Fusion protein peptide linkers and uses thereof

Abstract

Provided herein are fusion protein peptide linkers and uses thereof. The fusion protein peptide linkers comprise glycine, serine, and glutamate and / or aspartate amino acids. Such fusion protein peptide linkers can, inter alia, promote the solubility, expression, monomeric expression and / or thermostability of a fusion protein, inhibit or prevent aggregation and / or toxicity of a fusion protein, and / or promote the intracellular degradation of a fusion protein.

Description

[0001] FUSION PROTEIN PEPTIDE LINKERS AND USES THEREOF TECHNICAL FIELD The present invention is in the field of protein engineering, in particular fusion protein engineering. More particularly, the present invention relates to peptide linkers for use in fusion proteins, preferably binding molecules such as single chain variable fragment (scFv), bi-specific T-cell engager (BiTE), single-domain antibody-based formats (Variable Heavy domain of Heavy chain, VHH, or Variable New Antigen Receptor, VNAR), and antibody fragments fused to T cell chimeric antigen receptors called CAR T cells. The invention also relates to the use of negatively charged peptide linkers to improve inter alia stability, solubility and expression yields for both intracellular and secreted fusion proteins, preferably binding molecules such as scFv, bispecific T-cell engager (BiTE), single-domain antibody-based formats (VHH or VNAR), any of these formats fused to an immunoglobulin Fc domain and proteins fused to T cell chimeric antigen receptors called CAR T cells. BACKGROUND ART Introduction Fusion proteins are well established in the art. One example of a fusion protein is a single chain variable fragment (scFv) which is a small recombinant antibody format, generated by the fusion of the variable heavy domain, also called VH, to the variable light domain (VL) (Bird et al., 1988). Both domains are fused C- to N-terminal with a conventional glycine serine repeat linker - GGGS (SEQ ID NO: 1) or GGGGS (SEQ ID NO: 2) - that confers flexibility and hydration to the resulting single chain protein. To date, this ~3.5 nm linker peptide is used in most of scFv constructs and contributes to preserve antibody binding functionality (Huston et al., 1988; Holt et al., 2000). ScFvs offer several advantages compared to full antibodies in some applications as they can be incorporated in the design of multispecific antibody formats, including but not limited to, di-, tri-, tetra-bodies, BiTE or engineered CAR-T cells (Jin et al., 2022; Aslan et al., 2022). The absence of a constant region, including the Fc domain which mediates bystander activation of the immune system and FcRn recycling (Kholodenko et al., 2019), distinguishes scFvs from full length antibodies. Thanks to their small size (~27 kDa) and lack of Fc domain, scFvs present a short half-life (few hours) and better tissue penetration (Li et al., 2016), making them valuable tools in imaging applications such as positron emission tomography (PET) or single-photon emission computed tomography (SPECT) (Dewulf et al., 2020; Yokota et al., 1992). However, the absence of constant domains in scFv format lowers drastically the stability of the protein, which is often prone to aggregation, thereby limiting shelf life, lowering its potency and increasing the risk of immunogenicity when injected to patient (Nelson, 2010). In 1988, the scFv format was proposed for the first time by Carlson as valuable for intracellular applications, due to its potential functionality in cells compared to less suitable Fabs or full antibody formats (Carlson, 1988). This type of intracellular antibody is also called an intrabody. Unfortunately, the majority of intracellularly expressed scFvs tends to misfold and aggregate in a reducing environment such as the cytosol (Kvam et al., 2010; Cardinale et al., 2003; Auf der Maur et al., 2004), mostly due to the disruption of the two conserved disulfide bonds that connect the two β-sheets in each of the VH and VL, leading to suboptimal domain association and thus, poor stability (Ewert et al., 2003; Seo et al., 2009). It has also been reported that proteins have minimal solubility in a pH environment near their isoelectric point (pI), where their net charge is close to zero (Loeb, 1921). Importantly, scFvs aggregation can disrupt normal cell physiology leading to toxicity and ultimately, cell death (Sibler et al., 2003). Currently, there are no universal methods available to improve scFv intracellular stability and solubility without complex protein engineering. Attempts to solve this problem were developed using different approaches but remain cumbersome. The first example of stabilization of intracellularly expressed protein came from Lawrence et al. (Lawrence et al., 2007) who engineered a low aggregation green fluorescent protein (GFP) by mutating protein surface amino acids to negatively charged counterparts. It was later demonstrated that the pI of intrabodies and the overall negative net charge are key for solubility in the cytosol (that has a pH ranging between 7.2 and 7.4) (Kvam et al., 2010). Furthermore, it has been shown that Complementarity Determining Region (CDR) content influences the solubility of intrabodies and that, out of a set of 8 intrabodies developed in different studies, only those with a pI under 6.7 had a good cytoplasmic solubility with a negative net charge ranging from -0.5 to -8.6. The other scFv intrabodies with a pI ranging from 7.3 to 8.5 had poor solubility, with a net charge ranging between -0.2 and +3.5. The potential success of solubilizing intrabodies by increasing their negative net charge could be explained by the cytoplasmic environment, known to be negatively charged due to the abundance of both mRNAs (Jakel et al., 2002) and proteins (Chan et al., 2006). Those molecules associate to positively charged proteins (such as scFv) and contribute to their aggregation. Recently, the report of Kabayama et al. (Kabayama et al., 2020) highlighted the effect of negative charge additions to an intrabody. In their study, they fused two different peptides to a scFv: a negatively charged FLAG®peptide repeated 3 times (referred as s3Flag) on the N-terminus, and a hemagglutinin (HA) peptide on the C-terminus. Using this strategy, they were able to show an improvement in scFv solubility. They recommended to assume that the cytosolic pH is 6.6 instead of 7.2-7.4 (the physiological pH range) to account for the pH variations in the cytosol (Ruffin et al., 2014; Salameh et al., 2014). Aside from solubility in the cytosol, another key property of intrabodies is their clearance. Early evidence of intracellular scFv-antigen complex degradation by lysosomal autophagy was found by Southwell et al. with huntingtin aggregates (Southwell et al., 2011). Later, Prof. Messer’s laboratory decreased the pI of their anti-alpha-synuclein intrabody candidates by fusing a negatively charged proteasomal peptide to the C-terminus (Joshi et al., 2012), composed of human influenza hemagglutinin tag peptide (HA) linked to the mouse ornithine decarboxylase PEST motif sequence. Their resulting scFv-HA-PEST constructs with improved solubility were identified as self-degrading and potentially active to engage proteasome-directed clearance of two antigen targets, namely huntingtin and alpha-synuclein (Joshi et al., 2012; Butler et al., 2011; Chatterjee et al., 2018). Nevertheless, this construct offers limited possibilities to improve properties of scFvs with an initial high isoelectric point (i.e. if the pI of the parental scFv molecule is too high, the protein will not be soluble enough). Also, the addition of negatively charged peptides to either N- or C-terminal extremities lack adaptive designs (e.g., fusion proteins) and could trigger immunogenicity. Arslan et al., 2022, studied the effect of flexible repetitive (GnS)nlinker and non-repetitive linker in anti-VEGF scFv. Kvam et al., 2010, studied the effect of charged peptide tags linked to scFv to enhance their solubility as intrabody format. Kabayama et al., 2020, studied the effect of negative charge addition to scFv N- and C-terminus to enhance their solubility as intrabody format. US20140377270 is directed to optimized anti-CD3 variable sequences for use in a variety of bispecific formats. Various positive and negative charged scFv linkers are described. WO2012040518 relates to carrier immunoglobulins that can be used for example as quality control or analytical standards. A range of optional linker moieties are described. WO2014018572 describes single chain Fab regions (scFab), in which the VH and VL polypeptides are connected by a first linker to form a single chain Fv (scFv). A range of linkers are described for connecting various parts of the molecules. WO2015168474 relates to “chimeric activators” including a targeting element that binds to a cell surface receptor, an activity element that binds to a distinct receptor on the same cell, and a linker connecting the two protein domains. A range of linkers is described. WO2018218076 describes modified antibodies of formula A-L-P, wherein A is an antibody or antibody fragment that binds to a target antigen, P is a peptide that reduces binding of A to the target antigen at physiological pH and does not reduce binding of A to the target antigen at acidic pH, and L is a linking moiety that connects A to P at physiological pH and in a tumour microenvironment and L is bound to A outside an antigen binding site. Various linking moieties L are described. WO2021174198 describes heteromeric complexes of scFv antibodies targeting the extracellular domains of BMP type I and II receptors. A variety of linkers to connect VH and VL within a scFv and to link scFvs together are described. WO2019154986 describes IL-12 linked EDB binding domains. Various linkers are described. SUMMARY OF THE INVENTION Currently, there are no universal methods available to improve fusion protein properties such as stability and solubility. Accordingly, it is an object of the present invention to provide novel peptide motifs and sequences that can be employed as a universal fusion protein linker to improve the stability, solubility and / or expression titer of fusion proteins, preferably binding molecules, such as scFv, bi- specific T-cell engager (BiTE), Dual Affinity Retargeting Antibodies (DART), Designed Ankyrin Repeat Proteins (DARPins), single-domain antibody-based formats (including VHH or VNAR), any of these formats fused to an immunoglobulin Fc domain and antigen binding domains incorporated in chimeric antigen receptors (CARs). The invention is generally applicable to improve fusion protein manufacturing processes, as detailed herein. Furthermore, the invention is generally applicable to improve intrabodies, which bind an intracellular target. The present invention provides novel peptide motifs and sequences employed as a fusion protein linker, preferably a binding molecule linker (such as a scFv linker, BiTE linker, single-domain antibody-based linker, multispecific antibody or multispecific antibody construct linker, and non-antibody scaffold linker) can, inter alia, lower the fusion protein pI and improve stability, solubility and expression titer while conserving or improving the affinity to the target. Importantly, these novel peptide motifs and sequences also promote intracellular clearance properties thanks to the inclusion of unexpected PEST-like sequence configuration. This makes feasible different clearance approaches of intracellular fusion proteins, preferably binding molecules such as scFvs, single-domain antibody-based formats, multispecific antibody constructs and non-antibody scaffold-based formats, through the proteasome and / or autophagy pathways. Accordingly, in one aspect of the invention, there is provided a fusion protein peptide linker comprising, consisting essentially of, or consisting of the amino acid sequence Z, wherein Z consists of n independent occurrence of an amino acid sequence Y, wherein each Y is independently selected from the amino acid sequence set forth as X1X2X3X4X5X6X7X8X9X10X11X12X13X14X15X16X17, wherein X1to X17are each independently selected from E, D, G, S or absent; at least one from X1to X17is G; at least one from X1to X17is E or D; at least one from X1 to X17 is S; n is 1 to 10; and Z is at least 16 amino acids in length. The linkers of the invention link two polypeptide domains to form a fusion protein. The linkers are peptide linkers and thus are composed of amino acids. They contain glycine and serine residues, which provide flexibility allowing for mobility of the connected polypeptides in the fusion protein. They also include negatively charged residues providing for a range of improved properties for the resultant fusion protein and its manufacture, as described herein. In another aspect, there is provided a fusion protein comprising two polypeptides linked by a fusion protein peptide linker of the invention. In another aspect, the fusion protein peptide linkers of the present invention promote the expression, stability and / or solubility, prevent or inhibit aggregation and / or toxicity, and / or promote degradation of fusion proteins, preferably binding molecules, such as scFv, bi-specific T-cell engager (BiTE), Dual Affinity Retargeting Antibodies (DART), Designed Ankyrin Repeat Proteins (DARPins), single- domain antibody-based formats (such as VHH or VNAR), any of these formats fused to an immunoglobulin Fc domain and antigen binding domains incorporated in chimeric antigen receptors (CARs). Also provided herein are methods for decreasing the pI of a fusion protein, for promoting or increasing the solubility of a fusion protein, for inhibiting, decreasing or preventing the toxicity of a fusion protein, for inhibiting, decreasing or preventing the aggregation of a fusion protein, for promoting or increasing intracellular degradation of a fusion protein, for promoting or increasing the expression of a fusion protein, for promoting or increasing the thermostability of a fusion protein, for promoting or increasing the monomeric expression of a fusion protein, for promoting, increasing or maintaining the target affinity of the fusion protein, or for improving or increasing the purification yield of a fusion protein. The invention is further directed, inter alia, to (i) a nucleic acid encoding a fusion protein of the present invention; (ii) an expression vector encoding a nucleic acid of the present invention; (iii) a host cell comprising a nucleic acid of the present invention; (iv) a cell-free expression system comprising a nucleic acid or expression vector of the present invention; (v) a method for producing a fusion protein of the present invention comprising the step of culturing the host cell of part (iii) or the cell-free expression system of part (iv) under conditions suitable for producing the fusion protein and isolating the fusion protein. According to a further aspect of the invention, there is provided a pharmaceutical composition comprising a nucleic acid encoding a fusion protein of the invention or expression vector encoding a fusion protein of the invention, and a pharmaceutically acceptable carrier and / or excipient. In a related aspect of the invention, there is provided a pharmaceutical composition comprising a fusion protein, wherein the fusion protein comprises a fusion protein peptide linker of the invention, and a pharmaceutically acceptable carrier and / or excipient. In one aspect there is provided a fusion protein comprising a fusion protein peptide linker of the invention, wherein the fusion protein is an intrabody. In one aspect, provided is the use of a fusion protein peptide linker of the invention in the preparation of a fusion protein. Also provided is a fusion protein peptide linker (e.g. for use in an intrabody) comprising, consisting essentially of, or consisting of an amino acid sequence Z, wherein Z consists of n independent occurrences of an amino acid sequence Y, wherein each Y is independently selected from the amino acid sequence set forth as X1X2X3X4X5X6X7X8X9X10X11X12X13X14X15X16X17, wherein X1is G; X2 to X6 are each independently selected from G or absent; X7is E or D; X8to X16are each independently selected from E, D or absent; X17is S; n is 1 to 10; and Z is at least 16 amino acids in length.DETAILED DESCRIPTION OF EMBODIMENTS OF THE INVENTION Definitions Accordingly, in the context of the present invention, the term “peptide” refers to a short chain of amino acids (usually from 2 to about 50 amino acid residues) linked by a peptide bond. The terms “polypeptide” and “protein” refer to a longer chain of amino acids linked by a peptide bond. Amino acids and their nomenclature are well known in the art with particular reference herein to glycine (Gly or G), aspartate (Asp or D), glutamate (Glu or E) and serine (Ser or S). Herein, aspartate (D) and glutamate (E) are referred to as negative or negatively charged amino acids. Accordingly, in the context of the present invention, the term “linker” (or “spacer”) relates to a moiety linking one or more separate moieties together through a covalent bond. The linker may consist of an amino acid sequence and be referred to as a peptide linker. A peptide linker is usually used to link two amino acid sequences - such as a peptide, polypeptide or protein - together. The linkers of the present invention may be used to fuse any peptide, polypeptide or protein amino acid sequences together (by peptide bond) to form a fusion protein. Such linker may therefore be defined as a “fusion protein linker” and specifically a “fusion protein peptide linker”. “Fusion proteins” are proteins created by linking two or more amino acid sequences such as peptides, polypeptides, protein domains or proteins. The two or more amino acid sequences may be linked together by a peptide bond, or by a linker. The linker may be a peptide linker or any suitable linker. Non-limiting examples of fusion proteins include peptide, polypeptide or protein of interest linked to carrier proteins such as Keyhole Limpet Hemocyanin (KLH), Cross-Reacting Material 197 (CRM197) or tetanus toxoid, or linked to tag such as 6xHis, Glutathione S-transferase (GST), Maltose-Binding Protein (MBP), or linked to a fluorescent tag such as green fluorescent protein (GFP), or linked to an enzyme such as a luciferase or a ligase, or linked to peptides or polypeptides recognized by blood- brain barrier (BBB) receptor to promote transport across the BBB, or linked to Fragment crystallizable region (Fc). Further non-limiting examples of fusion proteins include binding molecules such as single chain variable fragment (scFv) or bi-specific T-cell engager (BiTE). A fusion protein may comprise non amino acid component including but not limited to prosthetic group (such as haems) or cofactor (such as metal ions). A fusion protein may comprise non amino acid components by conjugation to a chemical group, a molecule such as a drug, a fluorescent moiety or a radionucleotide. The term “binding molecule” refers to molecules, usually comprising one or more polypeptide(s), that specifically recognizes and binds a target molecule. Non-limiting examples of binding molecules include antibodies and antigen-binding fragments thereof, such as single chain variable fragment (scFv), which term includes bifunctional and multispecific scFvs, scFab, bi-specific T-cell engager (BiTE), Dual Affinity Retargeting Antibodies (DART), Designed Ankyrin Repeat Proteins (DARPins), any non-antibody scaffold-based formats, single-domain antibody-based formats (including VH only, VL only, VHH and VNARS), any of these formats fused together and / or fused to an immunoglobulin Fc domain, and / or fused to a non-immunoglobulin fusion protein, and antigen binding domains incorporated in chimeric antigen receptors (CARs). In general, the term "antibody" is used herein in the broadest sense and encompasses various antibody structures, including but not limited to monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), fully human antibodies and antibody fragments so long as they exhibit the desired antigen-binding activity. The antibodies may also be chimeric antibodies (especially mouse Heavy Chain Variable Region / Domain (VH) and Light Chain Variable Region / Domain (VL) fused with human constant domains), recombinant antibodies, antigen-binding fragments of recombinant antibodies, humanized antibodies, or fully human antibodies. An "antigen-binding fragment" of an antibody, or “functional fragment thereof” refers to a molecule other than an intact, or full-length, antibody that comprises a portion of an intact, or full-length, antibody and that binds (fully or partially) the antigen to which the intact, or full-length, antibody binds. Examples of antibody fragments include but are not limited to Fv, Fab, Fab', Fab' -SH, F(ab')2, diabodies, linear antibodies, single-chain antibody molecules (e.g. scFv) and multi-specific antibodies formed from antibody fragments. The term also encompasses single domain antibodies, i.e. an antibody fragment consisting of a single monomeric variable antibody domain (e.g. VHH, VNARs or human single domain antibody). Antigen-binding fragments may also be referred to as “functional fragments” as they retain the binding function of the original antibody from which they are derived. “Single-chain Fv” or “scFv” antibody fragments have, in the context of the invention, the VH and VL domains of an antibody, wherein these domains are present in a single polypeptide chain. Generally, the scFv polypeptide further comprises a peptide linker between the VH and VL domains which enables the scFv to form the desired structure for antigen binding. The VH and VL domains may be in any order to form a scFv (the VL may be in N-terminal and the VH in C-terminal of the amino acid sequence or vice-versa). Techniques to produce single chain antibodies are known in the art (e.g., in Plückthun, 1994). An "Fc" region contains two heavy chain fragments comprising the CH2 and CH3 domains of an antibody. The two heavy chain fragments are held together by two or more disulfide bonds and by hydrophobic interactions of the CH3 domains. A "Fab fragment" contains one light chain (VL + CL), a portion of one heavy chain that contains the heavy variable domain (VH), the constant CH1 domain, and a short part of the hinge region between the CH1 and CH2 domains, such that an interchain disulfide bond can be formed between the two chains. A "F(ab')2 fragment" contains two light chains (VL + CL), a portion of two heavy chains (VH + CH1) and the hinge region between the CH1 and CH2 domains, such that interchain disulfide bonds can be formed between 1) the light chain and heavy chain and 2) the two heavy chains. A F(ab')2fragment is thus composed of two Fab fragments held together by disulfide bonds. The "Fv region" comprises the variable regions from both the heavy and light chains but lacks the constant regions. A “multispecific antibody” as used herein is a binding molecule comprising more than one antigen- binding site that may bind different epitopes on the same target antigen or different epitopes on different target antigens. A multispecific antibody can be bispecific (comprising two antigen-binding sites targeting different epitopes), trispecific (comprising three antigen-binding sites targeting different epitopes), tetraspecific (comprising four antigen-binding sites targeting different epitopes), etc. Multispecific antibodies comprises many different protein constructs, including but not limited to, bispecific antibody conjugates (such as F(ab’)2 or CovX-Body), hybrid bispecific IgGs, variable domain only bispecific antibodies (such as Diabody, DART, BiTE, tandem scFv, triplebody, tandem VHH, triple VHH), Fab fusion proteins (such as Fab-scFv also known as bibody, Fab-scFv2 also known as tribody, Fab-Fv, Fab-VHH), non-immunoglobulin fusion proteins (scFv2-albumin, ImmTAC also known as TCR-scFv, miniantibody, DNL Fab4-IgG), Fc-modified IgG (such as CrossMab, DuetMab, Duobody, LUZ-Y), appended and Fc-modified IgG (such as IgG(kih)-scFab, scFab-Fc(kih)-scFv, DVI-Ig (four-in-one), half DVD-Ig, CrossMab-Fab), modified Fc and CH3 fusion proteins (such as scFv-Fc also known as SEEDbody, scFv-Fc (CH3 charge pairs or HA-TF), Fab-scFv-Fc (BEAT or kih), DART-Fc, TriFabs), appended IgGs – HC fusions (such as IgG-HC- scFv, IgG CrossFab, tandem Fab-IgG), appended IgGs – LC fusions, appended IgGs – HC and LC fusions (such as DVD-Ig, TVD-Ig, CODV-Ig, scFv4-Ig, Zybody), Fc fusions (such as Di-diabody, taFv-Fc, HCAb-VHH), CH3 fusions, IgE / IgM fusions, F(ab’)2 fusions, CH1 / CL fusion proteins, and modified IgGs (such as DAF, DutaMab). Non-limiting examples of multispecific antibody can be found in Weidle et al. (2013), Spiess et al. (2015), Biswas et al. (2023) and Brinkmann and Kontermann (2017). The term “intrabody” (or “intracellular antibody”) relates to an antibody, antibody fragments, or any non-antibody scaffold-based formats, not secreted by the cell in which it is expressed and able to reach subcellular compartments, including but not limited to, the nucleus, the endoplasmic reticulum, the cytoplasm, the Golgi apparatus and the mitochondria. An intrabody may incorporate a suitable signal peptide and / or retention sequence to enable targeting to specific subcellular compartments (see Marschall et al., 2015 for examples of signal peptides suitable for use with an intrabody). Thus, the vectors of the invention may encode such signal peptides and / or retention sequences. An intrabody binds, and typically specifically binds, to one or more intracellular target antigens. An intrabody thus may be used to inhibit, promote or modulate various intracellular processes or pathways linked to the targeted antigen(s). Non-limiting examples of intracellular processes or pathways an intrabody may inhibit, promote or modulate include transcription, translation, cell survival, cell proliferation, cell metabolism, gene replacement, oxidative stress, inflammation, aging or protein aggregation. An intrabody may be used to inhibit, promote or modulate pathological cellular processes or pathways. Non-limiting examples comprise binding to misfolded and / or aggregated proteins to prevent, block or inhibit aggregation or further aggregation and cytotoxicity, or promote degradation of the target. In particular, an intrabody may be used as a therapeutic agent for the treatment of conditions, diseases or disorders in a subject in need thereof. Intrabodies can take many forms, including but not limited to scFv, scFab, bifunctional and multispecific scFv, any non-antibody scaffold-based formats, single- domain antibody-based formats (including VH only, VL only, VHH and VNARS), any of these formats fused together and / or fused to an immunoglobulin Fc domain, and / or fused to a non- immunoglobulin fusion protein. Antibodies, antibody constructs, antibody fragments, antibody derivatives (all being Ig-derived) to be employed in accordance with the invention or their corresponding immunoglobulin chain(s) can be further modified using conventional techniques known in the art, for example, by using amino acid deletion(s), insertion(s), substitution(s), addition(s), and / or recombination(s) and / or any other modification(s) known in the art either alone or in combination. Methods for introducing such modifications in the DNA sequence underlying the amino acid sequence of an immunoglobulin chain are well known to the person skilled in the art; see, e.g., Sambrook et al. 2nd(1989) and 3rdedition (2001). The term “Ig-derived domain” particularly relates to (poly)peptide constructs comprising at least one CDR. Fragments or derivatives of the recited Ig-derived domains define (poly)peptides which are parts of the above antibody molecules and / or which are modified by chemical / biochemical or molecular biological methods. Corresponding methods are known in the art and described inter alia in laboratory manuals (see Sambrook et al., 2nd edition (1989) and 3rd edition (2001); Gerhardt, (1994); Lefkovits, (1997); Golemis, (2002)). The terms "protein solubility” or “soluble protein”, in the context of the present invention, are used to define the ability of a protein (that can be a monomer, dimer or higher order functional oligomer) to adopt and / or conserve a correctly folded structure in solution. Alternatively, the terms “protein insolubility” or “insoluble protein” are used to define the inability of a protein to adopt and / or conserve a correctly folded structure in solution, resulting in the protein oligomerization and / or aggregation and inability of the protein to be functional. The solubility / insolubility of a protein is determined by intrinsic and extrinsic factors. Intrinsic factors include, but are not limited to, the protein amino acid sequence, 3D structure and post-translational modification(s). Extrinsic factors include, but are not limited to, the solution ionic strength, composition, pH and temperature, the concentration of macromolecules (such as proteins and nucleic acids) in solution, and the interaction with said macromolecules (such as chaperone(s) or prion seed(s)) that may improve or impair the protein solubility. The solubility of a protein may thus vary in the numerous in vivo and in vitro environments it may be expressed or present in. Noteworthy, the protein translation process and the environment in which the protein is translated are critical for the resulting solubility of a protein as the proper folded 3D structure of the protein is usually acquired during translation. It should be noted that a protein may be soluble if present in a certain environment but would be insoluble if it had been translated in the very same environment, as the interaction with the environment is different between a mature protein with an acquired 3D folded structure (and possible post-translational modifications) and a protein being translated and acquiring its 3D folded structure. Accordingly, the terms “intracellular solubility” or “intracellularly soluble” relates to the ability of a protein to adopt a correctly folded structure when expressed (i.e. translated) in a cell and conserve said correctly folded structure in the intracellular environment. Noteworthy, the promoter strength and the expression / translation rate of the protein may impact the intracellular solubility of proteins, especially in over-expression paradigm with strong constitutive promoters, as a higher expression rate result in higher protein production and thus intracellular protein concentration, and stress on the translation process and machinery. The terms “isoelectric point” or “pI” refer to the pH at which a molecule (such as a protein) carries no net electrical charge or is electrically neutral, i.e. the overall charge of the protein is null. A molecule (such as a protein) has minimum solubility in water or salt solutions at the pH that corresponds to their pI and often precipitate out of solution. A protein having a lower pI (i.e. more acidic) than the pH of the solution is negatively charged while a protein having a higher pI (i.e. more basic) than the pH of the solution is positively charged. The terms “protein stability” or “stable protein”, in the context of the present invention, are used to define the ability of a protein (that can be a monomer, dimer or higher order functional oligomer) to conserve a properly folded 3D structure and remain functional. The stability of a protein is determined by intrinsic and extrinsic factors. Intrinsic factors include, but are not limited to, the protein amino acid sequence, 3D structure and post-translational modification(s). Extrinsic factors include, but are not limited to, the solution ionic strength, composition, pH and temperature, the concentration of macromolecules (such as proteins and nucleic acids) in solution, and the interaction with said macromolecules (such as chaperone(s) or prion seed(s)) that may improve or impair the protein stability. The stability of a protein may thus vary in the numerous in vivo and in vitro environments in which it may be present. Noteworthy, the protein translation process and the environment in which the protein is translated are critical for the resulting stability of a protein as the proper folded 3D structure of the protein is usually acquired during translation. Accordingly, the term “protein thermostability” and “thermostable protein” relates to the ability of a protein to conserve a properly folded 3D structure and remain functional at a given temperature. The thermostability of a protein may be determined by measuring the melting temperature (Tm) of the protein, i.e. the temperature at which 50% of the protein is unfolded or denatured. Reference can be made to Example 8 for methods to measure thermostability. The terms “cellular toxicity” or “cytotoxicity”, relates to the ability of a process or a substance (i.e. a stressor) to damage a cell, potentially resulting in cell death. Non-limiting examples of cytotoxic stressors include misfolded proteins and protein aggregates. The term “cell viability” relates to the ability of a cell to maintain a state of survival. The cytotoxicity of a stressor may be determined by quantifying cell viability. Methods and assays to quantify cell viability are well known in the art and reference can be made to Example 4 of the present application or Strober, (2015). The terms “protein degradation” or “protein clearance”, in the context of the present invention, relates to the process by which proteins are eliminated from the intracellular space, usually by proteolysis. Intracellular pathways responsible for the degradation / clearance (and recycling) of proteins include but are not limited to the ubiquitin-proteasome system (UPS) and autophagy-lysosome pathway (ALP) (Dikic, 2017; Trelford et al., 2021; Franic et al., 2021; Yin et al., 2020; Pohl et al., 2019), the latter including chaperone-mediated autophagy (CMA). A variety of signals can promote / induce protein degradation / clearance by intracellular degradation pathways such as the presence of certain peptide signal / motif. Non-limiting examples of peptide signal / motif promoting intracellular degradation include PEST motifs, KFERQ amino acid sequence, tripartite motif protein family (TRIM) and immunoglobulin Fc (Rogers et al., 1986; Rechsteiner et al., 1996; Kirchner et al., 2019; Catarino et al., 2017). The terms “dimeric” or “oligomeric”, in the context of the present invention, relates to the ability of certain proteins to bind together through non-covalent bond to form dimers and / or higher order oligomers. Antibodies and antibody fragments are able to form dimers or higher order oligomers depending on the environment in which they are expressed or present, their amino acid sequence and their structure. For instance, scFv can exist both in monomeric or dimeric format and stay functional (i.e. bind its target). The ability to control the protein expression as monomer, dimer or higher order oligomer is desirable as the protein stability, activity and function may change. The term “vector” is well known in the art, and in the context of the invention refers to any particle used to transport (by transduction or transfection) a polynucleotide(s) or polypeptide(s) into a host cell. This definition includes both non-viral and viral vectors. The viral or non-viral vectors may target any cell or tissue to deliver and / or express a fusion protein comprising a fusion protein peptide linker of the present invention. Non-viral vectors include, but are not limited to, organic nanomaterials such as, liposomes, exosomes, dendrimers, and micelles or inorganic nanomaterials such as gold nanoparticles, silica nanoparticles and carbon nanotubes. The non-viral vectors may express or present a peptide, small molecule (SME), antibody or antibody fragment thereof, protein, nanoparticle, lipid, oligonucleotide, aptamer or cationic molecule on the vector surface that targets the vector to the desired cell or tissue. Viral vectors include, but are not limited to, wild-type viruses and engineered (e.g. modified) viruses. Non-limiting examples of viral vectors include adeno-associated virus (AAV), adenovirus, retrovirus, rhinovirus, lentivirus, hepatitis, HSV and any virus-like particle. The AAV can be of any suitable serotypes, examples of which include, but are not limited to, AAV serotype 1 (AAV1), AAV serotype 2 (AAV2), AAV serotype 3 (AAV3), AAV serotype 4 (AAV4), AAV serotype 5 (AAV5), AAV serotype 6 (AAV6), AAV serotype 7 (AAV7), AAV serotype 8 (AAV8), AAV serotype 9 (AAV9), AAV serotype 10 (AAV10), AAV serotype 11 (AAV11), or AAV serotype 12 (AAV12), AAVrh.10 (AAV rhesus isolate10), or any other wild type serotypes or engineered AAVs (such as AAV-BR1 engineered from AAV2; AAV-S, AAV-F, AAV-PHP.eB or AAV9-PHP- V, AAV.CAP-B10, AAV.CAP-B22, AAV.CAP-Mac, VCAP-103, or bCap 1 engineered from AAV9; VCAP-100 engineered from AAV5; or AAV1RX, AAV1R6 or AAV1R7 engineered from AAV1). The vectors as described herein can be administered to a subject by any conventional route, including injection or by gradual infusion over time. The administration may be via parenteral administration. The administration may, for example, be by infusion or by intrathecal, intra-cisternal, intracerebroventricular, intraparenchymal, intrastriatal, intranasal, intravitreous, subcutaneous, or