compound VGFR(1 / 2)-mFc-FSD1, which contains native FSD1, neutralized activin A, myostatin, and GDF11 signaling with IC50s in the low nM range when tested over a concentration range up to 1 μM, and exhibited inhibitory effects on growth factor signaling at compound concentrations above 8.2 nM (compare Figures 4B, 5B, and 6B with Figure 21). However, when FSD1 mutations (Q124A or E126A) were applied to the type 2 design instead of native FSD1, the neutralizing effect on activin A was reduced (extrapolated to the low μM range) or abolished (myostatin and GDF11) over the tested concentration range (Figure 21). The commercially available counterpart, VGFR(1 / 2)-hFc (aflibercept), did not neutralize activin A signaling over a concentration range up to 1 μM.

[0234] Conclusion: FSD1 as a monomer or dimer (FSD1-FSD1) had no neutralizing effect on the signaling activity of activin A, myostatin, or GDF11 in a concentration range up to at least 1 μM. Consistent with this, type II designed compounds based on the native FSD1 platform did not exhibit neutralizing activity against activin A, myostatin, or GDF11 when tested in a concentration range up to 8.2 nM. However, when the concentration range moved above 8.2 nM, type II designed 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 affinity effect of the Fc dimer for native FSD1. In the type 2 design, replacement of native FSD1 with selected FSD1 mutations (FSD1(Q124A), FSD1(E126A), or FSD1(Q124A and E126A)) further reduced neutralization of activin A signaling (IC50 in the low μM range) and abolished neutralization of myostatin and GDF11 signaling over the range of concentrations tested. The mutations were selected based on structural analysis of the crystal structure of follistatin complexed with myostatin. Here, Gln124 and Glu126 from FSD1 were identified as key residues directly interacting with myostatin. The type 1 design compound, anti-vimentin-nanobody-FSD1, began to enhance pSmad2 / 3 signaling at approximately 100 nM. The interaction between vimentin and Smad2 / 3 has been reported, with phosphorylated vimentin enhancing pSmad2 / 3 signaling (Ref. 13). If FSD1 confers the ability to translocate its fusion partner (anti-vimentin-nanobody) to the cytosol, this may affect the interaction between vimentin and Smad2 / 3, leading to the observed enhancement of pSmad2 / 3 signaling. Type 4 designed compounds containing the full-length FST moiety retained growth factor neutralizing activity.

[0235] Example 6: Surface Plasmon Resonance the purpose: The purpose of this example was to quantify the affinity of the compounds produced for TNFα and VEGF.

[0236] Materials and Methods: Surface plasmon resonance (SPR) experiments were performed using a Biacore3000 (Cytiva). CM5 chips for capturing compounds were prepared according to the instructions in 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 and SEQ ID NO: 31) were captured at 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, noncovalently 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). A flow rate of 30 μl / min was applied for all steps of the experiment. Binding analysis was performed at 25°C, and data were collected at a rate of 1 Hz. Recorded signals were referenced using BIAevaluation 4.1.1 software (Cytiva). The signal from the in-line reference flow cell was subtracted, and the signal from a blank run (0 nM analyte) was similarly subtracted.

[0237] result: Both VGFR(1 / 2)-mFc-FSD1 and VGFR(1 / 2) / FST291-mFc exhibited very strong and nearly irreversible binding to VEGF (Figure 7). Similarly, TNFR2-mFc-FSD1 and TNFR2 / FST291-mFc exhibited very strong and nearly irreversible binding to TNFα. These affinities were consistent with the VEGF / TNFα affinities of the commercially available therapeutic agents VGFR(1 / 2)-hFc (aflibercept) and TNFR2-hFc (etanercept). Full-length FST315 (SEQ ID NO: 29), in which the heparin-binding site was mutated, showed no affinity for either VEGF or TNFα.

[0238] Conclusion: VGFR(1 / 2)-mFc-FSD1, VGFR(1 / 2) / FST291-mFc, TNFR2-mFc-FSD1, and TNFR2 / FST291-mFc have similar ligand affinities to their commercially available counterparts.

[0239] Example 7: ECM extract binding experiments the purpose: The purpose of this example was to estimate the binding affinity of the claimed platform to extracellular matrix (ECM) extracts and demonstrate the lack of binding to commercially available therapeutic agents.

[0240] Materials and Methods: Binding to ECM was quantified using a modified version of a previously described in vitro colorimetric assay based on ECM extract-coated plates (Corning, BioCoat Matrigel 354607) (reference 5). The ECM contains various heparan sulfates, which immobilize test compounds throughout the wash cycle depending on the strength of the compound's interaction with heparan sulfate. Briefly, plates were blocked overnight with 100 μL of 100% starting block (Thermo Scientific, 37543) per well. Test compounds were serially diluted in blocking buffer at concentrations starting at 60 nM or 120 nM and added to the plates in triplicate (50 μL). Plates were incubated for 2 h at room temperature and then washed four times with 200 μL of Tris-buffered saline containing 0.1% Tween 20 and 10% starting block. A 2-h primary incubation step was performed with 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 per well). Where relevant, incubation with a single primary antibody was used instead of the mixture. Binding of biotinylated anti-human IgG antibodies to commercially available therapeutic agents, VGFR(1 / 2)-hFc (aflibercept) or TNFR2-hFc (etanercept), was verified using direct ELISA at the same concentrations. A 1-h secondary incubation step was performed with streptavidin conjugated to horseradish peroxidase (streptavidin-HRP, 1:400 dilution, R&D Systems, DY998) (50 μL per well). Primary antibodies and streptavidin-HRP were diluted in 100% StartingBlock, and after each incubation step, plates were washed four times with 200 μL of wash solution. TMB substrate solution (Thermo Scientific, N301) was added (100 μL per well), and color development was monitored visually and stopped with an equal volume of stop solution (Thermo Scientific, N600). The color reaction was evaluated at a wavelength of 450 nm using a plate reader (PerkinElmer, EnSpire 2300).The background was determined by color development in blocking buffer. Data were analyzed and the half maximal effective concentration (EC50) was calculated using three-parameter, weighted, and unconstrained nonlinear regression (GraphPad, Prism version 9.4.1). The calculated EC50 was a relative measure of the strength of compound binding to ECM. The highest plateau of each curve did not represent affinity to ECM, but was a function of the degree to which the primary antibody recognized the test compound and the duration of the color reaction.

[0241] result: The OD450 curves of FSD1, FSD1-FSD1, and FSD1-FSD1-FSD1 showed a shift to the left, indicating increased ECM affinity for each module in the presence of FSD1, with EC50 values ​​ranging from 0.08 nM to 1 nM (Figure 22A). The largest curve shift was observed with one or two FSD1 modules. Accordingly, the second-type designed compounds VGFR(1 / 2)-mFc-FSD1 or TNFR2-mFc-FSD1 showed EC50 values ​​of less than 0.5 nM (Figure 8B-C). Native FST288 (SEQ ID NO: 27) showed an EC50 in the 1 nM range (Figure 8A), whereas the Type 4 designed compounds VGFR(1 / 2) / FST291-mFc or TNFR2 / FST291-mFc, which contain a single full-length FST moiety, showed an EC50 of approximately 15 nM (Figure 8B-C). FST315dHBS-mFc (lacking the heparin-binding site, SEQ ID NO: 30 and SEQ ID NO: 31) had a low but measurable ECM affinity with an EC50 of approximately 80 nM (Figure 8A). No ECM binding was demonstrated by the commercially available therapeutics VGFR(1 / 2)-hFc (aflibercept, Figure 8B) or TNFR2-hFc (etanercept, Figure 8C). To demonstrate the utility of the Type 3 design, FSD1(KTC) was covalently conjugated to adalimumab, a commercially available anti-TNFα antibody (described in Example 4). Fractions with different heparin affinities were collected during heparin affinity chromatography, indicating that various numbers of FSD1 modules were conjugated to adalimumab. Based on increasing heparin affinity, the fractions were marked as low, medium, or high adalimumab-FSD1. The exact number of FSD1(KTC) modules conjugated to adalimumab in each fraction is unknown and likely represents a continuum. Therefore, the exact molecular weight could not be determined, and thus the EC50 value could not be determined. However, similar to that observed for multimeric FSD1, the OD450 curve of the adalimumab-FSD1 fraction showed a leftward shift from low to high, indicating increased ECM affinity (Figure 22B). Adalimumab without the platform of the present invention exhibited limited binding to ECM in some cases (Figure 22B).Similar to the type 2 designed proteins utilizing native FSD1 and VGFR(1 / 2)-mFc-FSD1, type 2 designed proteins utilizing FSD1 mutations, including VGFR(1 / 2)-mFc-FSD1(Q124A), VGFR(1 / 2)-mFc-FSD1(E126A), and VGFR(1 / 2)-mFc-FSD1(Q124A, E126A), also exhibited binding to ECM (Figure 22C).

[0242] Conclusion: When used as a platform, FSD1, either as a standalone monomer, multimer, or as part of a full-length FST, confers binding capacity to compounds in ECM extracts, remaining there even after 12 wash cycles. ECM affinity increased with the number of FSD1 sites in the compound. Type 2 designed proteins utilizing FSD1 mutations also demonstrated binding to ECM extracts. FSD1-FSD1 or type 2 designed compounds exhibited 3- to 7-fold lower EC50 values ​​than FSD1 and native FST288, and 30- to 100-fold lower EC50 values ​​than type 4 designed compounds.

[0243] Example 8: In vivo estimation of half-lives of Type 2 and Type 4 designed compounds in mouse skeletal muscle using IVIS the purpose: The purpose of this example was to use fluorescence to estimate the half-life of Type 2 and Type 4 Design Compounds in skeletal muscle of mice.

[0244] Materials and Methods: Compound labeling: Compounds were fluorescently labeled using a near-infrared label, NIR730 (Sigma-Aldrich, 92315), according to the manufacturer's protocol, and compound concentrations were measured using a Pierce Coomassie (Bradford) protein assay kit (Thermo Scientific, 23200).

[0245] Animal experiments 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). Under anesthesia, the hair on the right hind limb was removed to expose the gastrocnemius muscle. A single dose of 25 μg of fluorescently labeled compound was administered intramuscularly in a volume of 5 μL into the gastrocnemius muscle. To address pain due to muscle tension, mice were administered an analgesic (5 mg / kg Carprofen) subcutaneously. Fluorescence was scanned immediately after injection and every 24 h thereafter using an IVIS Spectrum (PerkinElmer) in vivo imaging system. The exposure time was set to 1 second for all scans (whole body, isolated muscle, and serum), and the excitation / emission wavelengths were 675 / 760 nm. Four days later, the mice were euthanized, the right gastrocnemius muscle was dissected, blood was collected, and serum was centrifuged. Gastrocnemius and serum samples were immediately scanned to assess fluorescence intensity. Data were analyzed using Living Image 4.3.1 (Caliper Life Sciences, Inc.).

[0246] result: In skeletal muscle, the type 2 designed compound VGFR(1 / 2)-mFc-FSD1 had a half-life of 63.5 hours (Figure 9A-B) and statistically significantly increased mean radiance in isolated gastrocnemius muscle by 17.4 times compared to the commercially available VGFR(1 / 2)-hFc (aflibercept) (Figure 9C-D). The type 4 designed compound VGFR(1 / 2) / FST291-mFc had a half-life of 8.3 hours (Figure 9A-B) and significantly increased mean radiance in gastrocnemius muscle compared to PBS, but showed no significant difference from VGFR(1 / 2)-hFc (aflibercept) (Figure 9C-D). Commercially available VGFR(1 / 2)-hFc (aflibercept) has a half-life of 7.5 hours and showed no significant difference in mean radiance in isolated skeletal muscle compared to PBS, indicating extremely low local abundance. Notably, no presence of type 2 compounds was detected in serum after 96 hours (Figure 9E-F). While some radiance was observed in the serum of mice treated with type 4 designed compounds, the levels were not significantly different from those in PBS-treated mice. Finally, significant radiance was observed in the serum of mice treated with commercially available VGFR(1 / 2)-hFc (aflibercept), indicating high systemic distribution.

[0247] Conclusion: In skeletal muscle, the type 2 designed compound VGFR(1 / 2)-mFc-FSD1 exhibited a half-life nearly 10 times longer than that of its commercially available counterpart, VGFR(1 / 2)-hFc (aflibercept). As a result, the type 2 designed compound exhibited significant radiance even 96 hours after injection into the gastrocnemius muscle. The type 4 designed compound was also detectable in skeletal muscle 96 hours later, but the radiance from VGFR(1 / 2)-hFc (aflibercept) was not different from that of PBS-treated samples. While VGFR(1 / 2)-hFc (aflibercept) in serum exhibited significant radiance, the radiance from the type 2 and type 4 designed compounds was not different from that of PBS-treated mice.

[0248] Example 9: In vivo estimation of half-life of Type 4 designed compounds in mouse ocular tissues using IVIS the purpose: The purpose of this example was to use fluorescence to estimate the half-life of Type 4 Design Compounds in mouse ocular tissue.

[0249] Materials and Methods: Compound labeling: Compounds were fluorescently labeled using a near-infrared label, NIR730 (Sigma-Aldrich, 92315), according to the manufacturer's protocol, and compound concentrations were measured using a Pierce Coomassie (Bradford) protein assay kit (Thermo Scientific, 23200).

[0250] Animal experiments Seven 12-week-old female Balb / c mice were divided into two groups: PBS (n = 3, vehicle) or active treatment (n = 4, treatment depending on the eye). In the PBS group, PBS was administered to both the left and right eyes of the mice. In the active treatment group, VGFR(1 / 2)-hFc (aflibercept) was administered to the left eye, and VGFR(1 / 2) / FST291 (a fourth-type compound) was administered to the right eye. Under anesthesia, pupils were dilated using tropicamide eye drops, and a single dose of 10 μg of fluorescently labeled compound was administered intravitreally in a volume of 1 μL. After injection, the eyes were treated with chloramphenicol, and the mice were given an analgesic (5 mg / kg Carprofen) subcutaneously to address pain caused by ocular tension. Mice were fluorescently scanned immediately, 48, 72, and 96 hours after injection using an IVIS Spectrum (PerkinElmer) in vivo imaging system. Exposure times were set at 0.5 seconds for in vivo scans and 20 seconds for ex vivo scans. Excitation / emission wavelengths were set at 675 / 760 nm. Mice were euthanized after 4 days, and both eyes were enucleated. Data were analyzed using Living Image 4.3.1 (Caliper Life Sciences, Inc.).

[0251] result: The fourth-type designed compound VGFR(1 / 2) / FST291-mFc in ocular tissues exhibited a half-life of 26.4 hours (Figure 10A) and a 5.3-fold higher mean radiance in enucleated eyes compared to commercially available VGFR(1 / 2)-hFc(aflibercept) (Figure 10B-C). VGFR(1 / 2)-hFc(aflibercept) had a half-life of 13.3 hours and did not exhibit significantly different radiance in enucleated eyes compared to PBS.

[0252] Conclusion: The Type IV designed compound VGFR(1 / 2) / FST291-mFc exhibited a half-life nearly twofold longer than that of its commercial counterpart, VGFR(1 / 2)-hFc (aflibercept). The Type IV designed compound remained traceable in eyes enucleated 96 hours after injection.

[0253] Example 10: Ex vivo estimation of half-life of Type 4 designed compounds using IVIS in normothermic machine perfused porcine kidneys the purpose: The purpose of this example was to use fluorescence to estimate the half-life of Type 4 designed compounds in ex vivo perfused porcine kidneys.

[0254] Materials and Methods: Compound labeling: Compounds were fluorescently labeled using a near-infrared label, NIR730 (Sigma-Aldrich, 92315), according to the manufacturer's protocol, and compound concentrations were measured using a Pierce Coomassie (Bradford) protein assay kit (Thermo Scientific, 23200).

[0255] Ex vivo delivery to the kidney during normothermic machine perfusion (NMP): Nephrectomy and red blood cell isolation were performed on female Danish Landrace pigs weighing approximately 60 kg as previously described (Reference 6). After nephrectomy, the kidneys were washed with isotonic saline (Fresenius Kabi) followed by Belzer UW cryopreservation solution (Bridge to Life, UK) and stored in Belzer UW cryopreservation solution at 4°C for approximately 16 hours. Prior to perfusion, the kidneys were washed with isotonic saline and connected to a standard perfusion apparatus. Two kidneys from the same pig were perfused with the erythroid perfusion solution (as previously described). (6) The kidneys were perfused for 6 hours under oxygenation at 37°C at a flow rate of 400–500 mL / min using a fluorochrome-free fluorochrome converter. One mg of a labeled type II designed compound containing TNFR2 / FST291-mFc or TNFR2-hFc (etanercept) was diluted in 10 mL of perfusion solution and administered via the arterial line at a slow infusion rate (60 mL / h) 5 min after the start of perfusion. At the end of the perfusion, the kidneys were washed with 1.5 L of isotonic saline to remove the perfusate. The kidneys were sectioned at the outer cortex and midline and analyzed using an IVIS Spectrum (PerkinElmer) in vivo imaging system. The exposure time was set to 8 s for midline sections and 6 s for outer cortical sections. The excitation / emission wavelengths were set to 675 / 760 nm. Data were analyzed using Living Image 4.3.1 (Caliper Life Sciences, Inc.). To demonstrate background fluorescence, an additional pig kidney was scanned from an independent experiment in which 10 mL of PBS in NMP was infused into the arterial line.

[0256] result: In ex vivo perfused porcine kidneys, the quaternary designed compound TNFR2 / FST291-mFc exhibited two-fold higher mean radiance in both midline sections (Figure 11A-B) and cortical sections (Figure 11C-D) compared to the commercially available TNFR2-hFc (etanercept).

[0257] Conclusion: When delivering therapeutic agents during ex vivo normothermic machine perfusion, the quaternary designed compound TNFR2 / FST291-mFc exhibited two-fold higher radiance than its commercial counterpart, TNFR2-hFc (etanercept), demonstrating higher exposure with the quaternary designed compound.

[0258] Example 11: In vitro cellular uptake test the purpose: The purpose of this example was to evaluate the in vitro cellular uptake of the platform claimed in this invention.

[0259] Materials and Methods: Compound labeling: Compounds were fluorescently labeled with ATTO488 (ATTO-TEC GmbH, no. AD488), a green fluorescent protein spectral label, according to the manufacturer's protocol. After labeling, compound concentrations were measured using a Pierce Coomassie (Bradford) protein assay kit (Thermo Scientific, 23200).

