Half-life modulation by a tag comprising at least one constant domain of an immunoglobulin light chain

Abstract

The present invention provides a method of modulating a molecular mass of a fusion polypeptide, wherein the fusion polypeptide comprises a target polypeptide fused to a tag suitable for affinity purification or detection of the target polypeptide, wherein the tag comprises at least one constant domain of an immunoglobulin light chain or orthologous polypeptide or fragment thereof and wherein the molecular mass and / or an isoelectric point of the fusion polypeptide is modulated by increasing the molecular mass of the tag via combining the one constant domain of an immunoglobulin light chain or orthologous polypeptide or fragment thereof with one or more further constant domain(s) of an immunoglobulin light chain or orthologous polypeptide(s) or fragment(s) thereof; wherein the constant domain of the immunoglobulin light chain or orthologous polypeptide or fragment thereof of the tag is monomeric; and wherein optionally the target polypeptide is fused to the tag via a first linker. The invention further provides a fusion polypeptide obtained or obtainable by this method. The invention also provides a tag and a fusion polypeptide comprising the tag.

Description

[0001] HALF-LIFE MODULATION BY A TAG COMPRISING AT LEAST ONE CONSTANT DOMAIN OF AN IMMUNOGLOBULIN LIGHT CHAIN

[0002] CROSS-REFERENCE TO RELATED APPLICATION

[0003] The present application claims the benefit of priority of EP Patent Application No.

[0004] 24179805.7 filed 4 June 2024 and of EP Patent Application No. 24179814.9 filed 4 June 2024, the content of which is hereby incorporated by reference in its entirety for all purposes.

[0005] TECHNICAL FIELD OF THE INVENTION

[0006] The present invention provides a method of modulating a molecular mass of a fusion polypeptide, wherein the fusion polypeptide comprises a target polypeptide fused to a tag suitable for affinity purification or detection of the target polypeptide, wherein the tag comprises at least one constant domain of an immunoglobulin light chain or orthologous polypeptide or fragment thereof and wherein the molecular mass of the fusion polypeptide is modulated by increasing the molecular mass of the tag via combining the one constant domain of an immunoglobulin light chain or orthologous polypeptide or fragment thereof with one or more further constant domain(s) of an immunoglobulin light chain or orthologous polypeptide(s) or fragment(s) thereof; wherein the constant domain of the immunoglobulin light chain or orthologous polypeptide or fragment thereof of the tag is monomeric; and wherein optionally the target polypeptide is fused to the tag via a first linker and a fusion polypeptide obtained or obtainable by this method. The invention further provides a tag suitable for affinity purification or detection of a fusion partner, wherein the fusion partner is a target polypeptide and wherein the tag comprises at least one constant domain of an immunoglobulin light chain or orthologous polypeptide or fragment thereof and optionally a first linker that fuses the tag to the target polypeptide, and wherein the constant domain of the immunoglobulin light chain or orthologous polypeptide or fragment thereof is monomeric. The invention also provides a fusion polypeptide comprising a target polypeptide and a tag suitable for affinity purification or detection of the target polypeptide, wherein the tag comprises at least one constant domain of an immunoglobulin light chain or orthologous polypeptide or fragment thereof and optionally a first linker that fuses the tag to the target polypeptide, and wherein the constant domain of the immunoglobulin light chain or orthologous polypeptide or fragment thereof is monomeric. Also, the invention provides a pharmaceutical composition comprising the fusion polypeptide and optionally a pharmaceutically acceptable carrier, diluent, or excipient. The invention provides a nucleic acid for expression of the fusion polypeptide comprising a nucleic acid sequence encoding the target polypeptide and a nucleic acid sequence encoding the tag, comprising a nucleic acid sequence encoding at least one constant domain of an immunoglobulin light chain or orthologous polypeptide or fragment thereof, and optionally a nucleic acid sequence encoding the peptide linker. The invention further provides a pharmaceutical composition comprising the nucleic acid and optionally a pharmaceutically acceptable carrier, diluent, or excipient. Furthermore, the invention provides a method for purifying an expressed fusion polypeptide, comprising providing the fusion polypeptide expressed by cells, optionally transfected with the nucleic acid, provision of a ligand directed to the tag of the fusion polypeptide, binding of the ligand to the tag, and eluting the fusion polypeptide from the ligand. Further, the invention provides a method of modulating the plasma half-time of a fusion polypeptide in an organism, wherein the fusion polypeptide is the product of the method mentioned above or wherein the fusion polypeptide is the fusion polypeptide mentioned above, and wherein the molecular mass of the fusion polypeptide is at least above the filtration limit of a kidney of an organism. Also, the invention provides a method of detecting the fusion polypeptide, comprising providing a sample, which may comprise the fusion polypeptide, and detecting the fusion polypeptide in the sample by using a detecting agent directed to the tag or the target polypeptide.

[0007] BACKGROUND ART

[0008] Recombinant proteins as biologies, but also as diagnostics have opened up entirely novel ways of how to diagnose and treat severe human diseases, most notably cancer. One class of proteins dominates this sector by a large margin: monoclonal antibodies (Carter et al., 2018). The high affinity and specificity with which antibodies can be raised against most targets is one ingredient to their success. A further ingredient is the fact that modern antibody formats also allow the targeting of multiple antigens simultaneously, e.g., bispecific T cell engagers that bind T cell surface proteins as well as cancer cell surface epitopes and thus allow to recruit T cells to certain cell populations (Carter et al., 2018, Spiess et al., 2015). Lastly, developability is essential when it comes to biologies. A further foundation that antibody developments rely on is thus their well-established production and purification process. Most therapeutic antibodies are produced in Chinese hamster ovary (CHO) cells which allow for disulfide bond formation and glycosylation in antibodies (Kunert et al., 2016). The purification of antibodies generally relies on the bacterially derived protein A, which binds to the antibody C-terminal fragment crystallizable (Fc) region with high affinity and specificity (Deisenhofer, 1981 , Deis et al., 2015). The Fc region of antibodies is not only a well- suited basis for purification, but also mediates effector functions, among others Fc receptor binding (Bauer-Smith et al., 2023). This can induce downstream functional effects like opsonization, but also contributes to the long half-life of antibodies in the organism via binding to the neonatal Fc receptor (FcRn) (Roopenian et al., 2007). If antibodies are taken up by cells, binding to FcRn induces recycling and release instead of lysosomal degradation, which otherwise is the default for proteins taken up via endocytosis by cells. This FcRn-dependent mechanism significantly extends the halflife of antibodies in the organism (Roopenian et al., 2003). Taking these considerations into account, it comes as no surprise that Fc fusions of other proteins than antibodies are used as biologies due to their simplified, standardized purification and long half-life mediated by Fc (Tan et al., 2018). However, using the Fc as a tag also comes with major drawbacks: The Fc contains several disulfide bonds and is glycosylated and thus demands for proper production hosts and conditions to avoid unwanted side reactions like misfolding, aggregation, or mis-oxidation - but also to control the biological functions of the glycan itself (Dalziel et al., 2014). Furthermore, the Fc is a homodimer, and fusing a protein to Fc thus will induce homodimerization which may not correspond to the desired assembly state of the fusion partner. Lastly, effector functions and extended half-life mediated by Fc may be undesired for certain drugs. Together, this has led to the development of several other technologies to improve production, stability, solubility, and / or half-life of non-antibody biologies. These fusions and modifications include human serum albumin (HSA) (Andersen et al., 2011 ), transferrin (Kim et al., 2010), maltose binding protein (MBP) (Lebendiker et al., 2011 ), carboxyterminal peptide (CTP) (Duijkers et al., 2002), PASylation (Schlapschy et al., 2013), or PEGylation (Jevsevar et al., 2010, Harris et al., 2003). Such approaches are generally needed for non-antibody biologies like cytokines and enzymes since these proteins lack a common structural element and may be intrinsically unstable and / or have a too short half-life in the body. In principle, these biologies open up modes of action that are not attainable by antibodies and are thus very attractive as next-generation biopharmaceuticals (Xue et al., 2021 ). The short half-life of biologies, especially those which are smaller than 70 kDa, is due to the glomerular filtration of the kidney. Furthermore, peptides and small proteins which are negatively charged are less readily filtrated by the kidney than positively charged peptides. To circumvent the fast elimination of peptides or proteins via glomerular filtration they are mostly fused to tags like the above-described Fc, HSA, transferrin, PAS, or PEG. The fusion could be accomplished during translation by using a DNA which encodes the fusion protein, e.g. if PASylation is used. The fusion could be made after translation by fusing the tag, e.g. PEG, to an amino acid of the protein (Strohl, 2015; Zaman et al., 2019). Despite progress in the field, a generic fusion tag that increases ease of production, can serve as an affinity handle for purification, and improves expression and biophysical characteristics while being functionally inert is still missing.

[0009] SUMMARY OF THE INVENTION

[0010] The present invention provides in a first aspect a method of modulating a molecular mass of a fusion polypeptide, wherein the fusion polypeptide comprises a target polypeptide fused to a tag suitable for affinity purification or detection of the target polypeptide, wherein the tag comprises at least one constant domain of an immunoglobulin light chain or orthologous polypeptide or fragment thereof and wherein the molecular mass of the fusion polypeptide is modulated by increasing the molecular mass of the tag via combining the one constant domain of an immunoglobulin light chain or orthologous polypeptide or fragment thereof with one or more further constant domain(s) of an immunoglobulin light chain or orthologous polypeptide(s) or fragment(s) thereof; wherein the constant domain of the immunoglobulin light chain or orthologous polypeptide or fragment thereof of the tag is monomeric; and wherein optionally the target polypeptide is fused to the tag via a first linker.

[0011] In a second aspect, the invention provides a fusion polypeptide obtainable or obtained by the method of the first aspect. In a third aspect, the invention provides a tag suitable for affinity purification or detection of a fusion partner, wherein the fusion partner is a target polypeptide and wherein the tag comprises at least one constant domain of an immunoglobulin light chain or orthologous polypeptide or fragment thereof and optionally a first linker that fuses the tag to the target polypeptide, and wherein the constant domain of the immunoglobulin light chain or orthologous polypeptide or fragment thereof is monomeric.

[0012] In a fourth aspect, the invention provides a fusion polypeptide comprising a target polypeptide and a tag suitable for affinity purification or detection of the target polypeptide, wherein the tag comprises at least one constant domain of an immunoglobulin light chain or orthologous polypeptide or fragment thereof and optionally a first linker that fuses the tag to the target polypeptide, and wherein the constant domain of the immunoglobulin light chain or orthologous polypeptide or fragment thereof is monomeric.

[0013] In a fifth aspect, the invention provides a pharmaceutical composition comprising a fusion polypeptide of the second and / or fourth aspect and optionally a pharmaceutically acceptable carrier, diluent, or excipient.

[0014] In a sixth aspect, the invention provides a nucleic acid for expression of the fusion polypeptide of the second and / or fourth aspect comprising a nucleic acid sequence encoding the target polypeptide of the first and third aspect and a nucleic acid sequence encoding the tag mentioned in the first and / or third aspect, comprising a nucleic acid sequence encoding at least one constant domain of an immunoglobulin light chain or orthologous polypeptide or fragment thereof, and optionally a nucleic acid sequence encoding the first peptide linker of the first, third or fourth aspect.

[0015] In a seventh aspect, the invention provides a pharmaceutical composition comprising the nucleic acid of the sixth aspect and optionally a pharmaceutically acceptable carrier, diluent, or excipient.

[0016] In an eighth aspect, the invention provides a method for purifying an expressed fusion polypeptide, comprising providing a fusion polypeptide of the second or fourth aspect expressed by cells, optionally transfected with a nucleic acid of the fifth aspect, provision of a ligand directed to the tag of the fusion polypeptide, binding of the ligand to the tag, and eluting the fusion polypeptide from the ligand.

[0017] In a ninth aspect, the invention provides, a method of modulating the plasma half-life of a fusion polypeptide in an organism, wherein the fusion polypeptide is the product of the method of the first aspect or wherein the fusion polypeptide is the fusion polypeptide of the second and / or fourth aspect, and wherein the molecular mass of the fusion polypeptide is at least above the filtration limit of a kidney of an organism.

[0018] In a tenth aspect, the invention provides, a method of detecting the fusion polypeptide of the second and / or fourth aspect, comprising providing a sample, which may comprise the fusion polypeptide, and detecting the fusion polypeptide in the sample by using a detecting agent directed to the tag or the target polypeptide.

[0019] In an eleventh aspect, the invention provides a method of detecting the fusion polypeptide of the second and / or fourth aspect in-vivo after administration to a mammal, wherein the fusion polypeptide is labeled by a fluorescence probe, a magnetic resonance detectable probe, an X-ray detectable probe, or a radioactive nuclide.

[0020] BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1. Establishment of the platform biotechnology i-Tag (the tag used in the inventive method and as part of the fusion polypeptide). A The CL domain-based platform biotechnology can be utilized for a standardized protein production workflow, starting with the CL domain fusion on DNA level, protein expression, and a one-step CL domain-based affinity purification. B Increased secretion titers were achieved via fusion of p28L162C’ACto the kappa (K) constant light chain (CL) domain (SEQ ID NOs: 33, 59) while no increase in secretion levels were achieved with the lambda (A) CL fusion constructs (SEQ ID NOs: 57, 58). Expi293TMcells were transiently transfected with constructs of five K or A CL domain repeats, alone (SEQ ID NOs: 59, 57) or fused to the model protein p28L162C’AC(SEQ ID NOs: 33, 58). Protein levels in medium samples were examined by reducing SDS-PAGE and Coomassie stain. Non-reducing samples confirmed proper formation of disulfide bonds by mobility shifts (see dotted line). C The number of K CL domains within the immunoglobulin domain-based tag (i- Tag) can be varied and fused to the respective protein of interest (schematics). For the model protein p28L162C ACfusions of 1 , 3, 5, or 7 K CL domains (SEQ ID NOs: 27, 38, 33, 40) were tested. After one-step affinity chromatography purification of Expi293FTMsupernatants with the CaptureSelect™ KappaXP column, i-Tag fusion samples were analyzed by reducing and non-reducing SDS-PAGE. Relative protein levels of the reduced samples were quantified, divided by the number of CL domains, and normalized to the i-Tagi fusion signal (SEQ ID NO: 27). D Expression levels increased up to 240-fold for six different proteins of medical relevance when fused to i-Tags. The i-Tags fusion constructs (orange bars) were compared to the untagged constructs (black bars). Expression levels were measured by ELISA of Expi293FTMsupernatants for p28L162C AC(dark bar, left) or, for the other constructs, by SDS-PAGE (light bars, right). Note that for some untagged constructs, no expression was observed, so that fold-increases are minimum values based on the Coomassie stain detection limit (see Methods for details).

[0022] Figure 2. Protein expression SDS-PAGE gels of untagged versus i-Tag5-fused proteins. Secretion levels of proteins were assessed by Coomassie stained SDS- PAGE. Supernatants were harvested 3 days after transfection of Expi293FTMcells. A bovine serum albumin (BSA) dilution series served as concentration standard to quantify secretion levels. The molecular weight of untagged proteins, i.e. A DLL3- 4xCD3 bispecific T-cell engager (BiTE) (SEQ ID NO: 51 , w / o signal sequence), B Darbepoetin alfa (SEQ ID NO: 42, w / o signal sequence), C Erythropoietin (SEQ ID NO: 45, w / o signal sequence), D Granulocyte-colony stimulating factor (GCSF) (SEQ ID NO: 48, w / o signal sequence), and E IL-15Ra(sushi)IL-15 (SEQ ID NO: 54, w / o signal sequence) is marked with a black arrow, the i-Tags-fused proteins (SEQ ID NOs: 53, 44, 47, 50, 56; w / o signal sequence each), according to their predicted molecular weight, with an orange arrow. MW, molecular weight.

[0023] Figure 3. The i-Tag5enables standardized purification and improves characteristics of proteins in a non-antibody format. A To test the i-Tag platform biotechnology, the inventors applied this system to four further proteins of medical relevance: DLL3-4xCD3 BiTE, Darbepoetin alfa, GCSF, and IL-15Ra(sushi)IL-15. Structural models based on crystal structures (PDB: 1 BLIY for Darbepoetin, PDB: 2D9Q for GCSF, PDB: 2Z3Q for IL-15Ra(sushi)IL-15) or Al-driven (ColabFold) structural modeling (DLL3-4xCD3 BiTE) are depicted in gray, with N- and C-terminus marked. B Purity and oxidation status after a one-step purification with the CaptureSelect™ KappaXP column via i-Tags or with the HisTrap HP column via Hise- tag were assessed by SDS-PAGE for the respective proteins. Schematics show fusion of protein of interest with i-Tags or Hise-tag, respectively. For comparison, Hiss-tagged proteins were purified by affinity purification, proteins fused to i-Tags purified by the kappa XP affinity column. Purity of proteins was analyzed via SDS-PAGE. Nonreducing protein samples showed mobility shifts whenever disulfide bridges are present within the proteins. For GCSF-Hise, the faint protein band is highlighted with an arrow. C Analytical ultracentrifugation (AUC) of both Hiss-tagged (red) and i-Tagged (black) proteins to analyze the assembly status of the purified proteins. Respective c (s) values were normalized to the highest signal in each plot. D Molecular weights from AUC measurements. The fractional ratio (f / fo) reflects the impact of the respective tag on the protein shape. E Dynamic light scattering measurements suggest improved quality of Darbepoetin alfa and IL-15Ra(sushi)IL-15 with the i-Tags versus Hise-tag, as shown by intensity plots and high-molecular weight species in Hise-tag protein samples.

[0024] Figure 4. Generally improved protein homogeneity of proteins with i-Tag5versus His6-tag. A Immunoblot against K CL confirms identity and integrity of the purified i- Tags-fused proteins (SEQ ID NOs: 44, 56, 50, 53; w / o signal sequence each). Analysis was performed under reducing conditions. B Under the same conditions as in A, Hise- tagged, purified proteins (SEQ ID NOs: 43, 55, 49, 52; w / o signal sequence each) were analyzed via aHise-immunoblot. The faint band for GCSF-Hise is marked by an arrowhead for better visualization. C Protein homogeneity was examined by HPLC of proteins with i-Tags and Hise-tag. Aggregated / assembled species (high-molecular weight species, HMW) were detected for both (see lines in the respective colors above the curves), thus the corresponding area under the curve in the chromatogram was quantified. D Quantification of HMW species of HPLC runs. Percentages depict the relative HMW fraction of the protein sample run. E The two species on Coomassie- stained SDS-PAGE gels of Hiss-tagged, purified IL-15Ra(sushi)-IL-15 were confirmed to be non-glycosylated and A / -glycosylated species by PNGase F and 0- Glycosidase / Neuraminidase digestion. Bands above 35 kDa, marked by asterisks represent deglycosylation enzymes. MW, molecular weight.

[0025] Figure 5. The i-Tag5is an elongated, well-folded and stable protein-tag. A The in silico modelled i-Tags structure (gray), a repetition of five K CL domains, is overlaid with rigid body models, based on the small-angle X-ray scattering (SAXS) data, and generated from core K Ci-domain structures and flexible linkers in between (shades of magenta). Both types of models show a protein tag of elongated shape. B Hydrogendeuterium exchange (HDX)-mass spectrometry (MS) measurement of i-Tags (SEQ ID NO: 59) and i-Tagi (SEQ ID NO: 2) reveals only minor differences between both proteins. The structure of a single K CL domain (taken from PDB ID: 3QCII) is colored according to the differences in fractional deuterium uptake between the i-Tagi and the i-Tags. The fractional deuterium uptake of the average K CL domain in the i-Tags was subtracted from the fractional uptake of the i-Tagi protein. Blue corresponds to a higher fractional deuterium uptake in the i-Tags structure, thus lower structural flexibility of i- Tagi compared to i-Tags, whereas red coloring depicts a higher structural flexibility of i-Tagi compared to theKCL domains in the i-Tags structure. C Analytical ultracentrifugation (AUC) measurement of i-Tags shows a mostly monomeric status of the purified i-Tags, with c (s) normalized to the highest signal. MW, molecular weight. Calc, calculated. D Differential scanning calorimetry (DSC) measurement of i-Tags reveals its melting temperature at around 51.3 °C after fitting (blue curve). E Ellman’s assay shows intact intramolecular disulfide bonds within every K CL domain of the i- Tags (SEQ ID NO: 59). IL-12|3C199Sis a mutant of the cytokine subunit IL-12|3 / p40, in which the cysteine 199, one of its free cysteines, is exchanged by a serine. This mutant has a single free Cys274 and serves as a positive control for the detection of a free cysteine in this assay. In contrast, the i-Tags does not show free cysteines, in the native structure and even under harsh denaturing conditions (5 M guanidinium hydrochloride, GdmCI), whereas the control shows approximately one free cysteine per molecule as expected.

[0026] Figure 6. Biophysical characterization of the i-Tag5(I). A Experimental SAXS data. From the comparison of the datasets collected from two different concentrations, concentration-dependent effects of the i-Tags protein solution may be excluded. All subsequent analyses were performed on the 3.5 mg / ml dataset owing to the better signal-to-noise ratio. B Guinier fit and residuals of the 3.5 mg / ml dataset. Due to the elongated nature of the protein, the sRgmax was chosen smaller than 1 .3. From this fit, the inventors obtained an Rgof (54 ± 1 ) A. C Pair distance distribution function of the 3.5 mg / ml dataset. D Guinier fit and residuals of the pair distance function. E Fits of the rigid body models calculated for the 3.5 mg / ml dataset with models’ Rgranging from 46.0 A to 47.0 A. The higher Rgfrom the Guinier analysis compared to the one from the rigid body models points to the presence of non-monomeric protein, as also seen in analytical ultracentrifugation data (Figure 3C). F Coverage plot of i-Tags shows peptides detected in hydrogen deuterium exchange (HDX) experiment.

[0027] Figure 7. Biophysical characterization of the i-Tag5(II). A Dynamic light scattering (DLS) data of i-Tags (SEQ ID NO: 59) confirm a stable, mostly monomeric behavior in graphs depicting intensity, volume, or number. B High-performance liquid chromatography (HPLC) run of i-Tags (SEQ ID NO: 59) shows monomeric species with a relative percentage of over 90% for the i-Tags (SEQ ID NO: 59) after one-step purification. HMW, high-molecular weight species. C Temperature-dependent far-UV circular dichroism (CD) spectroscopy measurement of i-Tags (SEQ ID NO: 59) at 232 nm results in an unfolding curve with a melting temperature of around 53.5 °C for the i-Tags. D Differential scanning calorimetry (DSC) curve for i-Tagged Darbepoetin alfa (SEQ ID NO: 44, w / o signal sequence) shows a melting temperature of 50.5 °C according to the sequential fitting model (blue). E Temperature-dependent unfolding curve measured by CD spectroscopy at 218 nm confirms melting temperature of around 51 °C for Darbepoetin alfa i-Tags (SEQ ID NO: 44, w / o signal sequence). F For Hiss-tagged Darbepoetin alfa (SEQ ID NO: 43, w / o signal sequence), its melting temperature of around 52 °C was detected by temperature-dependent CD spectroscopy measurement at 218 nm.

[0028] Figure 8. Functionality of i-Tagged proteins is preserved, while stability is improved. A A HeLa STAT1 bioluminescence assay confirmed the preserved activity of untagged versus i-Tags-fused p28L162C’ACprotein (SEQ ID NOs: 22, 33) from Expi293FTMsupernatants. Mock ( / ), supernatants of untransfected Expi293FTMcells, served as negative control in this activity assay. HeLa cells were stimulated with adjusted volumes of Expi293FTMsupernatants, for which protein concentrations were measured and normalized before HeLa STAT1 cell stimulation. B DLL3-4xCD3 BiTE with an i-Tags (SEQ ID NO: 53) (orange; each oval depicting aKCL domain) binds to its two target proteins, DLL3 (green) and CD3s (yellow). Binding was confirmed by coimmunoprecipitation (co-IP) experiment, in which i-Tags-fused BiTE binding to the two Hise-tagged target proteins was analyzed in single (1 , 2) or combined (1 +2) addition of the target protein to the BiTE by anti-Hise IP and subsequent reducing SDS-PAGE. C Purified Darbepoetin alfa, which is a glycosylation mutant of Erythropoietin (Egrie et al., 2001 ), fused to i-Tags (SEQ ID NO: 44, w / o signal sequence) is detected in the Erythropoietin ELISA. D Remaining activity of i-Tags-fused p28L162C AC(SEQ ID NO: 33) after incubation at 50 °C for 30 min was assessed by the HeLa STAT1 bioluminescence assay. In contrast to i-Tags-fused p28L162C AC(SEQ ID NO: 33), no functionality was measured for the untagged p28L162C’AC(SEQ ID NO: 22) after incubation at 50 °C for 30 min.

[0029] Figure 9. Schematic of i-Tag biotechnology enabled half-life by design. Different numbers (x) of the K CL domain (i-Tagx) can be fused to the protein of interest. Thereby its molecular weight is designed in that way that its in vivo half-life can be adjusted to one’s specific needs. Note that the number of fused domains can also be larger than seven.

[0030] Figure 10. N- as well as C-terminal i-Tag5fusion enables a standardized protein production in mammalian cells. A Secretion levels of N- as well as C-terminal i- Tagged p28L162C AC(SEQ ID NO: 41 , 33) were assessed by Coomassie stained SDS- PAGE. Supernatants were harvested after transfection of Expi293FTMcells (load) and purified with the CaptureSelect™ KappaXP column via i-Tags (purified protein). A bovine serum albumin (BSA) dilution series served as concentration standard to quantify protein levels. MW, molecular weight. B Expression levels were significantly increased when fused to i-Tags with no detectable p28L162C’ACexpression when untagged (SEQ ID NO: 22). C Quantification of the Coomassie gel (A) shows that the standardized purification via CaptureSelect™ KappaXP column resulted in a very high purity of above 95% for N- as well as C-terminally i-Tagged p28L162C’AC(SEQ ID NO: 41 , 33). D THP-1 cell stimulation and subsequent CXCL10 ELISA proved that the functionality of i-Tagged p28L162C ACis preserved. Figure 11. Increased expression titer and preserved functionality are obtained regardless of a linker that fuses the i-Tag to p28L162C AC. A Secretion levels of p28L162c, AC by transfected Expi293FTMcells were increased by i-Tagi or i-Tags fusion, regardless of whether or which linker (sequences in green) between the protein and i- Tag was used (SEQ ID NOs: 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35). n = 6, ± SD. B Functionality of i-Tagged p28L162C ACwith all linkers tested was preserved. A HeLa STAT1 bioluminescence assay was used to determine functionality of i-Tags- fused p28L162C ACprotein, without or with linker (SEQ ID NOs: 26, 32, 33, 34, 35), from Expi293FTMsupernatants compared to mock, which served as negative control in this activity assay. HeLa cells were stimulated with adjusted volumes of Expi293FTMsupernatants, for which protein concentrations were measured and normalized before HeLa STAT1 cell stimulation, n = 2, ± SD.