intramuscular route. The vector may be administered by intravenous injection or intravenous infusion. For the avoidance of doubt, as used herein the term "treatment" includes the therapeutic treatment, as well as the symptomatic treatment and the prophylaxis of a condition. Use of the term "treat", "treating", or "treatment of" (and grammatical variations thereof) means that the severity of a subject's condition is reduced, at least partially improved, or ameliorated and / or that some alleviation, mitigation or decrease in at least one clinical symptom is achieved and / or there is a delay in the progression of the disease or disorder. As used herein the term "subject" refers to an individual, e.g., a mammal such as a human, having or at risk of having a specified condition, disease, disorder or symptom present. The subject may be a subject in need of treatment. The subject may have received treatment for the condition, disease, disorder or symptom. Alternatively, the subject has not been treated prior to treatment. As used herein, the term “pathological protein” refers to a protein associated with a condition, disease or disorder, including but not limited to proteins associated with proteinopathies, or toxins. Non- limiting examples of pathological proteins include amyloid, prion or botulinum toxin. As used herein, the term “cell surface receptor” refers to a membrane / transmembrane protein receptor in the plasma membrane of a cell. Non-limiting examples of cell surface receptors include ion channel, enzyme-linked receptors (such as receptor tyrosine kinases), transporter protein (such as GLUTs or transferrin receptors) or G protein-coupled receptors. As used herein, the term “tumour-specific antigen” (TSA) refers to a protein specifically produced by tumour cells. The term “tumour-associated antigen” (TAA) refers to a protein associated with tumour cells. Reference can be made to Tan & Zhang 2008 for non-limiting examples of TSAs and TAAs. Invention embodiments According to one aspect of the invention, there is provided a fusion protein peptide linker (i.e. one composed of amino acids) comprising, consisting essentially of, or consisting of the amino acid sequence Z, wherein Z consists of n independent occurrence of an amino acid sequence Y, wherein each Y is independently selected from the amino acid sequence set forth as X1X2X3X4X5X6X7X8X9X10X11X12X13X14X15X16X17, wherein X1to X17are each independently selected from E, D, G, S or absent; at least one from X1to X17is G; at least one from X1to X17is E or D; at least one from X1 to X17 is S; n is 1 to 10; and Z is at least 16 amino acids in length. In one embodiment, the fusion protein peptide linker consists of the amino acid sequence Z. In some embodiments, the fusion protein peptide linker comprises, consists essentially of, or consists of the amino acid sequence Z, wherein Z consists of n independent occurrence of an amino acid sequence Y, wherein each Y is independently selected from the amino acid sequence set forth as X1X2X3X4X5X6X7X8X9X10X11X12X13X14X15X16X17, wherein X1 to X17 are each independently selected from E, D, G, S or absent; at least 30% of Z amino acids are G; at least 15% of Z amino acids are D and / or E; from 10% to 20% of Z amino acids are S; n is 1 to 10; and Z is at least 16 amino acids in length. In an embodiment, the fusion protein peptide linker comprises, consists essentially of, or consists of the amino acid sequence Z, wherein Z consists of n independent occurrence of an amino acid sequence Y, wherein each Y is independently selected from the amino acid sequence set forth as X1X2X3X4X5X6X7X8X9X10X11X12X13X14X15X16X17, wherein X1 to X17 are each independently selected from E, D, G, S or absent; n is 1 to 10; from 30 to 70% of Z amino acids are G; from 15 to 55% of Z amino acids are D and / or E; from 10% to 20% of Z amino acids are S; and Z is at least 16 amino acids in length. In a preferred embodiment, the fusion protein peptide linker comprises, consists essentially of, or consists of the amino acid sequence Z, wherein Z consists of n independent occurrence of an amino acid sequence Y, wherein each Y is independently selected from the amino acid sequence set forth as X1X2X3X4X5X6X7X8X9X10X11X12X13X14X15X16X17, wherein X1 to X17 are each independently selected from E, D, G, S or absent; n is 1 to 10; from 30 to 50% of Z amino acids are G; from 35 to 55% of Z amino acids are D and / or E; from 10% to 15% of Z amino acids are S; and Z is at least 16 amino acids in length. In a more preferred embodiment, the fusion protein peptide linker comprises, consists essentially of, or consists of the amino acid sequence Z, wherein Z consists of n independent occurrence of an amino acid sequence Y, wherein each Y is independently selected from the amino acid sequence set forth as X1X2X3X4X5X6X7X8X9X10X11X12X13X14X15X16X17, wherein X1to X17are each independently selected from E, D, G, S or absent; n is 1 to 10; at least 35 % of Z amino acids are D and / or E; Z is at least 24 amino acids in length, preferably Z is from 24 to 40 amino acids in length. For the avoidance of doubt, when the proportion of different types of amino acid in a sequence are defined by percentages, the combined percentages total 100%. Z contains only G, S, D and / or E amino acids. For example, when a sequence is defined as at least 30% of Z amino acids are G; at least 15% of Z amino acids are D and / or E; and from 10% to 20% of Z amino acids are S, the combination totals 100%. So, if there are 10% S amino acids and 50% D (with no E) amino acids there will be 40% G amino acids. The amino acids that make up the fusion protein peptide linker of the invention may alternatively be defined using a ratio. The ratios may refer to the fusion protein peptide linker Z amino acid sequence and / or the component Y amino acid sequence. In one embodiment, the ratio of G : E and / or D : S amino acids is selected from 4:1:1, 4:2:1, 4:3:1, 4:4:1, 4:5:1, 4:6:1, 4:7:1, 4:8:1, 4:9:1, 4:10:1, 2:2.5 or higher:1, or 16:21:8. In some embodiments, the fusion protein peptide linker comprises, consists essentially of, or consists of the amino acid sequence Z, wherein Z is at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 26, at least 27, at least 28, at least 29, at least 30, at least 31, at least 32 amino acids in length. In a preferred embodiment, the amino acid sequence Z is at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, or at least 24 amino acids. In a more preferred embodiment, the amino acid sequence Z is at least 24 amino acids. In some embodiments, the amino acid sequence Z is no more than 40, no more than 45, no more than 50, no more than 60, no more than 70, no more than 80, no more than 90 or no more than 100 amino acids in length. In some embodiments, the amino acid sequence Z is no more than 40, no more than 45, or no more than 50 amino acids in length. In further embodiments, the fusion protein peptide linker does not contain additional amino acids over the amino acid sequence of Z, therefore the maximum length is the same for both the fusion protein peptide linker and Z. In some embodiments, the fusion protein peptide linker comprises, consists essentially of, or consists of the amino acid sequence Z, wherein Z is from 16 to 100 amino acids, from 16 to 90 amino acids, from 16 to 80 amino acids, from 16 to 70 amino acids, from 16 to 60 amino acids, from 16 to 50 amino acids, from 16 to 45 amino acids or from 16 to 40 amino acids in length. In other embodiments, the amino acid sequence Z is from 17 to 100 amino acids, from 17 to 90 amino acids, from 17 to 80 amino acids, from 17 to 70 amino acids, from 17 to 60 amino acids, from 17 to 50 amino acids, from 17 to 45 amino acids or from 17 to 40 amino acids. In some other embodiments, the amino acid sequence Z is from 18 to 100 amino acids, from 18 to 90 amino acids, from 18 to 80 amino acids, from 18 to 70 amino acids, from 18 to 60 amino acids, from 18 to 50 amino acids, from 18 to 45 amino acids or from 18 to 40 amino acids. In yet other embodiments, the amino acid sequence Z is from 19 to 100 amino acids, from 19 to 90 amino acids, from 19 to 80 amino acids, from 19 to 70 amino acids, from 19 to 60 amino acids, from 19 to 50 amino acids, from 19 to 45 amino acids or from 19 to 40 amino acids. In still other embodiments, the amino acid sequence Z is from 20 to 100 amino acids, from 20 to 90 amino acids, from 20 to 80 amino acids, from 20 to 70 amino acids, from 20 to 60 amino acids, from 20 to 50 amino acids, from 20 to 45 amino acids or from 20 to 40 amino acids. In yet other embodiments, the amino acid sequence Z is from 22 to 100 amino acids, from 22 to 90 amino acids, from 22 to 80 amino acids, from 22 to 70 amino acids, from 22 to 60 amino acids, from 22 to 50 amino acids, from 22 to 45 amino acids or from 22 to 40 amino acids. In yet other embodiments, the amino acid sequence Z is from 24 to 100 amino acids, from 24 to 90 amino acids, from 24 to 80 amino acids, from 24 to 70 amino acids, from 24 to 60 amino acids, from 24 to 50 amino acids, from 24 to 45 amino acids or from 24 to 40 amino acids. In yet other embodiments, the amino acid sequence Z is from 28 to 100 amino acids, from 28 to 90 amino acids, from 28 to 80 amino acids, from 28 to 70 amino acids, from 28 to 60 amino acids, from 28 to 50 amino acids, from 28 to 45 amino acids or from 28 to 40 amino acids. In yet other embodiments, the amino acid sequence Z is from 32 to 100 amino acids, from 32 to 90 amino acids, from 32 to 80 amino acids, from 32 to 70 amino acids, from 32 to 60 amino acids, from 32 to 50 amino acids, from 32 to 45 amino acids or from 32 to 40 amino acids. In a preferred embodiment, the amino acid sequence Z is from 16 to 50 amino acids, from 17 to 50 amino acids, from 18 to 50 amino acids, from 19 to 50 amino acids, from 20 to 50 amino acids, from 24 to 50 amino acids, or from 32 to 50 amino acids. In a more preferred embodiment, the amino acid sequence Z is from 18 to 40 amino acids, from 19 to 40 amino acids, from 20 to 40 amino acids, from 24 to 40 amino acids, or from 32 to 40 amino acids. In an even more preferred embodiment, the amino acid sequence Z is from 24 to 40 amino acids. According to the invention, the fusion protein peptide linker is composed of an amino acid sequence comprising, consisting essentially of, or consisting of the amino acid sequence Z as defined herein. In embodiments where the fusion protein peptide linker comprises the amino acid sequence Z, there may be additional amino acids N and / or C terminal to the Z amino acid sequence. In embodiments where the fusion protein peptide linker consists essentially of the amino acid sequence Z, there may be no more than 1-5 additional amino acids N and / or C terminal to the Z amino acid sequence. In one embodiment, the fusion protein peptide linker is 18 amino acids or more in length, 21 amino acids or more in length, 24 amino acids or more in length, 28 amino acids or more in length, 32 amino acids or more in length, 40 amino acids or more in length, 45 amino acids or more in length or 50 amino acids or more in length. In another embodiment, the fusion protein peptide linker is 50 amino acids or less, 45 amino acids or less, 40 amino acids or less, 32 amino acids or less, 28 amino acids or less, 24 amino acids or less, 21 amino acids or less, or 18 amino acids or less. In some embodiments, the fusion protein peptide linker comprises, consists essentially of, or consists of the amino acid sequence Z, wherein Z consists of 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 independent occurrence(s) of the amino acid sequence Y. In other embodiments, the amino acid sequence Z consists of 2, 3, 4, 5 or 6 independent occurrences of the amino acid sequence Y. In a preferred embodiment, the amino acid sequence Z consists of 2, 3 or 4 independent occurrences of the amino acid sequence Y. In a more preferred embodiment, the amino acid sequence Z consists of 3 or 4 independent occurrences of the amino acid sequence Y. According to the invention, the fusion protein peptide linker has n independent occurrences of an amino acid sequence Y as defined herein. n is 1 to 10. n may be 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 occurrences. In alternative embodiments n may be 1 or more, 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more or 10 or more occurrences. In some embodiments, the fusion protein peptide linker comprises, consists essentially of, or consists of the amino acid sequence Z, wherein Z consists of at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8 or at least 9 independent occurrences of the amino acid sequence Y. In one embodiment, Z consists of 2, 3, 4, 5, 6, 7, 8 or 9 independent occurrences of the amino acid sequence Y. In a preferred embodiment, the amino acid sequence Z consists of at least 2, at least 3 or at least 4 independent occurrences of the amino acid sequence Y. In a more preferred embodiment, the amino acid sequence Z consists of at least 2 independent occurrences of the amino acid sequence Y. In some embodiments, the fusion protein peptide linker consists of an amino acid sequence comprising, consisting essentially of, or consisting of the amino acid sequence Z, wherein Z consists of n independent occurrence of an amino acid sequence Y, wherein each occurrence of the amino acid sequence Y differs by 1, 2 or up to 3 amino acids when compared to one selected Y or when compared across all defined Ys. In some embodiments, the fusion protein peptide linker comprises, consists essentially of, or consists of the amino acid sequence Z, wherein Z consists of n independent occurrences of an amino acid sequence Y, wherein the amino acid sequence Y is the same for each of the n occurrences of Y. Thus, Y is repeated n times. This applies, for example, when n is 2 to 10. In alternative embodiments, each independent occurrence of Y may have a different sequence (order) of amino acids. As an example of the same sequence (order), Y1is GGGDGGDS (SEQ ID NO: 92) and Y2is GGGDGGDS (SEQ ID NO: 92) so that the relevant part of the sequence in Z (Y with n = 2) is GGGDGGDSGGGDGGDS (SEQ ID NO: 93). As an example of a different sequence (order), Y1 is GGGDGDGS (SEQ ID NO: 94) and Y2 is GGGDGGDS (SEQ ID NO: 92) so that the relevant part of the sequence in Z (Y with n = 2) is GGGDGDGSGGGDGGDS (SEQ ID NO: 95). In some embodiments, Z consists of n independent occurrences of an amino acid sequence Y, wherein each independent occurrence of Y has the same sequence (order), for example, GGDSGGDSGGDSGGDS (G2DS)4(SEQ ID NO: 96). In some embodiments, the fusion protein peptide linker comprises, consists essentially of, or consists of the amino acid sequence Z, wherein at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55% or at least 60% of Z amino acids are D and / or E. In other embodiments, at least 35%, at least 37.5%, at least 40%, at least 42.5%, at least 45%, at least 47.5%, or at least 50% of Z amino acids are D and / or E. In a preferred embodiment, at least 35% of Z amino acids are D and / or E. In a more preferred embodiment, at least 40% of Z amino acids are D and / or E. In an even more preferred embodiment, at least 50% of Z amino acids are D or E. In some embodiments, the fusion protein peptide linker comprises, consists essentially of, or consists of the amino acid sequence Z, wherein Z consists of n independent occurrence of an amino acid sequence Y, wherein each Y is independently selected from the amino acid sequence set forth as X1X2X3X4X5X6X7X8X9X10X11X12X13X14X15X16X17, wherein X1 is G; X2 to X6 are each independently selected from G or absent; X7 is E or D; X8 to X16 are each independently selected from E, D or absent; X17is S; n is 1 to 10; and Z is at least 16 amino acids in length. In some embodiments, X1and X2are G. In other embodiments, X1to X3are G. In yet other embodiments, X1to X4are G. In some embodiment, X5and X6are absent. In some embodiments, X14to X16are absent. In other embodiments, X12to X16are absent. In some embodiments, X7and X8are each independently selected from E or D. In other embodiments, X7 to X9 are each independently selected from E or D. In some embodiments, X1 and X2 are G; X7 and X8 are each independently selected from E or D; and X15and X16are absent. In other embodiments, X1to X3are G; X7and X8are each independently selected from E or D; and X6and X14to X16are absent. In yet other embodiments, X1to X3are G; X7to X9are each independently selected from E or D; and X6and X14to X16are absent. In some embodiments, X1 to X4 are G; X7 is E or D; X8 to X11 are each independently selected from E, D or absent; and X5 to X6 and X12 to X16 are absent. In other embodiments, X1 to X4 are G; X7 to X9 are each independently selected from E or D; X10 to X11 are each independently selected from E, D or absent; and X5to X6and X12to X16are absent. In some embodiments, the fusion protein peptide linker comprises, consists essentially of, or consists of the amino acid sequence Z, wherein Z consists of n independent occurrence of an amino acid sequence Y, wherein each Y is independently selected from the amino acid sequence set forth as X1X2X3X4X5X6X7X8X9X10X11X12X13X14X15X16X17, wherein X1and X2are G; X3to X6are each independently selected from G or absent; X7is E or D; X8 to X16 are each independently selected from E, D or absent; X17 is S;n is 1 to 10; and Z is at least 16 amino acids in length. In some embodiments, the fusion protein peptide linker comprises, consists essentially of, or consists of the amino acid sequence Z, wherein Z consists of n independent occurrence of an amino acid sequence Y, wherein each Y is independently selected from the amino acid sequence set forth as X1X2X3X4X5X6X7X8X9X10X11X12X13X14X15X16X17, wherein X1to X3are G; X4 to X6 are each independently selected from G or absent; X7 is E or D; X8 to X16 are each independently selected from E, D or absent; X17 is S;n is 1 to 10; and Z is at least 16 amino acids in length. In some embodiments, the fusion protein peptide linker comprises, consists essentially of, or consists of the amino acid sequence Z, wherein Z consists of n independent occurrence of an amino acid sequence Y, wherein each Y is independently selected from the amino acid sequence set forth as X1X2X3X4X5X6X7X8X9X10X11X12X13X14X15X16X17, wherein X1to X4are G; X5and X6are each independently selected from G or absent; X7 is E or D; X8 to X16 are each independently selected from E, D or absent; X17 is S;n is 1 to 10; and Z is at least 16 amino acids in length. In some embodiments, the fusion protein peptide linker comprises, consists essentially of, or consists of the amino acid sequence Z, wherein Z consists of n independent occurrence of an amino acid sequence Y, wherein each Y is independently selected from the amino acid sequence set forth as X1X2X3X4X5X6X7X8X9X10X11X12X13X14X15X16X17, wherein X1is G; X2to X4are each independently selected from G or absent; X5 and X6 are absent; X7is E or D; X8to X16are each independently selected from E, D or absent; X17is S; n is 2 to 10; and Z is at least 16 amino acids in length. In some embodiments, the fusion protein peptide linker comprises, consists essentially of, or consists of the amino acid sequence Z, wherein Z consists of n independent occurrences of an amino acid sequence Y, wherein each Y is independently selected from the amino acid sequence set forth as X1X2X3X4X5X6X7X8X9X10X11X12X13X14X15X16X17, wherein X1 is G; X2 to X6 are each independently selected from G or absent; X7 is E or D; X8to X13are each independently selected from E, D or absent; X14to X16are absent; X17is S; n is 2 to 10; and Z is at least 16 amino acids in length. In some embodiments, the fusion protein peptide linker comprises, consists essentially of, or consists of the amino acid sequence Z, wherein Z consists of n independent occurrences of an amino acid sequence Y, wherein each Y is independently selected from the amino acid sequence set forth as X1X2X3X4X5X6X7X8X9X10X11X12X13X14X15X16X17, wherein X1 is G; X2 to X6 are each independently selected from G or absent; X7 is E or D; X8to X11are each independently selected from E, D or absent; X12to X16are absent; X1is S; n is 2 to 10; and Z is at least 16 amino acids in length. In some embodiments, the fusion protein peptide linker comprises, consists essentially of, or consists of the amino acid sequence Z, wherein Z consists of n independent occurrences of an amino acid sequence Y, wherein each Y is independently selected from the amino acid sequence set forth as X1X2X3X4X5X6X7X8X9X10X11X12X13X14X15X16X17, wherein X1is G; X2to X6are each independently selected from G or absent; X7and X8are each independently selected from E or D; X9 to X16 are each independently selected from E, D or absent; X17 is S;n is 1 to 10; and Z is at least 16 amino acids in length. In some embodiments, the fusion protein peptide linker comprises, consists essentially of, or consists of the amino acid sequence Z, wherein Z consists of n independent occurrences of an amino acid sequence Y, wherein each Y is independently selected from the amino acid sequence set forth as X1X2X3X4X5X6X7X8X9X10X11X12X13X14X15X16X17, wherein X1 is G; X2to X6are each independently selected from G or absent; X7to X9are each independently selected from E or D; X10to X16are each independently selected from E, D or absent; X17 is S;n is 1 to 10; and Z is at least 16 amino acids in length. In an embodiment, the fusion protein peptide linker comprises, consists essentially of, or consists of the amino acid sequence Z, wherein Z consists of n independent occurrences of an amino acid sequence Y, wherein each Y is independently selected from the amino acid sequence set forth as X1X2X3X4X5X6X7X8X9X10X11X12X13X14X15X16X17, wherein X1and X2are G; X3to X6are each independently selected from G or absent; X7and X8are each independently selected from E or D; X9 to X14 are each independently selected from E, D or absent; X15 and X16 are absent; X17 is S;n is 2 to 10; and Z is at least 16 amino acids in length. In an embodiment, the fusion protein peptide linker comprises, consists essentially of, or consists of the amino acid sequence Z, wherein Z consists of n independent occurrences of an amino acid sequence Y, wherein each Y is independently selected from the amino acid sequence set forth as X1X2X3X4X5X6X7X8X9X10X11X12X13X14X15X16X17, wherein X1 to X3 are G; X4and X5are each independently selected from G or absent; X7and X8are each independently selected from E or D; X9to X13are each independently selected from E, D or absent; X6 and X14 to X16 are absent; X17 is S;n is 2 to 10; and Z is at least 16 amino acids in length. In an embodiment, the fusion protein peptide linker comprises, consists essentially of, or consists of the amino acid sequence Z, wherein Z consists of n independent occurrences of an amino acid sequence Y, wherein each Y is independently selected from the amino acid sequence set forth as X1X2X3X4X5X6X7X8X9X10X11X12X13X14X15X16X17, wherein X1 to X3 are G; X4 and X5 are each independently selected from G or absent; X7to X9are each independently selected from E or D; X10to X13are each independently selected from E, D or absent; X6 and X14 to X16 are absent; X17is S; n is 2 to 10; and Z is at least 16 amino acids in length. In an embodiment, the fusion protein peptide linker comprises, consists essentially of, or consists of the amino acid sequence Z, wherein Z consists of n independent occurrences of an amino acid sequence Y, wherein each Y is independently selected from the amino acid sequence set forth as X1X2X3X4X5X6X7X8X9X10X11X12X13X14X15X16X17, wherein X1to X3are G; X4and X5are each independently selected from G or absent; X7 to X9 are each independently selected from E or D; X10 and X11 are each independently selected from E, D or absent; X6 and X12 to X16 are absent; X17 is S; n is 2 to 10; and Z is at least 16 amino acids in length. In an embodiment, the fusion protein peptide linker comprises, consists essentially of, or consists of the amino acid sequence Z, wherein Z consists of n independent occurrences of an amino acid sequence Y, wherein each Y is independently selected from the amino acid sequence set forth as X1X2X3X4X5X6X7X8X9X10X11X12X13X14X15X16X17, wherein X1to X4are G; X5to X6and X12to X16are absent; X7is E or D; X8 to X11 are each independently selected from E, D or absent; X17 is S; and n is 2 to 10; and Z is at least 16 amino acids in length. In a preferred embodiment, the fusion protein peptide linker comprises, consists essentially of, or consists of the amino acid sequence Z, wherein Z consists of n independent occurrences of an amino acid sequence Y, wherein each Y is independently selected from the amino acid sequence set forth as X1X2X3X4X5X6X7X8X9X10X11X12X13X14X15X16X17, wherein X1to X4are G; X5 to X6 and X12 to X16 are absent; X7 is E or D; X8 to X11 are each independently selected from E, D or absent; X17 is S; and n is 3 or 4; and Z is at least 16 amino acids in length. Such a fusion protein peptide linker may be represented by the amino acid sequence of SEQ ID NO: 87 and the formula (G4(E / D)1-5S)3-4. In a more preferred embodiment, the fusion protein peptide linker comprises, consists essentially of, or consists of the amino acid sequence Z, wherein Z consists of n independent occurrences of an amino acid sequence Y, wherein each Y is independently selected from the amino acid sequence set forth as X1X2X3X4X5X6X7X8X9X10X11X12X13X14X15X16X17, wherein X1to X4are G; X5 to X6 and X12 to X16 are absent; X7 is E or D; X8 to X11 are each independently selected from E, D or absent; X17 is S; and n is 3 or 4; the amino acid sequence Y is the same for each of the n occurrences of Y; and Z is at least 16 amino acids in length. Such a fusion protein peptide linker may be represented by the amino acid sequence of SEQ ID NO: 89 and the formula (G4(E / D)1-5S)3-4. In an embodiment, the fusion protein peptide linker comprises, consists essentially of, or consists of the amino acid sequence Z, wherein Z consists of n independent occurrences of an amino acid sequence Y, wherein each Y is independently selected from the amino acid sequence set forth as X1X2X3X4X5X6X7X8X9X10X11X12X13X14X15X16X17, wherein X1to X4are G; X5 to X6 and X12 to X16 are absent; X7to X9are each independently selected from E or D; X10to X11are each independently selected from E, D or absent; X17is S; and n is 2 to 10; and Z is at least 16 amino acids in length. In a preferred embodiment, the fusion protein peptide linker comprises, consists essentially of, or consists of the amino acid sequence Z, wherein Z consists of n independent occurrences of an amino acid sequence Y, wherein each Y is independently selected from the amino acid sequence set forth as X1X2X3X4X5X6X7X8X9X10X11X12X13X14X15X16X17, wherein X1 to X4 are G; X5 to X6 and X12 to X16 are absent; X7 to X9 are each independently selected from E or D; X10to X11are each independently selected from E, D or absent; X17is S; and n is 3 or 4; and Z is at least 16 amino acids in length. Such a fusion protein peptide linker may be represented by the amino acid sequence of SEQ ID NO: 88 and the formula (G4(E / D)3-5S)3-4. In a more preferred embodiment, the fusion protein peptide linker comprises, consists essentially of, or consists of the amino acid sequence Z, wherein Z consists of n independent occurrences of an amino acid sequence Y, wherein each Y is independently selected from the amino acid sequence set forth as X1X2X3X4X5X6X7X8X9X10X11X12X13X14X15X16X17, wherein X1 to X4 are G; X5 to X6 and X12 to X16 are absent; X7 to X9 are each independently selected from E or D; X10to X11are each independently selected from E, D or absent; X17is S; and n is 3 or 4; the amino acid sequence Y is the same for each of the n occurrences of Y; and Z is at least 16 amino acids in length. Such a fusion protein peptide linker may be represented by the amino acid sequence of SEQ ID NO: 90 and the formula (G4(E / D)3-5S)3-4. In some embodiments, when the fusion protein peptide linker consists of an amino acid sequence Z, Z consists of n independent occurrence of an amino acid sequence Y as defined herein, followed by the amino acids GGS. In one embodiment, the fusion protein peptide linker comprises, consists essentially of, or consists of (G2DE2S)7G2S (SEQ ID NO: 16). In some embodiments, the fusion protein peptide linker comprises, consists essentially of, or consists of the amino acid sequence Z, wherein Z is any one of SEQ ID NO: 6 to SEQ ID NO: 15, SEQ ID NO: 17 to SEQ ID NO: 26, SEQ ID NO: 48 to SEQ ID NO: 54, SEQ ID NO:69 to SEQ ID NO: 73, SEQ ID NO: 87 to SEQ ID NO: 91, SEQ ID NO: 98 or SEQ ID NO: 101. In other embodiments, the Z amino acid sequence is any one of SEQ ID NO: 48 to SEQ ID NO: 54. In other embodiments, the Z amino acid sequence is any one of SEQ ID NO: 69 to SEQ ID NO: 73. In an embodiment, the Z amino acid sequence is G4E5SG3E5S (SEQ ID NO: 91). In a preferred embodiment, the Z amino acid sequence is any one of SEQ ID NO: 87 to SEQ ID NO: 90. In a more preferred embodiment, the Z amino acid sequence is any one of (G4ES)3 (SEQ ID NO: 6), (G4E2S)3 (SEQ ID NO: 7), (G4E3S)3 (SEQ ID NO: 8), (G4E4S)3 (SEQ ID NO: 9), (G4E5S)3 (SEQ ID NO: 10), (G4ES)4 (SEQ ID NO: 11), (G4E2S)4 (SEQ ID NO: 12), (G4E3S)4 (SEQ ID NO: 13), (G4E4S)4 (SEQ ID NO: 14), (G4E5S)4 (SEQ ID NO: 15), (G4DS)3 (SEQ ID NO: 17), (G4D2S)3 (SEQ ID NO: 18), (G4D3S)3(SEQ ID NO: 19), (G4D4S)3(SEQ ID NO: 20), (G4D5S)3(SEQ ID NO: 21), (G4DS)4(SEQ ID NO: 22), (G4D2S)4(SEQ ID NO: 23), (G4D3S)4(SEQ ID NO: 24), (G4D4S)4(SEQ ID NO: 25), (G4D5S)4(SEQ ID NO: 26), G4E5SG3E5S (SEQ ID NO: 91), G5E4S(G4E4S)3(SEQ ID NO: 98) or (G2DE2S)7 (SEQ ID NO: 101). In an even more preferred embodiment, the Z amino acid sequence is any one of (G4ES)3 (SEQ ID NO: 6), (G4E2S)3 (SEQ ID NO: 7), (G4E3S)3 (SEQ ID NO: 8), (G4ES)4 (SEQ ID NO: 11), (G4E2S)4 (SEQ ID NO: 12), (G4E3S)4 (SEQ ID NO: 13), (G4E4S)4 (SEQ ID NO: 14), (G4E5S)4 (SEQ ID NO: 15), (G4D3S)4 (SEQ ID NO: 24), (G4D4S)4 (SEQ ID NO: 25), (G4D5S)4(SEQ ID NO: 26). In one embodiment, the Z amino acid sequence is (G4E3S)4(SEQ ID NO: 13). In some embodiments, the fusion protein peptide linker consists of an amino acid sequence comprising, consisting essentially of, or consisting of the amino acid sequence Z as defined herein. When the fusion protein peptide linker comprises amino acid sequence Z, it may contain further amino acids in addition to those defined as Z. Preferably, however, the fusion protein peptide linker does not contain any further amino acids in addition to those defined by Z. In some embodiments, the fusion protein peptide linker consists of any one of SEQ ID NO: 6 to SEQ ID NO: 26, SEQ ID NO: 48 to SEQ ID NO: 54, SEQ ID NO:69 to SEQ ID NO: 73, SEQ ID NO: 87 to SEQ ID NO: 91, SEQ ID NO: 98 or SEQ ID NO: 101. In some embodiments, the fusion protein peptide linker consists of any one of SEQ ID NO: 6 to SEQ ID NO: 26, SEQ ID NO: 48 to SEQ ID NO: 54, SEQ ID NO:69 to SEQ ID NO: 73, SEQ ID NO: 87 to SEQ ID NO: 91, SEQ ID NO: 98, or SEQ ID NO: 101 to SEQ ID NO: 108. In a preferred embodiment, the fusion protein peptide linker consists of any one of SEQ ID NO: 87 to SEQ ID NO: 90. In a more preferred embodiment, the fusion protein peptide linker consists of any one of (G4ES)3 (SEQ ID NO: 6), (G4E2S)3 (SEQ ID NO: 7), (G4E3S)3 (SEQ ID NO: 8), (G4E4S)3 (SEQ ID NO: 9), (G4E5S)3 (SEQ ID NO: 10), (G4ES)4(SEQ ID NO: 11), (G4E2S)4(SEQ ID NO: 12), (G4E3S)4(SEQ ID NO: 13), (G4E4S)4(SEQ ID NO: 14), (G4E5S)4(SEQ ID NO: 15), (G4DS)3(SEQ ID NO: 17), (G4D2S)3(SEQ ID NO: 18), (G4D3S)3(SEQ ID NO: 19), (G4D4S)3(SEQ ID NO: 20), (G4D5S)3(SEQ ID NO: 21), (G4DS)4 (SEQ ID NO: 22), (G4D2S)4 (SEQ ID NO: 23), (G4D3S)4 (SEQ ID NO: 24), (G4D4S)4 (SEQ ID NO: 25), (G4D5S)4 (SEQ ID NO: 26), G4E5SG3E5S (SEQ ID NO: 91), G5E4S(G4E4S)3 (SEQ ID NO: 98) or (G2DE2S)7 (SEQ ID NO: 101). In an even more preferred embodiment, the fusion protein peptide linker consists of any one of (G4ES)3 (SEQ ID NO: 6), (G4E2S)3 (SEQ ID NO: 7), (G4E3S)3(SEQ ID NO: 8), (G4ES)4(SEQ ID NO: 11), (G4E2S)4(SEQ ID NO: 12), (G4E3S)4(SEQ ID NO: 13), (G4E4S)4(SEQ ID NO: 14), (G4E5S)4(SEQ ID NO: 15), (G4D3S)4(SEQ ID NO: 24), (G4D4S)4(SEQ ID NO: 25), (G4D5S)4(SEQ ID NO: 26), G4E5SG3E5S (SEQ ID NO: 91), G5E4S(G4E4S)3 (SEQ ID NO: 98) or (G2DE2S)7 (SEQ ID NO: 101). In one embodiment, the fusion protein peptide linker is (G4E3S)4 (SEQ ID NO: 13). In some embodiments, the fusion protein peptide linker has a total of 2, 3, 4 or 5 negatively charged amino acids (D and / or E) for each occurrence of Y. The negatively charged amino acids may all be D, all be E or may be a mixture of D and E. In one embodiment, the fusion protein peptide linker comprises a PEST-like motif, optionally wherein PEST-like motif provides a PEST value of 5 or more to the fusion protein. PEST values for fusion protein or fusion protein peptide linker comprising PEST-like motifs may be calculated with the Emboss epestfind algorithm (Rogers et al., 1986). Reference can be made to Example 3, Table 3, final column for calculated PEST values. According to Examples 3 and 12, sequence analyses with the Emboss epestfind algorithm (Rogers et al., 1986; Rechsteiner et al., 1996) identified an unexpected PEST-like motif in the charged linkers (G4E3S)3 (SEQ ID NO: 8) +6.99, (G4E2S)4 (SEQ ID NO: 12) +5.45, (G4E3S)4 (SEQ ID NO: 13) +8.99 and (G2DE2S)7G2S (SEQ ID NO: 16) +15.51 (Table 3, last two columns) and in the charged linkers A to H of Table 11, with PEST values ranging from +8.63 to +22.64. According to another aspect of the invention, there is provided a fusion protein comprising two polypeptides linked by a fusion protein peptide linker of the invention. In some embodiments, the fusion protein is a binding molecule. The binding molecule may be monovalent (binds to a single binding site) and / or monospecific (binds to a single target molecule). Alternatively, the binding molecule may be bivalent (binds to two binding sites) and / or bispecific (binds to two different target molecules). The binding molecule may be multivalent and / or multispecific. In preferred embodiments, the fusion protein (which is a binding molecule) is an antibody or antigen-binding fragment thereof. The fusion protein peptide linker is preferably used to link two antigen binding regions of an antibody or antigen-binding fragment thereof. For example, the fusion protein peptide linker may be used to link a VH and a VL domain (to form a scFv). As another example, the fusion protein peptide linker may be used to link two single domain antibodies (e.g. two VHHs). The fusion protein is preferably an intrabody that binds to an intracellular target. In some embodiments, the fusion protein comprises or consists of a scFv, a bi-specific T-cell engager (BiTE), a Dual Affinity Retargeting Antibodies (DART), a Designed Ankyrin Repeat Proteins (DARPins), a single-domain antibody, a chimeric antigen receptor (CAR), an antibody or antigen-binding fragment thereof fused to an immunoglobulin Fc domain. In some embodiments, the fusion protein is an antibody or antigen-binding fragment thereof. In a particular embodiment, the fusion protein is an antibody or antigen binding fragment that binds to a pathological protein, a cell surface receptor, a tumour-associated antigen or tumour-specific antigen. In a preferred embodiment, the fusion protein is an intrabody that binds to an intracellular target. In some embodiments, the fusion protein comprises or consists of a scFv, a VHH or a bi-specific T- cell engager (BiTE). In an embodiment, the fusion protein comprises single domain antibodies, wherein the fusion protein peptide linker links two single domain antibodies. The single domain antibodies can be the same or different. They can be of any type, e.g. VHH, VNAR or human single domain antibody. In another embodiment, the fusion protein comprises scFvs, wherein the fusion protein peptide linker links two scFvs. In yet another embodiment, the fusion protein comprises a scFv and a single domain antibody (e.g. a VHH), wherein the fusion protein peptide linker links the scFv and the single domain antibody (e.g. VHH). In a preferred embodiment, the fusion protein comprises two single domain antibodies (e.g. VHHs), wherein the fusion protein peptide linker links the two single domain antibodies (e.g. VHHs). In a more preferred embodiment, the fusion protein consists of a scFv, wherein the fusion protein peptide linker links a Heavy Chain Variable Region (VH) and a Light Chain Variable Region (VL) of a scFv. In the context of a CAR, the linker is typically comprised in the antigen binding domain of the CAR. For example, the antigen binding domain may comprise a scFv and the linker links the VH and VL domains of the scFv. However, alternative ligand binding domains are used in CARs such as VHH domains and the linker may be used to link the VHH domains to one another. As would be appreciated by the skilled person, the specified components of the fusion protein can be identified as the “two polypeptides” linked by the fusion protein peptide linker of the invention. All embodiments described previously relating to fusion proteins are equally applicable to these further embodiments. In some embodiments, the fusion protein peptide linker exhibits at least one of the following features: a) promotes the solubility of the fusion protein; and / or