[0260] Cellular uptake test: Briefly, HEK293 cells were seeded on glass coverslips (Thorlabs, CG15NH) treated with poly-D-lysine (ThermoFisher, A3890401) to promote adhesion. Cells were seeded in DMEM (ThermoFisher, 11995073) supplemented with 10% fetal bovine serum (FBS). After 24 hours, cells were washed with PBS, and compounds (1 μM) were added to 100% DMEM without FBS. Control samples using 100% DMEM without compounds were also included. After 3 and 18 hours of incubation, cells were washed three times with PBS. The cells were then incubated with a mixture of Hoechst 33342 (3 μg / mL) (ThermoFisher, H3570) and LysoTracker Red DND-99 (75 nM) (ThermoFisher, L7528) for 30 minutes to visualize cell nuclei and lysosomes, respectively. Afterwards, the cells were 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 two GaAsP detectors and one Airyscan detector, and Zen Blue Edition software (Carl Zeiss Microscopy GmbH, version 2.5). Confocal images were acquired with a PlanApo x63 oil objective with a NA of 1.4. For routine confocal imaging, a 488 nm diode laser was used to excite ATTO488 at 2.20% power (3 h samples) or 0.45% power (18 h samples) with detection between 510 and 575 nm. A 405 nm diode laser was used to excite Hoechst33342 at 0.50% power (both 3 h and 18 h samples) with detection between 400 and 510 nm. A 561 nm diode laser was used to excite LysoTracker Red DND-99 at 2.60% power (both 3 h and 18 h samples) with detection between 575 and 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). All scans were performed with a pixel time of 1.84 μs, averaging four times in line-by-line repetition mode and average intensity. For Airyscan super-resolution imaging, a 488 nm diode laser was used to excite ATTO488 at 0.03% power and detection was performed between 490 and 580 nm. A 405 nm diode laser was used to excite Hoechst33342 at 0.10% power and detection was performed between 400 and 469 nm. Images were processed using Fji (v2.14.0 / 1.54f). All images were adjusted for equal contrast and brightness for ATTO488 and Lysotracker Red DND-99 signals within each time point. The Hoechst 33342 signal was adjusted separately for contrast and brightness in the 3-hour sample and similarly in the 18-hour sample.

[0261] result: In HEK293 cells, a strong fluorescent signal from ATTO488 indicated that the Type 1 design compound, anti-vimentin nanobody-FSD1, was internalized by the cells after 3 hours of incubation (Figure 16). In contrast, almost no ATTO488 signal was observed in cells treated with anti-vimentin nanobody after 3 hours of incubation (Figure 16). The ATTO488 signal from anti-vimentin nanobody-FSD1 at 3 hours appeared in a punctate pattern characteristic of endocytosis. After 18 hours of incubation, the punctate ATTO488 signal in anti-vimentin nanobody-FSD1-treated cells was replaced by fluorescent signals that appeared as interwoven threads (or filaments) that curved and extended throughout the cells (Figure 12A and Figure 17). In contrast, little or no ATTO488 signal was observed in cells treated with anti-vimentin nanobody or DMEM alone (Figure 12A). In cells treated with anti-vimentin nanobody-FSD1, there was no clear overlap between the lysosomal (Lysotracker Red DND-99) and ATTO488 signals, indicating that type 1 compounds were not located within the lysosomal unit (Figure 12A and Figure 16). The filament-like structures observed in cells treated with anti-vimentin nanobody-FSD1 indicated that type 1 compounds escaped endosomal uptake and translocated to the cytosol, where they bound to vimentin, an intermediate filament protein that forms part of the cytoskeleton. Furthermore, the nuclei of cells treated with anti-vimentin nanobody-FSD1 showed significantly higher ATTO488 fluorescence intensity, indicating that type 1 compounds not only translocated to the cytosol but also into the cell nucleus. In orthogonal projections, it is possible to visualize the x- and y-sections simultaneously (Figure 12B). Here, ATTO488 fluorescence was observed to be located not only at the membrane surface but also between the nucleus and the surface in both the x- and y-sections. This further supported the notion that anti-vimentin-nanobody-FSD1 is present in the cytosol itself, and not just associated with the cell membrane.In anti-vimentin nanobody- or DMEM-treated cells, there were no nuclei or filament-like structures visualized by ATTO488 fluorescence, indicating that the anti-vimentin nanobody was not internalized by cells without the type 1 design of this platform.

[0262] Conclusion: The platform of the present invention, exemplified herein by the Type 1 designed compound anti-vimentin-nanobody-FSD1, was taken up by cells and allowed visualization of vimentin filaments in the cytosol. Type 1 designed compounds were also observed to enter the cell nucleus. Collectively, these data demonstrated that the platform of the present invention can confer the ability to enter the cytosol and cell nucleus to therapeutic or diagnostic agents.

[0263] Example 12: Preparation of FSD1 variants the purpose: The purpose of this example was to apply structural analysis to generate FSD1 mutants lacking neutralizing activity against growth factors, activin A, myostatin, and GDF11.

[0264] Materials and Methods: The crystal structure of the follistatin-myostatin complex (rcsbID:3HH2) was applied to structural analysis of the interaction between FSD1 and myostatin. We found that Glu126 (glutamic acid at amino acid residue 126) of FSD1 forms a salt bridge with Lys39 (lysine at amino acid residue 39) of myostatin. Furthermore, we found that Gln124 (glutamine at amino acid residue 124) of FSD1 forms a hydrogen bond with Asn83 (asparagine at amino acid residue 83) of myostatin. Apart from these specific interactions, no salt bridges or hydrogen bonds were observed between FSD1 and myostatin. We mutated Glu126 and / or Gln124 of FSD1 at these positions to alanine to obtain the variant FSD1 domains E126A, Q124A, and Q124AE126A, respectively, used herein, to disrupt the interaction and thereby prevent FSD1 from binding to and neutralizing the effects of myostatin. Mutations were introduced by site-directed mutagenesis using the InFusion HD cloning kit (Clontech).

[0265] The residues in GDF11 corresponding to Lys39 and Asn83 in myostatin are conserved, and we hypothesized that a similar interaction exists between GDF11 and FSD1. In activin A, the residues corresponding to Lys39 and Asn83 in myostatin are His36 and Ser90. Therefore, the interaction between FSD1 and activin A is expected to be weaker.

[0266] conclusion In conclusion, we tested these hypotheses by mutating Glu126 and Gln124 of FSD1 to alanine, preventing the interaction of FSD1 with the growth factors activin A, myostatin, and GDF11, resulting in the variant FSD1 domains E126A, Q124A, and Q124AE126A used herein.

[0267] Example 13: In vivo demonstration of ECM binding by Type 2 and Type 4 designed compounds the purpose: The objective of this example was to demonstrate ECM binding in vivo and visualize the increased local presence of Type 2 or Type 4 designed compounds in tissues compared to non-FSD1 fused compounds.

[0268] Materials and Methods: This example was based on material collected from animals treated 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) was intramuscularly injected into the right gastrocnemius muscle of C57BL / 6 mice (n = 3 per group). The compounds were fluorescently labeled with NIR730. Four days after injection, the gastrocnemius muscles were dissected and immersion-fixed in 4% formaldehyde for 48 hours. The gastrocnemius muscles were dehydrated with ethanol, embedded in paraffin, and cut into serial sections at a thickness of 2 μm using a microtome. Serial sections were stained with Sirius Red (commonly used to visualize connective tissue) or left unstained and mounted on microscope slides in DAPI-containing mounting medium (P36962, ProLong Diamond Antifade Mountant with DAPI). Slides were imaged using an Olympus VS120 slide scanner equipped with a SpectraX light engine and a Semrock pentafilter (DAPI / FITC / Cy3 / Cy5 / Cy7 Penta LED HC Filter Set, F68-050) and a Hamamatsu ORCA-FLASH 4.0 V2 (QE 82%) camera. Images were captured using a ×20 Air objective (numerical aperture 0.75). DAPI was excited with a 395 / 25 bandpass filter, and emission was detected with a 425 / 50 bandpass filter. The exposure time was 5 ms. NIR730 was excited with a 730 / 40 bandpass filter, and emission was detected with an 800 / 100 bandpass filter, with an exposure time of 150 ms. Images were processed using Fji (v2.14.0 / 1.54f). All images were adjusted equally for contrast and brightness relative to DAPI, except for VGFR(1 / 2)-mFc-FSD1, which had a stronger signal and was processed with less adjustment. All images were adjusted equally for NIR730 signal.

[0269] result: In skeletal muscle, the presence of the type 2 designed compound VGFR(1 / 2)-mFc-FSD1 was easily visualized by the NIR730 fluorescent signal (Figure 13A). In contrast, no NIR730 signal was observed in PBS-treated muscle. Weak NIR730 signals were detected in both muscle tissue treated with VGFR(1 / 2) / FST291-mFc (type 4 designed compound) and VGFR(1 / 2)-hFc (aflibercept). Strong signals from VGFR(1 / 2)-mFc-FSD1 were present in two distinct compartments. The first compartment, which showed the strongest signal, was identical to the distribution of linear connective tissue structures within the muscle visualized by Sirius Red staining. The second compartment formed a network of fluorescent signals throughout the muscle parenchyma (Figure 13B, enlarged region 1) and the vascular tissue penetrating the muscle (Figure 13B, enlarged region 2). The NIR730 signal from the parenchyma was emitted either from muscle fibers under the sarcolemma (subsarcolemmal region) or adjacent to the sarcolemma on the extracellular surface. The NIR730 signal from vascular tissue allowed visualization of all three layers of the vasculature (intima, media, and adventitia). Similar to the parenchyma, the VGFR(1 / 2)-mFc-FSD1 signal was emitted from either the inner cells or the extracellular compartments of the vasculature, or both.

[0270] Conclusion: ECM is a major component of connective tissue and likely serves as a reservoir for the platform claimed in the present invention. Thus, the local abundance of the Type 2 designed compound VGFR(1 / 2)-mFc-FSD1 in skeletal muscle was significantly increased, as indicated by the compound's strong NIR730 fluorescence signal. In contrast, both VGFR(1 / 2) / FST291-mFc (Type 4 designed compound) and VGFR(1 / 2)-hFc (aflibercept) showed limited NIR730 signals, indicating little or no presence of these compounds 4 days after administration.

[0271] Example 14: In vivo evaluation of half-life of Type 2 and Type 4 Design Compounds in mouse ocular tissues using fluorescent fundus imaging.

[0272] the purpose: The purpose of this example was to evaluate the half-life of Type 2 and Type 4 Design Compounds in mouse ocular tissues using fluorescein ophthalmoscopy.

[0273] Materials and Methods: Compound labeling: Compounds were fluorescently labeled with ATTO488 (ATTO-TEC GmbH, AD488), a green fluorescent protein spectral label, according to the manufacturer's protocol. After labeling, compound concentrations were measured using a Pierce Coomassie (Bradford) protein assay kit (Thermo Scientific, 23200).

[0274] Animal experiments Eight 12-week-old female BALB / c mice were divided into four groups (two mice per group): 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). Under anesthesia, pupils were dilated with tropicamide (mydriacyl, 0.5%) eye drops. A single 10 μg dose of fluorescently labeled compound was administered intravitreally by an experienced surgeon in a volume of 1 μL. After injection, chloramphenicol gel (chloramphenicol, 1%) was applied to the eye to prevent infection, and mice received subcutaneous analgesic (carprofen, 5 mg / kg) after the procedure. Immediately after intravitreal administration, baseline fluorescence fundus imaging (Micron IV, Phoenix Research Laboratories, OR, USA) was performed with an exposure time of 200 ms to confirm accurate compound delivery and fluorescent signal. Four days after treatment, mice were anesthetized, and fluorescence fundus imaging was performed again with an exposure time of 500 ms. Fluorescence intensity of the images was quantified using Fiji (v2.14.0 / 1.54f).

[0275] result: Fluorescence fundus examination of the eyes at baseline revealed no statistically significant differences in mean fluorescence intensity between the groups (Figure 14A). However, 4 days after treatment, eyes treated with VGFR(1 / 2)-mFc-FSD1 (a type 2 compound) had significantly higher mean fluorescence intensity than eyes treated with VGFR(1 / 2) / FST291-mFc (a type 4 compound) or VGFR(1 / 2)-hFc (aflibercept) (Figure 14B-C). The fluorescence signal from VGFR(1 / 2) / FST291-mFc or VGFR(1 / 2)-hFc (aflibercept)-treated eyes was not different from that of PBS-treated eyes or other eyes after 4 days of treatment (Figure 14C).

[0276] Conclusion: In ocular tissues, the type 2 designed compound VGFR(1 / 2)-mFc-FSD1 exhibited a superior intraocular half-life profile to the type 4 designed compounds VGFR(1 / 2) / FST291-mFc and VGFR(1 / 2)-hFc (aflibercept). This was supported by the observation of significantly higher fluorescence signals in eyes treated with VGFR(1 / 2)-mFc-FSD1 compared to the other compounds 4 days after treatment.

[0277] Example 15: In vivo efficacy of Type II designed compounds in exudative age-related macular degeneration disease models the purpose: The purpose of this example was to investigate the efficacy of Type II designed compounds in a choroidal neovascularization (CNV) disease model of exudative age-related macular degeneration (exudative AMD) in mice.

[0278] Materials and Methods: Animal experiments 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 (a type 2 compound, n = 8), and VGFR(1 / 2)-hFc (aflibercept, Bayer AG, Leverkusen, Germany, n = 8). Under anesthesia, pupils were dilated with tropicamide (midriacyl, 0.5%) eye drops. Laser induction of CNV was performed using an image-guided laser system (Micron IV, Phoenix Research Laboratories, OR, USA) according to the method reported by Gong et al. (7). The laser settings were: wavelength 532 nm, power 240 mW, exposure time 70 ms, and size 50 μm. Four laser ablations were applied in a clockwise direction (at 12, 3, 6, and 9 o'clock) in the eye. The distance between two laser ablations and the distance between the laser ablations and the optic nerve was twice the diameter of the optic disc of the optic nerve. After CNV induction, a trained surgeon blinded to the compound administered a single intravitreal injection of 10 μg of compound in a volume of 1 μL. PBS was used as the compound vehicle. After injection, chloramphenicol gel (chloramphenicol, 1%) was applied to the eye to prevent infection, and mice were administered a subcutaneous analgesic (carprofen, 5 mg / kg) after the procedure. One week after CNV induction and compound administration, mice were euthanized, and the eyes were enucleated and fixed in 4% paraformaldehyde for 2 hours at room temperature, then washed with PBS.

[0279] Retinal pigment epithelium / choroid flat mount and immunohistochemical staining Retinal pigment epithelium (RPE) / choroid flat mounts were prepared as described by Askoeta et al. (Reference 8). Flat mounts were permeabilized and blocked in PBB buffer (1x PBS, 4% BSA [Millipore, 81-068-3], and 0.5% Triton X-100 [Millipore, 1.08603]) in 96-well plates for 2 hours at 4°C. To visualize the vasculature, samples were incubated overnight at 4°C with a rat anti-mouse CD31 primary antibody (BD Pharmingen, 557355) diluted 1:100 in PBB and biotin-conjugated isolectin GS-IB4 (Invitrogen, I21414) diluted 1:100 in PBB. After washing four times in PBS-X wash solution (1x PBS and 0.5% Triton X-100), the samples were incubated with a secondary goat anti-rat IgG antibody conjugated to Alexa Fluor 568 (Invitrogen, A-11077) diluted 1:500 in PBB and streptavidin conjugated to Alexa Fluor 405 (Invitrogen, S32351) diluted 1:100 in PBB for 2 hours at room temperature, followed by six washes with PBS-X. RPE / choroid flatmounts were mounted on microscope slides and imaged using an Olympus VS120 slide scanner equipped with a SpectraX light engine and a Semrock pentafilter (DAPI / FITC / Cy3 / Cy5 / Cy7 Penta LED HC Filter Set, F68-050) and a Hamamatsu ORCA-FLASH 4.0 V2 camera (QE 82%). Images were taken using a x20 Air objective (numerical aperture 0.75). Alexa Fluor 405 was excited with a 395 / 25 bandpass filter and emission was detected with a 425 / 50 bandpass filter, with an exposure time of 10 ms. Alexa Fluor 568 was excited with a 575 / 25 bandpass filter and emission was detected with a 600 / 60 bandpass filter, with an exposure time of 10 ms. Images were processed using Fji (v2.14.0 / 1.54f).To quantify the average CNV lesion area per eye, samples were adjusted equally for contrast and brightness, and a global threshold was applied. Five samples were visually thresholded to determine the optimal lower and upper cutoff values. During quantification, assessors were blinded to the samples. CNV lesions were excluded before unblinding, based on the recommendations of Gong et al. (7).

[0280] result: In a mouse CNV disease model of exudative AMD, immunohistochemical staining of RPE / choroid flat mounts with CD31 and isolectin provided two complementary approaches for visualizing CNV lesions (Figure 15A). Based on both CD31 and isolectin staining, eyes treated with either VGFR(1 / 2)-mFc-FSD1 (type 2 compound) or VGFR(1 / 2)-hFc (aflibercept) showed a statistically significant reduction in CNV lesion area compared to eyes treated with the mouse IgG2a isotype control (Figure 15B). No significant difference was observed in CNV lesion area estimated by both CD31 and isolectin staining between VGFR(1 / 2)-mFc-FSD1-treated eyes and VGFR(1 / 2)-hFc (aflibercept)-treated eyes.

[0281] Conclusion: In a CNV disease model of exudative AMD in mice, the second-type designed compound VGFR(1 / 2)-mFc-FSD1 showed the same effect of reducing CNV lesion area as its commercial counterpart VGFR(1 / 2)-hFc (aflibercept). These results indicated that the platform of the present invention, exemplified herein by the second-type designed compound, does not inhibit the ligand neutralization effect of the treatment site (i.e., VGFR(1 / 2)). In this experimental setting, both compounds were administered on the same day as CNV induction, so it was not expected that the therapeutic effect of VGFR(1 / 2)-mFc-FSD1 would be greater than that of VGFR(1 / 2)-hFc (aflibercept).

[0282] Example 16: In vivo cellular uptake test the purpose: The purpose of this example was to evaluate the in vivo cellular uptake of the platform claimed in this invention.

[0283] Materials and Methods: Compound labeling: Compounds were fluorescently labeled with ATTO488 (ATTO-TEC GmbH, no. AD488), a green fluorescent protein spectral label, according to the manufacturer's protocol. After labeling, compound concentrations were measured using a Pierce Coomassie (Bradford) protein assay kit (Thermo Scientific, 23200).