[0031] Figure 12. Standardized protein production and protein functionality are obtained by i-Tag5fusion regardless of an interdomain linker. A Protein expression as well as purification of i-Tags fusion constructs are comparable with and without GAGAG-interdomain linkers between theKCL domains of the i-Tags (SEQ ID NOs: 33, 39). B Functionality of i-Tagged p28L162C’ACprotein is unaffected of interdomain linkers, as proven by THP-1 assay and CXCL10 readout.

[0032] Figure 13. The i-Tag2construct is stable in fetal bovine serum at 37 °C. An i-Tag2 protein sample (without linker) (SEQ ID NO: 71 ) was mixed 1 :10 in fetal bovine serum (FBS) and incubated at 37 °C for 97 hours. Immunoprecipitation (IP) was conducted with an antibody against the human kappa CL domain. Input as well as IP samples show no decrease in protein levels on the anti-K CL Western Blot.

[0033] Figure 14. Partial proteolysis experiment with trypsin shows increased stability of i-Tag2protein without interdomain-linker compared to linker constructs. A Protein samples of the constructs KCL-KCL (i-Tag2(without linker)) (SEQ ID NO: 71 ), KCL-(GGGGS)2-KCL(i-Tag2(with (GGGGS)2-linker)) (SEQ ID NO: 72), KCL-

[0034] A(EAAAK)2A-KCL (i-Tag2(with A(EAAAK)2A-linker)) (SEQ ID NO: 73), KCL-

[0035] GSPKSCDKTHTCPPCPAPGSEIK-KCL (i-Tag2(with

[0036] GSPKSCDKTHTCPPCPAPGSEIK-linker)) (SEQ ID NO: 74), and KCL- GS(GGGGS)2GEIK-KCL (i-Tag2(with GS(GGGGS)2GEIK-linker)) (SEQ ID NO: 75) were incubated with 1 :50 (w / w) Trypsin at 37 °C for up to 60 min. Samples taken after indicated time were analyzed on Coomassie-stained SDS-PAGE gels. Trypsin-induced protein degradation results in a decrease in protein levels of the full-length constructs at around 25 kDa and increase of protein degradation bands below 25 kDa. B Full length protein levels were quantified (fraction uncleaved) and depict the increased stability of i-Tag2 (KCL-KCL) above protein constructs with interdomain linker, n = 2, ± SD.

[0037] DETAILED DESCRIPTION OF THE INVENTION

[0038] In general, a protein-tag should ideally be inert, improve expression, and be amenable to simple purification procedures. An enabling platform biotechnology that in addition enhances the quality attributes of the protein of interest, such as stability, provides further significant advantages for the production process as well as the product per se. Also, most biopharmaceuticals are produced as secreted proteins from the medium of mammalian cells.

[0039] The inventors surprisingly found that a constant domain of an immunoglobulin light chain, especially a constant domain of a kappa immunoglobulin light chain, is a versatile tag e.g. suitable for affinity purification or detection of a fusion polypeptide wherein the tag is fused to a target polypeptide.

[0040] Previous studies of the inventors showed that a constant domain of the antibody light chain (CL) does not significantly engage chaperones in the endoplasmic reticulum (ER), where secretory proteins pass through and acquire their structure (Behnke et al., 2016). Thus, the constant domain undergoes a particularly robust folding process in a given cell. Furthermore, the CL domain is resistant against misfolding (Feige et al., 2008). Also, antibody light chains, that contain the CL domain, are naturally secreted in high quantities from human B cells without any harm to the human body (Nakano et al., 2006). Furthermore, the inventors found that CL domains could be fused to each other. Thereby it is possible to modulate the molecular mass and also the isoelectric point (pl) of the protein-tag and of a fusion protein wherein a protein is fused to this tag.

[0041] Thus, the invention provides in a first aspect a method of modulating a molecular mass of a fusion polypeptide, wherein the fusion polypeptide comprises a target polypeptide fused to a tag suitable for affinity purification or detection of the target polypeptide, wherein the tag comprises at least one constant domain of an immunoglobulin light chain or orthologous polypeptide or fragment thereof and wherein the molecular mass of the fusion polypeptide is modulated by increasing the molecular mass of the tag via combining the one constant domain of an immunoglobulin light chain or orthologous polypeptide or fragment thereof with one or more further constant domain(s) of an immunoglobulin light chain or orthologous polypeptide(s) or fragment(s) thereof; wherein the constant domain of the immunoglobulin light chain or orthologous polypeptide or fragment thereof of the tag is monomeric; and wherein optionally the target polypeptide is fused to the tag via a first linker.

[0042] In general, the molecular mass of the fusion polypeptide is modulateable or modulated by changing the molecular mass of the tag via combining the one constant domain of an immunoglobulin light chain or orthologous polypeptide or fragment thereof with one or more further constant domain(s) of an immunoglobulin light chain or orthologous polypeptide(s) or fragment(s) thereof;

[0043] The term “modulateable” means that the molecular mass of the fusion polypeptide could be modulated by the fusion of the tag to the target polypeptide.

[0044] It is to mention that the first linker is not necessary for the production and functionality of the fusion polypeptide as clearly shown in Example 6 and Figure 11 .

[0045] By modulating the molecular mass of the fusion polypeptide, its molecular mass could be raised above 70 kDa and thereby prolonging its serum half-life.

[0046] The term “constant domain of an immunoglobulin light chain” also includes any constant domain of orthologous immunoglobulin light chains and any fragments of the constant domain of the immunoglobulin light chain or of an orthologous immunoglobulin light chain. Further included are constant domains of immunoglobulin light chain-like sequences and kappa immunoglobulin light chain-like sequences.

[0047] The term “i-Tag” as used throughout the description and the examples means a constant domain of a human kappa immunoglobulin light chain. In the case more than one constant domain of a human kappa immunoglobulin light chain are included in the used tag, the number of constant domains is mentioned as an integer. For example, the term “i-Tags” means that five constant domains of a human kappa immunoglobulin light chain are present in the tag.

[0048] The term “molecular mass” is used synonymous with the expression molecular weight.

[0049] The inventive method may further comprise the steps: a) determining the molecular mass of the target polypeptide, b) (optional) determining the molecular mass of one constant domain of an immunoglobulin light chain or orthologous polypeptide or fragment thereof, c) calculating how many constant domains of an immunoglobulin light chain or orthologous polypeptides or fragments thereof are needed in addition to the molecular mass of the target polypeptide (step a) for reaching a desired molecular mass of the fusion polypeptide, and d) creating the tag to be fused to the target polypeptide by combining the necessary number of constant domains of an immunoglobulin light chain or orthologous polypeptides or fragments thereof according to the result of step (c).

[0050] The desired molecular mass of the fusion polypeptide may be below or above the filtration limit of a kidney of a given organism, depending on whether a plasma half-life of the fusion polypeptide should be prolonged or shortened. The desired molecular mass of the fusion polypeptide may be below or at least 50 kDa or 70 kDa.

[0051] The tag used in the inventive method may comprise two or more constant domains of the immunoglobulin light chain or orthologous polypeptides or fragments thereof. Optionally the constant domains or orthologous polypeptides or fragments thereof could be connected to each other by a second linker, e.g. a GAGAG-linker (for example cf. SEQ ID NO: 39). However, such a linker is not mandatory as shown in Example 7 and Figure 12.

[0052] In the inventive method, the fusion polypeptide may be expressed under conditions allowing a formation of one or more disulfide bond(s).

[0053] In the inventive method the constant domain of the immunoglobulin light chain or orthologous polypeptide or fragment thereof of the tag may be a constant domain of a kappa immunoglobulin light chain or orthologous polypeptide or fragment thereof.

[0054] The term “constant domain of a kappa immunoglobulin light chain” also includes any constant domain of orthologous kappa immunoglobulin light chains and any fragments of the constant domain of the kappa immunoglobulin light chain or of an orthologous kappa immunoglobulin light chain. Further included are constant domains of kappa immunoglobulin light chain-like sequences.

[0055] Furthermore, the constant domain of the immunoglobulin light chain or fragment thereof comprised in the tag used in the inventive method could be a mammalian immunoglobulin light chain or orthologous polypeptide or fragment thereof, preferred a constant domain of a primate immunoglobulin light chain or fragment thereof and most preferred a constant domain of a human immunoglobulin light chain or fragment thereof.

[0056] Also, the constant domain of the kappa immunoglobulin light chain or fragment thereof comprised in the tag used in the inventive method may be a constant domain of a mammalian kappa immunoglobulin light chain or orthologous polypeptide or fragment thereof, preferred a constant domain of a primate kappa immunoglobulin light chain or fragment thereof, and most preferred a constant domain of a human kappa immunoglobulin light chain or fragment thereof.

[0057] The constant domain of the immunoglobulin light chain contains one internal disulfide bond that is important for its efficient folding (Feige et al., 2007). The constant domain further contains a C-terminal cysteine residue, which normally engages with the antibody heavy chain CH1 domain to form a covalent bond in the antigen-binding Fab fragment (Huber et al., 1976).

[0058] In the preferred tag used in the method, the C-terminal cysteine of the constant domain of the immunoglobulin light chain or of the kappa immunoglobulin light chain, which is the cysteine which is not part of the intramolecular disulfide-bridge of the constant domain, may be deleted. It is to be understood, that the C-terminal cysteine is deleted only in the case that the C-terminal cysteine is present in the used peptide sequence of the constant domain of the immunoglobulin light chain.

[0059] This deletion is used to avoid any mis-oxidation of the tag.

[0060] Furthermore, the constant domain of the human kappa immunoglobulin light chain without the C-terminal cysteine comprised in the tag used in the inventive method has a molecular mass of 11.6 kDa and a predicted isoelectric point of 6.1. Using the inventive method, the molecular mass of the fusion polypeptide comprising the target polypeptide, e.g. G-CSF having a molecular mass of 19.6 kDa, could be modulated by fusing the tag comprising 5 constant domains of the human kappa immunoglobulin light chain without the C-terminal cysteine (equals about 58 kDa) to the target polypeptide resulting in a fusion polypeptide with a molecular mass of 77.6 kDa (cf. Figure 2D).

[0061] Furthermore, the constant domain of the kappa immunoglobulin light chain or orthologous polypeptide or fragment thereof comprised in the tag used in the inventive method may be selected from a group consisting of a constant domain of a mouse kappa immunoglobulin light chain, a constant domain of a rat kappa immunoglobulin light chain, a constant domain of a dog kappa immunoglobulin light chain, a constant domain of a rabbit kappa light chain, a constant domain of a pig kappa immunoglobulin light chain, a constant domain of a monkey kappa immunoglobulin light chain, and preferably the constant domain of the human kappa immunoglobulin light chain.

[0062] The tag used in the inventive method may also comprise the constant domain of the kappa immunoglobulin light chain which comprises or consists of a peptide sequence selected from a group consisting of the following peptide sequences: - constant domain of human kappa immunoglobulin light chain (SEQ ID NO: 1 ),

[0063] - constant domain of human kappa immunoglobulin light chain without the C-terminal cysteine (SEQ ID NO: 2),

[0064] - constant domain of mouse kappa immunoglobulin light chain (SEQ ID NO: 4),

[0065] - constant domain of mouse kappa immunoglobulin light chain without the C-terminal cysteine (SEQ ID NO: 5),

[0066] - constant domain of rat kappa immunoglobulin light chain (SEQ ID NO: 6),

[0067] - constant domain of rat kappa immunoglobulin light chain without the C-terminal cysteine (SEQ ID NO: 7),

[0068] - constant domain of rabbit kappa-b4 immunoglobulin light chain (KACB) according to UniProt entry: P01839 (SEQ ID NO: 8),

[0069] - constant domain of rabbit kappa-b4 immunoglobulin light chain (KACB) according to UniProt entry: P01839, without the C-terminal cysteine (SEQ ID NO: 9),

[0070] - constant domain of rabbit kappa-b4 immunoglobulin light chain (KAC4) according to UniProt entry: P01840 (SEQ ID NO: 10),

[0071] - constant domain of rabbit kappa-b4 immunoglobulin light chain (KAC4) according to UniProt entry: P01840, without the C-terminal cysteine (SEQ ID NO: 11),

[0072] - constant domain of rabbit kappa-b5 immunoglobulin light chain (KAC5) according to UniProt entry: P01841 (SEQ ID NO: 12),

[0073] - constant domain of rabbit kappa-b5 immunoglobulin light chain (KAC5) according to UniProt entry: P01841 , without the C-terminal cysteine (SEQ ID NO: 13), - constant domain of rabbit kappa-b5 immunoglobulin light chain (KAC6) according to UniProt entry: P03984 (SEQ ID NO: 14),

[0074] - constant domain of rabbit kappa-b5 immunoglobulin light chain (KAC6) according to UniProt entry: P03984, without the C-terminal cysteine (SEQ ID NO: 15),

[0075] - constant domain of dog kappa immunoglobulin light chain (SEQ ID NO: 16),

[0076] - constant domain of pig kappa immunoglobulin light chain (SEQ ID NO: 17),

[0077] - constant domain of monkey kappa immunoglobulin light chain (SEQ ID NO: 18), and

[0078] - constant domain of monkey kappa immunoglobulin light chain without the C-terminal cysteine (SEQ ID NO: 19).

[0079] The constant domain of human kappa immunoglobulin light chain (SEQ ID NO: 1 ) and the constant domain of human kappa immunoglobulin light chain without the C-terminal cysteine (SEQ ID NO: 2) are preferred peptide sequences comprised in the tag used the inventive method.

[0080] The used constant domain of the immunoglobulin light chain or orthologous polypeptide or fragment thereof comprised in the tag used in the inventive method is monomeric, as shown in Example 3 and Figures 5 and 7.

[0081] Due to the deleted C-terminal cysteine, the constant domain comprised in the tag used in the inventive method would not build a disulfide bridge with other cysteines present outside the tag. Therefore, no dimers or multimers connected by disulfide bridge(s) would be formed which may result in unwanted aggregates, especially when the disulfide bridges are formed between the C-terminal cysteine of the constant domain and a cysteine of the target polypeptide, which on the other hand, may be a suitable first linker. This feature is also beneficial for the linkage of more than one constant domain in the tag in an ordered manner by using linkers.

[0082] In the method the first linker, used for fusing the tag to the target polypeptide, and / or the second linker, used for fusing several constant domains comprised in the tag, may be a cleavable or a non-cleavable linker. Furthermore, the first linker may be a cleavable linker and the second linker may be a non-cleavable linker or the first linker may be a non-cleavable linker and the second linker may be a cleavable linker. A cleavable linker could be used if it is desired to cleave the tag and the target polypeptide at a defined position after purification of the fusion polypeptide. A non- cleavable linker may be used, if e.g. the tag is further used for detection of the fusion polypeptide or is necessary to enhance the stability of the fusion polypeptide.

[0083] Suitable linkers are known to the skilled person (reviewed e.g. in Su et al., 2021 ). Examples of such linkers are a G4-linker (SEQ ID NO: 60), a Gs-linker (SEQ ID NO: 61 ), a GGGS-linker (SEQ ID NO: 62), a GGGGS-linker (SEQ ID NO: 63), a A(EAAAK)4ALEA(EAAAK)4A-linker (SEQ ID NO: 69), a PAPAP-linker (SEQ ID NO: 64), a APAPAPA-linker (SEQ ID NO: 65), a APAPAPAPAPKPA-I inker (SEQ ID NO: 66), a APAPAPAPAPAPA-I inker (SEQ ID NO: 67), a A(EAAAK)4A-linker (SEQ ID NO: 68), and a A(EAAAK)2A-linker (SEQ IDS NO: 78), or one or more disulfide bridge(s).

[0084] It should be emphasized that neither the first linker nor the second linker is absolutely necessary for the production or functionality of the fusion protein as shown in Examples 6 and 7 and Figures 11 and 12.

[0085] Furthermore, example 8 and Figure 14 show that the tag without the second linker between several constant domains comprised in the tag is more stable in and trypsin solution than the tag with one of the linker sequences GGGGSGGGGS (SEQ ID NO: 77), GSPKSCDKTHTCPPCPAPGSEIK (SEQ ID NO: 79), or GSGGGGSGGGGSGEIK (SEQ ID NO: 80).

[0086] In the inventive method the tag may be fused to the C-terminus or to the N-terminus of the target polypeptide.

[0087] The tag may also be fused to the C-terminus and the N-terminus of the target polypeptide in the fusion polypeptide.

[0088] Due to the tag used in the method, the fusion polypeptide may be expressed and / or secreted at a higher level by the used cells and has a higher stability compared to other used tags or the untagged target polypeptide. Thus, an expression of the fusion polypeptide is higher than the expression of the untagged target polypeptide, as shown in Figure 11 A. The expression of the fusion polypeptide may be, for example, fivefold higher than the expression of the untagged target polypeptide.

[0089] Remarkably, the expression of the fusion polypeptide comprising more than one tag, e.g. 5 tags, is higher than the expression with one tag as shown in Figures 1 C and 11 A.

[0090] The expressed inventive fusion polypeptide may further have a higher homogeneity compared to a fusion polypeptide comprising a His-tag and the target polypeptide.

[0091] Also, a function of the target polypeptide, e.g. the enzymatic activity of the target polypeptide, should not be markedly reduced by the tag used in the method.

[0092] Thus, the function of the target polypeptide with the tag in the fusion polypeptide may be substantially comparable to a function of the target polypeptide without the tag.

[0093] Additionally, the fusion polypeptide may have an isoelectric point (pl) different from the untagged target polypeptide. The constant domain, as part of the tag used in the inventive method, has a predicted isoelectric point (pl) of 6.1 (according to Expasy ProtParam). As a result, the pl of the fusion polypeptide may change compared to the untagged target polypeptide with the numbers of constant domains present in the tag. Thus, the solubility of the fusion polypeptide as well as its binding to ion exchange columns can be modified compared to the untagged target polypeptide.

[0094] The invention further provides, in a second aspect, a fusion polypeptide obtained or obtainable by the method of the first aspect, described above.

[0095] In a third aspect the invention provides a tag suitable for affinity purification or detection of a fusion partner, wherein the fusion partner is a target polypeptide and wherein the tag comprises at least one constant domain of an immunoglobulin light chain or orthologous polypeptide or fragment thereof and optionally a first linker that fuses the tag to the target polypeptide, wherein the constant domain of the immunoglobulin light chain or orthologous polypeptide or fragment thereof is monomeric.

[0096] It is to emphasize, that this tag is the tag preferably or exclusively used in the method described above in the first aspect.

[0097] The term “constant domain of an immunoglobulin light chain” also includes any constant domain of orthologous immunoglobulin light chains and any fragments of the constant domain of the immunoglobulin light chain or of an orthologous immunoglobulin light chain. Further included are constant domains of immunoglobulin light chain-like sequences and kappa immunoglobulin light chain-like sequences.

[0098] The term “i-Tag” as used throughout the description and the examples means a constant domain of a human kappa immunoglobulin light chain. In the case more than one constant domain of a human kappa immunoglobulin light chain are included in the used tag, the number of constant domains is mentioned as an integer. For example, the term “i-Tags” means that five constant domains of a human kappa immunoglobulin light chain are present in the tag.

[0099] The tag may also comprise more than one constant domain. The constant domains are then linked together by e.g. a suitable second linker.

[0100] The tag comprising one or more constant domain(s) may be for example used to prolong the serum half-life of a fusion polypeptide comprising the tag and the target polypeptide in that the size of the fusion polypeptide is raised such that it would be no longer filtrated by a kidney. Thus, the fusion polypeptide having the tag with one or more constant domain(s), or depending on the molecular mass of the target polypeptide with more than one constant domain, may have a size of more than 70 kDa.

[0101] In the tag the constant domain of the immunoglobulin light chain or orthologous polypeptide or fragment thereof may be a constant domain of a kappa immunoglobulin light chain or orthologous polypeptide or fragment thereof. The term “constant domain of a kappa immunoglobulin light chain” also includes any constant domain of orthologous kappa immunoglobulin light chains and any fragments of the constant domain of the kappa immunoglobulin light chain or of an orthologous kappa immunoglobulin light chain. Further included are constant domains of kappa immunoglobulin light chain-like sequences.

[0102] Furthermore, the constant domain of the immunoglobulin light chain or fragment thereof used in the tag of the invention could be a mammalian immunoglobulin light chain or orthologous polypeptide or fragment thereof, preferred a constant domain of a primate immunoglobulin light chain or fragment thereof and most preferred a constant domain of a human immunoglobulin light chain or fragment thereof.

[0103] Also, the constant domain of the kappa immunoglobulin light chain or fragment thereof used in the tag of the invention may be a constant domain of a mammalian kappa immunoglobulin light chain or orthologous polypeptide or fragment thereof, preferred a constant domain of a primate kappa immunoglobulin light chain or fragment thereof, and most preferred a constant domain of a human kappa immunoglobulin light chain or fragment thereof.

[0104] The constant domain of the immunoglobulin light chain contains one internal disulfide bond that is important for its efficient folding (Feige et al., 2007). The constant domain further contains a C-terminal cysteine residue, which normally engages with the antibody heavy chain CH1 domain to form a covalent bond in the antigen-binding Fab fragment (Huber et al., 1976).

[0105] In the inventive tag, the C-terminal cysteine of the constant domain of the immunoglobulin light chain or of the kappa immunoglobulin light chain, which is the cysteine which is not part of the intramolecular disulfide-bridge of the constant domain, may be deleted. It is to be understood, that the C-terminal cysteine is deleted only in the case that the C-terminal cysteine is present in the used peptide sequence of the constant domain of the immunoglobulin light chain. This deletion is used to avoid any mis-oxidation of the tag. Furthermore, the constant domain of the kappa immunoglobulin light chain or orthologous polypeptide or fragment thereof used in the inventive tag may be selected from a group consisting of a constant domain of a mouse kappa immunoglobulin light chain, a constant domain of a rat kappa immunoglobulin light chain, a constant domain of a dog kappa immunoglobulin light chain, a constant domain of a rabbit kappa light chain, a constant domain of a pig kappa immunoglobulin light chain, a constant domain of a monkey kappa immunoglobulin light chain, and preferably the constant domain of the human kappa immunoglobulin light chain.

[0106] The inventive tag may also comprise the constant domain of the kappa immunoglobulin light chain which comprises or consists of a peptide sequence selected from a group consisting of the following peptide sequences: constant domain of human kappa immunoglobulin light chain (SEQ ID NO: 1 ), constant domain of human kappa immunoglobulin light chain without the C- terminal cysteine (SEQ ID NO: 2), constant domain of mouse kappa immunoglobulin light chain (SEQ ID NO: 4), constant domain of mouse kappa immunoglobulin light chain without the C- terminal cysteine (SEQ ID NO: 5), constant domain of rat kappa immunoglobulin light chain (SEQ ID NO: 6), constant domain of rat kappa immunoglobulin light chain without the C-terminal cysteine (SEQ ID NO: 7), constant domain of rabbit kappa-b4 immunoglobulin light chain (KACB) to UniProt entry: P01839 (SEQ ID NO: 8), constant domain of rabbit kappa-b4 immunoglobulin light chain (KACB) according to UniProt entry: P01839, without the C-terminal cysteine (SEQ ID NO: 9), constant domain of rabbit kappa-b4 immunoglobulin light chain (KAC4) according to UniProt entry: P01840 (SEQ ID NO: 10), constant domain of rabbit kappa-b4 immunoglobulin light chain (KAC4) according to UniProt entry: P01840, without the C-terminal cysteine (SEQ ID NO: 11 ), constant domain of rabbit kappa-b5 immunoglobulin light chain (KAC5) according to UniProt entry: P01841 (SEQ ID NO: 12), constant domain of rabbit kappa-b5 immunoglobulin light chain (KAC5) according to UniProt entry: P01841 , without the C-terminal cysteine (SEQ ID NO: 13), constant domain of rabbit kappa-b5 immunoglobulin light chain (KAC6) according to UniProt entry: P03984 (SEQ ID NO: 14), constant domain of rabbit kappa-b5 immunoglobulin light chain (KAC6) according to UniProt entry: P03984, without the C-terminal cysteine (SEQ ID NO: 15), constant domain of dog kappa immunoglobulin light chain (SEQ ID NO: 16), constant domain of pig kappa immunoglobulin light chain (SEQ ID NO: 17), constant domain of monkey kappa immunoglobulin light chain (SEQ ID NO: 18), and constant domain of monkey kappa immunoglobulin light chain without the C- terminal cysteine (SEQ ID NO: 19). The constant domain of human kappa immunoglobulin light chain (SEQ ID NO: 1 ) and the constant domain of human kappa immunoglobulin light chain without the C-terminal cysteine (SEQ ID NO: 2) are preferred peptide sequences.

[0107] In the inventive tag the used constant domain of the immunoglobulin light chain or orthologous polypeptide or fragment thereof is monomeric.

[0108] Due to the deleted C-terminal cysteine, the constant domain used in the inventive tag would not build a disulfide bridge with other cysteines present outside the tag. Therefore, no dimers or multimers connected by disulfide bridge(s) would be formed which may result in unwanted aggregates, especially when the disulfide bridges are formed between the C-terminal cysteine of the constant domain and a cysteine of the target polypeptide. This feature is also beneficial for the linkage of more than one constant domain in the tag in an ordered manner by using linkers.

[0109] The inventive tag may further comprise the first linker that fuses the tag to the target polypeptide.

[0110] The first linker and / or second linker may be cleavable or not cleavable. A cleavable linker could be used if it is desired to cleave the tag and the target polypeptide at a defined position after purification of the fusion polypeptide. A non-cleavable linker may be used, if e.g. the tag is further used for detection of the fusion polypeptide or is necessary to enhance the stability of the fusion polypeptide.

[0111] Linker suitable as first linker and / or second linker are known to the skilled person (reviewed e.g. in Su et al., 2021 ). Examples of such linkers are a G4-linker (SEQ ID NO: 60), a G5-linker (SEQ ID NO: 61 ), a GGGS-linker (SEQ ID NO: 62), a GGGGS- linker (SEQ ID NO: 63), a A(EAAAK)4ALEA(EAAAK)4A-linker (SEQ ID NO: 69), a PAPAP-linker (SEQ ID NO: 64), a APAPAPA-linker (SEQ ID NO: 65), a APAPAPAPAPKPA-linker (SEQ ID NO: 66), a APAPAPAPAPAPA-linker (SEQ ID NO: 67), a A(EAAAK)4A-linker (SEQ ID NO: 68), and a A(EAAAK)2A-linker (SEQ IDS NO: 78), or one or more disulfide bridge(s).