b) inhibits or prevents the toxicity of the fusion protein; and / or c) inhibits or prevents the aggregation of the fusion protein; and / or d) promotes the expression yield of the fusion protein; and / or e) promotes the intracellular degradation of the fusion protein; and / or f) promotes the monomeric expression of the fusion protein; and / or g) promotes the thermostability of the fusion protein; and / or h) improves the purification yield of the fusion protein; and / or i) promotes or maintains the target binding affinity of the fusion protein. In some embodiments, the fusion protein peptide linker exhibits at least one of the following features: a) increases the solubility of the fusion protein; and / or b) decreases or prevents the toxicity of the fusion protein; and / or c) decreases or prevents the aggregation of the fusion protein; and / or d) increases the expression yield of the fusion protein; and / or e) increases the intracellular degradation of the fusion protein; and / or f) increases the monomeric expression of the fusion protein; g) increases the thermostability of the fusion protein; and / or h) increases the purification yield of the fusion protein; and / or i) increases or maintains the target binding affinity of the fusion protein. The increase or decrease is relative to a corresponding fusion protein comprising a fusion protein peptide linker consisting of Gly and Ser amino acids (i.e. with no negatively charged amino acids, the negatively charged amino acids being omitted). In preferred embodiments, the relevant comparator may be the (G4S)3 peptide linker (SEQ ID NO: 3) or the (G4S)4 peptide linker (SEQ ID NO: 4). Reference can be made to Examples 2, 3, 4, 5, 7, 8, 9 and 10 which provide examples of methods to measure solubility, toxicity, aggregation, intracellular degradation, expression, monomeric expression, thermostability, purification yield and target binding affinity of a protein. In some embodiments, the fusion protein peptide linker decreases the pI of the fusion protein. The decrease is relative to the pI of a corresponding fusion protein comprising a fusion protein linker consisting of Gly and Ser amino acids (i.e. with no negatively charged amino acids, the negatively charged amino acids being omitted). In preferred embodiments, the relevant comparator may be the (G4S)3peptide linker (SEQ ID NO: 3) or the (G4S)4peptide linker (SEQ ID NO: 4). In one aspect, the fusion proteins of the invention are fusion proteins comprising two polypeptides linked by a fusion protein peptide linker according to the invention, wherein the fusion protein has a pI equal to or below 6. The fusion protein peptide linker according to the invention has negatively charged amino acids. The inclusion of the fusion protein peptide linker according to the invention into the fusion protein contributes towards the pI of the fusion protein so that the fusion protein pI is equal to or below 6. In other words, if you remove the fusion protein peptide linker of the invention with its negative amino acids then the fusion protein has a pI above 6. As exemplified herein, the fusion protein peptide linkers of the invention decrease the pI of the fusion protein providing improved properties, for example improved thermostability, solubility, stability and / or expression titer. Exemplary fusion proteins include clinical-stage antibodies Pexelizumab, FMC63, Vobarilizumab and Blinatumomab. Each of these antibodies have conventional glycine-serine linkers and they each have a pI above 6 (see Table 10). In one embodiment the fusion protein is Pexelizumab, FMC63, Vobarilizumab or Blinatumomab, comprises a fusion protein peptide linker of the invention and has a pI equal to or below 6. The fusion protein peptide linker of the invention may have 4 or more glutamate residues per repeating Gly-Ser motif. The fusion protein peptide linker of the invention may be (G4E4S)3(SEQ ID NO: 9) or G4E5SG3E5S (SEQ ID NO: 91). In one embodiment, provided is Pexelizumab with a fusion protein peptide linker comprising or consisting of (G4E4S)3 (SEQ ID NO: 9). In one embodiment, provided is FMC63 with a fusion protein peptide linker comprising or consisting of (G4E4S)3 (SEQ ID NO: 9). In one embodiment, provided is Vobarilizumab with a fusion protein peptide linker comprising or consisting of G4E5SG3E5S (SEQ ID NO: 91). In one embodiment, provided is Blinatumomab with a fusion protein peptide linker comprising or consisting of (G4E4S)3(SEQ ID NO: 9). Also provided herein are methods for decreasing the pI of a fusion protein, for promoting or increasing the solubility of a fusion protein, for inhibiting, decreasing or preventing the toxicity of a fusion protein, for inhibiting, decreasing or preventing the aggregation of a fusion protein, for promoting or increasing intracellular degradation of a fusion protein, for promoting or increasing the expression of a fusion protein, for promoting or increasing the thermostability of a fusion protein, for promoting or increasing the monomeric expression of a fusion protein, for promoting, increasing or maintaining the target binding affinity of a fusion protein, or for improving or increasing the purification yield of a fusion protein. These methods can also be specified as uses. Thus, the invention also provides for use of a fusion protein peptide linker of the invention for decreasing the pI of a fusion protein, for promoting or increasing the solubility of a fusion protein, for inhibiting, decreasing or preventing the toxicity of a fusion protein, for inhibiting, decreasing or preventing the aggregation of a fusion protein, for promoting or increasing intracellular degradation of a fusion protein, for promoting or increasing the expression of a fusion protein, for promoting or increasing the thermostability of a fusion protein, for promoting or increasing the monomeric expression of a fusion protein, for promoting, increasing or maintaining the target binding affinity of a fusion protein, or for improving or increasing the purification yield of a fusion protein. In an embodiment, a method for decreasing the pI of a fusion protein comprising two polypeptides linked by a fusion protein peptide linker is provided, the method comprising introducing negatively charged amino acids in the fusion protein peptide linker to obtain a fusion protein with a fusion protein peptide linker of the invention and a fusion protein with a pI equal to or below 6 or reduce the pI further below 6. Similarly, the invention provides for the use of a negatively charged fusion protein peptide linker for decreasing the pI of a fusion protein linked by the linker to a value of 6 or less, or for reducing the pI of a fusion protein further below 6. The negative amino acids may be introduced, for example, as insertions or substitutions of non-negative amino acids in the fusion protein peptide linker. In an embodiment, the method comprises introducing negatively charged amino acids in the fusion protein peptide linker to obtain a fusion protein with a pI equal to or below 5.5. In an embodiment, the method comprises introducing negatively charged amino acids in the fusion protein peptide linker to obtain a fusion protein with a pI from 3 to 6. In a preferred embodiment, the method comprises introducing negatively charged amino acids in the fusion protein peptide linker to obtain a fusion protein with a pI from 3.5 to 5.5. In a more preferred embodiment, the method comprises introducing negatively charged amino acids in the fusion protein peptide linker to obtain a fusion protein with a pI from 4.5 to 5.5. In an embodiment, the negatively charged amino acids are Glu and / or Asp amino acids. In an embodiment, the decrease in the pI of the fusion protein (i) promotes or increases the solubility of the fusion protein and / or (ii) inhibits, decreases or prevents the toxicity of the fusion protein and / or (iii) inhibits, decreases or prevents the aggregation of the fusion protein and / or (iv) promotes or increases the expression of the fusion protein and / or (v) promotes or increases the thermostability of the fusion protein and / or (vi) promotes or increases the monomeric expression of the fusion protein and / or (vii) promotes, increases or maintains the target binding affinity of the fusion protein and / or (viii) promotes or increases the purification yield of the fusion protein. Reference can be made to Example 3 for methods to measure the pI of a protein. In an embodiment, a method for decreasing the pI of a fusion protein having a pI greater than 6 and comprising two polypeptides linked by a fusion protein peptide linker is provided, the method comprising introducing negatively charged amino acids in the fusion protein peptide linker to obtain a fusion protein with a fusion protein peptide linker of the invention and a fusion protein with a pI equal to or below 6. Similarly, the invention provides for the use of a negatively charged fusion protein peptide linker for decreasing the pI of a fusion protein having a pI greater than 6 linked by the linker to a value of 6 or less. The negative amino acids may be introduced, for example, as insertions or substitutions of non-negative amino acids in the fusion protein peptide linker. In an embodiment, the method comprises introducing negatively charged amino acids in the fusion protein peptide linker to obtain a fusion protein with a pI equal to or below 5.5. In an embodiment, the method comprises introducing negatively charged amino acids in the fusion protein peptide linker to obtain a fusion protein with a pI from 3 to 6. In a preferred embodiment, the method comprises introducing negatively charged amino acids in the fusion protein peptide linker to obtain a fusion protein with a pI from 3.5 to 5.5. In a more preferred embodiment, the method comprises introducing negatively charged amino acids in the fusion protein peptide linker to obtain a fusion protein with a pI from 4.5 to 5.5. In an embodiment, the fusion protein having a pI greater than 6 comprises two polypeptides linked by a fusion protein peptide linker consisting of Gly and Ser amino acids. In an embodiment, the negatively charged amino acids are Glu and / or Asp amino acids. In an embodiment, the decrease in the pI of the fusion protein (i) promotes or increases the solubility of the fusion protein and / or (ii) inhibits, decreases or prevents the toxicity of the fusion protein and / or (iii) inhibits, decreases or prevents the aggregation of the fusion protein and / or (iv) promotes or increases the expression of the fusion protein and / or (v) promotes or increases the thermostability of the fusion protein and / or (vi) promotes or increases the monomeric expression of the fusion protein and / or (vii) promotes, increases or maintains the target binding affinity of the fusion protein and / or (viii) promotes or increases the purification yield of the fusion protein. Reference can be made to Example 3 for methods to measure the pI of a protein. In an embodiment, a method for promoting the solubility of a fusion protein is provided, the method comprising providing a fusion protein comprising two polypeptides linked by the fusion protein peptide linker of the invention and expressing the fusion protein in a cell or a cell-free expression system. Similarly, the invention provides for the use of a fusion protein peptide linker of the invention for promoting the solubility of a fusion protein. In an embodiment, the fusion protein is a binding molecule, as described herein. In a preferred embodiment, the fusion protein is an antibody or antigen-binding fragment thereof, as described herein. In a more preferred embodiment, the fusion protein comprises or consists of a scFv, a bi-specific T-cell engager (BiTE), a Dual Affinity Retargeting Antibodies (DART), a Designed Ankyrin Repeat Proteins (DARPins), a single-domain antibody, a chimeric antigen receptor (CAR), an antibody or antigen-binding fragment thereof fused to an immunoglobulin Fc domain. In an embodiment, a method for increasing the solubility of a fusion protein is provided, the method comprising providing a fusion protein comprising two polypeptides linked by the fusion protein peptide linker of the invention and expressing the fusion protein in a cell or a cell-free expression system. Similarly, the invention provides for the use of a fusion protein peptide linker of the invention for increasing the solubility of a fusion protein. The increase in solubility is relative to a corresponding fusion protein comprising a fusion protein peptide linker consisting of Gly and Ser amino acids, as described herein. In an embodiment, the fusion protein is a binding molecule, as described herein. In a preferred embodiment, the fusion protein is an antibody or antigen-binding fragment thereof, as described herein. In a more preferred embodiment, the fusion protein comprises or consists of a scFv, a bi-specific T-cell engager (BiTE), a Dual Affinity Retargeting Antibodies (DART), a Designed Ankyrin Repeat Proteins (DARPins), a single-domain antibody, a chimeric antigen receptor (CAR), an antibody or antigen-binding fragment thereof fused to an immunoglobulin Fc domain. Reference can be made to Examples 2 and 3 for methods to measure solubility. In an embodiment, a method for inhibiting or preventing the toxicity of a fusion protein is provided, the method comprising providing a fusion protein comprising two polypeptides linked by the fusion protein peptide linker of the invention and expressing the fusion protein in a cell or a cell-free expression system. Similarly, the invention provides for the use of a fusion protein peptide linker of the invention for inhibiting or preventing the toxicity of a fusion protein. In an embodiment, the fusion protein is a binding molecule, as described herein. In a preferred embodiment, the fusion protein is an antibody or antigen-binding fragment thereof, as described herein. In a more preferred embodiment, the fusion protein comprises or consists of a scFv, a bi-specific T-cell engager (BiTE), a Dual Affinity Retargeting Antibodies (DART), a Designed Ankyrin Repeat Proteins (DARPins), a single-domain antibody, a chimeric antigen receptor (CAR), an antibody or antigen-binding fragment thereof fused to an immunoglobulin Fc domain. In an embodiment, a method for decreasing or preventing the toxicity of a fusion protein is provided, the method comprising providing a fusion protein comprising two polypeptides linked by the fusion protein peptide linker of the invention and expressing the fusion protein in a cell or a cell- free expression system. Similarly, the invention provides for the use of a fusion protein peptide linker of the invention for decreasing or preventing the toxicity of a fusion protein. The decrease in toxicity is relative to a corresponding fusion protein comprising a fusion protein peptide linker consisting of Gly and Ser amino acids, as described herein. In an embodiment, the fusion protein is a binding molecule, as described herein. In a preferred embodiment, the fusion protein is an antibody or antigen-binding fragment thereof, as described herein. In a more preferred embodiment, the fusion protein comprises or consists of a scFv, a bi-specific T-cell engager (BiTE), a Dual Affinity Retargeting Antibodies (DART), a Designed Ankyrin Repeat Proteins (DARPins), a single-domain antibody, a chimeric antigen receptor (CAR), an antibody or antigen-binding fragment thereof fused to an immunoglobulin Fc domain. Reference can be made to Example 4 for methods to measure toxicity. In an embodiment, a method for inhibiting or preventing the aggregation of a fusion protein is provided, the method comprising providing a fusion protein comprising two polypeptides linked by the fusion protein peptide linker of the invention and expressing the fusion protein in a cell or a cell- free expression system. Similarly, the invention provides for the use of a fusion protein peptide linker of the invention for inhibiting or preventing the aggregation of a fusion protein. In an embodiment, the fusion protein is a binding molecule, as described herein. In a preferred embodiment, the fusion protein is an antibody or antigen-binding fragment thereof, as described herein. In a more preferred embodiment, the fusion protein comprises or consists of a scFv, a bi- specific T-cell engager (BiTE), a Dual Affinity Retargeting Antibodies (DART), a Designed Ankyrin Repeat Proteins (DARPins), a single-domain antibody, a chimeric antigen receptor (CAR), an antibody or antigen-binding fragment thereof fused to an immunoglobulin Fc domain. In an embodiment, a method for decreasing or preventing the aggregation of a fusion protein is provided, the method comprising providing a fusion protein comprising two polypeptides linked by the fusion protein peptide linker of the invention and expressing the fusion protein in a cell or a cell- free expression system. Similarly, the invention provides for the use of a fusion protein peptide linker of the invention for decreasing or preventing the aggregation of a fusion protein. The decrease in aggregation is relative to a corresponding fusion protein comprising a fusion protein peptide linker consisting of Gly and Ser amino acids, as described herein. In an embodiment, the fusion protein is a binding molecule, as described herein. In a preferred embodiment, the fusion protein is an antibody or antigen-binding fragment thereof, as described herein. In a more preferred embodiment, the fusion protein comprises or consists of a scFv, a bi-specific T-cell engager (BiTE), a Dual Affinity Retargeting Antibodies (DART), a Designed Ankyrin Repeat Proteins (DARPins), a single-domain antibody, a chimeric antigen receptor (CAR), an antibody or antigen-binding fragment thereof fused to an immunoglobulin Fc domain. Reference can be made to Examples 5, 7 and 9 for methods to measure aggregation. In an embodiment, a method for promoting the intracellular degradation of a fusion protein is provided, the method comprising providing a fusion protein comprising two polypeptides linked by the fusion protein peptide linker of the invention and expressing the fusion protein in a cell or a cell- free expression system. Similarly, the invention provides for the use of a fusion protein peptide linker of the invention for promoting the intracellular degradation of a fusion protein. In an embodiment, the fusion protein is a binding molecule, as described herein. In a preferred embodiment, the fusion protein is an antibody or antigen-binding fragment thereof, as described herein. In a more preferred embodiment, the fusion protein comprises or consists of a scFv, a bi- specific T-cell engager (BiTE), a Dual Affinity Retargeting Antibodies (DART), a Designed Ankyrin Repeat Proteins (DARPins), a single-domain antibody, a chimeric antigen receptor (CAR), an antibody or antigen-binding fragment thereof fused to an immunoglobulin Fc domain. In an embodiment, a method for increasing the intracellular degradation of a fusion protein is provided, the method comprising providing a fusion protein comprising two polypeptides linked by the fusion protein peptide linker of the invention and expressing the fusion protein in a cell or a cell- free expression system. Similarly, the invention provides for the use of a fusion protein peptide linker of the invention for increasing the intracellular degradation of a fusion protein. The increase in intracellular degradation is relative to a corresponding fusion protein comprising a fusion protein peptide linker consisting of Gly and Ser amino acids, as described herein. In an embodiment, the fusion protein is a binding molecule, as described herein. In a preferred embodiment, the fusion protein is an antibody or antigen-binding fragment thereof, as described herein. In a more preferred embodiment, the fusion protein comprises or consists of a scFv, a bi-specific T-cell engager (BiTE), a Dual Affinity Retargeting Antibodies (DART), a Designed Ankyrin Repeat Proteins (DARPins), a single-domain antibody, a chimeric antigen receptor (CAR), an antibody or antigen-binding fragment thereof fused to an immunoglobulin Fc domain. Reference can be made to Example 10 for methods to measure intracellular degradation. In an embodiment, a method for promoting the expression of a fusion protein is provided, the method comprising providing a fusion protein comprising two polypeptides linked by the fusion protein peptide linker of the invention and expressing the fusion protein in a cell or a cell-free expression system. Similarly, the invention provides for the use of a fusion protein peptide linker of the invention for promoting the expression of a fusion protein. In an embodiment, the fusion protein is a binding molecule, as described herein. In a preferred embodiment, the fusion protein is an antibody or antigen-binding fragment thereof, as described herein. In a more preferred embodiment, the fusion protein comprises or consists of a scFv, a bi-specific T-cell engager (BiTE), a Dual Affinity Retargeting Antibodies (DART), a Designed Ankyrin Repeat Proteins (DARPins), a single- domain antibody, a chimeric antigen receptor (CAR), an antibody or antigen-binding fragment thereof fused to an immunoglobulin Fc domain. In an embodiment, a method for increasing the expression of a fusion protein is provided, the method comprising providing a fusion protein comprising two polypeptides linked by the fusion protein peptide linker of the invention and expressing the fusion protein in a cell or a cell-free expression system. Similarly, the invention provides for the use of a fusion protein peptide linker of the invention for increasing the expression of a fusion protein. The increase in expression is relative to a corresponding fusion protein comprising a fusion protein peptide linker consisting of Gly and Ser amino acids, as described herein. In an embodiment, the fusion protein is a binding molecule, as described herein. In a preferred embodiment, the fusion protein is an antibody or antigen-binding fragment thereof, as described herein. In a more preferred embodiment, the fusion protein comprises or consists of a scFv, a bi-specific T-cell engager (BiTE), a Dual Affinity Retargeting Antibodies (DART), a Designed Ankyrin Repeat Proteins (DARPins), a single-domain antibody, a chimeric antigen receptor (CAR), an antibody or antigen-binding fragment thereof fused to an immunoglobulin Fc domain. Reference can be made to Examples 7 and 9 for methods to measure expression. In an embodiment, a method for promoting the monomeric expression of a fusion protein is provided, the method comprising providing a fusion protein comprising two polypeptides linked by the fusion protein peptide linker of the invention and expressing the fusion protein in a cell or a cell- free expression system. Similarly, the invention provides for the use of a fusion protein peptide linker of the invention for promoting the monomeric expression of a fusion protein. In an embodiment, the fusion protein is a binding molecule, as described herein. In a preferred embodiment, the fusion protein is an antibody or antigen-binding fragment thereof, as described herein. In a more preferred embodiment, the fusion protein consists of a scFv, wherein the fusion protein peptide linker links the Heavy Chain Variable Region (VH) and Light Chain Variable Region (VL) of the scFv. In an embodiment, a method for increasing the monomeric expression of a fusion protein is provided, the method comprising providing a fusion protein comprising two polypeptides linked by the fusion protein peptide linker of the invention and expressing the fusion protein in a cell or a cell- free expression system. Similarly, the invention provides for the use of a fusion protein peptide linker of the invention for increasing the monomeric expression of a fusion protein. The increase in monomeric expression is relative to a corresponding fusion protein comprising a fusion protein peptide linker consisting of Gly and Ser amino acids, as described herein. In an embodiment, the fusion protein is a binding molecule, as described herein. In a preferred embodiment, the fusion protein is an antibody or antigen-binding fragment thereof, as described herein. In a more preferred embodiment, the fusion protein consists of a scFv, wherein the fusion protein peptide linker links the Heavy Chain Variable Region (VH) and Light Chain Variable Region (VL) of the scFv. Reference can be made to Examples 7 and 9 for methods to measure monomeric expression. In an embodiment, a method for promoting the thermostability of a fusion protein is provided, the method comprising providing a fusion protein comprising two polypeptides linked by the fusion protein peptide linker of the invention and expressing the fusion protein in a cell or a cell-free expression system. Similarly, the invention provides for the use of a fusion protein peptide linker of the invention for promoting the thermostability of a fusion protein. In an embodiment, the fusion protein is a binding molecule, as described herein. In a preferred embodiment, the fusion protein is an antibody or antigen-binding fragment thereof, as described herein. In a more preferred embodiment, the fusion protein comprises or consists of a scFv, a bi-specific T-cell engager (BiTE), a Dual Affinity Retargeting Antibodies (DART), a Designed Ankyrin Repeat Proteins (DARPins), a single-domain antibody, a chimeric antigen receptor (CAR), an antibody or antigen-binding fragment thereof fused to an immunoglobulin Fc domain. In an embodiment, a method for increasing the thermostability of a fusion protein is provided, the method comprising providing a fusion protein comprising two polypeptides linked by the fusion protein peptide linker of the invention and expressing the fusion protein in a cell or a cell-free expression system. Similarly, the invention provides for the use of a fusion protein peptide linker of the invention for increasing the thermostability of a fusion protein. The increase in thermostability is relative to a corresponding fusion protein comprising a fusion protein peptide linker consisting of Gly and Ser amino acids, as described herein. In an embodiment, the fusion protein is a binding molecule, as described herein. In a preferred embodiment, the fusion protein is an antibody or antigen-binding fragment thereof, as described herein. In a more preferred embodiment, the fusion protein comprises or consists of a scFv, a bi-specific T-cell engager (BiTE), a Dual Affinity Retargeting Antibodies (DART), a Designed Ankyrin Repeat Proteins (DARPins), a single-domain antibody, a chimeric antigen receptor (CAR), an antibody or antigen-binding fragment thereof fused to an immunoglobulin Fc domain. Reference can be made to Examples 8 and 9 for methods to measure thermostability. In an embodiment, a method for promoting the target binding affinity of a fusion protein is provided, the method comprising providing a fusion protein comprising two polypeptides linked by the fusion protein peptide linker of the invention and expressing the fusion protein in a cell or a cell- free expression system. Similarly, the invention provides for the use of a fusion protein peptide linker of the invention for promoting the target binding affinity of a fusion protein. In an embodiment, the fusion protein is a binding molecule, as described herein. In a preferred embodiment, the fusion protein is an antibody or antigen-binding fragment thereof, as described herein. In a more preferred embodiment, the fusion protein comprises or consists of a scFv, a bi- specific T-cell engager (BiTE), a Dual Affinity Retargeting Antibodies (DART), a Designed Ankyrin Repeat Proteins (DARPins), a single-domain antibody, a chimeric antigen receptor (CAR), an antibody or antigen-binding fragment thereof fused to an immunoglobulin Fc domain. In an even more preferred embodiment, the fusion protein consists of a scFv, wherein the fusion protein peptide linker links the Heavy Chain Variable Region (VH) and Light Chain Variable Region (VL) of the scFv. In an embodiment, a method for increasing the target binding affinity of a fusion protein is provided, the method comprising providing a fusion protein comprising two polypeptides linked by the fusion protein peptide linker of the invention and expressing the fusion protein in a cell or a cell-free expression system. Similarly, the invention provides for the use of a fusion protein peptide linker of the invention for increasing the target binding affinity of a fusion protein. The increase in target binding affinity is relative to a corresponding fusion protein comprising a fusion protein peptide linker consisting of Gly and Ser amino acids, as described herein. In an embodiment, the fusion protein is a binding molecule, as described herein. In a preferred embodiment, the fusion protein is an antibody or antigen-binding fragment thereof, as described herein. In a more preferred embodiment, the fusion protein comprises or consists of a scFv, a bi-specific T-cell engager (BiTE), a Dual Affinity Retargeting Antibodies (DART), a Designed Ankyrin Repeat Proteins (DARPins), a single-domain antibody, a chimeric antigen receptor (CAR), an antibody or antigen-binding fragment thereof fused to an immunoglobulin Fc domain. In an even more preferred embodiment, the fusion protein consists of a scFv, wherein the fusion protein peptide linker links the Heavy Chain Variable Region (VH) and Light Chain Variable Region (VL) of the scFv. Reference can be made to Example 8 for methods to measure target binding affinity. In an embodiment, a method for improving the purification yield of a fusion protein is provided, the method comprising providing a fusion protein comprising two polypeptides linked by the fusion protein peptide linker of the invention, expressing the fusion protein in a cell or a cell-free expression system and isolating the fusion protein by performing a separation technique based on the fusion protein charge. Similarly, the invention provides for the use of a fusion protein peptide linker of the invention for improving the purification yield of a fusion protein. In an embodiment, the fusion protein is a binding molecule, as described herein. In a preferred embodiment, the fusion protein is an antibody or antigen-binding fragment thereof, as described herein. In a more preferred embodiment, the fusion protein comprises or consists of a scFv, a bi-specific T-cell engager (BiTE), a Dual Affinity Retargeting Antibodies (DART), a Designed Ankyrin Repeat Proteins (DARPins), a single-domain antibody, a chimeric antigen receptor (CAR), an antibody or antigen-binding fragment thereof fused to an immunoglobulin Fc domain. In an embodiment, a method for increasing the purification yield of a fusion protein is provided, the method comprising providing a fusion protein comprising two polypeptides linked by the fusion protein peptide linker of the invention, expressing the fusion protein in a cell or a cell-free expression system and isolating the fusion protein by performing a separation technique based on the fusion protein charge. Similarly, the invention provides for the use of a fusion protein peptide linker of the invention for increasing the purification yield of a fusion protein. The increase in the purification yield is relative to a corresponding fusion protein comprising a fusion protein peptide linker consisting of Gly and Ser amino acids, as described herein. In an embodiment, the fusion protein is a binding molecule, as described herein. In a preferred embodiment, the fusion protein is an antibody or antigen-binding fragment thereof, as described herein. In a more preferred embodiment, the fusion protein comprises or consists of a scFv, a bi-specific T-cell engager (BiTE), a Dual Affinity Retargeting Antibodies (DART), a Designed Ankyrin Repeat Proteins (DARPins), a single-domain antibody, a chimeric antigen receptor (CAR), an antibody or antigen-binding fragment thereof fused to an immunoglobulin Fc domain. Reference can be made to Examples 1 and 7 for purification methods and methods to measure the purification yield. The fusion protein peptide linkers of the invention thus provide several advantages applicable to fusion protein manufacturing. Noteworthy, promoting or increasing protein solubility, inhibiting, decreasing or preventing protein aggregation, promoting or increasing protein expression (i.e. protein titer), improving or increasing protein purification (i.e. purified protein yield), and / or promoting or increasing protein thermostability may allow higher protein concentrations to be achieved and lower buffer volumes required, increase protein stability and facilitate buffer formulations (i.e. reduce buffer formulation complexity). In the context of drug product formulation, it enables longer drug product shelf life, lower drug product volume and / or higher drug product concentration allowing lower doses to patients. In the context of gene therapy, or cell therapy comprising a fusion protein (e.g. CAR-T therapy), the reduction or absence of protein aggregation or degradation products (due to the fusion protein peptide linker of the invention) could contribute to lower immunogenic prevalence and anti-drug or inhibitory antibodies to the protein transgene product. Moreover, the unexpectedly high protein titers obtained with the method described herein are especially relevant to manufacturing processes where it can considerably reduce the volume of cell culture, cell media and bioreactor size, and therefore the associated manufacturing costs. Several other advantages are to be considered with higher protein expression titers. For example, expressed proteins can be monitored either in early bioreactor in process control time points or manufacturing stages and tested for functionality, prior to cell harvesting and purification, for protein quality and comparability. In addition, the higher expression titers improve the feasibility of downstream processing such as the reduction of cell harvest volume, the filtration of the harvest, purification, filtration and concentration of the purified fusion protein substance. In the context of gene therapy, the fusion protein peptide linker can increase the fusion protein expression titer and thereby lower the vector doses. Importantly, the enhanced stability and thermostability increases feasibility success rate of drug product fill and finish procedures by lowering protein loss and particulate occurrence rate in the finished drug product, but also potential immunogenicity responses to the drug due to aggregates or particles. Noteworthy, the fusion protein peptide linkers of the invention provide unique possibilities to improve protein purification by ion exchange chromatography (IEX) from the harvest (or other purification techniques), by increasing the salt concentration / molarity to release the retained protein and allowing earlier release of poorly retained protein contaminants. Overall, host cell protein contamination is reduced in the purification process. The fusion protein peptide linkers of the invention enable other possibilities in separation steps such as hydrophobic, reverse phase and other resin types. The forecasted main advantage of using ion exchanger resin with the fusion protein peptide linkers of the invention is the reduction of purification steps and thereby, the associated important increase of purified fusion protein yield, lowering the manufacturing cost. Importantly, promoting or increasing monomeric expression (resulting in an increased monomeric content of protein) improves manufacturing batch-to-batch reproducibility and more accurate in process control feasibility in the upstream and downstream protein production processes. In the context of gene therapy or cell therapy comprising a fusion protein (e.g. CAR-T therapy), predominantly monomeric fusion protein content could prevent potential short- or long-term immunogenic issues. The invention is further directed, inter alia, to (i) a nucleic acid encoding a fusion protein of the present invention; (ii) a expression vector encoding a nucleic acid of the present invention; (iii) a host cell comprising a nucleic acid of the present invention; (iv) a cell-free expression system comprising a nucleic acid or expression vector of the present invention; (v) a method for producing a fusion protein of the present invention comprising the step of culturing the host cell of part (iii) or the cell-free expression system of part (iv) under conditions suitable for producing the fusion protein and isolating the fusion protein. Suitable host cells for cloning or expression of fusion protein-encoding vectors include prokaryotic or eukaryotic cells described herein. For example, fusion proteins that are binding molecules such as antibody or antigen-binding fragment thereof may be produced in bacteria, in particular when glycosylation and Fc effector function are not needed. For expression of antibody fragments and polypeptides in bacteria, see, e.g., U.S. Patent Nos. 5,648,237, 5,789,199, and 5,840,523. (See also Charlton, Methods in Molecular Biology, Val.248 (B.K.C. Lo, ed., Humana Press, Totowa, NJ, 2003), pp. 245-254, describing expression of antibody fragments in E. coli.) After expression, the fusion proteins may be isolated from the bacterial cell paste in a soluble fraction and