Internalizing binding molecules that target receptors involved in cell proliferation or cell differentiation

Abstract

The present invention relates to the field of binding molecules comprising at least one single variable antibody domain that targets a receptor present on myofibroblasts and / or hepatic stellate cells (HSCs). The invention also relates to binding molecules comprising at least two single variable antibody domains, each targeting a receptor on HSCs and / or myofibroblasts. The invention further relates to nucleic acids encoding such binding molecules, host cells for the expression of such binding molecules and methods for preparing such binding molecules. The invention further relates to pharmaceutical compositions comprising such binding molecules and the use of such binding molecules and / or compositions, particularly for prophylactic, therapeutic or diagnostic purposes.

Description

[Technical field]

[0001] The present invention relates to the field of binding molecules comprising at least one single variable antibody domain, which targets a receptor present on myofibroblasts and / or hepatic stellate cells (HSC). The present invention also relates to binding molecules comprising at least two single variable antibody domains, each targeting a receptor on HSC and / or on myofibroblasts. The present invention further relates to nucleic acids encoding such binding molecules, host cells for the expression of such binding molecules and methods for preparing such binding molecules. The present invention further relates to pharmaceutical compositions comprising such binding molecules, and to the use of such binding molecules and / or compositions, in particular for prophylactic, therapeutic or diagnostic purposes.

[0002] 2. Background of the Invention chronic liver disease Chronic liver disease (CLD) in the clinical setting is a disease process of the liver that involves a process of progressive destruction and regeneration of the liver parenchyma leading to liver fibrosis (LF) and cirrhosis (LC), conditions characterized by fibrotic scarring, nodular regeneration of the liver parenchyma, and elevated portal vein pressure. CLD can be caused by viral infection (e.g., HCV), metabolic syndrome (e.g., NASH), or alcoholism. LF is characterized by the accumulation of extracellular matrix (ECM) proteins, tissue contraction, and consequently altered blood flow. These symptoms are caused by the activation of HSCs, which are pericytes found in the space of Disse (the small area between the sinusoids and the hepatocytes). Generally speaking, pericytes are contractile cells that wrap around endothelial cells that line capillaries and venules throughout the body. Upon activation, HSCs transdifferentiate into myofibroblasts and begin to secrete large amounts of ECM proteins and profibrogenic cytokines. If left untreated, accumulation of ECM proteins causes LF to progress to LC, which ranks as the 12th leading cause of death worldwide.

[0003] Treatment for CLD Cures targeting the primary causes of liver disease currently represent the most effective strategy, but only exist for a few etiologies. Liver transplantation is generally the only treatment available for patients with end-stage chronic liver disease. Given the high levels of morbidity and mortality, there is a clear need to develop (further) therapeutic approaches to complement or potentially replace current treatment methods. Research conducted on CLD over the last few decades has improved understanding of the pathophysiology of the disease and allowed the development of novel agents that target the underlying disease process. To date, there are no specific pharmacological therapies approved for CLD, and although there are newer therapies currently undergoing evaluation, research into these treatments is in a relatively early stage.

[0004] An alternative to current therapies is the reduction and eventual disappearance of portal hypertension and / or fibrotic scar tissue in the liver. In theory, antifibrotic therapies could target various aspects of fibrogenesis, including inhibition of matrix deposition and collagen synthesis and the breakdown of newly generated fibrotic tissue. Newly synthesized collagen is thought to be more susceptible to degradation than old collagen, but animal studies have shown that even advanced cirrhosis is reversible, and human data suggest the same. Recent advances in understanding the pathogenesis of liver fibrosis are now leading to novel therapeutic approaches for liver fibrosis. Drugs with "antifibrotic activity" have been studied in clinical trials but have proven ineffective, especially due to their systemic toxicity. The therapeutic application of these (often small) molecules, such as Rho-kinase inhibitors and JAK-2 kinase inhibitors, is hampered by their intractable pharmacodynamic and kinetic profiles. Thus, improved therapies and diagnostics for CLD, and in particular for liver fibrosis, are needed.

[0005] Summary of the Invention The present invention provides such an alternative and improved therapeutic method or diagnosis in the form of a binding molecule comprising at least one single variable antibody domain and at least one diagnostic and / or therapeutic molecule, where said at least one single variable antibody domain is capable of specifically binding to a transmembrane receptor expressed on hepatic stellate cells (HSCs) and / or on myofibroblasts.

[0006] Such binding molecules preferably allow targeting of activated hepatic stellate cells or myofibroblasts through binding to platelet-derived growth factor receptor-beta (PDGFRB) or insulin-like growth factor 2 receptor (IGF2R), both of which are highly expressed on such cells. The binding molecules are preferably linked to toxins or drugs, for example via linkers comprising Pt(II) transition metal complexes. In particular, when the drug is a kinase inhibitor, such as RHO-kinase, JAK-2 inhibitors, or neprilysin inhibitors, the binding molecules of the invention preferably provide relaxation of activated HSCs or myofibroblasts, which relaxation attenuates, for example, liver fibrosis. The use of the linkers reduces the (off-target) toxicity commonly observed with these kinase inhibitors.

[0007] In liver disease, selective delivery of drugs to (activated) cells, such as hepatic stellate cells (HSCs), which play a crucial role in the onset, maintenance and progression of cirrhosis, is an interesting goal to pursue. It is possible that effective drug treatment of liver disease can be achieved using targeting of receptors such as PDGFRB and IGF2R, which are upregulated in activated HSCs. In the present invention, targeting of PDGFRB or IGF2R is considered to be an important tool, since said targeting induces the internalisation of proteinaceous molecules, including, for example, rho-kinase inhibitors, JAK-2 kinase inhibitors or neprilysin inhibitors.

[0008] The invention further provides a nucleic acid encoding at least a part of a binding molecule of the invention and a host cell for expression of at least a part of a binding molecule of the invention comprising a nucleic acid of the invention. Also provided is a method for producing a binding molecule of the invention, comprising the steps of culturing a host cell of the invention, allowing expression of at least a part of said binding molecule, harvesting said binding molecule and coupling said therapeutic or diagnostic molecule to said part of said binding molecule, optionally by means of a linker as defined above.

[0009] Since the binding molecules of the invention, including therapeutic molecules, are particularly suitable for, for example, the treatment of fibrosis or cancer, the invention further provides a pharmaceutical composition comprising at least one binding molecule of the invention and at least one pharma- ceutical acceptable excipient. Such a pharmaceutical composition may further comprise at least one other compound useful in the treatment of chronic liver disease. The pharmaceutical composition of the invention is particularly useful as an adjunct treatment of variceal bleeding in the presence of portal hypertension or for use in the treatment of liver disease.

[0010] Since the binding molecules of the invention, including diagnostic molecules, are particularly suitable for diagnosing, for example, liver cirrhosis, the invention provides diagnostic compositions comprising at least one binding molecule of the invention, including a diagnostic molecule, preferably an imaging agent, and a diluent and / or excipient. [Brief description of the drawings]

[0011] [Figure 1] (A) Overview of the research process from generating a VHH library from blood samples of immunized llamas, to (B) performing phage display selection, and (C) selecting clones for screening (Figure taken from Schoonooghe S. et al, Immunobiology 2012,217:1266-1272). [Diagram 2] Binding of IGF2R-selected VHHs to the human IGF2R ectodomain. [Diagram 3]Phage output from the first selection round of PDGFRB libraries for the human PDGFRB ectodomain (libraries 152 and 153) and the rat PDGFRB ectodomain (libraries 154 and 155). [Figure 4] Phage output from the second selection round of the PDGFRB library on SCC VII cells transfected with either human PDGFRB (libraries 152 and 153) or rat PDGFRB (libraries 154 and 155). [Diagram 5] Amino acid sequences of human PDGFRB VHHs (CDRs underlined). Affinities are measured with respect to the human PDGFRB ectodomain (ECD), SCC VII cells transfected with the human PDGFRB receptor (SCC-hPDGFRB) and a human hepatic stellate cell line (LX-2). Data from competition assays are included to classify VHHs in different epitope groups. [Figure 6] Amino acid sequence of rat PDGFRB VHH (CDRs underlined). Affinities are measured with respect to rat PDGFRB ectodomain (ECD) and squamous cell carcinoma cells transfected with rat PDGFRB receptor (SCC-rPDGFRB). Data from competition assays are included to classify VHHs in different epitope groups. [Figure 7] Schematic representation of expression vector carrying VHH insert with peptide tail and purification tag. [Figure 8]Construction and characterization of bivalent and biparatopic VHH constructs directed against human IGF2R. A. Schematic of monovalent, bivalent and biparatopic VHH formats. In bivalent and biparatopic formats, two VHHs are fused with a flexible (Gly-Gly-Gly-Gly-Ser)3 linker (GGGGSGGGGSGGGGS; SEQ ID NO: 369). All formats contain a C-terminal cysteine ​​for site-specific conjugation to thiol-reactive probes and a hexahistidine tag (HHHHHH; SEQ ID NO: 370) or EPEA tag (EPEA; SEQ ID NO: 371) for affinity chromatography purification. B. Binding of anti-human IGF2R VHH constructs to recombinant human IGF2R ectodomain. C. Bmax and KD+95% confidence interval for selected VHHs. [Figure 9] Binding of selected VHHs on SCC VII cells transfected with either the human or rat PDGFRB receptor fused to GFP (SCC-hP or SCC-rP). VHHs are detected using anti-VHH rabbit serum and donkey anti-rabbit secondary antibody labeled with IRDye800CW. [Figure 10] Competitive enzyme-linked immunosorbent assay (ELISA) using VHHs directed against human IGF2R. [Figure 11] Competitive enzyme-linked immunosorbent assay (ELISA) assay with SP02P and SP26P for PDGFRB ECD. [Figure 12] 13F11-13E8 (F11E8) binding to human or rat IGF2R-expressing SCC cells. [Figure 13] Binding of 13F11-13E8-ABD (F11E8-ABD) to IGF2R and serum albumin. A) Binding assay on recombinant human IGF2R ECD. B) Binding assay on serum albumin from various species. C) Serum levels of F11E8-ABD and F11E8 in rats after intravenous injection. Serum concentrations as assessed by ELISA are shown. LOD: approximately 20 nM. [Figure 14]Binding of 13F11-13E8-ABD conjugated to Alexa Fluor 488 (FE-A-A488) to activated primary rat hepatic stellate cells. [Figure 15] Binding assays using PDGFRB VHH. A) Binding assay using human PDGFRB specific VHH on human PDGFRB ECD. B) Binding assay using biparatopic SP28-SP26P (SP28-26P) on human PDGFRB, rat PDGFRB and human IGF2R expressing target cells. C) Binding assay using biparatopic SP02P-SP26P on human PDGFRB, rat PDGFRB and human IGF2R expressing target cells. [Figure 16] Internalization of 13F11 and 13F11-13E8 in IGF2R expressing target cells, magnification 63x. Scale bar is 30 μm. A. A549 (IGF2R positive). B. NIH 3T3 2.2 transiently transfected with human IGF2R. C. Mock transfected NIH 3T3 2.2. D. Acid wash control for A549 cells. [Figure 17] Kinetics of internalization of 13F11-13E8 (F11E8) and 13E8-13F11 (E8F11) for human or rat IGF2R-expressing SCC cells. [Figure 18] Internalization in LX-2 cells of monovalent (13F11) and biparatopic (13F11-13E8 and 13E8-13F11) VHHs conjugated to the pH-sensitive pHrodo red dye. [Figure 19] Kinetics of internalization of SP02P-SP26P for human PDGFRB-expressing SCC cells and LX-2 cells. [Figure 20] Contraction assay for activated hepatic stellate cells (HSC). Measured area of ​​gel after 6 days of incubation with NDC is shown. Contraction of LX-2 cells without any inhibitor was set as 0% relaxation, while 1 μM BDM was set as 100% relaxation. Note: No contraction inhibition was observed with the medium. [Figure 21]Immunofluorescence binding of 13F11-13E8-ABD (F11E8-ABD) to cirrhotic (BDL) and healthy (SHAM) rat liver sections. [Figure 22] Expression of IGF2R in various tissues from cirrhotic (BDL) and healthy (SHAM) rats. [Figure 23] Changes in portal pressure (PP) and mean arterial pressure (MAP) in BDL rats as a function of time (min) after dosing for A) 13F11-13E8-ABD-Y27632 (test conjugate) and B) 13F11-13E8-ABD-ethyl (sham conjugate). [Figure 24] In vivo targeting of IGF2R by 13F11-13E8-ABD-Y27632 (test conjugate) in BLD and SHAM rats. A) In situ detection of injected test conjugate. Liver cryosections from BDL (top) and SHAM (bottom) rats probed for the presence of test conjugate with a VHH-specific detection antibody (green). Test conjugate is clearly detected in the cell layer covering the obstructed sinusoids in the cirrhotic liver of BDL rats and not in the healthy liver of SHAM rats. B) Distribution of test conjugate versus (tissue) distribution of IGF2R. The same cryosections were also stained for IGF2R using the fluorophore conjugate F11E8 (red). The test conjugate shows co-localization with IGF2R (green with red: yellow). Notably, more deeply located regions showed IGF2R expression (red) in the absence of test conjugate, suggesting that tissue penetration was still ongoing and not complete at the end of the single time in-life study. Consistent with earlier observations, IGF2R was not detectable in the liver of SHAM rats.

[0012] Detailed Description of the Invention First, it should be noted that all steps, methods, and techniques that are not specifically detailed can be performed essentially in a manner known per se, as would be apparent to one skilled in the art, unless otherwise stated. Reference is made to standard handbooks and common general knowledge referred to herein, which describe techniques related to protein engineering, such as affinity maturation and other techniques to improve the specificity and other desired properties of the binding moieties present in the binding molecules of the invention, such as immunoglobulin single variable domain antibodies (ISVDs), variable domains of heavy chains (VHs), variable domains of heavy chain-only antibodies (VHHs), domain antibodies (dAbs), or single domain antibodies (sdAbs). It should be noted that, as used herein, the singular forms "a", "an", and "the" encompass plural content, unless the context clearly indicates otherwise. Those skilled in the art will recognize or be able to ascertain, using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the present invention. The term "and / or" whenever used herein includes "and", "or" and "all or any other combination of the elements connected to said term". The terms "about" or "approximately" as used herein mean within 25%, preferably within 20%, more preferably within 15%, and most preferably within 10% of a given value or range. Throughout this specification and the accompanying claims, unless the context requires otherwise, the word "comprises" and variations such as "comprises (third person singular present tense)" and "comprising" are understood to include the meaning of the inclusion of the indicated feature but not the exclusion of any other feature. As used herein, the word "comprising" can be substituted with the words "containing" or "including" or "having". In contrast, the word "consisting of" in combination with a feature or features means the inclusion of the mentioned feature or features but also the exclusion of any other feature or features.

[0013] In a first aspect, the present invention provides a binding molecule comprising at least one single variable antibody domain and at least one diagnostic or therapeutic molecule, wherein the at least one single variable antibody domain is capable of specifically binding to a transmembrane receptor expressed on activated HSCs and / or myofibroblasts, preferably characterized by increased proliferation, contractility, chemotaxis, and enhanced extracellular matrix (ECM) protein production (e.g. collagen). For a complete description of how to preferably determine whether HSCs are activated or not, see Tsucuchida et al. (Mechanisms of hepatic stellate cell activation), Nature Reviews Gastroenterology & Hepatology, 2017).

[0014] The therapeutic molecule contained in the binding molecule of the present invention preferably induces HSC relaxation, which is a sign of reduced HSC activation, thereby reducing the fibrosis process initiated by primary liver disease. One example of such a therapeutic molecule is Y27632, a small molecule kinase inhibitor that has been shown in the examples of the present invention to induce HSC relaxation. In particular, the binding molecule of the present invention has been shown to specifically interact with a receptor on activated HSC. Research has shown proof of concept that the binding molecule of the present invention can be used to treat patients with liver fibrosis. Research has also demonstrated that the binding molecule of the present invention does not interfere with platelet activity.

[0015] The binding molecule of the present invention must be capable of specifically binding to a transmembrane receptor expressed on hepatic stellate cells, in particular activated hepatic stellate cells, and / or myofibroblasts. Examples of receptors expressed in sufficient amounts on activated HSCs and / or myofibroblasts are, for example, PDGFRB and IGF2R. Thus, in a preferred embodiment, the binding molecule of the present invention is provided, in which the transmembrane receptor is PDGFRB or IGF2R.