[0112] It should be emphasized that neither the first linker nor the second linker is absolutely necessary for the production or functionality of the fusion protein as shown in Examples 6 and 7 and Figures 11 and 12. Furthermore, example 8 and Figure 14 show that the tag without the second linker between several constant domains comprised in the tag is more stable in a trypsin solution than the tag with one of the linker sequences GGGGSGGGGS (SEQ ID NO: 77), GSPKSCDKTHTCPPCPAPGSEIK (SEQ ID NO: 79), or GSGGGGSGGGGSGEIK (SEQ ID NO: 80).

[0113] The inventive tag may be suitable to be fused to the C-terminus of the target polypeptide.

[0114] The inventive tag may be suitable to be fused to the N-terminus of the target polypeptide.

[0115] The inventive tag may also be suitable to be fused to the C-terminus and the N- terminus of the target polypeptide.

[0116] Thus, the inventive tag could be fused to one or both ends of the target polypeptides depending on the desired position of the tag in the resulting fusion polypeptide.

[0117] Furthermore, the inventive tag is stable as shown in Example 3 and Figures 5 and 7.

[0118] In further aspects, the invention provides a use of the tag as described before, wherein the tag comprises at least three constant domains of the immunoglobulin light chain or orthologous polypeptides or fragments thereof for increasing the expression of a fusion polypeptide comprising the target polypeptide and the tag.

[0119] It is shown in Figures 1 C and 11A that, as an example, the expression of the fusion polypeptide comprising more than one tag, e.g. 5 tags, is higher than the expression with one tag.

[0120] The invention also provides a use of the tag as described before, wherein the tag comprises one or more constant domain(s) of the immunoglobulin light chain or orthologous polypeptides or fragments thereof for increasing the molecular mass of a fusion polypeptide comprising the target polypeptide and the tag. In a fourth aspect, the invention provides a fusion polypeptide comprising a target polypeptide and a tag suitable for affinity purification or detection of the target polypeptide, wherein the tag comprises at least one constant domain of an immunoglobulin light chain or orthologous polypeptide or fragment thereof and optionally a first linker that fuses the tag to the target polypeptide.

[0121] It should be emphasized that the first linker is not absolutely necessary for the production or functionality of the fusion protein as shown in Example 6 and Figure 11 .

[0122] It is possible to increase the molecular mass of the target polypeptide by fusion with the tag, even if the tag comprises one constant domain of the immunoglobulin light chain or orthologous polypeptide or fragment thereof. The fusion of the target polypeptide with the tag results in the fusion polypeptide with the molecular mass of the target polypeptide plus the optional first linker plus the tag, namely the number of constant domains of the immunoglobulin light chain or orthologous polypeptide or fragment thereof and the number of optional second linkers between two or more constant domains of the immunoglobulin light chain or orthologous polypeptide or fragment thereof, if applicable. Thus, it is clear that the molecular mass of the target polypeptide is increased by the fusion with the tag.

[0123] The fusion polypeptide may have a molecular mass below 50 kDa or 70 kDa. In the alternative, the fusion polypeptide may have a molecular mass of at least 50 kDa or of at least 70 kDa.

[0124] Since the tag used in the method of the first aspect is the same as used in the fusion polypeptide of the third aspect, any disclosure regarding the first aspect above is included herein, except the description dictates otherwise.

[0125] The term “constant domain of an immunoglobulin light chain” also includes any constant domain of orthologous immunoglobulin light chains and any fragments of the constant domain of the immunoglobulin light chain or of an orthologous immunoglobulin light chain. Further included are constant domains of immunoglobulin light chain-like sequences. The term “i-Tag” as used throughout the description and the examples means a constant domain of a human kappa immunoglobulin light chain. In the case more than one constant domain of a human kappa immunoglobulin light chain are included in the used tag, the number of constant domains is mentioned as an integer. For example, the term “i-Tag5” means that five constant domains of a human kappa immunoglobulin light chain are present in the tag.

[0126] The term “constant domain of a kappa immunoglobulin light chain” also includes any constant domain of orthologous kappa immunoglobulin light chains and any fragments of the constant domain of the kappa immunoglobulin light chain or of an orthologous kappa immunoglobulin light chain. Further included are constant domains of kappa immunoglobulin light chain-like sequences.

[0127] The tag of the fusion polypeptide may comprise more than one constant domain, as mentioned above. The constant domains may then be linked together by e.g. a suitable second linker. However, the second linker is not mandatory as shown e.g. in Example 7 and Figure 12.

[0128] The tag comprising one or more constant domain(s) may be for example used to prolong the serum half-life of a fusion polypeptide comprising the tag and the target polypeptide in that the size of the fusion polypeptide is raised such that it would be no longer filtrated by a kidney. Thus, the fusion polypeptide having the tag with one or more constant domain(s), or depending on the molecular mass of the target polypeptide with more than one constant domain, may have a size of more than 70 kDa.

[0129] The constant domain of the immunoglobulin light chain contains one internal disulfide bond that is important for its efficient folding (Feige et al., 2007). Therefore, the production under conditions that allow for disulfide bond formation are indicated (e.g. the ER of eukaryotic cells including yeast and mammalian cells or the periplasm of bacteria). Thus, the fusion polypeptide may be expressed under conditions allowing a formation of a disulfide bond. Therefore, the fusion polypeptide may comprise a peptide signal sequence for targeting the fusion protein to be expressed to a cellular region (e.g. the ER of eukaryotic cells including yeast and mammalian cells or the periplasm of bacteria) in which a formation of disulfide bonds is possible.

[0130] In the tag of the fusion polypeptide the constant domain of the immunoglobulin light chain or orthologous polypeptide or fragment thereof may be a constant domain of a kappa immunoglobulin light chain or orthologous polypeptide or fragment thereof.

[0131] Furthermore, the constant domain of the immunoglobulin light chain or fragment thereof used in the tag of the inventive fusion polypeptide could be a mammalian immunoglobulin light chain or orthologous polypeptide or fragment thereof, preferred a constant domain of a primate immunoglobulin light chain or fragment thereof and most preferred a constant domain of a human immunoglobulin light chain or fragment thereof.

[0132] Also, the constant domain of the kappa immunoglobulin light chain or fragment thereof used in the tag of the inventive fusion polypeptide may be a constant domain of a mammalian kappa immunoglobulin light chain or orthologous polypeptide or fragment thereof, preferred a constant domain of a primate kappa immunoglobulin light chain or fragment thereof, and most preferred a constant domain of a human kappa immunoglobulin light chain or fragment thereof.

[0133] The constant domain of the immunoglobulin light chain contains one internal disulfide bond that is important for its efficient folding (Feige et al., 2007). The constant domain further contains a C-terminal cysteine residue, which normally engages with the antibody heavy chain CH1 domain to form a covalent bond in the antigen-binding Fab fragment (Huber et al., 1976).

[0134] In the tag of the fusion polypeptide, the C-terminal cysteine of the constant domain of the immunoglobulin light chain or of the kappa immunoglobulin light chain, which is the cysteine which is not part of the intramolecular disulfide-bridge of the constant domain, may be deleted. It is to be understood, that the C-terminal cysteine is deleted only in the case that the C-terminal cysteine is present in the used peptide sequence of the constant domain of the immunoglobulin light chain. This deletion is used to avoid any mis-oxidation of the tag.

[0135] Furthermore, the constant domain of the kappa immunoglobulin light chain or orthologous polypeptide or fragment thereof used in the tag of the fusion polypeptide may be selected from a group consisting of a constant domain of a mouse kappa immunoglobulin light chain, a constant domain of a rat kappa immunoglobulin light chain, a constant domain of a dog kappa immunoglobulin light chain, a constant domain of a rabbit kappa light chain, a constant domain of a pig kappa immunoglobulin light chain, a constant domain of a monkey kappa immunoglobulin light chain, and preferably the constant domain of the human kappa immunoglobulin light chain.

[0136] The tag of the fusion polypeptide may also comprise the constant domain of the kappa immunoglobulin light chain which comprises or consists of a peptide sequence selected from a group consisting of the following peptide sequences:

[0137] - constant domain of human kappa immunoglobulin light chain (SEQ ID NO: 1 ),

[0138] - constant domain of human kappa immunoglobulin light chain without the C-terminal cysteine (SEQ ID NO: 2),

[0139] - constant domain of mouse kappa immunoglobulin light chain (SEQ ID NO: 4),

[0140] - constant domain of mouse kappa immunoglobulin light chain without the C-terminal cysteine (SEQ ID NO: 5),

[0141] - constant domain of rat kappa immunoglobulin light chain (SEQ ID NO: 6),

[0142] - constant domain of rat kappa immunoglobulin light chain without the C-terminal cysteine (SEQ ID NO: 7),

[0143] - constant domain of rabbit kappa-b4 immunoglobulin light chain (KACB) according to UniProt entry: P01839 (SEQ ID NO: 8), - constant domain of rabbit kappa-b4 immunoglobulin light chain (KACB) according to UniProt entry: P01839, without the C-terminal cysteine (SEQ ID NO: 9),

[0144] - constant domain of rabbit kappa-b4 immunoglobulin light chain (KAC4) according to UniProt entry: P01840 (SEQ ID NO: 10),

[0145] - constant domain of rabbit kappa-b4 immunoglobulin light chain (KAC4) according to UniProt entry: P01840, without the C-terminal cysteine (SEQ ID NO: 11 ),

[0146] - constant domain of rabbit kappa-b5 immunoglobulin light chain (KAC5) according to UniProt entry: P01841 (SEQ ID NO: 12),

[0147] - constant domain of rabbit kappa-b5 immunoglobulin light chain (KAC5) according to UniProt entry: P01841 , without the C-terminal cysteine (SEQ ID NO: 13),

[0148] - constant domain of rabbit kappa-b5 immunoglobulin light chain (KAC6) according to UniProt entry: P03984 (SEQ ID NO: 14),

[0149] - constant domain of rabbit kappa-b5 immunoglobulin light chain (KAC6) according to UniProt entry: P03984, without the C-terminal cysteine (SEQ ID NO: 15),

[0150] - constant domain of dog kappa immunoglobulin light chain (SEQ ID NO: 16),

[0151] - constant domain of pig kappa immunoglobulin light chain (SEQ ID NO: 17),

[0152] - constant domain of monkey kappa immunoglobulin light chain (SEQ ID NO: 18), and

[0153] - constant domain of monkey kappa immunoglobulin light chain without the C-terminal cysteine (SEQ ID NO: 19).

[0154] The constant domain of human kappa immunoglobulin light chain (SEQ ID NO: 1 ) and the constant domain of human kappa immunoglobulin light chain without the C-terminal cysteine (SEQ ID NO: 2) are preferred peptide sequences. In the tag of the fusion polypeptide the used constant domain of the immunoglobulin light chain or orthologous polypeptide or fragment thereof is, as shown in Example 3 and Figures 5 and 7.

[0155] Due to the deleted C-terminal cysteine, the constant domain used in the tag would not build a disulfide bridge with other cysteines present outside the tag. Therefore, no dimers or multimers connected by disulfide bridge(s) would be formed which may result in unwanted aggregates, especially when the disulfide bridges are formed between the C-terminal cysteine of the constant domain and a cysteine of the target polypeptide. This feature is also beneficial for the linkage of more than one constant domain in the tag in an ordered manner by using linkers.

[0156] The tag may further comprise the first linker that fuses the tag to the target polypeptide. In the fusion polypeptide, the tag may be fused via the first linker with the target polypeptide. Furthermore, several constant domains within the tag could be fused via a second linker.

[0157] The first linker and / or the second linker may be cleavable or not cleavable. A cleavable linker could be used if it is desired to cleave the tag and the target polypeptide at a defined position after purification of the fusion polypeptide. A non-cleavable linker may be used, if e.g. the tag is further used for detection of the fusion polypeptide or is necessary to enhance the stability of the fusion polypeptide. Furthermore, the first linker may be a cleavable linker and the second linker may be a non-cleavable linker or the first linker may be a non-cleavable linker and the second linker may be a cleavable linker. Suitable linkers are known to the skilled person (reviewed e.g. in Su et al., 2021 ). Examples of such linkers are a G4-linker (SEQ ID NO: 60), a G5-linker (SEQ ID NO: 61 ), a GGGS-linker (SEQ ID NO: 62), a GGGGS-linker (SEQ ID NO: 63), a A(EAAAK)4ALEA(EAAAK)4A-linker (SEQ ID NO: 69), a PAPAP-linker (SEQ ID NO: 64), a APAPAPA-linker (SEQ ID NO: 65), a APAPAPAPAPKPA-I inker (SEQ ID NO: 66), a APAPAPAPAPAPA-I inker (SEQ ID NO: 67), a A(EAAAK)4A-linker (SEQ ID NO: 68), and a A(EAAAK)2A-linker (SEQ IDS NO: 78), or one or more disulfide bridge(s).

[0158] It should be emphasized that neither the first linker nor the second linker is absolutely necessary for the production or functionality of the fusion protein as shown in Examples 6 and 7 and Figures 11 and 12. Furthermore, example 8 and Figure 14 show that the tag without the second linker between several constant domains comprised in the tag is more stable in a trypsin solution than the tag with one of the linker sequences GGGGSGGGGS (SEQ ID NO: 77), GSPKSCDKTHTCPPCPAPGSEIK (SEQ ID NO: 79), or GSGGGGSGGGGSGEIK (SEQ ID NO: 80).

[0159] In the fusion polypeptide, the tag may be fused to the C-terminus or to the N-terminus of the target polypeptide.

[0160] The tag may also be fused to the C-terminus and the N-terminus of the target polypeptide in the fusion polypeptide.

[0161] Thus, the tag could be fused to one or both ends of the target polypeptides depending on the desired position of the tag in the resulting fusion polypeptide.

[0162] Furthermore, the tag is stable as shown in Example 3 and Figures 5 and 7.

[0163] Due to the tag, the inventive fusion polypeptide of the second aspect and / or third aspect is expressed and / or secreted at a higher level by the used cells and has a higher stability compared to other used tags or the untagged target polypeptide.

[0164] Thus, an expression of the fusion polypeptide is higher than the expression of the untagged target polypeptide, as shown in Figure 11 A. The expression of the fusion polypeptide may be, for example, fivefold higher than the expression of the untagged target polypeptide.

[0165] Remarkably, the expression of the fusion polypeptide comprising more than one tag, e.g. 5 tags, is higher than the expression with one tag as shown in Figures 1 C and 11 A.

[0166] The expressed inventive fusion polypeptide may further have a higher homogeneity compared to a fusion polypeptide comprising a His-tag and the target polypeptide. Also, a function of the target polypeptide, e.g. the enzymatic activity of the target polypeptide, should not be markedly reduced by the used tag.

[0167] Thus, the function of the target polypeptide with the tag in the fusion polypeptide may be substantially comparable to a function of the target polypeptide without the tag.

[0168] Additionally, the fusion polypeptide may have an isoelectric point (pl) different from the untagged target polypeptide. The constant domain, as part of the tag, has a predicted pl of 6.1 (according to Expasy ProtParam). As a result, the pl of the fusion polypeptide may change compared to the untagged target polypeptide with the numbers of constant domains present in the tag. Thus, the solubility of the fusion polypeptide as well as its binding to ion exchange columns can be modified compared to the untagged target polypeptide.

[0169] Furthermore, since specific nanobodies against the constant domain, which is part of the tag, are available, the tag and / or the fusion polypeptide containing the tag could also be used to immobilize fusion proteins on e.g. a surface in immune assays.

[0170] Further the inventive fusion polypeptide is stable due to the stability of the tag as mentioned above.

[0171] Not limiting examples of the target polypeptide may be Darbepoetin alfa (SEQ ID NO: 43), Erythropoietin (SEQ ID NO: 45), Granulocyte-Colony Stimulating Factor (SEQ ID NO: 48), an antibody, an interleukin-15 construct, or an alpha subunit of interleukin 27 comprising at least one mutation. The antibody of this example may be a bi-specific T- cell engager (BiTE) antibody construct targeting DLL3 and CD3 (SEQ ID NO: 51 ). The interleukin-15 construct of this example may be an interleukin-15 receptor alpha subunit (sushi domain) linked by a (GGGGS)e-linker (SEQ ID NO: 70) to interleukin-15 (SEQ ID NO: 55). The alpha subunit of interleukin 27 of this example may be comprised at least one mutation is p28L162C AC(SEQ ID NO: 22). To be absolutely clear, these are only exemplary target polypeptides. Every other polypeptide may be suitable to be used as target polypeptide within the disclosed invention. The target polypeptide of the fusion polypeptide of the first, second and / or third aspect may be a polypeptide used in the pharmaceutical industry. Thus, the target polypeptide may be e.g. an enzyme.

[0172] The target polypeptide of the fusion polypeptide of the first, second and / or third aspect may also be a polypeptide used for in-vitro diagnostics. This target polypeptide may bind to a structure present in a biological sample, wherein the binding may indicate that the structure is pathologically altered or not. Therefore, the fusion polypeptide may be further labeled e.g. with a biotin, a fluorescence probe, or a radioactive nuclide. Also, a labeled antibody directed to the tag of the fusion polypeptide may be used, e.g. in an ELISA. This includes the use of the fusion polypeptide in an in-vitro diagnostic procedure.

[0173] The target polypeptide of the fusion polypeptide of the first, second and / or third aspect may also be a polypeptide used for a medical indication and / or in-vivo diagnostics. This target polypeptide may be used as a therapeutic in a medical indication. Thus, the target polypeptide may compensate a reduction or loss of a physiological enzymatic activity or a physiologic polypeptide. As not limiting examples the target polypeptide may be a polypeptide useful in blood coagulation or may be insulin. Furthermore, the target polypeptide may bind to pathologically altered structures of a cell, e.g. a cancer cell, and may transport a drug or a radioactive nuclide, bound to the tag, to the cell. Also, the target polypeptide may be a polypeptide used for in-vitro diagnostics. Therein, the fusion polypeptide labeled with a detecting agent may be administered to a patient, the target polypeptide of the administered fusion polypeptide may bind to pathologically altered structures of a given tissue or cell, and the binding could be detected by detecting the detecting agent which may be bound to the tag of the fusion polypeptide. The detecting agent may be any useful substance used for in-vivo diagnostics, e.g. a radioactive nuclide or a paramagnetic substance for use in in-vivo (magnetic resonance) imaging. This includes the use of the fusion polypeptide for the manufacture of medicament for use in a medical indication and / or in in-vivo diagnostics.

[0174] Thus, also the fusion polypeptide of the invention may be used for a medical indication. The fusion polypeptide may also be for the use as a medicament. This includes the use of the fusion polypeptide as a medicament.

[0175] The target polypeptide of the fusion polypeptide of the first, second and / or third aspect may also be a polypeptide used in biotechnology methods. The polypeptide may be e.g. an enzyme used in laboratory methods, like an enzyme for nucleic acid or protein cleavage. This includes the use of the fusion polypeptide in biotechnology methods.

[0176] The target polypeptide of the fusion polypeptide of the first, second and / or third aspect may also be a polypeptide used in the chemical industry. Thus, the polypeptide may be a polypeptide present in a cleaning agent, or a polypeptide that is used in any other production method or product of the chemical industry. This includes the use of the fusion polypeptide in the chemical industry.

[0177] The target polypeptide of the fusion polypeptide of the first, second and / or third aspect may also be a polypeptide used in the production of food, e.g. the polypeptide may be an enzyme used for the production of cheese or yoghurt. This includes the use of the fusion polypeptide in the food production.

[0178] One preferred fusion polypeptide of the invention comprises as the target polypeptide an IL27a (L162C) (AC) protein, also mentioned as p28L162C’AC(SEQ ID NO: 22) throughout this application wherein position 162 relates to the IL27a protein including the N-terminal signal sequence, and wherein AC means a deletion of amino acids H229 to P243 relating to the IL27a protein including the N-terminal signal sequence. In the International Patent Application PCT / EP2020 / 081877, published as WO 2021 / 094435 A2, such a deletion is mentioned in Figure 13. The relating amino acid sequence is SEQ ID NO: 11 (including the signal sequence) of PCT / EP2020 / 081877. This target polypeptide could be fused to the tag by e.g. using the PAPAP-linker (SEQ ID NOs: 27, 33) (see also the Example section).

[0179] In a fifth aspect, the invention provides a pharmaceutical composition comprising a fusion polypeptide of the second and / or fourth aspect and optionally a pharmaceutically acceptable carrier, diluent, or excipient. In a sixth aspect, the invention provides a nucleic acid for expression of the fusion polypeptide of the second and / or fourth aspect comprising a nucleic acid sequence encoding the target polypeptide of the first and / or fourth aspect and a nucleic acid sequence encoding the tag mentioned in the first and / or fourth aspect, comprising a nucleic acid sequence encoding at least one constant domain of an immunoglobulin light chain or orthologous polypeptide or fragment thereof, and optionally a nucleic acid sequence encoding the first peptide linker of the first and / or fourth aspect.

[0180] Thus, any disclosure regarding the first, second, third, fourth, and fifth aspect above is included herein, except the description dictates otherwise.

[0181] The term “nucleic acid” includes DNA, RNA, and any further molecule containing a nucleic acid (e.g. a protein after binding to a nucleic acid), independent of the length of the nucleic acid.

[0182] The term “constant domain of an immunoglobulin light chain” also includes any constant domain of orthologous immunoglobulin light chains and any fragments of the constant domain of the immunoglobulin light chain or of an orthologous immunoglobulin light chain. Further included are constant domains of immunoglobulin light chain-like sequences.

[0183] The term “constant domain of a kappa immunoglobulin light chain” also includes any constant domain of orthologous kappa immunoglobulin light chains and any fragments of the constant domain of the kappa immunoglobulin light chain or of an orthologous kappa immunoglobulin light chain. Further included are constant domains of kappa immunoglobulin light chain-like sequences.

[0184] The inventive nucleic acid may further comprise the nucleic acid sequence encoding the tag comprising a nucleic acid sequence encoding two or more constant domains of an immunoglobulin light chain or orthologous polypeptides or fragments thereof, which are optionally connected to each other by a nucleic acid sequence encoding a second peptide linker. The inventive nucleic acid may further comprise a nucleic acid sequence encoding a peptide signal sequence for targeting the fusion protein to be expressed to a cellular region in which a formation of disulfide bonds is possible.

[0185] The nucleic acid sequence encoding the constant domain of the immunoglobulin light chain or orthologous polypeptide or fragment thereof of the inventive nucleic acid may be a nucleic acid sequence encoding a constant domain of a kappa immunoglobulin light chain or orthologous polypeptide or fragment thereof.

[0186] The nucleic acid sequence encoding the constant domain of the immunoglobulin light chain or fragment thereof of the inventive nucleic acid may be a nucleic acid sequence encoding a constant domain of a mammal immunoglobulin light chain or orthologous polypeptide or fragment thereof, preferred a nucleic acid sequence encoding a constant domain of a primate immunoglobulin light chain or fragment thereof and most preferred a nucleic acid sequence encoding a constant domain of a human immunoglobulin light chain or fragment thereof.

[0187] The nucleic acid sequence encoding the constant domain of the kappa immunoglobulin light chain or fragment thereof of the inventive nucleic acid may be a nucleic acid sequence encoding a constant domain of a mammal kappa immunoglobulin light chain or orthologous polypeptide or fragment thereof, preferred a nucleic acid sequence encoding a constant domain of a primate kappa immunoglobulin light chain or fragment thereof, and most preferred a nucleic acid sequence encoding a constant domain of a human kappa immunoglobulin light chain or fragment thereof.

[0188] The nucleic acid sequence encoding the constant domain of the immunoglobulin light chain or fragment thereof of the inventive nucleic acid may be a nucleic acid sequence encoding the constant domain of the kappa immunoglobulin light chain without a C- terminal cysteine of the constant domain of the kappa immunoglobulin light chain or fragment thereof.

[0189] The nucleic acid sequence encoding the constant domain of the kappa immunoglobulin light chain or fragment thereof of the inventive nucleic acid may be selected from a group consisting of a nucleic acid sequence encoding a constant domain of a mouse kappa immunoglobulin light chain, a nucleic acid sequence encoding a constant domain of a rat kappa immunoglobulin light chain, a nucleic acid sequence encoding a constant domain of a rabbit kappa immunoglobulin light chain, a nucleic acid sequence encoding a constant domain of a dog kappa immunoglobulin light chain, a nucleic acid sequence encoding a constant domain of a pig kappa immunoglobulin light chain, a nucleic acid sequence encoding a constant domain of a monkey kappa immunoglobulin light chain, and preferably the nucleic acid sequence encoding the constant domain of the human kappa immunoglobulin light chain.

[0190] The nucleic acid sequences encoding the constant domain of the kappa immunoglobulin light chain of the inventive nucleic acid could be deduced from the amino acid sequences of the constant domain of the kappa immunoglobulin light chains mentioned above (cf. first, second, third and fourth aspect). The nucleic acid sequence may be further codon optimized depending on the expressing cell (see e.g. the paragraph “Constructs” in the EXAMPLES section). Thus, suitable codon optimized nucleic acid sequence encoding one of the constant domains of the kappa immunoglobulin light chains could be prepared by the skilled person using known methods.

[0191] The inventive nucleic acid may comprise the nucleic acid sequence encoding the first and second peptide linker, wherein the peptide linkers to be expressed may a cleavable or a non-cleavable peptide linker as described above in the first, third, and fourth aspect.

[0192] The nucleic acid sequence encoding the tag, and optional the nucleic acid sequence encoding the peptide linker, of the inventive nucleic acid may be at the 3'-end of the nucleic acid sequence encoding the target polypeptide such that the tag is fused to the C-terminus of the target polypeptide in the expressed fusion polypeptide.

[0193] The nucleic acid sequence encoding the tag, and optional the nucleic acid sequence encoding the peptide linker, of the inventive nucleic acid may be at the 5'-end of the nucleic acid sequence encoding the target polypeptide such that the tag is fused to the N-terminus of the target polypeptide in the expressed fusion polypeptide. The nucleic acid sequence encoding the tag, and optional the nucleic acid sequence encoding the peptide linker, of the inventive nucleic acid may be at the 5'-end and the 3'-end of the nucleic acid sequence encoding the target polypeptide such that the tag is fused to the N-terminus and the C-terminus of the target polypeptide in the expressed fusion polypeptide.