can be further purified. In addition to prokaryotes, eukaryotic microbes such as filamentous fungi or yeast are suitable cloning or expression hosts for fusion protein-encoding vectors, including fungi and yeast strains whose glycosylation pathways have been "humanized". See Gerngross (2014) and Li et al. (2006). Suitable host cells for the expression of fusion proteins are also derived from multicellular organisms (invertebrates and vertebrates). Examples of invertebrate cells include plant and insect cells. Numerous baculoviral strains have been identified which may be used in conjunction with insect cells, particularly for transfection of Spodoptera frugiperda cells. Plant cell cultures can also be utilized as hosts. See, e.g., US Patent Nos. 5,959,177, 6,040,498, 6,420,548, 7,125,978, and 6,417,429 (describing PLANTIBODIES™ technology for producing antibodies in transgenic plants). Vertebrate cells may also be used as hosts. For example, mammalian cell lines that are adapted to grow in suspension may be useful. Other examples of useful mammalian host cell lines are macaque kidney CVl line transformed by SV40 (COS-7); human embryonic kidney line (293 or 293 cells as described, e.g., in Graham et al. (1977)); baby hamster kidney cells (BHK); mouse Sertoli cells (TM4 cells as described, e.g., in Mather, (1980)); macaque kidney cells (CV l); African green macaque kidney cells (VERO-76); human cervical carcinoma cells (HeLa); canine kidney cells (MDCK); buffalo rat liver cells (BRL 3A); human lung cells (WI38); human liver cells (Hep G2); mouse mammary tumour (MMT 060562); TRI cells, as described, e.g., in Mather et al., (1982); MRC 5 cells; and FS4 cells. Other useful mammalian host cell lines include Chinese hamster ovary (CHO) cells, including DHFR CHO cells (Urlaub et al., (1980)); and myeloma cell lines such as YO, NSO and Sp2 / 0. For a review of certain mammalian host cell lines suitable for production of fusion proteins that are binding molecules, in particular antibodies, see, e.g., Yazaki and Wu, Methods in Molecular Biology, Val. 248 (B.K.C. Lo, ed., Humana Press, Totowa, NJ), pp. 255-268 (2003). The expression vectors of the invention are recombinant vectors comprising a nucleic acid of the invention (that encodes a fusion protein of the invention). The expression vectors may direct expression of a fusion protein that is retained intracellularly. Thus, for example, the fusion proteins may be expressed such that they do not contain a secretion signal peptide. The expression vectors of the invention may encode signal peptides and / or retention sequences to enable targeting to specific subcellular compartments (see Marschall et al., 2015 for examples of signal peptides suitable for use with an intrabody). Expression vectors encoding fusion proteins to be expressed and retained intracellularly, particularly intrabodies, are known in the art. In some embodiments, the expression vector described herein is a wild-type or engineered viral vector. In a further embodiment, the expression vector is an adeno-associated virus (AAV), an adenovirus or a lentivirus. In yet a further embodiments, the expression vector is an AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAVrh.10, AAV-BR1, AAV-S, AAV-F, AAV-PHP.eB, AAV9-PHP-V, AAV.CAP-B10, AAV.CAP-B22, AAV.CAP-Mac, AAV VCAP-103, AAV bCap 1, AAV VCAP-100, AAV1RX, AAV1R6 or AAV1R7. According to a further aspect of the invention, there is provided a pharmaceutical composition comprising a nucleic acid or expression vector encoding a fusion protein of the invention and a pharmaceutically acceptable carrier and / or excipient. Similarly, according to a further aspect of the invention, there is provided a pharmaceutical composition comprising a fusion protein of the invention and a pharmaceutically acceptable carrier and / or excipient. Pharmaceutically acceptable carriers and excipients can be selected with regard to the intended route of administration and standard pharmaceutical practice. They are well known in the pharmaceutical art and are described, for example, in Remington's Pharmaceutical Sciences, 15th or 18th Ed. (Alfonso R. Gennaro, ed.; Mack Publishing Company, Easton, PA, 1990); Remington: the Science and Practice of Pharmacy 19th Ed. (Lippincott, Williams & Wilkins, 1995); Handbook of Pharmaceutical Excipients, 3rd Ed. (Arthur H. Kibbe, ed.; Amer. Pharmaceutical Assoc, 1999); Pharmaceutical Codex: Principles and Practice of Pharmaceutics 12th Ed. (Walter Lund ed.; Pharmaceutical Press, London, 1994); The United States Pharmacopeia: The National Formulary (United States Pharmacopeial Convention); Fiedler’s “Lexikon der Hilfstoffe” 5th Ed., Edition Cantor Verlag Aulendorf 2002; “The Handbook of Pharmaceutical Excipients”, 4th Ed., American Pharmaceuticals Association, 2003; and Goodman and Gilman's: the Pharmacological Basis of Therapeutics (Louis S. Goodman and Lee E. Limbird, eds.; McGraw Hill, 1992), the disclosures of which are hereby incorporated by reference. In another aspect, the invention provides a fusion protein, a pharmaceutical composition or an expression vector (as described herein) for use in therapy. Thus, the fusion protein, pharmaceutical composition or expression vector may be for use in a method of treatment of a condition, disease or disorder. In a similar aspect, the invention provides a method of treating a condition, disease or disorder in a subject in need thereof, the method comprising administering a fusion protein, pharmaceutical composition or expression vector (as described herein) to the subject. In an embodiment, the fusion protein is a binding molecule and binds a pathological protein, a cell surface receptor, a tumour-associated antigen or tumour-specific antigen. In a preferred embodiment, the fusion protein is an antibody and may be an intrabody. In an embodiment, the binding molecule binds TAR DNA-Binding Protein 43 (TDP-43), amyloid- beta, alpha-synuclein, Tau, apoptosis-associated speck-like protein containing a CARD (ASC) or huntingtin protein (HTT). In a further embodiment, the binding molecule binds TDP-43, and the condition, disease or disorder is associated with pathological TDP-43 or a TDP-43 proteinopathy. In yet a further embodiment, the condition, disease or disorder associated with pathological TDP-43 or TDP-43 proteinopathy is Frontotemporal dementia (FTD), amyotrophic lateral sclerosis (ALS), Alzheimer's disease (AD), Parkinson's disease (PD), Chronic Traumatic Encephalopathy (CTE), limbic-predominant age-related TDP-43 encephalopathy (LATE), or multiple sclerosis. The linkers of the invention provide a combination of advantageous properties that is particularly useful in the context of intrabodies that bind, preferably specifically bind, to an intracellular target. In one embodiment, the invention therefore provides a fusion protein comprising a fusion protein peptide linker of the invention, wherein the fusion protein is an intrabody. In one embodiment, the intrabody is a scFv and the fusion protein peptide linker links the VH and VL domains. In one embodiment, the fusion protein peptide linker: a) promotes or increases the solubility of the intrabody; b) inhibits, decreases or prevents the toxicity of the intrabody; c) inhibits, decreases or prevents the aggregation of the intrabody; d) promotes or increases the expression yield of the intrabody; e) promotes or increases the intracellular degradation of the intrabody; f) promotes or increases the monomeric expression of the intrabody; g) promotes or increases the thermostability of the intrabody; i) improves or increases the purification yield of the intrabody; and / or h) promotes, increases or maintains the target binding affinity of the intrabody; each relative to a corresponding intrabody comprising a peptide linker consisting of Gly and Ser amino acids, as described herein, preferably a (G4S)3 peptide linker (SEQ ID NO: 3). In one aspect, there is provided a pharmaceutical composition comprising a nucleic acid or expression vector encoding a fusion protein of the invention, and a pharmaceutically acceptable carrier and / or excipient. In one embodiment, the fusion protein is an intrabody. In one aspect, there is provided a fusion protein of the invention or a pharmaceutical composition of the invention for use in therapy, preferably for use in the treatment of a neurodegenerative disorder. In one embodiment, the fusion protein is an intrabody. In one aspect, the invention provides the use of a fusion protein peptide linker of the invention in the preparation of a fusion protein, preferably wherein the fusion protein is an intrabody. In one embodiment of the use of a fusion protein peptide linker of the invention in the preparation of a fusion protein, preferably wherein the fusion protein is an intrabody, the fusion protein or intrabody is a scFv and the fusion protein peptide linker links the VH and VL domains. In one embodiment of the use of a fusion protein peptide linker of the invention in the preparation of a fusion protein, preferably wherein the fusion protein is an intrabody, the fusion protein peptide linker: a) promotes or increases the solubility of the fusion protein, preferably intrabody; b) inhibits, decreases or prevents the toxicity of the fusion protein, preferably intrabody; c) inhibits, decreases or prevents the aggregation of the fusion protein, preferably intrabody; d) promotes or increases the expression yield of the fusion protein, preferably intrabody; e) promotes or increases the intracellular degradation of the fusion protein, preferably intrabody; f) promotes or increases the monomeric expression of the fusion protein, preferably intrabody; g) promotes or increases the thermostability of the fusion protein, preferably intrabody; i) improves or increases the purification yield of the fusion protein, preferably intrabody; and / or h) promotes, increases or maintains the target binding affinity of the fusion protein, preferably intrabody; each relative to a corresponding fusion protein, preferably intrabody, comprising a peptide linker consisting of Gly and Ser amino acids, as described herein, preferably a (G4S)3peptide linker (SEQ ID NO: 3). Numbered embodiments The invention will now be described by way of numbered embodiments. 1. A fusion protein peptide linker comprising, consisting essentially of, or consisting of the amino acid sequence Z, wherein Z consists of n independent occurrence of an amino acid sequence Y, wherein each Y is independently selected from the amino acid sequence set forth as X1X2X3X4X5X6X7X8X9X10X11X12X13X14X15X16X17, wherein X1 to X17 are each independently selected from E, D, G, S or absent; at least one from X1 to X17 is G; at least one from X1 to X17 is E or D; at least one from X1 to X17 is S; n is 1 to 10; and Z is at least 16 amino acids in length. 2. The fusion protein peptide linker of embodiment 1, wherein at least 30% of Z amino acids are G; at least 15% of Z amino acids are D and / or E; and from 10% to 20% of Z amino acids are S. 3. The fusion protein peptide linker of embodiment 1 or 2, wherein from 30 to 70% of Z amino acids are G; from 15 to 55% of Z amino acids are D and / or E; and from 10% to 20% of Z amino acids are S. 4. The fusion protein peptide linker of any one of the previous embodiments, wherein from 30 to 50% of Z amino acids are G; from 35 to 55% of Z amino acids are D and / or E; and from 10% to 15% of Z amino acids are S. 5. The fusion protein peptide linker of any one of the previous embodiments, wherein Z is no more than 50 amino acids in length. 6. The fusion protein peptide linker of any one of the previous embodiments, wherein Z is from 18 to 45 amino acids in length. 7. The fusion protein peptide linker of any one of the previous embodiments, wherein n is at least 2. 8. The fusion protein peptide linker of any one of the previous embodiments, wherein n is at least 3, at least 4, preferably n is 3 or 4. 9. The fusion protein peptide linker of embodiment 7, wherein the amino acid sequence Y is the same for each of the n occurrences of Y. 10. The fusion protein peptide linker of any one of the previous embodiments, wherein at least 35% of Z amino acids are D and / or E. 11. The fusion protein peptide linker of any one of the previous embodiments, wherein at least 40% of Z amino acids are D and / or E. 12. The fusion protein peptide linker of any one of the previous embodiments, wherein at least 50% of Z amino acids are D and / or E. 13. The fusion protein peptide linker of any one of the previous embodiments, wherein each Y is independently selected from the amino acid sequence set forth as X1X2X3X4X5X6X7X8X9X10X11X12X13X14X15X16X17, wherein X1is G; X2 to X6 are each independently selected from G or absent; X7 is E or D; X8 to X16 are each independently selected from E, D or absent; and X17 is S. 14. The fusion protein peptide linker of embodiment 13, wherein X1and X2are G. 15. The fusion protein peptide linker of embodiment 13 or 14, wherein X1to X3are G. 16. The fusion protein peptide linker of any one of embodiments 13 to 15, wherein X1to X4are G. 17. The fusion protein peptide linker of any one of embodiments 13 to 16, wherein X5 and X6 are absent. 18. The fusion protein peptide linker of any one of embodiments 13 to 17, wherein X14 to X16 are absent. 19. The fusion protein peptide linker of any one of embodiments 13 to 18, wherein X12to X16are absent. 20. The fusion protein peptide linker of any one of embodiments 13 to 19, wherein X7 and X8 are each independently selected from E or D. 21. The fusion protein peptide linker of any one of embodiments 13 to 20, wherein X7to X9are each independently selected from E or D. 22. The fusion protein peptide linker of any one of embodiments 13 to 21, wherein X1 and X2 are G; X7 and X8 are each independently selected from E or D; and X15 and X16 are absent. 23. The fusion protein peptide linker of any one of embodiments 13 to 22, wherein X1to X3are G; X7and X8are each independently selected from E or D; and X6 and X14 to X16 are absent. 24. The fusion protein peptide linker of any one of embodiments 13 to 23, wherein X1 to X3 are G; X7 to X9 are each independently selected from E or D; and X6and X14to X16are absent. 25. The fusion protein peptide linker of any one of embodiments 13 to 24, wherein X1to X3are G; X7 to X9 are each independently selected from E or D; and X6 and X12 to X16 are absent. 26. The fusion protein peptide linker of any one of embodiments 13 to 19, wherein X1 to X4 are G; X7is E or D; X8to X11are each independently selected from E, D or absent; and X5to X6and X12to X16are absent. 27. The fusion protein peptide linker of any one of embodiments 13 to 26, wherein X1 to X4 are G; X7 to X9 are each independently selected from E or D; X10 to X11 are each independently selected from E, D or absent; and X5to X6and X12to X16are absent. 28. The fusion protein peptide linker of any one of embodiments 13 to 19 or 26, wherein X1to X4are G; X7 is E or D; X8to X11are each independently selected from E, D or absent; X5to X6and X12to X16are absent; n is 3 or 4; and the amino acid sequence Y is the same for each of the n occurrences of Y. The fusion protein peptide linker of any one of embodiments 13 to 28, wherein X1 to X4 are G; X7 to X9 are each independently selected from E or D; X10to X11are each independently selected from E, D or absent; X5to X6and X12to X16are absent; n is 3 or 4; and the amino acid sequence Y is the same for each of the n occurrences of Y. The fusion protein peptide linker of any one of embodiments 13 to 19 or 26, wherein each Y is independently selected from the amino acid sequence set forth as (G4(E / D)1-5S) and n is 3 or 4 (SEQ ID NO: 87 to SEQ ID NO: 90). The fusion protein peptide linker of any one of embodiments 13 to 27 or 30, wherein each Y is independently selected from the amino acid sequence set forth as (G4(E / D)3-5S) and n is 3 or 4 (SEQ ID NO: 88 and SEQ ID NO: 90). The fusion protein peptide linker of any one of embodiments 13 to 19, 26, 28 or 30 wherein each Y is independently selected from the amino acid sequence set forth as (G4(E / D)1-5S), n is 3 or 4 and the amino acid sequence Y is the same for each of the n occurrences of Y (SEQ ID NO: 89). The fusion protein peptide linker of any one of embodiments 13 to 32, wherein each Y is independently selected from the amino acid sequence set forth as (G4(E / D)3-5S), n is 3 or 4 and the amino acid sequence Y is the same for each of the n occurrences of Y (SEQ ID NO: 90). The fusion protein peptide linker of any one of embodiments 13 to 18, wherein Z amino acid sequence is any one of SEQ ID NO: 48 to SEQ ID NO: 54. The fusion protein peptide linker of any one of embodiments 13 to 19, or 26, wherein Z amino acid sequence is any one of SEQ ID NO: 87 to SEQ ID NO: 90. The fusion protein peptide linker of any one of embodiments 13 to 19, 26, 28 or 30, wherein Z amino acid sequence is any one of SEQ ID NO: 69 to SEQ ID NO: 73. 37. The fusion protein peptide linker of any one of embodiments 13 to 19, 26, 28 or 30, wherein Z amino acid sequence is any one of (G4ES)3(SEQ ID NO: 6), (G4E2S)3(SEQ ID NO: 7), (G4E3S)3(SEQ ID NO: 8), (G4E4S)3(SEQ ID NO: 9), (G4E5S)3(SEQ ID NO: 10), (G4ES)4(SEQ ID NO: 11), (G4E2S)4(SEQ ID NO: 12), (G4E3S)4(SEQ ID NO: 13), (G4E4S)4(SEQ ID NO: 14), (G4E5S)4 (SEQ ID NO: 15), (G4DS)3 (SEQ ID NO: 17), (G4D2S)3 (SEQ ID NO: 18), (G4D3S)3 (SEQ ID NO: 19), (G4D4S)3 (SEQ ID NO: 20), (G4D5S)3 (SEQ ID NO: 21), (G4DS)4 (SEQ ID NO: 22), (G4D2S)4 (SEQ ID NO: 23), (G4D3S)4 (SEQ ID NO: 24), (G4D4S)4 (SEQ ID NO: 25), (G4D5S)4 (SEQ ID NO: 26). 38. The fusion protein peptide linker of any one of embodiments 13 to 19, 26, 28, 30, 32 or 37, wherein Z amino acid sequence is any one of (G4ES)3(SEQ ID NO: 6), (G4E2S)3(SEQ ID NO: 7), (G4E3S)3(SEQ ID NO: 8), (G4ES)4(SEQ ID NO: 11), (G4E2S)4(SEQ ID NO: 12), (G4E3S)4 (SEQ ID NO: 13), (G4E4S)4 (SEQ ID NO: 14), (G4E5S)4 (SEQ ID NO: 15), (G4D3S)4 (SEQ ID NO: 24), (G4D4S)4 (SEQ ID NO: 25), (G4D5S)4 (SEQ ID NO: 26), preferably (G4E3S)4 (SEQ ID NO: 13). 39. A fusion protein comprising two polypeptides linked by a fusion protein peptide linker according to any one of the previous embodiments. 40. The fusion protein of embodiment 39, wherein the fusion protein is a binding molecule. 41. The fusion protein of embodiment 39 or 40, wherein the fusion protein is an antibody or antigen-binding fragment thereof, preferably an intrabody that binds to an intracellular target. 42. The fusion protein of any one of embodiments 39 to 40, wherein the fusion protein comprises or consists of a scFv, a bi-specific T-cell engager (BiTE), a Dual Affinity Retargeting Antibodies (DART), a Designed Ankyrin Repeat Proteins (DARPins), a single- domain antibody, a chimeric antigen receptor (CAR), or an antibody or antigen-binding fragment thereof fused to an immunoglobulin Fc domain. 43. The fusion protein of any one of embodiments 39 to 42, wherein the fusion protein comprises or consists of a scFv, a single domain antibody, preferably a VHH, or a bi-specific T-cell engager (BiTE). 44. The fusion protein of any one of embodiments 39 to 43, wherein the fusion protein peptide linker links a Heavy Chain Variable Region (VH) and a Light Chain Variable Region (VL) of a scFv. 45. The fusion protein of any one of embodiments 39 to 43, wherein the fusion protein peptide linker links two single domain antibodies. 46. The fusion protein of any one of embodiments 39 to 43, wherein the fusion protein peptide linker links two scFvs. 47. The fusion protein of any one of embodiments 39 to 43, wherein the fusion protein peptide linker links a scFv and a single domain antibody, preferably a VHH. 48. The fusion protein of any one of embodiments 39 to 43 or 45, wherein the fusion protein peptide linker links two VHH. 49. The fusion protein of any one of embodiments 39 to 48, wherein the fusion protein peptide linker exhibits at least one of the following features: a) promotes or increases the solubility of the fusion protein; and / or b) inhibits, decreases or prevents the toxicity of the fusion protein; and / or c) inhibits, decreases or prevents the aggregation of the fusion protein; and / or d) promotes or increases the expression yield of the fusion protein; and / or e) promotes or increases the intracellular degradation of the fusion protein; and / or f) promotes or increases the monomeric expression of the fusion protein; and / or g) promotes or increases the thermostability of the fusion protein; and / or i) improves or increases the purification yield of the fusion protein; and / or h) promotes, increases or maintains the target binding affinity of the fusion protein relative to a corresponding fusion protein comprising a fusion protein linker consisting of Gly and Ser amino acids, preferably a (G4S)3 peptide linker (SEQ ID NO: 3). 50. The fusion protein of any one of embodiments 39 to 49, wherein the fusion protein peptide linker decreases the pI of the fusion protein relative to the pI of a corresponding fusion protein comprising a fusion protein peptide linker consisting of Gly and Ser amino acids. 51. A method for decreasing the pI of a fusion protein comprising two polypeptides linked by a fusion protein linker, the method comprising introducing negatively charged amino acids in the fusion protein linker to obtain a fusion protein with a pI equal to or below 6. 52. Use of a fusion protein peptide linker of any one of embodiments 1 to 38 for decreasing the pI of a fusion protein to a value of 6 or less. 53. The method of embodiment 51 or use according to embodiment 52, wherein the pI is equal to or below 5.5. 54. The method of embodiment 51 or 53, or use according to embodiment 52 or 53, wherein the pI is from 4.5 to 5.5. 55. A method for promoting or increasing the solubility of a fusion protein, the method comprising providing a fusion protein comprising two polypeptides linked by the fusion protein peptide linker of any one of embodiments 1 to 38 and expressing the fusion protein in a cell or a cell-free expression system. 56. A method for inhibiting, decreasing or preventing the toxicity of a fusion protein, the method comprising providing a fusion protein comprising two polypeptides linked by the fusion protein peptide linker of any one of embodiments 1 to 38 and expressing the fusion protein in a cell or a cell-free expression system. 57. A method for inhibiting, decreasing or preventing the aggregation of a fusion protein, the method comprising providing a fusion protein comprising two polypeptides linked by the fusion protein peptide linker of any one of embodiments 1 to 38 and expressing the fusion protein in a cell or a cell-free expression system. 58. A method for promoting or increasing intracellular degradation of a fusion protein, the method comprising providing a fusion protein comprising two polypeptides linked by the fusion protein peptide linker of any one of embodiments 1 to 38 and expressing the fusion protein in a cell or a cell-free expression system. 59. A method for promoting or increasing the expression of a fusion protein, the method comprising providing a fusion protein comprising two polypeptides linked by the fusion protein peptide linker of any one of embodiments 1 to 38 and expressing the fusion protein in a cell or a cell-free expression system. 60. A method for promoting or increasing the thermostability of a fusion protein, the method comprising providing a fusion protein comprising two polypeptides linked by the fusion protein peptide linker of any one of embodiments 1 to 38 and expressing the fusion protein in a cell or a cell-free expression system. 61. A method for promoting or increasing the monomeric expression of a fusion protein, the method comprising providing a fusion protein comprising two polypeptides linked by the fusion protein peptide linker of any one of embodiments 1 to 38 and expressing the fusion protein in a cell or a cell-free expression system. 62. A method for improving or increasing the purification yield of a fusion protein, the method comprising providing a fusion protein comprising two polypeptides linked by the fusion protein peptide linker of any one of embodiments 1 to 38, expressing the fusion protein in a cell or a cell-free expression system and isolating the fusion protein by performing a separation technique based on the fusion protein charge. 63. A method for promoting or increasing the target binding affinity of a fusion protein, the method comprising providing a fusion protein comprising two polypeptides linked by the fusion protein peptide linker of any one of embodiments 1 to 38 and expressing the fusion protein in a cell or a cell-free expression system. 64. Use of a fusion protein peptide linker of any one of embodiments 1 to 38 for promoting or increasing the solubility of a fusion protein. 65. Use of a fusion protein peptide linker of any one of embodiments 1 to 38 for inhibiting, decreasing or preventing the toxicity of a fusion protein. 66. Use of a fusion protein peptide linker of any one of embodiments 1 to 38 for inhibiting, decreasing or preventing the aggregation of a fusion protein. 67. Use of a fusion protein peptide linker of any one of embodiments 1 to 38 for promoting or increasing intracellular degradation of a fusion protein. 68. Use of a fusion protein peptide linker of any one of embodiments 1 to 38 for promoting or increasing the expression of a fusion protein. 69. Use of a fusion protein peptide linker of any one of embodiments 1 to 38 for promoting or increasing the thermostability of a fusion protein. 70. Use of a fusion protein peptide linker of any one of embodiments 1 to 38 for promoting or increasing the monomeric expression of a fusion protein. 71. Use of a fusion protein peptide linker of any one of embodiments 1 to 38 method for improving or increasing the purification yield of a fusion protein. 72. Use of a fusion protein peptide linker of any one of embodiments 1 to 38 for promoting or increasing the target binding affinity of a fusion protein. 73. The method of any one of embodiments 51 or 53 to 63, or use according to any one of embodiments 52 to 54 or 64 to 72, wherein the fusion protein is a binding molecule. 74. The method of any one of embodiments 51, 53 to 63 or 73, or use according to any one of embodiments 52 to 54 or 64 to 73 wherein the fusion protein is an antibody or antigen- binding fragment thereof, preferably an intrabody that binds to an intracellular target. 75. The method of any one of embodiments 51, 53 to 63, 73 or 74, or use according to any one of embodiments 52 to 54 or 64 to 74, wherein the fusion protein comprises or consists of a scFv, a bi-specific T-cell engager (BiTE), a Dual Affinity Retargeting Antibodies (DART), a Designed Ankyrin Repeat Proteins (DARPins), a single-domain antibody, a chimeric antigen receptor (CAR), an antibody or antigen-binding fragment thereof fused to an immunoglobulin Fc domain. 76. The method of any one of embodiments 51, 53 to 63 or 73 to 75, or use according to any one of embodiments 52 to 54 or 64 to 75, wherein the fusion protein consists of a scFv. 77. A nucleic acid encoding the fusion protein of any one of embodiments 39 to 50. 78. An expression vector comprising the nucleic acid of embodiment 77. 79. A host cell comprising the nucleic acid of embodiment 77 or the expression vector of embodiment 78. 80. A cell-free expression system comprising the nucleic acid of embodiment 77, or the expression vector of embodiment 78. 81. A method for producing the fusion protein of any one of embodiments 39 to 50, comprising the steps of: a) culturing the host cell of embodiment 79 or the cell-free expression of embodiment 80 under conditions suitable for producing the fusion protein, and b) isolating the fusion protein. 82. The expression vector of embodiment 78, wherein the vector is a wild-type or engineered viral vector. 83. The expression vector of embodiment 78 or 82, wherein the vector is an adeno-associated virus (AAV), an adenovirus or a lentivirus. 84. The expression vector of embodiment 78, 82 or 83, wherein the vector is an AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAVrh.10, AAV-BR1, AAV-S, AAV-F, AAV-PHP.eB, AAV9-PHP-V, AAV.CAP-B10, AAV.CAP-B22, AAV.CAP-Mac, AAV VCAP-103, AAV bCap 1, AAV VCAP-100, AAV1RX, AAV1R6 or AAV1R7. 85. A pharmaceutical composition comprising the fusion protein of any one of embodiments 39 to 50, nucleic acid of embodiment 77 or an expression vector of any one of embodiments 78 or 82 to 84 and a pharmaceutically acceptable carrier and / or excipient. 86. The fusion protein of any one of embodiments 39 to 50, nucleic acid of embodiment 77, expression vector of any one of embodiments 78 or 82 to 84 or the pharmaceutical composition of embodiment 85 for use in therapy. 87. The fusion protein, nucleic acid, expression vector or pharmaceutical composition for use according to embodiment 86, wherein the fusion protein is a binding molecule and binds a pathological protein, a cell surface receptor, a tumour-associated antigen or tumour-specific antigen, preferably an intracellular pathological protein. 88. The fusion protein, nucleic acid, expression vector or pharmaceutical composition for use according to embodiment 87, wherein the binding molecule binds TAR DNA-Binding Protein 43 (TDP-43), amyloid-beta, alpha-synuclein, Tau, apoptosis-associated speck-like protein containing a CARD (ASC) or huntingtin protein (HTT). 89. The fusion protein, nucleic acid, expression vector or pharmaceutical composition for use according to embodiment 88, wherein the binding molecule binds TDP-43, and the condition, disease or disorder is associated with pathological TDP-43 or a TDP-43 proteinopathy. 90. The fusion protein, nucleic acid, expression vector or pharmaceutical composition for use according to embodiment 89, wherein the condition, disease or disorder associated with pathological TDP-43 or TDP-43 proteinopathy is Frontotemporal dementia (FTD), amyotrophic lateral sclerosis (ALS), Alzheimer's disease (AD), Parkinson's disease (PD), Chronic Traumatic Encephalopathy (CTE), limbic-predominant age-related TDP-43 encephalopathy (LATE), or multiple sclerosis. Numbered clauses The invention will now be described by way of numbered clauses. 1. A fusion protein comprising a fusion protein peptide linker, wherein the fusion protein is an intrabody that binds an intracellular target, and wherein the fusion protein peptide linker comprises, consists essentially of, or consists of an amino acid sequence Z, wherein Z consists of n independent occurrences of an amino acid sequence Y, wherein each Y is independently selected from the amino acid sequence set forth as X1X2X3X4X5X6X7X8X9X10X11X12X13X14X15X16X17, wherein X1 to X17 are each independently selected from E, D, G, S or absent; at least one from X1 to X17 is G; at least one from X1 to X17 is E or D; at least one from X1to X17is S; n is 1 to 10; and Z is at least 16 amino acids in length. 2. Use of a fusion protein peptide linker in the preparation of a fusion protein, wherein the fusion protein is an intrabody that binds to an intracellular target, and wherein the fusion protein peptide linker comprises, consists essentially of, or consists of an amino acid sequence Z, wherein Z consists of n independent occurrences of an amino acid sequence Y, wherein each Y is independently selected from the amino acid sequence set forth as X1X2X3X4X5X6X7X8X9X10X11X12X13X14X15X16X17, wherein X1to X17are each independently selected from E, D, G, S or absent; at least one from X1to X17is G; at least one from X1to X17is E or D; at least one from X1 to X17 is S; n is 1 to 10; and Z is at least 16 amino acids in length. 3. The fusion protein of clause 1 or use of clause 2 wherein the fusion protein is a scFv and the fusion protein peptide linker links the VH and VL domains. 4. The fusion protein of clause 1 or 3 or use of clause 2 or 3 wherein the intracellular target is TDP-43, amyloid-beta, alpha-synuclein, Tau, apoptosis-associated speck-like protein containing a CARD (ASC) or huntingtin protein (HTT). 5. The fusion protein of any one of clauses 1, 3 or 4 or use of any one of clauses 2 to 4 wherein the fusion protein peptide linker is further characterised in that: at least 30% of Z amino acids are G, such as from 30%-70%, preferably from 30%-50%; at least 15% of Z amino acids are D and / or E, such as from 15%-55%, preferably from 35%- 55%; and from 10% to 20% of Z amino acids are S, preferably from 10%-15%. 6. The fusion protein of any one of clauses 1 or 3 to 5 or use of any one of clauses 2 to 5 wherein the amino acid sequence Z is no more than 50 amino acids in length, preferably from 18 to 45 amino acids.7. The fusion protein of any one of clauses 1 or 3 to 6 or use of any one of clauses 2 to 6 wherein n is at least 2, at least 3, at least 4, and preferably is 3 or 4. 8. The fusion protein of any one of clauses 1 or 3 to 7 or use of any one of clauses 2 to 7 wherein the amino acid sequence Y is the same for each of the n occurrences of Y. A fusion protein peptide linker for use in an intrabody that binds an intracellular target, wherein the fusion protein peptide linker comprises, consists essentially of, or consists of an amino acid sequence Z, wherein Z consists of n independent occurrences of an amino acid sequence Y, wherein each Y is independently selected from the amino acid sequence set forth as X1X2X3X4X5X6X7X8X9X10X11X12X13X14X15X16X17, wherein X1 is G; X2 to X6 are each independently selected from G or absent; X7 is E or D; X8to X16are each independently selected from E, D or absent; X17is S; n is 1 to 10; and Z is at least 16 amino acids in length.The fusion protein of any one of clauses 1 or 3 to 8, use of any one of clauses 2 to 8, or fusion protein peptide linker of clause 9, wherein: a) X1 and X2 are G; b) X1to X3are G; c) X1to X4are G; d) X5and X6are absent; e) X14 to X16 are absent; f) X12 to X16 are absent; g) X7 and X8 are each independently selected from E or D; and / or h) X7 to X9 are each independently selected from E or D. The fusion protein of any one of clauses 1 or 3 to 8 or 10, use of any one of clauses 2 to 8 or 10, or fusion protein peptide linker of clause 9 or 10, wherein: a) X1and X2are G; X7 and X8 are each independently selected from E or D; and X15 and X16 are absent; b) X1 to X3 are G; X7 and X8 are each independently selected from E or D; and X6and X14to X16are absent; c) X1 to X3 are G; X7to X9are each independently selected from E or D; and X6and X14to X16are absent; d) X1to X3are G; X7 to X9 are each independently selected from E or D; and X6 and X12 to X16 are absent; e) X1 to X4 are G; X7 is E or D; X8to X11are each independently selected from E, D or absent; and X5to X6and X12to X16are absent; f) X1to X4are G; X7 to X9 are each independently selected from E or D; X10 to X11 are each independently selected from E, D or absent; and X5 to X6 and X12 to X16 are absent;g) X1 to X4 are G; X7is E or D; X8to X11are each independently selected from E, D or absent; X5to X6and X12to X16are absent; n is 3 or 4; and the amino acid sequence Y is the same for each of the n occurrences of Y; h) X1to X4are G; X7to X9are each independently selected from E or D; X10to X11are each independently selected from E, D or absent; X5 to X6 and X12 to X16 are absent;n is 3 or 4; and the amino acid sequence Y is the same for each of the n occurrences of Y. The fusion protein of any one of clauses 1 or 3 to 8 or 10 to 11, use of any one of clauses 2 to 8 or 10 to 11, or fusion protein peptide linker of any one of clauses 9 to 11, wherein Z amino acid sequence is any one of SEQ ID NO: 6 to SEQ ID NO: 15, SEQ ID NO: 17 to SEQ ID NO: 26, SEQ ID NO: 48 to SEQ ID NO: 54, SEQ ID NO: 69 to SEQ ID NO: 73, or SEQ ID NO: 87 to SEQ ID NO: 91. 13. The fusion protein of any one of clauses 1 or 3 to 8 or 10 to 12, use of any one of clauses 2 to 8 or 10 to 12, or fusion protein peptide linker of any one of clauses 9 to 12, wherein the fusion protein peptide linker: a) promotes or increases the solubility of the fusion protein; b) inhibits, decreases or prevents the toxicity of the fusion protein; c) inhibits, decreases or prevents the aggregation of the fusion protein; d) promotes or increases the expression yield of the fusion protein; e) promotes or increases the intracellular degradation of the fusion protein; f) promotes or increases the monomeric expression of the fusion protein; g) promotes or increases the thermostability of the fusion protein; i) improves or increases the purification yield of the fusion protein; and / or h) promotes, increases or maintains the target binding affinity of the fusion protein; each relative to a corresponding fusion protein comprising a peptide linker consisting of Gly and Ser amino acids, preferably a (G4S)3 peptide linker (SEQ ID NO: 3). 14. A pharmaceutical composition comprising a nucleic acid or expression vector encoding the fusion protein of any one of clauses 1 or 3 to 8 or 10 to 13 or a fusion protein comprising the fusion protein peptide linker of any one of clauses 9 to 13, and a pharmaceutically acceptable carrier and / or excipient. 15. A fusion protein according to any one of clauses 1 or 3 to 8 or 10 to 13, a fusion protein comprising a fusion protein peptide linker according to any one of clauses 913 or a pharmaceutical composition according to clause 14 for use in therapy, preferably for use in the treatment of a neurodegenerative disorder.