[0016] PDGF is one of many growth factors that regulate cell growth and division. In particular, PDGF plays an important role in angiogenesis, the growth of blood vessels from pre-existing vascular tissue, mitogenesis, i.e., proliferation, of mesenchymal cells such as fibroblasts, osteoblasts, tenocytes, vascular smooth muscle cells, and mesenchymal stem cells, as well as chemotaxis, the direct migration of mesenchymal cells. PDGF is a dimeric glycoprotein that can be composed of two A subunits (PDGF-AA), two B subunits (PDGF-BB), or one of each (PDGF-AB). PDGF is a potent mitogen (a protein that induces mitosis, encouraging cells to begin cell division) for cells of stromal origin, including fibroblasts, smooth muscle cells, and glial cells. In both mice and humans, the PDGF signaling network consists of five ligands, PDGF-AA, PDGF-BB, PDGF-AB, PDGF-CC, and PDGF-DD, and two receptors, PDGFR-alpha (PDGFRA) and PDGFR-beta (PDGFRB). All PDGFs function as secreted disulfide-linked homodimers, but only PDGFA and PDGFB can generate functional heterodimers. PDGF is synthesized in excess upon activation of HSCs, which may be the case in conditions of alcoholism or hepatitis. Thus, fibroblast activation and cell division occur, leading to connective tissue, which is especially prevalent in wound healing. PDGF is a required element in this process. Essentially, PDGF can cause cells to skip the G1 checkpoint to divide. In monocyte-macrophages and fibroblasts, exogenously administered PDGF stimulates chemotaxis, proliferation, and gene expression, significantly increasing the influx of inflammatory cells and fibroblasts, promoting extracellular matrix and collagen production, and thus reducing the time for the healing process to occur. Quiescent HSCs express PDGFRA, whereas upon activation, HSCs switch to PDGFRB expression. HSCs become activated by hepatocyte injury and death, resulting in fibrogenesis.The mannose 6-phosphate / insulin-like growth factor-2 receptor (M6P / IGF2R) plays a key role in early fibrogenesis by participating in the activation of latent transforming PDGFB, a potent inducer of matrix proteins in activated HSCs that give rise to hepatic scarring. IGF2R is a transmembrane glycoprotein involved in the clearance of extracellular ligands (IGF-II), activating extracellular ligands (TGFβ), and in the sorting of newly synthesized M6P-tagged lysosomal enzymes from the trans-Golgi network (TGN) to lysosomes. For this function, IGF2R cycles between sorting endosomes, endocytic recycling compartments, the TGN, late endosomes, and the plasma membrane (Maxfield & McGraw, Nat Rev Mol Cell Biol. 2004 Feb;5(2):121-32).

[0017] The binding molecules of the present invention can be used in various fibrotic diseases in which myofibroblasts expressing one of the above-mentioned transmembrane receptors play a role, such as, for example, liver fibrosis, pulmonary fibrosis, or renal fibrosis. In the examples, the present invention provides proof of principle in (activated) HSCs. However, in liver fibrogenesis, activated hepatic stellate cells (HSCs) are thought to transdifferentiate into myofibroblasts that express abundant amounts of PDGFRB and IGF2R (Yuchang Li, et al. Mesothelial-mesenchymal transition in the liver. PNAS (2013), 110: 2324-2329). Another major source of hepatic myofibroblasts in fibrotic livers, both in experimental models of liver fibrosis and in patients suffering from liver disease, are portal fibroblasts (Jun Xu, et al. The types of hepatic myofibroblasts contributing to liver fibrosis of different etiologies. Frontiers in pharmacology (2014), 5:167). Thus, in a preferred embodiment, a binding molecule of the invention is provided, in which the myofibroblast is a hepatic myofibroblast. Preferably, the fibroblast is derived from HSC or portal fibroblast, more preferably from HSC. "Derived from" in this context refers to the differentiation of an originator cell (HSC or portal fibroblast) into a myofibroblast. The term "myofibroblast" preferably encompasses myofibroblast-like cells, which are in transition from HSC or portal fibroblast to myofibroblast and may have an intermediate phenotype between originator cells and myofibroblasts.Quiescent HSCs generally express the neuronal markers desmin, glial fibrillary associated protein (GFAP), synaptophysin, synemin, and the nerve growth factor receptor p75 (Geerts A. (2001). History, heterogeneity, developmental biology, and functions of quiescent hepatic stellate cells. Semin. Liver Dis. 21 311-335 10.1055 / s-2001-17550; Bataller R., Brenner DA (2005). Liver fibrosis. J. Clin. Invest. 115, 209-218). Furthermore, Nr1d2, Adipor1, Adpf, Dbp, Prei4, and Foxj1 were identified as unique markers associated with the HSC quiescent phenotype (Liu X., Xu J., Brenner DA, Kisseleva T. (2013). Reversibility of Liver Fibrosis and Inactivation of Fibrogenic Myofibroblasts. Curr. Pathobiol. Rep. 1, 209-214). In response to fibrotic liver injury and release of TGFβ1, quiescent HSCs rapidly undergo activation. Quiescent HSCs downregulate vitamin A-containing lipid droplets and neural markers and differentiate into activated HSCs / myofibroblasts that express type I collagen and α-SMA (Bataller and Brenner, 2005; Forbes SJ, Parola M. (2011). Liver fibrogenic cells. Best Pract. Res. Clin. Gastroenterol. 25, 207-217).Quiescent HSCs also upregulate the production of matrix metalloproteinases MMPs, particularly MMP13 and their inhibitors TIMPs (Uchinami H., Seki E., Brenner DA, D'Armiento J. (2006). Loss of MMP 13 attenuates murine hepatic injury and fibrosis during cholestasis. Hepatology 44, 420-429). Furthermore, activated HSCs upregulate Crlf1, Spp1, Lox, LoxL2, IL-17Ra, Fosl1, and Folr1, genes uniquely associated with an activated HSC / myofibroblast phenotype (Liu et al., Reversibility of Liver Fibrosis and Inactivation of Fibrogenic Myofibroblasts. Curr. Pathobiol. Rep. 2013,1:209-214).

[0018] In particular, when the binding molecule comprises a therapeutic molecule acting inside the cell, it is preferred that the binding molecule is taken up by the cell, for example through ligand or antibody internalization. Ligand internalization is defined as a receptor-mediated endocytosis process in which a cell takes up an extracellular molecule (e.g. a (natural) ligand or an antibody) only if the extracellular molecule binds to its specific receptor protein on the surface of the cell. Endocytosis is a process in which a cell internalizes non-particulate materials, such as proteins or polysaccharides, by phagocytosing them. Thus, in a preferred embodiment, a binding molecule of the invention is provided which is capable of inducing ligand or antibody internalization upon binding of the binding molecule to a receptor.

[0019] In a preferred embodiment, a binding molecule of the present invention is provided, in which the binding molecule comprises an additional single variable antibody domain capable of specifically binding to a transmembrane receptor expressed on HSCs and / or myofibroblasts. Such a binding molecule may comprise two single variable antibody domains capable of binding to two different antigens, for example epitopes on the same antigen, i.e. two different epitopes on one receptor. The latter are also called biparatopic binding molecules. The binding molecule of the present invention also comprises two single variable antibody domains, both specific for the same epitope. Such binding molecules, also known as bivalent binding molecules, cannot bind to two epitopes on a single receptor (since generally there are no two epitope sequences present in the sequence of one receptor (unless it is a dimer)), but can bind to the same epitope sequence on two receptors of the same type, for example two PDGF receptors that are present in the vicinity of each other. The effect is that the bivalent binding molecule crosslinks the two receptors, thereby inducing internalization of the receptors and the binding molecules they bind. In the case of biparatopic binding molecules, binding leads to receptor oligomerization, which further enhances receptor internalization. Thus, in a preferred embodiment, a binding molecule of the invention is provided, in which the binding molecule is a multiparatopic, preferably biparatopic binding molecule. Alternatively, or in conjunction with multiparatopic specificity, it is preferred that the binding molecule is a multivalent, preferably bivalent binding molecule. Alternatively, or in conjunction with multiparatopic specificity, bivalent specificity, or both, it is preferred that the binding molecule is a multispecific, preferably bispecific binding molecule. Thus, a binding molecule of the invention can combine multiple single variable antibody domains, where for example two are bivalent with respect to each other and further biparatopic and / or bispecific with respect to a third, fourth, fifth, or even sixth single variable antibody domain. Although a binding molecule of the invention can comprise more than six single variable antibody domains, it is preferred that a binding molecule of the invention comprises two to six, preferably two to four, more preferably two single variable antibody domains, as defined previously and below.This is because it is easier to produce and has shown superior results in animal models.

[0020] The single variable antibody domains present in the binding molecules of the invention can be any type of single variable antibody domain. Classical single variable antibody domains are variable domains of (parts of) antibodies, such as (single) domain antibodies or immunoglobulin single variable domain antibodies. Preferably, the at least one single variable antibody domain and / or the further single variable antibody domain are independently selected from the group consisting of immunoglobulin single variable domain antibodies (ISVD), variable domains of heavy chains (VH), variable domains of heavy chain-only antibodies (VHH), domain antibodies (dAbs) and single domain antibodies (sdAbs). More preferably, the at least one single variable antibody domain and the further single variable antibody domain are both variable domains of single variable domain antibodies, preferably of heavy chain-only antibodies. These (parts of) smaller antibodies are preferred, since, in particular in tandem formats (e.g. bivalent or biparatopic), the antibodies exhibit high internalization capabilities and optimal tissue penetration due to their smaller size.

[0021] The term "immunoglobulin single variable domain" ("ISVD") is used interchangeably with "single variable domain" and defines a molecule in which an antigen-binding site resides on and is generated by a single immunoglobulin domain. This sets immunoglobulin single variable domains apart from "conventional" immunoglobulins or fragments thereof, in which two immunoglobulin domains, in particular two variable domains, interact to generate an antigen-binding site. Usually, in conventional immunoglobulins, a heavy chain variable domain (VH) and a light chain variable domain (VL) interact to generate an antigen-binding site. In this case, the complementarity determining regions (CDRs) of both VH and VL contribute to the antigen-binding site, i.e. a total of six CDRs are involved in generating the antigen-binding site. In contrast, the binding site of an immunoglobulin single variable domain is generated by a single VH or VL domain. Thus, the antigen-binding site of an immunoglobulin single variable domain is generated by no more than three CDRs. The term "immunoglobulin single variable domain" therefore does not include conventional immunoglobulins or fragments thereof, which require the interaction of at least two variable domains for the generation of an antigen-binding site. This is also the case for the aspects of the invention "comprising" or "containing" an immunoglobulin single variable domain. Thus, a binding molecule or composition "comprising" or "containing" an immunoglobulin single variable domain can relate for example to a construct comprising more than one immunoglobulin single variable domain. Alternatively, further components other than the immunoglobulin single variable domain may be present, for example auxiliary agents of various kinds, protein tags, colorants, dyes, etc. However, these terms do not include fragments of conventional immunoglobulins, in which the antigen-binding site is generated by the single variable domain. In general, a single variable domain is an amino acid sequence that essentially consists of four framework regions (FR1 to FR4, respectively) and three complementarity determining regions (CDR1 to CDR3, respectively). Such single variable domains and fragments most preferably comprise an immunoglobulin fold or are capable of generating an immunoglobulin fold under appropriate conditions.Thus, a single variable domain may, for example, comprise a light chain variable domain sequence (e.g. a VL-sequence) or a suitable fragment thereof; or a heavy chain variable domain sequence (e.g. a VH-sequence or a VHH-sequence) or a suitable fragment thereof, as long as it is capable of generating a single antigen-binding unit (i.e. a functional antigen-binding unit essentially consisting of a single variable domain, which does not need to interact with another variable domain, e.g. through VH / VL interactions, to generate a functional antigen-binding domain, as is the case for variable domains present in e.g. conventional antibodies and scFv fragments). In one aspect of the invention, the immunoglobulin single variable domain is a light chain variable domain sequence (e.g. a VL-sequence) or a heavy chain variable domain sequence (e.g. a VH-sequence), more specifically the immunoglobulin single variable domain may be a heavy chain variable domain sequence derived from a conventional four-chain antibody or a heavy chain variable domain sequence derived from a heavy chain antibody (e.g. a VHH). For a general description of heavy chain antibodies and their variable domains, reference is made in particular to the prior art cited herein, as well as to the prior art mentioned on page 59 of WO 08 / 020079 and to the list of references mentioned on pages 41-43 of WO 06 / 040153, which are incorporated herein by reference. As described in these references, VHH sequences and partially humanized VHH sequences may be characterized in particular by the presence of one or more "hallmark residues" in one or more of the framework sequences.

[0022] Further description of VHHs, including humanization of VHHs as well as other modifications, parts or fragments, derivatives or "VHH fusions", multivalent constructs (including some non-limiting examples of linker sequences), and various modifications that increase the serum half-life of VHHs and their preparations can be found, for example, in WO 08 / 101985 and WO 08 / 142164. One example is, for example, the presence of a binding domain with affinity for human serum albumin.

[0023] Thus, to increase the half-life of a binding molecule of the invention, said binding molecule preferably comprises one or more other groups, residues, moieties or binding units that increase said half-life compared to a binding molecule without such one or more other groups, residues, moieties or binding units, said groups, residues, moieties or binding units also being referred to as half-life extenders.

[0024] The half-life extenders to be used in the binding molecules of the invention increase the serum half-life of the binding molecules in humans by at least 1 hour, preferably at least 2 hours or more. The half-life extenders may increase the half-life by more than 6 hours, for example more than 12 hours, or even more than 24 hours, 48 ​​hours or 72 hours. Particularly preferred binding units to be used in the binding molecules of the invention are albumin binding domains, albumin binding VHHs or antibody Fc tails or fragments thereof.

[0025] Regarding "dAbs" or "sdAbs", see Ward et al. 1989 (Nature 341(6242):544-6), Holt et al. 2003 (Trends Biotechnol. 21(11):484-490) and WO 04 / 068820, WO 06 / 030220, WO 06 / 003388.

[0026] Also included in the present invention are single variable domains from certain species of sharks (e.g. the so-called "IgNAR domains", see WO 05 / 18629) and mice (e.g. the so-called humabodies of Crescendo Biologics) that are not of mammalian origin and therefore less suitable for the present invention. The amino acid sequence and structure of an immunoglobulin sequence, and in particular an immunoglobulin single variable domain, may be considered to comprise, but is not limited to, four framework regions or "FRs", referred to in the art and herein as "framework region 1" or "FR1", "framework region 2" or "FR2", "framework region 3" or "FR3" and "framework region 4" or "FR4", respectively. These framework regions are interrupted by three complementarity determining regions or "CDRs", referred to in the art as "complementarity determining region 1" or "CDR1", "complementarity determining region 2" or "CDR2" and "complementarity determining region 3" or "CDR3", respectively. The total number of amino acid residues in an immunoglobulin single variable domain may be in the region of 110-120, preferably 112-115, most preferably 113. It should be noted, however, that parts, fragments, analogues or derivatives of immunoglobulin single variable domains are not particularly limited with respect to their length and / or size, as long as such parts, fragments, analogues or derivatives meet the further requirements outlined herein and are preferably suitable for the purposes described herein. Thus, in the sense of the present invention, the term "immunoglobulin single variable domain" or "single variable antibody domain" includes peptides derived from a non-human source, preferably from the family Camelidae, preferably as camelid heavy chain antibodies. An "immunoglobulin single variable domain" or "single variable antibody domain" may be humanized, as already described in WO 08 / 101985 and WO 08 / 142164. Furthermore, the term includes binding molecules derived from non-camelid sources, e.g. mouse or human, that have been "camelized", as already described in WO 08 / 101985 and WO 08 / 142164.The term "immunoglobulin single variable domain" encompasses immunoglobulin sequences of various origins, including mouse, rat, rabbit, donkey, human and camelid immunoglobulin sequences. The term also encompasses fully human, humanized or chimeric immunoglobulin sequences. For example, the term includes camelid immunoglobulin sequences and humanized camelid immunoglobulin sequences or camelidized immunoglobulin single variable domains, such as camelid dAbs as described by Ward et al. (see WO 94 / 04678 and Davies and Riechmann 1994, Febs Lett. 339:285 and 1996, Protein Engineering 9:531). As already mentioned above, the at least one binding molecule is capable of specifically binding to the extracellular domain of a receptor expressed on HSCs and / or myofibroblasts. Preferably, the second single variable antibody domain, if present, is also capable of specifically binding to a transmembrane receptor expressed on HSCs and / or myofibroblasts. Particularly preferred are binding molecules of the invention which comprise at least two single variable antibody domains capable of specifically binding to PDGFRB or which comprise at least two single variable antibody domains capable of specifically binding to IGF2R.

[0027] Less preferred are binding molecules of the invention which comprise at least one single variable antibody domain capable of specific binding to PDGFRB and at least one single variable antibody domain capable of specific binding to IGF2R.

[0028] In one preferred embodiment, a binding molecule of the invention is provided, wherein binding of one of the single variable antibody domains to its antigen modulates binding of the other single variable antibody domain to its antigen, preferably resulting in cluster-induced endocytosis and rapid receptor internalization.

[0029] The present invention provides several VHHs which in the examples show good binding to PDGFRB and internalization of PDGFRB. In a preferred embodiment, a binding molecule of the present invention is provided which comprises at least one single variable antibody domain capable of competing for specific binding to PDGFRB with a single domain antibody having a sequence selected from any one of the sequences SP02P or SP05P or SP14P or SP12P, as shown in Table 1 as SEQ ID NO: 9, SEQ ID NO: 25, SEQ ID NO: 81, and SEQ ID NO: 65, respectively. In a more preferred embodiment, the binding molecule comprises at least a second single variable antibody domain capable of competing with a single domain antibody having one of the sequences SP02P or SP05P or SP14P or SP12P, wherein preferably both the first single variable antibody domain and the second single variable antibody domain do not compete with the same single domain antibody having one of the sequences SP02P or SP05P or SP14P or SP12P. Alternatively, and also preferably, the binding molecule comprises at least two single variable antibody domains, where preferably the first single variable antibody domain and the second single variable antibody domain both compete with the same single domain antibody having one of the sequences SP02P or SP05P or SP14P or SP12P.