[0194] Furthermore, the nucleic acid sequence encoding the target polypeptide of the inventive nucleic acid may encode a polypeptide used in the pharmaceutical industry. The polypeptide may be the same as the respective polypeptide described in the fourth aspect.

[0195] The nucleic acid sequence encoding the target polypeptide of the inventive nucleic acid may encode a polypeptide used for in-vitro diagnostics. The polypeptide may be the same as the respective polypeptide described in the fourth aspect.

[0196] The nucleic acid sequence encoding the target polypeptide of the inventive nucleic acid may encode polypeptide for use as a medicament. The polypeptide may be the same as the respective polypeptide described in the fourth aspect.

[0197] The nucleic acid sequence encoding the target polypeptide of the inventive nucleic acid may encode a polypeptide used for a medical indication. The polypeptide may be the same as the respective polypeptide described in the fourth aspect.

[0198] The nucleic acid sequence encoding the target polypeptide of the inventive nucleic acid may encode a polypeptide for use as a medicament. The polypeptide may be the same as the respective polypeptide described in the fourth aspect.

[0199] The nucleic acid of the invention may be further used as a medicament, e.g. as RNA. The RNA may be suitable enveloped for entry in a cell.

[0200] The nucleic acid sequence encoding the target polypeptide of the inventive nucleic acid may encode a polypeptide used in the chemical industry or food production. The polypeptide may be the same as the respective polypeptide described in the fourth aspect. The nucleic acid sequence encoding the target polypeptide of the inventive nucleic acid may encode a polypeptide used in biotechnology methods. The polypeptide may be the same as the respective polypeptide described in the fourth aspect.

[0201] In a seventh aspect, the invention provides a pharmaceutical composition comprising the nucleic acid of the sixth aspect and optionally a pharmaceutically acceptable carrier, diluent, or excipient.

[0202] In an eighth aspect, the invention provides a method for purifying an expressed fusion polypeptide, comprising providing a fusion polypeptide of the second or fourth aspect expressed by cells, optionally transfected with a nucleic acid of the sixth aspect, provision of a ligand directed to the tag of the fusion polypeptide, binding of the ligand to the tag, and eluting the fusion polypeptide from the ligand.

[0203] The ligand of the inventive method of the eighth aspect may be directed to the constant domain of the immunoglobulin light chain of the tag.

[0204] Ligands to the constant domain of the immunoglobulin light chain are known and established, known ligands could be used for the purification of the expressed fusion polypeptide by binding to the tag of the fusion polypeptide. Furthermore, it is also possible to use now established GMP certified ligands for the purification of the fusion polypeptide which paves the way for the production of biopharmaceuticals. However, if the ligand to the respective constant domain used is not commercially available, the skilled person could produce such ligand by readily available methods.

[0205] It is to mention that the purification of the expressed fusion polypeptide is enhanced when more than one, e.g. five, constant domains of the antibody light chain or orthologous polypeptide or fragment thereof are used within the tag.

[0206] The ligand of the inventive method of the eighth aspect may be an antibody or an antibody fragment, optionally coupled to beads, further optionally coupled to magnetic beads. The inventive method of the eighth aspect may further comprise the washing of the fusion polypeptide bound to the ligand with a suitable buffer.

[0207] The expressed fusion polypeptide purified by inventive method of the seventh aspect is the fusion polypeptide of the second or third aspect. Thus, the disclosure of the second aspect and / or fourth aspect is herein included. Therefore, the expressed fusion polypeptide may comprise a polypeptide used in the chemical industry or food production as target polypeptide of the fusion polypeptide of the second aspect and / or fourth aspect.

[0208] Also, the expressed fusion polypeptide may comprise a polypeptide used in biotechnology methods as target polypeptide of the fusion polypeptide of the second aspect and / or fourth aspect.

[0209] The expressed fusion polypeptide may comprise a polypeptide used in the pharmaceutical industry as target polypeptide of the fusion polypeptide of the second aspect and / or fourth aspect.

[0210] The expressed fusion polypeptide may further comprise a polypeptide for use in a medical indication as target polypeptide of the fusion polypeptide of the second aspect and / or fourth aspect.

[0211] The expressed fusion polypeptide may comprise a polypeptide for in-vitro diagnostic procedures as target polypeptide of the fusion polypeptide of the second aspect and / or fourth aspect.

[0212] In a ninth aspect, the invention provides, a method of modulating the plasma half-time of a fusion polypeptide in an organism, wherein the method comprises a) determining the molecular mass of the target polypeptide, wherein the target polypeptide is equal to the target polypeptide as described above in the other aspects, b) (optional) determining the molecular mass of one constant domain of an immunoglobulin light chain or orthologous polypeptide or fragment thereof, c) calculating how many constant domains of an immunoglobulin light chain or orthologous polypeptides or fragments thereof are needed in addition to the molecular mass of the target polypeptide (step a) for reaching a desired molecular mass of the fusion polypeptide, and d) creating the tag to be fused to the target polypeptide by combining the necessary number of constant domains of an immunoglobulin light chain or orthologous polypeptides or fragments thereof according to the result of step (c).

[0213] The desired molecular mass of the fusion polypeptide could be below or above a filtration limit of a kidney of a given organism or, optionally, below or at least 50 kDa or 70 kDa.

[0214] In addition, it is also possible that the target polypeptide in this method already comprises a tag comprising one or more constant domains of an immunoglobulin light chain or orthologous polypeptides or fragments thereof and therefore is already a fusion polypeptide. For increasing the plasma half-life of this fusion polypeptide, one or more constant domains of an immunoglobulin light chain or orthologous polypeptides or fragments thereof could be added to the tag according to the steps b to d of the method described before so that the resulting fusion polypeptide has a molecular mass of 50 kDa or 70 kDa or more.

[0215] Preferably, the molecular mass of the fusion polypeptide is at least 50 kDa or at least 70 kDa or alternatively below 50 kDa or 70 kDa; depending on whether the plasma half-life of the fusion polypeptide should be prolonged or shortened.

[0216] This method could also be used for increasing the plasma half-life of the target polypeptide. Thus, the method described before used for increasing the plasma halftime of the target polypeptide by generating the fusion polypeptide according to the method described before.

[0217] The disclosure regarding to the method of the first aspect and to fusion polypeptide of the second aspect and / or fourth aspect is included herein, except the description dictates otherwise. In a tenth aspect, the invention provides, a method of detecting the fusion polypeptide of the second aspect and / or fourth aspect, comprising providing a sample, which may comprise the fusion polypeptide, and detecting the fusion polypeptide in the sample by using a detecting agent directed to the tag or the target polypeptide.

[0218] Since the tenth aspect is also directed to the second aspect and / or fourth aspect, the disclosure of the second aspect and fourth aspect is herein included.

[0219] The detecting agent used in the method of detecting may be labeled with biotin, a fluorescence probe, or a radioactive nuclide.

[0220] The detecting agent may optionally be an antibody or an antibody fragment.

[0221] The sample used in this method of detecting could be taken during the production process of the fusion polypeptide of the second aspect or the fourth aspect or the pharmaceutical composition of the fifth aspect.

[0222] Using a sample taken during the production process, this method could be used as a surveillance method of or during a production process.

[0223] In an alternative, the sample used in the method of detecting may be a biological sample of a mammal, to which the fusion polypeptide or the pharmaceutical composition has been administered.

[0224] The mammal could be a non-human mammal, preferably be a mouse, a rat, a rabbit, a dog, a cat, a pig, or a monkey.

[0225] The mammal could also be a human being.

[0226] The biological sample mentioned before may be tissue, urine, feces, or blood of the non-human mammal.

[0227] In an eleventh aspect, the invention provides a method of detecting the fusion polypeptide of the second aspect and / or fourth aspect in-vivo after administration to a mammal, wherein the fusion polypeptide is labeled by a fluorescence probe, a magnetic resonance detectable probe, an X-ray detectable probe or a radioactive nuclide. The mammal of this method of detecting may be a non-human mammal, preferably a mouse, a rat, a rabbit, a dog, a cat, a pig, or a monkey.

[0228] The mammal of this method of detecting may be a human being.

[0229] Taken together, with the method of the first aspect, it is possible to modulate the molecular mass of a fusion polypeptide by only modulating the number of constant domains comprised in the tag. The use of this method using the versatile protein fusion tag described in the first aspect should extend the serum half-life of non-antibody biopharmaceuticals and diagnostic proteins, the fusion polypeptides of the second or third aspect due to raising the molecular mass of the fusion protein and circumvent renal clearance. It is further possible to modulate the isoelectric point of a fusion polypeptide by addition of constant domains according to any one of the above aspects for improving or enabling of purification of the fusion polypeptide and / or the target polypeptide.

[0230] The description of the sequences, shown in the sequence listing and as used in the context of the present invention, is as follows:

[0231] SEQ ID NO: 1 shows the constant domain of human kappa immunoglobulin light chain, as recited in UniProt entry: P01834

[0232] SEQ ID NO: 2 shows the constant domain of human kappa immunoglobulin light chain, without the C-terminal cysteine

[0233] SEQ ID NO: 3 shows the DNA sequence of the constant domain of human kappa immunoglobulin light chain, including a sequence of a human p28, IL-27alpha peptide signal sequence, without the C-terminal cysteine

[0234] SEQ ID NO: 4 shows the constant domain of mouse kappa immunoglobulin light chain, as recited in UniProt entry: P01837

[0235] SEQ ID NO: 5 shows the constant domain of mouse kappa immunoglobulin light chain, without the C-terminal cysteine

[0236] SEQ ID NO: 6 shows the constant domain of rat kappa immunoglobulin light chain, as recited in GenBank entry: AAA91898.1 , without the signal peptide SEQ ID NO: 7 shows the constant domain of rat kappa immunoglobulin light chain, without the C-terminal cysteine

[0237] SEQ ID NO: 8 shows the constant domain of rabbit kappa-b4 immunoglobulin light chain (KACB), as recited in UniProt entry: P01839

[0238] SEQ ID NO: 9 shows the constant domain of rabbit kappa-b4 immunoglobulin light chain (KACB) according to UniProt entry: P01839, without the C- terminal cysteine

[0239] SEQ ID NO: 10 shows the constant domain of rabbit kappa-b4 immunoglobulin light chain (KAC4), as recited in UniProt entry: P01840

[0240] SEQ ID NO: 11 shows the constant domain of rabbit kappa-b4 immunoglobulin light chain (KAC4) according to UniProt entry: P01840, without the C- terminal cysteine

[0241] SEQ ID NO: 12 shows the constant domain of rabbit kappa-b5 immunoglobulin light chain (KAC5), as recited in UniProt entry: P01841

[0242] SEQ ID NO: 13 shows the constant domain of rabbit kappa-b5 immunoglobulin light chain (KAC5) according to UniProt entry: P01841 , without the C- terminal cysteine

[0243] SEQ ID NO: 14 shows the constant domain of rabbit kappa-b5 immunoglobulin light chain (KAC6), as recited in UniProt entry: P03984

[0244] SEQ ID NO: 15 shows the constant domain of rabbit kappa-b5 immunoglobulin light chain (KAC6) according to UniProt entry: P03984, without the C- terminal cysteine

[0245] SEQ ID NO: 16 shows the constant domain of dog kappa immunoglobulin light chain, as recited in UniProt entry: A0A8C0KWW5

[0246] SEQ ID NO: 17 shows the constant domain of pig kappa immunoglobulin light chain, as recited in GenBank entry: AHB17990.1

[0247] SEQ ID NO: 18 shows the constant domain of monkey kappa immunoglobulin light chain, as recited in UniProt entry: A0A5F8A7T3

[0248] SEQ ID NO: 19 shows the constant domain of monkey kappa immunoglobulin light chain, without the C-terminal cysteine

[0249] SEQ ID NO: 20 shows the constant domain of human immunoglobulin light chain without the C-terminal cysteine, including a human p28, IL-27alpha signal sequence

[0250] SEQ ID NO: 21 shows the human p28, IL-27alpha signal sequence SEQ ID NO: 22 shows the p28L162C ACprotein (p28 L162C, dC)

[0251] SEQ ID NO: 23 shows the p28L162C’ACprotein (p28 L162C, dC) fused to one constant domain of human kappa immunoglobulin light chain, w / o C-terminal cysteine

[0252] SEQ ID NO: 24 shows the p28L162C’ACprotein (p28 L162C, dC) fused to one constant domain of human kappa immunoglobulin light chain, w / o C-terminal cysteine, via the GGGS-linker

[0253] SEQ ID NO: 25 shows the p28L162C’ACprotein (p28 L162C, dC) fused to one constant domain of human kappa immunoglobulin light chain, w / o C-terminal cysteine, via the A(EAAAK)4ALEA(EAAAK)4A-linker

[0254] SEQ ID NO: 26 shows the p28L162C’ACprotein (p28 L162C, dC) fused to five constant domain of human kappa immunoglobulin light chain, w / o C-terminal cysteine, via the A(EAAAK)4ALEA(EAAAK)4A-linker

[0255] SEQ ID NO: 27 shows the p28L162C’ACprotein (p28 L162C, dC) fused to one constant domain of human kappa immunoglobulin light chain, w / o C-terminal cysteine, via the PAPAP-linker

[0256] SEQ ID NO: 28 shows the p28L162C’ACprotein (p28 L162C, dC) fused to one constant domain of human kappa immunoglobulin light chain, w / o C-terminal cysteine, via the APAPAPA-linker

[0257] SEQ ID NO: 29 shows the p28L162C’ACprotein (p28 L162C, dC) fused to one constant domain of human kappa immunoglobulin light chain, w / o C-terminal cysteine, via the APAPAPAPAPKPA-linker

[0258] SEQ ID NO: 30 shows the p28L162C’ACprotein (p28 L162C, dC) fused to one constant domain of human kappa immunoglobulin light chain, w / o C-terminal cysteine, via the APAPAPAPAPAPA-linker

[0259] SEQ ID NO: 31 shows the p28L162C’ACprotein (p28 L162C, dC) fused to one constant domain of human kappa immunoglobulin light chain, w / o C-terminal cysteine, via the A(EAAAK)4A-linker

[0260] SEQ ID NO: 32 shows the p28L162C’ACprotein (p28 L162C, dC) fused to five constant domain of human kappa immunoglobulin light chain, w / o C-terminal cysteine

[0261] SEQ ID NO: 33 shows the p28L162C’ACprotein (p28 L162C, dC) fused to five constant domain of human kappa immunoglobulin light chain, w / o C-terminal cysteine, via the PAPAP-linker SEQ ID NO: 34 shows the p28L162C’ACprotein (p28 L162C, dC) fused to five constant domain of human kappa immunoglobulin light chain, w / o C-terminal cysteine, via the APAPAPA-linker

[0262] SEQ ID NO: 35 shows the p28L162C’ACprotein (p28 L162C, dC) fused to five constant domain of human kappa immunoglobulin light chain, w / o C-terminal cysteine, via the APAPAPAPAPKPA-linker

[0263] SEQ ID NO: 36 shows the p28L162C’ACprotein (p28 L162C, dC) fused to five constant domain of human kappa immunoglobulin light chain, w / o C-terminal cysteine, via the APAPAPAPAPAPA-linker

[0264] SEQ ID NO: 37 shows the p28L162C’ACprotein (p28 L162C, dC) fused to five constant domain of human kappa immunoglobulin light chain, w / o C-terminal cysteine, via the A(EAAAK)4A-linker

[0265] SEQ ID NO: 38 shows the p28L162C’ACprotein (p28 L162C, dC) fused to three constant domain of human kappa immunoglobulin light chain, w / o C- terminal cysteine, via the PAPAP-linker

[0266] SEQ ID NO: 39 shows the p28L162C’ACprotein (p28 L162C, dC) fused to five constant domain of human kappa immunoglobulin light chain, w / o C-terminal cysteine, via the PAPAP-linker, wherein each constant domain is fused to another constant domain via a GAGAG-linker

[0267] SEQ ID NO: 40 shows the p28L162C’ACprotein (p28 L162C, dC) fused to seven constant domain of human kappa immunoglobulin light chain, w / o C- terminal cysteine, via the PAPAP-linker

[0268] SEQ ID NO: 41 shows five constant domain of human kappa immunoglobulin light chain, w / o C-terminal cysteine fused to the N-terminus of the p28L162c, AC protein(p28 L162C, dC), via the PAPAP-linker

[0269] SEQ ID NO: 42 shows Darbepoetin alfa, including N-terminal signal sequence

[0270] SEQ ID NO: 43 shows Darbepoetin alfa fused to a Hise-tag via a GGGGG-linker, including N-terminal signal sequence

[0271] SEQ ID NO: 44 shows Darbepoetin alfa fused to five constant domains of human kappa immunoglobulin light chain, w / o C-terminal cysteine via the GGGGG-linker, including N-terminal signal sequence

[0272] SEQ ID NO: 45 shows Erythropoietin, including N-terminal signal sequence

[0273] SEQ ID NO: 46 shows Erythropoietin fused to a Hise-tag via a GGGGG-linker, including N-terminal signal sequence SEQ ID NO: 47 shows Erythropoietin fused to five constant domains of human kappa immunoglobulin light chain, w / o C-terminal cysteine via the GGGGG- linker, including N-terminal signal sequence

[0274] SEQ ID NO: 48 shows Granulocyte-Colony Stimulating Factor, including N-terminal signal sequence

[0275] SEQ ID NO: 49 shows Granulocyte-Colony Stimulating Factor fused to a Hise-tag via a GGGGG-linker, including N-terminal signal sequence

[0276] SEQ ID NO: 50 shows Granulocyte-Colony Stimulating Factor fused to five constant domains of human kappa immunoglobulin light chain, w / o C-terminal cysteine via the GGGGG-linker, including N-terminal signal sequence

[0277] SEQ ID NO: 51 shows bi-specific T-cell engager (BiTE) antibody construct targeting

[0278] DLL3 and CD3 (DLL3-4 x I2C scFv), including human p28 signal sequence

[0279] SEQ ID NO: 52 shows bi-specific T-cell engager (BiTE) antibody construct targeting DLL3 and CD3 (DLL3-4 x I2C scFv) fused to a Hise-tag via a GGGGG-linker, including N-terminal signal sequence

[0280] SEQ ID NO: 53 shows bi-specific T-cell engager (BiTE) antibody construct targeting DLL3 and CD3 (DLL3-4 x I2C scFv) fused to five constant domains of human kappa immunoglobulin light chain, w / o C-terminal cysteine via the GGGGG-linker, including N-terminal signal sequence

[0281] SEQ ID NO: 54 shows IL-15Ra(sushi)IL-15, including N-terminal signal sequence

[0282] SEQ ID NO: 55 shows IL-15Ra(sushi)IL-15 fused to a Hise-tag via a GGGGS-linker, including N-terminal signal sequence

[0283] SEQ ID NO: 56 shows IL-15Ra(sushi)IL-15 fused to five constant domains of human kappa immunoglobulin light chain, w / o C-terminal cysteine via the GGGGS-linker, including N-terminal signal sequence

[0284] SEQ ID NO: 57 shows five constant domains of human lambda immunoglobulin light chain, w / o C-terminal cysteine and serine

[0285] SEQ ID NO: 58 shows the p28L162C’ACprotein (p28 L162C, dC) fused to five constant domain of human lambda immunoglobulin light chain, w / o C-terminal cysteine, via the PAPAP-linker

[0286] SEQ ID NO: 59 shows five constant domains of human kappa immunoglobulin light chain, w / o C-terminal cysteine SEQ ID NO: 60 shows the amino acid sequence GGGG (G4-linker)

[0287] SEQ ID NO: 61 shows the amino acid sequence GGGGG (G5-linker)

[0288] SEQ ID NO: 62 shows the amino acid sequence GGGS (GGGS-linker)

[0289] SEQ ID NO: 63 shows the amino acid sequence GGGGS (GGGGS-linker)

[0290] SEQ ID NO: 64 shows the amino acid sequence PAPAP (PAPAP-linker)

[0291] SEQ ID NO: 65 shows the amino acid sequence APAPAPA (APAPAPA-linker)

[0292] SEQ ID NO: 66 shows the amino acid sequence APAPAPAPAPKPA

[0293] (APAPAPAPAPKPA-linker)

[0294] SEQ ID NO: 67 shows the amino acid sequence APAPAPAPAPAPA

[0295] (APAPAPAPAPAPA-linker)

[0296] SEQ ID NO: 68 shows the amino acid sequence AEAAAKEAAAKEAAAKEAAAKA (A(EAAAK)4A-linker)

[0297] SEQ ID NO: 69 shows the amino acid sequence AEAAAKEAAAKEAAAKEAAAKALEAEAAAKEAAAKEAAAKEAAAK

[0298] A (A(EAAAK)4ALEA(EAAAK)4A-linker)

[0299] SEQ ID NO: 70 shows the amino acid sequence

[0300] GGGGSGGGGSGGGGSGGGGSGGGGSGGGGS ((GGGGS)6- linker)

[0301] SEQ ID NO: 71 shows two constant domains of human kappa immunoglobulin light chain, w / o C-terminal cysteine, including N-terminal signal sequence

[0302] SEQ ID NO: 72 shows two constant domains of human kappa immunoglobulin light chain, w / o C-terminal cysteine, linked by (GGGGS)2, including signal sequence

[0303] SEQ ID NO: 73 shows two constant domains of human kappa immunoglobulin light chain, w / o C-terminal cysteine, linked by A(EAAAK)2A, including signal sequence

[0304] SEQ ID NO: 74 shows the amino acid sequence of two constant domains of human kappa immunoglobulin light chain, w / o C-terminal cysteine, linked by GSPKSCDKTHTCPPCPAPGSEIK, including signal sequence (in italic)

[0305] SEQ ID NO: 75 shows the amino acid sequence of two constant domains of human kappa immunoglobulin light chain, w / o C-terminal cysteine, linked by GS(GGGGS)2GEIK, including signal sequence SEQ ID NO: 76 shows the amino acid sequence of two constant domains of human kappa immunoglobulin light chain, w / o C-terminal cysteine, linked by PAPAP, including signal sequence

[0306] SEQ ID NO: 77 shows the amino acid sequence GGGGSGGGGS ((GGGGS)2- linker)

[0307] SEQ ID NO: 78 shows the amino acid sequence AEAAAKEAAAKA (A(EAAAK)2A- linker)

[0308] SEQ ID NO: 79 shows the amino acid sequence GSPKSCDKTHTCPPCPAPGSEIK

[0309] SEQ ID NO: 80 shows the amino acid sequence GSGGGGSGGGGSGEIK (GS(GGGGS)2GEIK-linker)

[0310] An overview of the SEQ ID NOs and detailed sequences, as used in the context of the present invention, is given in Table 1.

[0311] It should be clear, that for expression of the soluble polypeptides, which sequences are detailed in the sequence protocol and in Table 1 , a suitable signal sequence (e.g. SEQ ID NO. 21 ) must be attached at the N-terminus of individual amino acid sequence (cf. e.g. SEQ ID NO. 20). On the other hand, it is clear, that the N-terminal signal sequence will be cut off during secretion of the polypeptide such that only the polypeptide without the signal sequence will be secreted by the respective cell.

[0312] Table 1 :

[0313]

[0314]

[0315]

[0316]

[0317] Notes: The amino acid sequences SEQ ID NOs 1 to 19, SEQ ID NOs 22 to 41 , SEQ ID NO: 57, and SEQ ID NO: 58 are without any signal peptide sequence; p28 L162C, dC = p28L162C AC; w / o = without

[0318] * * * * *

[0319] It is noted that as used herein, the singular forms “a”, “an”, and “the”, include plural references unless the context clearly indicates otherwise. Thus, for example, reference to “a reagent” includes one or more of such different reagents and reference to “the method” includes reference to equivalent steps and methods known to those of ordinary skill in the art that could be modified or substituted for the methods described herein.

[0320] Unless otherwise indicated, the term "at least" preceding a series of elements is to be understood to refer to every element in the series. Those skilled in the art will recognize or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the present invention.

[0321] The term "and / or", wherever used herein, includes the meaning of "and", "or" and "all or any other combination of the elements connected by said term".

[0322] The term “less than” or in turn “more than” does not include the concrete number.

[0323] For example, “less than 20” means less than the number indicated. Similarly, “more than” or “greater than” means more than or greater than the indicated number, e.g. “more than 80 %” means more than or greater than the indicated number of 80 %.

[0324] Throughout this specification and the claims which follow, unless the context requires otherwise, the word “comprise”, and variations such as “comprises” and “comprising”, will be understood to imply the inclusion of a stated integer or step or group of integers or steps, but not the exclusion of any other integer or step or group of integer or step. When used herein the term “comprising” can be substituted with the term “containing” or “including” or sometimes, when used herein, with the term “having”. When used herein, “consisting of' excludes any element, step, or ingredient not specified.

[0325] The term “including” means “including but not limited to”, “Including” and “including but not limited to” are used interchangeably.

[0326] It should be understood that this invention is not limited to the particular methodology, protocols, material, reagents, and substances, etc., described herein and as such can vary. The terminology used herein is for the purpose of describing particular embodiments, only, and is not intended to limit the scope of the present invention, which is defined solely by the claims. All publications cited throughout the text of this specification (including all patents, patent application, scientific publications, instructions, etc.), whether supra or infra, are hereby incorporated by reference in their entirety. Nothing herein is to be construed as an admission that the invention is not entitled to antedate such disclosure by virtue of prior invention. To the extent the material incorporated by reference contradicts or is inconsistent with this specification, the specification will supersede any such material.

[0327] The content of all documents and patent documents cited herein is incorporated by reference in their entirety.

[0328] The invention is further characterized by the following items:

[0329] 1 . A method of modulating a molecular mass and / or an isoelectric point of a fusion polypeptide, wherein the fusion polypeptide comprises a target polypeptide fused to a tag suitable for affinity purification or detection of the target polypeptide, wherein the tag comprises at least one constant domain of an immunoglobulin light chain or orthologous polypeptide or fragment thereof and wherein the molecular mass of the fusion polypeptide is modulateable or modulated by changing the molecular mass of the tag via combining the one constant domain of an immunoglobulin light chain or orthologous polypeptide or fragment thereof with one or more further constant domain(s) of an immunoglobulin light chain or orthologous polypeptide(s) or fragment(s) thereof; and wherein optionally the target polypeptide is fused to the tag via a first linker.