[0284] Animal experiments Two 12-week-old female Balb / c mice were assigned and injected with either anti-vimentin nanobody-FSD1 (type 1 compound, n = 1) or anti-vimentin nanobody (n = 1). Under anesthesia, the hair on the right hind limb was removed to expose the gastrocnemius muscle. A single dose of 8 μg of fluorescently labeled compound was administered intramuscularly in a volume of 5 μL into the gastrocnemius muscle. To address pain due to muscle tension, mice were administered an analgesic (5 mg / kg Carprofen) subcutaneously. 18 hours after injection, the gastrocnemius muscles were dissected and immersion-fixed in 4% formaldehyde. The gastrocnemius muscles were dehydrated with ethanol, embedded in paraffin, and cut at a thickness of 4 μm using a microtome. Sections were left unstained and mounted on microscope slides using DAPI-containing mounting medium (P36962, ProLong Diamond Antifade Mountant with DAPI). Confocal imaging was performed using a Zeiss LSM800 laser scanning confocal microscope (Carl Zeiss Microscopy GmbH, Oberkochen, Germany) equipped with two GaAsP detectors and one Airyscan detector, and Zen Blue Edition software (Carl Zeiss Microscopy GmbH, version 2.5). Confocal images were acquired with a PlanApo x63 oil objective with a NA of 1.4. ATTO488 was excited using a 488 nm diode laser at 1.30% power and detected at wavelengths between 510 and 575 nm. DAPI was excited using a 405 nm diode laser at 1.00% power and detected at wavelengths between 400 and 510 nm. For each sample, Z-stacks were recorded at 0.190 μm intervals. All scans were averaged four times using a pixel time of 1.10 μs, line-by-line repetition mode, and average intensity method. Images were processed using Fji (v2.14.0 / 1.54f). Contrast and brightness were adjusted equally for ATTO488 and DAPI for all images.

[0285] result: In skeletal muscle, the presence of the Type 1 designed compound, anti-vimentin-nanobody-FSD1, was easily visualized by the ATTO488 fluorescent signal (Figure 18). In contrast, no ATTO488 signal was detected in muscles injected with anti-vimentin-nanobody. The signal from anti-vimentin-nanobody-FSD1 was most prominent in cells present in connective tissue, some of which exhibited the spindle-shaped morphology characteristic of fibroblasts. The fluorescent ATTO488 signal resembled interwoven thread-like (or filament) structures that extended throughout the cell and curved toward the cell nucleus (DAPI staining). No distinct fluorescent features were detected in the muscle parenchyma.

[0286] Conclusion: The platform of the present invention, exemplified herein by the Type 1 designed compound anti-vimentin-nanobody-FSD1, was internalized by cells and allowed visualization of vimentin filaments. This is only possible if the compound enters the cytosol after cellular uptake. In contrast, no ATTO488 signal was detected in specimens injected with anti-vimentin-nanobody, indicating that the nanobody alone cannot enter the cytoplasm without the platform of the present invention. Vimentin, an intermediate filament protein, is known to be an indicator of cells of mesenchymal origin, including fibroblasts, although not expressed in all cell types (References 9 and 10). No clear ATTO488 signal was detected in muscle parenchyma injected with anti-vimentin-nanobody-FSD1, consistent with the fact that vimentin is not expressed in mature, healthy skeletal muscle fibers (Reference 11). Therefore, anti-vimentin-nanobody-FSD1 internalized by the parenchyma may be dispersed throughout the cytosol of skeletal muscle fibers and may not be concentrated at the target protein, vimentin. Skeletal muscle fiber volume is significantly larger than many other cell types, and in this study, the fluorescent ATTO488 signal from the parenchyma was likely diluted beyond the detection limit. Collectively, these data demonstrated that the platform of the present invention can confer therapeutic or diagnostic agents the ability to enter the cytosol and potentially the cell nucleus.

[0287] Example 17: Neutralization of glucocorticoid signaling the purpose: The purpose of this example was to quantitatively evaluate the neutralization of glucocorticoid signaling in compounds designed to capture and neutralize glucocorticoids based on the Type II or Type IV design of the platform of the present invention.

[0288] Materials and Methods: 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 reporter gene bioassay for glucocorticoid response element (GRE) activation (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 passaged into stable cell lines that exhibited 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 proteins at concentrations ranging from 2 μM. After 16–20 h, cells were lysed (Promega, Glo Cell Lysis Buffer, E2661), luciferase substrate was added (Promega, Steady-Glo Luciferase Assay System, E2520), and luminescence signals were analyzed using a plate reader (PerkinElmer, EnSpire 2300). Six or more positive controls (glucocorticoid without inhibitor) and negative controls (no glucocorticoid and no inhibitor) were included. Data were analyzed and the half-maximal inhibitory concentration (IC50) was calculated using three-parameter, weighted, and unconstrained nonlinear regression (GraphPad, Prism version 9.4.1).

[0289] result: Compounds containing CBG fused to Type 2 (CBG-mFc-FSD1) or Type 4 (CBG / FST291-mFc) designs of our platform were tested over a range of concentrations up to 2 μM and neutralized both endogenous glucocorticoid signaling (cortisol) or synthetic glucocorticoid signaling (prednisolone 21-hemisuccinate) with IC50 values ​​in the low-to-mid nM range. Type 2 design compounds have two CBG moieties and therefore exhibit approximately twice the ligand affinity of Type 4 design compounds with a single CBG moiety.

[0290] Conclusion: CBG, which normally acts as a serum glucocorticoid transport or buffering protein, can be utilized for glucocorticoid neutralization, as exemplified herein using both Type 2 and Type 4 designs of the platform of the present invention. By fusing CBG with the platform of the present invention, localized glucocorticoid neutralizing compounds were developed. Because the platform of the present invention remained relatively localized in the injected tissue (depending on the number of FSD1 modules, as shown in Examples 7 and 8), systemic effects of the glucocorticoid neutralizing compounds are not expected.

[0291] Example 18: Degradation of intracellular proteins using the platform of the present invention the purpose: The purpose of this example was to demonstrate the utility of the platform of the present invention for degrading intracellular proteins using a proteolysis-targeting chimeric molecule (PROTAC) strategy.

[0292] Materials and Methods: Creation of PROTAC nanobodies: Anti-vimentin nanobody and anti-vimentin nanobody-FSD1 (type 1 compounds) were conjugated with (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 (Blowpharm, BP-25703). Briefly, each compound was incubated with the VHL ligand for 1 hour in a buffer containing 500 mM KCl, 20 mM Hepes, and pH 8.3. The buffer was then exchanged with PBS, and the protein concentration was measured using a NanoDrop 2000c (Thermo Scientific, ND-2000C).

[0293] Vimentin degradation in vitro: HEK293 cells were suspended in DMEM medium (ThermoFisher, 11995073) supplemented with 2% FBS and seeded at 600,000 cells per well of a Nunclon Delta-treated 6-well plate (ThermoFisher, 140685). PROTAC nanobody compounds were added (500 nM) at the time of seeding, and a compound-free control condition was included (DMEM control). 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 removed, the pellet 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 of RIPA buffer (ThermoFisher, 89901) with 1 mM phenylmethanesulfonyl fluoride (PMSF, Sigma-Aldrich, 93482). The resulting homogenate was incubated at 4°C with shaking at 750 RPM for 30 minutes and then centrifuged at 16,000 × g for 20 minutes at 4°C. The supernatant (lysate) was stored at -20°C, the lysate was diluted 1 / 10 with PBS, and the protein concentration was measured using a Pierce Coomassie (Bradford) protein assay kit (Thermo Scientific, 23200).

[0294] Western Blot: For Western blotting, 16 μg of total protein was subjected to unstained SDS-PAGE and analyzed 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 with a primary rabbit polyclonal anti-vimentin antibody (Invitrogen, PA5-27231) diluted 1:10,000 in 5% skim milk. After five washes in Tris-buffered saline with 0.1% Tween 20 (TBST), a 1-hour incubation step was performed with a secondary goat anti-rabbit IgG-peroxidase antibody (Sigma-Aldrich, A0545) diluted 1:5,000 in 5% skim milk. After five washes in TBST, the blots were imaged using an Invitrogen iBright FL1500 imaging system (Invitrogen, A44241) with a 14-minute exposure. Background-adjusted vimentin band intensity was quantified using iBright analysis software (version 1.8.1). Next, Western blots for glyceraldehyde 3-phosphate dehydrogenase (GAPDH) as a loading control were performed on the same membrane. The same procedure was followed using a primary mouse monoclonal anti-GAPDH antibody (Invitrogen, MA5-15738) diluted 1:1,000 and a secondary goat anti-mouse IgG peroxidase antibody (Sigma-Aldrich, A2554) diluted 1:5,000. The blots were imaged with a 35-second exposure, and background-adjusted GAPDH band intensity was quantified.

[0295] result: Treatment of HEK293 cells with anti-vimentin nanobody-FSD1 conjugated to a VHL ligand (type 1 compound) for 24 hours significantly reduced vimentin levels (GAPDH normalized) compared to lysates from DMEM control or cells treated with a VHL-conjugated anti-vimentin nanobody (Figure 24A-B). Anti-vimentin nanobody (without FSD1) did not reduce vimentin levels compared to DMEM control. To ensure data consistency, vimentin band intensity was also quantified using Fiji and normalized to unstained total protein estimates (instead of GAPDH intensity). Similar results were obtained in this analysis (data not shown).

[0296] Conclusion: When the platform of the present invention, exemplified herein by Type 1 designed compounds, was used in a PROTAC strategy, it promoted the degradation of vimentin (a constitutively expressed intracellular protein). In contrast, an anti-vimentin nanobody bound to a VHL ligand but without the platform of the present invention did not degrade vimentin. These results demonstrated that the platform of the present invention can confer the ability to enter the cytosol to a therapeutic or diagnostic agent. Thus, the platform of the present invention can be used, for example, by recombinantly fusing or chemically conjugating a nanobody to an intracellular target protein, and then fusing or conjugating the compound (nanobody FSD1) to an E3 ligase ligand to degrade intracellular proteins.

[0297] References 1. Kober, L. et al. (2013) Optimized signal peptides for the development of high expressing CHO cell lines. Biotechnol. Bioeng. 110, 1164-1173 2. Choi, H.-J. et al. (2013) A Heterodimeric Fc-Based Bispecific Antibody Simultaneously Targeting VEGFR-2 and Met Exhibits Potent Antitumor Activity. 3. Dennler, S. et al. (1998) Direct binding of Smad3 and Smad4 to critical TGF beta-inducible elements in the promoter of human plasminogen activator inhibitor-type 1 gene. EMBO J. 17, 3091-3100 4. Lodberg, A. et al. (2019) A follistatin-based molecule increases muscle and bone mass without affecting the red blood cell count in mice. FASEB J. 33, 6001-6010 5. Pearsall, R. S. et al. (2019) Follistatin-based ligand trap ACE-083 induces localized hypertrophy of skeletal muscle with functional improvement in models of neuromuscular disease. Sci. Rep. 9, 11392 6. Lohmann, S. et al. (2021) Mesenchymal stromal cell treatment of donor kidneys during ex vivo normothermic machine perfusion: A porcine renal autotransplantation study. Am. J. Transplant 21, 2348-2359 7. Gong, Y. et al. (2015) Optimization of an Image-Guided Laser-Induced Choroidal Neovascularization Model in Mice. PLoS One 10 8. Askou, A. L. et al. (2017) Suppression of Choroidal Neovascularization in Mice by Subretinal Delivery of Multigenic Lentiviral Vectors Encoding Anti-Angiogenic MicroRNAs. Hum. Gene Ther. Methods 28, 222-233 9. Ostrowska-Podhorodecka, Z. et al. (2022) Impact of Vimentin on Regulation of Cell Signaling and Matrix Remodeling. Front Cell Dev Biol 10, 869069. 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). 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 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 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