[0030] The term "compete" means that in a competition assay, for example as described in Examples 5 and 8 of the present invention, the addition of the binding molecule induces a significant decrease in the binding of the given VHH, i.e. the VHH for which the competition is determined. "Significant" in this context is preferably a decrease of more than 5%, preferably more than 10%, when 250 nM of the binding molecule is added to 10 nM of fluorescently labeled VHH in an ELISA. Example 8 shows, for example, that 10 nM of labeled 13F11 and 250 nM of unlabeled 13E8 show a lesser fluorescence intensity of less than 5% when bound to the human IGF2R ectodomain in an ELISA, whereas a substantially greater decrease in fluorescence intensity is observed when 10 nM of labeled 13F11 and 250 nM of unlabeled 13A8, 13C11, 13G10 or 13A12 compete for the same or overlapping epitope. From these data, it can be concluded that 13F11 and 13E8 do not compete with each other, whereas 13A8, 13C11, 13G10 and 13A12 compete with 13F11 for binding to IGF2R. When determining whether a binding molecule competes with a given VHH, the same test conditions and thresholds apply for binding molecules directed against PDGFRB. [Table 1-1] [Table 1-2] [Table 1-3] [Table 1-4] [Table 1-5] [Table 1-6]

[0031] The present invention further provides several VHHs which in the examples show good binding to IGF2R and internalization of IGF2R. In a preferred embodiment, a binding molecule of the present invention is provided which comprises at least one single variable antibody domain capable of competing with a single domain antibody having the sequence of 13E8 or 13F11, as shown in Table 2 as SEQ ID NO: 249 and SEQ ID NO: 273, respectively, for specific binding to IGF2R. In a more preferred embodiment, the binding molecule comprises at least a second single variable antibody domain capable of competing with a single domain antibody having the sequence of 13E8 or 13F11, where preferably both the first and second antibodies do not compete with the same single domain antibody having the sequence of 13E8 or 13F11. Alternatively, and also preferably, the binding molecule comprises at least two single variable antibody domains, where preferably both the first and second single variable antibody domains compete with the same single domain antibody having one of the sequences of 13E8 or 13F11. [Table 2-1] [Table 2-2] [Table 2-3] [Table 2-4]

[0032] In a preferred embodiment, a binding molecule of the invention is provided, wherein the binding molecule comprises at least one single variable antibody domain capable of specifically binding to PDGFRB, wherein the at least one single variable antibody domain comprises a CDR1 sequence according to SEQ ID NO: 83, a CDR2 sequence according to SEQ ID NO: 85 and a CDR3 sequence according to SEQ ID NO: 87; or a CDR1 sequence according to SEQ ID NO: 3, a CDR2 sequence according to SEQ ID NO: 5 and a CDR3 sequence according to SEQ ID NO: 7; or a CDR1 sequence according to SEQ ID NO: 11, a CDR2 sequence according to SEQ ID NO: 13 and a CDR3 sequence according to SEQ ID NO: 15; or a CDR1 sequence according to SEQ ID NO: 16, a CDR2 sequence according to SEQ ID NO: 17 and a CDR3 sequence according to SEQ ID NO: 19. or the CDR1 sequence according to SEQ ID NO: 35, the CDR2 sequence according to SEQ ID NO: 37 and the CDR3 sequence according to SEQ ID NO: 39; or the CDR1 sequence according to SEQ ID NO: 75, the CDR2 sequence according to SEQ ID NO: 77 and the CDR3 sequence according to SEQ ID NO: 79; or the CDR1 sequence according to SEQ ID NO: 27, the CDR2 sequence according to SEQ ID NO: 29 and the CDR3 sequence according to SEQ ID NO: 31; or the CDR1 sequence according to SEQ ID NO: 43, the CDR2 sequence according to SEQ ID NO: 45 and the CDR3 sequence according to SEQ ID NO: 47; or the CDR1 sequence according to SEQ ID NO: 51, the CDR2 sequence according to SEQ ID NO: 53 and the CDR3 sequence according to SEQ ID NO: 5 or a CDR1 sequence according to SEQ ID NO: 115, a CDR2 sequence according to SEQ ID NO: 117 and a CDR3 sequence according to SEQ ID NO: 119; or a CDR1 sequence according to SEQ ID NO: 67, a CDR2 sequence according to SEQ ID NO: 69 and a CDR3 sequence according to SEQ ID NO: 71, or a combination of CDR1, CDR2 and CDR3 sequences, wherein independently, at most four, more preferably at most three, more preferably at most two and most preferably at most one amino acid has been conservatively substituted, preferably independently, by the amino acid Substitutions are selected from: Ala to Gly or Ser; Arg to Lys; Asn to Gln or His; Asp to Glu; Cys to Ser; Gln to Asn; Glu to Asp; Gly to Ala or Pro; His to Asn or Gln; Ile to Leu or Val; Leu to Ile or Val; Lys to Arg, Gln or Glu; Met to Leu, Tyr or Ile; Phe to Met, Leu or Tyr; Ser to Thr; Thr to Ser; Trp to Tyr; Try to Trp or Phe; and Val to Ile or Leu.

[0033] Preferably, a binding molecule of the invention comprises at least one single variable antibody domain capable of specifically binding to IGF2R, wherein the at least one single variable antibody domain comprises a CDR1 sequence according to SEQ ID NO: 251, a CDR2 sequence according to SEQ ID NO: 253 and a CDR3 sequence according to SEQ ID NO: 255; or a CDR1 sequence according to SEQ ID NO: 275, a CDR2 sequence according to SEQ ID NO: 277 and a CDR3 sequence according to SEQ ID NO: 279; or a CDR1 sequence according to SEQ ID NO: 259, a CDR2 sequence according to SEQ ID NO: 261 and a CDR3 sequence according to SEQ ID NO: 263; or a CDR1 sequence according to SEQ ID NO: 291, a CDR2 sequence according to SEQ ID NO: 293 and a CDR3 sequence according to SEQ ID NO: 295; or a CDR1 sequence according to SEQ ID NO: 299, a CDR2 sequence according to SEQ ID NO: 301 and a CDR3 sequence according to SEQ ID NO: 302; or a CDR1 sequence according to SEQ ID NO: 307, a CDR2 sequence according to SEQ ID NO: 309 and a CDR3 sequence according to SEQ ID NO: and any of the CDR1, CDR2 and CDR3 sequences according to sequence number 311, or a combination of CDR1, CDR2 and CDR3 sequences, wherein independently, at most four, more preferably at most three, more preferably at most two and most preferably at most one amino acid is conservatively substituted, preferably independently selected from the following amino acid substitutions: Ala to Gly or Ser; Arg to Lys; Asn to Gln or His; Asp to Gly; to lu; Cys to Ser; Gln to Asn; Glu to Asp; Gly to Ala or Pro; His to Asn or Gln; Ile to Leu or Val; Leu to Ile or Val; Lys to Arg, Gln or Glu; Met to Leu, Tyr or Ile; Phe to Met, Leu or Tyr; Ser to Thr; Thr to Ser; Trp to Tyr; Try to Trp or Phe; and Val to Ile or Leu.

[0034] Preferably, a binding molecule of the invention comprises at least one single variable antibody domain capable of specifically binding to PDGFRB, wherein the at least one single variable antibody domain comprises or consists of any one of SEQ ID NO:81, SEQ ID NO:1, SEQ ID NO:9, SEQ ID NO:33, SEQ ID NO:73, SEQ ID NO:25, SEQ ID NO:41, SEQ ID NO:49, SEQ ID NO:65, or SEQ ID NO:113.

[0035] Preferably, a binding molecule of the invention comprises at least one single variable antibody domain capable of specifically binding to IGF2R, wherein at least one single variable antibody domain comprises or consists of any one of SEQ ID NO:249, SEQ ID NO:273, SEQ ID NO:257, SEQ ID NO:289, SEQ ID NO:297, or SEQ ID NO:305.

[0036] In one preferred aspect, a binding molecule of the invention is provided which comprises at least two single variable antibody domains, wherein at least one single variable antibody domain comprises or consists of SEQ ID NO:81; and wherein at least one single variable antibody domain comprises or consists of any one of SEQ ID NO:1, SEQ ID NO:9, SEQ ID NO:33, SEQ ID NO:73, SEQ ID NO:25, SEQ ID NO:41, SEQ ID NO:49, SEQ ID NO:65, or SEQ ID NO:113; or wherein at least one single variable antibody domain comprises any one of SEQ ID NO:1, SEQ ID NO:9, SEQ ID NO:33, SEQ ID NO:73, SEQ ID NO:25, SEQ ID NO:41, SEQ ID NO:49, SEQ ID NO:65, or SEQ ID NO:113; or consisting of any one of SEQ ID NO:1, SEQ ID NO:9, SEQ ID NO:33, or SEQ ID NO:73, with at least one single variable antibody domain comprising any one of SEQ ID NO:25, SEQ ID NO:41, SEQ ID NO:49, SEQ ID NO:65, or SEQ ID NO:113, or consisting of any one of SEQ ID NO:25, SEQ ID NO:41, SEQ ID NO:49, SEQ ID NO:65, or SEQ ID NO:113, or consisting of any one of SEQ ID NO:25, SEQ ID NO:41, SEQ ID NO:49, SEQ ID NO:65, or SEQ ID NO:113. Such molecules encompass biparatopic anti-PDGFRB binding molecules.

[0037] Preferably, a binding molecule of the invention comprises at least two single variable antibody domains, wherein at least one single variable antibody domain comprises or consists of SEQ ID NO:249 and at least one single variable antibody domain comprises or consists of any one of SEQ ID NO:273, SEQ ID NO:257, SEQ ID NO:289, SEQ ID NO:297, or SEQ ID NO:305. Such molecules include biparatopic anti-IGF2R binding molecules.

[0038] Preferably, a binding molecule of the invention comprises at least two single variable antibody domains, wherein at least one single variable antibody domain comprises or consists of any one of SEQ ID NO:81, SEQ ID NO:1, SEQ ID NO:9, SEQ ID NO:33, SEQ ID NO:73, SEQ ID NO:25, SEQ ID NO:41, SEQ ID NO:49, SEQ ID NO:65, or SEQ ID NO:113, and at least one single variable antibody domain comprises or consists of any one of SEQ ID NO:249, SEQ ID NO:273, SEQ ID NO:257, SEQ ID NO:289, SEQ ID NO:297, or SEQ ID NO:305. Such binding molecules encompass bispecific binding molecules capable of binding one epitope on PDGFRB and one epitope on IGF2R.

[0039] For purposes of comparing two or more amino acid sequences, the percentage of "sequence identity" or "sequence similarity" (also referred to herein as "amino acid identity") between a first amino acid sequence and a second amino acid sequence may be calculated by dividing the number of amino acid residues in the first amino acid sequence that are identical to the amino acid residues at the corresponding positions in the second amino acid sequence by the total number of amino acid residues in the first amino acid sequence, and multiplying by 100%, where each deletion, insertion, substitution or addition of an amino acid residue in the second amino acid sequence compared to the first amino acid sequence is considered as a difference at a single amino acid residue (position), i.e., an "amino acid difference" as defined herein. Alternatively, the degree of sequence identity between two amino acid sequences may be calculated using known computer algorithms, as described above for determining the degree of sequence identity for nucleotide sequences, as well as using standard settings. Usually, for the purpose of determining the percentage of "sequence identity" between two amino acid sequences according to the calculation method outlined above, the amino acid sequence having the greatest number of amino acid residues is taken as the "first" amino acid sequence, and the other amino acid sequence is taken as the "second" amino acid sequence. In determining the degree of sequence identity between two amino acid sequences, the skilled artisan may also take into account so-called "conservative" amino acid substitutions, which may be generally described as amino acid substitutions in which an amino acid residue is replaced with another amino acid residue of similar chemical structure and which have little or essentially no effect on the function, activity or other biological properties of the binding molecule. Such conservative amino acid substitutions are well known to those skilled in the art, for example from WO 04 / 037999, GB-A-3 357 768, WO 98 / 49185, WO 00 / 46383 and WO 01 / 09300, and (preferred) types and / or combinations of such substitutions may be selected on the basis of the appropriate teachings from WO 04 / 037999 and WO 98 / 49185 and from the further references cited therein.Such conservative substitutions are preferably those in which one amino acid residue within the following groups (a) to (e) is replaced with another amino acid residue within the same group: (a) small aliphatic non-polar or slightly polar residues: Ala, Val, Leu, Pro, Ile and Gly; (b) polar negatively charged residues and their (uncharged) amides: Asp, Asn, Glu and Gln; (c) polar positively charged residues: His, Arg and Lys; (d) large aliphatic non-polar residues: Met, Ser, Thr, Sec and Cys; and (e) aromatic residues: Phe, Tyr and Trp. Particularly preferred conservative substitutions are: Ala to Gly or Ser; Arg to Lys; Asn to Gln or His; Asp to Glu; Cys to Ser; Gln to Asn; Glu to Asp; Gly to Ala or Pro; His to Asn or Gln; Ile to Leu or Val; Leu to Ile or Val; Lys to Arg, Gln, or Glu; Met to Leu, Tyr or Ile; Phe to Met, Leu or Tyr; Ser to Thr; Thr to Ser; Trp to Tyr; Try to Trp or Phe; and Val to Ile or Leu.

[0040] Any amino acid substitutions applied to the binding molecules described herein may also be based on the analysis of the frequency of amino acid variations between homologous proteins of various species developed by Schulz et al., Principles of Protein Structure, Springer-Verlag, 1978, the structure generating potential analysis developed by Chou and Fasman, Biochemistry 13:211, 1974 and Adv. Enzymol., 47:45-149, 1978, and the analysis of hydrophobicity patterns in proteins developed by Eisenberg et al., Proc. Natl. Acad. Sci. USA 81:140-144, 1984; Kyte & Doolittle; J Molec. Biol. 157:105-132, 1981, and Goldman et al., Ann. Rev. Biophys. Chem. 15:321-353, 1986. all of which are incorporated herein by reference in their entireties.

[0041] The present invention also encompasses optimized variants of these amino acid sequences. In general, "optimized variants" of the amino acid sequences of the present invention are variants that contain one or more beneficial substitutions, such as substitutions that increase i) the degree of "humanization", ii) chemical stability, and / or iii) the level of expression.

[0042] In one preferred embodiment, a binding molecule of the invention is provided that comprises at least two single variable antibody domains, where the at least two single variable antibody domains are separated by a linker amino acid sequence. In many cases, a simple Gly-Ser linker of 4 to 15 amino acids may be sufficient, but longer or more complex linkers may be used if greater flexibility of the amino acid chain is desired. Preferred linkers are (Gly4Ser)n, (GSTSGS)n or any other linker that provides flexibility for protein folding. The binding domains may be separated only by a linker, but other useful amino acid sequences may be introduced between the binding domains or at the N- or C-terminus of the first or final binding domain sequence, respectively. Thus, in one embodiment, a binding molecule of the invention is provided that further comprises an amino acid sequence encoding a linker. Such a linker sequence preferably provides flexibility within the molecule to increase the distance that can be bridged by the binding molecule to link (bridge) two epitopes. Furthermore, such linkers may reduce steric hindrance that may arise when one of the single variable antibody domains is bound to its first target and a second single variable antibody domain must find and bind to a second target.

[0043] It has been observed that the binding molecules of the invention are internalized when the single variable antibody domains within the molecule bind to their targets with sufficient affinity. Thus, in a preferred embodiment, at least one single variable antibody domain and, if present, further single variable antibody domains bind, independently of each other, to their respective receptors with a dissociation constant (K D The binding molecule of the present invention is capable of specifically binding at 10E-5 to 10E-12 mol / L or less, preferably 10E-7 to 10E-12 mol / L or less, and more preferably 10E-9 to 10E-12 mol / L.

[0044] In the context of the present invention, "binding to and / or having affinity for" a particular antigen has the meaning useful in the art, e.g., as understood in the context of antibodies and their respective antigens. In certain aspects of the invention, the term "binding to and / or having affinity for" means that at least one single variable antibody domain specifically interacts with the antigen. The term "specificity" refers to the number of different types of antigens or antigenic determinants to which a particular immunoglobulin sequence, antigen-binding portion or antigen-binding molecule (e.g., a binding molecule of the present invention) can bind. The specificity of an antigen-binding molecule can be determined based on its affinity and / or avidity for a target molecule(s) relative to its affinity and / or avidity for non-target molecules. The equilibrium constant (K D Affinity, represented by K ), is a measure of the binding strength between an antigenic determinant and an antigen-binding site on an antigen-binding protein, and K D The smaller the value of K, the stronger the binding strength between the antigenic determinant and the antigen-binding molecule (alternatively, affinity is also expressed as 1 / K D The affinity constant (K A ) can be expressed as K D Methods for determining affinity will be apparent to those skilled in the art and include, for example, the techniques mentioned herein. Affinity represents the strength or stability of a molecular interaction. Affinity is expressed as K, which has units of mol / liter (or M). D Affinity is also commonly given by the equilibrium dissociation constant, 1 / K D and has units of liters / mol, A As used herein, the stability of an interaction between two molecules (e.g., an amino acid sequence, immunoglobulin sequence, or binding molecule of the invention and its intended target) can be expressed primarily as the K D is expressed in terms of the value K A =1 / K D Considering the relationship between the molecular interactions, the strength of the molecular interactions is expressed as K D By specifying the value, the corresponding K AIt will be apparent to one skilled in the art that the above-mentioned techniques can be used to calculate the value of

[0045] K D The value characterizes the strength of the molecular interaction in a thermodynamic sense as well, since it is related to the free energy of binding. As will be clear to the skilled person (e.g. based on the further disclosures herein), the affinity can be determined in a manner known per se, depending on the specific antigen of interest. The avidity gives a measure of the overall strength of the antibody-antigen complex. Avidity depends on three main parameters: the affinity of the antibody for the epitope (see above), the binding valency of both the antibody and the antigen, and the structural sequence of the interacting moieties. Avidity relates both to the affinity between the antigenic determinant and its antigen binding site on the antigen-binding molecule, and to the number of suitable binding sites present on the antigen-binding molecule. Usually, the immunoglobulin sequences of the invention (e.g. the amino acid sequences, ISVDs and / or binding molecules of the invention) bind to their antigens with 10E-5 to 10E-12 moles / liter or less, preferably 10E-7 to 10E-12 moles / liter or less, more preferably 10E-9 to 10E-12 moles / liter. Any K greater than 10E-5M D The K value is generally considered to indicate non-specific binding. Preferably, the monovalent immunoglobulin sequence of the present invention binds to the desired antigen with an affinity of less than 500 nM, preferably less than 200 nM, more preferably less than 10 nM, and most preferably less than 500 pM. The binding specificity and binding affinity of an antigen-binding protein or antigen-binding protein to an antigen or antigenic determinant can be determined in any suitable manner known per se, including, for example, Scatchard analysis and / or competitive binding assays, such as radioimmunoassays (RIA), enzyme-linked immunosorbent assays (ELISA) and sandwich competition assays, and their different variants known per se in the art, as well as other techniques mentioned herein. The K value for a biological interaction, such as the binding of a binding molecule of the present invention to a cell-associated antigen as defined herein, that is considered to be significant (e.g., specific), is generally considered to be non-specific binding. Dis usually in the range of 10E-10M (0.1 nM) to 10E-6M (1 μM).