[0330] 2. The method of item 1 , wherein the method comprises a) determining the molecular mass of the target polypeptide, b) (optional) determining the molecular mass of one constant domain of an immunoglobulin light chain or orthologous polypeptide or fragment thereof, c) calculating how many constant domains of an immunoglobulin light chain or orthologous polypeptides or fragments thereof are needed in addition to the molecular mass of the target polypeptide (step a) for reaching a desired molecular mass of the fusion polypeptide, and d) creating the tag to be fused to the target polypeptide by combining the necessary number of constant domains of an immunoglobulin light chain or orthologous polypeptides or fragments thereof according to the result of step (c), wherein the desired molecular mass of the fusion polypeptide is optional above or below a filtration limit of a kidney of a given organism or, optionally, below or at least 50 kDa or 70 kDa. The method of any one of the preceding items, wherein the tag comprises two or more constant domains of the immunoglobulin light chain or orthologous polypeptides or fragments thereof and wherein optionally the constant domains or orthologous polypeptides or fragments thereof are connected to each other by a second linker. The method of any one of the preceding items, wherein the fusion polypeptide is expressed under conditions allowing a formation of one or more disulfide bond(s). The method of any one of the preceding items, wherein the constant domain of the immunoglobulin light chain or orthologous polypeptide or fragment thereof of the tag is a constant domain of a kappa immunoglobulin light chain or orthologous polypeptide or fragment thereof. The method of any one of the preceding items, wherein the constant domain of the immunoglobulin light chain or orthologous polypeptide or fragment thereof of the tag is a constant domain of a mammal immunoglobulin light chain or fragment thereof, preferred a constant domain of a primate immunoglobulin light chain or fragment thereof and most preferred a constant domain of a human immunoglobulin light chain or fragment thereof or wherein the constant domain of the kappa immunoglobulin light chain or orthologous polypeptide or fragment thereof of the tag is a constant domain of a mammal kappa immunoglobulin light chain or fragment thereof, preferred a constant domain of a primate kappa immunoglobulin light chain or fragment thereof, and most preferred a constant domain of a human kappa immunoglobulin light chain or fragment thereof. The method of any one of items 5 or 6, wherein a C-terminal cysteine of the constant domain of the kappa immunoglobulin light chain of the tag is deleted if present in the peptide sequence of the constant domain of the kappa immunoglobulin light chain of the tag. The method of any one of items 5 to 7, wherein the constant domain of the kappa immunoglobulin light chain or orthologous polypeptide or fragment thereof of the tag is selected from a group consisting of a constant domain of a mouse kappa immunoglobulin light chain, a constant domain of a rat kappa immunoglobulin light chain, a constant domain of a rabbit kappa immunoglobulin light chain, a constant domain of a dog kappa immunoglobulin light chain, a constant domain of a pig kappa immunoglobulin light chain, a constant domain of a monkey kappa immunoglobulin light chain, and preferably the constant domain of the human kappa immunoglobulin light chain. The method of any one of items 5 to 8, wherein the constant domain of the kappa immunoglobulin light chain of the tag comprises or consists of a peptide sequence selected from a group consisting of the constant domain of human kappa immunoglobulin light chain (SEQ ID NO: 1 ), the constant domain of human kappa immunoglobulin light chain without the C-terminal cysteine (SEQ ID NO: 2), the constant domain of mouse kappa immunoglobulin light chain (SEQ ID NO: 4), the constant domain of mouse kappa immunoglobulin light chain without the C- terminal cysteine (SEQ ID NO: 5), the constant domain of rat kappa immunoglobulin light chain (SEQ ID NO: 6), the constant domain of rat kappa immunoglobulin light chain without the C-terminal cysteine (SEQ ID NO: 7), the constant domain of rabbit kappa-b4 immunoglobulin light chain (KACB) according to UniProt entry: P01839 (SEQ ID NO: 8), the constant domain of rabbit kappa- b4 immunoglobulin light chain (KACB) according to UniProt entry: P01839, without the C-terminal cysteine (SEQ ID NO: 9), the constant domain of rabbit kappa-b4 immunoglobulin light chain (KAC4) according to UniProt entry: P01840 (SEQ ID NO: 10), the constant domain of rabbit kappa-b4 immunoglobulin light chain (KAC4) according to UniProt entry: P01840, without the C-terminal cysteine (SEQ ID NO: 11 ), the constant domain of rabbit kappa-b5 immunoglobulin light chain (KAC5) according to UniProt entry: P01841 (SEQ ID NO: 12), the constant domain of rabbit kappa-b5 immunoglobulin light chain (KAC5) according to UniProt entry: P01841 , without the C-terminal cysteine (SEQ ID NO: 13), the constant domain of rabbit kappa-b5 immunoglobulin light chain (KAC6) according to UniProt entry: P03984 (SEQ ID NO: 14), the constant domain of rabbit kappa- b5 immunoglobulin light chain (KAC6) according to UniProt entry: P03984, without the C-terminal cysteine (SEQ ID NO: 15), the constant domain of dog kappa immunoglobulin light chain (SEQ ID NO: 16), the constant domain of pig kappa immunoglobulin light chain (SEQ ID NO: 17), the constant domain of monkey kappa immunoglobulin light chain (SEQ ID NO: 18), and the constant domain of monkey kappa immunoglobulin light chain without the C-terminal cysteine (SEQ ID NO: 19). The method of any one of the preceding items, wherein the constant domain of the immunoglobulin light chain or orthologous polypeptide or fragment thereof of the tag is monomeric. The method of any one of the preceding items, wherein the first linker and / or the second linker are / is a cleavable linker. The method of any one of the items 1 to 10, wherein the first linker and / or the second linker are / is a non-cleavable linker. The method of any one of the preceding items, wherein the first linker of the fusion polypeptide is a cleavable linker and the second linker in the tag is a non- cleavable linker or wherein the first linker is a non-cleavable linker, and the second linker is a cleavable linker. The method of any one of the preceding items, wherein the tag is fused to the C- terminus of the target polypeptide. The method of any one of items 1 to 13, wherein the tag is fused to the N-terminus of the target polypeptide. The method of any one of items 1 to 13, wherein the tag is fused to the C-terminus and to the N-terminus of the target polypeptide. The method of any one of the preceding items, wherein the expression of the fusion polypeptide is higher than of the untagged target polypeptide. The method of any one of the preceding items, wherein a function of the target polypeptide with the tag is substantially comparable to a function of the target polypeptide without the tag. A fusion polypeptide obtained or obtainable by a method of any one of items 1 to 18. A fusion polypeptide comprising a target polypeptide and a tag suitable for affinity purification or detection of the target polypeptide, wherein the tag comprises at least one constant domain of an immunoglobulin light chain or orthologous polypeptide or fragment thereof and optionally a first linker that fuses the tag to the target polypeptide. The fusion polypeptide of item 20, wherein the fusion polypeptide has a molecular mass below 50 kDa. The fusion polypeptide of item 20, wherein the fusion polypeptide has a molecular mass of at least 50 kDa or 70 kDa. The fusion polypeptide of any one of items 20 to 22, wherein the tag is the tag according to any one of items 3 to 10. The fusion polypeptide of any one of items 20 to 23, wherein the first and / or second linker is / are a linker of any one of items 11 to 13. The fusion polypeptide of any one of items 20 to 24, wherein the tag is fused to the target polypeptide according to any one of items 14 to 16. The fusion polypeptide of any one of items 20 to 25, wherein the expression of the fusion polypeptide is higher than of the untagged target polypeptide. The fusion polypeptide of any one of items 20 to 26, wherein a function of the target polypeptide with the tag is substantially comparable to a function of the target polypeptide without the tag. The method of any one of items 1 to 18 and / or the fusion polypeptide of any one of items 19 to 27, wherein the target polypeptide is a polypeptide used in the pharmaceutical industry. The method of any one of items 1 to 18 and / or the fusion polypeptide of any one of items 19 to 27, wherein the target polypeptide is a polypeptide used in in-vitro diagnostic procedures. The method of any one of items 1 to 18 and / or the fusion polypeptide of any one of items 19 to 27, wherein the target polypeptide is used for a medical indication. The fusion polypeptide of any one of items 19 to 27 for use as a medicament. The fusion polypeptide of any one of items 19 to 27 for use in a medical indication. The method of any one of items 1 to 18 and / or the fusion polypeptide of any one of items 19 to 27, wherein the target polypeptide is used in biotechnology methods. The method of any one of items 1 to 18 and / or the fusion polypeptide of any one of items 19 to 27, wherein the target polypeptide is used in the chemical industry or in food production. Use of the method of any one of items 1 to 18 and / or the fusion polypeptide of any one of items 19 to 27 in the chemical industry or in food production. Use of the method of any one of items 1 to 18 and / or the fusion polypeptide of any one of items 19 to 27 in biotechnology methods. Use of the fusion polypeptide of any one of items 19 to 27 as a medicament. Use of the method of any one of items 1 to 18 and / or the fusion polypeptide of any one of items 19 to 27 for the manufacture of a medicament. Use of the fusion polypeptide of any one of items 19 to 27 in an in-vitro diagnostic procedure. Use of the method of any one of items 1 to 18 and / or the fusion polypeptide of any one of items 19 to 27 for the manufacture of medicament for use in a medical indication. A pharmaceutical composition comprising a fusion polypeptide of any one of items 19 to 27 and optionally a pharmaceutically acceptable carrier, diluent, or excipient. A nucleic acid for expression of the fusion polypeptide of any one of items 1 or 19 to 34 comprising a nucleic acid sequence encoding the target polypeptide of any one of items 1 or 20 to 34 and a nucleic acid sequence encoding the tag of any one of items 1 or 3 to 10 or 20, comprising a nucleic acid sequence encoding at least one constant domain of an immunoglobulin light chain or orthologous polypeptide or fragment thereof, and optionally a nucleic acid sequence encoding the first peptide linker of any one of items 1 or 20. he nucleic acid of item 42, wherein the nucleic acid sequence encoding the tag comprises a nucleic acid sequence encoding two or more constant domains of an immunoglobulin light chain or orthologous polypeptides or fragments thereof, which are optionally connected to each other by a nucleic acid sequence encoding a second peptide linker. The nucleic acid of any one of items 42 or 43, wherein the nucleic acid further comprises a nucleic acid sequence encoding a peptide signal sequence. The nucleic acid of any one of items 42 to 44, wherein the nucleic acid sequence encoding the constant domain of the immunoglobulin light chain or orthologous polypeptide or fragment thereof is a nucleic acid sequence encoding the constant domain of the immunoglobulin light chain or orthologous polypeptide or fragment of any one of items 5 to 10. The nucleic acid of any one of items 42 to 44, wherein the nucleic acid sequences encoding the first peptide linker and / or the second peptide linker encode the first peptide linker and / or the second peptide linker of any one of items 11 to 13. The nucleic acid of any one of items 42 to 46, wherein the nucleic acid sequence encoding the tag, and optional the nucleic acid sequence encoding the first peptide linker, is at the 3'-end of the nucleic acid sequence encoding the target polypeptide such that the tag is fused to the C-terminus of the target polypeptide in the fusion polypeptide. The nucleic acid of any one of items 42 to 46, wherein the nucleic acid sequence encoding the tag, and optional the nucleic acid sequence encoding the first peptide linker, is at the 5'-end of the nucleic acid sequence encoding the target polypeptide such that the tag is fused to the N-terminus of the target polypeptide in the fusion polypeptide. The nucleic acid of any one of items 42 to 46, wherein the nucleic acid sequence encoding the tag, and optional the nucleic acid sequence encoding the first peptide linker, is at the 5'-end and the 3'-end of the nucleic acid sequence encoding the target polypeptide such that the tag is fused to the N-terminus and the C-terminus of the target polypeptide in the fusion polypeptide. The nucleic acid of any one of items 42 to 49, wherein the nucleic acid sequence encoding the target polypeptide is encoding a polypeptide used in the pharmaceutical industry. The nucleic acid of any one of items 42 to 49, wherein the nucleic acid sequence encoding the target polypeptide is encoding a polypeptide used for in-vitro diagnostics. The nucleic acid of any one of items 42 to 49, wherein the nucleic acid sequence encoding the target polypeptide is encoding a polypeptide used for a medical indication. The nucleic acid of any one of items 42 to 49, wherein the nucleic acid sequence encoding the target polypeptide is encoding a polypeptide for use as a medicament. The nucleic acid of any one of items 42 to 49, wherein the nucleic acid sequence encoding the target polypeptide is encoding for a polypeptide used in the chemical industry. The nucleic acid of any one of items 42 to 49, wherein the nucleic acid sequence encoding the target polypeptide is encoding for a polypeptide used in biotechnology methods. The nucleic acid of any one of items 42 to 49 for use as a medicament. A pharmaceutical composition comprising the nucleic acid of any one of items 42 to 49 and optionally a pharmaceutically acceptable carrier, diluent, or excipient. A method for purifying an expressed fusion polypeptide, comprising providing a fusion polypeptide of any one of items 19 to 40 expressed by cells, optionally transfected with a nucleic acid of any one of items 42 to 49, provision of a ligand directed to the tag of the fusion polypeptide, binding of the ligand to the tag, and eluting the fusion polypeptide from the ligand. The method of item 58, wherein the ligand is directed to the constant domain of the immunoglobulin light chain of the tag. The method of any one of items 58 or 59, wherein the ligand is an antibody or an antibody fragment, optionally coupled to beads, further optionally coupled to magnetic beads. The method of any one of items 58 to 60, wherein the fusion polypeptide bound to the ligand is washed with a suitable buffer. A method of modulating the plasma half-time of a fusion polypeptide in an organism, wherein the fusion polypeptide is the product of the method of any one of items 1 to 18 or 28 to 40 or wherein the fusion polypeptide is the fusion polypeptide of any one of items 19 to 41 , and wherein the molecular mass of the fusion polypeptide is at least above or below the filtration limit of a kidney of an organism. The method of item 62, wherein the molecular mass of the fusion polypeptide is at least 50 kDa or at least 70 kDa. The method of item 62, wherein the molecular mass of the fusion polypeptide is below 50 kDa or below 70 kDa. A method for detecting the fusion polypeptide of any one of items 19 to 41 , comprising providing a sample, which may comprise the fusion polypeptide, and detecting the fusion polypeptide in the sample by using a detecting agent directed to the tag or the target polypeptide. The method of item 65, wherein the detecting agent is labeled with biotin, a fluorescence probe, a magnetic resonance detectable probe or a radioactive nuclide and optionally wherein the detecting agent is an antibody or an antibody fragment. The method of any one of items 65 or 66, wherein the sample is taken during the production process of the fusion polypeptide of any one of items 19 to 39 or the pharmaceutical composition of item 40. 68. The method of any one of items 65 or 66, wherein the sample is a biological sample of a mammal, to which the fusion polypeptide or the pharmaceutical composition was administered.

[0331] 69. The method of item 68, wherein the mammal is a non-human mammal, preferably a mouse, a rat, a rabbit, a dog, a cat, a pig, or a monkey.

[0332] 70. The method of item 67, wherein the mammal is a human being.

[0333] 71. The method of any one of items 68 to 70, wherein the biological sample is a tissue, urine, feces, or blood.

[0334] 72. A method of detecting the fusion polypeptide of any one of items 19 to 40 in-vivo after administration to a mammal, wherein the fusion polypeptide is labeled by a fluorescence probe, a magnetic resonance detectable probe, an x-ray detectable probe or a radioactive nuclide.

[0335] 73. The method of item 72, wherein the mammal is a non-human mammal, preferably a mouse, a rat, a rabbit, a dog, a cat, a pig, or a monkey.

[0336] 74. The method of item 72, wherein the mammal is a human being.

[0337] The invention is also characterized by the following further items:

[0338] 1 . A tag suitable for affinity purification or detection of a fusion partner, wherein the fusion partner is a target polypeptide and wherein the tag comprises at least one constant domain of an immunoglobulin light chain or orthologous polypeptide or fragment thereof and optionally a linker that fuses the tag to the target polypeptide.

[0339] 2. The tag of item 1 , wherein the constant domain of the immunoglobulin light chain or orthologous polypeptide or fragment thereof is a constant domain of a kappa immunoglobulin light chain or orthologous polypeptide or fragment thereof. 3. The tag of any one of the preceding items, wherein the constant domain of the immunoglobulin light chain or fragment thereof is a constant domain of a mammalian immunoglobulin light chain or orthologous polypeptide or fragment thereof, preferred a constant domain of a primate immunoglobulin light chain or fragment thereof and most preferred a constant domain of a human immunoglobulin light chain or fragment thereof or wherein the constant domain of the kappa immunoglobulin light chain or fragment thereof is a constant domain of a mammalian kappa immunoglobulin light chain or orthologous polypeptide or fragment thereof, preferred a constant domain of a primate kappa immunoglobulin light chain or fragment thereof, and most preferred a constant domain of a human kappa immunoglobulin light chain or fragment thereof.

[0340] 4. The tag of any one of items 1 to 3, wherein a C-terminal cysteine of the constant domain of the immunoglobulin light chain is deleted if present in the peptide sequence of the constant domain of the immunoglobulin light chain.

[0341] 5. The tag of any one of items 2 to 4, wherein the constant domain of the kappa immunoglobulin light chain or fragment thereof is selected from a group consisting of a constant domain of a mouse kappa immunoglobulin light chain, a constant domain of a rat kappa immunoglobulin light chain, a constant domain of a dog kappa immunoglobulin light chain, a constant domain of a rabbit kappa light chain, a constant domain of a pig kappa immunoglobulin light chain, a constant domain of a monkey kappa immunoglobulin light chain, and preferably the constant domain of the human kappa immunoglobulin light chain.

[0342] 6. The tag of any one of items 2 to 5, wherein the constant domain of the kappa immunoglobulin light chain comprises or consists of a peptide sequence selected from a group consisting of the constant domain of human kappa immunoglobulin light chain (SEQ ID NO: 1 ), the constant domain of human kappa immunoglobulin light chain without the C-terminal cysteine (SEQ ID NO: 2), the constant domain of mouse kappa immunoglobulin light chain (SEQ ID NO: 4), the constant domain of mouse kappa immunoglobulin light chain without the C-terminal cysteine (SEQ ID NO: 5), the constant domain of rat kappa immunoglobulin light chain (SEQ ID NO: 6), the constant domain of rat kappa immunoglobulin light chain without the C-terminal cysteine (SEQ ID NO: 7), the constant domain of rabbit kappa-b4 immunoglobulin light chain (KACB) according to UniProt entry: P01839 (SEQ ID NO: 8), the constant domain of rabbit kappa-b4 immunoglobulin light chain (KACB) according to UniProt entry: P01839, without the C-terminal cysteine (SEQ ID NO: 9), the constant domain of rabbit kappa- b4 immunoglobulin light chain (KAC4) according to UniProt entry: P01840 (SEQ ID NO: 10), the constant domain of rabbit kappa-b4 immunoglobulin light chain (KAC4) according to UniProt entry: P01840, without the C-terminal cysteine (SEQ ID NO: 11 ), the constant domain of rabbit kappa-b5 immunoglobulin light chain (KAC5) according to UniProt entry: P01841 (SEQ ID NO: 12), the constant domain of rabbit kappa-b5 immunoglobulin light chain (KAC5) according to UniProt entry: P01841 , without the C- terminal cysteine (SEQ ID NO: 13), the constant domain of rabbit kappa-b5 immunoglobulin light chain (KAC6) according to UniProt entry: P03984 (SEQ ID NO: 14), the constant domain of rabbit kappa-b5 immunoglobulin light chain (KAC6) according to UniProt entry: P03984, without the C-terminal cysteine (SEQ ID NO: 15), the constant domain of dog kappa immunoglobulin light chain (SEQ ID NO: 16), the constant domain of pig kappa immunoglobulin light chain (SEQ ID NO: 17), the constant domain of monkey kappa immunoglobulin light chain (SEQ ID NO: 18), and the constant domain of monkey kappa immunoglobulin light chain without the C- terminal cysteine (SEQ ID NO: 19).

[0343] 7. The tag of any one of items 4 to 6, wherein the constant domain of the immunoglobulin light chain or orthologous polypeptide or fragment thereof is monomeric.

[0344] 8. The tag of any one of the preceding items, wherein the linker comprises an amino acid sequence which is cleavable.

[0345] 9. The tag of any one of items 1 to 7, wherein the linker comprises an amino acid sequence which is not cleavable.

[0346] 10. The tag of any one of the preceding items, wherein the tag is suitable to be fused to the C-terminus of the target polypeptide.

[0347] 11 . The tag of any one of items 1 to 9, wherein the tag is suitable to be fused to the N-terminus of the target polypeptide. 12. The tag of any one of items 1 to 9, wherein the tag is suitable to be fused to the C-terminus and to the N-terminus of the target polypeptide.

[0348] 13. A fusion polypeptide comprising a target polypeptide and a tag suitable for affinity purification or detection of the target polypeptide, wherein the tag comprises at least one constant domain of an immunoglobulin light chain or orthologous polypeptide or fragment thereof and optionally a linker that fuses the tag to the target polypeptide.

[0349] 14. The fusion polypeptide of item 13, wherein the fusion polypeptide is expressed under conditions allowing a formation of a disulfide bond.

[0350] 15. The fusion polypeptide of item 13 or 14, wherein the constant domain of the immunoglobulin light chain or orthologous polypeptide or fragment thereof is a constant domain of a kappa immunoglobulin light chain or orthologous polypeptide or fragment thereof.

[0351] 16. The fusion polypeptide of any one of items 13 to 15, wherein the constant domain of the immunoglobulin light chain or fragment thereof is a constant domain of a mammalian immunoglobulin light chain or orthologous polypeptide or fragment thereof, preferred a constant domain of a primate immunoglobulin light chain or fragment thereof and most preferred a constant domain of a human immunoglobulin light chain or fragment thereof or wherein the constant domain of the kappa immunoglobulin light chain or fragment thereof is a constant domain of a mammal kappa immunoglobulin light chain or orthologous polypeptide or fragment thereof, preferred a constant domain of a primate kappa immunoglobulin light chain or fragment thereof, and most preferred a constant domain of a human kappa immunoglobulin light chain or fragment thereof.

[0352] 17. The fusion polypeptide of any one of items 15 or 16, wherein a C-terminal cysteine of the constant domain of the kappa immunoglobulin light chain is deleted if present in the peptide sequence of the constant domain of the kappa immunoglobulin light chain. 18. The fusion polypeptide of any one of items 15 to 17, wherein the constant domain of the kappa immunoglobulin light chain or fragment thereof is selected from a group consisting of a constant domain of a mouse kappa immunoglobulin light chain, a constant domain of a rat kappa immunoglobulin light chain, a constant domain of a rabbit kappa immunoglobulin light chain, a constant domain of a dog kappa immunoglobulin light chain, a constant domain of a pig kappa immunoglobulin light chain, a constant domain of a monkey kappa immunoglobulin light chain, and preferably the constant domain of the human kappa immunoglobulin light chain.

[0353] 19. The fusion polypeptide of any one of items 15 to 18, wherein the constant domain of the kappa immunoglobulin light chain comprises or consists of a peptide sequence selected from a group consisting of the constant domain of human kappa immunoglobulin light chain (SEQ ID NO: 1 ), the constant domain of human kappa immunoglobulin light chain without the C-terminal cysteine (SEQ ID NO: 2), the constant domain of mouse kappa immunoglobulin light chain (SEQ ID NO: 4), the constant domain of mouse kappa immunoglobulin light chain without the C-terminal cysteine (SEQ ID NO: 5), the constant domain of rat kappa immunoglobulin light chain (SEQ ID NO: 6), the constant domain of rat kappa immunoglobulin light chain without the C-terminal cysteine (SEQ ID NO: 7), the constant domain of rabbit kappa-b4 immunoglobulin light chain (KACB) according to UniProt entry: P01839 (SEQ ID NO: 8), the constant domain of rabbit kappa-b4 immunoglobulin light chain (KACB) according to UniProt entry: P01839, without the C-terminal cysteine (SEQ ID NO: 9), the constant domain of rabbit kappa-b4 immunoglobulin light chain (KAC4) according to UniProt entry: P01840 (SEQ ID NO: 10), the constant domain of rabbit kappa-b4 immunoglobulin light chain (KAC4) according to UniProt entry: P01840, without the C- terminal cysteine (SEQ ID NO: 11 ), the constant domain of rabbit kappa-b5 immunoglobulin light chain (KAC5) according to UniProt entry: P01841 (SEQ ID NO: 12), the constant domain of rabbit kappa-b5 immunoglobulin light chain (KAC5) according to UniProt entry: P01841 , without the C-terminal cysteine (SEQ ID NO: 13), the constant domain of rabbit kappa-b5 immunoglobulin light chain (KAC6) according to UniProt entry: P03984 (SEQ ID NO: 14), the constant domain of rabbit kappa-b5 immunoglobulin light chain (KAC6) according to UniProt entry: P03984, without the C- terminal cysteine (SEQ ID NO: 15), the constant domain of dog kappa immunoglobulin light chain (SEQ ID NO: 16), the constant domain of pig kappa immunoglobulin light chain (SEQ ID NO: 17), the constant domain of monkey kappa immunoglobulin light chain (SEQ ID NO: 18), and the constant domain of monkey kappa immunoglobulin light chain without the C-terminal cysteine (SEQ ID NO: 19).

[0354] 20. The fusion polypeptide of any one of items 13 to 19, wherein the constant domain of the immunoglobulin light chain or orthologous polypeptide or fragment thereof is monomeric.

[0355] 21. The fusion polypeptide of any one of items 13 to 20, wherein the linker is a cleavable linker.

[0356] 22. The fusion polypeptide of any one of items 13 to 20, wherein the linker is a non- cleavable linker.

[0357] 23. The fusion polypeptide of any one of items 13 to 22, wherein the tag is fused to the C-terminus of the target polypeptide.

[0358] 24. The fusion polypeptide of any one of items 13 to 22, wherein the tag is fused to the N-terminus of the target polypeptide.

[0359] 25. The fusion polypeptide of any one of items 13 to 22, wherein the tag is fused to the C-terminus and to the N-terminus of the target polypeptide.

[0360] 26. The fusion polypeptide of any one of items 13 to 25, wherein an expression of the fusion polypeptide is higher than the expression of the untagged target polypeptide.

[0361] 27. The fusion polypeptide of any one of items 13 to 26, wherein a function of the target polypeptide with the tag is substantially comparable to a function of the target polypeptide without the tag.

[0362] 28. The fusion polypeptide of any one of items 13 to 27, wherein the target polypeptide is a polypeptide used in the pharmaceutical industry. 29. The fusion polypeptide of any one of items 13 to 27, wherein the target polypeptide is a polypeptide used for in-vitro diagnostics.