[0002] Sequence Listing Amino acid sequence Z Sequence features Description of exemplary embodiment Formula SEQ ID NO GGGS - - (G3S) 1 GGGGS - - (G4S) 2 GGGGSGGGGSGGGGS - - (G4S)33 GGGGSGGGGSGGGGS - - (G4S)4 4 GGGGS KFERQ - - - 5 GGGGESGGGGESGGG - - (G4ES)36 GES GGGGEESGGGGEESGG - - (G4E2S)37 GGEES GGGGEEESGGGGEEES - - (G4E3S)3 8 GGGGEEES GGGGEEEESGGGGEEE - - (G4E4S)3 9 ESGGGGEEEES GGGGEEEEESGGGGEE - - (G4E5S)3 10 EEESGGGGEEEEES GGGGESGGGGESGGG - - (G4ES)411 GESGGGGES

[0003] GGGGEESGGGGEESGG - - (G4E2S)4 12 GGEESGGGGEES GGGGEEESGGGGEEES - - (G4E3S)413 GGGGEEESGGGGEEES GGGGEEEESGGGGEEE - - (G4E4S)414 ESGGGGEEEESGGGGE EEES GGGGEEEEESGGGGEE - - (G4E5S)415 EEESGGGGEEEEESGG GGEEEEES GGDEESGGDEESGGDE - - (G2DE2S)7G2S 16 ESGGDEESGGDEESGG DEESGGDEESGGS GGGGDSGGGGDSGGG - - (G4DS)3 17 GDS GGGGDDSGGGGDDSG - - (G4D2S)3 18 GGGDDS GGGGDDDSGGGGDDD - - (G4D3S)3 19 SGGGGDDDS GGGGDDDDSGGGGDD - - (G4D4S)320 DDSGGGGDDDDS

[0004] GGGGDDDDDSGGGGD - - (G4D5S)3 21 DDDDSGGGGDDDDDS GGGGDSGGGGDSGGG - - (G4DS)422 GDSGGGGDS GGGGDDSGGGGDDSG - - (G4D2S)423 GGGDDSGGGGDDS GGGGDDDSGGGGDDD - - (G4D3S)4 24 SGGGGDDDSGGGGDD DS GGGGDDDDSGGGGDD - - (G4D4S)4 25 DDSGGGGDDDDSGGG GDDDDS GGGGDDDDDSGGGGD - - (G4D5S)4 26 DDDDSGGGGDDDDDS GGGGDDDDDS GGGGXSGGGGXSGGG 1) X can be E or D Z consists of n independent occurrences of an amino acid (G4(E / D)1S)3-8 48 GXSGGGGXSGGGGXS 2) Up to 5 copies of sequence Y, wherein each Y is independently selected from GGGGXS can be the amino acid sequence set forth as GGGGXSGGGGXSGGG deleted X1X2X3X4X5X6X7X8X9X10X11X12X13X14X15X16X17, wherein X1to X4are G; GXS X5 to X6 and X8 to X16 are absent; X7is E or D; X17 is S; and n is 3 to 8.