[0046] K D Also, k off The dissociation rate constant of the complex, k on It is expressed as a ratio of the rate of dissociation to its rate of dissociation (i.e., K D =k off / k on , and K A =k on / k off The affinity of the molecular interaction between two molecules can be measured by various techniques known per se, such as the well-known surface plasmon resonance (SPR) biosensor technique (see, for example, Ober et al., Intern. Immunology, 13, 1551-1559, 2001), in which one molecule is immobilized on a biosensor chip and the other molecule passes over the immobilized molecule under flow conditions; k on Measurement value, k off measurements, and therefore K D (or K A ) values. This can be performed, for example, using the well-known Biocore instrument. Affinity measurements for transmembrane receptors expressed by cells are preferably performed by LISA.

[0047] In addition, the measured K D The apparent K can be increased if the measurement process affects in some way the intrinsic binding affinity of the molecule involved, for example, by artifacts associated with the coating on the biosensor of one molecule. D Also, the apparent K Dcan be measured when one molecule contains more than one recognition site for the other molecule. In such a situation, the affinity measured may be influenced by the avidity of the interaction by the two molecules. Another approach that can be used to assess affinity is the two-step ELISA procedure of Friguet et al. (J. Immunol. Methods, 77, 305-19, 1985). This method establishes a solution-phase binding equilibrium measurement, avoiding possible artifacts associated with the adsorption of one of the molecules on a support such as plastic. However, K D Accurate measurement of K can be very labor intensive and therefore often results in an apparent K D A value is determined to assess the binding strength of two molecules. As long as all measurements are performed in a consistent manner (e.g., by keeping the assay conditions unchanged), the apparent K D Measurements are based on true K D Therefore, in this document, K D and apparent K D It should be noted that all claims, disputes and dispute resolution processes should be treated with equal importance or relevance.

[0048] Also, the therapeutic or diagnostic molecule, if present, is preferably attached to the remainder of the binding molecule by a linker and / or spacer. The linker is attached to the targeting moiety, such as the binding molecule of the present invention, using strategies for either random / heterogeneous or site-specific conjugation of drugs. The former strategy is mainly based on traditional active ester or maleimide chemistry for drug conjugation to lysine or (hinge region) cysteine ​​residues, respectively, whereas the latter utilizes chemical residues that are introduced at specific sites of engineered "modified targeting moieties" genetically, enzymatically, or by other means. The single variable antibody domains may contain cysteine ​​or histidine residues on the C-terminus or N-terminus portion. Site-specific conjugation to the targeting moiety results in a homogenous mixture with minimal batch-to-batch variability. The linker may be cleavable or non-cleavable. Both types of linkers are assumed to be stable in circulation, but based on detailed analysis of their integrity in blood and on the toxicity profile of certain ADCs, stability is a concern for currently approved ADCs. In the case of ADCs with cleavable linkers, after internalization by target cells, the cleavable linker as well as the antibody will be degraded in lysosomes, making the drug freely available to exert its toxic activity. In the case of ADCs with non-cleavable linkers, only the antibody will be degraded in lysosomes, releasing the drug-linker complex. The above insights pave the way for linkers to be endowed with tunable physicochemical functions that can modify the properties of ADCs as well as drugs in beneficial ways, for example, by making the ADC production process simpler and cheaper and / or by widening the therapeutic window. Next, given the presence of a stable bond between the drug and the mAb, an ideal linker would (1) improve the solubility of the drug and the ADC, making conjugation of the drug to the mAb more efficient, (2) improve cellular trafficking and the interaction between the drug and its cellular target, (3) reduce efflux of the drug by tumor cells via multidrug resistance proteins, and (4) prevent uptake of the drug-linker complex by healthy cells.Because most of the conventional organic linkers provide suboptimal ADCs, extensive research into new conjugation technologies has been initiated in the last few years.

[0049] The present invention provides a specific linker, further referred to as "Lx", which comprises a functional platinum (II) complex having two reactive groups, such as, but not limited to, those described in International Patent Application Publication No. 2013103301. The first reactive group of Lx can be replaced by a kinase inhibitor, and the second reactive group by a targeting moiety, such as a binding molecule of the present invention. By using Lx, the immunoreactivity of the conjugated binding molecule remains substantially the same as the unconjugated natural binding molecule. This is particularly important if the immunoreactivity of the binding molecule remains high enough to allow the delivery of the conjugated drug as a therapeutic compound in place in the body. Furthermore, the targeting moiety used can be customized to the patient's specific needs, since it does not take much time to bind the binding molecule to LX, and the reaction conditions for this coupling are mild and very independent of the functional moiety used. Due to the mild reaction conditions, the binding molecule remains in excellent condition and retains its original (unconjugated) immunoreactivity. The present invention provides a technology that allows for rapid, efficient, stable and site-specific conjugation of small therapeutic or diagnostic molecules to binding molecules. Biparatopic polypeptides conjugated through Lx have shown excellent relaxant activity in cell models. The Lx linker is resistant to degradation in blood while still allowing release of the payload at the target. Furthermore, Lx allows for the conjugation of drugs of various chemical species, unlike other systems. As mentioned above, targeting drugs to HSCs via binding molecules is conceptually attractive for increasing specificity, reducing systemic toxicity and enabling therapeutic use of compounds that are in principle less suitable or unsuitable as systemic drugs, for example with respect to their toxicity profile. This requires stable site-specific coupling of drugs to the binding molecules of the present invention to target upregulated receptors exposed on the outer membrane of activated HSCs. Thus, the linker and / or spacer preferably comprises or consists of a transition metal complex. In a preferred embodiment, the transition metal complex comprises Pt(II).In a further preferred embodiment, the linker comprises a cis-platinum(II) complex, more preferably a cis-platinum(II) complex comprising an inert bidentate moiety, wherein the bidentate moiety is preferably ethane-1,2-diamine.

[0050] As mentioned above, inhibiting the cytoskeletal contraction of activated HSCs or myofibroblasts attenuates fibrosis. Thus, in a preferred embodiment, a binding molecule of the invention is provided, wherein the therapeutic molecule is capable of inhibiting the cytoskeletal contraction of activated HSCs. For example, kinase inhibitors can inhibit the contraction of the cytoskeleton of activated HSCs. The targeted kinases are involved in a signaling pathway called the renin-angiotensin system (RAS), which plays a role in liver fibrosis and portal hypertension. The RAS plays a central role in regulating blood pressure by regulating vascular smooth muscle tone. Two kinases involved in the RAS are the tyrosine kinase Janus kinase 2 (JAK2) and Pho-related coiled-coil-containing protein kinase (ROCK). Expression of these protein kinases has been associated with portal hypertension in human cirrhosis.

[0051] Portal hypertension is present in all stages of liver fibrosis and cirrhosis. In cirrhosis, activated HSCs contract around the sinusoids where they reside. This causes portal hypertension, where blood flow is obstructed through the portal system in the liver. Within the RAS, JAK2 becomes phosphorylated and activates Arhgef1, a nucleotide exchange factor responsible for activating the small GTPase RhoA, which in turn activates its downstream effector ROCK. ROCK is a regulator of the actomyosin cytoskeleton that promotes contractile force generation. ROCK phosphorylates and thereby inactivates myosin light chain phosphatase (MLCP), leading to increased myosin light chain phosphorylation and contraction. Thus, the use of Y27632, a ROCK inhibitor, causes a decrease in RAS-induced HSC contraction. JAK2 then plays a role in activating ROCK, so its inhibition by SB1518 (also called pacritinib, which is a JAK2 inhibitor) can help relax HSCs. These small molecules are highly toxic and non-specific, and therefore suffer from dose-limiting toxicities that make them unsuitable for systemic administration. The binding molecules of the present invention carrying an effective amount of a conjugated small molecule drug are designed to overcome the limitations of systemic administration of small molecules. Preferably, the therapeutic molecule is a kinase inhibitor, preferably selected from the group consisting of Rho-kinase inhibitors, JAK-2 inhibitors and neprilysin inhibitors, more preferably selected from the group consisting of Y27632, SB1518 and LBQ657. In another preferred embodiment, the therapeutic molecule is a toxin, such as a taxane, anthracycline, vinca alkaloid, calicheamicin, maytansinoid, auristatin, preferably auristatin F and CC 10065 analogs.

[0052] Usually, the binding to a transmembrane receptor induces an intracellular signaling cascade. For example, the binding of PDGF to PDGFRB induces phosphorylation of the receptor itself and other proteins, thereby engaging intracellular signaling pathways that trigger cellular responses such as migration and proliferation. Since the purpose of the binding molecule of the present invention is the transport of its payload into the cytoplasm of the target cell, and not the activation of the receptor used for transport, it is preferred that the binding of the binding molecule to the extracellular domain of the transmembrane receptor does not induce the intracellular signaling cascade of the receptor. It is further preferred that the binding of the binding molecule to the transmembrane receptor causes receptor-mediated internalization, endocytosis and release of the drug or toxin in the endolysosomal compartment.

[0053] In one preferred embodiment, a binding molecule of the invention is provided, wherein the binding molecule comprises an N- or C-terminal cysteine ​​or histidine residue, preferably an N- or C-terminal cysteine, that serves the purpose of conjugation to a linker molecule. In one embodiment, the invention provides a biparatopic binding molecule comprising at least two binding molecules of the invention.

[0054] As described above, the binding molecules of the present invention, including therapeutic molecules, are useful for delivering therapeutic molecules to the cytoplasm of target cells, such as fibrotic hepatocytes, thereby allowing the treatment of liver fibrosis. Thus, the present invention provides the internalizing binding molecules of the present invention for use as medicines. The terms "treat", "treating" or "treatment" refer to administering a therapy in an amount, manner and / or form effective to improve a condition, symptom or parameter associated with a disease or to prevent the progression of a disease, at a statistically significant level or at a level detectable by a person skilled in the art. In therapeutic applications, the treatment may improve, cure, maintain or reduce the duration of a disease or condition in a subject. In therapeutic applications, the subject may have partial or complete manifestations of symptoms. Typically, the treatment improves a disease or condition in a subject or prevents the disease or condition from worsening to a level detectable by a physician. As used herein, the terms "prevent" or "preventing" refer to relieving the symptoms of the disorder referred to. In particular, the term encompasses the full range of therapeutically positive effects of administering a binding molecule of the invention to a subject, including reduction, alleviation or relief from CLD-related disorders, such as esophageal variceal bleeding, and symptoms thereof. The term "prevention" encompasses preventing or postponing the onset of disease, preventing or postponing the onset of symptoms, and / or reducing the severity of such symptoms that develop or are expected to develop. These further encompass improving existing symptoms, preventing further symptoms, and improving or preventing the underlying cause of symptoms. As used herein, the terms "subject" and "patient" are used interchangeably. As used herein, the terms "subject" and "subjects" refer to animals, e.g., mammals, including non-primates (e.g., cows, pigs, horses, donkeys, goats, camels, cats, dogs, guinea pigs, rats, mice, sheep) and primates (e.g., monkeys, e.g., cynomolgus monkeys, gibbons, orangutans, gorillas, chimpanzees, and humans). A "patient" preferably refers to a human.The patients may include elderly, adults, adolescents and children of any age, such as children ranging from 2 to under 12 years of age, adolescents ranging from 12 to under 18 years of age, adults ranging from 18 to under 65 years of age, and elderly people aged 85 years and older.

[0055] Preferably, such medicament is for use in a method relating to the prevention and / or treatment of a disease or disorder associated with or characterised by increased expression of PDGFRB and / or IGF2R.

[0056] Preferably, such medicament is for use in a method for preventing and / or treating liver cirrhosis, liver fibrosis and / or portal hypertension. For example, portal hypertension (PH) is present in liver fibrosis and in all stages of liver cirrhosis. In liver cirrhosis, activated HSCs contract around the sinusoids where they reside. This causes PH, where blood flow is blocked through the portal system in the liver. The portal system is composed of the portal vein, which mixes large amounts of blood from the stomach, intestines, spleen and pancreas, and branches through smaller blood vessels to move the large amount of blood throughout the liver. When blood vessels in the liver are blocked due to liver damage, blood cannot flow properly through the liver, resulting in high blood pressure. When PH worsens and blood flow is blocked through the portal vein, blood is redirected through smaller blood vessels, such as the blood vessels in the esophagus, which are not designed to carry such large amounts of blood. This causes enlarged veins (varices) in the distal esophagus and proximal stomach, which are at increased risk of rupture (variceal hemorrhage), causing severe to fatal internal bleeding. The binding molecules of the invention are particularly useful for targeting cells that cause or exacerbate liver cirrhosis, liver fibrosis and / or portal hypertension.

[0057] Preferably, such medicament is for use in a method relating to the prevention and / or treatment of a disease or disorder associated with esophageal varices and / or hemorrhoids.Preferably, such medicament is for use in a method relating to the prevention and / or treatment of a disease or disorder characterised by variceal and external bleeding.

[0058] In one aspect, the invention provides a nucleic acid encoding at least a part of a binding molecule of the invention. One advantage of the invention is the ease of production of the molecules of the invention and their simplicity, while the selection for a single nucleic acid encoding essentially all essential functions allows for the relatively easy addition of other functionalities in the resulting binding molecule (as long as there is room in the selected expression vector). Such a nucleic acid, when transfected in a suitable host cell, allows for the production of a binding molecule of the invention. Thus, the invention provides a host cell for the expression of at least a part of a binding molecule of the invention, comprising a nucleic acid of the invention. In one aspect, the invention provides a method for producing a binding molecule of the invention, comprising the steps of culturing a host cell of the invention, allowing the expression of at least a part of said binding molecule, harvesting said binding molecule, and coupling said therapeutic or diagnostic molecule to said part of said binding molecule, optionally by means of a linker as defined hereinbefore.

[0059] The binding molecules of the invention can be produced by any commonly used method. Typical examples include recombinant expression in a suitable host system, such as bacteria, yeast or mammalian cells. The binding molecules of the invention undergo a suitable purification regime before being formulated according to the invention. Generally, the binding molecules of the invention are produced by living host cells genetically engineered to produce the binding molecules. Methods for genetically engineering cells to produce proteins are well known in the art. See, for example, Ausubel et al., eds. (1990), Current Protocols in Molecular Biology (Wiley, New York). Such methods involve introducing a nucleic acid encoding the binding molecule and allowing expression of the binding molecule into a living host cell. These host cells can be bacterial, fungal or animal cells grown in culture. Bacterial host cells include, but are not limited to, Escherichia coli cells. Examples of suitable E. coli strains include BL21(D3), HB101, DH5a, GM2929, JM109, KW251, NM538, IMM539, and any E. coli strain that cannot cleave foreign DNA. E. coli strain BL21(D3) is preferred. Fungal host cells that can be used include, but are not limited to, Saccharomyces cerevisiae, Pichia pastoris, and Aspergillus cells. A few examples of animal cell lines that can be used are CHO, VERO, BHK, HeLa, Cos, MDCK, HEK293, 3T3, and WI38. New animal cell lines can be established (e.g., by transformation, viral infection, and / or selection) using methods well known to those skilled in the art. Optionally, the binding molecule can be secreted into the culture medium by the host cell. In some aspects, the binding molecules can be produced in bacterial cells, for example, in E. coli cells.