[0363] 30. The fusion polypeptide of any one of items 13 to 27, wherein the target polypeptide is used for a medical indication and / or in-vivo diagnostics.

[0364] 31. The fusion polypeptide of any one of items 13 to 27 for use in a medical indication.

[0365] 32. The fusion polypeptide of any one of items 13 to 27 or 30 to 31 for use as a medicament.

[0366] 33. The fusion polypeptide of any one of items 13 to 27, wherein the target polypeptide is a polypeptide used in biotechnology methods.

[0367] 34. The fusion polypeptide of any one of items 13 to 27, wherein the target polypeptide is used in the chemical industry or food production.

[0368] 35. Use of the fusion polypeptide of any one of items 13 to 27 in biotechnology methods.

[0369] 36. Use of the fusion polypeptide of any one of items 13 to 27 in the chemical industry or food production.

[0370] 37. Use of the fusion polypeptide of any one of items 13 to 27 as a medicament.

[0371] 38. Use of a fusion polypeptide of any one of items 13 to 27 for the manufacture of a medicament.

[0372] 39. Use of the fusion polypeptide of any one of items 13 to 27 in an in-vitro diagnostic procedure.

[0373] 40. Use of the fusion polypeptide of any one of items 13 to 27 for the manufacture of medicament for use in a medical indication and / or in in-vivo diagnostics. 41. A pharmaceutical composition comprising a fusion polypeptide of any one of items 13 to 32 or 37 to 38 and optionally a pharmaceutically acceptable carrier, diluent, or excipient.

[0374] 42. A nucleic acid for expression of the fusion polypeptide of any one of items 13 to 40 comprising a nucleic acid sequence encoding the target polypeptide of any one of items 1 to 40 and a nucleic acid sequence encoding the tag of any one of items 1 to 12, comprising a nucleic acid sequence encoding at least one constant domain of an immunoglobulin light chain or orthologous polypeptide or fragment thereof, and optionally a nucleic acid sequence encoding the peptide linker of item 1 .

[0375] 43. The nucleic acid of item 42, wherein the nucleic acid further comprises a nucleic acid sequence encoding a peptide signal sequence.

[0376] 44. The nucleic acid of any one of items 42 or 43, wherein the nucleic acid sequence encoding the constant domain of the immunoglobulin light chain or orthologous polypeptide or fragment thereof is a nucleic acid sequence encoding a constant domain of a kappa immunoglobulin light chain or orthologous polypeptide or fragment thereof.

[0377] 45. The nucleic acid of any one of items 42 to 44, wherein the nucleic acid sequence encoding the constant domain of the immunoglobulin light chain or fragment thereof is a nucleic acid sequence encoding a constant domain of a mammal immunoglobulin light chain or orthologous polypeptide or fragment thereof, preferred a nucleic acid sequence encoding a constant domain of a primate immunoglobulin light chain or fragment thereof and most preferred a nucleic acid sequence encoding a constant domain of a human immunoglobulin light chain or fragment thereof or wherein the nucleic acid sequence encoding the constant domain of the kappa immunoglobulin light chain or fragment thereof is a nucleic acid sequence encoding a constant domain of a mammal kappa immunoglobulin light chain or orthologous polypeptide or fragment thereof, preferred a nucleic acid sequence encoding a constant domain of a primate kappa immunoglobulin light chain or fragment thereof, and most preferred a nucleic acid sequence encoding a constant domain of a human kappa immunoglobulin light chain or fragment thereof.

[0378] 46. The nucleic acid of any one of items 42 to 45, wherein the nucleic acid sequence encoding the constant domain of the immunoglobulin light chain or fragment thereof is a nucleic acid sequence encoding the constant domain of the kappa immunoglobulin light chain without a C-terminal cysteine of the constant domain of the kappa immunoglobulin light chain or fragment thereof.

[0379] 47. The nucleic acid of any one of items 44 to 46, wherein the nucleic acid sequence encoding the constant domain of the kappa immunoglobulin light chain or fragment thereof is selected from a group consisting of a nucleic acid sequence encoding a constant domain of a mouse kappa immunoglobulin light chain, a nucleic acid sequence encoding a constant domain of a rat kappa immunoglobulin light chain, a nucleic acid sequence encoding a constant domain of a rabbit kappa immunoglobulin light chain, a nucleic acid sequence encoding a constant domain of a dog kappa immunoglobulin light chain, a nucleic acid sequence encoding a constant domain of a pig kappa immunoglobulin light chain, a nucleic acid sequence encoding a constant domain of a monkey kappa immunoglobulin light chain, and preferably the nucleic acid sequence encoding the constant domain of the human kappa immunoglobulin light chain.

[0380] 48. The nucleic acid of any one of items 42 to 47, wherein the nucleic acid sequence encoding the peptide linker encodes the peptide linker according to item 8.

[0381] 49. The nucleic acid of any one of items 42 to 47, wherein the nucleic acid sequence encoding the peptide linker encodes the peptide linker according to item 9.

[0382] 50. The nucleic acid of any one of items 42 to 49, wherein the nucleic acid sequence encoding the tag, and optional the nucleic acid sequence encoding the peptide linker, is at the 3'-end of the nucleic acid sequence encoding the target polypeptide such that the tag is fused to the C-terminus of the target polypeptide in the fusion polypeptide. 51 . The nucleic acid of any one of items 42 to 49, wherein the nucleic acid sequence encoding the tag, and optional the nucleic acid sequence encoding the peptide linker, is at the 5'-end of the nucleic acid sequence encoding the target polypeptide such that the tag is fused to the N-terminus of the target polypeptide in the fusion polypeptide.

[0383] 52. The nucleic acid of any one of items 42 to 49, wherein the nucleic acid sequence encoding the tag, and optional the nucleic acid sequence encoding the peptide linker, is at the 5'-end and the 3'-end of the nucleic acid sequence encoding the target polypeptide such that the tag is fused to the N-terminus and the C-terminus of the target polypeptide in the fusion polypeptide.

[0384] 53. The nucleic acid of any one of items 42 to 52, wherein the nucleic acid sequence encoding the target polypeptide is encoding a polypeptide used in the pharmaceutical industry.

[0385] 54. The nucleic acid of any one of items 42 to 52, wherein the nucleic acid sequence encoding the target polypeptide is encoding a polypeptide used for in-vitro diagnostics.

[0386] 55. The nucleic acid of any one of items 42 to 52, wherein the nucleic acid sequence encoding the target polypeptide is encoding a polypeptide used for a medical indication.

[0387] 56. The nucleic acid of any one of items 42 to 52, wherein the nucleic acid sequence encoding the target polypeptide is encoding a polypeptide for use as a medicament.

[0388] 57. The nucleic acid of any one of items 42 to 52 for use as a medicament.

[0389] 58. The nucleic acid of any one of items 42 to 52, wherein the nucleic acid sequence encoding the target polypeptide is encoding for a polypeptide used in the chemical industry or food production.

[0390] 59. The nucleic acid of any one of items 42 to 52, wherein the nucleic acid sequence encoding the target polypeptide is encoding for a polypeptide used in biotechnology methods. 60. A pharmaceutical composition comprising the nucleic acid of any one of items

[0391] 42 to 52 and optionally a pharmaceutically acceptable carrier, diluent, or excipient.

[0392] 61 . A method for purifying an expressed fusion polypeptide, comprising providing a fusion polypeptide of any one of items 13 to 41 expressed by cells, optionally transfected with a nucleic acid of any one of items 42 to 58, provision of a ligand directed to the tag of the fusion polypeptide, binding of the ligand to the tag, and eluting the fusion polypeptide from the ligand.

[0393] 62. The method of item 61 , wherein the ligand is directed to the constant domain of the immunoglobulin light chain of the tag.

[0394] 63. The method of any one of items 61 or 62, wherein the ligand is an antibody or an antibody fragment, optionally coupled to beads, further optionally coupled to magnetic beads.

[0395] 64. The method of any one of items 61 to 63, wherein the fusion polypeptide bound to the ligand is washed with a suitable buffer.

[0396] 65. The method of any one of items 61 to 64, wherein the expressed fusion polypeptide comprises a polypeptide for in-vitro diagnostic procedures as target polypeptide of the fusion polypeptide of any one of items 13 to 27 or 29 or 39.

[0397] 66. The method of any one of items 61 to 64, wherein the expressed fusion polypeptide comprises a polypeptide used in the pharmaceutical industry as target polypeptide of the fusion polypeptide of any one of items 13 to 28.

[0398] 67. The method of any one of items 61 to 64, wherein the expressed fusion polypeptide comprises a polypeptide for use in a medical indication as target polypeptide of the fusion polypeptide of any one of items 13 to 27 or 30 to 31 .

[0399] 68. The method of any one of items 61 to 64, wherein the expressed fusion polypeptide comprises a polypeptide used in the chemical industry or food production as target polypeptide of the fusion polypeptide of any one of items 13 to 27 or 34 or 36.

[0400] 69. The method of any one of items 61 to 64, wherein the expressed fusion polypeptide comprises a polypeptide used in biotechnology methods as target polypeptide of the fusion polypeptide of any one of items 13 to 27 or 33 or 35.

[0401] 70. A method of detecting the fusion polypeptide of any one of items 13 to 27, comprising providing a sample, which may comprise the fusion polypeptide, and detecting the fusion polypeptide in the sample by using a detecting agent directed to the tag or the target polypeptide.

[0402] 71 . The method of detecting of item 70, wherein the detecting agent is labeled with biotin, a fluorescence probe, or a radioactive nuclide and optionally wherein the detecting agent is an antibody or an antibody fragment.

[0403] 72. The method of detecting of any one of items 70 or 71 , wherein the sample is taken during the production process of the fusion polypeptide of any one of items 13 to 40 or the pharmaceutical composition of item 41 .

[0404] 73. The method of detecting of any one of items 70 or 71 , wherein the sample is a biological sample of a mammal, to which the fusion polypeptide or the pharmaceutical composition was administered.

[0405] 74. The method of detecting of item 73, wherein the mammal is a non-human mammal, preferably a mouse, a rat, a rabbit, a dog, a cat, a pig, or a monkey.

[0406] 75. The method of detecting of item 73, wherein the mammal is a human being.

[0407] 76. The method of detecting any one of items 73 to 75, wherein the biological sample is a tissue, urine, feces, or blood.

[0408] 77. A method of detecting the fusion polypeptide of any one of items 13 to 27 in-vivo after administration to a mammal, wherein the fusion polypeptide is labeled by a fluorescence probe, a magnetic resonance detectable probe, an x-ray detectable probe or a radioactive nuclide.

[0409] 78. The method of detecting of item 77, wherein the mammal is a non-human mammal, preferably a mouse, a rat, a rabbit, a dog, a cat, a pig, or a monkey.

[0410] 79. The method of detecting of item 77, wherein the mammal is a human being.

[0411] A better understanding of the present invention and of its advantages is illustrated by the following examples, offered for illustrative purposes only. The examples are not intended to limit the scope of the present invention in any way.

[0412] EXAMPLES

[0413] Material and Methods:

[0414] Constructs.

[0415] Constructs for mammalian protein expression were synthesized human codon optimized by GeneArt (Thermo Fisher Scientific) in the pcDNA3.4 TOPO vector (Gibco). For the tag (= i-Tag) human immunoglobulin lambda constant 2 domain (UniProt accession number: PODOY2) as well as the human immunoglobulin kappa constant domain (UniProt accession number: P01834) (SEQ ID NO: 1 ) and repeats thereof were used, the human IL-27a / p28 (UniProt accession number: Q8NEV9) signal sequence (SEQ ID NO: 21 ) was used as N-terminal signal sequence. An exemplary used DNA sequence for the constant domain of the human immunoglobulin kappa light chain including the N-terminal signal sequence and without a codon for the C-terminal cysteine was SEQ ID NO: 3, which encodes the amino acid sequence MGQTAGDLG RLSLLLLPLLLYQAGY GRTVAAPSVFIFPPSDEQLKSGTASVVCL LNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHK VYACEVTHQGLSSPVTKSFNRGE (signal sequence in italic) (SEQ ID NO: 20). For i- Tag2 constructs (repeat of two human immunoglobulin kappa constant domains), a signal sequence of an antibody chain was used as N-terminal signal sequence. Darbepoetin alfa (Aranesp; DrugBank accession number: DB00012) (SEQ ID NO: 42), Erythropoietin (EPO; UniProt accession number: P01588) (SEQ ID NO: 45), Granulocyte-Colony Stimulating Factor (GCSF; UniProt accession number: P09919) (SEQ ID NO: 48), the bi-specific T-cell engager (BiTE) antibody construct targeting DLL3 and CD3 (DLL3-4 x I2C scFv, patent number: US20170037130A1 , SEQ-ID 517) (SEQ ID NO: 51 ) were all linked to a tag containing five constant domains (=i-Tags) by a Gs-linker (SEQ ID NOs: 44, 47, 50, 53, respectively). IL-15Ra (sushi domain) linked by a (GGGGS)6linker to IL-15 (D30N, E64Q, N65D) (IL-15Ra(sushi)IL-15; patent number: WQ2021155042) (SEQ ID NO: 54) was fused to i-Tags by a GGGGS-linker (SEQ ID NO: 56). For DLL3-4xCD3 BiTE, the human IL-27a / p28 signal sequence was fused N-terminally to the protein sequence, for the other proteins, endogenous signal sequences were used. p28L162C’AC(SEQ ID NO: 22) was fused to the i-Tags by a PAPAP-linker (SEQ ID NO: 27). Both, for constructs without a Tag-fusion and for constructs with a Hise-tag, stop codons or six histidine residues, respectively, were introduced by site-directed mutagenesis PCR using Pfu DNA polymerase (Promega) with the help of custom oligos (Sigma-Aldrich). All constructs were sequenced.

[0416] Molecular dynamics simulation and structural modelling.

[0417] For structural modeling of the tag containing five constant domains (i-Tags), the C- terminus of the p28L162C’ACmodel structure was covalently connected to the N- terminus of five repeats of a human immunoglobulin kappa constant domain (PDB: 3QCU)-based structure (SEQ ID NO: 32). The resulting structure was processed with the leap module of the amber18 package to prepare simulation systems (Case et al., 2014). This includes fixing the disulfide bonds, solvation with the TIP3P water model in a rectangular box with minimum distance of 1 nm between box boundary and solute, as well as electrostatic neutralization with sodium ions (Jorgensen et al., 1983). Afterwards, the system was energy-minimized in 2000 steps of steepest descent with the sander module, followed by an equilibration consisting of eight simulation stages. In these stages, the system was heated up to 300 K and a pressure of 1 bar was introduced using the Berendsen thermostat and barostat (Berendsen et al., 1984). The force constant for positional restraints were reduced from 2500.0 to 10.0 kcal / mol / nm2along the 8 stages. Each simulation stage consisted of 100,000 steps. The output structure was then simulated for 30 ns without any restraints using an integration timestep of 2 fs. The production run as well as equilibration simulation were performed with the pmemd.cuda module (Case et al., 2014). In the last step, the p28L162C ACstructure was removed from the structural model. Based on crystal structures from PDB database, structures of Darbepoetin alfa (Erythropoietin; PDB: 1 BUY), GCSF (PDB: 2D9Q), and IL-15Ra(sushi)IL-15 (IL-15 with IL-15Ra; PDB: 2Z3Q) were adjusted by replacement of single amino acids or modelling of missing loops in silico and subsequent steepest decent energy minimization using Yasara Structure (www.yasara.org). ColabFold v1.5.3 (Mirdita et al., 2022) was used for a structural model of DLL3-4xCD3 BiTE. Structures were depicted with PyMOL (PyMOL Molecular Graphics System, Version 2.5.4, Schrodinger, LLC, www.pymol.org).

[0418] Cell culture, protein production and purification.

[0419] Expi293FTMcells (Gibco) were cultured and transfected according to the manufacturer’s protocol. After expression, medium was centrifuged twice (first, at 5,000 g for 30 min and second, at 20,000 g for 15 min, both at 4 °C). Protein expression and purity were assessed by supplementing 0.2 volumes of 5x Laemmli buffer (0.3125 M Tris / HCI pH 6.8, 10% SDS, 50% glycerol, bromphenol blue) containing either 10% (v / v) [3-mercaptoethanol ([3-Me) for reducing SDS-polyacrylamide gel electrophoresis (PAGE) or 100 mM A / -ethylmaleimide (NEM) for non-reducing SDS-PAGE. For purification, the supernatant was supplemented with Roche complete Protease Inhibitor w / o EDTA (Roche Diagnostics). For the Hise-tagged proteins, the sample was applied to a HisTrap HP column (Cytiva) in phosphate buffered saline (PBS; pH 7.4). Elution was performed by performing a step gradient up to 500 mM imidazole in PBS (pH 7.4). The i-Tag fusion constructs were purified by application to a CaptureSelect™ KappaXP pre-packed column (Thermo Fisher). Purification was performed according to the manufacturer’s instructions, using 10 mM sodium phosphate (pH 7.4) as equilibration buffer and 100 mM sodium acetate (pH 3.0) for protein elution, with 1 M Tris buffer (pH 8.0) for neutralization of elution fractions. Both, purified Hise-tagged and i-Tags fusion proteins were dialyzed o / n in 20 mM HEPES, 150 mM NaCI (pH 7.4) before further characterization. Expression and purification of IL-12|3C199Sfrom mammalian cells was described previously (Meier et al., 2019).

[0420] Deglycosylation assays.

[0421] To assess the glycosylation status of produced proteins, samples were deglycosylated with PNGase F (SERVA) or a mix of O-Glycosidase and a2-2,6,8 Neuraminidase (New England Biolabs) according to the manufacturers’ protocols. Samples were supplemented with 0.2 volumes of 5x Laemmli buffer containing 10% [3-Me and analyzed by SDS-PAGE.

[0422] Co-Immunoprecipitation experiments.

[0423] Purified, i-Tags fused BiTE (SEQ ID NO: 53) was incubated with recombinant human DLL3 (abeam; ab255797) and / or human CD3 epsilon (ACRBCDE-H5256), both Hise- tagged, rotating samples at 300 rpm, 25 °C for 45 min. Co-immunoprecipitation (co-IP) experiments were then performed with HIS-Select® Nickel Affinity Gel (Sigma-Aldrich, P6611 ). 25 pl beads were added to the protein mixtures, rotated for 1 hour at 4 °C, and beads were washed three times with PBS (5,000 x g, 1 min). Elution of immunoprecipitated proteins from beads was performed with PBS with 500 mM imidazole (pH 7.4). 5x Laemmli buffer containing 10% [3-Me was added and samples were boiled for 5 min at 95°C.

[0424] Protein stability assessment of i-Tag2constructs in fetal bovine serum.

[0425] Purified, i-Tag2 protein constructs were incubated 1 :10 (v / v) in fetal bovine serum (FBS, Gibco, 10437-028) at a final concentration of 9.4 pg / ml at 37 °C. Input samples were taken after 0, 6, 24, and 96 hours of incubation. 5x Laemmli buffer containing 10% [3- Me was added and samples were boiled for 5 min at 95°C.

[0426] Immunoprecipitation (IP) experiments were performed with K CL-antibody (monoclonal anti-kappa light chain, 1 :5,000; EPR5367-8, abeam). After 1 hour rotation of 50 pl sample with 1 pl antibody at 4 °C, 25 pl Pierce™ Protein A / G Agarose beads were added to the protein samples, rotated for 1 hour at 4 °C, and beads were washed twice with RIPA buffer (50 mM Tris / HCI pH 7.5, 150 mM NaCI, 1 % Nonidet P40, 0.5% sodium deoxycholate, 0.1 % SDS) (5,000 x g, 1 min). Elution of immunoprecipitated proteins from beads was performed with 50 pl 2x Laemmli buffer containing 10% [3-Me and boiling for 5 min at 95°C.

[0427] Immunoblotting experiments.

[0428] For immunoblots, samples were separated on 12% or 15% SDS-PAGE gels, transferred to polyvinylidene difluoride (PVDF) membranes by blotting overnight (o / n) at 30 V (4 °C). After blocking the membrane with 5% (w / v) skim milk powder in Trisbuffered saline (25 mM Tris / HCI, pH 7.5, 150 mM NaCI; TBS) containing 0.05% (v / v) Tween-20 (M-TBST), primary antibody was added and incubated o / n at 4 °C (monoclonal anti-kappa light chain, 1 :5,000; EPR5367-8, abeam). Anti-rabbit HRP- conjugated secondary antibody (Santa Cruz Biotechnology, 1 :10,000 in M-TBST) was used to detect the proteins. For anti-His immunoblots, blots were incubated in monoclonal HRP-conjugated 6*His, Hise-tag antibody (HRP-66005, Proteintech, 1 :10,000 in M-TBST) for 1 hour before detection. ECL prime reagent (Cytiva) and Fusion FX7 Edge V0.7 imager (Vilber Lourmat) were used for detection.

[0429] Partial proteolysis experiments with trypsin.

[0430] Partial proteolysis experiments were conducted at 37 °C with i-Tag2 protein samples at a concentration of 60 pg / ml. Trypsin (Pierce™ Trypsin Protease, MS-grade, Thermo Fisher) was added at a ratio of 1 :50 (w / w). Samples were taken after the indicated time, 5x Laemmli buffer containing 5x protease inhibitor (Roche) was added and samples were boiled at 95°C for 5 min and analyzed by SDS-PAGE with Coomassie staining.

[0431] Analytical ultracentrifugation.

[0432] Sedimentation velocity analytical ultracentrifugation (AUC) measurements were performed on a Beckman Coulter Optima™ analytical ultracentrifuge (Beckman Coulter, Brea, CA, USA), equipped with absorbance optics. For each measurement, 350 pl of 0.6-6.3 pM protein sample in 20 mM HEPES, 150 mM NaCI (pH 7.4) were loaded into a standard 12 mm double-sector epon-filled centerpiece, covered with quartz windows, alongside the reference buffer. Samples were measured in an An-50 Ti rotor at 20 °C, 42,000 rpm (with an initial test run at 3,000 rpm). Detection was conducted at 235 nm with 250 scans and a radial step size of 0.001 cm. Resulting sedimentation velocity profiles were analyzed using SedFit software with a non-model based continuous Svedberg distribution method (c(s)) (Dam et al., 2005).

[0433] High performance liquid chromatography.

[0434] Protein quality of purified proteins was assessed by high performance liquid chromatography (HPLC) on the Agilent 1260 Infinity II instrument with a Superdex 200 Increase 5 / 150 GL (Cytiva) column, using the AdvanceBio SEC 130 A Protein Standard (Agilent Technologies). Runs were performed in 20 mM HEPES, 150 mM NaCI, pH 7.4, as running buffer and detection was done at 280 nm. Analysis was performed with the OpenLab Data Analysis software (version 2.5, Agilent Technologies).

[0435] Small-angle X-ray scattering.

[0436] Small-angle X-ray scattering (SAXS) experiments were performed on a Rigaku BioSAXS OO instrument attached to a Rigaku HF007 microfocus rotating anode generator with a copper target (40 kV, 30 mA). Transmissions were measured with a photodiode beam stop. The scattering vector calibration was done with a silver behenate sample. Samples with concentrations of 1 , 2, and 3.5 mg / ml, as well as respective buffer (20 mM HEPES, 150 mM NaCI, pH 7.4) were measured in using an automated sample changer (capillary volume approx. 70 pl). A typical measurement consisted of eight 900 s frames, which were compared to check for radiation damage, for a total measurement duration of 7200 s. Sample temperature was controlled using a Julabo F25-MA thermostat with a specified temperature stability of ±0.02 K. Circular averaging and solvent subtraction were done using the SAXSLab software (version 3.1 .1 ). Further data analysis was carried out using PRIMUS (Manalastas-Cantos et al., 2021 ), GNOM (Svergun, 1992) and CORAL (Petoukhov et al., 2012) from the ATSAS software package (by EMBL Hamburg, version 3.0.0-3). For the rigid body models, core residues were assumed as rigid, and flexible linkers were added to achieve the correct total number of residues for each domain (the respective assumed cores are indicated in Figure 5E). The first and last domains were kept complete up to the real N- and C-terminus, respectively. Otherwise, default CORAL settings were used.

[0437] Dynamic light scattering.

[0438] To evaluate the homogeneity of proteins, dynamic light scattering (DLS) was used. Each sample was diluted to 2 pM in 20 mM HEPES, 150 mM NaCI (pH 7.4) and centrifuged for one hour, 20,000 g at 4 °C. Samples were measured in an UV-cuvette micro (VWR) with the Zetasizer Nano ZS90 (Malvern Panalytical) at 21 °C. Protein size and homogeneity was evaluated by averaging three measurements of each sample and analysis using the Zetasizer software.

[0439] Differential scanning calorimetry.

[0440] Temperature-dependent unfolding of proteins was measured by differential scanning calorimetry (DSC) at a scan rate of 60 °C / h on the Microcal™ PEAQ System (Malvern Panalytical). Proteins were used at concentrations of 8.8 pM (i-Tags) or 4.6 pM (Darbepoetin alfa with i-Tags fusion (SEQ ID NO: 44)) in 20 mM HEPES, 150 mM NaCI (pH 7.4). Data was fitted with the sequential fit model.

[0441] Circular dichroism spectroscopy.

[0442] Far-UV circular dichroism (CD) was recorded with a Jasco J-1500 CD spectrophotometer (Jasco) at temperatures from 20-90 °C, using a 1 mm quartz cuvette for temperature transitions. 4.3 pM protein (i-Tags, Darbepoetin alfa- i-Tags (SEQ ID NO: 44)) or 12.2 pM protein (Darbepoetin alfa-Hise-tag (SEQ ID NO: 43)) in 20 mM HEPES, 150 mM NaCI (pH 7.4) was heated at a heating rate of 60°C / h, and recorded at 218 nm or 232 nm, as stated in the respective figure legends.

[0443] Hydrogen-deuterium exchange mass spectrometry.