[0005] GGGGXXSGGGGXXSG 1) X can be E or D Z consists of n independent occurrences of an amino acid (G4(E / D)2S)2-7 49 2) Up to 5 copies of sequence Y, wherein each Y is independently selected from GGGXXSGGGGXXSGG GGGGXXS can be the amino acid sequence set forth as GGXXSGGGGXXSGGG deleted X1X2X3X4X5X6X7X8X9X10X11X12X13X14X15X16X17, wherein GXXS X1to X4are G; X5 to X6 and X9 to X16 are absent; X7and X8are each independently selected from E or D; X17 is S; and n is 2 to 7. GGGGXXXSGGGGXXX 1) X can be E or D Z consists of n independent occurrences of an amino acid (G4(E / D)3S)2-6 50 SGGGGXXXSGGGGXX 2) Up to 4 copies of sequence Y, wherein each Y is independently selected from GGGGXXXS can be the amino acid sequence set forth as XSGGGGXXXSGGGGX deleted X1X2X3X4X5X6X7X8X9X10X11X12X13X14X15X16X17, wherein XXS X1 to X4 are G; X5 to X6 and X10 to X16 are absent; X7to X9are each independently selected from E or D; X17 is S; and n is 2 to 6. GGGGXXXXSGGGGXX 1) X can be E or D Z consists of n independent occurrences of an amino acid (G4(E / D)4S)2-5 51 XXSGGGGXXXXSGGG 2) Up to 3 copies of sequence Y, wherein each Y is independently selected from GGGGXXXXS can the amino acid sequence set forth as GXXXXSGGGGXXXXS be deleted X1X2X3X4X5X6X7X8X9X10X11X12X13X14X15X16X17, wherein X1to X4are G; X5 to X6 and X11 to X16 are absent; X7to X10are each independently selected from E or D; X17 is S; and n is 2 to 5.

[0006] GGGGXXXXXSGGGGX 1) X can be E or D Z consists of n independent occurrences of an amino acid (G4(E / D)5S)2-5 52 2) Up to 3 copies of sequence Y, wherein each Y is independently selected from XXXXSGGGGXXXXXS the amino acid sequence set forth as GGGGXXXXXSGGGGX GGGGXXXXXS can X1X2X3X4X5X6X7X8X9X10X11X12X13X14X15X16X17, wherein XXXXS be deleted X1to X4are G; X5 to X6 and X12 to X16 are absent; X7to X11are each independently selected from E or D; X17 is S; and n is 2 to 5. GGGGXXXXXXSGGGG 1) X can be E or D Z consists of n independent occurrences of an amino acid (G4(E / D)6S)2-4 53 XXXXXXSGGGGXXXX 2) Up to 2 copies of sequence Y, wherein each Y is independently selected from the amino acid sequence set f XSGGGGXXXXXXS GGG orth as X GXXXXXXS X1X2X3X4X5X6X7X8X9X10X11X12X13X14X15X16X17, wherein can be deleted X1 to X4 are G; X5 to X6 and X13 to X16 are absent; X7to X12are each independently selected from E or D; X17 is S; and n is 2 to 4. GGGGXXXXXXXSGGG 1) X can be E or D Z consists of n independent occurrences of an amino acid (G4(E / D)7S)2-4 54 GXXXXXXXSGGGGXX 2) Up to 2 copies of sequence Y, wherein each Y is independently selected from GGGGXXXXX the amino acid sequence set forth as XXXXXSGGGGXXXXX XXS X1X2X3X4X5X6X7X8X9X10X11X12X13X14X15X16X17, wherein can be deleted X1to X4are G; XXS X5 to X6 and X14 to X16 are absent; X7to X13are each independently selected from E or D; X17 is S; and n is 2 to 4.

[0007] GGGGXSGGGGXSGGG 1) X can be E or D Z consists of n independent occurrences of an amino acid (G4(E / D)1S)3-4 69 2) 1 copy of sequence Y, wherein each Y is independently selected from GXSGGGGXS GGGGXS can be the amino acid sequence set forth as deleted X1X2X3X4X5X6X7X8X9X10X11X12X13X14X15X16X17, wherein X1to X4are G; X5 to X6 and X8 to X16 are absent; X7 is E or D; X17 is S; and n is 3 or 4. GGGGXXSGGGGXXSG 1) X can be E or D Z consists of n independent occurrences of an amino acid (G4(E / D)2S)3-4 70 GGGXXSGGGGXXS 2) 1 copy of sequence Y, wherein each Y is independently selected from GGGGXXS can be the amino acid sequence set forth as deleted X1X2X3X4X5X6X7X8X9X10X11X12X13X14X15X16X17, wherein X1 to X4 are G; X5 to X6 and X9 to X16 are absent; X7and X8are each independently selected from E or D; X17is S; and n is 3 or 4. GGGGXXXSGGGGXXX 1) X can be E or D Z consists of n independent occurrences of an amino acid (G4(E / D)3S)3-4 71 2) 1 copy of sequence Y, wherein each Y is independently selected from SGGGGXXXSGGGGXX GGGGXXXS can be the amino acid sequence set forth as XS deleted X1X2X3X4X5X6X7X8X9X10X11X12X13X14X15X16X17, wherein X1 to X4 are G; X5to X6and X10to X16are absent; X7to X9are each independently selected from E or D; X17 is S; and n is 3 or 4.

[0008] GGGGXXXXSGGGGXX 1) X can be E or D Z consists of n independent occurrences of an amino acid (G4(E / D)4S)3-4 72 2) 1 copy of sequence Y, wherein each Y is independently selected from XXSGGGGXXXXSGGG GGGGXXXXS can the amino acid sequence set forth as GXXXXS be deleted X1X2X3X4X5X6X7X8X9X10X11X12X13X14X15X16X17, wherein X1to X4are G; X5 to X6 and X11 to X16 are absent; X7 to X10 are each independently selected from E or D; X17 is S; and n is 3 or 4. GGGGXXXXXSGGGGX 1) X can be E or D Z consists of n independent occurrences of an amino acid (G4(E / D)5S)3-4 73 XXXXSGGGGXXXXXS 2) 1 copy of sequence Y, wherein each Y is independently selected from GGGGXXXXXS can the amino acid sequence set forth as GGGGXXXXXS be deleted X1X2X3X4X5X6X7X8X9X10X11X12X13X14X15X16X17, wherein X1 to X4 are G; X5 to X6 and X12 to X16 are absent; X7to X11are each independently selected from E or D; X17is S; and n is 3 or 4. GGGGXXXXXSGGGGX 1) X in position 5, 15, Z consists of n independent occurrences of an amino acid (G4(E / D)1-5S)3-4 87 25 and 35 can be E or sequence Y, wherein each Y is independently selected from XXXXSGGGGXXXXXS D the amino acid sequence set forth as GGGGXXXXXS 2) X in position 6 to 9, X1X2X3X4X5X6X7X8X9X10X11X12X13X14X15X16X17, wherein X to X are G; 16 to 19, 26 to 29 and 1 4 X5to X6and X12to X16are absent; 36 to 39 can be E, D or X7 is E or D; X8to X11are each independently selected from E, D or absent absent; 3) 1 copy of X17 is S; and GGGGXXXXXS can n is 3 or 4. be deleted

[0009] GGGGXXXXXSGGGGX 1) X in position 5 to 7, Z consists of n independent occurrences of an amino acid (G4(E / D)3-5S)3-4 88 15 to 17, 25 to 27 and sequence Y, wherein each Y is independently selected from XXXXSGGGGXXXXXS 35 to 37 can be E or D the amino acid sequence set forth as GGGGXXXXXS 2) X in position 8 to 9, X1X2X3X4X5X6X7X8X9X10X11X12X13X14X15X16X17, wherein 18 to 19, 28 to 29 and X1to X4are G; X5 to X6 and X12 to X16 are absent; 38 to 39 can be E, D or X7to X9are each independently selected from E or D; absent X10 to X11 are each independently selected from E, D or absent; 3) 1 copy of X17 is S; and GGGGXXXXXS can n is 3 or 4. be deleted GGGGXXXXXSGGGGX 1) X in position 5, 15, Z consists of n independent occurrences of an amino acid (G4(E / D)1-5S)3-489 XXXXSGGGGXXXXXS 25 and 35 can be E or sequence Y, wherein each Y is independently selected from D as long as they are the amino acid sequence set forth as GGGGXXXXXS the same in all these X1X2X3X4X5X6X7X8X9X10X11X12X13X14X15X16X17, wherein positions X1 to X4 are G; 2) X in position 6, 16, X5to X6and X12to X16are absent; 26 and 36 can be E, D X7 is E or D; or absent as long as X8to X11are each independently selected from E, D or they are the same in absent; all these positions X17is S; 3) X in position 7, 17, n is 3 or 4; and 27 and 37 can be E, D the amino acid sequence Y is the same for each of the n or absent as long as occurrences of Y. they are the same in all these positions 4) X in position 8, 18, 28 and 38 can be E, D or absent as long as they are the same in all these positions 5) X in position 9, 19, 29 and 39 can be E, D

[0010] or absent as long as they are the same in all these positions 6) 1 copy of GGGGXXXXXS can be deleted GGGGXXXXXSGGGGX 1) X in position 5, 15, Z consists of n independent occurrences of an amino acid (G4(E / D)3-5S)3-4 90 XXXXSGGGGXXXXXS 25 and 35 can be E or sequence Y, wherein each Y is independently selected from D as long as they are the amino acid sequence set forth as GGGGXXXXXS the same in all these X1X2X3X4X5X6X7X8X9X10X11X12X13X14X15X16X17, wherein positions X1to X4are G; 2) X in position 6, 16, X5 to X6 and X12 to X16 are absent; 26 and 36 can be E or X7to X9are each independently selected from E or D; D as long as they are X10 to X11 are each independently selected from E, D or the same in all these absent; positions X17 is S; 3) X in position 7, 17, n is 3 or 4; and 27 and 37 can be E or the amino acid sequence Y is the same for each of the n D as long as they are occurrences of Y. the same in all these positions 4) X in position 8, 18, 28 and 38 can be E, D or absent as long as they are the same in all these positions 5) X in position 9, 19, 29 and 39 can be E, D or absent as long as they are the same in all these positions 6) 1 copy of GGGGXXXXXS can be deleted

[0011] GGGGEEEEESGGGEEE - - G4E5SG3E5S 91 EES GGGDGGDS - - G3DG2DS 92 GGGDGGDSGGGDGGD - - (G3DG2DS)293 S GGGDGDGS - - G3DGDGS 94 GGGDGDGSGGGDGGD - - G3DGDGSG3D 95 S G2DS GGDSGGDSGGDSGGDS - - (G2DS)496 GGGGGSGGGGSGGGG - - G5S(G4S)3 97 SGGGGS GGGGGEEEESGGGGEE - - G5E4S(G4E4S)3 98 EESGGGGEEEESGGGG Linker H EEEES GGGGSGGGS - - G4SG3S 99 GGSGGSGGSGGSGG - - (G2S)4G2100 GGDEESGGDEESGGDE - - (G2DE2S)7 101 ESGGDEESGGDEESGG DEESGGDEES GEDEDEDEDEDSGDED Linker A 102 EDEDEDESGEDEDEDE DEDS

[0012] GGGGGGGGGGGGEDS Linker B 103 EDGGGEESDEESESDES EGGSGEGEGSGDGEGG Linker C 104 DSEGGEDGGSGDEGEG SEDEGGGSEEEGGGGG Linker D 105 SEEEGGGGSEEGDGGG EESGGEESGGEESSGG Linker E 106 DDDGGSDESGEEEGSS GGEEEDEESGGGGGGD Linker F 107 EEEEESGGGGGEEEED ESGGGEEEEEES GEDEEDGDEDEESSGE Linker G 108 EDDEGEEEEESSGEDD EEGDEEEESS

[0013] BRIEF DESCRIPTION OF FIGURES Figure 1. Size exclusion chromatographs of purified anti-TDP-43 antibody formats: parental IgG (~145 kDa), Fab (~48 kDa) and scFv WT (~28 kDa). mAu: milli-absorbance unit. mL: millilitre. Figure 2. Western blot of cytoplasmic soluble (S) and non-soluble (NS) protein (recovered by centrifugation) fractions from CHO expressing the scFv WT construct. Arrow indicates the non- soluble / non-degraded scFv corresponding to ~28 kDa. Figure 3A and 3B. Western blot of soluble (S) and non-soluble (NS) protein fractions from CHO cells expressing the scFv constructs of Table 3. Figure 3C. Band density integration of Western blot of Figure 3A and 3B depicting the percentage of the different scFv constructs of Table 3 in soluble and non-soluble protein fractions. Figure 4A and 4B. Graph representing the % of viable CHO cells on day 1, 2, 4 and 5 following transfections with one of 15 scFv constructs of Table 3. Dotted line at 80% represents the viability of un-transfected (mock) cells at day 5. Figure 5. Fluorescence microscope images of QBI-293 cells transfected with scFv constructs and immunostained with an anti-Flag antibody. Arrows indicate scFv aggregates. Figure 6A. Fluorescence microscope images of GFP-TDP-43-NLSm QBI-293 cells transfected with scFv constructs expressing cytoplasmic GFP-TDP-43 under doxycycline induction. Column 1: GFP- TDP-43 fluorescence. Column 2: scFv immunostaining with anti-Flag or anti-His-tag antibody. Scale bars represent 20 µm. Arrows indicate scFv aggregates. Figure 6B. Fluorescence microscope image of Proximity Ligation Assay (PLA) using both monoclonal anti-Flag and anti-GFP antibodies. A “dot” represents interaction / complexes between the scFvs and target GFP-TDP-43-NLSm that are also indicated with white arrows. Scale bar represents 20 µm. Figure 7. Fluorescence microscope image of intrabody nuclear translocation assay in rat primary neurons transduced with anti-TDP-43 scFv-(G4E3S)4-c-myc-Flag construct without (1st column) and with (2nd column) GFP-TDP-43-NLSm co-transduction. scFv-(G4E3S)4-c-myc-Flag is detected by anti-Flag antibody (1st row), GFP-TDP-43-NLSm by the GFP fluorescence (2nd row), cell nuclei by Hoechst staining (3rd row) and neurons by anti-MAP2 antibody (4th row). Scale bar represents 100 µm. Figure 8. Coomassie blue staining of proteins separated by SDS-PAGE highlighting CHO-secreted anti-TDP-43 scFvs in harvest raw (H) and His-tag-purified (P) cell supernatant. Arrows indicate examples of scFv bands expected at approximately 28-32kDa. Except where otherwise indicated, all scFv constructs have a VH-VL configuration. Figure 9A and 9B. Size exclusion chromatographs of purified scFv constructs from Table 8. Except where otherwise indicated, all scFv constructs have a VH-VL configuration. mAu: milli-absorbance unit. mL: millilitre. Figure 10. Evidence of charged linker PEST-like functionality. ScFv expression induction with a single 10 ng / mL doxycycline dose at time 0 hours and degradation as a function of time. Total CHO cells protein extract separated by SDS-PAGE 12-4% / Western blot. Band integration using anti-Flag antibodies for the scFvs and anti-beta tubulin antibodies for normalization of scFv quantities. Figure 11. Graph representing the purified antibody titer of clinical stage antibodies with the original glycine-serine linker and redesigned with a negatively charged linker according to the invention. Data is represented as the mean. Figure 12. FMC63 scFv (B) with the original glycine-serine linker at 0.4 mg / ml in a storage tube and (A) redesigned with a negatively charged linker of the invention at 1.5 mg / ml in a storage tube, after a single freeze / thaw cycle. Figure 13. Size exclusion chromatographs of purified scFv constructs from Table 13. HMW: High Molecular Weight. mAu: milli-absorbance unit. mL: millilitre. Figure 13A shows results for linkers: (G4S)3; (G4E3S)4; and linkers A-D. Figure 13B shows results for linkers E-H. EXAMPLES Example 1. Expression quality of secreted scFv The quality of the scFv was compared to the conventional full IgG antibody and the derived fragment antigen-binding (Fab) consisting of VH-CH1 and VL-CL domains. To this end, all 3 antibody formats were produced from an antibody binding to the TDP-43 protein (transactive response DNA binding protein 43 kDa). The scFv was designed from the variable heavy (VH) and variable light (VL) chain domains of the anti-TDP-43 antibody linked with a conventional GGGGSGGGGSGGGGS linker (SEQ ID NO: 3), also known as (G4S)3 linker. A cMyc-tag and a His-tag were added to the scFv for detection and purification purposes, and the construct was named scFv Wild Type (WT). IgG, Fab and scFv WT constructs were cloned into the standard pCDNATM3.4 expression vector bearing a CMV promoter and WPRE regulatory sequence and transfected in ExpiCHO-STMcells, cultured following the supplier conditions. Of note, all clones including the scFv WT expression vector comprised a mouse immunoglobulin kappa chain (Igk) secretion peptide to promote scFv WT secretion. Cells were harvested 12 days after transfection by centrifugation (3000 rpm, 5 min) and cell supernatants were separated, filtrated through a 0.2 µm pore filter and stored at 4°C until further purification. IgG was purified using protein A affinity resin whereas Fab and scFv were purified by immobilized metal-ion affinity chromatography (IMAC, cobalt-based) resin, dialyzed in PBS buffer and quantified by OD280nmusing their respective coefficient of extinction. The purified scFv WT titer was about 13-fold lower than Fab and 30-fold lower than IgG antibody (Table 1, second column), suggesting a poor stability of the scFv format. The different antibody formats were then characterized by size exclusion chromatography for degradation and aggregation (low and high molecular weight species). Peak elution volume of chromatographs (Figure 1) corresponded to expected molecular weight for the different antibody formats (IgG 145 kDa; Fab 48 kDa; scFv 28 kDa). However, the results showed a substantial amount of degraded low molecular weight (LMW) species only present for the scFv antibody format. In addition, scFv dimers were present in the Superdex increase 75 chromatography analysis. To confirm antibody functionality, their binding affinity towards full length human TDP-43 was assessed by surface plasmon resonance (SPR). In brief, measurements were performed at room temperature in PBS buffer with a Biacore T200 instrument. Soluble recombinant human TDP-43 monomers (5 µg / mL in 10 mM sodium acetate pH 4.5) were coupled covalently to Series SCM5 sensor chips previously activated with the supplier amine coupling kit, using a flow rate of 30 µL / min. The resulting sensor chips were regenerated for 30 seconds in 10 mM glycine-HCl pH 1.7 to remove unbound material. Antibodies were diluted in PBS buffer and then tested at the concentrations of 0, 1.2, 3.7, 11.1, 33.3, 100 and 300 nM. The samples were then monitored respectively for 1) association for 300 seconds, 2) dissociation for 600 seconds and 3) two regeneration cycles for 30 seconds followed by 300 seconds stabilization period after each concentration injection. Data were then analysed with the Biacore T200 Evaluation Software. In this assay setting and data analysis, the IgG format presented a KDof 3.7 nM for soluble TDP-43, while the Fab and scFv displayed a comparable KDof 12.9 nM and 13.9 nM, respectively (Table 1). Noteworthy, the lower Fab and scFv affinity was expected and can be explained by their monovalence (one binding moiety per antigen- binding molecule) compared to the IgG bivalence (two binding moieties per antigen-binding molecule). Table 1. Comparison of antibody formats: titer and affinity to soluble recombinant human TDP-43 monomer Kon: association rate; Koff: dissociation rate; KD: equilibrium dissociation constant Altogether, the scFv format presented a poorer protein quality (degradation and aggregation) characterized by a lower expression yield compared to the Fab and IgG antibodies while exhibiting a similar affinity to TDP-43 than the Fab fragment. Example 2. Expression quality of scFv intrabodies The scFv quality was further investigated as an intrabody format. CHO cells were transfected as described in Example 1 with the scFv WT construct cloned without the Igk secretion peptide in a pAAV expression vector designed for adeno-associated virus capsid vectorization. The pAAV expression vector comprises a pUC origin of replication, ampicillin resistance gene, 5’ and 3’ ITR regions on the extremities of the gene construct, a WPRE and a bovine growth hormone polyadenylation signal (BGH pA) on the 3’ end of the gene construct. Two hundred microliters of suspension culture of CHO cells were harvested by centrifugation (3000 rpm, 5 min) 5 days after transfection and the supernatant was discarded. The resulting cell pellet was resuspended and lysed in 50 µL M-PER®buffer at 4°C supplemented by protease inhibitor cocktail (HaltTM, Thermofisher cat # 78438) and incubated on ice for 20 min. The cell lysate was then centrifuged at 8000 rpm for 5 min at 4°C and the supernatant containing soluble proteins was transferred to a fresh tube. The lysed cell pellet was resuspended with 800 µL PBS and centrifuged (8000 rpm, 5 min). The resulting pellet was then resuspended in 50 µL PBS. Ten microliters of both soluble and insoluble fractions were then analysed by SDS-PAGE 12-4% and Western blot. In brief, the proteins separated by SDS-PAGE were transferred to a nitrocellulose membrane using standard procedures. The resulting membrane was then saturated with Intercept®PBS blocking buffer for 1h at room temperature, under gentle shaking. Western blot was then revealed using a mouse anti-His tag antibody incubated for 1h as above at room temperature (Anti-6X His tag His.H8, Abcam cat# ab18184, diluted 1000X). The membrane was washed twice in PBS supplemented by 0.1% Tween for 5 min. The resulting membrane was incubated for 1h as above with labelled anti-mouse IgG (donkey anti-mouse, IRDye®680RD, LI-COR cat#926- 68072, diluted 10’000X). Finally, the membrane was washed twice in PBS supplemented by 0.1% Tween and analysed with the Odyssey imaging system. The Western blot (Figure 2) showed that scFv WT was only retrieved in the non-soluble protein pellet at the expected molecular weight of ~28 kDa (arrow in Figure 2). No scFv could be detected in the soluble fraction. Moreover, lower and higher molecular weight bands indicated scFv WT degradation and aggregation, respectively. This result suggested that the scFv format is unstable in cell cytosol, a finding consistent with previous reports (Kvam et al., 2010; Cardinale et al., 2003; Auf der Maur et al., 2004). Example 3. Optimization of linkers in scFv intrabodies improves scFv solubility Conventional glycine-serine linkers (G4S)3 (GGGGSGGGGSGGGGS; SEQ ID NO: 3) and (G4S)4 (GGGGSGGGGSGGGGSGGGGS; SEQ ID NO: 4) were modified with the insertion of the negatively charged amino acid glutamate in order to decrease the scFv pI and increase its solubility. From one up to five glutamate residues were inserted after the 4 glycines to preserve flexibility and prior to the serine to enhance hydration resulting in the linker sequences of Table 2. In an attempt to further decrease the pI, a larger charged linker was designed where 1 aspartate and 2 glutamates were inserted between 2 glycines and a serine in a total of 7 repeats, resulting in (G2DE2S)7G2S linker (last entry of Table 2). Table 2. Amino acid sequences of negatively charged scFv linkers Amino acid sequence Formula SEQ ID NO GGGGESGGGGESGGGGES (G4ES)3 6 GGGGEESGGGGEESGGGGEES (G4E2S)37 GGGGEEESGGGGEEESGGGGEEES (G4E3S)3 8 GGGGEEEESGGGGEEEESGGGGEEEES (G4E4S)3 9 GGGGEEEEESGGGGEEEEESGGGGEEEEES (G4E5S)310 GGGGESGGGGESGGGGESGGGGES (G4ES)4 11 GGGGEESGGGGEESGGGGEESGGGGEES (G4E2S)4 12 GGGGEEESGGGGEEESGGGGEEESGGGGEEES (G4E3S)413 GGGGEEEESGGGGEEEESGGGGEEEESGGGGEEEES (G4E4S)4 14 GGGGEEEEESGGGGEEEEESGGGGEEEEESGGGGEEEEES (G4E5S)415 GGDEESGGDEESGGDEESGGDEESGGDEESGGDEESGGDEE (G2DE2S)7G2S 16 SGGS Some of these new linkers, as well as the conventional (G4S)4 linker, were cloned in the same anti- TDP-43 scFv WT from Example 1 and 2 in place of the (G4S)3linker. Moreover, a Flag-tag (Hopp et al., 1988) was fused to the scFv C-terminals for detection and purification purposes in replacement of the His-tag, while the cMyc-tag was conserved. Additional constructs were also explored, for instance with the addition of the s3Flag-tag developed by Kabayama et al (2020), or following the findings of Messer et al (Kvam et al., 2010; Butler et al., 2011 & 2012), with the addition of peptide tags promoting cellular clearance by the proteasome such as hPEST KIR2.1 (Qile et al., 2019), hPEST- Hax1 (Li et al., 2012) or by the lysosome such as KFERQ (SEQ ID NO: 5). A construct comprising a KFERQ sequence directly inserted in the middle of the second G4repeat of the (G4S)3linker was also designed. All the constructs are described in Table 3 along with the in silico prediction of their different properties. Table 3. scFv intrabodies solubility and clearance properties The following protein parameters were calculated using the Expasy website (Gasteiger et al., 2005): isoelectric point (pI), net charge, grand average of hydropathicity (GRAVY) (Kyte et al., 1982) score, aliphatic index (AI), insolubility index (Ins. Ind.) and molecular weight (MW). PEST values were calculated with the Emboss epestfind algorithm (Rogers et al., 1986). Chaperone- mediated autophagy (CMA). The in silico analysis showed the scFv WT (first entry of Table 3) had a basic pI of 8.33 and a positive net charge, which are values consistent with the poor cytosolic solubility of Example 1 and 2. The grand average of hydropathicity (GRAVY) (Kyte et al., 1982) score was closer to zero and aliphatic index (AI, non-hydrated area) indicated a higher hydrophobicity compared to other constructs. In the case of the negatively charged PEST constructs, the pIs were acidic but the insolubility index remained closer to the scFv WT, i.e. close to 40 (40 being the lower limit for solubility at neutral pH). In contrast, the insertion of glutamates in the linker provided acidic pIs, but also improved the insolubility index up to greater than 50 and GRAVY scores with a decrease of more than 0.1 unit. Sequence analyses with the Emboss epestfind algorithm (Rogers et al., 1986; Rechsteiner et al., 1996) identified an unexpected PEST-like motif in the charged linkers (G4E3S)3(SEQ ID NO: 8), (G4E2S)4(SEQ ID NO: 12), (G4E3S)4(SEQ ID NO: 13) and (G2DE2S)7G2S (SEQ ID NO: 16) (Table 3, last two columns). The in silico analysis suggested the scFv comprising negatively charged linkers present an improved theoretical solubility profile compared to the scFv with conventional linkers. The in silico predictions were tested in vitro and the constructs of Table 3 were expressed as intrabody format in CHO cells as described in Example 2. Noteworthy, CHO cells were grown first at 37°C for about 18-20h and then at 32°C for an additional 24h in order to increase the intracellular expression yield and promote poor scFv construct expression (degradation, aggregation). Two hundred microliters of CHO cell suspension culture was lysed 2 days after transfection using the same protocol as in Example 2. Expressed proteins were analysed by SDS-PAGE 12-4% and Western blot using anti-His-tag and anti-Flag antibodies. Anti-Flag mouse monoclonal antibody was diluted 5000X (M2, Merck cat# F3165). Resulting blots are shown in Figure 3A and 3B while the band density integration ranked from the least to the most soluble scFv construct is shown in Figure 3C. As in Example 2, the scFv WT was mostly retrieved in the non-soluble fraction, along with the scFv comprising the conventional (G4S)3and (G4S)4linkers. The highest cytosolic solubility was found in linkers with 3 glutamate repeats reaching up to 70% solubility (Figure 3B and 3C). These in vitro results were in line with the in silico predictions and confirmed the potential of charged linkers to improve the solubility profile of scFv intrabodies. Example 4. Negatively charged linkers prevent scFv intrabodies toxicity As reported by Sibler et al. (2003) and others, unstable and poorly soluble scFv intrabodies can be toxic and induce cell death. Consequently, cell viability was evaluated in CHO cells transfected with the scFv constructs of Table 3, following the same protocol as in Example 3. Cell viability was monitored by collecting 20 µL of cell suspension culture per well mixed directly with the same volume of trypan blue within a period of 5 days. Cell viability was substantially affected by scFv constructs comprising the conventional (G4S)3linker apart from the scFv constructs comprising the s3Flag or hPEST KIR2.1 tag (Figure 4A). All cells transfected with scFv constructs comprising a negatively charged linker retained a cell viability close to 80% on day 5 (dashed line in Figure 4A and 4B) similar to the non-transfected mock cell control. Noteworthy, cell viability reflected the solubility of the tested scFv constructs except for the one comprising a (G4S)4linker that surprisingly retained a cell viability above 80% on day 5. One hypothesis is that the reducing environment of the cell cytosol likely breaks intrabody disulfide bonds and can lead to scFv aggregation, cell toxicity and ultimately, cell death. These results confirmed that scFv intrabodies comprising a negatively charged linker are soluble in the cytosol and are not cytotoxic. Example 5. Aggregation of scFv intrabodies in QBI-293 cells The scFv constructs from Table 3 were expressed in adherent QBI-293 cells (HEK derivate) (Porta et al., 2018) to further test the quality of their expression as intrabodies. QBI-293 cells were cultured and maintained in DMEM medium supplemented by 10% tetracycline-free FBS at 37°C, 5% CO2 and 1% penicillin / streptomycin. Cells were then plated at a density of 5 x 104cells / mL in cell culture- treated poly-D-lysine coated 96-well plates (50 µL cell per well). In brief, transfections were performed by serum-starving cells one day after seeding with OPTIMEM medium (ThermoFisher, #11058021) at 37°C. After that, cells received a mixture of lipofectamine 2000 (ThermoFisher, #11668500) and 1 µg / mL plasmid DNA in OPTIMEM medium. Cells were then incubated for 6h at 37°C, 5% CO2 and the medium was changed to DMEM medium supplemented by 10% tetracycline- free FBS. Forty eight hours post-transfection, QBI293 cells were fixed for immunocytochemistry (ICC) analyses with 50 µL of PBS supplemented by 3.7% paraformaldehyde (PFA) for 20 minutes at room temperature and then gently washed 3 times with PBS. After that, cells and their corresponding plate well were blocked in PBS supplemented with 3% BSA, 0.1% Triton X-100 for 1h at room temperature. Primary anti-Flag M2 antibodies were diluted 500X in the same solution and incubated overnight at 4°C. Cells were washed twice in PBS and then incubated with the secondary anti-mouse antibodies (Alexa647 goat α-mouse, Thermofisher cat# R37121) in PBS supplemented by 1% BSA and 0.1% Triton X-100 for 2h at 4°C. After two PBS washes, 2 drops / mL of DAPI staining solution were added to each well. ICC pictures of expressed cytosolic scFv constructs were obtained with a Leica Thunder microscope equipped with a monochrome scientific CMOS camera. As observed previously in CHO cells for scFv WT, scFv-(G4S)3-cMyc-Flag was poorly soluble and aggregated in the cytosol similarly to the scFv-PEST KIR2.1-Flag construct (Figure 5, white arrows). The scFv-(G4S)3-cMyc-s3Flag-Flag was confirmed with higher solubility as expected from Example 3. In contrast, there was virtually no aggregation in cells transfected with scFv constructs comprising a negatively charged linker. These cells presented a homogeneous spread of scFv in the cytosol ((G4E3S)4and (G2DE2S)7G2S shown in Figure 5). These results demonstrated that negatively charged linkers improve scFv cytosolic solubility and reduce their aggregation. Of note, scFv-PEST KIR2.1-Flag and scFv-(G4S)3-cMyc-s3Flag-Flag presented a similar pI (5.33 vs 5.39) but showed a different propensity to aggregation (Figure 5). This result suggests that, although an important factor, the pI alone is not sufficient for predicting scFv intrabody solubility. Example 6. Target engagement of anti-TDP-43 scFvs intrabodies in vitro Intracellular target engagement of the anti-TDP-43 scFvs constructs was investigated in QBI-293 cells. TDP-43 is a protein primarily located in the nucleus in healthy physiological conditions but has been shown to accumulate and aggregate in the cytosol in some neurodegenerative diseases, e.g. frontotemporal dementia (FTD) (Meneses et al., 2021). A stable QBI-293 cell line was engineered for doxycycline inducible expression of a cytosolic-retained and -modified human TDP-43, as described by Porta et al. 2018. For this, the green fluorescent protein (GFP) was fused to TDP-43 N-terminus. In addition, the nuclear localisation signal (NLS) of the TDP-43 protein was mutated to prevent its nuclear import (NLSm) and promote accumulation within the cell cytosol (Porta et al., 2018). The resulting expressed protein is called GFP-TDP-43-NLSm. QBI-293 cells were grown as described in Example 5. Every two passages the medium was supplemented with 400 µg / mL G418 (Corning, #61234RG) and 2 µg / mL puromycin (Sigma Aldrich, #P8833) for selective maintenance of iGFP- NLSm QBI293 cells. Cells were transfected as described in Example 5 with scFv-(G4S)3-c-myc-His (=scFv WT), scFv-(G4S)3-c-myc-Flag, scFv-(G4S)3-c-myc-s3Flag-Flag or scFv-(G4E3S)4-c-myc- Flag. Six hours after transfection, the medium was replaced with DMEM medium supplemented by 10% tetracycline-free FBS. In the meantime, GFP-TDP-43-NLSm expression was induced by supplementing the cell medium with 1 µg / mL doxycycline (Sigma Aldrich, #D9891-5G). Cells were then grown for no longer than 2 days, fixed and immunostained with an anti-Flag or anti-His antibody as described in Example 5. The anti-Flag / His staining showed that the scFv-(G4E3S)4-cmyc-Flag construct presented no aggregation, consistent with Example 5. This was similar for the scFv-(G4S)3-c-myc-s3Flag-Flag construct (Figure 6A). In contrast, the scFv-(G4S)3-c-myc-Flag and scFv-(G4S)3-c-myc-Flag had visible aggregates (indicated by white arrows in Figure 6A). ScFv-immunostained cell pictures (using anti-His and anti-Flag antibodies) were merged to their counterparts having GFP-TDP-43-NLSm fluorescence. The resulting data showed colocalized signals between the scFvs with negatively charged linkers or scFv-c-myc-s3Flag-Flag and the cytoplasmic GFP-TDP-43 suggestive of target engagement of the intracellular soluble anti-TDP-43 scFvs. In order to confirm the target engagement, we used a proximity ligation assay (PLA) labelling (Kaushik et al., 2018) to detect the scFv / GFP-TDP-43 interaction using complementary polynucleotides bound to monoclonal antibodies against either the Flag or GFP. The proximity of both labelled polynucleotides allows hybridization and ligation that is then amplified with a DNA polymerase incorporating fluorescent nucleotides. In brief, the PLA experiment was performed following Duolink® protocol and adapted to 96-well microplates. The secondary probes were The Duolink® In Situ PLA® Probe Anti-Mouse PLUS (Sigma Aldrich, #DUO92001-100RXN) and the Duolink® In Situ PLA® Probe Anti-Rabbit MINUS (Sigma Aldrich, #DUO92005-100RXN). The scFv-TDP-43 complexes were then detected using the Duolink® In Situ Detection Reagents FarRed (Sigma Aldrich, #DUO92013-100RXN) with Duolink® In Situ Wash Buffer for Fluorescence (Sigma Aldrich, #DUO82049) and cell imaging was performed with a Leica microscope with either 20X or 40X magnification. Microscopic analysis showed PLA signals, confirming the target engagement in vitro between the scFv intrabodies and target GFP-TDP-43-NLSm (Figure 6B, dots pointed by white arrows). While the standard scFv-(G4S)3-c-myc-Flag generated very little signal (few dots), a striking target engagement improvement was observed for the charged linker construction scFv-(G4E3S)4-c-myc- Flag (many more dots in Figure 6B). In conclusion, the enhanced stability and solubility provided by the negatively charged linkers to the scFv molecule leads to a higher target engagement compared to scFv constructs with conventional (G4S)3 linker. Cellular target engagement of scFv-(G4E3S)4-c-myc-Flag construct was further evaluated with an in vitro nuclear translocation assay using rat primary neurons mimicking physiological conditions (Figure 7). In brief, rat primary cells were obtained by dissection from rat pup brain and grown at a density of 30K per well (96 well microplates) for 5 days at 37°C, 5% CO2, in 100µL neurobasal medium supplemented with B27™ (ThermoFisher, cat: 17504044). Two pAAV constructs, bearing the human synapsin 1 promoter (SYN1, functional in rat neurons) were vectorized in an adeno- associated virus 6, namely AAV6_SYN1_scFv-(G4E3S)4-c-myc-Flag and AAV6_SYN1_GFP-TDP- 43-NLSm. Rat primary neurons were transduced from day 5 with a multiplicity-of-infection (MOI) of 10K (10’000 genome copies per cell) of either the scFv-(G4E3S)4-c-myc-Flag alone or in combination with GFP-TDP-43-NLSm. Cells were then incubated for 9 additional days. After that, cells were fixed in 3.7% PFA as described in Example 5. The resulting GFP-TDP-43-NLSm and scFv- (G4E3S)4-c-myc-Flag (detected by anti-Flag antibody) expression and cellular localization were monitored by fluorescence microscopy as above. When expressed alone, the scFv-(G4E3S)4-c-myc-Flag localized almost exclusively in the nucleus (Figure 7, first column) where most of TDP-43 resides in physiological conditions, as described elsewhere (37), suggesting scFv-(G4E3S)4-c-myc-Flag transferred from the cytosol to the nucleus to engage its target. In contrast, rat primary neurons co-transduced with both scFv-(G4E3S)4-c-myc-Flag and GFP-TDP-43-NLSm constructs (Figure 7, second column) showed scFv-(G4E3S)4-c-myc-Flag primarily co-localized with GFP-TDP-43-NLSm in the cytosol. These results confirmed scFv- (G4E3S)4-c-myc-Flag target engagement with both rat and human TDP-43. Example 7. Expression and quality of secreted anti-TDP-43 scFvs with negatively charged linkers in vitro The expression and quality of secreted scFv with negatively charged linkers was investigated in vitro with CHO cells. For this, the most relevant and promising gene constructs (listed in Table 4) were subcloned in the pAAV expression vector with the coding sequence comprising a His-tag and the mouse Igk secretion peptide on the 5’ end and transfected in CHO cells. Cell supernatants were collected 12 days later and filtered through a 0.2 µm pore filter. After that, cell supernatants were dialyzed in tubes with a cut off pore membrane of 10 kDa, in PBS buffer for a total of 3 buffer exchanges at 4°C to provide an optimal resin capture. Secreted scFv-His were purified with the TALON® Superflow™ histidine-tagged protein purification resin as recommended by the manufacturer. In brief, the resin was packed in chromatography columns and equilibrated in PBS pH 8.0 supplemented by 163 mM NaCl to reach a final concentration of ~300 mM. Dialyzed cell supernatants were adjusted to pH 8.0, supplemented by 163 mM NaCl and then directly applied to the resin that was then washed by 10 column volumes (CV) using equilibration buffer supplemented by 5 mM imidazole. After that, proteins were eluted in approximately 2 CV with equilibration buffer supplemented by 150 mM imidazole. Resulting purified proteins were dialyzed in tubes with a cut off pore of 10 kDa, in PBS buffer pH 7.4 overnight, 4°C and then quantified using their respectively calculated 280 nm coefficient of extinction. Filtrated cell supernatant or His-tag-purified scFvs were analysed by separation on SDS-PAGE followed by Coomassie blue staining. In brief, protein samples were heated in loading buffer at 95°C in the absence or presence of 5 mM dithiothreitol. The samples were then separated through bis-Tris SDS-PAGE with an acrylamide gradient ranging from 12 to 4% under 120-130 volts. Finally, gels were stained with Coomassie blue. All scFv preparations separated by SDS-PAGE presented a band, corresponding to the expected molecular weight of scFv, i.e. 28 kDa (Figure 8, exemplified by arrows for scFv-(G4S)3-c-myc-His and scFv-(G4E3S)4-c-myc-Flag-His). However, the results showed a substantial amount of degraded LMW species (ranging between ~15 and ~23 kDa) for the scFv-(G4S)3-c-myc-Flag-His and scFv- (G4S)3-PEST KIR2.1-Flag-His construct, and to a lesser extent for the scFv-(G4S)3-c-myc-His construct, while no scFv degradation was observed for the scFv constructs with a negatively charged linker. The total yields of purified scFvs comprising a charged linker were approximately 10- to 20-fold higher than scFv constructs with conventional (G4S)3 linker (Table 4). Of note, comparison of the scFvs with the (G4E3S)4 negatively charged linker suggested the VL-VH order results in higher titer (Table 4) while presenting the same pI. Similarly, scFv-(G4S)3-PEST KIR2.1-Flag-His construct presented a comparable titer to scFv-(G4S)3-c-myc-Flag-His, while having a much lower pI, suggesting pI is an important but not predominant factor of scFv stability.