[0298] Sequence Listing SEQ ID NO: 1: Human FSD1 ETCENVDCGPGKKCRMNKKNKPRCVCAPDCSNITWKGPVCGLDGKTYRNECALLKARCKEQPELEVQYQGRCK SEQ ID NO: 2: Human FSD1-FSD1 ETCENVDCGPGKKCRMNKKNKPRCVCAPDCSNITWKGPVCGLDGKTYRNECALLKARCKEQPELEVQYQGRCKETCENVDCGPGKKCRMNKKNKPRCVCAPDCSNITWKGPVCGLDGKTYRNECALLKARCKEQPELEVQYQGRCK SEQ ID NO: 3: Human FSD1 as a single entity produced in E. coli, including the initiation methionine (M) amino acid - FSD1 is METCENVDCGPGKKCRMNKKNKPRCVCAPDCSNITWKGPVCGLDGKTYRNECALLKARCKEQPELEVQYQGRCKETCENVDCGPGKKCRMNKKNKPRCVCAPDCSNITWKGPVCGLDGKTYRNECALLKARCKEQPELEVQYQGRCK SEQ ID NO: 4: Polynucleotide sequence encoding human FSD1 - FSD1 as a single entity produced in E. coli, including the initiation methionine (M) amino acid ATGGAGACATGCGAGAACGTGGATTGTGGACCAGGCAAGAAGTGCCGGATGAACAAGAAGAACAAGCCCAGATGCGTGTGCGCTCCTGACTGCAGCAACATCACCTGGAAGGGACCCGTGTGCGGCCTGGATGGCAAGACATACCGGAATGAGTGCGCCCTGCTGAAGGCTAGGTGTAAGGAGCAGCCTGAGCTGGAGGTGCAGTATCAGGGCCGGTGCA AGGAGACATGCGAGAACGTGGATTGTGGACCAGGCAAGAAGTGCCGGATGAACAAGAAGAACAAGCCCAGATGCGTGTGCGCTCCTGACTGCAGCAACATCACCTGGAAGGGACCCGTGTGCGGCCTGGATGGCAAGACATACCGGAATGAGTGCGCCCTGCTGAAGGCTAGGTGTAAGGAGCAGCCTGAGCTGGAGGTGCAGTATCAGGGCCGGTGCAAG SEQ ID NO: 5: Anti-vimentin-nanobody-FSD1. Expressed in E. coli and therefore contains the start methionine (M) amino acid. MQVQLVESGGGLVQSGGSLTLTCAASGFTFSAASMRWVRQVPGKGLEWVATIDGTGANSYYSESAKGRFTISRDNARNTLYLQMNNLKPDDTAVYYCANFGRNYWGKGTQVTVSSETCENVDCGPGKKCRMNKKNKPRCVCAPDCSNITWKGPVCGLDGKTYRNECALLKARCKEQPELEVQYQGRCK SEQ ID NO: 6: Polynucleotide encoding anti-vimentin-nanobody-FSD1. Expressed in E. coli and therefore includes the initial methionine (M) amino acid. ATGCAAGTACAGCTAGTTGAATCAGGTGGAGGGTTGGTGCAAAGCGGTGGTTCTCTGACCTTGACGTGCGCGGCGTCTGGCTTCACCTTCAGCGCTGCCTCCATGCGTTGGGTACGTCAGGTGCCGGGTAAGGGCCTTGAG TGGGTCGCAACCATCGACGGTACTGGTGCAAACAGCTATTACAGCGAGTCGGCGAAAGGCCGTTTTACGATCAGCCGTGATAATGCGCGTAACACCTTATATCTGCAGATGAATAACCTGAAACCGGATGACACCGCAGTT TACTACTGCGCGAACTTTGGTCGCAACTATTGGGGTAAGGGCACGCAAGTGACCGTTTCCTCCGAAACCTGTGAAAATGTCGATTGCGGTCCGGGCAAAAAGTGCCGTATGAACAAGAAGAACAAGCCGCGTTGTGTTTGC GCTCCGGACTGTAGCAATATTACCTGGAAAGGCCCAGTGTGCGGCCTGGACGGCAAAACCTACCGCAACGAATGTGCGCTGCTGAAAGCCAGATGCAAAGAGCAGCCGGAACTGGAGGTTCAATATCAGGGTCGCTGCAAG SEQ ID NO: 7: VGFR(1 / 2)-mFc-FSD1. Serum albumin preproprotein signal peptide is shown in bold, mouse IgG2A is underlined, and FSD1 is double underlined. MKWVTFISLLFLFSSAYSSDTGRPFVEMYSEIPEIIIHMTEGRELVIPCRVTSPNITVTLKKFPLDTLIPDGKRIIWDSRKGFIISNATYKEIGLLTCEATVNGHLYKTNYL THRQTNTIIDVVLSPSHGIELSVGEKLVLNCTARTELNVGIDFNWEYPSSKHQHKKLVNRDLKTQSGSEMKKFLSTLTIDGVTRSDQGLYTCAASSGLMTKKNSTFVRVHEK PRGPTIKPCPPCKCPAPNLLGGPSVFIFPPKIKDVLMISLSPIVTCVVVDVSEDDPDVQISWFVNNVEVHTAQTQTHREDYNSTLRVVSALPIQHQDWMSGKEFKCKVNNKDLPAPIERTISKPKGSVRAPQVYVLPPPEEEMTKKQVTLTC MVTDFMPEDIYVEWTNNGKTELNYKNTEPVLDSDGSYFMYSKLRVEKKNWVERNSYSCSVVHEGLHNHHTTKSFSRTPGKETCENVDCGPGKKCRMNKKNKPRCVCAPDCSNITWKGPVCGLDGKTYRNECALLKARCKEQPELEVQYQGRCK SEQ ID NO: 8: Polynucleotide sequence encoding VGFR(1 / 2)-mFc-FSD1 SEQ ID NO: 9: TNFR2-mFc-FSD1 Serum albumin preproprotein signal peptide is shown in bold, mouse IgG2A is underlined, and FSD1 is double underlined. MKWVTFISLLFLFSSAYSLPAQVAFTPYAPEPGSTCRLREYYDQTAQMCCSKCSPGQHAKVFCTKTSDTVCDSCEDSTYTQLWNWVPECLSCGSRCSSDQVETQACTREQNRICTCRPGWYCALSK QEGCRLCAPLRKCRPGFGVARPGTETSDVVCKPCAPGTFSNTTSSTDICRPHQICNVVAIPGNASMDAVCTSTSPTRSMAPGAVHLPQPVSTRSQHTQPTPEPSTAPSTSFLLPMGPSPPAEGSTGD PRGPTIKPCPPCKCPAPNLLGGPSVFIFPPKIKDVLMISLSPIVTCVVVDVSEDDPDVQISWFVNNVEVHTAQTQTHREDYNSTLRVVSALPIQHQDWMSGKEFKCKVNNKDLPAPIERTISKPKGSVRAPQVYVLPPPEEEMTKKQVTLTC MVTDFMPEDIYVEWTNNGKTELNYKNTEPVLDSDGSYFMYSKLRVEKKNWVERNSYSCSVVHEGLHNHHTTKSFSRTPGKETCENVDCGPGKKCRMNKKNKPRCVCAPDCSNITWKGPVCGLDGKTYRNECALLKARCKEQPELEVQYQGRCK SEQ ID NO: 10: Polynucleotide encoding TNFR2-mFc-FSD1 SEQ ID NO: 11: FST291-mFc knob-in-hole A chain (used with either VGFR(1 / 2) B chain, TNFR2 B chain, or CBG B chain). The serum albumin preproprotein signal peptide is shown in bold, and the A chain of mouse IgG2A is underlined. MKWVTFISLLFLFSSAYSGNCWLRQAKNGRCQVLYKTELSKEECCSTGRLSTSWTEEDVNDNTLFKWMIFNGGAPNCIPCKETCENVDCPGGKKCRMNKKNKPRCVCAPDCSNITWKGPVCGLDGKTYRNECALLKARCKEQPELEVQYQGRCK KTCRDVFCPGSSTCVVDQTNNAYCVTCNRICPEPASSEQYLCGNDGVTYSSACHLRKATCLLGRSIGLAYEGKCIKAKSCEDIQCTGGKKCLWDFKVGRGRCSLCDELCPDSKSDEPVCASDNATYASECAMKEAACSSGVLLEVKHSGSCNSIS PRGPTIKPCPPCKCPAPNLLGGPSVFIFPPKIKDVLMISLSPIVTCVVVDVSEDDPDVQISWFVNNVEVHTAQTQTHREDYNSTLRVVSALPIQHQDWMSGKEFKCKVNNKDLPAP IERTISKPKGSVRAPQVYVLPPPEEEMTEKQVTLTCMVTDFMPEDIYVEWTNNGKTELNYKNTEPVLDSDGSYFMYSWLRVEKKNWVERNSYSCSVVHEGLHNHHTTKSFSRTPGK SEQ ID NO: 12: Polynucleotide encoding FST291-mFc knob-in-hole A chain (used with either VGFR(1 / 2) B chain, TNFR2 B chain, or CBG B chain) 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 B chain of mouse IgG2A is underlined. The C-terminal thrombin site and his tag are double underlined. MKWVTFISLLFLFSSAYSSDTGRPFVEMYSEIPEIIIHMTEGRELVIPCRVTSPNITVTLKKFPLDTLIPDGKRIIWDSRKGFIISNATYKEIGLLTCEATVNGHLYKTNYL THRQTNTIIDVVLSPSHGIELSVGEKLVLNCTARTELNVGIDFNWEYPSSKHQHKKLVNRDLKTQSGSEMKKFLSTLTIDGVTRSDQGLYTCAASSGLMTKKNSTFVRVHEK PRGPTIKPCPPCKCPAPNLLGGPSVFIFPPKIKDVLMISLSPIVTCVVVDVSEDDPDVQISWFVNNVEVHTAQTQTHREDYNSTLRVVSALPIQHQDWMSGKEFKCKVNNKDLPAPIERTISKPKGSVRAPRVYVLPPPEEEMTKKQVTLTCMVTDFMPEDIYVEWTNNGKTELNYKNTEPVLVSDGSYTMYSKLRVEKKNWVERNSYSCSVVHEGLHNHHTTKSFSRTPGKLVPRGSHHHHHH SEQ ID NO: 14: Polynucleotide encoding VGFR(1 / 2)-mFc knob-in-hole B chain. SEQ ID NO: 15: TNFR2-mFc knob-in-hole B chain. The serum albumin preproprotein signal peptide is shown in bold and the B chain of mouse IgG2A is underlined. The C-terminal thrombin site and his tag are double underlined. MKWVTFISLLFLFSSAYSLPAQVAFTPYAPEPGSTCRLREYYDQTAQMCCSKCSPGQHAKVFCTKTSDTVCDSCEDSTYTQLWNWVPECLSCGSRCSSDQVETQACTREQNRICTCRPGWYCALSK QEGCRLCAPLRKCRPGFGVARPGTETSDVVCKPCAPGTFSNTTSSTDICRPHQICNVVAIPGNASMDAVCTSTSPTRSMAPGAVHLPQPVSTRSQHTQPTPEPSTAPSTSFLLPMGPSPPAEGSTGD PRGPTIKPCPPCKCPAPNLLGGPSVFIFPPKIKDVLMISLSPIVTCVVVDVSEDDPDVQISWFVNNVEVHTAQTQTHREDYNSTLRVVSALPIQHQDWMSGKEFKCKVNNKDLPAPIERTISKPKGSVRAPRVYVLPPPEEEMTKKQVTLTCMVTDFMPEDIYVEWTNNGKTELNYKNTEPVLVSDGSYTMYSKLRVEKKNWVERNSYSCSVVHEGLHNHHTTKSFSRTPGKLVPRGSHHHHHH SEQ ID NO: 16: Polynucleotide encoding TNFR2-mFc knob-in-hole B chain. SEQ ID NO: 17: CBG-mFc knob-in-hole B chain. The serum albumin preproprotein signal peptide is shown in bold and the B chain of mouse IgG2A is underlined. The C-terminal thrombin site and his tag are double underlined. MKWVTFISLLFLFSSAYSMDPNAAYVNMSNHHRGLASANVDFAFSLYKHLVALSPKKNIFISPVSISMALAMLSLGTCGHTRAQLLQGLGFNLTERSETEIHQGFQHLHQLFAKSDTSLEMTMGNALFLDGSLELLESFSADIKHYYESEVLAMNFQDWATASRQINSYVKNKTQGKIVDLFSGLDSPAILVLVNYIFFK GTWTQPFDLASTREENFYVDETTVVKVPMMLQSSTISYLHDSELPCQLVQMNYVGNGTVFFILPDKGKMNTVIAALSRDTINRWSAGLTSSQVDLYIPKV TISGVYDLGDVLEEMGIADLFTNQANFSRITQDAQLKSSKVVHKAVLQLNEEGVDTAGSTGVTLNLTSKPIILRFNQPFIIMIFDHFTWSSLFLARVMNPV PRGPTIKPCPPCKCPAPNLLGGPSVFIFPPKIKDVLMISLSPIVTCVVVDVSEDDPDVQISWFVNNVEVHTAQTQTHREDYNSTLRVVSALPIQHQDWMSGKEFKCKVNNKDLPAPIERTISKPKGSVRAPRVYVLPPPEEEMTKKQVTLTCMVTDFMPEDIYVEWTNNGKTELNYKNTEPVLVSDGSYTMYSKLRVEKKNWVERNSYSCSVVHEGLHNHHTTKSFSRTPGKLVPRGSHHHHHH SEQ ID NO: 18: Polynucleotide encoding CBG-mFc knob-in-hole B chain. SEQ ID NO: 19: Part of the extracellular domain of native human VGFR1 that binds to VEGF (immunoglobulin-like type 2) SDTGRPFVEMYSEIPEIIIHMTEGRELVIPCRVTSPNITVTLKKFPLDTLIPDGKRIIWDSRKGFIISNATYKEIGLLTCEATVNGHLYKTNYLTHRQTNTII SEQ ID NO: 20: Part of the extracellular domain of native human VGFR2 that binds to VEGF (immunoglobulin-like type 3) DVVLSPSHGIELSVGEKLVLNCTARTELNVGIDFNWEYPSSKHQHKKLVNRDLKTQSGSEMKKFLSTLTIDGVTRSDQGLYTCAASSGLMTKKNSTFVRVHEK SEQ ID NO: 21: Native human TNFR2 ectodomain (TNFα binding domain) LPAQVAFTPYAPEPGSTCRLREYYDQTAQMCCSKCSPGQHAKVFCTKTSDVCDSCEDSTYTQLWNWVPECLSCGSRCSSDQVETQACTREQNRICTCRPGWYCALSKQEGCRLCAP LRKCRPGFGVARPGTETSDVVCKPCAPGTFSNTTSSTDICRPHQICNVVAIPGNASMDAVCTSTSPTRSMAPGAVHLPQPVSTRSQHTQPTPEPSTAPSTSFLLPMGPSPPAEGSTGD SEQ ID NO: 22: Native human CBG. The native signal peptide is shown in bold. MPLLLYTCLLWLPTSGLWTVQAMDPNAAYVNMSNHHRGLASANVDFAFSLYKHLVALSPKKNIFISPVSISMALAMLSLGTCGHTRAQLLQGLGFNLTERSETEIHQGFQHLHQLFAKSDTSLEMTMGNALFLDGSLELLESFSADIKHYYESEVLAMNFQDWATASRQINSYVKNKTQGKIVDLFSGLDSPAILVLVNYIF FKGTWTQPFDLASTREENFYVDETTVVKVPMMLQSSTISYLHDSELPCQLVQMNYVGNGTVFFILPDKGKMNTVIAALSRDTINRWSAGLTSSQVDLYIPK VTISGVYDLGDVLEEMGIADLFTNQANFSRITQDAQLKSSKVVHKAVLQLNEEGVDTAGSTGVTLNLTSKPIILRFNQPFIIMIFDHFTWSSLFLARVMNPV SEQ ID NO: 23: Human FSD1 (KTC) ETCENVDCGPGKKCRMNKKNKPRCVCAPDCSNITWKGPVCGLDGKTYRNECALLKARCKEQPELEVQYQGRCKKTC SEQ ID NO: 24: Human FSD1 as a single entity produced in E. coli - FSD1(KTC) including the initial methionine (M) amino acid METCENVDCGPGKKCRMNKKNKPRCVCAPDCSNITWKGPVCGLDGKTYRNECALLKARCKEQPELEVQYQGRCKKTC SEQ ID NO: 25: Polynucleotide encoding human FSD1 (KTC) as a single entity produced in E. coli, including the initial methionine (M) amino acid ATGGAGACATGCGAGAACGTGGATTGTGGACCAGGCAAGAAGTGCCGGATGAACAAGAAGAACAAGCCCAGATGCGTGTGCGCTCCTGACTGCAGCAACATCACCTGGAAGGGACCCGTGTGCGGCCTGGATGGCAAGACATACCGGAATGAGTGCGCCCTGCTGAAGGCTAGGTGTAAGGAGCAGCCTGAGCTGGAGGTGCAGTATCAGGGCCGGTGCAAGAAAACATGT SEQ ID NO: 26: Native human follistatin 291. The native signal peptide is shown in bold. MVRARHQPGGLCLLLLLLCQFMEDRSAQAGNCWLRQAKNGRCQVLYKTELSKEECCSTGRLSTSWTEEDVNDNTLFKWMIFNGGAPNCIPCKETCENVDCGPGKKCRMNKKNKPRCVCAPDCSNITWKGPVCGLDGKTYRNECALLKARCKEQPELEVQY QGRCKKTCRDVFCPGSSTCVVDQTNNAYCVTCNRICPEPASSEQYLCGNDGVTYSSACHLRKATCLLGRSIGLAYEGKCIKAKKSCEDIQCTGGKKCLWDFKVGRGRCSLCDELCPDSKSDEPVCASDNATYASECAMKEAACSSGVLLEVKHSGSCNSIS SEQ ID NO: 27: Native human follistatin 288. The native signal peptide is shown in bold. MVRARHQPGGLCLLLLLLCQFMEDRSAQAGNCWLRQAKNGRCQVLYKTELSKEECCSTGRLSTSWTEEDVNDNTLFKWMIFNGGAPNCIPCKETCENVDCGPGKKCRMNKKNKPRCVCAPDCSNITWKGPVCGLDGKTYRNECALLKARCKEQPELEV QYQGRCKKTCRDVFCPGSSSTCVVDQTNNAYCVTCNRICPEPASSEQYLCGNDGVTYSSACHLRKATCLLGRSIGLAYEGKCIKAKKSCEDIQCTGGKKCLWDFKVGRGRCSLCDELCPDSKSDEPVCASDNATYASECAMKEAACSSGVLLEVKHSGSCN SEQ ID NO: 28: Native human follistatin 315. The native signal peptide is shown in bold. MVRARHQPGGLCLLLLLLCQFMEDRSAQAGNCWLRQAKNGRCQVLYKTELSKEECCSTGRLSTSWTEEDVNDNTLFKWMIFNGGAPNCIPCKETCENVDCGPGKKCRMNKKNKPRCVCAPDCSNITWKGPVCGLDGKTYRNECALLKARCKEQPELEVQYQGRCKKTCRDVF CPGSSTCVVDQTNNAYCVTCNRICPEPASSEQYLCGNDGVTYSSACHLRKATCLLGRSIGLAYEGKCIKAKSCEDIQCTGGKKCLWDFKVGRGRCSLCDELCPDSKSDEPVCASDNATYASECAMKEAACSSGVLLEVKHSGSCNSISEDTEEEEEDEDQDYSFPISSILEW SEQ ID NO: 29: Human follistatin 315 in which the heparin binding sequence (HBS) of FSD1 has been replaced by a structurally related sequence. The native signal peptide is shown in bold. MVRARHQPGGLCLLLLLLCQFMEDRSAQAGNCWLRQAKNGRCQVLYKTELSKEECCSTGRLSTSWTEEDVNDNTLFKWMIFNGGAPNCIPCKETCENVDCGPGSTCVVDQTNNPRCVCAPDCSNITWKGPVCGLDGKTYRNECALLKARCKEQPELEVQYQGRCKKTCRDVF CPGSSTCVVDQTNNAYCVTCNRICPEPASSEQYLCGNDGVTYSSACHLRKATCLLGRSIGLAYEGKCIKAKSCEDIQCTGGKKCLWDFKVGRGRCSLCDELCPDSKSDEPVCASDNATYASECAMKEAACSSGVLLEVKHSGSCNSISEDTEEEEEDEDQDYSFPISSILEW SEQ ID NO: 30: Human follistatin 315 fused to a mouse IgG2A Fc fragment (underlined), in which 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 shown in bold. MVRARHQPGGLCLLLLLLCQFMEDRSAQAGNCWLRQAKNGRCQVLYKTELSKEECCSTGRLSTSWTEEDVNDNTLFKWMIFNGGAPNCIPCKETCENVDCGPGSTCVVDQTNNPRCVCAPDCSNITWKGPVCGLDGKTYRNECALLKARCKEQPELEVQYQGRCKKTCRDVF CPGSSTCVVDQTNNAYCVTCNRICPEPASSEQYLCGNDGVTYSSACHLRKATCLLGRSIGLAYEGKCIKAKSCEDIQCTGGKKCLWDFKVGRGRCSLCDELCPDSKSDEPVCASDNATYASECAMKEAACSSGVLLEVKHSGSCNSISEDTEEEEEDEDQDYSFPISSILEW PRGPTIKPCPPCKCPAPNLLGGPSVFIFPPKIKDVLMISLSPIVTCVVVDVSEDDPDVQISWFVNNVEVHTAQTQTHREDYNSTLRVVSALPIQHQDWMSGKEFKCKVNNKDLPAPIERTISKPKGSVRAPQVYVLPPPEEEMTKKQVTLTCMVTDFMPEDIYVEWTNNGKTELNYKNTEPVLDSDGSYFMYSKLRVEKKNWVERNSYSCSVVHEGLHNHHTTKSFSRTPGK SEQ ID NO: 31: Polynucleotide sequence encoding human follistatin 315 fused to a mouse IgG2A Fc fragment, in which the heparin binding sequence (HBS) of FSD1 has been replaced by a structurally related sequence. SEQ ID NO: 32 Human FSD1 as a single entity produced in E. coli contains an initial methionine (M) amino acid. METCENVDCGPGKKCRMNKKNKPRCVCAPDCSNITWKGPVCGLDGKTYRNECALLKARCKEQPELEVQYQGRCK SEQ ID NO: 33: Polynucleotide encoding human FSD1 as a single entity produced in E. coli, including an initial methionine (M) amino acid ATGGAGACATGCGAGAACGTGGATTGTGGACCAGGCAAGAAGTGCCGGATGAACAAGAAGAACAAGCCCAGATGCGTGTGCGCTCCTGACTGCAGCAACATCACCTGGAAGGGACCCGTGTGCGGCCTGGATGGCAAGACATACCGGAATGAGTGCGCCCTGCTGAAGGCTAGGTGTAAGGAGCAGCCTGAGCTGGAGGTGCAGTATCAGGGCCGGTGCAAG SEQ ID NO: 34: Anti-vimentin nanobody. Expressed in E. coli and therefore includes the initiating methionine (M) amino acid. The C-terminal thrombin site and his tag are double underlined. MQVQLVESGGGLVQSGGSLTLTCAASGFTFSAASMRWVRQVPGKGLEWVATIDGTGANSYYSESAKGRFTISRDNARNTLYLQMNNLKPDDTAVYYCANFGRNYWGKGTQVTVSS LVPRGSHHHHHH SEQ ID NO: 35: Polynucleotide encoding anti-vimentin-nanobody, expressed in E. coli and therefore includes the initial methionine (M) amino acid. ATGCAAGTACAGCTAGTTGAATCAGGTGGAGGGTTGGTGCAAAGCGGTGGTTCTCTGACCTTGACGTGCGCGGCGTCTGGCTTCACCTTCAGCGCTGCCTCCATGCGTTGGGTACGTCAGGTGCCGGGTAAGGGCCTTGAGTGGGTCGCAACCATCGACGGTACTGGTGCAAACAGCTATTACAGCGAGT CGGCGAAAGGCCGTTTTACGATCAGCCGTGATAATGCGCGTAACACCTTATATCTGCAGATGAATAACCTGAAACCGGATGACACCGCAGTTTACTACTGCGCGAACTTTGGTCGCAACTATTGGGGTAAGGGCACGCAAGTGACCGTTTCCTCCCTAGTACCCAGGGGTAGCCACCACCACCACCACCAC SEQ ID NO: 36: Heparin-binding domain of human FSD1 KKCRMNKKNKPR SEQ ID NO: 37: Mouse IgG2A Fc PRGPTIKPCPPCKCPAPNLLGGPSVFIFPPKIKDVLMISLSPIVTCVVVDVSEDDPDVQISWFVNNVEVHTAQTQTHREDYNSTLRVVSALPIQHQDWMSGKEFKCKVNNKDLPAP IERTISKPKGSVRAPQVYVLPPPEEEMTKKQVTLTCMVTDFMPEDIYVEWTNNGKTELNYKNTEPVLDSDGSYFMYSKLRVEKKNWVERNSYSCSVVHEGLHNHHTTKSFSRTPGK SEQ ID NO: 38: Mouse IgG2A Fc knob-in-hole A chain PRGPTIKPCPPCKCPAPNLLGGPSVFIFPPKIKDVLMISLSPIVTCVVVDVSEDDPDVQISWFVNNVEVHTAQTQTHREDYNSTLRVVSALPIQHQDWMSGKEFKCKVNNKDLPAP IERTISKPKGSVRAPQVYVLPPPEEEMTEKQVTLTCMVTDFMPEDIYVEWTNNGKTELNYKNTEPVLDSDGSYFMYSWLRVEKKNWVERNSYSCSVVHEGLHNHHTTKSFSRTPGK SEQ ID NO: 39: Mouse IgG2A Fc knob-in-hole B chain PRGPTIKPCPPCKCPAPNLLGGPSVFIFPPKIKDVLMISLSPIVTCVVVDVSEDDPDVQISWFVNNVEVHTAQTQTHREDYNSTLRVVSALPIQHQDWMSGKEFKCKVNNKDLPAP IERTISKPKGSVRAPRVYVLPPPEEEMTKKQVTLTCMVTDFMPEDIYVEWTNNGKTELNYKNTEPVLVSDGSYTMYSKLRVEKKNWVERNSYSCSVVHEGLHNHHTTKSFSRTPGK SEQ ID NO: 40: Human VGFR(1 / 2) SDTGRPFVEMYSEIPEIIIHMTEGRELVIPCRVTSPNITVTLKKFPLDTLIPDGKRIIWDSRKGFIISNATYKEIGLLTCEATVNGHLYKTNYLTHRQTNTIIDVVLSPSHGIELSVGEKLVLNCTARTELNVGIDFNWEYPSSKHQHKKLVNRDLKTQSGSEMKKFLSTLTIDGVTRSDQGLYTCAASSGLMTKKNSTFVRVHEK SEQ ID NO: 41: Human FSD2 KTCRDVFCPGSSTCVVDQTNNAYCVTCNRICPEPASSEQYLCGNDGVTYSSACHLRKATCLLGRSIGLAYEGKCI SEQ ID NO: 42: Human FSD3 KAKSCEDIQCTGGKKCLWDFKVGRGRCSLCDELCPDSKSDEPVCASDNATYASECAMKEAACSSGVLLEVKHSGSCN SEQ ID NO: 43: Human follistatin N-terminal domain GNCWLRQAKNGRCQVLYKTELSKEECCSTGRLSTSWTEEDVNDNTLFKWMIFNGGAPNCIPCK SEQ ID NO: 44: Human activin A (inhibitory beta A chain) (signal peptide shown in bold) MPLLWLRGFLLASCWIIVRSSTPPGSEGHSAAPDCPSCALAALPKDVPNSQPEMVEAVKKHILNMLHLKKRPDVTQPVPKAALLNAIRKLHVGKVGENGYVEIEDD IGRRAEMNELMEQTSEIITFAESGTARKTLHFEISKEGSDLSVVERAEVWLFLKVPKANRTRTKVTIRLFQQQKHPQGSLDTGEEAEEVGLKGERSELLLSEKVVDA RKSTWHVFPVSSSIQRLLDQGKSSLDVRIACEQCQESGASLVLLGKKKKEEEGEGKKKGGGEGGAGADEEKEQSHRPFLMLQARQSEDHPHRRRRRGLECDGKVN ICCKKQFFVSFKDIGWNDWIIAPSGYHANYCEGECPSHIAGTSGSSLSFHSTVINHYRMRGHSPFANLKSCCVPTKLRPMSMLYYDDGQNIIKKDIQNMIVEECGCS SEQ ID NO: 45: Myostatin (also known as growth differentiation factor 8) (signal peptide shown in bold) MQKLQLCVYIYLFMLIVAGPVDLNENSEQKENVEKEGLCNACTWRQNTKSSRIEAIKIQILSKLRLETAPNISKDVIRQLLPKAPPLRELIDQYDVQRDDSSDGSLEDDDYHATTETIITMPTESDFLMQVDGKPKCCFFKFSSKIQYNKVVKAQLWIYLRPVETPTTVFVQILRLIKPMKDGTRYT GIRSLKLDMNPGTGIWQSIDVKTVLQNWLKQPESNLGIEIKALDENGHDLAVTFPGPGEDGLNPFLEVKVTDTPKRSRRDFGLDCDEHSTESRCCRYPLTVDFEAFGWDWIIAPKRYKANYCSGECEFVFLQKYPHTHLVHQANPRGSAGPCCTPTKMSPINMLYFNGKEQIIYGKIPAMVVDRCGCS SEQ ID NO: 46: Growth Differentiation Factor 11 (GDF11) MVLAAPLLLGFLLLALELRPRGEAAEGPAAAAAAAAAAAAAGVGGERSSRPAPSVAPEPDGCPVCVWRQHSRELRLESIKSQILSKLRLKEAPNISREVVKQLLPKAPPLQQILDLHDFQGDALQPEDFLEEDEYHATTETVISMAQETDPAVQTDGSPLCCHFHFSPKVMFTKVLKAQLWVYLRPVPRPATVYLQILRLKPL TGEGTAGGGGGGRRHIRIRSLKIELHSRSGHWQSIDFKQVLHSWFRQPQSNWGIEINAFDPSGTDLAVTSLGPGAEGLHPFMELRVLENTKRSRRNLGLDCDEHSSESRCCRYPLTVDFEAFGWDWIIAPKRYKANYCSGQCEYMFMQKYPHTHLVQQANPRGSAGPCCTPTKMSPINMLYFNDKQQIIYGKIPGMVVDRCGCS SEQ ID NO: 47: Human FSD1 (Q124A) ETCENVDCGPGKKCRMNKKNKPRCVCAPDCSNITWKGPVCGLDGKTYRNECALLKARCKEAPELEVQYQGRCK SEQ ID NO: 48: Human FSD1 (Q124A) as a single entity produced in E. coli, including the initial methionine (M) amino acid METCENVDCGPGKKCRMNKKNKPRCVCAPDCSNITWKGPVCGLDGKTYRNECALLKARCKEAPELEVQYQGRCK SEQ ID NO: 49: Polynucleotide sequence encoding human FSD1 (Q124A) as a single entity produced in E. coli, including the initial methionine (M) amino acid ATGGAGACATGCGAGAACGTGGATTGTGGACCAGGCAAGAAGTGCCGGATGAACAAGAAGAACAAGCCCAGATGCGTGTGCGCTCCTGACTGCAGCAACATCACCTGGAAGGGACCCGTGTGCGGCCTGGATGGCAAGACATACCGGAATGAGTGCGCCCTGCTGAAGGCTAGGTGTAAGGAGGCGCCTGAGCTGGAGGTGCAGTATCAGGGCCGGTGCAAG SEQ ID NO: 50: Human FSD1 (E126A) ETCENVDCGPGKKCRMNKKNKPRCVCAPDCSNITWKGPVCGLDGKTYRNECALLKARCKEQPALEVQYQGRCK SEQ ID NO: 51: Human FSD1 (E126A) as a single entity produced in E. coli, including the initial methionine (M) amino acid METCENVDCGPGKKCRMNKKNKPRCVCAPDCSNITWKGPVCGLDGKTYRNECALLKARCKEQPALEVQYQGRCK SEQ ID NO: 52: Polynucleotide sequence encoding human FSD1 (E126A) as a single entity produced in E. coli, including the initial methionine (M) amino acid ATGGAGACATGCGAGAACGTGGATTGTGGACCAGGCAAGAAGTGCCGGATGAACAAGAAGAACAAGCCCAGATGCGTGTGCGCTCCTGACTGCAGCAACATCACCTGGAAGGGACCCGTGTGCGGCCTGGATGGCAAGACATACCGGAATGAGTGCGCCCTGCTGAAGGCTAGGTGTAAGGAGCAGCCTGCGCTGGAGGTGCAGTATCAGGGCCGGTGCAAG SEQ ID NO: 53: Human FSD1 (Q124A, E126A) ETCENVDCGPGKKCRMNKKNKPRCVCAPDCSNITWKGPVCGLDGKTYRNECALLKARCKEAPALEVQYQGRCK SEQ ID NO: 54: Human FSD1 (Q124A, E126A) as a single entity produced in E. coli, including the initiating methionine (M) amino acid METCENVDCGPGKKCRMNKKNKPRCVCAPDCSNITWKGPVCGLDGKTYRNECALLKARCKEAPALEVQYQGRCK SEQ ID NO: 55: Polynucleotide sequence encoding human FSD1 (Q124A, E126A) as a single entity produced in E. coli, including the initiating methionine (M) amino acid ATGGAGACATGCGAGAACGTGGATTGTGGACCAGGCAAGAAGTGCCGGATGAACAAGAAGAACAAGCCCAGATGCGTGTGCGCTCCTGACTGCAGCAACATCACCTGGAAGGGACCCGTGTGCGGCCTGGATGGCAAGACATACCGGAATGAGTGCGCCCTGCTGAAGGCTAGGTGTAAGGAGGCGCCTGCGCTGGAGGTGCAGTATCAGGGCCGGTGCAAG SEQ ID NO: 56: VGFR(1 / 2)-mFc-FSD1(Q124A). Serum albumin preproprotein signal peptide is shown in bold, mouse IgG2A is underlined, and FSD1(Q124A) is double underlined. MKWVTFISLLFLFSSAYSSDTGRPFVEMYSEIPEIIIHMTEGRELVIPCRVTSPNITVTLKKFPLDTLIPDGKRIIWDSRKGFIISNATYKEIGLLTCEATVNGHLYKTNYL THRQTNTIIDVVLSPSHGIELSVGEKLVLNCTARTELNVGIDFNWEYPSSKHQHKKLVNRDLKTQSGSEMKKFLSTLTIDGVTRSDQGLYTCAASSGLMTKKNSTFVRVHEK PRGPTIKPCPPCKCPAPNLLGGPSVFIFPPKIKDVLMISLSPIVTCVVVDVSEDDPDVQISWFVNNVEVHTAQTQTHREDYNSTLRVVSALPIQHQDWMSGKEFKCKVNNKDLPAPIERTISKPKGSVRAPQVYVLPPPEEEMTKKQVTLTCMVTDFMPEDIYVEWTNNGKTELNYKNTEPVLDSDGSYFMYSKLRVEKKNWVERNSYSCSVVHEGLHNHHTTKSFSRTPGKETCENVDCGPGKKCRMNKKNKPRCVCAPDCSNITWKGPVCGLDGKTYRNECALLKARCKEAPELEVQYQGRCK SEQ ID NO: 57: Polynucleotide sequence encoding VGFR(1 / 2)-mFc-FSD1(Q124A) SEQ ID NO: 58: VGFR(1 / 2)-mFc-FSD1(E126A). Serum albumin preproprotein signal peptide is shown in bold, mouse IgG2A is underlined, and FSD1(E126A) is double underlined. MKWVTFISLLFLFSSAYSSDTGRPFVEMYSEIPEIIIHMTEGRELVIPCRVTSPNITVTLKKFPLDTLIPDGKRIIWDSRKGFIISNATYKEIGLLTCEATVNGHLYKTNYL THRQTNTIIDVVLSPSHGIELSVGEKLVLNCTARTELNVGIDFNWEYPSSKHQHKKLVNRDLKTQSGSEMKKFLSTLTIDGVTRSDQGLYTCAASSGLMTKKNSTFVRVHEK PRGPTIKPCPPCKCPAPNLLGGPSVFIFPPKIKDVLMISLSPIVTCVVVDVSEDDPDVQISWFVNNVEVHTAQTQTHREDYNSTLRVVSALPIQHQDWMSGKEFKCKVNNKDLPAPIERTISKPKGSVRAPQVYVLPPPEEEMTKKQVTLTCMVTDFMPEDIYVEWTNNGKTELNYKNTEPVLDSDGSYFMYSKLRVEKKNWVERNSYSCSVVHEGLHNHHTTKSFSRTPGKETCENVDCGPGKKCRMNKKNKPRCVCAPDCSNITWKGPVCGLDGKTYRNECALLKARCKEQPALEVQYQGRCK SEQ ID NO: 59: Polynucleotide sequence encoding VGFR(1 / 2)-mFc-FSD1(E126A) SEQ ID NO: 60: VGFR(1 / 2)-mFc-FSD1(Q124A, E126A). Serum albumin preproprotein signal peptide is shown in bold, mouse IgG2A is underlined, and FSD1(Q124A, E126A) is double underlined. MKWVTFISLLFLFSSAYSSDTGRPFVEMYSEIPEIIIHMTEGRELVIPCRVTSPNITVTLKKFPLDTLIPDGKRIIWDSRKGFIISNATYKEIGLLTCEATVNGHLYKTNYL THRQTNTIIDVVLSPSHGIELSVGEKLVLNCTARTELNVGIDFNWEYPSSKHQHKKLVNRDLKTQSGSEMKKFLSTLTIDGVTRSDQGLYTCAASSGLMTKKNSTFVRVHEK PRGPTIKPCPPCKCPAPNLLGGPSVFIFPPKIKDVLMISLSPIVTCVVVDVSEDDPDVQISWFVNNVEVHTAQTQTHREDYNSTLRVVSALPIQHQDWMSGKEFKCKVNNKDLPAPIERTISKPKGSVRAPQVYVLPPPEEEMTKKQVTLTCMVTDFMPEDIYVEWTNNGKTELNYKNTEPVLDSDGSYFMYSKLRVEKKNWVERNSYSCSVVHEGLHNHHTTKSFSRTPGKETCENVDCGPGKKCRMNKKNKPRCVCAPDCSNITWKGPVCGLDGKTYRNECALLKARCKEAPALEVQYQGRCK SEQ ID NO: 61: Polynucleotide sequence encoding VGFR(1 / 2)-mFc-FSD1 (Q124A, E126A) SEQ ID NO: 62: CBG-mFc-FSD1. Serum albumin preproprotein signal peptide is shown in bold, mouse IgG2A is underlined, and FSD1 is double underlined. MKWVTFISLLFLFSSAYSMDPNAAYVNMSNHHRGLASANVDFAFSLYKHLVALSPKKNIFISPVSISMALAMLSLGTCGHTRAQLLQGLGFNLTERSETEIHQGFQHLHQLFAKSDTSLEMTMGNALFLDGSLELLESFSADIKHYYESEVLAMNFQDWATASRQINSYVKNKTQGKIVDLFSGLDSPAILVLVNYIFFK GTWTQPFDLASTREENFYVDETTVVKVPMMLQSSTISYLHDSELPCQLVQMNYVGNGTVFFILPDKGKMNTVIAALSRDTINRWSAGLTSSQVDLYIPKV TISGVYDLGDVLEEMGIADLFTNQANFSRITQDAQLKSSKVVHKAVLQLNEEGVDTAGSTGVTLNLTSKPIILRFNQPFIIMIFDHFTWSSLFLARVMNPV PRGPTIKPCPPCKCPAPNLLGGPSVFIFPPKIKDVLMISLSPIVTCVVVDVSEDDPDVQISWFVNNVEVHTAQTQTHREDYNSTLRVVSALPIQHQDWMSGKEFKCKVNNKDLPAPIERTISKPKGSVRAPQVYVLPPPEEEMTKKQVTLTCMVTDFMPEDIYVEWTNNGKTELNYKNTEPVLDSDGSYFMYSKLRVEKKNWVERNSYSCSVVHEGLHNHHTTKSFSRTPGKETCENVDCGPGKKCRMNKKNKPRCVCAPDCSNITWKGPVCGLDGKTYRNECALLKARCKEQPELEVQYQGRCK SEQ ID NO: 63: Polynucleotide sequence encoding CBG-mFc-FSD1. SEQ ID NO: 64: FSD1 (KTC) (Q124A) ETCENVDCGPGKKCRMNKKNKPRCVCAPDCSNITWKGPVCGLDGKTYRNECALLKARCKEAPELEVQYQGRCKKTC SEQ ID NO: 65: FSD1 (KTC) (E126A) ETCENVDCGPGKKCRMNKKNKPRCVCAPDCSNITWKGPVCGLDGKTYRNECALLKARCKEQPALEVQYQGRCKKTC SEQ ID NO: 66: FSD1 (KTC) (Q124A, E126A) ETCENVDCGPGKKCRMNKKNKPRCVCAPDCSNITWKGPVCGLDGKTYRNECALLKARCKEAPALEVQYQGRCKKTC SEQ ID NO: 67: Serum albumin preproprotein signal peptide MKWVTFISLLFLFSSAYS item