[0060] In one embodiment, the binding molecule is expressed in yeast cells such as Pichia (see, e.g., Powers et al., J Immunol Methods 251:123-35 (2001)), Hansenula, or Saccharomyces. Exemplary mammalian host cells for expressing clonal antibodies or antigen-binding fragments thereof include Chinese hamster ovary cells (CHO cells) (including dhfr-CHO cells, described in Urlaub and Chasin, Proc. Natl. Acad. Sci. USA 77:4216-4220 (1980), used with the DHF selectable marker, e.g., as described in Kaufman and Sharp, Mol. Biol. 159:601-621 (1982)), lymphoid cell lines, e.g., NS0 myeloma cells and SP2 cells, COS cells, and cells from transgenic animals, e.g., transgenic mammals. For example, the cells are mammary epithelial cells. In addition to nucleic acid sequences encoding the binding molecule, recombinant expression vectors may carry additional sequences, such as sequences that regulate replication of the vector in host cells (e.g., origins of replication) and selectable marker genes. The selectable marker gene facilitates selection of host cells into which the vector has been introduced (see, e.g., U.S. Pat. Nos. 4,399,216, 4,634,665, and 5,179,017). For example, the selectable marker gene usually confers resistance to drugs, such as G418, hygromycin, or methotrexate, on the host cell into which the vector has been introduced. Standard molecular biology techniques can be used to prepare the recombinant expression vector, transfect the host cells, select for transformants, culture the host cells, and recover the antibody molecule from the culture medium. For example, the binding molecules of the invention can be isolated by affinity chromatography. In one embodiment, the binding molecules of the invention are purified as described in WO 10 / 058550.In an exemplary embodiment, the binding molecule is purified from one or more contaminants by contacting a mixture of the binding molecule and the contaminant(s) with a protein A-based support and / or an ion exchange support under conditions that allow the binding molecule to bind or adsorb to the support, removing the one or more contaminants by washing the bound support under conditions that allow the binding molecule to remain bound to the support, and selectively eluting the binding molecule from the support by eluting the adsorbed binding molecule with an elution buffer. The binding molecule of the present invention can also be produced by a transgenic animal. For example, U.S. Pat. No. 5,849,992 describes a method for expressing an antibody in the mammary gland of a transgenic mammal. A transgene is constructed that includes a milk-specific promoter and a nucleic acid encoding the antibody molecule and a signal sequence for secretion. Milk produced by females of such transgenic mammals includes the signal domain of interest in secreted form therein. The antibody molecule can be purified from the milk or, depending on the application, can be used directly. The present invention includes a method for producing a formulation as defined herein.

[0061] The present invention further provides a pharmaceutical composition comprising at least one binding molecule of the invention and at least one pharma- ceutically acceptable excipient.

[0062] The binding molecules of the invention may be administered or used for administration in the form of liquid solutions (e.g., injectable and infusible solutions). Such compositions may be administered by parenteral forms (e.g., subcutaneous, intraperitoneal or intramuscular injection) or by inhalation. The phrases "parenteral administration" and "administered parenterally" as used herein refer to forms of administration other than enteral and topical administration, usually by injection, and include subcutaneous (sc) or intramuscular administration as well as intravenous (iv), intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, intratracheal, subcuticular, subcapsular, subarachnoid, intraspinal, epidural, and intrasternal injections and infusions. Preferably, the second or further dose of the binding molecules of the invention described herein is administered subcutaneously or orally for sustained release and thus sustained effect. Preferably, especially in acute situations, the binding molecules of the invention are administered orally, since they are transported via organ circulation directly to the diseased liver after uptake in the stomach. The present invention also provides formulations of binding molecules comprising at least one immunoglobulin single variable domain against PDGF or IGF (also called "pharmaceutical compositions of the invention") that are stable and preferably suitable for pharmaceutical use, including the preparation of medicaments. The term "pharmaceutical composition" refers to a preparation that is present in a form that allows the biological activity of the active ingredient (the binding molecule of the invention) to be effective and does not contain additional components that are unacceptably toxic to the subject to whom the formulation is administered. Such formulations are preferably sterile. "Pharmaceutically acceptable" excipients (vehicles, additives) are those that may reasonably be administered to a mammalian subject so as to provide an effective dose of the active ingredient employed.

[0063] The term "excipient" as used herein refers to an inert substance commonly used as a diluent, vehicle, preservative, lyoprotectant, surfactant, binder, carrier or stabilizer for a compound that confers beneficial physical properties to the formulation. Those skilled in the art are familiar with excipients suitable for pharmaceutical purposes that may have specific functions in the formulation, such as lyoprotection, stabilization, preservatives, etc.

[0064] Non-limiting examples of agents that may be co-formulated with the binding molecules of the present invention include, for example, supportive therapy with adjunctive treatments (e.g., corticosteroids such as (methyl)prednisolone or (methyl)prednisone, diuretics, albumin, vitamin K, antibiotics and nutritional therapy, and systemic antihypertensive agents such as beta-blockers) and red blood cell transfusions. Such combination therapy may advantageously utilize lower doses of the administered therapeutic agent, thus avoiding possible toxicities or complications associated with various monotherapies. In one aspect, the present invention relates to combination therapy of the binding molecules of the present invention with systemic antihypertensive treatments. Preferably, the combination therapy is provided until portal vein pressure is normalized. Portal vein pressure measurements are useful for confirming the diagnosis and monitoring the course of the disease and the possible need for further treatment. The efficacy or dosing regimen of any particular proteinaceous molecule of the present invention may be determined by methods available to those skilled in the art. Briefly, during clinical trials, patients may be observed by medical personnel and the disease state is evaluated by any combination of criteria. Improvement in the patient's disease state is determined based on these criteria at multiple time points, and the combination of these determinations for a patient population is plotted to assess the effectiveness of the treatment. In exemplary embodiments, the assessment of efficacy may be measured by any or all of the criteria described below: Treatment response over time Number of subjects with complete remission Number of subjects with EVH exacerbations and time to first EVH exacerbation -Improvement of organ dysfunction and CLD-related signs and symptoms All-cause mortality during the study drug treatment period (including tapering) · Determination of biomarkers for CLD.

[0065] The dosage of the binding molecules of the invention should be established by animal studies and clinical studies in so-called ascending dose experiments. Usually, the dosage is comparable to the current antibody dosage (at the molar level, the weight of the molecules of the invention may differ from the weight of the antibody). Usually, such dosage is between 3 mg / kg and 15 mg / kg (body weight) or between 25 mg and 1000 mg per dose.

[0066] Especially in the more chronic stages of liver disease, the first application of the binding molecule of the invention is likely to be (at least initially) in combination with other treatments (standard care). Therefore, the present invention also provides a pharmaceutical composition comprising the binding molecule of the invention and a conventional therapy, e.g. an antihypertensive drug such as propranolol or a surgical procedure to stop bleeding from varices in the esophagus. Furthermore, the present invention also provides a pharmaceutical composition comprising the binding molecule of the invention for use in adjunctive treatment, e.g. in liver disease. Furthermore, the present invention also provides a pharmaceutical composition comprising the binding molecule of the invention for use in complex medical treatment of liver disease.

[0067] Therefore, an ideal therapeutic compound should allow the delivery of effector compounds that are much more potent than currently used molecules such as propranolol, and have a therapeutic window that is too narrow to allow their use as free drugs in other situations.Preferably, the stable conjugation of the drug to the targeting moiety avoids systemic toxicity due to the release of the compound in the circulation (which would otherwise be toxic).When the molecule is internalized by HSCs, the drug should be released, allowing it to inactivate HSCs.

[0068] Preferably, the pharmaceutical composition further comprises at least one other compound useful for the treatment of chronic liver disease. The pharmaceutical composition of the present invention is preferably for use as an adjunct treatment of variceal bleeding in the presence of portal hypertension and / or for use in the treatment of liver disease. The binding molecules of the present invention, including diagnostic molecules, are particularly useful, for example, for the diagnosis of liver fibrosis. Thus, in a preferred embodiment, the binding molecules of the present invention, including diagnostic molecules, are provided, where the diagnostic molecules are imaging agents. Diagnostic compositions are further provided, comprising at least one binding molecule of the present invention, including diagnostic molecules, and a diluent and / or excipient.

[0069] In a particularly preferred embodiment of the invention, the binding molecule of the invention comprises at least two single variable domains, which are capable of specifically binding, independently of each other, to the PDGFRB or IGF2R receptors of hepatic stellate cells and / or myofibroblasts, which are linked to a therapeutic molecule selected from a kinase inhibitor, such as a Rho-kinase inhibitor, a Jak-2 kinase inhibitor, a neprilysin inhibitor or an angiotensin II receptor antagonist, via a linker comprising a cis-platinum(II) complex. Preferably, the cis-platinum(II) complex of the linker comprises an inert bidentate moiety, most preferably ethane-1,2-diamine. Furthermore, preferred single variable domains which specifically bind to the IGF2R receptor comprise a CDR1 sequence according to SEQ ID NO: 275, a CDR2 sequence according to SEQ ID NO: 277 and a CDR3 sequence according to SEQ ID NO: 279, or comprise a CDR1 sequence according to SEQ ID NO: 251, a CDR2 sequence according to SEQ ID NO: 253 and a CDR3 sequence according to SEQ ID NO: 255 (i.e. according to 13F11 and 13E8, respectively), and preferred single variable domains which specifically bind to the PDGFRB receptor comprise a CDR1 sequence according to SEQ ID NO: 11, a CDR2 sequence according to SEQ ID NO: 13 and a CDR3 sequence according to SEQ ID NO: 15, or a CDR1 sequence according to SEQ ID NO: 155, a CDR2 sequence according to SEQ ID NO: 157 and a CDR3 sequence according to SEQ ID NO: 159 (i.e. according to SP02P and SP26P, respectively). Furthermore, said binding molecules preferably also comprise a so-called half-life extender, which is preferably selected from an albumin binding domain, an albumin binding VHH or an antibody Fc tail or a fragment thereof. In the following examples, inter alia, certain species of these preferred embodiments are described in more detail.

[0070] example (Example 1): Selection of human antibody fragments specific for IGF2R or PDGFRB. Llama immunization, cell harvest, phage VHH selection and library construction. VHH selection based on target affinity and HSC internalization: IGF2R (rat and human). The general procedure for generating and characterizing antigen-specific VHHs is depicted in FIG.

[0071] 1.1 IGF2R Our aim was to identify VHHs that bind specifically and with high affinity to the receptor IGF2R on human and rat hepatic stellate cells (HSCs). The starting material for the study was a previously generated llama VHH cDNA library. The library consisted of a large pool of VHH sequences extracted from peripheral blood mononuclear cells of a llama immunized with human IGF2R (hIGF2R)-expressing A549 cells. The library size was approximately 10, as assessed by colony titration upon DNA transformation. 8 The number of colony forming units (cfu) was 100.

[0072] Phages were prepared from the A549 VHH library and subjected to two rounds of selection against the recombinant human extracellular domain of IGF2R (hIGF2R-ECD). 11 Colony forming units (cfu) of phages were incubated on various concentrations of target, eluted, amplified, and then used for the next round of selection. The phage output after the second selection round was clearly higher than the first round (1.2 × 10 9 pcs and 3.3×10 7 ), demonstrating that VHH binders were amplified.

[0073] After the final selection round, 94 phage clones were randomly selected and tested for binding to hIGF2R-ECD in phage ELISA. Clones with high binding affinity were then further characterized by their restriction patterns using HinfI digestion and classified according to their restriction patterns. From each category, a number of clones were selected and the amino acid sequences of these binders were identified (Table 2). This selection resulted in a panel of 15 unique hIGF2R binders all derived from the A549 library (Figure 2).

[0074] 1.2 PDGFRB Selection of VHHs binding specifically and with high affinity to the receptor PDGFRB (rat and human) was initiated by immunization of llamas. For each receptor, two llamas were injected five times, four times with the extracellular domain (ECD) and once with SCC VII cells transfected with PDGFRB, according to the immunization schedule in Table 3. It should be noted that the two llamas immunized with rPDGFRB were also immunized with SCC VII cells transfected with rIGF2R for the generation of VHHs binding specifically and with high affinity to rIGF2R. Eight days after the last immunization, blood was collected, peripheral blood lymphocytes (PBLs) were purified and their RNA was extracted. [Table 3]

[0075] PDGFRB libraries (for both human and rat PDGFRB) were created according to the following procedure. Extracted RNA (40 μg, 4 reactions of 10 μg each) was transcribed into cDNA using a reverse transcriptase kit (Invitrogen), both according to the manufacturer's protocol, and the cDNA was cleaned up using a QIAquick PCR purification kit (Qiagen). The IGH fragment (conventional heavy chain) was amplified using primers annealing at the leader sequence region and the CH2 region. The PCR product was loaded onto a 1% agarose gel, after which a 700 bp fragment was excised from the gel and purified using a QIAquick kit (Qiagen) according to the manufacturer's protocol. 80 ng of purified DNA was then used as template for nested PCR to introduce restriction sites (final volume 800 μL). The amplified fragment was cleaned up using a QIAquick PCR purification kit according to the manufacturer's protocol and eluted in 120 μL of elution buffer. The eluted DNA was digested with BstEII and SfiI, then the sample was run on a 1.5% TAE-agarose gel and the 400 bp fragment was isolated from the gel. The DNA fragment was isolated using a QIAquick cleanup kit (Qiagen) according to the manufacturer's protocol and eluted in 100 μL of elution buffer. The DNA fragment (330 ng) was then ligated into a phagemid vector (pHEN1 derivative, 1 μg) and transformed into TG1 bacterial cells. Prior to ligation, the phagemid vector was prepared by digestion with SfiI and BstEII followed by dephosphorylation with FastAP.

[0076] The number of transformants was calculated from the dilution of plated out TG1 cells (8 mL). The titer of the library (Table 4) was calculated by counting the colonies at the highest dilution and using the following formula: Library size = (amount of colonies) x (dilution) x 8 mL / 0.005 mL (volume spotted). Insert frequencies were determined by picking 24 different clones from each library transformation and performing colony PCR. The insert frequencies were 100% for library 1, library 2, and library 4, and 96% for library 3. [Table 4]

[0077] Phages were prepared from the library and subjected to two rounds of selection. The first selection round was performed on the extracellular domain (hPDGFRB-ECD or rPDGFRB-ECD). Phages were incubated on various concentrations of target, eluted, amplified and subsequently used for the next round of selection. The phage output of the first selection round is displayed in FIG. 3. The second selection round was performed on SCC VII cells transfected with either hPDGFRB or rPDGFRB for selection of phages that bind and internalize. An acid wash was used to separate phages that only bind to PDGFRB from phages that internalize via PDGFRB after binding. This selection round was performed using the output from the first round from wells with the lowest ECD density. The phage output of the second selection round is displayed in FIG. 4.

[0078] After the second selection round, 94 internalizing phage clones were randomly selected and tested for binding to PDGFRB-ECD in a phage ELISA. Clones with high binding affinity were then further characterized by restriction pattern analysis using HinfI digestion. Clones were classified according to their restriction patterns, unique clones from each category were selected, and the amino acid sequences of these binders were identified, resulting in 18 unique hPDGFRB-binding VHHs and 11 unique rPDGFRB-binding VHHs (Table 1, Figures 5-6).

[0079] (Example 2): Genetic design for the production of binding molecules containing VHH To produce VHHs, the genes of the selected VHHs were cloned into pET-21 or pET-28 vectors containing the following sequences in the insert of the expression vector (Figure 7): A PelB sequence to induce the transport of the produced protein to the periplasm in bacterial cells; A T7 promoter and terminator and a lac operon in between to allow induction of the VHH protein upon addition with IPTG; Ampicillin or Kanamycin resistance genes for resistance to the antibiotics ampicillin or kanamycin, respectively; One free C-terminal cysteine ​​available for site-selective conjugation of a semi-final or maleimide-modified moiety containing a payload such as a dye or a kinase inhibitor; A histidine, EPEA or other affinity purification tag and a thrombin cleavage site are used to remove the purification tag if necessary.

[0080] Example 3: Production of monomeric, dimeric and multimeric binding molecules containing VHH domains E. coli BL21-DE3 Codonplus (Stratagene) was heat shock transformed with VHH-encoding plasmid DNA (Example 2) and grown in the presence of the appropriate antibiotic. A single colony was picked and used to inoculate 10 mL of 2xYT (supplemented with 2% (w / v) glucose, 35 μg / mL chloramphenicol, and 100 μg / mL ampicillin or 30 μg / mL kanamycin) and the inoculated medium was incubated overnight at 37°C and 180 rpm. The overnight culture was then diluted 1 / 100 in 900 mL of Terrific Broth (supplemented with 100 mL of KPO buffer, 0.1% (w / v) glucose, and 100 μg / mL ampicillin or 30 μg / mL kanamycin) and the OD 600The culture was incubated at 37°C and 180 rpm until the β-actin ratio reached a value of 0.5-0.8, after which 1 mL of 1 M IPTG was added to the culture to induce protein production, and incubation was continued overnight at 25°C and 180 rpm. The bacterial cells were harvested by spinning down the culture at 4700 rpm for 15 min at 4°C, after which the bacterial cells were resuspended in PBS (30 mL PBS per 800 mL bacterial culture). The periplasmic contents containing the VHHs were released from the cells by freeze-thawing the suspension twice, after which the suspension was spun down at 4°C and 4700 rpm. The VHHs were purified using immobilized metal affinity chromatography (IMAC) purification on Talon metal affinity resin (Clontech) (0.75 mL per L bacterial culture). The resin was pre-washed three times with PBS, after which the resin was added to the periplasm and incubated for 30 min at 4° C. and 15 rpm. The resin was separated from the periplasm by spinning down the suspension for 3 min at 4° C. and 900 rpm and washed with 0.05% (v / v) TWEEN 20 in PBS, followed by two washing steps with PBS. The resin was then added to a Poly-Prep chromatography column (Bio-Rad), after which non-specifically bound proteins were pre-eluted with 1 mL of 15 mM imidazole in PBS, and the protein of interest was then eluted with 150 mM imidazole in PBS. Protein concentrations of collected fractions were measured at 280 nm (setting 1 Ab = 1 mg / mL) on a NanoDrop 1000 spectrophotometer (TermoFisher Scientific) and fractions containing VHH were pooled and dialyzed against a solution of 1 mM TCEP-HCl in PBS overnight at 4°C using 3.5 kDa MWCO SnakeSkin Dialysis tubes (TermoFisher Scientific). Purified protein was aliquoted and stored at -20°C. All clones were successfully purified as evidenced by a single clear band of approximately 15 KDa on SDS-PAGE.