[0014] Table 4. Comparison of secreted purified scFv constructs titers The scFv constructs are by default with the Heavy Chain Variable Region (VH) first, followed by the linker, the Light Chain Variable Region (VL) and the peptide Tags. The scFv constructs with the VL before the VH are indicated by VL-VH in the third column. These results confirmed that negatively charged linkers can prevent scFv degradation, improve scFv expression titer, stability and thus, improve the overall quality of scFv expression. Further analysis was conducted to investigate the presence of high molecular weight aggregates and dimeric forms (as reported elsewhere for scFvs (Nelson, 2010)). His-tag purified proteins were separated in 4°C PBS buffer by size exclusion chromatography with the Superdex increase S200 resin having a globular protein separation range from 600 to 10 kDa with an exclusion size limit of 1300 kDa. The size exclusion chromatography confirmed the presence of high molecular weight species in scFv constructs (Table 5). The scFv constructs comprising the conventional (G4S)3linker presented extremely large (>700 kDa) soluble protein aggregates, which represented less than 10% of the total purified proteins as well as a substantial percentage of dimers (> 50%). Of note, no analysis on high molecular weight aggregates could be conducted for the scFv-(G4S)3-c-myc-His construct due to the chosen chromatographic conditions. The scFv constructs comprising a negatively charged linker presented high molecular weight aggregates under 150kDa and a higher percentage of monomer than the constructs with the conventional (G4S)3 linker. Remarkably, the scFv-(G4E3S)4-c-myc-Flag-His construct comprising the VL-VH order was up to 92% monomeric form, while presenting no aggregation (Table 5). Table 5. Comparison of secreted purified scFv constructs monomeric content and aggregation The scFv constructs are by default with the Heavy Chain Variable Region (VH) first, followed by the linker, the Light Chain Variable Region (VL) and the peptide Tags. The scFv constructs with the VL before the VH are indicated by VL-VH in the third column. HMW: High Molecular Weight. kDa: kilo Dalton. These results confirmed that negatively charged linkers can reduce scFv aggregation and promote scFv expression and secretion in its monomeric form. Moreover, the VL-VH order can further reduce high molecular weight species and increase monomer percentage substantially. Example 8. Target binding affinity and thermal stability of secreted anti-TDP-43 scFvs with negatively charged linkers in vitro The scFv constructs from Example 7 (listed in Table 4 and 5) were further evaluated for their binding affinity to human TDP-43 as well as for their thermostability as an indicator of functionality and proper protein folding, respectively. Binding affinity was evaluated by bio-layer interferometry (BLI), Octet®. In brief, all steps were performed at 30°C, under 1000 rpm shaking speed. First, streptavidin A biosensor tips were coated for 10 min with 100 nM C-terminal TDP-43 peptide dissolved in reaction buffer consisting of PBS pH 7.4 supplemented by 0.1% bovine serum albumin and 0.02% Tween. Biosensor tips were then regenerated in 50 mM glycine pH 2.0 for 30 seconds prior to be used. Purified protein samples were prepared in reaction buffer using 2-fold serial dilutions with concentrations starting from 250 nM down to 1.953 nM. Diluted samples were associated to the coated biosensor tips for 15 min and then dissociated in reaction buffer for 10 min. Finally, biosensor tips were regenerated as above prior to repeating the experiment with other purified scFvs. Curves were integrated and the resulting KD, Kon and Koff were calculated using the Octet analysis software. Thermostability was determined by differential scanning fluorimetry (DSF) using the Protein Thermal Shift™ Dye Kit (Applied Biosystems, cat# 4461146) following the supplier recommendations, in 96 well microplate using a qPCR thermocycler system. In brief, 5 µg samples were tested in a final volume of 20 µL supplier dye reagent with a thermal increase starting at 25°C and consecutive steps of 0.05°C per seconds up to 99.9°C. All scFv constructs presented a comparable KD of approximately ~8 nM, except for the scFv constructs with (G4E3S)4 (with VL-VH orientation) and (G2DE2S)7G2S negatively charged linkers that showed 24.2 and 17.9 nM KD, respectively. Several hypotheses can explain this discrepancy in binding affinity, such as the increase in scFv monomeric form (resulting in more monovalent than bivalent binding) or change in the scFv VH and VL bonding and thus conformation of the scFv. Nevertheless, these results were consistent with the affinity measured in Table 1 of Example 1 and the scFvs retained nM affinity for their target antigen. Regarding thermostability, scFv constructs with negatively charged linker presented approximately 2 to 3°C higher melting temperature than the scFvs with the conventional (G4S)3 linker. These results confirmed negatively charged linkers improve secreted scFv thermal stability while retaining their target binding affinity.

[0015] Table 6. Comparison of scFv constructs target affinity and thermostability Kon: association rate; Koff dissociation rate; KD: binding affinity; Tm: melting temperature. Example 9. Effect of additional negative charges on scFv negatively charged linkers and secreted scFv characterization The effect of further negative charges in the negatively charged linkers was evaluated, as well as replacement of the glutamate residues by aspartates, and inversion of VH-VL by VL-VH in the scFv constructs. For this, 13 different secreted scFv pAAV constructs were prepared (listed in Table 7), all comprising a Flag followed by a C-terminal His-Tag and expressed under the CMV promoter. Gene constructs were subcloned as above with the mouse Igk secretion peptide coding sequence on the 5’ end. Proteins were expressed in CHO cells for 12 days and then purified nearly to homogeneity using an IMAC resin, dialyzed in PBS and quantified using their calculated coefficient of extinction at 280 nm, as in Example 7. Resulting purified proteins were compared for the expression titers, aggregates and binding affinity to TDP-43.

[0016] Table 7. Comparison of secreted purified scFv constructs titers ScFv constructs with negatively charged linkers presented at least 3-fold higher titers than the constructs with a classical GS linker (Table 7). The scFv titers increased with the number of negatively charged residues in the negatively charged linkers, with scFv-(G4E5S)4-c-myc-Flag-His reaching a titer of about 200 mg / L. Interestingly and consistently with Example 7, the VL-VH order provided higher titer than the VH-VL format. Purified proteins were then analysed by size exclusion chromatography for monomeric, dimeric, and high molecular weight content with the Superdex increase S200 in 4°C PBS buffer as in Example 1. All scFv constructs comprising a negatively charged linker showed a higher % of monomeric scFv compared to the construct comprising the conventional (G4S)3 linker. Consistent with Example 7, the scFv construct comprising the VL-VH order and the (G4E3S)4 charged linker presented a high content of monomeric scFv (92% monomers, Table 8, Figure 9A). The results suggest that an increase in the number of glutamate or aspartate residues in the negatively charged linker provides a higher % of monomeric scFv, with all scFv constructs comprising a negatively charged linker with at least 4 glutamate or aspartate residues in their motif reaching more than 90% of scFv monomers, and no high molecular weight aggregates (Table 8, Figure 9B). The supplementary negative charges decreased the scFv pI, but also increased the length of the linker and thereby molecular weight as seen in the chromatograms with monomers excluded in a smaller elution volume (Figure 9A and 9B). It is also possible that the longer negatively charged linker stabilized the scFv and VL / VH presentation folding. To confirm that all scFv constructs were functional, their affinity to TDP-43 was evaluated by BLI as in Example 8. Interestingly, the resulting KDs highlighted ~5- to 10-fold lower values for scFvs having at least 4 glutamate or aspartate residues in the linker motif (Table 8). Finally, the KD values indicate that the newly design scFv approach with negatively charged linkers retains and possibly enhances their functionality, along with an improved solubility and stability, increase in secretion titer and significantly lower aggregation. In addition, newly purified scFvs thermostability was tested by DSF using the same procedure as in Example 8 and was compared to previously purified scFvs. Overall, the increased thermostability provided by the negatively charged linkers and additional charges was similar and ranged from 3 to 4.5°C compared to scFvs with a conventional GS linker. There were no clear differences between glutamate and aspartate repetitions, either with 3, 4 or 5 negative charge repeats, indicating that at least 3 negative charge repeats are sufficient to improve thermostability by up to 4.5°C. Finally, the VL-VH order increased the melting temperature (Tm) of ~2°C for the scFvs comprising the (G4E3S)3and (G4E3S)4linkers suggesting this orientation result in more thermostable scFv. Of note, in this case the VL-VH order generally resulted in improved scFv features (higher expression titer, higher % of monomeric expression, reduced aggregation and increased thermostability). Table 8. Comparison of purified scFvs monomeric content and affinity to target pI: Isoelectric point; HMW: High Molecular Weight; KD: binding affinity. Example 10. PEST-like functionality of the charged linkers. The next step was to monitor and possibly enhance the cellular clearance of scFvs through either the proteasome or lysosome cell mediated autophagy (CMA) pathways. In the case of the CMA pathway, it is known that alpha-synuclein, Tau, TDP-43 and other proteins can be metabolized intracellularly thanks to the presence of KFERQ sequences (SEQ ID NO: 5), but not their aggregates due to poorly accessible KFERQ sites (caused by hindrance; see Kaushik et al., 2018; Huang et al., 2014). To this end, representative scFv candidates were subcloned in the same pAAV expression vector as in Example 2 but driven by the doxycycline inducible TRE3G promoter (Gossen et al., 1992; Loew et al., 2010; Loetscher et al., 1991). The aim was to monitor cytosolic expressed proteins over time following doxycycline induction. For this, scFv-(G4S)4-Flag was used as a poorly soluble / aggregating control and scFv-(G4E3S)4-Flag as a soluble form with a predicted PEST-like motif thanks to the negatively charged linkers comprising at least 3 glutamate repeats (see Table 3). Two additional scFv constructs were tested with KFERQ-hPEST KIR2.1 (30) or KFERQ-PEST mODC (mouse ornithine decarboxylase PEST sequence) (Joshi et al., 2012; Loetscher et al., 1991) sequences fused to the C- terminal of scFv-(G4E3S)4-Flag. The KFERQ sequence was included prior to the PEST sequences in order to increase the probability of scFv cell clearance by cell mediated autophagy. Furthermore, and as for the above, both constructs would benefit from the PEST-like sequence of the negatively charged linker. To identify a time frame where scFv clearance could be monitored, triplicates of transfected CHO cells were induced with a single dose of doxycycline of 10 ng / mL. This concentration was found by titration as non-saturating and in the early linear range for induced scFv expression. Because doxycycline half-life is 24h, it was considered that expression induction was ended or very poor after 48h. As shown in Figure 10, total cellular proteins were analysed for scFv presence by SDS-PAGE followed by Western blot after 24, 48 and 72h and the scFv band intensities were integrated as performed in Example 3. Beta-tubulin was used to normalize the protein content of the same samples using rabbit polyclonal anti-beta tubulin antibody (Abcam, cat# ab6046A), diluted 2000X. Consistent with previous Examples, the scFv-(G4S)4-Flag was not cleared from the cytoplasm, which led to rapid aggregation. In contrast, scFv-(G4E3S)4-Flag was significantly cleared after 72h, with a mean 24h / 72h clearance ratio of ~1.8. The improved clearance of the scFv construct with the negatively charged linker may be explained by its better solubility and the presence of the PEST-like motif. Finally, both scFv-(G4E3S)4-Flag-KFERQ-PEST constructs were cleared significantly within 72h, with again, a tendency for faster mean clearance ratios of 2.5 for KFERQ-hPEST KIR2.1 and 3.2 for KFERQ-PEST mODC compared to the scFv-(G4E3S)4-c-myc-Flag. These results confirmed the functionality of the negatively charged linker PEST-like motif to promote degradation of the scFv. Example 11. Expression and properties of clinical-stage antibodies redesigned with negatively charged linkers of the invention The properties of the negatively charged linkers were further confirmed in antibodies having different targets and formats. Five clinical-stage antibodies comprising conventional glycine-serine linkers were modified to comprise negatively charged linkers according to the invention as set out in Table 9. Corresponding negatively charged linkers were designed by introducing 4 or 5 glutamate residues per repeating Gly-Ser motif. Table 9. Clinical antibodies and linkers *Brolucizumab is a FDA-approved drug. **FMC63 scFv is part of the chimeric antigen receptor of the FDA-approved cell therapy axicabtagene ciloleucel. ***Blinatumomab is a FDA-approved drug comprising a (G4S)3 and a (G2S)4G2 (SEQ ID NO: 100) glycine-serine linker, only (G4S)3 was replaced with a negatively charged linker. The antibodies comprising either their original glycine-serine linker or the corresponding negatively charged linker were produced with a C-terminal His-tag in CHO cells and purified as described in Example 7. The properties of the antibodies and antibody titers in cell culture media were determined as described in previous Examples (in silico pI determination as described in Example 3, antibody titer as described in Example 7, thermostability as described in Example 8) and are reported in Table 10 and Figure 11.

[0017] Table 10. Comparison of clinical-stage antibodies properties and titers The replacement of the conventional glycine-serine linker with a negatively charged linker according to the invention resulted in a decrease in pI ranging from -1.32 to -3.27, with overall pIs under 6.0. Pexelizumab, FMC63, Vobarilizumab and Blinatumomab redesigned with a negatively charged linker according to the invention showed an improved thermostability (+1.5 to +4.8 °C) and an increase in purified antibody titer from 1.4-fold (Vobarilizumab) to 70.5-fold (Pexelizumab). Advantageously, FMC63 redesigned with a negatively charged linker according to the invention showed an improved stability and solubility after a single freeze / thaw cycle (Figure 12A) compared to FMC63 with the original glycine-serine linker that displayed visible aggregates despite a lower concentration in solution (Figure 12B). As expected, Brolucizumab redesigned with a negatively charged linker according to the invention showed similar thermostability and purified titer compared to Brolucizumab with the original glycine- serine linker. These results support that the acidic pI of Brolucizumab with the original glycine-serine linker was already sufficiently low (i.e. under 6) to provide an optimal thermostability and expression level, such that a further decrease in pI from the introduction of negatively charged linker did not result in a further improvement of these properties. In contrast, Pexelizumab, FMC63, Vobarilizumab and Blinatumomab (which all presented a pI above 6 with the original glycine-serine linker) showed a substantially improved thermostability and expression titer when redesigned with a negatively charged linker of the invention which decreased the pI under 6. These results further demonstrate that the use of a negatively charged linker according to the invention can improve the thermostability, solubility, stability and expression titer of fusion proteins. Example 12. Evaluation of distribution and percentage of G, E / D, S amino acids in negatively charged fusion protein linkers The aim of this study was to evaluate the effect of amino acid distribution and percentage within the negatively charged fusion protein linker sequences on the biophysical properties of scFv constructs comprising the anti-TDP-43 antibody VH and VL from Examples 1 to 10. To this end, a series of novel linker variants were designed, introduced into the scFv constructs, and assessed based on multiple performance metrics, including expression titers, thermostability, and binding affinity to a peptide of the TDP-43 protein comprising the scFv epitope. Linker compositions were engineered to include a varying percentage and distribution of glutamate (E), aspartate (D), glycine (G) and serine (S) amino acid residues. The evaluated linker sequences are presented in Table 11. All negatively charged fusion protein linkers decreased the scFv pIs from 8.33 (with the conventional (G4S)3 linker) to 5.5 or less. Remarkably, all the negatively charged linkers had a PEST value higher than 5, indicating potential faster proteasome clearance if produced as intracellular antibodies. The highest PEST scores were attributed to linker A and G with respectively 22.64 and 22.48.