[0299] 1. A fusion protein or conjugated protein, (i) one or more polypeptide(s) (P1) comprising or consisting of at least one FSD1 domain comprising or consisting of a polypeptide having at least 70%, such as 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; (ii) Fusion or conjugated proteins comprising a therapeutic or diagnostic agent.

[0300] 2. (i) one or more polypeptide(s) (P1) comprising or consisting of at least one FSD1 domain having at least 70%, such as 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; (ii) a therapeutic or diagnostic agent;

[0301] 3. (i) one or more polypeptide(s) (P1) comprising or consisting of at least one FSD1 domain comprising or consisting of a polypeptide having at least 70%, such as 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; (ii) a therapeutic or diagnostic agent comprising or consisting of an immunoglobulin domain; 2. The fusion protein or conjugate protein of item 1, wherein the one or more polypeptide(s) (P1)(i) is / are linked to the C-terminus of the immunoglobulin Fc domain.

[0302] 4. The fusion protein or conjugated protein according to item 3, wherein the immunoglobulin is selected from the group consisting of IgG, IgA, IgM, IgE, and IgD.

[0303] 5. (i) one or more polypeptide(s) (P1) comprising or consisting of at least one FSD1 domain having at least 70%, such as 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; (ii) a therapeutic or diagnostic agent comprising or consisting of an immunoglobulin domain; 10. The fusion protein or conjugate protein of any one of the preceding items, wherein said one or more polypeptide(s) (P1)(i) is / are attached to the C-terminus of said immunoglobulin Fc domain, preferably said immunoglobulin is an IgG.

[0304] 6. The fusion protein or conjugate protein according to any one of items 3 to 5, wherein the immunoglobulin is an IgG and the IgG Fc domain comprises or consists of an IgG Fc homodimer or an IgG Fc heterodimer.

[0305] 7. The fusion protein or conjugate protein according to item 6, wherein the fusion protein or conjugate protein comprises or consists of an IgG Fc homodimer, each monomer of the IgG Fc homodimer being linked to the C-terminus of at least one polypeptide (P1) comprising or consisting of at least one FSD1 domain comprising or consisting of a polypeptide having at least 70%, such as at least 80%, for example at least 90%, such as at least 95%, for example at least 99% sequence identity to SEQ ID NO: 1, and each monomer of the IgG Fc homodimer being linked to the N-terminus of the same or a different therapeutic or diagnostic moiety.