[0081] (Example 4): Characterization of monomeric VHH domains. 4.1 IGF2R Fifteen unique hIGF2R binders (Example 1.1, Figure 2) were produced and purified as described in Examples 2 and 3. Apparent affinities were determined by binding assays for hIGF2R-ECD. Briefly, 3-fold serial VHH dilutions starting from 1 μM were added onto plates coated with hIGF2R-ECD. After 2 hours of incubation, plates were washed and bound VHHs were detected using a VHH-specific IgG antibody followed by an IRD800CW-conjugated IgG-specific antibody. The results of the binding assay are displayed in Figure 2. VHH 13F11 was also produced with a C-terminal free cysteine ​​and conjugated to Alexa Fluor 647 using maleimide chemistry (13F11-A647 in Figure 8A, conjugation method as described in Example 9.2). A titration ELISA (direct reading at 647 nm) with this construct was performed on hIGF2R-ECD to determine whether affinity was affected after site-specific conjugation (FIG. 8B). Using a site-specific binding assay, K D and B. max was calculated in Graphpad (version 8.3) (Figure 8C). The calculated affinity of 13F11-A647 was 1.00 ± 0.61 nM (K D ±SEM, n=2), which was calculated affinity for unconjugated 13F11 (0.23 ± 0.05 nM (K D ±SEM, n=2), suggesting that site-specific conjugation to the C-terminal free end causes a small, but acceptable, decrease in affinity.

[0082] 4.1 PDGFRB Eighteen unique hPDGFRB binders and eleven unique rPDGFRB binders (Example 1.2, Figures 5 and 6) were produced and purified as described in Examples 2 and 3, and the apparent affinities were determined by binding assays for hPDGFRB-ECD and rPDGFRB-ECD, respectively. The results are summarized in Figures 5 and 6 for hPDGFRB VHH and rPDGFRB VHH, respectively. The hPDGFRB and rPDGFRB binding VHHs with high binding affinity for ECD were then tested for binding to hPDGFRB and rPDGFRB transfected SCC VII cells in Figures 9A and 9B, respectively. In addition, the binding affinity of the hPDGFRB VHH human was also determined in the human hepatic stellate cell (HSC) line LX-2 (Figure 5).

[0083] Example 5: Determination of VHH binding to non-overlapping receptor epitopes to allow construction of biparatopic binding molecules 5.1 IGF2R VHH From a panel of 15 hIGF2R specific binders (Figure 2), VHHs 13A8, 13A12, 13C11, 13E8, 13F11 and 13G10 were selected for further characterization. A competition assay was performed to determine whether the selected VHHs recognize distinct epitopes on hIGF2R. For this, a non-saturating amount (10 nM) of 13F11 conjugated to Alexa Fluor 647 (13F11-A647, conjugation method as described in Example 9.2) was incubated with immobilized hIGF2R-ECD in the presence of unconjugated competitor (250 nM) (Figure 10). Incubation of hIGF2R-ECD with 13F11-A647+13F11 was used as a positive control, and incubation of hIGF2R-ECD with 13F11-A647 alone was used to determine the maximum fluorescence intensity. VHH 13A8 showed complete competition with VHH 13F11, demonstrating that these two VHHs recognize (at least partially) overlapping epitopes. Of the other VHHs, only 13E8 did not compete with 13F11, demonstrating that 13F11 and 13E8 recognize non-overlapping epitopes. Thus, the two VHHs qualify as components for biparatopic VHH construction.

[0084] 5.2 PDGFRB VHH A competitive ELISA was performed to determine whether the hPDGFRB (Figure 5) and rPDGFRB (Figure 6) specific binders recognize distinct epitopes on hPDGFRB or rPDGFRB, respectively. The hPDGFRB binding VHHs SP02P, SP05P, SP12P and SP14P as well as the rPDGFRB binder SP26P were conjugated to IRDye800CW (VHH-IRDye800CW, conjugation method as described in Example 9.2) and the conjugated VHHs were mixed with unconjugated competitors in three-fold serial dilutions, respectively, and incubated with immobilized hIGF2R-ECD. If increasing concentrations of unconjugated VHHs decrease the fluorescent signal of VHH-IRDye800CW, then the two VHHs share (part of) the same epitope. The epitopes determined by competition assays for hPDGFRB-binding VHHs and rPDGFRB VHHs are summarized in Figures 5 and 6, respectively. Additionally, binding epitope competition between hPDGFRB-binding VHH SP02P and rPDGFRB-binding VHH SP26P was performed using methods similar to those described in Section 5.1. No competition was observed between the two VHHs, qualifying them as building blocks for biparatopic binding molecules.

[0085] Example 6: Construction and characterization of biparatopic VHHs 6.1 IGF2R VHHs 13F11 and 13E8 were selected for the design of biparatopic and bivalent VHH constructs based on their high affinity and binding to non-overlapping receptor epitopes. The two VHHs were fused with a flexible linker consisting of three Gly-Gly-Gly-Gly-Ser repeats (Figure 8A). The sequences were codon-optimized for expression in E. coli and obtained as synthetic DNA fragments from GeneArt. The DNA fragments encoding the VHHs were cloned into a vector pET28-based vector introducing a C-terminal free cysteine ​​for site-specific conjugation and a His6 tag for IMAC purification (Figure 8A). The constructs were produced and purified as described in Example 3. The constructs were conjugated to Alexa Fluor 647 (conjugation method as described in Example 9.2) and a titration ELISA for recombinant hIGF2R-ECD (as described in Example 4.1) was performed to determine the K D and B. max was determined (calculated in Graphpad (version 8.3) using single-site specific binding analysis) (FIG. 8C). The calculated affinities for 13F11, 13F11-13F11, 13F11-13E8, and 13E8-13F11 were 1.00±0.61 nM (K D ± SEM, n = 2), 3.10 ± 0.87 nM, 2.5 ± 1.87 nM, and 2.5 ± 1.56 nM (K D ± SEM, n = 2) (Figure 8C).

[0086] To determine whether 13F11-13E8 is cross-reactive to rIGF2R, the construct was conjugated to IRDye800CW (13F11-13E8-IRDye800CW, conjugation method as described in Example 9.2) and binding affinity was determined using a binding assay on SCC VII cells overexpressing either human or rat IGF2R (Figure 12). 13F11-13E8-IRDye800CW binds to both human and rat IGF2R with comparable affinity (<10 nM), as shown in Figure 12, confirming cross-reactivity.

[0087] 13F11-13E8 was then genetically re-engineered to include an in vivo half-life extender in the form of an albumin binding domain (ABD) as described in Johansson et al. (Structure, specificity, and mode of interaction for bacterial albumin-binding modules, J. Biol. Chem. 2002(277):8114-8120) and subsequently produced and purified as described in Example 3. 13F11-13E8-ABD was then conjugated to IRDye800CW (F11E8-ABD-IRD800, conjugation method as described in Example 9.2) to determine the binding affinity of the biparatopic VHH as well as the ABD unit. For F11-E8-ABD-IRD800, low nanomolar binding affinity to hIGF2R-ECD was observed, indicating that the addition of the ABD domain had no effect on the binding of biparatopic F11-E8 to hIGF2R (Figure 13A). The binding affinity (K D The binding affinity for mouse serum albumin (MSA) and human serum albumin (HSA) was approximately 200 nM. No binding was observed for bovine serum albumin (BSA) (FIG. 13B).

[0088] The cross-reactivity of F11-E8-ABD was further evaluated in binding experiments to primary activated rat hepatic stellate cells (HSCs) by conjugating the construct to Alexa Fluor 488 (FE-A-A488, conjugation method as described in Example 9.2). Activated HSCs were obtained from perfused livers of cirrhotic bile duct ligated (BDL) rats and HSCs were isolated using density gradient centrifugation. Cells were kept in culture for 6 days to allow complete activation and were subsequently stained with 50 nM FE-A-A488. Activation was determined by further staining with alpha-smooth muscle actin (a-SMA). FE-A-A488 bound to rat HSCs as seen in Figure 14, and activation was confirmed by the presence of long actin polymers, or so-called stress fibers (Figure 14).

[0089] 6.2 PDGFRB VHHs SP02P, SP12P, SP14P and SP26P were selected for the design of biparatopic VHH constructs based on their high affinity and binding to non-overlapping hPDGRB epitopes and were produced in a similar manner as described in Example 6.1 (Figure 15A). The binding affinity of the biparatopic constructs was determined in a periplasmic ELISA assay (Figure 15A). VHHs SP26P and SP28P were selected for the design of biparatopic VHH constructs based on their high affinity and binding to non-overlapping rPDGRB epitopes and were produced in a similar manner as described in Example 6.1. The resulting biparatopic SP28P-SP26P was conjugated to IRDye800CW (SP28P-SP26P-IRDye800CW, conjugation method as described in Example 9.2) and subjected to binding assays on SSC VII cells transfected with hPDGFRB, rPDGFRB or hIGF2R (Figure 15B). The construct showed very high affinity for rPDGFRB (K<1 nM). D), whereas the binding affinity to hPDGFRB is several orders of magnitude lower. To generate a biparatopic VHH construct that is cross-reactive with respect to hPDGFRB and rPDGFRB, VHH SP02P binding to hPDGFRB and VHH SP026P binding to rPDGFRB were selected to generate a biparatopic construct SP02P-SP26P in a manner similar to that described in Example 6.1 (Figure 15C). The construct SP02P-SP26P was conjugated to IRDye800CW (SP02P-SP26P-IRDye800CW, conjugation method as described in Example 9.2) and subjected to binding assays on SSC VII cells transfected with hPDGFRB, rPDGFRB or hIGF2R (Figure 15C). The binding affinity for both hPDGFRB and rPDGFRB is less than 5 nM, making the biparatopic SP02P-SP26P particularly promising for use in the binding molecules of the invention.

[0090] Example 7: Improved in vivo pharmacokinetics of 13F11-13E8 in rats by addition of an albumin binding domain The in vivo pharmacokinetic properties of 13F11-13E8-ABD were compared to 13F11-13E8 without the ABD moiety in healthy Sprague-Dawley rats. Briefly, rats were dosed intravenously at approximately 3 mg / kg (body weight) and blood samples were taken at various time points to determine the concentration of the VHH construct using ELISA on immobilized hIGF2R-ECD. 13F11-13E8-ABD peak serum levels (C max ) exceeded 1000 nM, as shown in FIG. 13C. Approximately 20 nmoles were injected into the animals, and this C max represented approximately 50% of the injected dose, with 50% of the injected dose still present in the circulation 1 h after dosing, indicating good bioavailability, whereas 13F11-13E8 was rapidly cleared from the circulation and was no longer detectable within 2 h after injection (FIG. 13C).

[0091] Example 8: Synthesis and analytical characterization of Lx semi-final conjugates and maleimide functionalized moieties with small molecule kinase inhibitors: Y27632, pacritinib, sacubitril (at), and losartan 8.1. Structures of Y27632-Lx (semi-final moieties) SFM and Y27632 maleimide functionalized moieties that can be utilized in the context of the present invention [ka] [ka] [ka]

[0092] 8.1.1. Synthesis and analytical characterization of Y27632-Lx-Cl (1a) [ka]

[0093] AgNO3 (85 mg, 500 μmol, 1.0 equiv.) was added to a suspension of PtCl2 (ethane-1,2-diamine) (LxCl2; 163 mg, 500 μmol, 1.0 equiv.) in dry DMF (24.8 mL). The mixture was stirred overnight at room temperature in the dark under an argon atmosphere. The suspension was then filtered through a 0.2 μm syringe filter to obtain a 20.2 mM stock solution of the activated Pt complex. The 20.2 mM stock solution of the activated Pt complex prepared above (12.4 mL, 250 μmol, 2.0 equiv.) was then added to a solution of Y27632×2HCl (40 mg, 125 μmol, 1.0 equiv.) in MilliQ water (14 mL) (pH adjusted to 6.95 using 1 M NaOH). The reaction mixture was stirred at 60° C. for 4.5 h in the dark under an argon atmosphere. The reaction mixture was then filtered through a 0.2 μm filter, 0.9% NaCl was added to the solution (1 mL), and then the solvent was removed under reduced pressure. The residue was dissolved in MilliQ water / MeOH (85:15, 6 mL) and filtered through a 0.2 μm syringe filter. Purification was performed by preparative reverse-phase HPLC (Grace Alltima C18 5 μm column, 22×250 mm; gradient: 15% to 35% MeOH / 0.1% TFA in water / 0.1% TFA within 36 min). The product fraction was lyophilized to give product 1a (41.8 mg, 43.8% yield) as a colorless solid. 1 H NMR (400 MHz, CD3OD): δ 8.57 - 8.47 (m, 2 H), 7.74 - 7.68 (m, 2 H), 6.09 - 5.78 (m, 2 H), 5.72 - 5.40 (m, 2 H), 3.21 - 3.11 (m, 1 H), 2.80 - 2.51 (m, 4 H), 2.48 - 2.37 (m, 1 H), 2.10 - 2.01 (m, 2 H), 1.98 - 1.84 (m, 2 H), 1.67 - 1.51 (m, 3 H), 1.32 - 1.15 (m, 5 H). 195 Pt NMR (86.0 MHz, CD3OD): δ -2512.

[0094] HPLC (Grace Alltima C18 5 μm column, 25 × 4.6 mm) showed the product to be 95.6% pure (retention time 15.8 min; gradient: 5% to 25% MeCN / 0.1% TFA in water / 0.1% TFA within 18 min; measured at a wavelength of 273 nm).

[0095] 8.1.2. Synthesis and Analytical Characterization of Y27632-Lx-I(1c) [ka] Y27632x2HCl (10 mg, 31 μmol, 1.0 equiv.) and Pt(ethane-1,2-diamine)I2 (LxI2; 14.90 mg, 29 μmol, 0.95 equiv.) in MilliQ water (125 μL) were dissolved in dry DMF (250 μL) and the reaction mixture was shaken for 48 h at 60° C. The reaction mixture was then diluted with 10 mM NaI in MilliQ / MeOH (1:1, 3 mL) and incubated at 25° C. for 1 h, after which the suspension was filtered through a 0.2 μm syringe filter. Purification was carried out by preparative reverse-phase HPLC (Grace Alltima C18 5 μm column, 22 × 250 mm; gradient: 15% to 50% B in 40 min, except for eluent A: 95 / 5 water / MeOH (+0.1% TFA) and eluent B: 5 / 95 water / MeOH (+0.1% TFA)). Product-containing fractions were collected and lyophilized to give product 1c (11.0 mg, 41.1% yield) as a yellow solid. HRMS (ESI + ) C 16 H 29 N5OPt [M+H] + 629.1065, measured value 629.1092.

[0096] HPLC (Grace Alltima C18 5 μm column, 25 × 4.6 mm) showed the product to be 99.1% pure (retention time 13.3 min; gradient: 5% to 50% MeCN / 0.1% TFA in water / 0.1% TFA within 20 min; measured at a wavelength of 273 nm).

[0097] 8.1.3. Synthesis and Analytical Characterization of Mal-PEG4-Val-Cit-PAB-Y27632 (1e) [ka]

[0098] Y27632x2HCl (18 mg, 56 μmol, 1.0 equiv.), Mal-PEG4-Val-Cit-PAB-PNP (49 mg, 56 μmol, 1.0 equiv.) and triethylamine (19.6 μL, 112 μmol, 2.0 equiv.) were dissolved in dry DMSO (1 mL) and the reaction mixture was stirred for 45 min at 25° C. The reaction mixture was then diluted with MilliQ / MeOH (1:1, 3 mL) and the suspension was then filtered through a 0.2 μm syringe filter. Purification was carried out by preparative reverse-phase HPLC (Grace Alltima C18 5 μm column, 22 × 250 mm; gradient: 20% to 40% B in 80 min, eluent A: 95 / 5 water / MeCN (+0.1% TFA) and eluent B: 5 / 95 water / MeCN (+0.1% TFA)). Product-containing fractions were collected and lyophilized to give product 1e (22.3 mg, 36.3% yield) as a yellow solid. HRMS (ESI + ) C 48 H 70 N9O 13 [M+H] + 980.5088, measured value 980.5093.

[0099] HPLC (Grace Alltima C18 5 μm column, 25 × 4.6 mm) showed the product to be 97.1% pure (retention time 10.1 min; gradient: 20% to 100% MeCN / 0.1% TFA in water / 0.1% TFA within 20 min; measured at a wavelength of 273 nm).