[0018] Table 11. Evaluated scFv linkers and scFv in silico properties The following protein parameters were calculated using the Expasy website (Gasteiger et al., 2005): isoelectric point (pI), net charge, grand average of hydropathicity (GRAVY) (Kyte et al., 1982) score, aliphatic index (AI), insolubility index (Ins. Ind.) and molecular weight (MW). PEST values were calculated with the Emboss epestfind algorithm (Rogers et al., 1986).

[0019] The scFvs with the negatively charged linkers A to H were compared to the scFvs with the (G4S)3 conventional linker and the (G4E3S)4negatively charged linker used in previous Examples. The scFvs comprised a C-terminal cMyc-His or Flag-cMyc-His tag and were expressed under the CMV promoter. A mouse Igκ secretion signal was added at the 5′ end for protein secretion. All new scFv gene constructs were designed in VH-VL orientation and cloned into pAAV plasmids. CHO cells were transfected in 25 mL medium and grown in shaking flasks at 32°C for 12 days. Cells were then harvested by centrifugation. Corresponding supernatants were recovered and filtered through 0.2 µm filters. The purification protocol of Example 9 was optimized to recover larger recombinant protein quantities by dialyzing the filtered cell supernatants 4 times prior to scFv capture, in PBS buffer at 4°C (5 h minimum per dialysis). Proteins were then purified using an IMAC resin, with a first wash in PBS pH 8.0 supplemented by 300 mM NaCl and 5 mM imidazole. Proteins were then eluted in the same buffer supplemented with 250 mM imidazole and finally dialyzed once in PBS at 4°C overnight. The resulting purified proteins were quantified using their calculated coefficient of extinction at 280 nm, as in Example 7. Purified proteins were then compared for expression titers, thermostability and binding affinity to a TDP-43 peptide. Evaluation of scFv purified titer The optimized purification protocol generally increased the purified protein titer for all scFvs constructs compared to previous Examples. The scFvs with negatively charged linkers consistently demonstrated improved titers compared to the conventional (G4S)3 linker, with titer increases ranging from 3.8- to 18-fold (Table 12). The scFv with the (G4E3S)4 linker demonstrated the third highest titer with 330 mg / L (Table 12), while linkers C and F reached noteworthy titers of 480 and 540 mg / L, respectively (Table 12). Regarding the amino acid distribution, linkers B, C, E, G and D having a random distribution of the G, S and E / D amino acids exhibited similar titers to the negatively charged linkers comprising (G- E / D-S) motif (linkers A, F, H and (G4E3S)4), with titers ranging from 480 mg / mL to 115 mg / mL and from 540 mg / mL to 205 mg / mL, respectively (Table 12). These results show that negatively charged linkers with random amino acids distribution perform similarly to negatively charged linkers bearing amino acids motifs. Table 12. Purified titers of scFv constructs with negatively charged linkers A to H Overall, the results confirm that negatively charged fusion protein linkers of varying lengths, amino acid distribution (random or motif-based) and percentage of E / D amino acid can improve fusion protein titer compared to conventional GS linkers. Evaluation of scFv monomeric content Purified proteins were separated by size exclusion chromatography to evaluate their folding quality as well as the presence of scFv monomers and higher molecular weight (HMW) forms. The fraction of monomeric (or dimeric or HMW) protein was calculated as the ratio of the monomer (or dimeric or HMW) peak area to the total area of the chromatogram, as measured by UV absorbance at 280 nm (Figure 13A and B). As previously observed, the scFvs comprising the conventional (G4S)3 linker were 66.6% monomers, 25% dimers and 8.3% HMW (Table 13). In contrast, the scFvs comprising the negatively charged linker A to H and (G4E3S)4, exhibited improved monomeric content compared to the conventional GS linker (Table 13). Apart from linker F, all negatively charged linkers resulted in scFv of at least 90% in monomeric form (Table 13). These results show that negatively charged linkers with random amino acids distribution perform similarly to negatively charged linkers bearing amino acids motifs. Table 13. Monomeric, dimeric and HMW content of scFv constructs with negatively charged linkers A to H HMW: high molecular weight. Overall, the results confirm that negatively charged fusion protein linkers of varying lengths, amino acid distribution (random or motif-based) and percentage of E / D amino acid can stabilize fusion proteins in monomeric form compared to conventional GS linkers, resulting in higher % of fusion protein monomeric form, in particular for scFv. Evaluation of scFv binding affinity and thermostability To determine whether the novel linker variants preserved the antigen-binding affinity of the anti-TDP- 43 scFv, biolayer interferometry (Octet system) was performed using the same protocol as previously described in Example 8. Furthermore, the thermostability of the corresponding scFvs was assessed using nano-differential scanning fluorimetry (nano-DSF), following the same methodology outlined in Example 9. The results are shown in Table 14. Table 14. Binding affinity and thermostability of scFv constructs with negatively charged linkers A to H KD: binding affinity. Tm: melting temperature, indicator of thermostability. Δ: difference to KD or Tm of scFv with conventional (G4S)3 linker. All scFv constructs presented similar or slightly higher KDthan the scFv with the conventional (G4S)3linker ranging from ~18 nM to 35 nM (Table 14). The slightly higher KD of scFvs comprising the linker (G₄E₃S)₄, B, C, D, E, G and H can be explained by the increase in scFv monomeric form (Table 13), resulting in more monovalent than bivalent binding. Regarding thermostability, scFv constructs with a negatively charged linker presented a higher melting temperature ranging from + 1 to + 5.6°C than the scFvs with the conventional (G4S)3linker, except for the scFv comprising linker F which retained a similar melting temperature. This finding is consistent with size-exclusion chromatography (SEC) data, which showed that the scFv comprising linker F had the lowest reduction in dimeric and HMW species compared to other scFvs with a negatively charged linkers (Table 13). Overall, these results confirm that negatively charged linkers of varying lengths, amino acid distribution (random or motif-based) and percentage of E / D amino acid can improve scFv thermal stability compared to conventional GS linkers, while retaining their target binding affinity. Summary Altogether, the results from Example 1 to 12 demonstrate that negatively charged linkers of varying lengths, of varying amino acid distribution (random or motif-based) and of varying E / D amino acids percentage can, inter alia, stabilize intracellular and secreted fusion proteins, improve their solubility, improve their expression titer, improve their percentage of monomeric form, improve intracellular clearance, and improve their thermostability compared to conventional GS linkers.

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In Seminars in Ophthalmology, vol.39, no.4, pp.251-260. Taylor & Francis, 2024. Unless defined otherwise, all technical and scientific terms used herein have the same meanings as commonly understood by one of ordinary skill in the art to which this invention belongs. All publications and patents specifically mentioned herein are incorporated by reference in their entirety for all purposes in connection with the invention. The present invention is not to be limited in scope by the specific embodiments described herein. Indeed, various modifications of the invention in addition to those described herein will become apparent to those skilled in the art from the foregoing description and accompanying figures. Such modifications are intended to fall within the scope of the appended claims. Moreover, all aspects and embodiments of the invention described herein are considered to be broadly applicable and combinable with any and all other consistent embodiments, including those taken from other aspects of the invention (including in isolation) as appropriate.

Claims

CLAIMS:

1. A fusion protein peptide linker comprising, consisting essentially of, or consisting of the amino acid sequence Z, wherein Z consists of n independent occurrence of an amino acid sequence Y, wherein each Y is independently selected from the amino acid sequence set forth as X1X2X3X4X5X6X7X8X9X10X11X12X13X14X15X16X17, wherein X1 to X17 are each independently selected from E, D, G, S or absent; at least one from X1 to X17 is G; at least one from X1to X17is E or D; at least one from X1to X17is S; n is 1 to 10; and Z is at least 16 amino acids in length.

2. The fusion protein peptide linker of claim 1, wherein at least 30% of Z amino acids are G; at least 15% of Z amino acids are D and / or E; and from 10% to 20% of Z amino acids are S.

3. The fusion protein peptide linker of claim 1 or 2, wherein from 30 to 70% of Z amino acids are G; from 15 to 55% of Z amino acids are D and / or E; and from 10% to 20% of Z amino acids are S.

4. The fusion protein peptide linker of any one of the previous claims, wherein from 30 to 50% of Z amino acids are G; from 35 to 55% of Z amino acids are D and / or E; and from 10% to 15% of Z amino acids are S.

5. The fusion protein peptide linker of any one of the previous claims, wherein Z is no more than 50 amino acids in length.

6. The fusion protein peptide linker of any one of the previous claims, wherein Z is from 18 to 45 amino acids in length, preferably from 24 to 40 amino acids in length.

7. The fusion protein peptide linker of any one of the previous claims, wherein n is at least 2.

8. The fusion protein peptide linker of any one of the previous claims, wherein n is at least 3, at least 4, preferably n is 3 or 4.

9. The fusion protein peptide linker of claims 7 or 8, wherein the amino acid sequence Y is the same for each of the n occurrences of Y.

10. The fusion protein peptide linker of any one of the previous claims, wherein at least 35% of Z amino acids are D and / or E.

11. The fusion protein peptide linker of any one of the previous claims, wherein at least 40% of Z amino acids are D and / or E.

12. The fusion protein peptide linker of any one of the previous claims, wherein at least 50% of Z amino acids are D and / or E.

13. The fusion protein peptide linker of any one of the previous claims, wherein each Y is independently selected from the amino acid sequence set forth as X1X2X3X4X5X6X7X8X9X10X11X12X13X14X15X16X17, wherein X1 is G; X2 to X6 are each independently selected from G or absent; X7 is E or D; X8 to X16 are each independently selected from E, D or absent; and X17is S.

14. The fusion protein peptide linker of claim 13, wherein X1and X2are G.

15. The fusion protein peptide linker of claim 13 or 14, wherein X1to X3are G.

16. The fusion protein peptide linker of any one of claims 13 to 15, wherein X1 to X4 are G.

17. The fusion protein peptide linker of any one of claims 13 to 16, wherein X5 and X6 are absent.

18. The fusion protein peptide linker of any one of claims 13 to 17, wherein X14 to X16 are absent.

19. The fusion protein peptide linker of any one of claims 13 to 18, wherein X12to X16are absent.

20. The fusion protein peptide linker of any one of claims 13 to 19, wherein X7 and X8 are each independently selected from E or D.

21. The fusion protein peptide linker of any one of claims 13 to 20, wherein X7 to X9 are each independently selected from E or D.

22. The fusion protein peptide linker of any one of claims 13 to 21, wherein X1and X2are G;X7 and X8 are each independently selected from E or D; and X15and X16are absent.

23. The fusion protein peptide linker of any one of claims 13 to 22, wherein X1to X3are G; X7 and X8 are each independently selected from E or D; and X6 and X14 to X16 are absent.

24. The fusion protein peptide linker of any one of claims 13 to 23, wherein X1 to X3 are G; X7to X9are each independently selected from E or D; and X6and X14to X16are absent.

25. The fusion protein peptide linker of any one of claims 13 to 24, wherein X1 to X3 are G; X7 to X9 are each independently selected from E or D; and X6 and X12 to X16 are absent.

26. The fusion protein peptide linker of any one of claims 13 to 19, wherein X1to X4are G; X7is E or D; X8to X11are each independently selected from E, D or absent; and X5 to X6 and X12 to X16 are absent.

27. The fusion protein peptide linker of any one of claims 13 to 26, wherein X1 to X4 are G; X7 to X9 are each independently selected from E or D; X10to X11are each independently selected from E, D or absent; and X5to X6and X12to X16are absent.

28. The fusion protein peptide linker of any one of claims 13 to 19 or 26, wherein X1 to X4 are G; X7 is E or D; X8 to X11 are each independently selected from E, D or absent; X5 to X6 and X12 to X16 are absent; n is 3 or 4; and the amino acid sequence Y is the same for each of the n occurrences of Y.

29. The fusion protein peptide linker of any one of claims 13 to 28, wherein X1to X4are G; X7to X9are each independently selected from E or D; X10to X11are each independently selected from E, D or absent; X5 to X6 and X12 to X16 are absent; n is 3 or 4; and the amino acid sequence Y is the same for each of the n occurrences of Y.

30. The fusion protein peptide linker of any one of claims 1 to 3, 5 to 9, 13 to 19 or 26, wherein each Y is independently selected from the amino acid sequence set forth as (G4(E / D)1-5S) and n is 3 or 4 (SEQ ID NO: 87 to SEQ ID NO: 90).

31. The fusion protein peptide linker of any one of claims 1 to 10, 13 to 27 or 30, wherein each Y is independently selected from the amino acid sequence set forth as (G4(E / D)3-5S) and n is 3 or 4 (SEQ ID NO: 88 and SEQ ID NO: 90).

32. The fusion protein peptide linker of any one of claims 1 to 3, 5 to 9, 13 to 19, 26, 28 or 30 wherein each Y is independently selected from the amino acid sequence set forth as (G4(E / D)1-5S), n is 3 or 4 and the amino acid sequence Y is the same for each of the n occurrences of Y (SEQ ID NO: 89).

33. The fusion protein peptide linker of any one of claims 1 to 10 or 13 to 32, wherein each Y is independently selected from the amino acid sequence set forth as (G4(E / D)3-5S), n is 3 or 4 and the amino acid sequence Y is the same for each of the n occurrences of Y (SEQ ID NO: 90).

34. The fusion protein peptide linker of any one of claims 1 to 3, 5, 7 to 9, 13 to 18, wherein Z amino acid sequence is any one of SEQ ID NO: 48 to SEQ ID NO:

54.

35. The fusion protein peptide linker of any one of claims 1 to 3, 5 to 9, 13 to 19, or 26, wherein Z amino acid sequence is any one of SEQ ID NO: 87 to SEQ ID NO:

90.

36. The fusion protein peptide linker of any one of claims 1 to 3, 5 to 9, 13 to 19, 26, 28 or 30, wherein Z amino acid sequence is any one of SEQ ID NO: 69 to SEQ ID NO:

73.

37. The fusion protein peptide linker of any one of claims 1 to 3, 5, 6, 8 or 13 to 14, wherein Z amino acid sequence is any one of (G4ES)3 (SEQ ID NO: 6), (G4E2S)3 (SEQ ID NO: 7), (G4E3S)3(SEQ ID NO: 8), (G4E4S)3(SEQ ID NO: 9), (G4E5S)3(SEQ ID NO: 10), (G4ES)4(SEQ ID NO: 11), (G4E2S)4(SEQ ID NO: 12), (G4E3S)4(SEQ ID NO: 13), (G4E4S)4(SEQ ID NO: 14), (G4E5S)4(SEQ ID NO: 15), (G4DS)3(SEQ ID NO: 17), (G4D2S)3(SEQ ID NO:18), (G4D3S)3 (SEQ ID NO: 19), (G4D4S)3 (SEQ ID NO: 20), (G4D5S)3 (SEQ ID NO: 21), (G4DS)4(SEQ ID NO: 22), (G4D2S)4(SEQ ID NO: 23), (G4D3S)4(SEQ ID NO: 24), (G4D4S)4(SEQ ID NO: 25), (G4D5S)4(SEQ ID NO: 26), G4E5SG3E5S (SEQ ID NO: 91), G5E4S(G4E4S)3(SEQ ID NO: 98) or (G2DE2S)7(SEQ ID NO: 101).

38. The fusion protein peptide linker of any one of claims 1 to 3, 5 to 9, 13 to 19, 26, 28, 30, 32 or 37, wherein Z amino acid sequence is any one of (G4ES)3 (SEQ ID NO: 6), (G4E2S)3 (SEQ ID NO: 7), (G4E3S)3 (SEQ ID NO: 8), (G4ES)4 (SEQ ID NO: 11), (G4E2S)4 (SEQ ID NO: 12), (G4E3S)4 (SEQ ID NO: 13), (G4E4S)4 (SEQ ID NO: 14), (G4E5S)4 (SEQ ID NO: 15), (G4D3S)4(SEQ ID NO: 24), (G4D4S)4(SEQ ID NO: 25), (G4D5S)4(SEQ ID NO: 26), preferably (G4E3S)4(SEQ ID NO: 13).

39. The fusion protein peptide linker of any one of claims 1 , 5, 6 or 8, wherein the fusion protein peptide linker consists of any one of SEQ ID NO: 6 to SEQ ID NO: 26, SEQ ID NO: 48 to SEQ ID NO: 54, SEQ ID NO:69 to SEQ ID NO: 73, SEQ ID NO: 87 to SEQ ID NO: 91, SEQ ID NO: 98, or SEQ ID NO: 101 to SEQ ID NO:

108.

40. A fusion protein comprising two polypeptides linked by a fusion protein peptide linker according to any one of the previous claims.

41. The fusion protein of claim 40, wherein the fusion protein is a binding molecule.

42. The fusion protein of claim 40 or 41, wherein the fusion protein is an antibody or antigen- binding fragment thereof, preferably an antibody or antigen binding fragment that binds to a pathological protein, a cell surface receptor, a tumour-associated antigen or tumour-specific antigen, more preferably an intrabody that binds to an intracellular target.

43. The fusion protein of any one of claims 40 to 41, wherein the fusion protein comprises or consists of a scFv, a bi-specific T-cell engager (BiTE), a Dual Affinity Retargeting Antibodies (DART), a Designed Ankyrin Repeat Proteins (DARPins), a single-domain antibody, a chimeric antigen receptor (CAR), or an antibody or antigen-binding fragment thereof fused to an immunoglobulin Fc domain.

44. The fusion protein of any one of claims 40 to 43, wherein the fusion protein comprises or consists of a scFv, a single domain antibody, preferably a VHH, or a bi-specific T-cell engager (BiTE).

45. The fusion protein of any one of claims 40 to 44, wherein the fusion protein peptide linker links a Heavy Chain Variable Region (VH) and a Light Chain Variable Region (VL) of a scFv.

46. The fusion protein of any one of claims 40 to 44, wherein the fusion protein peptide linker links two single domain antibodies.

47. The fusion protein of any one of claims 40 to 44, wherein the fusion protein peptide linker links two scFvs.

48. The fusion protein of any one of claims 40 to 44, wherein the fusion protein peptide linker links a scFv and a single domain antibody, preferably a VHH.

49. The fusion protein of any one of claims 40 to 44 or 46, wherein the fusion protein peptide linker links two VHH.

50. The fusion protein of any one of claims 40 to 49, wherein the fusion protein peptide linker exhibits at least one of the following features: a) promotes or increases the solubility of the fusion protein; and / or b) inhibits, decreases or prevents the toxicity of the fusion protein; and / or c) inhibits, decreases or prevents the aggregation of the fusion protein; and / or d) promotes or increases the expression yield of the fusion protein; and / or e) promotes or increases the intracellular degradation of the fusion protein; and / or f) promotes or increases the monomeric expression of the fusion protein; and / or g) promotes or increases the thermostability of the fusion protein; and / or i) improves or increases the purification yield of the fusion protein; and / or h) promotes, increases or maintains the target binding affinity of the fusion protein relative to a corresponding fusion protein comprising a fusion protein linker consisting of Gly and Ser amino acids, preferably a (G4S)3 peptide linker (SEQ ID NO: 3).

51. The fusion protein of any one of claims 40 to 50, wherein the fusion protein peptide linker decreases the pI of the fusion protein relative to the pI of a corresponding fusion protein comprising a fusion protein peptide linker consisting of Gly and Ser amino acids.

52. A method for decreasing the pI of a fusion protein comprising two polypeptides linked by a fusion protein linker, the method comprising introducing negatively charged amino acids in the fusion protein linker to obtain a fusion protein with a pI equal to or below 6.

53. The method of claim 52, wherein the negatively charged amino acids are Glu and / or Asp.

54. Use of a fusion protein peptide linker of any one of claims 1 to 39 for decreasing the pI of a fusion protein to a value of 6 or less.

55. The method of claim 52 or 53, or use according to claim 54, wherein the pI is equal to or below 5.5.

56. The method of claim 52, 53 or 55, or use according to claim 54 or 55, wherein the pI is from 4.5 to 5.

5.

57. A method for promoting or increasing the solubility of a fusion protein, the method comprising providing a fusion protein comprising two polypeptides linked by the fusion protein peptide linker of any one of claims 1 to 39 and expressing the fusion protein in a cell or a cell-free expression system.

58. A method for inhibiting, decreasing or preventing the toxicity of a fusion protein, the method comprising providing a fusion protein comprising two polypeptides linked by the fusion protein peptide linker of any one of claims 1 to 39 and expressing the fusion protein in a cell or a cell-free expression system.

59. A method for inhibiting, decreasing or preventing the aggregation of a fusion protein, the method comprising providing a fusion protein comprising two polypeptides linked by the fusion protein peptide linker of any one of claims 1 to 39 and expressing the fusion protein in a cell or a cell-free expression system.

60. A method for promoting or increasing intracellular degradation of a fusion protein, the method comprising providing a fusion protein comprising two polypeptides linked by the fusion protein peptide linker of any one of claims 1 to 39 and expressing the fusion protein in a cell or a cell-free expression system.

61. A method for promoting or increasing the expression of a fusion protein, the method comprising providing a fusion protein comprising two polypeptides linked by the fusion protein peptide linker of any one of claims 1 to 39 and expressing the fusion protein in a cell or a cell-free expression system.

62. A method for promoting or increasing the thermostability of a fusion protein, the method comprising providing a fusion protein comprising two polypeptides linked by the fusion protein peptide linker of any one of claims 1 to 39 and expressing the fusion protein in a cell or a cell-free expression system.

63. A method for promoting or increasing the monomeric expression of a fusion protein, the method comprising providing a fusion protein comprising two polypeptides linked by the fusion protein peptide linker of any one of claims 1 to 39 and expressing the fusion protein in a cell or a cell-free expression system.

64. A method for improving or increasing the purification yield of a fusion protein, the method comprising providing a fusion protein comprising two polypeptides linked by the fusionprotein peptide linker of any one of claims 1 to 39, expressing the fusion protein in a cell or a cell-free expression system and isolating the fusion protein by performing a separation technique based on the fusion protein charge.

65. A method for promoting or increasing the target binding affinity of a fusion protein, the method comprising providing a fusion protein comprising two polypeptides linked by the fusion protein peptide linker of any one of claims 1 to 39 and expressing the fusion protein in a cell or a cell-free expression system.

66. Use of a fusion protein peptide linker of any one of claims 1 to 39 for promoting or increasing the solubility of a fusion protein.

67. Use of a fusion protein peptide linker of any one of claims 1 to 39 for inhibiting, decreasing or preventing the toxicity of a fusion protein.

68. Use of a fusion protein peptide linker of any one of claims 1 to 39 for inhibiting, decreasing or preventing the aggregation of a fusion protein.

69. Use of a fusion protein peptide linker of any one of claims 1 to 39 for promoting or increasing intracellular degradation of a fusion protein.

70. Use of a fusion protein peptide linker of any one of claims 1 to 39 for promoting or increasing the expression of a fusion protein.

71. Use of a fusion protein peptide linker of any one of claims 1 to 39 for promoting or increasing the thermostability of a fusion protein.

72. Use of a fusion protein peptide linker of any one of claims 1 to 39 for promoting or increasing the monomeric expression of a fusion protein.

73. Use of a fusion protein peptide linker of any one of claims 1 to 39 method for improving or increasing the purification yield of a fusion protein.

74. Use of a fusion protein peptide linker of any one of claims 1 to 39 for promoting or increasing the target binding affinity of a fusion protein.

75. The method of any one of claims 52, 53 or 55 to 65, or use according to any one of claims 54 to 56 or 66 to 74, wherein the fusion protein is a binding molecule.

76. The method of any one of claims 52, 53, 55 to 65 or 75, or use according to any one of claims 54 to 56 or 66 to 75, wherein the fusion protein is an antibody or antigen-binding fragment thereof, preferably an intrabody that binds to an intracellular target.

77. The method of any one of claims 52, 53, 55 to 65, 75 or 76, or use according to any one of claims 54 to 56 or 66 to 76, wherein the fusion protein comprises or consists of a scFv, a bi-specific T-cell engager (BiTE), a Dual Affinity Retargeting Antibodies (DART), a Designed Ankyrin Repeat Proteins (DARPins), a single-domain antibody, a chimeric antigen receptor (CAR), an antibody or antigen-binding fragment thereof fused to an immunoglobulin Fc domain.

78. The method of any one of claims 52, 53, 55 to 65 or 75 to 77, or use according to any one of claims 54 to 56 or 66 to 77, wherein the fusion protein consists of a scFv.

79. A nucleic acid encoding the fusion protein of any one of claims 40 to 51.

80. An expression vector comprising the nucleic acid of claim 79.

81. A host cell comprising the nucleic acid of claim 79 or the expression vector of claim 80.

82. A cell-free expression system comprising the nucleic acid of claim 79, or the expression vector of claim 80.

83. A method for producing the fusion protein of any one of claims 40 to 51, comprising the steps of: a) culturing the host cell of claim 81 or the cell-free expression of claim 82 under conditions suitable for producing the fusion protein, and b) isolating the fusion protein.

84. The expression vector of claim 80, wherein the vector is a wild-type or engineered viral vector.

85. The expression vector of claim 80 or 84, wherein the vector is an adeno-associated virus (AAV), an adenovirus or a lentivirus.

86. The expression vector of claim 80, 84 or 85, wherein the vector is an AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAVrh.10, AAV- BR1, AAV-S, AAV-F, AAV-PHP.eB, AAV9-PHP-V, AAV.CAP-B10, AAV.CAP-B22, AAV.CAP-Mac, AAV VCAP-103, AAV bCap 1, AAV VCAP-100, AAV1RX, AAV1R6 or AAV1R7.

87. A pharmaceutical composition comprising the fusion protein of any one of claims 40 to 51, nucleic acid of claim 79 or an expression vector of any one of claims 80 or 84 to 86 and a pharmaceutically acceptable carrier and / or excipient.

88. The fusion protein of any one of claims 40 to 51, nucleic acid of claim 79, expression vector of any one of claims 80 or 84 to 86 or the pharmaceutical composition of claim 87 for use in therapy.

89. The fusion protein, nucleic acid, expression vector or pharmaceutical composition for use according to claim 88, wherein the fusion protein is a binding molecule and binds a pathological protein, a cell surface receptor, a tumour-associated antigen or tumour-specific antigen, preferably an intracellular pathological protein.

90. The fusion protein, nucleic acid, expression vector or pharmaceutical composition for use according to claim 89, wherein the binding molecule binds TAR DNA-Binding Protein 43 (TDP-43), amyloid-beta, alpha-synuclein, Tau, apoptosis-associated speck-like protein containing a CARD (ASC) or huntingtin protein (HTT).

91. The fusion protein, nucleic acid, expression vector or pharmaceutical composition for use according to claim 90, wherein the binding molecule binds TDP-43, and the condition, disease or disorder is associated with pathological TDP-43 or a TDP-43 proteinopathy.

92. The fusion protein, nucleic acid, expression vector or pharmaceutical composition for use according to claim 91, wherein the condition, disease or disorder associated with pathological TDP-43 or TDP-43 proteinopathy is Frontotemporal dementia (FTD), amyotrophic lateral sclerosis (ALS), Alzheimer's disease (AD), Parkinson's disease (PD), Chronic Traumatic Encephalopathy (CTE), limbic-predominant age-related TDP-43 encephalopathy (LATE), or multiple sclerosis.

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