[0444] Hydrogen / deuterium exchange (HDX) mass spectrometry (MS) experiments were performed using an ACQUITY LIPLC M-class system equipped with automated HDX technology (Waters, Milford, MA, USA). HDX kinetics were determined in technical triplicates, taking data points at 0, 10, 60, 600, 1800, and 7200 s at 20 °C. At each data point, 3 pl of a solution of 30 pM protein (i-Tagi [one constant domain] or i-Tags [5 constant domains]) were diluted automatically 1 :20 into 99.9% D2O-containing 20 mM HEPES, 150 mM NaCI, pH 7.4 or the respective H2O-containing reference buffer. The reaction mixture was quenched by the 1 :1 addition of 200 mM KH2PO4, 200 mM Na2HPO4, pH 2.3 (titrated with HCI), containing 4 M guanidine hydrochloride and 200 mM TCEP at 1 °C and 50 pl of the resulting sample were subjected to on-column peptic digest on a Waters Enzymate BEH pepsin column 2.1 x 30 mm at 20 °C. Peptides were separated by reverse phase chromatography at 0 °C using a Waters Acquity UPLC C18, 1 .7 pm, 2.1 x 5.0 mm, 130 A trapping column and a Waters Acquity UPLC BEH C18, 1.7 pm, 1 x 100 mm, 130 A separation column. For separation, a gradient increasing the acetonitrile concentration stepwise from 5-35% in 6 min, from 35-40% in 1 min and from 40-95% in 1 min was applied and the eluted peptides were analyzed using an in-line Synapt G2-S QTOF HDMS mass spectrometer (Waters, Milford, MA, USA). UPLC was performed in protonated solvents (0.1 % formic acid), allowing deuterium to be replaced with hydrogen from side chains and amino / carboxyl termini that exchange much faster than backbone amide linkages (Englander and Kallenbach, 1983). All experiments were performed in duplicates. Deuterium levels were not corrected for back exchange and are therefore reported as relative deuterium levels (Wales et al., 2006). The use of an automated system handling all samples at identical conditions avoids the need for back exchange correction. MS data were collected over an m / z range of 100-2000. Mass accuracy was ensured by calibration with Glu-fibrino peptide B (Waters, Milford, MA, USA) and peptides were identified by MSEramping the collision energy automatically from 20-50 V. Data were analyzed in PLGS 3.0.3 and DynamX 3.0 software packages (Waters, Milford, MA, USA).

[0445] Ellman’s Assay.

[0446] Protein samples were diluted in reaction buffer (100 mM sodium phosphate, 1 mM EDTA, pH 8.0) or denaturing reaction buffer (5 M guanidinium hydrochloride, 100 mM sodium phosphate, 1 mM EDTA, pH 8.0). Ellman’s solution (4 mg / ml, 5,5'-Dithiobis(2- nitrobenzoic acid) in reaction buffer) was added 1 :25 to the protein and incubated for 15 minutes at room temperature. Absorbance was measured at 412 nm with a CLARIOstar® platereader (BMG Labtech).

[0447] HeLa STATI assay.

[0448] HeLa STAT1 cells (Signosis, SL-0004-FP) were used to measure STAT1 phosphorylation by bioluminescence. For one 96-well plate (Sigma-Aldrich, SIAL0596), 1 x 106HeLa cells or 1 x 104cells / well were seeded and incubated over night at 37 °C and 5% CO2. Thereafter, the cells were stimulated with supernatants containing p28L162C ACand incubated for 18 h. After incubation, the cells were lysed using lysis buffer (Promega, E1500). Then, Luciferase Assay reagent (Promega, E1500) was added, and the bioluminescence signal was measured at 560 nm in a plate reader (BMG Labtech CLARIOstar®). To assess stability of p28L162C AC, HeLa STAT1 activation tests were performed after incubation of the protein at 4°C or 50 °C for 30 min.

[0449] TH P-1 assay.

[0450] 2 x 105THP-1 cells / ml were seeded in RPMI medium containing 10% heat-inactivated FCS, 1 % antibiotic / antimycotic, and 25 nM PMA for differentiation. After 48 h incubation, cells were washed with PBS and fresh RPMI medium without PMA was added. The next day, before adding p28L162C-fusion variants, the medium was aspirated and 100 pL fresh RPMI without PMA was added. Cells were then stimulated with p28L162C-fusion variants (100 nM, 30 nM, 10 nM, 3 nM, 1 nM, 0.3 nM and 0.01 nM) for 24 h and then harvested at 300 x g for 15 min at 4 °C. The supernatants were used in an ELISA to quantify CXCL10 levels.

[0451] ELISA.

[0452] Concentrations of p28L162C ACwere measured by IL-27 ELISA (abeam, ab2667812) according to the manufacturer’s protocol. Darbepoetin alfa variants were quantified by Human Erythropoietin DuoSet ELISA (R&D Systems). The CXCL10 ELISA (R&D, DIP100) was performed according to the manufacturer's instructions. Absorption signals were measured in a plate reader (BMG Labtech CLARIOstar®).

[0453] Quantification, statistics, and illustrations.

[0454] Coomassie-stained SDS-PAGE gels were imaged with Gel Doc™ EZ Imaging System with Image Lab 5.2.1 (BioRad) and were quantified using Imaged (Fiji). If no bands were visible, the quantification, thus the x-fold increase, was calculated by assuming that the total amount of protein was just below the detection limit of the Coomassie stain (0.1 pg / band) (Brunelle and Green, 2014), and thus 0.1 pg / band were assumed. Statistical analyses and graph illustration were performed using Prism (GraphPad Software). Applied statistical test types and experimental sample sizes are stated in the figure legends. Icons in Figure 1A were created by and adapted from BioRender.com.

[0455] Experimental examples:

[0456] Example 1: Establishment of a platform biotechnology using the tag

[0457] A protein-tag should ideally be inert, improve expression, and be amenable to simple purification procedures. An enabling platform biotechnology that in addition enhances the quality attributes of the protein of interest, such as stability, provides further significant advantages for the production process as well as the product per se. Previous studies demonstrate that the constant domain of the antibody light chain (CL) combines these desired features. Most biopharmaceuticals are produced as secreted proteins from the medium of mammalian cells. Previous work has shown that CL does not significantly engage chaperones in the endoplasmic reticulum (ER), where secretory proteins pass through and acquire their structure (Behnke et al., 2016, Adams et al., 2019). This argues for a particularly robust folding process of the CL domain in cells. Furthermore, the CL domain is resistant against misfolding (Feige et al., 2008). Antibody light chains, that contain the CL domain, are naturally secreted in high quantities from human B cells without any harm to the human body (Nakano et al., 2006). Lastly, different e.g. nanobody-based, GMP-compatible purification procedures exist that allow the purification of antibodies via binding of their CL domain. This should allow for the simple purification of any protein where the CL domain is fused to (Figure 1 A). Humans contain two types of antibody light chains, which differ in the CL domain: the K or A subtype (Hill et al., 1966, Das et al., 2008). The inventors thus first compared the effect of fusing either K or A CL domains to an engineered human interleukin (IL) 27a / p28 subunit. The inventors used a variant of this IL subunit that contains a stabilizing disulfide bond (p28L162C(Muller et al., 2019)) and additionally a C-terminal truncation (AC): p28L162C AC, which abrogates O-glycosylation at C-terminal sites (Bohnacker et al., 2020) (SEQ ID NO: 22). IL-27a has immunomodulatory properties (Min et al., 2021 ) and thus is an attractive molecule for biomedical applications. However, like many proteins in a non-antibody format IL-27a is difficult to produce (Muller et al., 2019). Therefore, IL-27a was an ideal test candidate for the fusion technology of the invention. When fused to five sequential K CL domains (e.g. SEQ ID NO: 33), strong expression and secretion of p28L162C’ACfrom Expi293FTMcells was observed, whereas the corresponding A CL fusion (SEQ ID NO: 58) showed no detectable secretion (Figure 1 B). The same was observed for either five sequential K or A CL domains alone (Figure 1 B) (SEQ ID NOs: 59, 57). The inventors thus continued testing different K CL fusion lengths, leveraging the modular character of the CL domain as a building block. Since the inventors first data indicated that the K CL protein is potentially suited as a platform enabling biotechnology, the inventors named it i-Tag (Figure 1 C), which is part of the tag. With one to seven K CL domains (i-Tagi, i-Tags, i- Tag5, i-Tag7) fused toP28L162C ACvia a PAPAP-linker (SEQ ID NOs: 27, 38, 33, 40), expression levels of the protein of interest were significantly increased. In each case, the i-Tag fusion enabled a simple one-step purification process of the protein of interest via a commercially available column with immobilized anti-K CL nanobodies (CaptureSelect™ KappaXP) (Figure 1 C). Building on these findings, the inventors next tested if the CL fusion tag (i-Tags) would also have beneficial effects on the secretion levels of other proteins. The inventors thus selected several other structurally diverse proteins of medical relevance in a non-antibody format and lacking a common structural element. Indeed, when the inventors tested five other proteins, DLL3-4xCD3 bi-specific T-cell engager (BiTE) (Raum, 2017), Darbepoetin alfa (Ibbotson etal, 2001 , Powell et al., 2002), Erythropoietin (Weiss, 2003), Granulocyte-Colony Stimulating Factor (GCSF; (Avaloscase et al., 1990)), and IL-15Ra(sushi)IL-15 (Bernett, 2018, Ungewickell, 2021 ) the i-Tags fusion (SEQ ID NOs: 53, 44, 47, 50, 56, respectively) increased in each case the secretion by at least 5-fold up to 240-fold (Figures 1 D and 2A-E). Together, the inventors' data demonstrate that adding a tag of immunoglobulin K CL domain repeats significantly increases the secretion of various biomedically relevant proteins from mammalian cells. The i-Tag thus warranted further investigations in terms of purification, biophysical characteristics, and functionality.

[0458] Example 2: The i-Tag as part of the tag used herein enables simple and standardized purification of proteins and at the same time improves their biophysical characteristics

[0459] Building on the significantly increased secretion of various proteins upon i-Tag fusion (Fig. 1 ), the inventors next tested if the different fusion proteins could be purified via a simple, standardized procedure. For this purpose, the inventors used the commercially available CaptureSelect™ KappaXP column. The affinity of these columns to the i-Tag is based on anti-K CL nanobodies. This enabled easy one-step purification of p28L162C’AC- i-Tag - fusions (Figure 1 C). For all i-Tags fusion proteins tested in addition top28Li62c, AC DLL3-4xCD3 BiTE, Darbepoetin alfa, GCSF, and IL-15RD(sushi)IL-15 (Figure 3A), 81 % to 99% purity could be obtained via this simple one-step purification (Figure 3B). Identities and integrities were confirmed by immunoblotting (Figure 4A). Selected proteins were alternatively fused to a C-terminal Hise-tag (SEQ ID NOs: 43, 46, 49, 52, 55, respectively) to enable their purification for comparison to the i-Tags fusions. Purity and expression levels were generally higher for the i-Tagged constructs (= constructs tagged with the tag used herein) than if a C-terminal Hise-tag was used (Figures 3B and 4B). All proteins with an i-Tag could be produced in the inventors’ studies, in contrast to the His-tag versions of the same proteins of interest (Figure 3B). For the three proteins where the inventors could obtain expression and purify proteins also in a Hise-tagged version (DLL3-4xCD3 BiTE, Darbepoetin alfa, and IL- 15Ra(sushi)IL-15) (SEQ ID NOs: 52, 43, 56) the inventors continued with comparative biophysical analyses of the purified proteins. Analytical ultracentrifugation showed that fusion proteins were generally sedimenting with the expected molecular weights. Only Darbepoetin alfa differed in the observed molecular weight distribution upon fusion with the i-Tags (SEQ ID NO: 44) in comparison with a Hise-tag (SEQ ID NO: 43) (Figures 3C and D). For the proteins tested, dynamic light scattering (DLS) revealed a more homogenous protein population of the i-Tags versus Hise-tag fused proteins Darbepoetin alfa and IL-15Ra(sushi)IL-15, whereas for DLL3-4xCD3 BiTE this was less the case (Figure 3E). For Darbepoetin alfa and IL-15Ra(sushi)IL-15, also high- performance liquid chromatography (HPLC) analyses revealed a more homogenous protein population for the i-Tagged proteins than for the respective Hiss-tagged proteins (Figures 4C and D), in accordance with the DLS data (Figures 3C and D). Heterogeneity on SDS-PAGE for IL-15RD(sushi)IL-15 was due to endogenous / V- glycosylation (Figure 4E).

[0460] Example 3: The i-Tag5as part of the tag used herein is stable and well-folded

[0461] Together the inventors’ data show that the K CL fusions increase protein expression and secretion, facilitate purification, and improve protein quality characteristics. Building on these findings, the inventors set out to characterize the i-Tags itself in more detail. The i-Tag is a designed construct obtained by fusing the naturally occurring K CL domain. Small-angle X-ray scattering (SAXS) allows to determine the shape of biomolecules in solution. Using SAXS, the inventors could show that the i-Tags was elongated, as predicted by computer simulations (Figures 5A and 6A-E). To assess if the K CL domains within the i-Tags behave similarly to a single free K CL domain, the inventors performed hydrogen-deuterium exchange mass spectrometry (HDX-MS) experiments. HDX-MS experiments allow to determine protein dynamics and flexibility based on the accessibility of its amide protons (Narang et al., 2020). Using this technique, the inventors could show that hydrogen / deuterium exchange of the K CL domain in the context of the i-Tags was almost identical to an individual domain (Figures 5B and 6F). Folding of the K CL domains within the i-Tags thus seems unaltered in comparison to the non-fused, single domain. Analytical ultracentrifugation measurements confirmed the mostly monomeric protein state of the i-Tags (Figure 5C). The same results were obtained with DLS and HPLC measurements (Figures 7G and H). Thus, the homogenous state and only small high-molecular weight content of the i-Tags was validated. DSC measurements revealed the i-Tags to be a stable protein with a melting temperature (Tm) of around 51 °C (Figure 5D). This result was consistent with temperature-dependent unfolding measured by CD spectroscopy (Figure 7I). Together this showed that the i-Tags thermally unfolds in a two-state process without populating detectable intermediates that may destabilize a fusion partner. In agreement with this, the melting temperature of i-Tagged Darbepoetin alfa (SEQ ID NO: 44) (51 °C, Figures 7J and K) corresponded to the Tmof i-Tags and was almost identical to the unfolding temperature of Hiss-tagged Darbepoetin alfa (SEQ ID NO: 43) (52 °C, Figure 7L). Concluding the in-depth characterization of the i-Tags, an Ellmann’s assay showed that within each CL domain within the five repeats, the intramolecular single disulfide bond was correctly formed (Figure 5E). Taken together, the i-Tags is a monomeric, stable, and well-folded protein. The unaltered characteristics of the CL domain within the i-Tags highlight its modular character.

[0462] Example 4: The i-Tag as part of the tag used herein does not alter functionality of the fusion protein

[0463] Key for any fusion tag is that it does not alter the functionality of the fusion partner in undesired ways. As opposed to e.g., the Fc fusion, no biological activities have been described for the CL domain yet, which is the building block of the i-Tag (part of the tag used herein). In addition to the other features analyzed above, the inert nature of the i-Tag is an important characteristic compared to other protein tags. The inventors thus focused on assessing if functions of the proteins of interest were maintained upon i- Tags fusion.

[0464] For p28L162C’ACthe inventors used a cell reporter assay that monitors STAT1 phosphorylation via bioluminescence upon binding of a functional cytokine to the corresponding receptor. In this assay, the i-Tags fusion did not compromise activity of p28L162c,Ac (Figure8A). To validate that the functionality of the DLL3-4xCD3 BiTE was maintained, the inventors used a co-immunoprecipitation assay. Here, the inventors assessed binding of the i-Tagged BiTE to its target proteins, CD3s and DLL3 (Figure 8B). After preincubation of the BiTE with one or both of its target proteins, the inventors utilized the Hise-tag of the targets for immunoprecipitation via an anti-Hise-antibody. Pulling on the respective target proteins allowed the inventors to detect the bound i- Tagged DLL3-4xCD3 BiTE by reducing SDS-PAGE, verifying the preserved functionality of the bispecific T-cell engager protein. For Darbepoetin alfa, the inventors used an ELISA that was developed for measuring Erythropoietin activity. In this assay, the i-Tagged Darbepoetin alfa fusion protein was detected. This suggests a correct structure of Darbepoetin alfa in the fusion construct (Figure 8C). Building on these findings, the inventors evaluated if activity of the fusion partners was not only maintained by the i-Tags fusion but if the biophysically well-behaving K CL domains may even stabilize certain fusion partners. Indeed, whereas p28L162C’ACcompletely lost its activity after incubation for 30 min at 50 °C, the activity of the i- Tagged p28L162C ACwas maintained (Figure 8D).

[0465] Taken together, for the different fusion partners tested, the i-Tags did not compromise activity. Instead, the i-Tags fusion can lead to a maintenance of activity upon thermal challenge.

[0466] Example 5: N- as well as C-terminal i-Tag5fusion enables a standardized protein production in mammalian cells

[0467] This experiment was done to further analyze, if the fusion of the inventive tag (i-Tag) to the N-terminus or the C-terminus of a target polypeptide makes any difference. As a tag, a fusion of five constant domains of the human kappa immunoglobulin light chain was used as mentioned above. The target polypeptide was the p28L162C ACprotein (SEQ ID NO: 22) (mentioned above). The inventors analyzed the levels of the secretion of the fusion polypeptides of transfected Expi293FTMcells by Coomassie stained SDS- PAGE. Supernatants were harvested after transfection of Expi293FTMcells (load) and purified with the CaptureSelect™ KappaXP column via i-Tags (purified protein). A bovine serum albumin (BSA) dilution series served as concentration standard to quantify protein levels. The expression levels of the N-terminal fusion polypeptide (SEQ ID NO: 41 ) and the C-terminal fusion polypeptide (SEQ ID NO: 33) are comparable and significantly increased compared to the expression level of the untagged target polypeptide (SEQ ID NO: 22) (Figures 10A and 9B). Quantification of the Coomassie gel (Figure 9A) shows that the standardized purification via CaptureSelect™ KappaXP column resulted in a very high purity of above 95% for both the N-terminally and the C-terminally tagged p28L162C AC(SEQ ID NOs: 41 , 33) (Figure 10C). For analyzing the functionality of the p28L162C’ACfused at the N-terminus or the C-terminus to the inventive tag THP-1 cells were stimulated with different concentrations of both fusion polypeptides and the CXCL10 concentration in the supernatant was measured with an ELISA as mentioned above. The ELISA showed that both, the N-terminally and the C-terminally tagged p28L162C’AC(SEQ ID NOs: 41 , 33), are functional fusion polypeptides (Figure 10D). Example 6: Increased expression titer and preserved functionality are obtained regardless of a linker that fuses the i-Tag to p28L162C’AC.

[0468] The inventors further analyzed the impact of different linkers between the inventive tag and the target polypeptide p28L162C ACand of two different sizes of the tag on the expression level of the fusion polypeptides. Therefore, Expi293FTMcells were transfected with constructs for the expression of fusion polypeptides comprising the inventive tag containing one or five constant domain(s) of the human kappa immunoglobulin light chain fused to the C-terminus of the p28L162C’ACpolypeptide without (SEQ ID NOs: 23, 32) or with a linker having one of the following amino acids sequences: GGGS (only for tag with one constant domain of the human kappa immunoglobulin light chain (SEQ ID NO: 24)), A(EAAAK)4ALEA(EAAAK)4A (SEQ ID NOs: 25, 26), PAPAP (SEQ ID NOs: 27, 33), APAPAPA (SEQ ID NOs: 28, 34), APAPAPAPAPKPA (SEQ ID-NOs: 29, 35), APAPAPAPAPAPA (only for tag with one constant domain of the human kappa immunoglobulin light chain (SEQ ID NO: 30)), A(EAAAK)4A (only for tag with one constant domain of the human kappa immunoglobulin light chain (SEQ ID NO: 31 )). As shown in Figure 1 1 A, the secretion of the fusion polypeptide (No. 3 to 15) is generally increased compared to the untagged p28L162c, AC polypeptide (SEQ ID NO: 22) (No. 2). Further, the secretion level of the fusion polypeptide comprising the tag with five constant domains (SEQ ID NOs: 32,

[0469] 26, 33, 34, 35) (No. 11 to 15) is generally higher than the secretion level of the fusion polypeptide comprising the tag with one constant domain (SEQ ID NOs: 23, 24, 25,

[0470] 27, 28, 29, 30, 31 ) (No. 3 to 10). For testing the functionality of tagged p28L162C’ACfusion polypeptide, the HeLa STAT1 bioluminescence assay was used. The supernatants of Expi293FTMcells transfected with constructs for secretion of fusion polypeptides containing the p28L162C ACprotein fused at the C-terminus to the inventive tag containing five constant domains of the human kappa immunoglobulin light chain without (SEQ ID NO: 32) or a linker having one of the following amino acids sequences: A(EAAAK)4ALEA(EAAAK)4A (SEQ ID NO: 26), PAPAP (SEQ ID NO: 33), APAPAPA (SEQ ID NO: 34), APAPAPAPAPKPA (SEQ ID NO: 35). The mock control of the above experiment served as negative control. The HeLa cells were stimulated with adjusted volumes of these supernatants, for which protein concentrations were measured and normalized before HeLa STAT1 cell stimulation and the resulting bioluminescence was measured. As shown in Figure 11 B, the secreted fusion polypeptides were functional regardless of if a linker was present or not. Example 7: Standardized protein production and protein functionality are obtained by i-Tag5fusion regardless of an interdomain linker.

[0471] In this example the inventors examined, if a linker between the constant domains of the human kappa immunoglobulin light chain comprised in the inventive tag has any impact on the functionality of the fusion polypeptide. The inventors transfected Expi293FTMcells with constructs for the expression of fusion polypeptides containing the p28L162C’ACprotein fused with the PAPAP-linker to the inventive tag which comprised five constant domains of the human kappa immunoglobulin light chain linked together without a linker (SEQ ID NO: 33) or five constant domains of the human kappa immunoglobulin light chain linked together via a GAGAG-linker (SEQ ID NO: 39). The supernatants of the transfected cells were harvested (load) and purified with the CaptureSelect™ KappaXP column via the inventive tag (purified protein) and analyzed on a Coomassie stained SDS-PAGE gel. The expression of the fusion polypeptides with or without a linker between the constant domains of the tag was equal, as shown in Figure 12A. Also, the function of both fusion polypeptides was equal as tested by THP-1 cell stimulation and subsequent CXCL10 ELISA and shown in Figure 12B.

[0472] Example 8: Stability of the i-Tag2in fetal bovine serum

[0473] To show the stability of the i-Tag2 without linker sequence between the constant domains of the human kappa immunoglobulin light chain parts of the i-Tag (without C- terminal cysteine), the i-Tag2 construct without linker sequence (SEQ ID NO: 71 ) was constructed and mixed 1 :10 in fetal bovine serum (FBS) and incubated at 37 °C for 97 hours. Immunoprecipitation (IP) was conducted with an antibody against the human kappa CL domain. The i-Tag2 construct is stable in FBS incubated at 37°C for 96 hours, as shown in Figure 13.

[0474] Example 9: Stability of the i-Tag2in a partial proteolysis experiment using trypsin To further investigate the stability of the i-Tag with and without linker sequences between the constant domains of the human kappa immunoglobulin light chain parts of the i-Tag (without C-terminal cysteine), the i-Tag2construct with and without linker sequences were incubated with 1 :50 (w / w) Trypsin at 37 °C for up to 60 min. Samples taken after indicated time were analyzed on Coomassie-stained SDS-PAGE gels. The constructs tested were KCL-KCL (i-Tag2 (without linker)) (SEQ ID NO: 71 ), KCL- (GGGGS)2-KCL (i-Tag2(with (GGGGS)2-linker)) (SEQ ID NO: 72), KCL- A(EAAAK)2A - KCL (i-Tag2(with A(EAAAK)2A-linker)) (SEQ ID NO: 73), KCL- GSPKSCDKTHTCPPCPAPGSEIK-KCL (i-Tag2(with

[0475] GSPKSCDKTHTCPPCPAPGSEIK-linker)) (SEQ ID NO: 74), and KCL- GS(GGGGS)2GEIK-KCL(i-Tag2(with GS(GGGGS)2GEIK-linker)) (SEQ ID NO: 75). The i-Tag2 without linker sequence is more stable over the incubation time of 60 minutes than the i-Tag2 proteins with linker sequences. It is clearly shown in Figure 14A that the i-Tag2 at about 25 kDa without linker sequence is still visible after 60 minutes incubation with trypsin whereas e.g. the construct i-Tag2 with A(EAAAK)2A- linker is only stable for about 5 minutes. After that, bands of degradation products are visible at the bottom end of the gel. To get a quantification, the uncleaved fraction of the full-length protein of each construct were quantified (cf. Figure 14A). The resulting graph clearly shows that the i-Tag2 without linker sequence is more stable in the used trypsin solution compared to the tested i-Tag2 constructs containing a linker sequence as shown in Figure 14B.

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Claims

CLAIMS1 . A method of modulating a molecular mass and / or an isoelectric point of a fusion polypeptide, wherein the fusion polypeptide comprises a target polypeptide fused to a tag suitable for affinity purification or detection of the target polypeptide, wherein the tag comprises at least one constant domain of an immunoglobulin light chain or orthologous polypeptide or fragment thereof and wherein the molecular mass of the fusion polypeptide is modulated by increasing the molecular mass of the tag via combining the one constant domain of an immunoglobulin light chain or orthologous polypeptide or fragment thereof with one or more further constant domain(s) of an immunoglobulin light chain or orthologous polypeptide(s) or fragment(s) thereof; wherein the constant domain of the immunoglobulin light chain or orthologous polypeptide or fragment thereof of the tag is monomeric; and wherein optionally the target polypeptide is fused to the tag via a first linker.

2. The method of claim 1 , wherein the method comprises a) determining the molecular mass of the target polypeptide, b) (optional) determining the molecular mass of one constant domain of an immunoglobulin light chain or orthologous polypeptide or fragment thereof, c) calculating how many constant domains of an immunoglobulin light chain or orthologous polypeptides or fragments thereof are needed in addition to the molecular mass of the target polypeptide (step a) for reaching a desired molecular mass of the fusion polypeptide, and d) creating the tag to be fused to the target polypeptide by combining the necessary number of constant domains of an immunoglobulin light chain or orthologous polypeptides or fragments thereof according to the result of step (c), wherein the desired molecular mass of the fusion polypeptide is above a filtration limit of a kidney of a given organism or, optionally, at least 50 kDa or 70 kDa.

3. The method of any one of the preceding claims, wherein the tag before and / or after modulating the molecular mass of the fusion polypeptide comprises two or more constant domains of the immunoglobulin light chain or orthologous polypeptides or fragments thereof and wherein optionally the constant domains or orthologous polypeptides or fragments thereof are connected to each other by a second linker.

4. The method of any one of the preceding claims, wherein the fusion polypeptide is expressed under conditions allowing a formation of one or more disulfide bond(s).