[0100] 8.1.4. Synthesis and Analytical Characterization of Mal-Val-Cit-PAB-Y27632 (1f) [ka]

[0101] Y27632x2HCl (20 mg, 62 μmol, 1.0 equiv.), Mal-Val-Cit-PAB-PNP (48 mg, 66 μmol, 1.05 equiv.) and triethylamine (22.2 μL, 125 μmol, 2.0 equiv.) were dissolved in dry DMSO (1 mL) and the reaction mixture was stirred at 25° C. for 45 min. The reaction mixture was then diluted with MilliQ / MeOH (1:1, 3 mL) and the suspension was then filtered through a 0.2 μm syringe filter. Purification was performed by preparative reversed-phase HPLC (Grace Alltima C18 5 μm column, 22×250 mm; gradient: 20% to 40% B within 80 min, eluent A: 95 / 5 water / MeCN (+0.1% TFA) and eluent B: 5 / 95 water / MeCN (+0.1% TFA)). The product-containing fractions were collected and lyophilized to give the product 1f (23.5 mg, 39.2% yield) as a yellow solid. HRMS (ESI + ): C 43 H 60 N9O9[M+H] + 846.4509, measured value 846.4472.

[0102] HPLC (Grace Alltima C18 5 μm column, 25 × 4.6 mm) showed the product to be 95.6% pure (retention time 10.5 min; gradient: 20% to 100% MeCN / 0.1% TFA in water / 0.1% TFA within 20 min; measured at a wavelength of 273 nm).

[0103] 8.2. Structure of Sacubitril-Lx SFM that can be utilized in the context of the present invention [ka] [ka] [ka]

[0104] 8.2.1. Synthesis and Analytical Characterization of Sacubitril-py-Lx-I (2c) [ka]

[0105] 8.2.1.1. Synthesis and Analytical Characterization of Sacubitril-py [ka]

[0106] Sacubitril hemicalcium (100 mg, 0.232 mmol, 1.0 equiv), pyridin-4-ylmethanol (30 mg, 0.279 mmol, 1.2 equiv) and DMAP (3 mg, 0.023 mmol, 0.1 equiv) were dissolved in dry DMF (1.5 mL). EDC×HCl (67 mg, 0.348 mmol, 1.5 equiv) and additional DMF (0.5 mL) were then added. The resulting mixture was stirred at room temperature under argon for 4 days. The reaction mixture was diluted with water (20 mL) and extracted with EtOAc (2×20 mL). The organic phase was dried over Na2SO4, filtered and the solvent was removed under reduced pressure. The residue was purified by column chromatography on silica (eluent: 0-3% MeOH / DCM) to give a colorless solid (73 mg, 62.4% yield). 1 H NMR (400 MHz, CDCl3): δ 8.58 (d, 2 H), 7.60 - 7.54 (m, 2 H), 7.54 - 7.49 (m, 2 H), 7.46 - 7.40 (m, 2 H), 7.37 - 7.30 (m, 1 H), 7.27 - 7.22 (m, 4 H), 5.62 (d, 1 H), 5.13 (s, 2 H), 4.31-4.19 (m, 1 H), 4.13 (q, 2 H), 2.89 - 2.80 (m, 2 H), 2.76 - 2.69 (m, 2 H), 2.66 - 2.50 (m, 2 H), 2.49 - 2.42 (m, 2 H), 1.99-1.90 (m, 1 H), 1.59-1.49 (m, 1 H), 1.24 (t, 3 H), 1.16 (d, 3 H). HRMS (ESI + ) C 30 H 35 N2O5[M+H] + Calculated value 503.2540, measured value 503.2599.

[0107] 8.2.1.2. Synthesis and Analytical Characterization of Sacubitril-py-Lx-I (2c) [ka]

[0108] Pt(ethane-1,2-diamine)I2 (LxI2; 76.0 mg, 149.2 μmol, 5 equiv.) was dissolved in a solution of sacubitril-py (15.0 mg, 29.8 μmol, 1.0 equiv.) in dry DMF (200 μL) and the reaction mixture was shaken at 25° C. for 22 h. The reaction mixture was then diluted with water / MeOH (1:1, 3 mL) and filtered through a 0.2 μm syringe filter. Purification was performed by preparative reversed-phase HPLC (Grace Alltima C18 5 μm column, 22×250 mm; gradient: 45% to 100% B within 40 min, with eluent A: 95 / 5 water / MeOH (+0.1% TFA) and eluent B: 5 / 95 water / MeOH (+0.1% TFA)). The product-containing fractions were collected and lyophilized to give a yellow solid (14 mg, 47.0% yield). HRMS (ESI + ) C 32 H 42 IN4O5 195 Pt [M] + Calculated value 884.1845, measured value 884.1856.

[0109] HPLC (Grace Alltima C18 5 μm column, 25 × 4.6 mm) showed the product to be 95.8% pure (retention time 15.9 min; gradient: 20% to 100% MeCN / 0.1% TFA in water / 0.1% TFA within 20 min; measured at a wavelength of 273 nm).

[0110] 8.2.2. Synthesis and Analytical Characterization of Sacubitril-CH2CH2NHCO-py-Lx-I (2o) [ka]

[0111] 8.2.2.1. Synthesis and analytical characterization of 2,3,5,6-tetrafluorophenyl 3-(pyridin-4-yl)propanoate [ka]

[0112] 3-(Pyridin-4-yl)propionic acid (500 mg, 3.3 mmol, 1.0 equiv), 2,3,5,6-tetrafluorophenol (604 mg, 3.6 mmol, 1.1 equiv) and EDC×HCl (761 mg, 4.0 mmol, 1.2 equiv) were dissolved in dry DCM (10 mL). The resulting mixture was stirred at room temperature for 16 h. The mixture was then extracted with 0.1 M HCl (2×), brine (1×), dried over Na2SO4, and the solvent was then removed under reduced pressure. The reaction yielded a white solid (530 mg, 47.7% yield). 1 H NMR (400 MHz, CDCl3): δ 8.49 (m, 2 H), 7.91 (m, 1 H), 7.35 (d, 2 H), 3.21 (t, 2 H), 3.02 (t, 2 H).

[0113] 8.2.2.2. Synthesis and Analytical Characterization of N-(Hydroxyethyl)-3-(pyridin-4-yl)propanamide [ka]

[0114] 3-(Pyridin-4-yl)propionic acid-TFP (478 mg, 1.6 mmol, 1.0 equiv), 2-amino-ethanol (98 mg, 1.6 mmol, 1.0 equiv) and DIPEA (222 μL, 1.6 mmol, 1.0 equiv) were dissolved in dry DCM (10 mL). The resulting mixture was stirred at room temperature for 1 h. The solvent was then removed under reduced pressure and the residue was purified by column chromatography on silica (eluent: 0-3% MeOH / DCM) to give a colorless solid (260 mg, 83.3% yield). 1H NMR (400 MHz, CD3OD): δ 8.36 - 8.44 (m, 2 H), 7.31 (d, 2 H), 3.54 (t, 2 H), 3.26 (t, 2 H), 2.97 (t, 2 H), 2.55 (t, 2 H).

[0115] 8.2.2.3. Synthesis and Analytical Characterization of Sacubitril-CH2CH2NHCO-py [ka]

[0116] Sacubitril hemicalcium (200 mg, 0.487 mmol, 1.0 eq.), N-(2-hydroxyethyl)-3-(pyridin-4-yl)propanamide (114 mg, 0.584 mmol, 1.2 eq.) and DMAP (6 mg, 0.049 mmol, 0.1 eq.) were dissolved in dry DMF (1.5 mL). EDC×HCl (140 mg, 0.751 mmol, 1.5 eq.) and additional DMF (0.5 mL) were then added. The resulting mixture was stirred at room temperature under an argon atmosphere for 20 h. The reaction mixture was diluted with water (20 mL) and extracted with EtOAc (2×20 mL). The organic phase was dried over Na2SO4, filtered and the solvent was removed under reduced pressure. The residue was purified by column chromatography on silica (eluent: 0-5% MeOH / DCM) to give a colorless solid (99 mg, 33.5% yield). HRMS (ESI + ) C 35 H 41 IN3O6[M+H] + Calculated value 588.3068, measured value 588.3083.

[0117] HPLC (Grace Alltima C18 5 μm column, 25 × 4.6 mm) showed the product to be 100% pure (retention time 14.0 min; gradient: 20% to 100% MeCN / 0.1% TFA in water / 0.1% TFA within 20 min; measured at a wavelength of 273 nm).

[0118] 8.2.2.4. Synthesis and analytical characterization of Sacubitril-CH2CH2NHCO-py-Lx-I (2o) [ka]

[0119] Pt(ethane-1,2-diamine)I2 (LxI2; 34.6 mg, 272.8 μmol, 4.0 equiv.) was dissolved in a solution of Sacubitril-CH2CH2NHCO-py (40.0 mg, 68.2 μmol, 1.0 equiv.) in dry DMF (416 μL) and the reaction mixture was shaken at 50 °C for 27 h. The reaction mixture was then diluted with water / MeOH (1:1, 3 mL) and filtered through a 0.2 μm syringe filter. Purification was performed by preparative reversed-phase HPLC (Grace Alltima C18 5 μm column, 22 × 250 mm; gradient: 65% to 90% B within 40 min, with eluent A: 95 / 5 water / MeOH (+0.1% TFA) and eluent B: 5 / 95 water / MeOH (+0.1% TFA)). The product-containing fractions were collected and lyophilized to give a yellow solid (26.7 mg, 36.2% yield). HRMS (ESI + ) C 36 H 49 IN5O6 195 Pt [M] + Calculated value 969.2370, measured value 969.2388.

[0120] HPLC (Grace Alltima C18 5 μm column, 25 × 4.6 mm) showed the product to be 96.4% pure (retention time 15.2 min; gradient: 20% to 100% MeCN / 0.1% TFA in water / 0.1% TFA within 20 min; measured at a wavelength of 273 nm).

[0121] 8.3. Structures of Pacritinib-Lx SFM and Maleimide-Functionalized Pacritinib Moieties that can be Utilized in the Context of the Invention [ka] [ka]

[0122] 8.3.1. Synthesis and Analytical Characterization of Pacritinib-Lx-I (3c) [ka]

[0123] Pacritinib (20 mg, 42.3 μmol, 1 equiv.) and Pt(ethane-1,2-diamine)I2 (LxI2; 96.9 mg, 190.4 μmol, 4.5 equiv.) were dissolved in dry DMF (250 μL) and the reaction mixture was shaken at 60° C. for 48 h. The reaction mixture was then diluted with water / MeOH (1:1, 3 mL) and filtered through a 0.2 μm syringe filter. Purification was performed by preparative reversed-phase HPLC (Grace Alltima C18 5 μm column, 22×250 mm; gradient: 45% to 80% B within 40 min, eluent A: 95 / 5 water / MeOH (+0.1% TFA) and eluent B: 5 / 95 water / MeOH (+0.1% TFA)). The product-containing fractions were collected and lyophilized to give compound 3c (13.1 mg, 28.6% yield) as a yellow solid. HRMS (ESI + ) C 30 H 41 IN6O3 195 Pt [M] 2+ Calculated value 427.5962, measured value 427.5999.

[0124] HPLC (Grace Alltima C18 5 μm column, 25 × 4.6 mm) showed the product to be 92.7% pure (retention time 10.7 min; gradient: 20% to 100% MeCN / 0.1% TFA in water / 0.1% TFA within 20 min; measured at a wavelength of 273 nm).

[0125] 8.3.2. Synthesis and Analytical Characterization of Mal-PEG4-Val-Cit-PAB-Pacritinib (3e) [ka]

[0126] 8.3.2.1. Synthesis and Analytical Characterization of Mal-PEG4-Val-Cit-PAB-Cl [ka]

[0127] Mal-PEG4-Val-Cit-OH (10 mg, 14.2 μmol, 1.0 equiv) was dissolved in DMF (200 μL) and cooled in an ice / water bath to 0° C. Thionyl chloride (1.9 mg, 15.6 μmol, 1.1 equiv) was then added and the mixture was stirred at 0° C. for 15 min, after which the solvent was removed under reduced pressure. HRMS (ESI + ) C 33 H 49 35 ClNO 10 [M+H] + Calculated value 725.3271, measured value 725.3298.

[0128] HPLC (Grace Alltima C18 5 μm column, 25×4.6 mm) showed the product to be >90% pure (retention time 9.6 min; gradient: 20% to 50% B in 20 min, eluent A: 95 / 5 water / MeCN (+0.1% TFA) and eluent B: 5 / 95 water / MeCN (+0.1% TFA). Measurement was performed at 273 nm).

[0129] 8.3.2.2. Synthesis and Analytical Characterization of Mal-PEG4-Val-Cit-PAB-Pacritinib (3e) [ka]

[0130] Pacritinib (6.7 mg, 14.2 μmol, 1.0 equiv.) was dissolved in DMF (300 μL) and added to Mal-PEG4-Val-Cit-Cl (10.3 mg, 14.2 μmol, 1.0 equiv.). DIPEA (4.6 mg, 35.5 μmol, 2.5 equiv.) and tetrabutylammonium iodide (1.0 mg, 2.8 μmol, 0.2 equiv.) were then added and the mixture was stirred at 60° C. for 24 h. Product formation was confirmed by HRMS. HRMS (ESI + ) C 61 H 81 N 10 O 13 [M+H] + Calculated value 1161.5979, measured value 1161.5886.

[0131] 8.4. Structure of Losartan-Lx SFM that can be utilized in the context of the present invention [ka]

[0132] 8.4.1. Synthesis and Analytical Characterization of Losartan-Lx-Cl (4a) [ka]

[0133] AgNO3 (26.1 mg, 153.3 μmol, 1.0 equiv.) was added to a suspension of PtCl2 (ethane-1,2-diamine) (LxCl2; 50 mg, 153.3 μmol, 1.0 equiv.) in dry DMF (8.3 mL) and stirred overnight at room temperature in the dark under an argon atmosphere. The suspension was then filtered through a 0.2 μm syringe filter to obtain a 18.5 mM stock solution of the activated Pt complex. The 18.5 mM stock solution of the activated Pt complex prepared above (1.76 mL, 32.5 μmol, 0.75 equiv.) was then added to a solution of losartan potassium (20 mg, 43.4 μmol, 1.0 equiv.) in dry DMF (500 μL). The reaction mixture was stirred at 22° C. for 1.5 h. Subsequently, 1M HCl (70.4 μL, 2 eq.) and 0.9% NaCl (1 mL) were added to the mixture, after which the solvent was removed under reduced pressure. The residue was dissolved in MilliQ water / MeOH (1:1, 3 mL) and filtered through a 0.2 μm syringe filter. Purification was carried out by preparative reverse-phase HPLC (Grace Alltima C18 5 μm column, 22×250 mm; gradient: 20% to 50% B in 40 min, except for eluent A: 95 / 5 water / MeCN (+0.1% TFA) and eluent B: 5 / 95 water / MeCN (+0.1% TFA)). The product-containing fractions were lyophilized to give product 4a (11 mg, 47.4% yield) as a colorless solid. HRMS (ESI + ) C 24 H 31 35 Cl2NO 195 Pt [M] + Calculated value 712.1641, measured value 712.1628.

[0134] HPLC (Grace Alltima C18 5 μm column, 25 × 4.6 mm) showed the product to be 94.3% pure (retention time 9.6 min; gradient: 5% to 25% MeCN / 0.1% TFA in water / 0.1% TFA within 18 min; measured at a wavelength of 273 nm).

[0135] Example 9: Conjugation of semi-final conjugates and maleimide-functionalized moieties to binding molecules of the invention. 9.1. Conjugation of Lx SFM to binding molecules Biparatopic 13F11-13E8 (approximate molecular weight 27.9 kD, 105 μL, 5 nmol, 1.33 mg / mL, 1.0 equiv.) was diluted with borate buffer (20 μL, 250 mM sodium borate, 250 mM NaCl, and 10 mM diethylenetriaminepentaacetic acid, pH 8.0) and HO (200 μL), after which a solution of tris(2-carboxyethyl)phosphine hydrochloride (TCEP×HCl; 2 μL, 10 mM in HO, 10 nmol, 2.0 equiv.) was added. The mixture was incubated at 37° C. for 2 h in a thermoshaker. At the same time, a solution of SFM 1c (10 μL, 5 mM in 20 mM NaI, 50 nmol, 10.0 eq.) was mixed with an aqueous solution of thiourea (10 μL, 20 mM) and incubated in a thermoshaker at 37° C. for 2 hours. Subsequently, the above-prepared solution of the binding molecule of the present invention and SMF treated with thiourea was mixed and incubated in a thermoshaker at 37° C. for 1 hour. The conjugate was purified by spin filtration using a 10 kDa MWCO filter (washed 4 times with PBS), after which the conjugate was reconstituted and stored in PBS. The Y27632 to 13F11-13E8 ratio was 1.0 as determined by SEC-MS.

[0136] 9.2. Conjugation of Maleimide-Functionalized Moieties to Binding Molecules Biparatopic 13F11-13E8 (approximate molecular weight 27.9 kD, 105 μL, 5 nmol, 1.33 mg / mL, 1.0 equiv.) was diluted with borate buffer (20 μL, 250 mM sodium borate, 250 mM NaCl, and 10 mM diethylenetriaminepentaacetic acid, pH 8.0) and HO (200 μL), followed by the addition of a solution of tris(2-carboxyethyl)phosphine hydrochloride (TCEP×HCl; 2 μL, 10 mM in HO, 120 nmol, 2.0 equiv.). The mixture was incubated for 2 hours at 37° C. in a thermoshaker. The solution of the binding molecule of the present invention prepared above was then mixed with maleimide-functionalized moiety 1e (2.5 μL, 10 mM in DMSO, 50 nmol, 10.0 equiv.) and incubated at 0° C. for 1 hour. The conjugate was purified by spin filtration using a 10 kDa MWCO filter (washed 4 times with PBS), after which the conjugate was reconstituted and stored in PBS. The Y27632 to 13F11-13E8 ratio was 1.0 as determined by SEC-MS.