[0306] 8. the fusion protein or conjugate protein comprises or consists of an IgG Fc homodimer, each monomer of the IgG Fc homodimer being linked to the C-terminus of at least one polypeptide (P1)(i) comprising at least one FSD1 domain comprising or consisting of a polypeptide having at least 70%, such as at least 80%, for example at least 90%, such as at least 95%, for example at least 99% sequence identity to SEQ ID NO: 1; 10. The fusion or conjugated protein of any one of the preceding items, wherein each monomer of said IgG Fc homodimer is N-terminally linked to the same or different therapeutic or diagnostic moieties.

[0307] 9. The fusion protein or conjugate protein according to item 6, wherein the fusion protein or conjugate protein comprises or consists of an IgG Fc heterodimer, each monomer of the IgG Fc heterodimer being linked to the C-terminus of at least one polypeptide (P1) comprising or consisting of at least one FSD1 domain comprising or consisting of a polypeptide having at least 70%, such as at least 80%, for example at least 90%, such as at least 95%, for example at least 99% sequence identity to SEQ ID NO: 1, and each monomer of the IgG Fc heterodimer being linked to the N-terminus of the same or a different therapeutic or diagnostic moiety.

[0308] 10. The fusion protein or conjugate protein comprises or consists of an IgG Fc heterodimer, wherein each monomer of the IgG Fc heterodimer is linked to the C-terminus of at least one polypeptide (P1)(i) comprising at least one FSD1 domain comprising or consisting of a polypeptide having at least 70%, such as 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; 10. The fusion or conjugated protein of any one of the preceding items, wherein each monomer of said IgG Fc heterodimer is N-terminally linked to the same or different therapeutic or diagnostic moieties.

[0309] 11. (i) one or more polypeptide(s) (P1)(i) comprising or consisting of at least one FSD1 domain comprising or consisting of a polypeptide having at least 70%, such as 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; (ii) a therapeutic or diagnostic agent, the therapeutic or diagnostic agent comprising or consisting of an IgG Fc heterodimer; 10. The fusion protein or conjugate protein of any one of the preceding items, wherein at least one said monomer of said IgG Fc heterodimer is attached to the N-terminus of said at least one polypeptide(s) (P1)(i), and wherein at least one said monomer of said IgG Fc heterodimer is attached to the N-terminus of the same or different therapeutic or diagnostic moiety.

[0310] 12. The fusion protein or conjugate protein according to any one of the preceding items, wherein at least one said monomer of said IgG Fc heterodimer is linked to the N-terminus of at least one of one or more polypeptide(s) (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, comprising or consisting of at least one FSD1 domain comprising or consisting of a polypeptide having at least 70%, such as at least 80%, for example at least 90%, such as at least 95%, for example at least 99% sequence identity to SEQ ID NO: 1.

[0311] 13. The therapeutic or diagnostic agent of any one of items 1-3, or the fusion protein or conjugated protein of any one of the preceding items, wherein at least one therapeutic or diagnostic moiety, the same as or different from that of any one of the preceding items, is one or more cytokine receptors.

[0312] 14. The therapeutic or diagnostic agent of any one of the preceding paragraphs, or the fusion protein or conjugated protein of any one of the preceding paragraphs, wherein at least one therapeutic or diagnostic moiety, the same or different from that of any one of the preceding paragraphs, comprises or consists of the ligand binding domain of TNFR2.

[0313] 15. The fusion protein or conjugate protein of any one of the preceding paragraphs, wherein the therapeutic or diagnostic agent of any one of the preceding paragraphs, or at least one therapeutic or diagnostic moiety the same as or different from that of any one of the preceding paragraphs, comprises or consists of the ligand binding domain of one or more growth factor receptors.

[0314] 16. The therapeutic or diagnostic agent of any one of the preceding paragraphs, or the fusion protein or conjugate protein of any one of the preceding paragraphs, wherein at least one therapeutic or diagnostic moiety, the same as or different from that of any one of the preceding paragraphs, comprises or consists of the ligand binding domain of VGFR1.

[0315] 17. The therapeutic or diagnostic agent of any one of the preceding paragraphs, or the fusion protein or conjugate protein of any one of the preceding paragraphs, wherein at least one therapeutic or diagnostic moiety, the same as or different from that of any one of the preceding paragraphs, comprises or consists of the ligand binding domain of VGFR2.

[0316] 18. The fusion protein or conjugate protein according to any one of the preceding items, wherein said one or more polypeptide(s) (P1) comprising or consisting of at least one FSD1 domain comprise or consist of a polypeptide having at least 70% sequence identity to SEQ ID NO: 26, such as at least 80% identity, such as at least 90% identity, for example at least 95% identity, such as at least 99% identity.

[0317] 19. (i) one or more polypeptide(s) (P1) comprising or consisting of at least one FSD1 domain comprising or consisting of a polypeptide having at least 70%, such as 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; (ii) a therapeutic or diagnostic agent, 2. The fusion protein or conjugated protein of item 1, wherein the one or more polypeptide(s) (P1)(i) and the therapeutic or diagnostic agent are linked by a covalent or non-covalent interaction.

[0318] 20. The fusion protein or conjugated protein according to item 19, wherein the one or more polypeptide(s) (P1)(i) and the therapeutic or diagnostic agent (ii) are covalently linked using click chemistry.

[0319] 21. The fusion protein or conjugated protein according to item 19, wherein the one or more polypeptide(s) (P1)(i) and the therapeutic or diagnostic agent (ii) are linked by a streptavidin-biotin non-covalent interaction.

[0320] 22. The fusion protein or conjugate protein of item 19, wherein the one or more polypeptide(s) (P1)(i) and the therapeutic or diagnostic agent (ii) are bound by a nanobody, e.g., an anti-Fc nanobody, having affinity for the therapeutic or diagnostic agent.

[0321] 23. (i) one or more polypeptide(s) (P1) comprising or consisting of at least one FSD1 domain having at least 70%, such as 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; (ii) a therapeutic or diagnostic agent, 10. The fusion protein or conjugated protein of any one of the preceding items, wherein the one or more polypeptide(s) (P1)(i) and the therapeutic or diagnostic agent (ii) are covalently linked, for example by click chemistry, or are linked by a non-covalent interaction such as streptavidin-biotin or a nanobody with affinity for the therapeutic or diagnostic agent (ii).

[0322] 24. The fusion protein or conjugated 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.

[0323] 25. The fusion protein or conjugated protein according to item 24, wherein the full-length antibody is a bispecific therapeutic antibody.

[0324] 26. The fusion protein or conjugate protein according to any one of the preceding items, wherein the one or more polypeptide(s) (P1)(i) comprises or 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, for example at least 99% similarity, to a sequence selected from the group consisting of SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:64, SEQ ID NO:65, and SEQ ID NO:66.

[0325] 27. The fusion protein or conjugate protein according to any one of the preceding items, wherein said one or more polypeptide(s) (P1)(i) comprises or consists of a sequence having at least 70% similarity, such as at least 80% similarity, for example at least 90% similarity, such as at least 95% similarity, for example at least 99% similarity, to a sequence selected from the group consisting of SEQ ID NO: 23 and SEQ ID NO: 24.

[0326] 28. The fusion protein or conjugate protein according to any one of the preceding items, wherein the one or more polypeptide(s) (P1) are encoded by a sequence comprising or consisting of a sequence having at least 70% similarity to SEQ ID NO: 25, such as at least 80% similarity, for example at least 90% similarity, such as at least 95% similarity, for example at least 99% similarity.

[0327] 29. A fusion protein or conjugate protein described in any one of the preceding items, wherein the one or more polypeptide(s) (P1)(i) does not neutralize the activity of myostatin (SEQ ID NO: 45), GDF11 (SEQ ID NO: 46), and / or activin A (SEQ ID NO: 44).

[0328] 30. The fusion protein or conjugated protein enhances the activity of myostatin (SEQ ID NO: 45), GDF11 (SEQ ID NO: 46), and / or activin A (SEQ ID NO: 44) by a maximum of at least 5 nM, such as at least 7.5 nM, for example at least 8 nM, such as at least 8.2 nM, for example at least 8.4 nM, such as at least 8.6 nM, for example at least 8.8 nM, such as at least 9 nM, for example at least 10 nM, such as at least 12 nM, for example at least 15 nM, such as at least 20 nM, for example at least 30 nM, such as at least 40 nM, for example at least 50 nM, such as at least 60 nM, for example at least 61 nM, such as at least 62 nM, for example at least 63 nM, such as at least 64 nM, for example at least 64 nM, such as at least 65 nM, for example at least 70 nM, such as at least 80 nM. 5. The fusion protein or conjugated protein of any one of the preceding items, wherein the fusion protein or conjugated protein does not neutralize in a concentration range of 100 nM, such as at least 100 nM, for example at least 150 nM, such as at least 200 nM, for example at least 500 nM, such as at least 600 nM, for example at least 700 nM, such as at least 750 nM, for example at least 800 nM, such as at least 820 nM, for example at least 821 nM, such as at least 822 nM, for example at least 830 nM, such as at least 1 μM, for example at least 2 μM, such as at least 3 μM, for example at least 4 μM, such as at least 5 μM, for example at least 6 μM, such as at least 7 μM, for example at least 8 μM, such as at least 9 μM, for example at least 10 μM, wherein said neutralization or absence is measured by a reporter gene bioassay of Smad2 / 3 phosphorylation signaling.

[0329] 31. A fusion protein or conjugate protein according to any one of the preceding items, wherein the fusion protein or conjugate protein does not neutralise the activity of myostatin, GDF11 and / or activin A in a concentration range of up to at least 5nM, such as at least 7.5nM, for example at least 8nM, at least 8.2nM, such as at least 8.4nM, for example at least 8.6nM, such as at least 8.8nM, for example at least 9nM, such as at least 10nM, for example at least 12nM, at least 15nM, such as at least 20nM, for example at least 30nM, such as at least 40nM, for example at least 50nM, such as at least 80nM, for example at least 100nM, such as at least 150nM, for example at least 200nM.

[0330] 32. said one or more polypeptide(s) (P1)(i) increase the activity of myostatin (SEQ ID NO: 45), GDF11 (SEQ ID NO: 46), and / or activin A (SEQ ID NO: 44) by up to at least 5 nM, such as at least 7.5 nM, for example at least 8 nM, such as at least 8.2 nM, for example at least 8.4 nM, such as at least 8.6 nM, for example at least 8.8 nM, such as at least 9 nM, for example at least 10 nM, such as at least 12 nM, for example at least 15 nM, such as at least 20 nM, for example at least 30 nM, such as at least 40 nM, for example at least 50 nM, such as at least 60 nM, for example at least 61 nM, such as at least 62 nM, for example at least 63 nM, such as at least 64 nM, for example at least 64 nM, such as at least 65 nM, for example at least 70 nM, 5. The fusion protein or conjugate protein of any one of the preceding items, wherein the fusion protein or conjugate protein does not neutralize in a concentration range of 80nM, such as at least 100nM, for example at least 150nM, such as at least 200nM, for example at least 500nM, such as at least 600nM, for example at least 700nM, such as at least 750nM, for example at least 800nM, such as at least 820nM, for example at least 821nM, such as at least 822nM, for example at least 830nM, such as at least 1μM, for example at least 2μM, such as at least 3μM, for example at least 4μM, such as at least 5μM, for example at least 6μM, such as at least 7μM, for example at least 8μM, such as at least 9μM, for example at least 10μM, wherein said neutralization or absence is measured by a reporter gene bioassay of Smad2 / 3 phosphorylation signaling.

[0331] 33. A fusion protein or conjugate protein according to any one of the preceding items, wherein said one or more polypeptide(s) (P1)(i) does not neutralise the activity of myostatin, GDF11 and / or activin A in a concentration range of up to at least 5 nM, such as at least 7.5 nM, for example at least 8 nM, at least 8.2 nM, such as at least 8.4 nM, for example at least 8.6 nM, such as at least 8.8 nM, for example at least 9 nM, such as at least 10 nM, for example at least 12 nM, at least 15 nM, such as at least 20 nM, for example at least 30 nM, such as at least 40 nM, for example at least 50 nM, such as at least 80 nM, for example at least 100 nM, such as at least 150 nM, for example at least 200 nM.

[0332] 34. The fusion protein or conjugated protein of any one of the preceding items, wherein the one or more polypeptide(s) (P1)(i) binds heparan sulfate.

[0333] 35. A fusion protein or conjugate protein according to any one of the preceding items, wherein the one or more polypeptide(s) (P1)(i) comprising at least one FSD1 domain are encoded by a sequence comprising or consisting of a sequence having at least 70% identity, such as at least 80% identity, for example at least 90% identity, such as at least 95% identity, for example at least 99% identity to SEQ ID NO: 33.

[0334] 36. The fusion protein or conjugate protein of any one of the preceding items, wherein the one or more polypeptide(s) (P1)(i) comprising at least one FSD1 domain comprises or consists of a sequence having at least 100% identity to SEQ ID NO: 1.

[0335] 37. The fusion protein or conjugate protein of any one of the preceding items, further comprising a linker between the one or more polypeptide(s) (P1) (i) comprising at least one FSD1 domain and the therapeutic or diagnostic agent (ii).

[0336] 38. The fusion protein or conjugated protein according to item 37, wherein the linker is a chemical linker.

[0337] 39. The fusion protein or conjugated protein of any one of the preceding paragraphs, wherein the therapeutic or diagnostic agent (ii) of any one of the preceding paragraphs, or the same or different therapeutic or diagnostic moiety as that of any one of the preceding paragraphs, is selected from the group consisting of a peptide, a protein, such as an antibody or fragment thereof, a nanobody, a glycoprotein, streptavidin, or an interleukin, a nucleic acid, and a small molecule.

[0338] 40. A fusion protein or conjugated protein according to any one of the preceding items, wherein the fusion protein or conjugated protein further comprises a detectable moiety.

[0339] 41. The fusion protein or conjugated 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.

[0340] 42. The therapeutic or diagnostic agent (ii) of any one of the preceding paragraphs, or a therapeutic or diagnostic moiety that is the same or different from any one of the preceding paragraphs, is a fusion protein or conjugated protein of any one of the preceding paragraphs that binds to a target selected from the group consisting of a differentiation antigen (CD) protein, a cytokine such as an interleukin, a growth factor such as a colony stimulating factor, an immune checkpoint protein, an angiogenic factor, a hemostatic factor, a chemotactic factor, a neurotrophic factor, an inflammatory protein, a tumor antigen, a bacterial protein, and a viral protein.

[0341] 43. The fusion protein or conjugate protein of item 42, wherein the therapeutic or diagnostic agent (ii) according to any one of the preceding items, or a therapeutic or diagnostic moiety that is the same or different from that according to any one 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.

[0342] 44. The therapeutic or diagnostic agent (ii) according to any one of the preceding paragraphs, or a therapeutic or diagnostic moiety which is the same or different from that according to any one of the preceding paragraphs, comprises or consists of a ligand binding domain of a protein having at least 70% similarity, such as at least 80% similarity, for example at least 90% similarity, such as at least 95% similarity, for example at least 99% similarity, to a sequence selected from the group consisting of SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, and SEQ ID NO: 22.

[0343] 45. The therapeutic or diagnostic agent (ii) of any one of the preceding paragraphs, or a therapeutic or diagnostic moiety that is the same or different from that of any one of the preceding paragraphs, comprising or consisting of an anti-vimentin nanobody, is a fusion protein or conjugated protein of any one of the preceding paragraphs.

[0344] 46. ​​The fusion protein according to item 45, wherein the anti-vimentin nanobody has at least 70% similarity, such as at least 80% similarity, for example at least 90% similarity, such as at least 95% similarity, for example at least 99% similarity to SEQ ID NO: 34.

[0345] 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, for example at least 90% similarity, such as at least 95% similarity, for example at least 99% similarity to SEQ ID NO: 35.

[0346] 48. A therapeutic or diagnostic agent (ii) according to any one of the preceding paragraphs, or a therapeutic or diagnostic moiety the same or different from those described in any one of the preceding paragraphs, (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) (b) an anti-vimentin nanobody having at least 70% similarity, such as at least 80% similarity, for example at least 90% similarity, such as at least 95% similarity, for example at least 99% similarity to SEQ ID NO: 34, and / or (c) A fusion protein or conjugate protein according to any one of the preceding items, comprising or consisting of a full-length antibody, such as an anti-TNFα full-length antibody.

[0347] 49. A fusion protein or conjugated protein according to any one of the preceding items, wherein the fusion protein or conjugated protein is ubiquitinated.

[0348] 50. The fusion protein or conjugated protein of any one of the preceding paragraphs, wherein the therapeutic or diagnostic agent (ii) of any one of the preceding paragraphs, or a therapeutic or diagnostic moiety that is the same or different from that of any one of the preceding paragraphs, binds to a target, and the fusion protein or conjugated protein of any one of the preceding paragraphs is capable of simultaneously binding to heparan sulfate and the target.

[0349] 51. The fusion protein or conjugated protein of any one of the preceding paragraphs, wherein the fusion protein or conjugated protein has a longer local half-life at the site of administration than the therapeutic or diagnostic agent (ii) of any one of the preceding paragraphs, or the same or different therapeutic or diagnostic moiety as described in any one of the preceding paragraphs alone.

[0350] 52. The fusion protein or conjugated protein of any one of the preceding paragraphs, wherein the fusion protein or conjugated protein enhances binding of the therapeutic or diagnostic agent (ii) of any one of the preceding paragraphs, or a therapeutic or diagnostic moiety that is the same or different from that of any one of the preceding paragraphs, to an extracellular matrix compartment compared to the therapeutic or diagnostic agent (ii) of any one of the preceding paragraphs, or a therapeutic or diagnostic moiety that is the same or different from that of any one of the preceding paragraphs, alone.

[0351] 53. A fusion protein or conjugated protein according to any one of the preceding paragraphs, wherein the fusion protein or conjugated protein enhances binding of the therapeutic or diagnostic agent (ii) according to any one of the preceding paragraphs, or a therapeutic or diagnostic moiety that is the same or different from any one of the preceding paragraphs, to a predetermined organ or tumor, compared to the therapeutic or diagnostic agent (ii) according to any one of the preceding paragraphs, or a therapeutic or diagnostic moiety that is the same or different from any one of the preceding paragraphs, alone.

[0352] 54. The fusion protein or conjugated protein according to any one of the preceding items, wherein the EC50 of said therapeutic or diagnostic agent (ii) or said same or different therapeutic or diagnostic moiety for a given organ or tumour is reduced by at least 2-fold, such as at least 5-fold, for example at least 10-fold, such as at least 20-fold, for example at least 50-fold, such as at least 80-fold, for example at least 100-fold, such as at least 250-fold, for example at least 500-fold, such as at least 1000-fold, for example at least 5000-fold, such as 10000-fold, for example 100000-fold, as compared to said therapeutic or diagnostic agent (ii) or said same or different therapeutic or diagnostic moiety alone.