5. The method of any one of the preceding claims, wherein the constant domain of the immunoglobulin light chain or orthologous polypeptide or fragment thereof of the tag is a constant domain of a kappa immunoglobulin light chain or orthologous polypeptide or fragment thereof.

6. The method of any one of the preceding claims, wherein the constant domain of the immunoglobulin light chain or orthologous polypeptide or fragment thereof of the tag is a constant domain of a mammal immunoglobulin light chain or fragment thereof, preferred a constant domain of a primate immunoglobulin light chain or fragment thereof and most preferred a constant domain of a human immunoglobulin light chain or fragment thereof or wherein the constant domain of the kappa immunoglobulin light chain or orthologous polypeptide or fragment thereof of the tag is a constant domain of a mammal kappa immunoglobulin light chain or fragment thereof, preferred a constant domain of a primate kappa immunoglobulin light chain or fragment thereof, and most preferred a constant domain of a human kappa immunoglobulin light chain or fragment thereof.

7. The method of any one of claims 5 or 6, wherein a C-terminal cysteine of the constant domain of the kappa immunoglobulin light chain of the tag is deleted if present in the peptide sequence of the constant domain of the kappa immunoglobulin light chain of the tag.

8. The method of any one of claims 5 to 7, wherein the constant domain of the kappa immunoglobulin light chain or orthologous polypeptide or fragment thereof of the tag is selected from a group consisting of a constant domain of a mouse kappa immunoglobulin light chain, a constant domain of a rat kappa immunoglobulin light chain, a constant domain of a rabbit kappa immunoglobulin light chain, a constant domain of a dog kappa immunoglobulin light chain, a constant domain of a pig kappa immunoglobulin light chain, a constant domain of a monkey kappa immunoglobulin light chain, and preferably the constant domain of the human kappa immunoglobulin light chain.

9. The method of any one of claims 5 to 8, wherein the constant domain of the kappa immunoglobulin light chain of the tag comprises or consists of a peptide sequence selected from a group consisting of the constant domain of human kappa immunoglobulin light chain (SEQ ID NO: 1 ), the constant domain of human kappa immunoglobulin light chain without the C-terminal cysteine (SEQ ID NO: 2), the constant domain of mouse kappa immunoglobulin light chain (SEQ ID NO: 4), the constant domain of mouse kappa immunoglobulin light chain without the C- terminal cysteine (SEQ ID NO: 5), the constant domain of rat kappa immunoglobulin light chain (SEQ ID NO: 6), the constant domain of rat kappa immunoglobulin light chain without the C-terminal cysteine (SEQ ID NO: 7), the constant domain of rabbit kappa-b4 immunoglobulin light chain (KACB) according to UniProt entry: P01839 (SEQ ID NO: 8), the constant domain of rabbit kappa- b4 immunoglobulin light chain (KACB) according to UniProt entry: P01839, without the C-terminal cysteine (SEQ ID NO: 9), the constant domain of rabbit kappa-b4 immunoglobulin light chain (KAC4) according to UniProt entry: P01840 (SEQ ID NO: 10), the constant domain of rabbit kappa-b4 immunoglobulin light chain (KAC4) according to UniProt entry: P01840, without the C-terminal cysteine (SEQ ID NO: 11 ), the constant domain of rabbit kappa-b5 immunoglobulin light chain (KAC5) according to UniProt entry: P01841 (SEQ ID NO: 12), the constant domain of rabbit kappa-b5 immunoglobulin light chain (KAC5) according to UniProt entry: P01841 , without the C-terminal cysteine (SEQ ID NO: 13), the constant domain of rabbit kappa-b5 immunoglobulin light chain (KAC6) according to UniProt entry: P03984 (SEQ ID NO: 14), the constant domain of rabbit kappa- b5 immunoglobulin light chain (KAC6) according to UniProt entry: P03984,without the C-terminal cysteine (SEQ ID NO: 15), the constant domain of dog kappa immunoglobulin light chain (SEQ ID NO: 16), the constant domain of pig kappa immunoglobulin light chain (SEQ ID NO: 17), the constant domain of monkey kappa immunoglobulin light chain (SEQ ID NO: 18), and the constant domain of monkey kappa immunoglobulin light chain without the C-terminal cysteine (SEQ ID NO: 19).

10. The method of any one of the preceding claims, wherein the first linker and / or the second linker are / is a cleavable linker.11 . The method of any one of the claims 1 to 9, wherein the first linker and / or the second linker are / is a non-cleavable linker.

12. The method of any one of the preceding claims, wherein the first linker of the fusion polypeptide is a cleavable linker and the second linker in the tag is a non- cleavable linker or wherein the first linker is a non-cleavable linker, and the second linker is a cleavable linker.

13. The method of any one of the preceding claims, wherein the tag is fused to the C-terminus of the target polypeptide.

14. The method of any one of claims 1 to 12, wherein the tag is fused to the N- terminus of the target polypeptide.

15. The method of any one of claims 1 to 12, wherein the tag is fused to the C- terminus and to the N-terminus of the target polypeptide.

16. The method of any one of the preceding claims, wherein the expression of the fusion polypeptide is higher than of the untagged target polypeptide.

17. The method of any one of the preceding claims, wherein a function of the target polypeptide with the tag is substantially comparable to a function of the target polypeptide without the tag.

18. A fusion polypeptide obtained or obtainable by a method of any one of claims 1 to 17.

19. A tag suitable for affinity purification or detection of a fusion partner, wherein the fusion partner is a target polypeptide and wherein the tag comprises at least one constant domain of an immunoglobulin light chain or orthologous polypeptide or fragment thereof and optionally a first linker that fuses the tag to the target polypeptide; and wherein the constant domain of the immunoglobulin light chain or orthologous polypeptide or fragment thereof is monomeric.

20. The tag of claim 19, wherein the constant domain of the immunoglobulin light chain or orthologous polypeptide or fragment thereof is a constant domain of a kappa immunoglobulin light chain or orthologous polypeptide or fragment thereof.

21. The tag of any one of the claims 19 or 20, wherein the constant domain of the immunoglobulin light chain or fragment thereof is a constant domain of a mammalian immunoglobulin light chain or orthologous polypeptide or fragment thereof, preferred a constant domain of a primate immunoglobulin light chain or fragment thereof and most preferred a constant domain of a human immunoglobulin light chain or fragment thereof or wherein the constant domain of the kappa immunoglobulin light chain or fragment thereof is a constant domain of a mammalian kappa immunoglobulin light chain or orthologous polypeptide or fragment thereof, preferred a constant domain of a primate kappa immunoglobulin light chain or fragment thereof, and most preferred a constant domain of a human kappa immunoglobulin light chain or fragment thereof.

22. The tag of any one of claims 19 to 21 , wherein a C-terminal cysteine of the constant domain of the immunoglobulin light chain is deleted if present in the peptide sequence of the constant domain of the immunoglobulin light chain.

23. The tag of any one of claims 20 to 22, wherein the constant domain of the kappa immunoglobulin light chain or fragment thereof is selected from a group consisting of a constant domain of a mouse kappa immunoglobulin light chain, a constant domain of a rat kappa immunoglobulin light chain, a constant domain ofa dog kappa immunoglobulin light chain, a constant domain of a rabbit kappa light chain, a constant domain of a pig kappa immunoglobulin light chain, a constant domain of a monkey kappa immunoglobulin light chain, and preferably the constant domain of the human kappa immunoglobulin light chain.

24. The tag of any one of claims 20 to 23, wherein the constant domain of the kappa immunoglobulin light chain comprises or consists of a peptide sequence selected from a group consisting of the constant domain of human kappa immunoglobulin light chain (SEQ ID NO: 1 ), the constant domain of human kappa immunoglobulin light chain without the C-terminal cysteine (SEQ ID NO: 2), the constant domain of mouse kappa immunoglobulin light chain (SEQ ID NO: 4), the constant domain of mouse kappa immunoglobulin light chain without the C-terminal cysteine (SEQ ID NO: 5), the constant domain of rat kappa immunoglobulin light chain (SEQ ID NO: 6), the constant domain of rat kappa immunoglobulin light chain without the C-terminal cysteine (SEQ ID NO: 7), the constant domain of rabbit kappa-b4 immunoglobulin light chain (KACB) according to UniProt entry: P01839 (SEQ ID NO: 8), the constant domain of rabbit kappa-b4 immunoglobulin light chain (KACB) according to UniProt entry: P01839, without the C-terminal cysteine (SEQ ID NO: 9), the constant domain of rabbit kappa-b4 immunoglobulin light chain (KAC4) according to UniProt entry: P01840 (SEQ ID NO: 10), the constant domain of rabbit kappa-b4 immunoglobulin light chain (KAC4) according to UniProt entry: P01840, without the C-terminal cysteine (SEQ ID NO: 11 ), the constant domain of rabbit kappa-b5 immunoglobulin light chain (KAC5) according to UniProt entry: P01841 (SEQ ID NO: 12), the constant domain of rabbit kappa- b5 immunoglobulin light chain (KAC5) according to UniProt entry: P01841 , without the C-terminal cysteine (SEQ ID NO: 13), the constant domain of rabbit kappa-b5 immunoglobulin light chain (KAC6) according to UniProt entry: P03984 (SEQ ID NO: 14), the constant domain of rabbit kappa-b5 immunoglobulin light chain (KAC6) according to UniProt entry: P03984, without the C-terminal cysteine (SEQ ID NO: 15), the constant domain of dog kappa immunoglobulin light chain (SEQ ID NO: 16), the constant domain of pig kappa immunoglobulin light chain (SEQ ID NO: 17), the constant domain of monkey kappa immunoglobulin light chain (SEQ ID NO: 18), and the constant domain of monkey kappa immunoglobulin light chain without the C-terminal cysteine (SEQ ID NO: 19).

25. The tag of any on of claims 19 to 24, wherein the tag comprises two or more constant domains of the immunoglobulin light chain or orthologous polypeptides or fragments thereof and wherein optionally the constant domains or orthologous polypeptides or fragments thereof are connected to each other by a second linker.

26. The tag of any one of the claims 19 to 25, wherein the first linker and / or the second linker comprises an amino acid sequence which is cleavable.

27. The tag of any one of claims 19 to 25, wherein the first linker and / or the second linker comprises an amino acid sequence which is not cleavable.

28. The tag of any one of claims 19 to 27, wherein the tag is suitable to be fused to the C-terminus of the target polypeptide.

29. The tag of any one of claims 19 to 27, wherein the tag is suitable to be fused to the N-terminus of the target polypeptide.

30. The tag of any one of claims 19 to 27, wherein the tag is suitable to be fused to the C-terminus and to the N-terminus of the target polypeptide.

31. Use of the tag of any one of claims 19 to 30 comprising at least three constant domains of the immunoglobulin light chain or orthologous polypeptides or fragments thereof for increasing the expression of a fusion polypeptide comprising the target polypeptide and the tag.

32. Use of the tag of any one of claims 19 to 30 comprising one or more constant domain(s) of the immunoglobulin light chain or orthologous polypeptides or fragments thereof for increasing the molecular mass of a fusion polypeptide comprising the target polypeptide and the tag.

33. A fusion polypeptide comprising a target polypeptide and a tag suitable for affinity purification or detection of the target polypeptide, wherein the tag comprises at least one constant domain of an immunoglobulin light chain or orthologouspolypeptide or fragment thereof and optionally a first linker that fuses the tag to the target polypeptide; wherein the constant domain of the immunoglobulin light chain or orthologous polypeptide or fragment thereof is monomeric.

34. The fusion polypeptide of claim 33, wherein the tag comprises two or more constant domains of the immunoglobulin light chain or orthologous polypeptides or fragments thereof and wherein optionally the constant domains or orthologous polypeptides or fragments thereof are connected to each other by a second linker.

35. The fusion polypeptide of any one of claims 33 or 34, wherein the fusion polypeptide is expressed under conditions allowing a formation of a disulfide bond.

36. The fusion polypeptide of any one of claims 33 to 35, wherein the constant domain of the immunoglobulin light chain or orthologous polypeptide or fragment thereof is a constant domain of a kappa immunoglobulin light chain or orthologous polypeptide or fragment thereof.

37. The fusion polypeptide of any one of claims 33 to 36, wherein the constant domain of the immunoglobulin light chain or fragment thereof is a constant domain of a mammalian immunoglobulin light chain or orthologous polypeptide or fragment thereof, preferred a constant domain of a primate immunoglobulin light chain or fragment thereof and most preferred a constant domain of a human immunoglobulin light chain or fragment thereof or wherein the constant domain of the kappa immunoglobulin light chain or fragment thereof is a constant domain of a mammal kappa immunoglobulin light chain or orthologous polypeptide or fragment thereof, preferred a constant domain of a primate kappa immunoglobulin light chain or fragment thereof, and most preferred a constant domain of a human kappa immunoglobulin light chain or fragment thereof.

38. The fusion polypeptide of any one of claims 36 or 37, wherein a C-terminal cysteine of the constant domain of the kappa immunoglobulin light chain is deleted if present in the peptide sequence of the constant domain of the kappa immunoglobulin light chain.

39. The fusion polypeptide of any one of claims 36 to 38, wherein the constant domain of the kappa immunoglobulin light chain or fragment thereof is selected from a group consisting of a constant domain of a mouse kappa immunoglobulin light chain, a constant domain of a rat kappa immunoglobulin light chain, a constant domain of a rabbit kappa immunoglobulin light chain, a constant domain of a dog kappa immunoglobulin light chain, a constant domain of a pig kappa immunoglobulin light chain, a constant domain of a monkey kappa immunoglobulin light chain, and preferably the constant domain of the human kappa immunoglobulin light chain.

40. The fusion polypeptide of any one of claims 36 to 39, wherein the constant domain of the kappa immunoglobulin light chain comprises or consists of a peptide sequence selected from a group consisting of the constant domain of human kappa immunoglobulin light chain (SEQ ID NO: 1 ), the constant domain of human kappa immunoglobulin light chain without the C-terminal cysteine (SEQ ID NO: 2), the constant domain of mouse kappa immunoglobulin light chain (SEQ ID NO: 4), the constant domain of mouse kappa immunoglobulin light chain without the C-terminal cysteine (SEQ ID NO: 5), the constant domain of rat kappa immunoglobulin light chain (SEQ ID NO: 6), the constant domain of rat kappa immunoglobulin light chain without the C-terminal cysteine (SEQ ID NO: 7), the constant domain of rabbit kappa-b4 immunoglobulin light chain (KACB) according to UniProt entry: P01839 (SEQ ID NO: 8), the constant domain of rabbit kappa- b4 immunoglobulin light chain (KACB) according to UniProt entry: P01839, without the C-terminal cysteine (SEQ ID NO: 9), the constant domain of rabbit kappa-b4 immunoglobulin light chain (KAC4) according to UniProt entry: P01840 (SEQ ID NO: 10), the constant domain of rabbit kappa-b4 immunoglobulin light chain (KAC4) according to UniProt entry: P01840, without the C-terminal cysteine (SEQ ID NO: 11 ), the constant domain of rabbit kappa-b5 immunoglobulin light chain (KAC5) according to UniProt entry: P01841 (SEQ ID NO: 12), the constant domain of rabbit kappa-b5 immunoglobulin light chain (KAC5) according to UniProt entry: P01841 , without the C-terminal cysteine (SEQ ID NO: 13), the constant domain of rabbit kappa-b5 immunoglobulin light chain (KAC6) according to UniProt entry: P03984 (SEQ ID NO: 14), the constant domain of rabbit kappa-b5 immunoglobulin light chain (KAC6) according to UniProt entry: P03984, without the C-terminal cysteine (SEQ ID NO: 15), the constant domain of dog kappa immunoglobulin light chain (SEQ ID NO: 16), the constant domain of pig kappa immunoglobulin light chain (SEQ ID NO: 17), the constant domain of monkey kappa immunoglobulin light chain (SEQ ID NO: 18), and the constant domain of monkey kappa immunoglobulin light chain without the C-terminal cysteine (SEQ ID NO: 19).41 . The fusion polypeptide of any one of claims 33 to 40, wherein the first linker and / or the second linker is / are a cleavable linker.

42. The fusion polypeptide of any one of claims 34 to 40, wherein the first linker and / or the second linker is / are a non-cleavable linker.

43. The fusion polypeptide of any one of claims 34 to 42, wherein the tag is fused to the C-terminus of the target polypeptide.

44. The fusion polypeptide of any one of claims 34 to 42, wherein the tag is fused to the N-terminus of the target polypeptide.

45. The fusion polypeptide of any one of claims 34 to 42, wherein the tag is fused to the C-terminus and to the N-terminus of the target polypeptide.

46. The fusion polypeptide of any one of claims 33 to 45, wherein the fusion polypeptide has a molecular mass of at least 50 kDa or 70 kDa.

47. The fusion polypeptide of any one of claims 33 to 46, wherein the expression of the fusion polypeptide is higher than of the untagged target polypeptide.

48. The fusion polypeptide of any one of claims 33 to 47, wherein the expression of the fusion polypeptides comprising the tag with at least 3 or more constant domains of the immunoglobulin light chain or orthologous polypeptides or fragments thereof is higher than the expression of the fusion polypeptidecomprising the tag with one constant domain of the immunoglobulin light chain or orthologous polypeptides or fragments thereof.

49. The fusion polypeptide of any one of claims 33 to 48, wherein a function of the target polypeptide with the tag is substantially comparable to a function of the target polypeptide without the tag.

50. The method of any one of claims 1 to 17 and / or the fusion polypeptide of any one of claims 18 or 33 to 49, wherein the target polypeptide is a polypeptide used in the pharmaceutical industry.51 . The method of any one of claims 1 to 17 and / or the fusion polypeptide of any one of claims 18 or 33 to 49, wherein the target polypeptide is a polypeptide used in in-vitro diagnostic procedures.

52. The method of any one of claims 1 to 17 and / or the fusion polypeptide of any one of claims 18 or 33 to 49, wherein the target polypeptide is used for a medical indication.

53. The fusion polypeptide of any one of claims 18 or 33 to 49 for use as a medicament.

54. The fusion polypeptide of any one of claims 18 or 33 to 49 for use in a medical indication.

55. The method of any one of claims 1 to 17 and / or the fusion polypeptide of any one of claims 18 or 33 to 49, wherein the target polypeptide is used in biotechnology methods.

56. The method of any one of claims 1 to 17 and / or the fusion polypeptide of any one of claims 18 or 33 to 49, wherein the target polypeptide is used in the chemical industry or in food production.

57. Use of the method of any one of claims 1 to 17 and / or the fusion polypeptide of any one of claims 18 or 33 to 49 in the chemical industry or in food production.

58. Use of the method of any one of claims 1 to 17 and / or the fusion polypeptide of any one of claims 18 or 33 to 49 in biotechnology methods.

59. Use of the fusion polypeptide of any one of claims 18 or 33 to 49 as a medicament.

60. Use of the method of any one of claims 1 to 17 and / or the fusion polypeptide of any one of claims 18 or 33 to 49 for the manufacture of a medicament.

61. Use of the fusion polypeptide of any one of claims 18 or 33 to 49 in an in-vitro diagnostic procedure.

62. Use of the method of any one of claims 1 to 17 and / or the fusion polypeptide of any one of claims 18 or 33 to 49 for the manufacture of medicament for use in a medical indication.

63. A pharmaceutical composition comprising a fusion polypeptide of any one of claims 18 or 33 to 49 and optionally a pharmaceutically acceptable carrier, diluent, or excipient.

64. A nucleic acid for expression of the fusion polypeptide of any one of claims 1 or 18 or 33 to 56 comprising a nucleic acid sequence encoding the target polypeptide of any one of claims 1 or 33 to 56 and a nucleic acid sequence encoding the tag of any one of claims 1 or 3 to 9 or 19 to 30 or 33, comprising a nucleic acid sequence encoding at least one constant domain of an immunoglobulin light chain or orthologous polypeptide or fragment thereof, and optionally a nucleic acid sequence encoding the first peptide linker of any one of claims 1 or 33.

65. The nucleic acid of claim 64, wherein the nucleic acid sequence encoding the tag comprises a nucleic acid sequence encoding two or more constant domains ofan immunoglobulin light chain or orthologous polypeptides or fragments thereof, which are optionally connected to each other by a nucleic acid sequence encoding a second peptide linker.

66. The nucleic acid of any one of claims 64 or 65, wherein the nucleic acid further comprises a nucleic acid sequence encoding a peptide signal sequence.

67. The nucleic acid of any one of claims 64 to 66, wherein the nucleic acid sequence encoding the constant domain of the immunoglobulin light chain or orthologous polypeptide or fragment thereof is a nucleic acid sequence encoding the constant domain of the immunoglobulin light chain or orthologous polypeptide or fragment of any one of claims 5 to 9 or 20 to 30.

68. The nucleic acid of any one of claims 64 to 67, wherein the nucleic acid sequences encoding the first peptide linker and / or the second peptide linker encode the first peptide linker and / or the second peptide linker of any one of claims 10 to 12 or 26 to 27.

69. The nucleic acid of any one of claims 64 to 68, wherein the nucleic acid sequence encoding the tag, and optional the nucleic acid sequence encoding the first peptide linker, is at the 3'-end of the nucleic acid sequence encoding the target polypeptide such that the tag is fused to the C-terminus of the target polypeptide in the fusion polypeptide.

70. The nucleic acid of any one of claims 64 to 68, wherein the nucleic acid sequence encoding the tag, and optional the nucleic acid sequence encoding the first peptide linker, is at the 5'-end of the nucleic acid sequence encoding the target polypeptide such that the tag is fused to the N-terminus of the target polypeptide in the fusion polypeptide.71 . The nucleic acid of any one of claims 64 to 68, wherein the nucleic acid sequence encoding the tag, and optional the nucleic acid sequence encoding the first peptide linker, is at the 5'-end and the 3'-end of the nucleic acid sequenceencoding the target polypeptide such that the tag is fused to the N-terminus and the C-terminus of the target polypeptide in the fusion polypeptide.

72. The nucleic acid of any one of claims 64 to 71 , wherein the nucleic acid sequence encoding the target polypeptide is encoding a polypeptide used in the pharmaceutical industry.

73. The nucleic acid of any one of claims 64 to 71 , wherein the nucleic acid sequence encoding the target polypeptide is encoding a polypeptide used for in-vitro diagnostics.

74. The nucleic acid of any one of claims 64 to 71 , wherein the nucleic acid sequence encoding the target polypeptide is encoding a polypeptide used for a medical indication.

75. The nucleic acid of any one of claims 64 to 71 , wherein the nucleic acid sequence encoding the target polypeptide is encoding a polypeptide for use as a medicament.

76. The nucleic acid of any one of claims 64 to 71 , wherein the nucleic acid sequence encoding the target polypeptide is encoding for a polypeptide used in the chemical industry.

77. The nucleic acid of any one of claims 64 to 71 , wherein the nucleic acid sequence encoding the target polypeptide is encoding for a polypeptide used in biotechnology methods.

78. The nucleic acid of any one of claims 64 to 71 for use as a medicament.

79. A pharmaceutical composition comprising the nucleic acid of any one of claims 64 to 71 and optionally a pharmaceutically acceptable carrier, diluent, or excipient.

80. A method for purifying an expressed fusion polypeptide, comprising providing a fusion polypeptide of any one of claims 18 or 33 to 62 expressed by cells, optionally transfected with a nucleic acid of any one of claims 64 to 71 , provision of a ligand directed to the tag of the fusion polypeptide, binding of the ligand to the tag, and eluting the fusion polypeptide from the ligand.81 . The method of claim 80, wherein the expressed fusion polypeptide comprises the tag which comprises more than one constant domain of the antibody light chain or orthologous polypeptide or fragment thereof.

82. The method of any one of claims 80 or 81 , wherein the ligand is directed to the constant domain of the immunoglobulin light chain of the tag.

83. The method of any one of claims 80 to 82, wherein the ligand is an antibody or an antibody fragment, optionally coupled to beads, further optionally coupled to magnetic beads.

84. The method of any one of claims 80 to 83, wherein the fusion polypeptide bound to the ligand is washed with a suitable buffer.

85. A method of modulating the plasma half-time of a fusion polypeptide in an organism, wherein the method comprises a) determining the molecular mass of the target polypeptide of any one of claims 1 to 19 or 33 to 56, b) (optional) determining the molecular mass of one constant domain of an immunoglobulin light chain or orthologous polypeptide or fragment thereof, c) calculating how many constant domains of an immunoglobulin light chain or orthologous polypeptides or fragments thereof are needed in addition to the molecular mass of the target polypeptide (step a) for reaching a desired molecular mass of the fusion polypeptide, and d) creating the tag to be fused to the target polypeptide by combining the necessary number of constant domains of an immunoglobulin light chain or orthologous polypeptides or fragments thereof according to the result of step (c),wherein the desired molecular mass of the fusion polypeptide is above a filtration limit of a kidney of a given organism or, optionally, at least 50 kDa or 70 kDa.

86. The method of claim 85, used for increasing the plasma half-time of the target polypeptide by generating the fusion polypeptide according to claim 85.

87. A method for detecting the fusion polypeptide of any one of claims 18 or 33 to 63, comprising providing a sample, which may comprise the fusion polypeptide, and detecting the fusion polypeptide in the sample by using a detecting agent directed to the tag or the target polypeptide.

88. The method of claim 87, wherein the detecting agent is labeled with biotin, a fluorescence probe, a magnetic resonance detectable probe or a radioactive nuclide and optionally wherein the detecting agent is an antibody or an antibody fragment.

89. The method of any one of claims 87 or 88, wherein the sample is taken during the production process of the fusion polypeptide of any one of claims 18 or 33 to 61 or the pharmaceutical composition of claim 63.

90. The method of any one of claims 87 or 89, wherein the sample is a biological sample of a mammal, to which the fusion polypeptide or the pharmaceutical composition has been administered.

91. The method of claim 90, wherein the mammal is a non-human mammal, preferably a mouse, a rat, a rabbit, a dog, a cat, a pig, or a monkey.

92. The method of claim 89, wherein the mammal is a human being.

93. The method of any one of claims 90 to 92, wherein the biological sample is a tissue, urine, feces, or blood.

94. A method of detecting the fusion polypeptide of any one of claims 18 or 33 to 62 in-vivo after administration to a mammal, wherein the fusion polypeptide islabeled by a fluorescence probe, a magnetic resonance detectable probe, an X- ray detectable probe or a radioactive nuclide.

95. The method of claim 94, wherein the mammal is a non-human mammal, preferably a mouse, a rat, a rabbit, a dog, a cat, a pig, or a monkey.

96. The method of claim 94, wherein the mammal is a human being.

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