[0137] Example 10: Internalization of VHH 10.1 IGF2R VHH To assess whether monovalent 13F11 and biparatopic 13F11-13E8 are internalized in hIGF2R expressing cells, A549 cells (Figure 16A) and NIH 3T3 2.2 cells transiently transfected with hIGF2R (Figure 16B) were incubated with 25 nM VHH conjugated to ALexa Fluor 647 (conjugation method as described in Example 9.2). To detect IGF2R expression in hIGF2R transfected cells NIH 3T3 2.2, a mouse anti-IGF2R mAb was included (Figure 16B, right column) and the mAb was detected with a goat anti-mouse mAb conjugated to Alexa Fluor 555. Additionally, mock transfected NIH 3T3 2.2 cells were incubated with 13F11 and 13F11-13E8 (Figure 16C). To ensure that only internalized VHH or mouse anti-IGF2R signals were present, an acid wash (0.2 M glycine-HCl, 150 mM NaCl, pH 2.3) was used to remove VHH or mouse anti-IGF2R bound to the target receptor on the cell surface. The efficiency of the acid wash procedure was verified by incubating hIGF2R-transfected NIH 3T3 2.2 cells with 13F11-13E8-A647 on ice and subsequently treating the cells with an acid wash while still on ice to prevent internalization (Figure 10D). Biparatopic 13F11-13E8 was clearly internalized in A549 cells, whereas no internalization was observed for monovalent 13F11 (Figure 16A). On the other hand, neither VHH was internalized in NIH 3T3 2.2 cells transiently transfected with hGF2R, although a higher intensity of staining was observed for 13F11-13E8 (Figure 16B). The difference in internalization is likely related to the difference in IGF2R expression levels on the target cells. As expected, neither VHH construct was internalized in mock-transfected NIH 3T3 2.2 cells (Figure 16C). Acid wash control experiments showed that the acid wash treatment was sufficient to remove all membrane-bound VHH (Figure 16D).In summary, biparatopic 13F11-13E8 is clearly internalized in cells expressing hIGF2R on the cell surface.

[0138] Internalization rates (endocytosis rate constant, k e ) was determined using a kinetic internalization assay for hIGF2R and rIGF2R transfected SCC-VII cells. Briefly, 10 nM of IRD800CW conjugates 13F11-13E8 or 13E8-13F11 (conjugation method as described in Example 9.2) were added to hIGF2R- or rIGF2R-expressing SCC cells and incubated at 37° C. for various time points up to 12 min. Internalization was then purposely stopped by placing the cells on ice. Subsequently, cells were washed with cold PBS and non-internalized VHH constructs (bound fraction) were separated from internalized constructs (internalized fraction) using an acid wash as described above, after which the fluorescence of both fractions was measured. Both constructs exhibited similar endocytosis rate constants (k ) in hIGF2R-expressing SCC cells. e Approximately 1.5 minutes -1 ) (FIG. 17). Internalization was also observed in rIGF2R-expressing SCC cells, as can be seen in FIG. 17, where a clear increase in fluorescence over time (k e Approximately 0.7 minutes -1 ).

[0139] The internalization of monovalent 13F11 and biparatopic 13F11-13E8 and 13E8-13F11 in survival-activated HSC LX-2 was visualized using pHrodo pH-dependent probe (ThermoFisher Scientific). The pH probe becomes more fluorescent due to the resulting decrease in the surrounding pH. Endosomes and lysosomes have a maintained pH of 6.5 and 4.5, respectively, compared to the cytoplasmic pH of 7.0, and therefore the internalization of the VHH-pHrodo construct can be visualized if it internalizes via the endosomal pathway. LX-2 cells were incubated with 50 nM of pHrodo-red conjugates 13F11, 13F11-13E8 and 13E8-13F11 (conjugation method as described in Example 9.2) at 37° C. for 2 hours. Images were taken at 5 min intervals using a wide-field PEXScope microscope (Nicon) equipped with an mCherry filter and a 40x oil objective. A more pronounced increase in intracellular fluorescence was observed from 15 min onwards for both biparatopic constructs 13F11-13E8 and 13E8-13F11 compared to the monovalent constructs (Figure 18). Biparatopic 13F11-13E8 showed a higher increase in fluorescence over a 40 min period compared to 13E8-13F11, indicating a higher rate of internalization. Furthermore, internalized 13F11-13E8 and 13E8-13F11 accumulate in perinuclear compartments (endosomes / lysosomes).

[0140] 10.2.PDGFRB VHH To determine the rate of internalization of the VHH construct SP02P-SP26P, a kinetic internalization assay was performed on hPDGFRB-expressing SCC cells and LX-2 cells using a method similar to that described for the IGF2R construct in Example 10.1. Internalization of SP02P-SP26P was observed in both hPDGFRB-expressing SCC cells and LX-2 cells (Figure 19).

[0141] Example 11: NDC-Lx-pacritinib induces relaxation of activated hepatic stellate cells (HSCs) in a contraction assay. The relaxation potency of the biparatopic VHH 13F11-13E8 conjugated to the JAK2 inhibitor pacritinib (3c) (conjugation method as described in Example 9.1) was evaluated in vitro using a contraction assay with the human HSC line LX-2. The wells of a 24-well plate were filled with 0.5 mL of BSA and left at 37° C. for 1 h, after which the wells were washed with PBS and dried. A mixture of 1 M NaOH, 10×PBS, sterile water, 2×DMEM, 0.2 M HEPES and 1 mg / mL collagen type I was then added to the wells and incubated at 37° C. for 1 h to allow collagen to gel. 24 h prior to the assay, the medium was replaced with DMEM supplemented with 2% FBS, and LX-2 cells were harvested and 2×10 6 cells were added per mL of medium (2% FCS in DMEM). 5 The cells were resuspended in 100 ml of 13F11-13E8-Lx-pacritinib, 500 μl were added to the gel and incubated for 3 hours at 37° C. After the cells were bound to the collagen gel, the medium was removed and replaced with 500 μl of fresh medium with 1 μM 13F11-13E8-Lx-pacritinib, 1 μM 13F11-13E8-Cy5 (conjugation method as described in Example 9.2, negative control) or 1 μM contraction inhibitor 2,3-butanedione monoxime (BDM, positive control). The collagen gel was detached from the side of the well by moving a pipette tip or spatula around the edge and the cells were incubated at 37° C. for up to 7 days. Every 24 hours, pictures of the collagen gel were taken at a fixed distance or height using a camera (JAI CV-A55-IR). The collagen gel area was quantified in pixels using Adobe Photoshop (CC 2017). The percentage of contraction inhibition was quantified by setting up gels incubated without any inhibitors for 0% contraction inhibition and gels incubated with 1 μM BDM (positive control) for 100% contraction inhibition. The construct 13F11-13E8-Lx-pacritinib was able to induce contraction inhibition up to 76%, whereas the nanobody construct without a kinase inhibitor (13F11-13E8-mal-Cy5) did not inhibit contraction (Figure 20).

[0142] Example 12: Targeted IGF2R expression in cirrhotic liver sections To assess whether IGF2R receptor expression is upregulated in cirrhotic liver, various cryosections (preparation procedure as outlined in Example 13) of tissues (liver, kidney, spleen, heart, ileum, brain) from both healthy (SHAM) and cirrhotic (BDL) rats were stained with 13F11-13E8-ABD conjugated to Alexa Fluor 647 (13F11-13E8-ABD-A647, conjugation method as described in Example 9.2). After cryosectioning, tissue sections were stained with 50 nM fluorescent 13F11-13E8-ABD-AF647 and incubated overnight at 4° C. Sections were subsequently washed, stained with DAPI, mounted using mowiol mounting medium (Merck) and imaged on a Zeiss LSM700 laser scanning confocal microscope with a 20x objective. 13F11-13E8-ABD-AF647 clearly bound to cells in liver from BDL rats, but not to cells in liver from SHAM rats (Figure 21), indicating a strong upregulation of IGF2R in cirrhotic liver. Furthermore, no staining or only slight staining could be observed in other tissues from BDL and SHAM rats, indicating that IGF2R expression is highly restricted to cirrhotic liver (Figure 22).

[0143] Example 13: Selective reduction of portal hypertension by targeting activated hepatic stellate cells in vivo using IGF2R binding molecules of the invention conjugated to small molecule kinase inhibitors To assess in vivo functionality, 13F11-13E8-ABD was conjugated to the Y27632 maleimide-modified functional moiety (1e) (test conjugate) as outlined in Example 9.2. To provide a competitor, a similar amount of 13F11-13E8-ABD was conjugated to N-ethylmaleimide in a comparable similar preparation (mock conjugate).

[0144] To establish animals exhibiting portal hypertension (Klein et al, Fibrosis: Methods and Protocols. Methods in Molecular Biology. 2017(1627):91-122.), Sprague-Dawley rats were subjected to bile duct ligation by midline rest under intraperitoneal ketamine / xylazine-induced anesthesia (BDL rats). To provide healthy controls, a second cohort of rats was subjected to the same surgical procedure, except for the actual ligation of the bile duct (SHAM rats). The operation was followed up with pain control medication (subcutaneous carprofen). Approximately 5 weeks after surgery, ketamine / xylazine-anesthetized rats were subjected to midline rest, after which one catheter was inserted into the portal vein via the ileal vein and one into the femoral artery. The catheters were connected to pressure recording devices for assessment of portal vein pressure and mean arterial pressure, respectively.

[0145] After catheterization and hemodynamic stabilization, rats were dosed intravenously via the lateral tail vein with 5 mg / kg test conjugate or sham conjugate and pressure was monitored continuously (Example 13.1). One hour after dosing, animals were sacrificed and tissues were then isolated for ex vivo analysis (Example 13.2).

[0146] 13.1. Selective reduction of portal pressure by IGF2R binding molecules of the invention conjugated to small molecule kinase inhibitors In BDL, infusion of the test conjugate, but not the sham conjugate, resulted in a gradual decrease in portal vein pressure (PP) while arterial pressure remained constant (MAP), indicating selective alleviation of portal hypertension (Figure 23A). In animals infused with the sham conjugate, both pressures remained constant, indicating that the reduction in portal pressure by the test conjugate was mediated by a small molecule kinase inhibitor (Figure 23B). In SHAM rats, neither the test nor sham conjugates affected portal or arterial pressure, indicating that the effects observed in BDL rats are specific to the diseased state.

[0147] 13.2. In vivo targeting of IGF2R by IGF2R binding molecules of the invention conjugated to small molecule kinase inhibitors After isolation, the livers from the test conjugate-injected BDL and SHAM rats were fixed with paraformaldehyde, immersed in 30% (w / v) sucrose in PBS, and sectioned using a cryomicrotome. To detect the test conjugate in situ, the tissue sections were stained with rabbit polyclonal anti-VHH antiserum that recognizes the 13F11-13E8 portion of the conjugate, followed by an anti-rabbit antibody conjugated to the fluorophore Alexa-488 for visualization by confocal fluorescence microscopy (see Example 12). As shown in Figure 24A, the test conjugate was detected in the liver of BDL rats, but not in the liver of SHAM rats. Furthermore, a second staining of the same sections with excess 13F11-13E8 conjugated to the fluorophore Alexa-647 (13F11-13E8-A647, conjugation method as described in Example 9.2) demonstrated that the distribution of the test conjugate in the liver of BDL rats overlapped with the (tissue) distribution of IGF2R (Figure 24B). These results indicate that the presence of the test conjugate in the liver was driven by the (cirrhosis-specific) expression of IGF2R, and that in vivo targeting of the molecular target was indeed achieved. It is noteworthy that a second staining using Alexa-647 conjugated 13F11-13E8 to detect IGF2R distribution showed that not all IGF2R molecules were reached by the test conjugate within only 1 hour of the in vivo study. It was likely that further accumulation of the test conjugate in BDL liver beyond this 1 hour time point occurred. [Sequence List Free Text]

[0148] SEQ ID NO:369: <223> Gly-Gly-Gly-Gly-Ser linker SEQ ID NO:370: <223> Hexahistidine tag SEQ ID NO:371: <223> EPEA Tag

Claims

1. A binding molecule comprising at least two single variable antibody domains and at least one diagnostic or therapeutic molecule, said at least two single variable antibody domains being capable of specifically binding, independently of one another, to a transmembrane receptor expressed on hepatic stellate cells (HSC) and / or myofibroblasts, said transmembrane receptor being the platelet-derived growth factor beta receptor (PDGFBR); and The at least two single variable antibody domains At least one single variable antibody domain comprises: CDR1 sequence according to SEQ ID NO:11 CDR2 sequence according to SEQ ID NO: 13 and CDR3 sequence according to SEQ ID NO: 15 Including, At least one single variable antibody domain comprises: CDR1 sequence according to SEQ ID NO: 155 CDR2 sequence according to SEQ ID NO: 157 and CDR3 sequence according to SEQ ID NO: 159 The binding molecule comprising:

2. The at least two single variable antibody domains are humanized or camelized and / or contain the following substitutions: i) Substitutions that enhance the degree of humanization ii) substitutions that enhance chemical stability; and / or iii) Substitutions that increase expression levels The binding molecule of claim 1, comprising one or more substitutions selected from:

3. 3. The binding molecule of claim 1 or 2, wherein the at least one single variable antibody domain and / or the at least two single variable antibody domains are independently selected from the group consisting of immunoglobulin single variable domain antibodies (ISVD), heavy chain variable domains (VH), heavy chain only antibody variable domains (VHH), domain antibodies (dAb) and single domain antibodies (sdAb).

4. The binding molecule of any one of claims 1 to 3, wherein the at least two single variable antibody domains are both single variable domain antibodies.

5. The binding molecule of any one of claims 1 to 4, wherein the at least two single variable antibody domains are both variable domains of a heavy chain-only antibody.

6. At least two single variable antibody domains, comprising or consisting of a single antibody domain having the sequence of SEQ ID NO:9 (SP02P), and comprising or consisting of a single antibody domain having the sequence of SEQ ID NO: 153 (SP26P); A binding molecule according to any one of claims 1 to 5.

7. The binding molecule of any of claims 1 to 6, wherein a therapeutic or diagnostic molecule is attached to the at least two single variable antibody domains by a linker and / or a spacer.

8. The binding molecule of claim 7 , wherein the linker and / or spacer comprises or consists of a transition metal complex.

9. The binding molecule of claim 8 , wherein the linker and / or spacer comprises a cis-platinum(II) complex.

10. The binding molecule of claim 9 , wherein the linker and / or spacer comprises an inert bidentate moiety.

11. The binding molecule of any one of claims 1 to 10, comprising a half-life extender.

12. The binding molecule of claim 11, wherein the half-life extender comprises or consists of one or more groups, residues, moieties or binding units that confer an increased half-life to the binding molecule compared to the binding molecule without the half-life extender.

13. 13. The binding molecule of claim 11 or 12, wherein the group, residue, moiety or binding unit that confers increased half-life to the binding molecule is an albumin binding domain, an albumin binding VHH or an antibody Fc tail or a fragment thereof.

14. The binding molecule of any one of claims 1 to 13, wherein the myofibroblast is a hepatic myofibroblast.

15. The binding molecule of any one of claims 1 to 14, wherein the myofibroblasts are derived from HSCs or portal vein fibroblasts.

16. The binding molecule according to any one of claims 1 to 15, wherein the binding of the binding molecule to a receptor is capable of inducing internalization of the ligand or antibody.

17. The binding molecule of any one of claims 1 to 16, wherein the at least two single variable antibody domains are separated by a linker amino acid sequence.

18. The binding molecule of any one of claims 1 to 17, wherein the therapeutic molecule is a kinase inhibitor.

19. The binding molecule of any of claims 1 to 18, wherein the therapeutic molecule is selected from a Rho kinase inhibitor, a JAK-2 inhibitor or a Neprilysin inhibitor, or an Angiotensin II receptor antagonist.

20. The binding molecule of any one of claims 1 to 19, wherein the therapeutic molecule is a toxin.

21. A binding molecule according to any one of claims 1 to 20 for use as a medicament.

22. A binding molecule according to any of claims 1 to 20 for the prevention and / or treatment of liver cirrhosis, liver fibrosis and / or portal hypertension.

23. A binding molecule according to any of claims 1 to 20 or 22 for the prevention and / or treatment of diseases or disorders associated with esophageal varices and / or hemorrhoids.

24. A binding molecule according to any of claims 1 to 20, 22 and 23 for the prevention and / or treatment of a disease or disorder characterised by internal or external variceal bleeding.

25. A nucleic acid encoding a binding molecule according to any one of claims 1 to 24.

26. A host cell for the expression of a binding molecule according to any one of claims 1 to 24, comprising a nucleic acid according to claim 25.

27. A pharmaceutical composition comprising at least one binding molecule according to any of claims 1 to 24 and at least one pharma- ceutically acceptable excipient.

28. 21. The binding molecule of any one of claims 1 to 20, comprising a diagnostic molecule, said diagnostic molecule being an imaging agent.

29. 29. A diagnostic composition comprising at least one binding molecule according to claim 28 and a diluent and / or excipient.

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