[0353] 55. The fusion protein or conjugated protein according to Item 54, wherein the predetermined organ is selected from the group consisting of a musculoskeletal organ, a digestive organ, a respiratory organ, a urinary organ, a reproductive organ, an endocrine organ, a circulatory organ, a nervous system organ, a hematopoietic organ, and an integumentary organ.

[0354] 56. The fusion protein or conjugated protein according to item 55, 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, joint or subcutaneous tissue such as synovial tissue.

[0355] 57. The fusion protein or conjugated protein of any of the preceding paragraphs, wherein the fusion protein or conjugated protein enhances cellular uptake of the therapeutic or diagnostic agent (ii) of any of the preceding paragraphs, or a therapeutic or diagnostic moiety that is the same or different from any of the preceding paragraphs, compared to the therapeutic or diagnostic agent (ii) of any of the preceding paragraphs, or the same or different therapeutic or diagnostic moiety from any of the preceding paragraphs, alone.

[0356] 58. The therapeutic or diagnostic agent (ii) according to any one of the preceding paragraphs, or a therapeutic or diagnostic moiety which is the same or different from that according to any one of the preceding paragraphs, is a fusion protein or conjugated protein according to any one of the preceding paragraphs which binds 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, for example at least five polypeptides (P1)(i), comprising or consisting of at least one FSD1 domain which comprises or consists of a polypeptide having at least 70%, such as at least 80%, for example at least 90%, such as at least 95%, for example at least 99% sequence identity to SEQ ID NO: 1.

[0357] 59. The fusion protein or conjugate protein according to any one of the preceding items, wherein each monomer of said IgG Fc homodimer or Fc heterodimer binds 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), comprising or consisting of at least one FSD1 domain comprising or consisting of a polypeptide having at least 70%, such as at least 80%, for example at least 90%, such as at least 95%, for example at least 99% sequence identity to SEQ ID NO: 1.

[0358] 60. A fusion protein or conjugate protein according to any one 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, for example at least three FSD1 domains, such as at least four FSD1 domains, for example at least five FSD1 domains, wherein at least one of the at least one polypeptides (P1)(i) comprises or consists of a polypeptide having at least 70%, such as 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.

[0359] 61. The fusion protein or conjugate protein according to item 60, wherein at least two monomers of the FSD1 domain multimer are linked by a linker.

[0360] 62. A fusion protein or conjugated protein according to any one of the preceding items, wherein the fusion protein or conjugated protein comprises or consists of a polypeptide having at least 70% sequence identity, such as at least 80% sequence identity, for example at least 90% sequence identity, such as at least 95% sequence identity, for example at least 99% sequence identity, to a sequence selected from the group consisting of SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, and SEQ ID NO:9.

[0361] 63. A fusion protein or conjugated protein according to any one of the preceding items, wherein the fusion protein or conjugated protein comprises or consists of a polypeptide having at least 70% sequence identity, such as at least 80% identity, for example at least 90% identity, such as at least 95% identity, for example at least 99% identity to SEQ ID NO: 11, and a polypeptide having at least 70% sequence identity, at least 80% sequence identity, such as at least 90% identity, for example at least 95% identity, for example at least 99% identity to a sequence selected from the group consisting of SEQ ID NO: 13, SEQ ID NO: 15, and SEQ ID NO: 17.

[0362] 64. The fusion protein or conjugated protein comprises: (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) A fusion protein or conjugate protein according to any one of the preceding items, comprising or consisting of a polypeptide having at least 70% sequence identity, such as at least 80% sequence identity, for example at least 90% sequence identity, such as at least 95% identity, for example 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, for example at least 90% sequence identity, such as at least 95% identity, for example at least 99% identity, to a sequence selected from the group consisting of SEQ ID NO: 13, SEQ ID NO: 15 and SEQ ID NO: 17.

[0363] 65. The fusion protein or composite protein of any one of the preceding items, wherein the fusion protein or composite protein does not comprise any other of the follistatin domains FSD2, FSD3, and / or the N-terminal follistatin domain, preferably the fusion protein or composite protein does not comprise any of human FSD2 of SEQ ID NO: 41, human FSD3 of SEQ ID NO: 42, and human follistatin N-terminal domain of SEQ ID NO: 43.

[0364] 66. The fusion protein or composite protein of any one of the preceding items, wherein the one or more polypeptides (P1)(i) do not comprise any other of the follistatin domains FSD2, FSD3, and / or N-terminal follistatin domains, preferably the fusion protein or composite protein does not comprise any of human FSD2 of SEQ ID NO: 41, human FSD3 of SEQ ID NO: 42, and human follistatin N-terminal domain of SEQ ID NO: 43.

[0365] 67. A fusion protein or conjugate protein according to any one of the preceding items, wherein (i) said at least one FSD1 domain of said 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%, for example at least 90%, for example at least 95%, such as at least 96%, for example at least 97%, for example 98%, for example at least 99% sequence identity to those SEQ ID NOs.

[0366] 68. The fusion protein or conjugated protein according to any one of the preceding items, wherein the fusion protein or conjugated protein further comprises a PROTAC linker, preferably 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.

[0367] 69. One or more polynucleotides which, upon expression, encode a fusion protein or conjugated protein according to any one of the preceding items.

[0368] 70. 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, for example at least 99% identity, to 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.

[0369] 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, for example at least 99% identity, to a combination of SEQ ID NO: 12 and at least one polynucleotide selected from the group consisting of SEQ ID NO: 14, SEQ ID NO: 16, and SEQ ID NO: 18.

[0370] 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 conjugate protein according to any one of items 1 to 68.

[0371] 73. One or more constructs or vectors encoding the fusion protein of any of the preceding items.

[0372] 74. A host cell comprising one or more polynucleotides according to any one of items 69 to 71 or one or more constructs or vectors according to any one of items 72 to 73.

[0373] 75. The host cell according to item 74, wherein the host cell is a mammalian cell.

[0374] 76. The host cell according to item 75, wherein the host cell is a human cell.

[0375] 77. The host cell described in item 76, wherein the host cell is a human embryonic kidney 293 cell (HEK293) cell.

[0376] 78. The host cell according to item 75, wherein the host cell is a Chinese hamster ovary (CHO) cell.

[0377] 79. A composition comprising a fusion protein or conjugated 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.

[0378] 80. The composition according to item 79, wherein the composition is a pharmaceutical composition.

[0379] 81. A fusion protein or conjugated 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 for use in medicine.

[0380] 82. A fusion protein or conjugated 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 for use in treating an ophthalmic disease, a tumor disease, or an inflammatory disease such as a rheumatic disease.

[0381] 83. The use according to item 82, wherein the ophthalmic disease is neovascular age-related macular degeneration (AMD).

[0382] 84. The use according to item 82, wherein the inflammatory disease is caused by organ transplantation.

[0383] 85. The use according to any one of items 81 to 82 and 84, wherein the subject is donating an organ or receiving an organ, and the organ to be donated or transplanted is selected from the group consisting of kidney, heart, lung, bone marrow, and liver.

[0384] 86. A method for treating a disease or condition, comprising administering to a subject an effective amount of a fusion protein or conjugated 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.

[0385] 87. A method for treating an ophthalmic disease, an inflammatory disease, or a neoplastic disease, comprising administering to a subject an effective amount of a fusion protein or conjugated 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.

[0386] 88. Use of a fusion protein or conjugated 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 the treatment of a disease or condition, such as an ophthalmic disease, a neoplastic disease, or an inflammatory disease.

[0387] 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, comprising systemically administering a fusion protein or conjugated 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.

[0388] 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, comprising locally administering a fusion protein or conjugated 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.

[0389] 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 conjugated 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 intraadiposely, intra-arterially, intra-articularly, intracranially, intradermally, intralesionally, intramuscularly, intranasally, intraocularly, intraosseously, intrapericardially, intraperitoneally, intrapleurally, intraprostatically, intrarectally, intrathecally, intratracheally, intratumorally, intraumbilically, intravaginally, intravenously, intravesically, intravitreally, or via liposomes.

[0390] 92. A method for increasing the local half-life at the site of administration of a therapeutic or diagnostic agent, the method comprising obtaining a fusion protein or conjugated 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 a therapeutic or diagnostic agent (ii) according to any one of items 1 to 68, or a therapeutic or diagnostic site which is the same or different from that according to any one of items 1 to 68, comprises or consists of said therapeutic or diagnostic agent.

[0391] 93. A method for increasing the local half-life in vivo of a therapeutic or diagnostic agent at a site of administration to a subject, the method comprising administering to the subject a fusion protein or conjugated 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) according to any one of items 1 to 68, or a therapeutic or diagnostic site which is the same or different from that according to any one of items 1 to 68, comprises or consists of said therapeutic or diagnostic agent.

[0392] 94. A method for extending the local half-life of a therapeutic or diagnostic agent in vivo, comprising: a) providing therapeutic or diagnostic agents; b) obtaining a fusion protein or a conjugated protein according to any one of items 1 to 68, wherein the therapeutic or diagnostic agent in step a) is a therapeutic or diagnostic agent (ii) according to any one of items 1 to 68, obtaining said fusion protein or conjugated protein, which extends the local half-life in vivo of said therapeutic or diagnostic agent.

[0393] 95. The method of any one of items 93 to 94, wherein the fusion protein or conjugated protein, or the composition is administered to a subject suffering from an ophthalmic disease, and the fusion protein or conjugated protein, or the composition is administered intravitreally, subretinaly, or suprachoroidally.

[0394] 96. The method of item 95, wherein the ophthalmic disease is wet AMD.

[0395] 97. The method according to item 93, wherein the fusion protein or conjugated protein or the composition is administered to an organ, preferably a kidney, heart, lung, bone marrow, or liver, even more preferably to a subject donating a kidney or receiving the same, or to the organ by extracorporeal perfusion, and the therapeutic or diagnostic agent comprises or consists of a compound that reduces inflammation.

[0396] 98. The method according to any one of items 92 to 97, wherein the local half-life of the therapeutic or diagnostic agent is extended at the administration site by at least 6 hours, such as at least 12 hours, for example at least 24 hours, such as at least 48 hours, for example at least 72 hours, such as at least 96 hours, at least 120 hours, such as at least 1 week, for example at least 2 weeks, such as at least 4 weeks, for example at least 8 weeks, such as at least 3 months, for example at least 6 months, such as at least 12 months.

[0397] 99. A method for enhancing 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 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) according to any one of items 1 to 68, or a therapeutic or diagnostic moiety identical or different to that according to any one of items 1 to 68, comprises or consists of said therapeutic or diagnostic agent.

[0398] 100. A method for enhancing binding of a therapeutic or diagnostic agent to a predetermined organ, comprising administering to a subject a fusion protein or conjugated 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) according to any one of items 1 to 68, or a therapeutic or diagnostic moiety identical or different to that according to any one of items 1 to 68, comprises or consists of said therapeutic or diagnostic agent.

[0399] 101. A method for increasing the cellular uptake of a therapeutic or diagnostic agent, comprising administering to a subject a fusion protein or conjugated 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) according to any one of items 1 to 68, or a therapeutic or diagnostic moiety identical or different to that according to any one of items 1 to 68, comprises or consists of said therapeutic or diagnostic agent.

[0400] 102. A method for degrading an intracellular protein, comprising the step of binding a fusion protein or conjugated protein described in any one of the preceding paragraphs to a ligand of an E3 ubiquitin ligase, wherein (ii) a therapeutic or diagnostic agent of the fusion protein or conjugated protein binds to the intracellular protein.

[0401] 103. The method of claim 102, wherein the ligand of the E3 ubiquitin ligase is (S,R,S)-AHPC-PEG8-NHS.

[0402] 104. The method of any one of items 102 to 103, wherein the E3 ubiquitin ligase is the von Hippel-Lindau (VHL) tumor suppressor protein.

[0403] 105. The method according to any one of items 102 to 104, wherein the therapeutic or diagnostic agent (ii) is an anti-vimentin nanobody.

[0404] 106. The method of any one of items 102 to 105, wherein the fusion protein or conjugated protein is a type 1 protein described herein, preferably, the fusion protein or conjugated protein is anti-vimentin-nanobody-FSD1.

[0405] 107. The method according to any one of items 102 to 106, wherein the method is carried out in vitro, in vivo, or ex vivo.

[0406] 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 non-human animal.

[0407] 109. The use according to any one of items 81 to 85 and 88 to 91, or the method according to any one of items 92 to 107, comprising systemically administering a fusion protein or conjugated 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.

[0408] 110. The use according to any one of items 81 to 85 and 88 to 91, or the method according to any one of items 92 to 107, comprising locally administering a fusion protein or conjugated 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.

[0409] 111. The use according to any one of items 81 to 85 and 88 to 91, or the method according to any one of items 92 to 107, wherein the fusion protein or conjugated 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, 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 intraadiposely, intra-arterially, intra-articularly, intracranially, intradermally, intralesionally, intramuscularly, intranasally, intraocularly, intraosseously, intrapericardially, intraperitoneally, intrapleurally, intraprostatically, intrarectally, intrathecally, intratracheally, intratumorally, intraumbilically, intravaginally, intravenously, intravesically, intravitreally, or via liposomes.

[0410] 112. The method of any one of items 101 to 111, wherein the therapeutic or diagnostic agent is transported to the cytosol, such as the cytoskeleton or nucleus.

[0411] 113. One or more constructs or vectors encoding one or more polypeptide(s) (P1)(i) of the fusion protein or conjugate protein of any one of the preceding items.

[0412] 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(Q124E126A) of SEQ ID NO: 53, or a variant thereof having at least 70%, such as at least 80%, for example at least 90%, such as at least 95%, for example at least 96%, such as at least 97%, for example 98%, such as at least 99% sequence identity thereto.

Claims

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

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

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

4. (i) one or more polypeptide(s) (P1) consisting of at least one FSD1 domain comprising or consisting of a polypeptide having at least 70%, such as at least 80%, for example at least 90%, such as at least 95%, for example at least 99% sequence identity to SEQ ID NO: 1; (ii) a therapeutic or diagnostic agent comprising or consisting of an immunoglobulin domain; 10. The fusion protein or conjugate protein according to any one of the preceding claims, wherein said one or more polypeptide(s) (P1)(i) is / are attached to the C-terminus of said immunoglobulin Fc domain, preferably said immunoglobulin is an IgG.

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

6. said fusion protein or conjugate protein comprises or consists of an IgG Fc homodimer, each monomer of said IgG Fc homodimer linked to the C-terminus of at least one polypeptide (P1)(i) comprising at least one FSD1 domain comprising or consisting of a polypeptide having at least 70%, such as at least 80%, for example at least 90%, such as at least 95%, for example at least 99% sequence identity to SEQ ID NO: 1; 10. The fusion or conjugate protein of any one of the preceding claims, wherein each monomer of said IgG Fc homodimer is N-terminally linked to the same or different therapeutic or diagnostic moieties.

7. said fusion protein or conjugate protein comprises or consists of an IgG Fc heterodimer, each monomer of said IgG Fc heterodimer linked to the C-terminus of at least one polypeptide (P1)(i) comprising at least one FSD1 domain comprising or consisting of a polypeptide having at least 70%, such as at least 80%, for example at least 90%, such as at least 95%, for example at least 99% sequence identity to SEQ ID NO: 1; 10. The fusion or conjugate protein of any one of the preceding claims, wherein each monomer of said IgG Fc heterodimer is N-terminally linked to the same or different therapeutic or diagnostic moieties.

8. (i) one or more polypeptide(s) (P1)(i) consisting of at least one FSD1 domain comprising or consisting of a polypeptide having at least 70%, such as at least 80%, for example at least 90%, such as at least 95%, for example at least 99% sequence identity to SEQ ID NO: 1; (ii) a therapeutic or diagnostic agent, wherein the therapeutic or diagnostic agent comprises or consists of an IgG Fc heterodimer; 10. The fusion protein or conjugate protein of any one of the preceding claims, wherein at least one said monomer of said IgG Fc heterodimer is attached to the N-terminus of said at least one polypeptide(s) (P1)(i) and at least one said monomer of said IgG Fc heterodimer is attached to the N-terminus of the same or different therapeutic or diagnostic moiety.

9. 10. The fusion protein or conjugate protein of any one of the preceding claims, wherein at least one said monomer of said IgG Fc heterodimer is linked to the N-terminus of at least one of one or more polypeptide(s) (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, comprising 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.

10. 10. A fusion protein or conjugated protein according to any one of the preceding claims, wherein the therapeutic or diagnostic agent according to any one of the preceding claims, or at least one therapeutic or diagnostic moiety which is the same as or different from that of any one of the preceding claims, is one or more cytokine receptors.

11. 10. A fusion protein or conjugate protein according to any one of the preceding claims, wherein the therapeutic or diagnostic agent according to any one of the preceding claims, or at least one therapeutic or diagnostic moiety which is the same as or different from that according to any one of the preceding claims, comprises or consists of the ligand binding domain of TNFR2.

12. 12. The fusion protein or conjugated protein of any one of claims 1 to 11, wherein the therapeutic or diagnostic agent of any one of the preceding claims, or at least one therapeutic or diagnostic moiety which is the same as or different from that of any one of the preceding claims, comprises or consists of the ligand binding domain of one or more growth factor receptors.

13. 13. The fusion protein or conjugated 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 therapeutic or diagnostic moiety which is the same as or different from that of any one of the preceding claims, comprises or consists of the ligand binding domain of VGFR1.

14. 14. The fusion protein or conjugate 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 therapeutic or diagnostic moiety which is the same as or different from that of any one of the preceding claims, comprises or consists of the ligand binding domain of VGFR2.

15. 10. A fusion protein or conjugate protein according to any one of the preceding claims, wherein said one or more polypeptide(s) (P1) consisting of at least one FSD1 domain comprises or consists of a polypeptide having at least 70% sequence identity to SEQ ID NO: 26, such as at least 80% identity, such as at least 90% identity, such as at least 95% identity, for example at least 99% identity.

16. (i) one or more polypeptide(s) (P1) consisting of at least one FSD1 domain comprising or consisting of a polypeptide having at least 70%, such as at least 80%, for example at least 90%, such as at least 95%, for example at least 99% sequence identity to SEQ ID NO: 1; (ii) a therapeutic or diagnostic agent; 10. The fusion protein or conjugated protein of any one of the preceding claims, wherein the one or more polypeptide(s) (P1)(i) and the therapeutic or diagnostic agent are linked by covalent or non-covalent interactions.

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

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

19. 17. The fusion protein or conjugated protein of claim 16, wherein the one or more polypeptide(s) (P1)(i) and the therapeutic or diagnostic agent (ii) are bound by a nanobody, e.g., an anti-Fc nanobody, having affinity for the therapeutic or diagnostic agent.

20. (i) one or more polypeptide(s) (P1) comprising at least one FSD1 domain comprising a polypeptide having at least 70%, such as at least 80%, for example at least 90%, such as at least 95%, for example at least 99% sequence identity to SEQ ID NO: 1; (ii) a therapeutic or diagnostic agent; 10. The fusion protein or conjugated protein of any one of the preceding claims, wherein said one or more polypeptide(s) (P1)(i) and said therapeutic or diagnostic agent (ii) are covalently linked, for example by click chemistry, or linked by a non-covalent interaction such as streptavidin-biotin or a nanobody with affinity for said therapeutic or diagnostic agent (ii).

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

23. 10. The fusion protein or conjugate protein according to any one of the preceding claims, wherein said one or more polypeptide(s) (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 the group consisting of SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:64, SEQ ID NO:65 and SEQ ID NO:

66.

24. 10. The fusion protein or conjugate protein according to any one of the preceding claims, wherein said one or more polypeptide(s) (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 the group consisting of SEQ ID NO: 23 and SEQ ID NO:

24.

25. 10. A fusion protein or conjugate protein according to any one of the preceding claims, wherein said one or more polypeptide(s) (P1) is encoded by a sequence comprising or consisting of a sequence having at least 70% similarity to SEQ ID NO: 25, such as at least 80% similarity, for example at least 90% similarity, such as at least 95% similarity, for example at least 99% similarity.

26. A fusion protein or conjugate protein according to any one of the preceding claims, wherein the one or more polypeptide(s) (P1)(i) does not neutralise the activity of myostatin (SEQ ID NO: 45), GDF11 (SEQ ID NO: 46), and / or activin A (SEQ ID NO: 44).

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

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

29. wherein said one or more polypeptide(s) (P1)(i) increases the activity of myostatin (SEQ ID NO: 45), GDF11 (SEQ ID NO: 46) and / or activin A (SEQ ID NO: 44) by up to at least 5 nM, such as at least 7.5 nM, for example at least 8 nM, such as at least 8.2 nM, for example at least 8.4 nM, such as at least 8.6 nM, for example at least 8.8 nM, such as at least 9 nM, for example at least 10 nM, such as at least 12 nM, for example at least 15 nM, such as at least 20 nM, for example at least 30 nM, such as at least 40 nM, for example at least 50 nM, such as at least 60 nM, for example at least 61 nM, such as at least 62 nM, for example at least 63 nM, such as at least 64 nM , such as at least 64nM, for example at least 65nM, such as at least 70nM, for example at least 80nM, such as at least 100nM, for example at least 150nM, such as at least 200nM, for example at least 500nM, such as at least 600nM, for example at least 700nM, such as at least 750nM, for example at least 800nM, such as at least 820nM, for example at least 821nM, such as at least 822nM, for example at least 830nM, such as at least 1μM, for example at least 2μM, wherein said neutralization or absence is measured by a reporter gene bioassay of Smad2 / 3 phosphorylation signaling.

30. 10. A fusion protein or conjugate protein according to any one of the preceding claims, wherein the one or more polypeptide(s) (P1)(i) does not neutralise the activity of myostatin, GDF11 and / or activin A in a concentration range of up to at least 5nM, such as at least 7.5nM, for example at least 8nM, at least 8.2nM, such as at least 8.4nM, for example at least 8.6nM, such as at least 8.8nM, for example at least 9nM, such as at least 10nM, for example at least 12nM, at least 15nM, such as at least 20nM, for example at least 30nM, such as at least 40nM, for example at least 50nM, such as at least 80nM, for example at least 100nM, such as at least 150nM, for example at least 200nM.

31. 10. The fusion or conjugated protein of any one of the preceding claims, wherein said one or more polypeptide(s) (P1)(i) binds heparan sulfate.

32. 10. A fusion protein or conjugate protein according to any one of the preceding claims, wherein said one or more polypeptide(s) (P1)(i) comprising at least one FSD1 domain are encoded by a sequence comprising or consisting of a sequence having at least 70% identity, such as at least 80% identity, such as at least 90% identity, such as at least 95% identity, for example at least 99% identity to SEQ ID NO:

33.

33. 10. A fusion protein or conjugate protein according to any one of the preceding claims, wherein said one or more polypeptide(s) (P1)(i) comprising at least one FSD1 domain comprises or consists of a sequence having at least 100% identity to SEQ ID NO:

1.

34. 10. The fusion protein or conjugate protein of any one of the preceding claims, further comprising a linker between said one or more polypeptide(s) (P1) (i) comprising at least one FSD1 domain and said therapeutic or diagnostic agent (ii).

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

36. 10. The fusion protein or conjugated protein of any one of the preceding claims, wherein the therapeutic or diagnostic agent (ii) or therapeutic or diagnostic moiety, which may be the same or different from that of any one of the preceding claims, is selected from the group consisting of a peptide, a protein, such as an antibody or fragment thereof, a nanobody, a glycoprotein, streptavidin, or an interleukin, a nucleic acid, and a small molecule.

37. A fusion or conjugated protein according to any one of the preceding claims, wherein the fusion or conjugated protein further comprises a detectable moiety.

38. 38. The fusion protein or conjugated 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. 10. The fusion protein or conjugate protein of any one of the preceding claims, wherein the therapeutic or diagnostic agent (ii) or therapeutic or diagnostic moiety, which may be the same or different from that of any one of the preceding claims, binds to a target selected from the group consisting of differentiation antigen (CD) proteins, cytokines, e.g., interleukins, growth factors, e.g., colony stimulating factors, immune checkpoint proteins, angiogenic factors, hemostatic factors, chemotactic factors, neurotrophic factors, inflammatory proteins, tumor antigens, bacterial proteins, and viral proteins.

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

41. 10. The fusion protein or conjugate protein of any one of the preceding claims, wherein the therapeutic or diagnostic agent (ii), or therapeutic or diagnostic moiety which is the same or different from that of any one 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, for example at least 90% similarity, such as at least 95% similarity, for example at least 99% similarity, to a sequence selected from the group consisting of SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21 and SEQ ID NO:

22.

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

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

34.

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 therapeutic or diagnostic agent (ii) or therapeutic or diagnostic moiety, which may be the same or different from that of any one of the preceding claims, (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 having at least 70% similarity, such as at least 80% similarity, for example at least 90% similarity, such as at least 95% similarity, for example at least 99% similarity to SEQ ID NO: 34, and / or (d) A fusion protein or conjugate protein according to any one of the preceding claims, comprising or consisting of a full-length antibody, such as an anti-TNFα full-length antibody.

46. A fusion or conjugated protein according to any one of the preceding claims, wherein the fusion or conjugated protein is ubiquitinated.

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

48. 10. The fusion protein or conjugated protein of any one of the preceding claims, wherein the fusion protein or conjugated protein has a longer local half-life at the site of administration than the therapeutic or diagnostic agent (ii) or a therapeutic or diagnostic moiety that is the same or different from that of any one of the preceding claims alone.

49. 10. The fusion or conjugated protein of any one of the preceding claims, wherein the fusion or conjugated protein enhances binding of the therapeutic or diagnostic agent (ii) or a therapeutic or diagnostic moiety that is the same or different from that of any one of the preceding claims to an extracellular matrix compartment compared to the therapeutic or diagnostic agent (ii) or a therapeutic or diagnostic moiety that is the same or different from that of any one of the preceding claims alone.

50. 10. The fusion or conjugated protein of any one of the preceding claims, wherein the fusion or conjugated protein enhances binding of the therapeutic or diagnostic agent (ii), or a therapeutic or diagnostic moiety that is the same or different from that of any one of the preceding claims, to a predetermined organ or tumor compared to the therapeutic or diagnostic agent (ii), or a therapeutic or diagnostic moiety that is the same or different from that of any one of the preceding claims, alone.

51. 10. The fusion protein or conjugated protein according to any one of the preceding claims, wherein the EC50 of said therapeutic or diagnostic agent (ii) or said same or different therapeutic or diagnostic moiety for a given organ or tumour is reduced by at least 2 fold, such as at least 5 fold, for example at least 10 fold, such as at least 20 fold, for example at least 50 fold, such as at least 80 fold, for example at least 100 fold, such as at least 250 fold, for example at least 500 fold, such as at least 1000 fold, for example at least 5000 fold, such as 10000 fold, for example 100000 fold, such as 100000 fold, compared to said therapeutic or diagnostic agent (ii) or said same or different therapeutic or diagnostic moiety alone.

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

53. 53. The fusion protein or conjugated 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, subcutaneous tissue such as joint or synovial tissue.

54. 10. A fusion protein or conjugated protein according to any one of the preceding claims, wherein the fusion protein or conjugated protein enhances cellular uptake of the therapeutic or diagnostic agent (ii) or a therapeutic or diagnostic moiety that is the same or different from that of any one of the preceding claims compared to the therapeutic or diagnostic agent (ii) or the same or different therapeutic or diagnostic moiety of any one of the preceding claims alone.

55. 1. A therapeutic or diagnostic agent (ii) according to any one of the preceding claims, or a therapeutic or diagnostic moiety which is identical or different to that according to any one of the preceding claims, comprising a fusion protein or conjugate protein according to any one of the preceding claims, which binds 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), comprising at least one FSD1 domain which comprises or consists of a polypeptide having at least 70%, such as at least 80%, for example at least 90%, such as at least 95%, for example at least 99% sequence identity to SEQ ID NO:

1.

56. 56. The fusion protein or conjugate protein of any one of claims 5 to 55, wherein each monomer of said IgG Fc homodimer or Fc heterodimer binds 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), 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.

57. 10. A fusion protein or conjugate protein according to any one of the preceding claims, wherein at least one of the at least one polypeptides (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, for example at least four FSD1 domains, such as at least five FSD1 domains, consisting of a polypeptide having at least 70%, such as at least 80%, for example at least 90%, such as at least 95%, for example at least 99% sequence identity to SEQ ID NO:

1.

58. 58. The fusion protein or conjugate protein of claim 57, wherein at least two monomers of the FSD1 domain multimer are linked by a linker.

59. 10. A fusion protein or conjugated protein according to any one of the preceding claims, wherein the fusion protein or conjugated 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 at least 99% sequence identity, to a sequence selected from the group consisting of SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7 and SEQ ID NO:

9.

60. 10. The fusion or conjugated protein according to any one of the preceding claims, comprising or consisting of a polypeptide having at least 70% sequence identity, such as at least 80% identity, for example at least 90% identity, such as at least 95% identity, for example 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, for example at least 90% identity, for example at least 95% identity, for example at least 99% identity to a sequence selected from the group consisting of SEQ ID NO: 13, SEQ ID NO: 15 and SEQ ID NO:

17.

61. The fusion protein or conjugated protein comprises: (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) A fusion protein or conjugate protein according to any one of the preceding claims, comprising or consisting of a polypeptide having at least 70% sequence identity, such as at least 80% sequence identity, for example at least 90% sequence identity, such as at least 95% identity, for example 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, for example at least 90% sequence identity, such as at least 95% identity, for example at least 99% identity, to a sequence selected from the group consisting of SEQ ID NO: 13, SEQ ID NO: 15 and SEQ ID NO:

17.

62. 10. The fusion protein or composite protein of any one of the preceding claims, wherein said fusion protein or composite protein does not comprise any other of the follistatin domains FSD2, FSD3 and / or N-terminal follistatin domains, preferably said fusion protein or composite protein does not comprise any of human FSD2 of SEQ ID NO: 41, human FSD3 of SEQ ID NO: 42, and human follistatin N-terminal domain of SEQ ID NO:

43.

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

43.

64. 10. The fusion protein or conjugate protein of any one of the preceding claims, wherein (i) said 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%, for example at least 90%, such as at least 95%, for example at least 96%, such as at least 97%, for example 98%, for example at least 99% sequence identity to those SEQ ID NOs.

65. 10. The fusion protein or conjugated protein of any one of the preceding claims, further comprising a PROTAC linker, preferably 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. 10. One or more polynucleotides which, upon expression, encode a fusion protein or conjugated protein according to any one of the preceding claims.

67. 67. The one or more polynucleotides of 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, to 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.

68. 67. The one or more polynucleotides of claim 66, wherein the one or more polynucleotides have at least 70% sequence identity, such as at least 80% sequence identity, for example at least 90% sequence identity, such as at least 95% identity, for example at least 99% identity, to a combination of SEQ ID NO: 12 and at least one polynucleotide selected from the group consisting of 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 conjugated protein according to any one of claims 1 to 65.

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

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

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

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

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

75. 73. The host cell of claim 72, wherein the host cell is a Chinese hamster ovary (CHO) cell.

76. 10. A composition comprising 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 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. 77. The composition of claim 76, which is a pharmaceutical composition.

78. 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 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 for use in medicine.

79. 78. 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 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 for use in the treatment of an ophthalmic disease, a neoplastic disease, or an inflammatory disease such as a rheumatic disease.

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

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

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

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

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

85. 10. Use of 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 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 in the manufacture of a medicament for the treatment of a disease or condition, such as an ophthalmic disease, a neoplastic 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 conjugated protein of any one of claims 1 to 65, one or more polynucleotides of any one of claims 66 to 68, one or more constructs or vectors of any one of claims 69 to 70, a host cell of any one of claims 71 to 75 or a composition of any one of claims 76 to 77 is administered systemically.

87. 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, comprising topically administering a fusion protein or conjugated protein of any one of claims 1 to 65, one or more polynucleotides of any one of claims 66 to 68, one or more constructs or vectors of any one of claims 69 to 70, a host cell of any one of claims 71 to 75 or a composition of any one of claims 76 to 77.

88. 85. 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 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 composition of any one of claims 76 to 77 is administered intraadiposely, intra-arterially, intra-articularly, intracranially, intradermally, intralesionally, intramuscularly, intranasally, intraocularly, intraosseously, intrapericardially, intraperitoneally, intrapleurally, intraprostatically, intrarectally, intrathecally, intratracheally, intratumorally, intraumbilically, intravaginally, intravenously, intravesically, intravitreally or via liposomes.

89. 10. A method of increasing the local half-life of a therapeutic or diagnostic agent at a site of administration, the method comprising obtaining 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 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 a therapeutic or diagnostic agent (ii) according to any one of claims 1 to 65, or a therapeutic or diagnostic site which is the same or different from that of claims 6 to 65, comprises or consists of said therapeutic or diagnostic agent.

90. 1. A method for increasing the local half-life of a therapeutic or diagnostic agent in vivo, comprising: a) providing a therapeutic or diagnostic agent; b) obtaining a fusion protein or conjugated protein according to any one of claims 1 to 65, wherein the therapeutic or diagnostic agent of step a) is a therapeutic or diagnostic agent (ii) according to any one of claims 1 to 65, obtaining said fusion protein or conjugated protein, which enhances the local half-life of said therapeutic or diagnostic agent in vivo.

91. 10. The method of any one of the preceding claims, further comprising administering the fusion protein or conjugated protein to a subject suffering from an ophthalmic disease, wherein the fusion protein or conjugated protein is administered intravitreally, subretinaly, or suprachoroidally.

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

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

94. 94. The method of any one of claims 89 to 93, wherein the local half-life of the therapeutic or diagnostic agent is extended at the administration site by at least 6 hours, such as at least 12 hours, for example at least 24 hours, such as at least 48 hours, for example at least 72 hours, such as at least 96 hours, at least 120 hours, such as at least 1 week, for example at least 2 weeks, such as at least 4 weeks, for example at least 8 weeks, such as at least 3 months, for example at least 6 months, such as at least 12 months.

95. 10. A method of enhancing 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 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 (ii) a therapeutic or diagnostic agent according to any one of claims 1 to 65, or a therapeutic or diagnostic moiety which is the same or different from that of any one of claims 6 to 65, comprises or consists of said therapeutic or diagnostic agent.

96. 10. A method of enhancing binding of a therapeutic or diagnostic agent to a predetermined organ, the method 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 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 a therapeutic or diagnostic moiety which is the same or different from that of any one of claims 6 to 65, comprises or consists of said therapeutic or diagnostic agent.

97. 10. A method of increasing the cellular uptake of a therapeutic or diagnostic agent, the method comprising administering to a subject a fusion protein or conjugated protein of any one of claims 1 to 65, one or more polynucleotides of any one of claims 66 to 68, one or more constructs or vectors of any one of claims 69 to 70, a host cell of any one of claims 71 to 75, or a 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 a therapeutic or diagnostic moiety which is the same or different from that of any one of claims 6 to 65, comprises or consists of said therapeutic or diagnostic agent.

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

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

100. 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 of any one of claims 98 to 100, wherein the therapeutic or diagnostic agent (ii) is an anti-vimentin-nanobody.

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

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

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

105. 103. The use of any one of claims 78 to 82 and 85 to 88, or the method of any one of claims 83 to 103, comprising systemic administration of a fusion protein or conjugated protein of any one of claims 1 to 65, one or more polynucleotides of any one of claims 66 to 68, one or more constructs or vectors of any one of claims 69 to 70, a host cell of any one of claims 71 to 75, or a composition of any one of claims 76 to 77.

106. 103. The use of any one of claims 78 to 82 and 85 to 88, or the method of any one of claims 83 to 103, comprising locally administering a fusion protein or conjugated protein of any one of claims 1 to 65, one or more polynucleotides of any one of claims 66 to 68, one or more constructs or vectors of any one of claims 69 to 70, a host cell of any one of claims 71 to 75, or a composition of any one of claims 76 to 77.

107. 103. The use of any one of claims 78 to 82 and 85 to 88, or the method of any one of claims 83 to 103, wherein 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 composition of any one of claims 76 to 77 is administered intraadiposely, intra-arterially, intra-articularly, intracranially, intradermally, intralesionally, intramuscularly, intranasally, intraocularly, intraosseously, intrapericardially, intraperitoneally, intrapleurally, intraprostatically, intrarectally, intrathecally, intratracheally, intratumorally, intraumbilically, intravaginally, intravenously, intravesically, intravitreally, or via liposomes.

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

109. 10. One or more constructs or vectors encoding one or more polypeptide(s) (P1)(i) of the fusion or conjugated protein according to any one of the preceding claims.

110. 1. 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 a variant thereof having at least 70%, such as at least 80%, for example at least 90%, such as at least 95%, for example at least 96%, such as at least 97%, for example 98%, such as at least 99% sequence identity thereto.

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